GENETIC MARKERS FOR RISK MANAGEMENT OF ATRIAL FIBRILLATION, ATRIAL FLUTTER, AND STROKE

Abstract

The invention relates to procedure and methods of determining a susceptibility to cardiac arrhythmia, including Atrial Fibrillation, Atrial Flutter and Stroke, by assessing the presence or absence of alleles at polymorphic markers found to be associated with Atrial Fibrillation, Atrial Flutter and Stroke. The invention further relates to kits encompassing reagents for assessing such markers, and diagnostic methods, uses and procedures for utilizing such susceptibility markers.

Description

BACKGROUND OF THE INVENTION

[0001] Cardiac arrhythmia is a group of medical conditions, in which the electrical activity of the heart is irregular, or is slower or faster than normal. Some arrhythmias are life-threatening, and can cause cardiac arrest or sudden death. Others cause, or predispose to, other aggravating symptoms or disease, including stroke. Fibrillation is a serious form of arrhythmia, in which the heart muscle presents with irregular or quivering motion due to lack of unity in the function of contractile cells. Fibrillation can affect the atrium (Atrial Fibrillation (AF) or Atrial Flutter (AFI)), or the ventricle (Ventricular Fibrillation (VF)).

[0002] Atrial fibrillation (AF) is an abnormal heart rhythm (cardiac arrhythmia) which involves the two small, upper heart chambers (the atria). Heart beats in a normal heart begin after electricity generated in the atria by the sinoatrial node spreads through the heart and causes contraction of the heart muscle and pumping of blood. In AF, the regular electrical impulses of the sinoatrial node are replaced by disorganized, rapid electrical impulses which result in irregular heart beats.

[0003] Atrial fibrillation is the most common cardiac arrhythmia. The risk of developing atrial fibrillation increases with age--AF affects four percent of individuals in their 80s. An individual may spontaneously alternate between AF and a normal rhythm (paroxysmal atrial fibrillation) or may continue with AF as the dominant cardiac rhythm without reversion to the normal rhythm (chronic atrial fibrillation). Atrial fibrillation is often asymptomatic, but may result in symptoms of palpitations, fainting, chest pain, or even heart failure. These symptoms are especially common when atrial fibrillation results in a heart rate which is either too fast or too slow. In addition, the erratic motion of the atria leads to blood stagnation (stasis) which increases the risk of blood clots that may travel from the heart to the brain and other areas. Thus, AF is an important risk factor for stroke, the most feared complication of atrial fibrillation.

[0004] The symptoms of atrial fibrillation may be treated with medications which slow the heart rate. Several medications as well as electrical cardioversion may be used to convert AF to a normal heart rhythm. Surgical and catheter-based therapies may also be used to prevent atrial fibrillation in certain individuals. People with AF are often given blood thinners such as warfarin to protect them from strokes.

[0005] Any patient with 2 or more identified episodes of atrial fibrillation is said to have recurrent atrial fibrillation. This is further classified into paroxysmal and persistent based on when the episode terminates without therapy. Atrial fibrillation is said to be paroxysmal when it terminates spontaneously within 7 days, most commonly within 24 hours. Persistent or chronic atrial fibrillation is AF established for more than seven days. Differentiation of paroxysmal from chronic or established AF is based on the history of recurrent episodes and the duration of the current episode of AF (Levy S., J Cardiovasc Electrophysiol. 8 Suppl, S78-82 (1998)).

[0006] Lone atrial fibrillation (LAF) is defined as atrial fibrillation in the absence of clinical or echocardiographic findings of cardiopulmonary disease.

[0007] Atrial fibrillation is usually accompanied by symptoms related to either the rapid heart rate or embolization. Rapid and irregular heart rates may be perceived as palpitations, exercise intolerance, and occasionally produce angina and congestive symptoms of shortness of breath or edema. Sometimes the arrhythmia will be identified with the onset of a stroke or a transient ischemic attack (TIA). It is not uncommon to identify atrial fibrillation on a routine physical examination or electrocardiogram (ECG/EKG), as it may be asymptomatic in some cases. Paroxysmal atrial fibrillation is the episodic occurrence of the arrhythmia and may be difficult to diagnose. Episodes may occur with sleep or with exercise, and their episodic nature may require prolonged ECG monitoring (e.g. a Holter monitor) for diagnosis.

[0008] Atrial fibrillation is diagnosed on an electrocardiogram, an investigation performed routinely whenever irregular heart beat is suspected. Characteristic findings include absence of P waves, unorganized electrical activity in their place and irregularity of R-R interval due to irregular conduction of impulses to the ventricles. If paroxysmal AF is suspected, episodes may be documented with the use of Holter monitoring (continuous ECG recording for 24 hours or longer).

[0009] While many cases of AF have no definite cause, it may be the result of various other problems (see below). Hence, renal function and electrolytes are routinely determined, as well as thyroid-stimulating hormone and a blood count. A chest X-ray is generally performed. In acute-onset AF associated with chest pain, cardiac troponins or other markers of damage to the heart muscle may be ordered. Coagulation studies (INR/aPTT) are usually performed, as anticoagulant medication may be commenced. A transesophageal echocardiogram may be indicated to identify any intracardiac thrombus (Fuster V., et al., Circulation.; 104, 2118-2150 (2001)).

[0010] Atrial Flutter (AFI) is characterized by an abnormal fast heart rhythm in the atria. Patients who present with atrial flutter commonly also experience Atrial Fibrillation and vice versa (Waldo, A., Progr Cardiovasc Disease, 48:41-56 (2005)). Mechanistically and biologically, AF and AFI are thus likely to be highly related.

[0011] AF (and AFI) is linked to several cardiac causes, but may occur in otherwise normal hearts. Known associations include: High blood pressure, Mitral stenosis (e.g. due to rheumatic heart disease or mitral valve prolapse), Mitral regurgitation, Heart surgery, Coronary artery disease, Hypertrophic cardiomyopathy, Excessive alcohol consumption ("binge drinking" or "holiday heart"), Hyperthyroidism, Hyperstimulation of the vagus nerve, usually by having large meals ("binge eating"), Lung pathology (such as pneumonia, lung cancer, pulmonary embolism, Sarcoidosis), Pericarditis, Intense emotional turmoil, and Congenital heart disease.

[0012] The normal electrical conduction system of the heart allows the impulse that is generated by the sinoatrial node (SA node) of the heart to be propagated to and stimulate the myocardium (muscle of the heart). When the myocardium is stimulated, it contracts. It is the ordered stimulation of the myocardium that allows efficient contraction of the heart, thereby allowing blood to be pumped to the body. In atrial fibrillation, the regular impulses produced by the sinus node to provide rhythmic contraction of the heart are overwhelmed by the rapid randomly generated discharges produced by larger areas of atrial tissue. An organized electrical impulse in the atrium produces atrial contraction; the lack of such an impulse, as in atrial fibrillation, produces stagnant blood flow, especially in the atrial appendage and predisposes to clotting. The dislodgement of a clot from the atrium results in an embolus, and the damage produced is related to where the circulation takes it. An embolus to the brain produces the most feared complication of atrial fibrillation, stroke, while an embolus may also lodge in the mesenteric circulation (the circulation supplying the abdominal organs) or digit, producing organ-specific damage.

[0013] Treatment of atrial fibrillation is directed by two main objectives: (i) prevent temporary circulatory instability; (ii) prevent stroke. The most common methods for achieving the former includes rate and rhythm control, while anticoagulation is usually the desired method for the latter (Prystowsky E. N., Am J Cardiol.; 85, 3D-11D (2000); van Walraven C, et al., Jama. 288, 2441-2448 (2002)). Common methods for rate control, i.e. for reducing heart rate to normal, include beta blockers (e.g., metotprolol), cardiac glycosides (e.g., digoxin) and calcium channel blockers (e.g., verapamil). All these medications work by slowing down the generation of pulses from the atria, and the conduction from the atria to the ventricles. Other drugs commonly used include quinidine, flecainide, propafenone, disopyramide, sotalol and amiodarone. Rhythm control can be achieved by electrical cardioversion, i.e. by applying DC electrical shock, or by chemical cardioversion, using drugs such as amiodarione, propafenone and flecainide.

[0014] Preventive measures for stroke include anticoagulants. Representative examples of anticoagulant agents are Dalteparin (e.g., Fragmin), Danaparoid (e.g., Orgaran), Enoxaparin (e.g., Lovenox), Heparin (various), Tinzaparin (e.g., Innohep), Warfarin (e.g., Coumadin). Some patients with lone atrial fibrillation are sometimes treated with aspirin or clopidogrel. There is evidence that aspirin and clopidogrel are effective when used together, but the combination is still inferior to warfarin (Connolly S., et al. Lancet.; 367, 1903-1912 (2006)).(2) The new anticoagulant ximelagatran has been shown to prevent stroke with equal efficacy as warfarin, without the difficult monitoring process associated with warfarin and with possibly fewer adverse haemorrhagic events. Unfortunately, ximegalatran and other similar anticoagulant drugs (commonly referred to as direct thrombin inhibitors), have yet to be widely licensed.

[0015] Determining who should and should not receive anti-coagulation with warfarin is not straightforward. The CHADS2 score is the best validated method of determining risk of stroke (and therefore who should be anticoagulated). The UK NICE guidelines have instead opted for an algorithm approach. The underlying problem is that if a patient has a yearly risk of stroke that is less than 2%, then the risks associated with taking warfarin outweigh the risk of getting a stroke (Gage B. F. et al. Stroke 29, 1083-1091 (1998))

[0016] Atrial fibrillation can sometimes be controlled with treatment. The natural tendency of atrial fibrillation, however, is to become a chronic condition. Chronic AF leads to an increased risk of death. Patients with atrial fibrillation are at significantly increased chance of stroke.

[0017] Atrial fibrillation is common among older adults. In developed countries, the number of patients with atrial fibrillation is likely to increase during the next 50 years, due to the growing proportion of elderly individuals (Go A. S. et al., Jama., 285, 2370-2375 (2001))(3). In the Framingham study the lifetime risk for development of AF is 1 in 4 for men and women 40 years of age and older. Lifetime risks for AF are high (1 in 6). According to data from the National Hospital Discharge Survey (1996-2001) on cases that included AF as a primary discharge diagnosis found that 45% of the patients are male, and that the mean age for men was 66.8 years and 74.6 for women. The racial breakdown for admissions was found to be 71.2% white, 5.6% black, 2% other races, and 20% not specified. Furthermore, African American patients were, on average, much younger than other races. The incidence in men ranged from 20.58/100,000 persons per year for patients ages 15-44 years to 1203/100,000 persons per years for those ages 85 and older. From 1996-2001, hospitalizations with AF as the first listed diagnosis, increased by 34%.

[0018] Stroke is a common and serious disease. Each year in the United States more than 600,000 individuals suffer a stroke and more than 160,000 die from stroke-related causes (Sacco, R. L. et al., Stroke 28, 1507-17 (1997)). Furthermore, over 300,000 individuals present with Transient Ischemic Attack, a mild form of stroke, every year in the US. In western countries stroke is the leading cause of severe disability and the third leading cause of death (Bonita, R., Lancet 339, 342-4 (1992)). The lifetime risk of those who reach the age of 40 exceeds 10%.

[0019] The clinical phenotype of stroke is complex but is broadly divided into ischemic (accounting for 80-90%) and hemorrhagic stroke (10-20%) (Caplan, L. R. Caplan's Stroke: A Clinical Approach, 1-556 (Butterworth-Heinemann, 2000)). Ischemic stroke is further subdivided into large vessel occlusive disease (referred to here as carotid stroke), usually due to atherosclerotic involvement of the common and internal carotid arteries, small vessel occlusive disease, thought to be a non-atherosclerotic narrowing of small end-arteries within the brain, and cardiogenic stroke due to blood clots arising from the heart usually on the background of atrial fibrillation or ischemic (atherosclerotic) heart disease (Adams, H. P., Jr. et al., Stroke 24, 35-41 (1993)). Therefore, it appears that stroke is not one disease but a heterogeneous group of disorders reflecting differences in the pathogenic mechanisms (Alberts, M. J. Genetics of Cerebrovascular Disease, 386 (Futura Publishing Company, Inc., New York, 1999); Hassan, A. & Markus, H. S. Brain 123, 1784-812 (2000)). However, all forms of stroke share risk factors such as hypertension, diabetes, hyperlipidemia, and smoking (Sacco, R. L. et al., Stroke 28, 1507-17 (1997); Leys, D. et al., J. Neurol. 249, 507-17 (2002)). Family history of stroke is also an independent risk factor suggesting the existence of genetic factors that may interact with environmental factors (Hassan, A. & Markus, H. S. Brain 123, 1784-812 (2000); Brass, L. M. & Alberts, M. J. Baillieres Clin. Neurol. 4, 221-45 (1995)).

[0020] The genetic determinants of the common forms of stroke are still largely unknown. There are examples of mutations in specific genes that cause rare Mendelian forms of stroke such as the Notch3 gene in CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarctions and leukoencephalopathy) (Tournier-Lasserve, E. et al., Nat. Genet. 3, 256-9 (1993); Joutel, A. et al., Nature 383, 707-10 (1996)), Cystatin C in the Icelandic type of hereditary cerebral hemorrhage with amyloidosis (Palsdottir, A. et al., Lancet 2, 603-4 (1988)), APP in the Dutch type of hereditary cerebral hemorrhage (Levy, E. et al., Science 248, 1124-6 (1990)) and the KRIT1 gene in patients with hereditary cavernous angioma (Gunel, M. et al., Proc. Natl. Acad. Sci. USA 92, 6620-4 (1995); Sahoo, T. et al., Hum. Mol. Genet. 8, 2325-33 (1999)). None of these rare forms of stroke occur on the background of atherosclerosis, and therefore, the corresponding genes are not likely to play roles in the common forms of stroke which most often occur with atherosclerosis.

[0021] It is very important for the health care system to develop strategies to prevent stroke. Once a stroke happens, irreversible cell death occurs in a significant portion of the brain supplied by the blood vessel affected by the stroke. Unfortunately, the neurons that die cannot be revived or replaced from a stem cell population. Therefore, there is a need to prevent strokes from happening in the first place. Although we already know of certain clinical risk factors that increase stroke risk (listed above), there is an unmet medical need to define the genetic factors involved in stroke to more precisely define stroke risk. Further, if predisposing alleles are common in the general population and the specificity of predicting a disease based on their presence is low, additional loci such as protective loci are needed for meaningful prediction of disposition of the disease state. There is also a great need for therapeutic agents for preventing the first stroke or further strokes in individuals who have suffered a previous stroke or transient ischemic attack.

[0022] AF is an independent risk factor for stroke, increasing risk about 5-fold. The risk for stroke attributable to AF increases with age. AF is responsible for about 15-20% of all strokes. AF is also an independent risk factor for stroke recurrence and stroke severity. A recent report showed people who had AF and were not treated with anticoagulants had a 2.1-fold increase in risk for recurrent stroke and a 2.4 fold increase in risk for recurrent severe stroke. People who have stroke caused by AF have been reported as 2.23 times more likely to be bedridden compared to those who have strokes from other causes.

[0023] There is a need for an understanding of the susceptibility factors leading to increased predisposition for AF and stroke. Identification of at-risk variants for AF can, for example, be useful for assessing which individuals are at particularly high risk for AF and subsequent stroke. Furthermore, preventive treatment can be administered to individuals suffering from AF and who are carriers of at-risk susceptibility variants for AF and/or stroke. Finally, identification of at-risk variants for AF and/or stroke can lead to the identification of new targets for drug therapy, as well as the development of novel therapeutic measures.

SUMMARY OF THE INVENTION

[0024] The present invention relates to the discovery that certain genetic markers have been shown to be associated with cardiac arrhythmia, in particular atrial fibrillation and atrial flutter, and stroke. This discovery can be utilized in a variety of methods, procedures, apparatus, media and kits, as described herein, relating to methods and procedures of diagnosis and/or determination of a susceptibility, methods of genotyping associated variants, methods of predicting response to therapeutic agents, methods of predicting prognosis, methods of monitoring progress of treatment, and systems and kits for use in such methods.

[0025] One aspect of the invention relates to a method of determining a susceptibility to cardiac arrhythmia or stroke in a human individual, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample from the individual, wherein the at least one polymorphic marker is selected from the polymorphic markers set forth in Table 5, and markers in linkage disequilibrium therewith, wherein determination of the presence or absence of the at least one allele is indicative of a susceptibility to cardiac arrhythmia or stroke in the individual. In one embodiment, the at least one polymorphic marker is located within the LD block C04, set forth in SEQ ID NO:50 herein. In another embodiment, the at least one polymorphic marker is selected from the markers set forth in Table 9, and markers in linkage disequilibrium therewith. In one embodiment, the at least one marker is selected from marker rs2220427 (SEQ ID NO:1) and marker rs10033464 (SEQ ID NO:41), and markers in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic marker is selected from the markers set forth in Table 19. In one embodiment, the method further comprises a step of assessing at least one haplotype comprising at least two polymorphic markers in the individual.

[0026] In another aspect, the invention relates to a method of determining a susceptibility to cardiac arrhythmia or stroke in a human individual, comprising determining whether at least one at-risk allele in at least one polymorphic marker is present in a genotype dataset derived from the individual, wherein the at least one polymorphic marker is selected from the markers set forth in Table 5, and markers in linkage disequilibrium therewith, and wherein determination of the presence of the at least one at-risk allele is indicative of increased susceptibility to cardiac arrhythmia or stroke in the individual.

[0027] The genotype dataset comprises in one embodiment information about marker identity, and the allelic status of the individual for the at least one polymorphic marker, i.e. information about the identity of the two alleles carried by the individual for the marker and/or information about whether an individual is a carrier of a particular at-risk allele for the at least one polymorphic marker. The genotype dataset may comprise allelic information about one or more marker, including two or more markers, three or more markers, five or more markers, one hundred or more markers, etc. In some embodiments, the genotype dataset comprises genotype information from a whole-genome assessment of the individual including hundreds of thousands of markers, or even one million or more markers.

[0028] The invention, in another aspect, relates to a procedure comprising a step of analyzing a nucleic acid from a human individual to determine the presence or absence of at least one allele of at least one polymorphic marker or haplotype associated with the genomic sequence with sequence as set forth in SEQ ID NO:50; and a step of determining the status of a genetic indicator of cardiac arrhythmia or stroke in the individual from the presence or absence of the at least one marker or haplotype. Thus the genotype and/or haplotype status of the individual is used as in indicator of cardiac arrhythmia, including atrial fibrillation and atrial flutter, as well as stroke, in the individual.

[0029] The invention also relates to a method of assessing a susceptibility to cardiac arrhythmia or stroke in a human individual, comprising screening a nucleic acid from the individual for at least one polymorphic marker or haplotype in SEQ ID NO:50 that correlates with increased occurrence of cardiac arrhythmia or stroke in a human population; wherein determination of the presence of an at-risk marker allele in the at least one polymorphism or an at-risk haplotype in the nucleic acid identifies the individual as having elevated susceptibility to cardiac arrhythmia and/or stroke, and wherein the absence of the at least one at-risk marker allele or at-risk haplotype in the nucleic acid identifies the individual as not having the elevated susceptibility.

[0030] The procedure or methods of the invention in one embodiment entail at least one polymorphic marker or haplotype comprising a contiguous nucleic acid fragment of LD block C04 as set forth in SEQ ID NO:50, or the complement thereof, wherein the fragment is less than 500 nucleotides in size and specifically hybridizes to a complimentary segment of LD block C04. In one embodiment, the fragment is more than 15 nucleotides and less than 400 nucleotides in size, and wherein the fragment specifically hybridizes to a complimentary segment of LD block C04 as set forth in SEQ ID NO:50.

[0031] In alternative embodiments, the susceptibility conferred by the polymorphic markers or haplotypes is decreased susceptibility, i.e. the markers and haplotypes of the invention confer decreased risk of an individual develops cardiac arrhythmia, including atrial fibrillation and atrial flutter, and/or stroke. In one such embodiment, the decreased susceptibility is characterized by an odds ratio (OR) or relative risk (RR) of less than 0.8. In another embodiment, the decreased susceptibility is characterized by an odds ratio (OR) of less than 0.7. In another embodiment, the decreased susceptibility is characterized by an OR or RR of less than 0.6. In another embodiment, the decreased susceptibility is characterized by OR or RR of less than 0.5. Other embodiments relate to other values for OR or RR including values of 0.9, 0.85, 0.75, 0.65, 0.55, etc.

[0032] Another aspect of the invention relates to a method of identification of a marker for use in assessing susceptibility to symptoms associated with cardiac arrhythmia and/or stroke in a human individual, the method comprising at least one polymorphic marker within SEQ ID NO:50, or at least one polymorphic marker in linkage disequilibrium with at least one marker within SEQ ID NO:50, determining the genotype status of a sample of individuals diagnosed with cardiac arrhythmia and/or stroke and the genotype status of a sample of control individuals, wherein a significant difference in frequency of at least one allele in at least one polymorphism in individuals diagnosed with cardiac arrhythmia and/or stroke as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing susceptibility to cardiac arrhythmia and/or stroke. In one embodiment, an increase in frequency of the at least one allele in the at least one polymorphism in individuals diagnosed with cardiac arrhythmia and/or stroke, as compared with the frequency of the at least one allele in the control sample, is indicative of the at least one polymorphism being useful for assessing increased susceptibility to cardiac arrhythmia. In another embodiment, a decrease in frequency of the at least one allele in the at least one polymorphism in individuals diagnosed with cardiac arrhythmia and/or stroke, as compared with the frequency of the at least one allele in the control sample, is indicative of the at least one polymorphism being useful for assessing decreased susceptibility to, or protection against, cardiac arrhythmia and/or stroke. In preferred embodiments, the significant difference in frequency is characterized by a statistical measure. In one embodiment, the statistical measure is a P-value. In particular embodiments, a significant P-value is less than 0.05, less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001, less than 0.0000001 or less than 0.00000001. In other embodiments, the significant difference is characterized by an odds ratio (OR) or relative risk (RR) with particular confidence interval (CE) values.

[0033] In another aspect, the invention relates to a method of genotyping a nucleic acid sample obtained from a human individual, comprising determining the presence or absence of at least one allele of at least one polymorphic marker predictive of increased risk of cardiac arrhythmia and/or stroke in the sample, wherein the at least one marker is selected from the markers set forth in Table 5, and markers in linkage disequilibrium therewith, and wherein determination of the presence or absence of the at least one allele of the at least one polymorphic marker is predictive of increased risk of cardiac arrhythmia and/or stroke in the individual. In one embodiment, genotyping is performed using a process selected from allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, nucleic acid sequencing, 5'-exonuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, and single-stranded conformation analysis. In a preferred embodiment, the process comprises allele-specific probe hybridization. The process of genotyping preferably comprises amplifying a segment of a nucleic acid that comprises the at least one polymorphic marker, by Polymerase Chain Reaction (PCR), using a nucleotide primer pair flanking the at least one polymorphic marker. In a preferred method of genotyping, the following steps are performed:

[0034] 1. contacting copies of the nucleic acid with a detection oligonucleotide probe and an enhancer oligonucleotide probe under conditions for specific hybridization of the oligonucleotide probe with the nucleic acid; wherein

[0035] a) the detection oligonucleotide probe is from 5-100 nucleotides in length and specifically hybridizes to a first segment of the nucleic acid whose nucleotide sequence is given by SEQ ID NO:50 that comprises at least one polymorphic site;

[0036] b) the detection oligonucleotide probe comprises a detectable label at its 3' terminus and a quenching moiety at its 5' terminus;

[0037] c) the enhancer oligonucleotide is from 5-100 nucleotides in length and is complementary to a second segment of the nucleotide sequence that is 5' relative to the oligonucleotide probe, such that the enhancer oligonucleotide is located 3' relative to the detection oligonucleotide probe when both oligonucleotides are hybridized to the nucleic acid; and

[0038] d) a single base gap exists between the first segment and the second segment, such that when the oligonucleotide probe and the enhancer oligonucleotide probe are both hybridized to the nucleic acid, a single base gap exists between the oligonucleotides;

[0039] 2. treating the nucleic acid with an endonuclease that will cleave the detectable label from the 3' terminus of the detection probe to release free detectable label when the detection probe is hybridized to the nucleic acid; and measuring free detectable label, wherein the presence of the free detectable label indicates that the detection probe specifically hybridizes to the first segment of the nucleic acid, and indicates the sequence of the polymorphic site as the complement of the detection probe.

[0040] A further aspect of the invention relates to a method of determining a susceptibility to cardiac arrhythmia or stroke in a human individual, the method comprising determining the identity of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one marker is selected from the group of markers associated with the PITX2 gene, wherein the presence of the at least one allele is indicative of a susceptibility to cardiac arrhythmia or stroke in the individual.

[0041] Some embodiments of the invention relate to a further step of assessing at least one additional biomarker for atrial fibrillation, atrial flutter or stroke, wherein combining the genetic information from the markers provides risk assessment for atrial fibrillation, atrial flutter or stroke. In some of these embodiments, the biomarker is a genetic marker or haplotype, i.e. genetic risk factors shown to be, or contemplated to be, related to increased or decreased risk of atrial fibrillation, atrial flutter or stroke. In other embodiments the biomarker is a protein biomarker. The protein biomarker is in some embodiments selected from fibrin D-dimer, prothrombin activation fragment 1.2 (F1.2), thrombin-antithrombin III complexes (TAT), fibrinopeptide A (FPA), lipoprotein-associated phospholipase A2 (Ip-PLA2), beta-thromboglobulin, platelet factor 4, P-selectin, von Willebrand Factor, pro-natriuretic peptide (BNP), matrix metalloproteinase-9 (MMP-9), PARK7, nucleoside diphosphate kinase (NDKA), tau, neuron-specific enolase, B-type neurotrophic growth factor, astroglial protein S-100b, glial fibrillary acidic protein, C-reactive protein, seum amyloid A, marix metalloproteinase-9, vascular and intracellular cell adhesion molecules, tumor necrosis factor alpha, and interleukins, including interleukin-1, -6, and -8). In one embodiment, the at least one biomarker includes progenitor cells. In particular embodiments, more than one biomarker is determined. In a preferred embodiment, the biomarker is measured in plasma from the individual. Other embodiments further relate to combining non-genetic information to make risk assessment, diagnosis, or prognosis of atrial fibrillation, atrial flutter or stroke in the individual. The non-genetic information can comprise age, age at onset of disease, gender, ethnicity, previous disease diagnosis, e.g., diagnosis of cardiag arrhythmia (e.g., atrial fibrillation) and stroke, medical history of the individual, family history of disease, biochemical measurements, and clinical measurements (e.g., blood pressure, serum lipid levels). Analysis of such combined information from various genetic markers, or genetic markers plus non-genetic markers is possible by methods known to those skilled in the art. In one embodiment, analysis is performed calculating overall risk by logistic regression.

[0042] The invention further relates to a method of diagnosing increased susceptibility of stroke in a human individual, comprising the steps of (a) determining whether the individual has experienced symptoms associated with Atrial Fibrillation, Atrial Flutter or a Transient Ischemic Attack; (b) determining whether a nucleic acid sample from the individual comprises at least one copy of an at-risk allele of at least one polymorphic marker selected from the markers set forth in Table 5, and markers in linkage disequilibrium therewith; wherein the presence of symptoms associated with Atrial Fibrillation, Atrial Flutter and/or Transient Ischemic Attack and the presence of the at least one copy of the at-risk allele is indicative of increased susceptibility of stroke.

[0043] The invention in a further aspect relates to a method of assessing an individual for probability of response to a therapeutic agent for preventing and/or ameliorating symptoms associated with cardiac arrhythmia and/or stroke, comprising: determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from the markers set forth in Table 9, and markers in linkage disequilibrium therewith, wherein determination of the presence of the at least one allele of the at least one marker is indicative of a probability of a positive response to the therapeutic agent for cardiac arrhythmia and/or stroke.

[0044] In one embodiment, the therapeutic agent is an anticoagulant, an anti-arrhythmic agent, a hear rate control agent, a cardioversion agent, or a heart rhythm control agent. In another embodiment, the therapeutic agent is selected from warfarin, heparin, low molecular weight heparins, factor Xa inhibitors, and thrombin inhibitors, sodium channel blockers, beta blockers, potassium channel blockers, and calcium channel blockers.

[0045] In another embodiment, the therapeutic agent is selected from warfaring, ximelagatran, heparin, enoxaparin, dalteparin, tinzaparin, ardeparin, nadroparin, reviparin, fondaparinux, idraparinux, lepirudin, bivalirudin, argatroban, danaparoid, disopyramide, moricizine, procainamide, quinidine, lidocaine, mexiletine, tocainide, phenytoin, encainide, flecainide, propafenone, ajmaline, cibenzoline, detajmium, esmolol, propranolol, metoprolol, alprenolol, atenolol, carvedilol, bisoprolol, acebutolol, nadolol, pindololol, labetalol, oxprenotol, penbutolol, timolol, betaxolol, cartelol, sotalol, levobunolol, amiodarone, azimilide, bretylium, dofetilide, tedisamil, ibutilide, sematilide, N-acetyl procainamide, nifekalant hydrochloride, vernakalant, ambasilide, verpamil, mibefradil, diltiazem, digoxin, adenosine, ibutilide, amiodarone, procainamide, profafenone and flecainide.

[0046] Yet another aspect of the invention relates to a method of predicting prognosis of an individual diagnosed with, cardiac arrhythmia and/or stroke, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from the markers set forth in Table 9, and markers in linkage disequilibrium therewith, wherein determination of the presence of the at least one allele is indicative of a worse prognosis of the cardiac arrhythmia and/or stroke in the individual.

[0047] Methods of monitoring progress of a treatment of an individual undergoing treatment for cardiac arrhythmia and/or stroke are also within scope of the invention, the methods comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from the markers set forth in Table 9, and markers in linkage disequilibrium therewith, wherein determination of the presence of the at least one allele is indicative of the treatment outcome of the individual.

[0048] In particular embodiments of the invention, e.g. in the various methods, uses, procedures, apparatus and kits of the invention, the cardiac arrhythmia phenotype is further characterized as being atrial fibrillation or atrial flutter. The inventors have determined that the risk conferred by the AF at-risk variants described herein is greater for individual with early age at onset than for individuals with late age at onset. Thus in one embodiment, the atrial fibrillation or atrial flutter is further characterized by an age of onset in the individual of less than 80 years. In another embodiment, the atrial fibrillation or atrial flutter is further characterized by an age of onset in the individual of less than 70 years. In yet another embodiment, the atrial fibrillation or atrial flutter is further characterized by an age of onset in the individual of less than 60 years. Other age cutoffs are possible in alternative embodiments of the invention, and are also contemplated, including, but not limited to, age cutoff of less than 75 years, less than 65 years, and less than 55 years. Furthermore, age at onset or diagnosis above age 55, 60, 65, 70, 75 or 80 are also contemplated and within scope of the invention, as are age ranges within which diagnosis or symptoms or onset of the disease occurs, including, but not limited to, age 50-80, age 55-75, age 60-80, age 65-75, etc.

[0049] In certain embodiments of the invention, the stroke is further characterized as ischemic stroke. In other embodiments, the stroke phenotype may be characterized as one or more of the ischemic stroke sub-phenotypes large artery atherosclerosis (LAA), cardioembolic stroke (CES) and small vessel disease (SVD).

[0050] In particular embodiments of the invention, linkage disequilibrium (LD) is defined by a specific quantitative cutoff. As described in detail herein, linkage disequilibrium can be quantitatively determined by measures such as r² and |D'|. As a consequence, certain embodiments of the invention relate to markers in linkage disequilibrium by a measure within a certain range specified by particular values of r² and/or |D'|. In one such embodiment, LD is characterized by numerical values for r² of greater than 0.1. In another embodiment, LD is characterized by numerical values for r² of greater than 0.1. In another embodiment, LD is characterized by numerical values for r² of greater than 0.5. In yet another embodiment, LD is characterized by numerical values for r² of greater than 0.8. Other cutoff values for r² are also contemplated, as described in more detail herein. In certain embodiments, LD is characterized by certain cutoff values for r² and/or |D'|. In one such embodiment, LD is characterized by values for r² and/or |D'| of greater than 0.2 and 0.8, respectively. Other combination and permutations of these or other measures of LD are possible to practice the invention, and are also contemplated and within scope of the invention.

[0051] The procedures, uses, or methods of the invention in some embodiments further comprise a step of administering to an individual determined to be at increased risk for developing cardiac arrhythmia or stroke a composition comprising at least one therapeutic agent effective to treat or prevent cardiac arrhythmia or stroke, or prevent symptoms associated with cardiac arrhythmia or stroke. Thus, the invention can be used to determine whether an individual is suitable for a particular treatment module.

[0052] Kits for use in the various methods and procedures described herein are also within scope of the invention. Thus, in one aspect, the invention relates to a kit for assessing susceptibility to cardiac arrhythmia and/or stroke in a human individual, the kit comprising reagents for selectively detecting at least one allele of at least one polymorphic marker in the genome of the individual, wherein the polymorphic marker is selected from the group consisting of the polymorphic markers within the segment whose sequence is set forth in SEQ ID NO:50, and markers in linkage disequilibrium therewith, and wherein the presence of the at least one allele is indicative of a susceptibility to cardiac arrhythmia and/or stroke.

[0053] In one embodiment, the at least one polymorphic marker is selected from the markers set forth in Table 5. In another embodiment, the at least one polymorphic marker is selected from the group of markers set forth in Table 9, and markers in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic marker is selected from marker rs2220427 (SEQ ID NO:1) and rs10033464 (SEQ ID NO:41), and markers in linkage disequilibrium therewith. In one preferred embodiment, the at least one polymorphic marker is selected from the markers set forth in Table 19. In another preferred embodiment, the at least one polymorphic marker is selected from D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51). In one embodiment, the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising the at least one polymorphic marker, a buffer and a detectable label.

[0054] In another embodiment, the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic nucleic acid segment obtained from the subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes one polymorphic marker, and wherein the fragment is at least 30 base pairs in size. The at least one oligonucleotide is in preferred embodiments completely complementary to the genome of the individual. In one embodiment, the oligonucleotide is about 18 to about 50 nucleotides in length. In another embodiment, the oligonucleotide is 20-30 nucleotides in length. In one preferred embodiment, the kit comprises:

[0055] a. a detection oligonucleotide probe that is from 5-100 nucleotides in length;

[0056] b. an enhancer oligonucleotide probe that is from 5-100 nucleotides in length; and

[0057] c. an endonuclease enzyme; wherein the detection oligonucleotide probe specifically hybridizes to a first segment of the nucleic acid whose nucleotide sequence is given by SEQ ID NO: 2 that comprises at least one polymorphic site; wherein the detection oligonucleotide probe comprises a detectable label at its 3' terminus and a quenching moiety at its 5' terminus; wherein the enhancer oligonucleotide is from 5-100 nucleotides in length and is complementary to a second segment of the nucleotide sequence that is 5' relative to the oligonucleotide probe, such that the enhancer oligonucleotide is located 3' relative to the detection oligonucleotide probe when both oligonucleotides are hybridized to the nucleic acid; wherein a single base gap exists between the first segment and the second segment, such that when the oligonucleotide probe and the enhancer oligonucleotide probe are both hybridized to the nucleic acid, a single base gap exists between the oligonucleotides; and wherein treating the nucleic acid with the endonuclease will cleave the detectable label from the 3' terminus of the detection probe to release free detectable label when the detection probe is hybridized to the nucleic acid.

[0058] The polymorphic markers described herein as predictive of risk of cardiac arrhythmia (e.g., AF and Atrial flutter) and stroke are useful as diagnostic markers. In aspect, the invention therefore relates to the use of an oligonucleotide probe in the manufacture of a diagnostic reagent for diagnosing and/or assessing susceptibility to cardiac arrhythmia and/or stroke in a human individual, wherein the probe hybridizes to a segment of a nucleic acid whose nucleotide sequence is given by SEQ ID NO:50 that comprises at least one polymorphic site, wherein the fragment is 15-500 nucleotides in length.

[0059] In one such embodiment, the polymorphic site is selected from the polymorphic markers set forth in Table 5, and polymorphisms in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic marker is selected from D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51),

[0060] Computer-readable medium for storing information about disease-associated markers as described herein are also within scope of the present invention. In one such aspect, the invention relates to a computer-readable medium on which is stored an identifier for at least one polymorphic marker; an indicator of the frequency of at least one allele of said at least one polymorphic marker in a plurality of individuals diagnosed with atrial fibrillation, atrial flutter and/or stroke; and an indicator of the frequency of the least one allele of said at least one polymorphic markers in a plurality of reference individuals; wherein the at least one polymorphic marker is selected from the polymorphic markers set forth in Table 5, and polymorphisms in linkage disequilibrium therewith. In a preferred embodiment, the at least one polymorphic marker is selected from D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51).

[0061] The invention also related to an apparatus for determining a genetic indicator for cardiac arrhythmia and/or stroke in a human individual, comprising: a computer readable memory; and a routine stored on the computer readable memory; wherein the routine is adapted to be executed on a processor to analyze genotype and/or haplotype data for at least one human individual with respect to at least one polymorphic marker selected from the markers set forth in Table 5, and markers in linkage disequilibrium therewith, and generate an output based on the marker or haplotype data, wherein the output comprises a risk measure of the at least one marker or haplotype as a genetic indicator of cardiac arrhythmia and/or stroke for the human individual. In a preferred embodiment, the routine further comprises determining an indicator of the frequency of at least one allele of at least one polymorphic marker and/or at least one haplotype in a plurality of individuals diagnosed with cardiac arrhythmia and/or stroke, and an indicator of the frequency of at the least one allele of at least one polymorphic marker or at least one haplotype in a plurality of reference individuals, and calculating a risk measure for the at least one allele and/or haplotype based thereupon; and wherein a risk measure for the individual is calculated based on a comparison of the at least one marker and/or haplotype status for the individual to the calculated risk for the at least one marker and/or haplotype information for the plurality of individuals diagnosed with atrial fibrillation, atrial flutter and/or stroke. In certain embodiments, the risk measure is characterized by an Odds Ratio (OR) or a Relative Risk (RR), as described in more detail herein.

[0062] The polymorphic markers discovered in the present invention as predictive of a susceptibility of cardiac arrhythmia and stroke, as described, as well as markers in linkage disequilibrium therewith, are all useful for practicing the various aspects of the present invention. Thus, although particular polymorphic markers were used by the present inventors do detect an association of a particular region on chromosome 4 to cardiac arrhythmia (e.g, atrial fibrillation and atrial flutter) and stroke, it is equally useful to assess markers in strong linkage disequilibrium with those markers. As a consequence, in one embodiment of the methods, uses, kits, procedures, apparatus and media of the invention, the at least one polymorphic marker or haplotype useful in the methods or procedure of the invention comprises at least one of the markers set forth in Table 5 (e.g., Table 5A and Table 5B) and markers in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic marker or haplotype comprises at least one of the markers set forth in Table 9, and markers in linkage disequilibrium therewith. In one embodiment, the at least one polymorphic marker or haplotype comprises at least one of the markers set forth in Table 5. In another embodiment, the at least one polymorphic marker or haplotype comprises at least one of the markers set forth in Table 9. In another embodiment, the at least one polymorphic marker is selected from the markers set forth in Table 4. In one embodiment, the at least one marker is selected from marker rs2220427 (SEQ ID NO:1) and marker rs10033464 (SEQ ID NO:41), and markers in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic marker is selected from the markers set forth in Table 19.

[0063] In one embodiment, the at least one marker or haplotype comprises at least one of markers D4S406 (SEQ ID NO:45), rs2723296 (SEQ ID NO:35), rs16997168 (SEQ ID NO:36), rs2723316 (SEQ ID NO:37), rs6419178 (SEQ ID NO:38), rs1448817 (SEQ ID NO:39), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs13105878 (SEQ ID NO: 40), rs10033464 (SEQ ID NO:41), rs13141190 (SEQ ID NO:42), rs3853444 (SEQ ID NO:43), and rs4576077 (SEQ ID NO:44). In another embodiment, the at least one marker or haplotype comprises at least one of the markers D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51), In yet another embodiment, the at least one marker is selected from rs10033464, rs2200733, rs13143308 and rs2220427, and markers in linkage disequilibrium therewith.

[0064] In a further embodiment, the presence of alleles-2, -4 and/or -8 of marker D4S406, allele G of marker rs2723296, allele T of marker rs16997168, allele T of marker rs2723316, allele A of marker rs6419178, allele G of marker rs1448817, allele A of marker rs2634073, allele T of marker rs2200733, allele T of marker rs2220427, allele C of marker rs13105878, allele T of marker rs10033464, allele A of marker rs13141190, allele A of marker rs3853444, and/or allele T of marker rs4576077 is indicative of increased susceptibility of cardiac arrhythmia or stroke in the individual.

[0065] In particular embodiments of the invention, the susceptibility conferred by the at-risk variant (i.e. a particular allele at a polymorphic marker (e.g, a SNP) or a particular haplotype) is increased susceptibility, i.e. the markers and haplotypes of the invention confer increased risk of an individual develops cardiac arrhtythmia, including atrial fibrillation and atrial flutter, and stroke. Susceptibility is typically characterized by the measure Odds Ratio (OR) or, alternatively, by a Relative Risk (RR). In one embodiment, the increased susceptibility is characterized by an odds ratio (OR) of at least 1.3. In another embodiment, the increased susceptibility is characterized by an odds ratio (OR) of at least 1.4. In another embodiment, the increased susceptibility characterized by an odds ratio (OR) of at least 1.5. In another embodiment, the increased susceptibility characterized by an odds ratio (OR) or relative risk (RR) of at least 1.6. In yet another embodiment, the increased susceptibility characterized by an odds ratio (OR) or relative risk (RR) of at least 1.8. Other embodiments relate to other values for OR, or comparable values for RR including values of 1.25, 1.35, 1.45, 1.55, etc.

[0066] Certain embodiments of the invention relate to Individuals of a particular ethnicity or ancestry. In one such embodiment, the human individual has ancestry selected from black African ethnicity, Asian ethnicity, Caucasian ethnicity, Hispanic ethnicity, and Arabic ethnicity. In particular embodiments, the ethnicity is self-reported. In other embodiments, ancestry is determined by the assessment of particular ethnicity-specific genetic markers.

BRIEF DESCRIPTION OF THE FIGURES

[0067] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention.

[0068] FIG. 1 Shows a plot of linkage disequilibrium (LD) in the region comprising variants of the present invention for the CEPH population (HapMap data). The LD block C04 (111,954,811-112,104,250 on Chromosome 4, NCBI Build 35 positions) is indicated on the Figure by a black box. The plot shows two measures of LD, i.e. D' in the upper and left part of FIG. 1 and r² in the lower and right part of the figure.

[0069] FIG. 2 Shows a schematic of the haplotype structure at the associated region within the LD block. The areas of the dark (left) circles are proportional to the haplotype frequencies of the haplotypes in Iceland and the areas of the light (right) circles are proportional to the haplotype frequencies in Hong Kong. The intermediary haplotype, shown in the middle of the graph, no longer exists with certainty in either of the two populations (its estimated frequency is less than 0.2% which is indistinguishable from genotyping errors).

[0070] FIG. 3 Is an overview of a 200 kb genomic neighborhood of rs2200733 and rs10033464. It includes predicted ESTs, the locations of the three main classes of equivalent SNPs in the CEU HapMap samples and an overview of the LD structure of the region in the various ethnic HapMap samples.

[0071] FIG. 4. Shows Northern Blot analysis of PITX2 expression in human heart and aorta.

[0072] The PITX2 cDNA clone HU3_p983E0327D was used as a probe and detected 1.8, 2 and 3 kb transcripts and 2.2 and 3 kb PITX2 transcripts in left atrium and aorta respectively. Lane 1: Fetal heart, lane 2: Whole heart, lane 3: Aorta, lane 4: Apex of the heart, lane 5: Left atrium, lane 6: Right atrium, lane 7: Left ventricle lane 8: Right ventricle. Blot probed with PITX2 cDNA clone (HU3_p983E0327D).

DETAILED DESCRIPTION OF THE INVENTION

[0073] A description of preferred embodiments of the invention follows.

DEFINITIONS

[0074] The following terms shall, in the present context, have the meaning as indicated:

[0075] Atrial fibrillation (AF), as described herein, refers to AF as commonly defined according to established medical criteria. AF classified by ICD-10 in class 148 and by ICD-9 in class 427.3

[0076] Atrial flutter (AFI), as described herein, refers to AFI as commonly defined according to established medical criteria. Afl is classified ICD-10 class 148 and by ICD-9 in class 427.32.

[0077] A "polymorphic marker", sometime referred to as a "marker", as described herein, refers to a genomic polymorphic site. Each polymorphic marker has at least two sequence variations characteristic of particular alleles at the polymorphic site. Thus, genetic association to a polymorphic marker implies that there is association to at least one specific allele of that particular polymorphic marker. The marker can comprise any allele of any variant type found in the genome, including SNPs, microsatellites, insertions, deletions, duplications and translocations.

[0078] An "allele" refers to the nucleotide sequence of a given locus (position) on a chromosome. A polymorphic marker allele thus refers to the composition (i.e., sequence) of the marker on a chromosome. Genomic DNA from an individual contains two alleles for any given polymorphic marker, representative of each copy of the marker on each chromosome.

[0079] A nucleotide position at which more than one sequence is possible in a population (either a natural population or a synthetic population, e.g., a library of synthetic molecules) is referred to herein as a "polymorphic site".

[0080] A "Single Nucleotide Polymorphism" or "SNP" is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides). The SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI).

[0081] A "variant", as described herein, refers to a segment of DNA that differs from the reference DNA. A "marker" or a "polymorphic marker", as defined herein, is a variant. Alleles that differ from the reference are referred to as "variant" alleles.

[0082] A "microsatellite" is a polymorphic marker that has multiple small repeats of bases that are 2-8 nucleotides in length (such as CA repeats) at a particular site, in which the number of repeat lengths varies in the general population. An "indel" is a common form of polymorphism comprising a small insertion or deletion that is typically only a few nucleotides long.

[0083] A "haplotype," as described herein, refers to a segment of genomic DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus. In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles.

[0084] The term "susceptibility", as described herein, encompasses both increased susceptibility and decreased susceptibility. Thus, particular polymorphic markers and/or haplotypes of the invention may be characteristic of increased susceptibility (i.e., increased risk) of atrial fibrillation or stroke, as characterized by a relative risk (RR) or odds ratio (OR) of greater than one. Alternatively, the markers and/or haplotypes of the invention are characteristic of decreased susceptibility (i.e., decreased risk) of atrial fibrillation or stroke, as characterized by a relative risk of less than one.

[0085] A "nucleic acid sample" is a sample obtained from an individuals that contains nucleic acid. In certain embodiments, i.e. the detection of specific polymorphic markers and/or haplotypes, the nucleic acid sample comprises genomic DNA. Such a nucleic acid sample can be obtained from any source that contains genomic DNA, including as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.

[0086] The term "atrial fibrillation and/or stroke therapeutic agent" refers to an agent that can be used to ameliorate or prevent symptoms associated with atrial fibrillation (AF), atrial flutter (AFI) or stroke, as described in more detail herein.

[0087] The term "cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke-associated nucleic acid", as described herein, refers to a nucleic acid that has been found to be associated to cardiac arrhythmia, e.g., atrial fibrillation (AF), atrial flutter (AFI) or stroke. This includes, but is not limited to, the markers and haplotypes described herein and markers and haplotypes in strong linkage disequilibrium (LD) therewith. In one embodiment, an atrial fibrillation, atrial flutter or stroke-associated nucleic acid refers to the LD-block C04 found to be associated with atrial fibrillation and stroke. In another embodiment, the atrial fibrillation, atrial flutter or stroke-associated nucleic acid refers to the PITX2 gene.

[0088] The term "LD Block C04", as described herein, refers to the Linkage Disequilibrium (LD) block on Chromosome 4 between position 111,954,811 and 112,104,250 of NCBI (National Center for Biotechnology Information) Build 35, with the genomic sequence as set forth in SEQ ID NO:50.

[0089] The term "fragment", as described herein, refers to a segment of a nucleic acid or protein sequence. Fragments are of size smaller than their reference point, i.e. a fragment of a reference nucleic acid molecule that is 1000 nucleotides in size is smaller than 1000 nucleotides in size. Nucleic acid fragments of the invention are commonly more than 5 nucleotides in size and typically more than 15 nucleotides in size, with an upper limit as defined by either their reference nucleotide or by the practical utility of the nucleotide fragment. For example, nucleotide fragments useful as hybridization probes in some embodiments of the invention are more than 15 nucleotides and less than about 500 nucleotides in size. Other size ranges will apply for other nucleotide fragments and protein or peptide fragments of the invention.

[0090] The term "PITX2", as described herein, refers to the paired-like homeodomain transcription factor 2 gene on chromosome 4q25. This gene is also referred to as pituitary homeobox 2 (PTX2), rieg bicoid-related homeobox transcription factor 1 (RIEG1), solurshin, and all-1 responsive gene 1 (ARP1).

[0091] The present invention relates to the observation that certain polymorphic markers on chromosome 4q25 of the human genome have been found to be associated with cardiac arrhythmia an stroke. In particular embodiments of the invention, polymorphic markers at chromosome 4q25 are associated with the cardiac arrhythmias Atrial fibrillation (AF) and Atrial flutter (AFI), and stroke. These observation have important and unforeseen implications for the development of diagnostic and therapeutics methods, uses, kits and systems, as described in further detail herein.

[0092] In a genome-wide scan for genetic variants conferring susceptibility to AF, several markers on chromosome 4q25 were found to be associated with AF. The most significant association was found for markers rs2220427 and rs2220733, both of which gave p-values close to 10^-9 (Table 2) for AF, and smaller, but nominally significant association to stroke (Table 3). A large number of markers were identified as perfect surrogates for these markers, including the microsatellite marker D4S406 (Table 1) and a number of SNP markers (Table 4).

[0093] Further refinement of the results revealed that the association signal appears to center, in genetic terms, to markers of rs2200733 and rs10033464 (Table 7) and markers in linkage disequilibrium with those markers (including, but not limited to, the SNP markers listed in Table 9).

[0094] The original observation in the Icelandic population was replicated in an independent Icelandic AF/AFI cohort, in a Swedish AF cohort, and in a US AF cohort (Table 7). When combined with the Icelandic samples, the association to rs2200733 was unequivocal (OR=1.72, P=3.3×10^-41), and the significance of rs10033464 was well beyond the threshold of genome-wide significance (OR=1.39, P=6.9×10^-11). Assuming the multiplicative model, the population attributable risk (PAR) of the two variants combined is approximately 20% in populations of European ancestry. Furthermore, the association replicated in a Chinese AF cohort from Hong Kong (Table 7).

[0095] The inventors have also found that age at diagnosis of AF/AFI for the Icelandic samples correlates with the two SNPs rs2200733 and rs10033464. Thus, diagnosis occurs 2.28 years earlier per T allele of rs2200733 and 1.10 years earlier per T allele of rs10033464 (joint P=1.29×10^-6). This effect is manifested by the association of the two variants being strongest in those diagnosed at a younger age, although the risk remains significant even in those diagnosed after reaching 80 years of age (Table 8). A similar age at onset effect is observed in the US cohort (Table 8).

[0096] The inventors have also observed a strong association between the variants and AFI, that appears to be even stronger than for AF. Thus is revealed by the association to the subset (N=116) of the Icelandic patients that have a diagnosis of AFI (OR=2.60, 95% confidence interval (CI)=1.83-3.68, P=7.5×10^-8 for rs2200733, OR=1.94, 95% CI=1.26-3.00, P=0.0028 for rs10033464). In fact, for rs2200733, the OR for these definite AFI cases is significantly higher than that for the cases with an AF phenotype (P=0.0026), and close to significantly higher for rs10033464 (P=0.084). These results that both AF and AFI have significant genetic risk factors that are illustrated by the association to SNPs rs2200733 and rs10033464.

[0097] The inventors have furthermore established that the variants associating with AF/AFI also associated with stroke, in particular ischemic stroke (Table 21). Marker rs2200733 replicated significantly in Ischemic stroke and in the Ischemic stroke (IS) subphenotype cardioembolic stroke (CES). Both this marker and marker rs10033464 were found, after genotyping additional Icelandic IS cases and controls (total 1,943 cases/25,708 controls) and four large IS case/control replication sets (4,294 cases/3,709 controls), to associate most strongly with the CES, of which AF is the primary cause, (rs2200733: OR=1.53, P=1.5×10^-12; rs10033464: OR=1.27, P=5.9×10^-4) (Table 21).

[0098] There is no known gene present in the LD block containing rs2200733 and rs10033464 (FIG. 3). The LD block contains one spliced EST (DA725631) and two single-exon ESTs (DB324364 and AF017091). RT-PCR of cDNA libraries from various tissues did not detect the expression of these ESTs (Table 16). The PITX2 gene located in the adjacent upstream LD block is the gene closest to the risk variants. Several markers within the LD block containing PITX2 gene are correlated to the markers showing association to AF and Afl, as shown in Table 18. It is therefore possible that variants within the PITX2 gene are the underlying causative variants. Alternatively, it is possible that the variants of the present invention, as described herein, affect the function, stability, expression, post-translational modification, splicing, message stability of PITX2, or by other means affect the gene so as to predispose to the symptoms associated with atrial fibrillation, atrial flutter and/or stroke. The protein encoded by this gene, the paired-like homeodomain transcription factor 2, is an interesting candidate for AF/AFI as it is known to play an important role in cardiac development by directing asymmetric morphogenesis of the heart (Franco, D., Trends Cardiovasc Med 13: 157-63 (2003)). Furthermore, in a mouse knockout model Pitx2 has been shown to suppress a default pathway for sinoatrial node formation in the left atrium. There is very little mRNA expression of PITX2 in all easily accessible tissues, such as blood and adipose tissue, hampering the study of correlation between genotypes and expression levels. The next gene upstream of PITX2 is ENPEP, an aminopeptidase responsible for the breakdown of angiotensin II in the vascular endothelium. This gene is expressed more widely, but the variants associated with AF showed no correlation to its expression in blood or adipose tissue. No other annotated genes are located within a 400 kb region upstream and 1.5 Mb regions downstream of the associated variants.

Assessment for Markers and Haplotypes

[0099] The genomic sequence within populations is not identical when individuals are compared. Rather, the genome exhibits sequence variability between individuals at many locations in the genome. Such variations in sequence are commonly referred to as polymorphisms, and there are many such sites within each genome For example, the human genome exhibits sequence variations which occur on average every 500 base pairs. The most common sequence variant consists of base variations at a single base position in the genome, and such sequence variants, or polymorphisms, are commonly called Single Nucleotide Polymorphisms ("SNPs"). These SNPs are believed to have occurred in a single mutational event, and therefore there are usually two possible alleles possible at each SNPsite; the original allele and the mutated allele. Due to natural genetic drift and possibly also selective pressure, the original mutation has resulted in a polymorphism characterized by a particular frequency of its alleles in any given population. Many other types of sequence variants are found in the human genome, including microsatellites, insertions, deletions, inversions and copy number variations. A polymorphic microsatellite has multiple small repeats of bases (such as CA repeats, TG on the complimentary strand) at a particular site in which the number of repeat lengths varies in the general population. In general terms, each version of the sequence with respect to the polymorphic site represents a specific allele of the polymorphic site. These sequence variants can all be referred to as polymorphisms, occurring at specific polymorphic sites characteristic of the sequence variant in question. In general terms, polymorphisms can comprise any number of specific alleles. Thus in one embodiment of the invention, the polymorphism is characterized by the presence of two or more alleles in any given population. In another embodiment, the polymorphism is characterized by the presence of three or more alleles. In other embodiments, the polymorphism is characterized by four or more alleles, five or more alleles, six or more alleles, seven or more alleles, nine or more alleles, or ten or more alleles. All such polymorphisms can be utilized in the methods and kits of the present invention, and are thus within the scope of the invention.

[0100] In some instances, reference is made to different alleles at a polymorphic site without choosing a reference allele. Alternatively, a reference sequence can be referred to for a particular polymorphic site. The reference allele is sometimes referred to as the "wild-type" allele and it usually is chosen as either the first sequenced allele or as the allele from a "non-affected" individual (e.g., an individual that does not display a trait or disease phenotype).

[0101] Alleles for SNP markers as referred to herein refer to the bases A, C, G or T as they occur at the polymorphic site in the SNP assay employed. The allele codes for SNPs used herein are as follows: 1=A, 2=C, 3=G, 4=T. The person skilled in the art will however realise that by assaying or reading the opposite DNA strand, the complementary allele can in each case be measured. Thus, for a polymorphic site (polymorphic marker) characterized by an A/G polymorphism, the assay employed may be designed to specifically detect the presence of one or both of the two bases possible, i.e. A and G. Alternatively, by designing an assay that is designed to detect the opposite strand on the DNA template, the presence of the complementary bases T and C can be measured. Quantitatively (for example, in terms of relative risk), identical results would be obtained from measurement of either DNA strand (+ strand or - strand).

[0102] Typically, a reference sequence is referred to for a particular sequence. Alleles that differ from the reference are sometimes referred to as "variant" alleles. A variant sequence, as used herein, refers to a sequence that differs from the reference sequence but is otherwise substantially similar. Alleles at the polymorphic genetic markers described herein are variants. Additional variants can include changes that affect a polypeptide. Sequence differences, when compared to a reference nucleotide sequence, can include the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of a reading frame; duplication of all or a part of a sequence; transposition; or a rearrangement of a nucleotide sequence. Such sequence changes can alter the polypeptide encoded by the nucleic acid. For example, if the change in the nucleic acid sequence causes a frame shift, the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide. Alternatively, a polymorphism associated with a disease or trait can be a synonymous change in one or more nucleotides (i.e., a change that does not result in a change in the amino acid sequence). Such a polymorphism can, for example, alter splice sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of an encoded polypeptide. It can also alter DNA to increase the possibility that structural changes, such as amplifications or deletions, occur at the somatic level. The polypeptide encoded by the reference nucleotide sequence is the "reference" polypeptide with a particular reference amino acid sequence, and polypeptides encoded by variant alleles are referred to as "variant" polypeptides with variant amino acid sequences. A sequence or a reference sequence can either represent the (+) or (-) direction of double stranded DNA. Such sequences are related as being the reverse complement of one another, as well known to the skilled person.

[0103] A haplotype refers to a segment of DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus. In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles, each allele corresponding to a specific polymorphic marker along the segment. Haplotypes can comprise a combination of various polymorphic markers, e.g., SNPs and microsatellites, having particular alleles at the polymorphic sites. The haplotypes thus comprise a combination of alleles at various genetic markers.

[0104] Detecting specific polymorphic markers and/or haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites. For example, standard techniques for genotyping for the presence of SNPs and/or microsatellite markers can be used, such as fluorescence-based techniques (Chen, X. et al., Genome Res. 9(5): 492-98 (1999)), utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. Specific methodologies available for SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPIex platforms (Applied Biosystems), mass spectrometry (e.g., MassARRAY system from Sequenom), minisequencing methods, real-time PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), Molecular Inversion Probe array technology (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays). By these or other methods available to the person skilled in the art, one or more alleles at polymorphic markers, including microsatellites, SNPs or other types of polymorphic markers, can be identified.

[0105] In certain methods described herein, an individual who is at an Increased susceptibility (i.e., increased risk) for any specific disease or trait under study, is an individual in whom at least one specific allele at one or more polymorphic marker or haplotype conferring increased susceptibility for the disease or trait is identified (i.e., at-risk marker alleles or haplotypes). In one aspect, the at-risk marker or haplotype is one that confers a significant increased risk (or susceptibility) of the disease or trait. In one embodiment, significance associated with a marker or haplotype is measured by a relative risk (RR). In another embodiment, significance associated with a marker or haplotye is measured by an odds ratio (OR). In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.2, including but not limited to: at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, 1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, at least 4.0, and at least 5.0. In a particular embodiment, a risk (relative risk and/or odds ratio) of at least 1.2 is significant. In another particular embodiment, a risk of at least 1.3 is significant. In yet another embodiment, a risk of at least 1.4 is significant. In a further embodiment, a relative risk of at least about 1.5 is significant. In another further embodiment, a significant increase in risk is at least about 1.7 is significant. However, other cutoffs are also contemplated, e.g. at least 1.15, 1.25, 1.35, and so on, and such cutoffs are also within scope of the present invention. In other embodiments, a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, and 500%. In one particular embodiment, a significant increase in risk is at least 20%. In other embodiments, a significant increase in risk is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and at least 100%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention.

[0106] An at-risk polymorphic marker or haplotype of the present invention is one where at least one allele of at least one marker or haplotype is more frequently present in an individual at risk for the disease or trait (e.g., cardiac arrhythmia or stroke) (affected), compared to the frequency of its presence in a comparison group (control), and wherein the presence of the marker or haplotype is indicative of susceptibility to the disease or trait. The control group may in one embodiment be a population sample, i.e. a random sample from the general population. In another embodiment, the control group is represented by a group of individuals who are disease-free. Such disease-free control may in one embodiment be characterized by the absence of one or more specific disease-associated symptoms. In another embodiment, the disease-free control group is characterized by the absence of one or more disease-specific risk factors. Such risk factors are in one embodiment at least one environmental risk factor. Representative environmental factors are natural products, minerals or other chemicals which are known to affect, or contemplated to affect, the risk of developing the specific disease or trait. Other environmental risk factors are risk factors related to lifestyle, including but not limited to food and drink habits, geographical location of main habitat, and occupational risk factors. In another embodiment, the risk factors are at least one genetic risk factor.

[0107] As an example of a simple test for correlation would be a Fisher-exact test on a two by two table. Given a cohort of chromosomes, the two by two table is constructed out of the number of chromosomes that include both of the markers or haplotypes, one of the markers or haplotypes but not the other and neither of the markers or haplotypes.

[0108] In other embodiments of the invention, an individual who is at a decreased susceptibility (i.e., at a decreased risk) for the disease or trait is an individual in whom at least one specific allele at one or more polymorphic marker or haplotype conferring decreased susceptibility for the disease or trait is identified. The marker alleles and/or haplotypes conferring decreased risk are also said to be protective. In one aspect, the protective marker or haplotype is one that confers a significant decreased risk (or susceptibility) of the disease or trait. In one embodiment, significant decreased risk is measured as a relative risk of less than 0.9, including but not limited to less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 and less than 0.1. In one particular embodiment, significant decreased risk is less than 0.7. In another embodiment, significant decreased risk is less than 0.5. In yet another embodiment, significant decreased risk is less than 0.3. In another embodiment, the decrease in risk (or susceptibility) is at least 20%, including but not limited to at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and at least 98%. In one particular embodiment, a significant decrease in risk is at least about 30%. In another embodiment, a significant decrease in risk at least about 50%. In another embodiment, the decrease in risk is at least about 70%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention.

[0109] The person skilled in the art will appreciate that for markers with two alleles present in the population being studied, and wherein one allele is found in increased frequency in a group of individuals with a trait or disease in the population, compared with controls, the other allele of the marker will be found in decreased frequency in the group of individuals with the trait or disease, compared with controls. In such a case, one allele of the marker (the one found in increased frequency in individuals with the trait or disease) will be the at-risk allele, while the other allele will be a protective allele.

Linkage Disequilibrium

[0110] The natural phenomenon of recombination, which occurs on average once for each chromosomal pair during each meiotic event, represents one way in which nature provides variations in sequence (and biological function by consequence). It has been discovered that recombination does not occur randombly in the genome; rather, there are large variations in the frequency of recombination rates, resulting in small regions of high recombination frequency (also called recombination hotspots) and larger regions of low recombination frequency, which are commonly referred to as Linkage Disequilibrium (LD) blocks (Myers, S. et al., Biochem Soc Trans 34:526-530 (2006); Jeffreys, A. J., et al., Nature Genet 29:217-222 (2001); May, C. A., et al., Nature Genet 31:272-275 (2002)).

[0111] Linkage Disequilibrium (LD) refers to a non-random assortment of two genetic elements. For example, if a particular genetic element (e.g., an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.25 (25%) and another element occurs at a frequency of 0.25 (25%), then the predicted occurrence of a person's having both elements is 0.125 (12.5%), assuming a random distribution of the elements. However, if it is discovered that the two elements occur together at a frequency higher than 0.125, then the elements are said to be in linkage disequilibrium, since they tend to be inherited together at a higher rate than what their independent frequencies of occurrence (e.g., allele or haplotype frequencies) would predict. Roughly speaking, LD is generally correlated with the frequency of recombination events between the two elements. Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurrence of each allele or haplotype in the population. For populations of diploids, e.g., human populations, individuals will typically have two alleles for each genetic element (e.g., a marker, haplotype or gene).

[0112] Many different measures have been proposed for assessing the strength of linkage disequilibrium (LD). Most capture the strength of association between pairs of biallelic sites. Two important pairwise measures of LD are r² (sometimes denoted Δ²) and |D'|. Both measures range from 0 (no disequilibrium) to 1 (`complete` disequilibrium), but their interpretation is slightly different. |D'| is defined in such a way that it is equal to 1 if just two or three of the possible haplotypes are present, and it is <1 if all four possible haplotypes are present. Therefore, a value of |D'| that is <1 indicates that historical recombination may have occurred between two sites (recurrent mutation can also cause |D'| to be <1, but for single nucleotide polymorphisms (SNPs) this is usually regarded as being less likely than recombination). The measure r² represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are present.

[0113] The r² measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r² and the sample size required to detect association between susceptibility loci and SNPs. These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or whether there is more or less LD in a region than predicted under a particular model). Measuring LD across a region is not straightforward, but one approach is to use the measure r, which was developed in population genetics. Roughly speaking, r measures how much recombination would be required under a particular population model to generate the LD that is seen in the data. This type of method can potentially also provide a statistically rigorous approach to the problem of determining whether LD data provide evidence for the presence of recombination hotspots. For the methods and procedures described herein, a significant r² value can be at least 0.1 such as at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1.0. In one preferred embodiment, the significant r² value can be at least 0.2. Alternatively, linkage disequilibrium as described herein, refers to linkage disequilibrium characterized by values of |D'| of at least 0.2, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99. Thus, linkage disequilibrium represents a correlation between alleles of distinct markers. It is measured by correlation coefficient or |D'| (r² up to 1.0 and |D'| up to 1.0). Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be determined in a collection of samples comprising individuals from more than one human population. In one embodiment of the invention, LD is determined in a sample from one or more of the HapMap populations (caucasian, african, japanese, chinese), as defined (http://www.hapmap.org). In one such embodiment, LD is determined in the CEU population of the HapMap samples. In another embodiment, LD is determined in the YRI population. In yet another embodiment, LD is determined in samples from the Icelandic population.

[0114] If all polymorphisms in the genome were identical at the population level, then every single one of them would need to be investigated in association studies. However, due to linkage disequilibrium between polymorphisms, tightly linked polymorphisms are strongly correlated, which reduces the number of polymorphisms that need to be investigated in an association study to observe a significant association. Another consequence of LD is that many polymorphisms may give an association signal due to the fact that these polymorphisms are strongly correlated.

[0115] Genomic LD maps have been generated across the genome, and such LD maps have been proposed to serve as framework for mapping disease-genes (Risch, N. & Merkiangas, K, Science 273:1516-1517 (1996); Maniatis, N., et al., Proc Natl Acad Sci USA 99:2228-2233 (2002); Reich, D E et al, Nature 411:199-204 (2001)).

[0116] It is now established that many portions of the human genome can be broken into series of discrete haplotype blocks containing a few common haplotypes; for these blocks, linkage disequilibrium data provides little evidence indicating recombination (see, e.g., Wall., J. D. and Pritchard, J. K., Nature Reviews Genetics 4:587-597 (2003); Daly, M. et al., Nature Genet. 29:229-232 (2001); Gabriel, S. B. et al., Science 296:2225-2229 (2002); Patil, N. et al., Science 294:1719-1723 (2001); Dawson, E. et al., Nature 418:544-548 (2002); Phillips, M. S. et al., Nature Genet. 33:382-387 (2003)).

[0117] There are two main methods for defining these haplotype blocks: Blocks can be defined as regions of DNA that have limited haplotype diversity (see, e.g., Daly, M. et al., Nature Genet. 29:229-232 (2001); Patil, N. et al., Science 294:1719-1723 (2001); Dawson, E. et al., Nature 418:544-548 (2002); Zhang, K. et al., Proc. Natl. Acad. Sci. USA 99:7335-7339 (2002)), or as regions between transition zones having extensive historical recombination, identified using linkage disequilibrium (see, e.g., Gabriel, S. B. et al., Science 296:2225-2229 (2002); Phillips, M. S. et al., Nature Genet. 33:382-387 (2003); Wang, N. et al., Am. J. Hum. Genet. 71:1227-1234 (2002); Stumpf, M. P., and Goldstein, D. B., Curr. Biol. 13:1-8 (2003)). More recently, a fine-scale map of recombination rates and corresponding hotspots across the human genome has been generated (Myers, S., et al., Science 310:321-32324 (2005); Myers, S. et al., Biochem Soc Trans 34:526530 (2006)). The map reveals the enormous variation in recombination across the genome, with recombination rates as high as 10-60 cM/Mb in hotspots, while closer to 0 in intervening regions, which thus represent regions of limited haplotype diversity and high LD. The map can therefore be used to define haplotype blocks/LD blocks as regions flanked by recombination hotspots. As used herein, the terms "haplotype block" or "LD block" includes blocks defined by any of the above described characteristics, or other alternative methods used by the person skilled in the art to define such regions.

[0118] Haplotype blocks can be used to map associations between phenotype and haplotype status, using single markers or haplotypes comprising a plurality of markers. The main haplotypes can be identified in each haplotype block, and then a set of "tagging" SNPs or markers (the smallest set of SNPs or markers needed to distinguish among the haplotypes) can then be identified. These tagging SNPs or markers can then be used in assessment of samples from groups of individuals, in order to identify association between phenotype and haplotype. If desired, neighboring haplotype blocks can be assessed concurrently, as there may also exist linkage disequilibrium among the haplotype blocks.

[0119] It has thus become apparent that for any given observed association to a polymorphic marker in the genome, it is likely that additional markers in the genome also show association. This is a natural consequence of the uneven distribution of LD across the genome, as observed by the large variation in recombination rates. The markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait, and as such are useful for use in the methods and kits of the present invention. One or more causative (functional) variants or mutations may reside within the region found to be associating to the disease or trait. Such variants may confer a higher relative risk (RR) or odds ratio (OR) than observed for the tagging markers used to detect the association. The present invention thus refers to the markers used for detecting association to the disease, as described herein, as well as markers in linkage disequilibrium with the markers. Thus, in certain embodiments of the invention, markers that are in LD with the markers and/or haplotypes of the invention, as described herein, may be used as surrogate markers. The surrogate markers have in one embodiment relative risk (RR) and/or odds ratio (OR) values smaller than for the markers or haplotypes initially found to be associating with the disease, as described herein. In other embodiments, the surrogate markers have RR or OR values greater than those initially determined for the markers initially found to be associating with the disease, as described herein. An example of such an embodiment would be a rare, or relatively rare (<10% allelic population frequency) variant in LD with a more common variant (>10% population frequency) initially found to be associating with the disease, such as the variants described herein. Identifying and using such markers for detecting the association discovered by the inventors as described herein can be performed by routine methods well known to the person skilled in the art, and are therefore within the scope of the present invention.

[0120] It is possible that certain polymorphic markers in linkage disequilibrium with the markers shown herein to be associated with cardiac arrhythmia (e.g., atrial fibrillation and atrial flutter) and stroke are located outside the physical boundaries of the LD block C04 as defined herein by the sequence set forth in SEQ ID NO:50. This is a consequence of the historical recombination rates in the region in question, which may have led to a region of strong LD (the LD block), with residual markers outside the block in LD with markers within the block. Such markers are also within scope of the present invention, as they are also useful for practicing the invention by virtue of their genetic relationship with the markers shown herein to be associated with cardiac arrhythmia and stroke. Examples of such markers are shown in Table 18 (rs7668322 (SEQ ID NO:46), rs2197815 (SEQ ID NO:47), rs6831623 (SEQ ID NO:48), rs2595110 (SEQ ID NO:49))

Determination of Haplotype Frequency

[0121] The frequencies of haplotypes in patient and control groups can be estimated using an expectation-maximization algorithm (Dempster A. et al., J. R. Stat. Soc. B, 39: 1-38 (1977)). An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase can be used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies. Using a likelihood approach, an alternative hypothesis is tested, where a candidate at-risk-haplotype, which can include the markers described herein, is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups. Likelihoods are maximized separately under both hypotheses and a corresponding 1-df likelihood ratio statistic is used to evaluate the statistical significance.

[0122] To look for at-risk and protective markers and haplotypes within a linkage region, for example, association of all possible combinations of genotyped markers is studied, provided those markers span a practical region. The combined patient and control groups can be randomly divided into two sets, equal in size to the original group of patients and controls. The marker and haplotype analysis is then repeated and the most significant p-value registered is determined. This randomization scheme can be repeated, for example, over 100 times to construct an empirical distribution of p-values. In a preferred embodiment, a p-value of <0.05 is indicative of an significant marker and/or haplotype association.

Haplotype Analysis

[0123] One general approach to haplotype analysis involves using likelihood-based inference applied to NEsted MOdels (Gretarsdottir S., et al., Nat. Genet. 35: 131-38 (2003)). The method is implemented in the program NEMO, which allows for many polymorphic markers, SNPs and microsatellites. The method and software are specifically designed for case-control studies where the purpose is to identify haplotype groups that confer different risks. It is also a tool for studying LD structures. In NEMO, maximum likelihood estimates, likelihood ratios and p-values are calculated directly, with the aid of the EM algorithm, for the observed data treating it as a missing-data problem.

[0124] Even though likelihood ratio tests based on likelihoods computed directly for the observed data, which have captured the information loss due to uncertainty in phase and missing genotypes, can be relied on to give valid p-values, it would still be of interest to know how much information had been lost due to the information being incomplete. The information measure for haplotype analysis is described in Nicolae and Kong (Technical Report 537, Department of Statistics, University of Statistics, University of Chicago; Biometrics, 60(2):368-75 (2004)) as a natural extension of information measures defined for linkage analysis, and is implemented in NEMO.

[0125] For single marker association to a disease, the Fisher exact test can be used to calculate two-sided p-values for each individual allele. Usually, all p-values are presented unadjusted for multiple comparisons unless specifically indicated. The presented frequencies (for microsatellites, SNPs and haplotypes) are allelic frequencies as opposed to carrier frequencies. To minimize any bias due the relatedness of the patients who were recruited as families for the linkage analysis, first and second-degree relatives can be eliminated from the patient list. Furthermore, the test can be repeated for association correcting for any remaining relatedness among the patients, by extending a variance adjustment procedure described in Risch, N. & Teng, J. (Genome Res., 8:1273-1288 (1998)), DNA pooling (ibid) for sibships so that it can be applied to general familial relationships, and present both adjusted and unadjusted p-values for comparison. The differences are in general very small as expected. To assess the significance of single-marker association corrected for multiple testing we can carry out a randomization test using the same genotype data. Cohorts of patients and controls can be randomized and the association analysis redone multiple times (e.g., up to 500,000 times) and the p-value is the fraction of replications that produced a p-value for some marker allele that is lower than or equal to the p-value we observed using the original patient and control cohorts.

[0126] For both single-marker and haplotype analyses, relative risk (RR) and the population attributable risk (PAR) can be calculated assuming a multiplicative model (haplotype relative risk model) (Terwilliger, J. D. & Ott, J., Hum. Hered. 42:337-46 (1992) and Falk, C. T. & Rubinstein, P, Ann. Hum. Genet. 51 (Pt 3):227-33 (1987)), i.e., that the risks of the two alleles/haplotypes a person carries multiply. For example, if RR is the risk of A relative to a, then the risk of a person homozygote AA will be RR times that of a heterozygote Aa and RR² times that of a homozygote aa. The multiplicative model has a nice property that simplifies analysis and computations--haplotypes are independent, i.e., in Hardy-Weinberg equilibrium, within the affected population as well as within the control population. As a consequence, haplotype counts of the affecteds and controls each have multinomial distributions, but with different haplotype frequencies under the alternative hypothesis. Specifically, for two haplotypes, h_i and h_j, risk(h_i)/risk(h_j)=(f_i/p_i)/(f_j/p_j), where f and p denote, respectively, frequencies in the affected population and in the control population. While there is some power loss if the true model is not multiplicative, the loss tends to be mild except for extreme cases. Most importantly, p-values are always valid since they are computed with respect to null hypothesis.

Linkage Disequilibrium Using NEMO

[0127] LD between pairs of markers can be calculated using the standard definition of D' and r² (Lewontin, R., Genetics 49:49-67 (1964); Hill, W. G. & Robertson, A. Theor. Appl. Genet. 22:226-231 (1968)). Using NEMO, frequencies of the two marker allele combinations are estimated by maximum likelihood and deviation from linkage equilibrium is evaluated by a likelihood ratio test. The definitions of D' and r² are extended to include microsatellites by averaging over the values for all possible allele combination of the two markers weighted by the marginal allele probabilities. When plotting all marker combination to elucidate the LD structure in a particular region, we plot D' in the upper left corner and the p-value in the lower right corner. In the LD plots the markers can be plotted equidistant rather than according to their physical location, if desired.

Risk Assessment and Diagnostics

[0128] As described herein, certain polymorphic markers and haplotypes comprising such markers are found to be useful for risk assessment of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) or stroke. Risk assessment can involve the use of the markers for diagnosing a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) or stroke. Particular alleles of polymorphic markers are found more frequently in individuals with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) or stroke, than in individuals without diagnosis of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) or stroke. Therefore, these marker alleles have predictive value for detecting cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, or a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) or stroke, in an individual. Tagging markers within haplotype blocks or LD blocks comprising at-risk markers, such as the markers of the present invention, can be used as surrogates for other markers and/or haplotypes within the haplotype block or LD block. Markers with values of r² equal to 1 are perfect surrogates for the at-risk variants, i.e. genotypes for one marker perfectly predicts genotypes for the other. Markers with smaller values of r² than 1 can also be surrogates for the at-risk variant, or alternatively represent variants with relative risk values as high or possibly even higher than the at-risk variant. The at-risk variant identified may not be the functional variant itself, but is in this instance in linkage disequilibrium with the true functional variant. The present invention encompasses the assessment of such surrogate markers for the markers as disclosed herein. Such markers are annotated, mapped and listed in public databases, as well known to the skilled person, or can alternatively be readily identified by sequencing the region or a part of the region identified by the markers of the present invention in a group of individuals, and identify polymorphisms in the resulting group of sequences. As a consequence, the person skilled in the art can readily and without undue experimentation genotype surrogate markers in linkage disequilibrium with the markers and/or haplotypes as described herein. The tagging or surrogate markers in LD with the at-risk variants detected, also have predictive value for detecting association to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke or a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) or stroke, in an individual. These tagging or surrogate markers that are in LD with the markers of the present invention can also include other markers that distinguish among haplotypes, as these similarly have predictive value for detecting susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.

[0129] The markers and haplotypes of the invention, e.g., the markers presented in Tables 5 and 9, as well as markers in linkage disequilibrium therewith, may be useful for risk assessment and diagnostic purposes for, either alone or in combination. Thus, even in cases where the increase in risk by individual markers is relatively modest, i.e. on the order of 10-30%, the association may have significant implications. Thus, relatively common variants may have significant contribution to the overall risk (Population Attributable Risk is high), or combination of markers can be used to define groups of individual who, based on the combined risk of the markers, is at significant combined risk of developing the disease.

[0130] Thus, in one embodiment of the invention, a plurality of variants (markers and/or haplotypes) is used for overall risk assessment. These variants are in one embodiment selected from the variants as disclosed herein. Other embodiments include the use of the variants of the present invention in combination with other variants known to be useful for diagnosing a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. In such embodiments, the genotype status of a plurality of markers and/or haplotypes is determined in an individual, and the status of the individual compared with the population frequency of the associated variants, or the frequency of the variants in clinically healthy subjects, such as age-matched and sex-matched subjects. Methods known in the art, such as multivariate analyses or joint risk analyses, may subsequently be used to determine the overall risk conferred based on the genotype status at the multiple loci. Assessment of risk based on such analysis may subsequently be used in the methods and kits of the invention, as described herein.

[0131] As described in the above, the haplotype block structure of the human genome has the effect that a large number of variants (markers and/or haplotypes) in linkage disequilibrium with the variant originally associated with a disease or trait may be used as surrogate markers for assessing association to the disease or trait. The number of such surrogate markers will depend on factors such as the historical recombination rate in the region, the mutational frequency in the region (i.e., the number of polymorphic sites or markers in the region), and the extent of LD (size of the LD block) in the region. These markers are usually located within the physical boundaries of the LD block or haplotype block in question as defined using the methods described herein, or by other methods known to the person skilled in the art. However, sometimes marker and haplotype association is found to extend beyond the physical boundaries of the haplotype block as defined. Such markers and/or haplotypes may in those cases be also used as surrogate markers and/or haplotypes for the markers and/or haplotypes physically residing within the haplotype block as defined. As a consequence, markers and haplotypes in LD (typically characterized by r² greater than 0.1, such as r² greater than 0.2, including r² greater than 0.3, also including r² greater than 0.4) with the markers and haplotypes of the present invention are also within the scope of the invention, even if they are physically located beyond the boundaries of the haplotype block as defined. This includes markers that are described herein (e.g., Tables 5 and 9), but may also include other markers that are in strong LD (characterized by r² greater than 0.1 or 0.2 and/or |D'|>0.8) with one or more of the markers listed in Tables 5 and 9.

[0132] For the SNP markers described herein, the opposite allele to the allele found to be in excess in patients (at-risk allele) is found in decreased frequency in cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke patients. These markers and haplotypes in LD and/or comprising such markers, are thus protective for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. i.e. they confer a decreased risk or susceptibility of individuals carrying these markers and/or haplotypes developing cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.

[0133] Certain variants of the present invention, including certain haplotypes comprise, in some cases, a combination of various genetic markers, e.g., SNPs and microsatellites. Detecting haplotypes can be accomplished by methods known in the art and/or described herein for detecting sequences at polymorphic sites. Furthermore, correlation between certain haplotypes or sets of markers and disease phenotype can be verified using standard techniques. A representative example of a simple test for correlation would be a Fisher-exact test on a two by two table.

[0134] In specific embodiments, a marker or haplotype found to be associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, (e.g., markers as listed in Table 5 (Tables 5A and 5B), Table 9 and/or Table 19, and markers in linkage disequilibrium therewith) is one in which the marker allele or haplotype is more frequently present in an individual at risk for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke (affected), compared to the frequency of its presence in a healthy individual (control), wherein the presence of the marker allele or haplotype is indicative of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. or a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. In other embodiments, at-risk markers in linkage disequilibrium with one or more markers found to be associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. (e.g., marker alleles as listed in Tables 5A and 5B, and markers in linkage disequilibrium therewith) are tagging markers that are more frequently present in an individual at risk for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke (affected), compared to the frequency of their presence in a healthy individual (control), wherein the presence of the tagging markers is indicative of increased susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. In a further embodiment, at-risk markers alleles (i.e. conferring increased susceptibility) in linkage disequilibrium with one or more markers found to be associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. (e.g., marker alleles as listed in Tables 5A and 5B and markers in linkage disequilibrium therewith), are markers comprising one or more allele that is more frequently present in an individual at risk for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke compared to the frequency of their presence in a healthy individual (control), wherein the presence of the markers is Indicative of increased susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.

Study Population

[0135] In a general sense, the methods and kits of the invention can be utilized from samples containing genomic DNA from any source, i.e. any individual. In preferred embodiments, the individual is a human individual. The individual can be an adult, child, or fetus. The present invention also provides for assessing markers and/or haplotypes in individuals who are members of a target population. Such a target population is in one embodiment a population or group of individuals at risk of developing the disease, based on other genetic factors, biomarkers, biophysical parameters (e.g., weight, BMD, blood pressure), or general health and/or lifestyle parameters (e.g., history of disease or related diseases, previous diagnosis of disease, family history of disease).

[0136] The invention provides for embodiments that include individuals from specific age subgroups, such as those over the age of 40, over age of 45, or over age of 50, 55, 60, 65, 70, 75, 80, or 85. Other embodiments of the invention pertain to other age groups, such as individuals aged less than 85, such as less than age 80, less than age 75, or less than age 70, 65, 60, 55, 50, 45, 40, 35, or age 30. Other embodiments relate to individuals with age at onset of the disease in any of the age ranges described in the above. It is also contemplated that a range of ages may be relevant in certain embodiments, such as age at onset at more than age 45 but less than age 60. Other age ranges are however also contemplated, including all age ranges bracketed by the age values listed in the above.

[0137] Other embodiments related to individuals with age at onset of the disease at particular age or age range. Thus, it is known that predisposing factors, genetic and non-genetic, can affect at what age an individual develops a disease. For cardiovascular disorders, including cardiac arrhythmias and stroke, common risk factors can influence if, and at what age, an individual develops the disease. Some embodiments of the invention therefore relate to age at onset or age at diagnosis of cardiac arrhythmia (e.g., atrial fibrillation and/or atrial flutter) or stroke in a certain age range. In one embodiment, the individuals at risk for developing cardiac arrhythmia (e.g., atrial fibrillation and/or atrial flutter) or stroke have age at onset or age at diagnosis over the age of 40. In other embodiments, the individuals have age at onset or age at diagnosis over age of 45, or over age of 50, 55, 60, 65, 70, 75, 80, or 85. Other embodiments of the invention pertain to individuals who have an age at onset or age at diagnosis at age less than 85, such as less than age 80, less than age 75, or less than age 70, 65, 60, 55, 50, 45, 40, 35, or age 30. One preferred embodiment includes individuals diagnosed with atrial fibrillation or atrial flutter or stroke below age 80. Another preferred embodiment relates to individuals diagnosed with atrial fibrillation or atrial flutter or stroke below age 70. Another preferred embodiment, relates to individuals diagnosed with atrial fibrillation or atrial flutter or stroke below age 60. Yet another preferred embodiment relates to individuals diagnosed with atrial fibrillation or atrial flutter or stroke below age 50. Other embodiments relate to individuals with age at onset of the disease in specific age ranges, described in the above. It is also contemplated that a range of ages may be relevant in certain embodiments, such as age at onset at more than age 45 but less than age 60, age at onset at age more than 60 and less than age 70, age at onset at age more than 70 and less than 80, or age at onset at age more than 60 and less than 80. Other age ranges are however also contemplated, including all age ranges bracketed by the age values listed in the above.

[0138] The invention furthermore relates to individuals of either sex, males or females. It also provides for embodiments that relate to human subjects that are from one or more human population including, but not limited to, Bantu, Mandenk, Yoruba, San, Mbuti Pygmy, Orcadian, Adygel, Russian, Sardinian, Tuscan, Mozabite, Bedouin, Druze, Palestinian, Balochi, Brahui, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash, Han, Dai, Daur, Hezhen, Lahu, Miao, Orogen, She, Tujia, Tu, Xibo, Yi, Mongolan, Naxi, Cambodian, Japanese, Yakut, Melanesian, Papuan, Karitianan, Surui, Colmbian, Maya and Pima. The invention also relates to European populations, American populations, Eurasian populations, Asian populations, Central/South Asian populations, East Asian populations, Middle Eastern populations, African populations, Hispanic populations, and Oceanian populations. European populations include, but are not limited to, Swedish, Norwegian, Finnish, Russian, Danish, Icelandic, Irish, Kelt, English, Scottish, Dutch, Belgian, French, German, Spanish, Portuguese, Italian, Polish, Bulgarian, Slavic, Serbian, Bosnian, Chech, Greek and Turkish populations.

[0139] In one preferred embodiment, the invention relates to populations that include black African ancestry such as populations comprising persons of African descent or lineage. Black African ancestry may be determined by self reporting as African-Americans, Afro-Americans, Black Americans, being a member of the black race or being a member of the negro race. For example, African Americans or Black Americans are those persons living in North America and having origins in any of the black racial groups of Africa. In another example, self-reported persons of black African ancestry may have at least one parent of black African ancestry or at least one grandparent of black African ancestry.

[0140] The racial contribution in individual subjects may also be determined by genetic analysis. Genetic analysis of ancestry may be carried out using unlinked microsatellite markers such as those set out in Smith et al. (Am J Hum Genet 74, 1001-13 (2004)).

[0141] In certain embodiments, the invention relates to markers and/or haplotypes identified in specific populations, as described in the above. The person skilled in the art will appreciate that measures of linkage disequilibrium (LD) may give different results when applied to different populations. This is due to different population history of different human populations as well as differential selective pressures that may have led to differences in LD in specific genomic regions. It is also well known to the person skilled in the art that certain markers, e.g. SNP markers, are polymorphic in one population but not in another. The person skilled in the art will however apply the methods available and as taught ??herein to practice the present invention in any given human population. This may include assessment of polymorphic markers in the LD region of the present invention, so as to identify those markers that give strongest association within the specific population. Thus, the at-risk variants of the present invention may reside on different haplotype background and in different frequencies in various human populations. However, utilizing methods known in the art and the markers of the present invention, the invention can be practiced in any given human population.

Utility of Genetic Testing

[0142] The person skilled in the art will appreciate and understand that the variants described herein in general do not, by themselves, provide an absolute identification of individuals who will develop cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. The variants described herein do however indicate increased and/or decreased likelihood that individuals carrying the at-risk or protective variants of the invention will develop symptoms associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke This information is however extremely valuable in itself, as outlined in more detail in the below, as it can be used to, for example, initiate preventive measures at an early stage, perform regular physical and/or mental exams to monitor the progress and/or appearance of symptoms, or to schedule exams at a regular interval to identify the condition in question, so as to be able to apply treatment at an early stage.

[0143] The knowledge about a genetic variant that confers a risk of developing cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke offers the opportunity to apply a genetic test to distinguish between individuals with increased risk of developing the disease (i.e. carriers of the at-risk variant) and those with decreased risk of developing the disease (i.e. carriers of the protective variant). The core values of genetic testing, for individuals belonging to both of the above mentioned groups, are the possibilities of being able to diagnose cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, or a predisposition to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke at an early stage and provide information to the clinician about prognosis of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke in order to be able to apply the most appropriate treatment.

[0144] Individuals with a family history of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke and carriers of at-risk variants may benefit from genetic testing since the knowledge of the presence of a genetic risk factor, or evidence for increased risk of being a carrier of one or more risk factors, may provide increased incentive for implementing a healthier lifestyle, by avoiding or minimizing known environmental risk factors for cardiovascular diseases related to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. Genetic testing of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke patients may furthermore give valuable information about the primary cause of the disease and can aid the clinician in selecting the best treatment options and medication for each individual.

[0145] The present invention furthermore relates to risk assessment for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, including determining whether an individual is at risk for developing cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. The polymorphic markers of the present invention can be used alone or in combination, as well as in combination with other factors, including other genetic risk factors or biomarkers, for risk assessment of an individual for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. Many factors known to affect the predisposition of an individual towards developing risk of cardiovascular disease are susceptibility factors for cardiac arrhythmias (e.g., atrial fibrillation or atrial flutter) and/or stroke, and are known to the person skilled in the art and can be utilized in such assessment. These include, but are not limited to, age, gender, smoking status, physical activity, waist-to-hip circumference ratio, family history of cardiac arrhythmia (in particular atrial fibrillation and/or atrial flutter) and/or stroke, previously diagnosed cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, obesity, hypertriglyceridemia, low HDL cholesterol, hypertension, elevated blood pressure, cholesterol levels, HDL cholesterol, LDL cholesterol, triglycerides, apolipoprotein AI and B levels, fibrinogen, ferritin, C-reactive protein and leukotriene levels. Particular biomarkers that have been associated with Atrial fibrillation/Atrial flutter and stroke are discussed in Allard et al. (Clin Chem 51:2043-2051 (2005) and Becker (J Thromb Thrombolys 19:71-75 (2005)). These include, but are not limited to, fibrin D-dimer, prothrombin activation fragment 1.2 (F1.2), thrombin-antithrombin III complexes (TAT), fibrinopeptide A (FPA), lipoprotein-associated phospholipase A2 (Ip-PLA2), beta-thromboglobulin, platelet factor 4, P-selectin, von Willebrand Factor, pro-natriuretic peptide (BNP), matrix metalloproteinase-9 (MMP-9), PARK7, nucleoside diphosphate kinase (NDKA), tau, neuron-specific enolase, B-type neurotrophic growth factor, astroglial protein S-100b, glial fibrillary acidic protein, C-reactive protein, seum amyloid A, marix metalloproteinase-9, vascular and intracellular cell adhesion molecules, tumor necrosis factor alpha, and interleukins, including interleukin-1, -6, and -8). Circulating progenitor cells have also been implicated as being useful biomarkers for AF. In particular embodiments, more than one biomarker is determined for an individual, and combined with results of a determination of at least one polymorphic marker as described herein. Preferably, biomarker is measured in plasma or serum from the individual. Alternatively, the biomarker is determined in other suitable tissues containing measurable amounts of the biomarker, and such embodiments are also within scope of the invention.

[0146] Methods known in the art can be used for overall risk assessment, including multivariate analyses or logistic regression.

[0147] Atrial fibrillation is a disease of great significance both to the individual patient and to the health care system as a whole. It can be a permanent condition but may also be paroxysmal and recurrent in which case it can be very challenging to diagnose. The most devastating complication of atrial fibrillation and atrial flutter is the occurrence of debilitating stroke. Importantly the risk of stroke is equal in permanent and paroxysmal atrial fibrillation. It has repeatedly been shown that therapy with warfarin anticoagulation can significantly reduce the risk of first or further episodes of stroke in the setting of atrial fibrillation. Therefor, anticoagulation with warfarin is standard therapy for almost all patients with atrial fibrillation for stroke-prevention, whether they have the permanent or paroxysmal type. The only patients for whom warfarin is not strongly recommended are those younger than 65 years old who are considered low-risk, i.e., they have no organic heart disease, including, neither hypertension no coronary artery disease, no previous history of stroke or transient ischemic attacks and no diabetes. This group has a lower risk of stroke and stroke-prevention with aspirin is recommended.

[0148] Due to the nature of paroxysmal atrial fibrillation it can be very difficult to diagnose. When the patient seeks medical attention due to disease-related symptoms, such as palpitations, chest pain, shortness of breath, dizziness, heart failure, transient ischemic attacks or even stroke, normal heart rhythm may already be restored precluding diagnosis of the arrhythmia. In these cases cardiac rhythm monitoring is frequently applied in the attempt to diagnose the condition. The cardiac rhythm is commonly monitored continuously for 24 to 48 hours. Unfortunately atrial fibrillation episodes are unpredictable and frequently missed by this approach. The opportunity to diagnose the arrhythmia, institute recommended therapy, and possibly prevent a debilitating first or recurrent stroke may be missed with devastating results to the patient. Prolonged and more complex cardiac rhythm monitoring measures are available and applied occasionally when the suspicion of atrial fibrillation is very strong. These tests are expensive, the diagnostic yield with current approach is often low, and they are used sparingly for this indication. In these circumstances additional risk stratification with genetic testing may be extremely helpful. Understanding that the individual in question carries either an at-risk or a protective genetic variant can be an invaluable contribution to diagnostic and/or treatment decision making. This way, in some cases, unnecessary testing and therapy may be avoided, and in other cases, with the help of more aggressive diagnostic approach, the arrhythmia may be diagnosed and/or proper therapy initiated and later complications of disease diminished.

Methods of the Invention

[0149] Methods for risk assessment of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke are described herein and are encompassed by the invention. The invention also encompasses methods of assessing an individual for probability of response to a therapeutic agent for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, as well as methods for predicting the effectiveness of a therapeutic agent to treat patients with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. Kits for assaying a sample from a subject to detect susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke are also encompassed by the invention.

Diagnostic and Screening Assays of the Invention

[0150] In certain embodiments, the present invention pertains to methods of diagnosing, or aiding in the diagnosis of, cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke or a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, by detecting particular alleles at genetic markers that appear more frequently in cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke subjects or subjects who are susceptible to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. In a particular embodiment, the invention is a method of diagnosing a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke by detecting at least one allele of at least one polymorphic marker (e.g., the markers described herein). The present invention describes methods whereby detection of particular alleles of particular markers or haplotypes is indicative of a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. Such prognostic or predictive assays can also be used to determine prophylactic treatment of a subject prior to the onset of symptoms of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.

[0151] The present invention pertains in some embodiments to methods of clinical applications of diagnosis, e.g., diagnosis performed by a medical professional, which may include an assessment or determination of genetic risk variants, and their interpretation. In other embodiments, the invention pertains to methods of risk assessment (or diagnosis) performed by a layman or a non-medical professional. Recent technological advances in genotyping technologies, including high-throughput genotyping of SNP markers, such as Molecular Inversion Probe array technology (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays) have made it possible for individuals to have their own genome assessed for large number of variations simultaneously, or up to one million SNPs. The resulting genotype information, made available to the individual, can be compared to information from the public scientific literature about disease or trait risk associated with various SNPs. The diagnostic application of disease-associated alleles as described herein, can thus be performed either by a health professional based on results of a clinical test or by a layman, or non-medical professional, including an individual providing service for performing an assessment of SNPs through SNP genotyping, either on an individual SNP basis or by large-scale high-throughput methods such as array technologies. In other words, the diagnosis or assessment of a susceptibility based on genetic risk can be made by health professionals, genetic counselors, genotype services providers or by the layman, based on information about his/her genotype and publications on various risk factors. In the present context, the term "diagnosing", and "diagnose a susceptibility", is meant to refer to any available diagnostic method, including those mentioned above.

[0152] In addition, in certain other embodiments, the present invention pertains to methods of diagnosing, or aiding in the diagnosis of, a decreased susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke by detecting particular genetic marker alleles or haplotypes that appear less frequently in cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke patients than in individual not diagnosed with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke or in the general population.

[0153] As described and exemplified herein, particular marker alleles or haplotypes (e.g. the markers and haplotypes as listed in Table 5 (Tables 5A and 5B) and markers in linkage disequilibrium therewith, e.g., the markers listed in Tables 4 and/or 9 markers in linkage disequilibrium therewith, e.g., the markers as set forth in Table 19) are associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. In one embodiment, the marker allele or haplotype is one that confers a significant risk or susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. In another embodiment, the invention relates to a method of diagnosing a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke in a human individual, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from the group consisting of the polymorphic markers listed in Tables 5A and 5B, and markers in linkage disequilibrium therewith. In another embodiment, the invention pertains to methods of diagnosing a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke in a human individual, by screening for at least one marker allele or haplotype as listed in Tables 5A and 5B or markers in linkage disequilibrium therewith. In another embodiment, the marker allele or haplotype is more frequently present in a subject having, or who is susceptible to, cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke (affected), as compared to the frequency of its presence in a healthy subject (control, such as population controls). In certain embodiments, the significance of association of the at least one marker allele or haplotype is characterized by a p value<0.05. In other embodiments, the significance of association is characterized by smaller p-values, such as <0.01, <0.001, <0.0001, <0.00001, <0.000001, <0.0000001, <0.00000001 or <0.000000001.

[0154] In these embodiments, the presence of the at least one marker allele or haplotype is indicative of a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. These diagnostic methods involve detecting the presence or absence of at least one marker allele or haplotype that is associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. The haplotypes described herein include combinations of alleles at various genetic markers (e.g., SNPs, microsatellites). The detection of the particular genetic marker alleles that make up the particular haplotypes can be performed by a variety of methods described herein and/or known in the art. For example, genetic markers can be detected at the nucleic acid level (e.g., by direct nucleotide sequencing or by other means known to the skilled in the art) or at the amino acid level if the genetic marker affects the coding sequence of a protein encoded by a cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke-associated nucleic acid (e.g., by protein sequencing or by immunoassays using antibodies that recognize such a protein). The marker alleles or haplotypes of the present invention correspond to fragments of a genomic DNA sequence associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. Such fragments encompass the DNA sequence of the polymorphic marker or haplotype in question, but may also include DNA segments in strong LD (linkage disequilibrium) with the marker or haplotype. In one embodiment, such segments comprises segments in LD with the marker or haplotype as determined by a value of r² greater than 0.2 and/or |D'|>0.8.

[0155] In one embodiment, diagnosis of a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke can be accomplished using hybridization methods, such as Southern analysis, Northern analysis, and/or in situ hybridizations (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, including all supplements). A biological sample from a test subject or individual (a "test sample") of genomic DNA, RNA, or cDNA is obtained from a subject suspected of having, being susceptible to, or predisposed for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke (the "test subject"). The subject can be an adult, child, or fetus. The test sample can be from any source that contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs. A test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling. The DNA, RNA, or cDNA sample is then examined. The presence of a specific marker allele can be indicated by sequence-specific hybridization of a nucleic acid probe specific for the particular allele. The presence of more than specific marker allele or a specific haplotype can be indicated by using several sequence-specific nucleic acid probes, each being specific for a particular allele. In one embodiment, a haplotype can be indicated by a single nucleic acid probe that is specific for the specific haplotype (i.e., hybridizes specifically to a DNA strand comprising the specific marker alleles characteristic of the haplotype). A sequence-specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA. A "nucleic acid probe", as used herein, can be a DNA probe or an RNA probe that hybridizes to a complementary sequence. One of skill in the art would know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample.

[0156] To diagnose a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, a hybridization sample is formed by contacting the test sample containing an atrial fibrillation and/or stroke-associated nucleic acid, such as a genomic DNA sample, with at least one nucleic acid probe. A non-limiting example of a probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe that is capable of hybridizing to mRNA or genomic DNA sequences described herein. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length that is sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA. The nucleotide acid probe may be up to 1000 or more nucleotides in length, including up to 500 nucleotides, 400 nucleotide, 300 nucleotides, 200 nucleotides or 100 nucleotides. Certain embodiments include nucleotide probes that are from 15 to 1000 nucleotides in length. Other embodiments pertain to use of nucleotide probes that are from 15 to 500 nucleotides in length, or from 15 to 400 nucleotides in length, or from 20 to 400 nucleotides in length. Other size ranges of the nucleotide probes of the invention are contemplated, as well known to the skilled person. In one embodiment, the nucleic acid probe can comprise all or a portion of the nucleotide sequence of LD Block C04, as described herein, optionally comprising at least one allele of a marker described herein, or at least one haplotype described herein, or the probe can be the complementary sequence of such a sequence. In a particular embodiment, the nucleic acid probe is a portion of the nucleotide sequence of LD Block C04 as set forth in SEQ ID NO:50 or, as described herein, optionally comprising at least one allele of a marker described herein, or at least one allele of one polymorphic marker or haplotype comprising at least one polymorphic marker described herein, or the probe can be the complementary sequence of such a sequence. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization can be performed by methods well known to the person skilled in the art (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, including all supplements). In one embodiment, hybridization refers to specific hybridization, i.e., hybridization with no mismatches (exact hybridization). In one embodiment, the hybridization conditions for specific hybridization are high stringency.

[0157] Specific hybridization, if present, is detected using standard methods. If specific hybridization occurs between the nucleic acid probe and the nucleic acid in the test sample, then the sample contains the allele that is complementary to the nucleotide that is present in the nucleic acid probe. The process can be repeated for any markers of the present invention, or markers that make up a haplotype of the present invention, or multiple probes can be used concurrently to detect more than one marker alleles at a time. It is also possible to design a single probe containing more than one marker alleles of a particular haplotype (e.g., a probe containing alleles complementary to 2, 3, 4, 5 or all of the markers that make up a particular haplotype). Detection of the particular markers of the haplotype in the sample is indicative that the source of the sample has the particular haplotype (e.g., a haplotype) and therefore is susceptible to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.

[0158] In another hybridization method, Northern analysis (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, supra) is used to identify the presence of a polymorphism associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. For Northern analysis, a test sample of RNA is obtained from the subject by appropriate means. As described herein, specific hybridization of a nucleic acid probe to RNA from the subject is indicative of a particular allele complementary to the probe. For representative examples of use of nucleic acid probes, see, for example, U.S. Pat. Nos. 5,288,611 and 4,851,330.

[0159] Additionally, or alternatively, a peptide nucleic acid (PNA) probe can be used in addition to, or instead of, a nucleic acid probe in the hybridization methods described herein. A PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P., et al., Bioconjug. Chem. 5:3-7 (1994)). The PNA probe can be designed to specifically hybridize to a molecule in a sample suspected of containing one or more of the marker alleles or haplotypes that are associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. Hybridization of the PNA probe is thus diagnostic for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke or a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.

[0160] In one embodiment of the invention, a test sample containing genomic DNA obtained from the subject is collected and the polymerase chain reaction (PCR) is used to amplify a fragment comprising one or more markers or haplotypes of the present invention. As described herein, identification of a particular marker allele or haplotype associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, can be accomplished using a variety of methods (e.g., sequence analysis, analysis by restriction digestion, specific hybridization, single stranded conformation polymorphism assays (SSCP), electrophoretic analysis, etc.). In another embodiment, diagnosis is accomplished by expression analysis using quantitative PCR (kinetic thermal cycling). This technique can, for example, utilize commercially available technologies, such as TaqMan® (Applied Biosystems, Foster City, Calif.). The technique can assess the presence of an alteration in the expression or composition of a polypeptide or spiking variant(s) that is encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. Further, the expression of the variant(s) can be quantified as physically or functionally different.

[0161] In another method of the invention, analysis by restriction digestion can be used to detect a particular allele if the allele results in the creation or elimination of a restriction site relative to a reference sequence. Restriction fragment length polymorphism (RFLP) analysis can be conducted, e.g., as described in Current Protocols in Molecular Biology, supra. The digestion pattern of the relevant DNA fragment indicates the presence or absence of the particular allele in the sample.

[0162] Sequence analysis can also be used to detect specific alleles or haplotypes associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke (e.g. the polymorphic markers of Table 5 (Tables 5A and 5B), Table 9 and/or Table 19). Therefore, in one embodiment, determination of the presence or absence of a particular marker alleles or haplotypes comprises sequence analysis of a test sample of DNA or RNA obtained from a subject or individual. PCR or other appropriate methods can be used to amplify a portion of a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, and the presence of a specific allele can then be detected directly by sequencing the polymorphic site (or multiple polymorphic sites in a haplotype) of the genomic DNA in the sample.

[0163] Allele-specific oligonucleotides can also be used to detect the presence of a particular allele in a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, (e.g. the polymorphic markers of Table 5 (Tables 5A and 5B), Table 9 and/or Table 19), through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, Saiki, R. et al., Nature, 324: 163-166 (1986)). An "allele-specific oligonucleotide" (also referred to herein as an "allele-specific oligonucleotide probe") is an oligonucleotide of approximately 10-500 base pairs, approximately 15-400 base pairs, approximately 15-200 base pairs, approximately 15-100 base pairs, approximately 15-50 base pairs, or approximately 15-30 base pairs, that specifically hybridizes to a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, and which contains a specific allele at a polymorphic site (e.g., a marker or haplotype as described herein). An allele-specific oligonucleotide probe that is specific for one or more particular a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke can be prepared using standard methods (see, e.g., Current Protocols in Molecular Biology, supra). PCR can be used to amplify the desired region. The DNA containing the amplified region can be dot-blotted using standard methods (see, e.g., Current Protocols in Molecular Biology, supra), and the blot can be contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified region can then be detected. Specific hybridization of an allele-specific oligonucleotide probe to DNA from the subject is indicative of a specific allele at a polymorphic site associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke (see, e.g., Gibbs, R. et al., Nucleic Acids Res., 17:2437-2448 (1989) and WO 93/22456).

[0164] With the addition of such analogs as locked nucleic acids (LNAs), the size of primers and probes can be reduced to as few as 8 bases. LNAs are a novel class of bicyclic DNA analogs in which the 2' and 4' positions in the furanose ring are joined via an O-methylene (oxy-LNA), S-methylene (thio-LNA), or amino methylene (amino-LNA) moiety. Common to all of these LNA variants is an affinity toward complementary nucleic acids, which is by far the highest reported for a DNA analog. For example, particular all oxy-LNA nonamers have been shown to have melting temperatures (T_m) of 64° C. and 74° C. when in complex with complementary DNA or RNA, respectively, as opposed to 28° C. for both DNA and RNA for the corresponding DNA nonamer. Substantial increases in T_m are also obtained when LNA monomers are used in combination with standard DNA or RNA monomers. For primers and probes, depending on where the LNA monomers are included (e.g., the 3' end, the 5' end, or in the middle), the T_m could be increased considerably.

[0165] In another embodiment, arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from a subject, can be used to identify polymorphisms in a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke (e.g. the polymorphic markers of Tables 5A and 5B and markers in linkage disequilibrium therewith). For example, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also described as "Genechips®," have been generally described in the art (see, e.g., U.S. Pat. No. 5,143,854, PCT Patent Publication Nos. WO 90/15070 and 92/10092). These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods, or by other methods known to the person skilled in the art (see, e.g., Fodor, S. et al., Science, 251:767-773 (1991); Pirrung et al., U.S. Pat. No. 5,143,854 (see also published PCT Application No. WO 90/15070); and Fodor. S. et al., published PCT Application No. WO 92/10092 and U.S. Pat. No. 5,424,186, the entire teachings of each of which are incorporated by reference herein). Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261; the entire teachings of which are incorporated by reference herein. In another example, linear arrays can be utilized. Additional descriptions of use of oligonucleotide arrays for detection of polymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and 5,837,832, the entire teachings of both of which are incorporated by reference herein.

[0166] Other methods of nucleic acid analysis that are available to those skilled in the art can be used to detect a particular allele at a polymorphic site associated with atrial fibrillation and/or stroke (e.g. the polymorphic markers of Table 5 (Tables 5A and 5B), Table 9 and/or Table 19). Representative methods include, for example, direct manual sequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA, 81: 1991-1995 (1988); Sanger, F., et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977); Beavis, et al., U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single-stranded conformation polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield, V., et al., Proc. Natl. Acad. Sci. USA, 86:232-236 (1989)), mobility shift analysis (Orita, M., et al., Proc. Natl. Acad. Sci. USA, 86:2766-2770 (1989)), restriction enzyme analysis (Flavell, R., et al., Cell, 15:25-41 (1978); Geever, R., et al., Proc. Natl. Acad. Sci. USA, 78:5081-5085 (1981)); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton, R., et al., Proc. Natl. Acad. Sci. USA, 85:4397-4401 (1985)); RNase protection assays (Myers, R., et al., Science, 230:1242-1246 (1985); use of polypeptides that recognize nucleotide mismatches, such as E. coli mutS protein; and allele-specific PCR.

[0167] In another embodiment of the invention, diagnosis of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke or a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke can be made by examining expression and/or composition of a polypeptide encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke in those instances where the genetic marker(s) or haplotype(s) of the present invention result in a change in the composition or expression of the polypeptide. Thus, diagnosis of a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke can be made by examining expression and/or composition of one of these polypeptides, or another polypeptide encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, in those instances where the genetic marker or haplotype of the present invention results in a change in the composition or expression of the polypeptide. The haplotypes and markers of the present invention that show association to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke may play a role through their effect on one or more of these nearby genes (e.g., the PITX2 gene). Possible mechanisms affecting these genes include, e.g., effects on transcription, effects on RNA splicing, alterations in relative amounts of alternative splice forms of mRNA, effects on RNA stability, effects on transport from the nucleus to cytoplasm, and effects on the efficiency and accuracy of translation.

[0168] Thus, in another embodiment, the variants (markers or haplotypes) of the invention showing association to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke affect the expression of a nearby gene. It is well known that regulatory element affecting gene expression may be located tenths or even hundreds of kilobases away from the promoter region of a gene. By assaying for the presence or absence of at least one allele of at least one polymorphic marker of the present invention, it is thus possible to assess the expression level of such nearby genes. It is thus contemplated that the detection of the markers or haplotypes of the present invention can be used for assessing expression for one or more genes that are linked to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke.

[0169] A variety of methods can be used for detecting protein expression levels, including enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence. A test sample from a subject is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. An alteration in expression of a polypeptide encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced). An alteration in the composition of a polypeptide encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke is an alteration in the qualitative polypeptide expression (e.g., expression of a mutant polypeptide or of a different splicing variant). In one embodiment, diagnosis of a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke is made by detecting a particular splicing variant encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, or a particular pattern of splicing variants.

[0170] Both such alterations (quantitative and qualitative) can also be present. An "alteration" in the polypeptide expression or composition, as used herein, refers to an alteration in expression or composition in a test sample, as compared to the expression or composition of the polypeptide in a control sample. A control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from a subject who is not affected by, and/or who does not have a susceptibility to, cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. In one embodiment, the control sample is from a subject that does not possess a marker allele or haplotype as described herein. Similarly, the presence of one or more different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample, can be indicative of a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample, can be indicative of a specific allele in the instance where the allele alters a splice site relative to the reference in the control sample. Various means of examining expression or composition of a polypeptide encoded by a nucleic acid are known to the person skilled in the art and can be used, including spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and immunoassays (e.g., David et al., U.S. Pat. No. 4,376,110) such as immunoblotting (see, e.g., Current Protocols in Molecular Biology, particularly chapter 10, supra).

[0171] For example, in one embodiment, an antibody (e.g., an antibody with a detectable label) that is capable of binding to a polypeptide encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke can be used. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a labeled secondary antibody (e.g., a fluorescently-labeled secondary antibody) and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.

[0172] In one embodiment of this method, the level or amount of polypeptide encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke in a test sample is compared with the level or amount of the polypeptide in a control sample. A level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of an alteration in the expression of the polypeptide encoded by the nucleic acid, and is diagnostic for a particular allele or haplotype responsible for causing the difference in expression. Alternatively, the composition of the polypeptide in a test sample is compared with the composition of the polypeptide in a control sample. In another embodiment, both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample.

[0173] In another embodiment, the diagnosis of a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke is made by detecting at least one marker or haplotypes of the present invention (e.g., associated alleles of the markers listed in Tables 5A and 5B, and markers in linkage disequilibrium therewith), in combination with an additional protein-based, RNA-based or DNA-based assay. The methods of the invention can also be used in combination with an analysis of a subject's family history and risk factors (e.g., environmental risk factors, lifestyle risk factors).

Kits

[0174] Kits useful in the methods and procedures of the invention comprise components useful in any of the methods described herein, including for example, hybridization probes, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies that bind to an altered polypeptide encoded by a nucleic acid of the invention as described herein (e.g., a genomic segment comprising at least one polymorphic marker and/or haplotype of the present invention) or to a non-altered (native) polypeptide encoded by a nucleic acid of the invention as described herein, means for amplification of a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, means for analyzing the nucleic acid sequence of a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, means for analyzing the amino acid sequence of a polypeptide encoded by a nucleic acid associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, etc. The kits can for example include necessary buffers, nucleic acid primers for amplifying nucleic acids of the invention (e.g., one or more of the polymorphic markers as described herein), and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase). Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke diagnostic assays.

[0175] In one embodiment, the invention is a kit for assaying a sample from a subject to detect the presence of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke or a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke in a subject, wherein the kit comprises reagents necessary for selectively detecting at least one allele of at least one polymorphism of the present invention in the genome of the individual. In a particular embodiment, the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising at least one polymorphism of the present invention. In another embodiment, the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes at least one polymorphism, wherein the polymorphism is selected from the group consisting of the polymorphisms as listed in Tables 5A and 5B and polymorphic markers in linkage disequilibrium therewith. In yet another embodiment the fragment is at least 20 base pairs in size. Such oligonucleotides or nucleic acids (e.g., oligonucleotide primers) can be designed using portions of the nucleic acid sequence flanking polymorphisms (e.g., SNPs or microsatellites) that are indicative of cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. In another embodiment, the kit comprises one or more labeled nucleic acids capable of allele-specific detection of one or more specific polymorphic markers or haplotypes associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, and reagents for detection of the label. Suitable labels include, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.

[0176] In particular embodiments, the polymorphic marker or haplotype to be detected by the reagents of the kit comprises one or more markers, two or more markers, three or more markers, four or more markers or five or more markers selected from the group consisting of the markers in Tables 5A and 5B. In another embodiment, the marker or haplotype to be detected comprises the markers listed in Tables 5A and 5B. In another embodiment, the marker or haplotype to be detected comprises the markers listed in Tables 4 and 9. In another embodiment, the marker or haplotype to be detected comprises at least one marker from the group of markers in strong linkage disequilibrium, as defined by values of r² greater than 0.2, to at least one of the group of markers consisting of the markers listed in Tables 5A and 5B. In another embodiment, the marker or haplotype to be detected comprises at least one marker from the markers in strong linkage disequilibrium, as defined by values of r² greater than 0.2, to at least one of the group of markers consisting of the markers listed in Tables 4 and 9. In another embodiment, the marker or haplotype to be detected comprises marker rs2220427 (SEQ ID NO:1) or marker rs1033464 (SEQ ID NO:41), or markers in linkage disequilibrium therewith. In another embodiment, the marker or haplotype to be detected comprises at least one of the markers set forth in Table 19. In another embodiment, the marker or haplotype to be detected comprises markers D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51) and markers in linkage disequilibrium therewith. In yet another embodiment, the marker or haplotype comprises the at-risk alleles-2, -4 and/or -8 in marker D4S406, allele A of marker rs2634073, allele T of marker rs2200733, allele T of marker rs2220427, allele T of marker rs10033464, and/or allele G of marker rs13143308. In one such embodiment, linkage disequilibrium is defined by values of r² greater than 0.1. In another such embodiment, linkage disequilibrium is defined by values of r² greater than 0.2.

[0177] In one preferred embodiment, the kit for detecting the markers of the invention comprises a detection oligonucleotide probe, that hybridizes to a segment of template DNA containing a SNP polymorphisms to be detected, an enhancer oligonucleotide probe and an endonuclease. As explained in the above, the detection oligonucleotide probe comprises a fluorescent moiety or group at its 3' terminus and a quencher at its 5° terminus, and an enhancer oligonucleotide, is employed, as described by Kutyavin et al. (Nucleic Acid Res. 34:e128 (2006)). The fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties. The detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected. Preferably, the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template. The gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV. The enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch. Thus, by measuring the fluorescence of the released fluorescent moiety, assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.

[0178] The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.

[0179] In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection, and primers for such amplification are included in the reagent kit. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.

[0180] Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G. The use of modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule. In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.

[0181] In one of such embodiments, the presence of the marker or haplotype is indicative of a susceptibility (increased susceptibility or decreased susceptibility) to atrial fibrillation and/or stroke. In another embodiment, the presence of the marker or haplotype is indicative of response to atrial fibrillation and/or stroke therapeutic agent. In another embodiment, the presence of the marker or haplotype is indicative of atrial fibrillation and/or stroke prognosis. In yet another embodiment, the presence of the marker or haplotype is indicative of progress of atrial fibrillation and/or stroke treatment. Such treatment may include intervention by surgery, medication or by other means (e.g., lifestyle changes).

Therapeutic Agents

[0182] Variants of the present invention (e.g., the markers and/or haplotypes of the invention, e.g., the markers listed in Tables 5A and 5B and/or Table 19) can be used to identify novel therapeutic targets for cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. For example, genes containing, or in linkage disequilibrium with, variants (markers and/or haplotypes) associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, or their products, as well as genes or their products that are directly or indirectly regulated by or interact with these variant genes or their products, can be targeted for the development of therapeutic agents to treat cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, or prevent or delay onset of symptoms associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. Therapeutic agents may comprise one or more of, for example, small non-protein and non-nucleic acid molecules, proteins, peptides, protein fragments, nucleic acids (DNA, RNA), PNA (peptide nucleic acids), or their derivatives or mimetics which can modulate the function and/or levels of the target genes or their gene products.

[0183] The nucleic acids and/or variants of the invention, or nucleic acids comprising their complementary sequence, may be used as antisense constructs to control gene expression in cells, tissues or organs. The methodology associated with antisense techniques is well known to the skilled artisan, and is described and reviewed in Antisense Drug Technology: Principles, Strategies, and Applications, Crooke, ed., Marcel Dekker Inc., New York (2001). In general, antisense nucleic acid molecules are designed to be complementary to a region of mRNA expressed by a gene, so that the antisense molecule hybridizes to the mRNA, thus blocking translation of the mRNA into protein. Several classes of antisense oligonucleotide are known to those skilled in the art, including cleavers and blockers. The former bind to target RNA sites, activate intracellular nucleases (e.g., RnaseH or Rnase L), that cleave the target RNA. Blockers bind to target RNA, inhibit protein translation by steric hindrance of the ribosomes. Examples of blockers include nucleic acids, morpholino compounds, locked nucleic acids and methylphosphonates (Thompson, Drug Discovery Today, 7:912-917 (2002)). Antisense oligonucleotides are useful directly as therapeutic agents, and are also useful for determining and validating gene function, for example by gene knock-out or gene knock-down experiments. Antisense technology is further described in Layery et al., Curr. Opin. Drug Discov. Devel. 6:561-569 (2003), Stephens et al., Curr. Opin. Mol. Ther. 5:118-122 (2003), Kurreck, Eur. J. Biochem. 270:1628-44 (2003), Dias et al., Mol. Cancer Ter. 1:347-55 (2002), Chen, Methods Mol. Med. 75:621-636 (2003), Wang et al., Curr. Cancer Drug Targets 1:177-96 (2001), and Bennett, Antisense Nucleic Acid Drug. Dev. 12:215-24 (2002)

[0184] The variants described herein can be used for the selection and design of antisense reagents that are specific for particular variants. Using information about the variants described herein, antisense oligonucleotides or other antisense molecules that specifically target mRNA molecules that contain one or more variants of the invention can be designed. In this manner, expression of mRNA molecules that contain one or more variant of the present invention (markers and/or haplotypes) can be inhibited or blocked. In one embodiment, the antisense molecules are designed to specifically bind a particular allelic form (i.e., one or several variants (alleles and/or haplotypes)) of the target nucleic acid, thereby inhibiting translation of a product originating from this specific allele or haplotype, but which do not bind other or alternate variants at the specific polymorphic sites of the target nucleic acid molecule.

[0185] As antisense molecules can be used to inactivate mRNA so as to inhibit gene expression, and thus protein expression, the molecules can be used to treat a disease or disorder, such as cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. The methodology can involve cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Such mRNA regions include, for example, protein-coding regions, in particular protein-coding regions corresponding to catalytic activity, substrate and/or ligand binding sites, or other functional domains of a protein.

[0186] The phenomenon of RNA interference (RNAi) has been actively studied for the last decade, since its original discovery in C. elegans (Fire et al., Nature 391:806-11 (1998)), and in recent years its potential use in treatment of human disease has been actively pursued (reviewed in Kim & Rossi, Nature Rev. Genet. 8:173-204 (2007)). RNA interference (RNAi), also called gene silencing, is based on using double-stranded RNA molecules (dsRNA) to turn off specific genes. In the cell, cytoplasmic double-stranded RNA molecules (dsRNA) are processed by cellular complexes into small interfering RNA (siRNA). The siRNA guide the targeting of a protein-RNA complex to specific sites on a target mRNA, leading to cleavage of the mRNA (Thompson, Drug Discovery Today, 7:912-917 (2002)). The siRNA molecules are typically about 20, 21, 22 or 23 nucleotides in length. Thus, one aspect of the invention relates to isolated nucleic acid molecules, and the use of those molecules for RNA interference, i.e. as small interfering RNA molecules (siRNA). In one embodiment, the isolated nucleic acid molecules are 18-26 nucleotides in length, preferably 19-25 nucleotides in length, more preferably 20-24 nucleotides in length, and more preferably 21, 22 or 23 nucleotides in length.

[0187] Another pathway for RNAi-mediated gene silencing originates in endogenously encoded primary microRNA (pri-miRNA) transcripts, which are processed in the cell to generate precursor miRNA (pre-miRNA). These miRNA molecules are exported from the nucleus to the cytoplasm, where they undergo processing to generate mature miRNA molecules (miRNA), which direct translational inhibition by recognizing target sites in the 3' untranslated regions of mRNAs, and subsequent mRNA degradation by processing P-bodies (reviewed in Kim & Rossi, Nature Rev. Genet. 8:173-204 (2007)).

[0188] Clinical applications of RNAi include the incorporation of synthetic siRNA duplexes, which preferably are approximately 20-23 nucleotides in size, and preferably have 3' overlaps of 2 nucleotides. Knockdown of gene expression is established by sequence-specific design for the target mRNA. Several commercial sites for optimal design and synthesis of such molecules are known to those skilled in the art.

[0189] Other applications provide longer siRNA molecules (typically 25-30 nucleotides in length, preferably about 27 nucleotides), as well as small hairpin RNAs (shRNAs; typically about 29 nucleotides in length). The latter are naturally expressed, as described in Amarzguioui et al. (FEBS Lett. 579:5974-81 (2005)). Chemically synthetic siRNAs and shRNAs are substrates for in vivo processing, and in some cases provide more potent gene-silencing than shorter designs (Kim et al., Nature Biotechnol. 23:222-226 (2005); Siolas et al., Nature Biotechnol. 23:227-231 (2005)). In general siRNAs provide for transient silencing of gene expression, because their intracellular concentration is diluted by subsequent cell divisions. By contrast, expressed shRNAs mediate long-term, stable knockdown of target transcrips, for as long as transcription of the shRNA takes place (Marques et al., Nature Biotechnol. 23:559-565 (2006); Brummelkamp et al., Science 296: 550-553 (2002)).

[0190] Since RNAi molecules, including siRNA, miRNA and shRNA, act in a sequence-dependent manner, the variants of the present invention (e.g., the markers and haplotypes associated with LD block C04, e.g., the markers listed in Tables 5A and 5B) can be used to design RNAi reagents that recognize specific nucleic acid molecules comprising specific alleles and/or haplotypes (e.g., the alleles and/or haplotypes of the present invention), while not recognizing nucleic acid molecules comprising other alleles or haplotypes. These RNAi reagents can thus recognize and destroy the target nucleic acid molecules. As with antisense reagents, RNAi reagents can be useful as therapeutic agents (i.e., for turning off disease-associated genes or disease-associated gene variants), but may also be useful for characterizing and validating gene function (e.g., by gene knock-out or gene knock-down experiments).

[0191] Delivery of RNAi may be performed by a range of methodologies known to those skilled in the art. Methods utilizing non-viral delivery include cholesterol, stable nucleic acid-lipid particle (SNALP), heavy-chain antibody fragment (Fab), aptamers and nanoparticles. Viral delivery methods include use of lentivirus, adenovirus and adeno-associated virus. The siRNA molecules are in some embodiments chemically modified to increase their stability. This can include modifications at the 2' position of the ribose, including 2'-O-methylpurines and 2'-fluoropyrimidines, which provide resistance to Rnase activity. Other chemical modifications are possible and known to those skilled in the art.

[0192] The following references provide a further summary of RNAi, and possibilities for targeting specific genes using RNAi: Kim & Rossi, Nat. Rev. Genet. 8:173-184 (2007), Chen & Rajewsky, Nat. Rev. Genet. 8: 93-103 (2007), Reynolds, et al., Nat. Biotechnol. 22:326-330 (2004), Chi et al., Proc. Natl. Acad. Sci. USA 100:6343-6346 (2003), Vickers et al., J. Biol. Chem. 278:7108-7118 (2003), Agami, Curr. Opin. Chem. Biol. 6:829-834 (2002), Layery, et al., Curr. Opin. Drug Discov. Devel. 6:561-569 (2003), Shi, Trends Genet. 19:9-12 (2003), Shuey et al., Drug Discov. Today 7:1040-46 (2002), McManus et al., Nat. Rev. Genet. 3:737-747 (2002), Xia et al., Nat. Biotechnol. 20:1006-10 (2002), Plasterk et al., curr. Opin. Genet. Dev. 10:562-7 (2000), Bosher et al., Nat. Cell Biol. 2:E31-6 (2000), and Hunter, Curr. Biol. 9:R440-442 (1999).

[0193] A genetic defect leading to increased predisposition or risk for development of a disease, including cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, or a defect causing the disease, may be corrected permanently by administering to a subject carrying the defect a nucleic acid fragment that incorporates a repair sequence that supplies the normal/wild-type nucleotide(s) at the site of the genetic defect. Such site-specific repair sequence may concompass an RNA/DNA oligonucleotide that operates to promote endogenous repair of a subject's genomic DNA. The administration of the repair sequence may be performed by an appropriate vehicle, such as a complex with polyethelenimine, encapsulated in anionic liposomes, a viral vector such as an adenovirus vector, or other pharmaceutical compositions suitable for promoting intracellular uptake of the administered nucleic acid. The genetic defect may then be overcome, since the chimeric oligonucleotides induce the incorporation of the normal sequence into the genome of the subject, leading to expression of the normal/wild-type gene product. The replacement is propagated, thus rendering a permanent repair and alleviation of the symptoms associated with the disease or condition.

[0194] The present invention provides methods for identifying compounds or agents that can be used to treat cardiac arrhythmia, e.g. atrial fibrillation and atrial flutter, and stroke. Thus, the variants of the invention are useful as targets for the identification and/or development of therapeutic agents. Such methods may include assaying the ability of an agent or compound to modulate the activity and/or expression of a nucleic acid that includes at least one of the variants (markers and/or haplotypes) of the present invention, or the encoded product of the nucleic acid. This in turn can be used to identify agents or compounds that inhibit or alter the undesired activity or expression of the encoded nucleic acid product. Assays for performing such experiments can be performed in cell-based systems or in cell-free systems, as known to the skilled person. Cell-based systems include cells naturally expressing the nucleic acid molecules of interest, or recombinant cells that have been genetically modified so as to express a certain desired nucleic acid molecule.

[0195] Variant gene expression in a patient can be assessed by expression of a variant-containing nucleic acid sequence (for example, a gene containing at least one variant of the present invention, which can be transcribed into RNA containing the at least one variant, and in turn translated into protein), or by altered expression of a normal/wild-type nucleic acid sequence due to variants affecting the level or pattern of expression of the normal transcripts, for example variants in the regulatory or control region of the gene. Assays for gene expression include direct nucleic acid assays (mRNA), assays for expressed protein levels, or assays of collateral compounds involved in a pathway, for example a signal pathway. Furthermore, the expression of genes that are up- or down-regulated in response to the signal pathway can also be assayed. One embodiment includes operably linking a reporter gene, such as luciferase, to the regulatory region of the gene(s) of interest.

[0196] Modulators of gene expression can in one embodiment be identified when a cell is contacted with a candidate compound or agent, and the expression of mRNA is determined. The expression level of mRNA in the presence of the candidate compound or agent is compared to the expression level in the absence of the compound or agent. Based on this comparison, candidate compounds or agents for treating disorders such as atrial fibrillation, atrial flutter and stroke can be identified as those modulating the gene expression of the variant gene. When expression of mRNA or the encoded protein is statistically significantly greater in the presence of the candidate compound or agent than in its absence, then the candidate compound or agent is identified as a stimulator or up-regulator of expression of the nucleic acid. When nucleic acid expression or protein level is statistically significantly less in the presence of the candidate compound or agent than in its absence, then the candidate compound is identified as an inhibitor or down-regulator of the nucleic acid expression.

[0197] The invention further provides methods of treatment using a compound identified through drug (compound and/or agent) screening as a gene modulator (i.e. stimulator and/or inhibitor of gene expression).

[0198] In a further aspect of the present invention, a pharmaceutical pack (kit) is provided, the pack comprising a therapeutic agent and a set of instructions for administration of the therapeutic agent to humans diagnostically tested for one or more variants of the present invention, as disclosed herein. The therapeutic agent can be a small molecule drug, an antibody, a peptide, an antisense or RNAi molecule, or other therapeutic molecules. In one embodiment, an individual identified as a carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent. In one such embodiment, an individual identified as a homozygous carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent. In another embodiment, an individual identified as a non-carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent.

Methods of Assessing Probability of Response to Therapeutic Agents, Methods of Monitoring Progress of Treatment and Methods of Treatment

[0199] As is known in the art, individuals can have differential responses to a particular therapy (e.g., a therapeutic agent or therapeutic method). Pharmacogenomics addresses the issue of how genetic variations (e.g., the variants (markers and/or haplotypes) of the present invention) affect drug response, due to altered drug disposition and/or abnormal or altered action of the drug. Thus, the basis of the differential response may be genetically determined in part. Clinical outcomes due to genetic variations affecting drug response may result in toxicity of the drug in certain individuals (e.g., carriers or non-carriers of the genetic variants of the present invention), or therapeutic failure of the drug. Therefore, the variants of the present invention may determine the manner in which a therapeutic agent and/or method acts on the body, or the way in which the body metabolizes the therapeutic agent.

[0200] Accordingly, in one embodiment, the presence of a particular allele at a polymorphic site or haplotype is indicative of a different, e.g. a different response rate, to a particular treatment modality. This means that a patient diagnosed with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, and carrying a certain allele at a polymorphic or haplotype of the present invention (e.g., the at-risk and protective alleles and/or haplotypes of the invention) would respond better to, or worse to, a specific therapeutic, drug and/or other therapy used to treat the disease. Therefore, the presence or absence of the marker allele or haplotype could aid in deciding what treatment should be used for a the patient. For example, for a newly diagnosed patient, the presence of a marker or haplotype of the present invention may be assessed (e.g., through testing DNA derived from a blood sample, as described herein). If the patient is positive for a marker allele or haplotype at (that is, at least one specific allele of the marker, or haplotype, is present), then the physician recommends one particular therapy, while if the patient is negative for the at least one allele of a marker, or a haplotype, then a different course of therapy may be recommended (which may include recommending that no immediate therapy, other than serial monitoring for progression of the disease, be performed). Thus, the patient's carrier status could be used to help determine whether a particular treatment modality should be administered. The value lies within the possibilities of being able to diagnose the disease at an early stage, to select the most appropriate treatment, and provide information to the clinician about prognosis/aggressiveness of the disease in order to be able to apply the most appropriate treatment.

Treatment of Atrial Fibrillation and Atrial Flutter is Generally Directed by Two Main Objectives: (i) to Prevent Stroke and (ii) to Treat Symptoms.

(i) Stroke Prevention

[0201] Anticoagulation is the therapy of choice for stroke prevention in atrial fibrillation and is indicated for the majority of patients with this arrhythmia. The only patients for whom anticoagulation is not strongly recommended are those younger than 65 years old who are considered low-risk, i.e., they have no organic heart disease, no hypertension, no previous history of stroke or transient ischemic attacks and no diabetes. This group as a whole has a lower risk of stroke and stroke prevention with aspirin is generally recommended. For all other patients, anticoagulation is indicated whether the atrial fibrillation is permanent, recurrent paroxysmal or recurrent persistent. It cannot be generalized how patients who present with their first episode of paroxysmal atrial fibrillation should be treated and the decision needs to be individualized for each patient. Anticoagulation is also indicated even when the patient with atrial fibrillation is felt to be maintained in sinus rhythm with antiarrhythmic therapy (rhythm controlled) since this type of therapy does not affect stroke risk.

Anticoagulants.

[0202] Anticoagulation is recommended in atrial fibrillation, as detailed above, for prevention of cardioembolism and stroke. The most widely studied oral anticoagulant is warfarin and this medication is universally recommended for chronic oral anticoagulation in atrial fibrillation. Warfarin has few side effects aside from the risk of bleeding but requires regular and careful monitoring of blood values during therapy (to measure the effect of the anticoagulation). The oral anticoagulant ximelagatran showed promise in stroke prevention in patients with atrial fibrillation and had the advantage of not requiring regular monitoring like warfarin. Ximelagatran was found however to cause unexplained liver injury and was withdrawn from the market in 2006. Several agents are available for intravenous and/or subcutaneous therapy, including heparin and the low molecular weight heparins (e.g. enoxaparin, dalteparin, tinzaparin, ardeparin, nadroparin and reviparin). These medications are recommended when rapid initiation of anticoagulation is necessary or if oral anticoagulation therapy has to be interrupted in high risk patients or for longer than one week in other patients for example due to a series of procedures. Other parenteral anticoagulants are available but not specifically recommended as therapy in atrial fibrillation; e.g., the factor Xa inhibitors fondaparinux and idraparinux, the thrombin-inhibitors lepirudin, bivalirudin and argatroban as well as danaparoid.

(ii) Symptom Control.

[0203] Medical and surgical therapy applied to control symptoms of atrial fibrillation is tailored to the individual patient and consists of heart rate and/or rhythm control with medications, radiofrequency ablation and/or surgery.

Antiarrhythmic Medications.

[0204] In general terms, antiarrhythmic agents are used to suppress abnormal rhythms of the heart that are characteristic of cardiac arrhythmias, including atrial fibrillation and atrial flutter. One classification of antiarrhythmic agents is the Vaughan Williams classification, in which five main categories of antiarrhythmic agents are defined. Class I agents are fast sodium channel blockers and are subclassified based on kinetics and strength of blockade as well as their effect on repolarization. Class Ia includes disopyramide, moricizine, procainamide and quinidine. Class Ib agents are lidocaine, mexiletine, tocainide, and phenytoin. Class Ic agents are encainide, flecainide, propafenone, ajmaline, cibenzoline and detajmium. Class II agents are beta blockers, they block the effects of catecholamines at beta-adrenergic receptors. Examples of beta blockers are esmolol, propranolol, metoprolol, alprenolol, atenolol, carvedilol, bisoprolol, acebutolol, nadolol, pindolol, labetalol, oxprenotol, penbutolol, timolol, betaxolol, cartelol, sotalol and levobunolol. Class III agents have mixed properties but are collectively potassium channel blockers and prolong repolarization. Medications in this category are amiodarone, azimilide, bretylium, dofetilide, tedisamil, ibutilide, sematilide, sotalol, N-acetyl procainamide, nifekalant hydrochloride, vernakalant and ambasilide. Class IV agents are calcium channel blockers and include verapamil, mibefradil and diltiazem. Finally, class V consists of miscellaneous antiarrhythmics and includes digoxin and adenosine.

Heart Rate Control

[0205] Pharmacologic measures for maintenance of heart rate control include beta blockers, calcium channel blockers and digoxin. All these medications slow the electrical conduction through the atrioventricular node and slow the ventricular rate response to the rapid atrial fibrillation. Some antiarrhythmics used primarily for rhythm control (see below) also slow the atrioventricular node conduction rate and thus the ventricular heart rate response. These include some class III and Ic medications such as amiodarone, sotalol and flecainide.

Cardioversion.

[0206] Cardioversion of the heart rhythm from atrial fibrillation or atrial flutter to sinus rhythm can be achieved electrically, with synchronized direct-current cardioversion, or with medications such as ibutilide, amiodarone, procainamide, propafenone and flecainide.

Heart Rhythm Control

[0207] Medications used for maintenance of sinus rhythm, i.e. rhythm control, include mainly antiarrhythmic medications from classes III, Ia and Ic. Examples are sotalol, amiodarone and dofetilide from class III, disopyramide, procainamide and quinidine from class Ia and flecinide and propafenone from class Ic. Treatment with these antiarrhythmic medications is complicated, can be hazardous, and should be directed by physicians specifically trained to use these medications. Many of the antiarrhythmics have serious side effects and should only be used in specific populations. For example, class Ic medications should not be used in patients with coronary artery disease and even if they can suppress atrial fibrillation, they can actually promote rapid ventricular response in atrial flutter. Class Ia medications can be used as last resort in patients without structural heart diseases. Sotalol (as most class III antiarrhythmics) can cause significant prolongation of the QT interval, specifically in patients with renal failure, and promote serious ventricular arrhythmias. Both sotalol and dofetilide as well as the Ia medications need to be initiated on an inpatient basis to monitor the QT interval. Although amiodarone is usually well tolerated and is widely used, amiodarone has many serious side effects with long-term therapy.

How Genetic Testing May Directly Affect Choice of Treatment

[0208] When individuals present with their first (diagnosed) episode of paroxysmal atrial fibrillation and either spontaneously convert to sinus rhythm or undergo electrical or chemical cardioversion less than 48 hours into the episode, the decision to initiate, or not to initiate, anticoagulation therapy, is individualized based on the risk profile of the patient in question and the managing physicians preference. This can be a difficult choice to make since committing the patient to anticoagulation therapy has a major impact on the patients life. Often the choice is made to withhold anticoagulation in such a situation and this may be of no significant consequence to the patient. On the other hand the patient may later develop a stroke and the opportunity of prevention may thus have been missed. In such circumstances, knowing that the patient is a carrier of the at-risk variant may be of great significance and support initiation of anticoagulation treatment.

[0209] Individuals who are diagnosed with atrial fibrillation under the age of 65 and are otherwise considered low risk for stroke, i.e. have no organic heart disease, no hypertension, no diabetes and no previous history of stroke, are generally treated with aspirin only for stroke-prevention and not anticoagulation. If such a patient is found to be carrier for the at-risk variants described herein, this could be considered support for initiating anticoagulation earlier than otherwise recommended. This would be a reasonable consideration since the results of stroke from atrial fibrillation can be devastating.

[0210] Ischemic stroke is generally classified into five subtypes based on suspected cause; large artery atherosclerosis, small artery occlusion, cardioembolism (majority due to atrial fibrillation), stroke of other determined cause and stroke of undetermined cause (either no cause found or more than 1 plausible cause). Importantly, strokes due to cardioembolism have the highest recurrence, are most disabling and are associated with the lowest survival. It is therefore imperative not to overlook atrial fibrillation as the major cause of stroke, particularly since treatment measures vary based on the subtype. Therefore, if an individual is diagnosed with stroke or a transient ischemic attack and a plausible cause is not identified despite standard work-up, knowing that the patient is a carrier of the at-risk variant may be of great value and support either initiation of anticoagulation treatment or more aggressive diagnostic testing in the attempt to diagnose atrial fibrillation.

[0211] Furthermore, the markers of the present invention can be used to increase power and effectiveness of clinical trials. Thus, individuals who are carriers of at least one at-risk variant of the present invention, i.e. individuals who are carriers of at least one allele of at least one polymorphic marker conferring increased risk of developing cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke may be more likely to respond to a particular treatment modality, e.g., as described in the above. In one embodiment, individuals who carry at-risk variants for gene(s) in a pathway and/or metabolic network for which a particular treatment (e.g., small molecule drug) is targeting, are more likely to be responders to the treatment. In another embodiment, individuals who carry at-risk variants for a gene, which expression and/or function is altered by the at-risk variant, are more likely to be responders to a treatment modality targeting that gene, its expression or its gene product. This application can improve the safety of clinical trials, but can also enhance the chance that a clinical trial will demonstrate statistically significant efficacy, which may be limited to a certain sub-group of the population. Thus, one possible outcome of such a trial is that carriers of certain genetic variants, e.g., the markers and haplotypes of the present invention, are statistically significantly likely to show positive response to the therapeutic agent, i.e. experience alleviation of symptoms associated with cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke when taking the therapeutic agent or drug as prescribed.

[0212] In a further aspect, the markers and haplotypes of the present invention can be used for targeting the selection of pharmaceutical agents for specific individuals. Personalized selection of treatment modalities, lifestyle changes or combination of the two, can be realized by the utilization of the at-risk variants of the present invention. Thus, the knowledge of an individual's status for particular markers of the present invention, can be useful for selection of treatment options that target genes or gene products affected by the at-risk variants of the invention. Certain combinations of variants may be suitable for one selection of treatment options, while other gene variant combinations may target other treatment options. Such combination of variant may include one variant, two variants, three variants, or four or more variants, as needed to determine with clinically reliable accuracy the selection of treatment module.

Computer-Implemented Applications

[0213] The present invention also relates to computer-implemented applications of the polymorphic markers and haplotypes described herein to be associated with cardiac arrhythmia (e.g., atrial fibrillation and atrial flutter) and stroke. Such applications can be useful for storing, manipulating or otherwise analyzing genotype data that is useful in the methods of the invention. One example pertains to storing genotype information derived from an individual on readable media, so as to be able to provide the genotype information to a third party (e.g., the individual), or for deriving information from the genotype data, e.g., by comparing the genotype data to information about genetic risk factors contributing to increased susceptibility to cardiac arrhythmia (e.g., atrial fibrillation and atrial flutter) and stroke, and reporting results based on such comparison.

[0214] One such aspect relates to computer-readable media. In general terms, such medium has capabilities of storing (i) identifier information for at least one polymorphic marker or a haplotye; (ii) an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in individuals with cardiac arrhythmia (e.g., atrial fibrillation and atrial flutter) and/or stroke; and an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in a reference population. The reference population can be a disease-free population of individuals. Alternatively, the reference population is a random sample from the general population, and is thus representative of the population at large. The frequency indicator may be a calculated frequency, a count of alleles and/or haplotype copies, or normalized or otherwise manipulated values of the actual frequencies that are suitable for the particular medium.

[0215] Additional information about the individual can be stored on the medium, such as ancestry information, information about sex, physical attributes or characteristics (including height and weight), biochemical measurements (such as blood pressure, blood lipid levels, fasting glucose levels, insulin response measurements), biomarker results, or other useful information that is desirable to store or manipulate in the context of the genotype status of a particular individual.

[0216] The invention furthermore relates to an apparatus that is suitable for determination or manipulation of genetic data useful for determining a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation and atrial flutter) and stroke in a human individual. Such an apparatus can include a computer-readable memory, a routine for manipulating data stored on the computer-readable memory, and a routine for generating an output that includes a measure of the genetic data. Such measure can include values such as allelic or haplotype frequencies, genotype counts, sex, age, phenotype information, values for odds ratio (OR) or relative risk (RR), population attributable risk (PAR), or other useful information that is either a direct statistic of the original genotype data or based on calculations based on the genetic data.

[0217] The above-described applications can all be practiced with the markers and haplotypes of the invention that have in more detail been described with respect to methods of assessing susceptibility to cardiac arrhythmia (e.g., atrial fibrillation and atrial flutter) and stroke. Thus, these applications can in general be reduced to practice using markers listed in Tables 5, Table 4, Table 9, and Table 19, and markers in linkage disequilibrium therewith. In one embodiment, the markers or haplotypes are present within the genomic segment whose sequences is set forth in SEQ ID NO:50. In another embodiment, the markers or haplotypes comprise at least one marker selected from the markers set forth in Table 19. In another embodiment, the markers and haplotypes comprise at least one marker selected from D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51), optionally including markers in linkage disequilibrium therewith. In one such embodiment, linkage disequilibrium is defined by numerical values for r² of greater than 0.1. In another such embodiment, linkage disequilibrium is defined by numerical values for r² of greater than 0.2. In another embodiment, the marker or haplotype comprises at least one allele selected from alleles-2, -4 and/or -8 in marker D4S406, allele A of marker rs2634073, allele T of marker rs2200733, allele T of marker rs2220427, allele T of marker rs10033464, and/or allele G of marker rs13143308

Nucleic Acids and Polypeptides

[0218] The nucleic acids and polypeptides described herein can be used in methods an kits of the present invention, as described in the above.

[0219] An "isolated" nucleic acid molecule, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library). For example, an isolated nucleic acid of the invention can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC). An isolated nucleic acid molecule of the invention can comprise at least about 50%, at least about 80% or at least about 90% (on a molar basis) of all macromolecular species present. With regard to genomic DNA, the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. For example, the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.

[0220] The nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant DNA contained in a vector is included in the definition of "isolated" as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells or heterologous organisms, as well as partially or substantially purified DNA molecules in solution. "Isolated" nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention. An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means. Such isolated nucleotide sequences are useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis or other hybridization techniques.

[0221] The invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g., nucleic acid molecules that specifically hybridize to a nucleotide sequence containing a polymorphic site associated with a marker or haplotype described herein). In one embodiment, the invention includes variants that hybridize under high stringency hybridization and wash conditions (e.g., for selective hybridization) to a nucleotide sequence that comprises the nucleotide sequence of LD Block C04 (SEQ ID NO:50). Such nucleic acid molecules can be detected and/or isolated by allele- or sequence-specific hybridization (e.g., under high stringency conditions). Stringency conditions and methods for nucleic acid hybridizations are well known to the skilled person (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. et al, John Wiley & Sons, (1998), and Kraus, M. and Aaronson, S., Methods Enzymol., 200:546-556 (1991), the entire teachings of which are incorporated by reference herein.

[0222] The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The nucleotides or amino acids at corresponding positions are then compared, and the percent Identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions×100). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0), as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. See the website on the world wide web at ncbi.nlm.nih.gov. In one embodiment, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied (e.g., W=5 or W=20).

[0223] Other examples include the algorithm of Myers and Miller, CABIOS (1989), ADVANCE and ADAM as described in Torellis, A. and Robotti, C., Comput. Appl. Biosci. 10:3-5 (1994); and FASTA described in Pearson, W. and Lipman, D., Proc. Natl. Acad. Sci. USA, 85:2444-48 (1988). In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, Cambridge, UK).

[0224] The present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid that comprises, or consists of, the nucleotide sequence of LD Block C04 (SEQ ID NO:50), or a nucleotide sequence comprising, or consisting of, the complement of the nucleotide sequence of LD Block C04 (SEQ ID NO:50), wherein the nucleotide sequence comprises at least one polymorphic allele contained in the markers and haplotypes described herein. The nucleic acid fragments of the invention are at least about 15, at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500, 1000, 10,000 or more nucleotides in length.

[0225] The nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein. "Probes" or "primers" are oligonucleotides that hybridize in a base-specific manner to a complementary strand of a nucleic acid molecule. In addition to DNA and RNA, such probes and primers include polypeptide nucleic acids (PNA), as described in Nielsen, P. et al., Science 254:1497-1500 (1991). A probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule. In one embodiment, the probe or primer comprises at least one allele of at least one polymorphic marker or at least one haplotype described herein, or the complement thereof. In particular embodiments, a probe or primer can comprise 100 or fewer nucleotides; for example, in certain embodiments from 6 to 50 nucleotides, or, for example, from 12 to 30 nucleotides. In other embodiments, the probe or primer is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. In another embodiment, the probe or primer is capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.

[0226] The nucleic acid molecules of the invention, such as those described above, can be identified and isolated using standard molecular biology techniques well known to the skilled person. The amplified DNA can be labeled (e.g., radiolabeled) and used as a probe for screening a cDNA library derived from human cells. The cDNA can be derived from mRNA and contained in a suitable vector. Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art-recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.

[0227] In general, the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels, and as chromosome markers that are labeled to map related gene positions. The nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke or a susceptibility to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke, and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample (e.g., subtractive hybridization). The nucleic acid sequences can further be used to derive primers for genetic fingerprinting, to raise anti-polypeptide antibodies using immunization techniques, and/or as an antigen to raise anti-DNA antibodies or elicit immune responses.

Antibodies

[0228] Polyclonal antibodies and/or monoclonal antibodies that specifically bind one form of the gene product but not to the other form of the gene product are also provided. Antibodies are also provided which bind a portion of either the variant or the reference gene product that contains the polymorphic site or sites. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen-binding sites that specifically bind an antigen. A molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. A monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.

[0229] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or a fragment thereof. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, Inc., pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al., (eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.

[0230] Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a monoclonal antibody to a polypeptide of the invention (see, e.g., Current Protocols in Immunology, supra; Galfre et al., Nature 266:55052 (1977); R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); and Lerner, Yale J. Biol. Med. 54:387-402 (1981)). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods that also would be useful.

[0231] Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP® Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., Bio/Technology 9: 1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas 3:81-85 (1992); Huse et al., Science 246: 1275-1281 (1989); and Griffiths et al., EMBO J. 12:725-734 (1993).

[0232] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0233] In general, antibodies of the invention (e.g., a monoclonal antibody) can be used to isolate a polypeptide of the invention by standard techniques, such as affinity chromatography or immunoprecipitation. A polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells. Moreover, an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. The antibody can be coupled to a detectable substance to facilitate its detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³1I, ³⁵S or ³H.

[0234] Antibodies may also be useful in pharmacogenomic analysis. In such embodiments, antibodies against variant proteins encoded by nucleic acids according to the invention, such as variant proteins that are encoded by nucleic acids that contain at least one polymorphic marker of the invention, can be used to identify individuals that require modified treatment modalities.

[0235] Antibodies can furthermore be useful for assessing expression of variant proteins in disease states, such as in active stages of a disease, or in an individual with a predisposition to a disease related to the function of the protein, in particular cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke. Antibodies specific for a variant protein of the present invention that is encoded by a nucleic acid that comprises at least one polymorphic marker or haplotype as described herein can be used to screen for the presence of the variant protein, for example to screen for a predisposition to cardiac arrhythmia (e.g., atrial fibrillation or atrial flutter) and/or stroke as indicated by the presence of the variant protein.

[0236] Antibodies can be used in other methods. Thus, antibodies are useful as diagnostic tools for evaluating proteins, such as variant proteins of the invention, in conjunction with analysis by electrophoretic mobility, isoelectric point, tryptic or other protease digest, or for use in other physical assays known to those skilled in the art. Antibodies may also be used in tissue typing. In one such embodiment, a specific variant protein has been correlated with expression in a specific tissue type, and antibodies specific for the variant protein can then be used to identify the specific tissue type.

[0237] Subcellular localization of proteins, including variant proteins, can also be determined using antibodies, and can be applied to assess aberrant subcellular localization of the protein in cells in various tissues. Such use can be applied in genetic testing, but also in monitoring a particular treatment modality. In the case where treatment is aimed at correcting the expression level or presence of the variant protein or aberrrant tissue distribution or developmental expression of the variant protein, antibodies specific for the variant protein or fragments thereof can be used to monitor therapeutic efficacy.

[0238] Antibodies are further useful for inhibiting variant protein function, for example by blocking the binding of a variant protein to a binding molecule or partner. Such uses can also be applied in a therapeutic context in which treatment involves inhibiting a variant protein's function. An antibody can be for example be used to block or competitively inhibit binding, thereby modulating (i.e., agonizing or antagonizing) the activity of the protein. Antibodies can be prepared against specific protein fragments containing sites required for specific function or against an intact protein that is associated with a cell or cell membrane. For administration in vivo, an antibody may be linked with an additional therapeutic payload, such as radionuclide, an enzyme, an immunogenic epitope, or a cytotoxic agent, including bacterial toxins (diphteria or plant toxins, such as ricin). The in vivo half-life of an antibody or a fragment thereof may be increased by pegylation through conjugation to polyethylene glycol.

[0239] The present invention further relates to kits for using antibodies in the methods described herein. This includes, but is not limited to, kits for detecting the presence of a variant protein in a test sample. One preferred embodiment comprises antibodies such as a labelled or labelable antibody and a compound or agent for detecting variant proteins in a biological sample, means for determining the amount or the presence and/or absence of variant protein in the sample, and means for comparing the amount of variant protein in the sample with a standard, as well as instructions for use of the kit.

[0240] The present invention will now be exemplified by the following non-limiting example.

EXEMPLIFICATION

Example 1

Identification of At-Risk Variants for Atrial Fibrillation on Chromosome 4

[0241] The following contains description of the identification of susceptibility factors found to be associated with atrial fibrillation and stroke through single-point analysis of SNP markers.

Methods.

[0242] The study was approved by the Data Protection Commission of Iceland and the National Bioethics Committee.

Icelandic AF Cohort.

[0243] The patients were all diagnosed with AF at the Landspitali University Hospital in Reykjavik, Iceland, from 1987 to 2003. Diagnoses were confirmed by a 12 lead electrocardiogram demonstrating no P waves and irregularly irregular R-R intervals. All ECGs were manually read by a cardiologist.

Icelandic Stroke Cohort.

[0244] The stroke cohort was derived from two major hospitals in Iceland and the Icelandic Heart Association. Patients with hemorrhagic stroke represented 6% of all patients (patients with the Icelandic type of hereditary cerebral hemorrhage with amyloidosis and patients with subarachnoid hemorrhage were excluded). Ischemic stroke accounted for 67% of the total patients and TIAs 27%. The distribution of stroke subtypes in this study is similar to that reported in other Caucasian populations (Mohr, J. P., et al., Neurology, 28:754-762 (1978); L. R. Caplan, In Stroke, A Clinical Approach (Butterworth-Heinemann, Stoneham, Mass., ed 3, (1993)).

Genotyping.

[0245] A genome-wide scan of 437 Icelandic individuals diagnosed with Atrial Fibrillation (AF) and 7406 population controls was performed using Infinium HumanHap300 SNP chips from Illumina for assaying approximately 317,000 single nucleotide polymorphisms (SNPs) on a single chip (Illumina, San Diego, Calif., USA), SNP genotyping for replication in other case-control cohorts was carried using the Centaurus platform (Nanogen). A total of 347 individuals diagnosed with Stroke and 7497 controls was also performed for SNPs within the LD Block found to be associated with Atrial Fibrillation.

Statistical Methods for Association Analysis.

[0246] For single marker association to atrial fibrillation or stroke, we used a likelihood ratio test to calculate a two-sided p-value for each allele. We calculated relative risk (RR) and population attributable risk (PAR) assuming a multiplicative model (C. T. Falk, P. Rubinstein, Ann Hum Genet 51 (Pt 3), 227 (1987); J. D. Terwilliger, J. Ott, Hum Hered 42, 337 (1992)). For the CEPH Caucasian HapMap data, we calculated LD between pairs of SNPs using the standard definition of D' (R. C. Lewontin, Genetics 50, 757 (1964)) and R² W. G. Hill, A. Robertson, Genetics 60, 615 (November, 1968). When plotting all SNP combinations to elucidate the LD structure in a particular region, we plotted D' in the upper left corner and p-values in the lower right corner. In the LD plots we present, the markers are plotted equidistantly rather than according to their physical positions.

Results

Genome-Wide Association Study

[0247] We successfully genotyped 437 Icelandic Atrial Fibriallation patients and 7406 population control individuals using the Illumina 330K chip. Association analysis was performed for single SNPs. The most significant association was found for markers rs2220427 and rs2220733, both of which give p-values close to 10^-9. The value for rs2220427 is significant after correcting for the number of tests performed, i.e. the association is significant at the genome-wide level.

[0248] There is an apparent excess of homozygotes in affected individuals. We reject both the multiplicative model (P=0.002) and the recessive model (P=0.001). The best fitting model gives risk 1.46 to heterozygous carriers and 5.17 to homozygous carriers. The (uncorrected) P-value comparing this full model to the null model of no association is 5.43e-11. These data show that individuals with two copies of the at-risk allele are at greater risk than expected based on a simple multiplicative model.

[0249] Fitting an age at onset model for all genotypes gives a P-value of 4.84e-5. Heterozygotes are estimated to have onset 1.4090 years earlier than non-carriers and homozygote carriers are estimated to have onset 9.6126 years earlier than non-carriers. This shows a significant effect of age at onset--individuals carrying the at-risk variant are at significant risk of developing AF at a younger age than individuals who are non-carriers of the at-risk allele.

[0250] Investigating markers in the vicinity of rs2220427, we realized that the microsatellite marker D4S406 can be used as a surrogate marker for rs2220427. In particular, alleles-2, -4 and -8 (with respect to the CEPH reference) were found to be sufficient to tag the SNP based on haplotype frequencies:

TABLE-US-00001 TABLE 1 Relationship between rs2220427 and D4S406 Haplotype Frequency MS allele SNP Allele 7.55E-05 -2 D4S406 2 rs2220427 0.000109727 16 D4S406 4 rs2220427 0.000148065 -6 D4S406 4 rs2220427 0.000149685 20 D4S406 2 rs2220427 0.000149756 -4 D4S406 2 rs2220427 0.000210154 8 D4S406 4 rs2220427 0.000225802 -8 D4S406 2 rs2220427 0.000227036 4 D4S406 4 rs2220427 0.000299371 18 D4S406 2 rs2220427 0.000899281 0 D4S406 4 rs2220427 0.00203518 -4 D4S406 4 rs2220427 0.00673851 -6 D4S406 2 rs2220427 0.0245484 2 D4S406 2 rs2220427 0.0394983 -2 D4S406 4 rs2220427 0.0422112 14 D4S406 2 rs2220427 0.0594303 0 D4S406 2 rs2220427 0.0762831 -8 D4S406 4 rs2220427 0.0855451 6 D4S406 2 rs2220427 0.0949753 12 D4S406 2 rs2220427 0.100105 16 D4S406 2 rs2220427 0.145942 4 D4S406 2 rs2220427 0.155838 8 D4S406 2 rs2220427 0.164354 10 D4S406 2 rs2220427

Thus, for individuals typed for the D4S406 marker but not rs222047, merging the -2, -4 and -8 alleles leads to a very good estimate of the frequency of the 4 allele of the SNP.

[0251] We analyzed an Icelandic replication cohort for AF, comprised of 1269 cases and 69,070 controls, in this fashion. The results are quite dramatic in that the association is accompanied by a p-value of 2.94e-14 and a relative risk (multiplicative model) of 1.53. Thus, our initial finding has been replicated in an independent Icelandic cohort.

TABLE-US-00002 TABLE 2 Association of AF patients to Chromosome 4 (LD Block C04). Shown are values for RR under the multiplicative model. Relative p-value Risk Aff freq Con freq Allele Marker 0.16839039 0.8473 0.902746 0.916352 3 rs10033464 0.16839039 1.1802 0.097254 0.083648 4 rs10033464 0.24275346 0.8746 0.098398 0.110939 1 rs13105878 0.24275346 1.1433 0.901602 0.889061 2 rs13105878 4.89E-06 1.3816 0.441648 0.364078 1 rs13141190 4.89E-06 0.7238 0.558352 0.635922 3 rs13141190 1.25E-06 0.6905 0.677346 0.752498 1 rs1448817 1.25E-06 1.4483 0.322654 0.247502 3 rs1448817 0.00995996 0.7903 0.811213 0.844653 2 rs16997168 0.00995996 1.2654 0.188787 0.155347 4 rs16997168 1.07E-09 0.5601 0.811213 0.884688 2 rs2200733 1.07E-09 1.7855 0.188787 0.115312 4 rs2200733 7.78E-10 0.557 0.810345 0.884673 2 rs2220427 7.78E-10 1.7953 0.189655 0.115327 4 rs2220427 9.75E-08 1.5803 0.236239 0.163692 1 rs2634073 9.75E-08 0.6328 0.763761 0.836308 3 rs2634073 0.96927011 0.9968 0.768879 0.769444 1 rs2723296 0.96927011 1.0032 0.231121 0.230556 3 rs2723296 0.03281713 0.8529 0.665904 0.700311 2 rs2723316 0.03281713 1.1724 0.334096 0.299689 4 rs2723316 0.01803327 1.1855 0.635632 0.595393 1 rs3853444 0.01803327 0.8435 0.364368 0.404607 3 rs3853444 0.40105752 0.9214 0.146789 0.15734 2 rs4576077 0.40105752 1.0853 0.853211 0.84266 4 rs4576077 0.93269621 1.0084 0.145309 0.144275 1 rs6419178 0.93269621 0.9917 0.854691 0.855725 3 rs6419178

TABLE-US-00003 TABLE 3 Association of Stroke to markers within LD Block C04 (SEQ ID NO: 50) Relative p-value Risk Aff freq Con freq Allele Marker 0.37701852 0.8872 0.90634 0.916022 3 rs10033464 0.37701852 1.1272 0.09366 0.083978 4 rs10033464 0.2838194 0.8717 0.097983 0.110807 1 rs13105878 0.2838194 1.1472 0.902017 0.889193 2 rs13105878 0.01534596 1.2123 0.412104 0.366378 1 rs13141190 0.01534596 0.8249 0.587896 0.633622 3 rs13141190 0.04856224 0.8418 0.716138 0.7498 1 rs1448817 0.04856224 1.1879 0.283862 0.2502 3 rs1448817 0.14450185 0.8599 0.822767 0.843717 2 rs16997168 0.14450185 1.1629 0.177233 0.156283 4 rs16997168 0.00374992 0.724 0.84438 0.882271 2 rs2200733 0.00374992 1.3812 0.15562 0.117729 4 rs2200733 0.0025713 0.7141 0.842566 0.882274 2 rs2220427 0.0025713 1.4003 0.157434 0.117726 4 rs2220427 0.01664881 1.2682 0.201729 0.166154 1 rs2634073 0.01664881 0.7885 0.798271 0.833846 3 rs2634073 0.58350156 0.9511 0.760807 0.769811 1 rs2723296 0.58350156 1.0514 0.239193 0.230189 3 rs2723296 0.03150475 0.8367 0.661383 0.700107 2 rs2723316 0.03150475 1.1952 0.338617 0.299893 4 rs2723316 0.16517516 1.1172 0.622832 0.596462 1 rs3853444 0.16517516 0.8951 0.377168 0.403538 3 rs3853444 0.24301926 0.8797 0.14121 0.157473 2 rs4576077 0.24301926 1.1367 0.85879 0.842527 4 rs4576077 0.19773377 1.1482 0.161383 0.143543 1 rs6419178 0.19773377 0.8709 0.838617 0.856457 3 rs6419178

TABLE-US-00004 TABLE 4 Markers in perfect linkage disequilibrium (r² = 1.0) with rs2220427 in the CEU population in the International HapMap data set (Individuals of European descent). Also shown are correlation with samples from Yuroba (Nigeria), and Asia (China and Japan)- cohort description is further documented on http://www.hapmap.org. SNP Allele CEU_R2 CEU_frq YRI_R2 YRI_frq ASIA_R2 ASIA_frq rs17042059 1 1 0.117647 0.500382 0.117647 0.473183 0.30814 rs4529121 1 1 0.116667 0.604601 0.1 0.539766 0.337079 rs4543199 2 1 0.116667 0.502036 0.116667 0.539766 0.337079 rs10019689 1 1 0.116667 0.128175 0.341667 0.664071 0.388889 rs4626276 2 1 0.116667 0.603474 0.10084 0.537439 0.333333 rs17042076 2 1 0.117647 0.128175 0.341667 0.664071 0.388889 rs11098089 2 1 0.117647 0.549368 0.108333 0.539766 0.337079 rs11930528 4 1 0.11017 0.120773 0.321429 0.662926 0.377907 rs17042098 1 1 0.116667 0.669219 0.092437 0.64297 0.355556 rs17042102 1 1 0.091743 NA NA 0.580822 0.302632 rs17042121 3 1 0.116667 0.736119 0.141667 0.639142 0.353933 rs10516563 3 1 0.109244 0.846743 0.128205 0.636329 0.364706 rs4605724 1 1 0.116667 0.748252 0.083333 0.645257 0.359551 rs2350269 4 1 0.11017 0.495806 0.098214 0.628257 0.346154 rs6533527 1 1 0.116667 0.425151 0.133333 0.804123 0.421348 rs17042144 2 1 0.119658 0.727891 0.077586 NA NA rs1906618 2 1 0.115044 NA NA NA NA rs1906617 2 1 0.116667 0.541206 0.183333 0.977348 0.454545 rs12646447 2 1 0.119658 1 0.108333 1 0.444444 rs12646754 4 1 0.119658 0.681842 0.11017 1 0.425 rs2129981 1 1 0.116667 1 0.108333 1 0.444444 rs12639654 4 1 0.116667 0.139505 0.016667 1 0.438202 rs6817105 2 1 0.117647 0.27862 0.299145 1 0.440476 rs17042171 1 1 0.109244 0.281063 0.302521 1 0.425287 rsl 906591 1 1 0.116667 1 0.108333 1 0.444444 rs1906592 3 1 0.109244 0.283489 0.3 1 0.446429 rs2200732 2 1 0.112069 0.272544 0.308334 1 0.449438 rs2200733 4 1 0.116667 0.276161 0.301724 1 0.445783 rs4611994 2 1 0.116667 0.272544 0.308334 1 0.449438 rs4540107 1 1 0.116667 0.27862 0.305085 1 0.44382 rs1906593 4 1 0.117647 0.285134 0.301724 1 0.438202 rsl 906596 2 1 0.121739 0.255864 0.330275 0.97478 0.448718

TABLE-US-00005 TABLE 5 A. SNP markers within LD Block C04 (Between 111, 954, 811 and 112, 104, 250 on C04; NCBI Build 35; SEQ ID NO: 50). Pos in SEQ Marker ID Pos Build 35 ID NO: 50 Type Strand rs1448824 111954811 1 A/G - rs1947189 111955221 411 A/G - rs1947188 111955479 669 C/G - rs1992927 111956353 1543 C/T - rs1470619 111957122 2312 A/G - rs1448823 111958486 3676 A/G - rs4834327 111958676 3866 A/T + rs1448822 111958702 3892 C/T - rs2044674 111959075 4265 A/G - rs28445748 111959470 4660 A/T + rs2595116 111959591 4781 C/T - rs2595115 111959725 4915 A/C - rs13120244 111961948 7138 A/G + rs2723296 111962087 7277 A/G + rs2723297 111962201 7391 A/T + rs10021211 111962246 7436 C/T + rs17042011 111962331 7521 C/T + rs2595114 111962791 7981 C/G - rs2595113 111962792 7982 C/G - rs2595112 111963368 8558 C/G - rs6831623 111964677 9867 C/T + rs6854883 111964919 10109 C/T + rs2255793 111965457 10647 A/G + rs2723298 111966089 11279 C/T + rs12505886 111966218 11408 A/T + rs28718263 111966220 11410 A/T + rs12501913 111966355 11545 A/C + rs13126974 111966385 11575 A/T + rs36194761 111966385 11575 A/T + rs28473341 111966486 11676 C/T + rs36160675 111967780 12970 G/T + rs13147139 111968764 13954 A/G + rs13147489 111968795 13985 C/T + rs13147299 111968812 14002 A/C + rs13147726 111968923 14113 C/T + rs13147730 111968926 14116 C/T + rs13147552 111968949 14139 A/G + rs13123918 111968996 14186 A/T + rs35610510 111970561 15751 C/T + rs36162200 111971480 16670 G/T + rs11098086 111971997 17187 C/T + rs4034950 111972120 17310 A/G - rs11724067 111972144 17334 A/G + rs2723299 111972436 17626 A/G + rs2723300 111972512 17702 A/G + rs13138211 111972606 17796 A/G + rs2595075 111973312 18502 C/T - rs2723301 111973731 18921 C/G + rs2595074 111974709 19899 A/T - rs2723302 111974736 19926 C/G + rs2723303 111974741 19931 A/G + rs2218698 111975356 20546 G/T - rs2218697 111975357 20547 C/T - rs2595073 111975436 20626 A/G - rs2723307 111975800 20990 A/T + rs1584430 111976043 21233 C/G - rs1584429 111976151 21341 C/G - rs1900828 111976526 21716 C/T - rs7672226 111976785 21975 C/T + rs1839189 111976971 22161 C/T - rs1579946 111977724 22914 A/G - rs12509115 111977892 23082 A/G + rs1579945 111978096 23286 A/T - rs7661383 111979181 24371 A/C + rs2122078 111979201 24391 A/G - rs2122077 111979254 24444 C/T - rs2723311 111979626 24816 A/G + rs7667461 111979738 24928 A/G + rs1448799 111980386 25576 C/T - rs1448798 111980789 25979 C/T - rs12650829 111980880 26070 A/G + rs2723312 111980956 26146 A/G + rs1900827 111981343 26533 A/G - rs6815628 111981980 27170 C/T + rs6838131 111981993 27183 A/G + rs6838139 111982000 27190 A/C + rs6838295 111982012 27202 C/T + rs4582211 111982043 27233 A/G + rs4353966 111982088 27278 A/T + rs6838536 111982144 27334 C/T + rs1375302 111983068 28258 C/T + rs1375303 111983069 28259 A/G + rs7699114 111983094 28284 C/T + rs2197814 111983098 28288 A/C + rs2218700 111983340 28530 A/G + rs969642 111983529 28719 C/T + rs17042020 111984067 29257 A/C + rs2595099 111984371 29561 A/C + rs4371683 111984371 29561 A/C + rs2595093 111984960 30150 C/T - rs17625509 111984998 30188 A/G + rs2723313 111985093 30283 A/G + rs2723314 111985111 30301 G/T + rs2595092 111985112 30302 A/G - rs2595091 111985223 30413 C/G - rs1375301 111985458 30648 A/G - rs2245595 111985715 30905 C/T + rs2595088 111985958 31148 C/T - rs981150 111986232 31422 C/T - rs16997168 111986643 31833 C/T + rs6812840 111986654 31844 A/T + rs16997169 111986685 31875 C/T + rs4527540 111986742 31932 C/T + rs2595078 111987397 32587 A/G + rs11098087 111987538 32728 C/T + rs6843456 111988165 33355 C/T + rs998101 111988219 33409 A/G - rs13120535 111989691 34881 A/G + rs17042026 111989978 35168 A/G + rs6840960 111991045 36235 C/T + rs2122079 111991108 36298 C/T + rs2166961 111991365 36555 CIT + rs2723316 111991891 37081 C/T + rs2595079 111992019 37209 A/G + rs7665126 111992019 37209 A/G + rs2595080 111992042 37232 A/G + rs12646859 111992237 37427 G/T + rs10222783 111992430 37620 C/T + rs12498380 111992563 37753 C/T + rs2595081 111992761 37951 C/T + rs2595087 111992896 38086 G/T +

rs2723317 111993104 38294 A/G + rs6419178 111993104 38294 A/G + rs13110876 111993625 38815 A/G + rs2595083 111993625 38815 A/G I rs7690164 111994069 39259 C/T i rs2595084 111994163 39353 A/G + rs2595085 111994377 39567 C/G + rs2595086 111994385 39575 C/T + rs2723318 111994576 39766 G/T + rs17042050 111994805 39995 C/T + rs9998222 111995088 40278 A/G + rs2723319 111995233 40423 A/T + rs2595087 111995380 40570 C/T + rs17042052 111995521 40711 A/T + rs28558677 111995664 40854 G/T + rs6817731 111995691 40881 A/C + rs2723320 111997050 42240 C/T + rs12644107 111997588 42778 C/T + rs28482179 111998237 43427 C/T + rs28759131 111998559 43749 C/T + rs1448817 111998657 43847 A/G + rs28526075 111998725 43915 A/G + rs17042059 111998790 43980 A/G + rs10014075 112000023 45213 G/T + rs10026140 112000455 45645 G/T + rs13351232 112000455 45645 G/T + rs7666806 112000477 45667 G/T + rs10028327 112000489 45679 G/T + rs12650941 112002415 47605 A/T + rs28650220 112002617 47807 C/T + rs13113361 112002671 47861 G/T + rs13113522 112002686 47876 G/T + rs4529121 112003159 48349 A/G + rs6831284 112003582 48777 G/T + rs10009621 112003846 49036 C/T + rs10021534 112003945 49135 C/T + rs10032150 112004222 49412 A/G + rs10024267 112004571 49761 C/T + rs10012705 112004726 49916 C/T + rs11943627 112005073 50263 C/T + rs4543199 112005744 50934 C/T + rs28410055 112006340 51530 A/G + rs7693227 112006532 51722 C/T + rs6852197 112006679 51869 A/G + rs12647316 112006855 52045 C/T + rs12647393 112006886 52076 G/T + rs10019645 112007248 52438 G/T + rs10019689 112007473 52663 A/C + rs4626276 112007593 52783 A/C + rs10022067 112007672 52862 C/T + rs4469143 112007678 52868 C/G + rs6836206 112007902 53092 C/T + rs13150693 112008086 53276 G/T + rs11737637 112008416 53606 C/T + rs5011975 112008427 53617 A/G + rs6811511 112008429 53619 A/C + rs4383676 112008437 53627 A/G + rs28392642 112009161 54351 C/T + rs17631468 112009386 54576 A/G + rs17042076 112009942 55132 C/T + rs4434326 112010480 55670 C/T + rs17042081 112010815 56005 G/T + rs4833436 112011350 56540 C/T + rs7679623 112011519 56709 A/C + rs11098088 112011728 56918 C/T + rs4530699 112011761 56951 A/T + rs11098089 112011830 57020 A/C + rs17042088 112012418 57608 C/T + rs12648785 112013496 58686 A/G + rs12639820 112013644 58834 C/T + rs10001807 112013708 58898 A/G + rs10024486 112013722 58912 G/T + rs12648889 112013890 59080 C/G + rs28376747 112013925 59115 A/G + rs11098090 112014012 59202 C/T + rs11944778 112014571 59761 A/G + rs7436333 112014951 60141 A/C + rs4307025 112015107 60297 A/T + rs4447925 112015252 60442 C/T + rs28523292 112015772 60962 C/T + rs28635581 112015858 61048 C/T + rs28508237 112016004 61194 C/T + rs28521134 112016167 61357 C/T + rs17042093 112017716 62906 C/G + rs11930438 112017749 62939 C/T + rs28542185 112017795 62985 C/T + rs11930528 112017798 62988 G/T + rs13121382 112020177 65367 G/T + rs7439625 112021082 66272 A/T + rs28501998 112021318 66508 A/T + rs10016838 112021718 66908 C/T + rs17042098 112021762 66952 A/G + rs10005076 112021953 67143 C/T + rs10027473 112022056 67246 A/G + rs2634073 112023387 68577 A/G - rs1906611 112023520 68710 A/G - rs1906610 112023521 68711 C/T - rs28446238 112024025 69215 A/C + rs1906609 112024055 69245 A/C - rs34916665 112025294 70484 G/T + rs17042102 112026230 71420 A/G + rs17042104 112026555 71745 C/T + rs10015819 112026628 71818 C/T + rs2634071 112026824 72014 A/G - rs10007386 112028021 73211 C/T + rs10007547 112028050 73240 A/G + rs12647522 112028465 73655 C/T + rs1906614 112028900 74090 A/G - rs2723335 112029230 74420 A/G + rs17042112 112029281 74471 C/T + rs17042115 112029414 74604 A/G + rs10013510 112029527 74717 C/G + rs11939057 112029755 74945 C/T + rs2634076 112029877 75067 A/G - rs2723293 112031377 76567 A/G + rs28494131 112032777 77967 C/T + rs2634075 112033582 78772 A/G - rs13121715 112034239 79429 G/T + rs2634074 112034645 79835 A/T - rs17042121 112034705 79895 A/G + rs10516563 112035326 80516 G/T + rs17042125 112035883 81073 A/G + rs13136439 112036254 81444 G/T + rs13114686 112036503 81693 C/T + rs36166388 112037782 82972 A/G + rs2450934 112038532 83722 A/C -

rs36138049 112040474 85664 A/C + rs36168695 112040495 85685 A/G + rs36129850 112040548 85738 A/G + rs12513264 112041966 87156 C/T + rs2882365 112041975 87165 A/G + rs36139649 112042072 87262 C/T + rs4033107 112042072 87262 C/T + rs36176419 112042092 87282 A/G + rs4033108 112042092 87282 A/G + rs4450997 112042160 87350 A/C + rs2350268 112042213 87403 A/G + rs4613627 112042225 87415 A/C + rs4033109 112042227 87417 C/T + rs4833443 112042247 87437 C/T + rs2723336 112042258 87448 C/T - rs4033111 112042302 87492 A/G + rs1807360 112042333 87523 C/T - rs4605724 112042685 87875 A/C + rs6856879 112043066 88256 A/T + rs6834418 112043172 88362 C/T + rs2466455 112043219 88409 A/G - rs6857810 112043220 88410 A/G + rs2634079 112043541 88731 C/G - rs28366840 112043710 88900 A/T + rs2350269 112044728 89918 C/T + rs7665409 112045070 90260 C/T + rs6533527 112045118 90308 A/C + rs12649717 112045283 90473 A/C + rs6822831 112045374 90564 A/G + rs35916701 112046074 91264 C/T + rs6829419 112046178 91368 C/T + rs2723334 112046356 91546 A/G - rs2634078 112046528 91718 C/T - rs12512819 112046597 91787 C/T + rs17042144 112047270 92460 C/T + rs2171594 112047908 93098 A/G - rs6842887 112048170 93360 A/G + rs2171593 112048375 93565 G/T - rs7690874 112048681 93871 A/G + rs17042145 112049101 94291 G/T + rs17042146 112049106 94296 C/T + rs9998815 112049904 95094 C/G + rs7683336 112051207 96397 C/T + rs17042150 112051452 96642 A/T + rs10016842 112051810 97000 C/T + rs10005432 112052219 97409 A/G + rs1906620 112052624 97814 C/T - rs1906619 112052670 97860 C/T - rs1906618 112053026 98216 C/T - rs1906617 112053418 98608 C/T - rs6847935 112054255 99445 A/T + rs6831873 112055138 100328 C/T + rs1906616 112055172 100362 C/T - rs6837901 112055712 100902 C/T + rs2723333 112056695 101885 C/T - rs12646447 112056930 102120 C/T + rs6820568 112057435 102625 C/T + rs1906615 112059402 104592 A/C - rs2634077 112061112 106302 A/G - rs7689774 112061114 106304 G/T + rs12646754 112061176 106366 C/T + rs35807830 112061497 106687 G/T + rs2129983 112061684 106874 C/T - rs2129982 112061747 106937 C/T - rs2129981 112061803 106993 A/C - rs6854111 112062140 107330 A/T + rs12639654 112062899 108089 C/T + rs4515229 112062985 108175 A/G + rs2129984 112063010 108200 C/T + rs6817105 112063372 108562 C/T + rs12503217 1120G3765 108955 C/T + rs2634070 112064016 109206 A/C + rs17042171 112065891 111081 A/C + rs7434417 112066042 111232 A/G + rs1906591 112066493 111683 A/G + rs1906592 112066608 111798 G/T + rs12510087 112066632 111822 A/G + rs7661554 112067221 112411 A/G + rs34796144 112067333 112523 A/C + rs2200732 112067646 112836 C/T + rs2200733 112067773 112963 C/T + rs17042175 112068571 113761 A/T + rs4611994 112068645 113835 C/T + rs4540107 112068706 113896 A/C + rs1906593 112069526 114716 C/T + rs4371684 112069651 114841 A/G + rs1906594 112069739 114929 A/G + rs1906595 112069788 114978 G/T + rs1906596 112069840 115030 C/T + rs6838775 112069908 115098 G/T + rs2129977 112070036 115226 A/G + rs2129978 112070158 115348 A/C + rs1906597 112070190 115380 G/T + rs1906598 112070229 115419 C/T + rs1906599 112070290 115480 C/T + rs1906600 112070480 115670 C/T + rs1906601 112070883 116073 C/T + rs1906602 112070927 116117 C/T + rs1906603 112071040 116230 C/T + rs28645285 112071426 116616 A/G + rs2171590 112071435 116625 C/T + rs6852357 112071939 117129 C/T + rs13143308 112072023 117213 G/T + rs2220427 112072493 117683 C/T + rs17632693 112072538 117728 C/T + rs11935917 112072850 118040 A/G + rs4833456 112073911 119101 C/T + rs12644625 112074117 119307 C/T + rs4400058 112074277 119467 A/G + rs1906604 112074452 119642 A/G + rs1906605 112074796 119986 C/T + rs13126975 112075129 120319 A/T + rs6837490 112075447 120637 C/T + rs6843082 112075671 120861 A/G + rs13105878 112075751 120941 A/C + rs6533528 112076843 122033 A/G + rs7692272 112076857 122047 G/T + rs2171591 112077012 122202 A/G + rs17042195 112077142 122332 C/G + rs11931959 112077289 122479 A/G + rs17042198 112077582 122772 G/T + rs10033464 112078365 123555 G/T + rs2171592 112078392 123582 C/T + rs13121924 112078423 123613 A/G + rs2129979 112078601 123791 G/T +

rs2350539 112078814 124004 G/T + rs1906606 112080996 126186 A/C + rs7672570 112081189 126379 C/T + rs4834418 112081408 126598 A/G + rs723364 112082075 127265 C/G - rs723363 112082105 127295 A/G - rs7697491 112083422 128612 A/T + rs13125644 112083505 128695 A/G + rs2350294 112084449 129639 A/G - rs2350293 112084451 129641 A/G - rs3855819 112084767 129957 C/G - rs2220428 112085064 130254 A/G + rs2220429 112085089 130279 A/C + rs11727566 112085934 131124 A/T + rs13141190 112086218 131408 A/G + rs4032976 112086371 131561 A/G - rs3866829 112086379 131569 A/C - rs7671348 112086408 131598 A/G + rs3866830 112086598 131788 C/G - rs6811267 112086942 132132 C/T + rs3853440 112087213 132403 C/T - rs3853441 112087344 132534 A/G - rs3853442 112087632 132822 C/T - rs3853443 112087733 132923 A/G - rs4124158 112087798 132988 C/T + rs4124159 112087847 133037 A/G + rs12506083 112088016 133206 A/C + rs34809282 112088051 133241 A/G + rs7683219 112088051 133241 A/G + rs7683618 112088259 133449 A/C + rs7683625 112088269 133459 A/G + rs7662050 112088325 133515 C/T + rs36183416 112088804 133994 C/T + rs4447926 112088804 133994 C/T + rs4594787 112088813 134003 A/G + rs10390275 112089009 134199 A/T + rs36179422 112089009 134199 A/T + rs10006659 112089030 134220 G/T + rs36181695 112089078 134268 A/G + rs7440730 112089078 134268 A/G + rs10006881 112089277 134467 C/T + rs36149087 112089277 134467 C/T + rs6533530 112089540 134730 C/T + rs6533531 112089569 134759 G/T + rs3866831 112089718 134908 C/T - rs4269241 112089833 135023 A/G + rs4032975 112089842 135032 A/C - rs4032974 112090140 135330 A/G - rs4124160 112090452 135642 A/G + rs3866832 112091304 136494 C/G - rs3853444 112091740 136930 A/G - rs7662345 112091766 136956 A/G + rs2350545 112092258 137448 C/T - rs17042215 112092562 137752 C/T + rs2003121 112093023 138213 C/T + rs880309 112093143 138333 A/G + rs9991046 112093346 138536 G/T + rs17042216 112094463 139653 C/T + rs17570669 112094486 139676 A/T + rs17042218 112094520 139710 A/G + rs17042223 112094922 140112 C/T + rs3866833 112095138 140328 C/T - rs17042224 112096509 141699 G/T + rs13130446 112096760 141950 C/T + rs10516564 112096896 142086 A/G + rs7686320 112097215 142405 A/T + rs7686499 112097282 142472 C/T + rs17042230 112097319 142509 C/T + rs4124161 112097459 142649 C/T + rs4576077 112098061 143251 C/T + rs4260600 112098098 143288 C/T + rs12644093 112098445 143635 A/G + rs4124162 112098593 143783 A/G + rs7674295 112099042 144232 A/G + rs11938968 112100356 145546 A/G + rs28601812 112101457 146647 A/C + rs4032983 112101551 146741 G/T + rs3866834 112101617 146807 A/G + rs6852021 112101716 146906 A/G + rs28580491 112102583 147773 C/T + rs13110989 112102671 147861 G/T + rs3866835 112102983 148173 C/T - rs4124163 112103203 148393 A/G + rs3866836 112103244 148434 A/G + rs17042238 112103458 148648 A/G + rs4124164 112104250 149440 C/T + B. Microsatellite markers within LD Block C04 (Between 111, 954, 811 and 112, 104, 250 on C04; NCBI Build 35; SEQ ID NO: 50). End Marker Start position position Forward primer Reverse Primer D4S193 112062811 112062911 ACAACCCCATTTGTGAAGAC TTTATAGAAAATTTAGCATGGA D4S2940 112070055 112070267 CTAAGTTGTGCAGCCATGAA TGGAACCACTTTTGCAGTAA D4S406 112076047 112076292 CTGGTTTTAAGGCATGTTTG TCCTCAGGGAGGTCTAATCA

TABLE-US-00006 TABLE 6 Key to sequences presented in sequence listing. SEQ ID NO Marker ID 1 rs2220427 2 rs17042059 3 rs4529121 4 rs4543199 5 rs10019689 6 rs4626276 7 rs17042076 8 rs11098089 9 rs11930528 10 rs17042098 11 rs17042102 12 rs17042121 13 rs10516563 14 rs4605724 15 rs2350269 16 rs6533527 17 rs17042144 18 rs1906618 19 rs1906617 20 rs12646447 21 rs12646754 22 rs2129981 23 rs12639654 24 rs6817105 25 rs17042171 26 rs1906591 27 rs2200732 28 rs2200733 29 rs4611994 30 rs4540107 31 rs1906593 32 rs1906596 33 rs2634073 34 rs1906592 35 rs2723296 36 rs16997168 37 rs2723316 38 rs6419178 39 rs1448817 40 rs13105878 41 rs10033464 42 rs13141190 43 rs3853444 44 rs4576077 45 D4S406 46 rs7668322 47 rs2197815 48 rs6831623 49 rs2595110 50 LD Block C04 51 rs13143308

Example 2

Characterization of AF Risk Variants

[0252] The following contains further description of the identification of variants conferring risk for atrial fibrillation on chromosome 4q25

[0253] Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in man and is characterized by chaotic electrical activity of the atria¹. It affects one in ten individuals over eighty, causes significant morbidity, and is an independent predictor of mortality². Recent studies have provided evidence of a genetic contribution to AF³-5. Mutations in potassium channel genes have been associated with familial AF^6-10 but account for only a small fraction of all AF cases^11,12. We performed a genome-wide association scan, followed by replication studies in three populations of European descent and a Chinese population from Hong Kong and find a strong association between two sequence variants on chromosome 4q25 to AF. Approximately 35% of individuals of European descent have at least one of the variants and the risk of AF increases by 1.72 and 1.39 per copy. The association to the stronger variant was replicated in the Chinese population, where it is carried by 75% of individuals and risk of AF is increased by 1.42 per copy. A stronger association was observed in individuals with typical atrial flutter (AFI). Both variants are adjacent to PITX2, which is known to play a critical role in left-right asymmetry of the heart¹³-15. We conducted a genome-wide association study using the Illumina Hap300 BeadChip on an Icelandic population with AF and/or AFI. 316,515 SNPs satisfying our quality criteria were tested individually for association to AF/AFI in a sample of 550 patients and 4,476 controls from Iceland. Three strongly correlated SNPs, all located within a single linkage disequilibrium (LD) block on chromosome 4q25, were the only SNPs found to be genome-wide significant after accounting for the 316,515 SNPs tested (P<0.05/316,515=1.58×10^-7): rs2200733 (OR=1.75; P=1.6×10^-19), rs2220427 (OR=1.75; P=1.9×10^-10) and rs2634073 (OR=1.60; P=2.1×10^-9). These results and all other results based on the Icelandic population were adjusted for the relatedness of individuals. The two most significant SNPs, rs2200733 and rs2220427, are perfect proxies for one another in the CEPH CEU HapMap¹⁶ dataset and are close to being perfect proxies for one another in the Icelandic dataset (D'=1, r²=0.999), therefore, only rs2200733 will be referred to in the following discussion. The correlation of rs2634073 to rs2200733 is weaker in the Icelandic dataset (D'=0.95, r²=0.605). Upon further study of the Illumina Hap300 SNPs in the vicinity of the first three SNPs and conditioning on the association to rs2200733, an association to a new SNP, rs10033464, was identified (OR=1.42; P=0.0024). After accounting for the association to rs2200733 and rs10033464, the association to rs2634073 was no longer significant (P=0.30). Henceforth, all association results for rs2200733 T and rs10033464 T, including those presented in Table 7, are based on comparison to the wild type haplotype which carries neither of the two at risk alleles, rather than comparison to the major alleles of each SNP separately. Specifically, odds-ratios for rs2200733 T and rs10033464 T are each computed conditionally and could be interpreted as the estimated relative risk of each variant compared to the wild-type. The at risk alleles T of rs2200733 and T of rs10033464 have estimated population allelic frequencies of 12.05% and 8.53% in Iceland, respectively, and are never observed together on the same chromosome, in the Icelandic dataset or in the CEU HapMap dataset. A third SNP, rs13143308, which has a minor allele that corresponds completely to chromosomes carrying either the T allele of rs2200733 or the T allele of rs10033464, was identified through the CEU HapMap dataset. FIG. 2 demonstrates the haplotype structure over the key SNPs of the associated region. Sets of SNPs, that are perfect proxies (i.e., perfect surrogates, r2=1.0 to the tagging SNP) of each of these three key SNPs in the CEU HapMap samples, are provided in Table 9 and relative locations displayed in FIG. 3. We emphasize that the SNPs named should be considered representatives of the haplotypes defined by the SNPs which they are equivalent to and are primarily chosen for the sake of convenience.

[0254] A microsatellite marker, D4S406, located in the same LD block as the two SNPs was identified. In Iceland, three of the four shortest alleles of D4S406 (-8, -4, and -2) combine to form a near perfect surrogate for the T allele of rs2200733 (D'=0.995, r²=0.98) and the two shortest remaining alleles (-6 and 0) form a good surrogate of the T allele of rs10033464 (D'=0.98, r²=0.75) (Table 10). None of the remaining (longer) alleles of D4S406 are associated to AF/AFI after accounting for the effect of the short alleles. For the replication of the original observation in Iceland the D4S406 genotypes were used to provide information when SNP genotypes were not available.

[0255] In an attempt to replicate our original discovery we analyzed an additional Icelandic samples consisting of 2,251 AF/AFI patients and 13,238 controls (Table 7). The association of both SNPs to AF/AFI was replicated in these samples (OR=1.64, P=2.7×10^-23 for rs2200733, OR=1.40, P=8.2×10^-8 for rs10033464) and both achieve genome-wide significance in the combined Icelandic samples (OR=1.68, P=1.9×10^-30 for rs2200733, OR=1.38, P=9.4×10^-9 for rs10033464). We also typed all the 18 Hap300 Illumina SNPs in the region around our signal in 404 of the additional AF cases and 2,036 of the additional controls. None of these SNPs remained significant after accounting for the association to rs2200733 and rs10033464 (Table 11).

[0256] In further attempts to replicate our results, we tested these variants for an association to AF in two populations of European ancestry, one from Sweden, consisting of 143 cases and 738 controls, and the other from the United States (U.S.), consisting of 636 cases and 804 controls (Table 7). The association to rs2200733 was strongly replicated in both populations (OR=2.01, P=0.00027 in Sweden, OR=1.84, P=9.8×10^-10 in the U.S.). The association to rs10033464 is weaker, but was nonetheless replicated in the Swedish population (OR=1.65, P=0.0087) and was nearly significant in the U.S. population (OR=1.30, P=0.052). When combined with the Icelandic samples, the association to rs2200733 was unequivocal (OR=1.72, P=3.3×10^-41), and the significance of rs10033464 was well beyond the threshold of genome-wide significance (OR=1.39, P=6.9×10^-11). Assuming the multiplicative model, the population attributable risk (PAR) of the two variants combined is approximately 20% in populations of European ancestry.

[0257] Finally, we attempted to replicate these signals in a Han Chinese population from Hong Kong consisting of 333 cases and 2,836 controls. The association to rs2200733 T was significantly replicated (OR=1.42, P=0.00064), but the association to rs10033464 T was not significant, although in the right direction (OR=1.08, P=0.55) (Table 7). Interestingly, the T allele of rs2200733 is much more frequent in the Chinese (allelic frequency in controls: 0.528) than in those of European descent (allelic frequency in controls: 0.098-0.139) (FIG. 2) which is reflected in a greater joint PAR of approximately 35%, even though the estimated risk is less. The LD block containing the two variants is more fragmented in the Chinese CHB and Japanese JPT HapMap samples than in the CEU HapMap samples (FIG. 3). We therefore analysed several markers in the Hong Kong population which were in perfect LD with rs2200733 in the CEU samples, but in imperfect LD in the CHB and JPT samples (Table 12). These markers had weaker apparent association to AF than rs2200733, suggesting that the functional variants driving the association is located in the approximately 20 kb region around the original rs2200733 variant and defined by the SNPs that remain equivalent to rs2200733 in the CHB and JPT samples (coloured red in FIG. 3).

[0258] For the initial Icelandic discovery samples, rs2200733 had a significantly higher OR than rs10033464 (P=0.041). This held true in the replication samples, and overall there is a significant difference in the risks associated with the two variants (P=0.00019 in the combined European samples and P=0.0099 in Hong Kong). When genotype-specific odds ratios were studied, some deviation away from the multiplicative model is detectable in the combined dataset (P=0.018 for European samples, see Table 13). Estimated risks of heterozygous carriers relative to non-carriers were similar, but homozygous carriers of rs2200733 T and rs10033464 T have estimated risks that were, respectively, higher and lower than that predicted by a multiplicative model. A similar trend was seen in the Hong Kong samples; although the sample size is too small to have power to detect such deviations with significance. In the combined populations of European descent the observed OR for individuals homozygous for rs2200733 T was 3.64 as compared to individuals homozygous for the wild type haplotype and 1.77 for the Chinese population demonstrating that these variants are important components in any predictive modeling of AF.

[0259] The age at diagnosis of AF/AFI for the Icelandic samples correlates with the two SNPs (diagnosis occurs 2.28 years earlier per T allele of rs2200733 and 1.10 years earlier per T allele of rs10033464, joint P=1.29×10^-6). The effect of the age at diagnosis was also evaluated by measuring the strength of association while stratifying by age at diagnosis. The association of the two variants is strongest in those diagnosed at a younger age, although the risk remains significant even in those diagnosed after reaching 80 years of age (Table 8). Information on age at diagnosis of AF was not available for the Swedish samples. The U.S. samples were comprised of two main groups, younger patients with either lone AF or AF and hypertension (HTN), and older AF cases who are mostly hemorrhagic and ischemic stroke patients. In both populations there is a clear trend towards a stronger association in younger AF cases than in older cases. Our analysis of the data did not suggest any differential association by sex (Table 8).

[0260] AF1 often accompanies AF, but can occur in isolation¹⁷. Interestingly we observed a strong association between the variants and the small subset (N=116) of the AF1 Icelandic patients (OR=2.60, 95% confidence interval (CI)=1.83-3.68, P=7.5×10^-8 for rs2200733, OR=1.94, 95% CI=1.26-3.00, P=0.0028 for rs10033464). Indeed, for rs2200733, the OR for these definite AFI cases is significantly higher than that for the cases with an AF phenotype (P=0.0026), and close to significantly higher for rs10033464 (P=0.084). Our results suggest that while these traits share genetic risk factors, AFI is less influenced by phenocopies than AF.

[0261] Neither variant showed a association to obesity, hypertension or myocardial infarction in the Icelandic samples, all known risk factors for AF (observed OR<1.1 in all instances, Table 14). Although these negative results do not exclude the possibility that the new variants associate with these phenotypes, they do suggest, along with the high risk in U.S. lone AF and earlier age at onset in carriers, that the new variants are not affecting risk of AF through these known risk factors.

[0262] There is no known gene present in the LD block containing rs2200733 and rs10033464 (FIG. 3). The LD block contains one spliced EST (DA725631) and two single-exon ESTs (DB324364 and AF017091). RT-PCR of cDNA libraries from various tissues did not detect the expression of these ESTs (Table 16). The PITX2 gene located in the adjacent upstream LD block is the gene closest to the risk variants. Several markers within the LD block containing the PITX2 gene are correlated to the markers showing association to AF and Afl, as shown in Table 18. The protein encoded by this gene, the paired-like homeodomain transcription factor 2, is an interesting candidate for AF/AFI as it is known to play an important role in cardiac development by directing asymmetric morphogenesis of the heart¹³. In a mouse knockout model Pitx2 was shown to suppress a default pathway for sinoatrial node formation in the left atrium^14,15. There is very little mRNA expression of PITX2 in all easily accessible tissues, such as blood and adipose tissue, hampering the study of correlation between genotypes and expression levels. The next gene upstream of PITX2 is ENPEP, an aminopeptidase responsible for the breakdown of angiotensin II in the vascular endothelium¹⁸. This gene is expressed more widely, but the variants associated with AF showed no correlation to its expression in blood or adipose tissue. No other annotated genes are located within a 400 kb region upstream and 1.5 Mb regions downstream of the associated variants.

[0263] In summary, we have identified two variants on chromosome 4q25 that are strongly associated with AF in three distinct populations of European descent. The stronger variant also replicates well in a Chinese population where it is much more common and has higher PAR than in populations of European descent. This association is particularly compelling in younger patients and in those with lone AF, but is also present in older patients with more commonly encountered forms of AF. Although the mechanism for this association is unknown, our results provide a foundation for further studies on the molecular underpinnings of AF.

Methods

Subjects

[0264] The Icelandic cases consisted of all patients diagnosed with AF and/or AFI at the two largest hospitals in the country from 1987 to 2005. The Swedish cases were recruited from 1996 to 2002 as a part of an ongoing genetic epidemiology study, the South Stockholm Ischemic Stroke Study. The U.S. cases were a mixture of stroke patients with a AF diagnosis and younger consecutive patients with lone AF or AF with a coexisting diagnosis of hypertension. The Hong Kong cases were a collection of stroke and diabetes patients with an AF diagnosis. The AF diagnosis was confirmed by a twelve lead electrocardiogram in all study populations.

The Icelandic controls were chosen at random from individuals who have participated in other genetic studies at deCODE, excluding first-degree relatives of patients and controls (Table 15). The Swedish controls were recruited from the same region as patients from blood donors (in 2001) and healthy volunteers (1990-1994). The U.S. controls were recruited from a large primary care practice and from patients participating in a hemorrhagic stroke study. The Hong Kong controls were individuals without an AF diagnosis.

Icelandic Study Population

[0265] This study initially included the all patients consenting to participation, which were diagnosed with AF and/or AFI (ICD 10 diagnosis 148 and ICD 9 diagnosis 427.3) at Landspitali University Hospital in Reykjavik, the only tertiary referral centre in Iceland, and at Akureyri Regional Hospital, the second largest hospital in the country, from 1987 to 2005. All diagnoses were confirmed by a twelve lead electrocardiogram (EKG) which was manually read by a cardiologist. All cases were included, regardless of whether the patients had clinical symptoms or not, except those diagnosed only immediately after open cardiac surgery.

[0266] A set of 550 cases were successfully genotyped according to our quality control criteria in a genome-wide SNP genotyping effort, using the Infinium II assay method and the Sentrix HumanHap300 BeadChip (Illumina, San Diego, Calif., USA). The mean age at diagnosis for this initial group of 550 patients (370 males and 180 females) was 72.5 (SD=11.0) years and the range was from 34.7-96.2 years. The validation group of 2,273 patients (1,359 males and 913 females) had a mean age at diagnosis of 70.5 (SD=13.0) and the range was from 16.8-100.6. The AF/AFI free controls (2,201 males and 2,275 females at the initial genome-wide screening with mean age 61.5 (SD=15.8) and 5,654 males and 7,597 females at the validation stage with mean age 61.9 (SD=18.4)) used in this study consisted of controls randomly selected from the Icelandic genealogical database and individuals from other ongoing related genetic studies at deCODE. Controls having first-degree relatives (siblings, parents or offspring) with AF/AFI, or a first-degree control relative, were excluded from the analysis.

Icelandic MI, Obesity and Hypertension Populations

[0267] Individuals who suffered an MI were identified from a registry of over 10,000 individuals who: a) had an MI before the age of 75 in Iceland in the years 1981 to 2002 and satisfy the MONICA criteria 9 (REF II), or had MI discharge diagnosis from the major hospitals in Reykjavik in the years 2003 and 2005. MI diagnoses of all individuals in the registry follow strict diagnostic criteria based on signs, symptoms, electrocardiograms, cardiac enzymes and necropsy findings. Genotype information was available for 2,462 males and 1,114 females, mean age 72.6 (SD=11.7). Body mass index (BMI) was measured for individuals participating in the cardiovascular atrial fibrillation and/or stroke (CVD) genetics program at deCODE (either patients with CVD, their first degree relatives or spouses). For the purpose of this study subjects with BMI>35 were defined as obese. Genotype information was available for 555 males and 1,046 females, mean age 53.2 (SD=16.1). Hypertensive patients included those who had attended the ambulatory hypertension clinic at the Landspitali, University Hospital in Iceland and/or had been given the diagnosis on discharge from the hospital. The diagnosis was verified by confirming that they were taking antihypertensivemedications as a treatment for hypertension. Genotype information was available for 1,293 males and 1,327 females, mean age 71.5 (SD=12.5). The study was approved by the Data Protection Commission of Iceland and the National Bioethics Committee of Iceland. Written informed consent was obtained from all patients, relatives and controls.

Swedish Study Population

[0268] Patients with ischemic stroke or TIA attending the stroke unit or the stroke outpatient clinic at Karolinska University Hospital, Huddinge unit in Stockholm, Sweden were recruited from 1996 to 2002 as part of an ongoing genetic epidemiology study, the South Stockholm Ischemic Stroke Study (SSISS). The study was approved by the Bioethics Committee of Karolinska Institutet (Dnr 286/96 and 08/02). AF diagnosis in the Swedish samples was based on a twelve lead EKG. The fraction of males in the Swedish AF cases was 46.2% and the mean age at stroke diagnosis for the Swedish AF cases was 74.4 (SD=8.7).

[0269] The Swedish controls used in this study are population-based controls recruited from the same region in central Sweden as the patients, representing the general population in this area. The individuals were either blood donors (recruited in 2001) or healthy volunteers (collected in 1990-1994) recruited by the clinical chemistry department at the Karolinska University Hospital to represent a normal reference population. The fraction of males in the Swedish controls was 59.7% and the mean age at recruitment for the Swedish controls was 43.1 (SD=12.3).

U.S. Study Population

[0270] U.S. subjects were enrolled in ongoing case-control and cohort studies at Massachusetts General Hospital (MGH) between January 1998 and July 2006. All aspects of these studies have been approved by the local Institutional Review Board. Subjects enrolled in the case-control study consisted of patients hospitalized with acute ischemic or hemorrhagic stroke confirmed by CT or MRI, admitted to a single acute care hospital. Of the 328 hemorrhagic stroke patients recruited 78 were diagnosed with AF and were used as cases for the current study, the remaining 250 were used as controls. 170 ischemic stroke patients had an AF diagnosis and were treated as cases but no ischemic stroke patients were treated as controls. Patients were excluded for primary subarachnoid hemorrhage and for intracerebral hemorrhage secondary to head trauma, tumor, vascular malformation, or vasculitis. 624 stroke-free controls were recruited from a large, primary care practice (>18 000 patients) serving the hospital catchment area as well as the hospital's Anticoagulation Management Service. 70 of the 624 individuals collected as controls were diagnosed with AF and treated cases for the purposes of the current study. 50.9% of all individuals used as controls were males and their mean age was 67.4 (SD=12.3). All subjects or an accompanying informant provided informed consent for participation in genetic studies and were interviewed prospectively regarding medical history, medications, social and family history. Presence or absence of atrial fibrillation was prospectively documented through interview and from review of medical records.

[0271] The second part of the U.S. subjects consisted of consecutive patients with lone AF or AF with coexisting diagnosis of hypertension referred to the arrhythmia service who provided written informed consent for participation in genetic. Inclusion criteria were AF documented by EKG, and an age less than or equal to 65 years. The exclusion criteria were structural heart atrial fibrillation and/or stroke as assessed by echocardiography, rheumatic heart atrial fibrillation and/or stroke, hyperthyroidism, myocardial infarction, or congestive heart failure. Each patient underwent a physical examination and a standardized interview to identify past medical conditions, medications, symptoms and possible triggers for initiation of AF. All patients were evaluated by twelve lead EKG, echocardiogram, and laboratory studies. EKGs and echocardiograms were interpreted using standard criteria.

Hong Kong Study Population

[0272] All subjects in the Hong Kong study population were of southern Han Chinese ancestry residing in Hong Kong. The cases consisted of 217 individuals (49.1% male, mean age 68.1 (SD=9.6)) selected from the Prince of Wales Hospital Diabetes Registry²³ and 116 subjects (30.2% male, mean age 76.1 (SD=10.9)) from the Stroke Registry²⁴. All subjects were diagnosed to have atrial fibrillation by EKG. The controls consisted of 2,836 subjects without evidence of AF. Informed consent was obtained for each participating subject. This study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.

Illumine Genome-Wide Genotyping

[0273] All Icelandic case- and control-samples were assayed with the Infinium HumanHap300 SNP chips (Illumina, SanDiego, Calif., USA), containing 317 503 haplotype tagging SNPs derived from phase I of the International HapMap project. Of the SNPs assayed on the chip, 162 SNPs generated no genotypes, and an additional 178 SNPs had yield lower than 90%. Forty-eight SNPs were monomorphic and 107 others nearly monomorphic (i.e. the minor allele frequency in the combined cohort of patients and controls was less than 0.001). An additional 475 SNPs showed very significant distortion from Hardy-Weinberg equilibrium in the controls (p<1×10^-10). Lastly, a few markers (n=18) were determined to have genotyping problems after investigation of particular regions and possible signals in several different on-going genome-wide association studies in house. Thus, the final analyses presented in the text utilizes 316,515 SNPs. Any samples with a call rate below 98% were excluded from the analysis.

Single SNP- and Microsatellite Genotyping.

[0274] SNP genotyping was carried out by the Centaurus (Nanogen) platform²⁵. The quality of each Centaurus SNP assay was evaluated by genotyping each assay in the CEU and/or YRI HapMap samples and comparing the results with the HapMap data. Assays with >1.5% mismatch rate were not used and a linkage disequilibrium (LD) test was used for markers known to be in LD.

Association Analysis

[0275] An attempt was made to genotype all participating individuals for rs2200733, rs4611994 (a perfect proxy for rs2200733), rs13143308, and rs6843082 (a perfect proxy for rs13143308). For each of the SNPs, yield was higher than 90% in every group. In addition genotypes for the D4S406 microsatellite were available for all Icelandic and Swedish subjects. Because of the redundancy in genotyping, observed genotypes reduced the amount of information lost due to missing genotypes through a likelihood approach we have used before²⁶. This ensured that results presented in the tables were always based on the same number of individuals, allowing meaningful comparisons of results. As data on rs10033464 was only directly available in the initial Icelandic discovery samples and in the HapMap project the rs2200733 C rs13143308 T haplotype was used to tag this SNP. This tagging was perfect in both the initial discovery samples and the CEPH CEU HapMap samples.

[0276] A likelihood procedure described in a previous, and implemented in the NEMO software, was used for the association analyses. We tested the association of an allele to each phenotype using a standard likelihood ratio statistic, which, if the subjects were unrelated, would have asymptotically a chi-square distribution, with one degree of freedom, under the null hypothesis. Allele-specific OR was calculated assuming a multiplicative model for the two chromosomes of an individual⁴. Results from multiple case-control groups were combined using a Mantel-Haenszel model⁵ in which the groups were allowed to have different allelic population frequencies, haplotypes and genotypes but were assumed to have common relative risks. There was no significant deviation from Hardy-Weinberg equilibrium (HWE) in any control group.

[0277] In Tables 7 and 8, P values for both rs2200733 and rs10033464 were computed based on comparison to the wild type rs2200733 C, rs13143308 G, rs10033464 G haplotype carrying neither of the at risk alleles. The corresponding conditional odds ratio for rs2200733 T is defined as [f(rs2200733 T)/f(WT)]/[p(rs2200733 T)/p(WT)] where WT denotes the wild-type haplotype, and f(•) and p(•) denote frequencies in cases and controls respectively. Under the multiplicative model and when the controls could be considered as population controls, this conditional odds ratio is the appropriate estimate of the relative risk of rs2200733 T versus the wild-type. Conditional odd-ratio for rs13143308 T is similarly defined and has a similar interpretation.

Correction for Relatedness and Genomic Control.

[0278] Some of the individuals in the Icelandic case-control groups were related to each other, causing the aforementioned chi-square test statistic to have a mean>1 and median>0.675²⁶. We estimated the inflation factor by using a previously described procedure where we simulated genotypes through the genealogy of 731,175 Icelanders³⁰. For the initial discovery samples, where genotypes for the 316,515 genome-wide scan SNPs were available, we also estimated the inflation factor by using genomic controls and calculating the average of the 316,515 chi-square statistics, and by computing the median of the 316,515 chi-square statistics and dividing it by 0.675²⁶ as describe previously^31,32. For these initial samples the inflation factors, estimated by our genealogy method and the two genomic control methods gave similar inflation factor estimates; 1.047, 1.058 and 1.054 respectively. The P values and confidence intervals presented are based on adjusting by the inflation factor estimated by the genealogy method.

PCR Screening of cDNA Libraries. To confirm the expression of the spliced ESTs (DA725631, DB324364 and AF017091) within the LD block we screened commercially available cDNA libraries and libraries generated at deCODE. The commercial libraries screened were heart (Clontech-639304), aorta (Clontech-639325) bone marrow (Clontech 7416-1), testis (Clontech 7414-1) and whole brain (BD 50598) Marathon Ready cDNA libraries. In addition cDNA libraries were constructed for whole blood and EBV-transformed human lymphoblastoid cells. Total RNA was isolated from the lymphoblastoid cell lines and whole blood, using the RNeasy RNA isolation kit from Qiagen (Cat. 75144) and the RNeasy RNA isolation from whole blood kit (Cat. 52304), respectively. cDNA libraries were prepared at deCODE using High Capacity cDNA Archive Kit with random primers (Applied Biosystems PN 4322171). PCR screening was carried out using the Advantage® 2 PCR Enzyme RT_PCR System (Clontech) according to manufacturers instructions and using PCR primers from Operon Biotechnologies. The PCR reactions were done in 10 μl volume at a final concentration of 3.5 μM of forward and reverse primers (Table 16), 2 mM dNTP, 1× Advantage 2 PCR buffer and 0.5 μl of cDNA library.

Northern Blot Analysis.

[0279] Commercial multiple tissue poly-A Northern blots were obtained from Clontech (Human Cardiovascular system, Cat. 636825).

Probes Used:

[0280] i) The PITX2 cDNA clone (HU3_p983E0327D), obtained from RZPD Deutsches Ressourcenzentrum fur Genomforschung GmbH, Germany http://www.rzpd.de/products/genomecube.shtml) (sequence verified, data not shown); ii) cDNA clone that corresponded to exons 1-12 of the ENPEP transcripts obtained from RT-PCR experiments. The ENPEP clone was sequence verified:

TABLE-US-00007 TCCTGCTCCAGCTTGTGGATATTTTGCAAAAAAGCTCTCCATCTGCCACAGTTGCAGTTCAGTGTTG AATGGCTCTGCTATTGTGACAATTCGGCCAAGGTTTCTGTTATTGAGTGTATATCTGTTGACTAGAT AGTCCCAGTTGAGTTGTATCCAATTCCAGGCCATGTTCTTCCCATAGCTGTTATATGAGATATATCG AATGACTGTAAACACATCCTGAGTTTTAATAAGGTTCGTGTCCTTGAGCAAATCCAAATACCTTGAC AAAAGAGTAACGTTCTTCACTGATGCTAATCCATACAGCAGTTTTTCTTTTTCTTGAGCTAATGAAGT TTTCTGGTATTGCTCAAGAGTGTAGTTCCATGAAATCTCATTGCCAGAGTTCTGCATCCCATACCGA TACACCAGAAGCCTGAGATTTACGGGAAGGCTTACAGTCCCATTTAGCCACTGCTCAAATAACGAG GAAGCATTGTTCAAGGCTTCTCTGTCTCCCATCTTGCACGCAAACCCTAACACGGAGGAACGGAGT AACTTTGTGACATGGTCTCCAGCATCATTCCATCCCAGAGAATCTGCAATAGGCTTCACTTGACCTT GGAAGTATTCCTCAATCATAGGATATAGCTCTTTATCATCTTCAAACATGCTAATGATGTAGGTTACA GCTGAAATTACTCTCTGCCATGGTAAAAAATTCTCTTCCCTTTTGAGATACTTGGTCAAGTTCAAAG CCACCTTATAATCTAGAAGTTGAGCTCTTGCCAAGGCAAAAGCATCATCAATAAGACTTGCACGATC TGCTGAAGAAAATGTCTTGTGGTTCAAGGAGAGCGCTGTAGCTATCGAGTCCCAAGTTGCTACTTC ATAATTTACACGATAAAACCCAATATGATCTGGGTTTATTTTGAGAAAAGCATTTCCACTAGGATTA GAGGAGTTCAAAGTGATTCCTTCTTTTTCTGACCTATTAAATAACACACTGCTTGTTATATTATCTTC AGTCCATTTAACTGGGATATTCCATGTATAACCAAGATCTGAAGGGGGCTGAGAAGGGTTAGCTCT TGGGTCCAACAAAAAGCGTTTCTGTGTGATGTTCTTGACACCGTTCACGTTAAGCACAGGATAACC CATCTgGTCTGGTCCAGGTGTCCATTACTTCTTTCACTGGTAGCCTACTTGCCTCTTCCAGTGCTGC CCAAAAAT

cDNA fragments were radiolabelled with [α-³²P]dCTP (specific activity6000 Ci/mmol), using the Megaprime labeling kit (GE Healthcare Cat. RPN 1607) and unincorporated nucleotides removed from the reaction using ProbeQuant G-50 microcolumns (GE Healthcare Cat. 27-5335-01). Membranes were pre-hybridized in Rapid-hyb buffer (GE Healthcare Cat. RPN 1635) for at least 30 minutes and subsequently hybridized with 100-300 ng of the labelled cDNA probe. Hybridizations were performed in Rapid-hyb buffer at 65° C. overnight. The labelled probes were heated for 5 minutes at 95° C. before addition to the filters in the pre-hybridization solution. After hybridization, the membranes were washed at low stringency in 2×SSC, 0.05% SDS at room temperature for 30-40 minutes followed by two high stringency washes in 0.1×SSC, 0.1% SDS at 50° C. for 40 minutes. The blots were immediately sealed and exposed to Kodak BioMax MR X-ray film (Cat. 8715187).

Surveying for Candidate Regulatory Variants in the AF Region

[0281] The UCSC browser was used to extracted positions of SNPs and conserved transcription factor binding sites (TFBS) for a 172.5 kb region around the SNPs associated with AF (hg release 17, chromosome 4, 111,942,401-112,114,901). The two tables were cross referenced and SNPs that landed in binding sites were further interrogated for LD with rs2220427 or rs6843082 in the HapMap data. This was done for releases 16, 17 and 18 of the human genome, but the results are reported in hg 17 coordinates. This yielded 3 SNPs that land in conserved binding sites for known transcription factors (Table 17). Note, this analysis only detects a limited sample of functional candidates as i) the AF haplotypes have not been sequenced fully, ii) several candidate SNPs are not typed in Hapmap and it is unknown whether they sit on the AF haplotypes, iii) polymorphisms in less conserved regions could be functional.

Evolutionary Conservation of Three TFBS

[0282] Utilizing the Multiz alignment in the UCSC genome enabled an assessment of the evolutionary conservation of the regions affected by these SNPs. In all three cases is the core part of the TF binding sites intact, but the positions affected are preserved to a different degree. The SOX5 affected by rs12510087 is least conserved in mammals but the second one (affected by rs2220427) is strikingly preserved (with the exception of Opossum it is maintained in all species to the chicken). The rs17042171 mutation is in last position in the core GGAAAA motif of the NFAT binding site. The conservation indicates that a G is preferred at this location, resulting in a GGAAAG motif.

Correlation Between Genotype and Expression of ENPEP

[0283] Blood was collected in the morning, between 8 and 10 am, after overnight fasting (from 9 pm) and RNA extracted within 2 hours from phlebotomy from 1,002 individuals. RNA isolation was performed using the RNeasy Midi Kit (QIAGEN GmbH, Hilden, Germany). Subcutaneous fat samples (5-10 cm³) were removed through a 3 cm incision at the bikini line (always from the same site to avoid site-specific variation) after local anesthesia using 10 ml of lidocaine-adrenalin (1%) from 673 individuals. Purification of the total RNA was performed with the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany).

[0284] Integrity of the total RNA was assessed through analysis on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, U.S., CA). Each labelled RNA sample including reference pools, 1,765 samples in total, was hybridized to a Human 25K array manufactured by Agilent Technologies. Array images were processed as described previously to obtain background noise, single-channel intensity and associated measurement error estimates¹¹. Expression changes between two samples were quantified as mean logarithm (log₁₀) expression ratio (MLR), i.e. expression ratios compared to background corrected intensity values for the two channels for each spot on the arrayl². The hybridizations went through standard QC process, i.e. signal to noise ratio, reproducibility and accuracy at spike-in compounds, comparing Cy3 to Cy5 intensities.

[0285] Neither associated SNP was correlated to the expression of ENPEP adjusted for age and sex in blood (P=0.90 and P=0.82 for rs2200733 and rs10033464, respectively) or adipose tissue (P=0.23 and P=0.37 for rs2200733 and rs10033464, respectively)

TABLE-US-00008 TABLE 7 Analysis of the association of rs2200733 and rs10033464 on chromosome 4q25 to AF/AFl. Sample rs2200733 T^a rs10033464 T^a, b (N cases/ OR OR Compa- Joint N controls) Freq.^c (95% CI) P Freq.^c (95% CI) P rison P^d PAR Iceland^e Discovery 0.191 1.84 2.0 × 10^-11 0.110 1.42 0.0024 0.041 0.216 (550/4,476) 0.114 (1.54-2.21) 0.080 (1.13-1.77) Replication 0.166 1.64 2.7 × 10^-23 0.108 1.40 8.2 × 10^-8 0.028 0.176 (2,251/13,238) 0.108 (1.49-1.81) 0.080 (1.24-1.58) Combined 0.171 1.68 1.9 × 10^-30 0.108 1.40 9.4 × 10^-9 0.0025 0.180 (2,801/17,714) 0.110 (1.53-1.83) 0.080 (1.25-1.55) Other European ancestry Sweden 0.179 2.01 0.00027 0.172 1.65 0.0087 0.41 0.272 (143/738) 0.098 (1.38-2.93) 0.111 (1.14-2.41) U.S. 0.229 1.84 9.8 × 10^-10 0.105 1.30 0.052 0.026 0.232 (636/804) 0.139 (1.51-2.23) 0.083 (1.00-1.69) Combined^f -- 1.88 1.2 × 10^-12 -- 1.41 0.0019 0.027 0.237 -- (1.58-2.23) -- (1.13-1.75) All European ancestry Combined^f -- 1.72 3.3 × 10^-41 -- 1.39 6.9 × 10^-11 0.00019 0.206 -- (1.59-1.86) -- (1.26-1.53) Hong Kong Hong Kong 0.605 1.42 0.00064 0.190 1.08 0.55 0.0099 0.346 (333/2,836) 0.528 (1.16-1.73) 0.218 (0.84-1.39) Each row contains the results from a joint analysis of two variants, rs2200733 T and rs10033464 T^b. The numbers of cases and controls (N) are shown for each case-control study and for each variant the allelic frequencies of the variant in cases and controls, the OR with a 95% CI and two- sided P values, are shown. In addition a P value for comparing the effect of the two variants and their joint population attributable risk (PAR) is reported. For example, the first row indicates that, for the initial Icelandic discovery samples, rs2200733 T has an estimated odds ratio (OR) of 1.84 (95% CI (1.54-2.21), P = 4.1 × 10^-11) vs the wild type (rs2200733 C, rs13143308 G, rs10033464 G haplotype), and rs10033464 T has an estimated OR of 1.42 (95% CI (1.13-1.77), P = 0.0024) vs the wild type. ^aResults of comparing rs2200733 T and rs10033464 T to the wild type rs2200733 C, rs13143308 G, rs10033464 G haplotype. ^bIn the Swedish and the U.S. samples rs10033464 T was tagged by the rs2200733 C, rs13143308 T haplotype ^cThe frequency in cases (above) and controls (below) ^dP value for comparing the ORs of rs2200733 T and rs10033464 T. ^eThe association analysis was adjusted for the relatedness of some of the individuals. ^fFor the combined study populations of European decent, the PAR was calculated by using the average, unweighted control frequency of the populations, while the OR and the P value were estimated using the Mantel-Haenszel model.

TABLE-US-00009 TABLE 8 Association by age at diagnosis in Iceland and by AF sub-phenotype in the U.S. Sample rs2200733^a rs10033464^a, b (N cases/ Male OR OR N controls) % Age ± SD (95% CI) (95% CI) P Sex P Iceland^c Diagn. ≦60 77.8 50.7 ± 8.4 2.12 1.69 6.3 × 10^-18 0.82 (510/17,714) (1.77-2.54) (1.34-2.12) Diagn. 60-70 66.2 65.6 ± 2.9 1.88 1.44 6.7 × 10^-15 0.58 (654/17,714) (1.60-2.21) (1.18-1.77) Diagn. 70-80 58.9 75. 0 ± 2.8 1.60 1.23 7.5 × 10^-11 0.96 (958/17,714) (1.39-1.84) (1.03-1.47) Diagn. >80 47.4 85.6 ± 4.2 1.20 1.31 0.0044 0.36 (679/17,714) (1.01-1.43) (1.08-1.60) U.S. Lone AF 81.7 46.1 ± 11.5 2.32 1.68 1.2 × 10^-10 0.46 (251/804) (1.80-2.99) (1.19-2.37) AF/HTN 74.6 54.5 ± 10.2 2.23 1.66 0.0010 0.54 (67/804) (1.43-3.48) (0.90-3.04) Other AF 52.8 75.2 ± 11.3 1.44 0.97 0.015 0.85 (318/804) (1.12-1.84) (0.69-1.37) Each row contains the results from a joint analysis of two variants, rs2200733 T and rs10033464 T^a. The numbers of cases and controls (N), the percentage of male cases, and the mean age (±SD) for cases, are shown for each case-control study. The OR, with a 95% CI, and P values are shown for each variant. In addition a joint P value for the combined effect of the two variants, and a joint P value for testing if there is a difference of the allelic frequency of the variants between the sexes within each sub-group of patients. ^aResults of comparing rs2200733 T and rs10033464 T to the wild type rs2200733 C, rs13143308 G, rs10033464 G haplotype. ^bIn the U.S. samples rs10033464 T was tagged by the rs2200733 C, rs13143308 T haplotype. ^cThe association analysis was adjusted for the relatedness of some of the individuals.

TABLE-US-00010 TABLE 9 SNPs equivalent to rs10033464, rs13143308 and rs2200733 in CEU HapMap data Tagging SNP SNP Build 35 location SEQ ID NO: 50 location rs12503217 rs10033464 112063765 108955 rs12510087 rs10033464 112066632 111822 rs6852357 rs10033464 112071939 117129 rs4400058 rs10033464 112074277 119467 rs10033464 rs10033464 112078365 123555 rs2171592 rs10033464 112078392 123582 rs2350539 rs10033464 112078814 124004 rs1906606 rs10033464 112080996 126186 rs723364 rs10033464 112082075 127265 rs2220429 rs10033464 112085089 130279 rs4032976 rs10033464 112086371 131561 rs3853440 rs10033464 112087213 132403 rs3853441 rs10033464 112087344 132534 rs3853442 rs10033464 112087632 132822 rs3853443 rs10033464 112087733 132923 rs4124158 rs10033464 112087798 132988 rs4124159 rs10033464 112087847 133037 rs12506083 rs10033464 112088016 133206 rs4032975 rs10033464 112089842 135032 rs4032974 rs10033464 112090140 135330 rs2634074 rs13143308 112034645 79835 rs2466455 rs13143308 112043219 88409 rs2723334 rs13143308 112046356 91546 rs1906616 rs13143308 112055172 100362 rs1906615 rs13143308 112059402 104592 rs2129983 rs13143308 112061684 106874 rs2129982 rs13143308 112061747 106937 rs1906599 rs13143308 112070290 115480 rs13143308 rs13143308 112072023 117213 rs6843082 rs13143308 112075671 120861 rs17042059 rs2200733 111998790 43980 rs4529121 rs2200733 112003159 48349 rs4543199 rs2200733 112005744 50934 rs12647316 rs2200733 112006855 52045 rs10019689 rs2200733 112007473 52663 rs4626276 rs2200733 112007593 52783 rs17042076 rs2200733 112009942 55132 rs11098089 rs2200733 112011830 57020 rs17042088 rs2200733 112012418 57608 rs11930528 rs2200733 112017798 62988 rs17042098 rs2200733 112021762 66952 rs17042102 rs2200733 112026230 71420 rs17042121 rs2200733 112034705 79895 rs10516563 rs2200733 112035326 80516 rs4605724 rs2200733 112042685 87875 rs2350269 rs2200733 112044728 89918 rs6533527 rs2200733 112045118 90308 rs17042144 rs2200733 112047270 92460 rs1906618 rs2200733 112053026 98216 rs1906617 rs2200733 112053418 98608 rs12646447 rs2200733 112056930 102120 rs12646754 rs2200733 112061176 106366 rs2129981 rs2200733 112061803 106993 rs12639654 rs2200733 112062899 108089 rs6817105 rs2200733 112063372 108562 rs17042171 rs2200733 112065891 111081 rs1906591 rs2200733 112066493 111683 rs1906592 rs2200733 112066608 111798 rs2200732 rs2200733 112067646 112836 rs2200733 rs2200733 112067773 112963 rs4611994 rs2200733 112068645 113835 rs4540107 rs2200733 112068706 113896 rs1906593 rs2200733 112069526 114716 rs1906596 rs2200733 112069840 115030 rs2220427 rs2200733 112072493 117683

TABLE-US-00011 TABLE 10 Haplotype structure (haplotypes with estimated frequency >0.1%) over key SNPs and the D4S406 microsatellite in Iceland Frequency D4S406 rs2200733 rs13143308 rs10033464 0.0800 -8 T T G 0.00647 -6 C T T 0.00225 -4 T T G 0.0415 -2 T T G 0.00108 0 T T G 0.0592 0 C T T 0.00679 2 C T T 0.0169 2 C G G 0.00923 4 C T T 0.135 4 C G G 0.0853 6 C G G 0.1587 8 C G G 0.163 10 C G G 0.0928 12 C G G 0.0398 14 C G G 0.101 16 C G G

TABLE-US-00012 TABLE 11 Association to all Hap300 IIlumina SNPs in a 200 kb region around rs2200733 and rs10033464 in an extended set of Icelandic AF/AFl cases and controls. Results have not been adjusted for relatedness of individuals. Adjusting for Also adjusting rs2220427 for rs10033464 SNP Location All. Freq OR P value OR P value OR P value rs4834295 111892810 G 0.817 1.0 0.27 1.0 0.39 1.03 0.63 rs2278782 111899758 C 0.883 1.0 0.79 0.9 0.70 0.99 0.93 rs2595110 111902927 T 0.637 1.0 0.13 1.0 1.0 1.01 0.89 rs976568 111908325 A 0.743 1.0 0.83 0.9 0.62 0.97 0.58 rs2197815 111924481 T 0.030 1.1 0.34 1.1 0.34 0.97 0.84 rs2723286 111940938 A 0.231 1.0 0.26 1.0 0.50 1.03 0.59 rs2723296 111962087 G 0.229 1.0 0.38 1.0 0.60 1.03 0.67 rs1699716 111986643 T 0.153 1.3 4.7 × 10^-5 0.9 0.59 0.95 0.53 rs2723316 111991891 T 0.297 1.2 1.9 × 10^-5 1.0 0.59 0.95 0.40 rs6419178 111993104 A 0.143 1.1 0.17 1.0 0.25 0.98 0.77 rs1448817 111998657 G 0.252 1.4 4.2 × 10.sup.- 1.1 0.035 1.06 0.46 rs2634073 112023387 A 0.167 1.6 2.4 × 10.sup.- 1.2 0.039 0.90 0.48 rs2200733 112067773 T 0.119 1.7 7.6 × 10.sup.- -- -- -- -- rs2220427 112072493 T 0.120 1.7 5.6 × 10.sup.- -- -- -- -- rs1310587 112075751 C 0.888 1.0 0.33 0.9 0.89 0.95 0.56 rs1003346 112078365 T 0.082 1.2 0.013 1.3 5.1 × 10^-4 -- -- rs1314119 112086218 A 0.368 1.3 2.0 × 10.sup.- 1.1 0.0067 1.08 0.29 rs3853444 112091740 A 0.604 1.1 0.053 1.0 0.45 1.06 0.24

TABLE-US-00013 TABLE 12 Association study of SNPs which are equivalent to rs2200733 in CEU HapMap samples in the Chinese samples from Hong Kong. SNP Location All. Freq OR P value HapMap D' HapMap R² rs11930528 112017798 T 0.472 1.27 0.011 0.91 0.66 rs17042121 112034705 G 0.418 1.32 0.0029 0.97 0.64 rs6533527 112045118 A 0.518 1.37 0.0014 0.95 0.79 rs1906617 112053418 C 0.524 1.35 0.0026 1.00 0.98 rs12639654 112062899 T 0.519 1.39 0.0012 1.00 1.00 rs2200733 112067773 T 0.516 1.42 6.4 × 10^-4 -- -- rs4611994 112068645 C 0.518 1.39 0.0012 1.00 1.00 The LD values reported are to rs2200733 in the combined CHB and JPT HapMap samples

TABLE-US-00014 TABLE 13 Association to AF/AFl by genotype Allelic RR Genotype RR 1 2 00 01 02 11 12 22 P value Iceland 1.68 1.38 1 1.55 1.36 3.42 2.47 1.58 0.12 Sweden 2.01 1.65 1 1.66 1.72 5.86 3.10 2.04 0.68 U.S. 1.84 1.30 1 1.63 1.40 4.86 2.31 0.90 0.25 Combined 1.71 1.38 1 1.56 1.37 3.64 2.44 1.43 0.018 Hong Kong 1.42 1.07 1 1.15 0.95 1.77 1.34 0.97 0.87 The three possible haplotypes are coded as 0 = rs2200733 C, rs13143308 G, rs10033464 G 1 = rs2200733 T, rs13143308 T, rs10033464 G 2 = rs2200733 C, rs13143308 T, rs10033464 T

TABLE-US-00015 TABLE 14 Association of various phenotypes, considered risk factors for AF to risk variants. Phenotype T rs2200733 T rs10033464 (N cases/N controls) OR P value OR P value Hypertension 1.08 0.11 1.05 0.37 (2,620/19,862) Myocardial infarction 1.05 0.26 1.04 0.49 (3,576/19,542) Obesity-BMI >35 0.96 0.51 1.00 1.00 (1,601/21,593)

TABLE-US-00016 TABLE 15 A summary of the source of the Icelandic controls. Note that individuals may come from multiple project and that some individuals may have been collected as relatives of probands. Frequency of Frequency of Source Project Count T rs2200733 T rs10033464 Discovery Controls Addiction 376 0.096 0.082 Anxiety 337 0.110 0.088 Breast Cancer 876 0.116 0.085 Colon Cancer 370 0.119 0.070 Infectious Disease 297 0.109 0.096 MI 454 0.104 0.076 Population Controls 389 0.099 0.077 Prostate Cancer 713 0.123 0.081 Schizophrenia 291 0.110 0.091 Type II Diabetes 551 0.102 0.078 Replication Controls Breast Cancer 228 0.122 0.074 Type II Diabetes 340 0.097 0.082 Alzheimer 459 0.107 0.061 Osteoarthritis 1,175 0.107 0.081 PAD 479 0.096 0.083 COPD 326 0.125 0.082 Stroke 414 0.092 0.069 Osteoporosis 1,155 0.109 0.072 MI 390 0.112 0.075 Hypertension 210 0.118 0.101 Depression 152 0.128 0.061 Asthma 538 0.106 0.076 Parkinson 173 0.102 0.058 Population Controls 305 0.105 0.097 Ankylosing Spondylitis 155 0.095 0.077 Sleep Apnea 422 0.118 0.074 AMD 442 0.101 0.067 Rheumatoid Arthritis 430 0.100 0.094 Lung Cancer 237 0.106 0.084 FCH 265 0.112 0.057 Longevity 392 0.09 0.077 Benign Prostatic Hyperplasia 245 0.101 0.058 Pre-eclampsia 262 0.129 0.083 Enuresis 249 0.104 0.087 Migrane 590 0.112 0.085 Myopia 353 0.123 0.085 Thyroid Cancer 104 0.121 0.097 ADHD 123 0.119 0.089 Prostate Cancer 580 0.117 0.073 Anxiety 546 0.121 0.096 Obesity 162 0.081 0.092 Endometriosis 258 0.106 0.084 Kidney Cancer 174 0.099 0.100 Melanoma 283 0.088 0.089 Addiction 201 0.138 0.098 Psoriasis 392 0.136 0.079 IBD 356 0.093 0.102

TABLE-US-00017 TABLE 16 Primers used for ESTs screening of cDNA libraries ESTs* Forward primer Reverse primer DA725631 AGTGGAGGCTGCCAGACTTC TGCACCACTCATCACCAACA DB324364 CCGAGGATGTCTTTAGTCTGCAA ATCATACAGCAGGAATGCAAACA AF017091 TGAGATTCCACATCCAACATCTTT TGGCAAACTTGATATTGTTCTTG *EST names are from NCBI BUILD 35

TABLE-US-00018 TABLE 17 SNPs that land in conserved TFBS in the region associated with AF. SNP Location Strand Ancestral Polym. TFBS TF start TF end rs17042171 112065890 + C NC NFAT 112065889 112065900 rs12510087 112066631 + A NG SOX5 112066632 112066641 rs2220427 112072492 + C C/T SOX5 112072483 112072493 Strand indicates the strand in genome alignment that the mutation lands in. Polym. is the two alleles of the polymorphism at this site.

TABLE-US-00019 TABLE 18 Markers in or near the PITX2 gene in LD with markers in the LD block C04. Shown are markers in or near PITX2 (marker 1) and their correlation to markers in LD block C04 (marker 2). Marker 1 Marker 2 D' r2 p-value rs7668322 rs10033464 0.46291 0.133423 0.000953 rs2197815 rs10033464 0.660377 0.300172 2.55E-06 rs6831623 rs2200733 1 0.02834 0.025473 rs2595110 rs2200733 0.699643 0.02996 0.067245

TABLE-US-00020 TABLE 19 Markers in linkage disequilibrium with marker rs2220427 and markerrs10033464 by values for r² of greater than 0.1. LD was calculated based on the HapMap CEU population sample. Pos in Pos in SEQ ID Marker 1 anchor D' r2 P-value B35 NO: 50 rs9994891 rs2220427 1 0.128329 0.002914 111149057 rs11568995 rs2220427 1 0.128329 0.002914 111255189 rs4698804 rs2220427 1 0.128329 0.002914 111297649 rs721413 rs2220427 1 0.128329 0.002914 111305212 rs10488883 rs2220427 1 0.128329 0.002914 111305486 rs6854883 rs2220427 0.788889 0.510189 4.17E-09 111964919 10109 rs2255793 rs2220427 1 0.245283 9.27E-09 111965457 10647 rs2723298 rs2220427 1 0.274924 8.39E-09 111966089 11279 rs2723300 rs2220427 1 0.236507 1.40E-08 111972512 17702 rs2723307 rs2220427 1 0.176558 2.98E-07 111975800 20990 rs1584429 rs2220427 1 0.245283 9.27E-09 111976151 21341 rs1448799 rs2220427 1 0.245283 9.27E-09 111980386 25576 rs1448798 rs2220427 1 0.245283 9.27E-09 111980789 25979 rs1900827 rs2220427 1 0.246741 8.60E-08 111981343 26533 rs2197814 rs2220427 1 0.240506 1.20E-08 111983098 28288 rs969642 rs2220427 1 0.245283 9.27E-09 111983529 28719 rs2595093 rs2220427 0.830131 0.513828 1.59E-10 111984960 30150 rs2245595 rs2220427 1 0.252078 6.90E-09 111985715 30905 rs2595088 rs2220427 1 0.254302 1.99E-08 111985958 31148 rs981150 rs2220427 1 0.245283 9.27E-09 111986232 31422 rs16997168 rs2220427 0.819277 0.507451 2.11E-10 111986643 31833 rs16997169 rs2220427 1 0.245283 9.27E-09 111986685 31875 rs4527540 rs2220427 1 0.245283 9.27E-09 111986742 31932 rs17042026 rs2220427 0.833488 0.554106 1.23E-11 111989978 35168 rs2723316 rs2220427 1 0.245283 9.27E-09 111991891 37081 rs2595081 rs2220427 0.832621 0.549261 3.07E-11 111992761 37951 rs2595085 rs2220427 1 0.242283 1.16E-08 111994377 39567 rs2723318 rs2220427 1 0.236507 1.40E-08 111994576 39766 rs1448817 rs2220427 1 0.296277 9.75E-10 111998657 43847 rs17042059 rs2220427 1 1 1.62E-20 111998790 43980 rs4529121 rs2220427 1 1 1.43E-20 112003159 48349 rs10032150 rs2220427 1 0.296277 9.75E-10 112004222 49412 rs4543199 rs2220427 1 1 1.43E-20 112005744 50934 rs12647316 rs2220427 1 1 1.43E-20 112006855 52045 rs12647393 rs2220427 1 0.917379 1.57E-15 112006886 52076 rs10019689 rs2220427 1 1 1.43E-20 112007473 52663 rs4626276 rs2220427 1 1 1.43E-20 112007593 52783 rs17042076 rs2220427 1 1 1.62E-20 112009942 55132 rs11098089 rs2220427 1 1 1.62E-20 112011830 57020 rs17042088 rs2220427 1 1 1.62E-20 112012418 57608 rs11944778 rs2220427 0.91509 0.811642 4.20E-12 112014571 59761 rs4307025 rs2220427 1 0.296277 9.75E-10 112015107 60297 rs11930528 rs2220427 1 1 1.42E-19 112017798 62988 rs17042098 rs2220427 1 1 1.43E-20 112021762 66952 rs2634073 rs2220427 1 0.523052 1.42E-12 112023387 68577 rs17042102 rs2220427 1 1 2.07E-16 112026230 71420 rs2634071 rs2220427 1 0.528302 2.16E-13 112026824 72014 rs2634074 rs2220427 1 0.433962 5.12E-12 112034645 79835 rs17042121 rs2220427 1 1 1.43E-20 112034705 79895 rs10516563 rs2220427 1 1 1.43E-20 112035326 80516 rs4605724 rs2220427 1 1 1.43E-20 112042685 87875 rs2466455 rs2220427 1 0.491956 5.72E-12 112043219 88409 rs2350269 rs2220427 1 1 1.42E-19 112044728 89918 rs6533527 rs2220427 1 1 1.43E-20 112045118 90308 rs2723334 rs2220427 1 0.43396 25.12E-12 112046356 91546 rs17042144 rs2220427 1 1 1.43E-20 112047270 92460 rs1906618 rs2220427 1 1 2.67E-19 112053026 98216 rs1906617 rs2220427 1 1 1.43E-20 112053418 98608 rs6847935 rs2220427 1 0.921053 1.10E-18 112054255 99445 rs1906616 rs2220427 1 0.433962 5.12E-12 112055172 100362 rs12646447 rs2220427 1 1 1.84E-20 112056930 102120 rs1906615 rs2220427 1 0.433962 5.12E-12 112059402 104592 rs12646754 rs2220427 1 1 2.08E-20 112061176 106366 rs2129983 rs2220427 1 0.428571 6.61E-12 112061684 106874 rs2129982 rs2220427 1 0.433962 5.12E-12 112061747 106937 rs2129981 rs2220427 1 1 1.43E-20 112061803 106993 rs12639654 rs2220427 1 1 1.43E-20 112062899 108089 rs6817105 rs2220427 1 1 1.62E-20 112063372 108562 rs17042171 rs2220427 1 1 1.43E-20 112065891 111081 rs1906591 rs2220427 1 1 1.43E-20 112066493 111683 rs1906592 rs2220427 1 1 1.26E-19 112066608 111798 rs2200732 rs2220427 1 1 1.85E-19 112067646 112836 rs2200733 rs2220427 1 1 1.43E-20 112067773 112963 rs4611994 rs2220427 1 1 1.43E-20 112068645 113835 rs4540107 rs2220427 1 1 1.43E-20 112068706 113896 rs1906593 rs2220427 1 1 1.62E-20 112069526 114716 rs1906596 rs2220427 1 1 2.68E-20 112069840 115030 rs1906599 rs2220427 1 0.433962 5.12E-12 112070290 115480 rs13143308 rs2220427 1 0.438445 5.36E-12 112072023 117213 rs6843082 rs2220427 1 0.433962 5.12E-12 112075671 120861 rs11931959 rs2220427 1 0.249653 7.85E-09 112077289 122479 rs13121924 rs2220427 1 0.156089 9.36E-07 112078423 123613 rs2129979 rs2220427 1 0.256789 5.78E-09 112078601 123791 rs723363 rs2220427 1 0.156089 9.36E-07 112082105 127295 rs7697491 rs2220427 1 0.154058 1.07E-06 112083422 128612 rs13141190 rs2220427 1 0.156089 9.36E-07 112086218 131408 rs6533530 rs2220427 1 0.156089 9.36E-07 112089540 134730 rs6533531 rs2220427 1 0.156089 9.36E-07 112089569 134759 rs3866831 rs2220427 1 0.156089 9.36E-07 112089718 134908 rs3866832 rs2220427 0.857992 0.109603 0.000186 112091304 136494 rs11098083 rs10033464 0.407276 0.12964 0.00205 111855920 rs11721423 rs10033464 0.365905 0.11129 0.003321 111858873 rs10005945 rs10033464 0.433962 0.101848 0.004763 111860013 rs7668322 rs10033464 0.46291 0.133423 0.000953 111906200 rs2197815 rs10033464 0.660377 0.300172 2.55E-06 111924481 rs6831623 rs10033464 1 0.511236 3.33E-10 111964677 9867 rs7661383 rs10033464 0.637611 0.21478 0.000011 111979181 24371 rs7667461 rs10033464 0.637611 0.21478 0.000011 111979738 24928 rs1900827 rs10033464 0.735008 0.134278 0.000368 111981343 26533 rs998101 rs10033464 0.635887 0.20914 0.000014 111988219 33409 rs12646859 rs10033464 0.719793 0.271646 0.000191 111992237 37427 rs12498380 rs10033464 0.60232 0.178165 0.000097 111992563 37753 rs7690164 rs10033464 0.496308 0.148782 0.005455 111994069 39259 rs11098090 rs10033464 0.551083 0.169161 0.000104 112014012 59202 rs2634073 rs10033464 0.640189 0.223694 8.35E-06 112023387 68577 rs2634071 rs10033464 0.637611 0.21478 0.000011 112026824 72014 rs2634074 rs10033464 1 0.433962 5.12E-12 112034645 79835 rs2466455 rs10033464 1 0.428256 1.77E-09 112043219 88409 rs2723334 rs10033464 1 0.433962 5.12E-12 112046356 91546 rs1906616 rs10033464 1 0.433962 5.12E-12 112055172 100362 rs1906615 rs10033464 1 0.433962 5.12E-12 112059402 104592 rs2129983 rs10033464 1 0.428571 6.61E-12 112061684 106874 rs2129982 rs10033464 1 0.433962 5.12E-12 112061747 106937 rs12503217 rs10033464 1 1 1.43E-20 112063765 108955 rs12510087 rs10033464 1 1 1.43E-20 112066632 111822 rs1906599 rs10033464 1 0.433962 5.12E-12 112070290 115480 rs6852357 rs10033464 1 1 1.43E-20 112071939 117129 rs13143308 rs10033464 1 0.421583 2.22E-11 112072023 117213 rs4833456 rs10033464 1 0.923858 6.67E-19 112073911 119101 rs4400058 rs10033464 1 1 1.43E-20 112074277 119467 rs6843082 rs10033464 1 0.433962 5.12E-12 112075671 120861 rs2171592 rs10033464 1 1 1.43E-20 112078392 123582 rs13121924 rs10033464 1 0.156089 9.36E-07 112078423 123613 rs2350539 rs10033464 1 1 1.43E-20 112078814 124004 rs1906606 rs10033464 1 1 1.43E-20 112080996 126186 rs723364 rs10033464 1 1 1.43E-20 112082075 127265 rs723363 rs10033464 1 0.156089 9.36E-07 112082105 127295 rs7697491 rs10033464 1 0.154058 1.07E-06 112083422 128612 rs2220429 rs10033464 1 1 1.43E-20 112085089 130279 rs13141190 rs10033464 1 0.156089 9.36E-07 112086218 131408 rs4032976 rs10033464 1 1 1.62E-20 112086371 131561 rs3853440 rs10033464 1 1 1.62E-20 112087213 132403 rs3853441 rs10033464 1 1 1.43E-20 112087344 132534 rs3853442 rs10033464 1 1 1.43E-20 112087632 132822 rs3853443 rs10033464 1 1 1.43E-20 112087733 132923 rs4124158 rs10033464 1 1 1.10E-17 112087798 132988 rs4124159 rs10033464 1 1 1.43E-20 112087847 133037 rs12506083 rs10033464 1 1 1.43E-20 112088016 133206 rs6533530 rs10033464 1 0.156089 9.36E-07 112089540 134730 rs6533531 rs10033464 1 0.156089 9.36E-07 112089569 134759 rs3866831 rs10033464 1 0.156089 9.36E-07 112089718 134908 rs4032975 rs10033464 1 1 7.06E-20 112089842 135032 rs4032974 rs10033464 1 1 1.43E-20 112090140 135330 rs3866832 rs10033464 1 0.148936 1.44E-06 112091304 136494 rs7654080 rs10033464 1 0.151515 0.002495 112585323

REFERENCES

[0286] 1. Go, A. S. et al. Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the AnTicoagulation and Risk Factors in Atrial Fibrillation (ATRIA) Study. Jama 285, 2370-5 (2001).

[0287] 2. Miyasaka, Y. et al. Secular trends in incidence of atrial fibrillation in Olmsted County, Minn., 1980 to 2000, and implications on the projections for future prevalence. Circulation 114, 119-25 (2006).

[0288] 3. Arnar, D. O. et al. Familial aggregation of atrial fibrillation in Iceland. Eur Heart J 27, 708-12 (2006).

[0289] 4. Fox, C. S. et al. Parental atrial fibrillation as a risk factor for atrial fibrillation in offspring. Jama 291, 2851-5 (2004).

[0290] 5. Ellinor, P. T., Yoerger, D. M., Ruskin, J. N. & MacRae, C. A. Familial aggregation in lone atrial fibrillation. Hum Genet 118, 179-84 (2005).

[0291] 6. Chen, Y. H. et al. KCNQ1 gain-of-function mutation in familial atrial fibrillation. Science 299, 251-4 (2003).

[0292] 7. Yang, Y. et al. Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation. Am J Hum Genet 75, 899-905 (2004).

[0293] 8. Xla, M. et al. A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation. Biochem Biophys Res Commun 332, 1012-9 (2005).

[0294] 9. Olson, T. M. et al. Kv1.5 channelopathy due to KCNA5 loss-of-function mutation causes human atrial fibrillation. Hum Mol Genet 15, 2185-91 (2006).

[0295] 10. Hong, K., Bjerregaard, P., Gussak, I. & Brugada, R. Short QT syndrome and atrial fibrillation caused by mutation in KCNH2. J Cardiovasc Electrophysiol 16, 394-6 (2005).

[0296] 11. Ellinor, P. T. et al. Mutations in the long QT gene, KCNQ1, are an uncommon cause of atrial fibrillation. Heart 90, 1487-8 (2004).

[0297] 12. Ellinor, P. T., Petrov-Kondratov, V. I., Zakharova, E., Nam, E. G. & MacRae, C. A. Potassium channel gene mutations rarely cause atrial fibrillation. BMC Med Genet 7, 70 (2006).

[0298] 13. Franco, D. & Campione, M. The role of Pitx2 during cardiac development. Linking left-right signaling and congenital heart atrial fibrillation and/or strokes. Trends Cardiovasc Med 13, 157-63 (2003).

[0299] 14. Faucourt, M., Houliston, E., Besnardeau, L., Kimelman, D. & Lepage, T. The pitx2 homeobox protein is required early for endoderm formation and nodal signaling. Dev Biol 229, 287-306 (2001).

[0300] 15. Mommersteeg, M. T. et al. Molecular Pathway for the Localized Formation of the Sinoatrial Node. Circ Res (2007).

[0301] 16. A haplotype map of the human genome. Nature 437, 1299-320 (2005).

[0302] 17. Waldo, A. L. The interrelationship between atrial fibrillation and atrial flutter. Prog Cardiovasc Dis 48, 41-56 (2005).

[0303] 18. Zini, S. et al. Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release. Proc Natl Acad Sci USA 93, 11968-73 (1996).

[0304] 19. Gretarsdottir, S. et al. The gene encoding phosphodiesterase 4D confers risk of ischemic stroke. Nat Genet 35, 131-8 (2003).

[0305] 20. Falk, C. T. & Rubinstein, P. Haplotype relative risks: an easy reliable way to construct a proper control sample for risk calculations. Ann Hum Genet 51 (Pt 3), 227-33 (1987).

[0306] 21. Mantel, N. & Haenszel, W. Statistical aspects of the analysis of data from retrospective studies of atrial fibrillation and/or stroke. J Natl Cancer Inst. 22, 719-48 (1959).

[0307] 22. Grant, S. F. et al. Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet 38, 320-3 (2006).

[0308] 23. Yang, X. et al. Development and validation of stroke risk equation for Hong Kong Chinese patients with type 2 diabetes: the Hong Kong Diabetes Registry. Diabetes Care 30, 65-70 (2007).

[0309] 24. Baum, L. et al. Methylenetetrahydrofolate reductase gene A222V polymorphism and risk of ischemic stroke. Clin Chem Lab Med 42, 1370-6 (2004).

[0310] 25. Kutyavin, I. V. et al. A novel endonuclease IV post-PCR genotyping system. Nucleic Acids Research 34, e128 (2006).

[0311] 26. Amundadottir, L. T. et al. A common variant associated with prostate cancer in European and African populations. Nat Genet 38, 652-8 (2006).

[0312] 27. Gretarsdottir, S. et al. The gene encoding phosphodiesterase 4D confers risk of ischemic stroke. Nat Genet 35, 131-8 (2003).

[0313] 28. Falk, C. T. & Rubinstein, P. Haplotype relative risks: an easy reliable way to construct a proper control sample for risk calculations. Ann Hum Genet 51 (Pt 3), 227-33 (1987).

[0314] 29. Mantel, N. & Haenszel, W. Statistical aspects of the analysis of data from retrospective studies of atrial fibrillation and/or stroke. J Natl Cancer Inst. 22, 719-48 (1959).

[0315] 30. Grant, S. F. et al. Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet 38, 320-3 (2006).

[0316] 31. Devlin, B. & Roeder, K. Genomic Control for association studies. Biometrics 55, 997-1004 (1999).

[0317] 32. Devlin, B., Bacanu, S.-A. & Roeder, K. Genomic control to the extreme. Nature Genetics 36, 1129-1130 (2004).

[0318] 33. Nomenclature and criteria for diagnosis of ischemic heart atrial fibrillation and/or stroke. Report of the Joint International Society and Federation of Cardiology/World Health Organization task force on standardization of clinical nomenclature. Circulation 59, 607-9 (1979).

[0319] 34. Alpert, J. S., Thygesen, K., Antman, E. & Bassand, J. P. Myocardial infarction redefined--a consensus document of The Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. J Am Coil Cardiol 36, 959-69 (2000).

[0320] 35. Monks, S. A. et al. Genetic inheritance of gene expression in human cell lines. Am J Hum Genet 75, 1094-105 (2004).

[0321] 36. Schadt, E. E. et al. Genetics of gene expression surveyed in maize, mouse and man. Nature 422, 297-302 (2003).

Example 3

Association of Chromosome 4 Variants to Ischemic Stroke

[0322] Stroke is a common cause of death and the leading cause of adult disability in Western societies. It is now also becoming a major health problem in low-income and middle-income countries due to population ageing and changes in modifiable risk factors for cardiovascular diseases¹. Stroke is not a single disease but a highly complex syndrome consisting of a group of heterogeneous disorders with many genetic and environmental risk factors^2,3. Studies on twins, family history and animal models^4-8 provide evidence for genetic contribution to the common forms of stroke but no major risk variant has yet been identified showing consistent results across populations.

[0323] Ischemic strokes (IS), accounting for the majority of cerebral insults (>80%), result from thrombosis or embolism leading to obstruction of cerebral arteries. Various pathophysiological mechanisms can cause IS but the most common ones are large artery atherosclerosis (LAA), cardioembolic stroke (CES) and small vessel disease (SVD)⁹.

Methods

Study Populations.

[0324] Iceland:

[0325] Icelandic stroke patients were recruited from a registry of over 4,000 individuals diagnosed with ischemic stroke or TIA at the only university hospital in Reykjavik, the Landspitali University Hospital, during the years 1993 to 2006. Stroke patients have been enrolled over the last nine years through the cardiovascular disease (CVD) genetics program at deCODE. Stroke diagnosis was clinically confirmed by neurologists (see below). The discovery cohort included 1,661 patients and when analysing the SNPs on 4q25 we used an additional set of 282 patients (mean age±SD: 77.2±11.3 years, 45% females). We used 25,708 controls (mean age±SD: 59.2±21.1 years, 59% females) from various genetic programs under study at Decode, including: abdominal aneurysm (250), atrial fibrillation (1,150), addiction (750), Alzheimer (350), anxiety (200), asthma (1300), COPD (850), colon cancer (200), deep vein thrombosis (550), dyslexia (200), infection diseases (250), longevity (400), lung cancer (750), myocardial infarction (2,400), migraine (1,100), peripheral artery disease (1,200), polycystic ovary syndrome (1,200), pre-eclampsia (700), prostate cancer (400), psoriasis (750), rheumatic arthritis (550), restless leg syndrome (350), and type 2 diabetes (400).

[0326] The study was approved by the Data Protection Commission of Iceland (DPC) and the National Bioethics Committee of Iceland. All participants gave informed consent.

[0327] Sweden:

[0328] Swedish patients with ischemic stroke attending the stroke unit or the stroke outpatient clinic at Karolinska University Hospital, Huddinge unit in Stockholm, Sweden, were recruited from 1996 to 2002 as part of an ongoing genetic epidemiology study, the South Stockholm Ischemic Stroke Study (SSISS) (mean age±SD: 67.3±11.8 years, 44% females). The Swedish controls used in this study are population-based controls recruited from the same region in central Sweden as the patients, representing the general population in this area. The individuals were either blood donors recruited at the Huddinge or Karolinska University Hospitals or healthy volunteers (recruited in 1990-1994) recruited by the Clinical Chemistry Department at the Karolinska University Hospital to represent a normal reference population (mean age±SD: 46.8115.9 years for controls from Huddinge hospital, 41% females, age information not available for blood donors recruited at the Karolinska hospital). The study was approved by the Bioethics Committee of Karolinska Institutet.

[0329] South-Germany:

[0330] The German population, herein referred to as Germany-S, consisted of IS patients consecutively recruited during the period 2001-2006 at the stroke unit of the Department of Neurology, Klinikum Grosshadern, University of Munich, Germany (mean age±SD: 65.3±13.7 years, 38% females). The control group consisted of age and gender matched individuals without a history of cardiovascular disease (mean age±SD: 62.7±10.9 years, 38% females). These were selected from the KORA S4 study, a community based epidemiological project near Munich²³. The study was approved by the local ethics committee and informed consent was obtained from all individuals (or relatives or legal guardians).

[0331] Westphalia Region, Germany:

[0332] The second German population, referred to as Germany-W, recruited ischemic stroke patients through hospitals participating in the regional Westphalian Stroke Register, located in the west of the country, during the period 2000-2003 (mean age±SD: 70.4±12.6 years, 53% females). Population controls without a self-reported history of stroke were drawn from the cross-sectional, prospective, population based Dortmund Health Study²⁴, conducted in the same region, and subsequently frequency matched to the cases (mean age±SD: 52.3±13.7 years, 53% females). Both studies were approved by the ethics committee of the University of Munster. All participants gave their informed consent.

[0333] SE-England, United Kingdom.

[0334] Ischemic stroke patients of European descent attending a cerebrovascular service were recruited 1995-2002. All cases were phenotyped by one experienced stroke neurologist with review of original imaging (mean age±SD: 64.6±12.7 years, 41% females). Community controls free of symptomatic cerebrovascular disease were also recruited by sampling family doctor lists from the same geographical region as the patients. Sampling was stratified to provide a similar distribution of age and gender as in the patient group (mean age±SD: 64.8±8.6 years, 41% females). The study was approved by local research ethics committees and informed consent was obtained from all participants.

Phenotyping.

[0335] Only patients with ischemic but not with hemorrhagic strokes were included in the study. All patients had clinically relevant diagnostic work-up performed, including brain imaging with computed tomography (CT) or/and magnetic resonance imaging (MRI) as well as ancillary diagnostic investigations including duplex ultrasonography of the carotid and vertebral arteries, echocardiography, Holter monitoring, MR-angiography, CT-angiography and blood tests. Patients with clinically confirmed Transient Ischemic Attack (TIA) were included in the Ischemic stroke group from Iceland, Germany-S and Sweden. Patients were classified into etiologic subtypes according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST)²⁵. This classification includes six categories: (1) large-artery occlusive disease (large vessel disease), (2) cardioembolism (cardiogenic stroke), (3) small vessel disease (lacunar stroke), (4) other determined etiology, (5) etiology unknown despite diagnostic efforts, or (6) more than one etiology. Patients classified into the TOAST categories 4-6 were excluded from the stroke population from Germany-W. In Iceland, patients were classified as having large-artery occlusive disease if stenosis was 70% which is a stricter criterion than usually used i.e. 50%. Classification of stroke patients into subtypes according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification system²⁵ in the Icelandic discovery and the four replication sample sets is listed in Table 1.

IIlumina Genome-Wide Genotyping.

[0336] All Icelandic cases and control samples were assayed with the Infinium HumanHap300 SNP chips (Illumina), containing 317,503 tagging SNPs derived from phase 1 of the International HapMap project. OF the SNPs assayed on the chip, 6,622 SNPs were excluded because they showed either (i) a call rate lower than 95% in cases or controls, (ii) minor allele frequency less than 1% in the population or (iii) significant distortion from Hardy-Weinberg equilibrium in the controls (P<1×10^-10). Any sample with yield<98% were excluded from the analysis. In the final analysis 310,881 SNPs were used.

Single SNP Genotyping.

[0337] Single-SNP genotyping for all 121 SNP was carried out at deCODE genetics in Reykjavik, Iceland using the Centaurus (Nanogen) platform²⁶. The quality of each SNP assay was evaluated by comparing the genotyping of the CEU HapMap samples with the publicly available HapMap data. All SNPs passed mismatch tests, linkage disequilibrium (LD) tests and were in Hardy-Weinberg equilibrium.

Association Analysis.

[0338] For association analysis a standard likelihood ratio statistics was used, as implemented in the NEMO software created at deCODE²⁷, to calculate two-sided P values and odds ratio (OR) for each individual allele, assuming a multiplicative model for risk, i.e., that the risk of the two alleles a person carries multiply. Allelic frequencies, rather than carrier frequencies are presented for the markers.

[0339] At the locus on chromosome 4q25, we analysed 3 SNPs, rs2200733, rs10033464 and rs13143308. The third SNP, rs13143308, is in high LD with both rs2200733 and rs10033464 (D'=0.99 for both) and has a minor allele that corresponds completely to chromosomes carrying either rs2200733 allele T or rs10033464 allele T. It was genotyped in all populations using a Centaurus assay, and was used to infer genotypes for those individuals who had missing data for either rs2200733 or rs10033464 on the Illumina Infinium platform. In Table 21 and Supplementary Table 22, P values and OR for both risk alleles rs2200733-T and rs10033464-T were computed on the basis of comparison with the wild-type rs2200733 allele C, rs13143308 allele G, rs10033464 allele G haplotype, which contains neither of the at-risk alleles¹¹.

[0340] For the Icelandic study groups, P values are given after adjustment for the relatedness of the subjects and other possible population stratification using the method of genomic control¹⁰. The inflation factors for the chi-squared statistics are estimated to be 1.07, 1.04, 1.06 and 1.02 for the genome-wide association analysis of the IS, CES, LAA of SVD patient groups respectively. With the additional cases and controls typed for the 4q locus, we estimated the inflation factors using simulations as previously described²⁸. The resulting inflation factors are 1.09, 1.03, 1.06, 1.05, 1.01, 1.00, 1.01 and 1.00, for the groups IS, CES, IS excl CES, LVD, SVD, other, unknown and more than one cause, respectively.

[0341] Due to the large number of controls used, the effective samples size after adjusting for the relatedness of the cases and controls corresponds to testing 2,690 IS patients and 2,690 controls. The corresponding effective sample sizes for the CES, LAA and SVD patients are 710, 417 and 467, respectively.

[0342] Results from multiple case-control groups were combined using a Mantel-Haenszel model in which the groups were allowed to have different population frequencies for alleles, haplotypes and genotypes but were assumed to have a common relative risk²⁹

Results

[0343] The association of variants within the LD Block C04 region to Ischemic Stroke was investigated. In order to investigate further the contribution of the two AF risk variants on 4q25, rs2200733 and rs10033464, to the risk of developing Ischemic Stroke and its subtypes, large artery atherosclerosis (LAA), cardioembolic stroke (CES) and small vessel disease (SVD), we genotyped marker rs2200733 and marker rs10033464 in Icelandic samples, and for replication purposes we also analyzed replication data sets in cohorts from South-Germany (1,181 cases and 1,189 controls, Germany-S), Sweden (1,032 cases and 1,387 controls), Westphalia region in Germany (1,388 cases and 1,106 controls, Germany-W), and United Kingdom (654 cases/676 controls, UK). The phenotype classification of the study cohorts is shown in Table 20.

TABLE-US-00021 TABLE 20 TOAST subclassification of genotyped stroke cases, n (%) Replication groups Discovery group United Iceland Germany-S Sweden Germany-W Kingdom Ischemic stroke 1943 1183 1066 1391 654 TOAST subtyping: 1443 1183 1061 1389 654 Cardioembolism 385 (45) 297 (38) 185 (37) 554 (40) 78 (18) Large artery 229 (27) 372 (47) 230 (46) 560 (40) 232 (55) atherosclerosis Small vessel disease 246 (29) 118 (15) 82 (16) 275 (20) 114 (27) other cause 42 67 56 not recruited 3 more than one cause 34 42 not recruited 40 unknown cause 507 329 466 not recruited 187 TOAST = Trial of Org 10172 in Acute Stroke Treatment.

[0344] Additional patients (282) and controls (14,893) from Iceland were also genotyped for these particular SNPs. The association test was done by comparing each SNP with the wild-type haplotype (see Methods). As shown in Table 21, rs2200733 conferred an increased risk of Ischemic Stroke in all sample sets, and the association with Ischemic Stroke was highly significant with a combined OR=1.26 (P=8.8×10^-11). For rs10033464, the association with Ischemic Stroke was not significant (OR=1.03, P=0.45). Both SNPs however, associated significantly with Cardiembolic Stroke and this risk was significantly greater than in the Ischemic Stroke group as a whole (rs2200733: OR=1.53, P=1.5×10^-12; rs10033464: OR=1.27, P=5.9×10^-4). This is as expected given the known contribution of Atrial Fibrillation to this subphenotype. By removing patients with Cardioembolic Stroke from the Ischemic Stroke group, the observed effect for both SNPs was weaker in the remaining Ischemic Stroke patients, but remained significant for the stronger variant (rs2200733: OR=1.18, P=1.5×10^-5, rs10033464: OR=0.96, P=0.39). Apart from Cardioembolic Stroke, Large Artery Atherosclerosis and stroke of undetermined cause were the only subphenotypes showing significant association with rs2200733 (OR=1.22, P=1.5×10^-3, Table 2 and OR=1.18, P=0.01). These results suggest that a significant portion of strokes classified as either cryptogenic stroke or large artery atherosclerosis may be due to undiagnosed, intermittent AF.

TABLE-US-00022 TABLE 21 Association between rs2200733 (allele T) and rs1033464 (allele T) and Ischemic stroke. rs2200733-T rs10033464-T Phenotype frequency frequency Study population (m/n) Controls Cases OR (95% CI) P Controls Cases OR (95% CI) P Ischemic stroke Iceland ( 25708/1943) 0.119 0.142 1.23 (1.11-1.36) 4.7 × 10^-5 0.082 0.085 1.07 (0.95-1.21) 0.28 Germany-S (1186/1183) 0.118 0.138 1.19 (1.00-1.41) 0.05 0.093 0.083 0.90 (0.73-1.10) 0.31 Germany-W (1107/1391) 0.114 0.146 1.34 (1.13-1.58) 7.0 × 10^-4 0.092 0.096 1.10 (0.91-1.33) 0.34 Sweden (740/1066) 0.098 0.121 1.27 (1.02-1.58) 0.03 0.113 0.111 1.01 (0.81-1.24) 0.96 UK (676/654) 0.087 0.119 1.43 (1.11-1.74) 0.0056 0.090 0.088 1.02 (0.78-1.33) 0.90 All groups (29417/6237) 0.107 0.133 1.26 (1.17-1.35) 8.8 × 10^-11 0.094 0.093 1.03 (0.95-1.12) 0.45 Cardioembolism Iceland ( 25708/385) 0.119 0.164 1.50 (1.22-1.85) 1.1 × 10^-4 0.082 0.105 1.39 (1.09-1.79) 0.009 Germany-S (1186/297) 0.118 0.175 1.61 (1.25-2.08) 2.5 × 10^-4 0.093 0.096 1.11 (0.81-1.52) 0.502 Germany-W (1107/554) 0.114 0.161 1.52 (123-1.88) 1.0 × 10^-4 0.092 0.104 1.22 (0.95-1.56) 0.113 Sweden (740/185) 0.098 0.149 1.67 (1.18-2.36) 4.0 × 10^-3 0.113 0.133 1.28 (0.90-1.82) 0.162 UK (676/78) 0.087 0.090 1.08 (0.60-1.95) 0.79 0.090 0.122 1.42 (0.83-2.43) 0.198 All groups( 29417/1499) 0.107 0.148 1.53 (1.36-1.72) 1.5 × 10^-12 0.094 0.112 1.27 (1.11-1.45) 5.9 × 10^-4 Ischemic stroke excl Cardioembolism Iceland (25708/1558) 0.119 0.136 1.17 (1.05-1.31) 0.01 0.082 0.081 1.00 (0.87-1.14) 0.95 Germany-S (1186/886) 0.118 0.125 1.06 (0.87-1.28) 0.57 0.093 0.078 0.83 (0.67-1.04) 0.11 Germany-W (1107/837) 0.114 0.136 122 (1.01-1.48) 0.04 0.092 0.091 1.02 (0.82-1.28) 0.84 Sweden (740/881) 0.098 0.115 1.19 (0.95-1.50) 0.13 0.113 0.106 0.95 (0.76-1.19) 0.66 UK (676/576) 0.087 0.123 1.48 (1.14-1.91) 0.003 0.090 0.083 0.96 (0.73-1.28) 0.80 All groups (29417/4738) 0.107 0.127 1.18 (1.10-1.28) 1.5 × 10^-5 0.094 0.088 0.96 (0.88-1.05) 0.39 Large artery atherosclerosis Iceland (25708/229) 0.119 0.157 1.41 (1.08-1.86) 0.012 0.082 0.096 1.25 (0.89-1.74) 0.19 Germany-S (1186/372) 0.118 0.117 0.96 (0.75-1.25) 0.78 0.093 0.071 0.74 (0.54-1.00) 0.05 Germany-W (1107/560) 0.114 0.140 1.28 (1.03-1.59) 0.03 0.092 0.100 1.14 (0.89-1.46) 0.30 Sweden (740/230) 0.098 0.094 0.94 (0.65-1.34) 0.72 0.113 0.096 0.82 (0.58-1.17) 0.27 UK (676/232) 0.087 0.138 1.66 (1.19-2.31) 3.0 × 10^-3 0.090 0.071 0.82 (0.55-1.23) 0.34 All groups (29417/1623) 0.107 0.129 1.22 (1.08-1.38) 1.5 × 10^-3 0.094 0.087 0.96 (0.83-1.11) 0.57 Small vessel disease Iceland (25708/246) 0.119 0.112 0.94 (0.71-1.24) 0.64 0.082 0.085 1.03 (0.75-1.42) 0.86 Germany-S (1186/118) 0.118 0.145 1.23 (0.83-1.83) 0.30 0.093 0.063 0.68 (0.40-1.14) 0.14 Germany-W (1107/275) 0.114 0.126 1.10 (0.83-1.47) 0.51 0.092 0.075 0.81 (0.57-1.14) 0.22 Sweden (740/82) 0.098 0.110 1.11 (0.66-1.88) 0.70 0.113 0.091 0.80 (0.46-1.37) 0.42 UK (676/114) 0.087 0.101 1.18 (0.73-1.91) 0.50 0.090 0.087 0.99 (0.60-1.63) 0.97 All groups (29417/835) 0.107 0.119 1.07 (0.91-1.26) 0.39 0.094 0.080 0.88 (0.73-1.05) 0.16 Association results for rs2200733 allele T and rs10033464 allele T for ischemic stroke and the subphenotypes; cardioembolic stroke, large artery atherosclerosis and small vessel disease, in five study populations. Also presented are the results for ischemic stroke after excluding patients with cardioembolism. Results for each phenotype are also included after combining the study populations using a Mantel-Haenszel model (All groups). Number of controls (m) and cases (n) is shown in parenthesis, the allelic frequencies in each group, the OR with a 95% CI and two-sided P value for comparison to the wild type haplotype (see Supplementary Methods). The results for the Icelandic population are adjusted for relatedness of the individuals.

TABLE-US-00023 TABLE 22 rs2200733-T rs10033464-T Phenotype frequency frequency Study population (m/n) Controls Cases OR (95% CI) P Controls Cases OR (95% CI) P Other cause Iceland (25708/42) 0.119 0.155 1.32 (0.72-2.45) 0.37 0.082 0.060 0.73 (0.31-1.75) 0.48 Germany-S (1186/67) 0.118 0.119 1.03 (0.60-1.77) 0.91 0.093 0.105 1.14 (0.64-2.04) 0.66 Sweden (740/56) 0.098 0.125 1.36 (0.74-2.50) 0.32 0.113 0.134 1.26 (0.70-2.26) 0.44 All groups (27634/168) 0.111 0.133 1.19 (0.85-1.66) 0.32 0.096 0.099 1.06 (0.74-1.54) 0.74 More than one cause Iceland (25708/34) 0.119 0.088 0.68 (0.31-1.52) 0.35 0.082 0.044 0.49 (0.17-1.39) 0.18 Sweden (740/42) 0.098 0.112 1.27 (0.61-2.66) 0.52 0.113 0.187 1.84 (0.99-3.41) 0.05 UK (676/40) 0.087 0.213 2.89 (1.54-5.39) 8.9 × 10^-4 0.090 0.088 1.15 (0.51-2.61) 0.74 All groups (27124/116) 0.101 0.138 1.48 (0.99-2.21) 0.06 0.095 0.106 121 (0.78-1.88) 0.41 Unknown cause Iceland (25708/507) 0.119 0.135 1.15 (0.95-1.38) 0.15 0.082 0.073 0.89 (0.70-1.13) 0.35 Germany-S (1186/329) 0.118 0.129 1.10 (0.85-1.44) 0.46 0.093 0.087 0.94 (0.69-1.28) 0.70 Sweden (740/466) 0.098 0.126 1.32 (1.01-1.71) 0.04 0.113 0.104 0.94 (0.72-1.23) 0.65 UK (760/187) 0.087 0.102 1.20 (0.81-1.78) 0.35 0.090 0.096 1.10 (0.74-1.64) 0.63 All groups (28310/1489) 0.105 0.123 1.18 (1.04-1.34) 0.01 0.095 0.090 0.94 (0.82-1.08) 0.41 Association results for rs2200733 allele T and rs10033464 allele T for the TOAST subphenotypes; other cause, more than one cause and unknown cause in three or four study populations. Results for each phenotype are also included after combining the study populations using a Mantel-Haenszel model (All groups). Number of controls (m) and cases (n) is shown in parenthesis, the allelic frequencies in each group, the OR with a 95% CI and two-sided P value for comparison to the wild type haplotype (see Supplementary Methods). The results for the Icelandic population are adjusted for relatedness of the individuals.

[0345] As discussed in the above (Example 2), the risk alleles of rs2200733 and rs10033464 correlate significantly with the age of diagnosis of Atrial Fibrillation. A non-significant trend in the same direction was observed in our study for the age at diagnosis of Cardioembolic Stroke (0.62 years per copy of T rs2200733, P=0.33, and 0.29 years per copy of T rs10033464, P=0.71, Table 23), suggesting that the observed age effect on AF may apply to Cardioembolic Stroke also, albeit being a weaker effect.

TABLE-US-00024 TABLE 23 Linear regression of age at diagnosis on the number of risk alleles of rs2200733 allele T and rs10033464 allele T. Shown are the regression coefficients and the corresponding two-sided P-values obtained using the age at diagnostics as a response (in years) and the number of at risk alleles as predictor variables. The sex was included as a covariate factor in all tests, and also the population in the test for all groups combined. Numbers of cases used in the analysis are shown in parenthesis (n). rs2200733-T rs2200733-T rs10033464-T rs10033464-T reg. coeff P reg. coeff P Ischemic Iceland (1830) 0.11 0.85 -0.35 0.62 Germany-S (1174) 0.29 0.73 0.85 0.43 Sweden (780) 0.56 0.56 1.11 0.23 Germany-W (1352) 0.17 0.80 1.21 0.14 UK (654) 0.24 0.83 0.47 0.71 All Groups (5790) 0.40 0.25 0.68 0.10 Cardioembolic Iceland (356) -1.52 0.16 -0.29 0.85 Germany-S (296) -1.72 0.21 -2.53 0.18 Sweden (173) -2.09 0.20 0.81 0.62 Germany-W (1352) -0.04 1.00 0.82 0.53 UK (78) 7.11 0.084 -5.29 0.16 All Groups (1441) -0.62 0.33 -0.29 0.71

[0346] Discussion

[0347] Through this study on 1661 Icelandic IS patients and 10815 controls and the follow-up replication in large and well characterized European Ischemic Stroke case/control sample sets we identified and validated a risk variant on chromosome 4q25, tagged by rs2200733, that associates with Iscemic Stroke. In our study, as expected, these variants associated most strongly with the subphenotype Cardioembolic Stroke, which is a major complication of Atrial Fibrillation. The risk that is observed in Ischemic Stroke patients without Cardioembolic Stroke is possibly due to an underdiagnosis of Atrial Fibrillation and thereby Cardioembolic Stroke, since Atrial Fibrillation is often asymptomatic or intermittent and can consequently be difficult to detect in stroke patients.

[0348] Up to 30% of Ischemic Stroke are caused by cardioembolism(5, 6) of which a large proportion occurs in the presence of Atrial Fibrillation(7, 8). Atrial Fibrillation is the most common sustained cardiac arrhythmia of man and its prevalence increases with age, affecting approximately 10% of those over 80 years of age (3, 9). As such, AF is one of the most powerful independent risk factors for stroke and on a population level, AF is associated with a fourfold to fivefold increase in the risk of stroke(3, 7, 8, 10). Moreover, Caridembolic Stroke is generally severe, reflected by greater disability, higher rates of stroke recurrence and higher mortality than in other subtypes of strokes (6, 11). Early detection of those at risk for AF is important in order to reduce the risk of suffering a future stroke. Clinical trials on stroke prevention in patients with AF have shown that anticoagulant medications (e.g. warfarin) reduce the risk of stroke substantially(7, 12) and is much more effective than anti-platelet agents such as aspirin and clopidogrel. Our results strongly suggest that a significant portion of stroke patients have undiagnosed atrial fibrillation and are classified either as cryptogenic stroke or as large vessel stroke. Such patients may have asymptomatic, intermittent AF that is not detected during routine workup of 24 to 48 hours of cardiac monitoring. This is supported by two studies of post-stroke patients who underwent another 4 to 7 days of ambulatory cardiac monitoring; the rates of intermittent AF previously undiagnosed were 5.6 and 14.3% (13, 14). Stroke patients with asymptomatic or intermittent AF would be inadequately treated if misdiagnosed instead as e.g. cryptogenic stroke or large vessel stroke since such patients are placed on an anti-platelet agent instead of warfarin. Therefore, these markers for AF may help determine which patient might benefit from prolonged cardiac monitoring as an outpatient to document the presence or absence of AF. Prospective studies are needed to determine whether these findings can be translated into better prevention or treatment for stroke.

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Sequence CWU 1

1

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gttttctagt cttcattctt gcaatacagt ttctgaatcc 180ttctaaaatg ggtattttct c 20113201DNAHomo sapiens 13ttgagaagcc ttctagatga tttgaaacag gtggtcctat gaccacactc tcatgatctc 60ttgcctcaac tggtgccttg atgctggtac tcattcatat yttgtcctta gacttgtaat 120ttaaaatttg cctgtttttt atggtaccca ttatgaattg gtacctctta ttcttagcag 180atatttttac tttttatttt t 20114200DNAHomo sapiens 14atacaaagct gtaaggggca agaggtacat gggccagccg taaagtttgt ttattcaaat 60ggagcttgtt gatctgtcat atttttcaag ttgagctact mtgccagggc aacattctac 120tgtattcaaa tcagctgttt tggaattggt acacctgcta gtgtctgttt ctgaacactg 180aatgagttgc cataagggag 20015201DNAHomo sapiens 15catatggact aggtacccac tcacctggat gactatttca caactttctt tcttttcaaa 60attccagtcc ctttcctcat tttcaatttt agctgattga ytttttttaa aatgagacat 120ttagaacaat ctagagagaa cttcccaaca ccatgtttga tcatcagcat tacattcctt 180tactcttcct tcccccctca a 20116201DNAHomo sapiens 16atgaacaagc ttttctcgaa acagttctgc catccagcac gatttttgtc ccctttgcaa 60caaagcccct tgaaggagtt gtctatactt cttgttttca mttccattcc tcacattctc 120tcttaaccca ttctaatcag ttaggcttct actgccctcc cctatggtct ccaatggcct 180ctactttgct aaagactagt t 20117201DNAHomo sapiens 17cttatactgt tccatgcata cagtttctca tctaatgagt agtattatag ataaatatct 60acaaataaag tgagacttgt taaatgctta gcaccatgac ygtcactttg tataatctta 120acaaatgtta gctattacaa ttataaccaa taatgataat tattaagttg ttagccagaa 180gaaagatatc ttggttatag c 20118201DNAHomo sapiens 18tctttttgag aaatgtgata agaatataca gagtgagaaa ttactagctg ttgtttgacc 60ttttgatttc tgcctctgta acccaaattt acaaagggtg ygtgcataca cacacacaca 120cacacataca cacacaagat ataccaaaaa caagtgtaat tcacatgaaa gaaattgtgg 180tgggtatcta gcattttata t 20119201DNAHomo sapiens 19aaaacagtag atatgatctc tcatgacttt agcaagattt tagatttagc ccagcatgat 60agagtcatca atgaactgga aacacagtat gggtcacaca yactattgag ggctgcacag 120ctattgagtg gttatcactg gctcagtgtc aaccgggtgg gaaatgaata gaaaatccca 180aatgtaaatt tgtttaacat t 20120201DNAHomo sapiens 20gtggttatca cagtacccaa taggtagttt ttctacccgt gcttctctcc ctccctgcca 60tagtagtcca cagtgcctat tgttcccatg tttatgtcta yacgtactca gtgcttagct 120ccctgttccc atgtttatgt ctgtatgtac tcagtgctta gctccctgtt cccgtgttta 180tgtctatatg tactcaatgc t 20121201DNAHomo sapiens 21ttgggtgttt taatttagtg attcttttga tgttttctgg gggatagaat ttccatttct 60ttctgatttt gtatcaacct tgaatgtgga attaaaatag yaaatctcta ctttgtattt 120tatatataaa taaaaattct acatattaca tatgtagctg atattaatat gtattatata 180cacaatataa ttttattaca t 20122201DNAHomo sapiens 22catccacagc catgaggcca cataacacaa gccctccagt ctcagctctg acaccacttt 60ctcagagagg cattccctga gcacctaaga ggcctctcac mcttaccctc cttattctct 120accctggctg ctttattctc tacctggttt ctatcttggc tcttatgaat tgcagttatt 180ttttatttgt ttatgaaatt g 20123201DNAHomo sapiens 23ttcttagtaa tttttataga aaatttagca tggatatcat acatctgtgt gtgtgtgtgt 60gtgtgtgtgt gtgtgtgtgt gtgtgcattc caagtcttca yaaatggggt tgtattataa 120atactgatat atacctggct ctttcactta atcagaaggc taagtgacca tctgtttgag 180tccctagaaa tatactgttt t 20124201DNAHomo sapiens 24tttacttttg attcagattg ataccttttg ctgattactt gtcattttat tttcatttct 60ttgaattgct tatttacatt aattgatcag ttttttatta ycttttttct ggtccttttc 120ttactaattt gtacaagcat tgtataaatt agaaaattag ctctcaactg tcaaaatatg 180atgccaacct tttctttttt g 20125199DNAHomo sapiens 25tttagagtta atactagaga agaatctcac ttgaaaacat ttcacctata tgtatgcggt 60gtcttggtag acagggtgcc tgaggacagt ggcatagcaa mtttccagtg aggtaattta 120atttgttaaa ttaaataatt agatttattc ctcatgttgc cttggggtga tgaggaggag 180ttaaggacat ggagaaaaa 19926199DNAHomo sapiens 26aaactgaaaa atagactcct attttgaaac ctagaaaaaa gttgtgcttt aaacttttct 60ctgtcaaatg agaattgctt aattcttata cttaaggaac rtgggaaatg aaaaggcaga 120atgtatagtg ttggtccttt gatggaattt ctgagaaaaa acaaactatt ccagatgatg 180actaaatcac atagtttta 19927199DNAHomo sapiens 27atgaactagt gcatcacact gactgcctgc tctgtgtcag gttctacttg aggccccaga 60tactagcaag ccctccaggt tctcacagtc taatgagatg yagcaatgta aacagctact 120ttttatatga tgtgaaaaaa cagaagtatt aatgaaatgc tgtgggaaca taaactaact 180agggagtagt aattctgcc 19928199DNAHomo sapiens 28tgatgtgaaa aaacagaagt attaatgaaa tgctgtggga acataaacta actagggagt 60agtaattctg ccttggtggt acttgggttt tgattttgat yagagaaaat tagaacaggt 120aatatttaag gtagttttga agaatgagta aaattttcca gaaagtttgg ggaatgtaat 180tcctggtaga gggaagcct 19929198DNAHomo sapiens 29aatcctggaa tccaggttaa gaattctttc tctgggcagt gagtgccact gacaatattt 60cagtaaaaga gggacaggag aaactttggt tatagaatga ygactgctgt aatttaaaca 120atgaaatgga atggggagag gctgaagtta accacatcat gcaggaggtt ttgggattat 180aaaataaaat agaacatg 19830201DNAHomo sapiens 30agtaaaagag ggacaggaga aactttggtt atagaatgat gactgctgta atttaaacaa 60tgaaatggaa tggggagagg ctgaagttaa ccacatcatg maggaggttt tgggattata 120aaataaaata gaacatggtg acacagaggt tgtgtgtggt gatcgaaagg gaacagaaaa 180tgattccaag gtttctaact t 20131201DNAHomo sapiens 31gattcaatag acagctattg aagagtgacg tgaaagtgac actgcaggag gtggaagaat 60aaacaagaaa aagtggagat tgcgaatcta gattacttct ytaagggatt ggttaggaaa 120agatagaaga ggagaaagag gcaaaaagaa tagggagatt gcttatttta ggacataaga 180gactagcaaa catggtggac t 20132201DNAHomo sapiens 32aaatataaat tatagcagca acatattgaa taattgcaac aagtataggt gttgcttatt 60tcatgtctat catctaacta ttaattttta ccagataaaa ytagtacatt agggaataca 120tttaatctac aatacaggaa agtcctgttg atatgatttt gcctgtgggc tgggtgactg 180tattagtagt gatgattttc t 20133401DNAHomo sapiens 33aattgccttc tgctggaggg taggaaaact ttcttgggag gtggttttac aaggaatggg 60tcagaacatt aaaggtcaga gaatgggaag gggcaatcag atagaagaaa tcatgggaat 120aaattcttag agggtaaaat acatggtaaa aatgggagtg gtgtgtagaa atatgagaat 180tggaacttat agtggaagat racattggaa atgtaaattg tagctggatt gttgaagtcc 240ataaagacaa cccaaggtct gagactttat tctgtaggca atagagagag cccctgaaag 300cttttcaggt gggaagtatt attatcagag ctgaactcta ggcagggcag gttcagagag 360gtgcactgat ggcaaaggaa gtacttgaag agcttgcaaa t 40134201DNAHomo sapiens 34gcagaatgta tagtgttggt cctttgatgg aatttctgag aaaaaacaaa ctattccaga 60tgatgactaa atcacatagt tttaaatctt ctgtagattt ytaatggttt tttttcaaaa 120acgcatattg ttcaatataa taaatatttg tagagtcagg aaaatgacaa attctttctt 180cctaacaatt ttacaatgaa a 20135400DNAHomo sapiens 35taatcactga gcctgctgta cttattctgt cagtcttttt gccaaaaaga tttcatgggt 60agcaatatca gaagcatttt tgtaattaaa ttattgtcgc tttactggaa aatctcctta 120catttgcaaa gggtgaatgt gttcttattt cccccactcc ctacttgact tgtagtttac 180gcaggcactt ttggctgcgt rcgtttccag cctaatggcc tccttaccgc aatattcatt 240ttggttgggt tactccagcc atcattttcc agagacagtc ataatccctc aatgatgaca 300gaagtcattt gatcttgggg aacaagaaat aaaacgagca acaataattt ctttaggacc 360attttacggg tttttaagag aataaattct gtgataagtt 40036400DNAHomo sapiens 36tctaggaccc tgggtgccca gccttctttg tacaggttcc tgttccttcc cttccagcct 60gcatctggct gacttcagcc tgcaaatgct tcagaacaac ctttcttgtg cagcatcttt 120gtgtgatggg ggaagcactg aagaagaagg taccacagtc tacctgcttg ggccttctgg 180tgattatttc ttcaactcag ytgtcttgat ctgagggaga gttttatgag ctaaaaatct 240gtcagtgttt ccatttacca agtagctatt caatcgtttt atagcctaac cattcccaat 300taccctcaat taaaacttgg cacatatgtt gacaacctta gatgcaattt tttaattgaa 360aattgttgat ggcttttgat tgaagtctaa aaccaaaagt 40037400DNAHomo sapiens 37ctctcttgct ttcccctgcc cttctggctt ccaccatgtg atgatgtatt aagaaggcac 60ttgacagctg ctggcacctt gatcttggac ttcccacctc cagaactctg agaaaataaa 120tttctgttct ttataattac ccagtctgtg atattctgtt ataggagcat aaatgaaata 180agacaccgat gtttttctta ygattagatg ggggttatgt gttttggata tgatgactac 240agaggtaaag tgcattatca tcacattata tcaagaatat gtactatcaa catgacttat 300cactgttgag tttaaccttg atcacctaat tgaggtagtg tttgtcagtt tactccacta 360actcattttt cctcctttct gaggtgtatt atttggaagg 40038400DNAHomo sapiens 38aaaccacaat gtgataccat ctcacactag tcagaatggc tattactaaa aagtcaaaaa 60caatagatgc tggcgaggct gcagagaaaa ggcttttata cactgttggt gggaatataa 120attagttcag ccactgtgga aagcagtttg gagatttctc aaagaactta aaacagagct 180accatttgat ccagtaccca raggaaaata aatagatcat tataaccaaa agacacaagc 240attcatatgt tcattgccac actattcaca atagcaaaga catggaatca acctgggtgc 300ccatcattgg tggactggat aaagaaaatg tgatatacac atatacacca tggaatactc 360tgcagcatta aaaagaataa catcatgttc tttgcagcaa 40039400DNAHomo sapiens 39ttgggcggct ctgtacctat cagaatgttt ctacaccctc ggtactgaag ctgaatgctg 60agaaggagga aaggcaaaga ggttgctgta cctcttgctc ttctcctgtc cccgttattc 120tacggctgaa ttaggtggct tttttcccct tcctatgtgg cagggacttt tcttgtgtcc 180tatcttgagt tcttcagact ratagtaaaa ttgacggtta agccagccgg actggctttt 240aatacattac tggaagttta agaaatttaa aaaatatttt ttctcttgac aaaccctgtt 300tttaaaatag atggaattgc tagggattcg gctggtgtta attgcatttt tctattcctg 360tgctatgtgt actaatcacc atcctatgcc cccttcacct 40040400DNAHomo sapiens 40tgcagggaac ataggtaagc ttagaatatg gctattttta actgaaactg tactgacttt 60ctttttgaag atgctgatac aacttctgac actgttggtg atgagtggtg cataacagcc 120gtttcagctc tgaggctatc aaatagtgaa agagaatctt ctacatacaa atcccaggac 180accagaacag ctcttaacag mcctcacttt cctgagtaag ctgaaggtgg ctaactaaaa 240ggcctgagag aaattactgc ctaaacaatg aaggaaggag aaaaccccca aggaagacaa 300tgtaaagatt aagtaacaag gtttcaattt gatacaatgt ggggaatttc agtttaattc 360taatttatac taatttctca taattgtctt gatcctttac 40041400DNAHomo sapiens 41agaataatca tacagcagga atgcaaacac gtgatttcca aagagaattt tattccatat 60ggtatttttc ccctaactaa gaattaattg caactttaaa gaaggtttga ttgtgtctat 120gaaaaacatt ttaactactg aggaattcta aatgacttca atttaaaatt ttcttttttt 180acattgttag agtcaagaaa kaagtgcttt catcaagctc tgagttacag aatttattag 240agtaaaggga agaggataac cttcagatgt cttctgtggt gaaatgttga agttaaaaat 300tttaaatgtt tgcagactaa agacatcctc ggctgtgagg cctaaaagaa taaacaaaat 360gtgacatgtt ttattttgtt acttaaccct tgtataaaaa 40042400DNAHomo sapiens 42atatataaag cagctctttt gagtatatgt tcaagtgtgc tatacctctc catccagtgc 60tctcttacaa atgctggatt tgggacttcc tgtcttaacc tgggatccca cattagggac 120ttcctctctt gacctgggat ccgcacattt cagattgcct gtagagcttt tccagccttt 180aagacaaact ctgaatccat raattgagcc agttgctgga aagctaaaat cctatctctg 240tcaaagtgga gcagagagac aaaggtgagt ccacaaaaac agattttctt acctcagagt 300gatgtgaagg acaaggtggc tccagtgtct aaactctggg agtgagagaa tagcgaagaa 360ctgcccctga atccagtaag ttaaagaaac ggcagtgcta 40043400DNAHomo sapiens 43tatatagtac tcatatattt cacaataatg tgactaagat gattgcctat ctgaaaaaat 60aaatgcccat tataccttgt gcatagaggg aattcaataa atatggagtt tgttaaaggg 120aactcagccg gcaactgtga aatcataagt taataaataa aatacaatga tgatttacca 180taatcaaatt agccagtaac rtatatacat attacaagtc agctctcata aaaaggcctt 240tgctactttt tctgacacaa ctgcatgatt ctttttattc agtgtacatt tatgtgataa 300actctcatta gagtctaaaa agtttgatat tccccttata aaacatacag aaggagagct 360cgttaaagta atggatttat ttaacaactg tttttttctt 40044400DNAHomo sapiens 44ttatatggaa tgtaaataag gacatgaaaa agaattgtaa aagtcaacca agagctctga 60tactaaattc attgaggaca ctgataccca ttgtcatgaa tggtgacact ggcactataa 120aaatcatttt atgtataaga atgactgtga caaagtttta atttttgaaa atatagttgt 180ataaggtttc tcatgtctgg yattttagct tttggtttga gaattatttt aaaagaatat 240aggacaattt agttactgga ttggtattca aactactgga cttactcaat attcttccat 300caaaatcaaa tccaaaagaa cagagattta ttctcatatc taataggaat ctagtagaca 360atgctaaaat tagtgaacta agtaaggtga atcaaagtta 40045246DNAHomo sapiens 45tcctcaggga ggtctaatca tatgttttct taaaccaaac tcttttcctt ttcattaggc 60tttcttgagg caattggcta attaatgata ataaatagtt attgagcatc acagatattc 120taaatcctcc agtgtcatac atttggaagg aaaaaagacc agtagataaa cttaggaaga 180gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtat gtgtaacaaa catgccttaa 240aaccag 24646399DNAHomo sapiens 46atattgagca acagccaccc tctctgggtc cctgcaaatg gtacccattt ttccaaccca 60cagctctagc tgctcaacca tttgagattt ggggtaacta cctgggggaa cagtgttcag 120atggcagtgg gagttaccac ctcacagtgg cctggggaag agaagagaaa gagattagag 180gagggggcat ttgctaaaak cactcaacga acatgctgtt aatgcttcct cacatttgca 240tgttactgcc acagttttcc taggtgtcac tgagtctcca gaaagcaact acttgccgaa 300ctaagtaaaa taaggagaat ggtatagcac atgtgtttgg agaaggggaa ggaagggtgg 360aatatgaaat tgagcataga tatccaggtc aggaaagaa 39947399DNAHomo sapiens 47gcccccaaga agtgatgtgt ctgctagaaa agccctgaac atagaatttc ctgactttgt 60ttttaattta gttctttcag gcatcacgct gcataaccag gtgtaactct ctaaaagtct 120ctatgacaga attttccatc tgttaaatta ggctaataat attttcatct ttttttaggg 180taaagatgtg aaatatttgk agaactctgg aaaacatgcc ccctactaat tagagctttt 240tgatgtgaca tcattttctt cagtacttgg agtttagtca atagatatac agtgtagctg 300tgaaattatg aagcatgaga atgcattacc aagggaccgt aggaggctct ttactgaaaa 360gtgtagctgg ctatatttct ggaagtaatt taaacatag 39948399DNAHomo sapiens 48acacagagca catagggctt gaagacctgt tatggggcat ggatgtgctt gacagggaag 60cagggaagaa gtagcaaggc aaccgggagc acttttgagt ttcctctctt tgccaggcat 120gagaagacca cttggtgtgc attttagatg gtgctgagat gggatcaaca aaggtggagt 180gtgcaaaaga tgagagacay tgggcaatgt ggaaatgaag aataggatta ttgctcggaa 240ggattatggt tgtgtttaat aggaataacc aaagcaaaca atgatcacat ttgttgtcct 300tcaccttgtc aaatagtgat ttaggggcta gggaagtata tcacctctga ggttgctaca 360acatcctttt ctgcccacct tctccagaac ttagtatta

39949399DNAHomo sapiens 49tttaatttct ttggaaatta aatttgcttg gaaacagtgc tataaagagt tgatgtctcc 60aaaggtgatt ttttttgttt tatataaata aggttttgct tttgctagtt gagcgcagtt 120ctaggctttt cgcccttagc tcacacacac cccttctgcc tgcttggact ttaatggctc 180aagacagcct tgagctcacy gggaaaagaa aatgactgtt aaaaattatc cttgaaattg 240gttatttggc aacattctta attgtatgga aattcattaa ggcatatttc atatataatt 300agctcaaggt tgttgattct acaggcttta tggatttaaa tctgattgat aataaagtaa 360acaagagagt cgaatttaaa gcgtggctct ctcgggtta 39950149440DNAHomo sapiens 50tcctttcttt tattaatgta gtgactgctt aggataggta actatcctaa gtaggagaga 60gaactgagaa atgtgcccat aataatcact ttcaaactat cattaaaaca gacaataact 120ctttcatcat tttttgtatc taaaggatta aattgataca atattcttac catgccaaaa 180agtacacaat tatttagaga gcagagatca tatgtttcca tgaaccagta tttccctatt 240gaatggtaat gtttctttta gtaacaaatg agttcttata cttaatatta gggcaagtgc 300ttgctacaaa atgtcagtaa tatattcatg aggtgaatca ctttggaaca gtcttttaag 360caatttctga gcttgtggct tgccctcttt agagtggcca gtgacttagt ccctcaaaaa 420tgaggatgca gttcaggtag aacaggagcc ctgaaattat acaaagtgct agaaagtatt 480aatagttgga gttgaattga ggagacaaga ggaaggcttg tcacaaatga ctttttagag 540ttgggtctgt ggcagaaaca gccagttgtt ccctggtatc tcctctctcc tgtttctatg 600gaagcagaat ttttagctgg gcctcttgtg caattggtgt atacatatga tggaattcta 660gccaataaga tattagcaag gttgctgtgt gcaacttcta gattgtgttc tcaaagggag 720aggatgtgcc tttctgttct cttcttgtcc ctgctgactg gaatgtgtta ggagaactgg 780agctgaggca gctgtgattg agcaaaaagc tagaaggagt ccaggatcct ggcactacag 840aactgctgta cttgcatgac ttctgggact gcacttgaga gagaaataca cttctatttt 900gttcaagaca ccactatttc aggtctgtct attacaaaag ccaaactgat ataccacaaa 960tacaggggac agagtccttc cattgggtca gtctccaagc cttttggttc agcaccacat 1020gtgattaact tctgggataa tgggactcac tttatcatca tcatcatcac caccagatac 1080ttccagctgg ggaggcatcc cttctcattc agttgtaggt ccaaactgaa tcctagcctc 1140cacccatatt tttctactgc taacctggta aaaaccttcc caggtatttg gaggctgtta 1200ttgactgaca aaattcatat taaaagccaa tctccattgt ggtggtatta ggaggtggtt 1260aatttagggg gtgattaggt catgaggaat ccacaatcat ggatgggatt aatgccctta 1320taaaagaggt taaagaaagc tgccttgttt tttctgccat ctgaagacac aaaaggcacc 1380atctatgagg gatgggtcct caccagacct ggcatctgct ggcaccttga tcttgcactt 1440cccagtctct tgaactgtaa gaaatacatt tctgttgttt ataaattact cagtcaaagg 1500tattttgctt tagcagccaa aatagattaa aacagagggg ataagtagat ttacaagata 1560aatactttaa gtattgagta gatttcacaa gactgatttt tcctgtccac aatcccttga 1620gcctccttca gacttcagta gaaagtactg acctccaaaa tctgaaacct tcaaagaaga 1680tgctctgctc ccactgtcat tttttaaagt ttattctttc tgaacctgag atcttgcata 1740gcaaagaacc tattctagat atttagtggg gtattggaag cattagaaat cgaacttatt 1800tgcatcatat ttatgtgatt caactacatt tatctgattt atctaactgg aagcccttgt 1860tttttgtatt tagattttaa tctctctgct ttgtttcttc atacagtgaa gtgtcagagg 1920caacatgttg gtagaagagc tctggtcagg aattaggaaa ctggagaact catcctgggt 1980gtgctgttta ccagcatggc tctctgaccc tcaattttcc cagtcaagtt gggattttaa 2040tattttctct cagtgcctca atttactatt ctgtgattaa ccctttggtg catggaaata 2100atgagaaaaa aagtgccttg aacatcatga agtcttatat caccgtaggt tagaaggagt 2160ctccgtgaat ttgcgctggc agtcttcgtg gtgaagttgg attgttaatt ctcagtcaca 2220aatgtgaact tgcgttcctg ccaacagatg ctctcagcaa gtttctcaat aatttatata 2280cttgtaccct tttctctgga caattgcctc ctcactttcc cccccaaata gctatctaga 2340tatctagata atttctttta agttaccttt agttttgtct ataaaagatg attttacaga 2400ttcaggtctt aagacatatc agtgtttacc caatggcaca taagaaatgt tttggtcatt 2460tctggattta agtatagtta ctctcctttg aaggcaaaca catgttcccc ttggataaag 2520gagttagtgt tggttgctcc ttcctttctg cttcctcact ttctattttc ctttttcata 2580tcatttgcta cccagtattg tcataatggg gtcacagtga agagcagcag ggacttagtc 2640tcactaactg ttgcacctcg ggcttggcac catgccaggc atctagcagt ttctcattcc 2700atacatgttg aatgaatgaa tgagcaaatg tgaaggataa attttctatc tgggacaggc 2760agtgtggcag taatacagta cagattagtg ttcagattgg ctggtgattg aatggcactg 2820agtcagtctt gcctgtgtca ctgaaactga gacctcagaa gtttagcttg atagaataga 2880catgggttag aggaaaaagt tactgatgtt ggagcaggaa atacagcatg gaaaacagaa 2940gtaaatgtct gtggaaagga gctggggagc ttgaaacagg tctggtgctg tgtcaagctt 3000actgctgcag ctctgggaaa tatcttgttc agaaccatac acttcaggct ctgtaagtaa 3060atcactgaga attatttatt tctgctgtgt ctttaggctt tttttttgtt tttggccatc 3120atataggaag agtggattcc tttagaatat gacattcata tcttcctctt ctcagacatt 3180tgaacaagta atcatctatc tcagaagcat tactggtttt ttttttagcc agacaaaact 3240taaaatatct tatctattac aaattgcttt caggataata atgttgttct gtaactttat 3300ttgatgactc ttcacagtaa gtagacaaat aatttttacg tgccaggtca tttaaaaaac 3360ccctcccgag tagggatctc tgtaggtaca taaaagtaaa caattaaggg aaattctcat 3420taagaaaaat gtctaataga attagagaga acctactttt tgctacttta attccatgct 3480cccctccccc agcttcaaca atgtcaccag gttgagaaac agaaggagaa aaagattaat 3540cattagtaaa aacacaccct tctgcccctc cccctggcat cctgaaattt gagtgagtca 3600aaaatatgta acatttaatc agaggagttg attttttccc attcgtgtac tatcccccct 3660tcctctctcc attccctttt gaaaggggaa ggtgattttt ttggcttcac tcatgtctag 3720tcaaccttta tcctctccct tctccctttt caatctctag ggtgtatttt ggagaaaacc 3780ttccccgacc ccccctttct ggcattcatt attttcttta cttttagaca ctaatggttt 3840caaaaccaat ctcaacacat gactcagtta ttttagaaag gactgtacta tatcttttta 3900gatccttttc aggcctcaca tacaaacttc caaatgtagg ctaaggacgt gttcctttct 3960ttgtatgttt tcctgtacca atttttcagt agttggttag ctggccaaaa acgtttattc 4020taataagtgt atcaaagtgg ctttctcccc atctcttact ccctcctttt ctttgttcct 4080tcctttcctt ttttcttccc tccctttctc tctttattct ttcttctctg gttttactct 4140cttccccact tagatgctca gaacttggat taggtgctaa gaataaaaaa aaatgatatg 4200ataaaatccc agccctcagg gaactcacaa ttatcgatta actgtgaatt cccttgcaat 4260ggaacaagtg tcctaaaagg ggggtggaat ggtgcccggg aaggaagacc agagaaagcc 4320ctactgccag aggtggaaat gggaggggcc ctaaaggagg aaagagcatg cagattttgc 4380aggggtggga tgggacagac aggcacatag aggaaacagc agattcagag ataaaaagaa 4440atgagaagtt tagggcaatg cagactggaa tagatatgga gatgagtcag agatgagacc 4500agaaaggtct tgaaaggcct gcttagggtt tagactttcg actgtaggca ctggagccgt 4560tgatgatttt gtggaataga gggatctgcc tggatggagt tagaacagag gggagcaggg 4620aagagaagaa acatagggcg acaaaataaa aatattgtga tagtctaggt aaacaattat 4680aaaggactga actaaggcaa tgggcagagg ctgaaactga ggagacgaac ctgaggccta 4740tttggaggtg cactggacag gacataatgg atgattagat gtggagtcga gggaggggga 4800cgagtctcca agtctagcca gctttctgtt tcctgacctg gtgccgttca ttgagatagg 4860taacactgaa gtgggagagg ttttgaggga ggaaagatga tgaattcttg gcactaattc 4920agactgtatc attaagctaa cgaaccatct tgccagtaga agctgttgct agaaagaaga 4980accaggaaag gcagttacat caacacagct cttctgatat attggtaggg agaaacttca 5040gaactctaat ggaaaataca tggattagaa atcaaataag tgattttttt gaaaaaaatt 5100gcaaaataca tatatagcta gtactttgca tgaaggtgaa aatactgatt aggcattagg 5160gttaaaataa cattaaggat gaattttatt agtatatttc caaaacattt ctgggagact 5220agggaactct atgccatacc tcctctaaat taaactgtac attatatgcc tcatttttct 5280cttatagctt ctatcactgg aaagcagctc agccatgtta tctttaaact tcatttctat 5340ggtaaagaga gctcttcatt tacaaaatat tttgtgatca ttaaactaca gaaacatgac 5400tagcattgtt aaatggtcaa tagctgagga cctacaaaat atctttaaaa agcctcaaag 5460catttgtgca ctgcgcctct agggctacat gctgcctaga gaaattatct aactagtttg 5520gcatctctcc agaagactgg ttaccaaact gcattgggtt catgctgaac tagcagttat 5580ttcacaagga ttttcactag tgctaatctg catgtgtgga attcctgttc agaggttgtt 5640tttctgagct gtttagttga gccagattac aaattgactc tgtttggtgt gaacagaaac 5700ctatttacaa tgcagatctc tcaagccaat tttctggtta aatttcatcc attaaatttt 5760ttctattctt atataaaaaa cgtgtctaca gggcatttac ctttttacat ttcaaattgt 5820aaagtggtga tgtggactgc cttgaaatct tggaaaagtt taaatttcct tctagaaatt 5880aatcataaga tcacatagta tctttaattt ttaaattgtt attaattaat ttatttattt 5940tagagatggg gtcttgctat attgcccagg ctgaactcaa gctcccggac tcaagcatcc 6000ttccttacca ctgtttttaa ttaattcata attataaaaa gaagtactaa aaattcattt 6060atatgcctaa gacaaacaga tttgtcggaa ttatgatttt aagtattggt aaattaggac 6120agatttctaa aatttatgtt catagagttt tgttgttgtt gttaagggac ccacttttat 6180gttggtcatt aatatgaatt aaatattact tcctcataga aaccctaatg atttttcttt 6240tggtcaaaca tagacctagg tttgtttctt agactatgct attaattttg tgtcatcaca 6300gaaagtagcc cagtcttatt ttgagattgg tttattttcc ctggacattt gggatgaata 6360gctcagcata acagattatt ctatatactt tagtttcaag catttaaagt ggagtccaag 6420ttttagcatc aggaaccaat taaatgatag ttaaatctga ctgaaaaatt aatggtccag 6480aacagaaact gtgtagcttt ttttttttca gcacttctgt tttaaataaa aatctggatg 6540cttcccagta aaacacaaag cgtcattaaa aaaaaagcag ttgaatataa ataaaaccac 6600acccagtctt aaaaatcatt taatgatgat tgtattctta gtctgctatt aaacacaagg 6660tgtttatcag ccttcatggg ctgctaagag tttggctaca aaacacatag cactgtgggg 6720caatgtagga ttgcagtgct actgtgcctc tttagggtga gtatggtttg ctcttcaatg 6780tgattattct atagtcaaat catctagcat gattgtgata gcatctgatt tgtgccatag 6840gcgtcaatca gatttatgct aatattttgt actaatgatc aatataacag cagagaaaag 6900tgttgatgtt cagcccactg acttaaacct tactaacggg ccttgacaga acatagtggc 6960cttagatttt atgtgagtag aagagttcag gcttgaacat ttggctgccg cttcaagcac 7020cctacacaga aagtcagtat agtaccttta atctggctct aattcaaacc tatcagtaat 7080cactgagcct gctgtactta ttctgtcagt ctttttgcca aaaagatttc atgggtagca 7140atatcagaag catttttgta attaaattat tgtcgcttta ctggaaaatc tccttacatt 7200tgcaaagggt gaatgtgttc ttatttcccc cactccctac ttgacttgta gtttacgcag 7260gcacttttgg ctgcgtgcgt ttccagccta atggcctcct taccgcaata ttcattttgg 7320ttgggttact ccagccatca ttttccagag acagtcataa tccctcaatg atgacagaag 7380tcatttgatc ttggggaaca agaaataaaa cgagcaacaa taatttcttt aggaccattt 7440tacgggtttt taagagaata aattctgtga taagttagac tctgtgcttc taacttgggt 7500cacagcatct taacattttt cctgcgggaa ctaaagagca tactgtacaa tatgaaaagt 7560agcctttcgt agaccgccac tcccttctct gagttctggt gtatcttatc acttctcacg 7620aataggactg ccattcggcg cttatatatc tgcctctttc atggcatctt aacttctttg 7680aggcccgtga cattagttac gtggttacgc agttgttagc gatgactgcc tggcccatag 7740caggtgctca ttgtatgctt gatgcatgag tggataaaaa tactcattct ggtcctgggc 7800cagatctatt tcttgctttc ctcagttttg gcctatgcct cagggaagct gaccccagta 7860gcgtgcattt ctcaggctcc cacacatcgg gcattaggaa gcagtggcag gagacagaag 7920ggaaggaaga agggaaaatc cagggtcttt ctctgctcga tctgcattgg ggtggggggg 7980cggggtgcac aacctttgag acttaagctc acgctggata ggctgcagtg attccaactt 8040cttttgggtg acaccattca ctgggctctg ataatagtgc ttccgccctt actcccgcag 8100cactgggagt ggctccctcg tcttactgat ttttggcttt cttcactttc cccagtttgg 8160tttctcagca attttatcac ctgtgtaacc cattccttgc acgaaattcc ttctgtttta 8220aatacttatc gtctgttcag tgttgtattg tcatacaaag ttgaaattac acctcccaac 8280cttaaacctt tttaataaag agggacttgc cttatacttt aggggcacat agttgcctgg 8340gatctttgct cacactgcag agtctccaaa gtgtacctac actgagggga gagttaagag 8400gttcctttgg ggatcttatg cctgcacttt ctggaaatac ctaatcctta catattgtga 8460taatattctt gcttttgtcc tttatttgga attattgccg catattttaa gcgtgttctt 8520atgatattag gggatatcct tccttcctaa tgtgaagggg catccccaat ctgccttagt 8580aatttgacat agtagggcta tagagtcaca gaacaactga gagtggctct tacagacaga 8640gaaaagacag gattctgtca ttggcagttt gtccaggttc tgaaggtgag aaaggcctag 8700atcggtttta aatgtatgta atttctttaa aaatttctgt catacactac gtagttgtat 8760agctcttgga ataagcacat ggagtggtga aattccccta gatagcaccc ccaaattctg 8820gcactttaaa attctttgta aactttcaca atgagctgaa agttaattca aacctacata 8880gattctatgc ctcccttttc tcatctttgt agctctcctt tattcttcac agggggatgg 8940gaagagaaag gaatccggct gacatcaaat tatgaatatt ggtttaagtt cttaacaagc 9000ttgcatttca catgcacagc taacccacaa aagacactgg attttccaag gggttagttt 9060caaatctaca agaatgaggg atttcagcac agccctctgt cttactttct tgcatggatt 9120tggccatgca gccaggcaat ggtggtgtgg ttagctgtct gttgtcggga aaggagtcat 9180ctaatggagg actccttaat aaaactcctt aataaaacgt gaaattttag tactgagtct 9240gatttttatc attcataatc ctgtgtttat tcaggacatt caaatgcttt tgggggagaa 9300aactgttatc tgtgtgggtt gaattgggct aaatgataaa aagaaattac cttactgaac 9360tacttatttc cacaagaatt tcagaacttt agatctaaaa gggatttaaa aaatcttctg 9420attcaatctt tttatttctt tcaggtaaga aaattcagac tcagacacat taagaacagc 9480tggcttgtag cagaatagag attaaaatct agaatgattt tcatcttgct acctttaatt 9540aaaaacttgt tactttcagt ttacgcacct tagctctcaa gcctgatttt ctttcctgta 9600tcttgacctt aaacgtatca ggtctggccc agattcttta tgggagacaa tcaaaaatga 9660aaagtgcaca cagagcacat agggcttgaa gacctgttat ggggcatgga tgtgcttgac 9720agggaagcag ggaagaagta gcaaggcaac cgggagcact tttgagtttc ctctctttgc 9780caggcatgag aagaccactt ggtgtgcatt ttagatggtg ctgagatggg atcaacaaag 9840gtggagtgtg caaaagatga gagacattgg gcaatgtgga aatgaagaat aggattattg 9900ctcggaagga ttatggttgt gtttaatagg aataaccaaa gcaaacaatg atcacatttg 9960ttgtccttca ccttgtcaaa tagtgattta ggggctaggg aagtatatca cctctgaggt 10020tgctacaaca tccttttctg cccaccttct ccagaactta gtattataac aatagacaca 10080tgaaagctcc agtttgctta aacaatagca tctctattta aatatctatt cactaaagtg 10140ataaatctcc tcatttggga aaggtaattt cttgtaagta caaggaaaca aacaagtatt 10200tggaatatga gctactgcta ttatttcaga gtaaataatt cttttttgtt ttcatttttt 10260tttttttaca tttttaacct agaatattac tccactgtat agtgaacatt tatggttaaa 10320ttttcccatg actttctatg ctataggcct cgattaggta ttctgttctc attcaataaa 10380agcttttcac tgaaaataaa tgtcattatg ttgtaatgag tttcaatatc tactgacctt 10440acctggattt tggagggtga gttggctgtg agcgattcag cacctcttta tccttccctt 10500gagacactaa ttactcatta aattttcttt ttatcctttt tgctatattt caaaaatgcc 10560aaatataaag atttttgaaa aatgagaaac tagtggaaat gtctgcaagt gtttctttta 10620acttaaaaat atagatagcc aagtgagaaa gatataatgc aaactttggg gtctttattt 10680tagctggtta tatctagatc tgactgaaat tgggtattcc gagtttcatc ctgaagcaaa 10740tttctatagc tgagttataa gtccctgaaa aatgaggttt atgatgaaaa tgttgaaaga 10800ccctcaactt taggacaagg cacaactata attttacagg cattaggcac ctaaaataac 10860tttgtatgaa gattactctg ggatttgtcc cccattgtgt agaaaatgct taatgttgaa 10920ctatatttta tgcacacaaa aagacttact tcataagaca aggagcacag ggattttcat 10980tccaatattc aacaatacta ttactaaagg gctctctcct tctactctca taattgagtg 11040atcttaagtg atatttaaaa gaaaatatca ttccgctaaa cttacatgta aagatgatat 11100gtggcagagc aatttaaaat tttcaagcag aatgtattcc agcaatgtat ttggtatgtc 11160atgaaactga atggatggat cataataatg tcttaatatt ctattctata gcaacaaaga 11220aaatgagtgt ttagttttgc ttgtgtagag caatagctga agtgcttata tctgagtctg 11280cagtgcaata aaaaatatat atatatattt ttttcttaga gaccttgctt ttattgattt 11340tggatacata cccaaaagtg ggattgtgcc tgttagattg catttctttt tttttaatta 11400attaatttat tttttaaatt attatacttt aagttttagg gtacatgtgc acattgtgca 11460ggttagttac atatgtatac atgtgccatg ctggtgtgct gcacccacta actcgtcatc 11520tagcattagg tatatctccc aatgctatcc cttcccctcc ccccacccca caacagtccc 11580cagagtatga tgttcccctt cctgtgtcca tgtgatctca ttgttcaatt cccacctatg 11640agtgagaata tgctgtgttt ggttttttgt tcttgtgata gtttactgag aatgatgatt 11700tccaatttca tccatgtccc tacaaaggac atgaactcat cattttttat ggctgcatag 11760tattccatgg tgtatatgtg ccacattttc ttaatccaga ctatcattgt tggacatttg 11820ggttggttcc aagtctttgc tattgtgaat aatgccgcaa taaacatatg tgtgtatgtg 11880tctttatagc agcatgattt atagtccttt gggtatatac ccagtaatgg gatggctggg 11940tcaaatggta tttctagttc tagacccctg aggaatcgcc acactgactt ccacaatggt 12000tgaactagtt tacagtccca ccaacagtgt aaaagtgttc ctatttctcc acatcctctc 12060cagcacctgt tgtttcctga ctttttaatg attgccattc taactggtgt gagatggtat 12120ctcattgtgg ttttgatttg catttctctg atggccagtg atggtgagca ttttttcatg 12180tgttttttgg ctgcataaat gtcttctttt gagaagtgtc tgttcatgtc cttcacccac 12240tttttgatgg ggttgttttt tttcttgtaa atttgtttga gttcattgta gattctggat 12300attagccctt tgtcagatga gtaggttgcg aaaattttct cccattctgt aggttgcctg 12360ttcactctga tggtagtttc ttttgctgtg cagaagctct ttagtttaat tagatcccat 12420ttgtcaattt tggcttttgt tgccattgct tttggtgttt tacacatgaa gtccttgccc 12480atgcctatgt cctgaatggt aatgcctagg ttttcttcta gggtttttat ggttttaggt 12540ctaatattta agtctttaat ccatcttgaa ttaatttttg tataaggtat aaggaaggga 12600tccagtttca gctttctcca taaggctagc cagttttccc agcaccattt attaaatagg 12660gaatcctttc cccattgctt gtttttctca ggtttgtcaa agatcagata gttgtagata 12720tgtggcgtta tttctgaggg ctctgttctg ttccattgat ctatatctct gttttggtac 12780cagtaccatg ctgttttggt tactgtagcc ttgtagtata gtttgaagtc aggtagtgtg 12840atgcctccag ctttgttctt ttggcttagg attgacttgg cgatgcgggg tcttttttgg 12900ttccatatga actttaaagt agttttttcc aattctgtga agaaaggcat tggtagcttg 12960atggggatgt cactgaatct gtaaattacc ttgggcagta tggccatttt cacaatattg 13020attcttccta cccatgagca tggaatgttc ttccatttgt ttgtatcctc ttttatttcc 13080ttgagcagcg gtttgtagtt ctccttgaag aggtccttca catcccttgt aagttggatt 13140cctaggtact ttattctctt tgaagcaatt gtgaatggga gttcactcat gatttggctc 13200tctgtttgtc tgttgttggt gtataggaat gcttgtgatt tttgtacatt gattttgtat 13260cctgagactt tgctgaagtt gcttatcagc ttaaggagat tttgggctga gacaatgggg 13320ttttctagat atacaatcat gtcatctgca gacagggaca atttgacttc ctcttttcct 13380aattgaatac cctttatttc cttctcctgc ctaattgccc tggccagaac ttccaacact 13440atgttgaata ggagtggtga gagagggcat ccctgtcttg tgccactttt caaagggaat 13500gcttccagtt tttgcccatt cagtatgata ttggccatgg gtttgtcata gatagctctt 13560attattttga aatatgtccc atcaatacct aatttattga gagtttttag catgaacggt 13620tgttgaattt tgtcaaaggc cttttctgca tctattgaga taatcatgtg gtttttgtct 13680ttggctcagt ttatatgctg gattacattt attgatttgc gtatattgaa ccagccttgc 13740atcccaggga tgaagcccac ttgatcatgg tgggtaagct ttttgatgtg ctgctggatt 13800cgttttgcca gtattttatt gaggattttt gcatcaatgt tcatcaagga tattggtcta 13860aaattctctt ttttggttgt gtctctgccc ggctttggta tcacaatgac gctggcctca 13920taaaatgagt tagggaggat tccttctttt tctattgatt ggaatagttt cagaaggaat 13980ggtaccagtt tctccttgta catctggtag aattcggctg tgaatccatc tggtcctgga 14040ctctttttgg ttggtaaaat tctctaaaat tctctttttt ggttgtgtct ctgcccggct 14100ttggtatcag aacgacgctg gcctcataaa atgagttaag gaggattccc tctttttcta 14160ttgattggaa tagtttcaga aggaatggta ccagttcctc cttgtacctc tggtagaatt 14220cggctgtgaa tccatctggt cctggactct ttttggttgg taagctattg attattgcca 14280caatttcaga gcctgttaat ggtctattca gagattcaac ttcttcctgg tttagtcttg 14340ggagaatgta tgtgtcaagg aatttatcca tttcttctag attttctagt ttatttgcgt 14400agaggtgttt gtagtattct ctgatggtag tttgtatttc tgtgggattg gtggtgatat 14460cccctttatc attttttatt gcatctattt gattcttctc tctttttttc tttattattc 14520ttgttagcgg tctatcaatt ttgttgatcc tttcaaaaaa ccagctcctg gattcattaa 14580ttttttgaag ggtttgttgt

gtctctattt ccttcagttc tgctctgatt ttagttattt 14640cttgccttct gctagctttt gaatgtgttt gctcttgctt ttctagttct tttaattgtg 14700atgttagggt gtcaattttg gatctttcct gctttctctt gtgggcattt agtgctataa 14760atttccctct acacactgct ttgaatgcat cccagagatt ctggtatgtt gtgtctttgt 14820tctcattggt ttcaaagaac atctttattt ctgccttcat ttcattatgt acccagtagt 14880cattcaggag caggttgttc agtttccatg tagttgagcg gttttgagtg agattcttaa 14940tcctgagttc tagtttgatt gcactgtagt ctgagagata gtttgttata atttctgttc 15000ttttacattt gctgaggaga gctttacttc caactatgtg gtcaattttg gaataggtgt 15060ggtgtggtgc tgaaaaaaat gtatattctg ttgatttggg gtggagagtt ctgtagatgt 15120ctattagttc tgcttggtgc agagctgagt tcaattcctg ggtatccttg ttgactttct 15180gtctcattga tctgtctaat gttgacagtg gggtgttaaa gtctcccatt attaatgtgt 15240gggagtctaa gtctctttgt aggtcactga ggacttgctt tatgaatctg ggtgctcctg 15300tattgggtgc atatatattt aggatagtta gttcttcttg ttgaattgat ccctttacca 15360ttatgtaatg gtcttctttg tctcttttga tctttgttgg tttaaagtct gttttatcag 15420agactaggat tgcaacccct gccttttttt gttttccatt tgcttggtag atcttcctcc 15480atcctttcat tttgagccta tgtgtatctc tgcacgtgag atgggtttcc tgaatacagc 15540acactgatgg gtcttgactc tttatccaat tttccagtct gtctttgaat tggagcattt 15600agtccatttt catctaaagt taatattgtt atgtgtgaat ttgatcccgt cattatgatg 15660ttagctggtt attttgctcg ttagttgatg tagtttcttc ctagtctgga tggtcttcac 15720attttggcat gattttgcag cggctggtac tggttgttcc tttccatgtt tagcgcttcc 15780tccaggagct cttttagggc aggcctggtg atgacaaaat ctctcagcat ttgcttgtct 15840gtaaagtatt ttatgtctcc ttcacttatg aagcttagtt tggctggata tgaaattgtg 15900ggttgaaaat tcttttcttt aagaatgttg aatattggcc cccactctct tctggcttgt 15960agggtttctg ccgagagatc tgctgttagt ctgatgggct tccctttgag ggtaacccga 16020cctttctctc tggctgccct taacattttt tccttcattt caactttggt gaatctgaca 16080attatgtgtc ttggagttgc tcttctcgag gagtatcttt gtggcattct ctgtatttcc 16140tgaatctgaa cgttggcctg cgttgctaga ttggggaaat tctcctggat aatatcctgc 16200agagtgtttt tcaacttggt tccattctcc ccatcacttt caggtacacc aatcagacgt 16260agatttggtc ttttcacata gtcccgtatt tcttggaggc tttgcttgtt tctttttatt 16320cttttttctc taaactttcc ttctcgcttc atttcattca tttcatcttc cattgctgat 16380accctttctt ccagttgatc gtattggctc ctgaggcttc tgcattcttc acgtagttct 16440tgagccttgg ttttcagctg catcagctcc tttaagcact tctctgtatt ggttattcta 16500gttatacatt cttctaaatt tttttcaaag ttttcaactc ctttgccttt ggtttgaatg 16560tcctcccgta gctcagagta atttgattgt ctgaagcctt cttctctcag cttgtcaaag 16620tcattctctg tccagctttg ttccattgct ggtgaggagc tgcgttcctg tggaggagga 16680gaggcgctct gatttttaga gtttccagtt tttctgttct gttttttccc catctttgtg 16740gttttatcta cttttgatct ttgatgatgg tgatatacag atgggttttt ggtgtggatg 16800tcctttctgt ttgttagttt tccttctaac agacaggacc ctcagctgca ggtctgttgg 16860agtaccctgc cgtgtgaggt gtcagtgtgc ccctgctgga gggtgcttcc cagttaggct 16920gctcgggggt caggggtcag ggacccactt gaggaggcag tctgcccgtt ctcagatctc 16980cagctgcgta ctgggagaac cactgctctc ttcaaagctg tcagacaggg ccatttaagt 17040ctgcagaggt tactgctgtc tttttgtttg tctgtgccct gcccccatag gtggagccta 17100cagaggcaag caggcctcct tgagctgtgg tgggctccac ccagtttgag cttcccggct 17160gctttgttta cctaagcaag cctgggtaat ggtgggcgcc cctcccccag cctcgctgcc 17220gccttgcagt ttgatctcag actgctgtgc tagcaatcag cgagactccg tggggtagga 17280ccctccgagc caggtgcggg atataatctc gtggtgcgcc attttttaag cccattggaa 17340aagcgcagta ttcgggtggg agtgacccga ttctccaggt gccgtcagtc acccctttct 17400ttgattgggt aagggaactc cctgaccctt tgcttcccga gtgaggcaat gcctcgccct 17460gcttcagctc gtgcacggtg cgcgcaccca ctgtcctgtg cccactgtct ggcacttcct 17520agtgagatga acccggtacc tcagatggaa atgcagaaat cacctgtctt ctgcgtcgct 17580caggctggga gctgtagact ggagctgttc ctattcggcc atcttagctc ctccaatatt 17640tttgtctaat gaaaaatccc taacactgga aaatccttgc ctatcttttg tttctttgtt 17700tatttttggc tttgtttgtt ttcagagaaa aaggaatgct taaaacaata acaaatagga 17760atgtttagat ttgacacaac tttcttggtg atttaggcca attttttttt taagaaggaa 17820aaatattgat taacttctaa acttaagata cctggtttgg gtataattca gatgaaaaca 17880taaaagcaag tttgctttaa aaaacctttt aaaaaagtta ttttttattg acaaaattta 17940tatatttatt gcatacaaca tggtgttttg aaatatgtat acattatgaa atggctaaaa 18000caagttactt aacataccat tacctcatat actctttttt tttttttttt gtggtggaaa 18060cacttaaaat ctactctctt ggcaattctc aagaatatag tgtattgtta ttaactatat 18120atacaataaa tctcttaaac ttcttccttc tgcttttgta tcctttgacc aacatcagcc 18180caaccccatc ctcccacccc cctcctctgg taaacatcat tctactctct actctatgag 18240ttcaactttt aaaaatttat taaaaaataa aagttagata atgtctttct tttctttttc 18300ttgatcagat tggctagagg ttatccattt tattatcatt taaaagcatt agctttgagc 18360tgggttgatt ttttgctatt ttttttctgt tttctattta ttgctttcta cttatttctt 18420tattatctct tcctcctgct tactttggtg taatttcttc tctttctgtt ttcttaagga 18480ggaagcttag atcctatatg tgtctcttat tttctaatac agttacttaa tgccatagaa 18540atcccccgaa gccctgcttt agctgtgtac tacaattttt gtttaggttt agtttttagt 18600ttcatttggt tcaaaatact ggtcttcttt gtgacttttt gacttatatt taatttagaa 18660atttgttaat ttccaagtat tggaggtttt caaggaattt ttcggatatt catttctgtt 18720taatttcctt gtgtcagagg ccatattttg tgtgatttca actcccttac attgttaaga 18780tttgttttat ggcccagagt ctaataaatt gtagtttaaa aatgtgtgtt ctactgttgt 18840tgggtggggt tggctggcag tgttggtcac atcttttata ttcttacgca ttttcattta 18900cttattctct tacttactga caggtttttt tgaactctct aaccacactt gtggattttt 18960ctatttcttc tttcatgcct accagttttc gcctgtgtgt tttaaagttc cattattagg 19020tacctacata tttagtattg ttatgtcttc ttgattaatt ttattccttt agcattattg 19080aatggtcctc tttatcttgg taatatttct tgtactgaaa tctacattgt ctttattata 19140aaatcactct ggtattctct tgattagtat ttgcatagct tttcccatcc ttttactttt 19200aataagtgtg gttctttcag gctgctttta ttttggtctt gcttcttaaa atccaaattt 19260tagtatgata atctctgatt tttgtgtgta gaccacttga atttaataca attattgaca 19320tgattgtaca cagatacaac agtttgctct ttttctgttt cttttctgct tctatttgtt 19380ctgtttcttt ttttctcttt tcatttgtat tgcatatgtt tatgatttca ttttaacaca 19440actattagct atatacatat ctttccaatc agagtgcacc tttaaatagt attgttctac 19500ttcatatgca gtctaagaac cttaaaatag tacactttta tttcctccct ccaattcttt 19560gtgctattgt cataaatttt acttctactc aggttataca tcctacaata cattgctact 19620gcttttgctt tcgttatttt taaataattt aaataactat aaaaaattaa ataattaaaa 19680attttaaata tttaccctaa ttctcctgtt ttgattctct caatttcttt gtgtagatcc 19740aaattcccat ctggtttgac catatccttt ctgcttgaag aacttcattt aatatttctt 19800gtggtacagg cattttggca ataaattctc tctgcttttg tttgtctgga aaaaattctt 19860tatttcaaag aaatagtttc attggatata tagattctta atttacagtt tttcactttt 19920ctgtacttta aagatggtac tccattgtct tctggtttgc ttatagtttc taataagaaa 19980tctgtgataa ttctttattt gtttttctgt atgtagtttt tttttccagc tactttcaag 20040atttttctct ctgtttttag tttttagcaa gttaactctg atgcttcttc ttcatttgtt 20100ttttaaattt tgttaatcct gtttgggttc cccaagtatc ttgcattatg cattatgttt 20160tttgttatct tttgtcagtt tagaaatgtg tgtgtatata tatacattta taaatacata 20220aatatatata tgaaaatgtg tgtgtatata tatttacata tacattttaa atgtatgtgt 20280gtatttacat atacatttta aatatatgtg tgtatatata cattcaaaat attttatata 20340tacatttaaa atactatata tatatatata aaaactatat atatatagtt tttctttttt 20400ttttgagaag gagtcttgct ctgttgccca ggctggggtt tagtggggca atctcggctc 20460actgcaaact ccacctcctg ggcccaagtg attttcctgc ctcagcctcc ccagtagctg 20520agattcaggt gccgaccacc acgcccggct aatttttgta tgttagtaca gatggtgttt 20580caccattttg gccaggctgg tcttgaactc ctgacctcaa gagattggcc cacctcagcc 20640tcccaaagtg ctgggattac aggtgtgagc caatgtgccc ggtctgaaat gtatattttt 20700aaaatacttt ttttcctgtc atgttttttc ttctcttttt aggaatctac ttatacaatt 20760aaactctctg agattgaccc aagccttctg agttaaaaaa aacaaaaact gtattttcta 20820ttcatgtttc actttggata gttactattt acctatcttt aattctttac tctgttgtgt 20880ttgctgataa aactgacaaa tgaatttcca tcttcgatat tgtgttttta atttctataa 20940tttgcatttg gctcttcttt atagtttcaa tctctgtgct gaaaatccta atttattgag 21000gcatgtttcc cattttttcc tcttgatcct ttaacatatt aattatgatc tcaaaaatgt 21060ccttgtctga tcgttctaat agctgaatca tctctggatc tggttctgtt gtctaatctc 21120gacaaaggtt ttttttttgg ttgtttttct tttcattttg tgcaaagtag attgtattat 21180tttttaaatt gtatattgac aaattatagt tgtatatatt tatataaatg gtcttatgac 21240ttttgaatac aatgtggaat gattaaacta agctaattaa catatctatt acttcaaata 21300tttaacttct tttgtcatga gaacattagg aatttattct cttagtgata cggaaaggta 21360caatatttaa ttattagcta tattcagcat gcggtactat tgatctaaaa aaaattaaac 21420tcattcctct tatctaactg agactttgta tcctttgact atcatctccg gatttgtgtc 21480tttgactatc atctcctgat tccccccaac cccctgcctc tggtaaccac cattctaccc 21540tctgcttcta tgagttcagt tgttttagat ttcacatata agtgagaaca tgcagtattt 21600atctttcaat gtttggctta tttcacttag cataatattc tctaattcta cccatgttgt 21660tccaaacaac agaatttctt tctttttaaa ggttgaatag tatttcattt tgtatatata 21720ccacactttc tttatccatt tattccttta tggacactta ggttgattcc ataatttggc 21780tattgtgaat agtgctgcaa tgaacatggg gatacaggca ggcatctctt caacacactg 21840atttttaaat ctttcgggta tcagaaattg tattactaaa tgatgtggta attctatttt 21900tagttttttg aggaacctcc ataccatgtt ccatgtttgt actaatttgc attcccacca 21960acagtgtaca aggattccct ttcctccaaa tccttgctaa cacctgtctt ttgttgcttt 22020catagtagcc attctaacag ttgtgaggtg ctatctcatt gtggttttaa tttgcatctc 22080tttaatgatt agtgatgttg agcatttttt catatatctg aagtcatttg tatgtcttct 22140tttgagaaat gtctatttag atcctttgcc catttcttaa ttgggtaatt tgttttcttt 22200ttagagggtt gtttgagttt cttatatatt ttatatattg atccattatt agatgtatgg 22260cttgcagata ttttctccca atccataggt tgtctcttca ctctgttaat tgtttccttt 22320gatgtgcttt ttattttaat gtaatcgcat ttgtctatct ttgcttttgt tgcctaacct 22380ttggggtcaa acctaaaaaa taatcgccta gaccaatgtt gtgtagtttc tcccctgtgt 22440ttttttttct agtagtttta cagttttaga tcttacattt atgtctttaa tctattttga 22500gttagttttt ctatgtggtg taaaataagg gtccaatttc attcttttcc ctatagacac 22560ccagttttcc caacaccatt tgttgttcca attggatatt cttggcacct ttgttgaaaa 22620tcaagagacc acagatgaat gtgttctttt ctggattctg tatagtgtcc cattggctgc 22680tatgtctatt tttatgccag taccatgctg ttttagttac tattgatttg tagtatagtt 22740tgaaatctaa tagtgtaatg cctccaacta ttttcttttt gcaaataatt gacttagtta 22800tttgtaattt ttttgagagg tttcataaga attttaaaaa ctttttctac atctgtgaga 22860agtgacatta aaattttgat agagtttgct ttgaacctgt aggtcacttt gggcagtatg 22920gacattttaa caatattaat ccttccaatt catgaacatg agagatattt ttatttattt 22980atgttttctt gaatttcttt cattaatgtt ttatagtttt tagtgacagg tctttcactt 23040cttaggttaa atttattcct aagtacattt ttttttgtag cgattgtaaa taagacggtt 23100ctcctgattt ctttttcaga aacttcattg ttagtgtaga gaaacactac tgatttttgt 23160gtcttgattt tgtatcctgc aactttactg aatttatcag ttctaagtat tttttctttt 23220ttatagaatc tttaggtttt tttttaatat ataagataac atggtctgaa aatagggaca 23280atttatctct tccgttttta tttccctgcc ttattgccct ggctgggaac tccagtacta 23340tattgagtag aagtggtaga aatgggcatc tttgtcttgt tccaggtctt agagaaacag 23400ttttcaactt ttctccattg aatatgatgt tagctatgaa cttgtcatat atgaccttta 23460ttgtattgag gtacatttct tccatatcta attttttgag agttttttta atcataaaaa 23520gatattaaat tttgtcaaat gcttttcctg tatctagtga gataatcata tgatttttat 23580ccttcatcct attaatgtga catattacat ttataaattt gtgtgtgtta agccatcttt 23640gcatcctagg aattagtccc aattgattgt agtgaataat tcttttaatg tgttgttgaa 23700ttcagtttgc tagtgttttg gtgacagctt ttgcatctag atttatcaag gatcttggcc 23760tgtagttttc ttttcttgtg atgtcctttt ctggctttgg tgtcaggata atgttggtct 23820tgtaaaagga gtttggaagt atttcctctg ctctgatttt ttggaacaat ttgtggagaa 23880ttggtattag gtctttaaat gtttgataga attcagcagt gaagccatca ggtcctgagc 23940tgtttttttt gatggaagac tttatattac agcttcaatc ttctcttgtt attggtctgt 24000ttggattttt atatttcttt gtgattcggc cttgataagt tgcatgtgtc tagaaatgta 24060ttcatttctt ctagataatc taattttttg gcacataatt gttcataata gtttcttatg 24120atcccttcat ttccatgatg tcagttgtaa tgtctcttct ttcatttctg attttgttta 24180tttgagtctt ttctcttttt ttcttggtta gtctagctaa agatttgttg attttgttta 24240tcttttcaaa aaaacccaac tcagtttcat tgatcttttt tattgctttt ctagtctcta 24300ttacatttat ttctgctcca gtcttcatta ttccattcct tctgctaaca tctaacaatt 24360acttttttgt ctggtaattt ttttgttgac tgtgaagctt tttttttttt ttttttttag 24420accagtagag tctgaagtaa gtagaattaa ctcctagaaa tggtctcctc ttttttcagg 24480ctgttaatgt agggaagttg agccaatctg gttagtagct gagctgggct ttttgttgct 24540aacattacct ttgtgcacta caggctgcag attcttctag ctctgggtgg ctgctacctt 24600gtgcttactt tgggacctaa agtgcaggaa ggtttttctg tgttcttgct tcactctcag 24660ctttcagcag accctaaata tctatgcata aacgggggtt ctttatgctc ttgcccctct 24720ctcagttgca tattgctagg tacaaagctt atggtaggga aagtggagat tttctttgtt 24780ccctggtctc agcctcagtc ttagttaggc cctatattct tggtcctctg tgatgagggc 24840tttatcacca ctcctgcctt tccctgacat agtagccata tcttaccttg catctgtcaa 24900aggtcttgaa taggagagac agtctttgtt cttcctcagt agtagaaggt ctctgccttc 24960tattagtaca gttgaataga tttcctgttg tctgcctacc ccaatgggcc aatggatttt 25020gctactagtt ctctctcagt agctgatggc tttgcctggg agtggaggtg tacagttttc 25080tatactctcc taacagcctc tcactcagac agcttttgtg tctaccatct cccagaagca 25140gtggatcttt gcctgggtag gagacttttc tgcttctctc tcagaggcaa aggcttttgc 25200tttatttgag agcaggatca gaaatgcaga tacagtttca accctatggt tcaccaaggg 25260ggaagagagg gctgcctcag atctcttgtc atgttccagt ctttcttgtg agcaacgagt 25320agagaatcat ggaaaacaac tgaagaatgc atgtggattc cccacgggtt tgatctgcca 25380agcagtctga attgtcctag cacacacttg gcttttgtaa aaaagtgttg cagttttctt 25440cttatccact tttacgttgg cagattcttc cttactcttc caaagatgga acagttcatt 25500atcccattta ttttcagaag ggtttattgc tctttggaat ttagttttag tttgcctgat 25560ttattttgta actctgtgat tttagctctg atgggcttaa gaagagtgat atttttgtag 25620attatctgcc tttttccttg ctgtaagggt aggagtgatg ttctcttgtg atgttttcaa 25680tcctaagagg agcagaactc catgtcatat gatttttagt atgtgagtgt ggcctaggag 25740ccagctttct caacaaacat aaaatactaa tataaaaaaa tggagattcc taagtctagt 25800ttttagtttg agtttgtatt gacagtttgg aattgatatc caattctacg accactaaaa 25860tgaaaagcac atccttttgg taataaaata attttgttgt agtaatgttt ttctttaaat 25920taaaaagatc tgaaacctag agtggcttct tttcatgtta attagggtga aaatcaggaa 25980ggatgtgata tactgtcatt tacttttact taacacttcc aattcatttt aataggatag 26040gaaatacttg gaattcagat caacacttcg ctgtctgttt gctgatgaac tgaaatgctt 26100atatgagaac agggattttt cccaaatgct tgaatttagt tccccatttc ccattatgta 26160agaaaacatt gttccttgtg gtaggctgaa aaatggcttt ccaaaagaca tttatgtccc 26220agtccctgga acctgtaaat gttacctttt tggaaaaggg tttttgcagg tgtgattgat 26280tacttaagga ttttgagatg gagagattat cctagattat ctggatgagc cctgaatgcg 26340atcacaaata ccttcgtgag aagagcagag ggaaattaga cacacagagc ggaaggtgga 26400tatgaagata aggtaggtac tggaatgatg cagtactcca caggaatgcc ggcagccacc 26460tggagcagga agacaggcaa ggagcagatt ctccactgga ggctccagag aatgtacggc 26520cctgctgaca cgctgatttt ggcccagtga agcagattca gacttctggc ttccagaact 26580gtgagataat aaatttccac tgtgttaagc caccatgtct gtagaaattt cttacagtga 26640ccataggaca ctaatacatg tatcttagtc tttaaaaaaa atctatttgt gactgggcga 26700ggtggctcat gcctgtaatc ccagcatttt gggaggccga ggcgggtgga tcacctgagg 26760tcaggagttc gagaacagcc tgaccaataa ggtgaaaccc tatctctacc aaaaatacaa 26820aaattagcca ggtgtggtgg catgtgcctg tagttccaga tacccgggag gctgagacag 26880gagaattgct tgaacctggg agggagaggt tgcagtgagc cgagatcaca ctactgcact 26940ccagcccggg cgacagagtg agactctgtc tcaaaaaaaa aaaaaaaaaa aaagatttgt 27000ttttatttta ttattaaaaa aattttgagt acataatagc tgtatatatt gtcttgacag 27060aagaatttta atttgtttca attatcttat ttgaaatgtc attgaataaa agtcagttgt 27120acaaagtatt tatttttaaa tttatttatt tatttttgag atggagtctt gctctgtcgc 27180ccaggctgga gtacagtggt acgatcttgg ctcactgcaa cctctgcctc ccgggttcaa 27240gcaagtctcc tgcctcagcc tcctgaatag ctgagacaat gggcatgtgc cactacaccc 27300agataatttg tatttttagt agagatgggg tttcgccatg ttggccaggc tggtcttgaa 27360ctcctggcct caagtgatcc acccgccttg gcccttcaga gtgctgagat tccaggtatg 27420agccactgag cccaggtgaa gtatttattt attatgatta cacccacaat tgcgaagttt 27480taaaagtgtt aactcaattt acttaaaagc aataaaacat tagagaaaat tatgcttggc 27540ctgagatgaa atacatatat ttacagatta ctcttatgag tgatttgttg gtagattttt 27600ggatgcttct cattaatcct gacatgaatt gacaccaatg gtcatacagt catacatttg 27660ggttcccacc aaaataatga cttaaaggga gtcacctatt cattgttgat tttcagacaa 27720cttgacactc cttctggggt gccctaagct tcaccacctg cacacaattg ggtcttgttt 27780actttctcag gtatcttcca gtcagttcac aagatccatt tatgagaaaa taagacaaag 27840tatttgattg tggactaaat agaatgaaaa taaaagaata aatggaaagt tctagaaatg 27900tttaactgcc taagaacatg gtgtattgat tatatttttc ttcttctaga cttattgtca 27960catattgtat tatgtatctc caccataatt tctgtccttt tcatcctgct ttataatttt 28020tggtgcatag cagttgtctt ctaccatatt atacaaataa ttttcattat gttttatatt 28080tatctatctc ttctaattcg acataagctc caagagggta ggatctttct ttttttcagt 28140cttgctccaa atgcctaaaa cagtgtctgg ggcgtactgg gcactcaata aatatttgct 28200tagcgaatga aggaatgact aattctacag aatataaaga catcaatagt tttctcccag 28260agaggtgtta gttgtgttaa attctaacct gttttaaaaa tttcaaagat aaaagaaaga 28320ctttaatgac ctagagttaa aaaaattacc cttgccacca ttttttcctg ataaactaaa 28380atgcttaaaa tgactcaatt gtctagagga ctctcatgat ctgaagttgt ctacctcttt 28440gatactccct cctaccactc tcaccacagc tcaggaggct tcagccaccc cagccaatac 28500acaaagcctt ttgccatgtt agggtgtttg cccttatcca tcccctagct tctaccccca 28560aatccttcat ggctggccct ttcatatcac tttgcctcag atcagatgtc acctcttcag 28620agaagactcc cataaccctt gaagccagta gttcccaact ggtggtaatt ttgtccctca 28680aaggacattg ggaaatatct ggagacagtt ttgtttgtcg cacctgggaa ggagaggttt 28740tgctactggg tctaatgggt aaatgggttg gggatgctgc taaacatcct acaatgcaca 28800cagcaacccc cacaacagag aattatccag cccaaaaagt cagcagtgcc aagattggga 28860aactctgccc tgggctaatg cagcccccac ctctgacact cactttcttg ccaccagtgt 28920ttcacttcct catagcactt cctgcaagtc aaaaatactt tattaatttg ttcactttct 28980tttgttctcg ctactctact agaatgtaag ctccatgaat gcaaaagcct tgtctgtctt 29040attcacgtat tgtcctcagt gcacctagta gacatctaat tacttattgc ctagatgaat 29100aaataaacaa tcatatactg accattttct ctccccacca cattattggt gttctcaact 29160gtctttttgg ctacaacttt gagatagttt tacttttcag catggcttct tttccttata 29220agtactttca cgaacacctg tggaagtcat tatacacggt taaacaacca cagactctaa 29280aatcagaaca tctgggttca gtcagactga tacggtttgc ctatgtcctc acctaaatct 29340catttcgaat tataatctga attgtaatcc ccaggtgttg agggagggac atggtgggag 29400gtgattggat catggaggcg gtttccccaa cactgttctc ctcatagtga gggagttctc 29460accagatctg atggttttaa aagtggcact ttcccctgtg ctcacttgct gtctcctgcc 29520accgtataag acgtaccttg cttcctcttc ttctttgggc atgattgtaa gtttcctgag 29580gtctctccag ccatgtggaa ctgtgagtca atcaaaccgc ttctctttat aaattactca 29640gtcttaggta gtatctttat

agtaatatga aaacagacta atacatagac ctttataatt 29700tactagttct gtaagaatct attgaagaag ttactttatt aactcttgga aaagttgctt 29760tatttttgtg gtccttagat ttccaaatcc tttagaagag ggtaatatta atacttcctc 29820aaagagctac tttgaagact aaacgataat gcacataaag cacctggcac atgaagcaca 29880tatatatttg tcattattat tattactact attattaatt tttattttat tatggtcaaa 29940cgtatataac ataaaattta ccatcccaac cattttacat gtacagttca atgcattaag 30000tgtaatcata ttgtgcaact gttattatta tttttatcct ctgaatctgt attttattgt 30060ttaagcttcc ctttgtgctg tgtctcctac tctcataata gtattcaaga tagatcctac 30120tgatgttgga tagaattaga tcgacccata attcttagat agtatgtacc atacatttta 30180ttgctcaatg catctgcatg cttaagggtg tacattttcc ctatgtattg aataggttat 30240ctgccatgtg tttatctctt cataagtgtt ttcccagtaa taatatagaa accttgtgct 30300tcggtttgaa tgtgtcttcc aaattttagg tgttgaatac ttaatcccca aatatatatg 30360ttgatggtat ttggaggtag ggcctttggg agataattag aattagataa gggcatgagg 30420atagggcctc catcatgtca ctggtggctt tataagaaga gaaagagaga cctgagctgg 30480tgtgctcttg cctttttgtc ctgtgatgcc ttctgtcatg ttatgatgca gtaagaaggc 30540cctcaccagg tgcagcttct tgactttgga cttccagaat tgtaagaatt gtaagaatta 30600aatgtctttt tgtcataact tagccagtct gtgatactgt gctatagtaa cagaaaacag 30660gataagacat tggcacacat tttatttctg actgtgtgcc caataagcat acccactctg 30720tatgtgtgaa atccttgcca ccttcaactt gatagtaaca agtcacagaa gatgaatatc 30780tagttttata atcctaaaat acaggcttaa tagcaaatca tgaaagtaaa gatttgatta 30840tagagattag aagaatagaa attgggtttt acagtaatcg aataactatt aagaatactt 30900caaacgcctt gaaagaatcc ttaaaatacc aaggtttaaa acaagatcta gatatggact 30960gagggttttt cccaaggaaa aaatatccta ctcacttaat taagcaattt ttaaaaattg 31020tcatacaact gtttaaatac agtctcttag gtttctaaaa taacaaatta cgtgggaaaa 31080ttagtgctaa gggctaagaa gaaacaaagg tgtttctcaa caatcacgtt tggattattt 31140taaaaatacg attctaatca ctccacaatt tagaaaaggc taatggtcta ttagtgacct 31200ttgctggaag cttttcaaga ggatagattt gttgtgatag atttcatctg tgaagtgaaa 31260ttttgcaatg taagttttat agtctttatt catgaaaact tctcccaaag taattgaaag 31320gctagacatc cagttatctg aaccagagtt ttataaatga gtgtgtgtag gctaggaaaa 31380aatttttata cacggagttt tgttataacc tcagtacacg ggtgaggcac attaaaaaaa 31440aaatcacctc attaggctgg gagcatacag cttccaaggg ggaaatgaat agtttggggg 31500ttcagtagta caatccaact aacattttgt ccaaggatcc atagataact agtagtcaac 31560catacctgaa acccaggtca tctgagctgg agacagtggc tttttctttc cctctaccct 31620ctcttactac tctctaggac cctgggtgcc cagccttctt tgtacaggtt cctgttcctt 31680cccttccagc ctgcatctgg ctgacttcag cctgcaaatg cttcagaaca acctttcttg 31740tgcagcatct ttgtgtgatg ggggaagcac tgaagaagaa ggtaccacag tctacctgct 31800tgggccttct ggtgattatt tcttcaactc agctgtcttg atctgaggga gagttttatg 31860agctaaaaat ctgtcagtgt ttccatttac caagtagcta ttcaatcgtt ttatagccta 31920accattccca attaccctca attaaaactt ggcacatatg ttgacaacct tagatgcaat 31980tttttaattg aaaattgttg atggcttttg attgaagtct aaaaccaaaa gtgcacaatt 32040cccatgattc agatctttgg tgtggttctc agattattta ctgtcagtta tgatcacatg 32100ataccaccac tttctaaagg ccagtaaatg tcatgggttg aaggcaggtt aaggcttggg 32160agaaaccttt ttcaagtgtc gtatggtcta tatttttgtt cataggcaga tctgagactc 32220aagtggcaga gggttatggg atgggaaaat gaagaatggt gtgttgtaaa aataaggaag 32280aattaaatga cttcagaatg tagagtggag atggcaaaat aagaacttgc attgctcagc 32340attattatct gaacacttgt ttaaggacag gcccagacct gtaaagaaat gtgaagcagg 32400acaatttgtt ttgagatcca cttttctttc tcatcccagc ctttttctgt ttcttcctac 32460gcttttccat cctctcttgt tttctatccc cagtgatgtc atatccacat tactggtatt 32520aaggagtttc tttatttttc ttttaatttt actttaagtt ctgggataca tgtgccgaac 32580atgcaagttt gttacatagg tatacatgtg ccatgtggtt tgctgcacct atcaacctat 32640catctaggtt ttaaaccgcg catgcatgca ttagatattc gtcctataca ttaaagagtt 32700tcctatttta gttccttatg gaaaaaacga tttcaagaat atttttggca tatcagaaca 32760tagattctag agtcttaata cctaaggata agacaagcct tgctaaaagt tatcactttt 32820tgaaaataaa attatcgctt catttcttct agcttccaac accaataagg gacagtcatg 32880aaacatataa gctatgcaaa attaccgaag tatgtaaatt ttgcactgaa aaatagtatc 32940attggatata tttaaaatgc gaactttgta tgcacttata aaattataaa aatggaagcc 33000aagaattaga taaattattt taaatacttt aagcatagtt gtaaacatta ggtacctgat 33060acagtttgaa tatttgtccc tgcccaaatc tcgtgttgaa gcattggagg tggggtctgg 33120tgggaggtgt ttgggtcatg gaggaggatc cctcatggct tggtgttgtc cttgctgtgg 33180tgagtgagtt ctcacaagat ctggttgtgt aaaagtgtgt ggcacctccc tgttaccctt 33240gctccactct caccatgaga gacgtctgct ctcccttcac cttctgccat gattgcaagc 33300ttcctggggc cttcccagaa gcagatgcca gtaccatgtt tcctgtacag cctgcagaac 33360agtgagccaa ttaaacctct tttcttataa attaccctgt ctcaggtacg ttatagcaat 33420gccagaatta cctaatacag aatcttacaa tggaaatata acagggactg ataaaagttt 33480tttccttatt tctaaagtaa tctgggacca tttttagagt ttgggtagtg gcatgtatag 33540tttaggagtc caaaaatatt aatatgaatt ctattttctg tatcattcca tgactgcttt 33600ggagtgtgaa ctatataaat gaaatgctca aaagcagaga aaaagttaga taattggtat 33660aattctccac acacaaaacc acctggccca aataactgaa gttgttgcta tagacaggaa 33720atgtaaatgt gcatgcaaca attttcttgg taaggctgaa agctttataa aaatattttt 33780tcccttaaat gctacaaaca tttttgcaag atcagttaaa aattatcatc catgtaaaag 33840ttcacattta agaaaagtca ttcattttgt ttaataaaat ttcttttact gatttaatta 33900attggcaggt agatgcccac tttctgtgtt gttctaaaac aaatacttta aaaatgtcat 33960ctagatacat ctctatgttt tatactcaat ctatcccctc tttagtaaat aaagtataat 34020taagattcca tcaaagtggt aaatggagat tatctacaat caatcttatg tacctatgtc 34080tgccaggttt tattcttgtt cctgttgcat catgcctttg agagaccatt tctttagatt 34140tcatttcatg tctgtcttag tgagtcactt ctgggacaat atattcttga gtgcctcgcg 34200agggccccca gttaaatgcc attttgtaat gagagtttct ttggtcactg gactagtctc 34260taaagttgtt aaagcctgca gaccttaaat acagtgctgg ctgatgcaac ttaattaaac 34320tacagcaaat tgaatttagg taataaaact ttgcttggat tttatggttg tgattgtctt 34380ttagttattt attttataag gatgataagc tatccatcct gtgactgcct agccattttt 34440gatgagtcag aattactctg ggctggtccc cacaaaggaa gatttggctt ttctgcacat 34500agtgaaaaat aaatatggtg cttaatctga gatatcatga tctttcagtc accccaagat 34560tcttctggtc tgtttctgca ccattcattg ggtgtacact taggtaataa aaacttattt 34620taatattgtc taagtgaaag ataatttatg cttatttcac aaaatttata tagtaatgat 34680atatatagga catttgaatt atctgttatt acactttccc gttgacatta acaatggatg 34740gttgacttta taagaagttt aaatttaact taattttcac atactagatg taaattaggt 34800tttctgttta gtctacctaa gggaatttat caaactagga gaaattacaa gggtttaagg 34860atgggttgta tgatgctgta gaatctgaga aatttgtgag ctccaattct agtgggtgta 34920taaaccccag taccaaaggc cggtgggtga gtcccaactt ccctctgcag cagatgattg 34980ttatttggag cagtggtctc aaggggacac cgtgactgtc acacacaaat aggatgtatg 35040agtggttaca gcatcctcga gaggtgtggt ccttgagata tgagcctggt tctagattta 35100aatattgact cctcctggac atctgaagga atgactgtag ttgctgttgg aactcccctc 35160aacggtagcc aaaaataatt gcagcaacag aggcaggttt attttaacca catgccatga 35220caggatggtc agcagtgatg cctacagata cttattaaag gtaccctgag aagtgagaat 35280gcccttgttt tccagtgcct ttccttttca caattgcaag gatgagaata agggctgacc 35340ttgtggtaat gacattttaa atgttctgag tctgaagact gctgtaaggt caagacgttt 35400tctcctagaa ctgagcaaac cacagtgtgg acttacttaa gaagatttgt ggcattgtta 35460agatctcttt ataatgaata tttatacttg caaaggattt atttctttcc aggatgccaa 35520gaaatatgtt gatccagatt ctctgagttt caccaaacta cgttcccttt taacattttt 35580ggaaaatgga tgaatttctt tctgtctcct gaatgttcgt tggtttgtaa ctgagcctat 35640ccacagcacc tctgcggtct ttcaggcaca ttagatcttg gtttttttac tgctttgtgc 35700tgtagggcag aaatgcatcc acaaaaggaa gtatattagg tatggaaaga cgctggctct 35760ttcaatcagg gatgagtctc tctcgatgct atcttctttc atctaataac tagatttggc 35820agtagtctca ggtgggctga aatgcaggtt tctgttgctt cttccaagtc tcttctgtgt 35880gaactagaga aattacttca aagctgtaga acttgtcaga tttccatcaa ggctgcagaa 35940ccagcttata acttttaaaa tgaactttta attttaggag aggtttagat ttacagaaaa 36000actgcaaaga tagtacagag agttcctata tagcctatac cctatttccc ctattactaa 36060catcttatat tagtatggta tatttattac aaatagtgaa cgagtattga tatatgagta 36120ttaacttcat actttattca gattttcttc gtttttacct gatatggtct gactgctggt 36180gttccactaa aattcgtgtg ttaaaatcta actcccagtg tattagtatt aagatgtggg 36240acatttggga ggcgactagg tcatgagagc agagctcctt gtcaatgaca ttagtgcctt 36300tataaaggag gcctaagaga ccttgttcac ttcttctatc atatgaggac atatataagg 36360taccatttac aagataggcc ctcacgtgac actgaatctg ctggcatctt gatcttagac 36420ttcccagcct ccagaactgt gagagataaa tttttgttgt ttataaatta accagtctat 36480attgttatag cggcccaaat ggactaagac attactaatt gttttgtttc caggatccca 36540ttgaggatac cccactgggt ctagttgtct tgtctccttt gactttcctt ggctatgaca 36600gtttttcaga ctttccttgt ttatgatgac cttgacaatt ttgaggagtg tcagttaggt 36660gttttgtaca attttcccct actagggttt gtctgattgt tatggcttga atgtgtcctc 36720cagaaattca tgtgttggaa acttaatccc caatgcaaca tcgttggaag gtggcaggtg 36780tttatgtcac gaaggggagg gaggtgttta tgtcacaaag gctctattct catgaatgga 36840ttaatgctgt tataacaagg ggatgtgagg tggcatcatt ctctcttgct ttcccctgcc 36900cttctggctt ccaccatgtg atgatgtatt aagaaggcac ttgacagctg ctggcacctt 36960gatcttggac ttcccacctc cagaactctg agaaaataaa tttctgttct ttataattac 37020ccagtctgtg atattctgtt ataggagcat aaatgaaata agacaccgat gtttttctta 37080tgattagatg ggggttatgt gttttggata tgatgactac agaggtaaag tgcattatca 37140tcacattata tcaagaatat gtactatcaa catgacttat cactgttgag tttaaccttg 37200atcacctaat tgaggtagtg tttgtcagtt tactccacta actcattttt cctcctttct 37260gaggtgtatt atttggaagg aagtaatttt aaacagccca catttaagaa gtgggaagtt 37320attctccacc tcctggaggg tggagtatct acataaatta ttagaatttt tctgcagggg 37380agatttgttt attctccccc atttacttat tcaatcattt atttatttat atcagtacag 37440actcagatat tgattttcca ctttgggtta taatctaata ctactttatt tattttattg 37500cacaaattat gctagctttg gctactggga gctctttcat ttggctcctg aaagttgccc 37560ctgccactgt gttttccttt ttttttgagc acttatcttt tttcttgcac taaaagatgc 37620tcctggctca tttcatatat ttcctaccca agtcctagag tcagccattt cttcaaagag 37680tcctgttcag atcagttttt aaacagctta gtttttacat tctctctaaa tcttcccttg 37740tgtttgtgta tgcgattatg ttaatgcttt ttgtttagtt ggcattgaaa taggtttttt 37800aaatcttatt tttttgacaa gtgagaccta attaaactga aaaacttgca cagtaaaaga 37860aatcatcaat agagtaaata gacaacttag ataatgggag aaaatattca taaactatgc 37920atctgacaaa ggtctactat ccagagactg caagaactta aattagcaag caaaaagcaa 37980cccattaaaa aatgggcaaa ggacatgaac agacacttct taaaagaaga catacaagca 38040gccaacaagc atgaaaaaat actcaacatc actaatcaga gaaatgcaaa tcgaaaccac 38100aatgtgatac catctcacac tagtcagaat ggctattact aaaaagtcaa aaacaataga 38160tgctggcgag gctgcagaga aaaggctttt atacactgtt ggtgggaata taaattagtt 38220cagccactgt ggaaagcagt ttggagattt ctcaaagaac ttaaaacaga gctaccattt 38280gatccagtac ccaaaggaaa ataaatagat cattataacc aaaagacaca agcattcata 38340tgttcattgc cacactattc acaatagcaa agacatggaa tcaacctggg tgcccatcat 38400tggtggactg gataaagaaa atgtgatata cacatataca ccatggaata ctctgcagca 38460ttaaaaagaa taacatcatg ttctttgcag caacatggat aaagcttgga ggtcataatc 38520gaaagaaatt aatgtgggaa tagaaaaccg aatactgcat attctcactt ataagtgaga 38580gctaaatatt gagcacacag ggacataaac ataggaacaa tagacactgt ggactactaa 38640agtgaggagg gagggagatg tgtgttgaaa aactacatat tggaggctgg gcatggtggc 38700ttatgcctgt aataccagca ctttgggagg caaaggtggg aaggtcactt gagtctaagc 38760attcgaaatc agcttgggca acatcgtgaa actctgtctc tacataaaat acgagaatta 38820gccaggcatg gtggcacatg tctatggttc cagctaatta agtggctgag gtgggaggat 38880ggcttgagcc catcctcaag ccatcaaggg tgcagtgagc caagattgtg ccactatact 38940ccagcctggg tgacacagtg agaccctgtt taaaaaacaa aaacaaaact acctattgaa 39000tactatgttc actacctggt acaatatacc catgtaacaa tcctgcgagg atttagatac 39060aagttaccct cttatctaaa ataaaagctg aaattaaaac aaaacagaaa acagagtaag 39120ttttaaaaaa tattaaagaa tttttttttt aatatagaga agtgcaaaca cttacaaata 39180ggggaattta gctgtcatcc agcttcaaca atatcacctt tttgttaatc ttatttcttg 39240tatatccgga tccacttacc cttatatccc aaacatcata ttatttcatc tataaatatt 39300tcagcacata tctctaagta ataatatctt aaaaaacata aatattatgt agataaaaga 39360aacaataaca atttgtttgg tgatagattg aaggtggtaa caattatttg actctttcat 39420cacatggtgg ggtctatgtt cctatcctta aatcttggca ggctctgtgg ctacaatatg 39480gcagaagtga aattgtacca gttttggggg ctacacctta agatcttcca cttattgcct 39540cttagaatac tttctctggg agccttgata cacgtgtaat ttcaactacc ttgagacagc 39600tgtggagagg catgtgttag tgctccagcc aacagtccca gctgagctta gccttctagc 39660catatctgca aaggcaccag atgtatgggt gaagccatct tggatgatcc agaccagccc 39720atctgccacc tcagttgaca acacgcagag cagacaaatt gtccagccta ctcctgttgc 39780aattgttaac tcaacaaaat gtgagatata acaaaatgat tatagtttaa ttcactaagt 39840tttggggtaa tttgttaagc agaaataaat aactaaaatg gtaggctttt aaaatttgca 39900ttcccagtgt ttctggatga gcagtgcatc ttttgggagt gtgagttagc ctgtcgtcaa 39960aggtcttttt gaaattgtgt tgtcccattt gccatgaaaa tatttttgat tatttcctac 40020ctttgttctt aatatgattt attcaattat atatttatat aatgcaattt ttaataatga 40080ctattacagc atttttgaac atttaccagt tgacttttcc aagctggtat gatctgactc 40140tagcacgttt ctgatggaca gtccctcctc gatcagtttc tgattttacc tcaatactca 40200gctatggtag aagagttaaa tgacctagtt tgggagtcag agttcagtta acattttggt 40260tttgtgactt actggtagtg tgagcttgag taagtgactt agtgtttcat cccgttgcct 40320catgtgcaaa atggggatca tcataactac cttaaaagtt tgctgtagaa gattgtatga 40380gggaaatatg aacagaactt tgtacattgt cagctctaat atttttataa ttctttggtg 40440ttcttgtaaa atatatgggc tcacataggt agtaattcta ttgacattca cagtaataaa 40500aagaaaggta gcctttcttg agaaaattag ggagattttc cctagtcttt ggtccttgtt 40560ttgactgtac tggggactct gggctttaat cttcacagag aggtgtggtg ggtgtgggtc 40620gtggcaacat gaaggcttgt gcatactttt attcaaaaag gatgttagca gaaagaagca 40680ccaactttcc tagcatgagg attcctgaca tgcccatctt ctgtgacacg gttgagaaag 40740tatatgattt cccttttgcg gactggcaaa cagaagataa aagagattac aactttcccc 40800aggattatag tagcaaggct ggaaaaaaaa tggcagaaat attcagtaca gctttttgtt 40860agtggattcg ctccaatacc cagaccatga aggttcttta atttccccca aactatactc 40920ttaaatgagg cctatttcag gcagagacac taatccagcc ctgaggtgga atacttcctc 40980ctcagcgccc ttctttgatg tgtagctgat atggaactga gtgccaacat atgtttgtac 41040aaatatattg gcaaatgtaa gcagcctact gctaattact acactctgaa cctcatgtga 41100agtttgaatg atgcctcaag gaatttgaat gtaagaaaat aatatatata catatttttc 41160aaatcttagc aaaaaagggt taacttcttt atggttgaaa ctggaagaca ctcgagaatt 41220atcttgtctt ctcaaacctt tggcaataac atgtttattt aaggaactca ggtcaaacag 41280ttgctagaaa agattaagtg ttttggacac ttttaaaata aaaaagggcc gcttaaatta 41340aaaaaaccca aaattgctta atattttaca aatgaaaata agaacaatat aatttaaaca 41400gatgctatac aaaacaacag agtactatag gccaaaggtc acaaattaaa gaccatttat 41460gggctgagaa caaagctact gggttcaaaa cataaagcga atactgtggg aacttcagag 41520gggcttgccc tgcctaaagg tattcaaatt caaataattt tggagtacta tgctggctta 41580gcataattgg gaaggccaga tcaggctttt gtactcaacg ttgtggtcct tgatttagac 41640aatattggtt aaaaagatgg tactttaatc agtttactac atatttcaaa ctaaaacatt 41700atttttaaac atgaattata tgaacacatt tcttctacta attttgtaaa tattaagggt 41760tgattttaac taaaccaaat ggagggtttt tttttaactt ttagtgatgg atatatagaa 41820ttattctgtt gatcagcttc tgaacatctc aaattttaaa tatagaaatg ttatcagaat 41880acttcctttt taagaatacg aaatgtttgc attctgtgga gatgtggaga aaactaaagc 41940caaaccgaag gagtggctta atctctattt ctgtgatgaa ttgacttctg ctagtcagat 42000gggaaaaaat tagaatgttt aatcactgtg tttaattgaa aggaatttgt aatgattcaa 42060tttggttatc tctatatcta gatatttaga ctaatttttt tctaatttgg tttagttaca 42120ggggagatgt ctaaattgat aatatagaaa aaacaaaagc agtgttattt ttttaaaatg 42180taagtaacgt agtagaataa attcaactag ctaacccaat aattaaccaa ctaagtaatt 42240tgataaaaat ttggtttagt ttcttaggac acctgtatta atgaaaagaa aaggtcgatt 42300tgtaattagt acttaagtgt tgttatttac atggttgttt ctaaatttcc tagtgtgctt 42360caacttagaa tttcttctta aatattttaa ataattttaa ataatttcct gaattgttat 42420taaaaattca ctttattaaa tttctttttc cctgtagtca ggaagagtta aataaatact 42480attaagtatg tgaatgtact tggtaaattg tacataatta gaacattgtg agtgacacca 42540ccacctttga aatctttaag tatcccatgc tgtgatgccc atgtcttatt cttccagctc 42600aattgctcta gtacacccac tgcaacaatt cttcaatgtc atagagacct ccagtccagg 42660gaccctgtgt attccatcca tcagtttcct cccgtcttca tttcctttct tgttcagcgt 42720aatgtcatgg tccatcatta ttattactcc cttgctaatg ccctaaacct ccttgctcct 42780cttttccttc ttcacatttg actggcaaaa gctcaaccct gattgaaccc aaccatcgcc 42840ttttcatgaa cgctgtttgg ggaaaaaatt cacacacgga ggctgactga tggctaaatt 42900tttggtctca aacttcacat aggctcaacc aaaatctaga aattcactga ttttttttcc 42960tcctgttttt gggacaaaat tttacacatt gtaacagagc aaaagctgct ggtggtggtg 43020gtgctgaagc agtagggtag gagaaactta gatataaatg taatgaatgg agtcttaatt 43080ttagatccca ttgctttagg taacttttgg gtgttttgtc ttctagatct cttcactttc 43140tctagctcct cttcatggat gatatctaga catatggggc agctaactta attttatgat 43200ggtgggtaag gaggcctcag atatgcttaa taaactggtc agctgctgtg tttgcggagg 43260aggaggggtc ccctatcttt actgatagtt ggcccttggc attggtaccc actgaggtat 43320gtccgctccc ctgtggcctt ttggaggtct tgtgtgtgca gctgtttctg aaatttgcag 43380tcttagctgt ttgtcctggt tgcatagatc ttgtacttat gtgtcccttt tttttctggt 43440gccggatctc ttttggttaa ctagccctct catcaccttt atgtccactc atactccaca 43500caagctttaa gaagccagtg gcatatgggt gctattggtt ctgtgcccat tggacattgg 43560gttctgtccc agtgagggac ttctgctaat ggtttatatc tcttctaggc tctgcctgga 43620gaagcagtcg gctagctcta atgttcttgg gcggctctgt acctatcaga atgtttctac 43680accctcggta ctgaagctga atgctgagaa ggaggaaagg caaagaggtt gctgtacctc 43740ttgctcttct cctgtccccg ttattctacg gctgaattag gtggcttttt tccccttcct 43800atgtggcagg gacttttctt gtgtcctatc ttgagttctt cagactaata gtaaaattga 43860cggttaagcc agccggactg gcttttaata cattactgga agtttaagaa atttaaaaaa 43920tattttttct cttgacaaac cctgttttta aaatagatgg aattgctagg gattcggctg 43980gtgttaattg catttttcta ttcctgtgct atgtgtacta atcaccatcc tatgccccct 44040tcacctgctc tctaagtccc ctgtagatga atgcctctga gtagaaagaa ggtcacgtct 44100atagagatgc aaaagagaaa agcacattaa tatatatcaa aaattgttat ccttgttaca 44160ctactaagtt tacttttcca ttctatgaga tgactacttt gtctgcttgg tccttagagc 44220tctcattact cttccccctc tttcttaccc agttctcagc agaagacccc atctcatgaa 44280tcactgagaa aaattatagt cattggagaa ctctcactgc tgagtctatc cataatctgc 44340atctttttct tctttacaga aaagatgtaa gaagagtcaa ttctactgaa gaccaatccc 44400tccagctttg cttgcgattc tatttcctct tatcttctaa acatttcagt ccttcccttt 44460atcttttaca ttatgagtgt ctttttctac tgaattattc tcatcagtat aagaatatgc 44520tgtagtattc ctctttttta ttgtagaact taataaaact ttatttattt aatttaaaaa 44580tttttaatat ttgtgggtac ataataggtg tacatattaa tggggtacat gagatgtttt 44640gatacaggca tgaaagatga aataatcaca tcatggaaaa tggggtatcc atcccctcaa 44700gtattcctct tcttgaagga

aaaaaaactc accccatatc ttccccaccc tcaatttact 44760aattcctttc tctgccaccc ttcctggcta agttctttag aaaagtagtc tatatgctgt 44820caccctttct atgcagtttc aaaccactca aatatggctt tcttctctct accatttcac 44880tgaaatcact tgtgccaaat ccaatgttta tgattatgct cttgtcttac ttaatttccc 44940ggtagcattt agcatagctg attattttct cttttgggaa aaaaatttcg atgtctctta 45000gcttccacac tcttctggct ttcttcttgt ctcattagca actcaatctc attctttact 45060ggcttttcct ctttttttgt aattttaatt ttatttaaat tattttcaat ttcaatagtt 45120tttggggaag aggtggtgtt tggttacatg gatacattct ttagtggtga tttctgagat 45180tctggtgcac ccgtcacctg agcagtgtac atggtagcca tcgtgtactc ttttaaaccg 45240caaccccacc catcctttcc cccaagtccc cagagtccat tatatcattc ttacgccttt 45300gcatcctcat agcttagctc ctatttacaa gtgaaaacat acgatgtttg ttctcccatt 45360cctgagttac ttcacttaga ataatgctct ccaatttcat ccaagttgat gtgaattcca 45420ctattttatt cctttttatg gctgagtagt attttatggt atatttacat atattatata 45480tatatgtata tatacacata attttcttta tccactggtt aattgcttga tgggcattta 45540ggctggttcc atatttttgc aattgggaat tgtgctgcta caaacatgca tgtgcaagtg 45600tcttttttat ataatgattt ctttttctgt gggtagaacc agtattggga ttgctggatc 45660aaatggtagt tctactctta gttctttaag gaatctccac actgttctcc atagtggtcg 45720taccagttta cattttctcc agcagtgtaa aagtgttcct ttcttaccac atccatgcta 45780acatctatta ctttttgatt tttaaattat gccattcttg caggagtaag atggtattgc 45840attgtgattt tgacttgcat ttccttgatc attagtgatg ttgagcattt tttcatatgt 45900gtttggccat ttgtctatct tcttttgaaa attgtctgtt catgtcctta gcccactttt 45960tgatgggatt atttgttttt tttccttgat gatttgtttg agttccttgt agattctgga 46020tactaatcct ttgtcagatg catagtttgt gaatatattc tcccactctg tgggttgtct 46080gtttactctg ctgattattt cttttgccgt acagaagctt tttagtttaa ttaagtccca 46140cttatttatc tttatttttg ttgcatttgt ttttgggttc ttggtcatga actctttgcc 46200taagccaatg tctagaagag tttttctgat gttatctcct agaattttta tggtttcagg 46260tcttatattt aagcctttga ttcatcttga gttgattttt gtataaggtg agagatgagg 46320atccagtttt attcttctac gtgtggcttg ccaattatcg cagcaccatt tgttgaacag 46380ggtgacaggg tgtcttttcc tcactttact tttttgtttg ctttgttgaa gattacttgg 46440ctgtaagtat ttgactttgt ttctgagttc tctattctgt ttcattggtc tacatgtgtg 46500cttttatact agtaccatgc tgttttggta actatagcct tgtagtagta tagtttgaag 46560tcaggtaaag tgatgcctcc agatttcttc tttttgctta gtcttgcttt ggctatgtgg 46620gccctttgtt tggttccata tgaattttag agttgttttt ttctagttct gtgaagaatg 46680atgatggtat tttgatggga attgcattga atttatagat tgtttttggc agtatggtca 46740tcttcatgtt attgattcta cctatccatg agcatggtat gtgtttccat ttgttagtgt 46800catctattgt ttctttcaac agtgttttgt agttttcctt gtagagatct ttgacgtctt 46860tcgttaggta tattcctaag tatttttatt tttttgcagc cattgtaaaa ggggttgagt 46920tcttgatttg attctcatct tgattcctgt tggtgtatag cagtgttact gatttatgta 46980tgttgatttt gtatcctgaa actttgctga attcatttgt cagatctagg agctttttgg 47040atgagtcttt agggttttct tcctatagga ccatatcatc agtgaatagc aacagtttga 47100cttcctcttt accaatttgg atgcccttta tttctttctc ttgtctgatt gttcaggcta 47160ggacttccag tactatgttg actagaaatg gtaaaagtgg gcatccttgt cttgttccac 47220ttctcagagg gagtgctttc aacttttcct cattcagtat aatgttggct atgggtttgt 47280catagatggc ttttgttacc ttaaagtatg tcctttcttt gctgattttg ctgaggattt 47340taatcataaa gggatgctgg attttgtcaa atgctctttt ctgcatctat tacgatgatc 47400atattatttt tgtttttaat tctgtttaat gtagtgtatc acatttattg acttgcatat 47460gttaagccat ccctgcctcc ctggtatgaa acccacttga ccatggctta ttagcttttt 47520gatatggtgt tggatttggt tggctagtat tttgttgagg atttttgcat ttatgtttat 47580cagggatatt ggtttgtagt tttctttttt tgttatgacc tttcctggtt ttggtattag 47640ggtgatactg gcttcataga ataatttagg gaggattccc tttttcttta tcttttggaa 47700tagtgtcaat agggttggta ccaactcttc ttcgaatatc tgatagaatt cagctgtgaa 47760tccatctggt cctgggcttt ttttgttggc aattttttta ttactgcttc aattctcact 47820acttcttatt ggtctgttca gagtttctat ttctgtctgg tttaatctag gaaggttgaa 47880tatttccagg aatttaccca tttcctctag gttttctagt ttgtgtgcat aacagtgttc 47940atagtagcct tgaataatct tttgtatttc tgtagtattg gttgtaatat ctcccatttc 48000atggcttttc ctcttaactc aaactctaaa tgttggccat cttcagggct tagtccagcc 48060tttatttttt tttaaatatt ttttccaaag ttccctctcc caactttatg actttaaata 48120ccacataatt agaattttcc acatttgtat ctcctcagac ttctcgtctg agctatggac 48180tcaaacattc aaactatctt cttgatgtct ctgtttgaag gaatcacagc catcttaaac 48240ttaacatggt taacacaatt attgatttcc tgtctctcat tccccagtaa tcctcttcag 48300tcttccatat cttagtaata gtaccatcat cctctaggtc cttaatctgt aaatctagta 48360gtctttctgg atttcccagc tacttctttt cagtgtcttg catggttact ttttttgatg 48420tattctttaa acagtggtgt tttggcttag catctactca cttttcattt gaatttttct 48480ccctaatcaa ttggatctac ttctatggct tcattgtcat catatgctga tgactttctg 48540aatttatctc taaccttgga tttgcttctg aaattgtggc ccttatattt aaatacttag 48600tcaacatctt tatttcgatg ttccacagga aacctcaaat tttcctatcc agaatggaat 48660ttgctacatt cccaataact catgcttcct tcttgtcccc tctctcagtg aataatatga 48720gcatctaata gagtcacaag ctagaatcct ggataccact cttggtaagt tgtccctttt 48780tcattcacat taaatccatc aggttttgtc acctcgatgt ctaaaatatg gcacaaattt 48840gttaactcat cttcctttct catgaaacta ccttggaaca ggtctttcta atttttgtct 48900agactgttgc agcagtctcc tcattttcat ttttactcac ttccattcta ttcttcaaga 48960agtcagaatg atttctgaga aagcaaataa gaccatgtca ttgtcctgtg taaagccatt 49020tattttaata ttatgcttca taaatctgca atcattttgt ttatccattt gtttatctat 49080gattgtcatc cctcattggg attcatgctc catgtgaaga aggttgtgtc tgcttatctt 49140ttgtttctta agcatcttgc acagtttccc aagcaccatt tcttaatgat aatagtaatg 49200atgaaaaaca acaaagggac tcagtaatca acatagaggt ctggtcacca gaaatcccaa 49260aatagtgcag ttcagttttg tactaaaatg acattttagt gaatttaaac taagacattt 49320tagtaaataa atctagtatg gaattccatt tttcagcttt gttttcaagg gattgtgaat 49380ctaggcacat gagtgagtag ttttccctgt tactctatat gaggcctaca gcttgctcaa 49440gcaagttcag gaccacagct aggccctttg aagaaatcca tatgtgtaga actctaacca 49500ggtcagacgt aagttacaga tattcttaaa gtgacttttt gtctttctgg ctggatatga 49560tactgtagtt tctgtaaaac aaaggaggac catttagttt caaaaataca atatgggtaa 49620atacaatata gataacttgg taataaaagg attttaattt aatttaggta tttatgttta 49680gataccagct actagtaaaa taccacctcc ccccacaaca aacacatata ctctagtagt 49740ttctaatgct tatagaaaaa tggacttaat gatttttgga ttaatctttc ttaattttca 49800tttgcttttc aatcttcttt gggaaaaggg taacctgtgt ctcaactttt tatgatgatc 49860aatttacaca tcatatatat atatattttt ggatatatac cctaatggga ttgctcggct 49920gattggtagt tctaagttct ttgagaaatc tccaaactgc tttccacact ggccaaacta 49980atttacattc ccaccagcaa tgtatataca catcataatt tgaatatgct atgttccaga 50040ttagttgcca gcaagaaggg gtgagatggg ggctaagaaa aagatgatag caatataaaa 50100agagatttga aataaatata tatatcagtt taaaaatata tatttattct gggtgatact 50160gatgcgtcaa tgtgagttca tcaattgtgt ggtaacaatg tatcacttcg gtgggggatg 50220ttggtaatgg ggaggcccac acatgagtgg aagcaaggga tatgtgggaa atctctgtat 50280cttccactca tctttgctat gaacctaaga ctgctctaca gaataaagtc tattaaaaaa 50340tatatatctt accagtggca ggggtgtggg tagagtggag agtggggcta gggagtttca 50400gggcataagg gttaagccgt gtcttcattc agatacccca tggaaccact gaagggcttt 50460cccaggtatc ctacagtagc atggcataca ctcaaggtta gtggagagaa ctcttaactg 50520gtaaaatgtg tgaattctgt gtttctaggc aattatgaac accaggtaaa acaatgtcag 50580ttctttaacc tcagaacctt tgcggagtgg tcagctaaag tgaactagac agtgactcag 50640gaaatctaag catttctgtt ttttttttct tcattatttt tgtgtcagaa acttggtggt 50700taattgtgta gttaacccgt ggctggctgt agagggattc tattctctaa atgcaatccc 50760atataaaatg agtcttacca gcttataaaa gaaaaagttc cctatatcat taatttttat 50820aagtgtaatc acatcagaaa tatactaatg gagttcttta ttgaaagaaa acatatataa 50880gaatcttctt aatataagaa taatatctgc tccatcaact gcatctcaaa atttgtgcta 50940ggatttagca agttgtacaa agaacacaag cctttatctc aataggtctg ggttcttttc 51000ccaattgtca ctggctgtgt gacatcaaga aaaactccta acattgcagg ggcacttttc 51060ttatctctag aaagagttgc actagatgat gcccaagaat ttctctggct ttagaatttt 51120ctgtttcatc ttccaaactt ggaaattcat tgattttgat gtttttatgg ttgcatgctc 51180atattatata accaagatga ctgtgtgcta aattaatgct aatccccagg actaaataaa 51240agttatttga aaactgtggt gcattgattg ccatctctta atgaaacttc cgggaccttg 51300attaacctag atgttcgttt ttgttgttgg agaatctggc tgttggtatg gtatcaatat 51360gcatggcaat aactttagaa ttattggtga gataagaaat agtttcaaaa tccatgtaac 51420tcctcttgtg cagtgaaagg aatactaaaa ttttaaagat acattttgaa ttttggcctt 51480acctttttaa tgactctgtg acattgctaa gtcacttaat ttctctgata tttaattttt 51540tgcgtctttg aagaaaggga taatgcctgt tctgcataca ttataagtcg actataagtg 51600aaaatagttt tataggagaa ttctctttga gttattgcaa aatgcaaaac attctaaaga 51660ttgtagttta atcttttgcc atggcaaaga taaattactt ccaactgcta attttgggaa 51720gcgagcaata atattttatg aatgtctctc tgggttggag acctgcagca tttgtaggaa 51780atcacataca ggcggcatca tttgataatt ctgctatcct gatcctgctt actgcaatat 51840ccatcagtag agtaagggta tgtctgagga atactgtatg gcactctgca ctcttcatag 51900aagtgattac ttgttgaaac aaaggctatg gcagacacag tatcatcaat cttctgactt 51960ggtacttcat ttctttgggt ctagtctttt ttatcctggc tctagcactt actggcatgt 52020gaacttgtaa catgctacct aaccctgatg agcttataaa aggaaaagtt gtatagatca 52080ttaattttta taaactaggc accagttttc ctctccctca aaaatggtag ctattgtatt 52140tgctactctt acattattat tctctaagtg cagagatttg caggcatata catcctaaag 52200aaagggtatt aaaagtccca aagtttataa atcactgctt tccccaaatc ctctattgcc 52260aatgggcatt attatttagg attagagatt actgagaata acagagaccc aaaatatact 52320gtcttaagta aaattgaaat ttatttctct gacactgaaa ggtttgtaga taagtagttt 52380aaggctgatg tggtgggtca gttccacaga gtcctcaggg ccccagctcc gtctattgtg 52440ttgctctttt gtgtgtggct cctaatttac caaggtcatc tcatggctca acatagttgc 52500tctagctcca gccatcaagt ccacattcta gctagcagaa aagaggaagg gacaacagaa 52560aaggaaaatg gtgtgcatta tttaaagaag gtccccagaa tgttgtataa aatttgtcca 52620ctggatgaaa atgtatatac ttaccaacaa aggaggttgt gacatatagt ctgtccatgt 52680tcatgaggaa gaggagaaca aatattggga aaaagctagc ttcttagaca caagaacaat 52740gacagaggca tttaaaaatg tgtcccagat aattgagaaa aaaaccagca aaaaaaggaa 52800ggggagtgaa cccgggaggc ggagcttgca gtgagccgag attgcgccac tgcagtccag 52860cctgggcgac agagcaagac tctgtctcaa aaaaaaaaaa aaaaaaagga aggggatata 52920tgtagctatg ttagtctgag ttaggtggtt attatgagga ttttgttgtt gttgttgtta 52980catgtaatta tcaaacatac cgaagcctaa tttgcagtct tgagcttttg tttttttagc 53040tgttattgag aggaaaacaa tctcctggat tcttttctca tcctggaagt atggatgcct 53100tctctagtaa gtaagtttat tttgctttca gtgaagcata tgtattacct ttaaaaaaac 53160attaattata ttctgttatt tttttgacta gaaaaccata tactttaaga aggatttaat 53220aacttggtcc ctgggtgtca atcaataaag cacgtgaata accagtgcaa agtggtcact 53280cggctgccca aggggaggtc atccaagaat gaaatcagtg taacaacagc tgaattgcat 53340ccttcatcaa atgcatgtaa aacagaccag aaagacatca attcccaatg ctggtatggg 53400tggtctttct actgcatgta taatttccaa tttaacaaag gttaatacca tgttgtaatt 53460tgaaatgtca aaataataaa tcctatgtgg gtaaactaga tggtggtttt aaaatttata 53520atgaatattc agttgaaata gtaaagtcat ttaaatcaca tcagcagagt ttttctcttt 53580ttcaaactag tgatacacac acacatatat atatatatat atatatatgt gtgtgtgtgt 53640gtgtgtgtgt gtatcactag tttataccag aatagatata tattctgggt aacagctttt 53700atgggtctga tatcagatca gataactaac atctgatacc ttgtctttta ggtcagcatg 53760gcccagtatg attggaggca atgacatatt attaagtaaa aaattctaga tagagtgttt 53820ccttttgaca gcttttaatc acctgttact ttctttcagt tttttattcc ttgaggtaac 53880ttattttaga ggctagtttg aatatttaat ctgttcttgt cttttactgt gaatttcctt 53940ctaaatttta attcacaata ttattcatat taaatctgtt cataagataa aatgtgttag 54000taatttcgtc tttttaaaat tgaatttatt ttaataattt tctttaatca aaaaattaaa 54060attccctcct aggaacaagg cacaaaaggc tcaagagctt caatttactt ctcttcttct 54120tctttttttt ttgtcttgtt tttttgtttt gtttatctta ttttacttta agttccagga 54180tacatgtgca gaacctgcag gtttgttaca taggtaaacg tgtgccatgg tggtttaatc 54240cccacatgca ttagctattt gtcctgctgc tctccctccc ctccccaccc cgacaggccc 54300tggtgtgtgt tgttgccctc cgtgtgtcca tgtgttctcg ctgttcagct tccacttatg 54360agagagaaca tacttctctt ctttacctat ggtaaattca cttttgatgg gtgtgcttgt 54420caatactgaa aatgttctcc attgctttta aaaaagaaat agtcacatca gcaaaatggc 54480tacctttcat tttgggtatt tcattactct tttttggata cctgttagtt taattcattt 54540aaacaatcat gtattgaaag gctacttggt tccaggtgtt tgccaagatt gggaaatata 54600aacatgagtg aagtatggtt cctgaccttg agaagttcag agtctagagt ctgaactttc 54660taatttgaat tgcttttaga aaacaaaaat agaatatcaa tcattctggg agatttaaat 54720gatggattta agataagaaa tgggtaattt aaacataata gaaagcaatg tagagatctg 54780ttttgtttta aatctctatc cttgtattga acttgcatgt gagttttcca tctctctata 54840ttactaatgt tccagaagaa gccgaccttg gcaacaaaaa gatcacttta ccaccaatat 54900attacaaaga ctgatttgga tgcaacttag aacctgtgaa aatctagtcc tcttttattt 54960actcaaagat cctttccatt ttacggtact ttgcctataa gaatagtgtt aaaattagat 55020gtgatatcca aatcttctga tttgggtagt ggtgcatttt cacccaaagc taaattgcaa 55080agaaagtgga caatgatcaa gcatagatat tgatcccata tttcttaacc tttatgaatc 55140tattatcttc caaaaatcca tttaggattt agatagaaag caggatttcc ttttgactct 55200acagtgtata aaacacagaa catctgagaa acagaaacat ccaaccttaa tcatgttcca 55260ctttaagtga aggtaaaagt aataaagttg tgttggtaca aatctttaat agaattttta 55320tgtaaatagg ggattcttat gttactttaa tgcaaaatag aaagaaaaca aatgaaattt 55380tatatgaaaa ttaaaaacct aagagtcaaa aggtagatgt atgtagaatc ctccacagtc 55440ggcctatcaa tgaattcctt tctcattttt ctcttggtct gcattcagca gtgtcatctt 55500ttctttcttt ctttactctc ccctttcttc tcctttgctt ccttctcagt ttcttgctcc 55560ctttctgcca gctttttgaa aatgccatac tgtaccccac ctttccctgg ttggactcct 55620ctgacttctg agagttcctc tggtaggaag tagtccagtg attgcctgtt gacttcacaa 55680ggaagagttc ttaaatatgc tgttttctta gcagcttgga gaaaagcaag tgaatggctt 55740tcatcttaga gggaaatctg tagggacaat gtgggtgcac ttccttgcat agagctaccg 55800tttgcactgc atgggctctg aggttctttt acttcccttt tcatacctct ttaagagagg 55860cccctagaga accaactctc tgatttttgt gcttgggaag ctcttggaaa actgatgttt 55920ttctccctga tgtaaagtca gtagtcttag gactgaaagc ttagattcca gctttgagct 55980ctgatttatt gttctctgat gtatgtggat ccagctagga ctttaatact tacttcagtt 56040catttcctgc attgatttct caggtaatct caagatgccc tataaggtcc ttgaaggctc 56100tgaaacccct gacgtcccag gcctatgcca ggtttgccct aaggaccttg gtcaatttcc 56160tttacaacca catcccagcc cctacactcc tgatcttggt cgtccttgta ttacttggta 56220atgctttctt tctattttat ttggacttgg cccttcagat gtcccagtgg ggtctcagca 56280tgttctacca ctggctaata aatgaggtgc tcagtgtagt gggaaaggtg taggttatga 56340aatcacacaa acttgtgttt gtgtcctagc tctaaacctt tctccctatg taatgtggac 56400aattcactat tttttttttt tttttgagat ggagtctcgc tttgttgccc aggctggagt 56460gcagtggcgt aatcttggct cactgcaagc tccacttcct aggttcacgc cattctcctg 56520cctcagcctc ccaagtggcc gggattacag gtgcccacca ccacgcccgg ctaatttttt 56580gtatttttag tagagatggg gtttcaccat gttagccagg atggtctcaa tctcctgacc 56640tcgtgaccca cccgccttgg cctcccaaag tgctgggatt acagacatga gccaccgcgc 56700ctggccaaca attcactatt taaccttagc ttccttagcc ataaaatggg cataataatt 56760tcaatcccct gggaattgtt gtgagaatta tatgaaatat tcacgtaaat cacctgcccc 56820tttgtctgaa agcagtggga gctcaatcaa gttgattccc tttctctcct ctgtatgtat 56880gcatgagtgt atgtgtacaa acatgcgagt atagatgtat gcgtgtgtgt atgcatacaa 56940gcatgcatgt atgcatgtta tcctgatgcc gcttttgtgt tctttttctg tttttctctc 57000ctgcaagaca tgtactctga cagttttctg tattttctgg ttgtttctga gatttaagaa 57060aacaagccat ttctgcagaa acccgcaaca aagggtaggt tgatttacat cagtatttgg 57120tcctaccaac tcttccagaa tagaactatg ttcccttttt atgctctaca actctggaga 57180taaagaaaca catgaaaaat aaccctagtt ctcaattcct ttagttattt ttcctggtat 57240ccattccctt aatatttcca aatggtaggt acttcttgat gtatctattt agacaccact 57300tattaactta cacttaacta taagatgagg agttaacttt tttcaactac cccacactcc 57360ccttcacagt ctatcacagg ttggggtaca aactactcaa tgttttcagt attatgactt 57420tgtagatgtt cactgaagtg aactagtaga gtactatgat aatttattct ttcttgaaca 57480atatattttc ccctggggtt aatatttcct ggaatgaaaa acattttctg tttaatttgt 57540ttaattttta atatatctac taaaagtcct tcccagatac agagatttgt tcatcagatc 57600aaagcctcct attaccccac aattaaatga atcagttaat atactgctgg cacacctcca 57660tttgttttca gtcttccaaa aatgtgttga taattcccac ccactgtttc ttctttctca 57720ctcgttcatt cctatgccat taaaaaaatt cctttattca ttaacatgta tgtttctgag 57780caggactgtg gggaggactg tggctggagt gggcatgttc agctggcttc cggggtttgc 57840tcttcgtgtg gacaacagtg ctggtggctg agctccacat cggaggcctg gcacaggtca 57900atctgtctca atgataatgc tgtgggacaa ggcttttgca gtgaactgaa gttctatccc 57960attaactccc atctactttt tctcttgtta tttagcatta tggagactat ttactttttg 58020tactcatttc ctgttgctgc tgcaataaaa atgaccacaa atttagtctt ttgaaacaac 58080cgcaattttt tatcttattg ttctggaggt cagaagttta aaaagggtgg gcagagctat 58140gtttcttctg agtgctctag gacaaatccg tttctttggt ctttccagct tttgaggcca 58200cttatattcc tcggctcatg gccccacatc actctaactt ctacttttat tgtgacaact 58260cctttcctga ctctgatctt ctgtcttcct ctttcatctt ttaaggaccc ttgtgattac 58320attgggtcca catggatatc cattgtccca tttggataat atggataatc cattgtccca 58380tttgaatata cttaacttag tcacacctgt aaagttcctt ctgccatgca agataactta 58440ttcacaggtt tccaggattg ggatgtggac atctttgggg gccattaatt ctgcttgcca 58500caatcttctg tactttacaa acatttgaac aaacgttatg tcttctgata tggtttggct 58560ctgtgtcaaa tctcatctct aattgtaatt gtcatgtgtc gagggaggga cctgtaatcc 58620ccatgtgtca ggggagggaa gtgatgggat catgggggcg atttacccta tgctcttctt 58680gtgatggtga gtgagttctc tcacgagatg tgacggtttt ataagcactg gcatttcgct 58740gcttgcactc actctctcct gtagcattgt gaagaagatg cctgcttccc cttttgcctt 58800ccatcatgat tttaagttcc ttggggcctc ccctgcaacg cagaactgtg agtcaattaa 58860accttttttt ttctttatga attacccagt ctcaggtatt tctttatagc agtgtgaaaa 58920ctgactaata aatctcctct aaaacttctt tttaggttat cataagaatt gttttcactt 58980gcttggtaat tgatgtggct tccagattca tcaccatcct tattgcctcc atctgctttt 59040actccacttt accaatgtct tttttaagta tcccaaactg aacatagata tgatcagact 59100caggaagatt caatgaagac aacatttccc tggttctgga cacctggctt ataaaacagc 59160tcaagaattc attattttcc tgggaacatt tttgactaat atcgatcttt tcaccaacta 59220taatccctca gttttttttc atgtaaagta ctggtgaggt ttttcccatc ctgtatttct 59280gtaggattgt taagttttaa aaatattaat gcgctatatt caagtcctat taggggtcag 59340aaactggaca ttgttacact taggtaagcc cagtgtcagc tgcctacatg tcctcagttc 59400ttcagctctc tggggtcaca ttttaaattt aaacaaatta ccatgggtcc tatctcaaga 59460gtggaggaac tcacatcttc cctcttaata tgttcaaggc ttcttgggga agttggcact 59520atgactggtc atctctggct aatcatctat ttctactgag ctctcaactc ccaatctgca 59580tcctatgttc cacgccactg tcactttttt gcattttgtt gtcctaggct ggatacctca 59640atttctatct ccaccaaagt tctaccaacc caggtttata tatgtagact tttactgaag 59700gtctgaagtt atgaaaagag atagcatgca gcttagatag taacaaaaca aaacaaaaat 59760gaatattaga cctcagaagt

atctttatca ttcttgcctc tgtgaactcc tcccagggcc 59820cattcctggc acagagccca ggttgacatc aggctggtgg attcctcccc ttctgctctc 59880tcaggctttc agggccaacc ttcccttctt tattttctgc tgcagagcca ggtgtcctca 59940ttggagactt cacctttttg ggggtaaaga aactatgaag gtatttggct gaacagaaat 60000agcttttcta cctataaagc aggggcgtac aatcttttgt cttccctggg cagcattgga 60060agaagaaaaa ttgtcttggg ccacacataa agtacactaa tgctgacagt agctgataag 60120ctaaaaaaaa aaaaatcaca caaaaaaaat ctcataatgt tttaagaaag tttacaaatt 60180tgtgttgggc tgtgtgtcag aaaagacttg agtcttgcta taaagagttc catcccagga 60240ttttaacata agaaaaaggt tcaacctttt ttccatttat ctctgtgaaa aattcatgtc 60300cttatgctag tttattataa tttttgaaag gcgattccac tgcacagtgt ttttgttctc 60360actcttagca ttctcatctt tttaaagcca caaatttcat agatattttt ctacattttc 60420ctgtaattat ttaaaattaa gcggttaatt ttaaataatt aactaatttt aaataaaata 60480attaatctta aaataggata gagaataaag agcaattcta taaactttac ttggtgttgg 60540tactagtacc tcaataaaaa ttttggcaaa aatgccacta cttttgtggc acaaggatag 60600aatgagaact tatgcatgtc aaatgccatg ctggattgaa gatatgcttt gctgaagaga 60660ttttctctta ggatttgaag agattttctc ttaggacaat tttatgttct tggtgttctc 60720tttttccttc taagatatgt ttttaacaat aaattataca tttttaaaga tttttttccc 60780tttaaaaaca aggcaattta ggctattata tttgtcaggg atcagagatg ttgactcccc 60840tgcaatgtaa actttcatga attagtttaa gaattaagac taaggcctgg tgcagtagct 60900cgcacctgta gtcccagtat tttggaggcc aatgctggca gattgcttga gtccaggaat 60960tcgagaccat ccttggccac atggtgcaac cccgtctcta caaaaaaata caaaaaaatt 61020agctggtgtg gtggtgcacc ctgtagtctc agctatgcag gaggctgaag tgggaggatc 61080acctgagtcc aggaagttga gggcgcaagt gagctgtgat ggcgccactg cactctagcc 61140cgggtggcag aatgagaccc tgtctcaaaa aaaaaaaaaa agaaactcaa ggctactatt 61200tattgcaaaa aagaaaagtg aagtgcagtt cattttttgt atctgtagct ataattttta 61260tataacatca aagtttaact ttctaatatg acctgagtag ctattttatg tgttctgtta 61320ttacttgaat tgctcacagg tctcagtaag ttaatacata aaatattaaa tgcagattca 61380aaatttggtc tgctcttgac tatgcagtgg aaatttataa cattttcttt catacaccct 61440agaattgaaa tggacatagc atctttaact ctatgagagc ttataaactc cttcgttaca 61500cagaactatt aatatatatt ttatatttat tatattttat gaaaataaca tatacatata 61560tttaaaaact tcatatttta caaaaagcaa ttattataaa ttatcagtct caaaataaag 61620attacaatgt gttactctga atttccagct acatgaataa tctagggtac ctgcaaggtt 61680ttgcttccca caatgtgcat cctacagttc ttggacccac agaagtcagc ctgtataaga 61740actgtgggat caaaatctga ccaaatacca tgaagctaaa gatatcgccc acaaaagtgg 61800tttttctctc tttcactaag aagaggaaag gctgtttgtg tttaagtgca tcgtgggggt 61860taaactgtgt cctcctaaaa cccccagtac tccagaatgt gaccttattt ggaagtaagt 61920gaggtcagta aggtggtgta ttggtcaact ggtatattcc aatacgactg gtgtccttat 61980aaaaaagaag aatttatttt tatttatttt ttttcaagac aaggtctcag tctgtcaccc 62040aggctggaat gcagtgatgc catcacaact tgactcactg cacccttgac ctcctgggct 62100gaagcgatcc tcttacctca gcctctgaat agctggaact aaaggtgcac gtcaccacac 62160ccagcttatt aattattatt attttttttt ggtagaggca gggtctcact attttgccca 62220ggctggtctc aaactcctgg actcaagtgg ttctcctgcc ttggcctccc aaagtgcttg 62280tattacaggc atgagccact acgcctggca aaagggaaaa tttacacaca gagacagaca 62340cacacacagg gagaatgcca tgtgaacaag aaggtagaga tcagtgtcat gtgtctataa 62400tctaaggaat gccaaagatt gccagcaact ccctagaaga tagaggacag gtctggggca 62460gatccttata gccctcagaa ggaatcagtc ctcccaacac tttgatcttg gactgctagg 62520ttctagaact gtgagataac acatttctaa agccacccac tttgctaaaa tgaagacttt 62580attaataatt tcttaactcc ctgatctatt tctaaactaa aatatggtgg gaaaacactt 62640tatttagggg gaaattttct tactacagga agaccgttgg tcaatattaa ttaacatatt 62700tttgtcaaat aaaccattcc ttttaaatgc caaatatcca tctggaaaag atacttctgt 62760gtcaggaagt caaattgacg tttaccaatg agtcttgtgg ttcttcttgg aagaactgtc 62820ttcttgtgaa tgaactgaat gtttatgaat gtggaccaga tccagaaatc agaaacgaat 62880gccctgggaa gactggggca gcaccgactc ccacaatccg ttccaccaat tcaccctacg 62940acacaaacta aacagcaagc aaacaaaatc ctctttggac tccacatgtt cccccctctg 63000ttctgttgca taaatagatt tttttataca aactatctat acacactctg cccgcttcct 63060cttcttcctt tcattcttca acgttttcca atctggcttt atccctggca ttcaattgaa 63120actgcccttg ccaagggtct aagactccat gccagcaaca tttccccctc atcttactcc 63180tcccattggt agcatttggc actgttaacc atttgctcct tgctggaatg cctctctggc 63240ttctgtgata ccatactccc cgggtcttca ccatatctta ctgaacattc ctctgtcttt 63300gctggctctt cctgctctat ctgttcttta tttgttgttc ctttgagctt tgccgtaggt 63360cctcctttct tttctgtttc tactgtctcc ctgggctgtc tcattcaatc tcatggattt 63420aaatattata tataggctga tgaattttct gagttccaga cttttatatc caactaccta 63480tctactcgac gtctgtattt gtaaatgtca ttgtatcttg taaacaacat cttccaaact 63540gaattcttta ttcccatgca accaagcctt tgtttcttcc aacactttat catctcagta 63600aaatccacca gaagtctaca agtcagccct tattcctctc ttcccctaat ttaccatcca 63660tttctctcca tctgcattcc tgctactata gcccaggcta ccatcattta atatggaact 63720aatttaaaca gcctctatct aatcactctt ctgcccctct tgccttcccc tccctcatta 63780attctccaca tagtatccag aaatatctgt atatatgaaa ataggatcat ttaattctcc 63840tgcttaaaac tcttctatga attcctattg cacttagatt aaaatccaga cctattctag 63900tgaccttcaa ggccctctgt aaccatgtcc ctgcctccct ctctacttgt agaacagcat 63960cctgggaccc ccattcctta ctcactgctt tccatcatac aattggcttg aatttcttga 64020atagaccaag gctttttctg tgtcaggaca ttttcacatc ttccttctgt caaaaatgtt 64080aacctaataa catggtataa ggaaagacat agttaaatca tattgtggac tgggccactt 64140gggaagctct ttttgtggca gggaagttgg gatgctctcc tggtccagtc atttggggtt 64200cagtgaagtg gagtcggcca ggtggaggat ttctaatcta tacatgtgta tggactgaaa 64260ctatagctgg tgggtggacc tgcagcatta aagagtcctc ataagggtga tttcatgcag 64320ataattattc cttggaaaga ggatatggtc aaaattcttt ccacttaaac cattgattcc 64380tttgagtttt atacccattc tgaggagaat tgatttagta acatataggt gaaaaagaca 64440tgctagaagc tgctttgcat gcaaaataat aagtgaaaat gaattgtatt aattgatgtc 64500cctgagatta ccctttgctt ttttaaaaaa ataatgcaac ttttaaatat atattttcca 64560atatcatagc ttttatttca gaccttttct tttgtgatca tattctcatg tcctaaactt 64620tagtcatttg catttcatct ttaggatttt tgaaatattt ctgtctcatt tgttttagtg 64680tgtatgtgtg agagtgtgtg tgtatttcta acttaatatt aacatctgga aaagcatgag 64740tttgatgttt taagttacac ctttctattt ataacaaaat gcataaataa taccataaat 64800tatttatcta ggtaccatgt aaaattgtga ttcatcagta tgtaagtacc actttttgaa 64860aaagctatta tataccatcc ttaaacttag ccctacttag ctgacataat ttgacaacac 64920atttacctca tttaattttg tcttatttaa gctaagatta aaaggaatta gtattttctg 64980agcatgcacc agttgaagac atcaaagata ctatggaaag catgaagcca ggctgcaatt 65040aaaattttgc cttatgtaac agaaaagatg taaaatatac cagtgaataa taaaggaatg 65100tagtttacca agttatatct atctctctat atttatgttt ggagttttgc ccttattgat 65160gaccctgtat ggaaaagact ggtcaatcct acttagcatt aatcctttgc acggggagac 65220tactcttgga gactgagtgt attttcagtc tttacaaatg atgaactatt gaatagatca 65280cacacacatg cagatgaacc caatttcatg tctaacagca tatattcatt ttatttcata 65340tattaatcta tatataaaat atgaaagttg cttttcaata gaatgatgga tggctgatga 65400tctatttatt attccttagt cttccttttg atgaactaca cctattcact actcagacaa 65460catagttcgg ttgggacctc tggtaataga acagatcaaa gttaattgga ctacatggga 65520attgaattga agatcgactt tgacctcatt agcacaatga tatagtcagc taaggttgta 65580agatccaaca ctcatggaaa cactcaaagg aaaaactcat ttcaaaggga agttggatgt 65640atggttaata taccatgcag ctttgtagat gaaagattaa aaaagaatta tagaactatc 65700agagagcaga tcagaaagca ggatttttat ctttgcatat atttgactca gtttcccaac 65760taatactaag tgttgggaaa atacttagga agcagggcta attcatcatg gtattagaag 65820atacggtcag acagaatgac actgcaaact gtcgtaatca ctctacaagc atcattgaaa 65880ctatgctaat tgcatctgta ccagtcacat aatgagaatg gccaatgcta tgcagtggat 65940taagggccaa ttgtaagtgg agaaggtaag aaaaccctgc agaggcatat ttaagttatg 66000cacattacca taatattatc acagaatgga aataaatagc aagccaacct tttctgaatg 66060cagtaaccag atactctggc tattgttgtt tgacatggat atttaacttc agcaaaaaga 66120ctatgttaaa tggtaaagtt aggaggaata ggatttccaa agcaacatgc tatagggggt 66180aatggaggct gcctttattt aggaacaaga tacaagtgta tagaagtctg tggagtgtat 66240ttggccacat ttgcagacac aggaaatagc atgagtgcag agtcatcaaa tggcaattct 66300ctatatagtt ctgaccacat ctgaaaactt gtctttcctc tttaaagaca ggaaacttca 66360gtttttcttc ccttccttct ctcttattcc ctgtttcctt ccttcaagac atgttattca 66420tccctatttt tttcaaaata attttattgt tgaattcttt ctccaatata tcttgacctt 66480gacttccctt gtttggtagc tatgagtttg tattaatgat atgctactgg taacaatggt 66540tcacattcac agtctaggct ggacctttga gtaagttgtc tgtcttaatc agtttgggct 66600actataacag aataccatag attgggtgac ttaaagaaca aacatttatt tctcacagtt 66660ctggaggctg ggaagtccaa gatcaaggtg tctgcagatt tggtgtctgg taggcgccca 66720cctcctcgtt cgtggatagc catcctctca ctatgtcctc acatggtaga aggaaggaga 66780gcactctctg gagtctattt ttaaagggca ctaatcccac tcatgagtgc tttgcctcca 66840tgacctaatt gcctccaaaa ggccctacct tcagctatca tcacattggg gggattagat 66900tctaacatat aaattttggg gggacacaaa catgcagtcc atagcagtgt cggttcagca 66960tgatttgagg aataggaatc ccctctagac tgcaggtacc cctgactgtt tacctaaaat 67020taaactatta gagattaatt tttctattca gttaatattt attaaatggc tattgcatgc 67080tatgccctgt gataggtaat aaaaataaaa tatgcaaaaa acaaagtata gtctttgtgt 67140cacggcagtt ttagttaacg caatagtagc agcaattatc ttttggactt ctaggctccc 67200attcaaaagc aaatatctct tggagtaaat atcaagactt tagccattaa cgctttagag 67260gggatttgac ttcaatctaa gaggaaggct ctgtagaagc agcttttgac ttaggcactt 67320ggaagctagg acactggatg agaggaaatg ctattttgct taaagcactg atttgcgggg 67380gtgcaaaagt ggtgagggtg acttgaagac ctgatctaca tactgagaaa taagaagcta 67440gaaccttcat ttatttgcca ttttttgccc attattattg tttagttgag gatgtgaaaa 67500ttagatttct gagtcactga aaattcacct cagttgtcct gttttagtca ttaacattgt 67560gttcattttt cagataatct gttatggctt ttcattttct aatgtatgct tcccaaaatt 67620gactattttt ttgactgttt acatattaat gcatattttc aaaaactata attttatctt 67680aagtgtgtaa gatcactata ctaacagatt tgagttagag tttgcaaaat ttttagctta 67740tttaccattt ttaatcacaa aaatatatgc tactttgaat atttacttct ttttatacca 67800atatctaaat aattcaagag ataaagaaat aaatagaata gaataaataa tagcaaatat 67860gttgaattca gtgactttag atcattgaat attttgatat aaaacaatgt ttttttttag 67920ttgataaaag cacaattgtt tgcaattata tgcttattat tttgattata ttatcttaat 67980gagttattat cctattgtat tattttggag cccagcacaa tttgtactac gtccagctaa 68040tcaaattggt tatttcccct gaactatggg ataggtaatc taaataaatt cacttagata 68100ttataatcat ggtaaagatc agtagcaaac ctttacaatg ttcaaattca aggccttcat 68160ctccatttgt cacattccac aaaacctccc cttccttctc tctgacttta ttgggactca 68220cctactcatt cattttcttc aaacctgcct ctttttttct ctcagttgac tatgttttgt 68280caattgtacc ataaagatgt tttttcatgc tcattggttc atttccatca tctctgacac 68340tgttataatt tagctcttta tttttttcaa ccagaaattt gcaagctctt caagtacttc 68400ctttgccatc agtgcacctc tctgaacctg ccctgcctag agttcagctc tgataataat 68460acttcccacc tgaaaagctt tcaggggctc tctctattgc ctacagaata aagtctcaga 68520ccttgggttg tctttatgga cttcaacaat ccagctacaa tttacatttc caatgttatc 68580ttccactata agttccaatt ctcatatttc tacacaccac tcccattttt accatgtatt 68640ttaccctcta agaatttatt cccatgattt cttctatctg attgcccctt cccattctct 68700gacctttaat gttctgaccc attccttgta aaaccacctc ccaagaaagt tttcctaccc 68760tccagcagaa ggcaatttcc cttttctctc tcctattatt tcataaatta tgaaataatt 68820ttatttcaga gtagctgaag ctgttctgtt agttaaatgt atccagacat gttattaatt 68880aggtgagggc atattatgct gtggtaacaa aaaactccac caaatcttaa tggcttagct 68940gtgctccatg ttgcagttca actttcaagg ctgcttttga cacctctatc tcaagaaatg 69000ctttcgcagt tgtcacagca gaaggagcta catgggaatc acacaccagc tcttaagtgc 69060ttctttctgc ctggaattga tacacatctc ttcactcata tttcattggc caaagcaagc 69120tacctttaag tcgatagaaa agtgtaaaac ctttgaatgt gcagaaaaga gagaaccaga 69180aatattggtg agcactatta attttttcct tcccaatctg tataccttgt ctttccttta 69240gttgtattat ttcattgggt aggacttcca ataaaatatt ggctaagagt ggtaagagat 69300aagagagaac atcctttttg tcttgttcct gatcttaggg ggaaagcaac tatttttttt 69360ttaaccattg aatatgatat taactgtggg cttttgcaga ttctctaaaa aaaaaaaaac 69420tcaagttgaa gaagctccta tctaatccta gtttgctgag aatttatatc ataaacggat 69480gttgagtttt gtcaaacttt tttactacat ttacggatat gatcatatga ttttaattta 69540ttctcttggt gagatggatt acattagcta atttctgcat gttgaaccag ccttgcatac 69600ctagaataaa ttccacatgg tcatgatata taattcattt tgtacattgt ttgattcaat 69660gtgttaatat tttattgagg attttcacat ctgtgttcat gagagatatt gacatatagt 69720tttcctgtct tgtaatatct ttgtctagtt ttggtatttg ggtaatgctg atcttataga 69780atgagttagg acgtgttccc tctgttttta ttttcaggaa tagattatag ggaatctgtt 69840accatttctt ccctaaatat ttggtagaat ttaccagtga aaccatcaga atctggtgct 69900ttctgctttg gaaggtaatt aattattgat tcaatttaaa acacaaatat aggcctattt 69960agatgattta tttccccttg tgtgagtttt cacagactgt gtctttcaag gaattggtct 70020atttcaccta ggttatcaaa tttgtaggcc tacagttatt cataatattc cttcattatc 70080cttttaatgt gtatgggatc tatagtgaat gttctcactt tcattcctgg tattagtaat 70140ttgggttttc cttctttttt tcttgcttag ttggtctgga ggtttatcaa tttcattgct 70200cctttcaaag aactagcttt tattttcatt tgttttctcg aatttttttt gaaaaatttc 70260tttgatttct gctctaattt ttattttttg tcttctgctt accttggatc taatttgttc 70320ttatttttcc agcttcctaa gatatcttag attattgatt ttagatattt cttcttttct 70380aataacacta ttaatttcta tcataatata tgtgaaaact atcgtagact gtacattttt 70440gaagtcagca tttgtgattt attcttcact gtatttttct aggtcaaaga gaagcaccca 70500taaactagtg ttttccaaac ctcagtcatt cccagtccat cttcacattt ttgccatagt 70560cttgtgccat ttatgataat acttaactaa ttcttaaaaa ggaatcactt ttaaaaaact 70620aaatcaacat ttttagaatg ggatttttaa tacaactata aatggaaatc aggatctctt 70680cccatgtagg aatcttgaaa aatatatatt aaactctata aaatatttac tgaaaatatg 70740aaatgaaaat attacttggc tactatctga agtcctctta aataccagag ttatctttgt 70800ggtattttgg aaatcagtga aacaaataca aatgaaagag gatacaaatg tatgccaaag 70860ttctacacca gagattttaa actttttact ttattttctt ggggttaaat ataggaactt 70920agggtaagca ttaatagaac ggttacctta atgtgaaggg aaaaaaatat tagatttata 70980gtaagaagac atgaatttaa atgccctgtt tttctacctc cttgctgcat gagacagaaa 71040gttccacaga atttttgttg gtagaaagaa tcaatgatgt tcgcttgatt atattgtcag 71100atgtaaatga gataaattac aattatgttt taaaattgtc tagttttatg ggaaaaggat 71160tatgattctg cacagatatg cctgtatcta cccttttata atgtagtatt caaagtgtag 71220cataggcagt tttttttaag caacatttgt gcaacttttt tttgaaatga gtacttgacc 71280aggggcatag gtagagttac taaatatcaa aaaacaagat tcatattaga gatcttttta 71340atgaaaatta gtggagaaaa aatttagggt aaggactgtt gcctaggaat atatttattt 71400ttatgatttc cttaaatacg aaaaaccaat tctttgcggg tgttcacagt tggagaatgg 71460ttgtaagttc tacctgctat tttggtaagt caaaccaata tagtcttatt catccctttt 71520tctattttcc aagtgttcat tttttgctcc ttttttattg ccagaatttt ctaaagagtt 71580atttcagaat tgcatacaaa caacatgatt caccattgtt tggtaccata taagcaaaaa 71640gccctaactg attaagtgct ttatccctta actgaaggat tgacttctga gccaccatac 71700taagtagttg gagtaagagg taaagtaaat tagcaaatac ttcatgagcc aaatgggagt 71760acctcataag ataaaaattt attttagtta aagttccttg aaatattttt ctaagaatca 71820agttttaaaa aaagatacat tcaggagatt atttaataca gaagcattgg aagattttgt 71880cctcattgat ataatgaagt cactctttgg gctggatgtc agtcactgta gtttttgagg 71940atacagaaat gtctgcattc tgaagtgtaa acaaaggagt aaataaattt attttttaca 72000tggcttgatg taatagtata gaatctagac atggataata gatgcaactc agtcaacatg 72060gataaccaaa gttcttaaaa ctacgctttc aatgaacaca tatcctttga gcaagactaa 72120taatgaggaa tgggagccag ctcctgtgat atttatgcaa ctactaaatt ctcactgaag 72180tcaatgggag tttgcttacg taagggctgc aaactttagc ctccagagat taaaggggaa 72240aaaaatcctt aaactctttc aacattaata ttgcctgtaa ggaatccagc catgacctaa 72300gccatggagc tttctgaacc tagcaagtag aagggtaaac agtaaacacc agttatttta 72360agcacaatct aatcagagtt caatgagaag caatattata tttgatctct aaggtattaa 72420tacttgtata tcactattag acatctttat gtagtccatt atccaaacaa tggcttaagt 72480ctgtggtatt taataaatca agtttccatg gccgtgagac tgagtgggag tggggatgaa 72540gccttttttc ttcatttttt tttcctcagg tgcaattctg tgttaatata agagaagtgt 72600ggccttcctt ctcatagcac taaaagtgag ataatccctg tgtaagaaat cagtaagtac 72660ggtctgctta atctagtccc agtgtgaaac tgttgacatt tgttcttttt tctatcatta 72720tgtgactggg cctgttttgt gctggattag gcacaaatct cctatgcagc acatttggca 72780tgttactagt agtttaactt cattaataat gtatgaagaa aatgtaatcc atgacaagga 72840agcaaagaaa agtatttttt tttttttttg cttctcccaa atcctttgga atgagtaatt 72900attcaacatt ttatgtttga tgttatattt tacaattcaa cttccatagt gatatttaaa 72960aaagaaactt tggcaaatgc ttgcaaaaaa cacacctttt acaattttaa atgtgattta 73020ctgatggcca gaacttgtta aacatagtag gaaattaaat atttattcat cttatttcat 73080tttcagggcc gtaaacgctc cttctgagtc attcccaata acaagaattt ctaccagtaa 73140agctattaac aggcatcaaa ataggggagt gctaaattaa gatgagattg taaaagcaaa 73200taagaacata cgcagactcg cataggagtg caaatgatcg tttctgattg aaatgtttat 73260agctaaatga gtttggctga attaaacaca aatgttccaa aagataagcc gtagctggtg 73320cttctttttt ctgtttttta agctgcttta cagacgaaaa tggaactata tttggaacaa 73380tgctttctgt ttttccatac tattgatatt tgtggaaagt cacaaaatgg cctaaggaag 73440ctaagctcgc cccaagcagt ggtcacttac aagtactttt gtactctgta ctcctgtcac 73500atttgggcga tcagagcaac agctggggag actttttcaa caaagatgag tgtcagataa 73560tcctgatgag attccacatc caacatcttt tgtaattatg tcacattcag ctgtaatgga 73620ataattcaag ctgaaagaac aagctttgat cctttcttaa acctttccct gtggactggc 73680tatctaaaag atttaaagat atttctgtta caagatctag tgtttcctca gagaagtcat 73740gcttctgaag catcgtgatc tacaagaaca atatcaagtt tgccaaacac atttctgaaa 73800gcatcgtgtt ttggggggag gggttgtatt taatgaagat atcaataata tgctatgctt 73860caattttcat ctaggtgatc aagattcatt ttcttgttct gtcatccaaa taggcagaca 73920gaaaagtgat tgaaatacat tatggagatg tgtcattgca catataaagc atctgtgtgc 73980aaattcatct ttttttatgc ctgtgcttat taagttgtgt tttaatagaa actgacctag 74040tgaaatacta gctatgttgt agaaattaaa aaataaagtc atcaagatac tagcaagttc 74100caaatttctc atctataggg gaatttttgt gcaaaatatt tatattttac ttttattagg 74160ttttgtatta aactaattaa acggacagat tgaacctaac acaagatttt tgcataatca 74220tcaagattag tcatgttaaa aatcactttt acttgtttat agtaatcata tttctccaag 74280ttatcattgt aattctttct gtattttact cttacattat tttttttctt catttctttt 74340tcctttctaa tcccaattat tatttttttc ctgagactaa attcgtcaca acaggaaaga 74400atgtgatggg gaagaaaacg tagacctgat tttaattttg acttgaagaa aattattact 74460aaaattaaaa ccagtgttag aaaataaaca ttcttcaaat ttaattttta aaagtgaatc 74520tgtatgactt ttaaagcatg aaggtttagc tgctgtttgg tttattgagc aaagaaaaga 74580taatcagcgt gtgaatgctc ccaaaggaaa atagcttgat ttggtagaaa tgaaaagagg 74640caagtctgta ggactaagtg gtacaaatat taaaactatt tttgcccaaa ccagtgaagt 74700aacctttaat gcatgtgata aaggtcatct ggaagatcat ctttcccaca gaacatttcc 74760tctatgcccc cattagggat agcgagtgat cccagtgcta aatagaacat gctctaacct 74820ctatttgtgt ctttagctta

cactttaata gtgttttcca ttaaaattat gtatttgtaa 74880atgaaacata attatagatg ttgttttatt tcatttaggt ctttaaatgg atgtctgatt 74940taatcttaat attctcagct ctgaagtagg tatcattatt actattttgt agatgagaaa 75000actgaggttc agagtgatta aaggactttc acaggtatga gacaatgctg ggactgaaaa 75060ttggagtttc tacttgaaag tttagtactc tttctgctgt actatggttt ctttttctga 75120tttggttaat ggtgggagag gaaacagtaa gtaatacagc cactcaaaac tatttttttc 75180tatacattat taattttact tgtaatttga taataatttt taaaaatcta aacttattgt 75240taagaatata ttttgtctct gttagaggga gttggctgaa gtggatggaa attgtactct 75300gactgtgact caccaaaaat attaaaggga acagagtaga catttccgtg cttgccttta 75360ggcctaaagt ttaggttgta actctacaga tatatattca aagggtttga atattcaatt 75420gaatatgact caattttctc ctcctttgtt aggtttggtt atttctgatg aaacgacaag 75480ttggttttta aacaagcgag aaatttagaa tattgaaaaa cctgaccact atcactgtcc 75540tccagaagag atgttccttg ctggtgaata tgacaaaggc cagtacacat ttccccaaaa 75600gataaaaagt ctggtgggct gacagatctt agatttttac ccggtaccca cttcaccgta 75660tataacttta cagtcttttt ccctttattt aaaatagaca ttttatcatg cctatcattt 75720acatattaga attgaacaca taggtcacat tcctaacaaa ttcatcaagc gagccttcct 75780ggaaattcta ggtctctaac attacttaat tcttgggaga caaaaaattc ccacaatata 75840taacccatat tatgtgtttt gggtttttat aagaaaataa gaaatgtgtg aattaaactt 75900attcccttat tggcagtatt tacctagtat tttcttgata tttttccaat tcagtatttt 75960caaagatgtt ttggggaaaa agtcctctgt attttatacc tttaaaacaa gttattggct 76020aaaaaattag aatcttctgt ttattgcagt tgactaggtt ttaatgcttt gtgtgtgagg 76080caaaaacacc cattttcttc attttagtca ccatataaat ccagggaagc actctagaga 76140tgcagctgtg gttttgagga caatttcaac atagctaatg gtacggcatg ttttactttt 76200taaacgtggc tgtagaaaaa tcactgtctt atattgcagt aactttttca tttagggagc 76260catatattta aagatgcatt tcattcatgg gaagcacaat ggtttatatg gtttatttat 76320tgtggagact gtctagaaaa ataagtctct gtcagtgaca catgcacaca catacgtgta 76380tatttatata gaacacacac actttttgtg ttatgtatac acacagtttt ttttcagtta 76440ttttaagggg tatgttaaaa acatatttta tcatttccaa gtacaagaaa agtagaataa 76500atagtattat aattctccgt gagctcatta tttaacctca ataactatca tgataccttg 76560tttcacgtat attccccaca ttactgaatt atttttaagc aaatcagaca taatatttca 76620tctgtaaata ttccatatat atctctaaaa gtaagagtgc ttccttaaat atgtaaaaca 76680atctactgac acacctgggc ttattaatca tagttgttca gatgtcatca aatttaaaag 76740acagactttt cacatttatg gaacagtcat tacatttttg gttacttata tattttttca 76800tttcaatcca caagagctaa taaagttaat tccctttctt aaactcacaa agtataaaat 76860agtggttagg agtataggct tgaaaaacta gaagtcaagg ccttcccttg acttttatta 76920gctacgtggc tgggggcaca ttaataagcc tcaatttcct catcagtaaa aatgggaaca 76980atactactta gatcatacca ttatgagtgc tgtatgattt atatatggaa agtggcctta 77040gcttagtgcc tggtatgtag gatggtcttt gtaaatgtga actatttttt gaaaacttgg 77100agttggaatt tgaacccagg tctgactgat cccaaagcct gcgacctttc cacagtgcca 77160tgctcaacta gcgtggtgac aacactggca gcaccccagc ctggacactc tttaaagagt 77220atttaatgtg tacttattaa gagtccactc cttgatagcc ccaaactttc catcatgatt 77280ggcaagctag tgactggaat tctttagttg tggcaatcac tggtcacaga aaacagctga 77340gcctaaacag gtggttctga aggtgagcag agccagcatg ggcatggaga aaggctgagc 77400tgaacaccct gtgggcagag gggtctgatg ggagaaaatg agacctgaga aatgtcccct 77460ccactcctca taaagaacaa gttgccagga gccagcattc tgcattcgtc ttaatctttt 77520agaggagaat tgttattctg agccatgtct tagatgccac aagaatgagg ccggtctttg 77580tgtttgattt gtgctcatga ttacagctct tgtgtcttcc aacttcattt ttttttacgt 77640caagaaatat tttagtctag attttgctca aagcagaaca cacacatcaa gttttgagac 77700tgattgtttg ccaaaatgtt gtggattaaa aaaaaagagc aggaatgcag cattttcacc 77760aagctagaaa atttagcaga ttgggccaaa gactgtacaa ttattaagca cctaatacag 77820atcaggagct gagggacata taaaagacta cttgtcccgt ccccccgggt tctcactgtg 77880gtaaggagta atagctgaag caggagactt ttgttgaatc tatgttatta ttgatgttta 77940gtgcttctac actttaaatt tttccacgat tatacaagcc tgtcctctct gtgcttcaga 78000agcaagtgat gaaagttaat tccttcttct attaattcaa caaacatcta ttgacctctt 78060actgtatgcc atgtactttt gaaagcactg gggccatggc aatgaacaaa acagacaaaa 78120aatctctccc ctcatggata acatggggag atagaaaata aacaagaaaa atataaagca 78180tgttagcaaa aaatgctaag aagaaaaata aagcagggca ggcgggttgg aaggttcaag 78240gagagttatg aaatgttaga taaggccgac agggaaggcg ttcctgagaa ggtgacttag 78300gaatagcgac ctgaaggaag tgaggggaca accatgcagg tgtctgagta tgaatgtttc 78360tagacaaaag tgacagcagc tgtaaagacc caaaagcaga ctgtgatggg cacattggaa 78420gaatggcaag gaggctcctg tgcccgagtg agggtaaaag gaaatgaagc aagggagaga 78480atgtgtgtac gcatcgtggc cttgggggcc acagtgagga tgcttgcatt aggctattgt 78540aggggtatga gcagagcaga ggcgtgatct tactgagact gtattagaat cacttctgtt 78600gctgtgtaca gaacagaggg ataatttgga caaggcgcct gatatgtagt aaggcaaaat 78660aagtgtaaat tgttattgct aattttacaa ttactgtggt ctttacctgt tgcttgactg 78720tgcctgttga atgaattgac aaggagattc atgaaacatg acacggtcac ttattttact 78780gaaatgacta cataaaaatg attaactctg ctacacatgt cttcttttat tttgaagtga 78840tgatctatag agagctggct caagcggaaa aattccttgt ggttgtggtc tatctcttga 78900gaaactttag gattaaattt ttattttgga tgcttaagtg tgtcaacttt aaattggcta 78960taagtagtat tttttaaaaa accaacataa attgtggcta gggagatcag agtccagaat 79020acctttagaa tattaaacaa atctatttat tgcccaaaac atatttctta acaaataaaa 79080caacatgtaa atgtacgtat gccagttatt cacaatgaat aatacagctt cgaattaaat 79140attagaatgt agttttctct attgttactt atagaaatac ttctgcagta tgtcctagtg 79200gaaaaattta aaaacatttc ttatagtagg tcttgttttt aaaaattctg gattagatag 79260tgatgctatg ccttgaatat atagtatatt ctaaacaaat tattattgcc attaaaattt 79320aaagggcaag aataaagaat aaaggtaata ctttgtggag acaatttaat gaaccaactt 79380ttctgtacat tttaatttaa atttatcaaa cgtttacatt tctctctgtt tctttctctc 79440tttttttttt ttgagaccaa gtctcactat gtcaccttct ttttcaacat gaacagattt 79500ataaatatgc tagaattcac atttcaatag aaataaatac ttgtgtatct ttatctatac 79560ttatctatca gaccatttat tttgttgtaa caaatccaat tttaagaaag ggtggctatt 79620agtcactgaa aacaatagtt ttctaggaga ctattccctg tagatcttca ctacactatc 79680cattttgttt ggcatttgat gctgtctgtg aaattcaaga atttcactta cagacgcctt 79740gtcaactttt cctcacttga ggtttcccac acccacttaa ctgtcagttg cttttcagta 79800aagcccaaat ttaatgaact tcaatttcct agatttcact taaaggtgga tgttgagaga 79860atgtagatgt agaaagaacc agaactggaa aactataaat ttagcaaaca cttaaacata 79920cactatttgg tagtgttttc tagtcttcat tcttgcaata cagtttctga atccttctaa 79980aatgggtatt ttctccttga aaagccaaaa aatgctttgc acatgtaaaa ccatttacag 80040ccacagatga aggaattacc ttcttattcc ttgttacttg caacaatttg aagagagcat 80100ataatagaaa aaatctttta aaaaagtatt gagatgaagg agggaaagtg taaccgtaaa 80160gatctgtgcc tatagattca attaaacaca ttcaaaagca aattctttgg aagaaattta 80220gattatgaaa tttgatgctg ggtcttgaca gaaacttgaa acgtagtaag gattagccca 80280atggcatcag ggctgcttga attctggtga gcataggaat ttcctgaatt acttaccaaa 80340aaacgtagct ccttggccta ccaccaggga ctctgattta gttggtctct ggtagagttt 80400atgcagaaat catgtttgag aagccttcta gatgatttga aacaggtggt cctatgacca 80460cactctcatg atctcttgcc tcaactggtg ccttgatgct ggtactcatt catattttgt 80520ccttagactt gtaatttaaa atttgcctgt tttttatggt acccattatg aattggtacc 80580tcttattctt agcagatatt tttacttttt atttttttag atgataaaaa gctgtaatcc 80640ataatctccc taaatgctgc ctccccttgc ctcagaaagt cattacatct tgctcttccc 80700tctttcttct tcccatctta gaggataatg catttcccac ttttttctaa gattgtattt 80760ctgcctattt tcttctattt acttgctcaa tgtctggttc tttcattgta ttcccagctc 80820catgagaaca cagactgtga ctgttttctt gatcattgtg tcttcagaat tgagcacagg 80880gcttatagac actcaggaaa tgtctgtgtt ctgaagccat gagagatagc ttgtcttcct 80940tttcctacga atctatttca tctgttattt ttttacctcc agttctccag cttcttctcc 81000ctaaactcca gaaccttacc tttgtgtatg ctctctcttt cctatggcta cttcttttta 81060agctacaaac atacatattt ctgaaaatta aaaactacat ttcccagtgt tatggtctga 81120atggttgtat cccctcaaaa ttcatatgtt gtagtcctaa ctcccaaggt gatagtatta 81180ggaggtgggg cctttgggga ggtattagat catgagagca gagccccatg aatgtgatta 81240gtgccttaga gaagaagccc aagggagctt ggtggccccg cccaccacat gaggacacag 81300tacgaaggca ccagctatga aaaattggga ccttatcaga tagtgaatct ttggatgtct 81360tgatctggga cttcacagat attacaactg agagaaataa attcctgttg tttattttat 81420tttattttta ttttctttta ttgttattat actttaagtt ttagggtaca tgtgcacaat 81480gtgcaggcta gttacatatg tatacatgtg ccatgctggt gtgctgcacc cattaactcg 81540tcatttagca ttaggtatat ctcctaatgc tatccctccc ccctcccccc accccacaac 81600agtccccaga gtgtgatgtt ccccttcctg tgtccatgtg ttctcattgt tcaattccca 81660cctatgagcg agaatatgcg gtgtttggtt ttctgttctt gcaatagttt actgagaatg 81720atgatttcca atttcatcca tgtccctaca aaggacatga actcatcatt ttttatggct 81780gcacagtatt ccatggtgta tatgtgccac attttcttaa tccagtctat cattgttgga 81840catttgggtt ggttccaagt ctttgctatt gtgaatagtg ccacaataaa catatgtgtg 81900catgtgtctt tatagcagca tgatttataa tcctttgggt atatacccag taatgggatg 81960gctgggtcaa atggtatttc tagttctaga tccctgagga atcgccacac tgacttccac 82020aatggttgaa ctagtttaca gtcccaccaa cagtgtaaaa gtgttcctat ttctccacat 82080cctctccagc acctgttgtt tcctgacttt ttaatgattg ccattctaac tggtgtgaga 82140tggtatctca ttgtggtttt gatttgcatt tctctgatgg ccagtgatga tgagcatttt 82200ttcatgtgtt ttttggctgc ataaatgtct tcttttgaga agtgtctgtt gatgtccttt 82260gcccactttt tgatggggct atttgttttt ttcttgtaaa tttgtttgag ttcattgtag 82320attctggata ttagcccttt gtcagatgag taggttgcaa aaattttctc caattttgta 82380ggttgcctgt tcactctgat ggtagtttct tttgctgtgc agaagctctt tagtttaatt 82440agatcccatt tgtcaatttt ggctttggtt gccattgctt ttggtgtttt agacatgaag 82500tccttgccca tgcctatgtc ctgaatggta atgcctaggt tttcttctag ggtttttatg 82560gttttaggtc taacgtttaa gtctttaatc catcttgaat taatttttgt ataaggtgta 82620aggaagggat ccagtttcat ctttctacat atggctagcc agttttccca gcaccattta 82680ttaaataggg aatcctttcc ccattgcttg tttttctcag gtttgtcaaa gatcagatag 82740ttgtagatat gtggcgttat ttctgagggc tctgttctgt tccattgatc tatatctctg 82800ttttggtacc agtaccatgc tgttttggtt actgtagcct tgtagtatag tttgaagtca 82860ggtagcgtga tgcctccagc tttgttcttt tggcttagga ttgacttggc gatgcggggt 82920cttttttggt tccatatgaa ctttaaagta gttttttcca attctgtgaa ggaagtcatt 82980ggtagcttga tggggttggc attgaatcta taaattacct tgggcagtat ggccatttcc 83040acgatattga ttcttcctac ccatgagcat ggaatgttct tccatttgtt tgtatcctct 83100tttatttcct tgagcagtgg tttgtagttc tccttgaaga ggtccttcat atcccttgta 83160agttggattc ctaggtattt tattctcttt gaagcaattg tgaatgggag ttcactcatg 83220atttggctct ctgtctgtta ttggtgtata agaatgcttg tgatttttgt acattgattt 83280tgtatcctga gattttgttg aagttgccta tcagcttaag gagattttgg gctgagagga 83340tggggttttc tagataaaca atcatgtcat ctggaaacag ggacaatgtg acttcctctt 83400ttcataattg aatacccttt atttccttct cctgcctgat tgccctggcc agaacttcca 83460acactatgtt gtataggagt ggtgagagag ggcatccctg tcttatgcca gttttcaaag 83520ggaatgcttc cagtttttgc ccattcagta tgatattggc tgtgggtttg tcatacatag 83580ctcttattat tttgagatat gtcccatcaa tacctaattt attgagagtt tttatcatga 83640agggttgttg aattttgtca aaggcctttt ctgcacctat tgagataatc atgcggtttt 83700tttctttggt tctgtttata ttctggatta catttattga tttgtgtata ttgaaccatc 83760cttgcctccc aggtatgaag cccacttgat catggtggag aagctttttg atgtgctgct 83820ggattcagtt tgccagtatt ttattgagga tttttgcatc aatgttcatc aaggatattg 83880gtctaaaatt ctcttttttg gttgtgtctc tgcccggctt tggtatcagg atggtgctgg 83940cctcataaaa tgagttaggg aggattccct ctttttctat tgattggaat agttttagaa 84000ggaatggtac caattcctcc ttgaacctct ggtagaattc ggctgtgaat ccatctggtc 84060ctggactctt tttggttggt aagctattga ttattgccac aatttcacag cctgttattg 84120gtctattcag agattcaact tcttcctggt ttagtcttgg gagggtgtat gtgtcgagga 84180atttatccat ttcttctaga ttttctagtt tatttgtgta gaggtgtttg tagtattctc 84240tgatggtagt ttgtatttct gtggggtcgg tggtgatatc ccctttatca ttttttattg 84300catctatttg attcttctct gttttcttct ttgttagtct tgctagtggt ctatcagttt 84360tgttgatcct ttcaaaaaac cagctcctgg attcattaat tttttgaagg gttttttgtg 84420tctctatttc cttcagttct tctctgattt tagttatttc ttgccttctg ctagcttttg 84480aatgtgtttg ctcttgcttt tctagttctt ttaattgtga tgttagggtg tcaattttgg 84540atctttcctg ctttctcttg tgggcattta gtgctataaa tttccatcta cacactgctt 84600tgaatgtgtc ccagagattc tggtatgttg tgtctctgtt ctccttggtt tcaaagaaca 84660tctttatttc tgccttcatt tcattatgta cccagtagtc attcaggagc aggttgttca 84720gtttccatgt agttgagcgg ttttgagtga gtttcttaat cctgagttct agtttgattg 84780cactgtggtc tgagagacag tttgttataa tttctgttct tttacatttg ctgaggagtg 84840ctttacttcc aactatgtgg tcaattttgg aataggtgtg gtgtggtgct gaaaaaaatg 84900tatattctgt tgatttgggg tggagagttc tgtagatgtc tattaggtct gcttggtgca 84960gagctgagtt caattcctgg gtattcttgt taactttctg tcacgttgat ctgtctaatg 85020ttgacagtgg ggtgttaaag tctcccatta ttattgtgtg ggagtctaag tctctttgta 85080ggtcactcag gacttgcttt atgaaactgg gtgctcctgt attgggtgca tatatattta 85140ggatagttag ctcttcttgt tgaattgatc cctttaccat tatgtaatgg ccttctttgt 85200ctcttttgat ctttgttggt ttaaagtctg ttttatcaga gactaggatt gcaacccctg 85260cctttttttg ttttccattt gctcgataga tcttcctcca tccttttatt ttgagcctat 85320gtgtgtctct gcacatgaga tgggtttcct gaatacagca cactgatggg tcttgactct 85380ttatccaatt ttccagtctg tgtcttttaa ttggagcatt tagtccattt tcatttaaag 85440ttaatattgt tatgtgtgaa tgtgatcctg tcgttttgat gttggctggt tattttgctc 85500gttagttgat gcagtttctt cctagcctcg atggtcttta caatttggca tgattttgca 85560gtggctggta ccggttgttc ctttccatgt ttagtgcttc cttcaggagt tcttttaggg 85620caggcctggt ggtgacaaaa tctctcagca tttgcttgtc tgtaaagtat tttatttctc 85680cttcgcttat gaagcttagt ttggctggat atgaaattct gggttgaaaa ttctttagga 85740atgttgaata ttggccccca ctctcttctc gcttgtagag tttctgccaa gagatctgct 85800gttagtctga tgggcttccc tttgtgggta acctgacctt tctctctggc tgcccttaac 85860attttttcct tcatttcaac tttgatgaat ctgacaatta tgtgtcttgg ggttgctctt 85920ctcgaggaat atctttgtgg cattctctgt atttcctgaa tctgaatgtt ggcctgcctt 85980gctagattga ggaagttctc ctggataata tcctgcagag tgttttccaa cttggttcca 86040tcctgcctgt cactttcagg taccccaatc agacgtagat ttggtctttt cacatagtcc 86100catatttctt ggaggctttg tttgtttctt tttattcttt tttctctaaa cttctcttct 86160cacttcattt cattcatttc atcttccatc actgataccc tttcttccag ttgatcgcat 86220cagctcctga ggcttctgca ttcttcacat agttctcgag ccttggcttt cagatccatc 86280agctccttta agcacttctc tgtattggtt attctagtta tacattcgtc taaatttttt 86340tcaaagtttt caacttgttt gcctttggtt tgaatttcct cctgtagctc ggagtagttt 86400gatcgtctga agccttcttc tctcaactca tcaaagtcat tctccgtcca gctttgttcc 86460gttgctggtg aggaactgcg ttcctttgga ggaggagagg cgctctgctt tttagagttt 86520ccagattttc tgctccgttt tttccccatc tttgtggttt tatctacttt tggtctttga 86580tgatggtgat gtacagatga gattttggtg tggatgtcct ttctgtttgt tagttttcct 86640tctaacagac aggaccctca gctgcaggtc tgttggagtt tgctagaggt ccactccaga 86700ccctgtttgc ctgggtaaca gctgcggtgg ctgcagaaca gcggattttt gtgaaccatg 86760aatgctgctg tctgatcgtt cctctggaag ttttgtctca gaggagtacc cggccgtgtg 86820aggtgtcagt ctgcccctac tggggggtgc ctcccagtta ggctgcttgg gggtcagggg 86880tcagggaccc tcttgaggag gcagtctgcc tgttctcaga tctccagctg cgtgctggga 86940caaccactgc tctcttcaaa gctgtcagac agggacattt aagtctgcag aggttactgc 87000tgtctttttg tttgtctgtg ccctgccccc agagatggag cctatagagg caggcaggcc 87060tccttgagct gtgttgggct ccacccagtt caagcttcct ggctgctttg tttacctaag 87120caagcctggg caatggtggg cgcccctccc ccagcctcgc tgccaccttg cagtttggtc 87180tcagactgct gtgctagcaa tcagtgagac tccataggcg taggaccctc tgagccatgt 87240gcgggatata atgtcctggt gcgctgtttt ttaagccggt cggaaaagcc cagtattagg 87300gtaggagtga cccgattttc caggtgccat ctgtcacccc tttctttgac taggaaacgg 87360aactccctga ccccttgcgc ttcctgagtg aggcaatgcc tcgccctgct tcggctcatg 87420cacggtgcac tgcacccact gacctgcacc cactgtctgg cactccctag tgagatgaac 87480ccagtacctc agatggaaat gcagaaatca cctgtcttct gcgttgctca ctctgggagc 87540tgtagaccag agctgttcct attcggccat cttggctgct tcccgccaaa tttctgttgt 87600ctataagcaa cccagtttat gctattttgt tatagaaacc tgaatagact aagtgactca 87660gtttcttatg ctatgtagtg tctttgttca gtaaaaacag ttagcgatgt cctgctattg 87720atattgaaaa aaggttgagt tattttcctt ctttcagtgg acatttggag gtctatacaa 87780agctgtaagg ggcaagaggt acatgggcca gccgtaaagt ttgtttattc aaatggagct 87840tgttgatctg tcatattttt caagttgagc tactctgcca gggcaacatt ctactgtatt 87900caaatcagct gttttggaat tggtacacct gctagtgtct gtttctgaac actgaatgag 87960ttgccataag ggagaaaata taattctctt catcaactag aaaatataat tcaaagtagc 88020aataagatct gtaagatacc tacaatcaat gtaataaaga acacatttat tttatgtgga 88080aaaatttaaa actgtactaa agataaataa taataatctt tagtaaactg tacgaaagga 88140aataaataat aatctttctg attagagaga catttcatgc ttctggctgg aatgacttaa 88200tattgtgaaa ataccacgta cttcttgaat taatctataa attcaatata atttcaatca 88260aaatatcagt tatatttttc gtgtactggg aagaataatt gttcacaaat aataaattta 88320aatttaataa caataaaaag tatacgtggg tgaatcttgc ttgctctatc aaatattgag 88380aaattagccc aagaacagac aaactgaccg acaaaatgga ataaaaagtg caaagacaga 88440ttcgtgtgta tgtgtaaata tatttaattt acaataaaat gacactgcaa gtcaaggggg 88500aaaagatgga ttattaggaa aatgttggga aaactggttt accatatgca gaaagataaa 88560cttgattgct aactaacagc attcaaaagt agactctaga tgtgacaagt aaaactatac 88620agttaacaga agaaacagta gaagaaagcc tttgatccta gagaatgatt tctcagactt 88680tcctggagaa tgatttctag aactttcaaa gaacaaactg taaggcaaat gttggtgaat 88740ttgtttatat ctaaactaat gatttctaat taatgaatgg aaaatgttaa gagctaacaa 88800aatgggtgca tctatatgtt caagatatta atatctaaaa acggtaaata tctagaatat 88860ataagaaata cttgcaaatt aataaggaaa agacaacaat ctcaacagaa aaatacataa 88920agcataagaa taagcaatgt acaaatattt taagaaacac aaaataatgc actttttttg 88980gcaagaaaac acacaatgca aaatcttaaa gacattagaa tgcctgtgga ggggaatttt 89040aatatttaaa aatagttatg taccatacta gattttggat attgaagaca gaaatgaatt 89100taattcctcc atgttcagct atgctaagca ataatgtttg aggaaaaaaa aacacatcct 89160aatagccata aattagatta taatagaaat cagtaggagg ccgtggcacc tgtctagata 89220tgtgctgctg aatacctgga cttgggtggt ggcaagggaa tggagtgatg ggtcagcagg 89280cttttgtatt ggcatggatg tttgagggaa tgaaataaat aaataaacat gagttttaaa 89340gccaagtgaa ggtccaattt ccagtagtac tgcttagaat catttgaact aattgttcaa 89400ctaaaaatgt tggataaact ttaaaaaaat tcttaaaatg taaacaactt ggcaagacat 89460aaagccattt cagatcaaaa tgtataattc ctttatccca ctgtgactcc caatctactg 89520atcctaccca catttcagtg tccctcaccc ccatgatatc ctaattttcc tctttgatta 89580cttaaattgc atagacaatt aatataaatc actcccttgt atatttcctt gattcctttg 89640tccttcttgg gttttactat agtggcttgg ctcaacttaa ttaaacccaa gtttctcccc 89700ctatacttgt gtagctgagc atggttgatg caaaacataa aacatacaac cacattgaat 89760ggcatcactt taaatttaac gttatcaagc aaacatactt tctgtcccta gtccattcat 89820atggactagg tacccactca cctggatgac tatttcacaa ctttctttct tttcaaaatt 89880ccagtccctt tcctcatttt

caattttagc tgattgactt tttttaaaat gagacattta 89940gaacaatcta gagagaactt cccaacacca tgtttgatca tcagcattac attcctttac 90000tcttccttcc cccctcaatg aaatatacat gcttttatca aaagccaatc cctctatttg 90060tgccgtagat gtgatccctt ctcacctttt ccatgacatt tctccagcaa ttctccccac 90120tgcttcttat gacatcaatt tttcacttta ctgaagtatt ctcctttagc atacaaatct 90180gctgttattt cttccatatg aaacaatatg aacaagcttt tctcgaaaca gttctgccat 90240ccagcacgat ttttgtcccc tttgcaacaa agccccttga aggagttgtc tatacttctt 90300gttttcactt ccattcctca cattctctct taacccattc taatcagtta ggcttctact 90360gccctcccct atggtctcca atggcctcta ctttgctaaa gactagttct tgcccctcat 90420tttagtcttg acccatcagt aacatttact tctgattatt gattgttcaa ttctttttaa 90480catatgcttt cttttggctt ctaggacatc acacactcct tttccctcct gctttgctgg 90540tcttctattc ctctgtcttc ttcactggtt ttttcttttt tctcgtatat tttagagttg 90600gaaagtatcc tagagtgttt tcgttttttt ctcttttttc tttgtactca ctatctcagt 90660gatcccgttc tcatctgatt ttgaggcctt gaataccatc tgtatgttta tgactcccaa 90720atttatatct tccgctcaat ctctttccaa ttagctactt gatattttta cttgtacatc 90780tgatagatta ttttaaactc aacatattaa aaacagaact tctgaaacct tccacactgt 90840caaacaaaca tataaaaagt ataacaatac tactttggtt gatgacaact caggccataa 90900gcctttgtct aatatttgac tattctctga ctcttatacc ccacatccca tactttatta 90960attgcattgg aaattcagtt tccatctatg taaagctcca tgttgatctg ctgcaaggat 91020agttgtgggc cccagtgcaa taaaaatgtg gggctcctga tagggaaggg aagtcaatga 91080ttccttctta cgggtccttc ttctaatcca tggtgaacag atgaccctcc atagattgtc 91140gcctcctctc tcagatgtgc ttggtacccg gattgggagt ggtaagaggc tcttgccaaa 91200ttgtccatga aatgctctgt gttgctgcca gcctagagta gagaatcact gccttgatct 91260gccctaagat atcacgtggt gtgcacccaa cactgaccct ctctgtgctt gcatccaggc 91320cctcttgggg ctggaaggta aacaacagaa tgtgagtctt tccttgccga aggtgagggt 91380gttagtggtc acagggatgg tgtagggagg aggaggctgg acagggccta ggtggccaag 91440aacccatatt aagttggtgg gaaggtggag aaatggtagg aggaggctat ccatgaatga 91500gtttccaacc acctgtgcat gctcccttgt cccatcaaac ttcacttaaa aatgcaaatt 91560cagatgatca gattcaatgg ataatcttag ccaagttatt taacttctct aagcctgttt 91620tccttagatc atcatttcaa tatggtggct gcagagtact aaattgcaaa tgcagggctt 91680ttttgagcat tgagtcttgt gtgactgcat tgattacgct cccatgaagc cagccctgat 91740ctggtgatat tgcctagcca aaaataagac tccatttaat gattgccttt tgcctttctt 91800actggtactt ctgacttctc ttccttctga ttgcccaatt ggcatctttt ctaggctgtc 91860aaattttacc cagagcctcc aggatttgtg atgacttcga agaaggtttg gactctgatc 91920tccgatcttc agttttcaga acccagttag ttatgagtta gggatggtct tttttttttg 91980cacgtaacac ttaaaagcat gaattttgaa gacaactaga ccagagttta agtcctgtgt 92040ttgctcttac ttgatgggtg aacttgggca agttatttaa cctcgctaag gcttgctttc 92100cttagatcat tcatttagaa gactgtgtgg ttaagagtgt atgctgtagg cagtctttaa 92160cctctctgtg cctcagtttc ttcctctgaa aatgggaatg ataatactac ctaccctata 92220ggattgtatt gaagattcag tgagttagta catgctaaag tacttggaaa atgctcagaa 92280caggctggca ccattgcaag ggtttagtaa atgctaacta ttctattcat ttattcattg 92340aaaaaatgtt tatgtagcac ttatactgtt ccatgcatac agtttctcat ctaatgagta 92400gtattataga taaatatcta caaataaagt gagacttgtt aaatgcttag caccatgact 92460gtcactttgt ataatcttaa caaatgttag ctattacaat tataaccaat aatgataatt 92520attaagttgt tagccagaag aaagatatct tggttatagc tgtttgatgg taatgagagg 92580tgaaagctgc gtggtaaaaa acatattcaa agaatatcct agtcctattc cttgaggaca 92640ttctattttt ccatgcagag tgagcagcat agatctcaga tgacaacttg gtctccctac 92700catttgatca agataaaatt gaattggacc ccctatctca taacctgcat acaccattag 92760acctcccaag atttcttcaa aagatccacc caaggattta tgctttcata atcctctttt 92820cttgtgagtg gttttttccc agcaacatgt ctaacaagga aagagaccac agatctaggt 92880tttctaaaag aataccctca cattagtgaa aaacatagaa tttagtgtat attttgcagg 92940agggcagaag aagcgttgat tacatggcac atggtagaac aaacaattat taaactccat 93000gttagtgatt cttgttcttc taaacccctc tgttactctg ctgccatact gggcattttt 93060tcccttcttt attgagcacc tccttccttt cacattgtct cttatccact ccaggaatcc 93120cccaattttc ctggaaaata attggatgtt ttctggctta cactcccaaa cttatattta 93180gacatttatc aagtgctcaa ctcatcaatg gccttctagt aatataattc attcttcaga 93240gaaaattcag tggtgaaata tttccttatt atatttgctt tatatgtttg gcaatatgta 93300ttccttcatt tacgtgttta tttgaaaagt atctgttgaa tgcaaccttt ggttcaggca 93360ttaggctttt agtaatatga tgggagtccc tgacctcatg ggtatttaat taaaaagtta 93420aaacatagat atacatatct atatcataca ctgacattgc tttggtgtta tagcatacat 93480tttaaaagag ccttgtcaga attaaacttt gtttttcagc tactctgtac tcactctctg 93540gtcctcccac ttagatctag gaaccgagta aggactggca gtatatgcaa tggggagtag 93600tggctggaag gtgagtctcc agatgacatc cgggatctag tgccttcatt tctctgatga 93660agccatgcaa ataaccataa gagaaaaagt taattgaggt gatgagtgga tgaagagagt 93720gagtcatggt taccaagaat aaaagtgctg agttgttgat gaggtacaga aataattagt 93780aacatattta caattgctga catttcctaa ctactttcag ctggttcttt ggtgtggact 93840gtatgaaact taataattgg tactcattta atcttaagta gggtccaaaa tgttcttctg 93900cttggtctga ttctttcttc ttttgttcaa actcctttga tagacaaaca gaggaaagag 93960aaggaggcaa ctgttccaag atagattctt ccccaataca gaaaagagag acaagaaaaa 94020tcataatttt caatgccata tatttgtatg taatgcagac caaacttctt gtttaaaaat 94080gataaagtaa gtcatgctta taaggctcat ggcttggtgt tttctcagca agaaaaacag 94140atccaaaggt tgatatagtc tagaggatct actagattga tcttaaaatg aaaaactata 94200taatgctaaa gaacaatgtt agaaaatcgt ctagataaaa agtagttcct ttctaaataa 94260aaaagctact tcccacatac gttctcttga ttatgctcca aattctaggt cctaaataca 94320attcaataaa tatatgaaca ataagcgttg aaatggaaga cgagcaatca ataatttatc 94380atagcccatt tacatgatct tgtagagtat gaacccagat atattgtttc cactaatcta 94440tggtactatc ttaatgaact aagaggagat atacagattc agtacgttag gccttcaaat 94500aacattttta aagcttcctt tttcttagtt tattgcaaaa ggaaatattg gttcctcctg 94560aatagatgta gtggttagat aaatttcagc actcaagaga ccaaaaaaaa aggccatgag 94620agaaaatatc tttcttagac tcacttaagc ttgttatctt ttggctgttg cctgtattct 94680tttgcattct ttagccacat tggttattca aacacagtga attaaagcca ttagttttag 94740ttttgatgat cttgtataga aggttagttg atacggggtt ttgcatgtaa aatcagggtt 94800agctgatatt cattctataa attcagcatt ttgtgagaat tttgatggag tcttaaagcc 94860ctttttaaat atttcattga attgatagga gtcttgtgta agtaaagaaa ctctattccc 94920aagctgatcc actgatttgt ggacttgtca cttggtagta gatacttgtt tttggcatca 94980catatccatt tacccttcat ctgggtaata agaggttaat gtttatttag aaatcaattt 95040cttccctatt tttagctaca cagttagaat tgactccact actgatccta catgtgtggt 95100ttgtgagttc aggcttgact cagagcttct ccttctggcc atgataatca gcatgaacat 95160atgattcggt cagagacaag aagacacaaa gagaattttg caggtgattg tgagaaattt 95220tttttcctat tggacttaaa tcgagtggga taaaatcaac aagaaaaatg cagagctcag 95280taatgtagaa aaactttgtc ctatgacgtt gcttcagccc tgaatccagc cagacatgcc 95340cccaaccagt cttaccttta gacttttcaa ttacaggagc caatacattt cctttttggt 95400taagccaaat ataggttggg ctttctctca cttgcaacca aaagagttct cattgacatg 95460ggcttcttct ttatcttctt ctttttttaa ttctaagaca aacttgagtg aatctgtttg 95520gatggtggct tgcctatcat tgttttcatc ttacccaagt tgaccacacc cacagagaca 95580tgctctgtta ttttcagttt actcagatga ttttttttta acataaagaa ttttctgcat 95640aaagaaaagc tttacaatgg tgctttgagt tgaattgtgt ggtctctgca tctcccttat 95700ttgcatgtgg aagtcctaat ttctagtgct tcagtgtggc cttatttgaa gggtctttac 95760agaagtaatc cagttgaaat gacatcatta gggtaggccc taatccaata tgactggcat 95820tcttataaaa aggggaaatt tgaatacaga cacacataca gggagaatgc catgcgaaga 95880tgccggaaga agatggccat ctgcaagccc aggagggagg cctgaaacag cacgttccct 95940cacagccctc agaagaaaac aaccctgtgg ataccttgat cctgaacttc tagactgtgg 96000aactgagaga caataatttt ccattgttta agccatctaa tgtgtgatac tttattacag 96060caacgctggc aaatgcatac aattggcaaa agggactcgc attaaaacct gagtgagtaa 96120ataaaaagaa gtacaatttt atttgattta atttgataga gcattcacta caagttgttg 96180ttatttgtag agctagaaat ggattaagaa agcacttggg aaaatcaagg atggcaaagt 96240tataaactaa ataaaggaag agattcaatg tatttaatat tcaaagctga tttcagggaa 96300gattgctttt ggcaagaaaa tgaatgcgcc atatttacag catgtgtggc aatttctatg 96360tttctatact tcgatattta gaaaattaat gtaatgtgat atggcatttc atgctttctt 96420aaaatatttc aaaacatata tttttctgca aatccttcaa taaaaaaact aagaaatttc 96480agaataatcc caatgtttat acattttcat tgctgccttt ctttaaaatc tgtcataaaa 96540taatcattaa aaattattac aattttcttg aattataatt gtcctaaacc acttactttc 96600ttttatggaa ctacaagaaa aaatataaaa acgccagtta aactcttact taaagccaaa 96660cccctttaca tttaaattct ggactaatag ccaaggattt agattcagag gttggctaat 96720tacaagaaag aaaacagaac tctagtgaaa ggtatcccta tcatttacat acatgtttat 96780attaatatgt gtatatatat gtatatccac acagacacaa agtattatat aaggtttgtt 96840ttaaaatgaa tatatttgat ttgaaaggct aaaaaagatt acagatctca gaatgttttg 96900tcacagaata gatatgatac ctccaaatta agtggacaaa tttttaagta gcctgatcta 96960tgaaaactat ataaatgtaa actttttctt actagatatt acataaaatg acagacaact 97020ctttaatttt cttaaaactc tgacacattt tacagttgtc tgtcttggtg ttggatgaag 97080gagcaaagag aatgctgttt tacagaggtt acatttctcc tactatatga cagagatttt 97140aaaaaaagta ttgtcctttt gccctccctc ttcaaagagt gaaatcctac tcataagcaa 97200aaattgattt tttaaaaata aaagacgtta ttgtttgccc tttctaccag aactgcatga 97260tattgccatg gtctcaattt atactccata tggtttgtag gaatatttca tataaagtat 97320ttgtgggaat tcttaggaaa aagatgtttt gccaatagac aataaaatac tcttcagttt 97380ccaaatttag agatcaaaag ttttatgtga ttcgttgcct ctagcatcta ttctatttca 97440tggtttgtca tattctaaca ttttgcatta tttaaaaaaa tctttcttag aatgaggctg 97500aataaagcat gcagaattgt ctgcttggaa attgtacaca tagcattaaa caacttttta 97560acacgttgtt gtgtgcttaa ttcctcgata atctcatcct aattatttac tgctttacat 97620ctttttattt tttaattttt taaaattaat acatagtatt ttgcatatgt atgagctaca 97680tgtgagtatt tgttacctgc atagaatatg taacaatcaa gtcagggtgt ctggggtgta 97740acttaccttg agttcttact tttatgtctt agtaatattc aagtcctctt ttctagctac 97800cttaaaatat acagtatatt attactaact atagtcaccc tactctgctt tagaacattg 97860gaacgtatta gtacttctgt ctgtttacat ccatttaaga acctgtcttc atccccctct 97920ccccaacctt catacccgtt cccagcctct atcctccacc tcccgttctt ttgtgttacc 97980cagggcagta cacaacttca aaggatatag cctgtgttaa attggaggac acacacacaa 98040gaacaacaac actttaagga aaagtgttct ttcactagcg gcttctgagt cttattaaag 98100gaaaatgaag tttctatata aaatgctaga tacccaccac aatttctttc atgtgaatta 98160cacttgtttt tggtatatct tgtgtgtgta tgtgtgtgtg tgtgtgtgta tgcacacacc 98220ctttgtaaat ttgggttaca gaggcagaaa tcaaaaggtc aaacaacagc tagtaatttc 98280tcactctgta tattcttatc acatttctca aaaagactgt actgttctat gtgatattga 98340tcttgattat ttctgtgaac acattttctt agttaataat atgcagaatt ctgactctat 98400tgtatctctt ctcttcattt ttgagaattt tagtgccaga aaggacattt attttatcca 98460gctgcaaact taaataagct cttcatttct ctaggtcact gatggcaaat gttaaacaaa 98520tttacatttg ggattttcta ttcatttccc acccggttga cactgagcca gtgataacca 98580ctcaatagct gtgcagccct caatagtatg tgtgacccat actgtgtttc cagttcattg 98640atgactctat catgctgggc taaatctaaa atcttgctaa agtcatgaga gatcatatct 98700actgttttcc ctttgtgtcc caagagtgac ttcctttgaa aaaatgtgaa aaagagtttt 98760cactgtagtc agtctagatg ttaaaacttt atttgagcac gagtttctgt aaaatagaac 98820ttgaagatcc tgtaaagtta aaacatatag gcacactttt tcttcctggt cactttgggc 98880ctgttctgta gcagtgtctg cccataattg gtatagtaaa ctgttctcta gttatgagct 98940tcttatcaag ctctcaggag tgcattgatt ctatttttgg ttaattttta attatttata 99000cttatgtcac taagctgatt aaagtggaga attaacctca agcttagccg agtggcagct 99060tctctcagtc aaagacaagg atactgactg gttgtcagaa tatttgggtt ctcttgtcaa 99120ttaccaccca aagctacatg ttctccagga agatccttta cctttatatc agtttcatca 99180tctgaaaggt gaagataaca atatgtattt tactgaacat gaagggcttt cccaactatc 99240aaatgagata aaatatatgt aagttgtttt aacactctaa atatttgaat tatttactta 99300ataacatatt tgcatttttt taggattcat atattagtaa catatttttc aaaataatac 99360cttaaacaat atgtttcacc atgataaaca tttccagatt ctgattatat attttctact 99420ttatggctga ggctatggtt taaaaatata ttgtgtgata tattgctaat aaattaaaat 99480ttttttattc atatgaattg gattgaaaag aaatgaaagc aagtatttca gtaattcaag 99540ataaaaacta ctcccatcca aactctccaa atttcttttg tgtatttgaa attgacttgg 99600aggaccaaag aactgatatt ttgatgttgc acataaagga atggaacaat aacatcaaat 99660tgtacttctg ttgaaaatat atatgtgaaa ttaaatgttt ttgtataatt aaattgtttt 99720gtaaaaaatt ttaatcttct attatttgtg ttttagtcaa attaaagtgt gtgtgtatac 99780catatccaga gaatgtattt cctctgaagt gatcaggaga tttcatttat ttccctatgt 99840taccttggag aaaattactt ttagaatagg taaaatgtaa ataaggggta gtgaaattaa 99900atttttactt gcctatgact tttcctacta tttactttat atcttatatt gtttaaacaa 99960gttaagtggg gaagaatagg aattattttg ggaacagcag agcaggtgaa ggtgtagggt 100020ttgggcagct ggtaagactt taacattttt atccttcaag tgtactagcc accttaaaat 100080tgcaaaatca cacttgccta gagcttggta atcatcatat gttaaagaaa catacttgac 100140atcaacctga gatcttttct ttatttaata atcttaaatt ttgcttgaat ttgagttaat 100200ttttcattga aaatttatat ctattttctt catctgttat gatttttttt ctgaaagtga 100260cattgtctct gttgataaat ctggaaataa aaatgttaag accagcaatg gctatttcta 100320agtttattct atagggaagc agaaataata gcttgggatt tataattact tctgaaataa 100380cttcatcctc tgaatactgt taattttttc tttttttgtt ttttattttt attgagacag 100440ggtctccctc tgtcgcccag gctggagtgc agtggctgga tcatggctca ctgtagcctt 100500ggcctcctgg gctcgaatga ttctctcagc tcagcttccc aagtagctgg gactacaggt 100560gcgcactacc atgcccggct aattttttat ttttattttt agtacagatg ggggtctccc 100620tatgttgccc aggctggtct tgaactcctg ggctcaagca atcctcctgc ttaaactcca 100680aaagtgctgg gtttacaggt gtgagtcaat tcaccttggc agtattttct acattctcac 100740ttacgagcac ttcattgcag tcataccatt ggaccatgag taatcttata aatgacatac 100800attattttac agtcctatta aaattgttta ctttctattt tagggaaata tttcataaat 100860gattatttca aatcaaagaa cttagtgaac agagtaacat atctaaaaat atgaaactcc 100920aggatgctgc cttctaaggg ttgagcaact aatatgttat ttatgttact actttgagct 100980ttctgttggg attttcataa tttagtatta tatattgtag aacaaaaatt agatggacaa 101040gcactctctt ggtccatgta atttattttt catcttattt cagtagcttt gtagaggact 101100atggttacaa taataataaa aataaactaa tctgaaacaa ttttaaagat gtaaagtttt 101160ccagagcatc aaaagcacgt tctgatagtc tacaaccact ctaggtggtg tggaaatgta 101220tataaacaga taaacaagtt ttgcatctgg gactttataa tttaaagtgg caggaaatga 101280aagaaacagg taggtaacag aaaaacatgg tattgatgtt gaatatgttt tcctacaggt 101340gtgtgtgtgt gtacgtgtac gtttggaagg tgaagaacat agtggggaga caggaagctc 101400agtactcagg cagatcagcc agataccgtc catgttgcaa ctaagttccc atcaacacca 101460ggcaaggata aacaatacta gggttatgga tgataactct agtcatttta aatagtattt 101520tttcctaatt attagctgaa taattagagg gaatataagt ttcccttggg tgtcctccta 101580gatgctgaag ctagtcacta agcagggtgc tgagagctgg atgcaggatc caagggccat 101640cacgctggac ccgtcttcac tcatgcccct tagctgaagt gctcagatcc acacatatca 101700ccggccagtg aaaacaggaa tagccaattg gacagcctac acatggttac ccatggctcc 101760aagtgcaagt gctccaaatt ataatgagga agctgcatct tcttttctga cctagccatg 101820ctgtgttatc tcccctacat tcttttggtt gtgactcaaa aacccaccca gattcaagag 101880gttaggatat aggctccatt tcttcttctt ttttaataac ttcaactttt tagattcaag 101940ggtcgtacat atgcaggttt gttacacagg tgcattgtgt gatgctgagg tttagggaat 102000ggatgatccc atcatccagg tggttatcac agtacccaat aggtagtttt tctacccgtg 102060cttctctccc tccctgccat agtagtccac agtgcctatt gttcccatgt ttatgtctat 102120acgtactcag tgcttagctc cctgttccca tgtttatgtc tgtatgtact cagtgcttag 102180ctccctgttc ccgtgtttat gtctatatgt actcaatgct tagttttaaa tgagaacacg 102240tggtatttgg ttttttgttc ctgtattaat tcatttagga taatggcctc cagctgcatc 102300catgttgctg caaagtacat gatttcattc tttttttata gctgcatagt atcccatggt 102360gtatatgtgc cacattttct ttatctagtc cactgttgat gggcatctag gttgattcca 102420catctttact attgtgcagc aatgaacata ggggtgcata tgtctttttt tttaaacaac 102480ttattttcct ttggatatat acccaataat gggattgttg gattgaatga tagttctgtt 102540ttaagttctt tgagaaatct ccaaactgct ttccacagtg gctgaactaa tttacattcc 102600cattaagaat gtataagcat tctgctttct ctgtaacctt gccaacacgt gttgtgtttt 102660tggcttttta ataatagcca ttctgactca tgtgagatgg tatatcattg tggttttgat 102720ttgcatttct ctgataatga gtgatgagaa gcagttttcc atatgtttgt tggccgctta 102780tgtgtctttt gagaagtgtc tgttaatgtc ctttcccatt tttaaatagg gctgtttgtt 102840tttttgcttg atgatttgtg taagttcctt atagaccctg gatattagac ctttgttgga 102900tgcatggttt gtgaatattt tctcccattc tgtaggttgt ctgtttagtc tgttgatggt 102960ttcttttgct gtgtgaagtc tctttagttt aattaggtcc cacttgtcga tttttgtttt 103020tgttgcaatg acttttggga acctagtcat aaattctttg ccaaggtcaa tgtcaagaag 103080agtatttcct aggttgtctt ctaggatttt tatagtttga gcagacttca cttcttaaag 103140ggagacatat caaggtcaca ttgtatatga gacatgggat gggaaatatt atagccatct 103200ttggaaaacg tgatgtacta tggagcttat gtaagtattt atatgcactc acatctgagg 103260taatctcttt cagtctcata aatttaatta aatgccatat acaaactaat gagttccaag 103320tctatatctc cagtcctgac ttccgccctg agttctagat ctatatattc aactgcctac 103380ttgagatttc cactgaatgt tatggcagag atttttagtt gtctcacaac atccaatctt 103440attctacagt gttataattt tggatgaaca catggtaact cagaataaag cttatcattt 103500acccctaccc tcactcctga tgtttggtgt agttatgtca caaaattttg gctaagagga 103560taaaagggag tgctgttgga cagcttctga gaccctttgc tcattcttct acctttctcc 103620attctgatgc ttggactaga gcttaatctt gaactaggag atgatgtcca cactccagat 103680ctcatagaga agaaagttgg aatgattttc ttcctccagg ctcttatgcg gaagagatta 103740aaaactctat cttatttaag ctacctaact tacagttaaa cctaatcatt tcagactgga 103800catggcaaaa cagaactcct aatttccctc tgttaatctg cctgttactt ggatgtcttc 103860atattcgtaa atcacaccca tattcactca attgcttaga aatctaggag ttaccactaa 103920ttccttcctt tctcttggag tccacatata tgacacatgt aaatcccaac atctctacct 103980ttaaaaacct aactacttct actatcatgg tttaagccga catcatcttt tacttaaact 104040actcctgtcc ctaactgtgt ctctgcttcc acttttgctt ccttataacc cataaataca 104100tcagattata tcactagctg cttcaaaacc atctactggc tttctgtcac acttgggaag 104160acatcttaaa ggcttaccat ggtcaaagaa tcctacacca tgtaagacct gggtatctcg 104220aatcccatct cctacctgtc tcctccttgc tcactccatt cccttcccag tgatcttctt 104280gttgtgcctt aaatattcct gcatcaaggc ctttgcaaat gcagttcttt ctgcctgata 104340ctccctcaaa tttctacttg gttcactcct ttactttctt aaaacttctg cttggatggt 104400acattgtcag aatgagcttt cttgactatt ctttctaaaa cttcactcta ctctctatca 104460ctctccactt cttacccagt gttatttcat ttattgctac ttaatgtgta tgtggtagat 104520gctgtgatgt gccacacaaa tttctcccct tcagaactga agtacacatt cctccagcta 104580cttggagtgt tggtggtact tcttagctga atgtcccact gagtcctgtt agccatgaaa 104640ctcttagcct tgatggaaga ctgttgcctt gcccatcttc acttcccctt tctggaggca 104700gcacacatct actggctgtt gatgtggaag aataaaagcc tggccatttt gcttcaatgg 104760agagccactc tgaagagccg cttcagcttc agagctcccc atgggacctg ctaaggctcc 104820tgtggagact catcacagcc catcttctcc ctttgccact cctgcttaat ttacttctcc 104880ctcagcactt cataggaatg ggctctgaga gtgcttctta gtaaatgtca gtctcctccc 104940tgaagaaaat aacctaggac

acatattttt acttgtctct tgtttattgt tgatatctcc 105000cagtagaatg caatctcaat gagggcaagg attttttatc agttttgttt gctactgaat 105060cccagtacct agaacacaga gcttcaaaaa tatttaataa ataaatggcc ttgtttagga 105120tgacaaaact agtaatcctt gaagctagag ttcaaactaa atttggaatt cagagctgtt 105180cctctctcac actataagtg gaatgcaggg tttctcaaca gaagaaccag ataatcttgg 105240accagataat tttttattgt gggagcactc ctgtgcctta taggatgctt agcaacatcc 105300ctggcctctg cctactagat gcctgtagca ttcccacctc acccccaagt tgtgataaca 105360aaaactgtct gcaggtattg acaggtattc ctcgggggca aaattgtgcc caattgaaaa 105420tcactggcct acggtggttt tgactgattg gcaataatat tccactcact aatattaaaa 105480agttctaata gtagttcatg tttcctgctg aaagtttgtt tctttggttg tttcacatcc 105540ataaatacac tttaatggag atctgatttc acagagtcat gtgaataact actgttttta 105600caaatgggga tggctagcat gattttcagg gctgatgtga gtgaagccac tgggtaacag 105660aaagaaacac atatgcactg ttataattag tgtgtctgtt ttaggctgca tatcagttta 105720cagagctttg cccatgtgtg ttgaagacaa aagaaagact ttaggaatga gaatccagca 105780atgaattgta ggatattttt gtcaaggtaa attaatcttc cttttgcttt catccttttg 105840tttcaattca ttcttattaa actttcatag aaaatatcct gtgtttttgt attatcatcc 105900atactacctt cttttattga aaaatccata aagtaaaggg aatcagtttt catgctgtct 105960ttgaaaacac cgggcactat gcttcagaat tatttttgtt gcttctgctg atgataatgg 106020ttacactata taacctcatg ccagatataa atagtgaagt gacaattata ctgcatacaa 106080tggaatgtat caccgttgct tttggcttca cggagtcctc tgtattcaca caaaagagtg 106140aagctactta caggattttc ggaaaaaaat gatgtacttt tccaaagaat ggtgggagaa 106200ggatatcctt tctcattagt caccagaaat gtgcttcctc cttccatgtc aaaggagaag 106260tagaattggg tgttttaatt tagtgattct tttgatgttt tctgggggat agaatttcca 106320tttctttctg attttgtatc aaccttgaat gtggaattaa aatagcaaat ctctactttg 106380tattttatat ataaataaaa attctacata ttacatatgt agctgatatt aatatgtatt 106440atatacacaa tataatttta ttacatatta tattattaat aatatatata tttccttttt 106500tcctcattag ttgggtatga ctttgatcaa gttatttaat cattttatgt ctcggtttcc 106560tcatgcgtaa aataggaata agaatagaat ttatctcata aagttgtttt ggacatggca 106620tgaatataat gcataggtac ttagaatatt atacacagtg tatacactca ataaacctag 106680caattgttaa tattatattc tttctctctc tctttctata tatatgcaca cacacataac 106740tatgattttt aatttatact acataggttt attgaatatt aattacgtgc catgatttta 106800ctaggtgagc attagaaaca tagtggtaga caagtttact ttggtccttc tgtcacagag 106860catacattcc aatgaggatg acaaaccaaa accaatttca taaacaaata aaaaataact 106920gcaattcata agagccaaga tagaaaccag gtagagaata aagcagccag ggtagagaat 106980aaggagggta agggtgagag gcctcttagg tgctcaggga atgcctctct gagaaagtgg 107040tgtcagagct gagactggag ggcttgtgtt atgtggcctc atggctgtgg atgaatgagt 107100acagttatca tggatttgcc tgaccaaatg caggtgacat atcttccata gctcttgtta 107160ttctccatat cccttttgcc atcacaatgg gaaatggggc ttagaaatat atacagacac 107220tttctcacca attgacttaa ctgggggcta accattaagt agtgaatccc ccagaccact 107280cacttttggt tagggtttga gttattttga gttagggttc atggaagtca tttgggatgt 107340gaaaatgcaa tcaggttttt tttttttttt ttttttgaga cggagtctcg ctctgtcacc 107400agtctggagt gcagtggtgc gatctcggct cactgcaagc tccgcctcct gggttcacgc 107460cattctcctg cctcagcctc ccgagtagct gggactacag gcgcccgcca ccacacccgg 107520ctaatttaat ttttttgtat ttttagtaga gacggggttt caccgtgtta gccaggatgg 107580tcttgatctc ctgacctcat gatccgcccg cctcggcctc ccaaagtgct gggattacag 107640gcgtgagtta ccgcacccgg cctatgcaat caggtatttg taataatgaa agcagtaatt 107700cagaaatatg ttaagcctga caaaagtctt ttcttgctgg cagctgcagc tgtctgggtc 107760cctagaggtt catcttactt actgaaatgt ggtttttaat tttaaaccaa gaaaaacaaa 107820actccaatgt atttgatttt tcaaaagaaa aagttattta acaagtcaaa atgaaagtaa 107880tatatgcata aagagtacag aaagtttaat atgagaacta aaactcacct ttttcttcag 107940gccaccactc cttgtcccca gtgatctctt caagggtaac cacttagatt cttagtaatt 108000tttatagaaa atttagcatg gatatcatac atctgtgtgt gtgtgtgtgt gtgtgtgtgt 108060gtgtgtgtgt gtgcattcca agtcttcaca aatggggttg tattataaat actgatatat 108120acctggctct ttcacttaat cagaaggcta agtgaccatc tgtttgagtc cctagaaata 108180tactgttttt ttatgattgt acaatgtctt tttttatgac agtatcacaa aatattgtat 108240tttagtctta tcaataagtc tttggattat tttcctgatt atttttatta taaataattc 108300tgtaaagaac attctagtca gtgggaatat tatctgttca gtaggctcaa tttctaacaa 108360tggaatcgct gagtcaaaga atgtttattc attgttgatt ttgatcatta ctaaatttta 108420ccaatctgta aggtgaaaac ttatatgtga tttgaatttt ttttactttt gattcagatt 108480gatacctttt gctgattact tgtcatttta ttttcatttc tttgaattgc ttatttacat 108540taattgatca gttttttatt atcttttttc tggtcctttt cttactaatt tgtacaagca 108600ttgtataaat tagaaaatta gctctcaact gtcaaaatat gatgccaacc ttttcttttt 108660tgttattgat cttttaactt tctatttttt ggcatttttc ttaatagttc cttggttttg 108720tgtcatgttt aggaaggcct ttcatattac aaaattaaag aaatatttcc ccatgttttc 108780ttttgataca tttatggttt cattttgtaa ttcaaaattt cagatccatc tggaatttat 108840tttggcataa ggagtcattt ccttttcaaa atgatgaagc agttgtcacc atattattta 108900ttgaataata tgttaatgac tcattattac tctaataaag tgtatggctt agatttcgga 108960atcagaaata actaaattca aatgttggtg tgaatgttct tttgttagat ttaatatagg 109020cttaatagtg tagagtcaca atatcttaac tgaagctctt ggcaccacat gcattatact 109080tcactatgaa ggatctctac ctgcattccc tggttgaacc tgctaattag gaggtttaca 109140gggtgtcagg tcatggtggc tgtcatctct tcctattggt atccactgaa atccccatat 109200ttcataaggt ttctggttgt tgatctactt agatctcttt ccaagccctg aatagtcttc 109260attctctcag atactctcag ctctcattct ctcagctact tctgaacccc ttcccttgcg 109320caggacatat ttctctattc ttctgaatct ttatgggtgg gactatgggg aactttctgc 109380agttatctca gacccctatt gcccagaatt tccatgcagc catctttatt acagtgttcc 109440ctgtggcagg ctggtgtctg gggatattct gtgaggctta ccgcctctct gacctccgcc 109500catggtatct ctcccttctc agatccacta aagttatctg gaggctccag tcttatcaac 109560catcctcttg ttcagacacc cagagcatct caaaccccgt ttttaccttt tgcatgctcc 109620tcttctttcc gtcccagtgg caaacttcag ctctgtctgg ggatggctgg gtaggaaaaa 109680ctggttctga gaaacttacc attaaagatc tttttgtcct atgcctggat atcagccttt 109740ggtaatccaa aggcaaggat ttaattcttt tgttctcttc actaaagtat caaccctacc 109800ctaaagcaag agacatctca aagtctttga tagggaagag acacaggtta agaaggaatc 109860actgaagaga aagcacatta gttaatatgt tggaattcta gtcctctatg ttactatgat 109920attcaatact atagaaaaac caactttcag agttaaaaaa aaagattaaa caaagccaaa 109980tttattactt ttccaggttt aataaaacaa ttttaagggg tttcatagtc atcattataa 110040aaaatgtcaa catagccatt caatgaactt agttggactt tagccaattg gtgaatatta 110100taaatacctg caaatatgtt tcattattag gtgttgccat cttggccctg cttcctacag 110160agttataaat tctctcatca ttgactcctg gtccacagaa tatctttgtt atacctaata 110220gaaacacccc taggtatcca ttattgtgat gtatttttcc ttttctcctc cagcattcca 110280atatcctggt ctctcatagt caaaattttc tattgggaga aagcaattga agggctgcct 110340taattattca gatttatttt tccttgaaat gtattttgaa gattttgatt ttggcagtaa 110400tgaatttcat acgaacattt tactggttat caaaataatt tgcaagaacc aactagggcc 110460ataatgttga ataaaaagta tttaaaatat tctatagtac attaagattt ttcacttgaa 110520attattatgc agtatgaaat ggataatttt tgactgcttg aaaatatttc agtcactaaa 110580ctaagtttag atttagattt cccaaagcca agaaaatgtt aacatgatgt tccacctaca 110640attttaggag gaagtctata taattcttga atttaaatgt ggatcactaa ttatagttat 110700aacactaaat tattcctgag tgggtttgtt tagtattcaa atgtcatccc agaggaggaa 110760tcttttatct atagagctgt catagataag caatataatt cgaaccctct ttctaactaa 110820aatttatttt taacttgaaa gaacacatca cactgcccac ccacttactc actgagaatt 110880gtgccagtgt gtggaatccc tgctacaaaa ttagctaaat tattttgcca ggaattcagg 110940gtaagctaag ttcattttgg accttgtcca acaaaattta tttagagtta atactagaga 111000agaatctcac ttgaaaacat ttcacctata tgtatgcggt gtcttggtag acagggtgcc 111060tgaggacagt ggcatagcaa ctttccagtg aggtaattta atttgttaaa ttaaataatt 111120agatttattc ctcatgttgc cttggggtga tgaggaggag ttaaggacat ggagaaaaaa 111180gggcacagta aattttggtg gttattttta agtctgatta atgatgtttt aactgcaggt 111240catagattta gccgatggac tttcgcaatt caggaaaact gtcagagaag actcattctc 111300actgaaggtt catttaggtt gtcagtagca aagaaaaaga tattgtacga agctgccttg 111360gaggcactcg cataaaaatt atgttaccat ttatctatag ttgcattgag ttattcttat 111420aaactgatga tactaccaac ccaaattgac aattcttgag aatacacaca cacacacgca 111480catgaaaagt tttcataaaa atgctgtttg ataatttgtg tcctgttatc tctaacaagt 111540aaacaggtaa aaataaaata ccttcatgcg gtgaagaagt ataaactgaa aaatagactc 111600ctattttgaa acctagaaaa aagttgtgct ttaaactttt ctctgtcaaa tgagaattgc 111660ttaattctta tacttaagga acgtgggaaa tgaaaaggca gaatgtatag tgttggtcct 111720ttgatggaat ttctgagaaa aaacaaacta ttccagatga tgactaaatc acatagtttt 111780aaatcttctg tagattttta atggtttttt ttcaaaaacg catattgttc aatataataa 111840atatttgtag agtcaggaaa atgacaaatt ctttcttcct aacaatttta caatgaaaag 111900taatggtagt ccaactaccg aaaattctaa gaaatttgtg gctttcagtt gtgactatga 111960caaagaaagt gactacagaa tgtgtcacat tccatcaaag tcactcaggg ctttgttact 112020ctttaaaaat gcttcttggt ggattcacaa atgtgcagta tgatacaaaa aagaaaaaaa 112080tcacaccagt tagtatcttt gctagagttt tgtagaaatt taagattatg ataatcccca 112140ccattcatcc ttttgaacca caaaattata gaaacttgat ttatactcta cagtatattt 112200tttgatggct taaagtatag aatgcaactt atttatctgt gattcttcta gaagggtagg 112260gaagagaaat cattttaaaa agtgtatcag aaaccaagct gagaagagtt gaggagcaca 112320tctgggctgg attcacgtct atgtaattca ctgtgagaat tcccattgaa ttacacagga 112380caataattag acccagagaa acagatcagt acttttctaa cttaacattt ttatcgtcat 112440ttcccccacc acacacactc ttttaagtag ttatttgcag gcttaccaat gataattttt 112500aagtatttga atatgttaaa ttaaatattc agttatgata gttaagtaaa taaagctgtt 112560gagttctgtc ttgcatgtac gactcccttt ctgactttca taaacattca gaagactcca 112620gctcattcag agaattttca ctaagaattt atttgctttt taaaatacag aaaagtcata 112680aaatagaaaa agtttaggca aagacttccc tttgcctgct tgggtggatg aatgaatgaa 112740ctagtgcatc acactgactg cctgctctgt gtcaggttct acttgaggcc ccagatacta 112800gcaagccctc caggttctca cagtctaatg agatgtagca atgtaaacag ctacttttta 112860tatgatgtga aaaaacagaa gtattaatga aatgctgtgg gaacataaac taactaggga 112920gtagtaattc tgccttggtg gtacttgggt tttgattttg atcagagaaa attagaacag 112980gtaatattta aggtagtttt gaagaatgag taaaattttc cagaaagttt ggggaatgta 113040attcctggta gagggaagcc tgtgaataaa tgcagtggca ggagcattca cagtgtgaga 113100ttctaaccgg tgtggacatg tagagagtca agtgtgaggc gatgaaggga gtgagagagg 113160agtaggtagc atggggacct ggaaggtagg tggaggccag atgaggaggt tctatcctca 113220gcattttttt cctctgtcct ctaggctcct tacctatttt gggtcatgga cctcttaggg 113280cagtggtttg caaaattcat tgcgtattag aatcacctac ggagatttaa aaaatcgttc 113340agaaacctag gccacatccc atagagcctc tgggaatgag atccaggact caataattta 113400aaaatctcca gatgctttca gtgtaaaata gtgcttctca gagtgtggtc ccatgatcag 113460cagcattagt atcagccaag aacttggtaa aaatgccaat tcccagacca aaattcagac 113520ctagtgaatt tgaaactatg atggggtcca gcaatctgtg ttttaaaaag ctctccagga 113580ggttttcatg tatgctaaag tttgagagtc actgtcatag agaatctaat gaagctgata 113640gtttttccag aacatgtacc gcatgttgaa ttttaaacat aattctaggt ggtagatgga 113700aattccctct gatccctttc cattttctcc cccaaatcct ggaatccagg ttaagaattc 113760tttctctggg cagtgagtgc cactgacaat atttcagtaa aagagggaca ggagaaactt 113820tggttataga atgatgactg ctgtaattta aacaatgaaa tggaatgggg agaggctgaa 113880gttaaccaca tcatgcagga ggttttggga ttataaaata aaatagaaca tggtgacaca 113940gaggttgtgt gtggtgatcg aaagggaaca gaaaatgatt ccaaggtttc taacttgagc 114000tcagagtgaa tgaaaactac ttgggagagg gactataaaa agagaaggct tggaggcaaa 114060taatggtaat tactggacat gtatggggag tgcattaaaa acaggtctag agcttagttg 114120agagatgtag gttgtggata actatatagc cacatatata aacaggtaga tgtagaccat 114180ttctcatcct ctgttgacat tttgggtctg ctaattcttc attttagagg gatattttgt 114240gcattttaag ctatttattg gcattccagc tctctatcca ctagatgcca gcagccaata 114300ccctctgcag tgatgacaac caaaaatgtc ttcagacatt gcctaatgtc ttttggtggc 114360aaaatcatcc cccattgaga accacaggtg taaatgaaac ctggcgagta gatgagatta 114420ttattgacta agtggagggc aagtggtgaa ggaggatcca ttaagtgttc ataaagattg 114480ggagaaagtg gcatcaagac agcacaaaaa ggctgagttc cccaaagaag gtggttgggt 114540atcaaacact acccaggaga ataagaattg agaagaggcc tttggatttg gtgatccggg 114600gattattgtg aaacagattc aatagacagc tattgaagag tgacgtgaaa gtgacactgc 114660aggaggtgga agaataaaca agaaaaagtg gagattgcga atctagatta cttctctaag 114720ggattggtta ggaaaagata gaagaggaga aagaggcaaa aagaataggg agattgctta 114780ttttaggaca taagagacta gcaaacatgg tggactgata gggaagtgcc agtgtttcca 114840gataaatata gagctttagg ctggcccaga atcttttctt tactaagagc tcaaaataca 114900gataatatta attatgaagg agagtatgga aatataaatt atagcagcaa catattgaat 114960aattgcaaca agtataggtg ttgcttattt catgtctatc atctaactat taatttttac 115020cagataaaac tagtacatta gggaatacat ttaatctaca atacaggaaa gtcctgttga 115080tatgattttg cctgtgggct gggtgactgt attagtagtg atgattttct tttttctttt 115140ttagcttctg tctttaatct tttcttttgc ttcttcataa ccagcatgag tgcaggggac 115200acacatacca tttggtacaa gccttatttg tcttgccttg ggtctggaac cacttttgca 115260gtaacacatt tagaaaagag aaatcaagag caaaaaaggc ttcaatatag tttatttttt 115320tttgccagta actttcttgt tgcacaacta ttatatatga aattattctt tctaaacatg 115380tgtgtgtgtg tgtgtgtgtg tgtgcacgta ctcatgtgtt tcagttccca ggttcttttc 115440atggctgcac aacttagctg caaggcctta ggtgccttat ttattctttc cagatttcat 115500gtccatcatc tgcacaatga gcagggggga atctatgtgc tttgaggttg tttctggctt 115560taaaattcta taattgtatg attttctttc tgcattatct ttgtttggtg agagcttgcc 115620ttaatctgat ctattccaga gcaaaatctc tcaggttgac ttgaagcttt aatttttact 115680ctggttttca cacctttttg tttattaggt cataagtaat gagctctgaa tgcttaagaa 115740cctgttcaag ttcacattag tagcttatca gggttgtaga tagagtgaaa acaaattcgg 115800gctttcctat tttctcattg cagtatttaa atccaggaga acatgttacc tatgaaaatg 115860tagaatcttt gcatgtctgt cttttcttca agagattaca aaacattttc caagtgtaat 115920aaatgaatcc tttccttatg aatcccattt gtaaacaact ggattgatag gttctaagca 115980ccttaataga gagtgatgtg tgagtttatt tctaccacta gatattgtaa ctgtgataaa 116040tgaatcaact atttataatg gtaagttgat tctccagaga ttgcatttaa atttattagt 116100ataatttgca ttagcactcc taactttaaa actcttctta aattccctca cccttgtgac 116160tgatactcag taggtgggaa ggattatcac aggggaatga ttggctagac aatgggttgt 116220cctaaataat atagtttttc ttgggataag aagtgaacaa agtttctttt gtcctggaag 116280ttaaatggga cttttttttc aatctaagat ataaattgca ttcctacatt aagttatgtg 116340tatggcaacg aaggcacttg cttatatttt taaggtaatg atttcattct ttgcatgaca 116400ttctgtccat tgaaattgag taaaatgttt tgttatataa aatttgttcc tcacagcacg 116460ctgactgtta ggtgttatag ttgtgtaaac tgagtccaag atattagcag gcttatgttt 116520acatggcaaa ttcgaagcag gattgctttg agatataaaa aacgttctga atatatgggt 116580gtattatcca ctcatccttt gactatgagt gggaaaggga aaagcgggag aaaaatagga 116640gaggggaaat gaagaaaggt acacagctga gtaaggagga ctaaagaggt atttacaccc 116700actgagaaaa caatgattta aaggtgctaa aaacaaactg gagaagtgtt cctacttcag 116760gatctggagg aaaaggaaaa gatttaaatc attttaagca aacatttcaa gcaggaccag 116820gacttcagtg aatactactc cagggctagc ttttattatc ttttcaattt tattattatg 116880acttattagc actggctgga ttcatacttt ttggagaatt atttaactgt tttgtgtcaa 116940catatctatg tataaaatct ggaatagtct aaaacttggg ggatggacca gtataaccac 117000agcttcagct cttcttcgtt atataaaaga gctggcagta cccttttttt gtttctcttc 117060tcatggcctt cttatttatc attttttcct ttctgctttt taaaaaaatc tagaatacat 117120ttctttatcc aaattctgta ttttcttctt aacagagctc caaacttaca ttctccatga 117180aaactttgat ggataaacca aatgcataaa tatctttgcc ttatttctaa cctctttgac 117240cctgatatat tcagttcata tcaacaagtt gtcattgtct tccagattgt ctagttatgt 117300cataaatatg tgtttcaact ccccaagtag gaactgattt tcctcctttt tgttcatctc 117360ctataacatt gagcacagtg ctgggcccca aatacttccg gaatgaatca tgacaagctg 117420aagactgtgt aaagataaaa agaaaatcat actgaagctc ttctggcaaa aggtcagttg 117480aaatttatga caaatgtcag tagctgaaga cgagatatgg gtgttgttcc cagatatgaa 117540agggagatga caccaacaca gagaaatgtg aagcaattgt tctgtattct taacagtctg 117600tgtataatcc taaatgatac acatgggttg gcatagtgtg ctcagtttga ttccagggat 117660atctcaggct tatttaacaa tgctaccctt agaacaacat tagcatacta atctatacta 117720ctggccgcga accacattcc ttggtatctt ttaactggat ggattgagac tattgcccca 117780gatagccagg ataatattta gcatggtggc ctaggatttt aaaagggcag tatcaggggg 117840gaaagtcatg gatttaaatc atttttcttt tccagggttc ttaccaacaa cacacccagt 117900aaaaaaaaaa aaaaaaaaaa atcatcttct gaagtttaaa tatctctttc ttttctcttg 117960cttgtgctgg attatcactc ctgtgcactt cactgagctt gaaaattgca aaagaaagac 118020acagcttccc caccccccag ctcctaaact actctgttta tagtcagttt gcctccctgg 118080atcttatgtt ttaacacatg ctgtagtgtt cagattgcaa acacagcgaa gggatgttta 118140aatcctaaaa acaaactgtt ttcattgcaa tgtgcaaaac agcattccat gaagacattt 118200caattaatat tagctgagtg aggggagtcc tctctctgtg gcctgttcag tgtttggcct 118260ctctctgctg agactgccaa ccagggtgct gccaattaat gccagctgtg atctattgtt 118320tgcttctgac acctgggaag tcccagatca tcagactcct gcaaacagca gctggcttag 118380tggaggctgc cagacttcgg ggattctgag atctctcagc gaaggtaggg ggccgtcttg 118440ttttagttaa ggggatagtc cggaatagcc agcttaaaac cccagatgac ttacattaaa 118500caacccccaa atttggggag gatcagccca agtctgtaag cctcgtatct tactctaggt 118560tcccataggg cagtctttta aaaaataaac aaatgttcct aatcctttct tataaagtcc 118620caacaagtcc ctgttatgct gtaaaggtat tttcaggact ccatacttct acaggaggtt 118680ctcaatttgg tccaattgtg ctataattta attttacacg tgttgaaaat gtgtatgccc 118740tttctttgcc taaaacccta gccttctatt acagtactca ctctgatctt cttactgata 118800agacctaaga gccaaccttt actgagtgct tattatgtgc taggcactga gccctttaca 118860agttttatca ttttatttca tttattttta ataagcatgt ttgatcatag taatgatgtc 118920aagtggatga aaagtcagtt gggtacaagg gtttcttttt ttaaccttca aatattattg 118980ttcttaaatg ataaccatgt tttattctga cttgggcccg ctttgaggaa gaacttgagc 119040aagttatgct gcctgactgt atttcactgt ggtggggttg ttagatgagc tgtctcattc 119100tttctttttt ggggggtttt gagagagttc tgatgttttt gcctagtcct ctggaattat 119160aaatgtcagg aagacagaag ctacctgtct gtgtcatatt agtgcccttc ctacaatctg 119220taaattctaa taaggccctg taggaatagc agaaaatacc atagactctt tctcctgaga 119280gagacaatgt tgagacgggt ggtaaccggc ccagtaagtc tggcccaaaa atatgactag 119340atcttctgtc tcctgatgcc ttctttgcat cctgaattct tttcatatgg atgcccttca 119400aagctggctg gtagccctcc ttctactcgg ggctcctagg cttcagggtt ggagcctgct 119460gatggtgtta aacacacagt gactttatgg tgctggtcat tgaggctaag gtgaagccag 119520ttgcacttga tttattcagt acatgattcc atgtctcaag aagtgaaaat ccagtgaact 119580gagcctgtgc cctagttctc attaacaact taatccaaat ccacttgaaa tgctcactct 119640tgaagtcgtc agtgatgatg gcatgatttg tttgctgagg tctgttagtt caataggata 119700attataaata gaatttataa acagttccga ataatactct atttcttgta aatcaatgta 119760aaatttccca aggagttgaa attaatttta cgtttgtgcg atatacaaat aagtgagagc 119820ccttcagtgt tcagtgctca gtgtgcaaaa ctacagagtc atctcacatg tttctttgaa 119880ataaactatg taaaactaag aaagtgggaa agaaaaatta gaacaaaaga tattctaggt 119940gatttcttat agtcatatca tgatcaggtt aatttatata agacacttga tacatttcat 120000ctacatcagc ttgtttagaa

gtctagagtc aaagccttct ctgatccaga cttggtggac 120060tatctgagca gaagaatgtg ctggcaggct gattgagcta tgtcatttta ttgagggttt 120120gatccaggaa agaattaaaa ccatactttg ctctaccccc tatacagtca tttagtacta 120180gaagtttgct tctataaaca aaaggccgtc atcacttctt cctctgttct ttagatcttt 120240gaaaatcaag ccaattcaca gtggccctat atgtaatttt tatagcctaa agaacatatc 120300agaagtatct gtctaatctc cctatccacc ctccactatt agatctagaa aaggcacgag 120360catcatataa aattcatgcc agaaaactat ctttatatta actataatta ttctaactat 120420aattattctg tgttggcatt tttcttctgc tgccctttat tacataagtg ggaaaactat 120480gattttaagt aaatgaaata gccactgaac taaaataaat gtgacttgat acatattctc 120540ttaatgaaat gtctgttgga gactagaata acgttttggg atactagcca agtcaaacct 120600taagactttt taggtttgac tgatgctagg gagctgctgg attctggaaa taaaacaaag 120660gtgttttaca cctgatagaa attattgaac atgtcgaaga caggttcaaa ggtattttca 120720tttcaactta aaaaaagtag tgcagggaac ataggtaagc ttagaatatg gctattttta 120780actgaaactg tactgacttt ctttttgaag atgctgatac aacttctgac actgttggtg 120840atgagtggtg cataacagcc gtttcagctc tgaggctatc aaatagtgaa agagaatctt 120900ctacatacaa atcccaggac accagaacag ctcttaacag ccctcacttt cctgagtaag 120960ctgaaggtgg ctaactaaaa ggcctgagag aaattactgc ctaaacaatg aaggaaggag 121020aaaaccccca aggaagacaa tgtaaagatt aagtaacaag gtttcaattt gatacaatgt 121080ggggaatttc agtttaattc taatttatac taatttctca taattgtctt gatcctttac 121140gtagagatcg gtttggataa aggaatcata gctatgagaa ggcagttagt tactcagtga 121200atccaaatgg caaattctct gatgagaaag gggatgtcct cagggaggtc taatcatatg 121260ttttcttaaa ccaaactctt ttccttttca ttaggctttc ttgaggcaat tggctaatta 121320atgataataa atagttattg agcatcacag atattctaaa tcctccagtg tcatacattt 121380ggaaggaaaa aagaccagta gataaactta ggaagagtgt gtgtgtgtgt gtgtgtgtgt 121440gtgtgtgtgt gtgtatgtgt aacaaacatg ccttaaaacc aggaattgtg gttgtaaact 121500gggaagacat acataaaaga tgggatggct taagtcacag agacagacag aaacttcact 121560ttgtgtgatc atgcagagct ctccttatat ataataggtg taagaataat catttccaca 121620tctgtatttg gactttggcc ccatctctta aatcagagtt ataaattctg tgaagctgat 121680taatcttatt ttatgggtag ctgtaaataa agagaaaggc tgtctttcag ccttacttaa 121740ggaaaaacaa gaaatctttg caaaatagaa atttgggaaa ggatgttgag agggtatgat 121800ggtaggtaca cgggagacat actgggtttg ctcttgttat ctagggaatg gaatgagggt 121860gtcttttcaa agcagggggt aataaaaaga gcttaggctt tggggtgcag cagctctgac 121920tttggatcct ggctctccac tgaccagctt tgtgactttg gacatgttac ttaacctctc 121980tgacgccaag ttttatttgt aaaaatggag ataataccca gattacagaa ttattttggg 122040aattagggat gattataaaa gtacacaggg atgattagta aagtacacag tgtttggcac 122100ttaataatcg gtagctgtta ttattgatat tactgtttga ttgcaaagct gaaaaggcct 122160cagaaatcac aatcagatgg ctcatgtgag atcatgatgg ggtcctctct tctgaaaaca 122220catgctactt aagtctgtga gaggaattat ggatgaattg tcacagatta aactttatga 122280ggtagctctc gttggtgcgt gatggtttgc actttgctat tcttcctttt cgcatgagag 122340aatctcatct ctggaatgaa atttacctgg atttatcaca agagcacatg cttcgagttt 122400gatttggatg cgatcacctg ttaccctgaa atgaaaactt ctttttcttg ttttgggaga 122460atctccctgg cttaattgaa gaagtttccc ctcaaagatt cattcagatt cactcagatc 122520agagggtaat cttctaattt aaaagccaat ttatgacgaa aatcctaatg caaagaggca 122580aacgtgtcta ctagagttgg gcacaataga tttgtgtagt tgtaacattt tttgctactt 122640tttttcccca agcaatctca aaaacaacaa caaaaaagta tgttaacaag actagttgaa 122700attaatactc tgctattaga gtgtacgcgg accggctcac tcccttcttt tccaccgtgc 122760tgcacgctgc agttcctaga agacacgaca cgctccttcc ttaacgaatt ccatccctga 122820aatcagtgac gattggcatc aggccaggtc gacttctttt ggactcctta caagaccttg 122880ttcgtatttt gtttttatta gttcttacta ttctaatttc tctcactgtc gcatttttca 122940ttggtttaag ctctcagcag tcttgctttc ttgccaactt aaaaacaaac aaataaaacc 123000aagttagcac tggggcgggt ttcccccaac tcacagcact aattgtataa tcaccactca 123060aaagtcaaca gactaagtcc aatttgtttt taaacacggg caactcttga gcctttgcat 123120ttggaaatgt ctttctgccc atttgttgga tttagaataa taaactgcct atgtaaatga 123180gagggtgcca gccttcagca gcccacaccc tcaaactgcc actcgacgat tcaaacatac 123240agttcttgag gaaaagcctg ttgtgaaagt taatggttat aaaaagcact tatttacatc 123300tgttttagtg tagccttgaa gagtgcaatt aattaagaag tttctggtgt ggtaagaata 123360atcatacagc aggaatgcaa acacgtgatt tccaaagaga attttattcc atatggtatt 123420tttcccctaa ctaagaatta attgcaactt taaagaaggt ttgattgtgt ctatgaaaaa 123480cattttaact actgaggaat tctaaatgac ttcaatttaa aattttcttt ttttacattg 123540ttagagtcaa gaaataagtg ctttcatcaa gctctgagtt acagaattta ttagagtaaa 123600gggaagagga taaccttcag atgtcttctg tggtgaaatg ttgaagttaa aaattttaaa 123660tgtttgcaga ctaaagacat cctcggctgt gaggcctaaa agaataaaca aaatgtgaca 123720tgttttattt tgttacttaa cccttgtata aaaaagcaca acaaaaaatg tatataaatc 123780atatttttat ttctttctga aaacacggac atggattttt ggtgatgatc ttctatgtag 123840tggcatcatg ccataggtta aagagtggtt agtgtgtatg caaatagctt tattttggta 123900tggcagtttc ttaaagcaga aagttttaaa ggtatacaac ataaattctg tgaagatatg 123960ttcttctcct tctgttgcaa tatgctacgc attccagtat tttgctgtag cccagcaatg 124020aacaatgcaa agagatgaag aggggaaaaa caaaaacatg gcagctgaaa gcaagactgg 124080tggagaaagg atgctcgttg ctgcctaggc acagaaagag aatgtgacat cctagaggag 124140ggaaaaagaa taaaaacaac ttggatacag gcagagaacc tatttctatc aaatgcagtt 124200acaacagttg ctgagaagaa aatttcaatc aataacaaag agctcttatc acatgcaact 124260ttaatttgat tggcatgaca tttccattat atgctaatga attacagatg atgagaacgg 124320ctgttgcctt ctgagtgcaa ttgaacattc atcaatcagt tacttaaatg ccagactgtc 124380tgaaatatta aaggaagttg ttgctatttt ggagatgaaa gtaaacaaac tggaaatctt 124440gaattaattg agaaaaggat acacgttgat aagtaggatt tggcaagtta ttacttctct 124500gagtggatgt cagtgcaggt atcttattct cagtggaata agtgtgtccc tgcaaagcta 124560actgaaaaac aaattctctt aagtgaatcc tgtttttctg ttgactagca ttatacaatt 124620attgactatt actctgggga aaaaaaggct gatcctagaa tgtaataaga tcactctcaa 124680gtatgcaaca tttttgacaa ttcaagttta aaggaaaata actatccact aaaatattaa 124740caataatcct caaaatagca ggaacattga aattatcaca attgagtaac agtactaaat 124800aaagttgttg caaaagaaaa atgcaaaaga aaaaacacat gatataaaat tactaaattt 124860tcatcctttc cccatcaaaa aaatccaaaa tgtttggagc tttagaattt taattaaggt 124920tttcatcagt ctcaagccac ctaaagatgt ctaagtttat tttttcatca tcctttccac 124980ttccctttta cttgtcctta gcattttact ctagatggtt ttcctattaa tttaaaaaat 125040agttatgctg ctttacatgt gaggaaatgg aacattcagc taagtgctaa aatatctaat 125100ctttgcaaaa cacgaatttt gttctgcttt ctgcagagac atggtcagct ggggtattta 125160ggtaatggca aagatgatca atttgcaaac tttttgtgct ataaaaattg atttgttacc 125220actacctcac accaattagg atggctgcta ttaaaaaagc cagaatgtaa caagtgttgg 125280caaagatgta gagaaattgg aacccttgtg cactgttggt aagaaagtaa catgccagct 125340tctgtggaaa acagcacgat agtttctcaa aaattaaaaa taggattacc ataatcataa 125400ttatctagca attctacctc tgcatatgta tcccacataa ttgaagagat atttatatac 125460ccatgctcat agtcttaaaa tatgctagtt ttggtggaaa acttgagtta aaatttctta 125520aatattgaaa cgtcttgtca aaactttcca agctattaac ttttcccttc ctgaagtctt 125580attcatatcc agccaaaatg tggaggcaac ccaagtgtcc actgactggt gagtggataa 125640acaaaatgtg gtctatacat acaacaaact attatccagt ctttcaaagg agggaattct 125700gacacatcat cagcatggat aatccttgag gacattatgc taagtgaaat aagccagtca 125760caaaggacaa atactgtagg attccattta tatgaggaac tagaatagac aaatttacaa 125820agacagaaag taaaatgatg gttgccaggg gctagagtga ggagggaatg ggaattactg 125880ttttatgggt ctgaagtttc tatctggaaa gatgaaagat gttctggaga tggatggtgg 125940tggtagttac acagcaatgt gaatgtactt agtgctcctg aactgtatat ttaaaataat 126000caagatggta aatgttatgt gatatgtaat taattaattc acaattaata atgaaatttt 126060aaaagttgta aaaaatggat ttgttgccaa atgactagat ttacgaatta aagcaatgtc 126120tgctatacta attaggatgt attactgaat caacacagtt ttttgagaat tattagactt 126180ttctgcaacg tacatagaga gaaaatgtca aatagatttg gctgcctctg ttcgaacaag 126240cagaggtggc actaaatgtt ccaaattcta ttcatatgag ggcacttctt cttaaaacac 126300ataactttat ttctttgtag ctctcttagt aaattctcca gagagtaaga gcaaaagctg 126360ggggtgtttg aggaatagcc aggggtctga gtgactggag aaacatgaag aaggtggggc 126420agagggagga ggttagttca gatgaagtca gagagccaac tagggaccat ggctttttcc 126480ttgcacagga tggggaaacc atggcagggt tttgaataga agagtattgt ggggtttttt 126540ttgtttgttt tttttgagac agagtctcac ttactctgtt gccctgtgca gtggtgcaat 126600ctcagctcac tgcaacgttg gcctcccagg ttcaagagat tctcctgcct cagcctcccg 126660agtagctggg attacaggca tgcaacacca caccctggat aatttttgaa tttttagtag 126720aggcgggggt ttcactgtgt tggccaggct ggtctcaaac tcctgacctc aagtgatctg 126780cccgcctctg cctcccaaag tcctgggatt acaggtgtga gccactgcac ccggccatgt 126840gatttgatat atgtttaaaa agtacacaaa ctgtgtacta caagggagat gaaatggaag 126900tgcagaaatg gaattctaga ggtgagagat tactttcacc tgggggaatc ctggaaacaa 126960aaatctgagg ctgagctaca gaggagaaag aaatgtattc cttttcacat ttgagtatgc 127020ttgagaatct ctaaacagca caactgcacc aagaattctc ttgcattatt actctgtact 127080attaatttgt ttgttgatag cagcaaatga gctttcactt actgagtcct gtgtgagccc 127140agactatttc agcacttaag agtccttcaa tgactacctc agaggtggat ctcagggtgt 127200ctggggctcc tatgcacata tgtttcttgt tgtttacaaa gctctttgga cttaatctga 127260atgagagttg ccctaaaaag ctggagacaa atgattagcc ataaagtttt atttttcaac 127320atttaatttt tagaataatt aatttaataa cagattttag ggcatgtatt ccttgctact 127380tagtggttta tgtctctttt aggagaatac ctcattatgt tctctatatt gtcacataag 127440agatgctggt tctgcttttg attctaggag tcagtttaag tgaccataga tgatatacgt 127500agtgtatatc atctagggct ggaaggtaat caattaacaa acggtttaaa ttgcttgggc 127560ctgaagattc tagagtttct gcaagaacaa aatcaagcct gtcttctata tccttgagaa 127620aaccttctat atttatagta gtgctttcct caaattcaac ctgactgtaa aaatgcatga 127680tgatgtgcca aataaaggaa gtgtgcgtgc acacacacac acacacaaaa ttagtaagaa 127740ttagtaacaa caaattctaa acacaagcct tgatatgcta aaaactataa tatggtaaaa 127800aaataaaaat aaaaaaatca aatgctttca gcttaaaatg ccctgccaag gtttttttct 127860tcaacccaaa atgctcactt aagtaatctt ctaccacaaa gttaaagcta tacagagtaa 127920acagaaatag aactcaactg tgcccttcct tggtatattc aaataacttt cttcaacact 127980tatgcaattc tgtgaattgg taacgtcatt gaaaatatgc tagttttggt ataagttgag 128040ttaaaatgtc tcaaatattg aaaagtcttg tcaaaagttt ccaaactatt ttttctctcc 128100ctgaagtctt tatttggtgc aaattcagca tttaaatatt tgtgctaatt taagttttca 128160ttacattttt tctttggaaa aaaaatttgg ttgaatgatt gactaattat aaaagatatg 128220gcccaaatta aaaataactg aagtttatga cattgaagga agatatgctt tatctctgta 128280ctctaaatac aattctattc tttttcctga aattatatct gtgaatttct gtattctttc 128340tattttgtta aaaaatttat ttgaaaaatg ctgaaaaacc caaaatgcaa aaactactta 128400gaattaaaca atttaggaaa caacacagat gtacaagtga gagccatccc taggcactgc 128460ccctgcttca ctaattctac aacagagtgg taacccagtt aacctatgtt tttcaatttt 128520catgtgttta taggcagtcc tatgtataca tatctaccaa acaactgact ctgctattca 128580ttggtcctgt aatttaactt cgtttttctt tatatctatg gctagctttc tatctatcta 128640attctatctt tctatctaat tctacctcag atgtcacagt ctggaggtct gctggggaaa 128700gggactacac ataaattttg cttggtttga gccatttaat gtgtttgaaa aaatggaatt 128760tgcatccctt ttgatgacag gccttctgat tcctattaat ttcactcaac tctttattat 128820catatctgct tagtgatctg aagcatttat atttgcggct tccaaataat attccattat 128880attgttatag tctcatttat ttagcccctc ctcatattgg aacttgaagt ggctcttcct 128940ccccaccttt ttttttatta ttaaaaacaa tgctgtcctg atcattctta agtacctact 129000ctatgttatt tctttggtat aaatttttga aagtggggtt gctcagttgt agaatattac 129060atatttaaaa cttttataga cagtgcaaat tactctaaaa cacaaaatta ccatttatac 129120ttccaggaat agtatatagg agttaaattt tcttataccc tttaccaata ggttgtatcg 129180acttgttaaa tttttttcat tctgaatagc gataaaatta ttatggcttt aaaaagtatt 129240ttttaaacta ttgatgaaat tgaccttttc atatgtttat tgctatatgt atatatatat 129300gtatatatat agtctgtgaa tttcacaatc ctatgctatg actgtttttc atatggataa 129360tctgtctgaa atgtaaaata ttaatctttg ctttaaatgt tgaagataac tttttttagt 129420ttttggatgt cttttaactt tgttctttta ccttttgaca tataggaatt taagatttgt 129480atttaattaa atttcttatg tgtcgcttta aaaacttctt tgtcttagta agtttgaggg 129540gcaatttatc aatttgtact cttatacagt gttggtggga atgtaaggaa tagtacacat 129600aagcaatggt aaaatttggc aaaaaaatcc catatataca cacacacaca cacacacaca 129660cacacacaca cacacacaca tgcatatatg gcatagcaca agaatgtgca attcacagaa 129720tatatattat atagcaacaa aatatggaaa gaggtcaatt attatatatg tgtagaaata 129780cacacagcac tgtttaacac agctttaagt gtaagaacaa agcccataag atgttcattt 129840cttcaatatt tagtaaatat atcaaacatt ttctgtacac aaagcacatt tgaaagcttg 129900aaacttaaaa tctaactagt ttctaaaata ccttaaaaaa tactgtggca tggaagctag 129960tgttcttttg gaattttcat tttcgattta tatgtgtttt ctgaaaacaa cagaacacat 130020acaacacctc tccctgccgt attgttgtat taagcaatgt tgggattgat gtgaagatta 130080aatcttcatg tctgccttta tctgtctcct acttttacat gtttgacctg tgaattgaac 130140tgattttgtt tacttctatt cttttatctc ttttatttgt gataaatttt caatattaat 130200ttacctaaag gaggtatctt atttgtaatt gaaatctcaa ccatagcagt aaggtgtgga 130260tttattctga ggatgataca atggcctaaa ttaatgtggc aatggattcc agcagttttt 130320ctgttttaag tttgaaaaac tttgtaagtc ttacttcttg atgatcagag aggttgttgt 130380gggttttgtg tggcacttct ctcccttaag ggatctgact gatagaatcg acgtttaaag 130440aaaagtacat gtgtgtgtat atacatatta tatataatat aatatatatt atatgttata 130500tagtatgtat tatatataat ataatatata tgttatatcg tatgtattat atataatata 130560tattatatgt tatatatatt atgtattata tatattatat acatgataat aatttgcaag 130620gagtgtcaac ataataaagc tttagtgcag catcctagag atatttgcag attaacacct 130680tgtttgctaa ttgtgtcagt gctcaactta agttgcaaaa tatgaatgta aagagtatga 130740gagtcttgca gatgggattg ttaaagcaaa catctgcaca tatgtttcag cttcagctca 130800tagaaataag gcttctactg tctctccata tggaaaatca ttgaagagag catatttgtc 130860ccctctcttg ctggcccact tttcactcct atctactcct ataaccagaa aaattatgta 130920tattatatat attatatatt atatgagata tataatatgt atatataata tgttacatat 130980tataacatat aatatgtata tataatatgt tatatattat aatatataat atgtattata 131040tattatataa tatgtattgt atattatatg tatatatata catatatatg tattatatat 131100tatatataat atgtattata tataatatat aatatgtatt atatatacta tatgcatatg 131160tattatatgt attatatatt atatataata tgtatataca cacacatata tataaagcag 131220ctcttttgag tatatgttca agtgtgctat acctctccat ccagtgctct cttacaaatg 131280ctggatttgg gacttcctgt cttaacctgg gatcccacat tagggacttc ctctcttgac 131340ctgggatccg cacatttcag attgcctgta gagcttttcc agcctttaag acaaactctg 131400aatccataaa ttgagccagt tgctggaaag ctaaaatcct atctctgtca aagtggagca 131460gagagacaaa ggtgagtcca caaaaacaga ttttcttacc tcagagtgat gtgaaggaca 131520aggtggctcc agtgtctaaa ctctgggagt gagagaatag cgaagaactg cccctgaatc 131580cagtaagtta aagaaacggc agtgctagcc tggttcctgc ttttgaggca cagtagccct 131640gcccattatt agcaaatgaa cccagctctt gtcaattgga tcaggaactc atttttctgg 131700gagtagatag gagtgaaaag caggccagca agagagggga caaaaatgct ctcttcaatg 131760attttccata tggagagaca gtagaagcct tatttctatg agctgaagct aaaacatatg 131820tggagatgtt tgctttaaca atcccatctg caagactctc atactcttta cattcatatt 131880ttgcaactta agttgagcac tgacacaatt agcaaacaag gtgttaatct gcaaatatct 131940ctaggatgct gcactaaagc tttattatgt tgacacttac tccttccaaa ttatttttaa 132000ttacagcaat agtaagaatg gacaatggaa accctaactg cattgtttta gttaattaaa 132060catgggtctc ccagttgtgt ttttgttact tgttgcatgc aggtaactat aggttagatg 132120ctttgggaac ataaatatga atatgatgga ctgtagctac agaaagatca taatacaata 132180caattattga gcatgaagct ttgaaattat actgcctgag tttgggtatt tgttgtccca 132240cttaatgtct ctacacttca gtttctttag ctataaaatg gggacgataa taaggttata 132300taaagattaa attagatagt atatataaag catttggcat agtgcctgga acttggtaag 132360tattcaaaaa atgtttgtta ctatggtaat gacgactgta ctatgactgc taccactatc 132420ggatagtgga tgaattactc aacattgact tccaaatcac ccttctgagg aatagagccg 132480taaatgttac ctacggtaaa agttgtcatg tgactctact gtcctgccca caattgattg 132540aactggcagt tggtacctac aaccaatgat tctgagttat cgtgttgaca tacttttgaa 132600tagtagaact tttgccaacc tcagagagac tgaagtctat tcacagttat agcaccagtg 132660actggctcac cttgggcacc atgtggctgg gtaattattt gtgatactca ttccaccttt 132720gtgctcatga gtcacggttc cagtactgaa ctgaaactag tctatgtctt cctcacagtc 132780acctacaccc agaatatccc atagcctacc catgcagttt agtgatttaa cccacaggca 132840ttgccttaat aatatgtttt gcagttgtgc ctgcagactt gagagtgact gaccatcaca 132900ggggggatta tttgccttct atttgagagc tgccacttcc tctaggaaag tcatgtatta 132960caatggatcc cagtgcttgt gtatgtgcac atgggatgga atgggaatga atgtgtcaga 133020attaccttca ctcccaacaa gacatcctga aaataccacg tgggtaagcc agtgatttat 133080tccattatga cttttatatt tccccttatt aaggttcaag attattcgta acatgggaag 133140tataattgca cagacaggta gagattttgg agtctttatt gtttagtata tagctaggcc 133200actgaaatat cgagatccta taagagtgtg gcttaaaaga acaaagctgg aggcatcacg 133260ctacctgact tcaaactata ctacaaggct acagtaacca aaacagcatg gtgctggtac 133320caaaacagag atatagacca atggaacaga acagagccct cagaaataat accacacatc 133380tacaaccatc tgctctttga caaacctgac aagaacaaga aatagggaaa ggattcccta 133440tttaataaat ggtgctggaa aaactggcta gccatatgta gaaagctgaa actggatcca 133500ttccttacac tttatacaaa aattaattca agatggatta aagacttaaa tgttagacct 133560aaaaccataa aaaccctaga agaaaaccta ggcattacca ttcaggacat aggcatgggc 133620aaggacttcg tgtctaaaac accaaaagca atggcaacaa aagccaaaat tgacaaatgg 133680gatctaatta aactaaagag cttctgcaca gcaaaagaaa ctaccatcag agtgaacagg 133740caacctacag aatgggagaa aatgtttgca atctactcat ctgacaaagg gctaatatcc 133800agaatctaca aagaattcaa acaaatttac aagaaaaaaa caaccgcatc aaaaagtggg 133860tgaaggatat gaacagacac ttctcaaaag aagacattta tacagccaaa agacacatga 133920aaaaatgctc atcatcactg gccatcagag aaatgcaaat caaaaccaca atgagatatc 133980atctcatacc agttagaatg gcgatcatca aaaagtcagg aaacaacagg tgctggagag 134040gatgtggaga aacaggaaca cttttacact gttggtggga ctgtaaacta gttcaaccat 134100tgtggaagct ggtgtggcga ttcctcaagg aactagaact agaaatacca tttgacccac 134160aaatcccatt actgggtata tacccaaagg attataaaac atgctgctat aaagacacat 134220gcacacgtat gtttattgca gcactattca caatagcaaa gacttggaac caacccaaat 134280gtccatcaat gatagactgg attaacaaaa tatggcacat atacaccatg gaatactatg 134340cagccataaa aaggatgagt tcatgtcctt tgtagggaca tggatgaagc tggaaaccat 134400cattctcagc atactattgc aaggactaaa aaccaaacac tgcatattct cactcatagg 134460tgggaattga acaatgagaa cacttggaca tgggaagggg aacatcacac accagggcct 134520gtcatggagt cgggggaggg ggagggatac attaggagat atacctaatg ttaaatgacg 134580agttaatggg tgcagcacac caacatggtg catgtataca tatgtaacaa acctgcatgt 134640tgtgcacatg taccctagaa cttaaagtgt aattaaaaga aaaaaaaaga ctgtggctta 134700aaaataagga attttatttc tttttcacat tagagttcca acatgacagg gaaaccatgt 134760tctttatgta catttaagga ctcagttttg ctcaaagtca tttttctgac ataccctagg 134820gaactgtcat catctgcatg gtctatgctg ggtcaccacc atgtctgtgt tcaggctcgt 134880ggaaggagga ataagaaagt gaaaatggca agcttgatgc taggagggaa gcttataggc 134940cagtcccaga agtggcaaag accacttccc ttcagttcca ctggtgggga cctagttcat 135000ggctgctctt caatggagag agactgagaa ttgtaggtac tctaactgca cagccatgtg 135060tcctagatga agaggagaag

gggtttttgg agggtaacct ccagtctcac cacagaggcc 135120ttgttgagaa acatgagatg tacaactgta ttatcaaaca ttaactgtga ccaggagaca 135180ctaagaaaat acctattctc actgagtttt tgggaaaaag cttgtgattt ccaaagatta 135240taactttata ctcaattaat atacttacat atacacatat tcataaatat attttgaata 135300aattctcaga gatgatattg caagacatac actgtaaatg tgggacatga tgtgacacac 135360tataacagtt gctgtactat aaggacttta gatttatgat ttgggatacc tgagatttag 135420tgctgtcact gccattaaat gggtaacttt gtccaatgtg tttaacattt ctgggtctca 135480ctgcccaaga tatcttaagt tttaacagta cagtcctgtc ctgttgattc tacctctata 135540atagatctta acagcatcca ttcttctggt cataattcct tccagagttt ataatccaac 135600ttcttttcct tctctagccc cactgaggtt attcctccca gattgatccc actgatgttc 135660ttccagtccc tagacctctc taaacgcttc ttgctcatga cctctgcatc tggtagtctc 135720tctgactgcc ccttggtctt ctctcttttg tgtggccatt tccttctcat ccttcaggtc 135780tcagctttga tgtctcttct ttagaggaac ttctcctacc actctgtcta aagtttttta 135840gtttatctca aactcagttt atttcttcaa ccatacttgg tattgtcttt tgtatctatt 135900tatttgtttt ctttctgttc ccctcattgc actattaact tcactgaact attaaggtaa 135960tgggatatat ctgtattttt catcctttta atcctatcac ctggcacagt gccctgcacc 136020tagctggtgc tcagtaaata tatgttgaat aaattaatac aggaatttag aaatgtgtgg 136080tctaaatagg taagtacatt atttaatgta attaaaaaat tcatacttct tcaaaatttt 136140atggaaaaac ttaaaaaaat cagcgaacct tattttgaaa caaatatttc ataatatgaa 136200tttctctcta ttaattgtgt ttctttttaa ttttagattt actatcctat ctatctttaa 136260atgttctttg atttgaatac ttctgcaatc cagttaagat ggaaggtttg ggggagagaa 136320ctcactatac ttaggctatc attttgtttt gtataatttt aatcatgaca gtctttatca 136380aatccatgtc tatttaaata tttattttct tttgcatatg ttgggatgga atatgttgtt 136440ttaattagcg gcaccgcttt cttgttgcta aatgtattgc tctggcattt gaccaaaagc 136500aaaacagaat caacacagtg taatctatgg aaagggaact gaatctcact gcacatagta 136560tttcatcagc tgtaggttaa gggatagtct atccagtaaa gaatgtatat agcaactata 136620cctctatact ccatgacagt cagagctgcc atttctggtt ctcagaagaa acttttttat 136680ttgacttgga tcagcctaca gctagatgac aataacataa aataaagttg aagaaaaaaa 136740cagttgttaa ataaatccat tactttaacg agctctcctt ctgtatgttt tataagggga 136800atatcaaact ttttagactc taatgagagt ttatcacata aatgtacact gaataaaaag 136860aatcatgcag ttgtgtcaga aaaagtagca aaggcctttt tatgagagct gacttgtaat 136920atgtatatat gttactggct aatttgatta tggtaaatca tcattgtatt ttatttatta 136980acttatgatt tcacagttgc cggctgagtt ccctttaaca aactccatat ttattgaatt 137040ccctctatgc acaaggtata atgggcattt attttttcag ataggcaatc atcttagtca 137100cattattgtg aaatatatga gtactatata attatcttca aaaggaaatc ctgaatattt 137160aatattaata tttaatgtca ttaaagtcaa atttatctct attgcctgtc tttttttttt 137220tttctaaagc agagcaattg aaagctgaaa ggtgaaaagg agtgggaaaa gctggttcac 137280catggctcaa acacagctca aactttaccc agcaatgagc tttctattgg cttgacttgg 137340aaaatacaat ttatatgtgt catgtaaaag gggaaatgat acatcagaac tttattataa 137400cactattctc tctaaagtgt aatttagcac aagatatgac ttgtaaaggt taccactaaa 137460actctatcaa aacatcattg ttttatatct gtgagatata aaatatccct gaaatcagga 137520agccttccca cagatttaca gattaactct aaactttaga tcttttttct cttttttagt 137580acatacatgc gtgtcactaa aatgtctgta tataacatgt gaaatattgt tactcaagaa 137640atttcttaag agcaggttaa ttttataaga caatggcctt atgtttctac gtataaaata 137700tctacaggcc ttctctgcta aaatcccagt tttgtttagt tccaagtaat gtgatagaag 137760ggtctattat aaaactattt gcaaatgtgg aagccgtaga ttttactatg gatttaagaa 137820gggagcttac tttaagattt tctggcatag ggtgtttttt tttcccttaa aaaaaaccat 137880atttaggaaa gtaaataggg agacaaaggc taagaaaaca aaaattattt aagattttag 137940gctgtacaaa taaaattgta gaaacaaaat tattaaaaat agcttactga ccaaattcag 138000ctgagattta cagccacacc ccacatcatc atagtatcat attggtaatt gtaacattct 138060tttcatatga aaaaaaatca agcacgtgta aagttatcct aattgaaaaa catttcagga 138120caatatgttt attacttctt attcatgtac attttcaatt acagcccaaa tttatacaca 138180tgttgcttgc aaaatgccat ttggatataa tatggtaaat tttaaaaacc acaaaatcct 138240tagctttata acacattcac agtaagtata aacctaagct atggcttagt atgtaccact 138300gctacctctg ggaaaaaaaa ataaaagtgt tggggaattt ttatgcaatg gattcagtta 138360gctcaatttt tattgaagat agcattaaaa aatccatagt taagcaatca ttactataat 138420agtgattgtt tttctaagtg ttgagagttt gggctgagag agagtgtatt tgaagacatc 138480taaaagatgt ttcctcctct ctggaaatta tatacttatg tagttgtttt tggttgactt 138540ttattgtaca attacatatt tattttaatt attgtgtagt tatatatata tatatatatg 138600cttctctttc ttatgtgtat ataaatgagg gaatattata atcactcttt tctgaatcat 138660ttcctcttta ctgactagat ggtaggtacc tctgggcctc ctaataaatg gaaacaaggg 138720ggaataatac acatcaagtc agtgtggaat ttggagaaaa cttatagttc taaatgcttg 138780tcaacatcat catgaaaatg ttaactgtgt ggccattgca tgtgatgcat tgctgcctca 138840cttataactt ctagaactct atagttgctc accaaccaca tccacacaga ctgacataaa 138900aggtatacac ccgacagcat aagcacatag tgagaaaata aaagaaataa taaaaaccta 138960cacagtagcc tggtgtatca ctattgggtg gtaactgaga gtctgtgaaa agtgtttcat 139020gtttagtcag ggtgaggcca ggaggagctt gtcaatgcct ttcagttgtc tagaaagact 139080tcatggagac agagattttt aagaagggag tgaagtcatt aagtttgcag agaaagagta 139140tgttaggtgt aaggtatgtg gtcatgaaga tggcttatta ttattagagt aagaagatta 139200aataaggaag gcaaagggta gatttgcttc ctgggagtgg cgtataaaaa cacaatgata 139260tgttttcatg agctaatgca agcaaaagcg attgatgagc agaaaaataa cctcagcaaa 139320agtgtcaaga atcaaataga gagaagtgta cctgagtggg agagagtcag ctgatattgt 139380ctagctcata ggattgagaa gagatatttg aggtgtgaag aaagttggga caggggttag 139440aaatgtcggg atgtacactg aagtagcttg aatatgaaac aagtaagaga ggaacaaaaa 139500ctcaagggga aaacatgtgt actgaaatat tccttataca tagagggaat ttttatcaaa 139560agctgtgcat tcatatgatt tggtctttga ggaattaatt ttaaagaact gttaaaatgt 139620gttctttgtg atccagtgta ggccacgggg aattgataat gttcctagaa gttgtaatca 139680gctcaattga gtaaagaccc tttaccctag gaccatatcc attatcatag cccaaaacac 139740ctccaagtgc ccattggcca tttggcccag ttctaggtac tgtgtagcca agcttttggg 139800acacagtttt caaaagcaga tcaaaatatt tttgaggcat agcaatcatc taatctgcct 139860gaagaattat gttaccgtga aaagtgatgt cttaaaccca taaatttgcg atgttagaat 139920gagtcatttt ttatatcctt tacaagcgaa aaagtactca ggcctgttga caaagcaaag 139980aaatggtgtc aaaaataatt ttaaaacctt ttgacctata aggtttaaaa gaattaggca 140040cagattcttt gctttaagcc tcttggtgac aagaaatatg gtactctgcc agactgtttg 140100attgacagta gcgtttgtag tttcagctgc taatacagta tgcttcttgt atattctggg 140160tgggtaaata tagaagacta tttgtcacct gggttgatgg attacaaata agtaagacaa 140220tgtgtttagg attctgaagc ataaatgaaa agtcaaaaac cttatattat ttacaatata 140280acgtttatgc tttcagattt gttatcccta ttacatcaat aaaaatgatt gggttttaaa 140340ataagcatgc aactcatttt taaccatctc ttttcattac atagttattt gctgcaagat 140400agtatcaatt aagcatagaa aaaaactgat tagaatgaag aacttattat agatagctgc 140460atttattttt aaagatcatg attcaagtat cttaaaattt ttccactcat aggacctatg 140520actaatgaca tgtttctcca cccatgaaaa gctggttgtt ttcagtacct aattaatgtt 140580caagcttatt gtacacactt atgcttagaa cggtggcatt tctgaaaaat cttcttggtc 140640aacagccctc taataatatt gatttctctt tgtacaacat tttgaatttg ctaaggtgtt 140700acaagtagta taacataatt tcaaaatgat atcgtgaatg gctcagtaat taatattttc 140760aaagaaccgt gctattcaag atgaaaagat tattaaggtg atattactgg gtctttggat 140820ttagaaagaa aatttgcttt ggaaagcaca ttatcacctt tcaggacaag aagcaaaatt 140880ggcattttta gcatgctact cttatcttta tttgctgata aagaaagaat ccagagtttt 140940tcaaaacttt aaataaaagc ctatggtgag gttcacatta cccaatgttt aaagaagaaa 141000taaaaactcc agacataaaa ggatgggaac aaaatagaaa caatagtctt gacattttat 141060tttagagctt ttaaaaaata ttactattca aaacaaaact caacatatga agtctatttg 141120ctttcatcta cataattaat acagaattat actgaagtac atggaagtgt aaaagctaat 141180gttctttaac ttcatgtcat aacactatca tttcataatg gatattcagc ttctttttga 141240gtaggttttc ttttgatgtc tatgtttgaa gaccacatat gctaaatggg aggaaacaca 141300cttaattctt attgttataa cttgtggttt ggtttagaga attgtgaaga ggcaaacagc 141360attttcttgt gaatctctgt tatgagataa ttgttactta aaactagctg catttgactg 141420tatgtgctat cagaataatg ggacaagtca gcttaagttg tattaagtaa attataattc 141480aatactttaa aatatatagt atttaaaaaa tcaatactat ataaattaga actctatgcc 141540aatagtacac tcttggttaa atacaacagt taaaaattct ttccatgtga atttatcttt 141600ctttataaat tgtttctatg tgctacaaaa attcttttat atttttttcc acaagcatag 141660aaccataatc cagacaggaa ttgttaattg ctacattatt gaatatcagt ttggttactg 141720acatgctcag agaaatgtta gattagttta taagcttaag ttggatggct gacaataaat 141780tctctgtata tcttgttaaa atttgaaacc agaggcttta agttgtatgt taggatttca 141840aactaatgag atcaaaccag aatagctaat aaactcagat atcaacttct tattttgttt 141900cttagaaaaa acagtgatta ggtattacaa tgttaaagat cttacagtcc agtctcttta 141960ctatatggat aaagaaatgg aggtgtgagg taaggaagta ctgtgatgaa ggtaacataa 142020cagtgagtgg ggcaatggaa ctcaaaagca atgctgcttt tactacacaa catggctact 142080aatggaccag gataatttaa cctagtattt cctggaactc tgtagtcagg attccctgat 142140tctcttcgca gaaatactac actatatgtt gcttacagat aatagtattt tggggataca 142200acaataacaa caaaaagaat ttttaattga aaaagcacaa tttgtagatc tttagcagac 142260acagaacatg gacaccaaag gaaccacctc tgtctttgct cttgacacat ttatggtaga 142320aagagtggag ggggggctat gaggcatttc cagctgcatt cactctttaa tcttacagga 142380gattaaaata caaagtattt ctcttatttt tgctcatgga gatgctttta agaagttaaa 142440aattgatgat gtttcatatt tgtgtagcgc ttattgtcta aataatttga aaacacttaa 142500gcaccattcg atttcatagt ttccaacaat ttgacacgac tctgagagaa agcactgagc 142560agcctagcca gtctcggggt gaacacccac agaacaagtt ttccaattta ggtaaaaatt 142620tagcctccta ggaatctacc aaatgaagtg caaggacaat tatatgaggt ataaattaaa 142680gcttgtgtac ccaattagga agacctcttg ttagtgttat tgttagtgct tcttgaaaac 142740atagaaattc ccattgcttt actcatgagt ttgtttgtgt tttgaatgtg atgtgttaac 142800tattaaaata tgaggtgtga aatattgtta tacaaatgta aatgattaca attttaatat 142860aataccttta aagtattgtc aggtagggtt aaattaattc acttgtgggt agtggtgaat 142920ctagagccag cctgtgcctc tagatttcta gttcattatc ctcttaagtt ccaagttagg 142980cgcactttat gacacacaca gaaattagaa gatgtttgca ggtttagaag ctgaagaaaa 143040tgaggggaca ttatatggaa tgtaaataag gacatgaaaa agaattgtaa aagtcaacca 143100agagctctga tactaaattc attgaggaca ctgataccca ttgtcatgaa tggtgacact 143160ggcactataa aaatcatttt atgtataaga atgactgtga caaagtttta atttttgaaa 143220atatagttgt ataaggtttc tcatgtctgg tattttagct tttggtttga gaattatttt 143280aaaagaatat aggacaattt agttactgga ttggtattca aactactgga cttactcaat 143340attcttccat caaaatcaaa tccaaaagaa cagagattta ttctcatatc taataggaat 143400ctagtagaca atgctaaaat tagtgaacta agtaaggtga atcaaagtta gaacccaaat 143460tatgttcaat gcaagcaaag ctttacttat tgcttttggt aagtacaatt caatgtgata 143520atgtatgtca attaattgaa gcagtctgat agatgagctt ctggtaaatg tacccaattt 143580tatatgagag actcagggtt ttacggactg tcaaagtaag acttagtaag aaatgcatcg 143640tgattaaatg agtatacgat gtgtgaagaa tgtttacacc agtacaagga aacctagttg 143700gtcatctgct agaaagtcaa attggcagtg gttgtttcta atgtatcttt ttcttgtgtg 143760atgtgtcata tgtataatga ttacatttta gataaagtga atggatctat caaacattta 143820tcagactcct tcactgtaag gtgctgtgga ggtgcctcat ttaaaccacc atagttaaac 143880ctcaaagagc agccctatgg atctggcaat tttattctca acttcgtttt agaggtgagg 143940aaattaaagc aaagacaact taagtaattt ttccaaggtt ctaagactgt gctcaaagac 144000aggattttaa ttccactgtc ttttttcttt ctatcacatc acacagtggt tgcaacttgc 144060ttttctgtaa atgtaatgaa ggaaacatcg cagaccttgt ataaaactca tctacagatt 144120ctttggtagt taggtctttt tagtgctgct ttgataatga attctggtaa aaaaaattct 144180tttataataa taaaatgttt ttaaaaatcc tccttgaatt ttctgaagtg caatagcaga 144240taacgcagct gtgaatgata aaaatacctc actgcactgt ttctatttgt cattgctcag 144300ctgaagagaa acagggccaa ctgcgaccat aactgagaga cttctgaaag ttattttcca 144360attttaatct accatatatt cagtggttac tttgtgccag ctagtaagcc agttagacac 144420tgtgtattca ttttttaaaa agtagtgtag ctttattatt ttctggtttt gccatttgaa 144480aaattattct cccccctccc cgcctacatc aggccatctt cctctcacac cactgaatca 144540gggaagaatc tgctaccgaa cagtagtatt tactgaccaa gagataactg tacctcattt 144600tgaaggtgat tcctctttct acctaaactg tattagtatt tttaaatctt caggtagtgt 144660atatcggaat cactagtgaa cttgtaaaaa ggcatatttt taagcaccac ctaagtattt 144720taaatcacta cattttaaat agcatcccag tttactgcat gtaaagaagg cttccctctt 144780gaaggaaagg catcttttcc acaaacagaa tctagagacc agttggggat cctagtggga 144840ggattgtttg gaacagaagc atcccgagaa tcgtaacttt ataggctttc cacctccaga 144900attatgcttt tctttttctt ttccttatct attcatttat ttattcatcc attcttcagg 144960catttatgat ttgctggcaa caatggataa gaagcagtct tggcaaataa ggagctcaca 145020gtctagcagg gagacataca catcattcct aaaaacagca tcacccttag gcccaggcaa 145080gaagaggccc tgccttggcc tgtgtgcttt agaaagctta ccttctggtc tgccatttgg 145140ctgtgcccct tgctatggga gtgggaattt ggagagacaa aataatgtaa tcaactgcag 145200ttcacgtctc ctagatgaca ggattatctg ctggtctagt ccccggcctg tgcctctttc 145260ctgagtccat tccttggact ctgccactgg tttgcacaca ccaaagccca agggatcgtc 145320caaataccta gctgtttgca gggacaatgg atggaacgtg gacagggctg agatatctat 145380gtgtacacat gtgaagctat tgtagtgtgg aatgaagata gaggtggcag gactggttca 145440gaagagaagc ttatgttctg ggattgaagg gtagatctcc cagtgtggca agttctggct 145500cagagaagta caaggagttt gagaattcta actttgaacc taaccgtcta gaagaaatat 145560ttcgcagggc aggaggagat aatatatttt atttaacagc ttgtcagctt gatttataac 145620ctttaaatat atgagtatat aacaagtgag cttccatttg tactgttgcc ccagaccctg 145680aaaatgttaa gggagggctg cacttggaat taagagagtc ctgaggagtg gagtcaccca 145740gcttgtctga atcaggcaac acttgagcta gatattaagc aatggtcaat tgggaattat 145800ttaggcaaga taaatggatc aaagatgaga gaaaatggca gggcaacgca gggcaggaca 145860tggaattgac aactgcattt aatgcacgag ccaaggcaaa aaggcagcaa atcgcatgaa 145920gtgtgtttgg gccttaaaat gcaagtagag cagagtttac taaaagctag ggagcttcca 145980cctttaggtt ttctcacttg aacagtccct gatgagttct gaggatttct aggggttgta 146040agggttttag gtagaaagag gatgccagat tgcaatctgg aatcatttct gtgtaaacat 146100ttctatttaa tcgcctaagg agatttcaaa gaaagagacc catattttta agtcccagta 146160tatgttgtga tttctttttt tgtcctagat aaatatttac ttttgtatct aattttgtat 146220cctttttctt caatagaatt ctctcctaaa ttataggtgc ctcagatctc acccaaagtg 146280gattcacctc tgagttcaat atatcttgga aagatgaggc tagagaaatt gcagagaacc 146340aggcttgatt ctttgcaggc cacagagcca tgagatttag tcagggaagt gacctgggta 146400tttatacatt gggtattttc cacatgatgg gccatttgga gacaatagac atattggaga 146460ttcttctaac aatcccacag agtaaaaatg tagttttgaa ccagggcagt ggagaaaaaa 146520tatttattga gtaaaattgg caagactgag tgtttgatta gaaatgcata gtggaagagt 146580tcaaagaaga gtttgcaatt tctaattttg aggtcttata aattacatcc agttttaatt 146640gtgttgagtt tatatctgca taggatatgc aagtagagac atccagcagt cagttaagag 146700tgtgattctg aagcttgggg gagacagctg ggctggctgg gaatatctat gaatgtgtgt 146760gtgtctgtgt gtgtagtgac ataccttcac acacacaaat acctatgtac atttgcatct 146820gcatttatat atacatgcat atctttttga gcccaaatat gtatttggga gttatcagta 146880tacagttgaa accaccagaa tgaatgaagt caatcaggaa aagcatttaa aatgggtaca 146940ccagagagct gatgacagaa acctgagaaa cacaaatgtt tcagtaacga acagaggaag 147000aggacaatag aaaggacaca gcaaagtgag aattttctca aaatcttaaa aatcatagga 147060gggcagtttt ttaaatgagg gaataattga cagtgtcaat aaagcaatga taattaaaaa 147120gtgcaatttg gtaactagga agtcattgga gattttctgg agtttaagag ggaatggaaa 147180gcagctttca gtgggatgag aagtgacttg gaaatgagga atggagacgg tatctttctt 147240gaagaataca gttactctta aaagttaaat tgtaaacata tgaaaaattg tttgtattct 147300gaagggaact ggaaatcaag tgaggtatct ctttttgtct ctctccattc tctatcttcg 147360ggctgagaga ggcttgggca tgctcacgat gactcatgaa gtcaggtccc agggggcagg 147420tatgattcaa gagccttatt gggactcact atctgctgtg catgaaggaa ataagatgat 147480catgatgctt acagtgaagt aaaagtgttg gtaggtggtg tttcaggctt tgctagagca 147540agatatgaaa acagtgagtg agtggcaaca aattactctg gtatgatttc ttcaggtgca 147600ctcattcctt tacacagcta atagatttga gtttggtatt cagctgggag gcataataga 147660ggttaatggg taatatggtt tggctgtgtc cccacctaaa tctcatcttg aattgtagtt 147720cccataattc ccataattgt gggtgggacg cagtgggaga taattgaatc atcggagtgg 147780tttcccccat actgttctcg tggtagggaa taagtctcat gagatctgat ggtttataag 147840gggaaactcc ttttgcttgg tttttttttt tttatatata tatatataaa gaggagttct 147900cctgcatatg ctctcttctt gcctgctgcc aggtaagaca tgactttgct tctccttgcc 147960ttctgccatg attgtgaggc ctccctagcc atgtgaactg tgagtcaatt aagccttttt 148020cctttataaa tacccagtct cgggtaagtc tttattagca gcatgagaac agactaatac 148080aatgggtaca ataaatggaa tgttctagag caaaaaggtc cccaaccttt ttggcaccag 148140ggtctggttt cgtggaagac agtttttcca ccaatggggg ttatagtttt gggatgattc 148200aagtgcatta tatttattgt gcactatatt tctattatta ttacactgta atatataagg 148260agataattat acaactcacc ataatgtaga atcagtggga gccttgagct tgtttcctgc 148320aattagatgg tcccatctga gggtgatagg agacagtgac agatcatcag gcattagatt 148380ctcataaaga gcatgcagca gagacccctc acatgtgcgg ttcacaagag ggtgtatgct 148440cctatgaaaa tctaatgccg cggctgatct gacaggaggt ggaactcggg tggtaatgca 148500aacgatgggg agcagctgta aatacagatg aagcttcact cacttaatgg ctgcccacct 148560cctgctgtgt ggccccctaa caggccacag actggtactg gtctgtggtc tgggggttgg 148620ggacccctgt tctagaggtt aaagaatact tagcaaatca acaatgagga ttgagagaga 148680aaccaagaga tagtcgacaa tctcagaaga acttgaaaag ctcaggactt tgataagtta 148740atggagaggg gtgaataaag aaaggatctg agggagctgg ggtggtagaa catggggtta 148800gttttgagag tttatgattg cagagatgta atgatgtgtg atgataacag aattgagaat 148860ctagctgaag tgttttgaag tctatggttg aaattggttg aaagtgaaaa ttattcaatt 148920taacaaggtt tagaaacctc agacagagaa taaaaaattc tagacttccc aagatacatt 148980taaaaaagat aatttcaact tctctttaga tttagggggt atgtgtgcag gcttattaca 149040tgggtatgtt atgtgtttgt ggtatggatg atcctgtcac ccaggtagtg agtatagtac 149100tcaataggta gttttttagc ccttactccc tcctctctct acccaccagt agccccaggt 149160atctattgtt tcgatctttg tatctatgtg tacccagtgt ttagcttcta ctcacaagtg 149220aaagtatgca atattcagtt ttctgcttct gtgttaattc acttagaata atggccttca 149280cctgcatcta tgttgctgca aaggatatga tttcattctt ttttatggct gggtagtatt 149340ctatgatgtc tgtgtaccac attttcttta tccaatctac agttgatggg cacctaagtc 149400atgtctttgc tattgtgcat aatgctgcag tgatgcaaac 14944051599DNAHomo sapiens 51gagaattatt taactgtttt gtgtcaacat atctatgtat aaaatctgga atagtctaaa 60acttggggga tggaccagta taaccacagc ttcagctctt cttcgttata taaaagagct 120ggcagtaccc tttttttgtt tctcttctca tggccttctt atttatcatt ttttcctttc 180tgctttttaa aaaaatctag aatacatttc tttatccaaa ttctgtattt tcttcttaac 240agagctccaa acttacattc tccatgaaaa ctttgatgga taaaccaaat gcataaatak 300ctttgcctta tttctaacct ctttgaccct gatatattca gttcatatca acaagttgtc 360attgtcttcc agattgtcta gttatgtcat aaatatgtgt ttcaactccc caagtaggaa 420ctgattttcc tcctttttgt tcatctccta taacattgag cacagtgctg ggccccaaat 480acttccggaa tgaatcatga caagctgaag actgtgtaaa gataaaaaga aaatcatact 540gaagctcttc tggcaaaagg tcagttgaaa tttatgacaa atgtcagtag ctgaagacg 5995220DNAArtificial SequenceSynthetic primer 52acaaccccat ttgtgaagac

205322DNAArtificial SequenceSynthetic primer 53tttatagaaa atttagcatg ga 225420DNAArtificial SequenceSynthetic primer 54ctaagttgtg cagccatgaa 205520DNAArtificial SequenceSynthetic primer 55tggaaccact tttgcagtaa 205619DNAArtificial SequenceSynthetic primer 56ctggttttaa ggcatgttg 195720DNAArtificial SequenceSynthetic primer 57tcctcaggga ggtctaatca 20581211DNAArtificial SequenceSynthetic polynucleotide 58tcctgctcca gcttgtggat attttgcaaa aaagctctcc atctgccaca gttgcagttc 60agtgttgaat ggctctgcta ttgtgacaat tcggccaagg tttctgttat tgagtgtata 120tctgttgact agatagtccc agttgagttg tatccaattc caggccatgt tcttcccata 180gctgttatat gagatatatc gaatgactgt aaacacatcc tgagttttaa taaggttcgt 240gtccttgagc aaatccaaat accttgacaa aagagtaacg ttcttcactg atgctaatcc 300atacagcagt ttttcttttt cttgagctaa tgaagtttct ggtattgctc aagagtgtag 360ttccatgaaa tctcattgcc agagttctgc atcccatacc gatacaccag aagcctgaga 420tttacgggaa ggcttacagt cccatttagc cactgctcaa ataacgagga agcattgttc 480aaggcttctc tgtctcccat cttgcacgca aaccctaaca cggaggaacg gagtaacttt 540gtgacatggt ctccagcatc attccatccc agagaatctg caataggctt cacttgacct 600tggaagtatt cctcaatcat aggatatagc tctttatcat cttcaaacat gctaatgatg 660taggttacag ctgaaattac tctctgccat ggtaaaaaat tctcttccct tttgagatac 720ttggtcaagt tcaaagccac cttataatct agaagttgag ctcttgccaa ggcaaaagca 780tcatcaataa gacttgcacg atctgctgaa gaaaatgtct tgtggttcaa ggagagcgct 840gtagctatcg agtcccaagt tgctacttca taatttacac gataaaaccc aatatgatct 900gggtttattt tgagaaaagc atttccacta ggattagagg agttcaaagt gattccttct 960ttttctgacc tattaaataa cacactgctt gttatattat cttcagtcca tttaactggg 1020atattccatg tataaccaag atctgaaggg ggctgagaag ggttagctct tgggtccaac 1080aaaaagcgtt tctgtgtgat gttcttgaca ccgttcacgt taagcacagg ataacccatg 1140tggtctggtc caggtgtcca ttacttcttt cactggtagc ctacttgcct cttccagtgc 1200tgcccaaaaa t 12115920DNAArtificial SequenceSynthetic primer 59agtggaggct gccagacttc 206020DNAArtificial SequenceSynthetic primer 60tgcaccactc atcaccaaca 206123DNAArtificial SequenceSynthetic primer 61ccgaggatgt ctttagtctg caa 236223DNAArtificial SequenceSynthetic primer 62atcatacagc aggaatgcaa aca 236324DNAArtificial SequenceSynthetic primer 63tgagattcca catccaacat cttt 246423DNAArtificial SequenceSynthetic primer 64tggcaaactt gatattgttc ttg 23

Claims

1. A method of determining a susceptibility to cardiac arrhythmia or stroke in a human individual, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample from the individual, wherein the at least one polymorphic marker is selected from the polymorphic markers set forth in Table 5, and markers in linkage disequilibrium therewith, and wherein determination of the presence or absence of the at least one allele is indicative of a susceptibility to cardiac arrhythmia or stroke in the individual.

2. (canceled)

3. The method of claim 1, wherein the at least one polymorphic marker is selected from the markers set forth in Table 9, and markers in linkage disequilibrium therewith.

4. The method of claim 1, wherein the at least one polymorphic marker is selected from D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51), and markers in linkage disequilibrium therewith.

5. The method of claim 1, wherein the presence of alleles-2, -4 and/or -8 in marker D4S406, allele A of marker rs2634073, allele T of marker rs2200733, allele T of marker rs2220427, allele T of marker rs10033464, and/or allele G of marker rs13143308 is indicative of increased susceptibility of cardiac arrhythmia or stroke in the individual.

6. The method of claim 1, wherein the susceptibility is increased susceptibility characterized by an odds ratio (OR) of at least 1.3.

7-25. (canceled)

26. A method of determining a susceptibility to cardiac arrhythmia or stroke in a human individual, the method comprising determining the identity of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one marker is selected from the group of markers associated with the PITX2 gene, wherein the presence of the at least one allele is indicative of a susceptibility to cardiac arrhythmia or stroke in the individual.

27. The method according to claim 26, wherein the at least one marker is selected from rs7668322 (SEQ ID NO:46), rs2197815 (SEQ ID NO:47), rs6831623 (SEQ ID NO:48) and rs259110 (SEQ ID NO:49), and markers in linkage disequilibrium therewith.

28. The method of claim 26, wherein the susceptibility is increased susceptibility.

29. (canceled)

30. The method of claim 1, wherein the cardiac arrhythmia is atrial fibrillation or atrial flutter.

31-33. (canceled)

34. The method of claim 1, wherein the stroke is ischemic stroke.

35. The method of claim 1, wherein linkage disequilibrium is characterized by numerical values for r² of greater than 0.2.

36-48. (canceled)

49. The method of claim 1, further comprising assessing at least one biomarker for atrial fibrillation, atrial flutter and/or stroke in a sample from the individual.

50. The method of claim 1, further comprising analyzing non-genetic information to make risk assessment, diagnosis, or prognosis of the individual.

51. The method of claim 50, wherein the non-genetic information is selected from age, age at onset of disease, gender, ethnicity, socioeconomic status, previous disease diagnosis, medical history of subject, family history of atrial fibrillation, atrial flutter and/or stroke, biochemical measurements, and clinical measurements.

52. The method of claim 49, further comprising calculating overall risk by logistic regression.

53-74. (canceled)

75. An apparatus for determining a genetic indicator for cardiac arrhythmia and/or stroke in a human individual, comprising: a computer readable memory; and a routine stored on the computer readable memory; wherein the routine is adapted to be executed on a processor to analyze genotype and/or haplotype data for at least one human individual with respect to at least one polymorphic marker selected from the markers set forth in Table 5, and markers in linkage disequilibrium therewith, and generate an output based on the marker or haplotype data, wherein the output comprises a risk measure of the at least one marker or haplotype as a genetic indicator of cardiac arrhythmia and/or stroke for the human individual.

76. The apparatus of claim 75, wherein the routine further comprises determining an indicator of the frequency of at least one allele of at least one polymorphic marker and/or at least one haplotype in a plurality of individuals diagnosed with cardiac arrhythmia and/or stroke, and an indicator of the frequency of at the least one allele of at least one polymorphic marker or at least one haplotype in a plurality of reference individuals, and calculating a risk measure for the at least one allele and/or haplotype based thereupon; and wherein a risk measure for the individual is calculated based on a comparison of the at least one marker and/or haplotype status for the individual to the calculated risk for the at least one marker and/or haplotype information for the plurality of individuals diagnosed with atrial fibrillation, atrial flutter and/or stroke.

77. The apparatus of claim 75, wherein the at least one polymorphic marker is selected from D4S406 (SEQ ID NO:45), rs2634073 (SEQ ID NO:33), rs2200733 (SEQ ID NO:28), rs2220427 (SEQ ID NO:1), rs10033464 (SEQ ID NO:41), and rs13143308 (SEQ ID NO:51).

78. The apparatus of claim 75, wherein the risk measure is characterized by an Odds Ratio (OR) or a Relative Risk (RR).

79. The apparatus or medium of claim 75, wherein linkage disequilibrium is characterized by values of r² of greater than 0.2.

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