MATERIALS AND METHODS FOR DETERMINING CANCER RISK

Abstract

This document relates to materials and methods involved in assessing inflammatory bowel disease patients at risk for developing cancer. For example, materials and methods for monitoring colorectal cancer risk in ulcerative colitis patients are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 16/021,700, filed Jun. 28, 2018, which is a continuation of U.S. application Ser. No. 13/272,044, filed Oct. 12, 2011, which claims the benefit of U.S. Provisional Application Ser. No. 61/392,342, filed Oct. 12, 2010. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

BACKGROUND

1. Technical Field

[0002] This document relates to materials and methods involved in assessing inflammatory bowel disease patients at risk for developing cancer. For example, this document relates to materials and methods for monitoring colorectal cancer risk in ulcerative colitis patients.

2. Background Information

[0003] Inflammatory bowel disease (IBD) refers to chronic diseases that cause inflammation in the intestine. The major types of IBD are Crohn's disease and ulcerative colitis (UC). Crohn's disease and UC differ in the location and nature of the inflammation. Crohn's disease can affect any part of the gastrointestinal tract, though it most commonly affects the terminal ileum and parts of the large intestine. Ulcerative colitis is an idiopathic inflammatory bowel disease characterized by chronic, relapsing mucosal inflammation primarily limited to the colon and rectum. Patients with longstanding and extensive IBD are at increased risk to develop colorectal cancer (CRC). Because of this, patients with IBD are advised to undergo surveillance colonoscopy and biopsy, every one to two years, wherein biopsy samples are histologically evaluated for the presence of pre-cancerous changes (colorectal dysplasia) or CRC.

SUMMARY

[0004] This document provides methods and materials for assessing inflammatory bowel disease patients at risk for developing cancer. For example, this document provides materials and methods that can be used to monitor colorectal cancer risk in ulcerative colitis patients. As described herein, markers (e.g., nucleic acid markers, polypeptide markers, epigenetic markers, or combinations thereof) can be used to screen UC patients to determine risk for developing CRC. Detection of such markers may allow a physician to more closely monitor those patients deemed to be at a higher risk of developing CRC.

[0005] Patients with UC have an increased risk of developing CRC as compared with the general population (Ekbom et al., N. Engl. J. Med., 323:1228-1233 (1990)). The exact mechanism by which the extent and duration of UC contribute to the pathogenesis of CRC is unclear, but studies measuring colonic inflammation and CRC risk have found a correlation between increased severity of histologic inflammation and risk for CRC (Rutter et al., Gastroenterology, 126(2):451-459 (2004) and Gupta et al., Gastroenterology, 133(4):1099-1105 (2007)). Rutter et al. assessed disease activity using a four-point grading scale ranging from 0 (inactive) to 3 (severely active) to quantify levels of neutrophil infiltration on hematoxylin and eosin-stained tissue sections (H&E).

[0006] This document is based, in part, on the discovery that hemotoxylin and eosin-stained tissue section (H&E) examinations alone may underestimate the level of disease activity present in the colonic tissue of patients with UC, and that patients with UC-CRC may have higher levels of disease activity at the tissue level even when they have what would currently be defined as inactive disease. For example, nucleic acid markers, epigenetic markers, polypeptide markers, or combinations of markers can be used to identify patients with higher levels of immune cell infiltrate associated with UC-CRC and can be detected even during what is currently defined as inactive disease (e.g., no neutrophil infiltration seen on H&E stained tissue slides). Measuring polypeptide levels of MPO (myeloperoxidase), and/or the methylation status of MINT1, COX-2, and/or RUNX3 nucleic acids in patient samples can provide useful information about the risk of developing colorectal cancer in inflammatory bowel disease patients. In some cases, genetic associations in TNF-alpha nucleic acids or in other biomolecules regulated by NF.kappa.B can provide additional useful information about cancer risk.

[0007] In general, one aspect of this document features a method for assessing a mammal diagnosed with inflammatory bowel disease for the presence of or an increased risk of developing colorectal cancer. The method comprises, or consists essentially of, determining whether or not the mammal comprises at least two markers from the group consisting of elevated MPO polypeptide levels, elevated RUNX3 methylation status, elevated MINT1 methylation status, and reduced COX-2 methylation status as compared to a normal control, wherein the presence of the at least two markers is indicative of an increased risk of developing colorectal cancer. The method can further comprise determining whether or not the mammal comprises at least one polymorphism in a TNF alpha nucleic acid, wherein the presence of the polymorphism is indicative of an increased risk of developing colorectal cancer. The inflammatory bowel disease can be ulcerative colitis. The determining step can comprise performing an immunoassay. The determining step can comprise performing methylation-specific PCR. The mammal can be a human.

[0008] In another aspect, this document features a method for assessing a mammal with histologically inactive inflammatory bowel disease for the presence of colorectal cancer or an increased risk of developing colorectal cancer. The method comprises, or consists essentially of, determining whether or not the mammal comprises the presence of at least two markers selected from a group consisting of the presence of at least one polymorphism in a TNF alpha nucleic acid, an elevated MPO polypeptide level, an elevated methylation level of a RUNX3 nucleic acid, an elevated methylation level of a MINT1 nucleic acid, and a reduced methylation level of a COX-2 nucleic acid as compared to a normal control, wherein the presence of the at least two markers is indicative of the presence of colorectal cancer or an increased risk of developing the colorectal cancer. The mammal can be assessed as having the increased risk of developing colorectal cancer and can be categorized as a mammal needing more frequent monitoring than a mammal assessed as not having the increased risk of developing colorectal cancer.

[0009] In another aspect, this document features a method for assessing a mammal diagnosed with inflammatory bowel disease for the presence of or an increased risk of developing colorectal cancer. The method comprises, or consists essentially of, (a) determining the methylation status in a RUNX3 nucleic acid and a COX-2 nucleic acid in the human, (b) classifying the human as having or as having an increased risk of developing the colorectal cancer if the RUNX3 nucleic acid methylation status is elevated and the COX-2 nucleic acid methylation status is reduced as compared to a normal control, and (c) classifying the human as not having or as not having at an increased risk of developing the colorectal cancer if the RUNX3 nucleic acid methylation status is not elevated and the COX-2 nucleic acid methylation status is not reduced. The inflammatory bowel disease can be histologically inactive. The method can further comprise determining the level of an MPO polypeptide in the mammal, wherein an elevated level of the MPO polypeptide is indicative of the presence of or an increased risk of developing colorectal cancer. The method can further comprise determining the methylation status of a MINT1 nucleic acid in the mammal, wherein an elevated level of the MINT1 nucleic acid methylation status is indicative of the presence of or an increased risk of developing the colorectal cancer. The method can further comprise determining whether or not the mammal contains a polymorphism in a nucleic acid encoding a TNF-alpha protein, wherein the presence of the polymorphism is associated with the presence of or an increased risk of the colorectal cancer. The polymorphism can be rs1800629.

[0010] In another aspect, this document features a method for assessing a biopsy sample from an ulcerative colitis patient having the presence of a polymorphism in a TNF-alpha nucleic acid. The method comprises, or consists essentially of, analyzing the biopsy sample for at least two markers selected from the group consisting of an MPO polypeptide level, methylation status of a RUNX3 nucleic acid, methylation status of a MINT1 nucleic acid, and methylation status of a COX-2 nucleic acid.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0012] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0013] FIG. 1 contains a sequence listing of human TNF-alpha nucleic acid promoter region (GenBank Accession No. AB048818; GI No. 13365764; SEQ ID NO: 1).

[0014] FIG. 2 contains a sequence listing of a human clone MINT1 colon cancer differentially methylated CpG island genomic sequence (GenBank Accession No. AF135501; GI No: 4914684; SEQ ID NO: 51).

[0015] FIG. 3 contains a sequence listing of human cyclooxygenase nucleic acid, promoter region and exon 1 (GenBank Accession No. AF044206; GI: 3282785; SEQ ID NO: 52).

[0016] FIG. 4 contains the 3' end of human runt-related transcription factor 3 coding and non-coding regions and a CpG island, complete sequence. (GenBank Accession No. AL023096; GI: 3900882; SEQ ID NO: 53).

[0017] FIG. 5 contains a sequence listing of a human TNF-alpha nucleic acid promoter region (SEQ ID NO: 4). The underlined regions represent the area of the -308 and -238 SNP, respectively, and the parenthetic bases indicate the polymorphism sites.

[0018] FIG. 6 is a graph of histologic disease activity and cell surface marker levels.

[0019] FIGS. 7A-7B contain data showing the association of MPO expression levels with a TNF-alpha polymorphism (FIG. 7A) and RUNX3 methylation status (FIG. 7B).

[0020] FIGS. 8A-8C contain Receiver operating characteristic (ROC) curves for combined markers. The area under the curve (AUC) for any combination of markers was higher than for TNF-.alpha. (72.0%), MPO (72.3%) or RUNX3 (66.9%) alone. The AUC for RUNX3 & MPO was 84.1, for TNF-.alpha. & MPO was 82.2% and for TNF-.alpha., MPO & RUNX3 was 88.1%.

DETAILED DESCRIPTION

[0021] This document provides materials and methods related to assessing inflammatory bowel disease patients at risk for developing cancer. For example, this document relates to materials and methods for monitoring colorectal cancer risk in ulcerative colitis patients.

[0022] In general, this document provides methods for determining the risk of inflammatory bowel disease patients of developing a cancer by determining the methylation status, genetic polymorphism status, or level of one or more biomolecules in a test sample from a mammal. The methylation status, genetic polymorphism status, or level of one or more biomolecules can be correlated with the presence of or the risk of developing cancer. Identifying cancers at an early stage can help a physician properly diagnose and treat a cancer patient. Typically, a properly diagnosed and treated cancer patient can experience an improvement in general health and survival.

[0023] As described herein, methods and materials to stratify risk of inflammatory bowel disease patients developing CRC have been identified that may identify patients at risk of developing CRC even in patients deemed to have histologically inactive disease (0 neutrophils on H&E). Patients found to have an increased risk of developing colorectal cancer may benefit from more intensive surveillance and/or different treatment strategies.

[0024] The term "biomolecule" as used herein refers to DNA, RNA, or polypeptides. This document provides methods for measuring biomolecules related to, without limitation, markers of immune cell infiltration into gastrointestinal tissue such as markers of neutrophil granulocytes (e.g., myeloperoxidase; MPO), T-cells (e.g., CD3), natural killer cells (e.g., CD16, CD56, CD8), B-cells (e.g., CD19, CD20), and macrophages (e.g., CD68). This document also provides methods for measuring biomolecules related to inflammatory markers (e.g., tumor necrosis factor alpha; TNF-alpha, cyclooxygenase 2; COX-2), runt-related transcription factors (e.g., RUNX3), and other factors such as Methylated-in-tumor 1 (MINT1). In some cases, this document provides methods for measuring biomolecules that are regulated by nuclear factor kappa beta (NF.kappa.B).

[0025] The term "marker level" as used herein refers to a test level of a biomolecule that is either altered or normal compared to a control level. The level of a particular biomolecule can be measured in a test sample from a mammal. The resulting test level then can be compared to a control level of the corresponding biomolecule. If a test level is altered compared to a control level, then the potential for the presence of or the risk of developing cancer in the mammal corresponding to that test sample can be classified as increased. For example, if the level of an MPO polypeptide measured in a colorectal biopsy sample from a patient is elevated compared to a control level of MPO polypeptide, then that patient can be classified as having an increased risk of developing colorectal cancer. In another example, if the methylation status of a MINT1 or RUNX3 nucleic acid measured in a colorectal tissue biopsy is elevated compared to a control level of MINT1 or RUNX3 methylation, then that patient can be classified as having an increased risk of developing colorectal cancer. In yet another example, if the methylation status of a COX-2 nucleic acid measured in a colorectal tissue biopsy is reduced compared to a control level of COX-2 methylation, then that patient can be classified as having an increased risk of developing colorectal cancer.

[0026] In some cases, if a test level is normal compared to a control level, then the risk of developing cancer in a patient corresponding to that test sample can be classified as decreased. For example, if the level of an MPO polypeptide measured in a colorectal biopsy sample is normal compared to a control level of MPO polypeptide, then the patient corresponding to that tissue biopsy sample can be classified as having a decreased risk of developing colorectal cancer.

[0027] In another embodiment, detecting the presence, absence, levels, or status of multiple biomarkers can be used to determine the risk for developing a cancer. In some cases, the presence of one or more polymorphisms in the promoter region of a TNF-alpha nucleic acid can be determined in combination with determining the methylation levels of one or more MINT1, COX-2, and RUNX3 nucleic acids and/or polypeptide levels of biomarkers associated with immune cell infiltration (e.g., MPO). In some cases, a combination of biomarkers that do not include TNF-alpha polymorphism detection can be used as described herein. For example, the presence of a polymorphism (e.g., -308G>A, -301G>A, -293C>T) in a TNF-alpha nucleic acid, an elevated level of an MPO polypeptide, and an elevated level of methylation in a RUNX3 nucleic acid in a sample or samples from a mammal can indicate that that mammal has an increased risk of developing colorectal cancer. In some cases, determining the presence or absence of a polymorphism in a nucleic acid of a biomolecule regulated by NF.kappa.B (e.g., IL1B) in combination with determining the methylation status of one or more MINT1, COX-2, and RUNX3 nucleic acids can be used to determine the risk for developing cancer. Other non-limiting examples of suitable combinations of markers include determining an MPO polypeptide level in a patient sample in combination with determining the methylation status of one or more RUNX3, MINT1, or COX-2 nucleic acids.

[0028] In some cases, the presence, absence, level, or status of one or more biomarkers can be determined prior to testing for the presence, absence, level, or status of additional biomarkers. For example, the presence of one or more polymorphisms in a promoter region of a TNF-alpha nucleic acid (or other biomarkers regulated by NF.kappa.B) can be determined in an initial screening assay from a patient with UC. Genomic screening tools such as single nucleotide polymorphism (SNP) analysis are particularly useful in inflammatory disease settings because these markers are not affected by disease activity and thus do not change over time. Patients determined to have a SNP present in a TNF alpha nucleic acid could then undergo additional testing to determine the status or levels of other biomarkers. For example, MPO polypeptide levels could be determined in a biopsy sample. In another example, methylation levels of one or more RUNX3, MINT1, and COX-2 nucleic acids can be determined in patients with a SNP present in a TNF alpha nucleic acid. Other non-limiting examples of suitable screening/reflex tests include determining MPO polypeptide levels in a blood or biopsy tissue sample followed by determining methylation status of one or more RUNX3, MINT1, and COX-2 nucleic acids in biopsy samples from patients with increased MPO polypeptide levels.

[0029] In some cases, it may be useful to determine the presence, absence, level, or status of one or more biomarkers in a patient that has previously been deemed to have histologically inactive disease (e.g., 0 neutrophils on H&E). For example, determining the presence or absence of a polymorphism in a nucleic acid of a biomolecule (e.g., TNF-alpha, IL1B), optionally in combination with determining the methylation status of one or more MINT1, COX-2, and RUNX3 nucleic acids in a biopsy sample from an inactive area of the colon (e.g., 0 neutrophils on H&E), can be used to determine the risk for developing cancer in a patient previously found to have histologically inactive disease. Other non-limiting examples of suitable combinations of markers include determining an MPO polypeptide level in a patient sample in combination with determining the methylation status of one or more RUNX3, MINT1, or COX-2 nucleic acids in a patient previously found to have histologically inactive disease.

[0030] Various types of samples can be used when measuring a biomolecule. Such samples include, without limitation, tissue samples, neoplastic tissue biopsies, non-neoplastic tissue biopsies, blood, plasma, serum, surgical waste, and whole organs. Biopsy specimens can be frozen, embedded, sectioned, and stained to identify regions of cellular infiltration. Samples can also include those that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides or polypeptides.

[0031] Various appropriate methods can be used to measure a biomolecule level in a sample. Such methods can vary depending on the type of biomolecule measured. For example, methods for measuring polypeptide levels include, without limitation, ELISA, immunohistochemistry, and immunofluorescence-based techniques. Such methods typically involve using antibodies having specific binding affinity for a particular polypeptide.

[0032] The term "antibody" as used herein refers to intact antibodies as well as antibody fragments that retain some ability to bind an epitope. Such fragments include, without limitation, Fab, F(ab')2, and Fv antibody fragments. The term "epitope" refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules (e.g., amino acid residues, amino acid-nucleic linkages) and usually have specific three dimensional structural characteristics as well as specific charge characteristics.

[0033] The antibodies provided herein can be any monoclonal or polyclonal antibody having specific binding affinity for an MPO polypeptide. "Specific binding affinity" refers to an antibody's ability to interact specifically with a particular polypeptide without significantly cross-reacting with other different polypeptides in the same environment. An antibody having specific binding affinity for MPO can interact with MPO polypeptides specifically in the presence of multiple different polypeptides, for example, multiple different markers expressed by neutrophils. MPO antibodies can have specific binding affinity for full-length or fragments of an MPO polypeptide from any suitable species, including, without limitation, mouse, rat, chimpanzee, and human. For example, MPO antibodies can have specific binding affinity for a full-length human MPO polypeptide or fragments of a human MPO polypeptide including.

[0034] Antibodies used for measuring polypeptide levels can include a detectable label. A detectably labeled antibody can refer to an antibody (or antibody fragment which retains binding specificity for a target polypeptide or epitope), having an attached detectable label. The detectable label is normally attached by-chemical conjugation, but where the label is a polypeptide, it could alternatively be attached by genetic engineering techniques. Methods for production of detectably labeled proteins are well known in the art. Detectable labels may be selected from a variety of such labels known in the art, including, but not limited to, radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate. Various detectable label/substrate pairs (e.g., horseradish peroxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin), methods for labeling antibodies, and methods for using labeled antibodies are well known in the art (see, for example, Harlow and Lane, eds. Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0035] MPO polypeptide levels in a colon tissue biopsy sample can, for example, be measured using a quantitative or semi-quantitative immunohistochemistry technique. For example, a section of a colorectal tissue biopsy sample can be treated with anti-MPO primary antibodies. Negative control sections can be incubated with pre-immune rabbit or mouse serum in lieu of primary antibodies. After antibody binding and subsequent washing, the primary antibodies can be detected with appropriate label-conjugated secondary antibodies (e.g., gold-conjugated or enzyme-conjugated antibodies). The label is then developed and quantitated using an image analysis system such as a computer-aided imaging system.

[0036] The resulting quantitated polypeptide levels can be correlated with the risk of having or developing colorectal cancer. Although samples can be processed individually, samples from different tissues or from a population of different patients can be processed simultaneously. Such processing methods include, without limitation, tissue microarrays as described elsewhere (Kononen et al., Nat. Med., 4:844-847 (1998)).

[0037] Immunofluorescence techniques represent another approach to measuring the level of a polypeptide. For example, MPO and CD68 polypeptides can be localized in the same colon biopsy sample section using polyclonal and monoclonal antibodies against MPO and CD68. The bound antibodies can be detected using different fluorescently conjugated antibodies. The levels of MPO and CD68 fluorescence can be quantitated using an image analysis system, and the resulting quantitated levels correlated with the risk of having or developing cancer.

[0038] Suitable antibodies for ELISA-, immunohistochemistry- and immunofluorescence-based methods can be obtained using standard techniques. In addition, commercially available antibodies to polypeptides associated with immune cell infiltration can be used.

[0039] As used herein, a "methylated nucleic acid marker" is a mammalian nucleic acid sequence that is methylated (e.g., hypermethylated or hypomethylated) in certain conditions (e.g., pre-cancer, cancer) as compared to the methylation status of the same mammalian nucleic acid under normal conditions (e.g., in an individual that does not have pre-cancer or cancer). In some cases, hypermethylated DNA markers can be particularly useful for detecting colorectal dysplasia or colorectal cancer. Such hypermethylated DNA markers can include, for example, CpG sequences from a methylated-in-tumor 1 (MINT1) nucleic acid and a runt-related transcript factor 3 (RUNX3) nucleic acid. In some cases, hypomethylated DNA markers can be particularly useful for detecting colorectal dysplasia or colon cancer. Such hypomethylated DNA markers can include, for example, CpG sequences from a cyclooxygenase 2 (COX-2) nucleic acid.

[0040] DNA methylation does not alter the coding function of a DNA, but has the potential to alter gene expression and thus can have profound developmental and genetic consequences. DNA methylation occurs at target cytosine residues that are found within CpG dinucleotides. The methylation reaction involves flipping a target cytosine out of an intact double helix to allow the transfer of a methyl group from S-adenosylmethionine to form 5-methylcytosine (Klimasauskas et al., Cell 76:357-369 (1994)). Areas of the genome containing long repeats of CpG dinucleotides are referred to as "CpG islands" (Bird, Nature 321:209-213 (1986) and Gardiner-Garden et al., J. Mol. Biol., 196:261-282 (1987)). CpG islands typically are between 0.2 to about 1 kb in length and are located upstream of many genes, but may also extend into gene coding regions.

[0041] Methylation of cytosine residues contained within CpG islands of certain genes typically correlates inversely with gene activity. For example, CpG islands of promotors are unmethylated if genes are expressed. Methylation can lead to decreased gene expression by a variety of mechanisms including, without limitation, disruption of local chromatin structure, inhibition of DNA binding by transcription factors, or by recruitment of proteins that interact specifically with methylated sequences and thus indirectly prevent transcription factor binding. Hypermethylation of CpG islands within tumor suppressor genes therefore can lead to progressive reduction of normal tumor suppressor expression, resulting in the selection of a population of cells having a selective growth advantage (i.e., neoplasm). Alterations in normal methylation processes also can be associated with genomic instability (see, e.g., Lengauer et al., Proc. Natl. Acad. Sci. USA, 94:2545-2550 (1997)). Such abnormal epigenetic changes may be found in many types of cancer and can therefore serve as potential markers for oncogenic transformation.

[0042] Any appropriate method can be used to detect a DNA methylation marker in a sample. Such methods can include isolating DNA from the sample, separating out one or more particular regions from the total DNA (e.g., CpG islands), subjecting the DNAs to bisulfite treatment, and determining whether the separated DNAs are abnormally methylated (e.g., hypermethylated). To analyze which residues within a DNA sample are methylated, the sequences of PCR products corresponding to samples treated with and without sodium bisulfite can be compared. The sequence from the untreated DNA will reveal the positions of all cytosine residues within the PCR product. Cytosines that were methylated will be converted to thymidine residues in the sequence of the bisulfite-treated DNA, while residues that were not methylated will be unaffected by bisulfite treatment.

[0043] In some cases, a test nucleic acid sample can be amplified with primers which amplify a sequence region known to include a CpG island region of interest. For example, primers specific for unmethylated and methylated nucleic acids such as those described in Example 1 can be used to amplify the sample DNA and determine the methylation status of the tested residues. In some cases, oligonucleotide primers can amplify a region of interest in a RUNX3, MINT 1, or COX-2 nucleic acid. For example, the methylated primers of SEQ ID NO: 37 and SEQ ID NO: 38 amplify a 129 base pair fragment (SEQ ID NO: 45) and unmethylated primers of SEQ ID NO: 39 and SEQ ID NO: 40 can be used to amplify a 159 base pair fragment (SEQ ID NO 46) in the promoter region of a RUNX3 nucleic acid (Table 1). In some cases, alternate oligonucleotide primer sequences could be used to amplify all or part of the RUNX3 nucleic acid fragment of SEQ ID NO: 45 or SEQ ID NO: 46 or any fragment of SEQ ID NO: 53 (FIG. 4) that when amplified, can be analyzed to determine the methylation status of a RUNX3 nucleic acid. A patient diagnosed with IBD and containing a hypermethlated RUNX3 nucleic acid (e.g. elevated methylation status) can be classified as being at a higher risk of having or developing colorectal cancer as compared to a corresponding patient not containing a hypermethylated RUNX3 nucleic acid.

[0044] In another example, the methylated primers of SEQ ID NO: 29 and SEQ ID NO: 30 amplify a 81 base pair fragment (SEQ ID NO: 47) and unmethylated primers of SEQ ID NO: 32 and SEQ ID NO: 33 can be used to amplify a 112 base pair fragment (SEQ ID NO 48) in the promoter region of a MINT1 nucleic acid. In some cases, alternate oligonucleotide primer sequences could be used to amplify all or part of the MINT1 nucleic acid fragment of SEQ ID NO: 47 or SEQ ID NO: 48 or any fragment of SEQ ID NO: 51 (FIG. 2) that when amplified, can be analyzed to determine the methylation status of a MINT1 nucleic acid. A patient diagnosed with IBD and containing a hypermethlated MINT1 nucleic acid (e.g. elevated methylation status) can be classified as being at a higher risk of having or developing colorectal cancer as compared to a corresponding patient not containing a hypermethylated MINT1 nucleic acid.

[0045] In yet another example, the methylated primers of SEQ ID NO: 13 and SEQ ID NO: 14 amplify a 142 base pair fragment (SEQ ID NO: 49) and the unmethylated primers of SEQ ID NO: 15 and SEQ ID NO: 16 can be used to amplify a 138 base pair fragment (SEQ ID NO 50) in the promoter region of a COX-2 nucleic acid. In some cases, alternate oligonucleotide primer sequences could be used to amplify all or part of the COX-2 nucleic acid fragment of SEQ ID NO: 49 or SEQ ID NO: 50 or any fragment of SEQ ID NO: 52 (FIG. 3) that when amplified, can be analyzed to determine the methylation status of a COX-2 nucleic acid. In some cases, a patient diagnosed with IBD and containing a hypomethylated COX-2 nucleic acid (e.g. reduced methylation status) can be classified as being at a higher risk of having or developing colorectal cancer as compared to a corresponding patient not containing a hypomethylated COX-2 nucleic acid.

[0046] Other non-limiting examples of nucleic acids where analyzing the methylation status may be useful in determining the risk of developing colorectal cancer in IBD patients include p16, p14, e-cadherin, estrogen receptor and HPP1.

TABLE-US-00001 TABLE 1 Methylation Assay Amplification Products SEQ ID Gene Status Nucleotide Sequence 45 RUNX3 Methyl- G TG GTGGT G AG A GT A A ated T GAG G A T GGG GGG G G T GGG A GG G TG G G T AG G G G TGTT T G AT TTTG G G G G G 46 RUNX3 Un- GGGTTTTA GG GTTTG G GTTTAG methyl- G G GTTGTTTT GTTTATTTTG G G ated G G GTAGGGGAAGG GGGGAGGGA GGTGTGAAG GG GGTTGGTGTTTGGGTTT A GGGAATA GTATAATAG GG GTTAGG G G GGGT 47 MINT1 Methyl- T GAAG G TGT TGG GT TAAGAGA ated GAG AAGAGAGGG TGGAGAG AGGGGAG G GGGG TGAGG T 48 MINT1 Un- TGGAGAGTAGGGGAG G GGGGTTGAGG methyl- TTTTTTGTTAG GTTTGTATTTTTTA GTT ated ATAA GTTTTTATTTAGTAAAAATTTTTTG GG GTTTGTTGTG GTTAGGTT 49 COX-2 Methyl- AGGGGATT TG G GGA T AGG ated G G T AGATT TGGAGAGGAAG AAG TGTT T TG T GGTAT AT AAGG GAT AGT AGAA TGG T T GG AAG G T GGG AAAGA TG G 50 COX-2 Un- GAGGGGATTTTTTG G GGATTTTAG methyl- GG GTTTAGATTTTTGGAGAGGAAGTTAA ated GTGTTTTTTTG GGTATTTTAT TTAAGG GATTAGTTTAGAATTGGTTTT G GAAG GTT GGGTAAAGA

[0047] It is noted that a single sample can be analyzed for one DNA methylation marker or for multiple DNA methylation markers. For example, a sample can be analyzed using assays that detect a panel of different DNA methylation markers. In addition, multiple samples can be collected from a single mammal and analyzed as described herein. In some cases, PCR techniques can be used to detect the presence or absence of a methylated mammalian nucleic acid marker. Cottrell et al describe appropriate methods of methylation-specific PCR (MSP) and other DNA methylation techniques (Ann N Y Acad Sci 2003 March; 983:120-30).

[0048] Purified nucleic acid fragments from a sample or samples can be analyzed to determine the presence or absence of one or more polymorphisms, such as single nucleotide polymorphisms (SNPs). For example, a sample can be analyzed to determine the presence or absence of a polymorphism identified as rs1800629 (-308G>A) which can be viewed in the single nucleotide polymorphism section of the NCBI website and the TNF-alpha sequences carrying the major alleles disclosed as SEQ ID NO:1 in the present document. It is noted that the minor allele (e.g. A) of this SNP is associated with higher risk of having or developing colorectal cancer, whereas the major allele (e.g. G) is associated with lower risk of developing colorectal cancer. In some cases, a test sample can be analyzed to determine the presence or absence of one or more polymorphisms such as -301G>A, and -293C>T in a TNF-alpha nucleic acid. The exact position of the aforementioned variants may vary from individual to individual or from species to species, e.g., by from 1 to about 10 base pairs. Further description of these and other TNF-alpha polymorphisms are provided elsewhere (Garrity-Park et al., Am. J. Gastroenterol., 103:407-415 (2008)). In some cases, polymorphisms may occur in the promoter region of a TNF-alpha nucleic acid. In some cases, polymorphisms may occur in the coding or non-coding regions of a TNF-alpha nucleic acid.

[0049] A mammal diagnosed with IBD and containing one or more polymorphisms in a TNF-alpha nucleic acid can be classified as being at a higher risk of having or developing colorectal cancer as compared to a corresponding mammal containing wild-type TNF-alpha nucleic acid at one or both alleles. For example, detection of the rs1800629 polymorphism in a sample from an IBD patient indicates that the patient is at a higher risk of having or developing colorectal cancer. Detection of this polymorphism allows selection of a monitoring schedule or treatment plan that is most likely to be effective in early diagnosis and prevention of CRC.

[0050] In some embodiments, genomic DNA or mRNA can be used to detect polymorphisms. If mRNA is used, a cDNA copy may first be made. Genomic DNA or mRNA is typically extracted from a biological sample, such as a peripheral blood sample or a tissue sample. Standard methods can be used to extract genomic DNA or mRNA from a biological sample, such as phenol extraction. In some cases, genomic DNA or mRNA can be extracted using a commercially available kit (e.g., from Qiagen, Chatsworth, Calif.; Promega, Madison, Wis.; or Gentra Systems, Minneapolis, Minn.).

[0051] Any appropriate method of analysis can be used to detect a polymorphism in a nucleic acid. Methods of analysis can include conventional Sanger based sequencing, pyrosequencing, next generation sequencing, allele specific PCR, allele-specific restriction digests, microarrays, single molecule sequencing, sequencing by synthesis, single strand conformation polymorphism (SSCP) detection, restriction length polymorphism (RFLP) analysis, denaturing high performance liquid chromatography (DHPLC), and the like. The aforementioned techniques are well known in the art. Detailed description of these techniques can be found in a variety of publications, including, e.g., "Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA" (1997) G. R. Taylor, ed., CRC Press, and references cited therein.

[0052] In some cases, a test nucleic acid sample can be amplified with primers which amplify a sequence region known to comprise the polymorphism(s) of interest. For example, oligonucleotide primers such as SEQ ID NO: 2 (ACCTGGTCCCCA-AAAGA) and SEQ ID NO: 3 (CGGGGATTTGGAAAGTTG) can be used to amplify a region of interest in a TNF-alpha nucleic acid. The primers of SEQ ID NO: 2 and SEQ ID NO: 3 amplify a 186 base pair fragment (SEQ ID NO: 4) in the promoter region of a TNF-alpha nucleic acid. In some cases, alternate oligonucleotide primer sequences could be used to amplify all or part of the TNF-alpha nucleic acid fragment of SEQ ID NO: 4 (FIG. 5) or any fragment of SEQ ID NO: 1 that when amplified, can be analyzed for association with increased TNF-alpha expression levels. The reference TNF-alpha promoter region nucleic acid sequence is provided in GenBank (Accession No. AB048818; GI No. 13365764); a portion of this sequence is provided in FIG. 1 and SEQ ID NO: 1.

[0053] In another example, commercially available kits can be used to amplify a region of interest. For example, a commercially available kit, such as a SNP genotyping kit from Applied Biosystems, can be used to amplify of region of interest in an Interleukin 1B (IL1B) nucleic acid. In some cases alternate kits or methods could be used to amplify all or part of an IL1B nucleic acid fragment that when amplified, can be analyzed for the presence or absence of a polymorphism identified as rs1143627 (-31T>C) which can be viewed in the single nucleotide polymorphism section of the NCBI website. It is noted that the T allele of this SNP is associated with higher risk of having or developing ulcerative colitis associated-colorectal cancer, whereas the C allele is associated with lower risk of developing ulcerative colitis associated-colorectal cancer. The exact position of the aforementioned variants may vary from individual to individual or from species to species, e.g., by from 1 to about 10 base pairs. In some cases, polymorphisms may occur in the promoter region of an IL1B nucleic acid. In some cases, polymorphisms may occur in the coding or non-coding regions of an IL1B nucleic acid.

[0054] A mammal diagnosed with IBD and containing one or more polymorphisms in an IL1B nucleic acid can be classified as being at a higher risk of having or developing colorectal cancer as compared to a corresponding mammal containing wild-type IL1B nucleic acid at one or both alleles. For example, detection of the rs1143627 polymorphism in a sample from an IBD patient indicates that the patient is at a higher risk of developing colorectal cancer. Detection of this polymorphism allows selection of a monitoring schedule or treatment plan that is most likely to be effective in early diagnosis and treatment of CRC. Other non-limiting examples of polymorphisms associated with a higher risk of developing colorectal cancer include SNP's found in an Interleukin-23 Receptor (IL-23R) nucleic acid such as rs10889677 (2284C>A) and rs1884444 (94G>T).

[0055] Polymorphisms in promoter sequences may affect gene expression. In some cases, serum levels of one or more of a TNF-alpha, IL1B, or IL-23R polypeptide can be measured to determine whether or not an IBD patient has an increased risk of developing CRC. For example, an IBD patient with an increased serum level of a TNF-alpha polypeptide as compared to a normal control may have an increased likelihood of developing CRC. Detection of an increased level of a TNF-alpha polypeptide allows selection of a monitoring schedule or treatment plan that is most likely to be effective in early diagnosis and treatment of CRC. Any known method for measuring polypeptide levels can be used, such as denaturing high performance liquid chromatography (DHPLC, Underhill et al. (1997) Genome Res. 7:996-1005), infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry (WO 99/57318), and combinations of such methods. Other useful detection techniques include, but are not limited to surface-enhanced laser desorption/ionization (SELDI) mass spectrometry, immunoassays, and array-based technologies. Other non-limiting examples of increased polypeptide levels associated with a higher risk of developing CRC include increased IL-23R and IL1B polypeptide levels.

[0056] It is understood that the term "specifically amplifies" refers to the ability of an oligonucleotide primer to interact specifically with a particular nucleic acid without significantly cross-reacting with other different nucleic acids in the same environment and facilitate or promote the amplification of that particular nucleic acid. Likewise, the term "specifically hybridizes" refers to the ability of an oligonucleotide probe to interact specifically with a particular nucleic acid without significantly cross-reacting with other different nucleic acids in the same environment and facilitate or promote the detection of that particular nucleic acid.

[0057] The term "elevated level" as used herein with respect to the level of an MPO polypeptide is any level that is above a median polypeptide level in a sample from a random population of mammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 mammals) that do not have UC-CRC. Elevated MPO polypeptide levels can be any level provided that the level is greater than a corresponding reference level. For example, an elevated level of MPO polypeptide can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than the reference level MPO polypeptide observed in a normal colon biopsy or blood sample. It is noted that a reference level can be any amount. For example, a reference level can be zero. In some cases, an elevated level of an MPO polypeptide can be any detectable level of an MPO polypeptide in a tissue biopsy sample.

[0058] The term "elevated level" as used herein with respect to the methylation status of MINT1 or RUNX3 nucleic acid is any methylation level that is above a median methylation level in a sample from a random population of mammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 mammals) that do not have UC-CRC. Elevated MINT1 or RUNX3 methylation levels can be any level provided that the level is greater than a corresponding reference level. For example, an elevated level of MINT1 or RUNX3 methylation can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than the reference level methylation observed in a normal colon biopsy sample. It is noted that a reference level can be any amount.

[0059] The term "reduced level" as used herein with respect to the level of COX-2 methylation status is any level that is below a median methylation level in a sample from a random population of mammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500 mammals) that do not have UC-CRC. Reduced COX-2 methylation levels can be any level provided that the level is lesser than a corresponding reference level. For example, a reduced level of COX-2 methylation can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold lesser than the reference level methylation observed in a normal colon biopsy sample. It is noted that a reference level can be any amount.

[0060] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, includes any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0061] This document provides kits that can be used to determine the level of one or more biomolecules in a sample. Kits can contain an oligonucleotide primer pair that specifically amplifies all or a portion of a target region of a nucleic acid. For example, kits can contain oligonucleotide primers that specifically amplify a TNF-alpha, COX-2, MINT1, or a RUNX3 nucleic acid. Target regions can be defined at any place along a TNF-alpha, COX-2, MINT1, or RUNX3 nucleic acid. For example, a target region can be defined by nucleotides 1-500 of the 5' portion of a TNF-alpha nucleic acid. In this case, a kit of the invention can contain an oligonucleotide primer pair that specifically amplifies all 500 nucleotides defining that target region, or a portion (e.g., nucleotides 80-188) of that target region.

[0062] Components and methods for producing kits are well known. Kits can contain multiple oligonucleotide primer pairs that specifically amplify TNF-alpha, COX-2, MINT1, or RUNX3-related nucleic acids, or probes that specifically hybridize TNF-alpha, COX-2, MINT1, or RUNX3-related nucleic acids. In addition, kits can contain antibodies for detecting MPO-related polypeptides. The kits provided herein also can contain a reference chart that indicates a reference level or baseline for MPO polypeptides or TNF-alpha, COX-2, MINT1, or RUNX3 nucleic acids. Kits can be configured in any type of design (e.g., microtiter plate design) and can be made of any type of material (e.g., plastic).

[0063] In some cases, a human may have a family history of primary sclerosing cholangitis (PSC) or CRC. Family history or relatives with PSC or CRC can be identified by examining medical records or family tree history. The methods provided in this document can also be used to identify CRC risk in relatives of affected mammals likely to have IBD or UC. Thus, these methods can facilitate decisions regarding the course of evaluation and treatment in humans with and without altered methylation in MINT1, COX-2, or RUNX3 nucleic acids, with and without polymorphisms in a TNF-alpha nucleic acid, or with and without increased MPO polypeptide levels.

[0064] This document also provides materials and methods to assist a medical professional in determining the risk of having or developing colorectal cancer in a mammal. Such a medical professional can be, for example, a physician, a nurse, a medical laboratory technologist, or a pharmacist. A person can be assisted by (1) determining the presence or absence of a nucleic acid polymorphism in a nucleic acid such as a TNF-alpha nucleic acid, determining the methylation status of a nucleic acids such as a RUNX3 nucleic acid in a test sample, and/or determining the level of a polypeptide such as an MPO polypeptide, and (2) communicating information about the presence, absence, or level of that marker to that medical professional.

[0065] After the presence, absence, level, or status of a particular biomolecule or biomolecules is reported, a medical professional can take one or more actions that can affect patient care. For example, a medical professional can record the results in a patient's medical record. In some cases, a medical professional can record that a patient is at an increased risk of developing colorectal cancer, or otherwise transform the patient's medical record to reflect the patient's medical condition. In some cases, a medical professional can review and evaluate a patient's entire medical record and assess multiple strategies for clinical intervention of a patient's condition. In some cases, a medical professional can recommend a change in therapy or a change in frequency or type of surveillance.

[0066] Any appropriate method can be used to communicate information to another person. For example, information can be given directly or indirectly to a person. In addition, any type of communication can be used to communicate the information. For example, mail, e mail, telephone, and face-to-face interactions can be used. The information also can be communicated to a person by making that information electronically available to the person. For example, the information can be communicated to a person by placing the information on a computer database such that the person can access the information. In addition, the information can be communicated to a hospital, clinic, or research facility at which the person is located.

[0067] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1--Methylation Status of Genes in Non-Neoplastic Mucosa from Patients with Ulcerative Colitis-Associated Colorectal Cancer

Patient Selection

[0068] The Mayo Clinic Institutional Review Board approved this work. The UC-CRC cases and UC controls analyzed herein were described elsewhere (Garrity-Park et al., Am. J. Gastroenterol., (2008) and Garrity-Park et al., Gut (2009)). UC-CRC cases were selected from a review of 274 patients identified from the Mayo Clinic centralized diagnostic index of medical records (1976-2006). These patients had inflammatory bowel disease (either Crohn's or UC) and CRC. Patients with Crohn's disease were excluded. Medical records for the remaining UC-CRC patients were reviewed to establish a date of disease onset. For each case, pathology slides from the surgical resection were also recalled to confirm the diagnosis of UC and identify the best tumor and non-adjacent, non-neoplastic block for DNA extraction. Patients who did not have UC confirmed by review of the pathology or whose duration of disease was less than 10 years were excluded. After these exclusions, 114 UC-CRC cases were included.

[0069] Potential UC controls were identified through the Mayo pathology index (1994-2006), which indicated the patient age, gender, and extent of UC as well as the presence of other confounding pathologies such as dysplasia. The final pool of potential UC controls for this work included UC patients who did not develop CRC, who underwent either colectomy or colonoscopy with biopsy at the Mayo Clinic, and who did not have prior dysplasia. The Mayo Clinic centralized diagnostic index of medical records was used with these remaining controls to establish a date of diagnosis. Patients with less than ten years between the date of UC diagnosis and either colectomy or date of last biopsy were excluded as were patients with a prior dysplasia diagnosis. From the remaining list, 181 controls were selected that were most closely matched to the UC-CRC cases with regard to gender, age, ethnicity, duration, and extent of UC. The surgical resection or biopsy specimens from these 181 controls were re-reviewed to confirm histologically the diagnosis of UC. After final review, 114 UC controls were included.

DNA Extraction

[0070] All formalin-fixed, paraffin-embedded (FFPE) blocks and hematoxylin and eosin-stained (H&E) slides were reviewed on all cases and controls to determine the inflammatory activity level (as assessed by neutrophil infiltrates) for all non-neoplastic sections. Each section was scored as normal, inactive (0), mildly active (1), moderate (2), or severe (3). A total of three different DNA extractions were then completed: 1) UC control, 2) UC-CRC non-neoplastic, non-adjacent, and 3) UC-CRC tumor. For non-adjacent, non-neoplastic UC-CRC cases and UC controls, DNA was extracted from all non-neoplastic paraffin tissue sections that showed evidence of chronic disease (scores 0-3; n=1-6 blocks/patient). For the tumor DNA extraction, only the section with confirmed CRC was used. Any sections scored as "normal colon" or any dysplastic lesions located away from the CRC in UC-CRC cases were excluded from all extractions. DNA was extracted using Gentra Puregene Tissue kit (Qiagen, Valencia, Calif.). DNA pellets were suspended in TE (10 mM Tris, pH=7.5, 0.1 mM EDTA, Integrated DNA Technologies, Coralville, Iowa) and quantified using Quant-iT.TM. PicoGreen.RTM. (Invitrogen, Carlsbad, Calif.).

Bisulfite Treatment/Methylation Specific Polymerase Chain Reaction (MSP)

[0071] Methylation status of each gene was determined using MSP after bisulfite treatment of 500 ng of DNA using the EZ DNA Methylation-Gold Kit.TM. (Zymo Research, Orange, Calif.) following standard protocols. Primers were designed using Methyl Primer Express v1.0 software (Applied Biosystems, Foster City, Calif.) (Table 2). Most of the proposed genes have multiple methylation sites. Therefore, whenever possible, sites chosen for evaluation were selected based on published studies that indicated that methylation in that area altered protein expression in situ. Primers were designed for the following genes: p16, p14, cyclooxygenase-2 (COX-2), e-cadherin, estrogen receptor (ER), HPP1, methylated-in-tumor 1 (MINT1), MINT31, RUNX3, and sodium solute symporter family 5 member 8 protein (SLC5A8). Unmethylated and methylated PCR reactions were carried out in separate, 25 .mu.L reactions.

[0072] Amplicons were run through ethidium-stained agarose gels and visualized using the BioRad Gel Doc.TM. (Bio-Rad, Hercules, Calif.). Positive and negative controls were included in each experimental set-up. A sample was considered positive if amplicon was produced using the methylated primer set. A sample was negative for methylation if amplicon was produced using only the unmethylated primer set. Samples that did not produce amplicons for either reaction were excluded from analyses. To ensure the specificity of each reaction and to validate the adequacy of the bisulfite modification, 25 methylated and 25 unmethylated amplicons were sequenced for each gene using the ABI PRISM.TM. (Applied Biosystems) after shrimp alkaline phosphatase (USB, Cleveland, Ohio) and exonuclease (USB) treatment of the amplicon. All of the MSP products demonstrated methylation of CpG sites. To test the sensitivity of each methylated/unmethylated assay, serial dilutions of positive control DNA (100% to 0%) were tested for each gene. All assays could detect a positive result with 5% positive control DNA.

TABLE-US-00002 TABLE 2 Methylation-specific PCR primers. Forward Reverse Size Gene (5' .fwdarw. 3') (5' .fwdarw. 3') (bp) Temp p16 Methyl- TGGGGCGGATCGCGT CGACCCCGAACCGC 140 60 ated GCGTT GACGGT (SEQ ID NO: 5) (SEQ ID NO: 6) Un- TGGGGTGGATTGTGT CCACCTCCAACAAT 172 60 methyl- GTGTTTGGT ACCCATACCT ated (SEQ ID NO: 7) (SEQ ID NO: 8) p14 Methyl- GGCGGCGAGAATATG ACGACGAACGGCCC 137 60 ated GTGC TAACG (SEQ ID NO: 9) (SEQ ID NO: 10) Un- TTGGTGTTAAAGGGT AAAAACCCTCACTC 126 60 methyl- GGT ACAA ated (SEQ ID NO: 11) (SEQ ID NO: 12) COX-2 Methyl- AGGGGATTTTTTGCG CGCAATCTTTACCC 142 55 ated TTTTC GAACGC (SEQ ID NO: 13) (SEQ ID NO: 14) Un- GAGGGGATTTTTTGT TCTTTACCCAAACA 138 60 methyl- GTTTTT CTTCCAA ated (SEQ ID NO: 15) (SEQ ID NO: 16) E-cadherin Methyl- TTAGAGGGTTATCGC ACCAAATAAACCCC 150 50 ated GTTTATGC GAAACACGG (SEQ ID NO: 17) (SEQ ID NO: 18) Un- TAATTTTAGGTTAGA CACAACCAATCAAC 97 63 methyl- GGGTTATTGT AACACA ated (SEQ ID NO: 19) (SEQ ID NO: 20) Estrogen receptor Methyl- CGTTCGGTTTTATCG AAAAACTCAAAAAC 138 55 ated GATTC CGACGA (SEQ ID NO: 21) (SEQ ID NO: 22) Un- TGAGTTGGAGTTTTT ACACATTAACAACA 149 60 methyl- GAATTGTTT ACCACA ated (SEQ ID NO: 23) (SEQ ID NO: 24) HPP1 Methyl- TTTCGGCGTAGTTTT ACTAAACATCCCGC 167 60 ated TTAGC GAACG (SEQ ID NO: 25) (SEQ ID NO: 26) Un- TGGTGTAGTTTTTTA ACAATAACAATAAC 127 60 methyl- GTGGATG ACCCAACA ated (SEQ ID NO: 27) (SEQ ID NO: 28) MINT1 Methyl- TTTCGAAGCGTTTGT CAAAAAACCTCAAC 81 55 ated TTGGC CCCGGG (SEQ ID NO: 29) (SEQ ID NO: 30) Un- TGGAGAGTAGGGGAG AACCTAACACACAA 112 60 methyl- TTTGT CAAACA ated (SEQ ID NO: 31) (SEQ ID NO: 32) MINT31 Methyl- TATTCGATTTATTTC CTACGAAAAATAAA 105 55 ated GTC CACG (SEQ ID NO: 33) (SEQ ID NO: 34) Un- GATTTTAATTTTTTG CTAAAACCATCACC 95 60 methyl- TGGTGGT CCTAAACA ated (SEQ ID NO: 35) (SEQ ID NO: 36) RUNX3 Methyl- CGTTTGCGTGGTTCG ACGACGACGACGAC 129 60 ated TTAGTAC GACA (SEQ ID NO: 37) (SEQ ID NO: 38) Un- TTGGGTTTTATGGTT ACCCAACACCCTAA 159 60 methyl- GTTTGTGT CAACCAC ated (SEQ ID NO: 39) (SEQ ID NO: 40) SLC5A8 Methyl- ACGGGGTATCGGTAT TACGATCATTCTAC 151 55 ated TTTC GACCG (SEQ ID NO: 41) (SEQ ID NO: 42) Un- GGTTATTTTGGTTGT CAAACACTACAATC 104 55 methyl- TATT ATTCTACA ated (SEQ ID NO: 43) (SEQ ID NO: 44) bp, base pairs (bases in bold indicate methylation sites); COX, cyclooxygenase; HPP, hyperplastic polyposis gene; MINT, methylated-in-tumor; RUNX, runt-related transcript factor; SLC5A8, sodium solute symporter family-5 member-8.

Inflammation Scoring

[0073] All H&E slides from each case or control were reviewed by a pathologist, and the histologic disease activity was scored as inactive, mild, moderate, or severe based on the percentage of neutrophils. Each histologic activity level was given a corresponding number, such that inactive sections were scored as 0 and mildly active, moderate, or severe sections were scored as 1, 2, or 3, respectively. To obtain the final inflammation score for each case or control extracted, the values for all sections included in the extraction were summed and then divided by the total number of sections used. For instance, if a non-adjacent, non-neoplastic extraction for a case had four sections that were inactive and two sections that were mildly active, the inflammation score would be 0.33. Scores were obtained for all non-adjacent, non-neoplastic extractions for cases and for all extractions for controls.

Statistics

[0074] For initial identification of potential genes, a univariate analysis using the Fisher's exact test was done to compare the prevalence of methylation for each gene in UC-CRC cases versus UC controls. For genes identified as significant, another Fisher's Exact test was performed to determine its significance when comparing non-neoplastic DNA from UC-CRC cases versus UC controls. A multivariable analysis was then done to test for interactions between the genes found to be significant in the non-neoplastic comparison. Finally, logistic regression modeling was performed to determine the additive effect of these significant genes.

Results

Patient Selection

[0075] The summary of patient characteristics is given in Table 3. There were no significant differences between cases and controls with regard to age, gender, family history of CRC, or duration n/extent of UC. Because there were no significant differences in distribution of disease extent between cases and controls (p=0.07), all subsequent analyses involving extent therefore used the broad categorization of extensive versus non-extensive (left-sided and proctitis) disease for cases and controls. Primary sclerosing cholangitis (PSC) was more prevalent in UC-CRC cases (p<0.0001). All cases and controls were Caucasian.

TABLE-US-00003 TABLE 3 Demographic and clinical features of 114 UC-CRC cases and 114 UC controls. Demographic/clinical UC-CRC UC, no CRC information (n = 114) (n = 114) P value Mean age at index date, 47.8 (26-82) 48.8 (24-77) 0.35 years (range).sup.a Gender, n (%) Female 36 (32) 36 (32) Male 78 (68) 78 (68) 1.00 Mean duration of UC at 20.3 (10-49) 19.5 (10-45) 0.37 index date, years (range) Maximal extent, n (%).sup.b Proctitis 8 (7.0) 7 (6) Left-sided 12 (10.5) 25 (22) Extensive 94 (82.5) 82 (72) 0.07.sup.c PSC, n (% yes) 31 (27.2) 4 (3.5) <0.0001 Family history of CRC, n (%) 19 (17) 15 (13) 0.58 .sup.aIndex date for UC-CRC was the age at CRC and for UC controls was the age at colectomy or most recent biopsy. .sup.bExtent based on histological assessment of involvement. .sup.c.chi..sup.2-Test, P value represents the comparison between cases and controls with extensive colitis vs. left-sided vs. proctitis. Values in bold are significant.

DNA Extraction and Location

[0076] Sixty percent of UC-CRC non-adjacent, non-neoplastic DNA extractions included a tissue section that was from the same segment of the colon in which the tumor arose, i.e. the tumor was in the ascending colon, and a different block without neoplasia was also available from the ascending colon. The majority of UC-CRC non-neoplastic and UC control DNA extractions included tissues obtained from both the left and right side of the colon (69% versus 74%, p=0.57). Three cases and two controls had tissue available from the rectum only. The majority of tissues used for UC-CRC case and UC control (218/228) extractions were obtained from resected colons. The remaining 10 patients had only biopsy samples available.

UC-CRC DNA Versus UC Control DNA

Univariate Analyses

[0077] To identify targets to investigate in non-adjacent, non-neoplastic regions of the UC-CRC colon, initial univariate analyses focused on the level of gene methylation in DNA extracted from tumor sections only as compared to UC controls. The majority of DNA from UC controls (between 96 to 109) and UC-CRC tumors (between 83 to 100) were successfully amplified for each target. Table 4 summarizes the results of these analyses for all 10 genes included in this study. The prevalence of gene methylation for p16, RUNX3, MINT1, MINT31, and HPP1 was significantly increased in UC-CRC cases versus controls. Conversely, COX-2 and e-cadherin were more frequently methylated in controls as compared to cases. The difference in methylation for ER, p14, and SLC5A8 was not significantly different between cases and controls.

TABLE-US-00004 TABLE 4 Univariate analyses of gene methylation status in UC-CRC cases (tumor sections) vs. UC controls Gene (.+-.for methylation) UC-CRC (%) UC controls (%) P value .sup.a (a) Methylated in UC-CRC cases p16 Negative 72 (84.7) 107 (100) Positive 13 (15.3) 0 (0) <0.0001 RUNX3 Negative 46 (55.4) 97 (93.3) Positive 37 (44.6) 7 (6.7) <0.0001 MINT1 Negative 46 (49.5) 87 (85.3) Positive 47 (50.5) 15 (14.7) <0.0001 MINT31 Negative 39 (40.6) 76 (79.2) Positive 57 (59.4) 20 (20.8) <0.0001 HPP1 Negative 19 (21.3) 53 (49.5) Positive 70 (78.7) 54 (50.5) 0.0001 ESR1 Negative 10 (10.8) 17 (15.9) Positive 83 (89.2) 90 (84.1) 0.31 p14 Negative 75 (81.5) 95 (88.0) Positive 17 (18.5) 13 (12.0) 0.24 SLC5A8 Negative 14 (14.7) 6 (5.8) Positive 81 (85.3) 97 (94.2) 0.06 (b) Methylated in UC controls COX-2 Negative 64 (66.7) 43 (39.4) Positive 32 (33.3) 66 (60.6) 0.0001 E-cadherin Negative 64 (64.0) 42 (38.9) Positive 36 (36.0) 66 (61.1) 0.0003 COX, cyclooxygenase; CRC, colorectal cancer; HPP, hyperplastic polyposis gene; MINT, methylated-in-tumor; RUNX, runt-related transcript factor; SLC5A8, sodium solute symporter family-5 member-8; UC, ulcerative colitis. .sup.a Calculated using Fisher's exact test. Values in bold are significant.

UC-CRC Non-Neoplastic DNA Versus UC-Control DNA

Univariate Analyses

[0078] Only genes that were significantly different between tumor and UC controls were tested for significance in non-adjacent, non-neoplastic normal tissue. The majority of DNA from UC controls (between 96 to 109) and non-adjacent, non-neoplastic areas from UC-CRC patients (between 66 to 88) were successfully amplified for each target. RUNX3, p16, MINT1, MINT31, e-cadherin, and COX-2 remained significantly associated with UC-CRC. The association involving HPP1 was no longer significant (Table 5).

TABLE-US-00005 TABLE 5 Univariate analyses of gene methylation status in UC-CRC cases (non-adjacent, non-neoplastic sections) vs. UC controls. Gene (.+-.for methylation) UC-CRC (%) UC controls (%) P value .sup.a (a) Methylated in UC-CRC cases p16 Negative 53 (80.3) 107 (100) Positive 13 (19.7) 0 (0) <0.0001 RUNX3 Negative 37 (49.3) 97 (93.3) Positive 38 (50.7) 7 (6.7) <0.0001 MINT1 Negative 48 (54.5) 87 (85.3) Positive 40 (45.5) 15 (14.7) <0.0001 MINT31 Negative 47 (54.7) 76 (79.2) Positive 39 (45.3) 20 (20.8) 0.0005 HPP1 Negative 27 (36.0) 53 (49.5) Positive 48 (64.0) 54 (50.5) 0.09 (b) Methylated in UC controls COX-2 Negative 54 (61.4) 43 (39.4) Positive 34 (38.6) 66 (60.6) 0.003 E-cadherin Negative 46 (55.4) 42 (38.9) Positive 37 (44.6) 66 (61.1) 0.03 COX, cyclooxygenase; CRC, colorectal cancer; HPP, hyperplastic polyposis gene; MINT, methylated-in-tumor; RUNX, runt-related transcript factor; SLC5A8, sodium solute symporter family-5 member-8; UC, ulcerative colitis. .sup.a Calculated using Fisher's exact test. Values in bold are significant.

Multivariable Analyses

[0079] Multivariable logistic regression was performed with the univariately significant genes (p16, RUNX3, MINT1, MINT31, e-cadherin, and COX-2) to determine if each gene was independently significant for UC-CRC. Table 6 indicates p-values and odds ratios for the three genes that remained significant in this analysis. Methylation of RUNX3 and MINT1 in non-neoplastic sections remained strongly associated with the presence of CRC. Conversely, unmethylated COX-2 was an indication of CRC.

TABLE-US-00006 TABLE 6 Multivariate analyses of UC-CRC (non-adjacent, non-neoplastic sections) vs. UC controls. Logistic regression Gene (.+-.for methylation) Odds ratio CI P value (a) Significantly methylated in UC-CRC cases RUNX3 12.6 4.4, 35.7 <0.0001 MINT1 9.0 3.4, 23.7 <0.0001 (b) Significantly methylated in UC controls COX-2 0.2 0.07, 0.4 0.0002 CI, confidence interval; COX, cyclooxygenase; CRC, colorectal cancer; HPP, hyperplastic polyposis gene; MINT, methylated-in-tumor; RUNX, runt-related transcript factor; UC, ulcerative colitis. Values in bold are significant.

[0080] Given the association of methylation with inflammation (Kundu et al., Mutat Res (2008)), a multivariable logistic regression also was performed that included RUNX3, MINT1, COX-2, and the inflammation score to determine if the increased incidence of methylation merely reflected a higher inflammation score in cases versus controls. Table 7A summarizes these findings. The p-values and odds ratios all remained highly significant even when the degree of inflammation, as determined by H&E, was incorporated into the logistic regression. Interestingly, greater inflammation as determined by neutrophils on H&E was not associated with UC-CRC.

[0081] Because cases and controls varied with regard to the presence of PSC, logistic regression also was performed to ensure that the significance of RUNX3, MINT1, and COX-2 was independent of PSC (Table 7B). Although PSC remained highly associated with UC-CRC, the methylation status of these three genes was still significant in this analysis.

[0082] Given that for the majority of the UC-CRC cases (60%) the non-adjacent, non-neoplastic DNA sample included tissue procured from the same region in which the tumor arose, analysis was performed to determine whether proximity to the tumor affected methylation status. The presence or absence of a non-neoplastic section from within the corresponding tumor region did not affect the prevalence of methylation for RUNX3, COX-2, or MINT1 (P=0.17, 0.69, and 0.23, respectively).

[0083] Although there was no significant difference between the UC-CRC non-neoplastic and UC controls with regard to inclusion of tissue from both the right and the left colon (P=0.57), tests were performed to determine whether this could have affected the methylation status of a given gene. It was found that the prevalence of altered methylation was not significantly different between DNA samples containing tissue sections from both the left and the right side of the colon and those that did not (P=0.24, 0.87, and 0.48 for COX-2, MINT1, and RUNX3, respectively).

[0084] Finally, to interrogate whether these alterations in methylation were specific to UC, the prevalence of altered gene methylation in a cohort of non-UC patients described elsewhere (Garrity-Park et al., Gut, 58:1226-1233 (2009)) was assessed. In brief, this cohort included biopsies taken from 60 non-UC normal patients that are a part of the average risk CRC screening population at the Mayo Clinic. These were frequency matched for age to the UC patients (both CRC and non-CRC controls) in this study (average age for UC group, 48 years, vs. 49 years for non-UC patients). For RUNX3, COX-2, and MINT1, there was no significant difference between the UC controls and non-UC patients (P=0.53, 0.21, and 0.70, respectively), but there was a significance between the UC-CRC cases and non-UC patients (P<0.0001, 0.001, and <0.001, respectively).

TABLE-US-00007 TABLE 7 Effect of inclusion of inflammation and PSC in the multivariable model of UC-CRC risk Odds ratio CI P value (a) Inflammation Inflammation score 0.3 0.1, 0.6 0.001 RUNX3 11.9 3.9, 36.0 <0.0001 MINT1 9.7 3.4, 27.7 <0.0001 COX-2 0.2 0.1, 0.5 0.002 (b) PSC PSC 9 2.2, 37.8 0.003 RUNX3 11.7 3.9, 35.3 <0.0001 MINT1 10.4 3.7, 28.8 <0.0001 COX-2 0.2 0.06, 0.4 0.0002 CI, confidence interval; COX, cyclooxygenase; CRC, colorectal cancer; MINT, methylated-in-tumor; PSC, primary sclerosing cholangitis; RUNX, runt-related transcript factor; UC, ulcerative colitis. Values in bold are significant.

Diagnostic Modeling

[0085] Logistic regression modeling was undertaken to determine if RUNX3, MINT1, and COX-2 interacted to have an additive effect, i.e. did the odds of having a synchronous CRC increase as the number of genes altered increased (Table 8). These analyses indicated that having both RUNX3 methylated and COX-2 unmethylated greatly increased the likelihood of a CRC elsewhere in the colon. This also was true of the concurrent presence of MINT1 methylation and COX-2 unmethylation, although the increase was not as dramatic. Although informative, it is important to note that the number of samples available for this analysis was small, as reflected by the wide confidence intervals.

TABLE-US-00008 TABLE 8 Logistic regression model of gene methylation on UC-CRC. Logistic Exact odds ratio CI Gene combination regression estimation method (M = methylated; Odds (StatExact) .sup.a U = unmethylated) Ratio 95% CI Exact 95% CI RUNX3 (M) + MINT1 1.0 Referent 1.0 Referent (U) + COX-2 (M) RUNX3 (M) + COX-2 (M) 4.4 0.8, 23.2 4.2 0.5, 29.6 MINT1 (M) + COX-2 (M) 6.1 1.7, 21.4 5.9 1.5, 26.8 RUNX3 (U) + MINT1 4.7 1.6, 14.0 4.6 1.4, 17.7 (U) + COX-2 (U) RUNX3 (M) + MINT1 .sup.b .sup.b .sup.b 29.5, .sup.b (M) + COX-2 (M) RUNX3 (M) + COX-2 (U) 61.2 6.2, 608.5 53.5 5.4, 2,833.0 MINT1 (M) + COX-2 (U) 17.6 2.5, 121.6 16.0 1.8, 219.4 RUNX3 (M) + MINT1 .sup.b .sup.b .sup.b 19.6, .sup.b (M) + COX-2 (U) .sup.a Used to establish the lower confidence interval of the effect. .sup.b Unable to calculate because there is a 0 in the control group.

Example 2--Myeloperoxidase as a Measure of Disease Activity in UC: Association with UC-CRC, TNF Polymorphism, and RUNX3

Patient Selection

[0086] The Mayo Clinic Institutional Review Board approved this work. Patients with UC for >10 years who developed CRC were identified from the Mayo Clinic centralized diagnostic index of medical records (1986-2006). For each case, pathology slides from the surgical resection were recalled to confirm the diagnosis of UC and to identify the best non-adjacent, non-neoplastic block for immunostaining. Complete patient chart reviews were completed on all UC-CRC cases. Patients who did not have UC confirmed by review of the pathology or whose duration of disease was less than 10 years as documented in the clinical chart were excluded. A total of 50 UC-CRC cases, representing a subset of the UC-CRC cases described elsewhere (Garrity-Park et al., Gut., 58:1226-1233 (2009); Garrity-Park et al., Am. J. Gastroenterol., 103(2):407-15 (2008); and Garrity-Park et al., Am. J. Gastroenterol., 107(7):1610-9 (2010)), were examined in this study. UC controls were identified through the Mayo pathology index (1994-2006), which indicated the patient age, gender, and extent of UC as well as any other confounding pathologies such as dysplasia. Complete patient chart reviews were completed on all potential UC-controls. All potential UC-controls included UC patients who did not develop CRC, who had greater than 10 years of disease, who underwent either colectomy or colonoscopy with biopsy at the Mayo Clinic, and who did not have prior dysplasia. The final selection of UC-controls was based on frequency matching to UC-CRC cases for age, gender, extent, and duration of UC. A representative non-neoplastic section for each control was selected for analyses. A total of 50 UC-controls, a subset of the UC-control group described elsewhere (Garrity-Park et al., Gut., 58:1226-1233 (2009); Garrity-Park et al., Am. J. Gastroenterol., 103(2):407-15 (2008); and Garrity-Park et al., Am. J. Gastroenterol., 107(7):1610-9 (2010)), were analyzed in this study.

H&E Scoring

[0087] A board certified pathologist reviewed and scored all sections. Histologic disease activity level was determined for the entire resection specimen using standard clinical methodologies utilizing H&E-stained sections. A disease activity score was assigned to each case or control using the following cut offs: 0 (inactive)--no neutrophils; 1 (mild)--rare neutrophils in crypt or surface epithelium; 2 (moderate)--neutrophils in up to 25% of crypts; and 3 (severe)--neutrophils in more than 25% of crypts.

[0088] For cases, slides from all available sections were examined to identify the best non-adjacent, non-neoplastic section for immunostaining. Whenever possible, this section was from an area distinct from where the tumor arose. Selection criteria also included 1) a well-oriented, full thickness section with generous amounts of mucosa for improved likelihood of informative IHC scoring and 2) a section reflective of overall disease state, i.e. if the patient had colitis to the hepatic flexure, sections were not chosen from the cecum.

[0089] For controls, slides from all available sections were examined to represent the best normal section for immunostaining. This included a well-oriented, full thickness section with generous amounts of mucosa that was reflective of the overall disease state.

Immunohistochemistry

[0090] Serial 4-micron sections were cut from each selected block. CD3 (DAKO, Carpinteria, Calif.), CD68 (DAKO), and MPO (Abcam, Cambridge, Mass.) antibodies were applied and developed using the DAKO Envision+system (DAKO) after heated antigen retrieval. Whole sections were then digitally scanned using the NanoZoomer (Hamamatsu, Bridgewater, N.J.). Scans were downloaded and analyzed using ImageJ software (available at http://rsbweb.nih.gov/ij/). Using 15 different tissue sections, optimal thresholding was established that accurately distinguished positive cellular area (stained brown) from negative area (stained purple). Once established, this threshold was used for all subsequent slides to avoid biasing results. A total of four to six areas of mucosa were measured to determine the % area positive (scans were analyzed at 5.times. magnification). The average of the areas was recorded and used for statistical analyses.

TNF-.alpha. Polymorphism and RUNX3 Data

[0091] Prior studies included runt-related transcription factor 3 (RUNX3) methylation status and single nucleotide polymorphism testing for TNF-.alpha. (Garrity-Park et al., Am. J. Gastroenterol., 103(2):407-15 (2008); and Garrity-Park et al., Am. J. Gastroenterol., 107(7):1610-9 (2010)). These previously derived data were used for associations in the current study. Cases and controls were selected without knowledge of TNF-.alpha. or RUNX3 status.

Statistical Analyses

[0092] Four statistical analyses were performed: 1) a Fisher exact test or Chi-square test was used to determine if the demographic/clinical selection criteria between UC-CRC and UC-control groups were appropriately matched; 2) a Fisher exact test was used to test for significant differences in % area between UC-CRC cases and UC-controls for CD3, CD68, and MPO, and for the association between the % area and the presence/absence of TNF-.alpha. SNP and RUNX3; 3) a Chi-square test was used to determine the association between case/control status and TNF-.alpha. SNP and RUNX3 methylation; and 4) logistic regression and receiver operating characteristic (ROC) analyses were performed to determine if the combination of any significant variables improved the prediction of case/control status.

Results

Patient and Sample Characteristics

[0093] UC-CRC cases and controls did not have any significant differences with regard to clinical characteristics (Table 9). Similarly, the UC-CRC or UC-control tissue sections selected for analyses were matched for location. However, UC-controls demonstrated significantly higher levels of histologic disease activity as determined by H&E than UC-CRC cases (Table 10).

TABLE-US-00009 TABLE 9 Characteristics of UC-CRC cases versus UC-controls. Cases Controls Characteristic (n = 50) (n = 50) p-value Average age, years (range) 49.4 (26-80) 50.8 (28-77) 0.55 Average duration of UC, years 20.6 (10-49) 20.9 (10-45) 0.86 (range) Extent of UC Extensive/pancolitis 82% 68% Left-sided 18% 32% 0.11 Gender Male 68% 66% Female 32% 34% 0.83 Ethnicity Caucasian 100% 100% 1.00

TABLE-US-00010 TABLE 10 Pathological characteristics of UC-CRC cases versus UC-controls. Cases Controls (n = 50) (n = 50) p-value* Non-neoplastic tissue location Rectum 21% 26% 0.12 Sigmoid 32% 19% Descending 15% 33% Transverse 9% 14% Ascending 9% 5% Cecum 15% 2% Histologic disease activity level of non-neoplastic tissue section 0 66% 41% 0.01 1 26% 35% 2 8% 10% 3 0% 14% *p-value calculated using chi-square test.

Activity Level Determined Using Cell Surface Markers

[0094] Analysis of all cases and controls indicated detectable staining of all three cell surface markers, demonstrating that current H&E scoring, in general, underestimates cellular infiltrate (FIG. 6). Determination of the possible significance of a given cell surface marker in discriminating between UC-CRC cases and UC-controls is summarized in Table 11. For cases, MPO staining was significantly higher than that of UC-controls regardless of H&E activity level (p<0.0001). There were limited UC-CRC cases with active disease as measured by H&E so to facilitate subgroup analyses, cases and controls were pooled and categorized as either inactive (H&E=0) or active (H&E=1, 2 or 3). The significance of MPO was maintained in subgroup analyses of inactive cases and controls (H&E score of 0, p=0.002) as well as those classified as active (H&E score of 1-3, p=0.02). CD68 staining was slightly elevated in the overall analysis of UC-CRC cases versus controls (p=0.04), but this finding did not persist in subgroup analyses. CD3 staining did not vary between UC-CRC cases and UC-controls. The % area of positive staining for FOXP3, a marker of T-regulatory cells (T.sub.reg) involved in suppression of inflammation (Kamikozuru et al., Clin. Exp. Immunol., 156(2):320-327 (2009); Yu et al., Inflamm. Bowel Dis., 13(2):191-199 (2007)), on a subset of cases and controls (n=25 for both) was subsequently investigated to see if this could account for the lack of difference in CD3. Analysis indicated that there was no significant difference (p=0.624, data not shown).

TABLE-US-00011 TABLE 11 Disease activity level versus cell surface markers in cases and controls. Activity CD68 p- MPO p- CD3 p- Level Cases Controls value Cases Controls value Cases Controls value All 2.668 2.031 0.04 6.06 3.41 <0.0001 3.58 3.44 0.70 0 2.76 1.95 0.14 6.44 2.77 0.002 3.65 2.69 0.07 1-3 2.49 2.09 0.25 5.74 3.85 0.02 3.45 3.94 0.43 Values in bold are significant.

MPO Expression Associated with Genetic and Epigenetic Changes

[0095] Increased MPO expression was significantly associated with the presence of the TNF-.alpha.-308 G>A SNP (5.95 vs 4.02, p=0.008) (FIG. 7A) as well as RUNX3 methylation (5.90 vs 4.30, p=0.03) (FIG. 7B). It is important to note that the RUNX3 methylation was detected in DNA extracted from non-adjacent, non-neoplastic of UC-CRC cases. Receiver operating characteristic (ROC) curves indicated that an analysis with combined markers was more informative than individual marker assessment (FIG. 7). The area under the curve (AUC) was higher for MPO combined with TNF-.alpha. and/or RUNX3. To further interrogate this association, logistic regression was performed. Odds ratios and p-values were either improved or remained highly significant in the presence of other variables (Table 12).

TABLE-US-00012 TABLE 12 Univariate and Multivariate analyses of variables. Odds Ratio CI* p-value Univariate MPO 1.38 1.17, 1.67 <0.0001 RUNX3 methylated 8.07 2.47, 36.58 0.0003 TNF-.alpha. 7.87 3.18, 21.36 <0.0001 Multivariate MPO&TNF-.alpha. MPO 1.36 1.13, 1.69 0.0005 TNF-.alpha. 7.15 2.66, 21.06 <0.0001 MPO&RUNX3 MPO 1.51 1.25, 1.90 <0.0001 RUNX3 15.90 4.20, 81.78 <0.0001 MPO, RUNX3 &TNF-.alpha. MPO 1.46 1.19, 1.87 <0.0001 RUNX3 14.29 3.46, 80.00 0.0001 TNF-.alpha. 6.60 2.22, 21.63 0.0006 *Confidence interval P-values in bold are significant.

Example 3--Nucleic Acid Markers and UC-CRC

Patient Selection

[0096] The Mayo Clinic Institutional Review Board approved this work. The UC-CRC cases and UC controls analyzed in this study have been described elsewhere (Garrity-Park et al., Am. J. Gastroenterol., 103(2):407-15 (2008); and Garrity-Park et al., Gut., 58:1226-1233 (2009)). UC-CRC cases were selected from a review of 274 patients identified from the Mayo Clinic centralized diagnostic index of medical records (1976-2006). These patients had inflammatory bowel disease (either Crohn's or UC) and CRC. Patients with Crohn's disease were excluded. Medical records for the remaining UC-CRC patients were reviewed to establish a date of disease onset. For each case, pathology slides from the surgical resection also were recalled to confirm the diagnosis of UC and identify the best block for DNA extraction. Patients who did not have UC confirmed by review of the pathology or whose duration of disease was less than 10 years were excluded. After these exclusions, 114 UC-CRC cases were included in the study. Potential UC controls were identified through the Mayo pathology index (1994-2006), which indicated the patient age, gender, and extent of UC as well as the presence of other confounding pathologies such as dysplasia. The final pool of potential UC controls for this study included UC patients who did not develop CRC, who underwent either colectomy or colonoscopy with biopsy at the Mayo Clinic, and who did not have prior dysplasia. The Mayo Clinic centralized diagnostic index of medical records was used with these remaining controls to establish a date of diagnosis. Patients with less than ten years between the date of UC diagnosis and either colectomy or date of last biopsy were excluded as were patients with a prior dysplasia diagnosis. From the remaining list, 181 controls were selected that were most closely matched to the UC-CRC cases with regard to gender, age, ethnicity, duration, and extent of UC. The surgical resection or biopsy specimens from these 181 controls were re-reviewed to histologically confirm the diagnosis of UC. After final review, 114 UC controls were included in this study.

DNA Extraction

[0097] DNA was extracted from formalin-fixed, paraffin-embedded tissues using a modified Gentra (Gentra Systems Inc., Minneapolis, Minn.) protocol, and DNA was suspended in TE (10 mM Tris/0.1 mM EDTA, Integrated DNA Technologies, Coralville, Iowa). Quantification of total DNA was performed using the Picogreen assay (Invitrogen, Portland, Oreg.).

Genotyping

[0098] Samples are interrogated for the presence of additional SNP's in the nucleic acid sequences outlined in Table 13 below. If possible, testing is completed using Taqman genotyping kits (Applied Biosystems; ABI) after optimization for use with formalin fixed, paraffin embedded DNA samples. The 7900HT real-time PCR system is used for evaluating each sample. If a kit is not available for a given SNP, testing is then completed using traditional PCR followed by sequencing as described elsewhere (Garrity-Park et al., Am. J. Gastroenterol., 103(2):407-15 (2008)).

Statistical Analysis

[0099] The ability of a SNP to delineate a case from a control is determined using either a Fisher Exact test or chi-square, as appropriate. Any significant SNP is further interrogated using logistic regression with all other significant SNPs and previously known clinical risk factors (i.e., PSC). Modeling is then performed to determine the best diagnostic paradigm for predicting CRC.

TABLE-US-00013 TABLE 13 Analysis of nucleic acid polymorphisms in UC-CRC cases vs. UC controls (SEQ ID NOS 54-87, respectively, in order of appearance) SNP(s) p- Context sequence Target Identified value (ABI) IL-1 IL-6 -174G > C (1800925) IL-6 -6337T > C IL-10 -1082G > A TCCTCTTACCTATCCCTAC (1800896) TTCCCC[T/C]TCCCAAAG AAGCCTTAGTAGTGTTG IL-10 -819C.T AGTGAGCAAACTGAGGCAC (1800871) AGAGAT[A/G]TTACATCA CCTGTACAAGGGTACAC IL-10 -592C > A CTTTCCAGAGACTGGCTTC (1800872) CTACAG[T/G]ACAGGCGG GGTCACAGGATGTGTTC IL-10 -627C > A IL-15 IL-18 TGFB TNF-.alpha. -308G > A (G19) TNF-.alpha. -238G > A (673) TNF-.alpha. -863C > A (1800630); TNF-.alpha. -857C > T TNF-.alpha. -301G > A TNF-.alpha. -293C > T IL-12 IL-15 IL-23 IL-23R 2284C > A >0.0001 TTTAATTTTAGCCATTCTT (10889677) CTGCCT[A/C]ATTTCTTA AAATTAGAGAATTAAGG IL-23R 94G > T =0.02 TTTTCCTGCTTCCAGACAT (1884444) GAATCA[G/T]GTCACTAT TCAATGGGATGCAGTAA IL-23R 1142G > A ATTGGGATATTTAACAGAT (11209026) CATTCC[A/G]AACTGGGT AGGTTTTTGCAGAATTT IL-7 NFKB DelATTG TLR1-10 IL-8 -251T > A TTATCTAGAAATAAAAAAG (4073) CATACA[A/T]TTGATAAT TCACCAAATTGTGGAGC IL-8 2767A > T IL-8 781C > T AACTCTAACTCTTTATATA (2227306) GGAAGT[C/T]GTTCAATG TTGTCAGTTATGACTGT IFNG IL-4 -168C > T TTAGCTTCTCCTGATAAAC (2070874) TAATTG[C/T]CTCACATT GTCACTGCAAATCGACA IL-4 -590C > T IL-4 -34C > T IL-4 -588C > T ACACCTAAACTTGGGAGAA (2243250) CATTGT[C/T]CCCCAGTG CTGGGGTAGGAGAGTCT IL-1.beta. -31T > C >0.0001 CCAGTTTCTCCCTCGCTGT (1143627) TTTTAT[G/A]GCTTTCAA AAGCAGAAGTAGGAGGC IL-1.beta. -571C > T IL-1.beta. 3953C > T CATAAGCCTCGTTATCCCA (114634) TGTGTC[G/A]AAGAAGAT AGGTTCTGAAATGTGGA IL-1.beta. -511C > T (3087258) IL-21 IL-17 -197G > A TGCCCTTCCCATTTTCCTT (2275913) CAGAAG[A/G]AGAGATTC TTCTATGACCTCATTGG TREM1 MPO MIP-1.alpha. MDR1 P16 RUNX3 COX2 MINT1 HPP1 MINT31 PPAR.gamma. 34C > G PPAR.gamma. 161C > T IL-1RA 86 bp repeat (Intron 2) IL-13 2044G > A TTAAAGAAACTTTTTCGCG (20541) AGGGAC[A/G]GTTCAACT GAAACTTCGAAAGCATC IL-13 -1112C > T GGTTTCTGGAGGACTTCTA (1800925); GGAAAA[C/T]GAGGGAAG AGCAGGAAAAGGCGACA IL-13 -1512A > C TLR1 R80T AACACTGATATCAAGATAC (5743611) TGGATT[C/G]TATTATGA GAAATTATCAAAATCCT TLR1 1602S (5743618) TLR2 R753Q (5743708); TLR2 GT repeat (Intron2); TLR2 P631H GCCTGGCTCCAGGCCAAAA (5743704) GGAAGC[A/C]CAGGAAAG CTCCCAGCAGGAACATC TLR3 N284I CTCACTATGCTCGATCTTT (5743316); CCTACA[A/T]CAACTTAA ATGTGGTTGGTAACGAT TLR3 L412F ACTTGCTCATTCTCCCTTA (3775291); CACATA[T/C]TCAACCTA ACCAAGAATAAAATCTC TLR3 908T > C TLR4 D299G GCATACTTAGACTACTACC (4986790) TCGATG[A/G]TATTATTG ACTTATTTAATTGTTTG TLR4 T399I TGTTCTCAAAGTGATTTTG (4986791) GGACAA[C/T]CAGCCTAA AGTATTTAGATCTGAGC TLR5 R392* TLR6 S249P TTGAGGGTAAAATTCAGTA (5743810) AGGTTG[A/G]ACCTCTGG TGAGTTCTGATAAAAAT TLR6 -1401A > G (5743795) TLR7 Ql1L TTTCCAATGTGGACACTGA (179008); AGAGAC[A/T]AATTCTTA TCCTTTTTAACATAATC TLR7 A448V AGTGAAGTTGGCTTCTGCT (5743781) CAAATG[C/T]CAGAACTT CTGTAGAAAGTTATGAA TLR7 T801T GGTTTGTCTGGTGGGTTAA (864058) CCATAC[A/G]GAGGTGAC TATTCCTTACCTGGCCA TLR8 M1V AATGAAAAATTAGAACAAC (3764880); AGAAAC[A/G]TGGTAAGC CACTTCTATTTCTTTAG TLR8 D118D AATCAAATGGCTTGAATAT (2159377) CACAGA[C/T]GGGGCATT CCTCAACCTAAAAAACC TLR8 L651L GTCTGGATTTATCCCTTAA (2407992) TAGGCT[C/G]AAGCACAT CCCAAATGAAGCATTCC TLR9 1174G > A TGTGTGAGTGGCCGGCCCC (352139); CAGCTC[C/T]ACCTCCAC CCACTCCACTTCATGGG TLR9 1635G > A AGCTGAGGTCCAGGGCCTC (352140); CAGTCG[C/T]GGTAGCTC CGTGAATGAGTGCTCGT TLR9 -1237T > C (5743836) TLR10 N241H AGCAATAGAACCGATGTCT (11096957); TAGCAT[T/G]TTCTAAAC TAAGATTTCGTTGCATT TLR10 I369L AGAGTTTTCAAGTGAGGCA (11096955) GTTGGA[T/G]AGTTCTTT TAAACAACTCGTCTGTT TLR10 I473T GAGATCAGTTAGAAAATTA (11466657); AATGCA[A/G]TATTTAGT TCTCGTAAGGCCATCAG TLR10 R525W AAATTTTTTAATTCACAGG (11466658) TACACC[A/G]GAATGGAT TTCTTCCCGCATTTAGA

Example 4--Early Appearance of Nucleic Acid Markers in UC-CRC Pateints

[0100] Biopsies from 10 UC-CRC cases and 10 UC-controls obtained between 10 and 24 months prior to the index date analyzed in the work described in the above Examples were tested for methylation of RUNX3 and MINT1. RUNX3 was more frequently methylated in UC-CRC cases than controls (80% versus 10%). MINT1 was also more frequently methylated in UC-CRC cases than controls (60% versus 0%). These results demonstrate that the methylation changes apparent at the time of CRC (index date) actually occurred prior to overt neoplasm.

Other Embodiments

[0101] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1

1

8711051DNAHomo sapiens 1gctgtctgct tgtgtgtgtg tgtctgggag tgagaacttc ccagtctatc taaggaatgg 60agggagggac agagggctca aagggagcaa gagctgtggg gagaacaaaa ggataagggc 120tcagagagct tcagggatat gtgatggact caccaggtga ggccgccaga ctgctgcagg 180ggaagcaaag gagaagctga gaagatgaag gaaaagtcag ggtctggagg ggcgggggtc 240agggagctcc tgggagatat ggccacatgt agcggctctg aggaatgggt tacaggagac 300ctctggggag atgtgaccac agcaatgggt aggagaatgt ccagggctat ggaagtcgag 360tatggggacc cccccttaac gaagacaggg ccatgtagag ggccccaggg agtgaaagag 420cctccaggac ctccaggtat ggaatacagg ggacgtttaa gaagatatgg ccacacactg 480gggccctgag aagtgagagc ttcatgaaaa aaatcaggga ccccagagtt ccttggaagc 540caagactgaa ccaagcatta tgagtctccg ggtcagaatg aaagaagagg gcctgcccca 600gtggggtctg tgaattcccg ggggtgattt cactccccgg ggctgtccca ggcttgtccc 660tgctacccgc acccagcctt tcctgaggcc tcaagcctgc caccaagccc ccagctcctt 720ctccccgcag ggcccaaaca caggcctcag gactcaacac agcttttccc tccaaccccg 780ttttctctcc ctcaacggac tcagctttct gaagcccctc ccagttctag ttctatcttt 840ttcctgcatc ctgtctggaa gttagaagga aacagaccac agacctggtc cccaaaagaa 900atggaggcaa taggttttga ggggcatggg gacggggttc agcctccagg gtcctacaca 960caaatcagtc agtggcccag aagacccccc tcggaatcag agcagggagg atggggagtg 1020tgaggggtat ccttgatgct tgtgtgtccc c 1051217DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 2acctggtccc caaaaga 17318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 3cggggatttg gaaagttg 184186DNAHomo sapiens 4acctggtccc caaaagaaat ggaggcaata ggttttgagg ggcatgggga cggggttcag 60cctccagggt cctacacaca aatcagtcag tggcccagaa gacccccctc ggaatcagag 120cagggaggat ggggagtgtg aggggtatcc ttgatgcttg tgtgtcccca actttccaaa 180tccccg 186520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 5tggggcggat cgcgtgcgtt 20620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 6cgaccccgaa ccgcgacggt 20724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 7tggggtggat tgtgtgtgtt tggt 24824DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 8ccacctccaa caatacccat acct 24919DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 9ggcggcgaga atatggtgc 191019DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 10acgacgaacg gccctaacg 191118DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 11ttggtgttaa agggtggt 181218DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 12aaaaaccctc actcacaa 181320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 13aggggatttt ttgcgttttc 201420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 14cgcaatcttt acccgaacgc 201521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 15gaggggattt tttgtgtttt t 211621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 16tctttaccca aacacttcca a 211723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 17ttagagggtt atcgcgttta tgc 231823DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 18accaaataaa ccccgaaaca cgg 231925DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 19taattttagg ttagagggtt attgt 252020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 20cacaaccaat caacaacaca 202120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 21cgttcggttt tatcggattc 202220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 22aaaaactcaa aaaccgacga 202324DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 23tgagttggag tttttgaatt gttt 242420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24acacattaac aacaaccaca 202520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 25tttcggcgta gttttttagc 202619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 26actaaacatc ccgcgaacg 192722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 27tggtgtagtt ttttagtgga tg 222822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 28acaataacaa taacacccaa ca 222920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 29tttcgaagcg tttgtttggc 203020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 30caaaaaacct caaccccggg 203120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31tggagagtag gggagtttgt 203220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 32aacctaacac acaacaaaca 203318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 33tattcgattt atttcgtc 183418DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 34ctacgaaaaa taaacacg 183522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 35gattttaatt ttttgtggtg gt 223622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 36ctaaaaccat cacccctaaa ca 223722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 37cgtttgcgtg gttcgttagt ac 223818DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38acgacgacga cgacgaca 183923DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39ttgggtttta tggttgtttg tgt 234021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 40acccaacacc ctaacaacca c 214119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 41acggggtatc ggtattttc 194219DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 42tacgatcatt ctacgaccg 194319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 43ggttattttg gttgttatt 194422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 44caaacactac aatcattcta ca 2245129DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 45cgtttgcgtg gttcgttagt acgtttatta tcgagcgtat ttcgggtcgg gcgcgttttt 60cgggttttac ggtcgtttgc gcgtttagcg cgtcgttgtt ttcgtttatt ttgtcgtcgt 120cgtcgtcgt 12946159DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 46ttgggtttta tggttgtttg tgtgtttagt gtgttgttgt ttttgtttat tttgttgttg 60ttgttgttgt aggggaaggt tggggaggga ggtgtgaagt ggtggttggt gtttgggttt 120atgggaatat gtataatagt ggttgttagg gtgttgggt 1594777DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 47tttcgaagcg tttgtttggc gtttaagaga gagtaagaga gggttggaga gtaggggagt 60tcgcggggtt gaggttt 7748112DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 48tggagagtag gggagtttgt ggggttgagg ttttttgtta gtgtttgtat tttttatgtt 60ataatgtttt tatttagtaa aaattttttg ggtgtttgtt gtgtgttagg tt 11249142DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 49aggggatttt ttgcgttttc ggattttagg gtcgtttaga tttttggaga ggaagttaag 60tgtttttttg ttttttttcg gtattttatt taaggcgatt agtttagaat tggttttcgg 120aagcgttcgg gtaaagattg cg 14250138DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 50gaggggattt tttgtgtttt tggattttag ggttgtttag atttttggag aggaagttaa 60gtgttttttt gttttttttt ggtattttat ttaaggtgat tagtttagaa ttggtttttg 120gaagtgtttg ggtaaaga 13851528DNAHomo sapiensmodified_base(332)..(332)a, c, t, g, unknown or othermodified_base(342)..(342)a, c, t, g, unknown or othermodified_base(407)..(407)a, c, t, g, unknown or othermodified_base(425)..(425)a, c, t, g, unknown or othermodified_base(432)..(432)a, c, t, g, unknown or othermodified_base(495)..(495)a, c, t, g, unknown or other 51cccgggctgg gtacctggac ctataccttc atagctgcct taggctcaac ttttcggcgg 60ggatccctct gcagacgtgc aggtggcggg agagcagagg tagccgcagt aagtgctgag 120agagcctgaa agaaacacca tgaattttca aactctccca catacattcc cgaagcgcct 180gtctggcgtc taagagagag caagagaggg ctggagagca ggggagcccg cggggctgag 240gctctttgtc agcgcctgca cttcctacgt tacaacgcct tcattcagca aaaacctttt 300gggcgcctgc tgtgcgccag gccaggcgaa gnagaccgag gntgtgaagc tcagagggga 360gagggaccaa tcgcagtaaa taagctaccg aggtaatctt agatggngat gagggcagga 420aaagncatca gncgacctct gacctttctc ttagggggtt ttccccttcc gcctgggttc 480tagaactggg aaganttttc tccagagcgt cgcggggagc gccccggg 528527273DNAHomo sapiens 52ggtacccagg ctggagtgca ctggtgtgat catagctcac taacctcgaa ctcctgggct 60taggcaatcc tcttgccttg gcctcccaaa gtgccaggat tacaggcatg agccaccaca 120gtggagctct caattctgat actaataatt tgtgtcttct ctttttttcc ttagcctgac 180tagagtaatt aactttatgt cttttaaaag aaccaccttt ttggttttac ccattttctt 240ttttgatttt ctgtttttga tttgattgat atctactcta attttttatt atttcttttc 300ctctgcttac tttgaattta attacttttc ttttttgtag tctcctaaaa tagaagctta 360tattattgat tttagatctt tcttcttttc tattacagca ctcaatgcta taaatttccc 420tctaagcatt gctttcactg catcctacaa tatttcaact ctattgttat ttagctcaaa 480agaggttctt aatttctatt gggatttctc tttgacccat gtgttattca gaagtgttcc 540gtgtgatctc caaatatttg ggagtttttc agctatcttt ctattaatca tttcttgttt 600aattctattg tggcctgaga gcatatattg tatgatttat attcttgtaa atgtgttaag 660gtgtgtctta tggtgcagaa tgcggtttat cttgctatat gttccttaga gaataatgta 720tgttctgctg ttattggata aagtagtcta tagatgtcag ttacatctcg ttgattaatg 780gtgctgttga gttcagctat gtcctaaatg attttctgtc tgctgtatct gtctatttct 840gacacaaggc tgttgaagtc tccaaccata ataatgaatt aatctatttt tctttgcagt 900tttatcaatt ttgtcttata tatattgatg ctccattgtt tggcacatac acattaagaa 960ttgttatgtc ttcttggaga atttaccttt ccataacatg taacatttcc ctttattcct 1020gataattttt cttgctcaaa agtttgccct gttggaaatt accagaacta ctctggcttt 1080atttgattag tgttagcatg ctctctcttt ctctattctt acacttttaa tgtatacttg 1140actttgtatt taaagtgggg ttcttataga aaacatatac ttggtagggt gggaagtaaa 1200ataaaaagaa atacttgggt attggtttga tccactctaa caatctctat gttttaattg 1260atgtatttag accattgata cttatttttt tatcctcatc cctgtgatta cccagagagc 1320tgcttaaatt gattattgat atagacaaat taataattaa tatctaccgt ttgttactgt 1380tttctatttt tcattgccct tactttctgc tcctattttt tgctcctttt tctgttaatt 1440taggttttga gttattttat atcattctat tttctctccc ttctcagcat atgaattatc 1500tttctttttg acttttttag tggctgccct gaaggttgca atgtacattt acaaccagtc 1560ccaatctcct ttcaaaaaac acaatactgt ttcatggcta gtgcaagtac ctaataataa 1620gaagtcactc ctaatttctt tctctcattc tttgtatctt tactgttatt catttcactt 1680gtacataagc tgtaatcttt caatacatta ttgctattat tatttcaaaa catgttatct 1740attatatcta tttaaaataa gaaaaatagg ccaggtgcag tggcttactc atgtaatccc 1800agcactttgg gagaccgatg gattgctaga gctcaggaat tcgagaccag cctgggcaac 1860atagtgaaac cctgtctcta ctaaaaatac aaaaaaaaaa attgctgggc atggtggcat 1920gggcctgtgg tcacagctac tcgggaggct gaggtgagag gattgcttga gcctgggagg 1980cagaggttgc agtgaaccaa aatcaagcta ctgcactcca gcctaagtga cagagtgaga 2040ccctgtctca aaaaaaaaat gaaagaatta tttttattta tcttcactta tttcttctct 2100aatgctcttt gtttctttag tatgtagatc caagtttcta acctgtatca tttttcttat 2160ctcaataact tcttttaaca tttctcacaa agcagatcta ctggccacag aatgcctcaa 2220ttttcatttg tctgagaaaa ccttatttct ccttcacttt tgaaagataa ttttgtaggg 2280tacagaattc taggttgtag gttttttccc ctcaaagtga aatatttcat tccactcttt 2340tcttctttgt atggtatctg agaagaagtc agatgtaatt cttatcatta ttacttaaaa 2400gattgcttct gttcctttct ctcttctcct tcccttcttt ccttctctgt atattacacc 2460ttttatagtt gccccatatt tcttagatat tatgttttgg ttttcttctg tgtttttttc 2520tttgattctc agttttagaa gtctctattt atatatctgc aatcgcaggg attctttcct 2580ctgccatgtc cagtctacta ataagccctt acagacattg ttgacttctg ttccagtgtt 2640tttgatctct agcatttctc tgattatttc ttggaattgc catctgtcta cttacattac 2700caacctattc ttgtgtgttg tcttatcata gtaattgcag ttgttttaat ttcataggta 2760ttgtaatttc aacatctcta ccatatttga cattgattct gatgcttgct ctgtcttatc 2820aagctatgtt tttgtctttt agtgtgactt ctaatttttt gttgaaagcc aggcatgatg 2880tactgagtga aagaaactca atacattgta atgtgacgat aagagttcag gggaagtgaa 2940gcattctata gtcctatagc aggtctcggc cttttagtga gcctgtgcct atgaacggtg 3000actttcaaca agtgcttttc attccactct tttcctgtcc ttaagtggga caagatcact 3060gggggggggc tagaattggg tatttccctt ctccaatgta gaagctaaag agagggctgg 3120agttgggtat ttttcttccc ctgtatggaa agctagaggc agttaaattt ggatattttc 3180cttcttctaa ttcagttagg ctgcgacaaa aatcccgaca gtttaggctc taatattata 3240aaataatttc tcttgagtat aggccttatt aagaacacta tactctgatg gagctgaggg 3300ggagttttct ctgatattca ctgcgagaac ctcgtagagc tccaggaagc aaaactcaca 3360aaagtgtggg agtcttccag aatttttcct ttgcagactt atctgcactg aacctccaga 3420aattcatcaa ttacagttca ggttttccta cccaggtact ggttttcatg gaggtttctg 3480cctgtgcatt tctgctccag taagttgttc ttcttgtatg gtctgtcttt caaatttttt 3540aagtagggtt atgacctgtc gcctcacttc tctgacagtt ctgagagtgt tgatttttca 3600gtttgcttag atttttactt gtttttagga tgaagtgaca atttccaagc tcctccctga 3660catgccagat cagaaactga aagtcctaag cctcatattc tgtgcgtggg tatgttcaca 3720tcctgcctgc tccagtgccc ccacctcaca ctctctttcc cttccttgtc cccttgtgag 3780atttctaggt ccaatacaaa gactgtgttc aactcattca actacttggc tcatctgagt 3840attataatga acaatcacaa aaaaaaatga agtaaaagaa aaatccatca aagaattgag 3900atatttgaga aaaagaaagg agatcagtgt tttataaaac ttagaaatag attttttaag 3960tgtttcttca ttgacttatg tgaaaggact tttcttaatt taacaaatta tgtgctttcg 4020tttatagcct caaaacttct tgtgtagcta agaatgggta aataatcagg ctttactaaa 4080ggactaacgt aaagatcttc tgtaagtaac atttctgcta ctcaaggaag agataaactt 4140catggcataa ccttgccaaa gtatactaag aataaccctg acacaaagct cttttttcag 4200ccaacatgcc atgaaagaaa gaagacaagg ggtgatctcc actctctaag tgaaccacta 4260aacccaccaa agaagaaacg agggaaatag aaagaggacc cttgcctgag ataatggatc 4320tgtatgtatg agtagtagaa ccctgctcaa agtacaagga agggaaaaaa aagttagttt 4380atttggaatt ttggacatta agagtcttta ttgttcattt tcttttaact cacatgaatg 4440gcttatcact tcaattaata aatatttcat ttcttttcaa tctatattca tgaaacaaat 4500ctgaaatgaa cagtgcaaca tgtgaatgtt tagaacatta taaaattaaa cacaaaatct 4560gtctggcaat cttcctagca tcttaggaaa aaagttgaca aaatttcaag cagcagaagg 4620gggcagtaaa actcaacaga aagctctgga agatttttaa gattcttcct tattttcttt 4680tcatgtagag tatttcccaa caaatttcag acgctaatag aaattttgta caacagatcc 4740atatatttgc ctaaaataga cacagaaaca ttgatatatg caaacatgag agctataagt 4800tttacatgat caaaaccttt tttttatggt acacaatagt cacagtactt ttccatataa 4860aacaggttta gtggtcttaa tttagtttgg cacatttaat acactcccat gaccagcatc 4920ccaaatgtac ctatccgttt tattttattg tctcagaatt gtcagttatt taataaatta 4980tgtaactttt ttccttatgc tcagatttgc acttctttct aaaactctgc ccatccttaa 5040agtcccagat tctccttgaa cttttttttt tgactttcca agtacatgga actcttcact 5100ctatcctgct atataagtga cagaatttcc actatgggat agatggagtt caattccttt 5160gagtttaaaa taatctaaat ataattattc cttatgccct gtttttccct cacttttgta 5220tccaaatctc ttttcagaca acagaacaat taatgtctga taaggaagac aatgatgatg 5280atcacttcaa aataagcttg aattcaggat tgtaatgtaa aattttagta ctctctcaca 5340gtatggattc taacatggct tctaacccaa actaacatta gtagctctaa ctataaactt 5400caaatttcag tagatgcaac ctactccttt aaaatgaaac agaagattga aattattaaa 5460ttatcaaaaa gaaaatgatc cacgctctta gttgaaattt catgtaagat tccatgcaat 5520aaataggagt gccataaatg gaatgatgaa atatgactag aggaggagaa aggcttccta 5580gatgagatgg aattttagtc atccgtgtct catgaagaat cagatgtgta cactaagcaa 5640aacagttaaa aaaaaaacct ccaagtgagt ctcttattta tttttttctt ataagacttc 5700tacaaattga ggtacctggt gtagttttat ttcaggtttt

atgctgtcat tttcctgtaa 5760tgctaaggac ttaggacata actgaatttt ctattttcca cttcttttct ggtgtgtgtg 5820tatatatata tgtatatata cacacacaca tatacatata tatattttta gtatctcacc 5880ctcacatgct cctccctgag cactacccat gatagatgtt aaacaaaagc aaagatgaaa 5940ttccaactgt taaaatctcc cttccatcta attaattcct catccaacta tgttccaaaa 6000cgagaataga aaattagccc caataagccc aggcaactga aaagtaaatg ctatgttgta 6060ctttgatcca tggtcacaac tcataatctt ggaaaagtgg acagaaaaga caaaagagtg 6120aactttaaaa ctcgaattta ttttaccagt atctcctatg aagggctagt aaccaaaata 6180atccacgcat cagggagaga aatgccttaa ggcatacgtt ttggacattt agcgtccctg 6240caaattctgg ccatcgccgc ttcctttgtc catcagaagg caggaaactt tatattggtg 6300acccgtggag ctcacattaa ctatttacag ggtaactgct taggaccagt attatgagga 6360gaatttacct ttcccgcctc tctttccaag aaacaaggag ggggtgaagg tacggagaac 6420agtatttctt ctgttgaaag caacttagct acaaagataa attacagcta tgtacactga 6480aggtagctat ttcattccac aaaataagag ttttttaaaa agctatgtat gtatgtcctg 6540catatagagc agatatacag cctattaagc gtcgtcacta aaacataaaa catgtcagcc 6600tttcttaacc ttactcgccc cagtctgtcc cgacgtgact tcctcgaccc tctaaagacg 6660tacagaccag acacggcggc ggcggcggga gaggggattc cctgcgcccc cggacctcag 6720ggccgctcag attcctggag aggaagccaa gtgtccttct gccctccccc ggtatcccat 6780ccaaggcgat cagtccagaa ctggctctcg gaagcgctcg ggcaaagact gcgaagaaga 6840aaagacatct ggcggaaacc tgtgcgcctg gggcggtgga actcggggag gagagggagg 6900gatcagacag gagagtgggg actaccccct ctgctcccaa attggggcag cttcctgggt 6960ttccgatttt ctcatttccg tgggtaaaaa accctgcccc caccgggctt acgcaatttt 7020tttaagggga gaggagggaa aaatttgtgg ggggtacgaa aaggcggaaa gaaacagtca 7080tttcgtcaca tgggcttggt tttcagtctt ataaaaagga aggttctctc ggttagcgac 7140caattgtcat acgacttgca gtgagcgtca ggagcacgtc caggaactcc tcagcagcgc 7200ctccttcagc tccacagcca gacgccctca gacagcaaag cctacccccc gcgccgcgcc 7260ctgcccgaag ctt 72735395241DNAHomo sapiens 53gatcacctga ggtcaagagt tggagaccag cctggccatc atggcaaaac cctgtctcta 60ctaaaaatac aaaaattagg agggcatggt ggctcatgcc tgtaatccca gctacttggg 120aagcagagta ggagaatcac ttgaacctgg gaggtggagg ttgcaatgag ccgagatcgt 180gccactgcac tccagcctgg gcgacagagc aatctccatc tcaagaaaaa aaaaaaagaa 240aagaaaagaa atgaagattc tttctcccct ttcctcccag tgctctcccc acaggaacga 300gacctgcgtg gtgtggggag cagctgaaga cttctcatct gcctttgtga atgccaattg 360tacacagcca ctactggaat cttactcatc gcagcaggag gcctggcttc cggcacaggt 420ggaatgaatg aaggaaggaa cggatgaatg aaaacaatga agctgtacag agcagtctgt 480ctccgagtgg gcagtggatc ctggaaaaca catcttcagc catccagagt gggaagatct 540ggatttggga tgtagccgct tcctccctgt gtgacctttg gcagatgtca tttacttttt 600ggaacttcag tttttccatc cgcaaaaagg ggatgctgcc tgcccagttc atgtcacaga 660cctgtgaagg tcaaaacaga aggcaggagg gagcatattc cataaaaggt acagcagccg 720ggcagagtgg cccatgcctg gaatcccagc caaggcagga ggattgctgg agaccagaag 780tttgagacaa gtatgggcaa aatagcaata cctcatctct aaaaaaaatt atttaaaaaa 840tcagctgggg ctgggtgcgg tggctcacct gtaatcccag tactttggga ggccaaggca 900ggcggatcac ttgaggccag gagttcaagc ccagcctagc caacatgatg aaaccccatc 960tctcctaaaa atagaaaaat tagccaggtg tagtggtgca cacctgtagt accactgcac 1020tccagcctgg gtgacaaagc gagactctgt ctcaacaaac aaacaaacaa acaaacaaaa 1080aaccagcgga gcatggtggc acacactgta gtcccagata cttgggtggc tgagtgggga 1140ggatggcttg agcccaggag gtccaggctg cagtgaacca cgatcgcacc actgcactcc 1200ggcctgggcc acagagtgag actctgtctc tacaaaagaa aataagaaag gaaggaagga 1260aggaaagaaa aaaaaaataa ataaaaatga aagataaagc atagggacgt ctctgagaca 1320aagagctgag tggaagaatc aagttaggac tcagcagcgc aggatggcga ggcttataat 1380tttaatcccc tcctcgattt ctttcgatga ggcaaaaaac agtcttggaa attactcacc 1440aaaccagcag tgggtgccag gagctcattc actttgtgtg tcattataat tttttgtaac 1500tagaatggat ggagcaacca ctttggtagt gaaaatattt taatatccgc atgtgcataa 1560agtgacacga ataacagttc ccgtttactg ggctctgatg ctgtagcagg ctgtggggat 1620ggctactgtg ctcattttac agacaaggaa actgaaaccc aggcaggcga agtggcttgc 1680ccaggctcac acagccagaa cgggatgaag caggttctga cctccaagca agactgactc 1740cagtggggaa ttatgggttc ccaaatgaca ctgatcacag caacaacggg caggacagga 1800caggtgactc agagcagact cctcatgcaa ggggagatgt tgcccagtgc cgagggcacc 1860ggggcagggt tcatgccttc ccctgggaga gcaagaggtt cagagtcaga aagactgggg 1920cttgtggtcc cagctctgcc actttctggt tgtgtaactt ctgccaaatc ccttcacccc 1980tccgagcctc aatgtgctca tatgcaaaag gggcgagtaa ccacctacct tgcaggcttg 2040tgtggactga gtgtgttacg gccatgaaaa caccatgtgc tctataaggt gcgctttatt 2100cattcctgaa gtgagcattt atcatgcacc cacgttcatg ccaagccctt ctctgggatc 2160tggggagaca gcagcaaaca cagcagatga ggtcctggtc cctggagtta ctttcaagtg 2220gttggagcca gataatcaac cgagaaagcc tgacacagtt cagacggcgt tgagtgccgt 2280ggagaaccca cgccggacag cgtgacggag cggcctcggg gctgggctac tgagcaaggg 2340aggggcctct ctgactttgt gatgtctgca cagaggctga gcggtgtggt gcaggtcagt 2400gatggaaagc tgtttatggg aagtgtcaag ggatagcccc aaggaaggga ggagctacag 2460cgggtgagga acagaaggct agggcaggga gaatgggcaa ggggcccacc gggcagtgcc 2520tgtgcactag aggtggtctc tagaggtggg aatgtctttg tggacacgtg tcctttgctt 2580aggacagcgg agagaggctt ccaggtctgg gtgggtgaga aagagggagg agctgtcagg 2640cagaaccatg gaggtaggtg gtaggaggta ggaggtaggt gggagggtga ggcacctgct 2700ctgagcccct tctccctggg caggaatggg gcatgtgggc agagcagagg gaagcagcgg 2760tgcaggaatg gccctgacct gcacagatgt gggaggaggt ccgcacgccc agagaggggc 2820tgagatcata ccaccaggga cctggtgttg gttccacaag aggctcaggg acacacttcc 2880agaattttga gagcacccct agcagaggca gggacttgga ctggttgacc tggctttccc 2940acaccctcaa aacctcaaaa tgtccaatgt ccatccactg atcatgatgg gtctttctag 3000aatgtcattt tctccccagt gcagttggtg agaggcattc tcacctcctc cctggagagt 3060ggggttcctc cttaccattg cctggtggtt gagctctagt ccttctgtct ggctggccgt 3120gtagccttgg gcaagccgct ccatctctct gtgcctctgt tgcctgggct gtaaacagaa 3180gtgagctaaa ggcaggcaga ccgaggtctg tgaccacgta ataactcata ctcagttcca 3240gaaatattca cccacagaag tgtctgggac aagcctggaa ggctgatcac accagccctc 3300cgggtgctgc tcgtggctga gagaacagaa gggagccctg tccaccatgg gaagctgctg 3360tttccatcac cagcctgggc tgtggtgcag aaagaaggaa ggggagtctg ggtggggcga 3420gggaggcagc aaagggcctg gaccttcgtg ggagcacgga cacacaggac agccattgtc 3480gagcttggac tgaccctact tggtgacgtt aagttctcaa gctccaagaa acagcatctg 3540agttcttgag ctcaatcttc ccaccaaaga aaatcataca caagtcccgg cgcaggggct 3600tcacagctca aagcatggtc tgtgtccaca tttcctgtgg tgggtcaggc cccactgcag 3660tcctgagcca gctctgcatt cccaccagag ccccaggaga tcagatgcgg ggtgaactct 3720gagaagcgct gctctagggc acaggtaggc tcattgcagc cttgtcccca gcgggaaaac 3780gcggtggacc tgcagcagtc agaggcaagg cacactgcaa gctccaggaa caggcaggac 3840ccgagaaacg taggtgggtg gaagcaggaa gaaggagaac ccagcgcaaa actgatgatg 3900catattaaaa acatgcacat ggcggctggg cgtggtggct cacgcctgta atcccaggac 3960tttgggaggc cgagatgggt ggatcatgag gtcaggattt cgagaccagc ctggccaaga 4020tggtgaaacc ccatctctac taaaaattca aaaaaattag ccgggcgcgg tggtgggcat 4080agggagactg aggcaggaga atcacttgag cccaggaggt ggaggttgca gtgagctgag 4140attgcaccat tgtactccag cctgggagac agagcaagac tcagtctcaa acaaaacaaa 4200acaaaacaaa acaaaaaaac atgcacatgg caaaatgaca taaaggagag tgttgggatg 4260gtgggagccg tattgtgtca atgtgaactc tcctgaacgt ggttattgtt cctggttatg 4320taagagcagc tccttgttct caggaggccc acggggaatg gtcctgacat gtgtaggtac 4380ttctatatgg ctcagcaaac aaaatttatg cagatactca gagagaaccc ctggagcaaa 4440atgttgacaa tctgtgagcc taggtaaagg ggatatggga ggtttgttgt tgttgttttt 4500tgttttttga gacggagtct cgatctgtca ctgaggctgg agtgcagtgg tacaatctct 4560gctcgctgca acctctgcct ctcaggttca agtaattctc gtgcctcaac ctcctgagca 4620tctgggacta caggtgcacg ccactacacc tggctaattt ttgtattttt agtagagacg 4680gggttttgct gtgttggcta ggttggtctt aaactcctga cctcaagtga tcctcctgct 4740tcggcctccc aaagtgctgg gattacaggt gtgagccact gtgcctggct aatttttata 4800tttttagtag agatggggtt ttgccatgtt ggccagaact cctggcctca agtgatctgc 4860ctgcctcggc ctcccaaaat gctgggatta caggcatgag ccactgcacc cagccggata 4920tgggagttta ttgcactgtc cacagagtga aggtgtggct cctggacttc ctccctcgtc 4980cagaggagag tcaggctgaa ccagccgggt cccaggagac caaaggagac cccccccccc 5040ccgccgccac taacaaacca cagagatttt tactgaaaat aagttttctt cctctcttct 5100tgtaattaca aagataaacc aagtttattg taaaaatgtg aaatgactaa gaagtgtata 5160aagcaaaaag caaagcgttt tttcaccttt gcccctcccc aattcttaac ctctgtggac 5220agcttggcag ccccttctag gcatttttct ttccaggtga aagttctgtc aatctttttt 5280cctcccagag ggagtcctgc acaatttatt gttcatatat tggggacagg tttccatggc 5340aaaactcaat ctgattcttt tttacttttt tttttttttt tgagacagag tctcgctctg 5400ttacccaggt tggaatgcag tgccatgatc tcggctcact gcaacctccg cctcccaggt 5460tcaagcaatt ctccggcctc agcctcctga gtggttggga ttacaggcac ctgccaccat 5520gcctggctat ttttgtattt ttagtagaga tgaggtttca ccgtgttggt caggctggtc 5580tagaactcct gatctcaagc aatccactca cctcggtctc ccaaagtgtt gggattaaag 5640gcgtgagcca ccgcccctgg ccctttctta ctttttaaat caagaactga aaacgacttt 5700atttactctt cttgggacat ggccacgccc atggaagtcc ccaaagtagg ctggacaggc 5760cacagcagca cccggagcag tggtggcagc tcctgttgag ctgccctcca gaagccagtt 5820ctgatgcgcg gcctcgccgg gggcctgaga acccctgttt cctgtgaggc tgggccaggg 5880acaggataac aagggaggca gaaaagagtg ctggcgggga gccaggaggc ctgggttcca 5940gccccggccc tgccgcttgc ctgctaggag cccttgagaa agtcagttcc cctgcctgaa 6000cctcagtctc ctcaccttca gatggagatg ccggcccaga gggtaccaga ggcctttcct 6060ggcttgcaaa caggatgcca gtccacaaag ccacagggtg agggtgcttc ccagttctct 6120gtgcttcgga acagcgtgct gctccggggg accttggaaa ggtgactggg ctcttctggc 6180ggtttggggt gggggttgta gtttgtgctc ccggatgttt gcccacgtgg gtggagcctg 6240cctgtctgtt gccccttaga gggaagttgg cagtaggatg ggttgggggg ccgtggatgt 6300tgggaggccc taaagctgag cccagactct caggcttggg aaggaccttc ccgatcagcc 6360ttctgtccat ggcttgaatt cctgtcttgt ggcatcagga aagacttatg tcttttaggg 6420tccaaccaag aaagcagaaa aacactcagg tgttgcagac agagggcctt catacaggga 6480gttagtcaca caggttatgg gagagccgag aagccgaaga gggtgtgatg agttaaccca 6540gagattaaca actgcgagaa accaccaccg ccccaggatg gagaagccag ggaggtggtg 6600gggttagcag atcctgggat cggggtcacc cagtaccagc caggggcttg tggcagagag 6660ctggagcaca gaggagacat ggctgctgcc gctgagctca cgaaggaaga cagggaaggg 6720gagggatacc cagcttctct catcacatgt gccccatctt cagtctcccc cagtgcctcg 6780ctttggcaga actcgctgaa aaacgcagcc tgcaggtatc agcccccacc ctgccctgaa 6840cacagagagg aacatatttg aggtcagagg cccaggactg gcccagtgac tgtgtttaaa 6900tgcttcgggc actggggagc tcactctcta actcttaact gtagaggtgg tggcaacagc 6960ctgttttgct ggcggctgag catgagcatt tggttcagaa taaggaagaa acattggttt 7020cctgtgactc cctacagaaa gatgaggggt ttgtcttggg agcagaagcc cccgtgtgtg 7080ttgctttgtg ctgtcatgtc tgccaggaag gcccttcccg ccctcccatc gcttgagacc 7140caattctccc agaaaacctt ccggcacctc catgtcgagg gatagtaccg tcatgtccgt 7200ccacagcacc tgcgcttcct tctctattta gcagaatgta gcgaacatta tgttcaacgt 7260ggacttctct gtttgttctg cctcattcat agggggcatg gagcttggag aacctgacgt 7320tgcctaccca gagctggctc taggttaaag agatgctcac taaggcacct atagtgtgcc 7380aggtctgcca aatgtttggc atgcattatc tagtttaatg ctcccaacaa ctccggaggt 7440tggtatgatt agcccatgcc tgctcagctg gagaatctga ggctcaaaag gagggtgtcc 7500aaggccactt ggctagtaag ggcagagcta ggattcgaac acagcctctt aaaggccgcg 7560ttccttagcc acggggccac gtggtcttgc cacagtgcag ctgggcccag ggtgggattg 7620tgtgaagtcc ctcactggga atgtttccag ctcagctcct ggtgcctcct cccctgtcct 7680ctgtccaaga ccacatgtca gccccttgaa ggcgaggcag ccattcccac agccacttct 7740ctattctgac atgaccaaga agcctggctg ggacagcagg tctgaccaca gattgacaga 7800tgtttccaca tgtggaagtg aggtttgagc ctcgatgtgc tgtttctgtg gttccctttt 7860cacgctttcc ttgggagatg tgtccagaca tggtctcatt gccctaatag gtttccatgt 7920ctgttgtgca cagtctttag actgtttaac aatcctgttc actggtagag cactgcccag 7980cttgcacaca gcactttctt atgcattggc tcagtggctc ttcccaacaa tcctgggact 8040tgggttcatt tactggatgg aggctcagag aggctaagta acaacagtga caaccattag 8100ttgccttttg cagatctgtc agcatgcctt gctcaaagag agacagaaac tgcccagtgc 8160acagtgtctc acttgatctt cacaatagcc ctgcaaggta gatattatta caacctctca 8220ttggaagtag ggaaactgag gctcagagag aataattgac ttacccaagg tcacacagcg 8280tcaaatccac acctagaacc catctcttgg tcttgactcc tggttcagtg ttccaagcaa 8340ctgttgagaa catccatcaa acttaaaaat atatatgact atatttcaac aaatctatga 8400catcattgaa tatagataca ccactctttt atatacccca ggaaatagaa atgctgtcag 8460ctacagtaag acacagtatt tctcatcaca tagaattttt ttattttaga ctaattaaaa 8520gagctctttc atatctgtat gcatcatata tatataagca ttatatatgc atatatataa 8580tgcatcatat atgcatcata tataagcata tatatgcata tatataatgc atcatatata 8640tgcatcatat atatgcttat atatgatgca tatataagca tatatatgca tatataagca 8700tatatgcata tatatatgta tctctctcag ttgtctgtac atagaaggaa aatttatctg 8760aaaataaact tatatgacat gaaatggatt tatgtgaaaa taaacttctt catattcaaa 8820atctaactga gtaactgggc gtggtggctc atgcctgtgt ccagcacttt gggaggtcaa 8880ggcaggtgga tcacttgagg ccaggagttt gagaccagct tgggcaacat ggcgaaactc 8940tgtctctaca aaaaatacaa aagttagcca ggtgtggtgg cagaggctgt agccccagct 9000acttgggagg ctggggcagg agagttgctt gaacccggga ggcggaggtt gcagtgagcc 9060aagattgtgc cactgcactc cagtctgggt gacagagtga gactctgtct taagaaaaaa 9120aaaaaaaaag acaaactctg agtgagcagt agaagcccag ctctttccca taagattgtt 9180ctccgcacca gcaaatgctg gcgatgaaga tttctccctc tctcttcaaa aatatgttag 9240agaggaaaag cgtgtttaca tataacaaag tacataaatt ataagtacac ttgatgaatt 9300tttatccacg ttaatattca tctagactca ccagtgcgtt ggagctggct cacattagca 9360ttgttaaaca ctcaggaatt ttgcaaactg gtttttaaat tgttggtcac ttaaaatcag 9420ctgcgggcca ggcgcagtgg ctcacacctg taattccaac actttgggag gccaaggcag 9480gaggactgct tgagcccagg agcttgagac cagcctgggc aacataggga gaccctgtct 9540ctacaaaaat atatatattt ttaaattagc cagatgtggt ggtgtgtgcc tgttaagttc 9600cagttacttg ggaggattgc ttgagcccag gagattgagg ctgcagtagg ctatgatgga 9660gctgctgcac tccagcctgt gtgacagagc gagacgccgt ctcaaaaaac aaaaacaaaa 9720accaaaccct agctgcaatg ggagtatttt acatcatgga aattggcaaa tgctacataa 9780tccagggctg tttttttccc ttcagaggtc tggtttactg gcccaccact gagtgtagct 9840actacccaga tggggagagc ttcccagcat cctagaagcc tcctttgtgg ctgtcccagc 9900ccctttccac caagggaaca actactggat gcggcatttc ctagaggtgg ctgagggcca 9960ctggcgctgg gctcctgcga ggggtttgcc attgtgcggg gctggcccac tttcgacgcc 10020catgggagga atgctgctaa acacgtccga tttaccacct cctccatccc gtacccacag 10080ccatttggtt cctagaggtt aaaagaacac tcctctattg tctccagggt ttccttccaa 10140gccgcagaat cccattgtcg atgtgacggt gtaagcgggc tgtgaccact ccctggagag 10200ggcctcctgc caacaattac tgtaagacac acccctattt cagagacact aaaatgtgaa 10260aaatcaagcc tcttagagtc accaaaatac agtatattgc cttttgaaga tctttactga 10320agcagtttcc tctgagaagc agcttgtctc catcattaag ccccggaaag cagatgagac 10380tgcagttcct ccgggctagc tgtctcagtg gtcacttcgc ccccagacag gtagcttctg 10440cccacttctc tcatggggca gccaagtgtt actacctctg gccccggcct ggaataagag 10500gaccagcagg ccgtgggaaa cctcagctct aataccaggc tgcttctgga cagtcctttc 10560tgggtgtgga taaagaccag gcttgtgccc tctggggacc gttcaaagca gtcttcaggg 10620tcggacctca gactcatccc tgtgatgatt gtttcaggtc ctagcgagtt actttcccaa 10680cctgtgagct tctgcaactg tgtttttttg ttttttgtta ttgttgtttg tttgttttaa 10740tatttttttc ttttcttttt tttttgagac agagtcttgc tctgttgccc aggctggagt 10800gcagtggtgc gatctcggct cactgcaacc tccacctcct gggttcaagc gattcttctg 10860cctcagcctc gcgagtagct gggattacag acgtgtgcca ccacaccagc taatctttgt 10920atttttagta gagacagggt ttcgccatgt tgcccagact agtctcaaac tcctgacctc 10980aagtgatcca cccacctcga cctcccaaag tgttgggact acaggggtga gccactgtgc 11040ctggccatgc aactgtgttt taatcacctt tgtgttccca aggccctgac atggaacaag 11100cacccagtaa gtatttgaat gaatgagcaa atgaaaggcc aggaagggag agcctttatt 11160ttgaagcctg cccgccgggc tgcccttggg aaagccactt tctgcaaaag tcacaggagc 11220aaatgagaca aagatgcaaa attgctctgc ctgagctgtg agggcttaac tgtgaatgtc 11280tttaggtgac cttcttggag acctcaagac cacccctctg tgacttggtt caggctgccc 11340tgctgtgatg cctgctgggg ccaaggcctg gatccctggg tggggtgggg tggggtgggg 11400cggggcgggg aggggcgggg cggggcgggg caggagtggc agcaggaagg atccggctga 11460gacttgccct ggggggccag ggaaggaggg tggcaggagg cagaatccac aaatgaagta 11520gatctggagc caggcagatc agcccttaga tataatctca gaaggggttg ggagaatgga 11580aggattttgt tgaggatgga gtgagagggt tggagggttg ggtatgttcc tgagcatatt 11640tccctgtcta tggggccatt cagagagaag cccacgtgct ccaggccagt ggtggagcct 11700tcaacgtgga gctggaagac ctgggctcga gtcccacctc tgccatgtcc catcctccct 11760catgactccc agtagttacc gccttctctg ggcctcagtt tccccaactg gaaattaaag 11820aaaattaccc tgttcctgca tcagatggtt ggttgtggat atcactgaaa tctcctcacg 11880tggtattgag ccgctgctct tggccagaca cagagcaatt tacatgaaat gattttcgaa 11940gtctggtccg gggaccagca gtgtcagcat cacttgggaa ctttgtcaga aatgcaaatt 12000atcgggctcc accccaacta ctctagaccc aaaaacaatt tttattttta tttttattta 12060ctttttagga tggagtcttg ctctgtcacc caggctggac tgcagtggtg caatctcagc 12120tcactgaaac ctctgcctcc tgggttcaag cgattctcct gcctcagcct cctgaatagc 12180tgggattaca ggcatgcacc acgacgccca gctaaatttt tttattttta gaagaggcag 12240ggtttcacca tgttggccag gtggtctcgc actcctaacc tcaggtgatc cacctgtctc 12300ggcctccaaa agtgctggga ttacaggcgt gagccacagc gccctgcccc aaggacaatt 12360tttaaatgat ataattcata tcccataaaa ttaacccttt aaagtgtgca gtgtggtggc 12420ttttagtatt atccaccagg tcatacaacc tattatcact aattccagaa tattttcatt 12480gctctcaaaa gaaaccttgt accatttagc agtgactccc cactcccctg tccctcagcc 12540cctgcaatca caaacctact tttcatctct atggatttgc ctattctgga cacttcatat 12600aaatggaatc atagaatatg tggtcttttc tttcacttag cataatgtct tcaaggttca 12660tccatattgt aatatgtatt agtacatgtt gtactgatgg aacatgtatg ttgtagcatg 12720tttcaccctt ttaaaaaatc ttctttttaa attaaaaaca tttttaaaat acattcaaag 12780atttttttag agtcgtttta gtttcacagc aaaattggga ggcaggtatg gagatttccc 12840tatgttccct gcccccacaa catacgcagc ctcccatcat taacatcccc caccagaatg 12900gaacaatttt aacaaccgat gaactgacat tgacacatca ttatcactgc aaatccatag 12960tttactttgg ggttcactgt taatgtagta cattctatgg gtttggacaa gcgtataatg 13020acacgtatct gtcatgatgg tatcatacaa agtattttca ctgccataaa aatcctctgt 13080gtgccaccta tttcttcctc ccatccccct aatccccggc aaccactgat ctttctacca 13140tctccacagt tttgcctttt ccagaatgtc attctcttag tccattttct gctgctataa 13200caaaatacca cagactgggt aatttataaa gaaaagagac ttactaggct cgtggttggg 13260gaaatccaag gttgaggggt tgcatctggt gagggccttc ttgctgtgtc ataacacggc 13320agagggcaag cgagctcacg gaacagagag aggaactcag actgaactca tctgtttatc 13380aggagcccac tcctgcgata actaaccccc tcccctaata atggtattaa tccattcaag 13440agagcagagc tctcatggcc

taatcacctc ttttttgttt gtttgttttt gttttcagac 13500agggtctcac tctgttgctt aggctggagt gcagtggcac aaccatagct cattgcagcc 13560ttgacctcgc aggctcaagt gatcctccta cctcaggctc ccaagtagtt gaaactatag 13620gcatgtacca ccatgcttgg ctaattttga aattttttta gagatgaggg cttgctatgt 13680ttcctaggct agtcttgaac tcctggactc aagtgatcct tctgcctcag cctcccaaag 13740tgctgggatt acaggtgtga ggcattgcgc ctggccctaa tcacatccta aaggtcttgt 13800ctctccacgc tgttacaatg gcaacgaaat ttcaacataa gttttggaaa agacattcaa 13860gccatagcat tccacctctg gcccaccaaa actcttgtct tccttgcata caaaataaca 13920ttcatcccat tccaatagcc ccaaagtttt aactcattcc agcaccgact caaaagactg 13980aagtccagag tctcatctaa atcagatatg gatgagactc aaagcatgac tcatgctgtg 14040gcaaattcct tccagttgtg agtctgcaaa atcaaaacaa gttatctact tccaaaatac 14100aatagtggga caggcatagg atagatgttc ccattccgaa agggagcaac aggaaaggag 14160aaaggagtaa caggccccaa agaagtgcaa aacccaaaag ggaaaacaag attaagtctt 14220aaagctggag aacaatctcc tttgactcca cgaccagcca cctgggcaca ctgggcagcc 14280ctgcctctac ggctttgcta ggctcagccc acacaatttt cacaggttgg gatctcatgc 14340ctgcagcttt cccaggctgc catcactcac tggcagctca acagttctgt ggtctggaga 14400gtggccccac ttccacggct gcagtaggca ttgccctagt gaggactctg tgcagtgcct 14460ctgatcccac acttccactc ggcatttacc taatagggct ttctgtcatg gctttgcccc 14520tgtggcaggt ttctgcctgg gccccctttt aaatctagtt gaaggtagcc atgcccccac 14580agctcttgca ttctgtgagc ttgcagacct aacaccatgt ggatgctgct aaagtttaaa 14640gcttgtacct cctggagcag caggttgagc tgcacctggg accacttaag ccacagccag 14700ggaagtcaag aggtgctgca ctggaatgat gggggcagag tcctgagatg gctctgggca 14760gtgagcctgt ggaggatgtc ccaggcatgt tccctgaaac cattctgctc tcctagagct 14820ctgggcccgt aataagagaa acagcccgga agagctctga aatgtctttg gagtctttcc 14880tccaaaggaa taacacctgg ctttcttcta tctagcctga tcttttaagt aaatggttgc 14940ttggccacac ccttagtatt cttttccgaa tgttattgct tttcactctt taggaggcca 15000ggctgtgagt tttcctttgc ttctctttta attataaatt ctgtctttaa gtcattcctt 15060tccttttgca tctcactgta tgtggttaaa aggagccatg cagcaacctg aatgctctgc 15120tgcttagctg tttcttccat cagatatccc cgttcattgc tcttcagtcc tgcactctat 15180aaagccctta gacataaaca cagttcagcc aaagtctttg ctactttgta acaaagatgg 15240cctttcctct tagtttccaa taccttgttc ctcatttctg tctgagacct cattagaatg 15300gcctttactg ttcatatttc tacggacatt ctggtcatga ccacttaaat aatcttcaag 15360aagatttagg ctgtccttag ttctagggcc ttcttttgag ccctcagcaa aattgctctt 15420aatgctccat tcacaggaat ctaggctttt tctagcctgc tcctacaaac tcttccagct 15480tctatccatt acccagttcc aaagcagctt ctacatgttc aagtatttgt catggcaaca 15540gctcctcttc tgtgcaccaa ttttctgttg ctataacaca ataccacagg ctgggtaatt 15600ttacgtatat atagaatata tatatagaat atatagaaaa aatatatatg tatgtatttt 15660atataataat actgagcatt gactcatggt tctacaggct gggaagtcca aggttgagga 15720actgcatctg gtgaggacct tcttgctgtg ccataacatg gcagaagggc caagagaaag 15780agaacagaaa tcaggctgaa ctcattcttt ttatcaggag cctacttcct agataactaa 15840ccaactgtca caataacagc attaatccat tcatgagggc agagctctca taacctaatc 15900accttttaaa ggtcttgcct ctcaacagtt actatggcaa ctaaacttca acatcagttt 15960tttgagggga ctttcaaaca atagcagtca tatattggaa tcacacagta tgtagccttt 16020tctgattggc ttctttcact tagtaatatg gatttaagtt tcctccattc ttttcatggc 16080ttgatagctc atttcttttt agtgctgaat aatagttcat tgtctggatg taccacagtt 16140aatccattta cctgctgaag gacatcctgg tttcttcttt tggcagcatg aaaaaagctg 16200ctataaacat ctgtgtgcag atttttgtgt gaacataagt tttcaactct tttctgtaaa 16260taccatggag tgtgattgct caatcatatg gtaaaagtat gtttagcttt atagaatgac 16320aatttacctt tcaaagtgac tgtactatgt gctaagtggg tgtactattt tacatttacg 16380tttacaacaa tgaaggaaag ttcctgttgc tccatatcct cctcagtgtt tggtgctgtt 16440tgtattctgt attttggcca ttctaataga tatgtagtat cccgttattt tagttttcat 16500tcccttgata acatgtgatg tagagtatct tttcttatgc ttatttgaca tctgtatatc 16560ttttttggtg aggtgtctat taaggtacat ggcccatttt ttaattgggt tgtttttttt 16620cttattgaga gctttaagag ttctttgtat attttggaca actgtctctt atcaaacatg 16680tcttttgcaa atattttctc ccagtttgtt gcatgtctgg ttattccctt gacattggct 16740ttcacaaaac agaagtttaa aaaatttttt taatgaattc cagctcattc attgtttatt 16800tcagcaataa tgctttcggt gttatacctg acaagtcatc accataccta aggtcatcta 16860gactttttcc tatgttgtct tctcaagagt tttacagttt tgcattttta atttagattt 16920atgaggtact ttgagttaac ttttgtggaa tgtataatgt ctgtgtctaa attcagtttt 16980tttggtatat ggatgtccag ttattcatat tttttaaaag atggattttt gcatgatatt 17040ttgaaaagac tgtctttgct ctattgcatt gtctttgctt ctttgtcaaa gattagttga 17100ctacatttat gtgggcctat gttgggctct ctattctgtt tattaatcta cttgtttatt 17160cttttgccaa taccacactg tcttgattag tatagcttta agtcttgaag attactatag 17220ctttaagtct tgaagactac tatagctagt aagtcttgaa gtcaggtagt gtctgtcctc 17280caactttgtt cttcctcagt attgtgttga ttattttgat ctttccctct tcatataaac 17340tttagaatca tttttcaata tccacaaaat aacatgctgg gattttgatt gggattgcac 17400tgaatctata gatcaggttg gggaaaactt atatcatgac aattttgaat cttcctatct 17460gtgaatatgg aatatctctt tatttattta gttcttcttt gattttgttc atcagagttt 17520tgtagctttc ctcatataaa tcttatatat atttacttag atttatacct aagtactttc 17580ttttattggg tgctaacgta aatggtattg tgttttaaat ttcaaatttc acttgcccat 17640tgctggtata taggaaagtg acagacttgt acaacaacct tatatcctac aatcttacta 17700taatcaccta ttagttccag agatttttgt gttgatttat ttggattttt ctacatagat 17760aatcatgtca tctacaaagg cagttttatt tcttccttcc caatcagtat aacttttatt 17820tcattttctt gccttattga gttagcttgg acttccagta tgatgttgaa aaggagtggt 17880gagaggaaac atccttgact tgttcctgat tttagtggga aagcttctag tttctcacca 17940taagtatggt gtttgctgta agttttttgt agattttttc atcaaataga ggaagttctc 18000ctcaattcct agtttactga gagttgttat atgaatgggt gttgaatttt gcgaaattat 18060ttttcttcat ctattgatat aatcatgggg tttttctttt ttagcttgtt catgtgatgg 18120ttatattaat ttattttcaa atcttgaacc agccttatat acccaggata aatctcactt 18180gataatgagg tataattctt tttatacatg gttggatttg atttgctagt aatttgttga 18240agatttttgc atctgtgttt atgagatata ttggtctgta gttttgtttt ttggtaatgt 18300ttttttatct ggttttgtta gtgctggact catagggtga gttagaaagt atttgctctg 18360cttctatcct ctgaaagtga ttgtagagaa ttggtataat ttcttcctta aatgtttggt 18420tgaacttacc agtgaactct tctctgcctg gtgccttctg ttttggaagg ttattaacta 18480ttgattcaat agatataggc ctattcagat tgtctatttc ttcttttatg agttttggca 18540aattgtgtct ttcacagagt tggtccattt cacccagatt atcaaatttc tgggcataga 18600gttcatagta ttcctttctt atcctttcaa tgtccatagg atctgtagtg atgtcccttc 18660tttaatttct gatattagta atttgtgttc cctctctttt tttcttagtc tggctataga 18720cttattgatt taattgatct tttcaaagaa tcagcttttg atttcattga ttatttaatt 18780ttcttttttc aatttcattg atttctgccc taatttttac tattcttttt tttcttctac 18840ttactttggc cttcttttcc tagtctgtta aggtggaaac ttagattatt gattttagat 18900ttttcttctt tcctaatata tgcatttgat gctataatct tccctcaaac cactgctttg 18960gcttatctta cacattttaa taagttgtgt tttaattttc atcaggtaaa aatattaaaa 19020ttctttttga gatttcttct ttgacccatg tattatttag aagtgttttg tttaatctcc 19080acatgttttg gaattttcta gttatctttc tgttattgat ttcttttaat tccattgttg 19140tctgcgagca gaagttgtat gatttctact ccttttaatt tgttaaggtg ggttttatgg 19200cccaaaatgt ggtcaaattc tttctgtttt atggcccaat aatattccat tgtatagata 19260tacaacattt tgtttatcta ctcatgagtt ggtggacatt ggggttgttt tcattttttg 19320ttaattccat tgtacactga acatacaatt cagtggtatt ttgtatgctc acaatgttgt 19380gcagccatca cctctatcta actccaaaac atttcatcaa ctcaaaggag atcttgaatc 19440cattaagcag ccactcctca tgtccctgct ctcaacccct ggcaaccact aatctgcttt 19500ctgtctccat gaatatagct attttggata cttcatttaa atggaatcat acaatatgtg 19560atcttttgta tctgacttct tttacttttc ataatgtttt caatgttcat ccatgttgat 19620agcattcctt tttagggctg aatactgttg tgttgcatgg atatactatg ttgtgtttat 19680ccattcatct actgatggac gtttgagttg tttccacttt tgctgtgtga atagtgctgc 19740tatgtatttg tactcattgt acacattgtg tacaaacatt tgttcgaata cctgttttca 19800attcttttgg agaattattt tcaattctag gagcagaact gctgggttat atggtatcat 19860tgtgaggaac tgccaagctg tttcccaaag tggctgaacc attttacatc cccaccagca 19920acatatgaga gttctaattt ctccacattc tcaccagtgc ttgttttcct ttcctttcct 19980ttcctttcct ttcctttcct ttcctttcct ttcctttcct ctctctctct ttctgtcttt 20040taaattatag ccattctagt ggatatgaaa gagtatctca ttgtggtttt gatttggatt 20100tttcaaatga ctaatgatgt tgagcatctt ttcatgtgct tcttggccat tgtatatctt 20160ctttgaaaaa atgtctgttc aagcattttg accattttta aattgggtta ttttgtcttt 20220ctgttgctga attgcaagag ttttttttat atgtcctgga ttctagatgc ttatcagata 20280aatgatttac aaacattttc tcccattatt cattatttgc tgtcattcca ttttcctttt 20340tttttttttt ctttcttaga cagggtctta ctctgtcacc caggctggag tgcagtggtg 20400caatcttggc tcactgccac ctccacctcc ccagctcaag cagtcctccc acctcagcct 20460ccccagtagc tgggactaca ggtgcacacc accatgctct gctaattttt atatttcttg 20520tagagatgaa gtttcactat gctgcccagg ctggtctcga actcctgagc tcaagtgatc 20580ctcctgcctc agcctctaaa agtgttggaa ttacaggcat gagccactgt gcccagcctc 20640attttatttt cttgatagtg tctttttttt ttttttgaga caaggtctca ctctgtcacc 20700caggctggag tacagtgaca tgattatagc tcactgtaac cttgaactct tgggctcaag 20760caatcctcct gactcagcct ctcaagcagc tagtacaaca ggtgtgtgcc accacgtctg 20820gctaactttt acattttttt gtagaggtgg agtcttgctg tgttgcccag gctggatctt 20880gatagtgttt tgttttgttt tttttagatg gagtttcact tttgttgccc aggctgaagt 20940gcaatgtgca attgcgcgat ctcggctcac agcaacctcc atctcccagg ttcaagtgat 21000tcttctgcct cagcctccca agtagctgtg attacattta tgcaccacca cgcctagcta 21060attttgcatt tttagtagag atggggtttc accatgttgg ccaggctagt caggtgatcc 21120gcctgcctca gcctcccaaa gtgctaggat tataggcgtg agccactgtg cctgggtgcc 21180tggccttgat agtgtttttt gattaactat ctacttttat tttgatgaaa tccaagttac 21240ccatctatgt atatactgag ctgaccctga gacatggcaa aatgtgtgaa gatggtactt 21300gagtgagtga agtttgggca atgttgtttc tagtgaattc tttctccttg gtggtttcct 21360ctggcctgtg gatatacttt attcagcaaa agatgccagg gctgagggga tatggcctct 21420ggtctgccac ccagagggta aacttggcaa gaccccagcc aggctccccc tcctctgctg 21480ttcctaaaca tgcattcatg gacaaggggt ctctggagca aaggagagtg actccttccc 21540tctcccgcaa ccttgcccac ttaccttgta gccagccact ccctcctctt tctgtaactg 21600ggcattggtc cagctgccag gcccaggacc tctcccattt agccagatgt agttccaaaa 21660acaactgcag cagtatttgg atacattttc cagcctgaac tagtggtgtt cctgtccata 21720gctgggatcc aggtttgttg cctctgggtg gggctacagc tccttacctc ctggaaggtt 21780gtggaagtgt ggtttccttt ttctcctttc tcttgtggaa cataagcatc tttccaagtc 21840cttctggcca gatgatgatg gtgtgagcct gtccgtctcc catcagtgct gaggggcctc 21900agatgctgcc tcttacctat aacccagatg ctcccaggtg tgtttatttc ctagagctgc 21960tgtgacacag agccacaaac tgggaggctc agaacaacag gcattgttcc ttgcacagtt 22020ctggaggctg gaagtccaaa atcaaggtgt tggcagggct ggttcctacg ggaggctctg 22080aggaagaatc tgttccaggc gctctcctgg ctcctggtgg ttgctggcaa tccttggagc 22140cccttgactt gtagatgcat cactccagtc tctgccttca tcttcacatg gcgttctccc 22200tttccctctg tctctgtgtc ttcttctcct catcttattg tcatattgga ttaagggcct 22260accctgcatc agtatggcct cgtcttagct aatttcatct gcagttaccc tatttccaaa 22320ggtcacattc tcaggttcta agaagtacat gaattttgag caggataatg tatggcccag 22380tgcaccaggc aatgccaagg gcatcactag gtaggaggct ggagatgact ccatttctgt 22440gagctcctcc ttggctcctc tgtgtctttg cttctaacag cctgtgcctg ccactctctc 22500tcgagggctc cccttgagct attagagggg ctttgtgtgc acaaaattca gacacacaca 22560cacacacacg cacatgcaca tgcacacaca catgcacaca tgcacacaca cacacatata 22620cacacacaca cagagccaga gtgcctggat attcgtgacc cctggagctg tcttaccatg 22680gtgatgactg acaggtgggc agggcacggt ggctcacacc tgtaattcca gcactttggg 22740aggccaaggc aggtggacca cctgaggtta ggagttcaag accagcctaa ccaacatggt 22800gaaaccttgt ctctactaaa aatagaaaaa aattagttgg gcatggtggc gcatgactgt 22860aacccagcta cttgggaggc tgaggcagga gaatcacttc aacctgggag gcggaggttg 22920caatgaaccg agatcacgcc attgcactca agcttgggca acaagagtga aactccatct 22980caaaaaaaaa aaaaaaaaaa aaaaaaagga atgactgaca ggtgcacatg cagaagcata 23040gaagcccaga tccctggcct gcagttgggc acaaactctg aggtgtaact tatactccgg 23100agccccccac aggtcagttt caactggcct caccctccat gtctagctcc ccctactctg 23160acactggctt gtcctgggag tacttcctta agaaatcact ttcatgtgaa ttctcttctc 23220aaagtctgct tctgggcagc ccaagctgaa acagatcccc atacctggag cctgctgcag 23280ccaggactga tatgcaggaa cccagcccag ggagccacaa agggatccac ctccccggat 23340ccaggggttc atgattcatg ggcgatggtg ctctgtaaat gggaaggccc tctgtgaaca 23400ctggggtgtt tgccacgcat tgtgctcaat tgtcccctct atgggcgggc cttccccaac 23460cacaccatcc aagatattct aatcttgtcc tttcaggctg ctgatcaact agttcaggag 23520tcactgtgga catgtcacac ttcttcctcc atgagatgga gatgaccaaa tctattcata 23580gttctgtgtg ccaacctatg aaccagacct gagcccctta cccctctgac agtcggcttc 23640aggaaatcgc catgaggcta caggtgtgtg ttgaggggtg ggtagagaca caacataagt 23700gggtggcgtg gggtctggca cacttcttca tgtaacccac ttgtacctgc tggacctgcc 23760agtctcaatc ccaaatatca ctgtagcatt tctctttttt ttatattatg acaaatactt 23820atttatttat ttatttattt atttatttat ttatttttta attttatttt aagttccagg 23880gtacatgtgc agaatgtgca ggtttgttac gtaggtaaat gtgtgccatg gtggcttgct 23940gcacctatca acccatcatc taagcattaa gcccagcatg cattagctat ttatcctgat 24000gctctccctc cccacgcacc tcctgaaagg ccccagtgtg tgttgttccc ccaccgtgtc 24060cttgtgttct cattgttcag ctcccactta tgagtgaaaa cacgtggtgt ttggttttct 24120gttcctgcat tagtttgctg agaataatgg ctcccagttc catccatgtc cctgcaaagg 24180acataatatc gttccttttt atggttgtat agtattccat ggtgtatgtg taccacattt 24240tctttatcca gtctatcatt gatgggtatt tggattgatt tcatgtcttt gctattgtga 24300atagtgcatt gtagcatttc cattgtacag tgggttactg ctgtgcctgc ctcacattag 24360gatttggtgg atctggtcat agccagctca cagagggaaa ctcagccagc atagttgctt 24420gatgtctcat ggtcaggctc tgagtctctg tagggttcag tagcatgcca gcaattgttt 24480ttcaaaagga gagtagttct ccactgcaga aaattttaga ggtctgtact gggactcttc 24540tactggggtt tgttaaaggc tccacccaag ttctttatct agcaccataa atcttctcag 24600tctcatggct agcagagcag ctcacactgc agcttggacc tatgcagcgt tctcttttgc 24660tttgtctcag aactgaaagc tttctgaatt gcctaataaa taggtcagag tagcattccc 24720aagtgtggta tatgctgctt tgaaattcaa ggagaacaaa gaaggtgggc ataaaacaac 24780agaaggacag tttctcgcag ctggggagat cagaagtctg aaaccaagat gttggcaggg 24840ctgacacggg caatacacga ggcccgtgta ttgcctcttc ctgcttctct tggctccaga 24900cattccttgg cttgtggctg catcactcca atctgcgtct gtggtcacat ggcctcctcc 24960tcttccctat gtgcctctgt tctgtatgtc tcttataagg acatttgcca ttacatttag 25020gacctgcctg catcatccaa gattacctcc ccatcttgag atccttaact gaattacatc 25080tgcaaagatc tgttttccaa ataaggtaat atccccatag gttctggaaa ttaggacatg 25140gacatatctt cgtggtgggg tgggggggtg ctttttcatc ctactgtatg gtagaggtgc 25200aaatgcagca acttgtcttt ttttctgaga ggggatggct tggcatgcct cagatcacag 25260gttccttaag atcttcatca atacggggga ccctgaattt tcagaggctt tatcttccac 25320tctctggtgt gcatacattt ttgttttgtt ttgttttgag atggagtctc actctgtcac 25380ccaggctgga gtgcagtggc acgatcttgg ctcactgaaa cctccaactc ctgggttcaa 25440accattctcc tgcctcagcc tcccaagtag ctgggatgac aggtgcccgc caccatgcat 25500ggctaatttt tgtattttta gtagagacag ggtttcacca tgttgaccag gctggcctcg 25560aacgcctcac ctcaggtgat ccacccacct cagcctccca aagtgctggg attacaagcg 25620taagccactg tgcccagcca tatattttat taaagcatct tgggttcttg ccatttttcc 25680tccttaggtt tagagcagca ggagcatggg agcaactgtc cagtgaaggg ggtctgttga 25740gaggctcacc cacagcatcc actgcagtgt ccttgatcat cttgacaccc cacgctacca 25800cccaggtccg tcatgttaac attgtgtgag cattggctgc aaactgctca catctgcccc 25860cttctctgga gaattgctct ctgccaaaca gtagacatct caccgtggag gttatgctcc 25920tttgggggtg tggcaagtct tgccaactga cttacctgag gacacaaaaa gtctgctatc 25980tggaggggac aagtcagtgc tgtaattaat gctccaaagg ccctcatgag accagaatga 26040ggctggcctc cagcccaggg atgtcataga ttaactttct ttctctgctg tgtcctgctt 26100ccctctttcc acttcccctg aaagtcctcc ccacaaaaat ccccacctct tgatctgctt 26160ctagggaaac ctgacctaag agattccttg ggtgtattag tctattctca cgctgctaat 26220aaaggcatac tcgagactgg gtaatttata aaggaaagag gtttaattga ctcacagttc 26280ccatggcagg ggaggcctca caatcatggt agaagagcaa ggaatgtctt acatggtggc 26340aggcaagaga ggatgagagc taagttaaag gggaaactcc ttataaaatc tcgtgagatt 26400tattcaatat cacaagaaca gtatgaggga aaccacctcc atgattcaat tagctcccac 26460tgggtccctc ccacaacgta tgggaattat gggagctaca attcaagatg agatttgggt 26520gaggatacag ccaaaacata tcattccctc cctagcccct cccaaatctt atgtcatcac 26580atttcaaaat caatcatgcc attccaacag tccctcaaag tcttaactca tttcagcatg 26640aactcaaaag ttcacagtcc aaagtctcat ctgaaacaag gtaaatccct tctgcctatg 26700cacctgtaaa atcaaaagca agttagttac ttcctggata aaatgggggt acagggattg 26760ggtaaataca gctgttccaa atgggagaaa ttggccaaaa caaagggact acaggcccca 26820tgcaagtcca aaatccagtg gggcagtcaa atattaaagt tccaaaatga tctcctttga 26880ctccatgtct cacatccagg tcacactgat gcaagaggtg ggttcccatg gtcttgggca 26940gctctgcccc tgtggcttca tggggtagag cctccctcct ggctaatttc acaggctggc 27000gttgagtatc tatggctttt ccagatgcac agtgcaacct gttggtgggt ctaccattct 27060gaggtctgga ggatgatggc cctttctcac agctccacta ggcagcaccc cagtggggac 27120tctatgtggg ggcttcaacc ccacatttct tttctgcact gccctagcag aggttctcca 27180tgagggcctc acccctgcag caaacttctg cctagacatc cagttacatc ctctgaaatc 27240taagcagagg ttcccaaacc tcaattcttg acttctgtgc acccacaggc acaataccac 27300atggaagctg ccaaggcttg gggcttccac ctctgaagcc acagcctgag ctgtaccttg 27360gcccctttta gatatgacta gagcaactgg gatgcagggc accaagtccc taggctgcac 27420agagcagtgg ggctctggac cccagcccat gaagccattt tgtccttcta agcctctggg 27480cctgtgatgg gaggggctgc cacaaagtct ctgttatgcc ctggagacat tttccccatt 27540gtcttggcga ttaacatttg gctcctcatt acttatgcaa atttctgcag caggcttgaa 27600tttctcctca gaatatggat ttttcttatc tattgcatca tcaggctgca acttttccaa 27660acttttatgc tctgcttccc cattaaacat aagttccaat tccaaaccat atctttgtga 27720atgaataaaa ctgaatgctt ttaacagtac ccaagtcacc tcttgaacac tttgctgcct 27780agaaatttct cccaccagat gccctaaatc atctctctca agttcaaaat gccaccagtc 27840tctttggtaa aacatagcaa cagtcacctt tgctcttcct ttgtcttctg ccatgactgt 27900gaggcctccc cagccatgtg gaacagagag tcaattaaat gataccatga tggaggatgg 27960agtgagggag tcctgaggct ggaccatgaa ggtgctctgc tgtccctgcc atgtaaactg 28020ctctagtgcc tctctgctgt tggatatcag gaagaaagga tttaccaaat tggtagctgc 28080ataccaaatt ccagagacag tgttgatctg ctgtagtcaa gatgccacaa ctggtacagt 28140gggtgaaact ggactatgcc tggctatgtt tatgtagtcc acagtcagct cctctgagag 28200accttccctg accatcttat ctaatgatgc cttccaactc ccagtcttcc tccatcatat 28260tctcctgttt tatttttttg tgtactgatt actgtctgta gccatgtgat ctatttattc 28320gtttatggcc tttctcccca attagggtgt aggctccagg ggaataagga cattgtgtga 28380cttgtttgca gctgcatccc aagcacccgc cactgtagta gatgcctaac caatgtgtgt 28440tgaatgaata aaagagcagg ccaatgttct tttgctcaaa gtagagggga agaaataggg 28500ttttctgtgg agattccaag

gcagaggcca tttctggggg tcactggagt gggagaaggc 28560aggtcaaggt gggttgtctt ccaggcagtg caaaccccct ggcctctgcc agctgctcac 28620tggccagtct gcttgttggg tctggcacag gcctcaagga aacataacat ttttaataaa 28680acctcagagt caataaaggc gaatggtcct gggtgcctct cctgccggcc ccagctgttg 28740actttagaag tcaagagagt ggggcgttgc ccaattctca tgtagtacag ggagatataa 28800gctggaaggg cctagcccat tttatatgaa aacaaaacaa aacaaaacaa aactcaccag 28860gccctggaaa gagtccacca ccagccagaa tcaaaggtcc attcagagcg acagagctcc 28920tcacattcgc cgctaatgaa aaccaaattt ctcatccctc tgagcatttc caggggctac 28980aaatggaagg ggctgcagag tctttggcca ccgctcccac caccgaaggg gccccactgt 29040gttaaaatag ttttatgata atataggcct tgtattttcc taatttcagg cgtcagtgat 29100ttaggacgga gttgttttca tggaaaaaga aatagaacct gtttgtggcg gggcaagact 29160gatgcctggg cagatattcc cactgtgggc atatttgggt aggggggtga gcctgccatg 29220aagaggctca gacctagctc cggggaggcc tcgttcatga agttccccgc cttgggcggg 29280gaagaatggg ctgggggttt ccagacagat tcagagacag tcacagtgac ttctgttttt 29340tgatttcatg ctttgtgaaa tcttagaatc acaactcaga aaggtagagg catccctctc 29400agacgcagag aaagggcctc tgttttttta aaaagacatt ttctcatttc tttttctttt 29460tttcctcccc cttgatcaat ctttataagc aagtatgtgt agaaatgtca tatttttttt 29520ttcttaaagt caacttgatt cttactttga gcctccaata cttttagttg gtaggaaact 29580taatattttc agcgactgct ctgccttcgt caggatcagg tggaattctg tccttgtttc 29640tcagttttgt tttgttttgt tttcagatgg aatctcactc tgttgtccag gctggagtgc 29700agtggcacaa actcagctca ctgcaacctc tgcctcctgg attcaagtga ttctcctgcc 29760tcagcctctt gagtagctgg gattacaggc atgtgccacc atgcctggct aatttttgta 29820tttttagtag agatggggtt tcactatgtt ggccaggctg gtctcgaact cctgacctca 29880ggtgatcctc ctgcctcagc ctcccaaagt gctgggatta caggcaggag ctaccgcacc 29940caacctgttt ctcagttttt tttcatctgt aagatgggga gaatgataat acatacctca 30000atgggctggg ttaaaaaatg gtgaaatatt tagaatagtg cctggcacag agtaagtatt 30060aactattatt attattattt ttattattcc agagataaag agaaggcatc aaacctagta 30120tgagggtatc agggaaggct acctggaaga ggtggtgttt cagctaatga cagatgaggt 30180agtccttgca tgattttgaa ctcctctgct tggacattta tgtctagaat ttgatatgct 30240ataccctgaa caagtgtgct aattttagaa aactgtaaag aagaaaacag aaaacagcca 30300taatcccatc tttgcattga tttcattctg gattaatttt atctctattt ttaactatat 30360taattgataa cctgtatacg cagtgtgtgt ctggctagaa aaatgtttat ttctaaaaag 30420tatttatata tttataggaa ataaagatct gaatggggga gaaaagccta aaaatattaa 30480cagtggttat ctttggaggg ggggattatg gctcattttt ctcgtgtgtt tgttggtaat 30540ccggactgtc tactttccct ctcatgatta tatattagtt tgtgtcattt aaaaatgtca 30600tttagtctgg gcatggtggc tcatcctgta atcccgacac tttgggaggc caaggtggaa 30660ggtttgcttg aggccaagag tttgaggcca gcctgggaaa cgtaacgagg ccctgcctct 30720aaaaaaaaaa ttagccaggt gtggtggtgc acacctgtag ttctagctcc ttgagaggcc 30780aaggcaggag ggaggatcac ttgagcccag gagttggagg ctgcaatgca ctccagtctg 30840ggtgacagag tgagaccctg tctaaaaata aaaactaaaa atattactta aaatgtaata 30900tatagaactc aggaaccgca gatggagagt ctcataatct ttatattttc agaccatgaa 30960ggagagtggg gtagcttggc cggactctga gcgtcctgga cccacaagtc tgagaggaga 31020ggctgcatgt ggcctctggt atggtcacat ggttctataa ggaaactgag gcaggacata 31080aggcttcact tgtgaagtgg tggagaggga gggggcaatt gccaactggg tgataataaa 31140gactattgtt aagaccttgc ccccagtggc acatgaaatg ccactaaccc tgagagattg 31200agagacattc aaacctgagg tttggggcat ggtgcccttc ctgtgacttt ggtgctaatg 31260atgtctaaga tacctcttag ctcctccctc tgtcattctg cacaggcttc tcctttgcct 31320ggactatttt agtagcctgt gaacaggtct ctggaccctc atccccagtc cgcaccatga 31380tggtgctccc atccaacaca tacatctccg cttggctccc ccgtgccact gacccctgac 31440atggatcctc tcccacctcc catcaccatt gcgccacttg ccccaccatc ctcccagctc 31500agcgacacct ggttctccag gcctttgcac atgggattcc ctcctgccac atctctgctt 31560gatccattcc tactcatctt tccatctgat ctcgggggag agacattttc tctgaggagc 31620ttggcttgtt tgccctgatc cacactgggc tggcacaacc tgctttcctg tctgtctgcc 31680ctgtgagatc cctgaggcca tggctgtgac ttgttcacct tgttcttggt gtctggcacc 31740tgagggtggg gtggggctct gtgtctggtg aatgagtgaa tgaattctgg ccaaggcctc 31800aaagacaccc agcccaatga gctgagtggg gtggtgtggc ccaaatggtg tgtttggaca 31860ccagagagcc cacattgctg ccagccgtca gggtgggcac aaaggagggt agtccaggcc 31920ggctccaggg ctgccgcact ccccttccca tgataggtcc cctgggccag gcccagggca 31980ggccctttct gtgggtgaat ataaatatat aaaacacaca gcgcactctt agctgcaaaa 32040ctaaaaatag gaagcgcggg atccggctcc ccaggcttcc ccagccactg gaccacacag 32100gtgtggctgc ggatgtcggg gcgatgtggc ccctcacccc tcccagctct ggagccctca 32160tggggaggaa tgaggggcat tttggatttc tgccaggaac agttcattct ttcactctgg 32220cctccctcct gcccccgtcc catttgacag ctcatttcat ttacgacccc aaaatgaacc 32280gacccactga ggtgtattct ctactcacgt ggccaggctg ggttgtttgg tgcagctgag 32340agctgccctc tgggccatgc tggggggctg catttatgcg ggggtgcagt ctggagcaga 32400ggagaggccg gggctgagga gggaggcagg gctgggtctg catccagccc tgccctcccc 32460tacccacggc actggcccca ccccgcgcca tctcctcaag ccctcaccag gccctaacgt 32520gggaatgtgt catctctggc ctgtagcctc actgccagga catccattgc tcagtgtaaa 32580agccaaagcc atccccatgg ccacagccca acagtggcag ggctgctcct aggggccggc 32640aaggcgggtc catctgggcc acttcacccc acaggaggcc acttctggga gcccccaggc 32700cacagccggc tctctgggtc catcattggg cccatctggg ccaacctcaa gctgtggggg 32760ctgaagaaac tggagggact caaagtccag cccagatcaa caggactcct agagcctcca 32820aagcggaatt ctggagtcca ggggctccca ggctgtggaa ctaaatggct tcctcaatct 32880gaactggctt ctacatgacc taagctcttg ctggtggctc aggggacatg ggtggggctg 32940ggcccagggt ccaggaggcc agggttgtaa aactatgaaa gtcaaccctg ccttcaagcc 33000aggtacaccc tgtcccaaag cagacgatta tggggtgtgg ggtcctactc cacacctggc 33060acacggccgg tgctcattca gcgtatgacc aaaaagggag actcagagag ggacagggac 33120ctcccactgc cacacggctc gggaagggaa aaccttcccc acatcaagac ctttagctgg 33180ccctttcggg aatgagtcac ctgaggttgg gaagttcttc ttaatacctt acctgaattc 33240ttgctgtaac caaggcagcc tctcctcacc cttgtccaaa ggggatgatg tccactctct 33300ccacctgctg cccagggatg ccgcccccta ttgccacctg caggctgctg tggctactgc 33360agcacttcct cccgcagccc tgggacctca ggcaagctga gacttctcgg gaccttgagt 33420ttccccttgg caagctgggt gtgctggttc gtgcctgcta gggtgcaggt gatggagatt 33480tgagtcagga actctggagg gcacagctcc tctccgatct gctgtggcac caaagtgtgc 33540ctggtgagga gtgctaccat cccctacaaa gtgaccccaa ataaatagaa acagttttgg 33600ccatgtagat gccgtttcag gaccaaccct ggcagaggct gcccagagca gaccaacaga 33660gaagttctgt agccacggct gaggtcctgt ccagagatgg acctgctgtc ttttgggtaa 33720aaggggatgc cgggctaggg aaatggaagc ttcttgttgg gagctgagtt tcaggcagac 33780caaccaaact gagcagggca aagctttggg agtggttttt gaagtcggtg ggcatcactc 33840aaaaataagg tctgttttgt aaaaatttcc cttcacaaat cccaactggc caggctctgg 33900gctgtgtgtt ggtacaaatc ccaagtggac caggcctcct tgctggcaag gtgggagggg 33960ggctgtcaag ccaggtcccc acaccatcac acccatgcta ccattgttgg gctgtggtcc 34020cagttcagcc atggacaacc ccagggagac atggaccttg atgacactcc ttctttgcac 34080cgcagttgtc tcatctgcaa aatgggggca ctgaagttgc cgcttactcc caatccccac 34140tgctgctagc ttgccacaga tcttgaaaca cgagcctcag aggggggttc tcaccaaggc 34200acttggactc tccctctgcc tctgccccct cccgaaatgt gaatctgagg aacaggcata 34260ggaattcctc ccaacacggc tgggaagact cacagcccgc tcatgattgg tggaagggtt 34320gtggcacttt gaagacctat ttgatgctct cctggggtcc cagccataca ggagcaggcc 34380tcaccggctg tcctgtggcc agggtggtct ctgcggccat tcctgaagaa gttggaagca 34440aggagatgaa ggtgctgggt gtctctgttc ctgttcctcc tgggcaacag caggaggtct 34500ccatcctctc ccccacccca ccccacctcc atgcatagcc ctagaaaccg ggcactggac 34560tccttccaca catctcagag ttatattatt gtaacaaatc agtcaaaatt ccattttaca 34620gttaaatagt acagaagaca gtttactgta caagcaagtt gtgcgttaaa aacaaacacc 34680aagcaaacga tagtgcaaag cagtttccac ccagctccat cctctcgcca gctctgggat 34740ggttttacat cagatgagtg cagcaggtgt cacacctcag catgacaata tgtcacaaaa 34800gattggtacc cactactgac aggctcacag taacactata tcaaaacgtc ttcctttcct 34860cgtgcttcct acatcagtgt gtttgcctag tacaacttta acgcagcctt gtaaataagg 34920acctactttt accagcccag gctgtctgta cccactttgg gccttacaga ctcagtacgg 34980ctgccgtcac tttttgtcag gggatggggg atggggtagg aagagcaatt tatttactat 35040ccctgcctct ccaggatcag gaagggttag taatctggga tgagactaca aagtgctggg 35100cactgggaac ccaaaggtgc ctcccacgct gacctgggac cagctataac cagagaacag 35160gagggaagaa actacaagga caatggattc atagctgctt cctaagaagg catggagagg 35220ccccttgggt gcagggagtc agcaactatt ttgaggagat ggagactgtt tttccagtga 35280ggacaggcca agagaaaccg cagcaggagg gaatggaata ggatcaccag aaggactgcc 35340taacctgcca gtgtgtctct ggtgtatggt tctggccgtt gtgacaggtt gggtggggac 35400agtgctctca cagagacaac caatgaagag cattttgtag ggcagatttc tgcatccaca 35460gaggccgagg cagaaagtta aaataccgca tgctgctagc ctttatgagt tcccttacgc 35520cttttctaag cctttctagg gccagagaac tctgatgtga gaatccatgc agcctggccc 35580ttgggcaagc gcccactttc ccattttgca aaattcaggg gcaagacttc acctcggaac 35640gcagtgcagc tgagctgcgg ctggagagcc ctttacagtg cttctgtcct gccacgcaca 35700gccagtactc cccacctctc acatcctgcc cacctccgcc cagcctcctg gacagatgtc 35760ctagagccac agaggagaga tgcccagcgt ctcatcagcg tttccctcgt cctcccaggg 35820gagcctgtgg tgcaggctgg tgggatgctg cctgatgggg agtgtagccc cttgagaaag 35880tattgagacc ctaataaccc cacaccctca ggaggcagct gggggtccgc gggggggaga 35940gggggcgggg atgttgctta taatcacaga gctatcataa tcacggaact atacctgtaa 36000gagaccttgt gtttgaaaac gttagattaa gcttcttttt ctaaaatcag ttttaaaaac 36060tgttttgttt tttttttgtt tttttgtttt tttttttttt ttgctcagga ctatttgctt 36120tcagagcaca aaacaggtta cagacaggtg tgtgccagga gtcgcaagat ttggctggat 36180cctcccagga ggcttggggg atggggcaca gcttggctgg acccaggggg gacagggaca 36240ttgatgtctg acccaaaatg atccctcacc tcaatgcctt ctgctaggac ctatctatct 36300ggccctcctg ttctctccac aaatggaatt atgagaccac ctaggggaaa ggggacctcc 36360tatcccccct cccccgccct gccaagagaa cagagagtgg atgcgttgag ctggtaaagt 36420gcatggagga gccggtctgt aggtgctttc ctgggtttaa gaacctgatg ccatagactc 36480atcttctctg gggcctggga cccgtgcggc tgggggaaag ccaacagtta ggaacggagg 36540ggaagctggg ctggggggac tggcttggat gtgttctcaa accatctctg ccagcagcgt 36600gctgggtcct gccccatctg tacaatgagg ggaaccccgc tcgagggtgg tgggggtggg 36660ggacactttc cagttctgac tcaaatctgt gtaaatgcag agggggctgg acccagggga 36720tgcaggggct ccaacgaagg tgcaggggtc gggaggtttc ccagggcctg aggcttatcc 36780tgtgggccaa tgctgcctct ctctggaaga gagatggcct ctgtcccagg agacatgggt 36840cccatgcagc actgggcata gctggagaca gtgaggtcct tccggggggg tggcaggagg 36900ctgattcccc acagaagtat gggatgagac ggccaggatc tgggccgggg gcagtatccc 36960gggccggggt gggggtggta acctatgcct ctgtacaagg atgtggctgc acagatgcag 37020ccagaggctc ccaccagctg ggaccaccct gggaccgaga ccaccctgga gcgcaggtcc 37080cattcccgcc cggagcctcg gagccggccc atcactggtc ttgaaggttg ttagggtccc 37140cgcctccagc gggaggagtc caccagggcg gtcagtaggg ccgccacacg gcctcatcca 37200tgcggcctgg cgtgctcagg gccgtgggtg agttgctgtg gctgccgtcg gcctccacgc 37260catcactctg gccgcccagg ctggggttca tgaggttgcc ggcggcgaca gaggcagcgc 37320tgctggtgca agaggccagc atgcgggtag gtgagcggtc gcccccactg ctgctgccgg 37380ccaccatgga gaactggtag gagccagagg atgtcccgta gtagaggtgg tagggggacg 37440ggttggcctg gaagggcccg ctctggttct gcggggcccc cgggtagggt ggcgggaggt 37500aggtatggtg gaagcggctg gtggccggca tgcccgccac gctgaggctg ctgatgctcg 37560tgcccgaggg cgtggcgctg taggggaagg cagctgacat ggccccggga taatgcatcc 37620tggggtctgg gaagcggctc tccgtgaggg ttggcagcgt ggggaaggag cggtcaaact 37680ggcgggggtc ggagaatggg ttcagttccg aggtgcctgg aggacagcag ggaagaggtc 37740agttccagct cgagacaacc ccaggagggc ttcctgaaga atgaccttgg gctctggttc 37800ccaaggccca tctgggggac ccctagttct agacctggct ctcctcttcc tgccctaggc 37860tgcccggggc ctcccccgcc aggactccga acacagacct gccgggaagc tggttggagc 37920gtgccccggg ccaagagggg ccatgggagc ccccccacag cagcaacaga acagaggagg 37980gggtctattc ttctttttaa atcctccttc ccagcctcgc agaggagagg cctaggatgc 38040ggtggtgggg ctgagggcag agtcagctca ggcctcccag cagccctgcc caggcaggtt 38100cctctcccca ccggcccatg ttaacagctg ggaaggccgt ggatgtgtaa agggctccaa 38160tgaccgtgtg agactgggag ttggaacccg cttttgaaga caagaaaatg gaggcagaga 38220gagagcaaga ctgagtctct gtggcagaga aaggactggt tctcatcaca aggcctctgc 38280tggggacaca cgtgcctctc ctgcccaggt gcagcacgcg gaggttctgt gcgctcacac 38340ctgggttgtg ggcactgagt tcacaggagc tctggcctcc acctcaccct gggcctgtgt 38400ctctggagcc gactcgtggc cacacagtga ctggatgcca ccctaacctg ccttggcagc 38460aaagtgagac agcagtcaga caaacttggg gacccagacc ccaacctggt cacagtgtcc 38520agcccagact ctgcccctgc tcccccagga atgtggcttc tcaatgggct ccaaggcaag 38580ggtgttccat cctctgtccc caacttttgt catcacagac ccccaaaacc tcagcattca 38640aaggggctca gggattgagt ctaacaccct tgatggggaa actgaggccc agacagggtg 38700aggcacttcc ctcagggtca cacagcacat tggacctggc acacaatcct agggcctctg 38760gtcctgagcc ccaacacgta cttgagaggg agctgtcccg tctttgaggc agcacaggat 38820caaggcttac tgtgtggctt ctggagccgg acagttatcc tggtttggac acttactagc 38880tttgtgtcct tgggcaagtc acttaacctc tctgcgcatc agtttcccca tataaaacat 38940gagacgataa cagttcatca ggattcagtt aattcacatc gagtacttag aatggcactg 39000ggcacagagc aggggtccat gaggctttgc aaggccactg tggctgtggt gtctcttact 39060ctgggtaccc aggagaactg gctcattcag ggccctgcca agttgaggcc ctggtgcagg 39120gcctcccttc tactctggca gccgggggag gtggatgagc ccccagcagt ggtccagagg 39180tgcagtctgt ccagcccagc aacccctctg tgtcacccac caaaggataa gggccggtgc 39240tagccggagt gggctctgcc tgccacgccg aggcttggct gaggacggag agctatgagc 39300ctgaggtgtg tgtgacttcg gctgggactt ggaacttctc ggggctttgg ggtcttccca 39360agtcagctgg ggtatgtttc cctcagcagc gtactctggc cctgggcgtg gatccgaacg 39420gagtgatgct cctggcttaa ggtaagaaga tgtggggaca gcagtctggg tggcgggggc 39480cttctgggac atctgggatg ttccctagta ggtcacttgg ctgtcccggc cccttgaggc 39540cgagagcctc cgaggcacct ggctgccagt tttcatctgg ggagcccctc ggggggagag 39600gtcctgttgc aggtgctggg cacgtcagca cagctgagat gggtggggtg gaagtgggtg 39660ctggccgcct gatgggaacc ccattctcaa gacgaaggaa acaaatgggg accgcaggat 39720acaacggcag gactgtgccc ctcagagctc acgcgggctg cagggcgctg ggctgggcct 39780ccctggacct gccaccatcc cctccagcct ctttcctcag ggccaccacc cctcctccgg 39840ggtggtgggg gaagtacctg ccctcagcac tccctcagac cccccagcag cttccttgga 39900gctcctgtac ccccaccctg cggcctcgca gccccaggaa acccgagctg cccggggcac 39960tgtcgagtgg ccaatcccaa cagtggaaag aaatgtttat tttcttctcc agattgtccg 40020ggctgctgca tggtggctga atgagccctt tcagctgtga gaagccccca ttgtgggcgg 40080ctgcggctgg gggctggggc tggggtatgg gaggtgctgg ggtctctgca ctgcttgcca 40140gtgaccaata ttggagggtc aaagcactta acaggcaccg agggaagtgg tggtggggtg 40200tcccaagggg gatccccagg agggagtccg agggcagagg gaggagggcc tgtgagagtg 40260acttcccaag cctaggtctg ccagcaaccc ctctttgtca gggacctcct tctccccact 40320tcacagatga gaaaactgag gctgagttta agtgacttgt ctaagatcat acagccaatg 40380cctggcagag cctgaattcc tagcctggtc cagctgactg cagagttcat gctcgccctg 40440tcctggtcat ccgaggccct ttctctcacc caaaggggat gggcctgagg atggagatgc 40500ctggctgcct gtggcccagt gctgtggggg gctagcgagg gactgggcca ggcctcagga 40560gggagcaggc agagaagcag aagtcagcca ctgccccaca caggctgggc tcctttcctc 40620ccagcccagg atggaagcag cagctgtgcc tgccgtgggg ccaggcattg attcccaagc 40680tgtgcccacc cagcagtgga tgggcagatg tgggctctcc ttccatgggg gctggtggac 40740aggaagccac tgttcacccc acctcctgga ttctggctcc cccgctgagc cccgatcccc 40800tggcctggct ctgtccatgg cagagaaagg ctggctctca ggctactgca cctcgacaga 40860tgctggccca tgggtagcag aagcagaggc agctacgcgg caggggtggg cgtgagcaca 40920gcgtgcaggg ctccttccgc tacctcttga gagcagacct ccaactcctg ggctcgagag 40980ctgagagtct caaatgcact agctcctggg ctcagagagg ctgggcctgg ggcttctccc 41040aaccttggcg tctcagcagg accaaggcca aaagtcctga gcccaggcca gaaggggagg 41100ggtcctctct tcacactgaa ggcctgcatc cagcccctgg ctgcagcact atgcctggaa 41160caatgtcagt agagagaccc agtcggcccc cacctcagcg tggcaccgga aaagggggtg 41220gggcaggcag accggttggc agccctgttc caggcccctt tatctgtccc ctcagaagta 41280cagaaagttc ttgggagcag gtactgtgga gactgtggac ctggtcacag atgggctgtg 41340tgacccgagg gtggctctga acctcttagg cctctcaatt cattcatctg ccaaggggtt 41400ctaaccaggc tctggggaat tgagaaagaa tgggcacagt ccgtgacggc agccagctgc 41460ctgcctctgt ccacccggcc accaagcacc cttggcaccc cacttagccc aagggccggc 41520tgtgcacaca gcctcccatg tccccagctc actgactgag agaacagagg agagatgcag 41580ccggcagccg tttagtgagc ggctactatg cgccaggcac ctcgatactc cagaagacct 41640gcctgaggcc tggctgcaac tgtgcttgct gtatccgtct aggcagtgga gatggagacc 41700ccagctcggt cttcccttcc acctcagctc ctcctgtttg ggaggatgct ctgggcaggg 41760tgggagacct ttcccaggaa tgctatgtgc ctctctaggg ttggaatgtc acttaacagt 41820gtgcaaagtt tgtgtgagta cagtaatgtc atttgaatgt catcccagcc ctgggtggag 41880gcatccgccc caatccactt tcagatgaaa aatcgcaggc tgtggggcag gggtggggaa 41940actgtacatg gcaggggcga gtctgtcacg gctccttgga caagtcatgc cccaatttta 42000ataggggcac tatggggtta accccatttc cccaggcaca gtgaactcct ggtatgcaga 42060tccctggggc caggcaccag gcatgtgtca gtaatgtcag tgtttgctga gtgaacgaat 42120gatggctagc acacagaaag cccacaggaa ccgtctgcag gtgccaatga gcaccagcag 42180ctcctctaca aaacaagggg gtgcagtgac tgatcttcgg acaggctttt ggtctggggc 42240agattggacc acatcgaggc cctccacccc cacctcaccc cgctgcagcc cctccctccg 42300tgccgtacct tggattgggg tctggggctg gctgctgaag tggcttgtgg tgctgagtga 42360gcctcggggg ctgggtgtgc tcggtgtcac ccgcatgcgc agccgttcca ggtccccaaa 42420gcggtcaggg aacggcttgg tctggtcctc cagcttctgc cggtgccctg cagagcacag 42480gaagcccatc agccgttgct tccccagagt ctcagtggag acagaaatgc ctcactctgc 42540tgggaagttc ttcctgaggt ctgaccttag gcctctgctg ggagaaccct gaggtcaccg 42600ccagcctctt cacagaggtt ttcaaaagac tttctgaaca gagaatggtc gttatgtgcc 42660accccacata tctaaacctc tacaacacac ggtgatccaa acctctacaa cacgcggtga 42720tctaaacctc tacaacacgc ggtgatctaa acctctacaa cacgcggtga tctaaacctc 42780tacaacacac tgtgatgtaa acctctacaa cacgcggtga tctaaacctc tacaacacac 42840ggtgatccaa acctctacaa cacacggtga tctaaacctc tacaacacac ggtgatctaa 42900acctctacaa cacactgtga tgtaaacctc tacaacacgc ggtgatctaa acctctacaa 42960cacactgtga tgtaaacctc tacaacacgc ggtgatctaa acctcaacca cacgcggtga 43020tctaaacctc tacaacacgc ggtgatctaa acctctacaa cacgcggtga tccgaacctc 43080aaccacacgc ggtgatccga acctctacga cacacggtga tccgaacctc tacgacacgc 43140ggtgatccga acctctacga cacgcggtga tccaaacctc tatgacacgc ggtgatccga 43200acctctacga cacgcggtga tccgaagctc tacgacacgc ggtgatccga acctctacga 43260cacgcggtga tctgaacctc tatgacacgc ggtgatctga acctctacga cacgcggtga 43320tccaaacctc tacgacatgt ggtgatccaa acctctacga cacacggtga tccaaacctc 43380tacaacacac tgtttggcag aagaggaaac tgagggccag gtgcagtggc ttacgcctat 43440aatctcagca ctttgggaga ctgagatggg aggatcagtt gaacccagga gtttgagatc 43500agcctgggca actatcgaga cccctgtctg tacaaaaatt aaaaaaaaaa aagaaaaaag 43560aaaaacttag ccaggtgggg

tggcacaagc ctgtagtccc agctactggg atgactgagg 43620caggaggatc acttgagccc aggaggtgga ggctgcagtg agctgattgt accactgcat 43680cccagtctgg gcaacggaac aaggacccta gatctaaaaa aaggaaactg aggcaacaga 43740catgagaaag tggctcatgc ccccaaggga ggcagggaga taacccagga gcactgccac 43800cctctgcctc ccagcatccc agcctgcctt gcacactgtc tcccatgtct acaagaacaa 43860tgggaggtgg ccccaggagg ggactgcagg cttttccagc cctaagtcac tctgggatcc 43920ccagaacatg ccttcttctc tctgggcctc aggcagaaaa ataactccac caggatgctg 43980ggcagaggtg tagggggctt gcatgaggga ctagacagcc atctctgcct ggaagctggg 44040gtcaggggac gagatgtcac acctggagaa aactgccagc attttccact ccctatctgc 44100cagagcccac acaggaagaa tcccagcctc acatccgagg actcagaggt gctgggaggg 44160tcaaggtggc caggctccca ccctcctgcg gcctgctgag gccgagggac acttctggag 44220tgatatcaag cttgcaggga cctcccccgc cacacacact tttttaatta ctaattttac 44280atttcacaag caacacgtga atatttacaa aaataaaagc attacagata aggctctgtc 44340caccgctcta agctcctctc cagagtcccc accgtgacca gtttctttcc agacattttt 44400catcttccat gaatggaaaa cgcaaagtag gcttctcatg gaactctctt gaacagcctc 44460agtccgggtg tgtcattctg taacttgctt tcttagtagc acaccttgga gctcaaactc 44520agtttcccta tctgcaaaat ggggacagta atccagcccc acagaaatga cggagttccc 44580ataaaaccct ggggatcctc cctgctacgg aagggattca acaagcatgg cgagaatgac 44640gctgctctcc ctctttcctg ctggccactg gaggcacagt tcactccgca gtcctctcca 44700cccacatttc agtccttctc aagcttcccc tttagttccc ttacatgcaa cactcccggg 44760aacgtccctg ttcgcacccc ctagtggctg cagcttctcc agggctgacc cgaggaaagg 44820acggctccct tggaggactg tgcactccag ggttcggctg atcaacccta acacggtcac 44880ggccattcta cctcacgtga cttggtggga ggctgccagt caggcagggt ggccaggccc 44940tcttttacaa gtaagagaac tgcaactgcg gagaggcgag gaagcttgtt ggaggccaca 45000cgccgaacaa ggggtgggat ttcctggacc tgggaccctt tagaaaagat ggaggctagg 45060catggtggct acgcctgtaa tcccagcgct ttgggaagcc gaggcgggcg gatcacctga 45120gtgaggtcag gagtttgaaa ccagcctgac caatatggtg aaaccccgtc tctactaaaa 45180gtacaaaaat tagccgggcg tggtggcggg cgcctatgat cccaactact tgggaggctg 45240aggcaggaga atcgcttgaa cccgggaggc ggaggttgca gtgagccaag atcacaccac 45300tgcactccag cctaggtgac agagcaagat tccatctcaa aaaaaaaaaa aaaaaaaaat 45360gtgggagggg gtaaggggga ggagaaggtt tgcctaaggc cttgggtcta gaatgacatg 45420tgcgttttct agtttggcag agggagaaga ggcaagatgt aggtgggagg taaaacagaa 45480ctcactgcgc ttcttggggc ctgaacaagt gattgctggg gactcgaaaa gtgggaaaag 45540cctgtcgacc actgtagggc atgaggggac agagcccgag gcctcgcatg ggcctgattt 45600ctgattttca gaatgggaac gcaatggatt ctggtacttg taggccccca agctccctac 45660gcatccctgt tggaagctca aacaggtatc tgggagcctc agaaagaaag cagggggcct 45720gggagccaca ggggctcagc cagtcaccaa gaaccaggga gccccacctt ctcctccaat 45780gaggccccag accaggaatc catgggacac ttggtggcag gaataagacc atttggtctc 45840tggccaggcc cccactgctg cctcccaggg cccttggcaa caaaggggaa aacatgggct 45900gggggcgagt ttagctggag ctggggctgc aaactcagat gcccatgggg atggggcaag 45960tcacgtgaac gaggcaagtg ggatgggtgg ggcctggagc ggatggggag gagatgcctt 46020gtggaaagca cctgactgct accctgagga gggcaggccc agtacggcca gagcttccaa 46080ttccagaccg agtcctcagc cctaacaggc cttggaggaa atgttgtcct tgctcctgag 46140gccactggaa atggcaggga gatgtggatg agctggggga caagtgagca gaagaatctt 46200aacaggcatg aagcctgccg ggtggacgtg gggaccacag actgatccga caacagcctg 46260agtgcaaagg atctgggtgt tttagtcagc cacaagcttg aagggagcca atagggctgg 46320tgcaaaacct caggccttgt tactttcgtc acaggagaat aaagtcccct gtttctgagt 46380cacaccatgt acaaagagtg tttacagtta ctcctgcggc ctggcggaag agggcggggt 46440tgacagccag cctgggttcg aggacgggtc ctgtcacttg cagtctggtg gcctcactca 46500agtcacactc ctttccccca ccccgagctg cggtgtcccc atcacactgt ctttgtgggt 46560tgaggtgctg gcctaaggtg tggacacttt ccagtcacgt gggtcaccac catcatgacc 46620atcgctttta tctctgctca tgcccaaagg aagcagaact atcatcccca tgctggagac 46680cggggtgtgg aggccaggat gctgaagtcg tgtgaggaac acagagcctg agcagcaagg 46740caggattcac acccggatcc cccgactcca agcccagggc tcttttcctg acactttccc 46800ttttgttccc attgtttaga cggggccacc gaggcttcac catgagaccg acgctgagcg 46860cctgttccgg gacccaggct gtgggtcagg taatctgacc ccggaaccca cgctcccacc 46920acatgctccc ttgccctccg tagggcagac ttcccggagg aggggagtcc aacagcactt 46980cggaaatagc ttccttgtta ctgtggaacg ctggagccac tgccagggag gggagagggg 47040agccaaggcg gccccacgtg gccagggcgc cagagagtct cagagccaca gggccagggc 47100tctcacactg ggaataggac agaagttcca gtgcctgagg aaagaagatg gtcttcagaa 47160aaagcctctt tcattcggtt acccagagca agagctgcgt ggggagctct ggctctaacc 47220cactccgtca ccttgggccc agtcctgcta cgcctcagtt tcccctctgc acagtctgct 47280cactgaggcc ccttcctttt gaaagtccct gatttaaggt agcaaagatc agccgctggt 47340cagaggggcc ccagaaagaa aagaaggcag ggctgctggc cccagggccc acccactacc 47400acttcttcct gctgctgctg ttcccagtat ttcttgaata ttctctgccc cccactcttc 47460agagcctcag ctcaggacag cctctgccag caatgctttc tgtcctgtga agcccagtcc 47520aggagcccct cctccaggaa gccccccttc tcccacagta gatccccatc acacctctgc 47580aggctagggc tgtcctttaa agcagtcgcc agcaggagtg gaaatcatca aaacggcagc 47640agatgcttgc tgggtgcttc ctccacggca gcgtctaagc agctgacaag caccatcttg 47700tttcgcctgg cagcatcccc ttgagtaatg tctgccacca tcctatcata tgggtgaaaa 47760aactgaggct ctggacagcc agtgagctca aggtcaagca gaagacacac agccacctgc 47820tctccctaga gcctgtgaaa acacatctat tgtggcggaa gagggcgggg ttcacagcca 47880gcctgggttc gatacatcaa tagatgtatc gggactccac aatagatgtt agggccccgt 47940ccagccccag gcccagagct gctatctgca ggcccaggag gataggactt gggagaggaa 48000gatgagaagg tctcagtgga ctccaccagg ggcccttccc tgctctgaag ctcagggttg 48060agagtgcaat ttccaatcat accctgctct agaccaccaa gtcactctct gcctctgggc 48120cacagtttcc acatctgtaa agtggttatc atactgtcta acccctgagg gtgccgatga 48180gctggaggac aggccacatg cttttaaaag cagaggactg agatggctgg ggaaagcccc 48240gcgttggccc tcagggcctg tcctggctgc tgtcagcctc cagctgctgg gctcagatca 48300gacagctcct ccagcatggc ctggattagt gtctatgacc ctcacttatg ggagggcaga 48360tcccagcctg cccctcccaa gggcccagtg gccccaagct cataccaggc agctctcacc 48420caccagtggt cactgtcttg ggcaagccac tcttgccttc tgggcctcag ctgtcttatc 48480tgcaaaatgg ggatcacacc tctaaccccc gagggtcagg aaaggtttca agaattacac 48540agcccaccag gccttggcct ttgaggaagg tgttctgggt tcccattctg acttggccat 48600ctgctcctag gcaaacagct cctctctgat gcgtctgtgc agtgggggtg acccacctca 48660caggcatatg ataaaggcca aagtgggagc aggaatgctg ggccccagcc agtctgggga 48720ctcaccaggg tcacgcagtg tgggagctag aggaccaggg ctggattctg ggttggcagc 48780tcctttacca ctgtccccag ggaatccttc cccaccacca gcctggccag cctggggtcc 48840tacccccgcc aggtacctga tgcttctggg ggaaccaaga gaccatcagg gttaccccct 48900tgcctccatg caggcccaac acaagcccct gtcataggag tggcaaccat tttagcaggc 48960atccatgatg tgccgggcac tgtgcaaggg gggccatgca tgtcgtctcc aagggtcata 49020tccctctgac aggctgtgac tatcaccccc gttttacaga tggaaaagtg gaggcacacg 49080gtcaaggtca cacggtgtgt ggcacccctg agattcaaac ctggaaaggt cacacatgga 49140gctcagctgc taaggtcatc gcttcccaag acctccatga gagaagagct gggtcacctg 49200gccgtaaggt ccagctggca agaggccagc tcagtgttca gcctcttggg aaaagcagag 49260tcgggcaggg ccacaggaac agcatcgtct gctggggaca gtgtgggctc caatgaccag 49320gcccgtcacc catctgaagc cactcggcag ccttcttggc cgcctggtgc ggctgtgacc 49380cagacacagc agccactgtc tacccagcag cagggtgggg cgccgggccc gaggccggct 49440ctgcggcctg tcaggagatt tacacccgac tcttaacagc ctcgcggaat cgcaggcggg 49500tgccgggcct ggggtggtct gctgtgaatc ggccccctgt gagcagatga aagccgggtc 49560ggtggctggg cagggaaacg ggctggccgg gggccagcgg gcagggaggc gagcggttcc 49620ctcccagggc tgcaagtggg gcttccagag gcctggggtt gattaggaga acccaggagg 49680tctgtggtta accccttccc tcctgctggg cagactccgc tagccctgcc cctagcgcag 49740gagacactcc tgggggttgt ggggatcttg ggagccaggg acctggagca gctgcctctc 49800ctcagcccag gaagaaacta cagaaactct aaggccttca aaggcccaac tgcgggctca 49860gggtcacttc tcctgcccac gccaaaccct cggcagccac actctgctgg ctgctcactt 49920caggcccctg ctcaaaggtc acctcttcag gaggcctccc cgccccatcc cttgttccat 49980cccttgcacg ctccactcct tctcccagct ttgtttttct tcataggact tcctactacc 50040cgaaatgaca ttaatgaatc atttgcttat tcatcaacga tttatggagc agctgtgaag 50100ggctcctgcc cacattctca gggtctagct ataccagggc ctggcaaacc agagcaaaga 50160actctgccct tgtagagcat aaacaacagg gggccgggtg cggtggctca cgcctgtagt 50220cccagcactt tgggaggctg aggtgggcgg atcacttgag gtcgggagtt caagactagc 50280ctggccaaca tggtgaaacc ctgtctctat taaaaataca aaaattagct gggtgtggtg 50340gcgtgtgcct gtaatcccag ctcctaggga ggctgaggca agagaatctc ctgaacctgg 50400gaggcggagg ttgctgtgag ccgagatctt gccactgcac tccagcctgg gcaacagagc 50460aagactccat ctcaaaaaac aaaacaaaac aaaatgggag aaatgaataa caaatgaaac 50520aaactatcgg actagatagc accttagaag gtggtagtgg taagtgctcg gggtaacctt 50580aaagccagga aggaaaaggg ggagaggtga ggaaggctgt gtgtgtgcca cttgaaacag 50640gcgggctgct gagaagtgca gaggctttag ggtgtgaagg agtgtgccat gcatctgggg 50700gtgtccgggg aggagtgttc cagatagaaa aaagagcagt gcaaaggccc ccgaggcagg 50760agtgtccctg gcaagttcaa agaccagcca ggataccagg gtggccagag caggatgtgg 50820gagggagggc agggggtaac gggcacaggc taggggggcg tgagggcctt tcccccaccg 50880tggtccatgc cagacttgcc aggtgtcacc gcccctcctg ctgggatcct ggacctggct 50940cagcaacctg cttcttaacc agcccccagt gactctgagg gacaccagca ctgagaacct 51000cagaaaccga ggccacacag gcaggaagcc accaagccag ccttcaaacc cagctggcca 51060cctggctgca ggccgggcac gctctgcagg gcaccagagg ggaacgaccc ggccacagaa 51120cccacagccg gcctcaggga tctacagatt cccagtcctt ggctcccagg accagcccct 51180actcccactt caccccacag cgggctcaga tttcagaggg tcggaggtgg caaaacagga 51240aaaaagccgg gaaaggaagt ccaggagcac aaaaggcctg taacaacctg tgaaggttgt 51300gggggcactt cctggggcca ggccccggta aactcagtca accttcacag cgactcccct 51360aggcagacac caataccatc catttgacag ctgagcacac tgaggtgaaa aggcccttcc 51420aagtggccct cacttcccgc agcccccggg tcggagcccc cagggtgtgc tgacagtcac 51480cttgggcaaa aggttttgcg ccctggcctc tatcctctcc tggggttgcc caagagatca 51540gttactgggg actttgcaca gggcctgacg caagggaggg ggttgctcag tgaccaggag 51600ccgctgagct ggtcccttca ctcttacaga tggggacgct gaggacccga aaggccaagg 51660atttgtccag ggccaaagac aaaggagtgg ggctgcaacc cagggtatgg ggggggacct 51720gatctcaggg ccaggatatg ccagggacag gaacaggcag gtcctaagga tgggggacct 51780agtagactgc cccccgactc catctctgct ctgttctgta aataaaacca ctgatccagc 51840cgctgccggg gcccagagag ggaggtcacc tgtctcaggt ggtgcagcaa gcctggcttc 51900tgacgccgtg ggtctccagg cccagcctct gtccctccct cttgttgcct cgtcctgagc 51960cacgcattta ccttccagct caccccagaa ggggccatct caggtctggg agacccaggc 52020agggaagagc aggcagggga ttctgctgga atctcccaca ggcagggctg agtctccatg 52080ctcatccagg ggtcccagca gggcagagtg ggcggctctg gggtgggctg ggctgagcat 52140ggagggctct cagaggggcc aaccttgccc ggtcccttgg atcttcccac caagcgtcaa 52200gaccccgtcc cgtgcctccc tctttctgga gtggctcccc tctttctgga gtggcttctg 52260agtgccgcat ccccacccag agcccaactg aggctcctgt ccatgctgac cctgcccctg 52320gagacatagg gcagggctgc cacctccttc aatggagact tgatacctgc acctctatta 52380ccaaggcagc cacccagctg ctgcccatga gagagctcac cgttgactaa tggtggtggt 52440gggagtgcag gaagggggct gggtactgag gacgacaaaa cgctgcggac ccagtgactc 52500atgggacccc tctgtgctac ggccacgtgc tgtccacatg tcgcccctga tctccaggtc 52560cgcagggtgg gtggcatcat cacacttcat ggaggaggga gctgaggccc agagaggtca 52620gtgacttgcc ctaggtcaca ctgcagataa cagccctggc taaagtgacg gatcccttgc 52680taacccccac cgctaagtgc tttctataga ttaagccact gtttcctcgc aatagcatca 52740tgaggtagct gcttgtgcga atatcatttt tcagttcagg aaactgaggc acggagatga 52800ctagcccaag gacccacagc caggaaggct ggcttggaaa ctgctctcta caccatggtg 52860gtctatggct catgagggct tcccagccat caccaccttg agactcctgg agtcactgat 52920ccagttctca gatgacaaaa ctgaggccac aaagaagaca tgacttgcct agggtcatga 52980agcccaaggc caagggcatg ggctggtcta tgtctgatct cagcaggagg gaaccagcag 53040gagtgtggcc agggcaagtg ctggctggga gctgacggtg caggcctgag gatgcgtgcc 53100ggggctcagg gctggcagag gtgaccctga gagccctgga gggaaactct tccagggctg 53160ctggactcag ctccaagcct ttcccaagtg gccagatgct gggatgggcc caggaattgg 53220atgatggggt gtcaggccca gctgactccc aagaagggag gggccagccc agggctaggc 53280ctcctgcccc aggcctcctg ccccaggcct gctcagccta gaatcttgcc tctgggaaga 53340ctgaagcctg gggcgccttc ctgctccttg cacagcatta ggtcctattc aggtacccaa 53400ctccctcagg cctggattct ctcctcactg gaacttgggt gacccctctg gctctgctgt 53460catcaagatc ccattcaata gtgactgcta aaaggtcttc taaactacaa agggtcacat 53520ttctgagaaa gagaggggtg ggccaacctt cagtgcacca agctgaaaat gccttgggga 53580ggtgggatgg agctcaggaa gctggctggc tctatttcat tcattcattc attcattcag 53640tcagtcagtc agtcagtcat tcattcattc tgtggacaca gagcctcagc ctaccctccc 53700acttccccag ccttaatctg accttcagca agcagagaga attaaacaca aactcgcttt 53760gatggaccag aactccctgc tcatagggtc tgggtgcccg gactctgggt gacctgagca 53820agtcacatgc taagattcaa agactcagtt tccaaggaag aggcctggcc tcacagccag 53880accagcccct gacttttgat cactcctgcc ctccatgcat ccctcagcca cccgcagaga 53940agctgggggc agagtaaagc aagcctggct caacctccac ccagaaacac acaagcaccc 54000gacaaatgcc atatctgaaa gctttctcca tccttttcct ttccttgact ccctcagtag 54060tctccatgga cagtcatctc cactcccagc ctcctcgctg gcctcccacg gtctcaggct 54120aagcccagag ggtttagggg tttgccagca ggcacgcagt gtgtgggggc acagagccaa 54180ggactgcaac cccccgagga gggctccatc tgtctgacct agctgctgtc cttcccgcac 54240tggaccctcc tcccccgcgc aggggctcag ggggctcggt ggcacttacg tctgggctcc 54300cggggtccgt ccacggtcac cttgatggct cggtggtagg tcgccacttg ggtggggttg 54360gtgaacacag tgatggtcag ggtgaaactc ttccctgggg agagtgggga atagaggcag 54420gtggttggca cctggagctt ccacaatacc ctgctctccc acctgtatct acccctggaa 54480gcccctaact gtcaagaagg ggcactctgt cctctttgaa catgggcaga agatagggct 54540ctgggtgaag ttcaagctct tgggcttggc attcaaggcc cctgggggtc tgacgccaac 54600tttgtcaacc ccccgcccca tgccgtgacc accctggctc atgttcccct cttcttggcc 54660tttctgctgt ctcttctatt cagagaccca cacgattttg tggtggggag caggatgggt 54720atattctatt ttctgaaagt aattggtgat ctttgtagaa aaattcaaga acatacaaaa 54780tataaataaa gaagaaaaga cacccccccc ccacggttcc actagctgga gatagacacc 54840gttaccattt ggtgttttcc ctttcagctc tttttgtatg ggtttgtata tttacacagt 54900cgcagtggta ctaaaataca gattttcata ctgttttttt tttcatttaa cctcacatca 54960gaagcacttt cccacgtcat taaaactcca taaacttcgt ttttaatggc tgcaaaatat 55020ttcaactcaa ggaagcctcc tcatctttta tttatctacc tccttactct cgggtattta 55080catcgttgct aatttcttat tgatgtgtgc agctggagct gaaaaaggac tgatttggga 55140gctgcagaca tttcttctgt agacacaact gttatttcca gaatgttcta tttttagata 55200gacatttggc tccaaagtct ccattcaaaa ttcctgagag gggaaaaaac ttttaaaata 55260ctactttttt ttttttttac catttaaaat aaaatgaaag tgaccttctg tttataaaaa 55320tctttgtctg catctctgct tatttcctta gaagagattc caagaagcgg tgagtgattt 55380cacggcagca gagggttggg acatattacg ggcgcggatc cctcttggag tgagatgact 55440ctccggagag atttagtcgt caccctcgcg tgtgaggctg cgtcacaccc cagggatgtg 55500tctatcaaga tggaagatct tttacacgct cttgattttg tttgcctttt tttctattac 55560tagtgagaat gaaacttttt atatgattat tatccatcat aatccaacac aaattactgc 55620ttcatgttct tttactttcc tgtgaaggtt ttagtgcctt ttaaaaattg ctatatatta 55680agcttgttaa tactttccat gctgtatttg tggccatcag tttccccggg cacaggcctg 55740cacattttgc cttcacacgc tgggtggttt ttcattttca cttctatttc tcgttcttct 55800atcgttttat gttcagacgg gtttctccgt gtagaaagca gtttatgaag atttactttc 55860gacagtcttc tctctacttt ctacagtgaa ttctctgatg tgtctgggag tttgggggtc 55920tgggtaagag tcctcctctc accctattct ctattacgat ccacagcctc atgctttatg 55980agattggtgg ccgggagcgg gggagatttg cggatccccc aagccagact ttatccccct 56040atccctgcct ctggatccca cgtacaggcc tgggaactcc ctgtgggtag gggccaatgg 56100tctcgcactc tcacctgtac cccagggctg gcacaggatg gtcaaggaga gaggctgccc 56160aagcgcatcc ctctggtgtc cccctgacac gcctccaaag tgagcaggta ggtttcaaca 56220gccccacgtt gcaggtggga gatgaagctc agggtggaga ccagtatctc acagttctct 56280ttgcatggcc gggtacttgt tagtcaactg atcaagtgaa aattctagcc ccagaggcag 56340gagaatccgg aacaaaatta aaccagccag gctgccagga gccatgccac aggacccaag 56400gccctctgag acaccagggg gaatttaaag ctcaagaccc actgagtgtc actccagctg 56460ggaaatgagg ggcttctctg gaagcctttt cctaagccag tcggctgagg cagggataga 56520aattctgact gcacttgccc ccggagcccc aggtcagaac agacctggtc tcccactctc 56580aggtcacagg ggccactttg tatgatttct ggaagcagaa gtgcagatgg tctagggaag 56640tgccaggcag atgcctcggg ctccctgccc gacccctcct actgcctttc ctcactctga 56700ggtcatttct ctgctggacc tctttctcct ccaaccagcc cagcactctc ctggggtccc 56760tgagcctctg accctgccag cattgtccag caccttcttg gttatgacgg ggagtttagg 56820cagacagccc agagccctag gggccagact ggagacacgg aggactaatg ggtcccagtg 56880ccctgccaca gggccccggg cccacagcag catttgaaag cttactaaaa ccctccttca 56940ggtcgcccac cttctcagtc aggccttccc tggtcacttt atctgaagta ggcattttta 57000attttaatta atttttttga gacaaggtct tgctctgtca cccaggttgg agtgcagtgg 57060catgatcata gctcactgca gcctggacct cccgggctca agtgatcctc ctgtctcagc 57120ctcctgagta gctgggacaa caggtgagcg ccaccatgcc cggctatttc tttttttccc 57180ttccttcttt tccttccctc ccttccttcc ttcctttcct ttcttttctt tctttccttt 57240ctttcttttt tttttttttc aagcttttac tatgtgccca ggctggtctt gaactcctgg 57300gctcaagtga tcctcctgcc ttggcctccc aaagtgttgg gattacagtc gtaaaccact 57360acacctggaa ggcattttta acttggctcc gtagagttga atgagcctga gaactagggt 57420aggaaaaaat tacaattgta ttgtccctaa cctctaactg aaatttagca tcactctcaa 57480gtacgagcgt aggcaacaaa ccacagaggt attatcagcc gtacctgtga ccttgtcacc 57540aacagacgtc acagatactt acatatcaca ttacagttgc tgcagattgc tctaaatatc 57600ttttatgctc atcacaactt caaaaccatg gttgtcatta ggcccaatgc tagatcttat 57660ttaatacatt gaataaagca gcacatttac cacaattttt aaagtatttt gctatgtttt 57720aatagaaatg gtttctattg taatactttg tatttgattt tataccttaa aaatatcatt 57780gttctgagaa aggtgtgcgg gcttcaccag ctatcagagg ggcccacagg gcaaaaaaaa 57840aaaaaaaaaa aaaaaaagcg ctaagcagct caacctgaag tatcacaggc cctaccactc 57900cctttctcta ttccctgcac ctgctggaat tttctcacaa tgcatatgct tttaataatc 57960catctactca ttttgtctcc ttctactaga ttataacctc cccaggggcc caagtttttg 58020tcttgttcat gcagtgtctc cagcccctag gacggcatcc ggcacagagt aggtgctcaa 58080caacatttgt taaataaatt aagggcagag ataatggctc ccattttgca cacaggtact 58140aacgtcccgc tcctgagaag tgagaagccc ccacccatac caggtagcaa accacatgcc 58200acccctgagg tcaccagcac tcctcggccg cttccaccag cttccacgcc tgtcaccacc 58260cctcccaggt acaaaggaga ggagtgtggg gcctaagagg aggagtgaga gggaggggca 58320ggagtcctgg acctcgggag acagggagcc tggggagcag gggtgggaga aagctgtctc 58380cctgagtgcc cctcagctac cccggccctg cccagctctc tctctgcctg gcagtggcaa 58440acccatccat ccctctctct cagcctctag atataactct gtgcaggagt cccaggcaaa 58500cctgcaatcc atcaggagcc caggaagtgt aaacccaggc tctctgaggg ctggccctgg 58560ttgcagggga gaagtcttgg tctgggaaat gggtttcctt tagggctcca gaaactcctc 58620caggacccat catcaaccag

ccggggtggc agcagggcct caggcaagtc cttgagcatt 58680ctctgcctgg gttcctatgt gtataaggtc cccgccccac ccacaggagc tgcatgggtg 58740gggggagggg acgtgtctca gtctcagggg acctcggggt tttctcagct tcagccaaga 58800agccattcat ctctccccca accagcggtt cccctcagcc tgcaccggca cactgcaccc 58860cgaatctctg tcgacacaca gttgcttttt aaccagttga tcacagctcg agagctcatg 58920tgcttttcat tttcacttag gccagtggcc gcctgctaga ggggcatttt tgggatttgt 58980ggtggcgtgt ggtcaacata gtgttggggt ggcactgcca gcgttagggg tggggtgcgt 59040gtatgtggtg ggggatgcca gcacccaacg ctgcccaggg tggtgaagat tcaattcttc 59100ctgggaggga aaaacttgct tataaaagtt ctctggctgg tcgcagtggc tcatgcctgt 59160aatcccaaca ctttgagagg ctgaggcagg aggatcgctt gagtccagga gttcaagacc 59220agcctgagca acacagtgaa caacaccccc atctctacaa caaataattt taaaaaatca 59280gctgagcatg gtggcgcatg cctatagtcc cagctattga ggtgggagga ctgcttgaga 59340ccaggaggtt gagactgcag tgatcgcacc actgcaccct ggcctgggcg acagagcgag 59400accttgtccc aaaaaaaagt aaaagaaaaa aaaattatct gagtcatgaa cctaactcag 59460ttttacataa aacaagggtt ttttttgtac ttttaatatc tactgaattt tccagaagga 59520aagacagttc tttttttttt tttaattttg ttcagcgctt tgccaacagg tgttgacaac 59580ttcagaaagt catggtattg gcagcaaggc caggttcaga ttgagccctg ccaccctgcc 59640tgttccctct gctgtgggct tctgcatgga gggcattcgt ccacctcatg gagtcctgtg 59700gccccaacgt ttacatattc aaatcagtgt tttattataa attactttcc ctttttttct 59760ccatcatagc tatggaataa catagtttgc aactgcatgt aaataggtag gtttcattat 59820ttatacattt caacgtagaa tagtaaggct tgatataaaa tatgtattgt aagaaaggct 59880cctcgtgtct ggcagggcag ggacctcagc cctaatcact gcaggagaca gcaatgacct 59940ggttttcctc ccttcctttt cttggttcac accttcagcc ctgttgttaa gagctctgtg 60000gtgttactgg gtgcgtgtct ttcatggaaa gccatcttcc tggaattcag acagaatgta 60060gaactaaaaa ttgaggcaac aagcagaggt ttccatcaga cttcttagtt ctggcagaag 60120tcaagagacc caggcaaggg ttctgggtcc caacccccag tcttaactcc caaagtgtcc 60180catctcctaa agtggcccag attgtcactg tcaaccactg actgttctct caggtgggaa 60240tttcccagtc agcaggatgg gcactgcaga tgtgtgtctg catgccagcg gacccggcac 60300cctccttcct ccctgccaac cgcctccacc tctcccactc agcagttcac accttctggg 60360tttcccccac ccccgcccaa accacacagt aatcagagaa tcagtggctg tcaccgctca 60420aagggacctc aaagtcctcc tccagtccca ggcatttgaa gtaacaaaat ctctaacatg 60480tatccagctc tcaatatgcg ccagctgata cacttgtgtc aatttcccta accttcccaa 60540aatctcatga ggtaggtacc attatcatcc ccatctcaca gatgaggaaa ctgaggcaca 60600gagtggttaa gtcatttgcc caatgtcatc cagcaagtca ttagcagagc tgggactcaa 60660acgcagggtg gctgatacta gaatgcaggc tctcaaagac ctcgagcctc tgaaggctga 60720acgccttagc cacagttcct cagacatcgg aactcctcct cagatcactt cctgcctccc 60780aggaccactg agactggtta tggacctctg agaggagatg gatgagagaa tggtttataa 60840actcagcctc ttgcatctcc cagagccaca gtcccagcct cggccattcc tgctacaagg 60900acaagctccc aaccaacgcc ttggaaaccc atttccctcc ctgcaggcct ggggaggggg 60960gctcaaggtc tgtgggcatg aaaaccccta aaaaaatcat tctcagtgtg cagaatggcc 61020agacaaggtc tcggtaactc agaaaatcgt cgtctcttct ctttctctcg cttcccagga 61080gagagagtgg gaagggagaa tcaagttcct gatgccttgc tgggctccca gatcgacagc 61140accttctgcc cgcctcgcaa caggcagcag ctatagtgct cctgacacat acctgggcta 61200gcagacctgg ccactgcccc gcagtcagca gagctcatca gccttgtctg ccaccgacca 61260aggaccagtg actgtcctct cagggttggg attaagtcgc aaagggtttg agagattggg 61320gatgacaaaa gggacttgga gactaattag gagcagcaat gaaagcttaa ttcataaaag 61380caaacatttt ccatccatca acctgcaacc agttaagggc accgtttgaa agaaatctgt 61440gtgtggggaa gggagccaac aggaacagga aatgtttgaa agaatgtaaa ctatttcagt 61500ttcataaaaa gtaacaagta aacagttatt acatgcaaat aatgtcctgg ttttaattaa 61560tgctgaaaag tcaaaatatg gctgacattt gtatgtatac atcgaacggc tggaaaggaa 61620aaaatggtgc ccagatgcct gtttcagagc ggggctggca gctcagaggg aactagaacc 61680ttgagaaggt cctgtttatt ggtgatgaaa agcacggttc tgcttcagcc acttcagcct 61740gctgtggagt tggggagcag agggaaccca gcttacttct taacaaagct agaggcgggc 61800ctggtgcttg ggaagggcga ctcccacttc agccacttct cgtaggcagg ctggtcttaa 61860agggccagtg gaccctcagg cctccgttcc acaggggcag ggtttccagg actttcccat 61920ccaggagtta agtgatgatg ggtttcaggt cccagaagcc tcccattcaa cagcccccca 61980cccccgtccc gccttccttc tgctgctcaa ggtcggtcag acaggcaggg tggcacaccc 62040gccttgactc tggggcagga gatggcagcc ttcgagctgt gctttccaac attcagctgc 62100gttagcttcc gttctagacc acctagggct caaaggcgct gggaaactgg gtctgggaga 62160ccacagctgg agagacagcc tcagagtgtg ggggatattc tgccccctat ggagagagtg 62220gctggggtgc ttgggcccca cagatcaggg acttgtcctg caaccgcctt gctgaaagac 62280ctataagctc cctttttgag cttgttaatc caccatctcc tgccagcatt ttttgtgaga 62340ccaggtgtgc ttaaccggga aagagggggt ggcatgaacg gtttcaggag ttggtaaacc 62400ctagaaactg ggagaaaatt gtctttttct ggcaagagac cataactttc ctcacctcct 62460caaagcgatc tgtaatatcc tacaggatta caaattgctg tttttagaca gagctgcatc 62520tggagacctg tttttcggga ttctaaggcc cctctttcaa cctccttccc tgctgcccct 62580gccattgcca atgctgaaat ggcgaggcct cccttccact tacctcgccc actgcggccc 62640acgaagcgaa ggtcgttgaa cctggccacc tggttcttca tgacggccga ggcattgcgc 62700agctcagcgg agtagttctc gtcattgcct gccatcacag tcaccaccgt accatccggc 62760acgtccccca atgccaccac ctgaagacac ggggcggggg gatgcagggg gacagcttag 62820aaaggaagag ggtgaccagg gaaaggaggg gaggggctgg gctgggcagc tcccccaggt 62880cccaggcaca ctgagtattt ctccaatgca gggtggagaa gaggcttaaa aacaataaag 62940accttccccc aaatatcacg aaaacaagaa gatggaatct cgagcttcca caccaaaatc 63000ctagatcaac tgcttacata aactgtgtcc caagaaatca tcctttcaat gaaatctaag 63060ccagagctgt gaatcagctc agtcactatg atgtggggtg cagttcccct gttgtcttcg 63120gctgcagcga aagaggaatc aacatgctcc tagcaacgaa gtctccaaat gagaaagagt 63180aacaacaata ataacaacag ggctgctacc cccactcaat ttatgcaaga gctgtttagg 63240gcatgaaatt tggccctgaa atgtggacca ggcccagttt attggcctct gcagagccta 63300aattcgttat gcagagaaaa tgcagaatgc aaaactcact ggtgttttga aaaaggccac 63360cagaaaaccc ctttaaagtg agagtggggc ttttgataat ggaaggatgc acctgccggg 63420aattgcagga tgggggtggc gatgtccccc taaacaccat ctcccccaaa tcccccaccc 63480ccaggagcac ggagaggcgg atgccttttg aaaaagaatc agactttaaa cagagtcaca 63540actatttaaa cgtggccgcc gcgtgcaggg actggggatc catatggtaa aaatttcaag 63600gagaaaatgt ttgggatctg attaagaaga ccagatttcc tgtcaacatc ctgtcttctt 63660ttaatttcaa agactccttt taagctccaa gtgacagtaa aacctccgat ctgacgatta 63720aagtcacacg ggcctcccgc ccctcccggc gagatttccc ccactggtat tttaagatgt 63780cacccgggag acctcaaaga gccactcttc ctttttttcc catttagagt cgtcttaatg 63840ggagcaggga cggcctcagc ttccagccac ctcgggcagc accaccccca gccgccggcc 63900cttcctgccc tgcccttttc tcacggcagc tgtgagaggt ttaggggaaa accgaggcgt 63960tttcgtttca tctcgctgcc cccttaaaaa aatgaaaatg aaacagtcgc ctactccctg 64020gcataaagaa aaaggtcctc taaatggctg ggggctgcca gggttagggg tcccccaatc 64080tcaactcgcc attcgggacg cataatatcc ccgagcaaac gtctggagag cagtgccccg 64140atcccggcct agcgccgtcc ggtaaaattt cggaagcccg agggtgtgag caggaagctt 64200ttgcgaagcg gcgcgggagg aggggtgctg gaggcggagg gtaggccctt tcaccgttcg 64260caccccaccc gcggtgtcct tgcccctgtc ccgggatcct cttctccgtt acccgcaggg 64320ctgtatctga gcgatccggg ttaggggggc gcaaaacccc atccgcccat ttccgcacca 64380acgtctctac gcaaggcgcc ccaaaaccca ggtggagcgg ggcaaccccg ttaaaagtca 64440ttcctgcagg gcgcatccaa aacggaacgc cgaggtcccg gagccgagcg cgcagccaga 64500ctgaaccggg tgcccgggtg tcgccgcggc gtctcgggca cctcccatcc ccactgctcc 64560cgaggctctg gctcccgcag ctcagacgcc cggagcccca gggccggcgc cctcccgccc 64620cgggtcccgc actcaccttg aaggcgacgg gcagcgtctt gttgcagcgc cagtgcgagg 64680gcagcacgga gcagaggaag ttggggctgt cggtgcgcac gagctcgcct gcgtggtccg 64740ccagcacgtc caccatcgag cgcacctcgg gccgggcgcg ccctccgggc cccacggccg 64800cctgcgcgct cagcgcgccg ctgttctcgc ccatcttgcc gccgccgccg ccgcagggga 64860aggccgggga gggaggtgtg aagcggcggc tggtgcttgg gtctacggga atacgcataa 64920cagcggccgt cagggcgccg ggcaggcgga gacggcgcgg cttcccccgg gggcggccgg 64980cgcgggcgcc tcctcggccg ccgctgccgc gagaagcggg aaagcagaag cggcggggcc 65040cgggcctcag ggcgcagggg gcggcgcccg gccactactc gccagggccc gcccgctgcg 65100aggcctcgct ggcccgacgg ccgcccgcag cctgcccggc tagtcccgca tcctcggcgc 65160gcggccccgc gtgcggccgc ccctcgtggc tgtcccggct gcctgggccg cggcggggcc 65220cgcgcggggc tgtgccgctg ccgccgcctc ccgccccgaa gctcgcccgc ggccgccccg 65280actccgcggc cgcagcccca gaacaaatcc tccagaatca agtggcgggg ccgcggccgc 65340ccgcgcgggg ttagtacccc cggggcccgc ggggcggggc tggcggagcg acgcgtcgca 65400cagccaatcg gcggagcccc catcgcgggc acctcggtgg cgttcgcggg gaggaacggg 65460gcctgccgga ggccgcccaa cggggagggg cggaaggcgc caccccgcgg aggaggcccc 65520agtgccacag cccagggccc ccgagagctc tgggagcccg gggcaaatgc tagaaatttg 65580cttagaacgt ccgggtccca cggaaggcgc ccttgccgcc ctctctcggg tcgtagctcc 65640ctgacgctgg ggcgcaaccc cttcgctcct cctccccgct ggccgcggcc gggcttcccc 65700agctcttgct gcttcgggcc tgtgacttct gcaaccccgg gctgggggcc gcggggtctc 65760agggccggtg acgccgcact gggagccgcc ccaaagaggt tactcacctc cctcgtcccg 65820cacattattc tgacccaaga gcctccaccc cacacgggat tttgcgcgtc gtccacgccc 65880ggccggcggc ctttgctgct cccagccctg cgcggctttg gtcccagcct cggtggcccc 65940tgtgccaaac cggggacagg cggaagggag tctcctaggg accctaagta gcctggggcc 66000aacaacccct ttcctctctg ctctcccctc aaaacaagtt tcaggatctt gcaggcctcg 66060cggcgtcgtt cttcgttgtg gcggcctgtg gctctttgaa aaacacgacg aggcctgcaa 66120aatgcgtttt tctttttttc ctttacgcat gtaaccacgg tcctgcatcg tgaaacggta 66180cgcgcgtcgg tggcaaaaga aaaacagcag tggctgcaaa gctaagggcc ctcgctttca 66240gaggagagaa ttttctttct ccatgcgggt ggaaagtggc ctctgcgggt ccaaccccac 66300ttcttcttgg gcccgtgcgc tccggctgcg ccgcagggac cgcggacagc ttcgccaagg 66360cactgcctgc ccgcccggct ccgggtcccc gctcccactc ccagccgcgt ggcccaacct 66420ctcctgggct tcactgcaaa tcaccccttc ctctcccgcc tcctaagtct gtcgagcaga 66480cctaggggcc ggctacagtt gggagggcaa cgggaaagat caagccacaa tcattccgaa 66540ttatcgcccc agacacctcc ctagactctg gggaacgaac gcgtgctgag cctccccgcc 66600gctttggaga cggggctaga ttttcgttgc ctccggctct cgacaggtgc aaaacaatga 66660attccaagcc tcggaagcaa agaagcttag gatccgacgg tggccgcaag atctcatcat 66720ggatctgacc cctgctcagc gcgcgccatt tcgtcgttgc caaacgaaat caagccccgc 66780gtgcgctcca ggggcgaagg actctggact caccccgacc accgggagag ctggccccta 66840cccacctcgg gacctcacag cacgccctca ggccgtgtcg aaaggaagga cggcaaaggt 66900cccttactga accttttaag agagcctgcg cctggcagtt gtcgattgcg gacccaggcc 66960cgcgcgccct cggacgcgct ggcacgagca gcagaactag aggaaagcga gtgatccagc 67020ctgggcgctc ccacctccgg gaacgtctcc gagaaggcgc agcgcgtcgt ggccaggtag 67080ggccctggcc gggggcgggc aacacgtgct gccctcgagc aggttgcggg accatgaccc 67140gctgtttcag gtggtggtaa attccatttg tcgaatggtt tcggtttgca ccgtgccctt 67200tgcttgttcc tccgcctgat ttctccctct ccgcttacga tgggttcaca gacaagtttc 67260cagagaatga gggactcttg tgggccctgg cacctggcgc agggcccggc acggctccgg 67320ctctccgtag ggcgctggct ccccgtgggc accagatcca agggaccagg gcggcggggg 67380gagggggggc gggtgcaggc ccttgggtcc ccagaccaag gtcgcggggc cgcctggcag 67440gcacagtggc gggagccgcc gctagttggc gcccgcgccc tgccagccgc ggaggtgcgg 67500gcccggccgg gctacagatg cgcgccagct gcggccccgg gtgcaggcgc ggcgaccgcc 67560cccgaggagc tgccctttcc ttgccatcca tgcggccagg tctcagacaa accgatggct 67620ttgtgtcaaa ccaaggccgc cttcctcacc tctgataaga tggacgcctt ctgtcttcgc 67680gttttcaggc acccggggaa gacccacaga acaggctagc ttgttcccaa tttccacctg 67740cttcctcccc atcccggacc gacaaaaatt gtcgtctgtt tgatgggagg gagaactccg 67800actcccccac ctggggcatg cagacaccct cgcccttccc cagttggcat ggaccgtcgt 67860cttttctccc tcttccatca gatcgatgga caaacaggcc agtttctccc cagtggcccc 67920cacctaagag caccctaagt tgtccacagc agggctagga agcagaaggt caggacactc 67980ccctacccta ccttgactta gagctgggta aacccagaac ccatccccgg gcaaatagag 68040ccagctcctt tgccccagga aggggattcg tctccctctg gcatttagga gtgctctcta 68100agtgcgttct tggcagtgag ggtgccgcct tcccagggca ggtgtgattc atgtggactc 68160tgtggcgcct gggcagggat ccccaggtat accagacaag gggcaggtgt gccctgggaa 68220accgcctaag aggtccatgg gctatggaag gagctggggt ccacagtccc tctgcctgag 68280cgtgtctttt tccctcaccc acagcgctct agggaaagtt gcctaaacct ctctgagcct 68340catttctttc atttgtaaag tggggcactc atagtggccc ttcatagaat tgtgtgtaaa 68400gtgcttagca caggcctggc acatggaggg tgctccagcc tccgggagcc atcactgtca 68460tgaaaaaata agacctctca atccttgctg ggggcctttg acccacccct cctctctctg 68520ggcctcacac ttccatctgt gaaatgtcca gttctcatat tcaaagctta ctaggactcc 68580aagccagtcc atgctgtcct gatccctcaa ttcgcccaca ggctgcctgg gggaggtaag 68640gactggctgt gacctacctc cacgtggagt cagctcatag cggggtttcc agcaaccatc 68700acagggcggc cagagctggg tctcgatgat tgcctgtctg accattcctc tcagaacctc 68760actttcgccc ccagccggcc gccctcctgt gggcagaccc tttcctgagt agcaactggg 68820cctcagcgga cactgccagg gaccccgttt ccttcccagg aggcctctgt tccccatatc 68880ccgaatcaca caggagccta gtccagcgaa gagagcagag gactctcttc tagaactgaa 68940aatttctccc agcctggccc taaatcccct gtccagaggg acccgtggtg aaacctatct 69000cctgcccagt gccctagaac tcaaagggga cattcatgcc cctcactgag cctcaatttc 69060ctcttctgtc aatggaggtc attctaacca ctccatttca cgggaggggg attaaggatt 69120ccctctagga ggggaggggc atcattgtga ttgatgatcg attgtttgaa gaaacagaaa 69180gaaaatgctg ctgagtaaac taggactcat ctgcatcctg atttcagata atgatctctg 69240aatatataag cgagaaatgt taatgaaaaa tggcaatata tctgggttga ggggttgtct 69300cctgtaggcc gggggtccag ctccagagag tccagctctg gggtcatcta tcctgggcag 69360cctctctgga aggattcaga atgtgtggga gcacaaatgt gcttctcaaa ttacagagat 69420ctttcttcct ttttggaaag ttccagactt ggaggggagg gagaaggagc aagggagagc 69480agggtggtga gggtgttagg acccagatgc tgcctgtgcg gtctgagact tttgcctggt 69540gtccacgctc ccctgagcct tggtccccga gggtaaaatg ggaagaacag taacagctgg 69600gggtgctgag gctttacctt gtgccaggcg ccgcacatgg gcattgctca tggtattcaa 69660tccccacggc gtcatatgtg gtaggtgtta tgcccatgta agcaaagagg aacgttgtcc 69720gaggtcagcc aggctagaga gggccagacc cgggttaaaa gtctgctctg gttcaaaatg 69780tggggcatga acgcatcacc tggccaagca tgtcagcact ctcctcctag tggctgagta 69840atgggaagag ctagcatcta gatacagagg aaagagctat tgtgatgggg agagggagct 69900gggtttggta aatcctgcta agcagccctg ggcttggaaa tcagtaaact cttcaaatct 69960gcagggagtc aggaaggact tgccagggtc attcgggagg gtcctgtgat agtcaaggtg 70020cacccaccac ctgctctcct ttggcctcag aaccagtctg cgaggaggca ggactggcag 70080tagtccccag tttacagatg ggaacactga ggcccagaaa ggggaaaggg cgtgatcagg 70140atctggaatg agctccagca aggccaggag caagcacctc gaggcaaaac gcagttggac 70200aggacctttg ccttgcagga gactgcagcc cagtcctggg cctcatacac tagcaccctg 70260atgccacatt cagtgcctct cgcccagggg aagtgctaat cagacgtgtt tccctctggg 70320cctcagtgtt tgcatctgaa tgcgggggtg cactttcaag gcccctctac atgccatgcg 70380ggttccatag gaccccaggg tttggttgtg acccgaggcc cctcctcccc acccacctcc 70440tctccacctc ccgcggggcg ccagctccct tgcgtccaca tgacctcgga tccttccacg 70500cccatcccca ccctgttctg caggtgggtg gtcagagggt gctctgcttt gaggatggga 70560gagagaaagg gaggcaagga cggagaaaag agacttcttt tgcgggagcg cagagcagaa 70620aaaccgtctc catcggttac cagggaaggg gtttctggtt tcagatccca tcacttggtg 70680gggccttcct accaccctcc ctgctactcg ctcttgtcat ctgtaaatca gggaaatact 70740tctggaagac agttatctgg tctgtgactt tgatcattgg tctatgacta ataattgccc 70800taattttttg aacacctgcc gcatgctggg agttttccgc caattgtcgc tcaccctcag 70860gtgcctctga agggcagaga ttttattctt tccatttcac agatggggaa acccaagctc 70920cgaaagtaaa gagcttttcc tctgtgggcc tcagaatctg agaagttcaa acaggttctc 70980aggagccctt ccagcacccc actcctcgat cagggagggg ctgtctgcac tctgaccgct 71040gctctcagcg cagagctctc catccaaagc agcaggtgcg tgcagagcta cctgccagca 71100gagccatcaa acacggactc ttctactggg agccatggag tggtgagaga gacctgggca 71160gcttggagcc aagggggctt ctgggaaaca tgtgcccttc ccccagggtg gggttcagct 71220ctggcgggca gggagagaaa gggctcttct gagtggctgt tgctttacac acatttttgc 71280ttcacagtat tcttagggag tagcgacagt tatcactccc attttacagg aaagaaaact 71340gaggcttaga gagctcaagt aacttgtcca agttggcacc actgggaaac cacaggggta 71400ggattccaac gaggcagcct ggccccagag cccatgttgc tgcccactac actctactct 71460tgtggactaa aaccagatgc tcagagttac agtcatggaa tagaattaga atcctggcag 71520aagaactgtg ggcaggattc ggaattttac aatgtcagac tcgaaagggc tctgagatat 71580caaatccaaa tccccatttc tcaaatgaca gaactgaggc ctaggaagga agagtctcac 71640tcaaggtcac agccagtgcc agggacagag tctgcacccc ctgcctctcc agctacctcc 71700cgctgactcc gcaccttcct ctctcgcagg ccctcctctc cccactgccc acccagcagc 71760ttctgggccc agccaggccc attagggatt ttccacctcc ccaaaaaggt cctgatgact 71820gtcagtcctt gtgaagcctt aattaatctc agaggccgat ggctcggagg agactggggg 71880ctttggcctt acgcagatga agattgcggc tctatttcat gtggtggtga aagaacgcct 71940cagacattcc tgccagcaat aaaagccaca tggctttcca gcatcgccct tggaaaagaa 72000aaaaaagtgc agccctttgc ggaaataaat caactatgtg ctgtacgcat ggcatgagat 72060acaaatgggc atacggaggt gggcaacagt cggtctttta tgccgcctct gatgtccact 72120gacagtggca gggccagcgg tcatggtccc agctgcaatc ctggggagag ggagtgaccc 72180ccagtgtggt gggggaagcc tcagcttctc cacctgaact ggatttgagc caccctagat 72240atcccagagg cagggccggc tttctggcct gtgacccatg cagtcgcaca gggccctggt 72300ctcagaaggg tcctgagctt gttttaatgc cctgccacca ctgccttgaa cttctgaata 72360cttgctcaac aaaggtcctg cgttttcatt ttgtactggg ccccccaaat tatatagcca 72420gtcctgacca caaatccacc cctcatcacc aattgtcacg tctctcctgg cccctgccat 72480gtacccaatc ccggggagta gggtttcttg agtgcctact agccagtttg cttatatcac 72540ctgagatgaa cttcagaatg actttgtgaa ttgggcagat gtggaaaatt gaggctcaga 72600gaggcttcca tatggcaagg aagcctagac ttgaactcag gtctccctga ctccaaagtg 72660agtgctctta gcagctctac attctgcatt atttcatctt caccatgccc agggggatgg 72720ggatacacac agttaggctg ctctattccc agataacaga aggcataact gaggccagag 72780aagtgaaggt tctcaagtca gtgtcaaacc gagggcctgg gcaacagtgg acctgggcct 72840ggatccatag ggctggggat ggagtctcag ttttatagtt gtttgtgcca cttgtaaatt 72900tattagctct ttccatgcag gtcactgcct tgagtctggt ctggaatgtg gctggagccc 72960taccctgtcc ccctccccca cagctctcca ttctaaacat ctggaagtcc ttccttgtgt 73020cttcttccac tctttcacgc tgcagttttc ctctgccacc ctcactggtt gggaagcagt 73080tggatctggc accttgataa actcaaaaga gtccaaattc ttgatgaaag ttggggctga 73140acagagccca tagattgcca tgtcctataa ccaggcctgg gcctaaggct catagagcca 73200actgctagat ccagggcagc catttccttg ttccttgctg ggtaaccttg agcaagtccc 73260ttccctctct ggccctcaga ctccccttca gggagataaa tgcattggac cacacctgag 73320ccccaggagg cctctctgtc ttcaacattc tagaattcca tattaatcta caacaggtct 73380gttcatttcc gcatctaata gctggggaaa ccgaggccca ggaaggatca gagatttgcc 73440caccgtcaca gaaggtgctt attgacaagt ggacttgact ctgaggctcc tgtcagctgg 73500cccggttgcc tctgcacaaa ctttcggagg atctggcctc agcatcagct cagcttgccc 73560ttgtcccgcc gcctttagcc caggtggtct gtcaggcacc ctcagtgtcc aggcctggaa 73620atcacagcta agagtccttg gcaggcaata aagttcctct tctatggctt gaatgtctcc 73680caaaagtcat acattaaaac

ttcaccccca ttgtgatggt attaagaggc agtggggggc 73740ctttcgggaa gtgattaagt ggtgaaggct ctgccctcat gaatggatta ggccctcttt 73800gcccttctga cttcaggaca caatgttctg tgtcctccgg aggacacagc cagaagacac 73860tgccttggaa acagggagtc caggacctca ccagatgcgg aacctgccag agccttgatc 73920ttggacttcc cagtctccag aaccatgtgt agtaagtttc tatttctcta tttataaatt 73980atcccgtctc aggtattttg ttacagcgac acagagtgaa ctaagacact ctctttagac 74040aaaagtgggc caggggatgg cagcaaccct tttctcccca atcgcatttg ggctgtgtca 74100gtgtttccgt aataaaggcc ccttttccag gggttataat ttggctggaa aatgaggagg 74160aaagaccaga ctccaggact ggaggggcac atgaagtagg aggctaggat gggaaaagtc 74220tccactggac cctgggcacg cagagtgcac acacacacgc acacacatct ataccctaca 74280tgtgtgcact cacacacagc acccacgctc atgggcacag tctctcacac attcactggc 74340agctcacacc cacatggaca agccctcatg gaggacagca ttgttacagt gcagccacag 74400gtgcaaacag ttaagtgcag gtgtgtgcaa agatgctcct aggagatgcc tctgtctgca 74460tcatcatgca tggacctatt ggtatagatg cgcagataga tgcacagata ggccccatta 74520tatgagtggt gtggacacac acatgggcag aaacccacat cacagctgtg taaacagcag 74580accattgtgt ggacaaatct ttacacacag aggcaggcat ggaatcaggg ctcagagctt 74640tggatttgtt ctacagagca gctctgggag gagtcgaacc ctggctctgg aagtttctgc 74700ttctcctcaa ttcagaggca tggactttct gggtggtttg cccccctggg gcttccaaac 74760cattccccag catctgagtt taacccgctc cctcattgtt cgatggggac aaggagagcc 74820tgtcttcctg gtccagagaa aggcagtggg aggggagaag tgggagggtt gcagctaggg 74880tgccccacgg cagcatgggt ggaagggcag ggcactagcc taggggccca gagacctgag 74940tttgggttta ggttgagatg ccctaggcca acacatggcc tctctgggct tcatcctgag 75000ccccctctgt tagggccatg tgacaccccc aggggcctca gcatggggaa gagcactgaa 75060accatgtcac atgatgaact attaaagcaa ctggagactt tgccctggag gagagcaggc 75120ttggggggta agagctcctc tggcagatct atgaagagct cccaggtggc agggaccata 75180tggatgctgg gggctccata ccaggaatag aaatattgag agctggcttg gaatagggac 75240acgtcccctc agaggtagag atcaagttga gaccaggata ttgtgcaggg agttcgagtg 75300ttagatgggg caggggccgg accagatact agtgtctcaa acgctcagct catagcaaac 75360acgtattgaa caaatgagag agcgactgca gagctccatt tctgagccaa tcatccgtga 75420ttcagagcat accagctctg ggttcccacc ttgccatctg catgaccttg gccctctcca 75480aacctcagtt tcctcatcta tgaaatgggg agaacaaatt atttccaaga gctccagcaa 75540gtcacatccc ctattgttgg tctttcaggt catcccagaa tttctgctct tataaataga 75600aaatgacatt gaaggtgaaa agcagacaga caagcaagag aatagttaat acaaaaatca 75660tagctaggcg tggtaacttg tgtctgtaat ctcagctact tggaagggtg aggtgggggg 75720atctacttga ggcctagagt tcaagactag cctgggcaac aaagtgagac tctgtatcta 75780ccaaaaaaaa aaaaaaaaat caggagagtg gtccccacca cttgccacct gtgatgagta 75840gggagaggga tgcagtcagg gaagggacac tggtgggagc cctaaggtcc cattagtgct 75900ttgtttttta aagccaggtg gtaggtagat agatgtctgc tttattcttc ttctttaaac 75960aatacttata ttttatacat tcttctgtac atgtatttta catgtttaaa aatattttaa 76020aggaaagcaa aagataaaat atagaaaaag ttcccctgcc ccaaacctct gaaaaaatgg 76080acaatatgct caaatgtgca taatatcgta caattattca tgatgcagca aagctgcact 76140gtttcatccg gatggtcctg tgtaccatca cactctcagt tgaatctctg caggcccttg 76200cagctgtcct catcatggca acccccacct aggtaagcac ttctaggtaa cagcccctgc 76260tgagcacgct ccccaagcac tcctcatggc cgaccagtag cccttcaggt atgtgtcagt 76320gggcccactt tacaggcaag gaagtccctt gcacctacat aggaaggggc agagctggga 76380tttgaaccag ctctgtcaat gccaaagttg tgcagcaacc tcacccgagg agccaggccc 76440cttgattata gtaactagcg ttatgtacac tcacacatgc tgttgaatcc ccggagccac 76500ttttgtatta ggtacattta tcatcatccc cattgtaaca gtaggacaac ggaggcatag 76560caaggtcagg aacgtgttca agttcacacc ctaggtgagt atcagagctg agccttgaac 76620ctcagcagcc tgatcccaga ttgtgtttcc tggcctggct gtgtggggag ctcagacttc 76680atggaaacaa aagacagaac ggtggctcca gggtccacag cggatcccaa gggaccagag 76740gccagcaggg gggttggctg gggttggagg atgctgccta ggagatctgc tcccagagtg 76800atgctagccc tgtgtgatga cctgagtccc cgcctcctta cagggtcatg gctgctgggg 76860aggtgctgag gctgtgggta cagccaaacg gagctagagc aggctttgga ctccctgcct 76920ggcaagtcca ggtgacaggc tcagacactg ggcactctgt catttgctgt tggcataagt 76980ttccactggc aggaatgtga catttatcac ctgagtgggc ttccagaagc ccactgaatg 77040tcctcagacc tggggtgggg ggccctctca ctgcctcacc tctgagcctt aatcaaatcc 77100agggtggctg gatgatctga aggccctttt agctcacgga ggcctgggct tgcccctgcc 77160cccatccgtg tcctcagggg aaaaggttcc cagtcctgcc cttagcagct ctgagcttag 77220atgagggggg gagatgagat ggaaaggaaa aggagaagta agaaacagac agaggaaaag 77280gagttggcac tagattgaag cagtcaacac acacttatta ggcacctagg gctattttag 77340gtgctgggga tacaagcaat ggaccagaaa gccatggagc tccttggggg ctttgattcc 77400agcagggcag acagaagaca gacaaggagg caaataaata agcacgccaa tatttgatag 77460tgtcttggga cactcaagaa aacagatggg ggtaaagtca gagagagtga ctgggtggat 77520ggggagagag gggctcttgg caatacttta gacagggtgg tcagggaagg tctgtcggag 77580caggtgacat gcgagctgag accagagggt tgagagggac ccaggaaggg agaagggggc 77640tggtaggagg gcccactgca cagtggaact ggctttaccc acctgccttc tccccactcc 77700tctgcactgc tagtatcccc agttcctaaa actcttactg ccctttctct ctgttgcttc 77760tcccacaaca gccctgagag ccagatgggg tgagcctaag tagcctgacc tgcagtgcag 77820gaaactgagg ctagagtggg gagggtcagc atcagcggtg ccctaatgcc aggacctgac 77880ccgggctccc gcctcccagc ctggtgctcc tcggagcctg cccattgcct ggcatgttat 77940tcaaccaccc cagtccaggc aggctgcagc cactgtggag ccagcccgtg ggcaccgctc 78000ctgagaggtc acaggctgga aatgtgggca gctgggtagg gtctaggagg gggcagcggc 78060tcaggactgg gcgggggtcc ggagcggaag gcgcccagcc ctgattggaa caaggtggca 78120gcaccgggag ccgagccggg tgtcattgat cttgcccggt gttccagcca ccaggcggga 78180ccagcgccgg gcagactgcc ggttttccca ggtgtgggga caccctgagg gaatgacttt 78240tcatgtggtt gtggggcagg catgccaccc agcacgtggg ggaggccagg gctttgggag 78300catgctggca gcagggtgga ggggggtgtc tggagactca gaatcccaca gccagcaaat 78360gtgaggctcc tggagacagg gtcatggact tgaggctctg agaccctgag gatgttagaa 78420tcttcatcgc agtagctccc actgatggtg tgctcacggt gccaggcacg gttctgagaa 78480ctcacacagc ttaactcttc atccttgctc catcctaaga gggggttctg tgatcatccc 78540cacttacagt tggggaaact gaggctcggc aaggttaagt agcctgccaa acacacagct 78600accaggtttt tgtcttagga aataagagcc ctggaacagt tggccagtgc ggagaggacc 78660cccgaagatc tctgaggcta gtccccttgt gtcggaaaaa caggtctgga gaggggatgt 78720gacgtgctgg ggcccagggg agtccaaagt caggactcat tcttccccca ggtcatcgtg 78780ggacctccgc tggtccctga atgtcaggcc ccctgagggc agggtcctca gccaggacct 78840aggctcccag atgttaccaa ccttaactga cagttttctg ctagcacaca ggaagttctt 78900ttgaacatct aacctgaatc cctcgtttgc agggaaagcc tcttccttct catcttgcag 78960tactctaaga agagtttggc ctttgatgtt agggaagatc accagttccc tggtgttgtg 79020ggaggtgaga ctgtgcccct ctctgccata aaatatctct ttactgtcca tcgctgggcc 79080taaacattag cgacttagcc cttggggcct tacagagttt cttattaaaa tgtgagtact 79140cctggaatgg gtgtcagctt agcaggacag ggtggtactt caggggcagg gcttgggggc 79200cgttgagggg caggagagag ctgattctcc ccttctagcc aggcttgatg gggtctacat 79260gacctgccac cctccacctc tctgacctca tctgcttcca ctctgcccct ccctcaccct 79320gctccagcca ccctgccttc aaatatgccc atcatactcc caccacaggg cctttgtctg 79380tgctgccctt tacctggaaa acccttccca ttctgtctgc ctggctcagc tacccacttc 79440attcaggtcc ctgctgcctc ctccaagagg ccttctctgg tctcctgtgg taggcagaat 79500aatggccaca gagatgtcca catcctaatc cccaaacctg tggctatgtt accttatatg 79560gcaaaaggga cttcgcagat gtgatgaagg ataaagactt tcagatggga gattatcctg 79620gattccccag gtgggcccca tatgatcaca aggatcctca cacatggaat agggaggcag 79680aaaaggacag tcggagggag atggggtgtg gaaactgatt agggaacctg agagatggca 79740gcgtgggaaa aacatggctc aaagctgtgg gctttgaagg tggaggaagg ggctatgagc 79800catggaaagc agacagcctt taggagctgg acaagacaag gaaacaaatt ctccaccaga 79860gcctccagca aggaacacag ccctgccctg accttgatct tggccaaggg agactcatag 79920agaatttctg atccctggaa ctgtaagatt ataaatgcat gtttttttta aggcactaaa 79980agtgggttaa tttatgatgg caggcatagg aaacgaatat gtctcccttc cttgattgac 80040agttcctcag cacatttatt agtgcctgat acacaatagc ttgacttatg aattgtctct 80100tttctctcac agaaggtcag ctgcaggagg gcagggattt tttgcttgct tggtgttaca 80160ttcgcagata gagttgtcac atttaacaaa tggaaatata aaacacccag ttaaattgaa 80220tttcagataa ataatgaact cctttttagt ataaagttgc cccaaatatt gcaattattt 80280atcgtttatc tgaaattaaa ataatttagg agtcttgtat tttatctggc gaattcatcc 80340ccaacacata aaaccattcc tgggtatgta ttaggatctc aataaatgtc tgttgaatga 80400gtgaaataga taagcaaatg aattcacatt aacttctagc ttaaaaaccc tctttggctc 80460ccagaatgcc tacagggtaa agtgtaaatc atagcaaatg ataaaagcag acagtttcat 80520agccgcttca atatgccaga cccgggctga gtgctttaca cttattaact cactcggttc 80580ttgcaataac ccaatgaggt ggttagccca ttttccagat gaggaaactg aggcccagga 80640ggcttagtaa cttgttcaaa gtcacataac cattgagcag cagagccagg caattccagg 80700cctttgaaaa cttggctcat ggaatgttct aatgttatct ccctacccat tcccctgaac 80760actgtggatc ctctctctct aacagccccc ggttcctttt cagggtatgt gcttccgcat 80820tctctgactg ctgaactcct cctcatacat caaagccctg tcctactttt ctctctttgt 80880gagaccatct ctaaaactcc caggaggact tggccccctc tctttcccct ccccaccatg 80940gccccttgtc tgaatgcgtt gtaaggactt gctattgtgt cctttacgtt ccctgactgt 81000gacctccctg agaacggaga tgggcccctt tcagctcttt ggcatggggc ttcaaactga 81060gtctggcttt aggggggttc ccagaagcat ttgagaatga atgaatgaat gaatgaatga 81120gtgagtgaat gaatggctga gtgaatgaac gaatgctgtt tgtttccact ctgggcctca 81180gtttcagagt ctataaaagg ggaagaacaa tcctgaaccg ccccccattc cttaaaacaa 81240aacacatttt tttctgatta taaaaataat acacattcat taaagaaaaa ttggaaaata 81300ataaaattat gaagaagaaa attaaaacca tccataatcc tgccacccag aacaatggtt 81360cccactgggt gttcagcctt ctgctcttct tactgtatgt atagatttat tatcttcttc 81420tcttccccgc cctccctccc ttctctcctt cctttttttt ttgagtgttg ggaacataca 81480gtatggggtc ctttaaacct gcttttgaaa tcccccaaca tgtgtgtatg tgtgttctcc 81540tgttttctta aagcctccct gatggcaggg gacaagtggc tgctagacaa gcctggtagg 81600ggccagggtg tggaaagccc attccccccc tactcatgga ccaccatagt tcccttgtga 81660tgggctgttt agggggtttc cagatttctg tcagggaaca ccctgcgtgg atgtcttagc 81720tgcatccctg gttccttcct taggacagat tctgggactg gggttgctga gaccagtata 81780gacactgaga ggctccggac accgccccac atcctcccca cagctgctct ccagccccac 81840tcccactggc agcctggact tctcagcttg gcagaaagcc gggggcagta ttccacctcc 81900tccagagaaa agccttgtct acacccaagg cctcactgat tccatccaac gctgaaaata 81960cccatttact cagctatttc tccacgttat ggtctaaggc ccagatgctt agttccaacc 82020aaaatagcca cagaggtcac tgtggtctga gggtcctctg gacctcaggc ctgtgtagac 82080atcatacttg gtaataatta acccacggag ctcaccacgt gccagaccct ctgcgtctct 82140gtgtgtcctc acggtaaccc tgaagctagg gatggctgtc tcccatctta caggtgagaa 82200cactgagggt gctccagaga gctgtgggcc ctaggccttg tcacctgggg gtgcaaaggc 82260gctgccccag tagtccggct gcctgctacc tggctgcctg ctacccaatg gggcaaagtc 82320ccagcactac cccgaaacaa ggacaagctt tggcctagaa gctgggcagc caggtcctgt 82380gcctgttttt tctccatatg tgccctgggc agctcaccct ctccgtctgt gaaatgggag 82440agcagggagt cagagacagc ctcctaggcc tgtggcggct ctgagcacag tgggtgcaga 82500tactcagtgc tgagtcaaag agagagagaa ccctgccaga tgggccagct caccacaagc 82560agccaagccc ggtctttgag gggttggagt gggcaggcct ttgaccaagg ctgagctagg 82620agggacctga cggccccaga tggaggctgg cccaccctgc cccagcagat gggaggctat 82680tttttaaccc cacaggaaga agaggacaga aatgattgca gggggttaac ctaggcccct 82740ggggacgctc cctgtttgca tcctcctttc cccacaaccc agagagctat gtgggcttca 82800cctggagttc cctgaatcct tctggagctc cccgcagatc acagccggag ctggcagggc 82860ctgagtggcc cctgtctgcc aggcgaggga cccagagccc agagaagttt gaccaggact 82920ggcttcctct gccctctctg ctgtgtgctt ccaccaggtg aggctgcctc ctccgcttct 82980acctttcttc ctggggtgac ctggggaggc cccaccttct cccgggccag tttccccatc 83040tgtcagacaa tgggaccctc cagacatcac tgaggcttct cccagctggg aaacctcctt 83100ctgagctggg gccctgactc tgtcacatca gtcctggatt cctggaggcc tcagccctcc 83160agaagcatcc acccagtgga caggagctcg tggcaggtgt ctggggaccc ccaggaagag 83220gaaggatttc ctggccagag ataagaagag cagcgtgggt aggggttaag catcctcccc 83280ctgcagcctc cctcagacca cgccaccagg tggcccttgg tccccccaaa aggagttcct 83340gaaaagtctg tgtctgttgc agcaggtgcg gcctgtgaag tgtgtgtatg cttgtgtgag 83400ggtggtgtgt gttcacatgc acatggtggg ggtgggcaca caaggcggga ggcctaacat 83460ggtggcaggg acagactttg gttgctgagc tgggacagcc tgtgacagag gccccagcac 83520acccgcaggt cttaccagaa accctcagat ggtgctggtc tgacctgaag gtgggcacat 83580gcagggaagg ggtacatgca ggacaggggt gcatgtgggg gagggccatg tacagggcag 83640gggtgcatgt ggaggagggg tacatgcagg atgggtgcat gtcggggagg gtcatgtgca 83700ggacggggtg catgtggggg gtgcgtgcaa gacagaggtg catgggagag aagggtttgt 83760acatggcagg ggtgcattgg gggtgcatgc agggcaggtg tgcatgtggg ggaggggcat 83820atccaggaca agggtacatg tggggaggcc acagggctca aatgctgtca gggcctctgg 83880gaagctggga ccccagtgaa tgcttgaggg gagccaactc tgcctgacct cctcttatga 83940ttgtctattt aaacaatact gtaaattaat cacattaatc gaacccacct ccctgcctcc 84000tgctgcttgc ccctgtgata caaataatat gagcacaatg aaaaatcttg gaaaatacag 84060aaaacacata aaaaatgtta aagcctgaag tcttataacc acagtgaaca ctgcgagtgt 84120ctttgagggg atgggggtct gcaggtcttc ttgatacaat cacattcatt cccacatact 84180ggagcatttc ccacgggcgg ctgtggagct gagcacttca ggtttgtctt agtgaattct 84240ctaacagcct gagagggagg tactgttatt ctccccattg tatggctgaa gaaacagcaa 84300aaaggaggtt aaatatcccc ctcagggtgt aagaagcaga gccaagattt gaatccaagt 84360ctggctaaat ggaaagtgca aatcgtccag cgtccagggc tgcactccag ccatgccccg 84420ccccccgtga gcagaccact catttattca ttcctccagg agcatttact gagcacctcc 84480tgtgactcag accctgccca gcacccacac caaggacttg gcggatgtga acgagacaga 84540gagaggcccc aacctgactc cccaagcggc cacaaactga gtcccaacct tgaccacagc 84600ttgatttcta gtccaagttg tcactgaccc ccactggcct tcatcactga ctgaactgtg 84660acctggcctc ctgcactctg cagtggcctc tgtgagcttt tcattccccg tgatgtgtgt 84720gaaagccaag gccagcagcc cacctcaccc agcctatcat ccacgcggcc tgggccaggg 84780aggccgtcag gagcccaccc accacctctg gcctgccact ctgggccagg cctctctgga 84840gcgggggttt ggccttggcc cttggcaccc tgcttggcag aagggtgggc cttggctcag 84900agcatggggc caccccagga ggggtcagca tagctgagct cagggtacct gtgggcgggg 84960cttccatgtc ccagggtcct cacactgcag cctcctcttt ttgcctgggc cctggaaccc 85020caggagaccc caggagccgt tgctccctcc tctcacttgc agaaactcaa cagggcagct 85080catctgagct cccccgatgc ctgcactgta tttctggggg tcctgcatgt ctctccaatc 85140ctcaggcagg gccagttacc tactttatag gacccagtgc aaaaagaaaa tacaggaccc 85200cttttcaaaa tgcaggaaca aaagtttttc ctttcttctg tggactctca acccacggtg 85260gtgtttttta tttgctgttc aatgtcacac gtacttggac ctggggagac ttgtgcagaa 85320agtgcagacc ctcacagatg ctcaggggcc accccaaaac ttggtgtgca gattccaacc 85380cctttctcct ccccatgcct gcctcagtgg agggcggcag tgcaggtagt gggctgctga 85440gaacccatcc ctggaggcag caggaggcag actggacccg ggccccaagt ccccaggcat 85500gctgcactag cccatcaggc ttcatttaca acacactaat tcagagcgaa aatgatccag 85560catttcaata tggcaactgc tgagcgttaa attcaagcac aggaggtggg ggggccaggt 85620agccctggaa catagcagtc tcacaggtgg ctgggcgtgg ggtggatctc tgttcttgga 85680gtagagggat gtggagtacc tccctctgct tggagtatct ggggtacctg gacaagcaca 85740gggggccatg aacagggcca tgcctgtgtg cctgcccctc gctcagaaga gggcacctga 85800cgggaatacc agggcatatc tgcaccatgc ccgggcagta ggcctgggca tgaccctgga 85860tcaggcagac ctgtagtagg tggaagggcc ccaggagagc tgaggagcct aggggagagg 85920aacccagagg tccctgccaa agtgcttgat gtgctgccgt aagaagggca gcataggccg 85980ggcgtggtgg ctcacgcctg taatcctagc accttgggag gctgaggtgg gtggatcacg 86040aggtcaggag attgagaccc tcctggataa catggggaaa ccctgtctct actaaaaata 86100caaaaattag ccggttgtgg tggtgcgtgc ctgtaatccc agctactcgg aaggctgagg 86160tagaagaatt gcttgaacca gggagttgga ggttgcagtg agccaagatc atgccactgc 86220actccagtct ggcaacagag agagactcca tctcaaaaaa aaaaaaaaaa aaataaggca 86280gcatgggtgc ctgctgagag agagagaaag aagctctttc cctgcatgtg ttgccatggg 86340attctggccc agctccctgg ggtgctctct gagctcagct ttggccctgt ccctctctct 86400ctgtgcctca atttctctaa ctatgcactg agcaaggaga agaccaccac acctcaagta 86460ccttctgcat gggccataca ctgagtttta tgaatctccc ctctcttgtt ccacaaatga 86520ttactggccc atttctcaga cgaggaaact gaagcccaga ggaggcaatg actcacccag 86580taagaaggtg gtggagctgg ttctgcctgg cttcccttca ccccttgagt cgctccagcc 86640tctctaggtt tgggtggagg acgtgggaac caagctcgtg ggggcaccac cagctcttgc 86700cagaaatggg gccaagagaa gaccaaggat gctccttgac ctgaggaaac gtccattaat 86760tcatagctac tgtgctttgg cgagccacgc aggctctgga tgcaggctgc ctgggtgggt 86820gacctgagca gatgccttaa tctctctggg gttcagtttt ctcatctgta aaataggcct 86880cataagagct tttgtcttat agggttgtga ggattaaatg agctaaggta tatcacttga 86940gcctgggagg cagagacttt agtgagcaag attatgccac tgcactccag cctggaagac 87000agagccagaa cctgtctcaa atacatatat aaacaaaatg agcaaaggta tggaaaacac 87060ttagacagtg gctgacatag agttaagagc tatgtaaatg tttactgcta atggaactat 87120ttaaaagttg agtcataatt tatattttct agactgtcaa ttacgaattg attcatttca 87180atgttgtgct tttccctttt gtatttagga tccagcaaat tttcctttga aatctcaata 87240caatttccta ggtccttgag aagataattt ccccgccccc acagtgctta tagcccatgg 87300tggatccaat agctctctct agagcagctt ttccaaaagt ggactttgca cacaccagcc 87360ccttccagat gcatcatctc accccaagag ataactcaat aaacagttga gcatacacta 87420ttttagatct ccatggccca acaaggtagc cattagcata tcaaagactc tgacaagtcc 87480tgcagcaaaa caccattgaa cattgtttga aacaaccaat cccaatcttg tttgaccaca 87540gagttccatt atttctgctc aacagctgat aacatctgaa cacacgttgg gagatgccac 87600cctcatttcc tgctttctag gaaatggcaa ggggagtcag agctgtgagg aacaccctct 87660cgcagggatg agtggctcca cctctacaga aatcatctcc agtcatgtgc accatcgcta 87720ggccattcct cctgttctca ccttccttgt ctgattcagc ccccacagcg gcctggagag 87780gtcactagca tcaatgtctc catcatacag atgaggaaat tgaggttcac aaaggttaag 87840tgggcacata gccagtaagt ggcagatccg gtagacaaac ccacagcttc tgattctaaa 87900ccccacattc gttcttctgt atgttgactg gaaaagtaaa aatagatcct attctaacag 87960gatcaatctt cccccatcat aggcttttaa aaaactcagg tatttttttt ttccggtagc 88020attgaatgct ttaaaaactt aaaattttta ctatctttct tttgattact aaagcagtac 88080gtgcttgtta tgtataaaac ttttcaaaca ttttgagttg aaaaatgaaa aaaaggcagg 88140gcgcggtggc tcaagcctgc aatcccatca ctttgggagg ctgaggcggg cggatcacga 88200ggtcaggaga tcgagaccat cctggctaac acagtgaaac cccgtctcta ctaaaaatac 88260aaaaattagc caggcgtagt ggcgggcacc tgtagtccca gctactcgag aggctgaggc 88320aggagaatgg cgtgaaccca ggaggcggag cttgcagtga gtgcgattgc gccactgcac 88380gccagcctgg gcgacagagc cagactccgt ctgaaaaaaa aaaaaaaaag aaaagaaaag 88440aaaagaaaaa tgaaaaaaaa aaaaaaacag atctgcatag ccctacaaag tagccattag 88500cttttaaatg aaaatactta aatgctatct aaatgaaaat agttaaaatg aaataagata 88560aaaaattcag ttcctcagtt acagtagcca cgtttcaagc gctcgggatt cacgtgcccc 88620tggtggctac tgtgttgggc agcacagaca tgggacatta ctatcatcac agagaatcct 88680acgggacggt gccgttctag attcttctta gacatatcct aacacctata caggttgatt 88740atccctaatt caaaaatcta

aaatctgaaa tgctccaaaa tccaaaactt ttttagggcc 88800aacatggtac tcaaaggaaa tgctcattgg agaattttgg attttggact gaagtataat 88860ccaactattc cgaaatctga caaaatcaga agtcctaaat ttgaatgctt ctggtcccag 88920ccatcttggg taagggatgt tcaacctgta atgactgtta atgtgtggtt tttttttttg 88980gagaccaggt cttgctctgt cgcccaagct agagtgcagt ggcacgatca tagttcactg 89040cagccttcac ctcttgagct caagttatcc tcctgcctca gcctcccaaa gtgttgggat 89100tacaggcggg agccgccatg ccccagccta ttgatattct tgttgaggtt ctcagacata 89160tctgcatggc ctcacacatg gaggaaagag acccacagag gcaaaaacaa gacatggggt 89220aaaaatagac tggaaggaaa cacaccgaat gatagtggtt ttttctgggt gttgagatta 89280ccgagcttat ttttaaattt cttagatcct tcaggtgttc tacaacgtaa aatgcagaca 89340gggtggggac gttggttgga gtcatgtttt ccctaatgtt cttactggtt ctaaaatctt 89400caagctatgc tctcacccaa ggcttcactt attattattt taacactgtg gattcataaa 89460gaatggaagc ccacacaagt ccagggaagg aaggaaaggc agacagaggc ttattttcag 89520gcctgggcag ttgcacaggg tcccttgctt agaagggcct catgcttggt ttcgtgttct 89580gtggtcgctg tcctgaaatt cttactgatt tttgaacaag ggatcctgta ttttcatttt 89640gcactgtgcc ctgaaaatca tgccgccgtc actagccctg ggattctccc caggacaggt 89700ttcccttcag ctgctctaag ccttcgccct tgtccttgtc caaccacgga cgtggccatc 89760cacggagccc tctacgtgcc tcagagcaag tgtgcttcgg ctgctcaggt gtgtgtctag 89820agactgataa aaacagggct cgtgagtggg tgcgggaggc ccctgtggtc tctgttcaca 89880cacgtaccta ccctcaaggc catgtctaca ctggcctatt tcagagaacc gccctgtgca 89940tcatgggatg ctttgcccca catcacggcc cagcttggtt cagtcctgga gccctgtgtc 90000ctgaagccat gaccaacccc aggcctggcc caccttcttc ctcagtctcc tctccctcaa 90060gccctccaca ggacccataa accttccatc tccatgtaat ccttttgtgt gatccttctt 90120ccataccttt gcccatgctg ttcactctgc ttgtaccagc aaggttcctt cctccccaga 90180tctgcccttt ctagacccat ctcagattcc accctttcta gaaagacttc aggaagtatt 90240tgagaagggc ctgaagatgc tgtggttgca tagaggagga tatgaatcac ctcgcttaga 90300ggtatcgggg agggctccat ggaggtggtg ccctcaggcc aaggaagaga agatatcttc 90360cgggcagaag ggagagtttg acgggctggt ttgatgtcag acagaccaca ggatgactca 90420aggtcctctt tcctcttctt aaacattagt tgcagcaagt cccaccatct gcctgagtct 90480gttttcatct ccacaatgga ggtggtgatg cccagcacac ggagctgtga tgaggattta 90540atggggaatc cagagcattt atggaagtgc caggaggcca agattctgca taggtaggaa 90600ggacctaagc cagaggggtg tgggtggcca gaggagagag cctaccttag tagggcttgg 90660taggctaagg tctgggctga atctcgaggc tctggagctt agaacagcat ctgcaactcc 90720ctggctgtcg gggttcaggc aaggactgcc tcctctctga gtgtcagttt ccccatctat 90780aaatggaaag ctgggacact gaaaaacact gggggggagg gtggttcctg aggcagtctc 90840tctccacaca tcacaggaat gtggcgcccc agggagaagg catctttgtc cattgtgttc 90900acttctgcat ctccagtgcc cagaacagtg cttgcatgca gtagatgctc aataaatgtt 90960cattgaatga atcagcagca accaattcgc ccacctcaca tcctaagtcc gctggggacg 91020taggcccacc tctccaggga aactgtctgc agattgggac cacacctcag gtcacaagca 91080tttcctgagc acctactgta tgcatggctc tgcgcagccc ggggcagacc cttgctccac 91140agcccgacag ggcagagcca aggaggcagg tgacagacac agtgatttgg gaagaagaac 91200agagagcagg gtggccagca gggccttgcc tgggtgggag caggccgttc ccaggctgga 91260gctcaggctg atgggagccc cagcttgcct gttcctggga gggtgggact gcctcttcct 91320ctttctttct ctgaaaacaa aaattgtttt cccttaaatt tacaagtatt aaaagtttgg 91380aaaatacaca ataatcaaaa gaatataaaa taaaggttac ctgccatcat ggcggaccac 91440acagtattaa ctactatgga cttttcagtg tttccctcta gtcttttttc tgaggtggct 91500ttgctctcag aggtggcttt ctctccccct gggaagggat atagctgctc tgtaggagat 91560gtgggggcac caggctctct ccatgggctc tttatcactt ctgacttgga ggtcctttct 91620ccaccccccc tggacctagc acctcttccc gagacacagg ggtgcgaaga gctgggggag 91680gtacgtcagc aggcctcccc tcctgcccct gcttacccca cggaggtggg gtgggacaga 91740actcaggctt gaggaaggag cactggaggc caagccacag gtgctggtcc tcagccctgg 91800tgcccaggca agttgggttt gggaataggg gcatgaccaa aatggacccc tcctttcctc 91860cccacccctt tccactccat ccgcctttcc ctttgcgttc tccaagcgtt ccgtccccca 91920gatgccctct gtttcctctg ctccagcctt ttaactcctc tcaacaaagg ccaaggaatc 91980aggcagactg tggacactca ggtgtgcatg atgggaaggg acctgcattt acaaaagctc 92040ccccccacag ggactgcatg tgctggtgga atctcagtca ccatgtccca cgttgaagga 92100tgtttggagg gggctgactt tggtcaccct tcaaatcata agttatatgc gtctgccctt 92160tccgcccaca cgtaagtctg aaccttttgc caaaaacact tttcccagac tcgaaagaaa 92220accccaaaga ggaacctaaa ccttcacgct gcctgcaccg attggaccgg agtgctcagg 92280ctcgcgccaa caagtgtttc aaagaagaag ccagacagtg aagggggaag tgagggagaa 92340gctggaaaac tctcaggctg accaatcgtc agcccattca ttcgttcatc catccatcca 92400tccatccatc catccatcca tccatccatc cattctttct ttctttcctc tatccattca 92460gtgagaaggg ctgaatatca cctgtgagcc tgcccctgct cccaagaaca ccctttggac 92520acctgtgggg tgagtagtga ggggcagcaa tggcagagca cccccaccgc ctggagggga 92580gaaagggaag ggctgggaag gctccccaag ggggcggcca tgaagctggg ccttgagaag 92640acaggccagg gtttctcacc ttccacatcc tgcttagagt cagacagaag gcttttgcag 92700aggaggagga acattaaata gtagtaattt ctggcattcc atgagggctt gtctgtgccg 92760ggccctgtgc tgtgcacgtg atacacacta cctcattaaa cctacccaac atgccttgag 92820ggggtgctat tcattttctc cattttacag gtaagaaaat gaaacacaga gaggtgaggc 92880acctctccca acgccacaca gcgaggaagt ggcagagcct ggctgcagct gaggcttata 92940gccgcatttt acattgcttt ctctccgaag agtgccttcc tttatccctg ggagccattg 93000acaaggggtc tgacagtccc tctagtcttg tgcctgctca gccctctcta gccctgaaaa 93060aaccagggct tggcgctgga gaaagagcag gagggtgaga tgtggaaaca tctgttgagt 93120ggcaggggat cacgctggcg cagaggggcc cgagccgatc aggaggccgg cctgtgccag 93180gccagtgctc cctgtgtacc aggtgccaca tgcggggctc agggtagggc cacagttgct 93240cctcccaacc accctttgag gtcagtgtta ctagcccatt ttacagagga ggaaactgag 93300accttaagag gtgaattaac atgtcaggtc acccagctac catgcagttt aagcctagat 93360tgttctgact cctaaactgt gtgcaaggcg gatgattgga ccccagggag gcaaggaaag 93420tcagttttcc tgctgctgaa ttcaatgttt tacaagacca cacacctctt tagacctcag 93480tttcgtcatg tatgaaatga ggaggggaac tctctgcctc cgggctctga tatgctcctg 93540gactgattca ctgttccttg ttcttgtgac ttctcaaagc aagaccagag tcccactccc 93600agccctaggc ccgagattcc catccccact gtgtccaggg gcttcaggag gtgctatttt 93660agggcagatg gcaaaggcct gggctgtaga tccactgagg gctaaaggca atctttcttc 93720cctccacccc tcccttcctt ccttccttct ctttacttcc actaagcaag gtagggaact 93780actcgctgag tctcagggca ggcccacgga ctgaagctca ggaggacagg gctccccagt 93840ggctgcaggt gtcccagcac tgactcctag cagagggggt gtttgggttc agtctggaag 93900attgggtgag attccccaac tacggggggt gggggcacac tctgggtggc agatttgagg 93960aagagtctgc agataaggca tccccaggag atggcaataa gagctggtgt tggggctgcc 94020ccactgaacc cagagcccag gcctgtttcc ccacccatgg aggagtccgg actggctcag 94080tggcaaggcc ggggtcagag gctgccactc tcctcctgcc tctcacagcc cgctggaagg 94140tcaggttttc aggctctgct tatccgtctc ccggcctcct ccctccaggt aaccgaggga 94200gcctccgctt tgatgcggcc acctccaggc ccaggcgtca atgagccctc tatatgacca 94260gtggggctgc tgggggcctc cagcccgcca gagtgggtgc ggtgaggcct ggacacacag 94320tcccgctgtg tggggtcggc tcatgcctgc ctagaccctg tgggcagtgg ggggctccta 94380ggaatgcttt tccagcctgg ggggcacttt ggacaggcag ggtggtctgg ggagacgggt 94440gtgtgcaggg cagcctcaga agccgccatc aaagggacct agcagacgtg gcgccaggca 94500agcgccatag tgggcacgga agggctggcg gtcagtctgt tcctctccca gggatggcgg 94560ggagggggag gccccatgga cacatgtgct cagggtgacc agccatcagg ggttgcctgg 94620gatgaagggg tttcctggga cgtggagctt tcagtgctaa aacagagagt cccctgttat 94680tggaacttcc tggaccttcg gaaaggatac agtgactgac ctctctggtc tgggcagcct 94740cctccctgtc cggtgacctc tgagtcagac catctcggcc agacctgccc agggccattt 94800tgtccacccc ctgcctccac acaggcctgc ctcataccca agagtccact ttccatttct 94860cccaggcatc ttcaggggag gagctgccgg ccaactccaa ccactgctag ggggacctcg 94920gccagaccca caccacgctc ccagccctcc ctgtggctcc cgagccagct catactttcc 94980tgcttccaca cctttgccca ggacgttcct tctgcctgga acatcctttc cctgtctttg 95040ttacctcttt acccttaggg acccagtttc caagtcactc ctccagagga cttgttctct 95100ctttcccaag gctgggctag tacccctctt tgaactcaca gccctggttc ttctcccaaa 95160aaacctttgt cacgccccag ggcaattttc tgttgaccca tctttttcta caccagatgg 95220tgagctctta ggatggagat c 952415451DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 54tcctcttacc tatccctact tccccytccc aaagaagcct tagtagtgtt g 515551DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 55agtgagcaaa ctgaggcaca gagatrttac atcacctgta caagggtaca c 515651DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 56ctttccagag actggcttcc tacagkacag gcggggtcac aggatgtgtt c 515751DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 57tttaatttta gccattcttc tgcctmattt cttaaaatta gagaattaag g 515851DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 58ttttcctgct tccagacatg aatcakgtca ctattcaatg ggatgcagta a 515951DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 59attgggatat ttaacagatc attccraact gggtaggttt ttgcagaatt t 516051DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 60ttatctagaa ataaaaaagc atacawttga taattcacca aattgtggag c 516151DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 61aactctaact ctttatatag gaagtygttc aatgttgtca gttatgactg t 516251DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 62ttagcttctc ctgataaact aattgyctca cattgtcact gcaaatcgac a 516351DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 63acacctaaac ttgggagaac attgtycccc agtgctgggg taggagagtc t 516451DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 64ccagtttctc cctcgctgtt tttatrgctt tcaaaagcag aagtaggagg c 516551DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 65cataagcctc gttatcccat gtgtcraaga agataggttc tgaaatgtgg a 516651DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 66tgcccttccc attttccttc agaagragag attcttctat gacctcattg g 516751DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 67ttaaagaaac tttttcgcga gggacrgttc aactgaaact tcgaaagcat c 516851DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 68ggtttctgga ggacttctag gaaaaygagg gaagagcagg aaaaggcgac a 516951DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 69aacactgata tcaagatact ggattstatt atgagaaatt atcaaaatcc t 517051DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 70gcctggctcc aggccaaaag gaagcmcagg aaagctccca gcaggaacat c 517151DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 71ctcactatgc tcgatctttc ctacawcaac ttaaatgtgg ttggtaacga t 517251DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 72acttgctcat tctcccttac acataytcaa cctaaccaag aataaaatct c 517351DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 73gcatacttag actactacct cgatgrtatt attgacttat ttaattgttt g 517451DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 74tgttctcaaa gtgattttgg gacaaycagc ctaaagtatt tagatctgag c 517551DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 75ttgagggtaa aattcagtaa ggttgracct ctggtgagtt ctgataaaaa t 517651DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 76tttccaatgt ggacactgaa gagacwaatt cttatccttt ttaacataat c 517751DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 77agtgaagttg gcttctgctc aaatgycaga acttctgtag aaagttatga a 517851DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 78ggtttgtctg gtgggttaac catacrgagg tgactattcc ttacctggcc a 517951DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 79aatgaaaaat tagaacaaca gaaacrtggt aagccacttc tatttcttta g 518051DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 80aatcaaatgg cttgaatatc acagaygggg cattcctcaa cctaaaaaac c 518151DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 81gtctggattt atcccttaat aggctsaagc acatcccaaa tgaagcattc c 518251DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 82tgtgtgagtg gccggccccc agctcyacct ccacccactc cacttcatgg g 518351DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 83agctgaggtc cagggcctcc agtcgyggta gctccgtgaa tgagtgctcg t 518451DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 84agcaatagaa ccgatgtctt agcatkttct aaactaagat ttcgttgcat t 518551DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 85agagttttca agtgaggcag ttggakagtt cttttaaaca actcgtctgt t 518651DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 86gagatcagtt agaaaattaa atgcartatt tagttctcgt aaggccatca g 518751DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 87aaatttttta attcacaggt acaccrgaat ggatttcttc ccgcatttag a 51

Claims

1. (canceled)

2. A method for conducting a beneficial colonoscopy and biopsy surveillance regimen on a human diagnosed with inflammatory bowel disease, wherein said method comprises: (a) detecting the presence of hypermethylated RUNX3 nucleic acid by performing a methylation specific polymerase chain reaction, using nucleic acid obtained from a colon sample of said human and treated with bisulfite, and (b) conducting more frequent colonoscopy and biopsy surveillance on said human than surveillance colonoscopy and biopsy every year based at least in part on said presence of said hypermethylated RUNX3 nucleic acid.

3. A method for conducting a beneficial colonoscopy and biopsy surveillance regimen, wherein said method comprises conducting more frequent colonoscopy and biopsy surveillance on a human that was diagnosed with inflammatory bowel disease and was identified as having the presence of hypermethylated RUNX3 nucleic acid by the performance of a methylation specific polymerase chain reaction, using nucleic acid obtained from a colon sample of said human and treated with bisulfite.