Means and Methods for Diagnosing and/or Treating a Subject at Risk of Developing Heart Failure

Abstract

The present invention relates to a method for identifying a subject at risk of developing heart failure, comprising: (a) determining the level of one or more biological markers in a biological sample of the subject; (b) comparing the level of the biological marker to a standard level of the same biological marker; and (c) determining whether the level of the marker is indicative of a risk for developing heart failure, wherein the biological marker is Kruppel-Like Factor 15 (KLF-15) and/or lysosomal integral membrane protein-2 (LIMP-2) and/or fragments and/or variants thereof, and/or wherein the biological marker is a gene coding for KLF 15 and/or LIMP-2, and/or fragments and/or variants thereof. The invention further relates to use of the KLF15 and/or LIMP-2 protein, and/or the gene coding for KLF15 and/or LIMP2, and/or fragments, and/or variants of the genes and/or proteins, for the preparation of a medicament for a prophylactic and/or a therapeutic medicament for prevention and/or treatment of heart failure.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 12/440,226, filed Aug. 25, 2009, now U.S. Pat. No. 8,153,376, issued Apr. 10, 2012, which is a national phase entry of PCT/EP2007/060173, filed Sep. 25, 2007, and published in English as International Patent Publication WO 2008/037720 A2 on Apr. 3, 2008, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 06121196.7, filed Sep. 25, 2006, and European Patent Application Serial No. 06121525.7, filed Sep. 29, 2006, the disclosure of each of the above-referenced priority documents is hereby incorporated herein by this reference in its entirety.

TECHNICAL FIELD

[0002] The present invention, in general, relates to the field of medicine, more specifically, the field of cardiology. The invention, in particular, relates to means and methods for diagnosing and/or treating subjects at risk of developing heart failure.

BACKGROUND

[0003] It is generally known that chronic cardiac loading, as occurs during long-standing hypertension, valvular disease or other chronic disorders like diabetes, induces cardiac hypertrophy, which is one of the most important risk factors for heart failure. Congestive heart failure (HF) is a common but severe and complex clinical syndrome, especially among elderly people, characterized by a diminished cardiac contractile function and decreased exercise tolerance. Symptoms of heart failure include, amongst others, pulmonary and peripheral edema, fatigue and/or dyspnea. Severe heart failure may also lead to reduced function in other organs because these are not receiving enough blood.

[0004] Not all hypertrophied hearts, however, will ultimately fail. Thus, while an important number of patients progress to developing life-threatening complications, others may remain stable for prolonged periods. Until now, the molecular changes that precede and herald this transition from hypertrophy towards heart failure are incompletely understood.

[0005] As early identification of patients at risk for developing hypertensive end organ damage, such as heart failure, may prevent rapid progression, it would be preferable to be able to identify (diagnose) those patients in which heart failure is likely to occur before it actually does so. Early diagnosed patients may thus be treated in order to prevent the onset of heart failure. In addition, it would be preferable to be able to identify those patients suffering from heart failure who are at risk for developing severe complications.

[0006] Current methods can reliably exclude the actual presence of heart failure, but cannot reliably prove the existence of heart failure, nor can these methods predict the outcome of established heart failure, or predict the occurrence of heart failure.

[0007] A need, therefore, exists for simple and reliable methods for predicting the likelihood of onset of heart failure and/or for predicting the outcome of already established heart failure. In addition, the development of means and methods for treating patients who are at risk of developing heart failure, before heart failure and/or its complications occur, would be of great clinical importance.

DISCLOSURE

[0008] The object of the present invention is to provide diagnostic methods by which patients can be identified who are at particular risk of developing heart failure and/or who are at particular risk to develop complications of heart failure. It is a further object of the present invention to provide means and methods for treating patients who are at risk of developing heart failure and/or who are at risk for developing complications of heart failure.

[0009] This objective is achieved by the present invention by providing a method for diagnosing a subject at risk of developing heart failure, comprising the steps of: [0010] (a) determining the level of one or more biological markers in a biological sample of the subject; [0011] (b) comparing the level of the biological marker(s) to a standard level of the same biological marker(s); and [0012] (c) determining whether the level of the biological marker(s) is indicative of a risk for developing heart failure, [0013] wherein the biological marker is lysosomal integral membrane protein-2 (LIMP-2) and/or Kruppel-Like Transcription Factor 15 (KLF15).

[0014] In the research that led to the present invention, a number of genes have been identified that are involved in the development of heart failure. The identified genes have been listed in Table 2. It has furthermore been demonstrated that specific polypeptides encoded by the genes are indeed mechanistically linked to heart failure. It has, in particular, been demonstrated that specific proteins encoded by the genes from Table 2 are involved in the molecular mechanisms that are responsible for the transition from cardiac hypertrophy towards heart failure, and thus can be used as a biological marker for identifying patients at risk of developing heart failure. In addition, these proteins, and/or the genes encoding these proteins, and/or polypeptide and/or polynucleotide fragments or variants of these proteins and/or genes, can be used as a target for treating those patients at risk.

[0015] According to the present invention, it has, in particular, been demonstrated that specific intercalated disc components, in particular, lysosomal integral membrane protein-2 (LIMP-2) and Kruppel-Like Transcription Factor 15 (KLF15) are involved in the molecular mechanisms that predict the transition from cardiac hypertrophy towards heart failure, and can suitably be used as biological markers (biomarkers) for the identification of individuals who are at risk of developing heart failure.

[0016] According to the present invention, it has thus been found that subjects at risk for developing heart failure can be identified by determining the level of one or more of the identified biological markers in a biological sample of the subject and comparing the level of the marker to a standard level. The standard level is derived from healthy subjects, i.e., the standard level is the level of the biological marker in the biological sample of healthy persons, i.e., persons free from cardiac disease. If the level of the biological marker tested is altered, e.g., elevated or reduced (depending on the specific biological marker concerned) compared to the standard level, the subject is at risk for developing HF and/or for developing severe complications of heart failure.

[0017] An early diagnosis of heart failure, preferably before clinical symptoms occur, is essential for, e.g., successfully addressing underlying diseases and/or preventing further myocardial dysfunction and clinical deterioration by, for example, treatment of the diagnosed patients.

[0018] In the research that led to the present invention, the gene expression profile of a large number of genes from hearts that were hypertrophied due to high blood pressure, but appeared functionally well and compensated by traditional techniques (echocardiography) but later proved to develop heart failure, were investigated. This expression profile was compared to the gene expression profile obtained from hearts that that were also hypertrophied due to high blood pressure and appeared equally functionally well and compensated by traditional techniques (echocardiography), but later proved NOT to develop heart failure and remained stable. This way, genes were identified that predicted the occurrence of later developing heart failure, which, according to the present invention, have been shown to be novel and crucial modulators of hypertrophy and the transition toward heart failure. These genes have been listed in Table 2. Subsequently, specific preferred biological markers, in particular, specific intercalated disc-related biological markers were identified. The intercalated disc (ID) forms the connection between cardiac myocytes making up the cardiac fibers in the heart. The intercalated disc thus is a specialized cell-cell junction providing mechanical and electrical coupling between the cells and supporting synchronized contraction of cardiac tissue.

[0019] According to the present invention, it has thus been demonstrated that increased cardiac expression of LIMP-2, as compared to standard levels of expression, identifies those hypertrophied hearts that are prone to progress to overt heart failure. Thus, while cardiac development is normal in LIMP-2 null mice (Gamp et al., 2003), hypertension induced dilated cardiomyopathy in these mice. It was shown that LIMP-2 binds to the vital cardiac adherens junction protein N-cadherin and is essential to secure proper interactions between N-cadherin and β-catenin. It has further been found that expression of LIMP-2 is increased in hypertrophied rat hearts that are on the brink of progressing to heart failure, thus suggesting that increased LIMP-2 expression by cardiac myocytes heralds their inability to normalize mechanical forces. As such, increased LIMP-2 expression may be seen as a desperate attempt of the myocyte to respond to worsening loading and be indicative of imminent failure. It has moreover been shown that LIMP-2 expression is significantly increased in patients with clinically severe pressure loading. By determining the level of LIMP-2 protein and/or the level of expression of the gene coding for LIMP-2 in hypertensive subjects, and comparing these level(s) with a standard level, and subsequently determining whether the level is indicative of a risk for developing heart failure, it thus is possible to identify in a very early stage the myocardium that is about to succumb to the pressure. In particular, an increased level of LIMP-2 protein and/or an increased level of LIMP-2 gene expression as compared to a standard level is indicative of a risk for developing heart failure and/or heart failure-related complications.

[0020] In the research that led to the present invention, it has further been shown that the gene coding for Kruppel-Like Factor 15 (KLF15) characterized hypertrophied hearts that quickly progressed to heart failure. This was confirmed by real-time PCR, which showed that KLF15 was down-regulated in compensated LVH, but that KLF-15 was significantly further suppressed in the hypertrophied hearts that quickly progressed to failure. It was further shown that KLF15 has a role in cardiac myocytes as a suppressor of cardiac hypertrophy. Determining the level of the KLF15 protein and/or the level of expression of the gene coding for KLF15 in hypertensive subjects, and comparing these levels to standard levels, thus also is useful for identifying in a very early stage those patients that are likely to develop heart failure. In the case of KLF15, a decreased level of KLF15 protein and/or decreased KLF15 gene expression in a biological sample, as compared to standard levels, is indicative for the development of heart failure.

[0021] The present invention relates both to in vivo methods, i.e., methods wherein the level of the biological marker is determined in a biological sample in vivo and to in vitro methods.

[0022] In a preferred embodiment of the invention, the level of the biological marker is determined in vitro in a biological sample obtained from an individual. For in vitro determining the level of the biological markers of the present invention, any suitable biological sample of any bodily fluid that may comprise a biological marker identified by this research may be used. Preferably, the biological sample is selected from the group consisting of blood, plasma, serum, or cardiac tissue. More preferably, the biological sample is a peripheral blood sample, or a plasma or serum sample derived from peripheral blood. Peripheral blood samples can, e.g., easily be taken from the patients and do not need complex invasive procedures such as catheterization. The biological sample may be processed according to well-known techniques to prepare the sample for testing.

[0023] For measuring the level of the biological markers of the invention, use can be made of conventional methods known in the art.

[0024] When the biological marker is a protein and/or a fragment and/or a variant thereof, several conventional methods for determining the level of a specific protein, and/or fragments and/or variants thereof, which are well-known to the skilled person, may be used. The level of the marker may, for example, be measured by using immunological assays, such as enzyme-linked immunosorbent assays (ELISA), thus providing a simple, reproducible and reliable method. Antibodies for use in such assays are available, and additional (polyclonal and monoclonal) antibodies may be developed using well-known standard techniques for developing antibodies. Other methods for measuring the level of the biological protein markers may furthermore include (immuno)histochemistry, Western blotting, flow-cytometry, RIA, competition assays, etc., and any combinations thereof. In vivo, the level of, for example, non-secreted proteins can be determined by labeling and tagging specific antibodies against one of the proteins of interest. This allows visualization of the amount of protein in the heart by so called "molecular imaging" techniques.

[0025] When the biological marker is a gene, and/or a polynucleotide fragment and/or variant thereof, e.g., DNA, cDNA, RNA, mRNA, etc., such as a gene coding for a specific protein, or mRNA that is transcribed, the biological marker can be measured in, e.g., cardiac biopsies, by, e.g., well-known molecular biological assays, such as in situ hybridization techniques using probes directed to the specific polynucleotides. Other nucleic acid-based assays that may be used according to the invention include RT-PCR, nucleic acid-based ELISA, Northern blotting, etc, and any combinations thereof.

[0026] In order to enhance the specificity and/or sensitivity of the diagnostic method, the method of the invention may include the detection of the level of one or more other (biological) markers, i.e., the detection of the biological markers of the present invention may suitably be combined with the detection of other markers that are indicative for the development of heart failure.

[0027] The present invention further relates to kits for performing the diagnostic methods as described above. The invention, in particular, relates to such diagnostic kits for identifying a subject at risk of developing heart failure, comprising means for receiving one or more biological samples of the subject, and means for determining the level of the biological marker(s) in the biological sample of the subject. Thus, a kit is provided that can be used as a reliable and easy diagnostic tool. The means for receiving the biological sample may, for example, comprise a well of a standard microtiter plate. The means for determining the level of an intercalated disc-related biological marker in the biological sample of the subject may, for example, comprise one or more specific antibodies, polynucleotide probes, primers, etc., suitable for detecting the biological marker(s), identified according to the present invention. The kits may further also comprise calibration means and instruction for use.

[0028] The invention also relates to the use of the biological markers of the present invention and/or fragments and/or functional variants thereof in a screening method for identifying compounds for the prevention and/or treatment of heart failure. In a particular embodiment, the method for identifying a compound for prevention and/or treatment of heart failure comprises: [0029] (a) contacting one or more compounds with a polypeptide encoded by a polynucleotide listed in Table 2, preferably KLF15 and/or LIMP-2, and/or fragments, and/or variants thereof; [0030] (b) determining the binding affinity of the compound to the polypeptide; [0031] (c) contacting a population of mammalian cells with the compound that exhibits a binding affinity of at least 10 micromolar; and [0032] (d) identifying the compound that is capable of prevention and/or treatment of heart failure.

[0033] The polypeptides to be tested in the screening method of the present invention may be tested in vitro, e.g., free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly, or in vivo.

[0034] To perform the methods, it is feasible to immobilize either the polypeptide of the present invention or the compound to facilitate separation of complexes from uncomplexed forms of the polypeptide, as well as to accommodate automation of the assay. Interaction (e.g., binding) of the polypeptide of the present invention with a compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

[0035] The binding affinity of the compound with the polypeptide can, e.g., be measured by methods known in the art, such as using surface plasma resonance biosensors (Biacore), by saturation binding analysis with a labeled compound (e.g., Scatchard and Lindmo analysis), via displacement reactions, by differential UV spectrophotometer, fluorescence polarization assay, Fluorometric Imaging Plate Reader (FLIPR®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer. The binding affinity of compounds can also be expressed in a dissociation constant (Kd) or as IC50 or EC50. The IC50 represents the concentration of a compound that is required for 50% inhibition of binding of another ligand to the polypeptide. The EC50 represents the concentration required for obtaining 50% of the maximum effect in vitro. The dissociation constant, Kd, is a measure of how well a ligand binds to the polypeptide; it is equivalent to the ligand concentration required to saturate exactly half of the binding sites on the polypeptide. Compounds with a high affinity binding have low Kd, IC50 and EC50 values, i.e., in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e., in the micromolar range.

[0036] The present invention also relates to the use of the genes and/or proteins listed in Table 2, preferably of the KLF15 and/or LIMP-2 gene and/or protein, for the preparation of a medicament for a prophylactic and/or therapeutic medicament for the prevention and/or treatment of heart failure.

[0037] Preferably, the present invention relates to the use of a modulator of the genes and/or proteins listed in Table 2, preferably of the KLF15 and/or LIMP-2 gene and/or protein, for the preparation of a prophylactic and/or therapeutic medicament for the prevention and/or treatment of heart failure.

[0038] In the present application, a modulator may be any compound that stimulates the expression of and/or increases the level of one or more of the biological markers that are found to be reduced according to the invention (e.g., an agonist), or any compound that suppresses the expression and/or reduces the level of one or more of the biological markers that are found to be increased according to the invention (e.g., an antagonist).

[0039] The medicament may be a protein-based molecule, such as, for example, an antibody directed against the protein marker, and/or fragments and/or variants thereof. The present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments and the products of a Fab expression library and Fv fragments and the products of an Fv expression library.

[0040] Alternatively, the medicament may be a nucleic acid-based molecule. The down-regulation of a gene can, for example, be achieved at the translational or transcriptional level using, e.g., antisense nucleic acids. Antisense nucleic acids are nucleic acids capable of specifically hybridizing with all or a part of a nucleic acid encoding a protein and/or the corresponding mRNA. The preparation of antisense nucleic acids, DNA encoding antisense RNAs, is known in the art. The medicament may also comprise small interfering (hairpin) RNA (siRNA). SiRNAs mediate the post-translational process of gene silencing by double-stranded DNA (dsDNA) that is homologous in sequence to the silenced RNA. The preparation of siRNAs is known in the art. Similarly, the up-regulation of a gene (or over-expression) may be achieved by several methods that are known in the art.

[0041] In a preferred embodiment of the present invention, the modulator is an inhibitor of TGFβ. According to the present invention, it has been shown that suppression of KLF-15 is a crucial step in the development of failure-prone forms of hypertrophy and that TGFβ strongly suppresses KLF-15. Inhibitors of TGFβ, which are currently being developed in different fields, thus may suitably be used for the development of a prophylactic and/or therapeutic medicament for the prevention and/or treatment of heart failure. Examples of suitable inhibitors of TGFβ that can be used according to the invention are TGFβ receptor inhibitors as made by Scios Inc., Los Angeles, Calif., U.S.A., who indicate on their website (world-wide web at sciosinc.com/scios/tgf): "Scios has developed novel and potent small molecule inhibitors designed to inhibit the action of TGF-beta at its receptor. These small molecules have been shown to be effective in reducing scar formation (fibrosis) when given orally to animals. Scios expects to advance two lead molecules, representing different chemical classes, into preclinical development that could potentially be used to treat disease conditions in patients with significant unmet medical needs."

[0042] The present invention further relates to the use of the proteins identified according to the invention for generating diagnostic means for use in (molecular) imaging of one or more of the identified proteins to assess the level of the protein and thus identify a subject at risk of developing heart failure. The diagnostic means may, for example, comprise labeled antibodies directed against the biological protein markers.

[0043] The present invention is further illustrated by the following figures and Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1: Increased expression of LIMP-2 in Ren-2 rats. FIG. 1A, A left ventricular cardiac biopsy was taken at age 10 weeks, when Ren-2 rats exhibit comparable cardiac hypertrophy and fractional shortening cannot distinguish rats that will progress to heart failure or stay compensated. Between 15 and 18 weeks of age, part of the Ren-2 rats developed heart failure and the remainder stayed compensated until sacrifice at 21 weeks of age. *, P<5e^-6. FIG. 1B, LIMP-2 mRNA was found by microarray analysis in 10-week-old hypertrophic Ren-2 rats to be specifically over-expressed in failure-prone rats (HF-prone LVH, n=4), as compared to the hypertrophied LVs that remained compensated (comp LVH, n=6) and to controls (n=4). FIG. 1C, LIMP-2 protein was up-regulated in end-stage failing Ren-2 rats (HF, n=9), as compared to compensated Ren-2 rats (comp, n=6). Both failing and compensated Ren-2 rats had significantly elevated levels of LIMP-2 protein as compared to control rats (n=6). *, P<0.05 versus control; **, P<0.01 versus control; $, P<0.05 versus comp; Mwm, molecular weight marker; au, arbitrary units.

[0045] FIG. 2: AngII-treated LIMP-2 KO (KO Ang) mice have dilated cardiomyopathy. FIG. 2A, WT Ang mice (n=14) significantly increased their LV weight, while KO Ang (n=14) mice did not (*, P<0.01 versus WT (n=8) and KO Ang). In KO Ang mice, individual myocytes failed to increase their volume (WT and KO, n=4; WT Ang and KO Ang, n=5; myocyte area (au): 264±42, versus 308±14 in WT Ang; *, P<0.01). Bars represent 50 μm. FIG. 2B, LIMP-2 KO (n=3) and WT (n=4) mice showed comparable blood pressure responses to AngII. FIG. 2C, AngII-treated WTs (n=8) and KOs (n=8) showed comparable increases in BNP and ANF mRNA expression (*, P<0.05 versus baseline (n=4)), while aska mRNA expression was induced to a significantly lesser extent in KO Ang mice, consistent with their reduced myocyte volume (*, P<0.05 versus KO (n=4); ^$, P<0.05 versus WT Ang). FIG. 2D, Baseline echocardiographic parameters were similar for WT (n=10) and KO (n=11) mice (day 0). After 14 and 28 days of AngII, wild-type LV walls were significantly hypertrophied, while knockouts did not show hypertrophy and were even dilated (*, P<0.005 versus baseline and versus KO Ang; $, P<0.005 versus baseline and vs WT Ang). FIG. 2E, Beta-adrenergic response to dobutamin was decreased in KO Ang mice (WT and KO, n=4; WT Ang, n=14; KO Ang, n=9; *, P<0.005). LVW/BW, LV weight corrected for body weight.

[0046] FIG. 3: AngII-treated LIMP-2 KO mice have massive interstitial fibrosis. Sirius red staining of LVs of AngII-treated LIMP-2 knockout (n=4) and wild-type (n=5) mice shows marked interstitial fibrosis in knockout mice (*, P<0.02 versus WT Ang and KO baseline), while both knockout and wild-type mice treated with AngII show similar degree of perivascular fibrosis. Bars represent 250 μm.

[0047] FIG. 4: AngII-treated LIMP-2 KO mice show myocyte disarray. Desmin-stained cardiac myocytes of AngII-treated LIMP-2 KO mice show disarray and have a disturbed internal structure, as shown by the higher and more capricious desmin-expression in these mice. Bars represent 250 μm.

[0048] FIG. 5: LIMP-2 expression is up-regulated in other forms of cardiac stress. FIG. 5A, In neonatal rat cardiac myocytes, 6 hours stretch elevated LIMP-2 mRNA expression (n=4 per group). LIMP-2 mRNA was also up-regulated in hypertrophic myocardium (FIG. 5B) from rats that had undergone exercise training for 10 weeks (5 days per week, n=6) as compared to non-hypertrophic control rats (n=7) and from patients suffering from aortic stenosis (LVH, n=20) as compared to non-hypertrophic control patients (n=7). *, P<0.05 versus control; **, P<0.01 versus control; LVH; LV hypertrophy.

[0049] FIG. 6: LIMP-2 is present at the plasma membrane of cardiac myocytes and is important for intercalated disc function. FIG. 6A, Paraffin-embedded tissue section of pressure-overloaded mouse LV immunostained with anti-LIMP-2 shows positive staining not only in intracellular compartments (*) but also on plasma membranes of cardiac myocytes (). Scale bar represents 250 μm. FIG. 6B, Immuno-electron microscopy with anti-LIMP-2 in pressure-overloaded rat LV tissue sections also shows the presence of LIMP-2 at the plasma membrane (). Scale bar represents 1 μm. FIG. 6C, Electron microscopy shows normal intercalated discs in AngII-treated wild-type mice, while in AngII-treated LIMP-2 KO mice, the intercalated discs have a higher degree of convolution and a higher concentration of adherens junctions (appreciate the dark black spots in the right panel), which is paralleled by the dilated cardiomyopathy in these mice. Bars represent 2 μm. M, mitochondrion; ID, intercalated disc; a, adherens junction; d, desmosome.

[0050] FIG. 7: LIMP-2 regulates cadherin distribution. FIG. 7A, LIMP-2 binds to cadherin in neonatal rat ventricular myocytes. LIMP-2 was immunoprecipitated (IP), and cadherin was immunoblotted (IB) in the total cell lysate (input), the supernatant (sup) and the precipitated protein lysate (IP). Part of the cadherin protein content of cardiac myocytes is bound by LIMP-2. FIG. 7B, Tissue sections of control subject and two heart failure patients were immunofluorescently stained with anti-pan-cadherin (red) and anti-LIMP-2 (green). Arrows () show co-localization of LIMP-2 and cadherin at the ID of cardiac myocytes. Bars represent 50 μm. FIG. 7C, Tissue sections of AngII-treated LIMP-2 knockout and wild-type LVs were immunostained with anti-pan-cadherin. In wild-type mice, the cadherin distribution is confined to the intercalated discs yielding a regular appearance of cadherin, while in LIMP-2 KO mice, the localization of cadherin has lost the typical pattern produced by a strict location at the intercalated disc. Bars represent 250 μm.

[0051] FIG. 8: LIMP-2 regulates intercalated disc integrity by regulating the binding of phosphorylated beta-catenin to cadherin. FIG. 8A, Immunoblot (IB) of lysates of neonatal rat cardiac myocytes that were treated either with shRNA against LIMP-2 (shLIMP-2) or with control shRNA. After 10 days of culture, cardiac myocytes show a 92% knockdown of LIMP-2 protein. Equal protein loading was confirmed by GAPDH. FIG. 8B, Immunoblot (IB) shows diminished levels of P-beta-catenin after immunoprecipitation (IP) with anti-pan-cadherin in shLIMP-2 lysates as compared to control lysates. Cadherin loading was comparable in control and shLIMP-2 IP-lysates. Phosphorylation of beta-catenin in total shLIMP-2 and control protein lysates was comparable. *, P=0.0006. FIG. 8C, Immunoblot showing the specificity of the immunoprecipitation with anti-pan-cadherin. When adding IgG instead of pan-cadherin antibody to the protein lysates, no P-beta-catenin is bound.

[0052] FIG. 9: FIG. 9A, KLF15 expression assessed by real-time CR in left ventricular biopsies from Ren-2 rats at the age of 10 weeks. After biopsy, rats were allowed to recover and followed to determine whether they would progress to failure or remain compensated. Expression of KLF15 is significantly down-regulated in hypertrophied hearts that remained compensated, but significantly further suppressed in the hypertrophied hearts that quickly progressed to overt failure, indicating that the level of KLF15 suppression identifies failure prone forms of cardiac hypertrophy. FIG. 9B, In situ hybridization for KLF15 in a normotensive control heart compared to hypertrophic myocardium. The widespread nuclear staining in the normal heart is lost in a large number of myocytes, while there is residual staining in non-myocyte nuclei, indicating that KLF15 expression occurs specifically in cardiac myocytes. FIG. 9C, Stable knock-down of KLF15 by lentiviral introduction of short hairpin RNA, induced expression of BNP in cultured NRVM. FIG. 9D is a graph depicting relative MEF2 luciferase activity.

[0053] FIG. 10: FIG. 10A, Addition of TGFβ (10 ng/ml) to cultured cardiac myocytes almost completely suppressed KLF15 mRNA expression. Stable knock-down of the TGFβ type I receptor by lentiviral introduction of short hairpin RNA abolished this effect, demonstrating that TGFβ via its TGFβ type I receptor is capable of suppressing KLF15 expression. FIG. 10B, Whole heart homogenate immunoblotted against the Tgfβ type I receptor shows a substantial and significant reduction in expression of Tgfβ type I receptor, but no compete loss of the receptor. FIG. 10C, Immunohistochemistry demonstrates that the myocyte specific activation of cre resulted in a clear and robust loss of TGFβ type I receptors from cardiac mycocytes when comparing WT hearts to the MerCreMer-TGFβ type I mice. FIG. 10D, Angiotensin II infusion induced a significant hypertrophic response in Wt mice, which was blunted in MerCreMer-TGFβ type I mice. FIG. 10E, Angiotensin II infusion induced a significant loss in fractional shortening as an indicator of loss of cardiac function, which was blunted in MerCreMer-TGFβ receptor mice. FIG. 10F, Angiotensin II infusion and subsequent hypertrophy induced a down-regulation of KLF15, which was blunted in the MerCreMer-TGFβ receptor mice.

[0054] FIG. 11: The upper panel shows significantly up-regulated KLF15 mRNA in the mouse heart after AAV9-KLF15 injection, compared to green fluorescent protein (AAV9-GFP) injection. **: p<0.05 comp to GFP group. *: p<0.05 compared to GFP group. #: p<0.05 compared to GFP+AngII group. The lower panel shows significantly less hypertrophy in the AAv9-KLF15 group after AngII stimulation compared to the AAV9-GFP group with AngII (#: p<0.05). Statistical analysis with student's t-test, n=3-5 animals/group.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLES

Example 1

Lysosomal Integral Membrane Protein-2 is a Novel Component of Intercalated Discs and Prevents Cardiomyopathy

Materials and Methods

Ren-2 Rats, Microarray Analysis and Immunoblotting

[0055] From 10-week-old Ren-2 and Sprague-Dawley (SD) rats (Mollegard, Lille Skensveld, Denmark), a biopsy of the LV was taken as described previously (Van Haaften et al., 2006). Rats were followed by serial echocardiography at 10, 12, 15, 16, 18, 19 and 21 weeks of age and sacrificed at 15-18 weeks upon clinical signs of heart failure (heart failure-prone/HF-prone rats) or at 21 weeks when clinical signs of failure had not appeared (compensated/comp rats). Total RNA was isolated from LV biopsies and amplified as previously described (Schroen et al., 2004; Heymans et al., 2005), hybridized to Affymetrix rat 230 2.0 GeneChips and analyzed with Microarray Analysis Suite Software 5.0. LV protein extracts (50 μg) were immunoblotted with polyclonal rabbit anti-LIMP-2 (Novus Biologicals, Littleton, Colo., 1:500) and polyclonal rabbit anti-GAPDH (Abcam, Leusden, Netherlands; 1:10,000).

LIMP-2 Knockout Mice, RNA Isolation and Quantitative PCR Analysis

[0056] Ten- to twelve-week-old male LIMP-2 KO and WT C57/B16 mice weighing 20-25 grams were used. To study blood pressure effects of AngII, arterial pressures were monitored during intravenous infusions at doses of 0.5, 1.5, 5, 15, and 50 ng per minute. To study development of LV hypertrophy, AngII (1.5 μg/g/day) was infused subcutaneously by osmotic minipump 2004 (Alzet osmotic pumps, Cupertino, Calif.) for 28 days.

[0057] Echocardiography was performed at day 0, day 14 and day 28. At day 28, mice were hemodynamically monitored (dP/dt) using Millar® under basal and dobutamin-stimulated conditions; afterward, LVs were removed. RNA was isolated with RNeasy mini kit (Qiagen, Valencia, Calif.) and SYBR Green quantitative PCR analysis was performed on a BioRad iCycler to determine BNP, ANF and alpha-skeletal actin (aska) expression (Table 1). LV sections were stained with hematoxylin-eosin (HE) and Picro serious red (SR) as described before (Junqueira et al., 1979), or were immunohistochemically stained with monoclonal mouse anti-pan-cadherin (Sigma, Saint Louis, USA; 1:500) and monoclonal mouse anti-human desmin (Dako Cytomation, Denmark, 1:50). Ultrastructural analysis was performed by transmission electron microscopy as described previously (Schroen et al., 2004).

LIMP-2 in Aortic Stenosis and Heart Failure Patients

[0058] RNA was isolated from transmural biopsies obtained from 20 aortic stenosis patients and seven non-hypertrophic control patients as described before (Heymans et al., 2005), and SYBR Green quantitative PCR analysis was performed to determine LIMP-2 expression (Table 1).

[0059] Double immunofluorescent stainings with rabbit anti-LIMP-2 (1:250, Cy2) and mouse anti-pan-cadherin (1:500, Cy3) were done on sections of one control subject and two patients that died of overt heart failure, as defined by an ejection fraction of less than 35%. Nuclear counterparts were stained with Topro-3 (Invitrogen, Breda, The Netherlands). Sections were imaged with a laser scanning confocal system (Leica, Rijswijk, The Netherlands), digitized at a final magnification of ×126 and analyzed with Leica Confocal Software. The ethics committees of the Academic Hospital Maastricht and of University Hospital Leuven approved the study, and all patients gave informed consent.

Cell Culture and Lentiviral Vector

[0060] A rat-LIMP-2 shRNA expressing lentiviral vector was generated by annealing complementary shLIMP-2 oligonucleotides (Table 1) and ligating them into HpaI XhoI digested pLL3.7 puro vector DNA (modified from a kind donation by Luk van Parijs, Massachusetts Institute of Technology, Cambridge, USA). Lentiviral production was performed by co-transfection of 3 μg shLIMP-2/pLL3.7 puro or empty pLL3.7puro and packaging vectors into 293FT cells by Lipofectamine 2000 (Invitrogen) and virus-containing supernatant was harvested after 48 hours.

[0061] Rat ventricular cardiac myocytes (RCMs) were isolated by enzymatic disassociation of 1- to 2-day-old neonatal rats as described previously (De Windt et al., 1997). For lentiviral infection, RCMs were plated on gelatinized six-well plates with 5*10⁵ cells per well, cultured overnight in DMEM/M199 (4:1) media supplemented with 10% horse serum, 5% newborn calf serum, glucose, gentamycin and AraC, and next day infected with shLIMP-2 or empty lentivirus, facilitated by Polybrene (Sigma). After puromycin selection (3 μg/ml), infection efficiencies were above 80%. After ten culture days, cellular protein was isolated for immunoprecipitation (IP) with anti-LIMP-2 (1:100), monoclonal mouse anti-pan-cadherin (Sigma, 1:100) or IgG. IP lysates were immunoblotted with monoclonal anti-pan-cadherin (1:5000), polyclonal anti-phospho-beta-catenin (Ser33/37/Thr41; Cell Signaling Technology, Danvers, Mass., USA, 1:1000) and monoclonal anti-beta-catenin (BD Transduction Laboratories, Franklin Lakes, USA, 1:1000).

[0062] For stretch experiments, RCMs were cultured on a collagen type I-coated silastic membrane (Specialty Manufacturing, Inc., USA) and subjected to static, equibiaxial stretch during a six-hour period. RNA was isolated with RNeasy mini kit (Qiagen) for LIMP-2 SYBR Green quantitative RT-PCR (Table 1).

[0063] All study protocols involving animal experiments were approved by the Animal Care and Use Committee of the Universiteit Maastricht, and were performed according to the official rules formulated in the Dutch law on care and use of experimental animals, highly similar to those of the NIH.

Statistical Analyses

[0064] Data are presented as average±SEM. The data for each study group were compared using Mann Whitney or student's t-test where appropriate. P<0.05 was considered to be statistically significant.

Results

[0065] In Table 2, a list is presented of genes differentially expressed in failure-prone as compared to compensated Ren-2 rats. The differential expression of these genes precedes the development of heart failure in Ren-2 rats, because it is derived from cardiac biopsies taken at 10 weeks of age, when all rats still have compensatory hypertrophy.

[0066] In Table 3, elaborate echocardiographic data are presented of LIMP-2 WT and KO mice at baseline, and after 14 and 28 days of AngII treatment.

Gene Expression Profile of Failure-Prone LV Hypertrophy

[0067] Cardiac biopsies in ten homozygous Ren-2 rats were obtained at a stage of compensated LV hypertrophy at 10 weeks of age. Four rats rapidly progressed toward heart failure within five weeks after the biopsy was taken, while the remaining six rats remained compensated for 11 weeks after biopsy (FIG. 1A). After linear T7 based amplification and subsequent Affymetrix 230 2.0 gene expression analysis in these biopsies (GEO number GSE4286), 143 differentially expressed genes that were up- or down-regulated only in the hypertrophied hearts that progressed towards heart failure were identified (Table 2). LIMP-2, a lysosomal membrane protein, was one of the up-regulated mRNAs in heart failure-prone rats (FIG. 1B), and of particular interest given its ability to interact with thrombospondins (TSP) 1 (Crombie et al., 1998) and 2 (data not shown), the latter has been shown earlier to be crucial in the transition from hypertrophy towards heart failure. FIG. 1C shows that LIMP-2 protein also has a role in end-stage heart failure in Ren-2 rats.

Angiotensin II Induces Dilated Cardiomyopathy in LIMP-2 Knockout Mice

[0068] Since loss-of-function mutations in lysosomal proteins have been linked to heart failure (Eskelinen et al., 2003; Nishino et al., 2000; Stypmann et al., 2002), the role of LIMP-2 in a mouse model of angiotensin II- (AngII-) induced hypertension was further investigated. AngII was given subcutaneously for four weeks to LIMP-2 knockout and control mice. AngII treatment resulted in a 30% increase in LV mass index in wild-type mice, but the hypertrophic response was attenuated in the AngII-treated LIMP-2 knockout mice (14% increase in LV mass index; P<0.01) (FIG. 2A). This was confirmed by measurement of individual cardiac myocyte area. LV myocyte area was significantly smaller in AngII-treated knockout mice than in AngII-treated WT controls (myocyte area in arbitrary units: 264±42 in AngII-treated knockout mice, versus 308±14 in AngII-treated wild-types; P<0.01). In addition, while AngII induced comparable increases in perivascular fibrosis in LIMP-2 knockout and wild-type mice (data not shown), AngII induced a massive interstitial fibrotic response in the LV of LIMP-2 knockout mice as opposed to wild-type control littermates (interstitial fibrosis: 15.0±6.0% in AngII-treated knockout mice versus 1.8±0.1% in AngII-treated controls; P<0.002) (FIG. 3).

[0069] Immunohistochemical staining for desmin showed myocyte disarray in AngII-treated LIMP-2 null mice (FIG. 4).

[0070] It was confirmed that AngII induced a similar blood pressure response in both wild-type and knockout mice (FIG. 2B). Despite decreased LV hypertrophy, LIMP-2 null mice demonstrated a normal response of the classical markers for hypertrophy Brain Natriuretic Peptide (BNP) and Atrial Natriuretic Factor (ANF) (FIG. 2C), suggesting that the hypertrophic gene expression program was normally initiated upon AngII treatment. In contrast, the structural cellular hypertrophy marker alpha-skeletal actin was induced to a significantly lesser extent in AngII-treated LIMP-2 knockouts as compared to AngII-treated wild-types (FIG. 2C), reflecting the reduced hypertrophic response of cardiac myocytes (Stilli et al., 2006). Serial echocardiography revealed that AngII induced significant cardiac dilatation in AngII-treated LIMP-2 null mice, whereas AngII-treated wild-types showed concentric LV hypertrophy without dilatation (FIG. 2D and Table 3). In addition, AngII induced loss of contractile reserve in LIMP-2 null mice as demonstrated by a reduced contractile response to dobutamine infusion (+dP/dt 79.8/second±5.1 in AngII-treated knockout mice versus 100.0/second±4.0 in AngII-treated controls; P<0.005) (FIG. 2E).

[0071] Taken together, in LIMP-2 null mice hypertension did not induce the normal hypertrophic response but rather dilated cardiomyopathy with reactive interstitial fibrosis and loss of cardiac function.

LIMP-2 Expression is Regulated by Cardiac Stress

[0072] The finding that AngII-treated LIMP-2 null mice failed to mount a hypertrophic response, yet normally induced expression of BNP and ANF suggested that LIMP-2 is a crucial part of the normal response to mechanical loading. Indeed, it was also shown that LIMP-2 expression increased significantly after cardiac myocyte stretch in vitro (P=0.02) and also increased in exercise-induced physiological hypertrophy (P=0.04) (FIGS. 5A and 5B). To ascertain that LIMP-2 is also involved in the human adaptation to cardiac pressure loading, the expression of LIMP-2 was analyzed by quantitative RT-PCR in cardiac biopsies of twenty aortic stenosis patients with overt cardiac hypertrophy and seven controls. This experiment showed a significant LIMP-2 up-regulation in the hypertrophic hearts of aortic stenosis patients as compared to controls by Mann-Whitney test (1.23-fold; P=2.3e^-4).

LIMP-2 is Present at the Cardiac Intercalated Disc

[0073] Next, the expression pattern of LIMP-2 in pressure-overloaded murine myocardium was analyzed by immunohistochemistry. The protein is expressed, as expected, in intracellular vacuole-shaped compartments of cardiac myocytes and endothelial cells, but was also found to be atypically distributed on the plasma membrane of cardiac myocytes (FIG. 6A). Immuno-electron microscopy confirmed this finding (FIG. 6B). Strikingly, electron-microscopy of AngII-treated LIMP-2 knockout and control left ventricular sections revealed abnormal morphology of the ID in LIMP-2 null mice, suggesting that LIMP-2 may be involved in normal ID biology. At cell-cell contacts, the membrane at the AngII-treated KO-ID showed a higher degree of convolution with a higher concentration of adherens junction proteins (FIG. 6C), indicative of disturbed ID architecture (Perriard et al., 2003). Since alterations in the ID have been shown to cause dilated cardiomyopathy (Perriard et al., 2003), it was surmised that LIMP-2 may be crucial for proper functioning of the ID.

[0074] Immunoprecipitation of neonatal rat cardiac myocytes protein showed that LIMP-2 physically interacts with N-cadherin, a vital constituent of adherens junctions (FIG. 7A). This finding was translated to the human situation as confocal microscopy of control as well as failing human myocardium confirmed the interaction between cadherin and LIMP-2 and showed that this interaction takes place at the site of the ID, where cadherin and LIMP-2 co-localize (FIG. 7B). This suggested that LIMP-2 may be important for proper ID function by mediating the role of cadherin. Indeed, histochemical analysis of cadherin in hearts of LIMP-2 null mice showed aberrant cadherin distribution (FIG. 7C), but normal distribution in AngII-treated wild-types. AngII-treated wild-type mice show cadherin expression at the contact sites between two longitudinal cardiac myocytes, while this expression is less organized and more diffuse in cardiac myocytes of AngII-treated LIMP-2 knockout mice. These data establish that LIMP-2 is crucial for the proper structural organization of the intercalated disc.

LIMP-2 Regulates Intercalated Disc Integrity

[0075] To identify which regulatory mechanism depends on LIMP-2, lentivirally introduced short-hairpin RNA against LIMP-2 (shLIMP-2) was used to obtain a separate model of LIMP-2 inactivation in neonatal rat cardiac myocytes. After ten days of culture, LIMP-2 protein expression was diminished by 92% in shLIMP-2-treated cardiac myocytes as compared to control-treated cardiac myocytes (FIG. 8A). It has been reported that the functional integrity of the intercalated disc depends on the proper interaction between P(Ser37)-β-catenin and cadherin. Therefore, it was investigated whether the absence of LIMP-2 affected the binding of P-β-catenin to cadherin.

[0076] Immunoprecipitation of cadherin in lysates of cardiac myocytes showed that knock-down of LIMP-2 indeed diminished the interaction between P-β-catenin and cadherin (FIG. 8B). Immunoprecipitation was specific for cadherin (FIG. 8C).

[0077] It was demonstrated in this study that the lysosomal protein LIMP-2 is an important and novel component of the cardiac myocyte intercalated disc, in particular, adherens junctions. According to the present invention, it has been shown that LIMP-2 binds to N-cadherin, and that LIMP-2 null mice develop dilated cardiomyopathy upon AngII-induced hypertension, accompanied by disturbed localization of N-cadherin in the heart. Confirming this in vitro, it was shown that knock-down of LIMP-2 in cultured myocytes disturbs interactions between N-cadherin and β-catenin. This suggests that LIMP-2, which was initially known as a lysosomal protein, is an important part of the intercalated disc.

LIMP-2 has a Role in the Heart During Pressure Overload

[0078] LIMP-2 stands out among ID proteins. Complete loss of other major constituents of the ID (cadherin, β-catenin, plakoglobin) results in lethal developmental cardiac derangements, suggesting that these components of the ID are essential for normal cardiac development. In contrast, according to the invention, it was found that LIMP-2 null mice have normal cardiac development, but that its loss only affects postnatal cardiac remodeling. This suggests that LIMP-2 represents a different type of ID protein, whose role is essential mainly under increased loading conditions. This specific role for LIMP-2 is underlined by the finding that expression of LIMP-2 further rises in hypertrophied rat hearts that are on the brink to progress to failure, which suggests that LIMP-2 expression particularly increases in cardiac myocytes that seem unable to normalize loading conditions. Taken together, it was suggested that LIMP-2 is a novel mediator of ID function, and represents a hitherto unidentified class of mediators that are essential for the ID and the myocyte to respond to increased loading conditions.

LIMP-2 Null Mice Respond Abnormally to Increased Loading

[0079] LIMP-2 increased particularly in those hypertrophied hearts that would later progress to failure, in comparison to the hypertrophied hearts that remained compensated. This indicates that cardiac LIMP-2 expression may be an early molecular sign of excessive loading. That LIMP-2 constitutes a defensive mechanism against excessive loading is suggested by the finding that when LIMP-2 null mice were subjected to pressure loading by chronic angiotensin II infusion, they developed cardiac dilatation and fibrosis, yet very little cardiac myocyte hypertrophy. Natriuretic peptides were normally induced, which suggests that the cardiac myocytes of LIMP-2 null mice do sense increased loading conditions, yet fail to mount an adequate hypertrophic response, as evidenced by the attenuated expression of alpha-skeletal actin. This suggests that LIMP-2 is essential for a normal response to cardiac loading, and that LIMP-2 expression is strongly increased when loading conditions exceed compensatory mechanisms. These findings were translated to the human situation, which showed that LIMP-2 is also robustly increased in patients with clinically severe pressure loading. Taken together, LIMP-2 is a novel constituent of the ID and seems to represent a novel type of ID protein, essential for the response to loading rather than for normal cardiac function.

Mechanisms of LIMP-2 Response to Loading

[0080] Intercalated disc abnormalities were documented in pressure-loaded LIMP-2 null mice, characteristic of a disturbed cardiac intercalated disc, which normally acts to organize adjoining myocytes. Remodeling of the intercalated disc has been shown previously during the transition from compensated LV hypertrophy towards heart failure, while structural perturbations of the intercalated disc have been linked to dilated cardiomyopathy in humans, hamsters and pigs. It was shown that LIMP-2 binds N-cadherin, suggesting a role for LIMP-2 via this ID constituent. Indeed, pressure-overloaded LIMP-2 KO mice show abnormal intercalated discs on electron microscopy and their N-cadherin distribution is disturbed, suggesting a defect in the adherens junctions. The strength of adherens junctions is determined by the binding affinity between N-cadherin and β-catenin (Gumbiner et al., 2000), which is regulated by phosphorylation of the latter. It was shown in vitro by knock down of LIMP-2 in cultured myocytes, that loss of LIMP-2 disturbs this N-cadherin/β-catenin complex. Given the linkage of adherens junctions to myofibrils, a loss of LIMP-2 is expected to lead to less efficient force transduction across the plasma membrane (Ferreira et al., 2002).

[0081] It has been suggested that LIMP-2 is essential for the proper binding of N-cadherin to β-catenin, and that this role is particularly important under loading conditions. However, the precise way by which LIMP-2 assures binding of N-cadherin to β-catenin remains to be elucidated. LIMP-2 contains two transmembrane domains, a cytoplasmic loop and two luminal glycosylated domains. It is known that lysosomal membrane proteins can shuttle between lysosomal and plasma membranes, where LIMP-2 can bind to TSP1 and TSP2 (data not shown). The latter is intriguing, as it has been documented earlier that TSP2 is also essential for the response to cardiac pressure loading and increases in failure-prone forms of LV hypertrophy (Schroen et al., 2004). This suggests that both LIMP-2 and TSP2 may be part of a complex that is needed for the cardiac myocyte to mount an adaptive response to loading.

Implications

[0082] According to the present invention, a novel role for LIMP2 has been uncovered as an important mediator of the ID when the myocardium faces increased loading conditions. Apart from this novel biological insight, the finding that expression of LIMP-2 rises in hypertrophied rat hearts that are on the brink to progress to failure, makes it tempting to speculate that increased LIMP-2 expression by cardiac myocytes demonstrates their inability to normalize loading conditions. As such, increased LIMP-2 expression may signify imminent failure. Since it has been shown that LIMP-2 expression is also robustly increased in patients with clinically severe pressure loading, and is located at the plasma membrane, LIMP-2 may be an attractive target for molecular imaging to identify already in a very early stage, the myocardium that is about to succumb to the pressure.

Example 2

TGF-Beta Promotes Cardiac Hypertrophy By Suppressing Kruppel-Like Factor 15, a Novel Inhibitor of Cardiac Hypertrophy

Materials and Methods

Transgenic Rats, Left Ventricular Biopsies and Hemodynamic Studies

[0083] Eighteen male homozygous Ren-2 rats and five age-matched Sprague-Dawley (SD) (Mollegard Breeding Center, Lille Skensveld, Denmark) were studied. Three Ren-2 rats were sacrificed at 10 to 12 weeks of age upon clinical signs of heart failure and excluded from the study. From the remaining healthy 15 Ren-2 rats and five SD controls, a biopsy of the left ventricle was taken at 10 weeks of age, as described previously. Rats were followed by serial echocardiography at 10, 12, 15, 16, 18, 19 and 21 weeks of age as described above. Nine Ren-2 rats were sacrificed at 15 to 18 weeks of age upon clinical signs of heart failure and designated "heart failure-prone" rats. The remaining six Ren-2 rats were monitored and sacrificed at 21 weeks when clinical signs of failure had not appeared, and they were designated "compensated" rats.

Microarray Analysis

[0084] Total RNA was isolated and amplified as previously described from LV biopsies taken at 10 weeks of age of four SD controls, of six rats that remained compensated and of four heart failure-prone rats. Amplified cRNA was then hybridized to Affymetrix rat 230 2.0 GeneChips. Gene transcript levels of SD controls, compensated and HF rats were determined with Microarray Analysis Suite Software version 5.0 (MAS5.0).

Lentiviral shKLF15 Production

[0085] Lentiviral vectors were generated by annealing complementary shKLF-15 oligonucleotides (sense 5'-GATGTACACCAAGAGCAGC-3' (SEQ ID NO:1) and antisense 5'-GCTGCTCTTGGTGTACAT-3' (SEQ ID NO:2)) and cloning them into digested pLL3.7 puro vector DNA (kindly donated by Luk van Parijs, Department of Biology, Massachusetts Institute of Technology, Cambridge, USA) using E. Coli DH5α-competent cells. Constructs were purified using Qiagen Plasmid Midi kit. 293FT cells were cultured in DMEM with 10% FCS, 2 mM L-Glut, 10 mM non-essential amino acids, 1 mM sodium pyruvate and pen/strep antibiotics. Lentiviral production was performed by co-transfection of 3 μg shKLF15 or shTgfb1/pLL3.7 puro or empty pLL3.7 puro and packaging vectors into 293FT cells by Lipofectamine 2000 (Invitrogen Life Technology, Breda, The Netherlands) and virus containing supernatant was harvested, filtrated and snap-frozen after 48 hours.

Neonatal Rat Ventricular Myocyte Experiments

[0086] Neonatal rat ventricular myocytes (NRVM) were isolated by enzymatic disassociation of one- to three-day-old neonatal rat hearts as previously described (Schroen et al., 2004). NRVMs were cultured in DMEM/M199 (4:1) media supplemented with 10% horse serum (HS), 5% newborn calf serum (NBCS), glucose, gentamycin and 2% antibiotic/antimycotic on a gelatinized six-well plate with 5*10⁵ cells per well. For shKLF15 infection, NRVM were cultured overnight and the next day infected with shKLF15 and an empty lentiviral control vector, facilitated by polybrene (Sigma). After 48 hours, cells were washed free of vector and placed under puromycin selection for another 48 hours. Then, cells were kept under quiescent conditions overnight in DMEM/M199 (4:1), glucose, gentamycin and 10% antibiotic/antimycotic. The next day, medium was replaced by medium containing DMEM/M199, glucose, gentamycin, 5% antibiotic/antimycotic, insulin, L-carnitin and BSA. After one hour, TGF-b (10 ng/ml medium) was added for 1 hour and whereafter RNA was isolated using the RNeasy mini protocol (Qiagen) for SYBR Green quantitative PCR with KLF15 or BNP primers (F 5'-GCT GCT TTG GGC AGA AGA TAG A-3' (SEQ ID NO:3) or R 5'-GCC AGG AGG TCT TCC TAA AAC A-3' (SEQ ID NO:4). Knock-down efficiency of shKLF15 is about 80% compared to levels in NRVM infected with an empty lentiviral vector.

MEF2 Luciferase Promotor Assay

[0087] NRVM were isolated as described above. Cells were cultured in DMEM/M199 (4:1) media supplemented with 10% horse serum (HS), 5% newborn calf serum (NBCS), glucose, gentamycin and 2% antibiotic/antimycotic on a gelatinized six-well plate with 5*10⁵ cells per well. For shKLF15 infection, NRVM were cultured overnight and the next day infected with shKLF15 and an empty lentiviral control vector, facilitated by polybrene (Sigma). After 48 hours, cells were washed free of vector and placed under puromycin selection for another 48 hours. A Mef2 reporter plasmid (pGL2-3×MEF2-luciferase) containing three Mef2 binding sites cloned upstream of the Tata-box and luciferase in the cells via transient transfection. Cells were washed and per well, 1.6 μg of the MEF2 construct was added together with Opti-MEM I media (Invitrogen) and lipofectamine 2000, and antibiotics free media. The next morning, cells were washed and placed under normal culture media for another two days. Cells were kept overnight under low serum conditions (see above) and the next morning AngII (x grams/nil) was added for 4 hours. The luciferase assay was performed using the Luciferase Assay Protocol (Promega).

Generation of Double Transgenic Mice

[0088] TGFβRI^f/f mice (C57B1/6 background) generated by flanking exon 3 of TGFβRI with lox-P site (Sohal et al., 2001) were crossed with mice (C57B1/6 FVB background) containing cre-recombinase under the control of α-MHC promoter (MerCreMer^cre/wt (Larsson et al.) to generate heterozygous double transgenic mice that contained TGFβRI^fl/wtcre genes. These mice were then back-crossed with TGFβRI^f/f mice resulting in a colony with TGFβRI^f/cre and TGFβRI^f/wcre in a mixed background of C57B1/6 FVB, and C57BL/6.

DNA Isolation and Genotyping

[0089] DNA was isolated from the mouse tail using genomic DNA purification kit (Promega) according to the manufacturer's instruction. We used PCR to assess the genotype of TβRI flox mice, using the three primers, 5-ATG AGT TAT TAG AAG TTG TTT (SEQ ID NO:5), 3'-ACC CTC TCA CTC TTC CTG AGT (SEQ ID NO:6), and 3'-GGA ACT GGG AAA GGA GAT AAC (SEQ ID NO:7) as previously described (Sohal et al., 2001).

Cre Recombination Protocol

[0090] To induce α-MHC-coupled cre recombinase in cardiomyocytes, adult TGFβRI^f/fcre and TGFβRI^f/wtcre double transgenic mice were treated with tamoxifen (Sigma) at a dose of 20 mg/kg per day for seven days by subcutaneous insertion of mini-osmotic pumps (ALZET, model 2001). A group of wild-type mice was treated with tamoxifen to check whether tamoxifen itself had any effects on cardiac morphology and function. Tamoxifen was dissolved in 10% ethanol and 90% polyethyleneglycol-400 followed by a brief sonification. Mice were allowed to recover for two weeks prior to treatment with Ang II or vehicle.

Subcutaneous Implantation of Mini-Osmotic Pumps and Ang II Infusion

[0091] Mice of either sex weighing 24-32 g were anesthetized with 2.5% isofluorane. Under sterile conditions, a midscapular incision was made, a pocket was created in the subcutaneous tissue by a blunt dissection and a mini-osmotic pump (ALZET model 2004; ALZA Corp., Palo Alto, Calif., USA) filled with saline or Ang II (0.5 mg/kg/day) was inserted. The contents of the mini-osmotic pump were delivered into the local subcutaneous space at a rate of 0.25 μl/hour for four weeks. In each group, seven to nine mice were recruited for experiments and at least five mice from each group completed the experiments. All the dropouts were due to death from anesthesia except three animals in which LV catheterization did not succeed.

Echocardiography

[0092] Transthoracic echocardiography was performed preoperatively and after four weeks of AngII infusion in wild-type TGFβRI-/- and TGFβRl-/+ mice under 2.5% isofluorane anesthesia. Standard views were obtained in 2D-echocardiography, end-diastolic and end-systolic internal diameters were measured and ejection fraction and fractional shortening were calculated.

Hemodynamic Measurements

[0093] Mice were anesthetized with intraperitoneal injection of urethan. A Millar (1.4 F) catheter (Millar Instruments Inc., Houston, Tex., USA) was placed in the right common carotid artery and advanced into the left ventricle for the measurement of left intraventricular pressure. Body temperature was maintained at 37° C. using a thermally controlled surgical table and monitored with a rectal probe. The mice were then allowed to stabilize for 30 minutes prior to hemodynamic measurements.

Tissue Procurement and Myocardial Morphometry

[0094] Following hemodynamic measurements, hearts were rapidly excised, washed in 0.9% sodium chloride solution, atria were removed and the ventricles were cut into pieces. For RNA and protein isolation, samples were snap frozen in liquid nitrogen and stored in -80° C. For histological analysis, left ventricles were fixed in paraformaldehyde (1%) and embedded in paraffin. For the visualization of total collagen, picrosirius staining was performed as described previously. P38 was localized by immunostaining using anti-P38 antibody according to the manufacturer's instruction (Cell Signaling Technology, Leusden, the Netherlands).

Protein Isolation and Western Blotting

[0095] Frozen ventricles were crushed and homogenized in radioimmunoassay buffer according to the standard protocol (SantaCruz Biotechnology, Leiden, the Netherlands). Western blotting was performed using specific antibodies against TβRI (1:1000), total and phospho (P)-Smad2, total and P-P38 (1:1000, Cell Signaling Technology, Leusden, the Netherlands), P-Smad3 (1:5000, a kind gift from Professor E. Leof and Dr. M. Wilkes, Mayo Clinic Cancer Research, Rochester, Minn., USA), collagen I (1:3000) and III (1:500) antibodies (Abeam, Leusden, the Netherlands).

TGFβ Type 1 Receptor Immunohistochemistry

[0096] Cardiac tissue sections were deparaffinized and rehydrated and antigen retrieval tissue was incubated overnight with the primary antibody (rabbit anti TGFβ receptor 1 (Santa Cruz SC-398) and subsequently the secondary antibody (Goat anti Rabbit-Biotine (DakoCytomation E0432), whereafter they were treated with Streptavidin-Horseradish Peroxidase (Renaissance TSA® Biotin System, Perkin Elmer Precisely, Tyramide Signal Amplification kit).

[0097] All study protocols described above involving animal experiments were approved by the Animal Care and Use Committee of the Maastricht University, and were performed according to the official rules formulated in the Dutch law on care and use of experimental animals, highly similar to those of the NIH.

Statistical Analyses

[0098] Data are shown as mean±SEM. Unpaired t-test was performed to compare the difference between the means of Ang II/TGFβ/shKLF15 and vehicle-treated animals and cells. P-values of ≦0.05 were considered statistically significant.

Results

[0099] It has previously been shown that the outbred homozygous hypertensive TGR(mRen2)27 rat (Ren-2) enables study of the transition from hypertrophy towards heart failure (Schroen et al., 2004). Myocardial biopsies obtained at the age of ten weeks were used to investigate whether altered gene expression can predict which rat later will later progress to heart failure.

[0100] Expression profiling of these biopsies revealed that suppression of the gene coding for Kruppel-Like Factor 15 (KLF15) characterized the hypertrophied hearts that would quickly progress to failure. This was confirmed by real-time PCR, which showed that KLF15 was down-regulated in compensated LVH, but that it was significantly further suppressed in the hypertrophied hearts that quickly progressed to failure (FIG. 9A). In situ hybridization showed that expression of KLF15 was particularly down-regulated in cardiac myocytes (FIG. 9B). These findings extend earlier observations that KLF15 is constitutively expressed in the heart, but down-regulated in hypertrophy. That more intense suppression of KLF15 preceded the transition toward heart failure has led to the suggestion that KLF15 has important protective properties. To explore the functional role of KLF15, a short hairpin RNA (shRNA) against KLF15 was stably introduced. Spontaneous expression of BNP, a molecular hallmark of the hypertrophy gene program, was induced more than ten-fold upon shRNA-mediated suppression of KLF15 in cultured cardiac myocytes (FIG. 9C). This suggests that the constitutive presence of KLF15 is important to prevent the expression of the hypertrophy gene program.

[0101] In a parallel study, it has been shown that KLF15 null mice develop hypertrophy and cardiac function loss upon pressure loading, underlining that constitutively expressed KLF15 is essential to protect against maladaptive forms of LVH.

[0102] To explore the mechanism by which KLF15 can repress the hypertrophy gene program, its role in activation of MEF2 was studied. MEF2 is a target for hypertrophic signaling conveyed by the calcineurin and the MAPK pathway and is recognized as one of the crucial transcriptional activators of the hypertrophy gene program. A MEF2 reporter construct was used to address whether altered levels of KLF15 affect MEF2 activity in cardiac myocytes. This reporter only weakly responds to stimulation by MEF2 (Creemers, Olson unpublished data). Indeed, only minor increases in MEF2 activity were observed in response to angiotensin II. However, knockdown of KLF15 significantly increased MEF2 activity (FIG. 9D), suggesting that KLF15 acts as a repressor of MEF2.

[0103] It was next sought to explore which mechanism suppresses KLF15 in cardiac myocytes. Therefore, known mediators of cardiac hypertrophy were screened for their ability to inhibit KLF15 expression in cardiac myocytes. In cultured cardiac myocytes, TGFβ very robustly suppressed KLF15, so that expression of KLF15 was almost completely abolished after addition of TGFβ. Knockdown of the TGFβ type I receptor by inhibitory RNA prevented the suppression of KLF15 by TGFβ (FIG. 10A), demonstrating that classical TGFβ signaling involving its type I receptor is essential for this effect.

[0104] Therefore, to address the regulation of KLF15 by the TGFβ type I receptor, in vivo mice carrying a floxed TGFβ receptor type I gene, combined with the MerCreMer allele, were generated, which allows activation of cre specifically in cardiac myocytes by administration of tamoxifen (Larsson et al., Sohal et al., 2001). This allowed deletion of the TGFβ type I receptor specifically in cardiac myocytes in adult mice, avoiding the developmental effects of an embryonic loss of the TGFβ I receptor. Hypertension was induced by chronic angiotensin II infusion as described above in these mice to provoke hypertrophy and down-regulation of KLF15. Western blotting of whole heart homogenate revealed a significant down-regulation of the TGFβ type I receptor (FIG. 10B). Immunohistochemistry confirmed the myocyte-specific down-regulation of the TGFβ type I receptor, and showed the expression of this receptor in other cell types explaining the residual signal of the TGFβ type I receptor found in the whole heart homogenate (FIG. 10C). Angiotensin II induced LVH in wild-type mice, but the development of LVH was prevented in the MerCreMer-TGFβ type I mice. While in WT mice angiotensin II decreased fractional shortening, fractional shortening remained preserved in the MerCreMer-TGFβ type I mice. This indicates that loss of the TGFβ type I receptor from cardiac myocytes can prevent hypertension-induced hypertrophy and function loss. As expected, the expression of KLF15 was suppressed in the hypertrophied hearts from WT mice, but this suppression was absent in the hearts of MerCreMer-TGFβ type I mice (FIG. 10F). This shows that the TGFβ type I receptor on cardiac myocytes is important for the development of hypertensive hypertrophy, and at the same time for the suppression of KLF15.

[0105] Taken together, KLF15 is the first Kruppel-Like Factor to have a role in cardiac myocytes as a suppressor of cardiac hypertrophy. KLF15 inhibits MEF2 and parallel work shows it inhibits other prohypertrophic transcription factors like GAT4 as well. Consequently, it is conceivable that loss of KLF15 very robustly induces hypertrophic gene expression and is related to an adverse outcome. Suppression of KLF15 may, therefore, be a novel and crucial step in the development of failure prone forms of hypertrophy. It has been shown that TGFβ very robustly can suppress KLF15. Inhibitors of TGFβ, which are currently being developed in different fields, thus may have unexpected therapeutic potential as to prevent cardiac hypertrophy from progressing toward heart failure.

Conclusion

[0106] The heart hypertrophies in response to loading and injury, which often progresses towards overt heart failure. According to the present invention, a novel mechanism in this process is unveiled, where the cytokine TGFβ suppresses a novel inhibitor of hypertrophy, Kruppel-Like Factor 15 (KLF-15). Loss of the TGFβ type I receptor in vivo and in vitro prevents the suppression of KLF-15 and the development of cardiac hypertrophy and failure. The finding that TGFβ can hinder this novel mechanism that suppresses cardiac hypertrophy, opens exciting possibilities for inhibition of TGFβ signaling to prevent adverse forms of cardiac hypertrophy.

Example 3

Kruppel-Like Factor 15, a Transcriptional Repressor of Cardiac Hypertrophy

[0107] According to the invention, it has been shown that the zinc-finger transcription factor, Kruppel-Like Factor 15 (KLF-15), is a potent transcriptional repressor of LV hypertrophy. Gene-targeting studies showed that KLF15 null mice develop normally, but in response to pressure overload, develop an exaggerated form of cardiac hypertrophy, characterized by increased heart weight, increased expression of hypertrophic genes, left ventricular cavity dilatation with increased myocyte size and reduced left ventricular systolic function. All together, these studies demonstrate a role for KLF15 in LV hypertrophy, in vivo.

[0108] Interestingly, KLF15 is down-regulated in several forms of pathological but not physiological hypertrophy, indicating that KLF15 is a regulator of pathological hypertrophy, but not of physiological hypertrophy. The fact that KLF15 counteracts hypertrophy and the additional observation that KLF15 is significantly down-regulated in pathological hypertrophy and heart failure led to the exciting possibility that interventions aimed at preventing the decrease of KLF15 levels could prevent or even reverse pathological growth.

In Vivo Experiment

[0109] To test the intriguing possibility that preventing the loss of KLF15 during pathological hypertrophy may limit pathological growth of the heart, KLF15 was over-expressed specifically in the mouse heart using recombinant adeno-associated virus (rAAV)-mediated gene delivery under the control of the cardiac troponin I promoter (Vandedriessche et al., 2007). In particular, rAAV9 vectors have been shown to achieve a robust increase of transgene expression in cardiac tissue for several weeks following intravenous administration.

[0110] Mice were intravenously injected with 1×10¹⁰ vg AAV9-KLF15 or AAV9-GFP, after which hypertrophy was induced by Angiotensin II (AngII) treatment (four weeks, through osmotic mini-pumps). As shown in FIG. 11 (upper panel), KLF15 was over-expressed in the heart. Strikingly, mice allocated to AAV9-KLF15 gene transfer developed significantly less hypertrophy upon AngII stimulation, compared to AngII-treated mice that received AAV9-GFP. (See FIG. 11, lower panel.) Together, these data show that forced expression of KLF-15 in cardiac myocytes suffices to reduce cardiac hypertrophy.

Conclusion

[0111] Loss of KLF15 is a vital step in the development of hypertrophy and the transition toward heart failure. The observation that cardiac over-expression of KLF15 inhibits the development of pathological hypertrophy opens exciting possibilities for strategies that prevent the down-regulation of KLF15 in vivo to prevent hypertrophy and subsequent heart failure.

TABLE-US-00001 TABLE 1 List of primers for SYBR Green PCR and for shLIMP-2 production Gene Primer Sequence Mouse-BNP F 5'-GTTTGGGCTGTAACGCACTGA-3' (SEQ ID NO: 8) R 5'-GAAAGAGACCCAGGCAGAGTCA-3' (SEQ ID NO: 9) Mouse-ANF F 5'-ATTGACAGGATTGGAGCCCAGAGT-3' (SEQ ID NO: 10) R 5'-TGACACACCACAAGGGCTTAGGAT-3' (SEQ ID NO: 11) Mouse-aska F 5'-TGAGACCACCTACAACAGCA-3' (SEQ ID NO: 12) R 5'-CCAGAGCTGTGATCTCCTTC-3' (SEQ ID NO: 13) Mouse-PPIA^a F 5'-CAAATGCTGGACCAAACACAA-3' (SEQ ID NO: 14) R 5'-GCCATCCAGCCATTCAGTCT-3' (SEQ ID NO: 15) Human-LIMP-2 F 5'-GTTTGGGCTGTAACGCACTGA-3' (SEQ ID NO: 16) R 5'-GAAAGAGACCCAGGCAGAGTCA-3' (SEQ ID NO: 17) Human-GAPDH^a F 5'-ACCCACTCCTCCACCTTTGAC-3' (SEQ ID NO: 18) R 5'-ACCCTGTTGCTGTAGCCAAATT-3' (SEQ ID NO: 19) Rat-LIMP-2 F 5'-TGCGTCCAAACAAGGAAGAAC-3' (SEQ ID NO: 20) R 5'-AATCTCTTGGCCCCTCTTAAAATAA-3' (SEQ ID NO: 21) Rat-PGK-1^a F 5'-CGGAGACACCGCCACTTG-3' (SEQ ID NO: 22) R 5'-AAGGCAGGAAAATACTAAACATTGC-3' (SEQ ID NO: 23) Rat-shLIMP-2^b Sense 5'-GGAAGAACATGAGTCATTT AAATGACTCATGTTCTTCCTTTTTC-3' (SEQ ID NO: 24) Anti- 5'-TCGAGAAAAAGGAAGAACATGAGTCATTT sense AAATGACTCATGTTCTTCC-3' (SEQ ID NO: 25) ^aHousekeeping genes: cyclophilin A (PPIA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK-1) ^bRat-shLIMP-2 oligonucleotides; hairpin structure in bold italic

TABLE-US-00002 TABLE 2 Genes that are differentially expressed in biopsies of failure-prone Ren-2 rats as compared to compensated Ren-2 rats. Fold Probe Set ID^a p-value^b Change^c Gene Name^d 1374153_at 0.000052 4.64 Transcribed locus 1393098_at 0.000074 -1.39 similar to Lethal giant larvae homolog 2 1374120_at 0.000116 -1.89 similar to KIAA1126 protein (predicted) 1382598_at 0.000201 1.49 heat shock factor 2 1385234_at 0.000237 -1.67 Transcribed locus 1389074_at 0.000515 1.36 FCH and double SH3 domains 2 (predicted) 1376763_at 0.000520 1.23 Transcribed locus 1369722_a_at 0.000529 -1.31 xylosyltransferase II 1377000_at 0.000593 -3.11 similar to junction-mediating and regulatory protein; p300 transcriptional cofactor JMY 1374560_at 0.000652 1.56 similar to RIKEN cDNA 3110038B19 1379466_at 0.000692 -1.50 Shadow of prion protein 1385586_at 0.000774 -1.41 Solute carrier family 2 (facilitated glucose transporter), member 6 (predicted) 1370265_at 0.000784 1.82 Arrestin, beta 2 1375827_at 0.001303 2.46 similar to zinc-finger protein 1370102_at 0.001335 -1.89 potassium intermediate/small conductance calcium-activated channel, subfamily N, member 1 1384938_at 0.001399 -1.28 Rho GTPase activating protein 1 (predicted) 1375415_at 0.001433 1.22 similar to U7 snRNP-specific Sm-like protein LSM10 1380961_at 0.001458 -1.59 CCAAT/enhancer binding protein (C/EBP), gamma 1392644_s_at 0.001475 -2.35 similar to RIKEN cDNA 2610019F03 1377928_at 0.001490 -1.95 similar to RIKEN cDNA 1810018L02 1368950_a_at 0.001579 -2.06 glutamate receptor, ionotropic, NMDA2D 1368553_at 0.001633 1.47 activin A receptor type II-like 1 1389569_at 0.001702 1.31 similar to BRIX (predicted) 1374495_at 0.001744 -1.30 LPS-responsive beige-like anchor (predicted) 1397694_at 0.001928 -2.94 potassium channel modulatory factor 1 (predicted)/// similar to RNA binding motif and ELMO domain 1 1397750_at 0.001951 1.36 Transcribed locus 1373541_at 0.002074 -1.29 Rho guanine nucleotide exchange factor (GEF) 17 (predicted) 1368998_at 0.002089 -1.93 NK6 transcription factor related, locus 1 (Drosophila) 1382183_at 0.002205 1.62 Transcribed locus 1381635_at 0.002217 -1.60 Collagen, type XVIII, alpha 1 1374536_at 0.002228 -1.15 LOC499569 1385494_at 0.002248 -2.24 similar to RIKEN cDNA 4930451A13 (predicted) 1390663_at 0.002310 -1.36 Transcribed locus 1395547_at 0.002465 -2.57 Guanine nucleotide binding protein, alpha q polypeptide 1390818_at 0.002500 -1.29 ATPase, Na+/K+ transporting, beta 3 polypeptide 1381396_s_at 0.002502 -2.02 Kruppel-Like Factor 15 1374216_at 0.002528 -1.25 Transcribed locus 1386857_at 0.002661 1.23 Stathmin 1 1372140_at 0.002774 -1.33 similar to chromosome 6 open reading frame 80; chemokine C-C motif receptor-like 1 adjacent (predicted) 1379930_at 0.002805 -3.02 Transcribed locus 1376179_at 0.002832 -1.51 Transcribed locus 1399140_at 0.002847 1.14 Transcribed locus 1387923_at 0.002922 -1.47 zinc finger protein 179 1372789_at 0.002997 1.35 zinc finger protein 1 1395400_at 0.003131 -2.65 similar to hypothetical protein MGC23280 1390600_at 0.003208 -1.42 alpha-2,6-sialyltransferase ST6GalNAc IV 1368024_at 0.003228 -1.32 Quiescin Q6 1387555_at 0.003235 -1.90 amiloride-sensitive cation channel 2, neuronal 1395863_at 0.003299 1.34 nuclear receptor subfamily 2, group F, member 2 1394609_at 0.003309 -1.58 actin-binding LIM protein 2 1381045_at 0.003350 1.54 Transcribed locus 1377956_at 0.003355 -1.67 Cofilin 2, muscle (predicted) 1392061_at 0.003497 -1.89 minichromosome maintenance deficient 10 (S. cerevisiae) (predicted) 1394986_at 0.003538 -1.42 Similar to KIAA1838 protein (predicted) 1397855_at 0.003753 2.68 Similar to cysteine sulfinic acid decarboxylase 1375291_at 0.003813 -1.76 Transcribed locus, strongly similar to NP_733751.1 myeloid/lymphoid or mixed-lineage leukemia 3; myeloid/lymphoid or mixed-lineage leukemia3; ALR-like protein [Homo sapiens] 1370720_at 0.003856 -2.07 Putative pheromone receptor VN6 1373533_at 0.003878 1.25 EST 1391763_at 0.003988 -1.33 Transcribed locus, weakly similar to XP_516348.1 PREDICTED: similar to glycerol-3-phosphate dehydrogenase 1-like [Pan troglodytes] 1379258_at 0.004084 1.30 kelch-like 5 (Drosophila) (predicted) 1372855_at 0.004101 1.80 Bromodomain containing 4 (predicted) 1377342_s_at 0.004128 1.46 Rapostlin 1372030_at 0.004292 -1.12 zinc finger, FYVE domain containing 21 (predicted) 1385789_at 0.004371 -2.09 EST 1378878_at 0.004442 -2.84 similar to hypothetical protein FLJ25530 (predicted) 1369748_at 0.004471 -1.63 serine (or cysteine) proteinase inhibitor, clade I, member 2 1385103_at 0.004588 -1.29 fused toes (predicted) 1384518_at 0.004619 -1.32 similar to PHD finger protein 14 isoform 1 1368323_at 0.004645 1.38 tissue factor pathway inhibitor 1387230_at 0.004717 -1.64 solute carrier family 12, member 3 1383457_at 0.005114 -1.86 similar to hypothetical protein DKFZp761N1114 1392702_at 0.005181 1.41 EST 1370866_at 0.005191 1.41 Ribosomal protein L41 1371958_at 0.005199 -1.36 poly(A) binding protein, nuclear 1 1390189_at 0.005346 1.15 similar to Zinc finger protein 277 1374029_at 0.005356 1.66 EST 1386917_at 0.005360 -1.50 Pyruvate carboxylase 1373245_at 0.005366 1.22 procollagen, type IV, alpha 1 (predicted) 1385709_x_at 0.005519 -1.24 Progressive ankylosis homolog (mouse) 1394995_at 0.005638 -1.83 Cobl-like 1 (predicted) 1383147_at 0.005643 1.31 Transcribed locus 1397631_at 0.005720 -1.34 Ubiquitin specific protease 8 (predicted) 1398290_at 0.005734 1.27 Potassium channel, subfamily K, member 13 1371572_at 0.005800 1.26 amyloid beta (A4) precursor protein 1379651_at 0.005994 -1.30 Forkhead box P1 (predicted) 1387215_at 0.006008 -1.51 alanine-glyoxylate aminotransferase 1379418_at 0.006108 -1.53 LOC501008 1375358_at 0.006302 1.38 Transcribed locus 1394163_at 0.006408 2.23 SNF related kinase 1372025_at 0.006485 -1.45 Paternally expressed 3 (predicted) 1386016_at 0.006532 -2.45 EST 1376964_at 0.006542 1.56 similar to Ofdl protein 1367750_at 0.006592 1.22 phosphoribosyl pyrophosphate synthetase-associated protein 1 1388755_at 0.006685 1.22 SEC23A (S. cerevisiae) (predicted) 1390854_at 0.006734 -1.41 transmembrane protein 24 (predicted) 1376014_at 0.006832 -1.55 Transcribed locus 1379754_at 0.006998 -1.31 Staufen, RNA binding protein, homolog 2 (Drosophila) 1395327_at 0.007147 1.29 Lysosomal integral membrane protein-2 (LIMP-2) 1373262_at 0.007220 1.27 Similar to 2310014H01Rik protein (predicted) 1391108_at 0.007232 2.13 Transcribed locus 1387938_at 0.007304 -2.17 brain and acute leukemia, cytoplasmic 1391819_at 0.007324 -1.68 similar to hypothetical protein ET (predicted) 1383216_at 0.007326 -1.44 Cohen syndrome homolog 1 (predicted) 1367744_at 0.007456 1.58 Melanoma antigen, family D, 2 1395869_at 0.007484 1.20 Similar to RIKEN cDNA A230063L24 gene 1384147_at 0.007519 1.43 eukaryotic translation initiation factor 1A (predicted) 1372476_at 0.007698 1.37 fatty acid desaturase 3 1387836_at 0.007703 1.62 Prenylated SNARE protein

1378958_at 0.007759 1.22 EST 1385885_at 0.007837 -1.48 Transcribed locus, moderately similar to XP_418312.1 PREDICTED: similar to Snf7 homologue associated with Alix 3 [Gallus gallus] 1372621_at 0.007920 1.44 similar to 2610027C15Rik protein (predicted) 1386228_at 0.007970 -2.39 Similar to PHD finger protein 20-like 1 isoform 1 1388798_at 0.007986 -1.26 ubiquitin-conjugating enzyme E2E 2 (UBC4/5 homolog, yeast) (predicted) 1373584_at 0.008209 -1.29 similar to hypothetical protein A430031N04 1383237_at 0.008308 1.25 Myoneurin (predicted) 1394721_at 0.008493 -1.38 similar to TTF-I interacting protein 5 1378315_at 0.008560 1.82 Transcribed locus 1381519_at 0.008621 -1.76 AT rich interactive domain 1A (Swi1 like) (predicted) 1376059_at 0.008765 -1.53 similar to hypothetical protein MGC38689 1388377_at 0.008789 1.10 coatomer protein complex subunit alpha (predicted) 1374247_at 0.008841 1.31 Stabilin 1 (predicted) 1373077_at 0.008994 1.39 similar to hypothetical protein D11Ertd497e (predicted) 1376484_at 0.009158 -1.56 Transcribed locus 1388910_at 0.009255 -1.13 mitochondrial ribosomal protein S24 (predicted) 1393574_at 0.009262 -1.55 Similar to Cc1-6 1393164_at 0.009350 -1.45 mitochondrial ribosomal protein S27 (predicted) 1373697_at 0.009502 1.66 myosin binding protein C, fast-type (predicted) 1390317_at 0.009509 2.80 Transcribed locus 1397642_at 0.009553 1.27 RAD50 homolog (S. cerevisiae) 1397352_at 0.009668 -2.04 Transcribed locus 1392557_at 0.009692 1.26 Transcribed locus 1384672_at 0.009749 -2.83 similar to RIKEN cDNA 4930434E21 1396724_at 0.009932 -1.29 EST 1393135_at 0.009960 -1.24 Transcribed locus 1388478_at 0.009986 1.27 Transcribed locus 1393318_at 0.010526 -1.32 EST 1394773_at 0.010614 -1.31 Transcribed locus, moderately similar to XP_580018.1 PREDICTED: hypothetical protein XP_580018 [Rattus norvegicus] 1397268_at 0.010662 -1.67 similar to solute carrier family 17 (sodium phosphate), member 4; Na/PO4 cotransporter 1371289_at 0.011056 -1.77 nitric oxide synthase 2, inducible 1390885_at 0.011168 1.34 Transcribed locus 1392299_at 0.011420 -1.56 C-terminal PDZ domain ligand of neuronal nitric oxide synthase 1394767_at 0.012694 -1.52 EST 1373120_at 0.019354 -1.40 Spermatogenesis associated 2 ^aProbe Set ID, Affymetrix probe set number ^bp-value, P < 0.05 is considered statistically significant ^cFold-change, fold-change in gene expression of failure-prone Ren-2 rats as compared to compensated Ren-2 rats. For example, negative sign means down-regulated in failure-prone Ren-2 rats. ^dGene Name, name of gene associated with the Probe Set ID

TABLE-US-00003 TABLE 3 Echocardiographic parameters in LIMP-2 KO and WT mice at baseline, and after 14 and 28 days of AngII treatment Baseline Day 14 Day 28 IVSd^a KO(n = 11) 0.10 ± 0.00 0.10 ± 0.01^$ 0.10 ± 0.01^$$ (cm) WT(n = 10) 0.10 ± 0.01 0.13 ± 0.01 0.13 ± 0.00* LVIDd^a KO(n = 11) 0.34 ± 0.01 0.38 ± 0.01^$$ 0.38 ± 0.01^$$,* (cm) WT(n = 10) 0.34 ± 0.01 0.33 ± 0.02 0.33 ± 0.01 LVPWd^a KO(n = 11) 0.10 ± 0.01 0.09 ± 0.01^$$ 0.09 ± 0.00^$$ (cm) WT(n = 10) 0.10 ± 0.00 0.12 ± 0.01 0.15 ± 0.01* LVAd^b KO(n = 11) 0.21 ± 0.01 0.27 ± 0.01^$$,* 0.28 ± 0.02^$,* (cm) WT(n = 10) 0.21 ± 0.01 0.23 ± 0.01 0.22 ± 0.00 LVLd^b KO(n = 11) 0.73 ± 0.01 0.85 ± 0.02^$,* 0.84 ± 0.03^$$,* (cm) WT(n = 10) 0.73 ± 0.01 0.79 ± 0.02.sup.,* 0.79 ± 0.02 FS^a KO(n = 11) 28.7 ± 1.9 20.1 ± 3.1 22.2 ± 3.0 (%) WT(n = 10) 27.4 ± 2.1 25.8 ± 1.9 25.5 ± 2.9 Averages ± SEM *P < 0.005 vs baseline KO or WT ^$P < 0.05 vs age-matched WT ^$$P < 0.001 vs age-matched WT ^aMeasured from short axis: IVSd, Interventricular septum in diastole; LVIDd, Left ventricular inner diameter in diastole; LVPWd, Left ventricular posterial wall thickness in diastole; FS (%), Percentage fractional shortening ^bMeasured from long axis: LVAd, Left ventricular area in diatole; LVLd, Left ventricular length in diastole

LITERATURE

[0112] Gamp et al., Hum. Mol. Genet. 12:631-646, 2003. [0113] Van Haaften et al., BMC Bioinformatics 7: online, 2006-09-21. [0114] Schroen et al., Circ. Res. 110:3121-3128, 2004. [0115] Heymans et al., Circulation 112:1136-1144, 2005. [0116] Junqueira et al., Histochem. J. 11:447-455, 1979. [0117] De Windt et al., J. Mol. Cell. Cardiol. 29:2095-2106, 1997. [0118] Crombie et al., J. Biol. Chem. 273:4855-4863, 1998. [0119] Eskelinen et al., Trends Cell. Biol. 13:137-145, 2003. [0120] Nishino et al., Nature 406:906-910, 2000. [0121] Stypmann et al., Proc. Natl. Acad. Sci. USA 99:6234-6239, 2002. [0122] Stilli et al., Exp. Physiol. 2006. [0123] Perriard et al., Trends Cardiovasc. Med. 13:30-38, 2003. [0124] Gumbiner, J. Cell. Biol. 148:399-404, 2000. [0125] Ferreira-Cornwell et al., J. Cell. Sci. 115:1623-1634, 2002. [0126] Sohal et al., Circ. Res. 89:20-25, 2001. [0127] Larsson et al., Embo. J. 20:1663-1673. [0128] Vandendriessche et al., J. Thromb. Haemost. 5(1):16-24, 2007.

Sequence CWU 1

30119DNAArtificialshKLF-15 sense oligonucleotide 1gatgtacacc aagagcagc 19218DNAArtificialshKLF-15 antisense oligonucleotide 2gctgctcttg gtgtacat 18322DNAArtificialFLF15 or BNP primer forward 3gctgctttgg gcagaagata ga 22422DNAArtificialFLF15 or BNP primer backward 4gccaggaggt cttcctaaaa ca 22521DNAArtificialPCR primer to assess the genotype of TbetaRI flox 5atgagttatt agaagttgtt t 21621DNAArtificialPCR primer to assess the genotype of TbetaRI flox 6accctctcac tcttcctgag t 21721DNAArtificialPCR primer to assess the genotype of TbetaRI flox 7ggaactggga aaggagataa c 21821DNAArtificialMouse BNP forward primer 8gtttgggctg taacgcactg a 21922DNAArtificialMouse BNP backward primer 9gaaagagacc caggcagagt ca 221024DNAArtificialMouse ANF forward primer 10attgacagga ttggagccca gagt 241124DNAArtificialMouse ANF backward primer 11tgacacacca caagggctta ggat 241220DNAArtificialMouse aska forward primer 12tgagaccacc tacaacagca 201320DNAArtificialMouse aska backward primer 13ccagagctgt gatctccttc 201421DNAArtificialMouse PPIA forward primer 14caaatgctgg accaaacaca a 211520DNAArtificialMouse PPIA backward primer 15gccatccagc cattcagtct 201621DNAArtificialHuman LIMP-2 forward primer 16gtttgggctg taacgcactg a 211722DNAArtificialHuman LIMP-2 backward primer 17gaaagagacc caggcagagt ca 221821DNAArtificialHuman GAPDH forward primer 18acccactcct ccacctttga c 211922DNAArtificialHuman LIMP-2 backward primer 19accctgttgc tgtagccaaa tt 222021DNAArtificialRat LIMP-2 forward primer 20tgcgtccaaa caaggaagaa c 212125DNAArtificialRat LIMP-2 backward primer 21aatctcttgg cccctcttaa aataa 252218DNAArtificialRat PGK-1 forward primer 22cggagacacc gccacttg 182325DNAArtificialRat PGK-1 backward primer 23aaggcaggaa aatactaaac attgc 252453DNAArtificialRat shLIMP-2 sense 24ggaagaacat gagtcatttg tcaagagaaa atgactcatg ttcttccttt ttc 532557DNAArtificialRat shLIMP-2 antisense 25tcgagaaaaa ggaagaacat gagtcatttt ctcttgacaa atgactcatg ttcttcc 57262543DNAHomo sapiens 26ccacgcgtcc ggcgacccgc gggccggcgg gcgatcgagc cagcgcagga cccgcggctc 60ggcccccggc cgccgccgga ccgagagtct agccgccgcc cccagcccag cccgcccggc 120cgcaggaccg ccggggcctg gccgccggtc cggcgtgcgc caagttcagc cgccaccggc 180acggccaggc cagcatggtg gaccacttac ttccagtgga cgagaacttc tcgtcgccaa 240aatgcccagt tgggtatctg ggtgataggc tggttggccg gcgggcatat cacatgctgc 300cctcacccgt ctctgaagat gacagcgatg cctccagccc ctgctcctgt tccagtcccg 360actctcaagc cctctgctcc tgctatggtg gaggcctggg caccgagagc caggacagca 420tcttggactt cctattgtcc caggccacgc tgggcagtgg cgggggcagc ggcagtagca 480ttggggccag cagtggcccc gtggcctggg ggccctggcg aagggcagcg gcccctgtga 540agggggagca tttctgcttg cccgagtttc ctttgggtga tcctgatgac gtcccacggc 600ccttccagcc taccctggag gagattgaag agtttctgga ggagaacatg gagcctggag 660tcaaggaggt ccctgagggc aacagcaagg acttggatgc ctgcagccag ctctcagctg 720ggccacacaa gagccacctc catcctgggt ccagcgggag agagcgctgt tcccctccac 780caggtggtgc cagtgcagga ggtgcccagg gcccaggtgg gggccccacg cctgatggcc 840ccatcccagt gttgctgcag atccagcccg tgcctgtgaa gcaggaatcg ggcacagggc 900ctgcctcccc tgggcaagcc ccagagaatg tcaaggttgc ccagctcctg gtcaacatcc 960aggggcagac cttcgcactc gtgccccagg tggtaccctc ctccaacttg aacctgccct 1020ccaagtttgt gcgcattgcc cctgtgccca ttgccgccaa gcctgttgga tcgggacccc 1080tggggcctgg ccctgccggt ctcctcatgg gccagaagtt ccccaagaac ccagccgcag 1140aactcatcaa aatgcacaaa tgtactttcc ctggctgcag caagatgtac accaaaagca 1200gccacctcaa ggcccacctg cgccggcaca cgggtgagaa gcccttcgcc tgcacctggc 1260caggctgcgg ctggaggttc tcgcgctctg acgagctgtc gcggcacagg cgctcgcact 1320caggtgtgaa gccgtaccag tgtcctgtgt gcgagaagaa gttcgcgcgg agcgaccacc 1380tctccaagca catcaaggtg caccgcttcc cgcggagcag ccgctccgtg cgctccgtga 1440actgaaagcg ccctgaaccc cagcctgtcc gtcaccccgg atccccaccc catccccatt 1500tttttaagca ataatttatt tgcctcctcc agagggacat ggcaatgtta ccagcccacc 1560ttctgaagcc tgggaggtgt gaacccaggg cccgccaacc gctgcctttc tcgggagtac 1620ttagagcctc gaacccgcgt ccctgggggc tgggccccag gcgcacgggg ctggaggcag 1680gccttcgtgc cttcgtgcct tcgtgccttc ccgcggtggc caggcctctg ctgcagccgc 1740tggttgcagg cagagttttg gggacctggc ccttctccca ctgggctccc ccatcctggg 1800ccaaggccag aactttagtg ctaggggaag atgaaatgtg cagttttgaa atgttgggtt 1860tccagagaga gtcatgctgg aggagaagga agtaggccag aagtccaggg ctgcactgtg 1920gtgtgagggt ggctttgtct aagatgcctg ctcagcatga tcaccagagg gtgtgggcag 1980gtccctggag cggggggggg gggggggggg ggcggaccgg gccgctgggc cctcatgtgg 2040gagagaggtg aaaagcgtcc cccactaggg ggctggcagt gcatgtgctt gagttaaatg 2100tgcagggcag acagagccag aagggcctgt acccaggggc tcgtcccctc ctccggtttc 2160ccagacaaat ccagacacca gcctttaggg tggccttggg aggagagggc caggctgtcc 2220tgggtgtgag agaactagat agagcctccc aaccctgatt tagaaatgca ttccttattt 2280tgtctagaaa ttaataaatg aactagcttg ttttgacagg tttatttcac atcctatgaa 2340tgtatgtaaa taaactgtac ataggtccat ccacataaaa tatcttttaa taacatatca 2400acatttgtgt aaatttgaaa tttaaaaaaa tctatgaagc tggtgtacat atgttacaat 2460tacgtatatt ttctttggtc cttcataaaa atatatttac tttgccaata aaaagaaaaa 2520gaactcacaa aaaaaaaaaa aaa 254327416PRTHomo sapiens 27Met Val Asp His Leu Leu Pro Val Asp Glu Asn Phe Ser Ser Pro Lys1 5 10 15Cys Pro Val Gly Tyr Leu Gly Asp Arg Leu Val Gly Arg Arg Ala Tyr 20 25 30His Met Leu Pro Ser Pro Val Ser Glu Asp Asp Ser Asp Ala Ser Ser 35 40 45Pro Cys Ser Cys Ser Ser Pro Asp Ser Gln Ala Leu Cys Ser Cys Tyr 50 55 60Gly Gly Gly Leu Gly Thr Glu Ser Gln Asp Ser Ile Leu Asp Phe Leu65 70 75 80Leu Ser Gln Ala Thr Leu Gly Ser Gly Gly Gly Ser Gly Ser Ser Ile 85 90 95Gly Ala Ser Ser Gly Pro Val Ala Trp Gly Pro Trp Arg Arg Ala Ala 100 105 110Ala Pro Val Lys Gly Glu His Phe Cys Leu Pro Glu Phe Pro Leu Gly 115 120 125Asp Pro Asp Asp Val Pro Arg Pro Phe Gln Pro Thr Leu Glu Glu Ile 130 135 140Glu Glu Phe Leu Glu Glu Asn Met Glu Pro Gly Val Lys Glu Val Pro145 150 155 160Glu Gly Asn Ser Lys Asp Leu Asp Ala Cys Ser Gln Leu Ser Ala Gly 165 170 175Pro His Lys Ser His Leu His Pro Gly Ser Ser Gly Arg Glu Arg Cys 180 185 190Ser Pro Pro Pro Gly Gly Ala Ser Ala Gly Gly Ala Gln Gly Pro Gly 195 200 205Gly Gly Pro Thr Pro Asp Gly Pro Ile Pro Val Leu Leu Gln Ile Gln 210 215 220Pro Val Pro Val Lys Gln Glu Ser Gly Thr Gly Pro Ala Ser Pro Gly225 230 235 240Gln Ala Pro Glu Asn Val Lys Val Ala Gln Leu Leu Val Asn Ile Gln 245 250 255Gly Gln Thr Phe Ala Leu Val Pro Gln Val Val Pro Ser Ser Asn Leu 260 265 270Asn Leu Pro Ser Lys Phe Val Arg Ile Ala Pro Val Pro Ile Ala Ala 275 280 285Lys Pro Val Gly Ser Gly Pro Leu Gly Pro Gly Pro Ala Gly Leu Leu 290 295 300Met Gly Gln Lys Phe Pro Lys Asn Pro Ala Ala Glu Leu Ile Lys Met305 310 315 320His Lys Cys Thr Phe Pro Gly Cys Ser Lys Met Tyr Thr Lys Ser Ser 325 330 335His Leu Lys Ala His Leu Arg Arg His Thr Gly Glu Lys Pro Phe Ala 340 345 350Cys Thr Trp Pro Gly Cys Gly Trp Arg Phe Ser Arg Ser Asp Glu Leu 355 360 365Ser Arg His Arg Arg Ser His Ser Gly Val Lys Pro Tyr Gln Cys Pro 370 375 380Val Cys Glu Lys Lys Phe Ala Arg Ser Asp His Leu Ser Lys His Ile385 390 395 400Lys Val His Arg Phe Pro Arg Ser Ser Arg Ser Val Arg Ser Val Asn 405 410 4152855142DNAHomo sapiens 28cctcgcgggg ttgcggcgag cccggcccgc gaacgtcacg tccctgcgcg ctccctgcac 60tctcccgagc tgcgctaggc gggcgccacg gctgcccggc gaaggaaacc gaaaccgagt 120ccgggcccgt ccctccgcgg ccccatccgc ccggtgcacc cggggccgcg ctcgccaggc 180cgcggagccc agagctgcgc gcacgaaccg tgcgccggga gggcgtgggc gtggcgccga 240agggtcccgg gtcttcgacg cctctgcggc ggctcctccc tccttgcagt tggatccctg 300gcgggtgcgg cccggcccgg cccgtgagcg gcgcacagaa tgggccgatg ctgcttctac 360acggcgggga cgttgtccct gctcctgctg gtgaccagcg tcacgctgct ggtggcccgg 420gtcttccaga aggctgtaga ccagagtatc gagaaggtga ggcggggcgg gctgtgtgtg 480tgttgtggag tcgtccagct caccctccca tccctgctgc tacatcttgt atgggaaaga 540ccagcactca ggcagaccct ccgcgctaga gctcttcttg cgtccctgtc ttccagggct 600tggtaggcga gggttaaaga aggatgagga aggatggagc cgactctgtt ccctttacag 660tgataagtaa ggctatggat cctgactttt aaaaaggcct tcggttgtgt tctgtcgttt 720gcatctctcc ccactccttc cgtctacacc ccaatctcag gtgggcccaa acttgtttcc 780ttcccacttc atgaagaaac agaactgaat gaaagacacc gcagaagagg caggttatgt 840tgtctgcttg ctgtagcctt tgaagtggtg tgcgcctggg gatgagacgt gggtgtgcgg 900tctattgtgt tcttgccctc ttgggctggg caggggcagt tggtagctgg gccagtcctt 960taggctgggt gacaattcat ccatcagtct ttgctgaacg ctgacttagt ggctttgtat 1020cttgtaccaa cagtctgtgt tgctttgtct caccatgaat tcttgttttt ctcaaacggc 1080cataattttc aggcacagat gcatttagat accctacaag aatctgaact tgtctcttcc 1140tctagatgat tgggctggag agccagtcct ttaagaagac ttcatctttt gatttcactg 1200gtcatttcat ctgccctttc ccccacatcc accaagctgt gtcaaaagag gaacaaaaga 1260gaattactgg gatcattttt tccttaaaaa agaaaaataa tcactggagc agcagccttt 1320cttgttagca tccataactg cacgcttcat ttctcccact ggtctgcact tgctgcggcc 1380agataaaaaa gcccaggcct agtctacttt gaatcactgc actctccagt cagtaaagct 1440attttattcc aggtggcaat ggatatcttc ctttaactga gtttccaaaa gtaattaagt 1500gagtacctga aatctggcag ttaaaggcag tttttcaaaa agcaccatgg caaagcataa 1560tgtaattttt aaagttcaca ttgccaaggg aagacaaggc ccactgtctg agcgtctcga 1620gggtcagttt cctttgagca atcatgttca aggaaaaaag aaactgaatc ctttctccac 1680tcctgtccat gtccaaatcc atggtgagct gtgtttgagg atgtaaactc ttgctttggg 1740tgactagagt aaagcaatcc aattatagta aatatgtgag ttttcttata tggccttgaa 1800aggctgtttc aaggagccct ataggatgag tgatcttccc cgtggttccc atatagccag 1860cccttagatt ctctgtgtgt ctccatttct ctgggtgagg gggtggctgt aaggcaactt 1920ggctggggcc tgagtttgtg ttgatatttc caagtgtttt tctccatgag gaatggactc 1980tgttctgata agtaatagta cagggaagtg gactttcaga gctgctcaat cccagctaag 2040gaggactgca cggtgtattg tatgaaacta aaggacaggg ctagtttcag tgggcaggtg 2100tggaaatact gttgtcatta gaggtaagtc acacatcgct attctgcttt ttcctggatt 2160acctgtaggg tggtaggtag catgggacag tgggaaagat tgaggatgtt acactcctgg 2220gcatgaaacc tagcttccac tatgtcccag ctcggtgacc gtgggctgtt taatcttact 2280tgattcatgt tttccccatc tgtaaatagg gataataagg actaacgatc acatctgctt 2340tgtcaaataa ttcttataac cttaaggtgt gatctttctg ggatatagtt agtgcttaat 2400aggtgatagc aactttattt ttattagaag ccacacaaag aaaatacgtg agatttggag 2460tcagattcca tatgttcaaa tcctatcctt gccagtataa gctttatgac ttacgtcaaa 2520taacttactt ggtcctcaat ttcattgctt gtaaaactgg ggatatgagc tgttagctaa 2580cttgggcaca gtgatgcatt taccatgaaa ctcgcaaagt taagctttgg gtctctcact 2640tgcacagacc cttgcgaagc cctgtacctc attctatatc ccaaattttc ttttttctta 2700agaaggaccc ttcaaattgt ataagcttca ggacccacaa aacctggatt tgtctctgca 2760tgtgtattat tgtaggacca agtataaagc acttagcata gtatcaggca cagaataggt 2820aatcaatatg tggcagtcat ctttattgcc actagtatta ctatgggaga attcattcat 2880aacttgattc taatgtagtt ataatataat ctactctgat atttccagtt ctttgttttt 2940tcatttgaag attgtctatg ccccgacttg tctatgtcaa aaagggaaga gggaaaaggg 3000atgaaattca aatgagtagg tgtgggaagc ttagtaagag aattcctatc caaaaagttg 3060tctcaaatga taattgtcct tgaaccctgc agtgaccaga ggtaactgaa gagagacttt 3120taattttggc cagtgaatat cgtggaagga accaagacaa ttttgtttca tttttggcct 3180cagtggtgac tgcctcgtat tgggcaaggc ctgtaattgt ttctgcttca attttctcat 3240ttgcaagaga ggcataatag tacttctctt tttcccacac cctcttccta ggggatattg 3300tgagcattaa taaggcattt ccttacaagc gtttgggcct tcatgaagag agagctttgc 3360agagacccag cagttccttt tcctccctta ggcctggaaa ggcaggagtt tacgttacat 3420ttcatctggt atcccagtga actgccttga cacctagcac agtgcctgag atacatatta 3480aatgcctctt cactcgcctg tgatcatttt aatagaaaga agagtgtcct gagaatcagc 3540agaactgaat caaacatttt ttttctgtcc ctaaggattt cttggactaa gctgaacaag 3600cacagtggca agttatttaa tccctcaaat cctaaggatc ctcattttga aaataggaac 3660aataaggata agaatactta actcagagtt gtcacaagaa tgaaataata tgatatctat 3720aaagcaccca gcacaatgcc tcgcacaatg cctgaacgtt aacagttgtt atgtctttag 3780ctatatttgt catctgtgaa ttgaaactta ttatacaagc tatggttcct cttagctcca 3840aacttctgag atcccatgac ctttaagagc agctatgcag ttcagaatat taagtttgcg 3900ctcctaatct ctgcagacgt gctggcaggt tgactgtctc ccagtaatag ggatgcagaa 3960ctggccacag tcagggctgg ccaacaaggg atgggatggc aagggaacca gtgggagact 4020gtttacacct ttgacttcct cttcacaggt caaggcagga ctgtagcatt aggtctcaga 4080gatgcaatga acaggacaaa tacacttccc cttcatctgg acctgaggct ctgggcaagt 4140cagtaccagc tggtttcttt tgggagccat agaaactctg gagctgttct accgattcct 4200caagaagtaa aatataggcc aggcgtggtg gctcacgcct gttatcctaa cactttggga 4260ggctgaatcg ggtggatcac aaggtcggga gttcgagacc agcctggcca atatggtgaa 4320accctgtctc tactaaaaat acagaaatta gctaggcatg gtggcaggtg cctgtagtcc 4380cagctactcg ggaggctgag gcaggagaat cgcttgaatc cgggaggcgg aggttgcagt 4440gagccaagat ggctcccttg aactccagcc tgggcgacag agcaagactc tgtctcaaaa 4500aaaataaaaa taaaaataaa aaaagaagta aaatgtaatc actgataacc agattgcttt 4560gattggtgac tatgggatct tgttttttaa agtcaaatca gcaaaacatc tttattgatg 4620tgtatgtaaa ggcattcagc tatatatcat tttaaaaggc tccttcttgc atatctacct 4680catctcctga gttgcctcta ttcaaagaag acttatctta gtctgagatt tttcttgatc 4740agaaaaaaac ctgtaaagat atcttaaatc tgttatctta cggtcaagga aattgaggct 4800cttggaatag aagtgactta tctaaagtca aatggagagg gaaggagaga gcgtgccagc 4860tagccttctg atcctgaacc cacttttccc atctcaagga tgaccataca tcccatttgc 4920ctggaatagt cctacatggt ttctgtgtac ctgttgtgct ggtgtaatca ctaacacccc 4980ctttcctcca aaagatttct gctttggaca ataaattcta tggccatccc tcacctctca 5040tgctacattg tccttacaga ccacaatgta tcccgcttct tggctctctt aaggagttga 5100ggcctactca aactagctct ggcaagagag gattcttttt tttgtttctg taaatctgga 5160aggggaagct catagaaatc aaagggcaag aaccacggca cagtaaggcc tcctgagagg 5220acaaccgaaa gtggaaagtc agcaggaagc aggcacactc tgcctcccac tttttcctct 5280gctttctcca ccaacaggct tcctctgttt actcagcctg gctctgtact catccgggct 5340tgctcagccc cgcctcagtg tgactttcca gctgttgacc tctcatagac atgcttcatc 5400tcctagtttc tcttttccaa gtttataacg gagcaatgtg attggctcag cacaggaaga 5460aactatagca ctgtaaacgg aggcagtgtc gttaatttcc ttgcaagatt ctctaactcc 5520taagcaaaat tatccaaatt ctttcctggc ttttagaaac acgatcttgt gatatcaaaa 5580tcatattaag tttattttag tgtcttacta attcatgttg actatgagtg cattccttat 5640taatgattaa gacaatatag taggctaaca gttgtcagca tagcccctag actctaggag 5700atgcatctaa atcacttaca aagatttaga aaataccaga ccccatccca gacttgctga 5760atcagactca ttaggaacag aactcaggtt aaagtgctta tcataaaatc cattactgga 5820ggcaggttgc tgtgggatcg taatctgcaa ttttgggaat atgcattttt aaaaaaggtg 5880tttatatttg tatagtgatt attttagacc atgtcttcat tccttaagat ggtaattctg 5940actgaggaga ttactactta taattactat taatctgcaa gtttacatta aaggaaatat 6000tatttggtga ggaggggaag attcctgcct gtggaaacta ctttgcttta ttgttaaata 6060aatccaagat ttcattctgt caggccacag cagaggggcc atctgttctg ccagacattt 6120gcttatttcc caggaagaat caggaggtgg aattataagc tcattgatga aaacatagga 6180tgttggccaa gttactagac attcctggcc atatcatcaa gatatgcact atctatacac 6240tatacactaa ggaagggacc atttttaatc caagctggta actggaaaaa acaaccaagg 6300aaaaataaag accactttag gtgtttgctt tctatatcta aatgcacttt tgggtacatg 6360ctttggcagt cattctttca tttgacaaat atttactgag tgtgtaccat ttgcaaggca 6420gtgtgctaag agctatggat ataacactaa acaagaggca gtctctaccc ttgcagattt 6480tataatccaa tttaaactcc tgggcccctc atattcttaa aaattattga ggaccccaaa 6540gaacttatgt ttccatacct attgatagtt acaatattag aaattgaaac tgatatgtat 6600ttttgagaaa tgtttaaaac agaagactac acaagcacat attccaatat gacatcatta 6660tatatcacgt aacctctgga agactccact ctacactcag agaaataata gaattaaaaa 6720cgcaaatgat accttagtat taaaatagtt ttgaccttgt gggcgcctcc taagaaggtc 6780ttcgggctcc ccaggggtcc tgagatcaca ctttgagaac tgttgatcta gttttcattt 6840aattattttt ttagaatatt tttaaaaata cataatatcc ctgttttata ttcctgtaag 6900gccagtttgg aagctatctt gttaatgttt tcaaacaacc aggattttgc ttggtgtgtc 6960tttgatttac tgtgtccttt tggctttcct gtgagtccag tgttaaaaat aggagtttgg 7020gccatcagta attccagttc cacaatctct tgtctttccc cgagtgctga tcaaagtaga 7080caagagcatt catatctcca taattgaatt actcagtaat tcccttctgg cctcagccct 7140gcggggattt tcaacccata tgacgtaatt tggtatttta atctagtatg aaagctctct 7200ttgaaggtag atgtcctgtc gtatttagtt ttgtgtcttt tcttactgcc aaatgcaatg 7260tcttattcaa aatagatggt cagtaagtgt tacttggatg aatagagaaa ataatttctt 7320tggaaatgat tttgtttgca ttttgttgtt tagtctcaca

ttttgcctca ctctgtaggc 7380cagtggtcac atgtttgtct catgaatgat ttataccttt aaaatctgga aacttcacgt 7440attggtccag atttccagct tctcttgaaa catgtggatg ttctttggta ctcgtcatac 7500caagttctgt gtggtagctg tctcggcaga aaagaggctg acccttagaa gagacctgcc 7560ttccctgttt cccactgtct tagtccattc aggctgctgt cacaaaatag catagactgg 7620gtggcttata aacaacagaa atttatttct tacagttctg gaggctagga agtccaagat 7680caaggccccg gcagatttgg ggtatggtga gggtccactg cctcatagat tatggtcttc 7740tcactctaac ctcacatggc aaatgagctc tctggggtac cttttatgag aactaatccc 7800attcgagagg gttctacgct cgtgacctaa tcacctccaa aggccccacc tcctaatctc 7860accaccttgg gggtaaggat atcaacaaag gagttttgag gggacataaa cattcagacc 7920acagcaccca ccgtcctttg cgcccttatt ggctcccaag gcccagtgaa ggtatccgtt 7980gttgtgcatg tgctgttgtg tttcttagag tagagaaatg tttctctata tctgacactc 8040cttaccaaaa gaaggaaaat gaaagatagc ttgagaggat catgtgttta acagaaatgg 8100gggagggcat gtttctctgt agaaatgaat attgctgctg gggtttgaca tgcaaacaaa 8160tgtacaaacc catggcctgt gtcactcatt tgccctagcc atctggcccc ttcagcatct 8220gaattttaca cactttccag agacagtgtg aggcagagtg ccattgttac actaatgtaa 8280tcactcatca caagtcccat tttttaaaat tcatttttta attgacaagt caaaattgta 8340catatttagg gcatgcaaca tgatgttttg agagagatat acatatagat atacatagtg 8400aaatggctaa atcaagctgt ttaacataca ccttacctca attacttatc tttgttttgt 8460ggtaagaaca caaaatctaa tctcagcatt tttcaaatat acaatatttt ggggttttat 8520tttattttat ttttgttttt gtttttgttt ttgttttgag atagggcttc tctctgtcac 8580ccaggctgga gtacagtgcc actaccatgg ctcactgcag ccttgacctc ctgggttcaa 8640gccatcctcc tgccttaaac tctaaagttg ctggaactac aggtgtgcac catgccaggc 8700taatttttaa atattttttt gtagagatgg ggtcttgctg tgttgcccag gctggtctca 8760aattcctgag cttaagcaat cctcccacct ttgcctccta aagtgctggg attacaggcg 8820tgagccactg tgcctggccc aatatattgt tatccactgt gatcaccatg atgtacatta 8880gatctcttga atttgtttat atcaaactga aattttgtgt cctttgacta acatctcacc 8940agtcctccca gcgcccacaa ttcccatttt aaggatattt tcttttaaca ctgtacttct 9000gataatcaac agtagtggaa gaggagacat agaaataggt aggtaatgaa aatattactt 9060atttcaattt tgagagatat aatcacaacc aaacaggttg caaaataatg agactggctg 9120tgcaagttac acacagaatg tgggccttgt ttccgtggcc ttatttctta tttattcaca 9180tctgaaactc ctctttctca cccgcctccc cactccccat atatgtatat atatattccc 9240ctcccgctcc caattttcac ctggttctat gtttaagttt ctatcggtag ctgctttttt 9300tccctttagg tcatcagttc tcaaacttgg ctgtacattt gtaatcacct agaaactttc 9360aaaaataagt tagtgggtat tttgtagcag gaggtttttt gggattgtca ttccaccata 9420ttgtcctgta tgtaatttat aaacaaagta cagttcaccc atgagctaca tttgaatctg 9480aactatcaga gctcattagt taaaattatg acagttgcaa aatgaaaatg agttaagcaa 9540cagtgacata aataaatgaa tgccaataca tttttactga gttaaaataa ggtcagggaa 9600taagagtaga atgttaatgg cagtttcttt cttttatgta aacagtggaa attacttaaa 9660ttactgcatg agtctgggtt gggtttgctt cgtttcaggg agcaatttta aaaaacaaag 9720caaatcaatt ggtgttttgc catttgatga gttttattaa tctggagtcc agtatacaat 9780taggccaata taccatccat acacaattag ggcaagttct ccaagttggt ctgcattatc 9840tctggatgaa tttagactgt gcagatggca acatggatgc ttaagttgtg tttaattatg 9900aagtagcact ttactgacct taaagaacaa ccagtagtgc tctcgaatcc cgaaactttc 9960cttaaaggta ttactgattc ccgtgaaagc ctaatggatt ccctgagtgc ttatttttat 10020cacttggaag tggcttacat caggacagct ggtttcacca ctctcatggt gttgccttcc 10080caaaagattg ctgttaggct gacttgggtt gtggctgtat taagtcatgt cagtgttcag 10140cagtcaagct aatccagcct gcatctgttc cttcatctgc aggtcaggtg tcagcctggc 10200cagcgactcc atgttcacta cacacagctc agagaggttg ttctcagttc tgtctctgac 10260cagccttctg tgtacccaaa tggaagctta tgctggttga agattctttt acttttgttt 10320taaaacagtc tcacttcata catggtaaaa actgttatgc ttctccagta attcaagaaa 10380tgcaagttca aacaagaaga tgctattttg ccactactga ggaagcaaag atttctgaaa 10440accttaaaca ccaatattgg gatggatatg ctgaaatggt tgctcataca ctgctttctg 10500gagcaacttg ttgatattta aaggctgatg agttcatgaa aactttttgt ctctagaccc 10560aataatccca atctaggagt ctattctaag aaattattca ctgatttggg ccaggtgtgg 10620tgcctcatgc ctgtagtccc agcactttgg gaggccgagg taggtggatc acttgaggcc 10680aggagttaga gaccagcctg gcccacatgg ttaaacccca tctctactaa aaatacaaaa 10740agtagctggg tgtagtggtg tgcacctgta atcccagcta ctccagtggc tgaggcatga 10800cagtcaaaag ttgcagtgag ccaagattgc accactgcat tccagcctga gtgacaaaag 10860caagactctg tctcaaaaaa agaaaacaaa attattcact aatttggaca aatatattca 10920agaacattta cttcagcatg aaagcaacac aaatgtccta tagtaaaaga atgattaaat 10980aaactgattt agtatatttt tttaaatata ctaatacagt ggattagtat acagacatta 11040aaataacagt ttcaaaacct atttaataac aaggataatc gtttataaaa gatgagtcaa 11100aaaatgatac aatacatatg atctcaattt tgtttaaaat acacatatat atgactagac 11160cagagggaaa taaacccata tgttaacagc gatagtgtgg attacagggg atttaaattt 11220tgttcctttt actttcttca ttgtccaaat tttctgtgac aaatataatg gaatgggtag 11280ggaatggatg gagtagcatt tacctgattt aaagtaacaa caggctgggc acggtggctc 11340acacctgtaa tctcaacact ttgggaggct gaggtgggca gatcacctga gatcaggagt 11400tcaagatcag cctgactaac atggtgaagc cctttctcta ctaaaaatac aaaattagcc 11460aggtgtggtc atgtgtgcct gtaatcccag ctactcgaga ggctgaggca ggagaatcgc 11520ttgaacccgg gaggcggagg ttgtggtgag ccgagtcatg ccactgcact ccagcctggg 11580caacagaatg agactccctc tcaaaaaata aataagtaac tgaataaata aaaaataaag 11640taacaacaat cagtagcatc atttgtgaat agaaattgga ggcaacacat gactttttct 11700gcacatttgt agtaatgtta tctgctactt taaacatctc ttctaatatg aatatagaaa 11760tttctgtaga ttgataattt atcataatat ctaggtattt ttcccttaga taaatgtgat 11820gttttattgc ttacaagtct agtttgttcc ttgttatatc aaggagtttt tagaattcac 11880caataaaatg agaaggtagg agatcttgtc taccattata tctccaatac ctagaacagt 11940ggcttagcac aaaggaattg ctaagttaat atttgtggaa agaatgaaag agtttcatca 12000gttttaaggt tgtgaaaatc ctatgctata cttgctatga aaaaggatgt tataaaaaat 12060tcattaaaac caaaattgct aaagacagat ctaacagact tagaactaat gtttgtgtgc 12120ctttggcata ttaattatgc ataaaaatag accctgctaa ttccaaacta gtcttaacca 12180tttatgaaag gaggccaaaa tgtctccttg aaaaataata tttgcagcta agaaagttac 12240tgtctcttct tgattctgca gtactgaaat tcagactttt tatttattca gatcaccttt 12300cttacatatt atttagaata tgcggcattt tgccacattg aagattttac caagaagaac 12360tgggtaaaca gcagaagatg aatgcctggc acagcgttgt gctctacgct gcggtatcat 12420ctgtttcttc attaattatt tattgtgtcc cttctgtgcc agaaaccatg ttatgtgctg 12480gggatccatg aaaaagatac agttcttagt atcagaccca atctgacact aaagactaat 12540gaacacattt tccttataaa acttgtgaag tttcatgaaa gcagagagaa aacacatagt 12600tcagaggggc cttctgagag atggcacttc tgaattgagc cctgaagccc aagtagaagt 12660gaaccaggtg ataagggccc tgcagagact tctgggtgaa gggcggtgag tgactcaggc 12720caaggcactg gctgagaact tgtgtggtgg gtgctaggta acagtagatg gttgcgtgtt 12780actggtgcat aaagtgccta ccaggaaaag acagcgacag gtgacgcagt tgcatcagat 12840cacaacaggt caaggactgt ggactttatc ctttgagcag gggtggagta tctgttccct 12900taggcacttt tgacaggctt ctctgaagcc tgagatggat gaagcatttg aaaggaaaaa 12960ggctggagag agggagacca gttaggagcc tacatacagt attgtcacag tccaagccaa 13020tgatgaaatg tgccttgggg tagaaatggg gaagaggagg aggtgaaatc agcaggactt 13080agtgatcaac taggggagaa aatcatgagt ccattttggt catgttgtct tggagcagcc 13140tgtgagatgt ttagatgggg gaattttatc acagatccat gctactggaa ggatctaaat 13200aatttcaggc ataagctgtt tgttaggctg aattactgag tcttgcacat aatttttcac 13260tccctgttac atgcatgttg gttttgtgtc cttagctaaa tttcttctta cagacagggc 13320ctatgcctgt ttgcagggca tagtccgggc acatagaaag ctctcagtag gtacatattg 13380ggtgattaac tgaaaagtat gttttttagt ccaattatta atttagctgt tgacactgtg 13440aattcaccat agaacttgat aattttccag tattttgttt tagtggtagt gccacagtat 13500tgtgcgtgga aaaattcatt atgttcgtct tggttaaaac taataagcaa cagttatatt 13560gtgactccac agtgccactc ttaatatcca gagtcacact gtaaaactga tttaaggcct 13620tatgttgctt tttcatttag tgtaactaag aagactcagc tgattacaag gtttgtctgg 13680tggctgtcat caccacagca gaaccctcgg tgaggaagat cagttgttcc caagccatgg 13740ctttctgttc agactggcaa agggcgcctt tgagggggac actagctcgt tgtatctggc 13800ggaatgaata acttcctgtg ccagacagtc cgcggtcttg aggctgaggt tattatttgt 13860tagtttctca ggccttcctt tgccgagcaa gctcctggga gtgcacactt gaatgccctg 13920aggatcagaa atagaagggg cagaaattaa aagtttaagt caaaacagag ccctctctgg 13980gccatcttgt ggcaggaggt tgaggtgctt tctttggacc cattagcata ctggggctgc 14040gcggctgcct ggaaactcta gagttgaact ctcccttttt cggttcctca cctgtgggct 14100ccttactagg agctgacagt gacctcacgc agctgcccta gtttcctttc ctccagatgg 14160cacgggtaac actcgaggaa accctgagag ttcagagtcc aacttatggc ttctccatag 14220tttcactgtc cctctgggct agagttgcca aataaaataa aatacaggac aaccagttaa 14280atttgaattt cagataacaa ataacatttt agtatgagtc tgtctcgtgt gatatttcgg 14340ttcagtccga tgtatcagtt gttaatgtcc tgccatattt atctctctga tgcacttttt 14400cttgaattat ttgaagtaag ttgtagatat cacacacttt gttcctaaat ccttcagcat 14460gcatctccta tgaataaaaa catttttgat gtagcccatg caatatttgg gacataatta 14520tattataaaa ttatttgttg tttatctgaa atacaaattt aactgggtat cccaggtttt 14580atttgctaaa tctggcaact ctaccctcag ggtgccatgg ggtggcagct gctggatggg 14640tgagtaaatc tgcactcctg gacttggtca gatttgattt catttactgg ctttgtgact 14700tgggacagcc agtgccacca tttctttgtc tgcaaaatgg ggatttttat tttgtagtgt 14760cagtgtaaaa ctcagagata aggtacatta taaagcccct agcatagcac ctgactttag 14820cagactcaca gtagattgca gctggcgtgg cagtaatggt aactgaatag taactgccat 14880tctctgagct gaaagtgaga aaataaaaat aaagacacac tgttttagaa atgttagcag 14940ctgtatttac aacttccctc tgttctctca ttccccatct ctcttcctac ttagtgcatt 15000cgcatgagca cacacacaca cacacacaca cacacacaca ctcactcact ctccagcacg 15060tgatctactc tgggattgac agagggtttc ctgacaaaca tccaacttgt aaggtggagg 15120aaaagagaca tatgaaagca gcaggaaaag ccaccactct tgagggcaag atttgaaggg 15180agtaaaaggg gctatgagga tttgtctgag tgctggtgac tcaactaaga agtggccagg 15240cagagaaggg ctgtgggggt ggagggagac agaggagaag ccccagctgt aacatggtct 15300gggttggtta tgactctggc atgctgccaa ggacagtttt tattgttctt gtttttcatt 15360tgctttgttt cattaagaag tcaaatgtac taaatgttta tttctatcct gaaagttgga 15420ggaacaagtt caagtcacac tgtttacgca tgtcagaaca gccaggttta cccttctttc 15480tggtaacccc ttctttctgg aaacccttcc acttgtcttt ggatttatgg ttgcctaaaa 15540gctggatgcc agcccagcaa gataaaccag ggaggttagc catctgttca tttatgtttg 15600ctgattcttt cagggttcct aattctgagg tgattttctt cacatgaaca aataaatgag 15660atctcaagtc ctttctcact ggaaacttca cacactatga tgcttgtcag gaaaaattag 15720gcaggaatac agagtgctca ctcaactgtc ctgacagtaa taggaagaga agtgcggtgg 15780agtcgagagt gggggcggct gctcctgggc ctttgcagca gagcagaccg cagctgaggc 15840agcatcaggc tcgcctttgt tttgctagaa cagaggaaag ccaagatgaa gtgagttctc 15900tttgtgtcca ttccttctcc ttgccttagg gtctatccat aacatgctac agaatctgag 15960tttcttagat tttcacttaa aaaaaaagat aagggaacat ttttttttct gcttacagaa 16020gtaatttatt tctggtgtaa aaatccaaac aaaagcaaca gtgagtggaa gactcctcct 16080aatccggacc tgaattataa ctgatacaaa ttctcgacac tcaaagttca cattttggtt 16140tgtttatttg cagtctgtgc ttttatatta tttttgtatt tcagccgttt ttacagtaaa 16200aatacaaggc ttttctccat taacagatag aggactgttg ggaataccag gtgttaggtt 16260tctgttaacc tcaggcgctg tatttatcag gcctctactg ccacggtgag taacaagtaa 16320ctccccagtc tcgttgctta taacaataat atgtgggtgt gcagccagct cagctggctc 16380agtggaacga ggctggactc caggcttctg gtccgcttta ggactagtct acacatctcc 16440gttttagaac ctaggctgaa ggatcagcag ctacctgtgg cctgctcttc tcttttccaa 16500gggtggcaac ttcaaggtgg cagatagaaa cttgccatgc atcttaaggc cttagctcca 16560aattgataca ttgccattcc acccacatcc tgttggtcaa atctagtcac ttggcctagc 16620ctgaagtcag tgaggcaggg agaatacttc acccttaggg gagtcatggt gtattaattt 16680gctagggctt cccgaatgaa tacaacagac tgggtggctt aagcaacaga aatttgtctc 16740atagttctgg atgctagaag tctgagatca aggtattggc agagttggtt tcttctgagg 16800cctgtcttct tggcttgtag agggctgtca gttctgcgtg ttttcacgtg gccttccctc 16860tgtgcttgtc tgtgtcccag tttcctcctc cagttaggac accggtccta ttagcagggc 16920ctaccctcat gacctcattt aaccttcatc agctctttaa agaccccgac tccaaataca 16980ggcacattgt gaggtattgg gggttaggac ttcagcttag gaatttagag tatggggggc 17040attattcatc ctgtaacaca gaacaggggt ggaaagtgca ggagggattg tgtgcagagt 17100gcagtctcca ttgtcagcta acctcagtga ggctaggcct cctggttttt gaaacgggga 17160taggcagttc cattttaggg tttttgggga tccaataaga aaatacattt aaagcccctg 17220gtggtggagg cggtagggat ggatgtggtt tgacttgaaa aaggaccaaa actggccttc 17280ttgggccctt ccccttgtta ttctctcatc tgggcaaact gggagtgtat ggtgcttttc 17340agaattgctt aacttctaca gaagatacac catccatgga atcttcaaaa ataacaaagc 17400cagcaggccc tttaggcact caatctgtgc cctaggtttt ggaaacacta tgcttcatgc 17460ttctgcccaa gtgctgtctt tagtaccctt cctttacaat tattaattaa ttaactagtt 17520aatttatttt gagacagagt cttgctctgt aacccaggct ggagtgcagt ggcacgatct 17580tggctcactg caacctccac ctcccaggtt caagcaagtc tactgcctca gcctcccgac 17640tagccaggat tacaggcacg caccaccaca cctggctgat ttttgtattt ttagtagaga 17700cggggtttca ccatgttgac caagcacgaa ctcttgacct caagtgatct gcccgccttg 17760gcctcccaga gtgttgggcc actgggcccg gcccctttac aaatatttat cgaaggcctt 17820ctctgactta ttagaactgt gggctctatt ggtgtaagat atggccctgt tctcaggaga 17880ctgcagttga atgcctgtga aatgattaaa atagtgatct aggaggtcag aatagggggc 17940gtcccttcct ctgcttcttg ggtttaaagc caactattgc cttacatttt tgtaaatctc 18000cttttggttt tttcctagaa aattgtgtta aggaatggta ctgaggcatt tgactcctgg 18060gagaagcccc ctctgcctgt gtatactcag ttctatttct tcaatgtcac caatccagag 18120gagatcctca gaggggagac ccctcgggtg gaagaagtgg ggccatacac ctacaggtaa 18180gtcttgacca cctcctgtct tgaaagaaat acagagtgac cccaatatgg cctgagtcct 18240tgcctcttcc caggagcaag gctccagggc tgtgtgggag cacactttct gatttcattg 18300tctgaaacgc tgctgatgtt ttgtatgtgt gtgtgaaacc tcagcagaga atggctttgt 18360agactgggcc aagtctcagc gtgttgtgtt ttgaagtgca gcggtttcct cctgagttgg 18420cagactgtga ttattaaacc atgaagggtt actccttacc ctgacccttt taaggttgcc 18480ccttgatatt cttattttat tttattttat ttttttaaga gacaaggtct ggctttgttg 18540cccaggctgg agtgcagtag agcagtcata gctcactgcc acctccaact cctgggctca 18600agtgatcctc ctgcctcaac ctccctagta gctgggattg caagtgttca ccaccacacc 18660cagataattt tttttgtaga gatgaggtct tgctatattg cccaggctgg tcttgaactc 18720ctgggctcag gtgattctcc cgcctcagcc tcccacagtg ttgggattac aggcgtgagc 18780accaccatgc ctggctactg cttgatattc ttaagtgact tttagggcct ccctccattt 18840ccattcccca cataactagt tttaaagcag ttccagaatt tacttttgtc tgagacagat 18900ctttctgttt cttacccgct ccttttctcc acctatcctg cgtataagca ataagcaagt 18960agcagaggct gttagagtct ttgctggtga ccaccaattc caaacactgg ggaatcagga 19020tccctttaat catttttaaa cgattagtag agggagacgc ttgaatcaga gcattttctc 19080tgttgcaatg ttcccctttt ccctattaca atacctcctc cctctcccat acaataatct 19140ttccaataaa gtctctcctt accaggttag aaaacaaatt aatagaggaa gcagacaaca 19200agatctttct ttttcaaaac tgctgaatga taaaaatgcc tgacaattct gttgtaatcg 19260attgctcaat aaattattgc tgactgattc atgtgctatg aatcagtact attctcagtg 19320gtgggaattc agagataaaa atcctagttc ctgccttggg tgctcatgct ctgactctgg 19380agtcagatga cccattttga ttcccgcctc tgcaacttac tagttctctg tgcttccatt 19440tccttatcta taaaatgggg ataaaaatag gacctcctcc acatggtttt tacaaagatt 19500gagtcaatga gtcatgtata aagtgcttag actagtgcct agcacataat tagtgatact 19560taagtgtttg ctcttactat tagttggatt aggctatttt tgcattgcta tgaagaaata 19620cctgagactg ggtaatttat aaagaaaaga ggtttaattg gctcatggtt ctgcaggctg 19680tacacacatg gcacactggc tctgctcggc ttctggggaa gcctcaagga gcttttactc 19740atggcagaag gcaaagcagg agcaggcacg ttacatggtg aaagcaggag caaggggtga 19800ggaggtgcca cacttttaca acaacaagat ctcgtgagaa gtcacccact gttgtgagga 19860cagcaccaaa ccttgaggga tcctccccta tgacccaaac agctcccacc tggccctacc 19920tccaacactg aagattacag ttcaacatga gatttggagg ggacatccaa actatatcat 19980tccacccctg gcctccaaat ttcatgtcct tctcacatgt taaaatataa tcatcccttc 20040tcaatagtcc cccaaagtct taactcattc cagcattaac tcaaaagtcc taagtcccag 20100gtctcatcta gaaatgaatt ccttctacct ataagcctgt aaaatcaaac aagttatttc 20160ctttcaagat acaatgggtg tatcagcatt gggcaaacat tcccatttga gaagggagaa 20220attggccata agaaaggggc tacaggcccc aagcaagtct gaaacccaac agggcagtta 20280ttaaatcata aagctccaaa atcatcttct ttgactccgt gtctcacatc cagagcacac 20340tggtgcaagg ggtgggctcc caaggcctta ggcagctccg cctcagtagc ttttttatgc 20400tgagattgca agctgatggt ggctctacca ttctggtgtc tcaaggacgg tggccttctt 20460cccacagctc cactaggcag tgcctcagtg gggactcttt gtggagcctt cagctccgca 20520tttcccctcc tcactgccct agtagagttt ctcttgaggg ctctgccctt ggagcaggct 20580tctgctgggg cacttaggct ttctcataca tcatctgaaa tctaggtgga ggatgccaag 20640cttccttcac tcttgcactc tgtgttcctg caggcttaac atcacatgga agctgccaaa 20700gcttatggct tgcaccctcc agaggagtgg cctgaactgt acctgagccc ctttgagcca 20760tgtctggagc tggagtggct gggatgtggg aaacagcaca gggaagtagt gcccctggcc 20820tggtccctga aaccactcag tcctcttagg tctcagggcc tgtgatggaa ggggctgtct 20880cagagatctc tgaaatgttt tcaaggcctt ttacccattg tcttgactct cagcacttgg 20940cttcttttta gttatgcaaa tcgttttagc aatggttgct ctacagcctg cttgaattta 21000tctccccaca aaaatatttt gttttgtttt gtttttgaga cagggtctct gttgccaggc 21060tagagtgcgg tggcatgatc atggctcact gcagccttga cctcctgggc tcaagggttc 21120cccctgcctc agcctcccaa gtatctagga ctacaggcaa gtgccaccat gcctggctaa 21180tttttaactt tttttttttt tgagacagag tctccctatg ttgcccagac tgcggaaatg 21240cttttctttc tctgccacat ggccaggttg caaattttcc aaacctctat gttctgcttc 21300ccttttaaaa ataaggtcca actttaagtc atttttttac tcccacatct gagtgtaggc 21360tgttagaagc agccaggcga catcttgaat gctttgctgc ctagaaatct ctgccagata 21420tcctaaatca tcactctcaa gttcaaactt ccacagatcc ctggggtgtg aacacaatgc 21480agccaagatc tttgctaagg cataacacat gtgaactttg ctccagtccc aataagttcc 21540tcatttccat ctgagacctt ttcagcctga acttcactgt ccatgtcact gtcagcaatt 21600tggtcacgat catttaatca gtctttaaga cattgcaaac tttccctcat cttcttgtct 21660tcttctgagc cctccaaact cttcccttct ctgcccatta cccagtttca aagctgcttc 21720cacattttcc agctatcttt atagcaatat ttgactctca gtaccaattt tctaaattag 21780gttgttctta cattgctata aagaaataac cttcctgggc atggtggctc atacctgtaa 21840tcctagcact ttgggaggcc aaggtgggtg gatcacttga ggtcaggagt ttgggaccag 21900tctggctaac acagcaaaac cccacctcta ctaaacatac aaagattagc ctggcatggt 21960ggtgcccacc tgtattccca gctagtcagg aggctaaggc aggagaattg cttgaacctg 22020ggaggcagag gttgcagtga gccgagaaca tgccactata ctccagcctg ggtgacagag 22080caaggccctg tctcaaaaaa aaagaagaaa gaaagaaaga cagggaggga gggagggagg 22140gaggggagag agagagaaag agagagaaga aagaaaagaa agagggagag acctgagact 22200gggtaattta cttaaaaaag aggtttaatt ggcctatagt tccacaggtt ctacaggaag 22260catgatgctg gcatctgctt ggcttctggg gaggcctcag gaaacttata atcatggtgg 22320aagtaagaga gggggagcag gcatgtcaca tggccagagc aggagcaaga gaggggagag 22380gtgccacact ttacaacagc cagatctcgt gagaactcac

tatctcgagg acagcactaa 22440gccatgagga atccttccca tgacccaaac acctaccagg ccccacctac aacattgggg 22500atcatatttc aacatgagat tttgagggga caccaaaact atattattgg tcttcgtaga 22560ttaattgttt tagtctgtac aagtcaatat gtagaattat aaagatgaac cctgttgcta 22620tatgaagaaa tctaagaaaa tacttgggtc ctcattttaa aaagccattt tgtatgtgtt 22680ctatgcctga tgaaacactg tgtactgggt agagaaagtt tgtattttaa gctctcccac 22740tggcaataat tagaagttga accaggatca gtatctaagt gtcccattgt ttgttgttgt 22800tgttattttt aaatgaaatt ccttaagatg tgatggagac tctgcaaact atccaaaata 22860gtgtcattcc aacatgttag caatatttag aattattaat gagatacttt acaatctttt 22920tttatactgt ctttgaaatc ctgtgtgcat tttacactta tagaacattt catttcagac 22980tagccacatt ttgaatgtgc aatagccaca tgtgtctagt ggctacccta ttagacagca 23040taattccaac catactccca tgattttttg tttgtttgtt tgtttgtttg tttgtttttg 23100agacagagtc tcattctgtc tcccaggctg gagtgcagtg gcgcgatctt ggctcactgc 23160aacctttgtc tcccaggttc aagtgattct tctgcctcag cctcccccgt agctgggatt 23220acaggcacgg ccaccacgcc tggctaattt tttgtatttt tagtagagac aaggtttcac 23280catgttgacc aggctggtct cgaactcctg gtctcaagtg atccacccgc cttggcctcc 23340caaagtgctg ggactacagg cgtgagccac catgcttggc cactcccatg atttataagc 23400tccccagaca cactgccttg ccagtattca tgctgttgct tcagtcaaga atgaattcct 23460cagcgctacc tctcttgacc ttcacatctt acttagtgca aatgctgtct cctcctgaga 23520cttgcctgac ttcggatact ctccctgtta catcttattt aaaatgtcaa gtagactggg 23580cacggtggct cacacctata atcccagcat tttggggggt tgaggtgggc agatcacttg 23640agcttaggag ttcgagacta ggctgggcaa catagcaaaa ctccgtcttt aacaaaaata 23700caaaaaatta gccaggcgtg gtggcatgtg cctgtggccc cagctacgtg ggaggctgag 23760gtgggagcat cgcttgaacc tgggaggtgg aggttgcagt gagctgagat tgcaccactg 23820cactccagcc tgggtaacag ggtaagaccc ccatctcaaa aaaaaaaaaa aaagtgtcaa 23880gtataccact ttatagcact tgtaaagtgg tatgttgatg tggtttggtt gtgtccccac 23940cagccaaatc tcagcttgaa ttgtagtttc cataatcccc acatgtcatg ggagggaccc 24000aggggggtaa tttaatcacg ggggcagcta ccctcatgct gttctcttga tactgagtta 24060gttctcacga gatctgatgg ttttataagg gactttttaa aaaacccttt tggttgggtg 24120tggtggctga tgcctatatt cccagtgctt tgggaagtca aggtaggcag atcacttgag 24180gtcaggagtt taagaccaat gtggccaaca taatgaaacc ctgtctctac taaaaataca 24240aaaagaaaaa aaaattagct gggtgtggtg gcacacaccg gctaaggcag gagaatcact 24300tgaaccccgg aggcagaggt tgcagtgagc tgagatcacg ccaccgaatg ccagcctggg 24360cgaccgagca agactttgtc tcaaacaaca acaacaacaa caacaacaga caaacaaaaa 24420cccttttgct gggcacttct ccttgctgct gccacgtgaa aaaggacgtg tttgtgtccc 24480cttctgccat gactgtaagt ttcctgaggc ctccccagcc atgctgagct gtgagtcaat 24540taaacctctt tcctttataa atgactcagt ctcaggtatg tccttattag cagcatgaga 24600acagactaat aaatatgtgc atccattatg acagtgacta attgcatttt aataattggc 24660ttaggtatct gtctttcttc ccaatgtact ggaagctcca tggggtgcag tgaccaagtc 24720ctttggtctc tgactgtcta ctatttaatg aatatttgtg gaatatgtaa attaaacatc 24780ttcaactgtg gtttaaattc ataggacaca tggaaaaact aacactatag gacacaatta 24840ttaaaaagta tttaaaagta atgacaaaaa acgcagttac tttgcaccca cctaatagcc 24900ggaaccatag gacttagttc aggaaaccca aatataggtt caagaaaggg gaatcaggcc 24960ctgaaaatga atggctttgt taggaatatg catatgtgca gtgcaggctg ctaagagctc 25020cttacagggc tgagtgggtc gtgagatctg ggatttgtag aaaaacatgg ctttttaggg 25080agctgtccaa accttctatg gggcctgtta gtctctacat gtgccttttg cggtcattca 25140ctggatttga tttctgtcac ttgtggcttt atgatgctat tgtttaggct ggataagaca 25200gaacattcca gaagcttctg ctccagtgtt ttctcccaac tgagtcagct gactccatca 25260attgtatgcc agggggcctg gggcaaagga aggggagggc atgtagcccc tttcgccagt 25320tagtacagat tggtgtggtg gtgggaaacc tctgagatgg ggaagggcca agcaagactg 25380tggagggcct aactcatctg acacaggatt tacagtggca catttcccaa acctttcaca 25440tactggggag ttcagaatag taatgttttt ggaaaaagat gatgggcatt ccaaatgttg 25500ataacagcca gtcagtctgg tttaaggaat gaagtgtatt atttacattt ttgtaaagct 25560gcagattgag cagccctgga atgggtctgt gaatacaagg ctcattggcc tcatgatcct 25620tttgctcagg gacatatttg tataaaattt gtatcaaagt tgtggtatcc catggcttct 25680gcagcctttt tgaggtttat tttttatctt gtcagatacc tttttctatt ttgcctacca 25740tttaacattt caagaattaa aaaagaaaac aatttgtcta agggcacttc caattaattt 25800gcgtatttct aagaagtatt tactaaattt acagtatccc gtgaaatatc ttattttggg 25860gtttcttagt tttgctgttt ggattattca tttctatttt aggcttgtat tatttaaaaa 25920gatgtaaggg ctagatctta agttatttta ttattcattg ataacaagta atactggatt 25980ttcaaatata atcaacatat ataatatata cagcattatc tttaagagtg tagactatat 26040tttatattat aaaaattaca catatatatg tgttccatat atataatcac taaagatcaa 26100cttgaaaatg cagtaaagtt ttgaacaaaa actactaatc catggcttca ctgtctcaaa 26160aataaccaat gctaactaac attttagtgg atatcttctt ggccattttt ccatggatga 26220gttttagatg gttatcatac tatgtacata tgcatgtaca tatacaaaat atacatattt 26280tttattcacc taatgttata gcataggatt tttcaatgtt atattttggt cttcatacaa 26340atgaatttta atgatgatta tatttaatag agtaaatgta ttactacact taactgttcc 26400tttcctgaac atgtagatcc gttttcattt tctcctctgg ttacctcgtg ggttgaatgt 26460tctcctcatg tttgccagcc agttgtgttt ccacactttt agttcttgtc ctttgtccat 26520ttgcttatct gggttctgga gtgttcatgg cactctgtga accttttatg tttcatttgg 26580ggcaactgct tttccattgt ttaccttgtc aatttttaaa atgtgttgta gtcaagtcta 26640ttctttttca acatttctat caggtctgag ctttaaaagt cttactccct tcaatggttt 26700gatgttcaag tcgattttct tatagttctg tttttccaca tttaatttct tcaggatcta 26760agtgtcttcc ttgcaattta gcaaaggcag gaattttgtt tatactgtct aattctgtcc 26820tattaaccct ttagttcttt aaccgtgtct gagccaagct gtataagaaa tttttttaac 26880ataaagagac aacaaaaaat ttaggaacac cgtgatcatt gtttccatta ttataggaga 26940atgctttctc cttttttcta aacttgcctc gcttttctct gatagtgttt ttagattggt 27000agctaaaact gtatttgttt tgccaaaata gaaacagatt ttgaaaaaaa aaatccttaa 27060actgctcaga agtaaaaaaa aatagaaaaa gatctctcac accatcttat catgcacaaa 27120aggttgtgac caggtatgtg tgagttttaa atgcacgtgg tacattaggt gaattgccta 27180cgtgagccta tgggtcactg cctggggaca gggagaccag ggagatattt ccatcttaag 27240gagtgaaact gtttcttgca cactgtcaac aagcgggtga gtcttctccc gaccctgctc 27300ttaccacttc ttctcagtat cctgctttgt atctacaggt tgctgccttc agttacctga 27360gcgggcagcg tactcaaagt taagggtaga ggctcctcag cctgtgcctt tggctggctc 27420cccttgcttt ccctcttctc tgtggtcacc ctaggacttc agtactcctt tcacgttgat 27480gtgttcatca ccccagcctc gcctggatct gtgatggtct tttaaagcgt gtcctcctcc 27540tccaggacca cagctttctc actccccaca tgggaatgct ttcatgcttg ctgcccacct 27600tcgccccacc gctgcctctg ttggaacacc cccttcaccc tcccacctgc agaagcccac 27660tctttccaga atcaagcaaa agtgtctttt gcaggagtcc ccttccagtt gccatccctc 27720cctcctctgt atgcccacag tgcgtttact gtgaaggagg tggcctctga aggcagtctc 27780ccatgctcta ctgtggattc ttggaggacc agaaccagga cttaatgtca caccatctca 27840agagcttagc acaagccgtt gcacatggta tcagtagcta atccctccta agcagcaggt 27900gtgccaggta gtgttgtaac tggatttatg tggatttcat tctcacaaca accctacaag 27960gtgagtacgt tattacctcc attttacaga tgaggaaact gaggcacaga agcagtgagt 28020aagatgaaac cagggttgaa tgttaaccct accagccaga tctcagtcta gtttgtatcc 28080agtgtatgcc caatcacttc ttaatagtaa ttctcaacag aggattaggt gaatgaagga 28140accaatatga ctgaaagata acgatactat gatagtatta tacgcatcca atttatcaaa 28200cacctctgag gaattagata taccgatgtt aattttccac ctaattaaaa ttattccttt 28260tgatttccac tccttatctc agtctattgt ttctaaaatt atttctaaaa ccaaccatga 28320attttcctgc ctgattaaat agttcatgga gcataactga accttgtctt aatgacaagg 28380accgtcatag taatgcacaa atgttatact atagtctgag attcattagc atctactaaa 28440taccagggac catgccgggc acttttaagt atagttcctt gattaattgc ctaggctgaa 28500aatttgattt gttttttgtt gttgtttttg ttttgttttt tcaaactctt ctttgtattt 28560ctgaaagttc atttggtctt ctttgatctt gtaggacagt aactgatcaa tatgcccctc 28620tctgcaggca ccagccttgg cttacacaaa agtttggctt tgaaatttcc tctcctcctc 28680catattccaa taaattactg tgaattaacc tctgtgtaaa atcaaggaat accctgaatg 28740agaggtgagg tcaccaggat tctaaacctg gccctgacac tttcgattgg tcctgaatcc 28800acatatcctt atctataaaa aggtggagtt aatgatttct aagtgaaggg tctttggtta 28860taagcaaaat aaactacctc tggctaattt aagtaaaacg ggaatctatt tgaaggctat 28920aaagtaggtt aacagaatca aggaaagcag cttggaagac aaaagcagga tgcagggcag 28980ctttaaagat ccaggtcacg agagtgtatt gatcatcaca tcatgtgtct gccactggga 29040ggaatcctgt cttttactga gttttcttgt ttttaaatta aaattttttt tttttttggt 29100agagacaaag tcttgctctg ttgctcatgc tggaatgcag tggcatgatc atagctcact 29160gtaacgtcaa actcctgggc tcaagccatc ctcccacctt agcctcccaa gtagctgaca 29220ctacaggtac atgtcaccac gcctggttct tttactgtgt tttctatctt gaggcactgt 29280gctcaggatt cagaggccag aggaaaggaa ttggttgagc tcaggtccca tagcaacctt 29340gactgatggt cttacacagc tgtacaatac aagagaggaa agcaaatcag ggctattacc 29400agcagaaagc ggaatggttg tctaacaaat gaaaagtcca ctgcaaacac tgaacttcaa 29460gctgtgtaag tggtttaaca cggaagtgtc tatcaaattt ttaaatatat tctttgtaaa 29520ctggaagggc atgtaattta tcattagatt tggctttggt tagaggcatt tgttagaggc 29580attagctggt ttcatcctac aggcgtttct taggtacctg ttaggagagg ttgtctcata 29640cagagagagg agcaccttgt gctaatgaga aagacttggg atcaagttct cactcttctg 29700cctgtgctct gtgtccccag ctgtgaagtg gggcaatact acatgctgta tttgtactac 29760atacttgtaa ggattaaata aaataaaata atgtgggtta aactctttga agagctatgc 29820aagggtcagc tatttatagg attagtgggc atagcttggt gcttagtgtg atatgatgtg 29880tgatatgtgt atatgtgata ggaagtatgc ttccttctat acacttgaca gagtatagga 29940tatagacatg tgatatgatg aaatatatat ttagtcttcc tccatttcct ggcatacagc 30000tcctaagatc cttggaatcg cttaagtgat aagtgtcttt ttgtatgcta gtgaattgac 30060tgatgactgg cagcctctat ggctcctgga tgggagctgg tcctgaaaga agaggacagg 30120attagagggt tgggactttc agccccaccc cccaacctcc caggagggga gaggggctga 30180aagttgggtt gatcacaaat ggccagtgat ttaatcaatc atgcctatgt aatgtagctt 30240ccataaaaaa ccaaaaggac tgaatttgga gagtttccag agaaccaaac atggggaggt 30300tcctggagag tagcgagcct gcagagggca tggaagttct gagctccttc ccctacacct 30360tgcccagtgc atctcttcat ctgtatactt tgtaatatcc tttataataa aatgtaagtg 30420tttccctgag tactctgggc tgttctagca aattaattga gcccaagcac agggggatgg 30480gaaccccaat ttatagccat tagttcagaa gcacaggtaa aacagcctgg ggcttttgat 30540tggcatagga ggtggaaggc agtctcaacc tgtagaatct gatgctatgt ccaagtagaa 30600agtatcagaa ttgaattgaa ttggaggaca ctgcagaatt attggtgcag agaaaacgcc 30660acacacttct tggtgaccag aagtctcaga agtcttagaa gtcttctgtg taagttgtta 30720cagtatgaga gcacagaaaa aaatggtttg ttttttctac tggagaagat acaaagactc 30780atctatttgt atgcatctat atgaatgcat ttattttctc atctgttttt attttaaagt 30840ctagaaggga tttaagaggt ataaagagat acaatataca gacagtcctt gacttatgat 30900ggttcaactt gaatgatgtt ttgacttcgc gattgtgtga aagtgatgta tttggcatgc 30960ccctccaatg acgatagagt tacatccaga taaacccatt gtaaattgga aagatactaa 31020gttgttgaat caaaaacaaa ctttcaactt acgatgtttt caacttatga tgggtttact 31080gggacataac tccatcatca ccgaggcgca tttttacgta ccagtttcag ccttcagctt 31140gcttgcagtc tacctgaggc tacatgataa tttcacaatc taaatgatgt cactcagaag 31200tttctaattg aatgctaaag acatgtacgg acatccacag gtgggtgaga aaaatccctg 31260tgctgctatg gttgggaaag gcctcagagg aggtgtgggt agcccagctg gccaatgcct 31320tcctaacctg tgctcttaaa ttagctctgt tcaatagaaa tattacatga accacatgtg 31380taatttaaga ttttaagatt ttctagtagc cacatttttt aaaaagtaga aggaagcagg 31440taaaattaat tttgataata tattttaatt ttacccatta taactgaaat agcatcattt 31500caatatataa tcaatataaa aaaaggttgg cactgttttt gaaattctgt atatatttta 31560cacttacagc aaatctcaat tccagttagc cacagtttaa gtactcagta gtcacatgtg 31620actagtggct accatgtcag acataataac cctagctggt ttaggaagaa ggaaagataa 31680gcaaagggaa agaaacaaaa gtcactggaa tttattgaac acctactatg taccaggcag 31740ggctagggct agggctagtg tgaggcattt gatgaacaaa agatcagatt ctgtttttat 31800ttaaaatttt gttcatcata aatttttggc ataaattttg gttttaataa atagtgcatt 31860aaactattta tcttgattac tacttgttta ggcacctctt acatcactca cactgactct 31920aggcccagcc ctggcaccag gcgctttcta agaactttct aatgcttcat ttgggggctc 31980taacttgaac caaactgtgc tgtatatcag aaacagagac cgggtgtggt agctcatgcc 32040tgtaatgcca gcattttgga aggtcgaggc aggaggatca cttgagtcca ggagttcaag 32100accagcctgg gtagcatagt aagacctcat ctctacaaaa aaataaaaca attagctggg 32160tgtggtggca tgcttctgta cttctagcta cttgggaggc tgaggtggga ggattgtttg 32220agcccaggag gctgaggctg cagtgagctg tagtcatgcc actgcactcc agcctgggca 32280acagagcgag accctatctc aaaaaaagaa acaggagtga actcctgtaa tttactaagc 32340cttgggcatt attttttaaa ataaaatttc aatttaattc aatgtagaaa caagaagaca 32400ttggtaaaca aattgaaatt caaactaaat ctgtgaacca aactgaaaat tttgaaaata 32460cttcaggacc aaaccataac ccaggagaga gttcatcggc tttgattccc ggtcaaggtg 32520accctcaact attgcacctc cctaatcagg gaagaaaaga aggaaaacaa agtttttgag 32580aagactctat cctaggcctc gtttccttcc cacattatag cttcgatctc cagcttttta 32640aggcttagta gctgcagtct ctatataatg ttatattggg aatgttatat tgaaaatata 32700acttggatgc tgaaagaaag gaaaagtctt gcttattaaa gtggtgtcta ttttcaaata 32760aaaccctttt ctttctttta gggaactcag aaacaaagca aatattcaat ttggagataa 32820tggaacaaca atatctgctg ttagcaacaa ggcctatgtt tttgaacgag accaatctgt 32880tggagaccct aaaattgact taattagaac attaaatatt cctgtattgg taagtaggca 32940ttttaatacc attaagttgg attttggaag aaaggaaaaa gctaagttaa tgctctttat 33000gattttacat ttaggagcca ttaagttatg ttgaaatata catatataaa aagaaaaata 33060cttcttttct tttctttaaa gtacatcttg gacttttttc taagatgtat caaataaggt 33120cttttcaaat gatgacaaaa ttcagatttt aggatttgtt ttcataattt cttcctgggt 33180gttcaggatt agcaattgtg caaataaata tgcccctagg attttttggt agctaataat 33240tttaaaaatg tttatttgcc ccttgttttc attcagaaat tctagcaatg aaatttgtat 33300accatttctt acttcagatt tttatgttgg attttatact ctcaaaattc ataaattgac 33360accagaaatc aatagactgg cagcctactg tgatttcagt gggctctgtt tataatggta 33420attctaccta ataacgacac ctattctaca tctattctat actgagaccc atgagacttt 33480cttttttttt aaattaaatt aatttattta ttattaattt tttttgagac agggtcttgc 33540tctgtcgccc aggctggagt gcagtcagtg gctccatcac agctcactga agcttctacc 33600tcctgggctc cagggatcct cctgtgtcgg ccagggactc caggcgtgaa ccactgttcc 33660tgggctggcc cagtaaacat tctaattcac ctacaccaac ccctgccgac tctcccaccc 33720taacccctag gtgaccgctc catatgcagc tggacactga tgatggaccc aggacctaga 33780cagtatgagg tccctgccac ctagtcccag gggtgtaggt cgggtttata atgagcctct 33840ggccctgctt aggcggaagg tagcgccacc ttgtggccaa agatgcccac atgtttacaa 33900gagcttttcc ccttttgtgg tgtctggata actcagagcc tgcaaatggt ctctttgtaa 33960atattacact tacaaaatgc agtactggga aaggacagag aagaggcttt gtcttccata 34020tgcagccaga aaggagctca ctgcctgatt tttcagctat ttaatctcaa cctttacatt 34080tatatttcca accccctttg ctatggggta gtaggagaga gtggatggag gtgctttgga 34140ggggatgctg ctgtcttaat atacctctgc tccgcagact gtcatagagt ggtcccaggt 34200gcacttcctc agggagatca tcgaggccat gttgaaagcc tatcagcaga agctctttgt 34260gactcacaca gttgacgaat tgctctgggg ctacaaagat gaaatcttgt cccttatcca 34320tgttttcagg cccgatatct ctccctattt tggcctattc tatgaggtaa gtagattttt 34380cttttcagaa cctctttttt tagctgacag tgggttgggg caccccaagc cagattaact 34440ttgagtttct tctagaaatt gcctagagtt aagttatctt atagagtgtg tgaaaaatgg 34500agtgtttttt ggaaagaaaa aaaacttgag gccgggtgcg gtggctaatg cctgtaattc 34560caggactttg ggagaccgag gtgggtggat catttgaggt taagactttg agaccagcct 34620ggccaacatg gcaaaactct atctctacta aaaatacaaa aattagccag ctgtggtgat 34680gtacacctgt aatctcacct actcaggaga ctgaggcagg taagttgcat gaacccagga 34740ggcagaggtt gcagtgagct gaaattacac cactgtactc cagccagggt gacagagcaa 34800gactctgtct aaaaaagcca aaaaaaaaac aaaaacaaaa acaaaaacaa aaaccctgga 34860gtgcagtgga tagttgggaa gttggtaaaa gataaaataa ccccaggaag aggattagat 34920taggggtgtg ctctgggatt gtgatgggtc tcctcagctt taaatttgta gctgttttct 34980ggctctgcct gttcaagagt cttaccctct tcatgtaatc ctgtagcatt ccagttttta 35040tggcaaacag caataattgt gtccctgggc cttcaaggtt atataaaacg tagaaattgt 35100taaaatagaa ttgtgagcaa gttttaaaaa attatatgtg tgagagacag gagtcctaaa 35160agtagggcct caccaactct gaatgcatca ttcgggagaa gcagaccagg caggagttcc 35220ctagagggtc ctgtctcaga ggaatacagc ttcacaggac cagaggaagc acagatttcc 35280tgtcaagccc tgactgaacc tcccttctag aaataatttc aggagagcct cctactgaga 35340gatattatgc aaaggttccc tcgaggccct tttgttccca gtggcttcaa tccatcagcc 35400aagtagatct tgttgctaat atctcactgt ttctcagata atttgcatta tataactcac 35460tttttttgtt tttacatcat tgaattcacc agactgatag ccacactatc ttatgtggtt 35520gtgaggattt catgagatga ttcatgcaaa aattttagaa cagagccagg aatattatac 35580tcaacaattg ttcactccta ttattataga ggagctacta cctgtgctat ttctgagaaa 35640tcatagctac atgcccaaat aaagaatttt aggaatgtag tgtctgtatg tgcccttaag 35700aagtataact tggagagaat atacaattga agaaacctta attgaaaata gggctatatt 35760aataccgtct tcttccagtg tagctattct ggtataattg gtcctggtcc tggacttagg 35820cttttaaatt tgttattatt gagaaataat tcacttacta taacatttac cattttaaaa 35880tgtacaattc agtagtcctt agtatagtca caaggttgtg caaccattac tgctaattcc 35940agaacatttt cattatccca aaaaagaaac tgcatactct cttagcagtc actcctattc 36000ctgtcttact ctcccaggcc ctggcaacca ctaatctact ttctggctct gtgaatttgc 36060atattctgga catctcatat aaatggagtc atataatata tgcccttctg tgtctgcttt 36120ttaaatttag tgtaatgttg tcagggttca tccatgttgt agcatgtatc agtgttccat 36180tcctttttat aactgaggaa tattccgttg tgtagatata ccacattttg cttatccatt 36240tatcagttga tggacatttg ggttgtgttc cttcttggct attattagaa gtgctgctat 36300gaatatttgt tcatacaagc ttttatatga acattgggta tataccttga agtggaattc 36360tcttgagtat atacataggt gtggaatttt ggagtcctat gaattctgtt taactttgtt 36420aagaactgac aaactgtttt ctgcagtgac tgcaacattt ttatattcct gctagcaatg 36480tataaaggtt ccaatttctc cacatcctca ccaacgctta ctatttttct tttttttttt 36540tttaattgcc ctcctagtgg gtgtgaagtg gtatctcatt gtggttttga tttgcatttc 36600cctaatgact aatgatgctg aacatgtttt tatgtgctct ctggccatta gtatattttc 36660ttttcagaaa tgtctcttca aatcctttgc ccattttaaa attgagctcc tactttttaa 36720aaccataaaa taataatatg gttaatatag tatttagcaa aatagtcact attttctggt 36780cctggttcag tcttgtgtag cagttaggcc ctagtaggac agttagatga agattaaatt 36840ttttagttac tgtatcagtt agggttgcca gagaaactga atcaagaggg tgtatatgta 36900gagatgagaa agagggagag aaagaaattt atattatgga attggcttat gtgattatgg 36960gagctggtaa gtctgaaatc catagagcag gccagcagct ggaaactcag gtaaaagttg 37020atgatgtggt cttgagtttg aaatctgtag ggtgggctgc aacctggaaa tccaggcaga 37080atttcgatgt tgcagtcttg aggagaattc ctcctctaag aaacctctgt ttttgcttgt 37140aaggctttga acagattgga gggggcccag ccactcatta ttgagggtaa cctcccttaa 37200agttaacgga ttgtagatat taatcacacc tacaaaatac cttcaccaca ccatctgggc 37260tagtgtttga ccaaacaact gggcaccata gtctagccaa gttgtcataa aattatcaca 37320gttactttaa atgatcatat tcacatattt tcagaaaaat gggactaatg atggagacta 37380tgtttttcta actggagaag acagttacct taactttaca aaaattgtgg aatggaatgg 37440gaaaacgtaa gtcaaatgat ttatcagtgg aattattttt

tcagttttca tttaaactac 37500atgtctactt ctacattgct taaaaatgtg tttagccttt tttggatgaa aaatataaaa 37560acatgtaaat agtcttgtaa ttattattct acaaatcaag atggtggtaa aacatcttac 37620agattaaata ttagtgattt gtagatttat catggccagg catgaattac aggccagtga 37680aaagtcccac tcttgcttag tagtcattga gtgatgtgta gtagctacag cacaaatact 37740tttcaaacca ccacatgttt tgggctattt ggccattgcc tctatgaaag ctgatttggg 37800ccagggacct actttttctc ccccttgatc taattaaaac tgttgtgtct gtgtatgaat 37860aagatactgt ggatagacag ctccagttaa atcttgcaaa gtcctgacca tttaagagaa 37920tctaaactca aatgtgatca tctttactct ttcttgtgct tatcaattat aggtcacttg 37980actggtggat aacagacaag tgcaatatga ttaatggaac agatggagat tcttttcacc 38040cactaataac caaagatgag gtcctttatg tcttcccatc tgacttttgc aggtaaaagc 38100tttaaagata agttcttttt caaaaataag gtctttcact gcaagaccac caaaagcatg 38160aggaatatat gcagacacac ttattatgca tggatattta tgcataatca gtgataaatg 38220ctgatggcac atcgagacag tatagtgtac tggttcacag cacagacttc tgactgttgg 38280tttgaatctt ggctctacca cttatagctg tgtgaccttg ggcaaattac ttaaccactc 38340tgtacctcac tttccttatt tgtaaaacaa agatagaaat tgttaactat gttagaggtt 38400gttatgagga caaatgagtt agtattagtc aaatacttag cccagggccc atcacacagt 38460aaccaatatt aagtgttggc tcttagttac tatgatatgc tctgtcaagc atcaactgac 38520ctggtctact tccagtcaat aggtagtctc tacatggaag ccaaataaaa gatactgttg 38580tccatatttc agcagagtgg tttcctcagg ggaggcagag aaggaatgga atcaagagca 38640ttacgtagac aggttcagtt atatccataa tataattttt atttgtctaa gtggcagata 38700caaaggtatt tgttagagta ttttatctct ttctggtgcc taaaattttt cattaaaaaa 38760tgctgtaggg gtggtttggt ttgtgcagtg aaaccaccta tgttattcac aaccttgcga 38820gagatgtatc aacagctctg gttcagagat gaaaaagtga ggcccagaag gctcttccgg 38880ccagccttca aaggaagggc tgaaggctca gtggtgactg tgtacatcaa agtggttagg 38940cactatgcag tgtaagcagt ggtcatatta catgggacta tgtttgaaat cagtaagaat 39000gaaaggcaga attacgttga actccattta aataaatgac tggggcaaaa tatggaatct 39060gatttttctt ggaggggggg attgagtatc aaagtgtgta ttccaataaa gtatctgtcc 39120aatgtgacac ttttcaagtg cctaagaccc tacgaggcca cggccatcgc tgcctgttct 39180accttagctt tggctctgcc gtgccctggc ttccctggct ttgccggtgg cccgtccaca 39240ctggcatatg tccatgcagg tctagagagg cctcttattt cttttttatg tgagtctcca 39300ttctaaatgt tatcatctta attttgtacc acatattgtt ttttcttgtt tggatgatat 39360caccgttgct acaataaaca ccttccacta caattcatct tctgctttat tacacgttga 39420gttgacagaa tgccttccaa acattccttt ctacttaccc tgtgagcttt tggccatgtt 39480gtacctgaga actgttgcta acttgcggat ttcgggcaga ttacactgac cagagtccac 39540attcactttt tattcttaca tcctaacagg tcagtgtata ttactttcag tgactatgag 39600agtgtacagg gactgcctgc ctttcggtat aaagttcctg cagaaatatt agccaatacg 39660tcagacaatg ccggcttctg tatacctgag ggaaactgcc tgggctcagg agttctgaat 39720gtcagcatct gcaagaatgg taagaactca gagaggggac atgatagggg tgtcaagaat 39780gcagaaggat tggagttcaa caaagaatat gtagctgggc gtagtggcgc acacctacag 39840tcccagctac taaggatgct gaggtgggat gatcacttga gcccaggagt tctcaagtcc 39900atcctgggca acatggcgag accactgtct ctaaaaaaca aaacaaacaa aaaagaatat 39960attgtcagcc caatagaaag atcagtttct ttcaagaaca atgtttcata acatggctat 40020tgaatggtaa aaaaaaatca tggcttttct gtgatgcttt ttatttctag aatgccatgt 40080tgtgtttttt tttctggaca gtggtttaga agtcttaaca gtaataatag ctcatgttta 40140ctaagcacct gatgtatgcc agtcactgtc ctaagtgctt tacatattta catgtagtta 40200atgcatgcaa aacattaact gatttcatgt tcaaaacaat gctaaggctg ggcacggtgg 40260ctcatgcttg tattcccagc actttgagag gccgagatgt gaggattgct tgaggccagg 40320ggttcaagat cagcctgggc aacatagcaa gaccctgtct ctacaaaaaa aaaaaaatta 40380aaaaattagc tgggtgtggc ggcacacacc tgtagttaca gctgctcagg aggctgaggc 40440agagagattg cttgaaccca ggagttcgag gttgcactgt gctatgattg tgctgctgca 40500ctccagcctg ggcaatagag tgattccgca cctccccacc aaaaaaaaaa aaaagctaaa 40560agaaaaagtg gacaactcag ttttaagaaa gtacgtatca tggcaaagat tctcagtgat 40620tttgaagcag agttagtttt aaaccctgac acaaatgtcc cagtgtttct taatgtcaga 40680gctactctgc tcctttccat gaatgaggca gaattatcag tggcacaact gacaaaacta 40740ttttcttcat cttgaagagt agttttaaca gccacatgtt gcagaattat tcacaatgaa 40800cttactagat agtagacatt cagcggcctg tttttataat tggggaaact agggctcaga 40860gagggaacgt gattttgatg cgactcctcc ctctgagcag atccctggcg gaactcaggg 40920tcttctcata tttgccctgt gattccctct tgggctgtaa ttcctcttta aaatttctct 40980ggagagctct ctgtgtagtc cctttctcct gaatagtaat gtgctcactt ttgctccctt 41040atctatctca ttttaaaatc tcctttcagg cctggcatgg tggcgtgtgc ctgtagaccc 41100agctactcag gaggctatgg tggaagggtc gtttgagccc agtttgattc tagcctgggc 41160aacatagcga gactccatct tttaaaaaag aaaaaaaaaa aaagaataag atatcatttt 41220ggaataagtc tgaagtcagc agtatacctc ctgatccttg tctgacaggg cagtgacccc 41280cacacctggc tttacatcag aattatctgg agagttatta aaaatatatt ggtgaggcat 41340ggtggatcat gcctgtaatc ctaacacttt aggaggctga agcaggaggc tcacttgagg 41400ccaagaattt agacctgcat gggcaacagg gagaccccat ctgaacaaaa aatttaaaaa 41460ttagctgggc atagtagcat acacctatag ttccagatac ccaggaggct gaggctggag 41520cgtaggaatt caaggctgca gtgagctatg attgcaacac tgcactccag cctgggcaac 41580agagtgagac ctggtctcaa aaaacaaaaa aattatatat atataaaaaa ttgtttatat 41640atacgtatat catatatatg tgtatatata catatatata tacaactgac aaaagtattt 41700ttcatcttga agactagttt taacagccac atgtatatat ttatgtatgt atatgttaat 41760atatttgtta atatatgagt atattaacaa attttatagt tatatataaa tatgcatata 41820tttgcatata cttatatttg ctatatttta atatatatgt atatagtata tatatatagg 41880tatacacaca cacgtgcgca agtgtgcaca cacactcatg gtgcacacac atgcatgtat 41940gtcccagggt cccatgttgg atataccagg gccccagaat ctacactttt aacaagtact 42000ccagctgatt tccataaaga tggcgtctag gtatttgggg accattgtgc aagggaacat 42060ggtacccttt acctggagat aaagaccaag ggtgaatggg aacaagttcc tggttgtgat 42120ctcccccatt gccttactgg taatagtcct gaaacagtgt ccctgccccc tcatctggtg 42180tgcatctggc tcaaagcaaa aatgtagata tattttctct tgctgttttt attaaattct 42240gtttccttaa gaagctgatg ccagaatcaa cagaagtatt ctactttaca catttaaact 42300tcttgctaca atccttttcc tcttccctct taacgtattc ccgaatttgg actgaagtga 42360aactccccag gattatcctc cagtgttttg aattctccag ggaaacattt tggatagcag 42420gatgagtgtt ctggagtcag gcagattggg tttgagtcct attctatttg ttggtaaatt 42480ggccaaagtt tattaaggag ggttccagac atcagttttc tcattaataa aatggaggtg 42540gttatagctt cttgaggttg tgtgagagtt agaaataata tatataaaac actcaggaca 42600gtagccccta ttaagccctc aacagattta aaaaatacat tttcaaggtc attataggtc 42660acaaagaagg aagtcatgcc tttgtggaac tattcagcag taaacagcaa aatgcatgtg 42720catgtgagag atgagggatg agttccttaa gccatgggct ggtgaatatg taagcaaatt 42780taaataactt gccgacaagc tcctttctcc aaaaatgtat ttccctggca gcaattgttg 42840ggtccctctc ttcttttcaa tagccagatc tagaataggt ttcctttagt aacttgtttc 42900cctatttggt tatatatata tatatatata tatatattta aatttacata agcacttatt 42960tggttcttaa aatgaatagg aaaggaaaaa ccccagaatc tttgtgatct ggttagaggc 43020tgggtgtata gcacaaccag gctaactctt aaaaaattat gtaggccagg tgtggtggct 43080caagcctgta atctcagcac tttgggaggc tgaggcgggc agatcacttg aggtaaggag 43140ttcaagacca gccttgccaa catggtgaaa cgctgtctct actaaaaata caaaaaaatt 43200atccgggtgt ggtggtgcat gcctgtagtc ccagctactt gggaggctga ggcaggagaa 43260ttgcttgaac ctgggaggcg gaggctgcag tgagctgaga ttatgcctgt ctttggtaaa 43320aacaaaacaa aacaaaacaa aaaaccacca aaaatttttt tttaatttta aaaaggacaa 43380gaaataaaaa gtcagtccac ttactgatag agaattgtag ttgaaactag tgctttgagg 43440tacaacaccc ttcattcact tagtattctg ccttcatagc ctgatggcta gctgtccctg 43500ggtaaatttt gtaacttcta tgaacctcac tttcttcaaa tgcaaagtga ggctaatagt 43560tctgacttta ctgggctgtt gggagcatta aataaaataa tgtgtatgaa gcacttatac 43620tttaacttca gtaaatattt gttgaagaat aaataattta gcctgaactt caatatataa 43680tgtggaataa aaaattgggg gtaaatgaga tgtcagaaaa tgaagtataa atcctgaact 43740aggaggtaaa ggatggtgtt ttagcctacc caagttaatt tatatgtagc ttttttgttt 43800ttctttagga accaggctgt ggccaggaac ttttatattc agctcagctt agattttaaa 43860aacagcatca ttcatttctg ttttccttca aatgcaggtg cacccatcat tatgtctttc 43920ccacactttt accaagcaga tgagaggttt gtttctgcca tagaaggcat gcacccaaat 43980caggaagacc atgagacatt tgtggacatt aatcctgtga gtacatactt cctcctggta 44040aaagagcata tgttttattc tgtctataca ttgttcacca gtttagtcct gagccttctt 44100taaaaatatc cttactttca tatacatatt caagtgggag aaattcagct cagtgctgat 44160ggaggcattg tctcatagcc ctgtaattag aaggctgaat tttgtggatt gaatagcaga 44220aatacgagca aacctgcctt taacaaaaag caacagtgaa aactcagtta ttggaaacta 44280ctgatgtacc catataacct ggtcaatctt ctgatgctag ccttggacct caacatatat 44340ttatttctcc ccttgggaca cagatcgata aggttaaaga tgtccctttg cttcctcact 44400gccaggaata ttttggttga aatatgaata tgtatattaa cctcagtcac ttttggcaaa 44460cttctttcta caggttattt gagagtttat tatattatta attgtaaagg tatcagatat 44520acatagagga gacatacaca cgtgtgtgtg tgtgtgtgtg tgtgtataat acacatttac 44580tcctgtattc atagacgaga aaaacaaaaa ccatgctaac catgtcttca tttttggaat 44640ttgaacatta tggaccaata aaaaaaaaaa acaagaacat tcttttagca cagtatttta 44700tgggtacttc acactcttag ctgcatacat tgcatttgta tctgagaaac cagtgatctt 44760atgctctggt gaataaaatc gtgtgtggtt ttctcccttt tctcttagcc atcagctgat 44820cagttccctt ggatcagcca aactgttagc agtgcttgtc accaagggac gaacacagtc 44880ttataacctt agtgcaaaga tggagctgcc ctaaggatga caagtgctgc agctgtgccc 44940catggctacg ctaggcgggc cctatactag gatgccactc tctggcagtg accaccaccc 45000tcttcttagc tctctggaca actgcctgga aatccagacc tgacaggcta tcttttaggc 45060ctccttcctg ttggatccct tctaaggctg tgctattcag agtggcagca tcctgggagc 45120ttgttagaaa tggccagtct actgaatcag agcctgcatt ttaacaggat tcccaaggac 45180ctacattcat actcatgttt gagacacact ggtctatcag ccacctaata caacccatgg 45240ttctcattct gttctgggga tcacttttac ttgtttccac tctgcctgtc acacatggta 45300atggtcacct tgtttatgag gactacacag aaatggtgct ctattgaaaa agttgtttgc 45360agtttaataa atcctaatgt ttccttttca cttctctgat ttgcagttga ctggaataat 45420cctaaaagca gccaagaggt tccaaatcaa catttatgtc aaaaaattag atgactttgt 45480gtaagttttg cttctttcta tggaggggac agcagttttg ctccattgcc cgtctggtgg 45540ggttgatgtc aggaggagta cagcctactt catctaagcc tgctcacccc tgcccttcag 45600ggatggggca tgatcagcat tttcatgggt gccttggtct tggaatttac ttgttttatt 45660taatcataca cttacagagt acttcccatg gatcaggtat tgttctcagc accttatgtg 45720gattcacata taaccttgta ttatccccat tttacagatg aggaaacaga agcagagtgt 45780ttaggtaact tggctaagcc ttactatcta gtcaactttt atccactcca ctaagccttt 45840ccctcatgca ccttgactct cttggtgtat tagactgttc ttgtgttgct ataaagaaat 45900acctggctgg gtacagtggc tcatgcctgt aatccagcac tttgggaggc caaggtgggt 45960ggatcacaag gtcaggagat caagaccatc ctggccaaca tggtgaaacc tcgtctctac 46020taaaaataca aaaattagct gggcatggtg gtgggtgcct ataatcccag ctattcggga 46080ggctgaggca agagaatcgc ttgaaccagg gagtcggagg tcacagtgag ccaagatggt 46140ggcactgcac tccagtctgg cgacagagca agactgtgtc tcaggaaaaa aaaaaaaagc 46200gggggggggg agggagggag ggaaaggaaa ggaaatacct gagattggtt aatttataaa 46260gagatttaat tggcttacag ttctgtaggc tgtacaggaa gcatggtgct ggcatctgct 46320cagtttctag ggatgcctca gggagctttt gctcatggca gaaggagaag caggaataaa 46380gcacgtcaca tggggtgagc aggagcaaga gagagaggag gggaggtgcc acacactttt 46440aaataacaga tctcatgata actcactatc acaaggacag caccaagcga atggtgctaa 46500accattcatg agaaatccac ccccatgatc cagtcactgc ctaccaagcc ccacctccaa 46560cactggggat tacatttcac cctgagattt gggggaacaa gtatccaaac tatatcactc 46620agagacttga gacagagtct cttcctcttc ctctctctct attcccactg atcccacttg 46680gggccagcca tagctctcat ttgtatagtt ataatctctt gagtctagcc ttacttcccc 46740atgctgcagc ttaatttgga ttccaaagta taatctcctg ctcagacttt tcaatgggaa 46800ttttttagat atgagaaaat tatgatataa tgttgagtga aataaacagg atgcaaaact 46860atgcataata tatttatcta taaaacttgg cttgtttacc tcattgaatt tgccagattt 46920aataccaggt gtcatggctc tttctaagaa gtaggtggtt tacaagaatg cattttgtca 46980tctgcatatc tatttccgag gcagtataat ttagtagtta agagctggaa tcttagaagc 47040aaagtggaat tacatttgtg gctccatcac ttacttgtgg tgatcctagg cctctctgag 47100tcataatttt ctcatctgta aacctgggac agtaatacct actgtaggtg actactctag 47160gaattgaatg agataatatc ccaaaaacac ccagcacaga gctggcacgt ggtacatgct 47220cagttggctc ttgttgcaaa gacagtactt cagtggccta gttacaaaac actctgagga 47280gtgacagttt tgttctgtcg actggagcct tccctgatga ctctaaggag actagtcatc 47340attttaagga atctgccttc agtatcttgt gttttgataa acgtacagca catggaattg 47400ctagacagac tggaatctaa cttttggggt acatgtttgc aaagataact aagaaatcag 47460caaattctgg gtaatttaag aaataagtgt actagaatct tcatataaat gtgtctgtcc 47520gggaaagtgt gccattgtga tgaggatgtt tctgaaacaa tctcatcctt tgtctttgta 47580gtgaaacggg agacattaga accatggttt tcccagtgat gtacctcaat gaggtaagtc 47640ctgagacgga gggagccacg ggtgttttca gactcagaaa aaatccatta tgatgtctac 47700tgttgtagtt acatgtaagt tatgacagaa gggcaaaaga tttttaaaac ctgttattcc 47760tcagaaaatt tcacttaaat gttcacatga atctgcctgt gtctgagagc tgatttggag 47820aaatgatgga aagaaatttt ccttctcaag aacactattt gggcaaaact cccttctggg 47880gagctgctca cgtaggaaac agaaacaggt aaaaggagcc attggcagac attgttgaag 47940cagggaaata gctcacactc agatagatgc cagctagccc tttgtctccc ccttctttga 48000aaccatgctg gtttaagaca cctatgataa attcttttct gaatcacata tcacgtttac 48060tgtagttata tctcctcagt ttctcctgct tccgcccagt ttgagggaaa aaaaaaaaac 48120gcaggttttt ttcttcaata aattcttgat tggcaaactt ctccttttta ttatttttgt 48180gtttcctaaa aatacttgct gtttgtagaa tattgaataa cagagattag caagaaaaag 48240aaaaattaaa atcactattc acaaaatctg tgtcagcctg ataatatttt tattttattt 48300tactcaatgt gttgtacaat tttttccaat aacaacttta aaaggtaatc taatgtttta 48360cattttataa aacatgtgct ctttataaaa ttcttagaaa tttttttaaa aattgattta 48420aaaagtaaat tactgataag tccctctcta tcagaggcag gtgcagttca gatgtcaact 48480cctgatattt tctggtcttg atattacata gttgaaatca tacgatagat acaattttat 48540atcttttttc tccatttagc attatattgg tattcacatt tacaaacttt taagggtttg 48600ttattacaat tctaagatga ggtttttatc gtttcatagg aaaaattcaa gcaatattta 48660ccaatttata ggatctcttt gcaattgctc ctgatcatcc atgttcctgt ttgtgggcag 48720gggcatgaaa gatctttttg aaaatgcaga agggcccttt tctgccaact tctgttcagt 48780agtggcatat ttgaagtccc tccaccccca acttctaatt tgcttttttg tgaaagacta 48840gattaagtcc aggtgtgtgg agtggaacct tgtatttttg ctgcctccta ggaagagcgt 48900gattattctc ttctctagaa aaataaaata ttagacctgg aagtctggaa gtgacattgg 48960agactgttgc cacaccacct ttttaccgag gaggaaactc tatccagtta ttggaaaaaa 49020tgtcttagaa cccaatccac tgactcggtc caatgaaaaa aaatgctttc ttttcttttt 49080tttttttttt tgagacaagg tctcactctc accctctaca gtggtgcgat tttggctcac 49140tgcagcctcg acttcccggg ctcaagtgat cctcccacct ctgccttcca agtagctgga 49200actacaggtg tgtgccacca tgcccggcta attttttttt tttttcctga gatggagtct 49260tgctctgttg cccaggctgg agtgcagtgg cccaatctca gctcacggca acctctgcct 49320cctgggttca agcaattctc gtgcctcggc ctcccaagta gctgggacta caggcgcccg 49380ccaccacccc tggctaattt ttgtattttt agcagagatg aggtttcacc atgttgaaac 49440tcatgacctc aagtgatctg cccatctcgg cctcccaaag tgctgggatt acagtcatga 49500gccaccatgc ccagccccag ctaatttttg tattttttgt agagaggtgg tttcaccaca 49560ttgctcaggc tggtctcaaa cttttgagct caagcaatcc gcctgccttg gcctcccaaa 49620gtgctgggat tataggcgtg tgcaactatg ccaggcctcc agtgcaaatt aagcctaaac 49680taggccagaa ttgttgctca ttatttctat ggaattaagc tcttcagata cctcactcat 49740tacactaggt ggtaattcct ctgctctgca tacactaagt gttattctag tacatttcct 49800tctatcagca gtctctggaa ttctagttag cccacaactt tgctgagcac tttctgtaga 49860gggctgtggt cctgattact atagatgtaa ccaagaaaga aattatactt tccacatacc 49920atttttgcag tgtttcattt atttattcat ttaatcagcc agcattaatc aagcacctac 49980tgtgtgctgg gtgctatacc cagtgtggag gtgactgagg ttaagtcttg ccttcaggga 50040cttcacagcc attgcaagac aaacagttac aatgcagagt gcaaggacaa tgatggaagt 50100agagagtgag atggaaactt agggaggagg cctttgacta ggcctgaggt agccagagaa 50160aacttttgga aggaagtgat gctgagctgg atttttaagg atgaattgga attatctgac 50220ataaaacaaa aggtgatact gtctcaatat ttctgaatac attttacctc ctttttccaa 50280tctattttaa gagtaaaaaa aatgtcttca ggagaagaca agatattaat gaataaatgt 50340cccttctgat tactctgact tataaatgga ttttcagtat gtttggtttg ctaacaggag 50400gacattccca aattgtatta aacaaaggaa agatatagca aaaaacagtt acacactgga 50460tgtttagtca tcaagaatac tattttcttg cctctccaga gtgttcacat tgataaagag 50520acggcgagtc gactgaagtc tatgattaac actactttga tcatcaccaa cataccctac 50580atcatcatgg cgctgggtgt gttctttggt ttggttttta cctggcttgc atgcaaagga 50640cagggatcca tggatgaggt gagaactggc tgaaggaact tcttccttac tggataactt 50700tacctgagga attcaactgt acttcactga agggctgtca gctggcttat taggataaat 50760tctgggattt tatgctgggc atagtgattt cgtgcttatt ttactgctgg accaaatggg 50820aagcaaagga agtgtgtcaa agaaggtgga gggttagaag gtaccatcta atatttgggg 50880aatagaatct ctttggatgt atcccctggg agttaacttt atgtctttga aaacaataac 50940ctgaaagaaa aggaagaata tataacagtt acacatctga tctttggtgt cagactgcct 51000gagttcaaat cccagttctg ccacttaaca gctatacgac cctagaaact gtgcttcagt 51060ttcttcatct gtaaaatggg ataataaaag cctcccttat gagtttttca tgaggattag 51120atgatatagt agacttaaag gacttggccc agtgttagca tatagtaaga gctcaataaa 51180ggtttaagta ttattaaata tgataaaaca gctccaagat tatagagcaa gacagccagg 51240gtcctgcttg gaaagaatcc ctgatgtatc cgaagatacc tatgatttag gtatcactgc 51300ataataaatg attggaacat gggtgtagtt actttcctgg aaggctagga taaagagttg 51360ggttcgcctt tccaagtgac cagcagccca gccaggaacc tgaatacctg caatgagcct 51420tttctgtaga agcccttctt aaattttctg cagcttcctt ttttttacaa cagtggcctt 51480ttttcctcca gtttcctcaa ctaagtgtcc aaggcaaatg tgaagtagaa aataaatacg 51540tgaaaccatc tgagtaaaat tagctgaatt tccctgactt ccatattctt ccattatttc 51600tcttatcccc tgccagtcac tttagcctga attagctgtg aggcaaacta ttttgccatt 51660tctatgtgtg gatcttccag tgtgacttta tgcagtcatt gaaattgtct taagcacaaa 51720tgaaacctca tggaaatgtt aattactgct cctacaatta accttcctca tatattcaac 51780aagcaaggaa ttattgagaa ctaatacctc tgagacacta ctccttagta ggtttcaaac 51840caggaaggag gcaagttgaa tagagtctaa gttttataat ccagatcaac acagagaagg 51900gtactgctct ggtattgagg gtgggagtgg tcagagaagg cctcctggaa aggttaggcc 51960tgctgaatct tcaagaacaa gcagcagccc aagaggggag gtgagtgaga tgagccacct 52020tcctataggt ctctcctcct tccatgcccc cactccccac ccaaactttt cacactgggg 52080agaagtttct gacatgaaca tctaacttgt tttctctctt ctgtgtttca gggaacagcg 52140gatgaaagag cacccctcat tcgaacctaa acattgcctt tgcttggtga agaaactgtg 52200tgagctgtcc tgacctggac gatgacgtgg ggaaaccctc cacctccttg caggcttgtt 52260gcctgttgaa agaaggaaaa agacacggcg ctggcaagtg ataggaacat tctggccaga 52320ggttaaagag caggctgaca tggctggcca ttaagcttta taaaatcatg tgggctctga 52380aattgttctt ttatgtgtct agcaagtatt taataaaccc ttgtatagta attttgttgt 52440tgttgggtgc tggtagctcc agaattttgt gaccactatt gtgggtaaaa tgtctctgca 52500tcacttgtta atgctactgg tctaacttca ttcagtatgc

ttcattcacc gaactttgtg 52560ctcaaaatgc gtatatacca ttttatgttg tattcctcca tttcacttgc aaaacagaag 52620taaataagag ttcgggaccc agggtaaaat ggtagcttca tccaatatat cattcaaatg 52680catctgattt ctaaaacata ttacatttta tgctgatctt cagttcataa ttcttccagg 52740aaaactcagt cttccaactg caataaaata ctgggtagaa tcaaatggga aaggggttgg 52800gtggggcaat acccatgagt tgatagtgat aagctcctaa ggatttttaa cttgtacttt 52860tgtgaacgaa gagaatgcat aaataatgtt ggtgaggata aagtacagat atttcatgta 52920gaattaattg ctagttatga tgcttgtgga tagttaactg tttttttttt agtcaaaatg 52980atcatgctac gaaaagatgc ttctgagaga atgtaatgag taactgattt ttcttcctga 53040gtcgcccttg ccaaatatgt tactgtatta attaatctaa tattgagtga ttatttgtaa 53100aattatgaat atgggaaatc catctatcta cagcctaagt tacacataag tttcagaaag 53160tctgattaga ctaaagagat atttcttctg ggacagccgc cttcttggta attttgaagt 53220tctttttaca agttccttcc tcagtttcag ttctttccag tgttttgtag ctcactgtca 53280ctcactgaat agagaaacgt gtgccctata cttcctgtga caatcatttt gctgacagaa 53340tgatggatgt ttaaaatatt gcacaaagta ctttaaagaa aggtctgtta ggaccagaag 53400cagagacacc acttttcaaa ggacttcttg gtttcagcat aacctaagac agggaattgg 53460gagccatcat atgtcacagt gttcagaatt caagcatatt taagggcatt ttctttgatt 53520ctcaaagttc agcattcatt ttgaattgag aagcctatac atttagctga caaagtgctt 53580atagaatttc ttaacaactg aaccattcaa aaggattttt tttgtttaaa actggatttc 53640aatgtaagca aatgaagaaa aaaatataga tttcatttcc atagcttctt atccctgtat 53700tgaggtaata aattgtttta ctgacaattt ttcctttttc tacactaaaa caatatgtga 53760tatatttccc ctcttgaaga ggcaattcat taaactctca aattttctat agaatcaaga 53820tagaaccttt agatactcca actcaccaaa atgtaaaaaa actaacaaaa atatttggtc 53880ttcaataatg ctaaatatct acatttttag aatttatcaa catttaacta gataattggg 53940catgtcttaa ttatgcatgt acttatccat actaataaaa ttgacaatgc tagtgcatac 54000ttattggttt agtcctatta tcaggatata atcatctgtg aggaggatat tttaaatact 54060gtaaatgata acagttaatg atatacacat ttagactgag ttgcacactg gcagggagac 54120caaaaacatt acttccatac ttgtgtcatg attctttttt ttttgagaga gtctcactct 54180gtcgccaggc tggagtacag tggcatgatc tcggctcact gcaacctctg cctcccgggt 54240tcaagcaatt ctcctgcctc agccacccaa gtagctggga ctacaggtgc gtgccaccac 54300gcccagctaa attttgtatt tttagtggag acggggtttc accatgttgg ccaggatggt 54360ctcaatctcc tgaccctgcg atctgcccac ctcagcctcc caaagtgctg ggattacagg 54420cgtaagccac cgggcctggc ctgttttatg attcttaata gttacttggt ttaaatcaca 54480tttgatacta tccttctgaa aagtctgaga cagatctaca aactacagtc aaaattatag 54540attaagagga atgaatgcac ctatttggct ttaagttgaa gatgaattat ttctcatgct 54600cattttcttg cggcagttat cttagaaaga cccccaaagg ctttgtgatt gtaagcactg 54660tcatgatcac agaatgcaag cttctggtac catgatcctc aacttagaga ggaagaaacc 54720aagacagaga gcttaactca cttctctcag ggaaaattag gagttgagca caggacagga 54780aatgggcttt gccactttta gctccaggct tttctaacca gacttgattt cctcatgttc 54840tagaaagatc actaatggtc aagtggaaca agcactacac gactaacccc tattggggtt 54900tttaacttaa gggaggctaa tttttaattt aaactgctcg agatatgagt tctgcaaaag 54960gtggtccgca tccttggccc tctggacatt atcactaaat tgcttgtgcc tgttaacaag 55020aatactgacc agaatgctct tcatgtagct tatacagttg gttcacttca tgcggttctt 55080gacatgttta tttctaccct taatgcaatg aaatgtttca ttaataaaaa accactttat 55140at 55142294748DNAHomo sapiens 29cctcgcgggg ttgcggcgag cccggcccgc gaacgtcacg tccctgcgcg ctccctgcac 60tctcccgagc tgcgctaggc gggcgccacg gctgcccggc gaaggaaacc gaaaccgagt 120ccgggcccgt ccctccgcgg ccccatccgc ccggtgcacc cggggccgcg ctcgccaggc 180cgcggagccc agagctgcgc gcacgaaccg tgcgccggga gggcgtgggc gtggcgccga 240agggtcccgg gtcttcgacg cctctgcggc ggctcctccc tccttgcagt tggatccctg 300gcgggtgcgg cccggcccgg cccgtgagcg gcgcacagaa tgggccgatg ctgcttctac 360acggcgggga cgttgtccct gctcctgctg gtgaccagcg tcacgctgct ggtggcccgg 420gtcttccaga aggctgtaga ccagagtatc gagaagaaaa ttgtgttaag gaatggtact 480gaggcatttg actcctggga gaagccccct ctgcctgtgt atactcagtt ctatttcttc 540aatgtcacca atccagagga gatcctcaga ggggagaccc ctcgggtgga agaagtgggg 600ccatacacct acagggaact cagaaacaaa gcaaatattc aatttggaga taatggaaca 660acaatatctg ctgttagcaa caaggcctat gtttttgaac gagaccaatc tgttggagac 720cctaaaattg acttaattag aacattaaat attcctgtat tgactgtcat agagtggtcc 780caggtgcact tcctcaggga gatcatcgag gccatgttga aagcctatca gcagaagctc 840tttgtgactc acacagttga cgaattgctc tggggctaca aagatgaaat cttgtccctt 900atccatgttt tcaggcccga tatctctccc tattttggcc tattctatga gaaaaatggg 960actaatgatg gagactatgt ttttctaact ggagaagaca gttaccttaa ctttacaaaa 1020attgtggaat ggaatgggaa aacgtcactt gactggtgga taacagacaa gtgcaatatg 1080attaatggaa cagatggaga ttcttttcac ccactaataa ccaaagatga ggtcctttat 1140gtcttcccat ctgacttttg caggtcagtg tatattactt tcagtgacta tgagagtgta 1200cagggactgc ctgcctttcg gtataaagtt cctgcagaaa tattagccaa tacgtcagac 1260aatgccggct tctgtatacc tgagggaaac tgcctgggct caggagttct gaatgtcagc 1320atctgcaaga atggtgcacc catcattatg tctttcccac acttttacca agcagatgag 1380aggtttgttt ctgccataga aggcatgcac ccaaatcagg aagaccatga gacatttgtg 1440gacattaatc ctttgactgg aataatccta aaagcagcca agaggttcca aatcaacatt 1500tatgtcaaaa aattagatga ctttgttgaa acgggagaca ttagaaccat ggttttccca 1560gtgatgtacc tcaatgagag tgttcacatt gataaagaga cggcgagtcg actgaagtct 1620atgattaaca ctactttgat catcaccaac ataccctaca tcatcatggc gctgggtgtg 1680ttctttggtt tggtttttac ctggcttgca tgcaaaggac agggatccat ggatgaggga 1740acagcggatg aaagagcacc cctcattcga acctaaacat tgcctttgct tggtgaagaa 1800actgtgtgag ctgtcctgac ctggacgatg acgtggggaa accctccacc tccttgcagg 1860cttgttgcct gttgaaagaa ggaaaaagac acggcgctgg caagtgatag gaacattctg 1920gccagaggtt aaagagcagg ctgacatggc tggccattaa gctttataaa atcatgtggg 1980ctctgaaatt gttcttttat gtgtctagca agtatttaat aaacccttgt atagtaattt 2040tgttgttgtt gggtgctggt agctccagaa ttttgtgacc actattgtgg gtaaaatgtc 2100tctgcatcac ttgttaatgc tactggtcta acttcattca gtatgcttca ttcaccgaac 2160tttgtgctca aaatgcgtat ataccatttt atgttgtatt cctccatttc acttgcaaaa 2220cagaagtaaa taagagttcg ggacccaggg taaaatggta gcttcatcca atatatcatt 2280caaatgcatc tgatttctaa aacatattac attttatgct gatcttcagt tcataattct 2340tccaggaaaa ctcagtcttc caactgcaat aaaatactgg gtagaatcaa atgggaaagg 2400ggttgggtgg ggcaataccc atgagttgat agtgataagc tcctaaggat ttttaacttg 2460tacttttgtg aacgaagaga atgcataaat aatgttggtg aggataaagt acagatattt 2520catgtagaat taattgctag ttatgatgct tgtggatagt taactgtttt ttttttagtc 2580aaaatgatca tgctacgaaa agatgcttct gagagaatgt aatgagtaac tgatttttct 2640tcctgagtcg cccttgccaa atatgttact gtattaatta atctaatatt gagtgattat 2700ttgtaaaatt atgaatatgg gaaatccatc tatctacagc ctaagttaca cataagtttc 2760agaaagtctg attagactaa agagatattt cttctgggac agccgccttc ttggtaattt 2820tgaagttctt tttacaagtt ccttcctcag tttcagttct ttccagtgtt ttgtagctca 2880ctgtcactca ctgaatagag aaacgtgtgc cctatacttc ctgtgacaat cattttgctg 2940acagaatgat ggatgtttaa aatattgcac aaagtacttt aaagaaaggt ctgttaggac 3000cagaagcaga gacaccactt ttcaaaggac ttcttggttt cagcataacc taagacaggg 3060aattgggagc catcatatgt cacagtgttc agaattcaag catatttaag ggcattttct 3120ttgattctca aagttcagca ttcattttga attgagaagc ctatacattt agctgacaaa 3180gtgcttatag aatttcttaa caactgaacc attcaaaagg attttttttg tttaaaactg 3240gatttcaatg taagcaaatg aagaaaaaaa tatagatttc atttccatag cttcttatcc 3300ctgtattgag gtaataaatt gttttactga caatttttcc tttttctaca ctaaaacaat 3360atgtgatata tttcccctct tgaagaggca attcattaaa ctctcaaatt ttctatagaa 3420tcaagataga acctttagat actccaactc accaaaatgt aaaaaaacta acaaaaatat 3480ttggtcttca ataatgctaa atatctacat ttttagaatt tatcaacatt taactagata 3540attgggcatg tcttaattat gcatgtactt atccatacta ataaaattga caatgctagt 3600gcatacttat tggtttagtc ctattatcag gatataatca tctgtgagga ggatatttta 3660aatactgtaa atgataacag ttaatgatat acacatttag actgagttgc acactggcag 3720ggagaccaaa aacattactt ccatacttgt gtcatgattc tttttttttt gagagagtct 3780cactctgtcg ccaggctgga gtacagtggc atgatctcgg ctcactgcaa cctctgcctc 3840ccgggttcaa gcaattctcc tgcctcagcc acccaagtag ctgggactac aggtgcgtgc 3900caccacgccc agctaaattt tgtattttta gtggagacgg ggtttcacca tgttggccag 3960gatggtctca atctcctgac cctgcgatct gcccacctca gcctcccaaa gtgctgggat 4020tacaggcgta agccaccggg cctggcctgt tttatgattc ttaatagtta cttggtttaa 4080atcacatttg atactatcct tctgaaaagt ctgagacaga tctacaaact acagtcaaaa 4140ttatagatta agaggaatga atgcacctat ttggctttaa gttgaagatg aattatttct 4200catgctcatt ttcttgcggc agttatctta gaaagacccc caaaggcttt gtgattgtaa 4260gcactgtcat gatcacagaa tgcaagcttc tggtaccatg atcctcaact tagagaggaa 4320gaaaccaaga cagagagctt aactcacttc tctcagggaa aattaggagt tgagcacagg 4380acaggaaatg ggctttgcca cttttagctc caggcttttc taaccagact tgatttcctc 4440atgttctaga aagatcacta atggtcaagt ggaacaagca ctacacgact aacccctatt 4500ggggttttta acttaaggga ggctaatttt taatttaaac tgctcgagat atgagttctg 4560caaaaggtgg tccgcatcct tggccctctg gacattatca ctaaattgct tgtgcctgtt 4620aacaagaata ctgaccagaa tgctcttcat gtagcttata cagttggttc acttcatgcg 4680gttcttgaca tgtttatttc tacccttaat gcaatgaaat gtttcattaa taaaaaacca 4740ctttatat 474830478PRTHomo sapiens 30Met Gly Arg Cys Cys Phe Tyr Thr Ala Gly Thr Leu Ser Leu Leu Leu1 5 10 15Leu Val Thr Ser Val Thr Leu Leu Val Ala Arg Val Phe Gln Lys Ala 20 25 30Val Asp Gln Ser Ile Glu Lys Lys Ile Val Leu Arg Asn Gly Thr Glu 35 40 45Ala Phe Asp Ser Trp Glu Lys Pro Pro Leu Pro Val Tyr Thr Gln Phe 50 55 60Tyr Phe Phe Asn Val Thr Asn Pro Glu Glu Ile Leu Arg Gly Glu Thr65 70 75 80Pro Arg Val Glu Glu Val Gly Pro Tyr Thr Tyr Arg Glu Leu Arg Asn 85 90 95Lys Ala Asn Ile Gln Phe Gly Asp Asn Gly Thr Thr Ile Ser Ala Val 100 105 110Ser Asn Lys Ala Tyr Val Phe Glu Arg Asp Gln Ser Val Gly Asp Pro 115 120 125Lys Ile Asp Leu Ile Arg Thr Leu Asn Ile Pro Val Leu Thr Val Ile 130 135 140Glu Trp Ser Gln Val His Phe Leu Arg Glu Ile Ile Glu Ala Met Leu145 150 155 160Lys Ala Tyr Gln Gln Lys Leu Phe Val Thr His Thr Val Asp Glu Leu 165 170 175Leu Trp Gly Tyr Lys Asp Glu Ile Leu Ser Leu Ile His Val Phe Arg 180 185 190Pro Asp Ile Ser Pro Tyr Phe Gly Leu Phe Tyr Glu Lys Asn Gly Thr 195 200 205Asn Asp Gly Asp Tyr Val Phe Leu Thr Gly Glu Asp Ser Tyr Leu Asn 210 215 220Phe Thr Lys Ile Val Glu Trp Asn Gly Lys Thr Ser Leu Asp Trp Trp225 230 235 240Ile Thr Asp Lys Cys Asn Met Ile Asn Gly Thr Asp Gly Asp Ser Phe 245 250 255His Pro Leu Ile Thr Lys Asp Glu Val Leu Tyr Val Phe Pro Ser Asp 260 265 270Phe Cys Arg Ser Val Tyr Ile Thr Phe Ser Asp Tyr Glu Ser Val Gln 275 280 285Gly Leu Pro Ala Phe Arg Tyr Lys Val Pro Ala Glu Ile Leu Ala Asn 290 295 300Thr Ser Asp Asn Ala Gly Phe Cys Ile Pro Glu Gly Asn Cys Leu Gly305 310 315 320Ser Gly Val Leu Asn Val Ser Ile Cys Lys Asn Gly Ala Pro Ile Ile 325 330 335Met Ser Phe Pro His Phe Tyr Gln Ala Asp Glu Arg Phe Val Ser Ala 340 345 350Ile Glu Gly Met His Pro Asn Gln Glu Asp His Glu Thr Phe Val Asp 355 360 365Ile Asn Pro Leu Thr Gly Ile Ile Leu Lys Ala Ala Lys Arg Phe Gln 370 375 380Ile Asn Ile Tyr Val Lys Lys Leu Asp Asp Phe Val Glu Thr Gly Asp385 390 395 400Ile Arg Thr Met Val Phe Pro Val Met Tyr Leu Asn Glu Ser Val His 405 410 415Ile Asp Lys Glu Thr Ala Ser Arg Leu Lys Ser Met Ile Asn Thr Thr 420 425 430Leu Ile Ile Thr Asn Ile Pro Tyr Ile Ile Met Ala Leu Gly Val Phe 435 440 445Phe Gly Leu Val Phe Thr Trp Leu Ala Cys Lys Gly Gln Gly Ser Met 450 455 460Asp Glu Gly Thr Ala Asp Glu Arg Ala Pro Leu Ile Arg Thr465 470 475

Claims

1.-11. (canceled)

12. A method for identifying a subject at risk of developing heart failure, comprising: (a) determining a level of one or more biological markers in a biological sample of said subject; (b) comparing the level of said biological marker to a standard level of the same biological marker; and (c) determining whether the level of the biological marker is indicative of a risk for developing heart failure, wherein the biological marker is a protein selected from the group consisting of amyloid beta (A4) precursor protein (APP), collagen type 4 alpha 1 (COL4A) and KLF15.

13. The method claimed in claim 12, wherein the method is performed in vitro.

14. The method claimed in claim 12, wherein the biological sample is selected from the group consisting of blood, plasma, serum, and cardiac tissue.

15. The method claimed in claim 13, wherein the biological sample is selected from the group consisting of blood, plasma, serum, and cardiac tissue.

16. The method claimed in claim 12, wherein an increased level of APP or an increased level of COL4A or a decreased level of KLF15, as compared to a standard level, is indicative of a risk of developing heart failure.

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