COMPOSITIONS AND METHODS FOR CHARACTERIZING AND TREATING MUSCULAR DYSTROPHY

Abstract

Compositions and methods for identifying new treatments for Facioscapulohumeral muscular dystrophy (FSHD), and uses thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of and claims priority to U.S. patent application Ser. No. 13/861,227, filed on Apr. 11, 2013, now U.S. Pat. No. 9,260,755, which claims the benefit of U.S. Provisional Patent Application No. 61/622,942, filed on Apr. 11, 2012. The entire contents are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0003] Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder caused by contractions of repetitive elements within the macrosatellite D4Z4 on chromosome 4q35. There is currently no effective treatment available for FSHD and clinical trials with novel therapeutics have been discouraged by the lack of a recognized mouse model. Clinical trials have also been discouraged by the fact that FSHD is a highly variable and slowly progressing disease whereas the efficacy of therapeutic interventions is ideally established over short periods of time. Therefore, molecular biomarkers of FSHD that could be used to assay responsiveness to therapy would greatly facilitate FSHD therapeutic development and clinical research. High-density oligonucleotide arrays reliably quantify the expression levels of thousands of genes simultaneously and enable identification of such biomarkers.

SUMMARY OF THE INVENTION

[0004] As described below, the present invention features panels of biomarkers useful in diagnosing muscular dystrophy (e.g., FSHD) in a subject, as well as cellular compositions and chimeric animals useful in drug screening.

[0005] Thus, in a first aspect, the invention provides methods for identifying a candidate compound for treatment of Facioscapulohumeral muscular dystrophy (FSHD). The methods include contacting a sample comprising a cell derived from an FSHD affected subject with a test compound; determining a level of expression of a gene selected from the group consisting of PRAMEF1, SLC34A2, TRIM49, TRIM43, CD177, NAAA, HSPA6, TC2N, CD34, ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, and SPATA17, or at least two genes selected from the group consisting of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, CD34, ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, and SPATA17 in the sample; and selecting as a candidate compound a test compound that reduces the level of expression of one or more of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, or CD34, or a test compound that increases the level of expression of one or more of ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, or SPATA17. In some embodiments, where expression of only a single gene is determined, that gene is not PRAMEF1 or TRIM43. In some embodiments where expression of only two genes is determined, the genes are not PRAMEF1 and TRIM43. Thus, in some embodiments, where PRAMEF1 or TRIM43 are determined, at least one other gene that is not PRAMEF1 or TRIM43 is also determined.

[0006] In some embodiments, the methods include determining a level of expression of at least one gene shown in Table 4 that is upregulated in FSHD, optionally wherein the gene is selected from the group consisting of PRAMEF1; TRIM43; SLC34A2; TRIM49 and CD34, in a sample comprising a cell from the subject; and determining a level of expression of at least one gene shown in Table 4 that is downregulated in FSHD, optionally wherein the gene is selected from the group consisting of PAX7; MYF5; ATP2A1; DAG1; and MRAP2; in the sample; and selecting as a candidate compound a test compound that reduces the level of expression of a gene shown in Table 4 that is upregulated in FSHD and increases the level of expression of a gene shown in Table 4 that is downregulated in FSHD.

[0007] In some embodiments, the methods include administering the selected candidate compound to an animal model of FSHD, wherein the animal model comprises at least one chimeric muscle tissue comprising cells from a subject affected with FSHD; performing an assay to determine a level of expression of at least one gene shown in Table 4; comparing the level of expression of the at least one gene to a reference level of expression that represents a level of expression in the absence of the candidate compound; and selecting as a candidate therapeutic compound a candidate compound that reduces the level of expression of a gene shown in Table 4 that is upregulated in FSHD and increases the level of expression of a gene shown in Table 4 that is downregulated in FSHD.

[0008] In some embodiments, the level of expression of a gene shown in Table 4 that is upregulated in FSHD is reduced to a level that is nearly or substantially the same as, i.e., not statistically significantly different from, a level in a control cell that is not derived from an FSHD affected subject, or an animal model that comprises at least one chimeric muscle tissue comprising cells from a control subject who is not affected with FSHD.

[0009] In some embodiments, the level of expression of a gene shown in Table 4 that is downregulated in FSHD is increased to a level that is nearly or substantially the same as, i.e., not statistically significantly different from, a level in a control cell that is not derived from an FSHD affected subject, or an animal model that comprises at least one chimeric muscle tissue comprising cells from a control subject who is not affected with FSHD.

[0010] In some embodiments, levels of expression are determined using quantitative PCR (qPCR).

[0011] In some embodiments, the control cell is derived from a first degree relative of the FSHD affected subject.

[0012] In another aspect, the invention provides methods (e.g., computer-implemented methods) for identifying a candidate compound for treatment of Facioscapulohumeral muscular dystrophy (FSHD). The methods include contacting a sample comprising a cell derived from an FSHD affected subject with a test compound; determining a level of expression of at least one gene shown in Table 4 that is upregulated in FSHD, optionally wherein the gene is selected from the group consisting of PRAMEF1; TRIM43; SLC34A2; TRIM49 and CD34, in the sample, to determine a value [GeneUP]; determining a level of expression of at least one gene shown in Table 4 that is downregulated in FSHD, optionally wherein the gene is selected from the group consisting of PAX7; MYF5; ATP2A1; DAG1; and MRAP2; in the sample, to determine a value [GeneDOWN]; using the value [GeneDOWN] and the value for [GeneUP] to calculate a classifier for the test compound; comparing the classifier to a reference classifier that represents a classifier in a cell that is from a control subject who is not affected with FSHD; and selecting as a candidate compound a test compound that has a classifier that is not statistically different from the reference classifier.

[0013] In some embodiments, [GeneUP] is a level of PRAMEF1 in the sample.

[0014] In some embodiments, [GeneDOWN] is a level of PAX7 in the sample.

[0015] In some embodiments, the classifier is calculated as:

[GeneUP]-[GeneDOWN]=classifier

[0016] In some embodiments, the test compound is an inhibitory nucleic acid.

[0017] In some embodiments, the methods include administering the selected candidate compound to an animal model of FSHD, wherein the animal model comprises at least one chimeric muscle tissue comprising cells from a subject affected with FSHD; performing an assay to determine a level of expression of at least one gene selected from the group consisting of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, CD34, ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, SPATA17; comparing the level of expression of the at least one gene to a reference level of expression that represents a level of expression in the absence of the candidate compound; selecting as a candidate therapeutic compound a candidate compound that reduces the level of expression of one or more of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, or CD34, and increases the level of expression one or more of ATP2A1, PAX7, MYF5, MRAP2, DAG1, CLYBL, CALCRL, ZNF445, or SPATA17.

[0018] In some embodiments, the methods include administering the selected candidate compound to an animal model of FSHD, wherein the animal model comprises at least one chimeric muscle tissue comprising cells from a subject affected with FSHD; evaluating an effect of the candidate compound on a biological function associated with FSHD in the animal model; and selecting as a candidate therapeutic compound a candidate compound that improves the biological function (i.e., effects a return to normal or near normal function) in the animal model.

[0019] In some embodiments, biological function is assayed using live cell imaging, muscle fiber turnover, the number of muscle stem cells, or biomarker expression.

[0020] In another aspect, the invention provides methods for treating FSHD in a subject, the method comprising administering to the subject one or more inhibitory nucleic acids targeting one or more of SLC34A2, TRIM49, TRIM43, CD177, NAAA, HSPA6, TC2N, or CD34. In an additional aspect, the invention provides methods for treating FSHD in a subject, the method comprising administering to the subject two or more inhibitory nucleic acids targeting two or more of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, or CD34. In some embodiments, the inhibitory nucleic acid is a double-stranded RNA, siRNA, shRNA, or antisense oligonucleotide, e.g., a morpholino oligonucleotide.

[0021] Also provided herein are inhibitory nucleic acids targeting SLC34A2, TRIM49, TRIM43, CD177, NAAA, HSPA6, TC2N, or CD34 for treating FSHD, and the use of such inhibitory nucleic acids for treating FSHD, as well as for the manufacture of a medicament for the treatment of FSHD.

[0022] In another aspect, the invention provides cell lines, e.g., shown in FIG. 1, optionally selected from the group consisting of cell lines designated 07A, 07U, 09A, 09U, 12A, 12U, 15A, 15B, 15V, 21B, or 21U, where A and B designate cells from genetically affected persons with FSHD, and U and V designate genetically unaffected family members of the persons with FSHD.

[0023] In another aspect, the invention provides kits including a plurality of cell lines, e.g., a pair or trio of cell lines, from a family cohort as shown in FIG. 1, wherein the kit includes at least one cell line from a genetically affected person with FSHD, and at least one cell line from a genetically unaffected family member, e.g., a first degree relative, of the person with FSHD. In some embodiments, the kit comprises pairs or trios of cell lines selected from the group consisting of: 07A, 07U; 09A, 09U; 12A, 12U; 15A, 15 B, 15V; and 21B, 21U; where A and B designate cells from genetically affected persons with FSHD, and U and V designate genetically unaffected family members of the persons with FSHD.

[0024] In one aspect, the invention features a panel of isolated biomarkers including a DUX4 nucleic acid molecule and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) additional biomarkers including any one or more of the biomarkers listed in Table 2 or 4.

[0025] In another aspect, the invention features a microarray containing at least a DUX4 nucleic acid molecule and one or more polynucleotides listed in Table 2 or 4 or their encoded polypeptides, or fragments thereof, fixed to a solid support. In one embodiment, the solid support is a membrane, bead, biochip, multiwell microtiter plate, or a resin.

[0026] In another aspect, the invention features a method of characterizing Facioscapulohumeral muscular dystrophy (FSHD) in a cell derived from an FSHD affected subject, the method involving determining the level of expression of one or more nucleic acid molecules listed in Table 2 or 4 or their encoded polypeptides in the cell relative to the level of expression of the nucleic acid molecules or polypeptides in a cell obtained from a first degree relative of the subject who does not have FSHD, thereby characterizing FSHD in the cell. In one embodiment, the method identifies the molecular biomarker profile of the cell. In another embodiment, the cells are in vitro or in vivo. In another embodiment, the FSHD subject is identified as having a contracted 4q D4Z4 region in combination with a 4 qA telomeric allele.

[0027] In another aspect, the invention features a set of cell cultures, containing one culture containing cells derived from a subject identified as having FSHD and at least one control culture containing cells derived from a first degree relative of the subject that does not have FSHD. In one embodiment, the set comprises two, three or four control cultures obtained from first degree relatives of the subject. In another embodiment, the cell cultures are enriched for myogenic cells. In another embodiment, the cells are isolated by selecting cells positive for human CD56. In another embodiment, the cells are obtained from skeletal muscle biopsies. In another embodiment, the biopsy is of a bicep or deltoid muscle. In another embodiment, the FSHD subject is identified as having a contracted 4q D4Z4 region in combination with a 4 qA telomeric allele, and the first degree relative does not have the contracted 4q D4Z4 region.

[0028] In another aspect, the invention features a collection containing two or more sets of the cell cultures of any previous aspect or any other aspect of the invention delineated herein, where each set comprises a culture containing cells obtained from a distinct FSHD affected subject and at least one control culture containing cells obtained from that FSHD affected subject's first degree relatives.

[0029] In another aspect, the invention features a method for identifying an FSHD biomarker, the method involving comparing the expression of one or more polynucleotides in cells derived from a subject having FSHD relative to the expression of the polynucleotide in control cells derived from a first degree relative of the subject, where an increase or decrease in the polynucleotides relative to the control identifies the polynucleotide as an FSHD biomarker.

[0030] In another aspect, the invention features a chimeric mouse containing at least one human cell derived from an FSHD affected subject or a first degree relative thereof.

[0031] In another aspect, the invention features a set of chimeric mice including one mouse containing a human cell of an FSHD affected subject, and at least one mouse containing a human cell derived from a first degree relative of the FSHD affected subject.

[0032] In another aspect, the invention features a method of identifying an agent that ameliorates FSHD in a subject in need thereof, the method involving contacting a cell derived from an FSHD affected subject with a candidate agent, and comparing the cell's biological function or the level of expression of a nucleic acid molecule of Table 2 or 4 with the biological activity or the level of expression of the nucleic acid molecule in a control cell, where an agent that normalizes the expression of the nucleic acid molecule or enhances biological function ameliorates FSHD. In one embodiment, the control cell is derived from a first degree relative of the affected.

[0033] In another aspect, the invention features a method of identifying an agent that ameliorates FSHD in a subject in need thereof, the method involving administering the agent to the chimeric mouse of any previous aspect, and comparing the biological function of a human cell of the mouse before and after treatment, where an agent that enhances the biological function of the cell is identified as ameliorating FSHD.

[0034] In another aspect, the invention features a method of identifying an agent that ameliorates FSHD in a subject in need thereof, the method involving administering the agent to the chimeric mouse of any previous aspect, and comparing the level of expression of a nucleic acid molecule of Table 2 or 4 in a human cell of the mouse relative to the level in an untreated control cell, where an agent that normalizes expression in the cell is identified as ameliorating FSHD.

[0035] In another aspect, the invention features a method of identifying an inhibitory nucleic acid that ameliorates FSHD in a subject in need thereof, the method involving contacting a cell derived from an FSHD affected subject with an inhibitory nucleic acid molecule that targets a polynucleotide over expressed in FSHD, and comparing the level of expression of the polynucleotide relative to the level in a reference, where an inhibitory nucleic acid molecule that reduces expression of the polynucleotide ameliorates FSHD.

[0036] In another aspect, the invention features a method of identifying an inhibitory nucleic acid that ameliorates FSHD in a subject in need thereof, the method involving contacting a cell derived from an FSHD affected subject with an inhibitory nucleic acid molecule that targets a polynucleotide over expressed in FSHD, and comparing the biological function of a human cell of the mouse before and after treatment, where an agent that enhances the biological function of the cell is identified as ameliorating FSHD.

[0037] In another aspect, the invention features a method of diagnosing a subject as having, or having a propensity to develop, Facioscapulohumeral muscular dystrophy (FSHD), the method involving determining the level of expression of one or more nucleic acid molecules listed in Table 2 or 4 or their encoded polypeptides in a biological sample of the subject relative to the level of expression of the nucleic acid molecules or polypeptides in a reference, where an alteration in the level of expression is indicative of FSHD.

[0038] In various embodiments of the previous aspects or any other aspect of the invention delineated herein, the panel includes polynucleotide or polypeptide biomarkers that are any one or more of DUX4, tripartite motif containing 43 (TRIM43), TRIM49, tandem C2 domains, nuclear (TC2N), PRAME family member 13 (PRAMEF13), PRAMEF2, PRAMEF1, solute carrier family 34 (SLC34A2), heat shock 70 kDa protein 6 (HSP70B), FLJ44674 protein, CD177, and chromosome 9 open reading frame 4 (C9orf4). In one embodiment, the panel includes or consists of DUX4 and one or more additional upregulated biomarkers selected from the group consisting of TRIM43, PRAMEF13, PRAMEF2, PRAMEF1, SLC34A2, TRIM49, CCNA1, and TNXA. In another embodiment, the panel comprises DUX4 and a downregulated biomarker selected from the group consisting of microRNA 30b (MIR30B), dystroglycan 1 (DAG1), melanocortin 2 receptor accessory protein (MRAP2), chromosome 9 open reading frame 153 (C9orf153), ATPase, Ca++transporting, cardiac (ATP2A1), citrate lyase beta like (CLYBL), calcitonin receptor-like (CALCRL), cytochrome P450, family 39, subfamily (CYP39A1), mastermind-like 3 (MAML3), adrenergic, beta, receptor kinase 2 (ADRBK2), Rho guanine nucleotide exchange factor (ARHGEF7), microRNA 95 (miR95), spermatogenesis associated 17 (SPATA17), islet cell autoantigen 1.69 kDa-like (ICA1L), GABRR1, gamma-aminobutyric acid (GABA) KIAAl217, zinc finger protein 445 (ZNF445), and chromosome 14 open reading frame 39 (C14orf39. In another embodiment, the panel comprises or consists of DUX4 and a downregulated biomarker selected from the group consisting of CALCRL, ATP2A1, MYLK4, E2F8, RGS13, MYOZ2, LRRC39, C6orf142, and MYOZ1. In other embodiments, the human cell is a skeletal muscle cell, muscle stem cell, or differentiated muscle fiber. In other embodiments, the human cells replace 1-100% (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) of the cells present in a muscle of the mouse. In one embodiment, the human cells replace cells present in the tibialis anterior. In still other embodiments, biological function is assayed using live cell imaging, muscle fiber turnover, the number of muscle stem cells, or biomarker expression.

[0039] The invention provides compositions and methods for characterizing FSHD in a subject, as well as compositions and methods useful in drug screening. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

DEFINITIONS

[0040] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0041] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0042] By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0043] By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels."

[0044] By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0045] By "biomarker" is meant a polypeptide, polynucleotide, or clinical criteria associated with a disease or condition. For example, an alteration in the presence, level of expression, or sequence of a biomarker may be associated with or diagnostic of a disease or condition.

[0046] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0047] By "cell culture" is meant a cell or cells in vitro. A cell culture includes a cell growing or capable of growing in vitro. Thus, a cell culture includes frozen cells capable of growth in vitro.

[0048] "Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

[0049] By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable. Exemplary methods used to detect a detectable label, include spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

[0050] By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0051] By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

[0052] The invention provides a number of targets that are useful for the development of highly specific drugs to treat a condition or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

[0053] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0054] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0055] By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

[0056] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0057] By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0058] By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0059] By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

[0060] By "Marker profile" is meant a characterization of the expression or expression level of two or more polypeptides or polynucleotides.

[0061] As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

[0062] "Primer set" means a set of oligonucleotides that may be used, for example, for PCR. A primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers. In particular embodiments, primers of the invention are useful in amplifying a gene listed in Table 2 or 4.

[0063] By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0064] By "reference" is meant a standard or control condition. For example, the level of a polynucleotide or polypeptide of the invention (e.g., a polynucleotide listed in Table 2 or 4 or the encoded polypeptide) in a subject that is not affected with FSHD, such as a first degree relative of the subject.

[0065] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

[0066] By "a set" is meant a group having more than one member. The group may be composed of 2, 4, 5, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, or 300 polypeptide, nucleic acid molecule, cell culture, or chimeric mouse members.

[0067] By "siRNA" is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity. In one embodiment, the invention provides siRNA that target a polynucleotide of the invention (e.g., a polynucleotide upregulated in FSHD).

[0068] By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention. In one embodiment, the invention provides antibodies against polypeptides, or fragments thereof, encoded by a gene listed in Table 2 or 4.

[0069] Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0070] For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30.degree. C., more preferably of at least about 37.degree. C., and most preferably of at least about 42.degree. C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30.degree. C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37.degree. C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42.degree. C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 .mu.g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0071] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25.degree. C., more preferably of at least about 42.degree. C., and even more preferably of at least about 68.degree. C. In a preferred embodiment, wash steps will occur at 25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0072] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0073] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e.sup.-3 and e.sup.-100 indicating a closely related sequence.

[0074] By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

[0075] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0076] As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0077] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0078] Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0079] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0080] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 shows a table of cell lines as described herein. Each cohort was composed of at least one affected individual with genetically and clinically verified FSHD (designated A or B), and at least one unaffected first degree relative with unshortened D4Z4 alleles and normal strength (designated U or V).

DETAILED DESCRIPTION OF THE INVENTION

[0082] The invention provides compositions and methods that are useful treating Facioscapulohumeral muscular dystrophy (FSHD), and methods of identifying new treatments for FSHD.

[0083] The invention is based, at least in part, on the discovery of genes whose expression is aberrantly regulated in cells derived from subjects having FSHD (e.g., genes listed in Tables 2 and 4). In certain embodiments, a subset of genes is identified whose expression is most robustly altered in FSHD affected subjects (e.g. 20 genes in Table 2 with smallest p-values among those genes upregulated in FSHD and 20 genes in Table 2 with smallest p-values among those genes downregulated in FSHD). Genes whose expression is altered in FSHD are useful as biomarkers in methods for diagnosing or characterizing FSHD. Thus, the invention provides panels comprising FSHD biomarkers, as well as polynucleotide and polypeptide microarrays comprising such biomarkers.

[0084] The discovery of FSHD biomarkers was made possible using a unique collection of cultured cells derived from the skeletal muscles of subjects affected by FSHD, as well as of their first degree relatives. These "FSHD paired cultures" provide a unique advantage not only in identifying genes that are aberrantly regulated in FSHD, but also in identifying and/or assessing the efficacy of therapeutic agents useful in ameliorating FSHD or symptoms thereof. These FSHD paired cultures provide a unique advantage over other cells derived from FSHD affected subjects because they control for familial relationships by comparing expression differences in related FSHD affected subjects and controls, thereby diminishing the effects of interindividual variation on gene expression. Therefore, the expression differences observed between FSHD and control muscles in these FSHD paired cultures likely reflect true pathogenic gene expression profiles suitable for developing into disease biomarkers. The invention further provides screening methods using collections of FSHD paired cultures to identify agents that modify the expression of genes and/or proteins that are aberrantly regulated in FSHD.

[0085] In other embodiments, the invention provides pairs of chimeric mice, wherein one member of the pair comprises cells derived from a subject affected by FSHD, and the other member of the pair comprises a cell derived from a first degree relative of the subject. In other embodiments, the invention provides two, three, four or more mice, where one mouse comprises cells from an FSHD affected subject, and the other mice comprises cells derived from one or more of the first degree relatives of that subject. Preferably, certain skeletal muscle cells of the mouse are derived from an FSHD subject or first degree relative of such a subject. Thus, the invention provides a mouse model that is uniquely suited for the identification and characterization of agents useful in treating and/or ameliorating FSHD, and or symptoms thereof.

[0086] In still other embodiments, the invention provides panels of biomarkers comprising at least 2, 3, 5, 10, 15, 20, or more of the genes listed in Table 2 or 4. In one embodiment, the panel comprises those genes identified as upregulated in FSHD. In another embodiment, the panel comprises those genes identified as downregulated in FSHD.

Facioscapulohumeral Muscular Dystrophy

[0087] Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular dystrophy affecting approximately 1 in 7,000-20,000 individuals. It is characterized by progressive weakness and wasting of facial, shoulder girdle and upper arm muscles from which the disease takes its name, and also trunk, hip and leg muscles in some patients. One of the hallmarks of FSHD is asymmetrical and selective degeneration of skeletal muscles. For example, biceps muscle is involved early and severely, whereas the proximal deltoid muscle is relatively spared. The underlying mechanism of this distinctive sparing of certain muscle types is unknown. In addition to muscle degeneration, abnormalities in retinal vasculature and hearing loss are observed in up to 49% and 64% respectively in some populations.

[0088] FSHD is caused by partial deletion of a critical number of repeat elements within the highly polymorphic macrosatellite D4Z4 on the subtelomeric region of chromosome 4q. In unaffected individuals, the D4Z4 array consists of 11 to 100 repeats (corresponding to EcoRI fragments of 41 to 350 kb), whereas FSHD patients carry 1 to 10 repeats (corresponding to EcoRI fragments of 10 to 35 kb). Longer residual repeat sizes are often associated with later onset or milder disease severity. In addition to reduction of the tandemly arranged D4Z4 3.3 kb repeat units, the disease causing deletions must occur on chromosomal allele 4 qA, whereas deletions on the equally common 4 qB allele do not result in FSHD. Although the genetic lesion responsible for 95% of FSHD cases was identified two decades ago, the molecular mechanisms leading to disease progression have long been controversial. The predominantly held position-effect variegation hypothesis proposed that contraction of the D4Z4 repeats induces derepression of one or more genes adjacent to D4Z4 with myopathic potential. Several genes (FRG1, FRG2, SLC25A4) residing in the vicinity of D4Z4 have been evaluated using various quantitative approaches by numerous studies but no consistent deregulation of these genes have been demonstrated in human muscle (Winokur et al., (2003) Hum Genet, 12, 2895-2907; Osborne (2007) Neurology, 68, 569-577; Masny et al., (2010) Eur J Hum Genet, 18, 448-456).

DUX4

[0089] Several studies have demonstrated the myopathic potential of DUX4, a gene located within each repeat element, in skeletal muscle cells. Overexpression of DUX4, as a result of chromatin relaxation within D4Z4, was initially proposed to induce toxicity to muscle cells, potentially leading to muscle degeneration in vivo. Subsequent studies demonstrated further evidence to support this finding. Recently, genetic analysis of rare families carrying translocations between 4q and 10q chromosomes identified single nucleotide polymorphisms (SNPs) in the pLAM region adjacent to the distal D4Z4 repeat that segregate with FSHD. These SNPs create a canonical polyadenylation signal on the permissive chromosomal allele, whereas the non-permissive alleles lack these SNPs. DUX4 transcripts expressed from the distal-most repeat extends into the pLAM sequence and are polyadenylated when the poly(A) signal SNPs are incorporated into the transcripts, thus increasing their intracellular stability. DUX4, a double homeodomain containing protein, shares similarities with transcription factors PAX3 and PAX7 and is proposed to interfere with transcriptional networks regulated by PAX3/7. It has yet to be determined whether DUX4 overexpression results in global gene misexpression, and in particular it is of considerable interest to determine whether the expression of PAX3/7 target genes are compromised in FSHD muscles, as these transcription factors play an important role in muscle development and maintenance. In view of these findings, agents that reduce DUX4 expression are of interest in treating FSHD and/or ameliorating symptoms associated with FSHD. The analysis of such agents has been hampered by the lack of suitable in vitro and in vivo models systems useful for assaying the efficacy of such agents on FSHD. Thus, the invention provides cell and animal models useful for analysing the agents that treat FSHD. In particular, FSHD paired cultures are useful for analysing the effect of such agents on the expression of genes that are aberrantly regulated in FSHD. In other embodiments, chimeric FSHD mice of the invention are useful for assaying the efficacy of such agents on muscle cells affected with FSHD. In particular, the invention provides methods for assaying the effects of agents that reduce DUX4 expression on genes that are aberrantly regulated in FSHD (e.g., genes listed in Table 2 or 4).

FSHD Cell Cultures and Collections

[0090] While the results reported herein provide specific examples of the isolation of muscle cells from subjects identified as having FSHD (or their first degree relatives) during the course of a muscle biopsy, the invention is not so limited. The unpurified source of cells for use in the methods of the invention may be any tissue known in the art obtained from an FSHD subject, although preferably, muscle cells derived from FSHD affected subjects are used. In various embodiments, cells of the invention are isolated from muscle tissue whose biological function is reduced in FSHD (e.g., adult biceps or deltoid skeletal muscles). In one embodiment, the FSHD subject is identified as having a contracted 4q D4Z4 region in combination with a 4 qA telomeric allele and the first degree relative is identified as lacking such genetic abnormalities.

[0091] The invention provides for the generation of primary muscle cell cultures. Such cultures are obtained by enzymatic dissociation of the tissue using, for example, collagenase IV, dispase and other enzymes known in the art. The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). Preferably, the cells are collected in a medium comprising fetal calf serum (FCS) or bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium. Dissociated cells are cultured under standard conditions using cell culture media (e.g., Ham's F10 medium supplemented with fetal bovine serum and/or chicken embryo extract) suitable for maintaining cultures of primary muscle cells. Examples of suitable media for incubating cells of the invention include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), RPMI media or other medias known in the art. The media may be supplemented with fetal calf serum (FCS) or fetal bovine serum (FBS), as well as antibiotics, growth factors, amino acids, inhibitors or the like, which is well within the general knowledge of the skilled artisan.

[0092] Cultures are expanded to increase cell number (e.g., to about 50%, 60%, 70%, 80% confluence). Cells are harvested and selected for myogenic cells using standard methods. Such methods include a positive selection for cells expressing one or more myogenic markers. Monoclonal antibodies are particularly useful for identifying markers associated with the desired cells. If desired, negative selection methods can be used in conjunction with the methods of the invention to reduce the number of irrelevant cells present in a population of cells selected for a myogenic phenotype.

[0093] In one approach, fluorescence-activated cell sorting (FACS) is carried out to identify cells that are positive for human CD56 (BD Biosciences), MYOD, PAX7, or MYFS. In another approach, magnetic-activated cell sorting (MACS) is used to select for the desired cell type. Other procedures which may be used for selection of cells of interest include, but are not limited to, fluorescence based cell sorting, density gradient centrifugation, flow cytometry, magnetic separation with antibody-coated magnetic beads, cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix or any other convenient technique.

[0094] CD56-positive myogenic cells obtained by FACS are incubated in culture. Cells derived from the skeletal muscles of subjects affected by FSHD, as well as of their first degree relatives are termed "FSHD paired cultures." In one embodiment, such paired cultures are useful in identifying markers that are aberrantly regulated in FSHD. In another embodiment, such cells are useful in identifying and/or assessing the efficacy of therapeutic agents useful in ameliorating FSHD or symptoms thereof. These FSHD paired cultures provide for the analysis of expression differences in related FSHD affected subjects and controls, thereby diminishing the effects of interindividual variation on gene expression.

[0095] Selected cells of the invention may be employed in methods of the invention following isolation and/or growth in vitro.

[0096] In one approach, the invention provides paired cell cultures, where one culture is derived from a subject having FSHD and the other culture is obtained from a first degree relative of the subject. Such paired cell cultures comprise skeletal muscle cells isolated from the subject or his relative during muscle biopsy. Such cells are then cultured in vitro to obtain sufficient cells for drug screening or marker expression analysis. The invention further provides a collection of such paired cell cultures. Desirably, the collection includes cell samples from two, three, four, five, six, seven, eight, nine, ten or more FSHD affected subjects and paired control cultures obtained from one or more of the subjects first degree relatives. In certain embodiments, the invention provides a frozen collection of cells suitable for paired culture. Frozen cell compositions typically comprise cryoprotective agents that provide for cell viability when the cells are frozen for a period of months or years and then subsequently thawed.

FSHD Chimeric Animals

[0097] The invention further provides chimeric animals that comprise human cells obtained from an FSHD affected. Preferably, the invention provides pairs of chimeric mice, wherein one member of the pair comprises human cells obtained from an FSHD affected and the other member of the pair comprises human cells obtained from a first degree relative of the FSHD affected.

[0098] In one embodiment, skeletal muscle cells of a mouse are injured or destroyed, for example, using cardiotoxin. The skeletal muscle cells of the injured mouse are replaced with at least about 10%, 20%, 30%, 50%, 75% or even 100% human cells derived from an FSHD subject. In one embodiment, the mouse's endogenous tibialis anterior is replaced, at least to some degree, with human muscle cells derived from an FSHD affected or a first degree relative thereof. If desired, such cells are genetically modified to express a detectable reporter (e.g., GFP, YFP, RFP, luciferase).

[0099] In one embodiment, the method provides chimeric animals, wherein one animal comprises cells of an FSHD affected and one or more other animals comprises cells of a first degree relative of the affected individual. Such chimeric animals are useful in identifying markers that are aberrantly regulated in FSHD. The invention provides a collection of such paired chimeric mice. Desirably, the collection includes cell samples from two, three, four, five, six, seven, eight, nine, ten or more FSHD affected subjects and paired control chimeric mice comprising cells obtained from one or more of the subjects' first degree relatives.

Diagnostics

[0100] The present invention features diagnostic assays for the detection of FSHD or the propensity to develop such conditions. In one embodiment, levels of any one or more of the markers listed in Table 2 or 4 are measured in a subject sample and used to characterize FSHD or the propensity to develop FSHD. In other embodiments, levels of markers listed in Table 2 or 4, are characterized in a subject sample. Standard methods may be used to measure levels of a marker in any biological sample. Biological samples include tissue samples (e.g., cell samples, biopsy samples) or biological fluid samples that include markers of the invention (e.g., blood, serum, plasma, urine). Methods for measuring levels of polypeptide biomarkers of the invention (e.g., markers listed in Table 2 or 4) include immunoassay, ELISA, western blotting and radioimmunoassay. The increase in marker levels may be altered (e.g., increased, decreased) by at least about 10%, 25%, 50%, 75% or more relative to levels of markers found in a corresponding control sample (e.g., samples obtained from a normal subject unaffected by FSHD). In one embodiment, any increase or decrease in a marker of the invention, i.e., a marker listed in Table 2 or 4, is indicative of FSHD.

[0101] Any suitable method can be used to detect one or more of the markers described herein. Successful practice of the invention can be achieved with one or a combination of methods that can detect and, preferably, quantify the markers. These methods include, without limitation, hybridization-based methods, including those employed in biochip arrays, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy. Expression levels of markers (e.g., polynucleotides or polypeptides) are compared by procedures well known in the art, such as RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, ELISA, microarray analysis, or colorimetric assays. Methods may further include, one or more of electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)n, quadrupole mass spectrometry, fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero.

[0102] Detection methods may include use of a biochip array. Biochip arrays useful in the invention include protein and polynucleotide arrays. One or more markers are captured on the biochip array and subjected to analysis to detect the level of the markers in a sample.

[0103] Markers may be captured with capture reagents immobilized to a solid support, such as a biochip, a multiwell microtiter plate, a resin, or a nitrocellulose membrane that is subsequently probed for the presence or level of a marker. Capture can be on a chromatographic surface or a biospecific surface. For example, a sample containing the markers, such as serum, may be used to contact the active surface of a biochip for a sufficient time to allow binding. Unbound molecules are washed from the surface using a suitable eluant, such as phosphate buffered saline. In general, the more stringent the eluant, the more tightly the proteins must be bound to be retained after the wash.

[0104] Upon capture on a biochip, analytes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method. In one embodiment, mass spectrometry, and in particular, SELDI, is used. Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Immunoassays in various formats (e.g., ELISA) are popular methods for detection of analytes captured on a solid phase. Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy.

[0105] Mass spectrometry (MS) is a well-known tool for analyzing chemical compounds. Thus, in one embodiment, the methods of the present invention comprise performing quantitative MS to measure the serum peptide marker. The method may be performed in an automated (Villanueva, et al., Nature Protocols (2006) 1(2):880-891) or semi-automated format. This can be accomplished, for example with MS operably linked to a liquid chromatography device (LC-MS/MS or LC-MS) or gas chromatography device (GC-MS or GC-MS/MS). Methods for performing MS are known in the field and have been disclosed, for example, in US Patent Application Publication Nos: 20050023454; 20050035286; U.S. Pat. No. 5,800,979 and references disclosed therein.

[0106] The protein fragments, whether they are peptides derived from the main chain of the protein or are residues of a side-chain, are collected on the collection layer. They may then be analyzed by a spectroscopic method based on matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI). The preferred procedure is MALDI with time of flight (TOF) analysis, known as MALDI-TOF MS. This involves forming a matrix on the membrane, e.g. as described in the literature, with an agent which absorbs the incident light strongly at the particular wavelength employed. The sample is excited by UV, or IR laser light into the vapour phase in the MALDI mass spectrometer. Ions are generated by the vaporization and form an ion plume. The ions are accelerated in an electric field and separated according to their time of travel along a given distance, giving a mass/charge (m/z) reading which is very accurate and sensitive. MALDI spectrometers are commercially available from PerSeptive Biosystems, Inc. (Frazingham, Mass., USA) and are described in the literature, e.g. M. Kussmann and P. Roepstorff, cited above.

[0107] Magnetic-based serum processing can be combined with traditional MALDI-TOF. Through this approach, improved peptide capture is achieved prior to matrix mixture and deposition of the sample on MALDI target plates. Accordingly, methods of peptide capture are enhanced through the use of derivatized magnetic bead based sample processing.

[0108] MALDI-TOF MS allows scanning of the fragments of many proteins at once. Thus, many proteins can be run simultaneously on a polyacrylamide gel, subjected to a method of the invention to produce an array of spots on the collecting membrane, and the array may be analyzed. Subsequently, automated output of the results is provided by using the ExPASy server, as at present used for MIDI-TOF MS and to generate the data in a form suitable for computers.

[0109] Other techniques for improving the mass accuracy and sensitivity of the MALDI-TOF MS can be used to analyze the fragments of protein obtained on the collection membrane. These include the use of delayed ion extraction, energy reflectors and ion-trap modules. In addition, post source decay and MS-MS analysis are useful to provide further structural analysis. With ESI, the sample is in the liquid phase and the analysis can be by ion-trap, TOF, single quadrupole or multi-quadrupole mass spectrometers. The use of such devices (other than a single quadrupole) allows MS-MS or MS' analysis to be performed. Tandem mass spectrometry allows multiple reactions to be monitored at the same time.

[0110] Capillary infusion may be employed to introduce the marker to a desired MS implementation, for instance, because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a MS with other separation techniques including gas chromatography (GC) and liquid chromatography (LC). GC and LC can serve to separate a solution into its different components prior to mass analysis. Such techniques are readily combined with MS, for instance. One variation of the technique is that high performance liquid chromatography (HPLC) can now be directly coupled to mass spectrometer for integrated sample separation/and mass spectrometer analysis.

[0111] Quadrupole mass analyzers may also be employed as needed to practice the invention. Fourier-transform ion cyclotron resonance (FTMS) can also be used for some invention embodiments. It offers high resolution and the ability of tandem MS experiments. FTMS is based on the principle of a charged particle orbiting in the presence of a magnetic field. Coupled to ESI and MALDI, FTMS offers high accuracy with errors as low as 0.001%.

[0112] In one embodiment, the marker qualification methods of the invention may further comprise identifying significant peaks from combined spectra. The methods may also further comprise searching for outlier spectra. In another embodiment, the method of the invention further comprises determining distant dependent K-nearest neighbors.

[0113] In another embodiment of the method of the invention, an ion mobility spectrometer can be used to detect and characterize FSHD markers. The principle of ion mobility spectrometry is based on different mobility of ions. Specifically, ions of a sample produced by ionization move at different rates, due to their difference in, e.g., mass, charge, or shape, through a tube under the influence of an electric field. The ions (typically in the form of a current) are registered at the detector which can then be used to identify a marker or other substances in a sample. One advantage of ion mobility spectrometry is that it can operate at atmospheric pressure.

[0114] In an additional embodiment of the methods of the present invention, multiple markers are measured. The use of multiple markers increases the predictive value of the test and provides greater utility in diagnosis, toxicology, patient stratification and patient monitoring. The process called "Pattern recognition" detects the patterns formed by multiple markers greatly improves the sensitivity and specificity of clinical proteomics for predictive medicine. Subtle variations in data from clinical samples indicate that certain patterns of protein expression can predict phenotypes such as the presence or absence of FSHD.

[0115] Expression levels of particular nucleic acids or polypeptides are correlated with FSHD, and thus are useful in diagnosis. Antibodies that bind a polypeptide described herein, oligonucleotides or longer fragments derived from a nucleic acid sequence described herein (e.g., one or more Markers listed in Table 2 or 4), or any other method known in the art may be used to monitor expression of a polynucleotide or polypeptide of interest. Detection of an alteration relative to a normal, reference sample can be used as a diagnostic indicator of FSHD. In particular embodiments, the expression of one or more Markers listed in Table 2 or 4 is indicative of FSHD or the propensity to develop FSHD. In other embodiments, a 2, 3, 4, 5, or 6-fold change in the level of a marker of the invention is indicative of FSHD. In yet another embodiment, an expression profile that characterizes alterations in the expression of two, three, four, five, ten, fifteen, twenty, thirty, or forty markers is correlated with a particular disease state (e.g., FSHD). Such correlations are indicative of FSHD or the propensity to develop FSHD. In one embodiment, FSHD can be monitored using the methods and compositions of the invention.

[0116] In one embodiment, the level of one or more markers is measured on at least two different occasions and an alteration in the levels as compared to normal reference levels over time is used as an indicator of FSHD or the propensity to develop FSHD. The level of marker in a subject having FSHD or the propensity to develop such a condition may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, or 90% or more relative to the level of such marker in a normal control. In general, levels of Markers listed in Table 2 or 4 are present at low or undetectable levels in a healthy subject (i.e., those who do not have and/or who will not develop FSHD). In one embodiment, a subject sample of a skeletal muscle (e.g., bicep) is collected prior to the onset of symptoms of FSHD or early on in the progression of FSHD.

[0117] The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein for a more accurate diagnosis of the presence or severity of FSHD.

[0118] The diagnostic methods described herein can also be used to monitor and manage FSHD, or to reliably distinguish FSHD from other degenerative diseases or diseases having symptoms that are similar to or overlap with FSHD.

[0119] As indicated above, the invention provides methods for aiding a muscular dystrophy (e.g., FSHD) diagnosis using one or more markers, as specified herein. These markers can be used alone, in combination with other markers in any set, or with entirely different markers in aiding human muscular dystrophy (e.g., FSHD) diagnosis. The markers are differentially present in samples of a human FSHD patient and a normal subject (e.g., first degree relative of an FSHD subject) in whom FSHD is undetectable. Therefore, detection of one or more of these markers in a person would provide useful information regarding the probability that the person may have FSHD or have a propensity to develop FSHD.

[0120] The detection of one or more peptide marker is then correlated with a probable diagnosis of FSHD. In some embodiments, the detection of the mere presence of a marker (e.g., a marker listed in Table 2 or 4), without quantifying the amount thereof, is useful and can be correlated with a probable diagnosis of FSHD. The measurement of markers may also involve quantifying the markers to correlate the detection of markers with a probable diagnosis of FSHD. Thus, if the amount of the markers detected in a subject being tested is different compared to a control amount (i.e., higher or lower than the control), then the subject being tested has a higher probability of having FSHD.

[0121] The correlation may take into account the amount of the marker or markers in the sample compared to a control amount of the marker or markers (e.g., in normal subjects). A control can be, e.g., the average or median amount of marker present in comparable samples of normal subjects. The control amount is measured under the same or substantially similar experimental conditions as in measuring the test amount. As a result, the control can be employed as a reference standard, where each result can be compared to that standard, rather than re-running a control.

[0122] Accordingly, a marker profile may be obtained from a subject sample and compared to a reference marker profile, so that it is possible to classify the subject as having or not having FSHD. The correlation may take into account the presence or absence of the markers in a test sample and the frequency of detection of the same markers in a control. The correlation may take into account both of such factors to facilitate determination of FSHD status.

[0123] In certain embodiments of the invention, the methods further comprise managing subject treatment based on the status.

[0124] The markers of the present invention have a number of other uses. For example, they can be used to identify agents useful in methods of treating or ameliorating FSHD. In yet another example, the markers can be used in heredity studies. For instance, certain markers associated with FSHD may be genetically associated with the disease. This can be determined by, e.g., analyzing samples from a population of human subjects whose families have a history of FSHD. The results can then be compared with data obtained from, e.g., subjects whose families do not have a history of FSHD. The markers that are genetically linked may be used as a tool to determine if a subject whose family has a history of FSHD is pre-disposed to having FSHD.

[0125] While individual markers are useful diagnostic markers, in some instances, a combination of markers provides greater predictive value than a single marker alone. The detection of a plurality of markers (or absence thereof, as the case may be) in a sample can increase the percentage of true positive and true negative diagnoses and decrease the percentage of false positive or false negative diagnoses. Thus, preferred methods of the present invention comprise the measurement of more than one marker.

Microarrays

[0126] As reported herein, a number of markers (e.g., a marker listed in Table 2 or 4) have been identified that are associated with FSHD. Methods for assaying the expression of these polypeptides are useful for characterizing FSHD. In particular, the invention provides diagnostic methods and compositions useful for identifying a polypeptide expression profile that identifies a subject as having or having a propensity to develop FSHD. Such assays can be used to measure an alteration in the level of a polypeptide.

[0127] The polypeptides and nucleic acid molecules of the invention are useful as hybridizable array elements in a microarray. The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci. 93:10614-10619, 1996), herein incorporated by reference. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res. 28: e3. i-e3. vii, 2000), MacBeath et al., (Science 289:1760-1763, 2000), Zhu et al. (Nature Genet. 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.

Protein Microarrays

[0128] Proteins (e.g., proteins encoded by genes listed in Table 2 or 4) may be analyzed using protein microarrays. Such arrays are useful in high-throughput low-cost screens to identify alterations in the expression or post-translation modification of a polypeptide of the invention, or a fragment thereof. In particular, such microarrays are useful to identify a protein whose expression is altered in FSHD. In one embodiment, a protein microarray of the invention binds a marker present in a subject sample and detects an alteration in the level of the marker. Typically, a protein microarray features a protein, or fragment thereof, bound to a solid support. Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer-based films (e.g., polystyrene), beads, or glass slides. For some applications, proteins (e.g., antibodies that bind a marker of the invention) are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer).

[0129] The protein microarray is hybridized with a detectable probe. Such probes can be polypeptide, nucleic acid molecules, antibodies, or small molecules. For some applications, polypeptide and nucleic acid molecule probes are derived from a biological sample taken from a patient, such as a homogenized tissue sample (e.g. a tissue sample obtained by muscle biopsy); or a cell isolated from a patient sample. Probes can also include antibodies, candidate peptides, nucleic acids, or small molecule compounds derived from a peptide, nucleic acid, or chemical library. Hybridization conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions. Such conditions are known to the skilled artisan and are described, for example, in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual. 1998, New York: Cold Spring Harbor Laboratories. After removal of non-specific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan.

Nucleic Acid Microarrays

[0130] To produce a nucleic acid microarray, oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.), incorporated herein by reference. Alternatively, a gridded array may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.

[0131] A nucleic acid molecule (e.g. RNA or DNA) derived from a biological sample may be used to produce a hybridization probe as described herein. The biological samples are generally derived from a patient, as a tissue sample (e.g. a tissue sample obtained by muscle biopsy). For some applications, cultured cells or other tissue preparations may be used. The mRNA is isolated according to standard methods, and cDNA is produced and used as a template to make complementary RNA suitable for hybridization. Such methods are known in the art. The RNA is amplified in the presence of fluorescent nucleotides, and the labeled probes are then incubated with the microarray to allow the probe sequence to hybridize to complementary oligonucleotides bound to the microarray.

[0132] Incubation conditions are adjusted such that hybridization occurs with precise complementary matches or with various degrees of less complementarity depending on the degree of stringency employed. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30 C., more preferably of at least about 37 C., and most preferably of at least about 42 C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30 C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37 C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42 C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 .mu.g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0133] The removal of nonhybridized probes may be accomplished, for example, by washing. The washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 C., more preferably of at least about 42.degree. C., and most preferably of at least about 68 C. In a preferred embodiment, wash steps will occur at 25 C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.

[0134] A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct nucleic acid sequences simultaneously (e.g., Heller et al., Proc. Natl. Acad. Sci. 94:2150-2155, 1997). Preferably, a scanner is used to determine the levels and patterns of fluorescence.

Diagnostic Kits

[0135] The invention provides kits for diagnosing or monitoring FSHD. In one embodiment, the kit includes a composition containing at least one agent that binds a polypeptide or polynucleotide whose expression is increased in FSHD. In another embodiment, the invention provides a kit that contains an agent that binds a nucleic acid molecule whose expression is altered in FSHD. In some embodiments, the kit comprises a sterile container which contains the binding agent; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0136] If desired the kit is provided together with instructions for using the kit to diagnose FSHD. The instructions will generally include information about the use of the composition for diagnosing a subject as having FSHD or having a propensity to develop FSHD. In other embodiments, the instructions include at least one of the following: description of the binding agent; warnings; indications; counter-indications; animal study data; clinical study data; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

Screening Assays

[0137] As discussed herein, the expression of genes listed in Tables 2 and 4 is altered in FSHD. Based on this discovery, compositions of the invention are useful for the high-throughput low-cost screening of candidate agents to identify those that modulate the expression of genes that are aberrantly expressed in FSHD. In one embodiment, the effects of candidate agents on genes expressed in Tables 2 and 4 are assayed using microarrays, cell compositions, and/or chimeric animals of the invention.

[0138] Those genes identified in Tables 2 or 4 whose expression is inappropriately increased in FSHD are targets for therapeutic intervention. The genes TRIM43 and PRAMEF1 are of particular interest. The inappropriate activation of one or more genes unregulated in FSHD likely contributes to the pathology observed in FSHD. Therefore, agents that reduce the expression of genes that are over-expressed in FSHD are useful in the methods of the invention. Such agents include, for example, inhibitory nucleic acids that reduce or eliminate the expression of such genes, as well as proteins (e.g., antibodies and fragments thereof) and small compounds that interfere with the expression or biological activity of the genes or the proteins that they encode. The present methods can be used to identify such agents.

[0139] Those genes identified in Table 2 or 4 whose expression is inappropriately decreased in FSHD are also targets for therapeutic intervention. Such agents include, for example, small compounds that increase the biological activity or expression of a gene listed in Table 2 or 4 or of the protein that gene encodes. In other embodiments, agents (e.g., expression vectors encoding proteins downregulated in FSHD) are useful to increase the expression of such genes, particularly in skeletal muscle. Such expression would be expected to ameliorate FSHD or symptoms associated with FSHD. The present methods can be used to identify such agents.

[0140] A number of methods are available for carrying out screening assays to identify candidate agents that reduce the expression of genes that are overexpressed in FSHD, or that increase the expression of a gene that is downregulated in FSHD. In one example, candidate agents are added at varying concentrations to the culture medium of cultured cells (e.g., FSHD paired cultures) expressing one of the nucleic acid sequences of the invention. Gene expression is then measured, for example, by microarray analysis, Northern blot analysis (Ausubel et al., supra), reverse transcriptase PCR, quantitative real-time PCR, or any other method known in the art using any appropriate fragment prepared from the nucleic acid molecule as a hybridization probe. The level of gene expression in the FSHD derived cells in the presence of the candidate agent is compared to the level measured in a control culture. In one embodiment, the control culture is a culture of FSHD derived cells that lack the agent. In another embodiment, the control culture is the paired culture of cells obtained from a first degree relative of the FSHD affected. An agent that normalizes or promotes the normalization of expression of aberrantly regulated genes is considered useful in the invention. Such an agent may be used, for example, as a therapeutic to treat FSHD in a human patient. An agent that "normalizes" the expression of an aberrantly regulated gene restores the expression of that gene to a level that is substantially normal. An agent that "promotes normalization" reduces the extent of the disregulation.

[0141] In one example, the effect of candidate agents is measured at the level of polypeptide production using the same general approach and standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific for a polypeptide encoded by a gene listed in Table 2 or 4. For example, immunoassays may be used to detect or monitor the expression of at least one of the polypeptides of the invention in an organism. Polyclonal or monoclonal antibodies, (produced as described above) that are capable of binding to such a polypeptide may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) to measure the level of the polypeptide. In some embodiments, a agent that normalizes or promotes normalization of the expression or biological activity of an aberrantly regulated polypeptide is considered useful. Again, such an agent may be used, for example, as a therapeutic to delay, ameliorate, or treat FSHD disorder, or the symptoms of FSHD, in a human patient.

[0142] In yet another working example, candidate agents may be screened for those that specifically bind to a polypeptide encoded by a gene listed in Table 2 or 4. The efficacy of such a candidate agent is dependent upon its ability to interact with such a polypeptide or a functional equivalent thereof. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra). In one embodiment, a candidate agent may be tested in vitro for its ability to specifically bind a polypeptide of the invention. In another embodiment, a candidate agent is tested for its ability to normalize or promote the normalization of the biological activity of a polypeptide described herein. The biological activity of a polypeptide may be assayed using any standard method.

[0143] In another example, a gene described herein (e.g., listed in Table 2 or 4) is expressed as a transcriptional or translational fusion with a detectable reporter, and expressed in an isolated cell (e.g., mammalian or insect cell) under the control of a heterologous promoter, such as an inducible promoter. The cell expressing the fusion protein is then contacted with a candidate agent, and the expression of the detectable reporter in that cell is compared to the expression of the detectable reporter in an untreated control cell. A candidate agent that alters (e.g., normalizes or promotes normalization) the expression of the detectable reporter is an agent that is useful for the treatment of FSHD. In preferred embodiments, the candidate agent increases the expression of a reporter gene fused to a gene that is downregulated in FSHD.

[0144] In one particular working example, a candidate agent that binds to a polypeptide encoded by a gene listed in Table 2 or 4 may be identified using a chromatography-based technique. For example, a recombinant polypeptide of the invention may be purified by standard techniques from cells engineered to express the polypeptide (e.g., those described above) and may be immobilized on a column. A solution of candidate agents is then passed through the column, and an agent specific for the polypeptide encoded by a nucleic acid molecule listed in Table 2 or 4 is identified on the basis of its ability to bind to the polypeptide and be immobilized on the column. To isolate the agent, the column is washed to remove non-specifically bound molecules, and the agent of interest is then released from the column and collected. Similar methods may be used to isolate an agent bound to a polypeptide microarray. Agents isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). In addition, these candidate agents may be tested for their ability to increase the activity of gene whose expression is downregulated in FSHD. Agents isolated by this approach may also be used, for example, as therapeutics to treat FSHD in a human patient. Agents that are identified as binding to a polypeptide of the invention with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention. Alternatively, any in vivo protein interaction detection system, for example, any two-hybrid assay may be utilized.

[0145] Potential agonists and antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid molecules, and antibodies that bind to a nucleic acid sequence or polypeptide of the invention. (e.g., those listed in Table 2 or 4). For those nucleic acid molecules or polypeptides whose expression is decreased in a patient having FSHD, agonists would be particularly useful in the methods of the invention. For those nucleic acid molecules or polypeptides whose expression is increased in a patient having FSHD, antagonists would be particularly useful in the methods of the invention.

[0146] Each of the DNA sequences identified herein may be used in the discovery and development of a therapeutic agent for the treatment of FSHD. The encoded protein, upon expression, can be used as a target for the screening of drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct sequences that promote the expression of the coding sequence of interest. Such sequences may be isolated by standard techniques (Ausubel et al., supra). Optionally, agents identified in any of the above-described assays may be confirmed as useful in cell culture or in a chimeric animal of the invention. Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.

Test Agents and Extracts

[0147] In general, agents capable of normalizing or promoting the normalization of expression of a gene listed in Table 2 or 4 are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries (e.g., Table 2 or 4), according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or agents is not critical to the screening procedure(s) of the invention. Agents used in screens may include known agents (for example, known therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or agents can be screened using the methods described herein. Examples of such extracts or agents include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic agents, as well as modification of existing agents. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical agents, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based agents. Synthetic agent libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural agents in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or agent is readily modified using standard chemical, physical, or biochemical methods.

[0148] In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.

[0149] When a crude extract is found to normalize or promote normalization of the activity of a polypeptide that is aberrantly regulated in FSHD, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that increases the expression or activity of the polypeptide. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, agents shown to be useful as therapeutics for the treatment of human FSHD are chemically modified according to methods known in the art.

Inhibitory Nucleic Acids

[0150] Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of a polypeptide that is overexpressed in FSHD (e.g., a polypeptide encoded by a gene listed in Table 2 or 4). Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes a polypeptide that is overexpressed in FSHD (e.g., antisense molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers).

[0151] MOE Gapmers

[0152] In one embodiment, the invention provides methods for characterizing the effects of RNaseH1-activating antisense oligonucleotides (ASO's) ("MOE gapmers") on markers of the invention. The RNAseH1 ASO chemistry provides for a 20 nucleotide phosphorothioate backbone (5-10-5 gapmer). In particular, the oligonucleotide comprises five nucleotides at each end with the 2'-O-(2-methoxyethyl) (MOE) modification and ten central deoxyribonucleotides for activation of RNase H1. In one embodiment, cells derived from an FSHD affected and paired control cells are contacted with ASO's targeting DUX4. The effect of the downregulation of DUX4 on markers of the invention (e.g., markers listed in Table 2 or 4) is assayed. In another embodiment, a marker of the invention (e.g., a marker upregulated in FSHD) is targeted, and the effect of such targeting is assessed on the levels of other markers of the invention.

[0153] siRNA

[0154] Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down-regulating gene expression (Zamore et al., Cell 101: 25-33; Elbashir et al., Nature 411: 494-498, 2001, hereby incorporated by reference). The therapeutic effectiveness of an sirNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38-39.2002).

[0155] Given the sequence of a target gene, siRNAs may be designed to inactivate that gene. Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically. The nucleic acid sequence of a gene can be used to design small interfering RNAs (siRNAs). The 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat FSHD.

[0156] The inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of expression. In one embodiment, expression of a gene listed in Table 2 or 4 is reduced in a skeletal muscle cell. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.

[0157] In one embodiment of the invention, a double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.

[0158] Small hairpin RNAs (shRNAs) comprise an RNA sequence having a stem-loop structure. A "stem-loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). The term "hairpin" is also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, the base-pairing can be exact, i.e. not include any mismatches. The multiple stem-loop structures can be linked to one another through a linker, such as, for example, a nucleic acid linker, other molecule, or some combination thereof.

[0159] As used herein, the term "small hairpin RNA" includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). While there may be some variation in range, a conventional stem-loop shRNA can comprise a stem ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. "shRNA" also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. In some instances the precursor miRNA molecule can include more than one stem-loop structure. MicroRNAs are endogenously encoded RNA molecules that are about 22-nucleotides long and generally expressed in a highly tissue- or developmental-stage-specific fashion and that post-transcriptionally regulate target genes. More than 200 distinct miRNAs have been identified in plants and animals. These small regulatory RNAs are believed to serve important biological functions by two prevailing modes of action: (1) by repressing the translation of target mRNAs, and (2) through RNA interference (RNAi), that is, cleavage and degradation of mRNAs. In the latter case, miRNAs function analogously to small interfering RNAs (siRNAs). Thus, one can design and express artificial miRNAs based on the features of existing miRNA genes.

[0160] shRNAs can be expressed from DNA vectors to provide sustained silencing and high yield delivery into almost any cell type. In some embodiments, the vector is a viral vector. Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations. Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells through any means known in the art. A producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein. Such retroviral vector particles then can be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a DNA replication protein.

[0161] Catalytic RNA molecules or ribozymes that include an antisense sequence of the present invention can be used to inhibit expression of a nucleic acid molecule in vivo (e.g., a nucleic acid molecule listed in Table 2 or 4). The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 A1, each of which is incorporated by reference.

[0162] Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8:183, 1992. Example of hairpin motifs are described by Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids Research, 18: 299, 1990. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

[0163] Essentially any method for introducing a nucleic acid construct into cells can be employed. Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct. A viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA. Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like. Thus the shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.

[0164] For expression within cells, DNA vectors, for example plasmid vectors comprising either an RNA polymerase II or RNA polymerase III promoter can be employed. Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters and in some cases, shRNAs are most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al., 2005, Nat. Genet. 39: 914-921). In some embodiments, expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters. Examples of useful promoters in the context of the invention are tetracycline-inducible promoters (including TRE-tight), IPTG-inducible promoters, tetracycline transactivator systems, and reverse tetracycline transactivator (rtTA) systems. Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types. A certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Patent Application PCT/US2003/030901 (Publication No. WO 2004-029219 A2) and Fewell et al., 2006, Drug Discovery Today 11: 975-982, for a description of inducible shRNA.

Modified Nucleic Acids

[0165] At least two types of oligonucleotides induce the cleavage of RNA by RNase H: polydeoxynucleotides with phosphodiester (PO) or phosphorothioate (PS) linkages. Although 2'-OMe-RNA sequences exhibit a high affinity for RNA targets, these sequences are not substrates for RNase H. A desirable oligonucleotide is one based on 2'-modified oligonucleotides containing oligodeoxynucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC.sub.50. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present invention may be used in conjunction with any technologies that may be developed, including covalently-closed multiple antisense (CMAS) oligonucleotides (Moon et al., Biochem J. 346:295-303, 2000; PCT Publication No. WO 00/61595), ribbon-type antisense (RiAS) oligonucleotides (Moon et al., J. Biol. Chem. 275:4647-4653, 2000; PCT Publication No. WO 00/61595), and large circular antisense oligonucleotides (U.S. Patent Application Publication No. US 2002/0168631 A1).

[0166] As is known in the art, a nucleoside is a nucleobase-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure; open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

[0167] Specific examples of preferred nucleobase oligomers useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, nucleobase oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleobase oligomers.

[0168] Nucleobase oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriest-ers, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.

[0169] Nucleobase oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts. Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

[0170] In other nucleobase oligomers, both the sugar and the internucleoside linkage, i.e., the backbone, are replaced with novel groups. The nucleobase units are maintained for hybridization with a gene listed in Table 2 or 4. One such nucleobase oligomer, is referred to as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Methods for making and using these nucleobase oligomers are described, for example, in "Peptide Nucleic Acids: Protocols and Applications" Ed. P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Representative United States patents that teach the preparation of PNAs include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0171] In particular embodiments of the invention, the nucleobase oligomers have phosphorothioate backbones and nucleosides with heteroatom backbones, and in particular --CH.sub.2--NH--O--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- (known as a methylene (methylimino) or MMI backbone), --CH.sub.2--O--N(CH.sub.3)--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2--, and --O--N(CH.sub.3)--CH.sub.2--CH.sub.2--. In some embodiments, the oligonucleotides have morpholino backbone structures described in U.S. Pat. No. 5,034,506.

[0172] Nucleobase oligomers may also contain one or more substituted sugar moieties. Nucleobase oligomers comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly preferred are O[(CH.sub.2).sub.nO].sub.nCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m are from 1 to about 10. Other preferred nucleobase oligomers include one of the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a nucleobase oligomer, or a group for improving the pharmacodynamic properties of an nucleobase oligomer, and other substituents having similar properties. Preferred modifications are 2'-O-methyl and 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE). Another desirable modification is 2'-dimethylaminooxyethoxy (i.e., O(CH.sub.2).sub.2ON(CH.sub.3).sub.2), also known as 2'-DMAOE. Other modifications include, 2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on an oligonucleotide or other nucleobase oligomer, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Nucleobase oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.

[0173] Nucleobase oligomers may also include nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyl uracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of an antisense oligonucleotide of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are desirable base substitutions, even more particularly when combined with 2'-O-methoxyethyl or 2'-O-methyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is herein incorporated by reference.

[0174] Another modification of a nucleobase oligomer of the invention involves chemically linking to the nucleobase oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553-6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let, 4:1053-1060, 1994), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 20:533-538: 1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 10:1111-1118, 1991; Kabanov et al., FEBS Lett., 259:327-330, 1990; Svinarchuk et al., Biochimie, 75:49-54, 1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 36:3651-3654, 1995; Shea et al., Nucl. Acids Res., 18:3777-3783, 1990), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 14:969-973, 1995), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 36:3651-3654, 1995), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1264:229-237, 1995), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 277:923-937, 1996. Representative United States patents that teach the preparation of such nucleobase oligomer conjugates include U.S. Pat. Nos. 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 4,835,263; 4,876,335; 4,904,582; 4,948,882; 4,958,013; 5,082,830; 5,109,124; 5,112,963; 5,118,802; 5,138,045; 5,214,136; 5,218,105; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,414,077; 5,416,203, 5,451,463; 5,486,603; 5,510,475; 5,512,439; 5,512,667; 5,514,785; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,565,552; 5,567,810; 5,574,142; 5,578,717; 5,578,718; 5,580,731; 5,585,481; 5,587,371; 5,591,584; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,608,046; and 5,688,941, each of which is herein incorporated by reference.

[0175] The present invention also includes nucleobase oligomers that are chimeric compounds. "Chimeric" nucleobase oligomers are nucleobase oligomers, particularly oligonucleotides, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide. These nucleobase oligomers typically contain at least one region where the nucleobase oligomer is modified to confer, upon the nucleobase oligomer, increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the nucleobase oligomer may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of nucleobase oligomer inhibition of gene expression. Consequently, comparable results can often be obtained with shorter nucleobase oligomers when chimeric nucleobase oligomers are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.

[0176] Chimeric nucleobase oligomers of the invention may be formed as composite structures of two or more nucleobase oligomers as described above. Such nucleobase oligomers, when oligonucleotides, have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.

[0177] The nucleobase oligomers used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0178] The nucleobase oligomers of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

Delivery of Polynucleotides

[0179] Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).

Polynucleotide Therapy for FSHD

[0180] Polynucleotide therapy is one therapeutic approach for preventing or ameliorating FSHD associated with the reduced expression of a nucleic acid molecule listed in Table 2 or 4. Such nucleic acid molecules can be delivered to cells that lack sufficient, normal protein expression or biological activity. The nucleic acid molecules must be delivered to those cells in a form in which they can be taken up by the cells and so that sufficient levels of protein can be produced to increase protein expression or function in a patient having FSHD.

[0181] Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, a full length gene (e.g., a nucleic acid molecule listed in Table 2 or 4), or a portion thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific for a target cell type of interest (e.g., a skeletal muscle cell). Promoters useful in the methods of the invention include, for example, myoD.

[0182] Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). Most preferably, a viral vector is used to administer the gene of interest (e.g., nucleic acid molecules listed in Table 2 or 4) systemically or to a skeletal muscle.

[0183] Non-viral approaches can also be employed for the introduction of therapeutic agent to a cell of an FSHD affected. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.

[0184] Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue.

[0185] cDNA expression for use in gene therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types, such as cells of the central nervous system or their associated glial cells, can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0186] Another therapeutic approach included in the invention involves administration of a recombinant therapeutic, such as a recombinant polypeptide encoded by a gene downregulated in FSHD. In one embodiment, the protein is either administered directly to a disease-affected tissue (for example, by injection into the muscle) or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the administered protein depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Generally, between 0.1 mg and 100 mg, is administered per day to an adult in any pharmaceutically acceptable formulation.

Pharmaceutical Therapeutics

[0187] The invention provides a simple means for identifying agents (including nucleic acid molecules, inhibitory nucleic acid molecules, peptides, small molecules, and mimetics) capable of acting as therapeutics for the treatment of FSHD. Accordingly, a chemical entity discovered to have medicinal value using the methods described herein is useful as a drug or as information for structural modification of existing agents, e.g., by rational drug design.

[0188] For therapeutic uses, the agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of FSHD therapeutic in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of FSHD. An agent is administered at a dosage that controls the clinical or physiological symptoms of FSHD as determined by clinical evaluation or by a diagnostic method of the invention that assays the expression of a nucleic acid molecule listed in Table 2 or 4, or the biological activity of a polypeptide encoded by such a nucleic acid molecule.

Formulation of Pharmaceutical Compositions

[0189] The administration of an agent for the treatment of FSHD may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing FSHD. The agent may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0190] Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the central nervous system or cerebrospinal fluid; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target FSHD by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., skeletal muscle cell) whose function is perturbed in FSHD. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.

[0191] Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the FSHD therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the FSHD therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

[0192] The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

[0193] Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active therapeutic (s), the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic (s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

[0194] As indicated above, the pharmaceutical compositions according to the invention may be in a form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

Solid Dosage Forms for Oral Use

[0195] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to the skilled artisan. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0196] The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.

[0197] The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active therapeutic substance). The coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of Pharmaceutical Technology, supra. Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

[0198] The present invention provides methods of treating FSHD or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to FSHD or a symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of a compound herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.

[0199] The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

[0200] As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0201] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0202] The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which muscular dystrophy may be implicated.

[0203] In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to FSHD, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.

[0204] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

EXAMPLES

[0205] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

Example 1

Microarray Analysis Identifies Genes that are Differentially Regulated in FSHD

[0206] Open muscle biopsy was performed on both the biceps and deltoid muscles of 6 FSHD affected and 5 unaffected subjects that are first degree relatives of the FSHD affected subjects. Characteristics of the donors are provided in Table 1.

TABLE-US-00001 TABLE 1 Clinical characteristics of FSHD subjects and unaffected donors. Age at Enroll- EcoRI/ Familial ment Blnl Deltoid Biceps Donor* Relations Gender (yrs) Allele Strength** Strength 07A proband F 18 29 kb 4+/5 at 90.degree. full 07U mother of F 49 34 kb full full proband (4qB), 53 kb (4qA) 09A proband F 31 25 kb 5/5 at 45.degree. 4+/5 09U mother of 09A F 57 47 kb full full 12A proband M 49 18 kb 4+/5 at 90.degree. 4+/5 12U sister of 12A F 45 >112 kb full full 15A proband M 67 28 kb 5/5 at 90.degree. 5/5 15B brother of 15A M 69 28 kb full full 15V sister of 15A F 60 107 kb full full 21B proband F 59 34 kb 5/5 4+/5 21U sister of 21B F 48 150 kb full full *Donors are designated by cohort (family) number (07, 09, etc.) followed by A, B for the FSHD donors or U,V for the unaffected 1.sup.st degree relative(s) of the FSHD subject(s) in the cohort. Each cohort was composed of at least one affected individual with genetically and clinically verified FSHD, and at least one unaffected first degree relative with unshortened D4Z4 alleles and normal strength. **Muscle strength is presented using a modified MRC scale where 5/5 is full strength for right/left sides.

[0207] Molecular diagnosis of FSHD was confirmed by the University of Iowa Diagnostic Laboratories and indicated that each donor with a clinical diagnosis of FSHD also had a contracted 4q D4Z4 region in combination with a 4 qA telomeric allele (Table 1).

Primary Cell Culture.

[0208] Primary muscle cell strains were established from open muscle biopsies following collagenase IV and dispase dissociation as previously described (Stadler et al., 2011). Cells were cultured at 37.degree. C. in 5% CO.sub.2 on 0.1% gelatin-coated dishes and propagated by daily feeding with HMP growth medium consisting of Ham's F10 medium (Cellgro) supplemented with 20% characterized fetal bovine serum (Hyclone), 0.5% chicken embryo extract, 1.2 mM CaCl.sub.2, and 1% antibiotics/antimycotics (Cellgro). Cultures were incubated until cells reached 50-70% confluence, at which time cells were harvested after dissociation with TrypLE Express (Gibco), counted, and expanded for fluorescence-activated cell sorting (FACS) or frozen storage.

[0209] The initial primary cultures were enriched for myogenic cells by using a FacsAria instrument (BD Biosciences) to select cells based on positive staining with APC-conjugated anti-human CD56 (BD Biosciences). For FACS, cells were trypsinized, counted, and collected by centrifugation, after which .about.1.times.10.sup.6 cells were resuspended in 0.1 ml 10% fetal bovine serum (Hyclone) in PBS and incubated with the CD56 antibody according to manufacturer's instructions. As a control, cells were incubated with APC-conjugated mouse IgG1 K isotype antibody (BD Biosciences). Cells were incubated for 30-60 min on ice, collected by centrifugation, washed twice with 10% fetal bovine serum in PBS, and resuspended in 0.5-1.0 ml 10% fetal bovine serum in PBS and subjected to FACS to select CD56-positive cells.

[0210] The CD56-positive populations of myogenic cells that were obtained by FACS were seeded on dishes coated with 0.1% gelatin (Sigma) and incubated at 37.degree. C. and 5% CO.sub.2, with each cell strain grown independently. Cells were propagated by daily feeding with HMP growth medium consisting of Ham's F10 medium (Cellgro) supplemented with 20% characterized FBS (Hyclone), 1% chicken embryo extract, 120 mM CaCl.sub.2, and 1% antibiotics/antimycotics (Cellgro). Cultures were incubated until cells reached 50-70% confluence, at which time cells were harvested after dissociation with TrypLE Express (Gibco), counted, and used for expansion or for frozen storage. For all experiments described here, cultures were examined at 20-35 population doublings after the initial isolation, which was at least 10-15 population doublings prior to loss of proliferative capacity.

[0211] FIG. 1 provides a table showing cell lines produced using these methods.

Primary Cell Cultures for RNA Isolation.

[0212] To initiate cultures, CD56-positive cells were seeded at .about.4000 cells/cm.sup.2 and cultured with daily feeding with LHCN growth medium consisting of 4:1 DMEM:Medium 199 supplemented with 15% characterized FBS (Hyclone), 0.02M HEPES (Sigma-Aldrich), 0.03 .mu.g/ml ZnSO.sub.4 (Sigma-Aldrich), 1.4 ug/ml Vitamin B12 (Sigma-Aldrich), 0.055 ug/ml dexamethasone (Sigma-Aldrich), 1% antibiotics/antimycotics (Cellgro), 2.5 ng/ml hepatocyte growth factor (Chemicon International) and 10 ng/ml basic fibroblast growth factor (Millipore). To induce differentiation, cells were propagated by daily feeding with LHCN growth medium until .about.95% confluent, at which time cultures were switched to a low serum differentiation medium (DM) consisting of 4:1 DMEM:Medium 199 supplemented with 2% horse serum (Hyclone), 2 mM L-glutamine (Gibco), 1% antibiotics/antimycotics (Cellgro), 10 mM HEPES (Gibco), and 1 mM sodium pyruvate (Gibco). For RNA isolation for microarray analysis, cultures were harvested at two different stages of culture: (1) after two days of proliferation in growth medium, at which point cells were sub-confluent (GM); (2) after four days in differentiation medium (DM). Cells were harvested by rinsing culture dishes 2.times. with PBS and removing the cells with cell lifters (Costar), after which the cells were collected by centrifugation, snap frozen in liquid nitrogen, and stored at -80.degree. C.

RNA Isolation and Microarray Analysis.

[0213] Total RNA was isolated from frozen cell pellets using 1 ml TRIzol reagent (Invitrogen). RNA concentration was quantified with UV absorption at 260 nm using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific) and the RNA integrity was assessed using the RNA 6000 Nano chip on the Agilent 2100 Bioanalyzer (Agilent Technologies). Gene expression profiling was carried out using the Affymetrix GeneChip Human Gene 1.0 ST arrays. The current format of these arrays interrogates 28,869 annotated genes in the human genome with approximately twenty six 25-mer oligonucleotide probes spread across the full length of the transcript. Microarray data was collected at Expression Analysis, Inc. (Durham, N.C.). Biotin-labeled target for the microarray experiment was prepared using 100 ng of total RNA and cDNA was synthesized using the GeneChip WT (Whole Transcript) Sense Target Labeling and Control Reagents kit as described by the manufacturer (Affymetrix). The sense cDNA was then fragmented by UDG (uracil DNA glycosylase) and APE 1 (apurinic/apyrimidic endonuclease 1) and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit (Affymetrix). Hybridization was performed using 5 micrograms of biotinylated target, which was incubated with the GeneChip Human Gene 1.0 ST array (Affymetrix) at 45.degree. C. for 16-20 hours. Following hybridization, non-specifically bound material was removed by washing and detection of specifically bound target was performed using the GeneChip Hybridization, Wash and Stain kit, and the GeneChip Fluidics Station 450 (Affymetrix). The arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix) and raw data was extracted from the scanned images and analyzed with the Affymetrix GeneChip Command Console Software (Affymetrix).

Microarray Data Analysis.

[0214] The raw array data was preprocessed and normalized using the Robust Multichip Average (RMA) method. This procedure includes background correction and quantile normalization of the arrays at the probe level, followed by robust summarization of expression at the transcript level. Differential expression between classes was calculated using linear models with the limma package from the Bioconductor project (Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, No. 1, Article 3). The linear model used was ".about.0+Class:Muscle:Medium+Cohort", where Class, Muscle, and Medium are each two-level factors with levels FSHD & Control; Biceps & Deltoid; and GM & DM, respectively; and the factor Cohort has one level for each cohort. The interaction terms (denoted ":") for the three two-level factors model changes between FSHD and Control expression levels that may vary for each of the four combinations of muscle type and medium, and the additive Cohort factor models different baseline expression levels for samples from different cohorts. To control for multiple hypothesis testing false discovery rates (FDRs) were computed based on the p-values from empirical Bayes moderated t-statistics for differential expression. The reported results are based on only those probesets annotated with Entrez gene IDs, and in cases of multiple probesets with the same Entrez ID on only the probeset with the largest interquartile range; after this filtering probesets corresponding to 19,983 genes were left. FSHD typically affects biceps more severely than deltoid, and differences between FSHD and control cell-cultures were stronger in DM than in GM.

[0215] Table 2 lists 142 genes for which the expression difference between FSHD and control biceps in DM had p-value at most 0.001, which corresponded to an FDR of 0.15. The columns labeled AvsU.DM.B.pval and AvsU.DM.B. gives the p-value and FDR, respectively, and the column labeled AvsU.DM.B.lfc gives the log 2(fold-change) between FSHD and control expression levels, with positive scores indicating higher expression in FSHD samples relative to controls, and negative scores indicating lower expression in FSHD samples relative to controls (Table 2).

TABLE-US-00002 TABLE 2 AvsU. AvsU.DM.B. AvsU.DM. Probeset SYMBOL UNIGENE ENTREZID REFSEQ GENENAME DM.B.lfc pval B.fdr 7933733 FAM13C Hs.499704 220965 NM_001001971.2 family with sequence -0.85 5.30E-07 0.011 similarity 13, member C 8153065 MIR30B NA 407030 NR_029666.1 microRNA 30b -0.9 1.70E-06 0.017 8079753 DAG1 Hs.76111 1605 NM_001165928.3 dystroglycan 1 -0.59 1.20E-05 0.071 (dystrophin-associated glycoprotein 1) 7910923 FMN2 Hs.24889 56776 NM_020066.4 formin 2 0.77 1.40E-05 0.071 7927876 TET1 Hs.567594 80312 NM_030625.2 tet oncogene 1 -0.65 1.80E-05 0.072 8075673 RBFOX2 Hs.282998 23543 NM_001031695.2 RNA binding protein, -0.43 4.10E-05 0.1 fox-1 homolog (C. elegans) 2 7980891 TC2N Hs.510262 123036 NM_001128595.1 tandem C2 domains, 0.72 5.20E-05 0.1 nuclear 8126770 CYP39A1 Hs.387367 51302 NM_016593.3 cytochrome P450, -0.51 6.60E-05 0.1 family 39, subfamily A, polypeptide 1 8054041 TRIM43 Hs.232026 129868 NM_138800.1 tripartite motif 2.59 7.30E-05 0.1 containing 43 8034099 MIR199A1 NA 406976 NR_029586.1 microRNA 199a-1 0.88 7.50E-05 0.1 8057578 CALCRL Hs.470882 10203 NM_005795.5 calcitonin receptor-like -1.17 8.40E-05 0.1 7898537 PAX7 Hs.113253 5081 NM_001135254.1 paired box 7 -0.75 8.40E-05 0.1 8084100 USP13 Hs.175322 8975 NM_003940.2 ubiquitin specific -0.75 8.40E-05 0.1 peptidase 13 (isopeptidase T-3) 7994463 ATP2A1 Hs.657344 487 NM_004320.4 ATPase, Ca++ -1.82 8.70E-05 0.1 transporting, cardiac muscle, fast twitch 1 7958174 TXNRD1 Hs.728817 7296 NM_001093771.2 thioredoxin reductase 0.49 8.80E-05 0.1 1 7982000 SNORD116- NA 100033438 NR_003340.2 small nucleolar RNA, -0.78 9.10E-05 0.1 26 C/D box 116-26 7973580 FITM1 Hs.128060 161247 NM_203402.2 fat storage-inducing -0.72 9.90E-05 0.1 transmembrane protein 1 7928661 MBL1P Hs.102310 8512 NR_002724.2 mannose-binding -0.67 1.00E-04 0.1 lectin (protein A) 1, pseudogene 8053984 ANKRD23 Hs.643430 200539 NM_144994.7 ankyrin repeat domain -0.45 0.00011 0.1 23 7941761 RHOD Hs.15114 29984 NM_014578.3 ras homolog gene 0.45 0.00011 0.1 family, member D 8072015 ADRBK2 Hs.657494 157 NM_005160.3 adrenergic, beta, -0.78 0.00012 0.1 receptor kinase 2 8027674 ZNF302 Hs.436350 55900 NM_001012320.1 zinc finger protein 302 -0.36 0.00012 0.1 8120961 MRAP2 Hs.370055 112609 NM_138409.2 melanocortin 2 -0.93 0.00013 0.1 receptor accessory protein 2 7960865 SLC2A3 Hs.419240 6515 NM_006931.2 solute carrier family 2 0.64 0.00013 0.1 (facilitated glucose transporter), member 3 7947052 IGSF22 Hs.434152 283284 NM_173588.3 immunoglobulin -0.58 0.00014 0.1 superfamily, member 22 8093665 GRK4 Hs.32959 2868 NM_001004056.1 G protein-coupled -0.46 0.00014 0.1 receptor kinase 4 8162132 C9orf153 Hs.632073 389766 NM_001010907.1 chromosome 9 open -0.52 0.00014 0.1 reading frame 153 8008664 AKAP1 Hs.463506 8165 NM_003488.3 A kinase (PRKA) -0.64 0.00015 0.11 anchor protein 1 8101086 NAAA Hs.437365 27163 NM_001042402.1 N-acylethanolamine 0.52 0.00016 0.11 acid amidase 7915261 TRIT1 Hs.356554 54802 NM_017646.4 tRNA -0.48 0.00016 0.11 isopentenyltransferase 1 8058570 C2orf67 Hs.282260 151050 NM_152519.2 chromosome 2 open -0.56 0.00018 0.11 reading frame 67 7912595 PRAMEF13 Hs.531192 400736 NM_001024661.1 PRAME family 1.52 0.00019 0.11 member 13 7978932 SOS2 Hs.291533 6655 NM_006939.2 son of sevenless -0.27 0.00019 0.11 homolog 2 (Drosophila) 8023121 ST8SIA5 Hs.465025 29906 NM_013305.4 ST8 alpha-N-acetyl- -0.61 0.00021 0.12 neuraminide alpha-2,8- sialyltransferase 5 7934945 PANK1 Hs.376351 53354 NM_138316.3 pantothenate kinase 1 -0.66 0.00021 0.12 7979483 C14orf39 Hs.335754 317761 NM_174978.2 chromosome 14 open -0.79 0.00022 0.12 reading frame 39 7923978 CD34 Hs.374990 947 NM_001025109.1 CD34 molecule 0.79 0.00023 0.12 7920552 KCNN3 Hs.490765 3782 NM_001204087.1 potassium -0.88 0.00024 0.12 intermediate/small conductance calcium- activated channel, subfamily N, member 3 8082003 EAF2 Hs.477325 55840 NM_018456.4 ELL associated factor -0.69 0.00024 0.12 2 8024518 ZNF555 Hs.47712 148254 NM_001172775.1 zinc finger protein 555 -0.82 0.00026 0.12 8151074 PDE7A Hs.527119 5150 NM_002603.3 phosphodiesterase 7A -0.61 0.00026 0.12 8130071 C15orf29 Hs.633566 79768 NM_024713.2 chromosome 15 open -0.76 0.00027 0.12 reading frame 29 8123584 MYLK4 Hs.127830 340156 NM_001012418.3 myosin light chain -1.18 0.00028 0.12 kinase family, member 4 7906764 HSPA6 Hs.654614 3310 NM_002155.3 heat shock 70kDa 0.49 0.00029 0.12 protein 6 (HSP70B') 7897987 PRAMEF2 Hs.104991 65122 NM_023014.1 PRAME family 1.59 0.00029 0.12 member 2 7926679 KIAA1217 Hs.445885 56243 NM_001098500.1 KIAA1217 -0.58 0.00031 0.12 8163733 CDK5RAP2 Hs.269560 55755 NM_001011649.2 CDK5 regulatory -0.4 0.00032 0.12 subunit associated protein 2 8050443 SMC6 Hs.526728 79677 NM_001142286.1 structural maintenance -0.5 0.00033 0.12 of chromosomes 6 7947110 E2F8 Hs.523526 79733 NM_024680.3 E2F transcription -1.38 0.00033 0.12 factor 8 8073943 ZBED4 Hs.475208 9889 NM_014838.2 zinc finger, BED-type -0.36 0.00034 0.12 containing 4 7958884 OAS1 Hs.524760 4938 NM_001032409.1 2',5'-oligoadenylate 0.63 0.00035 0.12 synthetase 1, 40/46kDa 8133477 GTF2IRD1 Hs.647056 9569 NM_001199207.1 GTF2I repeat domain -0.51 0.00035 0.12 containing 1 7944955 PKNOX2 Hs.278564 63876 NM_022062.2 PBX/knotted 1 -0.62 0.00036 0.12 homeobox 2 8020068 ANKRD12 Hs.464585 23253 NM_001083625.2 ankyrin repeat domain -0.43 0.00037 0.12 12 7983704 GLDN Hs.526441 342035 NM_181789.2 gliomedin -0.45 0.00037 0.12 8131803 IL6 Hs.654458 3569 NM_000600.3 interleukin 6 1.12 0.00037 0.12 (interferon, beta 2) 7909730 KCNK2 Hs.497745 3776 NM_001017424.2 potassium channel, 1.18 0.00037 0.12 subfamily K, member 2 7908397 RGS13 Hs.497220 6003 NM_002927.4 regulator of G-protein -1.02 0.00037 0.12 signaling 13 8072170 KREMEN1 Hs.229335 83999 NM_001039570.2 kringle containing -0.53 0.00037 0.12 transmembrane protein 1 8002020 TPPP3 Hs.534458 51673 NM_015964.2 tubulin -0.61 0.00039 0.12 polymerization- promoting protein family member 3 7897978 PRAMEF1 Hs.454859 65121 NM_023013.2 PRAME family 1.33 0.00039 0.12 member 1 7909545 TRAF5 Hs.523930 7188 NM_001033910.2 TNF receptor- -0.64 0.00039 0.12 associated factor 5 8094441 SLC34A2 Hs.479372 10568 NM_001177998.1 solute carrier family 2.23 4.00E-04 0.12 34 (sodium phosphate), member 2 8137670 PDGFA Hs.535898 5154 NM_002607.5 platelet-derived -0.58 4.00E-04 0.12 growth factor alpha polypeptide 8086482 ZNF445 Hs.250481 353274 NM_181489.5 zinc finger protein 445 -0.31 0.00041 0.12 7964646 PPM1H Hs.435479 57460 NM_020700.1 protein phosphatase, -0.42 0.00041 0.12 Mg2+/Mn2+ dependent, 1H 8027312 ZNF429 Hs.572567 353088 NM_001001415.2 zinc finger protein 429 -0.58 0.00042 0.12 7969815 CLYBL Hs.655642 171425 NM_206808.2 citrate lyase beta like -0.57 0.00043 0.12 8099302 MIR95 NA 407052 NR_029511.1 microRNA 95 -1 0.00045 0.12 7971653 DLEU2 Hs.547964 8847 NR_002612.1 deleted in lymphocytic -0.53 0.00045 0.12 leukemia 2 (non- protein coding) 8069991 TCP10L Hs.728804 140290 NM_144659.5 t-complex 10 (mouse)- -0.4 0.00047 0.12 like 7970111 ARHGEF7 Hs.508738 8874 NM_001113511.1 Rho guanine -0.4 0.00047 0.12 nucleotide exchange factor (GEF) 7 7995440 FLJ44674 Hs.514338 400535 XR_041153.1 FLJ44674 protein 0.35 5.00E-04 0.12 7898211 DDI2 Hs.718857 84301 NM_032341.4 DNA-damage -0.48 5.00E-04 0.12 inducible 1 homolog 2 (S. cerevisiae) 8163109 C9orf4 Hs.347537 23732 NM_014334.2 chromosome 9 open 0.4 0.00052 0.12 reading frame 4 7918552 C1orf183 Hs.193406 55924 NM_019099.4 chromosome 1 open -0.43 0.00052 0.12 reading frame 183 7960850 SLC2A14 Hs.655169 144195 NM_153449.2 solute carrier family 2 0.49 0.00053 0.12 (facilitated glucose transporter), member 14 8050658 ATAD2B Hs.467862 54454 NM_017552.2 ATPase family, AAA -0.33 0.00053 0.12 domain containing 2B 8124502 ZNF184 Hs.158174 7738 NM_007149.2 zinc finger protein 184 -0.35 0.00053 0.12 8060813 MCM8 Hs.597484 84515 NM_032485.4 minichromosome -0.39 0.00053 0.12 maintenance complex component 8 8097086 MYOZ2 Hs.381047 51778 NM_016599.4 myozenin 2 -1.2 0.00054 0.12

8044008 IL1RL2 Hs.659863 8808 NM_003854.2 interleukin 1 receptor 0.38 0.00054 0.12 like 2 8054664 ZC3H8 Hs.418416 84524 NM_032494.2 zinc finger CCCH-type -0.4 0.00055 0.12 containing 8 8097256 FGF2 Hs.284244 2247 NM_002006.4 fibroblast growth 0.88 0.00056 0.12 factor 2 (basic) 8100312 LRRC66 Hs.661450 339977 NM_001024611.1 leucine rich repeat -0.77 0.00056 0.12 containing 66 8102352 PITX2 Hs.643588 5308 NM_000325.5 paired-like -0.53 0.00056 0.12 homeodomain 2 8015590 STAT5B Hs.595276 6777 NM_012448.3 signal transducer and -0.54 0.00056 0.12 activator of transcription 5B 8069348 PCNT Hs.474069 5116 NM_006031.5 pericentrin -0.34 0.00057 0.12 8136235 CPA1 Hs.2879 1357 NM_001868.2 carboxypeptidase A1 -0.4 0.00058 0.12 (pancreatic) 7968883 C13orf31 Hs.210586 144811 NM_001128303.1 chromosome 13 open 0.94 0.00058 0.12 reading frame 31 7950955 TRIM49 Hs.534218 57093 NM_020358.2 tripartite motif 1.68 0.00058 0.12 containing 49 7957126 KCNMB4 Hs.525529 27345 NM_014505.5 potassium large -0.82 0.00059 0.12 conductance calcium- activated channel, subfamily M, beta member 4 8102862 MAML3 Hs.586165 55534 NM_018717.4 mastermind-like 3 -0.54 0.00059 0.12 (Drosophila) 7951781 C11orf71 Hs.715083 54494 NM_019021.3 chromosome 11 open -0.37 6.00E-04 0.12 reading frame 71 7909768 SPATA17 Hs.171130 128153 NM_138796.2 spermatogenesis -0.49 0.00061 0.12 associated 17 8094778 UCHL1 Hs.518731 7345 NM_004181.4 ubiquitin carboxyl- 0.77 0.00061 0.12 terminal esterase L1 (ubiquitin thiolesterase) 8164580 PTGES Hs.146688 9536 NM_004878.4 prostaglandin E 0.94 0.00061 0.12 synthase 8104163 LRRC14B Hs.683662 389257 NM_001080478.1 leucine rich repeat -0.57 0.00062 0.12 containing 14B 8036406 ZNF571 Hs.590944 51276 NM_016536.3 zinc finger protein 571 -0.41 0.00062 0.12 8128087 GABRR1 Hs.99927 2569 NM_002042.4 gamma-aminobutyric -0.63 0.00063 0.12 acid (GABA) receptor, rho 1 8028219 ZNF420 Hs.444992 147923 NM_144689.3 zinc finger protein 420 -0.39 0.00064 0.13 8057771 STAT4 Hs.80642 6775 NM_003151.3 signal transducer and 0.67 0.00067 0.13 activator of transcription 4 8058350 ICA1L Hs.516629 130026 NM_138468.4 islet cell autoantigen -0.46 0.00068 0.13 1,69kDa-like 8018922 CYTH1 Hs.191215 9267 NM_004762.2 cytohesin 1 -0.47 0.00068 0.13 7968637 CCNA1 Hs.417050 8900 NM_001111045.1 cyclin A1 2.12 0.00073 0.14 7974771 C14orf135 Hs.509499 64430 NM_022495.5 chromosome 14 open -0.37 0.00076 0.14 reading frame 135 7907790 CEP350 Hs.413045 9857 NM_014810.4 centrosomal protein -0.44 0.00076 0.14 350kDa 8030823 IGLON5 Hs.546636 402665 NM_001101372.1 IgLON family member 0.45 0.00077 0.14 5 7917996 LRRC39 Hs.44277 127495 NM_144620.3 leucine rich repeat -1.23 0.00078 0.14 containing 39 7905986 FDPS Hs.335918 2224 NM_001135821.1 farnesyl diphosphate 0.59 0.00079 0.14 synthase 8097867 KIAA0922 Hs.205572 23240 NM_001131007.1 KIAA0922 -0.4 0.00079 0.14 8120300 C6orf142 Hs.449276 90523 NM_138569.2 chromosome 6 open -1.71 0.00079 0.14 reading frame 142 8068220 C21orf49 Hs.54725 54067 NR_024622.1 chromosome 21 open -0.43 8.00E-04 0.14 reading frame 49 8037298 CD177 Hs.232165 57126 NM_020406.2 CD177 molecule 0.83 8.00E-04 0.14 8148501 PTP4A3 Hs.43666 11156 NM_007079.2 protein tyrosine -0.93 0.00081 0.14 phosphatase type IVA, member 3 7954012 LOH12CR1 Hs.720779 118426 NM_058169.3 loss of heterozygosity, -0.43 0.00084 0.14 12, chromosomal region 1 8108708 PCDHB7 Hs.203830 56129 NM_018940.2 protocadherin beta 7 -0.37 0.00085 0.14 8116595 WRNIP1 Hs.236828 56897 NM_020135.2 Werner helicase -0.31 0.00085 0.14 interacting protein 1 7934434 MYOZ1 Hs.238756 58529 NM_021245.3 myozenin 1 -1.55 0.00085 0.14 8024909 KDM4B Hs.654816 23030 NM_015015.2 lysine (K)-specific -0.26 0.00086 0.14 demethylase 4B 8144812 PCM1 Hs.491148 5108 NM_006197.3 pericentriolar material -0.33 0.00086 0.14 1 7933092 ZNF248 Hs.528423 57209 NM_021045.2 zinc finger protein 248 -0.5 0.00086 0.14 7928705 TSPAN14 Hs.310453 81619 NM_001128309.1 tetraspanin 14 -0.62 0.00086 0.14 8151457 HEY1 Hs.234434 23462 NM_001040708.1 hairy/enhancer-of-split -0.58 0.00087 0.14 related with YRPW motif 1 7934442 SYNPO2L Hs.645273 79933 NM_001114133.1 synaptopodin 2-like -0.78 0.00088 0.14 8033241 CD70 Hs.501497 970 NM_001252.3 CD70 molecule 0.32 0.00088 0.14 7921955 RXRG Hs.26550 6258 NM_006917.4 retinoid X receptor, -0.58 0.00089 0.14 gamma 8167603 CLCN5 Hs.166486 1184 NM_000084.3 chloride channel 5 -0.57 9.00E-04 0.14 8089647 KIAA2018 Hs.632570 205717 NM_001009899.2 KIAA2018 -0.4 0.00091 0.14 8139160 FAM183B Hs.144075 340286 NR_028347.1 acyloxyacyl hydrolase 0.35 0.00091 0.14 (neutrophil) 7957379 MYF5 Hs.178023 4617 NM_005593.2 myogenic factor 5 -0.91 0.00094 0.14 8144082 C7orf13 Hs.647014 129790 NR_026865.1 chromosome 7 open -0.39 0.00095 0.14 reading frame 13 7986004 ZNF774 Hs.55307 342132 NM_001004309.2 zinc finger protein 774 -0.28 0.00095 0.14 8045198 CFC1B Hs.503733 653275 NM_001079530.1 cripto, FRL-1, cryptic 0.26 0.00096 0.14 family 1B 8125289 TNXA Hs.708061 7146 NR_001284.2 tenascin XA 1.87 0.00097 0.14 pseudogene 7915277 MYCL1 Hs.437922 4610 NM_001033081.2 v-myc -0.77 0.00098 0.14 myelocytomatosis viral oncogene homolog 1, lung carcinoma derived (avian) 8002303 NQO1 Hs.406515 1728 NM_000903.2 NAD(P)H 0.67 0.001 0.15 dehydrogenase, quinone 1 8033362 INSR Hs.465744 3643 NM_000208.2 insulin receptor -0.59 0.001 0.15 8025672 SLC44A2 Hs.534560 57153 NM_001145056.1 solute carrier family -0.35 0.001 0.15 44, member 2 7965510 TMCC3 Hs.370410 57458 NM_020698.2 transmembrane and -0.42 0.001 0.15 coiled-coil domain family 3 8118644 RPS18 Hs.627414 6222 NM_022551.2 ribosomal protein S18 0.34 0.001 0.15 7940824 NAA40 Hs.523753 79829 NM_024771.2 N(alpha)- -0.33 0.001 0.15 acetyltransferase 40, NatD catalytic subunit, homolog (S. cerevisiae)

Example 2

A Humanized Mouse Model of FSHD

[0216] Both FSHD- and control-derived myoblasts from multiple cohorts (described in Homma et al., European Journal of Human Genetics (2012) 20, 404-410) engrafted and formed human muscle fibers after 30 days in vivo. All mouse experiments were performed using BBRI IACUC-approved protocols. Nonobese diabetic Rag1 and IL2r.gamma. null (NOD-Rag1 null IL2r null or RAG, Jax stock number 007799) mice were used as recipients for human cell transplantations. Adult muscle, composed of multinucleated terminally differentiated myofibers, has a very low rate of cellular turnover under normal conditions. However, it has a remarkable capacity to regenerate in response to injury due to the presence of quiescent satellite cells. A regenerating muscle, which is in the process of incorporating newly differentiating cells, provides a favourable environment to receive a cell graft. Recipient tibialis anterior (TA) muscles were injected with 10 .mu.M cardiotoxin to induce a muscle degeneration/regeneration cycle. 1.times.10.sup.6FSHD myoblasts (from five different family cohorts), maintained in culture between 15 and 20 population doublings, were injected into surgically-exposed TA muscles 6 hours after cardiotoxin injection; following surgery, mice were monitored for recovery from anaesthesia and provided analgesics as required. Mice were sacrificed 4 weeks after transplantation and injected TA muscles, as well as non-injected gastrocnemius muscles were dissected and frozen in nitrogen-cooled isopentane. Entire muscle samples were cut into 10 .mu.m transverse cryostat sections and analyzed by immunofluorescence.

[0217] Visualization of engrafted fibers was performed via immunofluorescence using antibodies against the human specific sarcolemmal protein spectrin and the human specific nuclear protein lamin A/C. As shown in FIG. 1, immunofluorescence using human specific antibodies demonstrated high engraftment efficiency. To date, 36 xenografted mice have been generated and investigated. Histological analyses have confirmed that injected human FSHD myoblasts participate in the regeneration of murine muscle to form "humanized" fibers within the host TA. Quantifications have revealed that engraftment rates of greater than 100,000 human nuclei can be achieved in host muscle. These engraftments are of a sufficient magnitude to conduct morphological and molecular phenotype analyses of xenografted muscles. It is hypothesized that prior irradiation of host mice enriches engraftment of human myoblasts.

Example 3

DUX4-Fl Expressing FSHD Cells Engraft

[0218] Five cell strains (described in Homma et al., European Journal of Human Genetics (2012) 20, 404-410) were used for engraftment studies. Recent breakthroughs in the field suggest that DUX4, a gene identified inside D4Z4 repeats, is inappropriately expressed in the muscles of patients with FSHD. The disease could arise though a toxic gain of function. The precise molecular and cellular pathological mechanism involving DUX4 remains to be uncovered. Recent studies described the detection of two DUX4 transcripts, a long form (or full-length, fl) and a short form, and while the role of the short form is still unclear, the long form was specifically detected in FSHD samples, suggesting a central role in the pathogenic mechanism.

[0219] Based on engraftability and expression of DUX4-fl, cell strain selection for engraftment was refined to consist of three strains derived from the biceps of patients affected by FSHD, and three cell strains from corresponding unaffected firstdegree relatives. DUX4-fl transcript and protein were detected in cultured, differentiated myotubes for each of the three FSHD cell strains, and was absent in each control. Two control cell strains possessed at least one permissive allele for the disease (4 qA), but repressed DUX4 transcription. The third control strain did not contain the permissive allele (i.e. was genotyped as 4 qB/4 qB), and was therefore an ideal negative control for these studies.

[0220] Current theory predicts that DUX4 is actively transcribed in an average of 1 out of a 1,000 FSHD-derived nuclei at a given time. Recent engraftment trials have established that over 100,000 human myonuclei can be integrated with murine muscle. Adapting current theory to the invention's xenograft model, DUX4 might be expressed in greater than 100 nuclei in sizeable xenografts. This represents an amount of DUX4 mRNA detectable using 55 cycles of nested PCR; therefore, DUX4 expression at these levels should be detectable in xenografts from FSHD-derived myoblasts. Currently the expression of DUX4 at the mRNA and/or protein level is being assessed in FSHD- and control-transplanted TAs.

Example 4

Xenograft Integration with the Murine Skeletal Muscle Environment: Innervation of Human Fibers

[0221] Injecting cultured human myoblasts into murine skeletal muscle imposes a drastic environmental change. The ability of human myoblasts to assimilate successfully with host muscle is one important feature of a disease model. Immunohistological assays have confirmed that injected myoblasts successfully adapted to the murine microenvironment and integrated with the host muscle. Innervation of engrafted fibers by the nervous system of the host is important to prevent atrophy. Immunohistology studies using antibodies against neurofilament and Synaptic Vesicle protein 2 (SV2) were used to visualize afferent murine neurons in transverse sections. SV2 immunofluorescence at the pre-synaptic cleft was coupled with bungarotoxin-rhodamine staining at corresponding post-synaptic acetylcholine receptors to demonstrate an active neuromuscular junction (FIG. 2). Neuromuscular junction dispersion was observed throughout the muscle in specific patterns, directly innervating fibers in their vicinity without appearing to discriminate between mouse and human. Neuromuscular junctions on human and mouse fibers had no noticeable morphological differences. It is likely that resulting human fibers are successfully integrating with the murine musculature and nervous system.

Example 5

Xenograft Integration with Murine Skeletal Muscle Environment: Satellite Cell Pool Replenishment

[0222] The ability of injected cells to contribute to long-term muscle regeneration is dependent upon their inclusion into the satellite cell pool of host muscle. Satellite cells are muscle progenitor cells located beneath the basal lamina of myofibers. They are activated in response to damage, causing them to proliferate and fuse to form new myofibers during the repair process. Satellite cells can be identified by the expression of the transcription factor PAX7 and their anatomical location beneath the basal lamina. Using antibodies against these distinct features coupled with human LaminA/C, human nuclei that express PAX7 were identified. This indicates that these cells have assumed a satellite cell identity (FIG. 3).

Example 6

Development of a Tracking Strategy to Follow the Transplant Over Time

[0223] In vivo imaging provides a powerful tool to track the growth and survival of implanted muscle cells over time. Lentiviral particles are highly efficient at infection and stable integration of a gene of interest into a cell system. Lentiviral particles expressing a firefly luciferase (Luc) reporter gene provide a simple, long-term cell tracking system. Live small animal in vivo imaging techniques can then be performed to follow the destiny of transplanted Luc+cells over time. These techniques have been used successfully to track the evolution of muscle cell transplantations. Accordingly, a commercial lentiviral vector carrying a luciferase reporter gene under the control of a CMV promoter (SABiosciences, FIG. 4) was used to develop stable Luc+FSHD and control myoblast cell lines.

[0224] To develop cell lines that could be tracked in vivo following engraftment, FSHD and their matching control cells were seeded on day 0 and lentivirus infection was performed on day 2 according to Manufacturer's directions. Cells were transduced using a 4-hour infection with a Multiplicity of Infection (MOI) of 50. Cells were further amplified and maintained in culture under proliferative conditions where they showed normal signs of proliferation and differentiation. In vitro luciferase assays demonstrated luciferase activity, confirming development of cell models that can be used to track the destiny of the engrafted cells in vivo using bioluminescence imaging techniques.

[0225] In short, these results demonstrate the successful engraftment of FSHD cells into murine muscle with high efficiency as well as the development of a method to track the implanted cells in vivo. Live whole animal imaging experiments will be carried out to investigate how engrafted FSHD cells survive and regenerate compared to controls, and to identify biomarkers specific to FSHD.

[0226] Luciferase-expressing FSHD cells are engrafted into injured TA muscles, and their growth and differentiation assayed over time in vivo using Bioluminescence Imaging (BLI). Cell number is assessed as the bioluminescence signal derived from constitutive luciferase activity, and the linearity, sensitivity, and reproducibility of the bioluminescence assay for quantifying cell numbers will be first validated both in vitro and in vivo.

[0227] For BLI studies, cell-transplanted animals are anesthetized prior to receiving an intraperitoneal injection of luciferin (15 mg/ml at a dose of 130 mg/kg body weight recommended) and assayed in an imaging chamber with a Xenogen device. Images are acquired continuously for 30 minutes, and the same mice are imaged repeatedly over time once a month for up to 6 months. It has been shown that the dynamics of muscle cell behavior during muscle repair can be followed using this imaging technique. In vivo BLI of same mice imaged repeatedly over time has established the ability of transplanted satellite cells to respond to serial injury with successive waves of progenitor expansion and regeneration of muscle fibers. The magnitude of the regeneration response to sequential cardiotoxin injection, as monitored by imaging luciferase activity, reflects the persistence and renewal of stem cells over time. The relative regenerative responses of FSHD versus control muscles over time will test whether satellite cell regenerative capacity is impaired as an FSHD disease mechanism.

[0228] Live in vivo imaging technologies provide a unique technology to evaluate the role of satellite cell regenerative potential and muscle fiber survival in FSHD disease progression. In addition to engraftment studies of affected FSHD subjects, gene expression and regeneration and survival aree evaluated in xenografts of myogenic cells from non-manifesting FSHD subjects (i.e. individuals with shortened D4Z4 arrays but no detectable signs of muscle weakness). While cell culture studies have suggested that these non-manifesting cells behave similarly to cells from subjects with clinically diagnosed FSHD (e.g. expression of DUX4-fl), it is possible that their in vivo characteristics will show reduced pathology, providing opportunities to investigate modifiers of disease progression.

Example 7

RNaseH1-Active Antisense Oligonucleotides (ASOs)

[0229] As indicated in Tables 2 and 4, certain markers are increased in subjects with FSHD relative to the levels of those markers in first degree unaffected subjects. Therapeutic effects are achieved by reducing the levels or biological activity of markers whose expression is upregulated in FSHD. In particular the invention provides targeted for degradation using RNaseH1-activating antisense oligonucleotides (ASO's) ("MOE gapmers"). The RNAseH1 ASO chemistry provides for a 20 nucleotide phosphorothioate backbone (5-10-5 gapmer). In particular, the oligonucleotide comprises five nucleotides at each end with the 2'-O-(2-methoxyethyl) (MOE) modification and ten central deoxyribonucleotides for activation of RNase H1.

[0230] For screening purposes, cell cultures of the invention are contacted with ASOs and the cells assayed for an amelioration of FSHD phenotype. In particular, the cells are assayed for an increase in the biological function of the cell or for an increase in the levels of one or more markers down-regulated in FSHD. In another embodiment, ASOs are administered to a chimeric mouse comprising a human FSHD cell. The chimeric mouse is then assayed for an increase in the biological activity of a human FSHD cell or an increase in the level of expression of a marker down-regulated in FSHD. In one embodiment, 25 mg/kg of the ASOs are administered by sub-cutaneous injection at least about 2.times. per week for 4 weeks or more.

[0231] In particular embodiments, the effects of ASOs on cells or chimeric mice of the invention are assayed using live cell imaging, muscle fiber turnover, or biomarker expression. In one embodiment, nude mice are treated to eliminate or reduce the number of muscle stem cells and/or differentiated muscle cell fibers and muscle stem cell replacement of muscle fiber turnover is assayed.

Example 8

Validation with qPCR

[0232] Of the 142 genes identified as candidate biomarkers in the microarray study described above, 18 genes (9 of which were up-regulated in FSHD vs. control myotubes and 9 which were down-regulated in FSHD vs. control myotubes) have now been evaluated on a larger collection of samples using quantitative real-time PCR (qPCR). The samples are derived from four of the five families from the microarray study and four additional families. Clinical information for the samples is given in Table 3. The qPCR experiments were performed using the BioMark 96.96 Dynamic Array (Fluidigm) platform with TaqMan Gene Expression Assays (Applied Biosystems).

TABLE-US-00003 TABLE 3 Samples used in qPCR study. Deltoid Biceps Familial EcoRI/ strength strength Subject relations Gender Age BlnI (kb) (R, L) (R, L) 01A proband M 42 >40, 18 4+, 5 4+, 3- 01U brother of 01A M 46 >40, >40 5, 5 5, 5 03A proband F 40 >40, 20 5, 5 4+, 4+ 03U sister of 03A F 42 157, 80 5, 5 5, 5 05A proband F 55 67, 25 5, 5 5, 5 05C brother of 05A M 49 67, 25 5, 5 5, 5 05V son of 05A M 18 67 5, 5 5, 5 09A proband F 31 >112, 25 5, 5 4+, 4+ 09U mother of 09A F 57 >112, 47 5, 5 5, 5 12A daughter of 12B F 22 63, 18 4+, 4+ 4+, 4+ 12U daughter of 12B F 24 >112, >112 5, 5 5, 5 15A proband M 66 >112, 28 5, 5 4+, 4+ 15V sister of 15A F 60 >145, 107 5, 5 5, 5 16A proband F 56 97, 20 5-, 5- 4-, 4+ 16U sister of 16A F 60 97, 93, 56 5, 5 5, 5 21B daughter of 21A F 59 26, 40 5, 5 4+, 4+ 21U daughter of 21A F 48 142, 63 5, 5 5, 5

The 18 genes assessed with qPCR are listed in Table 4 below, along with their log(base 2) fold-change (LFC) between FSHD and control myotubes and the associated statistical significance (P-value) of this difference using qPCR. Table 2 also includes columns for the LFC and P-value from the original microarray study for comparison.

TABLE-US-00004 TABLE 4 Genes tested with qPCR. LFC P-value LFC P-value Gene (qPCR) (qPCR) (microarray) (microarray) PRAMEF1 15.36* 0.008* 1.33 3.90E-04 TRIM43 12.77* 0.008* 2.59 7.30E-05 SLC34A2 11.30* 0.008* 2.23 4.00E-04 TRIM49 11.72* 0.008* 1.68 5.80E-04 TC2N 2.96 0.002 0.72 5.20E-05 DAG1 -0.73 0.002 -0.59 1.20E-05 PAX7 -1.79 0.027 -0.75 8.40E-05 CLYBL -0.5 0.03 -0.57 4.30E-04 MYF5 -1.72 0.068 -0.91 9.40E-04 ZNF445 -0.35 0.069 -0.31 4.10E-04 ATP2A1 -1.94 0.076 -1.82 8.70E-05 CD34 3.29 0.082 0.79 2.30E-04 MRAP2 -0.88 0.129 -0.93 1.30E-04 NAAA 0.36 0.154 0.52 1.60E-04 CALCRL -0.36 0.342 -1.17 8.40E-05 HSPA6 0.83 0.38 0.49 2.90E-04 SPATA17 -0.04 0.763 -0.49 6.10E-04 CD177 0.09 0.88 0.83 8.00E-04 Log(base 2) fold-change (LFC) for FSHD vs. control myotubes and the associated p-values are shown for qPCR and also for the original microarray study. Negative values indicate that the gene is down regulated in FSHD. Asterisks (*) in qPCR columns indicate that the transcript was not detected in at least one sample. In these cases the LFC may be inaccurate and a non-parametric sign test rather than a t-test was used for computing the p-value.

Cycle threshold (Ct) values for each gene in each sample were computed as the median Ct value of three technical qPCR replicates, and were then normalized by additive scaling of all Cts for each sample so that the average Ct of three reference genes M6PR, HPRT 1, and PPIA was identical across samples (and equal to the un-normalized mean of these three genes across all samples). Transcripts of four genes (PRAMEF1, TRIM43, SLC34A2, TRIM49, highlighted in Table 4) were not detected with qPCR in one or more of the samples. In these cases the normalized Ct value was set to 40, which represents 2 0.67=1.6-fold lower transcript abundance than the highest observed Ct of 39.33. The LFC estimates may be inaccurate for these genes, and these estimates are flagged with asterisks in the LFC column. Also, because this treatment of non-detected transcripts may violate the assumption of normality in t-tests, non-parametric sign tests were used on the paired (by family) differences between FSHD and control myotubes for these cases, indicated by asterisks in the p-value column. Multiple FSHD samples in a single family were replaced by their median value. In this test non-detected transcripts are considered to have lower expression than detected transcripts, but results do not otherwise depend on the precise Ct value assigned the non-detected transcripts. For genes that were detected in all the samples, p-values are bases on t-tests of the contrast FSHD vs. control from linear models with additive fixed effects for FSHD status and for family. This generalized a usual paired t-test by accommodating families with more than one FSHD subject.

[0233] All 9 genes that were up-regulated in FSHD in the microarray study were also up-regulated in the qPCR study (positive LFC in both cases), and all 9 genes that were down-regulated in FSHD in the microarray study were also down-regulated in the qPCR study (positive LFC in both cases). This overall concordance is directionally of change is significantly better than random (p=3.8e-6 by binomial test), and 6 of the genes individually showed significant differences between FSHD and control myotubes in the qPCR study at the p<0.01 level. Note that in the microarray analysis, to moderate the effect of outliers when ranking the more-than 20,000 genes, a statistical model with a pooled estimate of variance across the myoblasts and myotubes derived from biceps and deltoid biopsies was used, which further shrunk estimates of variance across different genes towards a common mean (by use of empirical Bayes moderated t-statistics). In the present qPCR analysis self-contained statistical tests were performed on myotubes derived from biceps, with no reference to myotube or deltoid samples, and sharing of information across genes. These factors may explain why more of the genes did not attain p<0.01 in the qPCR study.

[0234] Note that for each of the six genes with p<0.01 in the qPCR study (PRAMEFL TRIM43, SLC34A2, TRIM49, TC2N, and DAG1) the FSHD vs. control paired differences showed the same direction for all of the cohorts: For the first five of these genes, each FSHD sample had a lower Ct value (higher expression) than its paired control sample, and for DAG1 each FSHD sample had a higher Ct value (lower expression) than its paired control sample. A stronger result held for PRAMEL TRIM43 and SLC34A2: for these three genes each FSHD sample had a lower Ct value (higher expression) than all of the control samples, not just the sample from the paired first-degree relative. This property is appealing for a biomarker since scores can then be assigned to individuals without the requirement of first-degree relatives as controls. However, the margin between the highest Ct values of FSHD samples and lowest Ct value of control samples was fairly small for these genes (0.56 Ct for TRIM43, 1.06 Cts for SLC34A2, 1.68 Cts for PRAMEF1).

[0235] It was then tested whether the difference of Ct values between two genes would provide discrimination between the FSHD and control samples with a larger margin, and thus more likely to generalize to other samples. The use of a simple difference rather than a more complex combination involving more genes makes the test simpler, and also removes the reliance on the choice of "housekeeping" gene(s), as these terms would cancel out so the difference is self-normalizing. The precise cutoffs for biomarkers would still depend on qPCR primers and efficiency of qPCR reactions, however, so should be recalibrated if these change.

[0236] Because the genes in the qPCR were selected on the basis of differential expression in the microarray study, assessing discriminants using the samples present in the microarrays will be biased. Moreover, searching over all pairs of genes introduces multiple hypotheses and the potential for overfitting. To address these issues, the pair of genes to use, and the cutoff on their difference to use as a discriminant, were selected based only on the qPCR data for the eight samples present in the microarray, so that the qPCR data for the nine samples not present in the microarray study could serve as an independent validation set. By examining all pairs of the 18 genes with qPCR data, the difference (Ct for PRAMEF1)-(Ct for PAX7) provided the maximum margin between FSHD and control samples, of 4.49 Cts. (Non-detected transcripts were assigned Ct of 40 during this maximization, and in application of the discriminant rule.)

[0237] The midpoint of the gap between FSHD and control samples for this difference was 7.05, yielding the discriminant rule of: classify as FSHD if (Ct for PRAMEF1)-(Ct for PAX7)<7.05, and classify as control otherwise. This rule correctly classified all nine samples (five FSHD and four control) that were not represented in the microarray experiment (and hence played no role in selecting the genes PRAMEF1 or PAX7, or the cutoff of 7.05). This is significantly better than random guessing (p=0.002 by binomial test). The margin between FSHD and control samples was slightly reduced when these additional nine samples were included, but was still 3.32 Ct, roughly twice the best margin (1.68) for Cts of any single gene when normalized by the reference genes M6PR, HPRT 1, and PPIA.

[0238] Note the there are other pairwise differences that give larger margins that 1.68, and in the above we have focused just on the single maximal example chosen using a subset of the samples to avoid multiple-hypothesis testing on the validation samples. Other pairs with large margin are typically differences between one gene up-regulated in FSHD vs. controls and one gene down regulated in FSHD vs. control.

Example 9

Using FSHD Biomarkers to Identify and Evaluate the Efficacy of Antisense Oligonucleotide-Morpholino Drugs Using FSHD Myogenic Cells and Xenograft Muscles

[0239] Antisense oligonucleotides conjugated to morpholinos are developed as inhibitors of the expression of FSHD disease genes, using cultured FSHD myogenic cells (prepared as described above in Example 1 and in Homma et al.) and FSHD xenograft muscle derived by engraftment and differentiation of FSHD myogenic cells into regenerating mouse muscles as described above. Antisense oligonucleotide mopholinos are designed that have nucleotide sequences designed to disrupt translation initiation, polyadenylation, and/or RNA splicing to knockdown expression of targeted FSHD disease mRNAs and block production of their encoded disease proteins. Specific antisense oligonucleotide drugs will first be tested by introduction into FSHD myogenic cells by electroporation or transfection with EndoPorter (Gene Tools). Drug-treated FSHD and control cells are monitored for evidence of cytotoxicity and changes in cell morphology, myofiber differentiation, and the expression of muscle protein biomarkers (desmin, MyoD, myogenin, MyHC). The efficacy of selected antisense oligonucleotides to block expression of targeted FSHD disease gene RNAs and proteins is evaluated by qPCR and immunoblotting assays. The efficacy of the antisense oligonucleotides as candidate FSHD drugs is evaluated by quantitative assays of the expression of FSHD disease biomarkers using qPCR, as established above. Promising candidate antisense FSHD drugs are identified by their activities to restore expression of FSHD biomarkers to levels produced by control cells derived from unaffected individuals.

[0240] Promising candidates are then tested in FSHD xenograft muscles by localized muscle injection and electroporation or systemic injection of antisense oligonucleotides, followed by qPCR assays of the expression of FSHD biomarkers and evaluation of hepatotoxic and immunostimulatory side effects over the time course of treatment. Antisense drugs with promising therapeutic value are identified by their activities to restore expression of FSHD biomarkers in both FSHD cells and xenograft muscles to levels observed in control myogenic cells and xenograft muscles derived from unaffected individuals.

OTHER EMBODIMENTS

[0241] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0242] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0243] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Sequence CWU 1

1

41188DNAHomo sapiens 1accaagtttc agttcatgta aacatcctac actcagctgt aatacatgga ttggctggga 60ggtggatgtt tacttcagct gacttgga 8825904DNAHomo sapiens 2aggcagaagc cggcggcgcg cggacagcca gtcggcgccg cgcggagctg gccgctggat 60tggctgcaac actcgcgtgt caggcggttg ctaggctccg gccgcgcgcc ccgcccttgc 120gctcagcgcc ctctcaccgc ccggtacgtg ctcgcgcgaa ggctgcggcg cggcgctcgc 180gcctcttagg cttggcggtg gcggcggcgg cagcttcgcg ccgaatcccc ggggagcggc 240ggtggcggcg tcctggggcc aggaggagcg aacacctgcc gcggtcctcc cgccggcgct 300gggcacacga ctgtctatcc cagatggctg cactggccta ggacagacgg ttcttccccc 360agtacgtcaa gaccctggag ggtgcaagct acagaacagc tgggctcctg cattcctccc 420agaccctgtc ttcagtagga caacaggcaa caaacttaaa tttgggtcaa gcgattcccc 480tgcctcagct tcctgagtag ctgggattac agatagccct gggatctcct gaatttccag 540gttgggctac tcaggcatgg attgactcct acagttcagg cggatggaga tcccaccaac 600ccacagctta attcttcaga tgctttatct tcatttctca tgtatcttca gttggcttct 660aatgctctgt gtgctccggg atggagcagg tgtgcagagg gtgagaaccc agctctggga 720ccaagtcact tgcttcctta cttagcaaga ctatcgactt gagcaaactt ggacctggga 780tgaggatgtc tgtgggcctc tcgctgctgc tgcccctctg ggggaggacc tttctcctcc 840tgctctctgt ggttatggct cagtcccact ggcccagtga accctcagag gctgtcaggg 900actgggaaaa ccagcttgag gcatccatgc actcagtgct ctcagacctc cacgaggctg 960ttcccacagt ggttggcatt cctgatggca cggctgtcgt cgggcgctca tttcgagtga 1020ccattccaac agatttgatt gcctccagtg gagatatcat caaggtatca gcggcaggga 1080aggaggcttt gccatcttgg ctgcactggg actcacagag ccacaccctg gagggcctcc 1140cccttgacac tgataagggt gtgcattaca tttcagtgag cgctacacgg ctgggggcca 1200acgggagcca catcccccag acctccagtg tgttctccat cgaggtctac cctgaagacc 1260acagtgagct gcagtcggtg aggacagcct ccccagaccc tggtgaggtg gtatcatctg 1320cctgtgctgc ggatgaacct gtgactgttt tgacggtgat tttggatgcc gacctcacca 1380agatgacccc aaagcaaagg attgacctcc tgcacaggat gcggagcttc tcagaagtag 1440agcttcacaa catgaaatta gtgccggtgg tgaataacag actatttgac atgtcggcct 1500tcatggctgg cccgggaaat gcaaaaaagg tggtggagaa tggggccctt ctctcctgga 1560agctgggctg ctccctgaac cagaacagtg tgcctgacat tcatggtgta gaggcccctg 1620ccagggaggg cgcaatgtct gctcagcttg gctaccctgt ggtgggttgg cacatcgcca 1680ataagaagcc ccctcttccc aaacgcgtcc ggaggcagat ccatgctaca cccacacctg 1740tcactgccat tgggccccca accacggcta tccaggagcc cccatccagg atcgtgccaa 1800cccccacatc tccagccatt gctcctccaa cagagaccat ggctcctcca gtcagggatc 1860ctgttcctgg gaaacccacg gtcaccatcc ggactcgagg cgccattatt caaaccccaa 1920ccctaggccc catccagcct actcgggtgt cagaagctgg caccacagtt cctggccaga 1980ttcgcccaac gatgaccatt cctggctatg tggagcctac tgcagttgct acccctccca 2040caaccaccac caagaagcca cgagtatcca caccaaaacc agcaacgcct tcaactgact 2100ccaccaccac cacgactcgc aggccaacca agaaaccacg gacaccccgg ccagtgcccc 2160gggtcaccac caaagtttcc atcaccagat tggaaactgc ctcaccgcct actcgtattc 2220gcaccaccac cagtggagtg ccccgtggcg gagaacccaa ccagcgccca gagctcaaga 2280accatattga cagggtagat gcctgggttg gcacctactt tgaggtgaag atcccgtcag 2340acactttcta tgaccatgag gacaccacca ctgacaagct gaagctgacc ctgaaactgc 2400gggagcagca gctggtgggc gagaagtcct gggtacagtt caacagcaac agccagctca 2460tgtatggcct tcccgacagc agccacgtgg gcaaacacga gtatttcatg catgccacag 2520acaagggggg cctgtcggct gtggatgcct tcgagatcca cgtccacagg cgcccccaag 2580gggatagggc tcctgcaagg ttcaaggcca agtttgtggg tgacccggca ctggtgttga 2640atgacatcca caagaagatt gccttggtaa agaaactggc cttcgccttt ggagaccgaa 2700actgtagcac catcaccctg cagaatatca cccggggctc catcgtggtg gaatggacca 2760acaacacact gcccttggag ccctgcccca aggagcagat cgctgggctg agccgccgga 2820tcgctgagga tgatggaaaa cctcggcctg ccttctccaa cgccctagag cctgacttta 2880aggccacaag catcactgtg acgggctctg gcagttgtcg gcacctacag tttatccctg 2940tggtaccacc caggagagtg ccctcagagg cgccgcccac agaagtgcct gacagggacc 3000ctgagaagag cagtgaggat gatgtctacc tgcacacagt cattccggcc gtggtggtcg 3060cagccatcct gctcattgct ggcatcattg ccatgatctg ctaccgcaag aagcggaagg 3120gcaagcttac ccttgaggac caggccacct tcatcaagaa gggggtgcct atcatctttg 3180cagacgaact ggacgactcc aagcccccac cctcctccag catgccactc attctgcagg 3240aggagaaggc tcccctaccc cctcctgagt accccaacca gagtgtgccc gagaccactc 3300ctctgaacca ggacaccatg ggagagtaca cgcccctgcg ggatgaggat cccaatgcgc 3360ctccctacca gcccccaccg cccttcacag cacccatgga gggcaagggc tcccgtccca 3420agaacatgac cccataccgg tcacctcctc cctatgtccc accttaaccc gcaagcgcct 3480gggtggaggc agggtagggc aggggcctgg agacgacatg gtgttgtctg tggagaccgg 3540tggcctgcag accattgccc accgggagcc gacacctgac ctagcacaca ctgacacagg 3600ggcctggaca agcccgccct ctctggtcct cccaaacccc aaagcagctg gagagacttt 3660ggggactttt ttatttttat tttttgccta acagcttttg gtttgttcat agagaattct 3720tcgcttcatt tttgatggct ggctctgaaa gcaccatgtg gagtggaggt ggagggagcg 3780aggaaccatg aatgaactcg caggcagtgc cgggcggccc cctggctctc tgcgttttgc 3840ctttaacact aactgtactg ttttttctat tcacgtgtgt ctagctgcag gatgtaacat 3900ggaaaacagt aactaaagat taaattcaaa ggactttcag aagttaaggt taagttttta 3960cgtttaatct gctgtttacc taaacttgta tgtataattt ttgggtgggt atggggaatt 4020gctttgctaa aaataagctc ccagggtgtt tcaaacttag agaagaccaa gggacagtat 4080tttttatcaa aggaatacta ttttttcaca ctacgtcaac ttggttgctc tgatacccca 4140gagcctgatt gggggcctcc cggccctggc tcacgccaag tccctggtgc tgggtttgct 4200ctcccgctgt tgccaggggc tggaagctgg aggggtctct tgggccatgg acatccccac 4260ttccagccca tgtacactag tggcccacga ccaaggggtc ttcatttcca tgaaaaaggg 4320actccaagag gcagtggtgg ctgtggcccc caactttggt gctccagggt gggccagctg 4380cttgtggggg cacctgggag gtcaaaggtc tccaccacat caacctattt tgttttaccc 4440tttttctgtg cattgttttt ttttttcctc ctaaaaggaa tatcacggtt ttttgaaaca 4500ctcagtgggg gacattttgg tgaagatgca atatttttat gtcatgtgat gctctttcct 4560cacttgacct tggccgcttt gtcctaacag tccacagtcc tgccccgacc caccccatcc 4620cttttctctg gcactccagt cccaggcctt gggcctgaac tactggaaaa ggtctggcgg 4680ctggggagga gtgccagcaa tagttcataa taaaaatctg ttagctctca aagctaattt 4740tttactaaag tttttataca gcctcaaatt gttttattaa aaaaaagatt taaaatggtg 4800atgcttacag cagtttgtac gagctcttaa gtgttgattc catggaactg acggctttgc 4860ttgttttgat tcttttcccc ctacttttcc taatggttta aattctggaa ttacactggg 4920gttcttttgc cttttttagc agaacatccg tccgtccatc tgcatctctg tcccatgact 4980caggggcgcc cactctgctt cgattctcct cctgtggaag aaaccatttt gagcatgact 5040tttcttgatg tctgaagcgt tattttgggt actttttagg gaggaatgcc tttcgcaata 5100atgtatccat tccctgattg agggtgggtg ggtggaccca ggctcccttt gcacacagag 5160cagctacttc taagccatat cgactgtttt gcagaggatt tgtgtgtgct gcctcaggag 5220gggagggctg gtaggagggg gggagaggtc tctgtcctac tgctctccag agggcatttc 5280cccttgcgcc ttctcccaca gggcccagcc cctctcccct gccccagtcc ccagggggta 5340ctctggagtg agcagtgccc ctgtggggga gcctgtaaat gcgggctcag tggaccactg 5400gtgactgggc tcatgcctcc aagtcagagt ttccctggtg ccccagagac aggagcacaa 5460gtgggatctg acctggtgag attatttctg atgacctcat caaaaaataa acaattccca 5520atgttccagg tgagggcttt gaaaggcctt ccaaacagct ccgtcgcccc tagcaactcc 5580accattgggc actgccatgc agagacgtgg ctggcccaga atggcctgtt gccatagcaa 5640ctggaggcga tggggcagtg aacagaataa caacagcaac aatgcctttg caggcagcct 5700gctcccctga gcgctgggct ggtgatggcc gttggactct gtgagatgga gagccaatct 5760cacattcaag tgttcaccaa ccactgatgt gtttttattt ccttctatat gattttaaga 5820tgtgttttct gcattctgta aagaaacata tcaaactaaa taaaagcagt gtctttatta 5880caacgcaaaa aaaaaaaaaa aaaa 590432229DNAHomo sapiens 3tccggcgccc cgcgccgcct ccgctgcggg tcgggagcgc gcgtctccgc cgcacctcgg 60atctaggagc tactcgcccg gccctgggcg gtgggaggcg gcggcggcgg cggcgctcgc 120gcacctcgga ggagccagga gccggaacca gggccgagcc cgcgggccgg ggctagccag 180ccggtcggag atgtccgccc agaggttaat ttctaacaga acctcccagc aatcggcatc 240taattctgat tacacctggg aatatgaata ttatgagatt ggaccagttt cctttgaagg 300actgaaggct cataaatatt ccattgtgat tggattttgg gttggtcttg cagtcttcgt 360gatttttatg ttttttgtgc tgaccttgct gaccaagaca ggagccccac accaagacaa 420tgcagagtcc tcagagaaga gattcagaat gaacagcttt gtgtcagact ttggaagacc 480tctggagcca gataaagtat tttctcgcca aggcaacgag gagtccaggt ctctctttca 540ctgctacatc aatgaggtgg aacgcttgga cagagccaaa gcttgtcacc agaccacagc 600ccttgacagt gacgtccaac tccaggaagc catcagaagc agtgggcagc cagaggagga 660gctgaacagg ctcatgaagt ttgacatccc caactttgtg aacacagacc agaactactt 720tggggaggat gatcttctga tttctgaacc acctattgtt ctggaaacta agccactttc 780ccagacctca cacaaagacc tggattgaga aacatgctct gtaaagggtc ttcctgaaga 840tgtggattct atctttatgt agcaagaaat ctacatccac caaaattgtg tgtgtttggg 900ggagagagag acatagagat agagacagag aggcagagaa gagacccctt tagaagagag 960ctgagctgat taagctgagt ggttttttgt tttgttttgt ttttgctttt taatacattt 1020ggagctttgg gagtattaaa gtatttacac caagcttgtc caacccgtgg catgtgtccc 1080aggacagctt tgaatgtggc ccaatacaaa ttttaaactt tattaaaaca tgagttttgt 1140tttttttttt gctatttttt ttaaagctcg tcagctatcg ttagtgttag tgtactttat 1200gtgtggccca agacaactct tcttccagtg tggcacaggg aagctaaaag attggacacc 1260tctgatttat actagctcgt tttgcttgtt gaaaaatttg gccaaatacc tattgtcagc 1320attcttgggt gaggattagc ctaccatgtt ctaatctggc cctgccacta ctatgctcta 1380cctttggtga gttgctttac ctctctgggc tgccccattt ttaactgtag gttgacaggt 1440ctagagtgat ccatcccacc tctaatattt tgtgaattta tgactttgcc ttcagatgag 1500gctgagctat acataaaaca gtataaacta gggtactgcc tcgtatctct tgtaggctct 1560ctcaaatctc tgtaccttcc acttaaccct aattgagcca agctttagtc aggggatctg 1620gttgtctacc agaatgtcag gagactcatc ttacacagtc atggtggcca atgtttctgg 1680tgggttgtgc tgaaacagct cttctgagaa cttccaacca cccatgctct aacctggaga 1740cagccatccc ctgcctcaga ataagtacca attcgtagta catgtatggt actcttgtcc 1800ccaagaaatg ttaggaagct tgtcagctga atgagaggag gtgccttctg ggtatctctg 1860tgttggtgta tctgtgccat tggctacaga acaagaaaaa tactatttgc catgctatta 1920ccttggcaga tgtgtaggtg atagtcatct ggctttgagc tgagatggtc agtgggttgt 1980aaattcccca ctagcagata ttcagggtgg cctgagttat gtaaacaagt gagcaacaca 2040gctttaattt catggaggaa tcaaagctgc acactggtat taaaacaact tgattttgcg 2100cacacagttg catgcatggc aagctgttaa cctctgggtg gcattttcat tatgaatttg 2160ttcaccacct gtcttgctta agctacaaaa taaatgcatt tgactgcaca gaaaaaaaaa 2220aaaaaaaaa 22294409DNAHomo sapiens 4gtgcagagac tcaagacaga tatgaccacg tgaagagaca actgtaaaac aagtggaatt 60agagaataaa atcccagcac gatgttcctc actggagaca ccagtccagc tgaggacaat 120agagaagcca cccttcctca atgttcactt ccagaattat atgcatgtat tgagaatttt 180aataaggaga gcaagaaatc aaatcttcta aaaatgcatg gtatttcact taacgaagca 240caggaagtac ttgctagaaa cctgaatgtc atgtcattca ccaggggcgc tgatgtgaga 300ggagatctcc aacctgttat cagtgtcaat aaaatgaaca agcctggaaa acatagaaag 360accccatctc caaaaataaa taaataaata aataaataaa taaaaatta 40953570DNAHomo sapiens 5ggccggctgc tcaagtggga cgggggtcag agctttgtgg agggaagaaa aacctggagg 60gggcaggaga gtaaaaagaa gaaacccagg cagacaggca gttggacaca ctgaggaaga 120ccccccacga gtgggaaccc cctggaagga acacaccggc cccggccccc aggaagggag 180cacaatggag gccgctcatg ctaaaaccac ggaggaatgt ttggcctatt ttggggtgag 240tgagaccacg ggcctcaccc cggaccaagt taagcggaat ctggagaaat acggcctcaa 300tgagctccct gctgaggaag ggaagaccct gtgggagctg gtgatagagc agtttgaaga 360cctcctggtg cggattctcc tcctggccgc atgcatttcc ttcgtgctgg cctggtttga 420ggaaggtgaa gagaccatca ctgcctttgt tgaacccttt gtcatcctct tgatcctcat 480tgccaatgcc atcgtggggg tttggcagga gcggaacgca gagaacgcca tcgaggccct 540gaaggagtat gagccagaga tggggaaggt ctaccgggct gaccgcaagt cagtgcaaag 600gatcaaggct cgggacatcg tccctgggga catcgtggag gtggctgtgg gggacaaagt 660ccctgcagac atccgaatcc tcgccatcaa atccaccacg ctgcgggttg accagtccat 720cctgacaggc gagtctgtat ctgtcatcaa acacacggag cccgttcctg acccccgagc 780tgtcaaccag gacaagaaga acatgctttt ctcgggcacc aacattgcag ccggcaaggc 840cttgggcatc gtggccacca ctggtgtggg caccgagatt gggaagatcc gagaccaaat 900ggctgccaca gaacaggaca agaccccctt gcagcagaag ctggatgagt ttggggagca 960gctctccaag gtcatctccc tcatctgtgt ggctgtctgg cttatcaaca ttggccactt 1020caacgacccc gtccatgggg gctcctggtt ccgcggggcc atctactact ttaagattgc 1080cgtggccttg gctgtggctg ccatccccga aggtcttcct gcagtcatca ccacctgcct 1140ggccctgggt acccgtcgga tggcaaagaa gaatgccatt gtaagaagct tgccctccgt 1200agagaccctg ggctgcacct ctgtcatctg ttccgacaag acaggcaccc tcaccaccaa 1260ccagatgtct gtctgcaaga tgtttatcat tgacaaggtg gatggggaca tctgcctcct 1320gaatgagttc tccatcaccg gctccactta cgctccagag ggagaggtct tgaagaatga 1380taagccagtc cggccagggc agtatgacgg gctggtggag ctggccacca tctgtgccct 1440ctgcaatgac tcctccttgg acttcaacga ggccaaaggt gtctatgaga aggtcggcga 1500ggccaccgag acagcactca ccaccctggt ggagaagatg aatgtgttca acacggatgt 1560gagaagcctc tcgaaggtgg agagagccaa cgcctgcaac tcggtgatcc gccagctaat 1620gaagaaggaa ttcaccctgg agttctcccg agacagaaag tccatgtctg tctattgctc 1680cccagccaaa tcttcccggg ctgctgtggg caacaagatg tttgtcaagg gtgcccctga 1740gggcgtcatc gaccgctgta actatgtgcg agttggcacc acccgggtgc cactgacggg 1800gccggtgaag gaaaagatca tggcggtgat caaggagtgg ggcactggcc gggacaccct 1860gcgctgcttg gccctggcca cccgggacac ccccccgaag cgagaggaaa tggtcctgga 1920tgactctgcc aggttcctgg agtatgagac ggacctgaca ttcgtgggtg tagtgggcat 1980gctggaccct ccgcgcaagg aggtcacggg ctccatccag ctgtgccgtg acgccgggat 2040ccgggtgatc atgatcactg gggacaacaa gggcacagcc attgccatct gccggcgaat 2100tggcatcttt ggggagaacg aggaggtggc cgatcgcgcc tacacgggcc gagagttcga 2160cgacctgccc ctggctgaac agcgggaagc ctgccgacgt gcctgctgct tcgcccgtgt 2220ggagccctcg cacaagtcca agattgtgga gtacctgcag tcctacgatg agatcacagc 2280catgacaggt gatggcgtca atgacgcccc tgccctgaag aaggctgaga ttggcattgc 2340catgggatct ggcactgccg tggccaagac tgcctctgag atggtgctgg ctgacgacaa 2400cttctccacc atcgtagctg ctgtggagga gggccgcgcc atctacaaca acatgaagca 2460gttcatccgc tacctcattt cctccaacgt gggcgaggtg gtctgtatct tcctgaccgc 2520tgccctgggg ctgcctgagg ccctgatccc ggtgcagctg ctatgggtga acttggtgac 2580cgacgggctc ccagccacag ccctgggctt caacccacca gacctggaca tcatggaccg 2640ccccccccgg agccccaagg agcccctcat cagtggctgg ctcttcttcc gctacatggc 2700aatcgggggc tatgtgggtg cagccaccgt gggagcagct gcctggtggt tcctgtacgc 2760tgaggatggg cctcatgtca actacagcca gctgactcac ttcatgcagt gcaccgagga 2820caacacccac tttgagggca tagactgtga ggtcttcgag gcccccgagc ccatgaccat 2880ggccctgtcc gtgctggtga ccatcgagat gtgcaatgca ctgaacagcc tgtccgagaa 2940ccagtccctg ctgcggatgc caccctgggt gaacatctgg ctgctgggct ccatctgcct 3000ctccatgtcc ctgcacttcc tcatcctcta tgttgacccc ctgccgatga tcttcaagct 3060ccgggccctg gacctcaccc agtggctcat ggtcctcaag atctcactgc cagtcattgg 3120gctcgacgaa atcctcaagt tcgttgctcg gaactaccta gagggataac tgttccccct 3180cctccatctc tgagcccgtg tcacagatcc agaagatgaa agaaggaagt gagcatcctt 3240ttgctctgtc ctccccaccc cgatagtgac acatcttcag gcagagctgt ggcacagacc 3300cccgtcctgt cccccacacc cgtgtcatgt gtctgtttat aaacatgtcc ccttcccttt 3360ccttccccct cggccacccg cctccctctc aaccttgtaa attccccttc ccaaccccga 3420ggggcttgca gggacaaggc gaccgactgc gctgagctgc ttatttattg aaaataaacg 3480acggaaaagt ctggccttgc ctctgtgcaa gcttggaggc ctgggtcgcc gctgtgaaaa 3540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 357065779DNAHomo sapiens 6aggcaaaggg cggggcgcgc gcgtcgggaa gatggcgcta cgtctgctgc ggagggcggc 60gcgcggagct gcggcggcgg cgctgctgag gctgaaagcg tctctagcag ctgatatccc 120cagacttgga tatagttcct catcccatca caagtacatc ccccggaggg cagtgcttta 180tgtacctgga aatgatgaaa agaaaataaa gaagattcca tccctgaatg tagattgtgc 240agtgctcgac tgtgaggatg gagtggctgc aaacaaaaag aatgaagctc gactgagaat 300tgtaaaaact cttgaagaca ttgatctggg ccctactgaa aaatgtgtga gagtcaactc 360agtttccagt ggtctggcgg aagaagacct agagaccctt ttgcaatccc gggtccttcc 420ttccagcctg atgctaccaa aggtggaaag tcctgaagaa atccagtggt ttgcagacaa 480attttcattc cacttaaaag gccgaaaact tgaacaacca atgaatttaa tcccttttgt 540ggaaactgca atgggtttgc tcaattttaa ggcagtgtgt gaagaaaccc tgaaggtcgg 600gcctcaagta ggtctctttc tagatgcagt cgtttttgga ggagaagact ttcgagccag 660cataggtgca acaagtagta aagaaaccct ggatattctc tacgcccggc aaaagattgt 720tgtcatagcg aaagcctttg gtctccaagc catagatctg gtgtacattg actttcgaga 780tggagctggg ctgcttagac agtcacgaga aggagccgcc atgggcttca ctggtaagca 840ggtgattcac cctaaccaaa ttgccgtggt ccaggagcag ttttctcctt cccctgaaaa 900aattaagtgg gctgaagaac tgattgctgc ctttaaagaa catcaacaat taggaaaggg 960ggcctttact ttccaaggga gtatgatcga catgccatta ctgaagcagg cccagaacac 1020tgttacgctt gccacctcca tcaaggaaaa atgatctgtt aaatgaagct gtcatcaggc 1080taaagggtat tgaagctgca gagggatcaa cttgtgcttg ccagaggacg ccaatgaagt 1140ttgaaacacc aacaatcaga gattttgttt ctgttcctca ttaaatcatg agcttttgtg 1200ccgagactct ggacgactgt tccttaagaa attaacagaa tgggaagttt taaactctac 1260accaaccttt tcatgaccta cacagcagca acgctgctac tcttagacaa acaccgcggg 1320gaaggctgtt ctgtttattt aaatttgtaa atagaaaaca gttgtttttt actttcattt 1380ttcacctcct cctacgccct tttggattat ttcctctgcg gcccctagca tgagccccaa 1440ccaggcctcc ccttttcccc acttctctca attcccacag gaagcccgag aggtgaggag 1500ctgaggttag acaccaggag aggcaccatc acacaaaagc gcggccgcag agtcccaccg 1560ccaccaggcg acccccaccc agagagggac agacatgcgg ggagccagca ccgggcaaga 1620tggctctggg gatcctcatt ctgtgaagac accaactcat ttctcaaaca caggatccag 1680gagacagata gctcctaaat ggagatggca catgctccgt ggggtccctc atagaggagt 1740gccaccctcc acactggcca cgctgggctg ccccagagcg gccagaaagg aaggtgggag 1800ctagccccat cctcactcag aggccggaag gaggaagatg gcatctcgcc aacttcagag 1860ccgaatggcc tctagccaca ctgcttccag accccagacg gggcagcagc agcagttccc 1920agaggagcac ccattgttgc agctaggacc caccaaggat gggactcctg gagtcaggtg 1980cacaccaggt aacccaggac cacgctgtgc acccccagtc tgcccctctg ctcagaacac 2040agagggatgg gaggatggct tggcagtggg aaggcaaaag aaggcctctc tcactctgcc 2100cctgccatac gcacccgctg agggtgttag aagcagtgga ggcagagctg gccctaagca 2160aaagaagaaa ataaacctca ccttgtcctt cctatgggca gccctcaccc caatctaact 2220aaccttccca tcctccagcc taatccaaag aagcccttgg cttgagggga tgaagggctt 2280gcgttctagt ctccacccca gccccacagg cagccagcca gccagcccca cacagagggg 2340cctcctcaag gcctcactgg gagcctcctg tccccagcca aggagcccta cagacctggg 2400gaaaagcagg cccctccgca aggcccctgg gtggtgcgga ggccggaccg gctaggcctc 2460ccgctgctgg ccactcccag gccacgggtt cctaggtccc aaccacattg gactgctcgc 2520cacgccactg ccctcatccc aaactgcttt gttcaaacaa aaccaccttg tttcagtctc

2580cccaaatgtc cagttcatta tcttcctcat aacccatggt ccctattagg gtaggatcat 2640gagactaaga cctatgagag ccacaaatct ggaccaaagt cccattccct gaacagagac 2700ccaaacgacg agggtcccag aagagagttc agtcctgatc agaaacccat ggtgccttag 2760tccttgaaga acaaaactct aggaagaaga tgctggaagg caaagggtat tccccctctt 2820tccccacccg cgtctctgtg gtgccatggg tgggcccagg taccctggga aaggtgggag 2880agcagtcaag ggtgggtgtg tgatctccac tgggctcact gggcaccccc gacacgggga 2940agtgagagct gcccgactcc tgagccaggc gtgggtgaca ggaagaggac cttgcagcta 3000atctgattca ttagaaacca tacctgttta tgttttgtgt agctcatcac aagccgctta 3060gccatatcac ccccgttatt aattcttggg gtctaaatta tgggtaacac tattaaaaca 3120ttatcagaac taatgagaaa caattactta gaaaatgagc cgggacaaga ctgagttggg 3180aacatcagtg gtgatcactg tagattagtt taataaatca tcaggtgcaa ggcaagactg 3240actgtatgta tgcaaagccc cgtcacggga aaatgaaatt gagatttttt ttttttgcat 3300aattttttca ttaatctcaa taggacccgt gtggtgtaga ttgctttatt cccctaatac 3360cttgcctgag gcgggggggg gggggggtgc ccaataatgt cttcattgtt ttactgaggt 3420cttaatgaca atctgtgaca acctcatttt aatgtataga ggattatatc aatacagtca 3480tgttttattg aaatggtgaa gggaactcca gtcagagtca ggcagtgtct gtgagcacaa 3540gttaggaagt ttctcggcat ccgggagcgt tcccgtctga tgtcccagtg ttagtcctcc 3600cggccgccct cccctcctct cctccaggga cggctgggaa aacagtcctg cgggatgaag 3660acttcaccgc ctccgatttg aatttgaaag taactccttc cgtggcatat ttcgctgggc 3720agatagaaca aaccatgtcg ttttcccccg tctctaaaat agacatatta ttatcattca 3780cacttttgca cccggtcgtt ttgcgggagt tcgggaaact gactttcttc attggggaca 3840ttgtaatttt ctgatgatgc cacgaggaga aaaaaaatac gggtttgttt taattggaag 3900gaccttccgc ttttatgatt tcggtttacc ttggaaaact gaatcttctg tgttttattt 3960ctttcctcta gtactagaaa agcaatgaat taattgcaca aaacaggttc tgagacggcc 4020cgcaggcccc gagctcgtgg acgcggccga gggtcgggtg tgacccgcgg agccgctgcc 4080aggcttccca gctcgtcttc gcggggaggc gggaggcagg accagacccc agcatttcag 4140cgtgaaagtc ttcgcctttc tttccgcgct gtctttcccg cgggcggagc ggcgtacctg 4200agcgcggtcc ccacggagga tcagtgactt tcccagaccc cgcgggcgag cccgcgcttg 4260ggacccggca gctctgcgcg ggctggtttt ggagggggtg gtgttcgttt gtttaaattc 4320cagttgttat ttggcagcat atcgccttcc gagtcagtaa gaattgccca cgacgtaaag 4380agcgacttgc aggaaggggc cgaagccgtc gttagcgccc gggcggcggc ggccactcga 4440accccgtttc cgccaagcgc gctgcaacct gcggggcgag ttcgttttgt tttgccaaaa 4500tcatttgggg acttctttgt cattcatgcc cttcctttta aatgattttt atttttttcc 4560actgtaagtg accggctggg tttgactttt gcttcttccg acggaagggc accgcgagcc 4620ggggtgggcg gccccgcggg caggaggagc gcggggtaca cgcggtggcc gcagacgccg 4680agccctgcgg agccccgagg cctcgtcgcc cgcgcccccg gtgcgcgcgg agccggggcc 4740gaggccgggc caggaggagt gtggcggccc aggaggcctg gactgtgggc cctgctcgcc 4800cgcccgccgc gggccgcccg gagcccgcgc gcctgtcgcg cagcccggct gtaatggtgg 4860cagatcaaag gcggccccgt gtccccgcgg agcccgggac aatcccgcgc ctttgtgcgc 4920tgttgctagg agcccgagaa actaagagaa agtgtcagga gcatgttaat cagactcgtt 4980acactgtaac aataacgtct ctctcgggtc tcccaggccc cagtaccccc gcgcaccctg 5040cgcgcaggcc ggacacctgc gcagggccct tgcgcccgcc ctggggtccc gccggccctg 5100gggtccctgc agccccgaat ccgcacccga gccacgcgga acgactagcc ccgaggggcc 5160ccgcaggctc ccggtgcagc tccctggtcg tggtctcctt gaccgaagcc ccggcctcac 5220accgcctggc cgcgagcccg agggacgcag ggacgatccc ggtcccctgc ttttcagtcc 5280tccagtcgaa tcgcccactt tgttcaatca cagtattcga atcaagagga aaatgaacat 5340tccctttatg gtagcttttt ggttatgagt tttggcaaaa ctgttaaatc aactttgccg 5400atttcccttg ggaatccctg gaggccactc taagtggtaa tcccaagttt aagaaggaaa 5460tggggaaatt ttgctgagag taaagatgtc gccgaacttt ttgaagggat ttgcttcatt 5520cattcattta gtcattcaac agacacctgt tgaacccgac tgggtgccag aggggccagt 5580cacaggactt attccagatg aacttttctt ttgaaattag aatgcccttg tggaagccaa 5640aggaggagca gaggcctaaa ataatgtcaa gtgtcaaagc aaaaagaagt gccattggct 5700actacagtgt gtgatgataa caagagaggt gcgaattagg aattaaaaac gtttgaaaaa 5760ctacaggcca aaaaaaaaa 577975006DNAHomo sapiens 7gctgctgatc acttacaatc tgacaacact tacaatctac tcagaacaac ctctctctct 60ccagcagaga gtgtcacctc ctgctttagg accatcaagc tctgctaact gaatctcatc 120ctaattgcag gatcacattg caaagctttc actctttccc accttgcttg tgggtaaatc 180tcttctgcgg aatctcagaa agtaaagttc catcctgaga atatttcaca aagaatttcc 240ttaagagctg gactgggtct tgacccctga atttaagaaa ttcttaaaga caatgtcaaa 300tatgatccaa gagaaaatgt gatttgagac tggagacaat tgtgcatatc gtctaataat 360aaaaacccat actagcctat agaaaacaat atttgaaaga ttgctaccac taaaaagaaa 420actactacaa cttgacaaga ctgctgcaaa cttcaatttg tcaaccacaa cttgacaagg 480ttgctataaa acaagattgc tacaacttct agtttatgtt atacagcata tttcattttg 540gcttaatgat ggagaaaaag tgtaccctgt attttctggt tctcttgcct ttttttatga 600ttcttgttac agcagaatta gaagagagtc ctgaggactc aattcagttg ggagttacta 660gaaataaaat catgacagct caatatgaat gttaccaaaa gattatgcaa gaccccattc 720aacaagcaga aggcgtttac tgcaacagaa cctgggatgg atggctctgc tggaacgatg 780ttgcagcagg aactgaatca atgcagctct gccctgatta ctttcaggac tttgatccat 840cagaaaaagt tacaaagatc tgtgaccaag atggaaactg gtttagacat ccagcaagca 900acagaacatg gacaaattat acccagtgta atgttaacac ccacgagaaa gtgaagactg 960cactaaattt gttttacctg accataattg gacacggatt gtctattgca tcactgctta 1020tctcgcttgg catattcttt tatttcaaga gcctaagttg ccaaaggatt accttacaca 1080aaaatctgtt cttctcattt gtttgtaact ctgttgtaac aatcattcac ctcactgcag 1140tggccaacaa ccaggcctta gtagccacaa atcctgttag ttgcaaagtg tcccagttca 1200ttcatcttta cctgatgggc tgtaattact tttggatgct ctgtgaaggc atttacctac 1260acacactcat tgtggtggcc gtgtttgcag agaagcaaca tttaatgtgg tattattttc 1320ttggctgggg atttccactg attcctgctt gtatacatgc cattgctaga agcttatatt 1380acaatgacaa ttgctggatc agttctgata cccatctcct ctacattatc catggcccaa 1440tttgtgctgc tttactggtg aatctttttt tcttgttaaa tattgtacgc gttctcatca 1500ccaagttaaa agttacacac caagcggaat ccaatctgta catgaaagct gtgagagcta 1560ctcttatctt ggtgccattg cttggcattg aatttgtgct gattccatgg cgacctgaag 1620gaaagattgc agaggaggta tatgactaca tcatgcacat ccttatgcac ttccagggtc 1680ttttggtctc taccattttc tgcttcttta atggagaggt tcaagcaatt ctgagaagaa 1740actggaatca atacaaaatc caatttggaa acagcttttc caactcagaa gctcttcgta 1800gtgcgtctta cacagtgtca acaatcagtg atggtccagg ttatagtcat gactgtccta 1860gtgaacactt aaatggaaaa agcatccatg atattgaaaa tgttctctta aaaccagaaa 1920atttatataa ttgaaaatag aaggatggtt gtctcactgt tttgtgcttc tcctaactca 1980aggacttgga cccatgactc tgtagccaga agacttcaat attaaatgac tttttgaatg 2040tcataaagaa gagccttcac atgaaattag tagtgtgttg ataagagtgt aacatccagc 2100tctatgtggg aaaaaagaaa tcctggtttg taatgtttgt cagtaaatac tcccactatg 2160cctgatgtga cgctactaac ctgacatcac caagtgtgga attggagaaa agcacaatca 2220acttttctga gctggtgtaa gccagttcca gcacaccatt gcatgaattc acaaacaaat 2280ggctgtaaaa ctaaacatac atgttgggca tgattctacc cttattgccc caagagacct 2340agctaaggtc tataaacatg aagggaaaat tagcttttag ttttaaaact ctttatccca 2400tcttgattgg ggcagttgac tttttttttg cccagagtgc cgtagtcctt tttgtaacta 2460ccctctcaaa tggacaatac cagaagtgaa ttatccctgc tggctttctt ttctctatga 2520aaagcaactg agtacaattg ttatgatcta ctcatttgct gacacatcag ttatatcttg 2580tggcatatcc attgtggaaa ctggatgaac aggatgtata atatgcaatc ctacttctat 2640atcattagga aaacatctta gttgatgcta caaaacacct tgtcaacctc ttcctgtctt 2700accaaacagt gggagggaat tcctagctgt aaatataaat tttgtccctt ccatttctac 2760tgtataaaca aattagcaat cattttatat aaagaaaatc aatgaaggat ttcttatttt 2820cttggaattt tgtaaaaaga aattgtgaaa aatgagcttg taaatactcc attattttat 2880tttatagtct caaatcaaat acatacaacc tatgtaattt ttaaagcaaa tatataatgc 2940aacaatgtgt gtatgttaat atctgatact gtatctgggc tgatttttta aataaaatag 3000agtctggaat gctatatttg gtaaatattt taaagacaac cagatgccag catcagaagt 3060ctgtttgaga actaagagaa cagaaacatc tatcataaga tatatttatt ttaaaaacac 3120aaggtcacta ttttattgaa tatatttgtt ttgataactc ataccttaat aataggtgtg 3180tttgacatat ttcttttttc attttgacaa tgaactcaca ttctaatcca gaaattttaa 3240acaactactg tgataaatac caatctgcta cttttataga ttttacccca ttaaaatatt 3300actttactga cttttactat gtgaagatat atagctttgg aaatgtccca ggctattcaa 3360gaaatataaa aaactagaag gatactatat ataccatata caatgcttta atattttaat 3420agagctactg tatataatac aaattaggga aatacttgaa tatatcattg agaaaaaatt 3480attgtcagat cttactgaat tattgtcaga ctttattaaa taaagataga agaaaacctt 3540gctaatgaat taaagtgaaa tttgcatggg attcagtttc tctaatgtta ttttccgctg 3600aaatctctaa agaacaagaa tgacttcaat tagtaaaagt caattttggg aaaagtcatg 3660ggtatctgtt ttttaagtgt gtcaatctga ttaaaatgga tgaaacaaat tactcatcat 3720aagttgtttc ttaagctgtc aatatgtcaa tagatggtga gttcagaact tatttcaaat 3780tgctaagaca aattatctaa attcgtaaga attaacatat agaatggtct ggtcagtaca 3840tttataattt atctatgcat gaaaaagtat tgttttgttt gaaacatgaa tttcatagca 3900agctgccata gaaaggaacg caggctgttc tagaccttca actgcctaaa ttatacaaaa 3960attcatttta ataaactcaa ttattagcta tttattattc aaagacccat atttaaatcc 4020tttgctgacc atgttgacat atatcagcct tcttctagac aaactgtcaa ctctcaacca 4080tcttgacagt agaagtgaca gtaaaaaatg ttgaatgatc agagattata ttaaaataaa 4140catgtaattt tcaagtattt ttgttgtgct tttataatat taattctaga tcagatttat 4200tttatagcca gggtttgtct gttgtagagt cttgaggcgt agcagtcatt catgattaat 4260cactgttagt tttgtaccca tatattttta gaatagtttt aaatgttaga tttctcaaaa 4320gctaaatgct acttaatatc tttgtatcat actcataaag caaagtaaat ctgacacttt 4380ttttaaagca aacttctttg ctgtcaaaaa aataaatttg gggaaatttc tagcttttaa 4440aatgtagatc tgcattttac tgtgattact tgtgaaagtc atattttaat tttctaaatt 4500ctaatttgtc attttatttc ctaaagttaa tttccaatgc atttattcat aaaatattca 4560ttctggaatg cagtgtttgt ttaaatgtaa tccaatgtat atagaattag tggtggctgt 4620agtgctgtat ttattgctta taattttttt taaatgtgaa cttactttta attttctctt 4680ggttttaatc tgctagtaga aaccactagt tatctgtaaa aatatattca agatattctg 4740atcaattata acaatttatg ttatgcctag agtatatctc tattttttga ttgtatgaaa 4800atattaaagt tatgagttaa agtttatttt cactgatatt tactacagtg ccaaataatc 4860taatttataa acataattct tacagtaatc aatgggatac ttctcaaaat taacaaatct 4920cttaacaaaa tatatctttt gccctcttta aagtcttcag taaaccagta aatgaattca 4980ataaaccaat taagaaaaaa aaaaaa 500682288DNAHomo sapiens 8gaagtggagg tgggagggag cgacaatgga aaaatcacct gaaaactggg acagaggaag 60gaagctacag ttacgaagga gagctgcaaa agttgcagca gaaaggttgg gagtcccgac 120aggttccgta gcccacagaa aagaagcaag ggacggcagg actgtttcac acttttctgc 180ttctggaagg tgctggacaa aaacatggaa ctaatttccc caacagtgat tataatcctg 240ggttgccttg ctctgttctt actccttcag cggaagaatt tgcgtagacc cccgtgcatc 300aagggctgga ttccttggat tggagttgga tttgagtttg ggaaagcccc tctagaattt 360atagagaaag caagaatcaa gtatggacca atatttacag tctttgctat gggaaaccga 420atgacctttg ttactgaaga agaaggaatt aatgtgtttc taaaatccaa aaaagtagat 480tttgaactag cagtgcaaaa tatcgtttat cgtacagcat caattccaaa gaatgtcttt 540ttagcactgc atgaaaaact ctatattatg ttgaaaggga aaatggggac tgtcaatctc 600catcagttta ctgggcaact gactgaagaa ttacatgaac aactggagaa tttaggcact 660catgggacaa tggacctgaa caacttagta agacatctcc tttatccagt cacagtgaat 720atgctcttta ataaaagttt gttttccaca aacaagaaaa aaatcaagga gttccatcag 780tattttcaag tttatgatga agattttgag tatgggtccc agttgccaga gtgtcttcta 840agaaactggt caaaatccaa aaagtggttc ctggaactgt ttgagaaaaa cattccagat 900ataaaagcat gtaaatctgc aaaagataat tccatgacat tattgcaagc tacgctggat 960attgtagaga cggaaacaag taaggaaaac tcacccaatt atgggctctt actgctttgg 1020gcttctctgt ctaatgctgt tcctgttgca ttttggacac ttgcatacgt cctttctcat 1080cctgatatcc acaaggccat tatggaaggc atatcttctg tgtttggcaa agcaggcaaa 1140gataagatta aagtgtctga ggatgacctg gagaatctcc ttctaattaa atggtgtgtt 1200ttggaaacca ttcgtttaaa agctcctggt gtcattacta gaaaagtggt gaagcctgtg 1260gaaattttga attacatcat tccttctggt gacttgttga tgttgtctcc attttggctg 1320catagaaatc caaagtattt tcctgagcct gaattgttca aacctgaacg ttggaaaaag 1380gcaaatttag agaagcactc tttcttggac tgcttcatgg catttggaag cgggaagttc 1440cagtgtcctg caaggtggtt tgctctgtta gaggttcaga tgtgtattat tttaatactt 1500tataaatatg actgtagtct tctggaccca ttacccaaac agagttatct ccatttggtg 1560ggtgtccccc agccggaagg gcaatgccga attgaatata aacaaagaat atgacatctg 1620ttgggcctca caaggaccag ggccttctgg aggagtggca ctaccccacc tggcagcacc 1680tagacctgag ctctacaaaa acacactgct tcactttgtt ttaggactta gttcaagaac 1740acattcaaat ggtgcatgtg tttggtatct tcaacagtag accaagaatc taacatcact 1800ctcagtaata tagagaccgg aatacatggt ttataggaaa tgatcaaatg atccaaaaaa 1860actccacatt ttttaagaag ttggaatttg atttcatgca taactgtatt aaaacattaa 1920atagaaataa tgtcatttga atgaaaatct tatcacatta aattcactgt gaaggcagca 1980tacttaaatt tttattttga aaagtctaaa aggcttagat ttttaaaatt taataattat 2040ttctacaaat tttctatttt tcttgaggtg attctcaact agcaattgga actcctaggc 2100tctattaaca taattcttta ttgtaaacgt atctaatgct aaaagtaata aaatggtagt 2160tttctgagac ctgtgaggac aggaatggtg tcttacattc atttctacac tttattatgc 2220tcaggattgc accttcttta cagagtatat tcaataaatg tatgttgatt gaaaaaaaaa 2280aaaaaaaa 228897086DNAHomo sapiens 9agcagcggcg gcagcggcag cccagccgag cgttaggtgc tgctctctgc gcggcgtttt 60gcaaaggact tcaccgatct acttttgcag tcgcctcgga ctgtccatgt gtttacttcc 120cccagcccga ggattcgata tctaggttcc tgtgaaatgc aactgagcag ccaaagtact 180ttgagaacac ggggcggcat aaacaccaaa acttttttgt ggaaggaaaa tgcaataagc 240aagcttgccg ttttccgatg cggtgtggag tgagtgtgtg tcgcgcgtgt ccgcactgga 300ggcatatgct tgtgtgtgta catggggtgt gtttttcggt atgtagggag aaaatgcttg 360ccaaccaccg gaaatctcct ggaatttatt agaaaataat ggattataaa aagaaggcaa 420gcaaggagcg gatctcccct tgagttgcaa cccgatttgc tgctggctca gtttgttgtg 480attctttttg ttgataggtg tctgatggta ttccgataac gttcccccct tttcttcccc 540ttgagctttt acagtttaaa aaaaggaaac aaaaaccacc ccaaaatctc cccccccgtt 600tttttcgccc cgtcgggatc gccgtttcca tccatgtgct tgcgtctccc ccgcgttcca 660cttaaactat tttaatcctt ggacccaagg aggaggctga taggggggtg gataaaaaaa 720gttcttccaa aatagtgtgc ccggggagca ggatggggga tttcgcagcc cccgctgctg 780ccgcgaatgg cagtagtatt tgcatcaaca gtagcctgaa cagcagcctc ggcggggccg 840ggatcggtgt gaataatact cccaatagta ctcccgctgc tccgagtagc aatcacccgg 900cagccggtgg atgcggcggc tccgggggcc ccggcggcgg ttcggcggcc gttcccaagc 960acagcaccgt ggtggagcgg ctccgccagc gcatcgaggg ctgccgtcgg caccacgtca 1020actgcgagaa caggtaccag caggctcagg tggagcagct ggagctggag cgccgggaca 1080ccgtgagcct ctaccagcgg accctggagc agagggccaa gaaatcgggc gccggcaccg 1140gcaaacagca gcacccgagc aaaccccagc aagatgcgga ggctgcctcg gcggagcaga 1200ggaaccacac gctgatcatg ctacaagaga ctgtgaaaag gaagttggaa ggagctcgat 1260caccacttaa tggagaccag cagaatggtg cttgtgatgg gaatttttct ccgactagca 1320aacgaattcg aaaggacatt tctgcgggga tggaagccat caacaatttg cccagtaaca 1380tgccactgcc ttcagcttct cctcttcacc aacttgacct gaaaccttct ttgcccttgc 1440agaacagtgg aactcacact cctgggcttc tagaagatct aagtaagaat ggtaggctcc 1500ctgagattaa acttcctgtc aacggttgca gtgacctgga ggatagcttc accatcttgc 1560agagcaaaga cctcaaacaa gaacctctcg atgaccctac ttgcatagac acatcagaaa 1620catctctttc aaatcagaac aagctgttct cagacattaa tctgaatgat caggagtggc 1680aagaattaat agatgaattg gccaacacgg ttcctgagga tgacatacag gacctgttca 1740acgaagactt tgaagagaag aaggagccag aattctcgca gccagcaact gagacccctc 1800tctcccagga gagtgcgagc gtgaagagcg acccctctca ctctcccttc gcacatgtct 1860ccatgggatc tccccaggcg aggccttctt cttctggtcc tcccttttct actgtctcca 1920cggccactag tttaccttct gttgccagca ctcccgcagc tccaaaccct gcaagctcac 1980cagcaaactg tgctgtccag tcccctcaaa ctccaaacca agcccacact ccaggccaag 2040ctccacctcg gcctggaaat ggttatctcc tgaatccggc agcagtgaca gtggccggtt 2100cagcgtcagg gcctgtggct gtgcccagct ctgacatgtc tccagcagaa cagctcaaac 2160agatggctgc acagcagcaa caaagggcca aactcatgca gcagaaacag caacagcaac 2220agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcaacag cactcaaatc 2280agacttcaaa ttggtctccc ttaggacctc cctctagtcc atatggagca gcttttactg 2340cagaaaaacc aaatagccca atgatgtacc cccaagcctt taacaaccaa aaccctatag 2400tgcctccaat ggcaaacaac ctgcagaaga caacaatgaa taactacctc cctcagaatc 2460acatgaatat gatcaatcag cagccaaata acttgggtac aaactcctta aacaaacagc 2520acaatattct gacttatggc aacactaaac ccctgaccca cttcaatgca gacctgagtc 2580agaggatgac accaccagtg gccaacccca acaaaaaccc cttgatgccg tatatccagc 2640agcagcaaca gcagcagcaa cagcaacagc agcagcagca gcagcagcag ccgccacctc 2700cacagctcca ggcccccagg gcacacctga gcgaagacca gaaacgcctg cttctcatga 2760agcagaaagg agtgatgaat cagcccatgg cttacgctgc acttccatcc cacggtcagg 2820agcagcatcc agttggactt ccccgaacca caggccccat gcagtcctcc gtgcccccag 2880gctcaggtgg catggtctca ggagccagtc ccgcaggccc cggcttcctg ggcagccagc 2940cccaagcagc catcatgaag cagatgctca ttgatcagcg ggcccagttg atagagcagc 3000agaagcaaca gttcctgcgg gagcaaaggc agcagcagca gcagcagcag cagattttgg 3060cggaacagca gttgcagcaa tcacatctac cccggcagca cctccagcca cagcggaatc 3120catacccagt gcagcaggtc aatcagtttc aaggttctcc ccaggatata gcagccgtaa 3180gaagccaagc agccctccag agcatgcgaa cgtcacggct gatggcacag aacgcaggca 3240tgatgggaat aggaccctcc cagaaccctg ggacgatggc caccgcagct gcgcagtcgg 3300agatgggact ggccccttat agcaccacgc ctaccagcca accaggaatg tacaatatga 3360gcacaggcat gacccaaatg ttgcagcatc caaaccaaag tggcatgagc atcacacata 3420accaagccca gggaccgagg caacctgcct ctgggcaggg ggttggaatg gtgagtggct 3480ttggtcagag catgctggtg aactcagcca ttacccagca acatccacag atgaaagggc 3540cagtaggcca ggccttgcct aggccccaag cccctccaag gctgcagagc cttatgggaa 3600cagtccagca aggagcacaa agctggcaac agaggagctt gcagggcatg cctgggagga 3660ctagtggaga attgggacca ttcaacaatg gcgccagcta ccctcttcaa gctgggcagc 3720cgagactgac caagcagcac ttcccacagg gactgagcca gtcagtcgtg gatgctaaca 3780cgggcacagt gaggaccctc aacccagctg ccatgggtcg gcagatgatg ccatcgctcc 3840cggggcagca aggcaccagc caggcgaggc caatggtcat gtctggcctg agccagggag 3900tcccaggcat gccagcgttc agccagcccc cagcacagca gcagataccc agtggcagct 3960ttgctccaag cagccagagc caagcctatg agcggaatgc ccctcaggac gtgtcataca 4020attacagtgg cgacggagct gggggttcct tccctggcct cccggacggt gcagaccttg 4080tggactccat catcaaaggc gggccagggg acgagtggat gcaggagctt gatgaattgt 4140ttggtaaccc ctaatcaaga gaggccccaa gatccacaac tcgagtggtt aaagcttaaa 4200aagtgaaaaa gaaacaggat gttgacccat ccttgttttt tgtttttttg acccacgtaa 4260actgagcaaa actgcagctg gctgacaatg gaagatccag gtgccaatcc acagccccac 4320caggcctcat ttcacctgat tttcacacag caatcgagat gagacgccat gcagatcccg

4380gctgcgagag agggagacac ccggaggagc aggtgggaag atgaagccgg ccagagcccc 4440tctgcccagc atgccctgtg atcgcctggc ccagcaggag ctgcttcagc cgagagggac 4500tattacccaa gagaggtatc ctcagcccct cctgccccag gtcgggagac agcagctttg 4560gagacacaaa agagacagag cctcagccag ggagagtgag tcccccagaa gaggctgggt 4620ggttgcacag gccaggtgca caggttggaa atgcactgaa ctctgggtgc cgagagatgt 4680aaggctttga gacatgctac tgaatttgga gggcaggcac gaagaacagt gagattgtca 4740aaaggagaca accacagatc ctacaggact gtctgtctcc tgccccatga tgaccctcag 4800gaattgcaaa ggctctgctg tcacaaggag agcaggctga gtttggagca gggtccatcc 4860ggcagtcctg ggacggcttc cctctgctgg tgcccctggt ggcagtccct ccaggtgggg 4920ctggagcctg ctggcgccca atacaaaacc catacatcca ggtgggtcac atctacttct 4980ggcggccgca gggcagggaa acccctactg gaccctgtgt gtctgccagc ctggagcctt 5040tgtctccagc cctgccttta ttcctccttg cctccacacc agcctcccct tgcttctcct 5100tacagactat ccaagaagtg aagcttatgt ctttagggag ccttgggcag agtccacata 5160aatgcaggaa gaacttagac aatgcctgaa atgcaaaggc gacactggag tcttctttct 5220ctaacgtgta gcgttgaatg aatatctgcc tggaaccaag agggctgctc tgatgtttgg 5280gagtcggttt tttgtgagcc acatctgata tttctgatat ccccaggaag gagtggcctg 5340gaggtcactg gttcaggctc cctttgggcg aaatcctggg agtgatgctc taaaaatcca 5400cctttcccat catccctact catcagaaag acaaatataa aatcccagag aggtggagga 5460gctaaaaaag caattgctcc accttacaaa tttggataga aaggagatgt agtttatttc 5520atatgggcaa agtagtcctc ttccaaagtc ctgtacagtt gttctctgca attgacgcac 5580atctgcccta agcgaaatct gtcagaagga atcaacaagg ctccttgcct cccctcccaa 5640tccccctttt ggaggacttg tggcttcggt gtcgtcctaa gtgagagtgg cgtgtgcttt 5700tttcctgtcc cctcctccct ccgtgtccta gacgctggct gccttctgtg cactcccagg 5760cagatcacta cggaagagtc ggagcctgtg gggttggact ggccacactc agtcctgaga 5820aggcgagttg ccatggaaag ctgggggcag aggtgttttt ggagaggagg cggcaggcaa 5880acattgcctt tgacttgctc tccgcgtacc cggggttgta gagctgctca ggaaggggca 5940ggatgtaagg ccagaggtgc ctggtgggtg agaagcccag gcaggggctg ggcgccctct 6000ccgaagaggt ggcagcaggg tgaccctgaa ctccccaaat ggggagtgat gccactgggg 6060aaactgagtg gatcaaagag atgaaaccaa aaaaaagcaa acaaacaaat gagaagacac 6120aaaacataat taccttttcc tgaaaggtac aggaaataaa tatataagca atgatgagaa 6180actggaggtg gctaatggaa gtgagacaga caggggtggg gggctccatt atcttttaaa 6240agcttcctcc aaatgctcag tactgggacc aactaatagg tagattttaa tatggtggtt 6300ttgttttggt tttgttttta ctacggtgct gatgtatatg taatgtctaa aaaaagttat 6360ttgtacataa gtttttacaa tactgcagat atcactgggt ctactatctg taaaaaatat 6420acatataaat atatatatac tgtttgttta aaatagagta tttttatttc attccttaac 6480tcatcatcac agcagtggta ttgcacttca gatgacatct aattactaat ttgtactgta 6540tgacctctgg caacttgctc cattttattc agatttttct agttttctgt ttttactttg 6600tacattgagc attgcttatt tccttttaag aaatgtacag aacgctgaaa tgtagaaatg 6660aagtgatgtt gacataccac ttttaaagaa aacaaaaaca aaaataaaag attatctgaa 6720tcaatccaaa gtatagttta ttttaagatc ctcactggag ctccaatctt aattaagcag 6780acatattatt cctgggtttt aattaactgc agttcttttc tcaaaatttt gatatcattc 6840tttaaacatt tatttaatgg gagagatcaa tcattcccag cctttgcatt caactgcaca 6900actgatgata ctttctgaat ttactgccgc actctgaagg atgtggcaaa gggaagggag 6960gaaagtcaac tttgcatgga acatagtgca atccatttgt agattcagta ccataacaat 7020tccatttcca actagttcat ctggaggtag ggaggctcca ctggaaaaat aaatattttt 7080gtcaaa 7086109068DNAHomo sapiens 10cggcgcgcgc gcggggcggg ggcgcgcgga ggggggggct gccccggggc ggccccccca 60ggtcggggcg cggcgggcgg cggcggcggg cgcgcgtccc gtccaggtcc ggagtaaccg 120ccgccgccgc cgccaaagct cgccaacatg gcggacctgg aggctgtgct ggccgatgtc 180agttacctga tggccatgga gaagagcaag gcgaccccgg ccgcccgcgc cagcaagagg 240atcgtcctgc cggagcccag tatccggagt gtgatgcaga agtaccttgc agagagaaat 300gaaataacct ttgacaagat tttcaatcag aaaattggtt tcttgctatt taaagatttt 360tgtttgaatg aaattaatga agctgtacct caggtgaagt tttatgaaga gataaaggaa 420tatgaaaaac ttgataatga ggaagaccgc ctttgcagaa gtcgacaaat ttatgatgcc 480tacatcatga aggaacttct ttcctgttca catcctttct caaagcaagc tgtagaacac 540gtacaaagtc atttatccaa gaaacaagtg acatcaactc tttttcagcc atacatagaa 600gaaatttgtg aaagccttcg aggtgacatt tttcaaaaat ttatggaaag tgacaagttc 660actagatttt gtcagtggaa aaacgttgaa ttaaatatcc atttgaccat gaatgagttc 720agtgtgcata ggattattgg acgaggagga ttcggggaag tttatggttg caggaaagca 780gacactggaa aaatgtatgc aatgaaatgc ttagataaga agaggatcaa aatgaaacaa 840ggagaaacat tagccttaaa tgaaagaatc atgttgtctc ttgtcagcac aggagactgt 900cctttcattg tatgtatgac ctatgccttc cataccccag ataaactctg cttcatcctg 960gatctgatga acgggggcga tttgcactac cacctttcac aacacggtgt gttctctgag 1020aaggagatgc ggttttatgc cactgaaatc attctgggtc tggaacacat gcacaatcgg 1080tttgttgtct acagagattt gaagccagca aatattctct tggatgaaca tggacacgca 1140agaatatcag atcttggtct tgcctgcgat ttttccaaaa agaagcctca tgcgagtgtt 1200ggcacccatg ggtacatggc tcccgaggtg ctgcagaagg ggacggccta tgacagcagt 1260gccgactggt tctccctggg ctgcatgctt ttcaaacttc tgagaggtca cagccctttc 1320agacaacata aaaccaaaga caagcatgaa attgaccgaa tgacactcac cgtgaatgtg 1380gaacttccag acaccttctc tcctgaactg aagtcccttt tggagggctt gcttcagcga 1440gacgttagca agcggctggg ctgtcacgga ggcggctcac aggaagtaaa agagcacagc 1500tttttcaaag gtgttgactg gcagcatgtc tacttacaaa agtacccacc acccttgatt 1560cctccccggg gagaagtcaa tgctgctgat gcctttgata ttggctcatt tgatgaagag 1620gataccaaag ggattaagct acttgattgc gaccaagaac tctacaagaa cttccctttg 1680gtcatctctg aacgctggca gcaagaagta acggaaacag tttatgaagc agtaaatgca 1740gacacagata aaatcgaggc caggaagaga gctaaaaata agcaacttgg ccacgaagaa 1800gattacgctc tggggaagga ctgtattatg cacgggtaca tgctgaaact gggaaaccca 1860tttctgactc agtggcagcg tcgctatttt tacctctttc caaatagact tgaatggaga 1920ggagagggag agtcccggca aaatttactg acaatggaac agattctctc tgtggaagaa 1980actcaaatta aagacaaaaa atgcattttg ttcagaataa aaggagggaa acaatttgtc 2040ttgcaatgtg agagtgatcc agagtttgtg cagtggaaga aagagttgaa cgaaaccttc 2100aaggaggccc agcggctatt gcgtcgtgcc ccgaagttcc tcaacaaacc tcggtcaggt 2160actgtggagc tcccaaagcc atccctctgt cacagaaaca gcaacggcct ctagcaccca 2220gaaacaggga gggtcctcga ggaggacaca ccagggtctc agccttttgg ggtgaacgag 2280gatgaggcat ctgatctatt cgctaccggg actcctccag gctcccgaga ggagtcggga 2340cccttcggct tggggtcagc tcagctccct gccttgtcac atttgtctgc attagaaact 2400actgaagaaa taaaagttct ttttctttgc tacacacttt ggtacctatg aacctagaac 2460ttgaagtgac tcctacttat cacgtaaatt tttatgtctg atatcaaaca catcttagac 2520tccccagaat ggaatttaaa gatgttcagt gttgggtaac agattgccct aagcattgcc 2580acatattctg tctagtcact gctgattttc tatgtctttg ctccatactg ctgggggatg 2640ggagagccac agtgtgtttc ttttgtgcac ttcgcaactg acttcttgtc ctggggttaa 2700aagttgaaga tattttctga tgatattaaa agttgaagat atttctgcac ttgggccctc 2760ctctgggagc cgcacccaca tgactgccct gcctctgacc agtctgttcc ggggccccct 2820cagccaggtg ggaatgacgg acacgtacta tccaagtgta tgggattaac taatcattga 2880aggcattcat ccgtccatca ttggaaagat ttacagtgat tctgaaggac aggccgtgga 2940gttttaggtt tcaggggcaa gagcagtttt caaaagtctt tgagtccagt gtgcacgagt 3000cgacaagcag tacctggcat gcaggagcac tcatgggtga gtccgtctca ggtctcgaca 3060attagcagtt gtgtgacagt cattctggtt ccttctgcct gaccctggga gacatatcag 3120taatggatgt acaaaagcag gtctgtttta tgtcttagta taatttcaga tgaattgtat 3180tgaaaaaatg ctgaggaatg aatgtgtcaa aatgggttaa ctgtgtatat tgactttcat 3240gtcgtcatgc atctgtcatg aatgaatgat actttgcact gggctgtacg acagtgagga 3300ccttagggca tgaagccttt ttcctggtcc cagcagcatc tgccctgtga agtttgtttt 3360ctcccactgc ctccaggccc cactgatacc cccaaataga tgctgggtta tgagaaccag 3420cgaaatcccc catgtcatca gtcttaaaaa aaaaatttta caaatccacg tatttgtccc 3480attcttggag tagttttagt gtatgtcttt acattaacta ctaacagtat aaataacttg 3540acatcgtaat tgtctgcatc ctgtccttga tatttttagc agttccaaat ctttgttttt 3600gtatttgttt gctgtgttca tgggcaaagt aagtactttt taatgcagtt attttgagag 3660tttggaagat aattaccaaa agggtccatt atttcataag agttactttg caaaaaaaaa 3720aatgtgggtt tttttttttg tctatctcaa ctactagttg gggtttaaat taacatacat 3780tttctactat ctgttatttc cagtgtggga ggagggatgt actacttaca tgcattctcc 3840ttatttaaaa aggaagaata gtattcaaat tctgttgaaa cacacacaca cacacacaca 3900cacacacaca cacactccag aagcagaaaa gccattgttc ttaaagagtg aatgtcttcc 3960cagccctggt taattatagc tgtgactgat gccgttcccg tctgcatctc aagctcatag 4020gttctcagca tgtgcagttg aggatgcgct gggcctcatg cctgttctag atctccagga 4080taaagggcct gctgttgact ccaccagggt ctgggcttag cgtctaatat ctcgtaccta 4140gggcgtgagc tgcacaaacg tgttcagaaa gattattcaa ctttcccata cttgttctaa 4200aattgagctg atccgcatct ctttcaaaaa ctagaatttc tgctctaaga atagaacata 4260aggctccact cccttttaga aaagatatat gaattggaaa atgctctgaa agtccttttg 4320cttcaaacaa aagtgtaaac ttttacactt ccccaactca catttgattt gtaatgatat 4380ggttgagaag tacatctaga tgtcatttat taaaagtgct ttgtaagact agattgagct 4440gtttctgagg gcggtcacca gttgtgttgg ggtctggttt gagtgccttc tgccaaaatg 4500ttgtgatgga ggtgtttctg cgaccagaca caggataccg ctgtgtctgc acccggttgc 4560ctgcatggcc agaggaaaag tcagttggat taaacatcat ggtatacttg gctgttgttt 4620ttttttaatt ttttaatttt ttgggatagg gcctcgctct gtcacccagg ctggagaaca 4680gtgggatgat catggctcac tgcagccttg aattcctagg ttcaagcaat cctcccacgt 4740cagcctcctg agtagctagg actacaggtg catgccacct ttcctggcta atttattttt 4800tgggtagaga tggggtcttg aactcttagg ctcaagtgat cctccttcct tggcctccca 4860aaatgctgga attagagatg taagccacca tgcccagcca tagtacttgg atgttttaga 4920aggttttcca agtattacat aattcctaga tgttcaccct tattacactc caactattaa 4980aaaggtcaaa attcagccta ttttttttca ttattttaga ttcctgtggt tgggatattt 5040taacattgat gagaaaaata attgaggttg atatttttac aaaatcatgc ggtaataagt 5100cttgatttca tgattcaaaa gaatcaataa agcctaaaaa taatagatta ctttaagctg 5160ctatgtaaga tatatatgga ataaattaaa aacctttgtg aattcaggtt tattattttt 5220aacctaaaac attctctttg gttcattcat cccctcatgt catgggggct cattggtttt 5280ccttctttgt catatttaag tatgattttt caacaaaact tctagaagtc agcttattat 5340gtcaccattc atgcaaagtg ctcatgcctc tgattggtcc attcactgac gtgacaattt 5400caggtcctat gtttaaaaag aaggggctgg ccgggcacga tggctcacgc ctataatccc 5460agcactttgg gaggccgaga ggggcggttc acgaggtcag gagattgaga ccatcctggt 5520tagcagagtg aaaccccgtc tctactaaaa atacaaataa aaattagccg ggcgtggtgg 5580cgggcgcctg tagtcccagc tacttgggag gctgaggcag gagaatggca tgaacccggg 5640aggcagagct tgcagtgagc cgagattgcg ccactgcact ccagcctggg cgacagagcg 5700agactctgtc tcaaaaaaaa aaaggagggg ggctaaatat ccagtgagat gcactgagga 5760aaggaagcat tttgctgaag acagcagcag caacaaacaa tggtctgttt gttgcaaaca 5820agatgtagct tgatttctgg tctgacatat gccatataca gatattagaa acgactgttt 5880gaaggccaca ctggtcatct acaaagtaat gtttaccaat tgacgacagg gatttaacta 5940gattaaaaag atcaaagtgt ggtttttctc tgctttttaa aatttcactc ggaatttgta 6000gctgggccaa ttcaacacat tttacttttc agtggaattg atttttctaa tgtttcagaa 6060ttttaacata tcaagaagaa aacaacgttc tcaaagtctg gcctctttag catgatgtaa 6120acctatagaa atgctttgaa atgtgctggt gtaagataag agttatcttg tatgatttaa 6180tcatatgcag tgttgtctca gttacgttca gggaaatgtt tctgtgtcat tcagagatgc 6240ttgatgaatt aacacctccc accctgagtg aggggttgac ttgttgggag atgatttggg 6300cttcactggg atctgtgaca ggtgggggct gggctgggtg tcacaaagag aatagtggta 6360gaaatcgggc gaaggaagaa agaagttact ggtaaaaatc attacaccat aaagcaccaa 6420ggaaataact gagttaaaat aggtgaagtt tcttttttcc cccctgtaac aggagagttt 6480tccttatgat aattattctg agacttggtc actttgtttt tgaatgtgga gctgctgaac 6540tcattcagaa gccatttgct gcctatcagg actttctgaa gaagttcttt tgcctctgcc 6600taccctctgg caccctccca tggaggcaca ggggacccag agctaaagca ttaccaggcc 6660atctccaaaa caccccgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 6720gcactttgca gcccccgagg tggagaggca gtgtctggat cactgtgaat gcattgcccc 6780attggtcagt tggggacact gttacaaatc cactgaagtc ctggtaaaac tgtcaagagt 6840aacaggcctc ttctgttcta ccctgctcac ttccacggtg agttaccagc ctgggcaaca 6900cagcaagacc ccatctctac aaaaaaaatt tttttaagta attaaccgtt taaatttttt 6960cctaaagatt taacatgatt tttccctcct atgtaaagtt tactggagag acttgaatta 7020cttaaattca tgttaatatg attttttttt aatccaggtc acattttaac aaagtttatt 7080atgaaacaaa tgaaatttga actctaaaat ggtactcctt ggcttcctca agtcacaatg 7140aactttatat tttctttgtc cttaaggact aagatagttg ttttatttca gccgaatcac 7200agagataacc actcctgcag gcccccacag ctggcccaaa ggggctgtct ttctgacctg 7260gctgtgttag cactgattga gaaacgcagg ctcccaaatt ttaaattgcc tttattaaaa 7320acacaaacta cagaaaatgg gttaagagta tacgcatttc atcaaacaca tataggggaa 7380aaaatccttc aatttagagt taaataactc agctttgtat agtagagtta gcgctccagt 7440atctaacaat ctcagaatca tctctgaaaa ctggtaacta tgcttccatt tttaattttg 7500tcctaaatat cagatgtctt tgatgtaagg gtagggaatg gagaaatatt ttcaattgtg 7560tatttgtatt acaaagaact tgaaatttac tttcttagtt gattatatta aatgatgtat 7620atattatatg tggtttataa gctcaacact ggccattttt ttagttttat tgttaaatgg 7680tatttttcta tgtttaatta taatagatct ggctttttct ggatagcata aagatcactg 7740aactatatat atataagaaa caagagttct attttagcac aaaggcattt tatattattt 7800attgaatcca taagtttgtt ttcgtcaaaa acattccata ttatttctgc tcctttttat 7860ttgtatagtt tgttatttaa agaaatggca gtccttcctg ttcttaatac aataaaattg 7920aaataatgca cctagtaatg tggccgacat ctcttctcac caccatggac tgttttcaac 7980aacagttgat cttctggtct gtgctgagag gcgcatgcat gtctttcgtc acgtcgggca 8040gcacacctgc tgtgaaatac tgctttcatc tacctcttca gaaggcttct tgcttgttga 8100caagtaccgc aaaggcttta ttctggactg gctatctcat aaaaggattt ctgtaagact 8160ttgcagtgtc attccctcag aacctaggtt tgtttctaaa gccacggtat tgtccaggag 8220cccctgtgtg tggggcaggt agctatccct cccatgtcat tagtaatcct ttaggattta 8280aggtacaact ggacagcatc attccttccc cttattgtgc caaatcccca ccatcagcct 8340tgccattgcc ttaagatttg attattgcac ccaattacct aaccactaaa cagaaaggcc 8400accttcactc tttgaaaaag gcaagctgtg cttagaaaca ctgcttttaa gagtagcaca 8460tttgagtgtg actttttccc cccttcacta tttcaaaatg gttttgaaat ggggtcttaa 8520aggtaagcgc cctcatacat gactgaaact ttgtgagagg tcttatattt gaatggaccc 8580ttaatgattt atgtgaaata gaatgaagtc ctgtctctgt gagagaacgt gcctcctcac 8640tcatttgtct ctgtctgttt tcatagccat caatatagta acatatttac tatattcttg 8700aatacccttg aagaaagaaa tccgttttct attgtgcatt gctatacgaa gtgaagccag 8760taaactagat actgtaaatc tagatattgt acctagacaa aatatcattg gttctatctc 8820tttttgtatc tgttgtgcca gggaaggttt ataatccctt ctcagtatac actcactagt 8880gcacgtctga aatagtatcc cacgggagat gctgctccac gtctgaggtc acctgccctg 8940tgtggggcac accaccgtca gcaccaccgt ttttacagtt actttggagc tgctagactg 9000gttttctgtg ttggtaaatt gcctatataa atctgaataa aaaggatctg tacaaaaaaa 9060aaaaaaaa 9068115069DNAHomo sapiens 11aaaaaagggt gaggagaagc agcggctgag cgggttggca tctggggcag cgggctcgct 60ccaggccgtc gggggccgct cgccagcgtc gcccgctgtg ttgggagcgc gggccgtggg 120cgtcgctcgg ccttgtccgc ggcgtccccg ctgccggcca cggcgctcag cgcttgtgct 180ctgtattgca ggtctacccc gagccccgga gcgagagcga gtgcctgagc aacatccgcg 240agttcctgcg cggctgcggg gcttccctgc ggctggagac gtttgatgca aatgatttgt 300atcaggggca gaattttaac aaggtcctca gttccttagt gactctaaat aaagtaacag 360cagacatcgg gctggggagt gactccgtgt gtgcccggcc ctcgtctcac cgcataaagt 420cttttgactc ccttggatca cagtctttgc acactcggac ttcaaaactg ttccagggcc 480agtatcggag tttggacatg accgataata gcaacaatca actggtagta agagcaaagt 540ttaacttcca gcagaccaat gaggacgagc tttccttctc aaaaggagac gtcatccatg 600tcacccgtgt ggaagaggga ggctggtggg agggcacact caacggccgg accggctggt 660tccccagcaa ctacgtgcgc gaggtcaagg ccagcgagaa gcctgtgtct cccaaatcag 720gaacactgaa gagccctccc aaaggatttg atacgactgc cataaacaaa agctattaca 780atgtggtgct acagaatatt ttagaaacag aaaatgaata ttctaaagaa cttcagactg 840tgctttcaac gtacctacgg ccattgcaga ccagtgagaa gttaagttca gcaaacattt 900catatttaat gggaaatcta gaagaaatat gttctttcca gcaaatgctc gtacagtctt 960tagaagaatg caccaagttg cccgaagctc agcagagagt cggaggctgc tttttaaacc 1020tgatgccaca gatgaaaacc ctgtacctca cgtattgtgc caatcaccct tctgcagtga 1080atgtcctcac ggaacacagt gaggagttgg gggagttcat ggagaccaaa ggtgccagca 1140gccctgggat tctcgtgctg accacgggcc tgagcaaacc cttcatgcgc ctggataaat 1200accctacgct gctcaaagag ctcgagagac acatggagga ttatcataca gatagacaag 1260atattcaaaa atccatggct gccttcaaaa acctttcagc ccaatgtcaa gaagtccgga 1320agaggaaaga gcttgagctg cagatcctga cggaagccat ccggaactgg gagggcgatg 1380acattaaaac tctgggcaac gtcacttaca tgtcccaggt cctgattcag tgtgccggaa 1440gtgaggaaaa gaatgaaaga tatcttctac tcttcccaaa tgttttgcta atgttgtctg 1500ccagtcctag gatgagtggc tttatctatc agggaaagct tccaacgaca ggaatgacaa 1560tcacaaagct tgaggacagt gaaaatcata gaaatgcatt tgaaatatca gggagcatga 1620ttgagcggat attagtgtcg tgcaacaacc agcaggatct gcaggaatgg gtggagcacc 1680tacagaagca aacgaaggtc acgtctgtgg gaaaccccac cataaagcct cattcagtgc 1740catctcatac cctcccctcc cacccggtca ctccgtccag caagcacgca gacagcaagc 1800ccgcgccgct gacgcccgcc taccacacgc tgccccaccc ctcccaccac ggcaccccgc 1860acaccaccat caactgggga cccctggagc ctccgaaaac acccaagccc tggagcctga 1920gctgcctgcg gcccgcgcct cccctccggc cctcagctgc tctctgctac aaggaggatc 1980ttagtaagag ccctaagacc atgaaaaagc tgctgcccaa gcgcaaacct gaacggaagc 2040cttcagatga ggagttcgcg tcccggaaaa gcacagctgc tttggaagaa gatgctcaga 2100ttctgaaagt cattgaagct tactgcacca gcgccaaaac aaggcaaaca ctcaattcaa 2160gttcacgcaa agaatctgct ccacaagttt tgcttccaga agaagagaaa attatagtgg 2220aagaaactaa aagtaatggt cagacagtga tagaagaaaa gagtcttgtg gataccgtat 2280atgcattaaa ggatgaagtt caagaattaa gacaggacaa caaaaagatg aagaaatctc 2340tagaggaaga acagagagcc cgcaaagacc tggagaagct ggtgaggaaa gtcctgaaga 2400acatgaatga tcctgcctgg gatgagacca atctataagg gatgtcctca gttctttctg 2460ttgaagacca gttctgaggt gaagctgggc acccctgacc caagtcgggg tgcactcagg 2520accacagggc agggctgggt ggggcgccac cttgctctct gtatatagaa aagctggagc 2580ttattctgcg aatggagacg atcaaaccat gactgatgaa tccagacagg agggattgac 2640tctgaggacc tgagctacat caatccactc tgtgaacatc tcagttacct cattctgcaa 2700taagttcagt gactgactaa aagtcttgtt tttccagact ttgaattgaa tatataaata 2760ttatatatac atgtttcttg taaatatccc attttgaatg catacctgtg gtggttctgt 2820ccgggctaat ccccatgcta gaatgtcctt tccagctacg tgaataagaa gtcccatgcc 2880cgcatccacc ggaagcagaa gcctggtgga tgcctggttc gttccgcagc accagggcct 2940ccaccgtgct gtggcagcac cccccatgtc ggtatttcta aataacctta tttatacctg 3000cagagataca cttcagtccc attcagaagt cttctcttaa agcagcatta cagtcccaga 3060cctgcgggtt tctgagggca acttgctggc tgacagactc agtcttgacc tcaaggaagg 3120cccatacggc actgccgcat

ccacctagag gtgtttgctc ttgtccgctg tctgagtact 3180gtgattctca gatgagtttg ctgcgttttg ggaggacaca gacggttctg tataggctag 3240ttcagtaaca acaaaataca ctgttttgtc ttccctcaaa gagagatctt actagaacct 3300gtaaatagaa tgtattattt attataagtc actgcagctg atgaaaacag atggaggcca 3360tgctgcaggc tgatactgat gggtggagtt ttgtcatcag gccagcctca tcccgaggtc 3420tcctccacca ttggccgtag ccagcaggct tcagtgctca ccgaaagtaa aatcccctcc 3480ttcagcaaga ataaagcaat atacacctta ggttccacta agtaacatag gcataagcag 3540ggaacgtttc ccccactgtg ttccagtgca gaggagacga agcctgtcct caccgcggct 3600cgctgggccc aggctggctc tggaaagcct gtgcggtcct gggcaggaag cccggcccgt 3660ggagcaggtt ttcgttctgc ttcagcaata aataagggtg accacaggga ctttgctttt 3720ggtttccttt cctgtgaaaa ggttggtttt aaagtgagat acacttttcc gtagaacaag 3780tgttctatct ttaaaaaccc aaattgcagc accgtggatt actggtctca gaacaactca 3840ttgcgcatca gatttgactc tctgattttc tgtctattgg ccaaattgcc ctttaactgc 3900acctgaatcc tttgtgtact gatgcctttg agctgggcac cttgggagag tgttgtgttg 3960ctgtttacgg ttcttccttg cccttgctaa ttacagtctc tggtgcccag caagcccctt 4020tggcttcctt ccgtgactgg tcacgttgtc tgcctgggct cagcgtggac ctgccccatg 4080ctgcagaacc tggcctcacc tggacttttc actagaattg ccagcttcct caacttagca 4140gatcattcac tcatgcgggc acaagcaaag atcaacactt tcttttttgg taagcttgag 4200ttttacaagt tattttttgg tgatgcgtaa gacattgcag tgggaaacca ttcaacttga 4260gtttattgga gtttgctgtt gtagcaggtt ttaactcagg aacaactctt gtctgatctc 4320tccgcccctc tgccgggagg cgacattaac tgtcctctcg gagccggtag cgttgctgtc 4380cgagtcccca ggacggatct cctgcagacc tgccttaatg ctcagatcga agtatttcac 4440aagaatactt gtgtttttaa cagcccttcc cctggacggt gcggccatga gggcctcatg 4500ttacggcatt gccttttctt tctgtggatc cagtatcttc ctcggctttt tagggagcag 4560gaaaaatgcg tctgagagca actcttttta aaaacctgcc ctgttgtata taactgtgtc 4620tgtttcaccg tgtgacctcc caagggggtg ggaacttgat ataaacgttt aaaggggcca 4680cgatttgccc gagggttact cctttgctct caccttgtat ggatgaggag atgaagccat 4740ttcttatcct gtagatgtga agcactttca gttttcagcg atgttggaat gtagcatcag 4800aagctcgttc cttcacactc agtggcgtct gtgcttgtcc acatgcgctg ggcgtctggg 4860accttgaatg cctgccctgg ttgtgtggac tccttaatgc caatcatttc ttcacttctc 4920tgggacaccc agggcgcctg ttgacaagtg tggagaaact cctaatttaa atgtcacaga 4980caatgtccta gtgttgacta ctacaatgtt gatgctacac tgttgtaatt attaaactga 5040ttatttttct tatgtcacaa aaaaaaaaa 50691281DNAHomo sapiens 12aacacagtgg gcactcaata aatgtctgtt gaattgaaat gcgttacatt caacgggtat 60ttattgagca cccactctgt g 81131251DNAHomo sapiens 13agttgtaaac ccaaggccaa gagaccatgg ccacgttagc ccggctgcaa gctaggtcgt 60cgactgtagg aaatcagtac tactttagga acagtgttgt agatccattt agaaaaaagg 120agaatgatgc agcagttaaa atccaaagct ggtttcgagg atgtcaagtt cgggcatata 180tcaggcattt aaacaggatt gtaacaatta ttcaaaaatg gtggagaagt ttcttaggca 240gaaagcaata tcaactaact gtgcaggtag catattatac tatgatgatg aatctctaca 300atgcaatggc tgtcaggatt cagagacgat ggcgaggcta tagggttcgg aagtacctct 360ttaattatta ttatttgaaa gagtacctga aagtcgtttc agagaccaat gatgcaatta 420ggaaggcact ggaggagttt gcagaaatga aagaaagaga agagaagaag gctaacctcg 480aaagggaaga gaagaaaaga gattaccaag cccgaaagat gcattacctc ctcagcacaa 540agcagattcc aggaatatac aattcaccct tcagaaaaga gcctgatcca tgggagctgc 600aattacaaaa ggcaaagcct ttaacacacc gaagacctaa agttaagcag aaggactcca 660ccagccttac tgattggcta gcttgtacaa gcgcccgttc ttttcctcgg tctgaaattc 720taccacctat taatagaaag caatgtcagg ggcccttccg agatatcacc gaagtattag 780aacaacgcta caggcctttg gagccaacgt tgcgggtggc agaaccaatc gatgagttaa 840agttggccag agaggagctc agaagagagg aatggctgca aaatgtaaat gacaatatgt 900ttttgccatt ttcttcatac cataaaaatg aaaagtacat cccatcaatg catttatcaa 960gcaagtatgg tcctatttct tacaaagaac aattccgaag tgaaaatcct aagaaatgga 1020tctgtgacaa ggatttccag actgtattac catcatttga gctcttctca aagtatggaa 1080aattatattc aaaagctgga cagattgtat aaaggcgtca gaagaagaaa ctgaagccat 1140ctgcatttta aaacttaaca gttctgaaag gaaaacacag atgaagatcc tgtaggaaat 1200atacttgcta tgattcaata aactataaaa ttttgaaaaa aaaaaaaaaa a 1251147996DNAHomo sapiens 14gcgacccgag gaggcggaag agcggcgccg gcgacgtact gtaagacgat attactttaa 60tcatcttcac atcagtattt atggaatagc cacaggtgcc tcatccttta gtaggagtta 120attatacatt tactggccga gtaaacatct ccgaatgtca ctccatggat tcctttgggc 180aacccagacc agaagataat cagtcagtag tcagaagaat gcaaaagaaa tactggaaaa 240ctaaacaggt ctttatcaaa gcaacaggaa aaaaagagga tgagcacttg gtggcgtctg 300atgctgaact ggatgctaaa cttgaggttt ttcactctgt tcaagagaca tgcactgaac 360ttctgaagat aatcgagaaa taccagctaa gactcaatgt tatatcagag gaagaaaatg 420agctagggct ctttttaaaa tttcaagcag aacgggatgc aactcaagct ggcaaaatga 480tggatgccac tggcaaggca ctttgttctt cagccaagca aagattggcc ctgtgtactc 540ctctgtctcg tctgaagcaa gaagtagcaa cattcagtca aagggcagta tctgatacct 600tgatgacaat taatcggatg gagcaggcac gcacagaata cagaggagct ctactgtgga 660tgaaagatgt atcccaagag ctggacccag acaccttaaa gcaaatggaa aagtttagaa 720aagtacagat gcaagtgaga aatagcaaag cttcttttga caagttaaag atggatgttt 780gtcagaaagt ggatttactt ggagctagtc gctgcaatat gctatctcat tcgctcacta 840cctaccagag aacactgctt ggattctgga agaaaacagc tcgaatgatg tcccaaattc 900atgaagcctg tattggcttt catccgtatg attttgtagc tctcaagcaa ctacaagaca 960cgccaagcaa gattagtgaa gacaataaag atgaacaaat aggcggtttt cttactgaac 1020agctcaataa gctagttttg tctgatgagg aagcaagctt tgagagtgaa caagcaaaca 1080aagatcacaa tgaaaaacat tctcaaatga gagaatttgg agcacctcag ttttctaact 1140ctgaaaatgt tgcaaaagat ctacctgtag attcattgga aggagaagat tttgagaagg 1200aattctcatt tctgaacaac ctcctaagtt ctggttcttc aagtactagt gaatttaccc 1260aagaatgcca gactgccttt gggagcccca gtgccagtct cacatcccag gagccttcca 1320tggggtctga gcccctcgct cattcttctc gattccttcc ttcacaactc tttgaccttg 1380gctttcatgt ggctggagcg ttcaacaact gggtctccca agaggaatca gaactttgtc 1440tttcacacac tgataaccag ccagtgcctt cacagagtcc aaagaaatta acaagatccc 1500ccaacaatgg caaccaagac atgtcagcct ggttcaatct gtttgcagac ttggatccac 1560tttcaaaccc agatgctatt ggacactcag atgatgaact tcttaatgct tgactgaagt 1620tataatgtca cttcagtggc cttgagacat caattttgca acgtatttcc ttcgtggaaa 1680ggatttagat tgtaacccgc acacaaaagc acggtgtttg tgaatataac acctgtcagc 1740caactttaga cagatggtaa agaccacatt tgaataagta cacatctttc atatcttgga 1800tttgcagctg ttggtactat gtggaaaata ttagaaactt ctatgtggaa aatattagaa 1860actacagagt ttgcgatatt tagatactga aatttatgtc aaaataacgg ctaggaataa 1920ttctgtcaat atggagttga gcttatttct ttggaaaccc ttttaagttg ccttgctggc 1980tgtgagaatt ttatatgtgg ataacaaaga tagatagata gcatgtaaat tgggttgtgg 2040tttggggtca gtttttaaat gaaatagtag cgaggaggat tttctgtttt ggaaaacacc 2100attagaacca gaccagcttt gttttgggtt agagagagta agatttgaga actcagtttg 2160ctttaatgaa atcacagaga aacttggtac ttgtttttct tcatttggag gctaaaatgt 2220aatgtttttt cattcataca aaataatgga cactccctaa ttccattatt aaatcttgaa 2280ggggaagtag caggataatt aatttgctaa gcccatcctc tgcagaaaca gaaaaatcta 2340tcttcccatc tcctaaaact cagaatgcac agtaatactt aaggcttgta caagtgtctt 2400cagacccact ttttcataca cttgctatat agtagtatgc agtatttata ttattcctga 2460aaataaaatg aggggagaat attccctaag caactggcaa tagtattcct gaaataccta 2520gaaatttcta tctgaatgag ggagacactt atgaacacct tatccttaca tatatttgca 2580tacttatctc atattttgtg acataattat ttaacccaga atactttctg gcagacatac 2640agaaagctct gtgtgatcaa taagggagtg tctcattttt ctacttccct ctttctgtgg 2700gtgacatgat ctgaggttct atttgattac taagcaaaat ctgttacccc tacagggttt 2760agaacctaag tattagagag gaaggctatt taatggaagt tagtgtaagc tgataaaaac 2820gtagctaccg tacacacaca tcaatcactc aatttcctgt ccttttaaat tgcccaccct 2880ttaattttga agcaatttcc caagtgtgtg tttgttttat atttgtcatc cagtccattg 2940catttccata agaagacatt ttgactggct gggtgcggtg gctcacgctt gtaatcccag 3000cactttggga ggctgaggca ggcggatcac gaggtcagga gatggagacc atcctggcta 3060acatggtgaa accccgtctc tactaaaaat acaaaaaatt agccaggcgt ggtggcacgt 3120gcctgtagtc ccagctactc cggaggctga ggcaggagaa tggcgtgaac ccgggaggcg 3180gagcttgcag tgagctgaga tggcgccact gcactccagc ctaggcaaca gagcaagact 3240ccgtctcaaa aaaaaaaaaa aaaaaaaaag acattttaac taagttattc acagtagctt 3300ccatgtgctc ttagttctat tctaaacagg cttatttaga aaaggattgc ttgtaatgtt 3360tgtcatggta catagaaaac attggaccag agtaggtaaa atgcagtcca tgtcccatcc 3420atagccatct acaatagtaa ctgcccacag gctctccaga aaactactac aatggccaag 3480tacagtatag gctggaaaga ccttatctga aggtcagaaa cattgactca gaaaaaaggt 3540atgaagtctt tccataaaat cttttcacaa tattactcct atttctttta gattttaatg 3600agccattact tatctcttca gaagacttaa gtcttccttt atactcagtg aaatttccca 3660gaatgtaata ctgtcactgt tctgccaagt tccaatcacc aagatcatga ttacgaatcc 3720caatctgaat tctataccca tggtgactct gatgctctca acttttgagt gcctcaaaaa 3780atgctaaaac tttggctggg catggtggct cacgcctgta atcccagcac tttgggaggc 3840catggcgggt ggaacatttg agggcatgag ttcgagacca gcctggccaa catggtgaaa 3900ccctgtctct actaaaaata caaaaattag ccaggtgtgg aggtacatgc ctgtaatccc 3960agctactcag gaggctgagg cagaagaatt gcttgaaccc aggcggcaga ggttgcagtg 4020agtcgagatt gcaccactac actccagcct gggcaacaga gagcgactct ctcaaaaaaa 4080aaaaaaaaaa aaaaaaaaaa agctaacttt atgtcttgag agtttgtacc atttttcttt 4140gtagtggtca ccttgctaat gcattagttc tgagatattt atctccctca catgtgtgca 4200aggaagtccc tgttatcgaa tacagatact ttaacaagca gacaaagcag aaacccaaag 4260tccatactag gaacacccta aattttccaa aagcaaaagt tctcccgaaa tggagacata 4320caagggactt ttattattct gttactagtt tctataacat ttcttctttc aacagagtat 4380atgtttccca tttaacccag agcaacatta acttccttag caagtccagt tctaacttcc 4440aacaagtcca accactgttt ttgaagagca tatcagtaac tatattaaga tgaaggtaac 4500cacattcgta ttttctcaag attagttatt tgaagctcag cagtttttgt ggtcagaaag 4560aaattttgct ctattaaacc aatactgcta atataaaaaa ccaccacact gaagaaacga 4620gggaaaggac gggataagca cagaacagag aatgactggt tgctttttgt ctcaatctag 4680ataatccatt caataagaag taaattaatt atccttaacc aatggtaggc tgagaacaac 4740cctcaaaata gatatttttt atgttaaatg gggagaaata tctatacttt atgttatact 4800ggataaaaat gtgttttaag tctaaaaaaa accagacgag ctaaactttg cctagtgtgt 4860ctacaaccat tttttaggag acgcaggaat accagggcat aataagatca gattggtgta 4920attttgtatg tttttgaaat ccttcattaa ttgtagaacc ttgatatgat tagaaacaaa 4980ctgtatttca acaaacaggt ttcagtattt gcacactgaa aaagtgtttt gtattttaac 5040tataaatatt tcacgtatct gtatagacca tctagaaatg tagaggtctt acagcattag 5100aacgaaggaa gtttacatgt gctctatcta tttttctgag cctcttttaa taaagattgc 5160aagaaggcat aaaacaagag tttgtttcct gaagttttta gtacaattat tgttttccta 5220ttcaaaaact tgggttttac ctcaagatca tagtattagg aaagtacatt gagttgatac 5280ggacatggga gaacgaaaat aaaaccaggg caattaatat ccttgtaagg ccaggcgcgg 5340tagctcacgc ctatataaca gcactttggg aggccaaggc aggcagatca cttgaagttg 5400ggagtttgag accaccctgg ccaacatggt gaaaccccat ctctatgaaa aatataaaaa 5460ttagctgggc atggtggcag atgcctgtaa tcccagctac ttgggaggct gaggcaggag 5520aatcgcttga acccgggagg tggaggttgc agtgagctga gatcaggcca tgcactccag 5580cctgggtgac agagtgagac tccgtctcaa aagaaaatcc ttgtgagatg aattcgttct 5640tatttcatat acaaggggac tatgtaagat atgggaaata atataatgta cgttatttat 5700gtaaatactt tcagtaacaa aaactaacaa atatcaaaaa tctgagccta gacacaaaca 5760attaaatata agccatagta tgtaacctga cttattgaag gcaggaataa aaagaagaga 5820gccagaattg attcagttat ttttgtcttc catagtgttg gcagggccct gcatttctct 5880accttgagca atgaagcagt cccagaattt tggaatatag aaattaggaa ggaaaaacga 5940actttaaaat attaatttag tagaactgaa agtaatgcat ttcatgcaac agtaaagtgc 6000ttaaacatgg caaagaaaac taagggacaa gataagaaaa atggttggta aagatgggtc 6060acatccagaa gcccaacata agctattttt ccatcttttt ctgtaccatt taaaagacta 6120ctaaagagtt caaaacaaac tgtccttggc atttaaagtc aaaacatggt tcttcactca 6180gtggtgtaaa ttaactactc attggccaaa gagtaaatac aaaaaggaga tgaagttctg 6240actggtgttg tgggtgagat tttgcagtgt atgtttttac atttgtagtg tacgttttaa 6300acttccggtt ttcttgctgc ctcaacatct ccacaaacag gctgtgagca cctggcccag 6360tgtgatatgg gtggcccctg ccacaagttt cccttgctac cttgcccatg acctagtaac 6420atttaaatgc accagtgaaa ttctcttttt cttgtgtgct tctccctgac ccaaaggctc 6480ctgcccatgt cttaggcctc tccgctcccc acctgcttgg ctgagccccc tcccatgtga 6540ccccctctca gcatgcagtg actggtctct ctaggacctg tgagtatatt acaacttttt 6600ttcctgtgtc tctcctgtga tatctcttgt ggctaaacct cactgaccat aacctaaaaa 6660aacacataca aaaaacatga aagaaaaagt tgtttcttga gctggcctgg acgaacgggg 6720agccaggggc tcgaccctgg ctgttggagg cgcagtgagg cctggtctcc ggctgccaga 6780ccacgctgag cggagcgcgc ggcaggctcg cctcagcgct gcggggaacg cgcgcgccgc 6840cgtccgcggt cgcccgtagg tgcctgcacg cgtcggggtc acggcctttg gccgacagga 6900gcgagaagac tcaggagccg ccccgcgcct tcgatccggc gctgctggag ttcctggtgt 6960gcccgctctc caagaagccg ctcagatatg aagcatcaac aaatgaattg attaatgaag 7020agttgagaat agcttatcca atcattgatg ggattcctaa tattatcata ttaggcagct 7080aggatgacac atcaaagaag caagaagtgg agcagcgcta gttcataatt taaaaaaata 7140aaacagccaa ctcttcttag taccatatac cttttaaaac acagtggcaa gtaataagcg 7200gaagagaaga atctttctgt ctctttctac gttgactgtt cttattccac tggtttattt 7260agcaggactg ttccactcag cctctgtaga agaaaacttc ccacagggct gcacttgcac 7320agctagcctt tgcttttaca gcctgctctt gcctattacc ataccggtgt atgtattctt 7380ccacctttgg acctggatgg ttattaaact cttcatgcat aactgatgca actagagtca 7440atatgctgta tatattaatg atagctcttg ggcatctatc tctgaaagct caaatggatg 7500gaatttagtt tgtgggaaag aggctttgct ttgagcatat caggcttagg actgtggacg 7560gcttaagttg cagacgcttc ttttattgta ctcttgttct gcccgtgttt tttgaaggct 7620ctgacataac tgctttatca gaagaaacat tttgacagtg tcttgttgga gataaacatc 7680cctaattgac atgtgatgac tacttcttat tccattcatc taagagtcat tgaaattttg 7740ttttctttgt ttgtttagct tcaaggtctt tggtagtcac atgttaggga tgactgaaat 7800aattccaaag gagtgatgtt ggaatagtcc ctctaaggga aagaaatgca tttgaacgaa 7860tgtgatataa aaccacataa tcaaatagaa atttcatgta cttacaaaaa tttagtttgt 7920aaaattacct tcatttcttt gacattaaat gcttatatta gcaataaaga tgttgacact 7980ttctcataaa aaattt 7996153174DNAHomo sapiens 15taataatggc cgtaagctta aaatagatcc agggaggagc tcattaacgt gaacatagaa 60agcagttccg cacctctggc cttactcctc ttggaaattg ctttggtcca tttttacttc 120cttttattcg acgcaccaga aaataagact tttaccaaca tttttactgc atttgacgat 180gaactaattt agaccggcta aaataattgt tccactggga cacaggaatt caacctcagt 240tcagaaaatc cctgacatct gacgtaggag gatttatagg tttagtggaa attgctttct 300cctgctctcc agattgcatc ctgtgggttg attttttttt tgcatgagta aacatccttc 360taataatgaa cagaccaata atgtcttaag agagaaaaag aacaatcttt tcctttttgc 420tgtttctgga gagagctgtt tgaatttgga aacccatgtt ggctgtccca aatatgagat 480ttggcatctt tcttttgtgg tggggatggg ttttggccac tgaaagcaga atgcactggc 540ccggaagaga agtccacgag atgtctaaga aaggcaggcc ccaaagacaa agacgagaag 600tacatgaaga tgcccacaag caagtcagcc caattctgag acgaagtcct gacatcacca 660aatcgcctct gacaaagtca gaacagcttc tgaggataga tgaccatgat ttcagcatga 720ggcctggctt tggaggccct gccattcctg ttggtgtgga tgtgcaggtg gagagtttgg 780atagcatctc agaggttgac atggacttta cgatgaccct ctacctgagg cactactgga 840aggacgagag gctgtctttt ccaagcacca acaacctcag catgacgttt gacggccggc 900tggtcaagaa gatctgggtc cctgacatgt ttttcgtgca ctccaaacgc tccttcatcc 960acgacaccac cacagacaac gtcatgttgc gggtccagcc tgatgggaaa gtgctctata 1020gtctcagggt tacagtaact gcaatgtgca acatggactt cagccgattt cccttggaca 1080cacaaacgtg ctctcttgaa attgaaagct atgcctatac agaagatgac ctcatgctgt 1140actggaaaaa gggcaatgac tccttaaaga cagatgaacg gatctcactc tcccagttcc 1200tcattcagga attccacacc accaccaaac tggctttcta cagcagcaca ggctggtaca 1260accgtctcta cattaatttc acgttgcgtc gccacatctt cttcttcttg ctccaaactt 1320atttccccgc taccctgatg gtcatgctgt cctgggtgtc cttctggatc gaccgcagag 1380ccgtgcctgc cagagtcccc ttaggtatca caacggtgct gaccatgtcc accatcatca 1440cgggcgtgaa tgcctccatg ccgcgcgtct cctacatcaa ggccgtggac atctacctct 1500gggtcagctt tgtgttcgtg ttcctctcgg tgctggagta tgcggccgtc aactacctga 1560ccactgtgca ggagaggaag gaacagaagc tgcgggagaa gcttccctgc accagcggat 1620tacctccgcc ccgcactgcg atgctggacg gcaactacag tgatggggag gtgaatgacc 1680tggacaacta catgccagag aatggagaga agcccgacag gatgatggtg cagctgaccc 1740tggcctcaga gaggagctcc ccacagagga aaagtcagag aagcagctat gtgagcatga 1800gaatcgacac ccacgccatt gataaatact ccaggatcat ctttccagca gcatacattt 1860tattcaattt aatatactgg tctattttct cctagatgct tgtaattcta caaatttcac 1920atttccatgg catgcactac agaaataact gtataatgaa aaagtattta aggatatggt 1980taaaaaaaaa tcccaggacc cacccatgtt ttcactatcc cttctgcagc tttccaaagc 2040tacattgacg agacacttac tggtttaatt tgcacttatt aaccatctat tgaatacaca 2100gcattatatt aggtgctgca ggaaatacga cactgtagcg actgatgtta gttgttaccc 2160agatcccctg gaaaagcaca ctaccagtgt tgtgggcaca tttagttcca cccgttagac 2220ccttgatgct attcacatga ataatttatt ttcctcaagt gtcattacat tgttcaggct 2280acgtgaactt ggaagcacct acaggccatt tgcatgaaat tcacatgcac ctaaatcctc 2340actttgacag aaactcatgc ttcagtttat aacctattac ctattttgta tgcgactcca 2400cctccgcatg tttattttaa taaaaggcaa tgataacatt cacattattt ttctttatat 2460gctgtggttc acaggcttta ccccttcaca agaaaagctc tttagattgg cgcaattgct 2520tctgattttg gtgaaatttt ccctggtagg gaaactttga agataagagt acacacatgc 2580attttgtctg ttgtgtcata gaggtaacta ggctagaaaa tttgtgttta aatgttccct 2640attttatata atcaccactt catgtttctt cttcttggag catgtccttg ttcaaagaga 2700agtgctttct cagtgatgtg atatcttcac tgaggaactt gggtagagaa tgatttcttc 2760tgcataaaca cttcaaggaa atacataatt tgggactact tgtaactcat tagaatgaga 2820aatactcaca tggtttctta agagaaaaag aacatcggaa agcaaaataa atgggaagat 2880atcactggac atctgcattt atactcgaaa taccagcatt ttctatggac cagaaaactg 2940ccatcaccta gaccacacag cccagatacc aggcagacgg atggcccaat ggcaactgat 3000gtcagggcat ggggtaaagg agagggttct aatctggtgt atcacttaaa aacagttatt 3060tatattatat atctgctata tagatcaacc tccaccaaac ttacccaaac agcatttgtt 3120ttatttgaaa ctcactttaa taaagtgaat tatatacaca aaaaaaaaaa aaaa 3174167773DNAHomo sapiens 16agagcctcgg tggtttgcag cagtggaacc aggcaggccc agttgtgggt aggagaggcc 60gtcacctgtt gaggcctccc ccccacaccc ccgcatcgcc ctgccctggc agagcccagc 120ccccagtccc cggagagcgc gcctgaggac ggacggacgg acggacggac agacctaggg 180acggagggcc aggggcaggg gagatccaag aggccccgcg ctggaatgca gttttctcgg 240gcgagggaga ctttgcaccg gagtggaaaa tagtttgggg tggggtttcg caccgtcccc 300tcctccccag ccccgggccc cctcccaggc gctttctggg agcttttaga actgcgctct 360gaagtttcca gagagcgagg agcttttgcg gcaggcagag

acaatggaag aaaatgaaag 420ccagaaatgt gagccgtgcc ttccttactc agcagacaga agacagatgc aggaacaagg 480caaaggcaat ctgcatgtaa catcaccaga agatgcagaa tgccgcagaa ccaaggaacg 540cctttctaat ggaaacagtc gtggttcagt ttccaagtct tcccgcaata tcccaaggag 600acacacccta ggggggcccc gaagttccaa ggaaatactg ggaatgcaaa catctgagat 660ggatcggaag agagaagcgt tcctagaaca tctgaagcag aagtaccccc accacgcctc 720tgcaatcatg ggtcaccaag agaggctgag agaccagaca aggagcccca aactgtctca 780cagtcctcaa ccacccagtc tgggtgaccc ggtcgagcat ttatcagaga cgtccgctga 840ttctttggaa gccatgtctg agggggatgc tccaacccct ttttccagag gcagccggac 900tcgtgcgagc cttcctgtgg tgaggtcaac caaccagacg aaagaaagat ctctgggggt 960tctctatctc cagtatggag atgaaaccaa gcagctcagg atgccgaatg aaatcacaag 1020tgcagacaca atccgtgctc tcttcgtaag tgcctttcca cagcagctca ccatgaaaat 1080gctggaatcg cccagtgtcg ccatttacat caaagatgaa agcagaaatg tctattatga 1140attaaatgat gtaaggaaca ttcaagacag atcactcctc aaagtgtaca acaaggatcc 1200tgcacatgcg tttaatcaca caccaaaaac tatgaatgga gacatgagga tgcagagaga 1260acttgtttat gcaagaggag atggccctgg ggcccctcgc cccggatcta ctgctcatcc 1320accccatgcg attccaaatt ccccaccgtc tactccagtg ccccattcca tgcccccctc 1380cccgtccaga attccttatg ggggcacccg ctccatggtt gttcctggca atgccaccat 1440ccccagggac agaatctcca gcctgccagt ctccagaccc atctctccaa gcccaagcgc 1500cattttagaa agaagagatg tcaagcctga tgaagacatg agtggcaaaa acattgcaat 1560gtacagaaat gagggtttct atgctgatcc ttacctttat cacgagggac ggatgagcat 1620agcctcatcc catggtggac acccactgga tgtccccgac cacatcattg catatcaccg 1680caccgccatc cggtcagcga gtgcttattg taacccctca atgcaagcgg aaatgcatat 1740ggaacaatca ctgtacagac agaaatcaag gaaatatccg gatagccatt tgcctacact 1800gggctccaaa acaccccctg cctctcctca cagagtcagt gacctgagga tgatagacat 1860gcacgctcac tataatgccc acggcccccc tcacaccatg cagccagacc gggcctctcc 1920gagccgccag gcctttaaaa aggagccagg caccttggtg tatatagaaa agccacggag 1980cgctgcagga ttatccagcc ttgtagacct cggccctcct ctaatggaga agcaagtttt 2040tgcctacagc acggcgacaa tacccaaaga cagagagacc agagagagga tgcaagccat 2100ggagaaacag attgccagtt taactggcct tgttcagtct gcgcttttta aagggcccat 2160tacaagttat agcaaagatg cgtctagcga gaaaatgatg aaaaccacag ccaacaggaa 2220ccacacagat agtgcaggaa cgccccatgt gtctggtggg aagatgctca gtgctctgga 2280gtccacggtg cctcccagcc agcctccacc tgtgggcacc tcagccatcc acatgagcct 2340gcttgagatg aggcggagcg tggcggaact caggctccag ctccagcaga tgcggcagct 2400ccagctgcag aaccaggagt tgctgagggc aatgatgaag aaggccgagc tggaaatcag 2460tggcaaagtg atggaaacaa tgaagagact ggaggatccc gtgcagcgac agcgcgtcct 2520agtggagcaa gagagacaaa aatatcttca tgaggaagag aagatcgtca agaagttgtg 2580cgagttggaa gactttgttg aagacttgaa gaaggactcc acggcagcca gccgattggt 2640tactctgaaa gacgtggaag acggggcttt cctcctgcgt caagtgggag aggctgtagc 2700taccctgaaa ggagaatttc caaccttaca aaacaagatg cgagccatcc tgcgcataga 2760agtggaggcc gtgcggtttc tgaaggagga gccacacaag ctggacagtc tcctgaagcg 2820tgtgcgcagc atgacagacg tcctgaccat gctgcggaga catgtcactg atgggctcct 2880gaaaggcacg gacgcagccc aagccgcaca gtacatggct atggaaaagg ccacagccgc 2940agaagtcctg aagagtcagg aggaggcagc ccacacctcc ggccagccct tccacagcac 3000aggtgcccct ggcgatgcga agtcggaagt ggtgcctttg tccggcatga tggttcgcca 3060cgcgcagagc tcccctgtgg tcatccagcc ctcccagcac tccgtggccc tgctgaaccc 3120tgctcagaac ttgcctcacg tggccagctc cccagccgtc ccccaggaag caacctccac 3180tctgcagatg tcgcaggctc cgcagtcccc acagataccc atgaatgggt ctgccatgca 3240gagcttgttc attgaagaaa tccacagtgt gagtgccaag aacagggcag tgtctatcga 3300gaaagcagaa aagaaatggg aggaaaaaag gcaaaatctg gatcactata atgggaaaga 3360gtttgagaag ctcctagaag aagctcaggc caatatcatg aagtcaatac caaatctgga 3420gatgccgcca gccacaggcc cactgccaag gggagatgcc ccagtggaca aggtggaact 3480ttcagaagat tctccaaatt cggaacagga cttggaaaag ctggggggaa agtcgccccc 3540tcctcctccg ccacctcctc gtcgaagcta cctgccagga tcgggactca ccaccacgag 3600gtcaggcgat gtggtctaca ccggcagaaa ggagaacatc accgctaagg caagcagtga 3660agatgctgga ccaagcccac agaccagagc tacaaaatat ccagcagagg agcctgcttc 3720agcctggacc ccatccccac cgcctgtcac cacctcctcc tcaaaggatg aggaggaaga 3780agaagaagaa ggagacaaaa taatggcaga actccaggca ttccagaagt gttcctttat 3840ggatgtaaat tcaaacagtc atgctgagcc atcccgggct gacagtcacg ttaaagacac 3900taggtcgggc gccacagtgc cacccaagga gaagaagaat ttggaatttt tccatgaaga 3960tgtacggaaa tctgatgttg aatatgaaaa tggcccccaa atggaattcc aaaaggttac 4020cacaggggct gtaagaccta gtgaccctcc taagtgggaa agaggaatgg agaatagtat 4080ttctgatgca tcaagaacat cagaatataa aactgagatc ataatgaagg aaaattccat 4140atccaatatg agtttactca gagacagtag aaactattcc caggaaactg tgcctaaggc 4200cagtttcggt ttctctggca ttagtccatt agaagatgaa ataaacaaag ggtctaaaat 4260ctcaggcctg caatactcta tacctgacac cgagaaccag acgctgaatt acggaaagac 4320aaaggagatg gaaaagcaaa atacggataa gtgtcacgtt tcctctcaca ctagactaac 4380agaatcaagc gtgcatgatt ttaaaacaga agatcaagag gttatcacga cagattttgg 4440ccaagttgtt ctaagaccca aggaggcaag gcatgctaac gtgaacccta atgaggatgg 4500agaatcaagt tcaagttctc ccactgaaga aaatgcagcc actgacaata ttgccttcat 4560gattaccgaa accactgtcc aggttctttc cagtggggag gtgcatgata ttgttagcca 4620aaagggagaa gacatacaga cggttaatat cgatgccaga aaagagatga ccccccgaca 4680agaagggact gacaatgagg atccagtcgt gtgcctggac aagaaaccag tgatcatcat 4740tttcgatgag cccatggaca tccggtctgc ctataagaga ctttcaacta tctttgagga 4800atgtgatgag gaattagaga gaatgatgat ggaggaaaag atagaggagg aggaagagga 4860ggaaaatggg gattctgtag tccagaataa taacacttcc cagatgtctc ataagaaggt 4920ggccccaggc aatcttagaa ccggacaaca ggtggaaaca aagtcacagc cacactccct 4980ggccacagag accagaaacc caggaggaca ggaaatgaac agaacggagc tgaacaagtt 5040cagccacgtg gattctccaa attcggaatg caagggtgag gacgcgaccg atgaccagtt 5100tgaaagcccc aagaaaaagt ttaaattcaa attccctaag aagcaactcg ccgctctcac 5160tcaagccatt cgcaccggaa ctaaaacagg gaagaagact ttgcaagtgg tagtctatga 5220agaagaggaa gaggatggca ccctgaaaca gcacaaagaa gccaagcgct tcgaaatcgc 5280taggtctcaa cctgaagaca cccctgaaaa cacagtgagg aggcaagagc agcccagcat 5340cgagagtaca tctccgattt caagaactga tgaaattaga aaaaacacct acagaacatt 5400ggatagcctg gagcagacca ttaaacagct cgaaaataca atcagtgaaa tgagtcccaa 5460agccctagtt gatacctcat gttcttccaa cagagattct gttgcaagtt catcccacat 5520agcccaagag gcctctcccc gacccttgct agttccggat gaaggtccca ctgccctaga 5580gccccctacg tcgatacctt cagcttcacg taagggctcc agcggggccc cacagacgag 5640caggatgcct gtccccatga gtgccaagaa cagacccgga accctggaca aacccggcaa 5700gcagtccaaa ctgcaggatc cccgccaata tcgtcaggct aatggaagtg ctaagaaatc 5760tggtggggac tttaagccta cttccccctc cttacctgct tctaagattc cagccctttc 5820tcccagctct gggaaaagca gttctctgcc ctcttctagt ggtgacagct ctaacctccc 5880taatccacct gctactaaac catcgattgc ttctaaccct ctcagccccc aaacaggacc 5940acctgctcac tctgcctccc tcatcccttc tgtctctaat ggctctttga agtttcagag 6000cctcactcat acaggtaaag gtcaccatct ttcattctca ccgcagagtc aaaatggccg 6060agcaccccct cctttgtcat tttcctcctc ccctccttct cctgcctcct ccgtctcact 6120gaatcaaggt gccaagggca ccaggaccat ccatactccc agcctcacca gctacaaggc 6180acagaatgga agttcaagca aagccacccc atccacagca aaagaaacct cttaaaggtc 6240aaatcctatt aggcacaagt cggagttaca tttaaaaaaa attaacagtc tacaacaact 6300gttttcacaa gagaatgtaa catattgctg tatcgtttga ggcttaatgc taaatatgtg 6360ctaaatactg gattaataga tttcagtaaa gctcgttcgt tttgtttggt tttcttttta 6420cctagttgct atagtgtcta cagtctatac tcaataccta taaaatgcag taagcatgtg 6480ttacagaaag aggttctggt gggagagaaa ggtgcgtgtg agacaggaga attgtcttaa 6540gcatataaaa catgtatgat tccagaattt tagtatgttt tgtataaaac tatttttcat 6600tacggagact agaagtgaac agagaattac acaagtgtga ctatacaaat tgtaaaacag 6660atactataat atttcctttt attttagtgt tatttagctt tattacagat ttctattttt 6720gtcaaaactt catggttcct ttcaagatct tttttgccaa aacattttga tactatagca 6780ttgtacattt gaaagtagtg ttctagacta taaaaccaat gaacttctac atgagcccta 6840cagacaggca tgtgtagaag gcaatttatc aaacctattg cactgccatg aaaagtgtgt 6900ataataattt gctagcccaa gcaagctagt tttctttgct tgcttctttt ctttcttttt 6960tccttccttt tttttttttt tttctttttt aacatgttga gattctctag ttgttttctt 7020tggcgtatct aaccccttct tttgttttct gagacctggt aacccacgct cttgcattgt 7080ggattttaaa atgtatactc tgtacggttc tgtaaaccga aaaacttttg taaatatata 7140aatatacata gacataaaaa tactgtatgt gacagcacat agagtagttt tcccacacca 7200aagttaattt ttatgcatgc tttaaaagta tatatcggga ccggcagaaa tggaagtatc 7260catacatttt taaaaagcaa caagtttgca cagctagagt gtttttgtaa ataaatgtat 7320ttgtataaca cagtcatgta atatacagaa ctataagcag agactttgca aaactaaata 7380aagggctgca tgcttattat tttttgtacc ttgtcactat aactacttcc tagtcaaaga 7440acgaaatgta actgttaccg agttaaatgt ttttccgctt tgagggatgt aaccacatcc 7500actcagagga cactactttt ctgaaagctc tggggtgact aatgatgagt tcctaataaa 7560ttaattgcaa gtgtggtgcc ttggatgtgg cctgttggct cgctttcttc tctgtggctt 7620atcaaggtgt agatgacaga aagcaaacct ggatacagag tttccaccct cagttcctgg 7680aggggctctt attattttct ctctttttaa aaaacttcca gtagaagtaa agtggaaata 7740aaatgtcttt atcaaaaaaa aaaaaaaaaa aaa 77731710264DNAHomo sapiens 17ggagccgccg ggaggtaggc gcgggacggg cggctgcggt ggcggcgggt ggaccgattg 60tcgctcggcg gcgggagtcg gtggagtctc gctctttcgc aaggctggag tgcaatggca 120tgatctcggc tcactgcagc ctccgcctcc tgggttcaag tgattcttct gcctcagcct 180cccgagtagc tgggactaca gaaatgctgg taatttcagt cacttgctga tgacttctga 240tgtcccagca aatgttggca ttgttgactg ctgagaagat acttctgagg acccacgtct 300aaggtggact tggttcgcac tcttctggtt agtccctgaa caggagtgat gcctccaggc 360aggtggcatg ctgcctatcc agctcaggcc cagtcttcga gggagcgagg gcggcttcag 420acagtaaaga aggaagaaga ggatgaaagc tatactccag tgcaggctgc caggccacag 480actctcaacc gccctggcca ggagctgttc cgccagctct tcagacagct tcgctaccat 540gagtcttcag ggcccctaga aactctgagc cggctccggg aactctgtcg ctggtggctg 600aggcctgacg ttctctccaa ggcacagatc ctagagctgc tggtgctgga acagttcctg 660agcatcctgc ctggggagct ccgggtttgg gtgcagcttc ataaccctga gagtggcgag 720gaggctgtgg ccttgctgga ggagctgcag agggaccttg atgggacatc ctggagggac 780ccgggccctg cccagagccc agatgtgcat tggatgggta caggagccct gcgatctgca 840cagatatggt cccttgcttc acctctcagg agcagctctg ctctggggga ccacctggag 900cctccctatg aaatagaagc acgtgacttc ctggctgggc aatccgatac tcctgctgcc 960cagatgcctg cccttttccc gagagagggg tgcccgggag accaggtaac accaaccagg 1020tccctgacag cccagctcca ggagaccatg actttcaagg atgtggaggt gaccttctcc 1080caggacgagt gggggtggct ggactctgct cagaggaacc tgtacaggga tgtgatgctg 1140gagaattata ggaacatggc ttccctggtg ggaccattca ccaaacctgc tctgatctcc 1200tggttggaag caagggagcc atggggcctg aacatgcagg cagctcagcc taaggggaat 1260ccagttgctg ctcctacagg agatgacctc cagagtaaaa caaacaaatt catcttaaat 1320caggaacctt tggaagaagc agaaacctta gctgtgtcat caggatgtcc tgcgacaagt 1380gtttctgagg gaattgggct cagagaatct tttcaacaga agagcaggca gaaggatcaa 1440tgtgaaaatc ccatacaagt aagagttaag aaagaagaga ccaatttcag tcacaggaca 1500ggaaaagact ctgaagtatc aggaagtaat agtcttgact taaaacatgt tacatatttg 1560agagtttctg gaagaaagga atcccttaaa catggctgtg gcaaacactt cagaatgagt 1620tcacaccact atgactacaa gaaatatggg aaggggctca gacacatgat tgggggcttc 1680agcctacatc agagaattca tagtggactg aaagggaaca aaaaggacgt gtgtggaaaa 1740gacttcagcc ttagctctca tcaccaacgt gggcagagtc ttcacacagt gggagtgtca 1800tttaagtgca gtgactgtgg aaggactttc agtcatagct cccatcttgc gtatcatcag 1860agacttcaca ctcaagagaa agcatttaaa tgtagggtgt gtgggaaagc cttccggtgg 1920agttccaact gtgcgcggca tgagaaaatt cacactggag tgaagcctta taaatgcgat 1980ttatgtgaga aagctttccg acgcctgtca gcctaccgtc tgcaccgaga aacccatgct 2040aagaagaaat ttcttgaatt gaatcagtat agggcagctc tcacctacag ctcagggttt 2100gatcatcatt tgggagacca aagtggggag aaactctttg actgcagcca gtgcaggaaa 2160tccttccact gtaagtcata tgttcttgaa catcaaagga ttcacaccca ggagaagccc 2220tataaatgta ccaaatgtag gaaaaccttt agatggagat caaactttac tcgtcatatg 2280aggttgcatg aggaggaaaa attctacaaa caagatgaat gtcgtgaagg cttcaggcaa 2340tctcctgact gcagtcagcc ccagggtgct cccgctgtgg agaaaacatt tctgtgtcag 2400cagtgtggga aaacttttac tagaaagaaa actctcgttg accaccagag aattcacaca 2460ggtgagaaac cttaccagtg tagcgattgt gggaaggact ttgcctatag gtcagccttt 2520attgttcata agaagaagca tgccatgaaa agaaaacctg agggcgggcc atcttttagt 2580caggacacag tgttccaggt tcctcagagc agtcactcca aagaggagcc ctacaaatgc 2640agccagtgtg gcaaggcctt ccgcaatcac tcattcctcc tcatccatca gagagttcac 2700actggagaga agccatataa gtgcagggag tgtgggaaag ccttcagatg gagttccaat 2760ctctaccgac atcagaggat tcactctctt caaaaacagt atgattgcca tgaaagtgaa 2820aagactccaa atgtggagcc aaaaatcctc actggtgaga aacgtttttg gtgtcaagaa 2880tgtgggaaaa cctttacacg taaaagaacc cttttagatc ataagggaat acacagtgga 2940gagaagcgct ataaatgtaa tctatgtggg aaatcttatg atagaaacta tcgccttgtt 3000aaccatcaga ggatccactc tacagagaga cctttcaaat gtcagtggtg tgggaaagag 3060ttcattggga gacataccct ttccagtcac cagaggaaac acaccagagc agcacaggct 3120gaacgtagcc cgcctgcacg gtcttcctct caggacacaa agttgagatt acagaagcta 3180aaaccaagtg aagagatgcc cctcgaagac tgcaaagaag cttgcagcca gagctccagg 3240ctcactggac tccaggacat aagcattggg aaaaagtgcc acaaatgcag catatgtggg 3300aaaactttta acaagagttc acaactcatt agccacaaga gatttcatac tcgagagagg 3360cccttcaaat gcagcaagtg tggaaagacc ttcaggtggt cttcgaacct ggctcggcat 3420atgaaaaacc atattagaga ttagcctggg acctgacagt gacagtgggg gtgggttctc 3480agtcccctgc tagagaaccc ttaattatag gcattgtgga gtaactttga taaagggccc 3540agccctttct gttttggaag ctagtgacag aatcccaagg atttgaaagc tcggggagtc 3600cccagcctgc ctgctaggat gtgacgctgg ggaagtgcag caccatgtcc tttggagccc 3660ttctggagac tccggcccct aggagtggcc tctgcaccat agcctgcggc tcccctattc 3720aggtctcctt ccacaactct gaagagagag accactgccc tttgtggttg gacagaatat 3780ctgtggcatc atgggctatg gctgctggaa aggggccagt gggatcctag atttgtcttc 3840aagtttggcc tgtggccatg cctattctgt tgactttaaa agcagcagca tcaagaactc 3900ctagccttcc caaatgcccc ctggggagtt ctggctgggg cttcagcctt cctggctggc 3960ttttggatat ctgctagggg gttagagtgg tctcagcggc aggtggagga gagcaggatg 4020ctgggctcaa gcgcttggcg tgtggatctc taccagtacc ctgttgccat ccccatccca 4080caggcctgca taggcagcag cggtccatct gtttaacaga aatgtgctga gcactcccat 4140acaccaggcg ctggtgtgtt tgccagagac ccagcaggga ccagaacaga tgaaaatcct 4200gccatcttgg atctttacag atgacaaaca aatgggatac gcagtattct ggatggtgat 4260gtgtgtagtg gacaaagtga atcagagaag agcagaggga gtgctggatt gggagatcag 4320tcgtgatttt agatagtctg gtcagggagg gcctcaactg agaaggtgaa atgtgagcaa 4380agacgtgaag gaagccaggg aggtagcagg tagctctcta gggagagttg caggcagagg 4440agattgggtc ttgactggtg tcctcaggaa ggaagcccct gggatggagt gcagggagcc 4500agctggaagg gaaggagagg ggccagggag gccacaggcc cggccatatg agcatcatgg 4560gtcattacgg gggcttgagc tggctcagag ggaggcaggg agcttttgga caattttgag 4620ttgaggagtg atgtggtttg aatctgaatt atattttata agggttaacc acagatccaa 4680ggactcagta aaagggtgaa aggcttctca tgcagttagc cttgctgcca gttcccataa 4740atcagactcc tccccactcc gtttctgaat tttctatagg atcctcggcc tcagcagata 4800taaacgctta ccattgttgt gttcatgtct ctagcccctg cacactgcag gcccctctca 4860gcttcacata cccctctcca tgtatgagat ctggtgctag caatcagatt caacccgaag 4920gaacaagtgc atgtgcctgc tgctctcagg agaaaaggca gcctgttctg ggaggagctc 4980cctcactggc ccacccactt gtgttgaagc aaaaagcaag aatcaattac ttaaaggcaa 5040aggagtgata ttgctaaagc atgtgagtaa tcattgcttc tgattattga agaggtcaga 5100ttatgaatga aaggataaaa tgttttaata gtagagcagt gtcctgacta taggggcccg 5160ttcatggagc tggtcatttc ttgaaatgag aagaaaccaa cctggaagat gtctttttca 5220gtagagtatg gaataatctt acctgctctg gattgccatg agcaggtgaa aatgaaggct 5280ctgatattgt gtggtggtgt cacaggatgg tgggggcggg gcagaagtct cctgttggtg 5340ttctgatgcc tgttcagctt tgtcctcagg aaggtttggt catcttggtt ctgccatcct 5400cttgccttcc tacggcagca gctgttcatg gcactgaaga tgtcccttca ctgtcccaaa 5460actggcacta gaagtggctg ggcagggggg ccagagtcat aggaagggtc ttggagtcat 5520cagatctggg ttccaggcat ggtgtgcctt ttcttggcag gattgatctt gagcaagtca 5580atgaacctct gcatcaattt ccttatgtgt gaagttagag tgcttggctg tgctacctga 5640ttcctgagat tggtgaaaga gggcttggtt taccaaggct ctccatcagg tggttgacct 5700ctaggaccag ggccctctca gaggtgcagc ttccatgtca gttggtgttg taaccatgga 5760aaagtccctt tccaatcaaa cttttagcac ccctttcccc ccatctctgc tccctttgcc 5820acaggatacc ttgtaaagtt cctgccccag tatagggata tttttactgt cttttttatt 5880ttttgagaca gggttgccca gggtggagtg cagtggcagg atcacagctt actgcagcct 5940tgacctccca ggctcaggtc ccatctgagc ctcccgagta gctggggcta caggtgtgtg 6000ccaccctgcc agctaatttt ttatattttt ttgtagagac aaaactttta tttttttttc 6060ccatgttgct catgctggtc tcaaactcct gggctcaagc aatccaccca cctctgcctc 6120ccaaagtgct gggattacag gcgtgagcca ctgtgcctga cctgtcattt ttatagaatg 6180aaatgtgctt atttctaaac attctaatca aattttaaaa tgtgcaaaga atgaagtgaa 6240aaactgaatt tttttttttt tttgatacgg agtctcactc tgttacccag gctagagtgc 6300agtggcgcga tctccgctca ctgcaagctc tgcctcctgg gctcacacca ttctcctgcc 6360tcagcctcct cagtagctgg gactacaagt gcctgccacc acgcctgggt tattttttat 6420atttttagta gagacggggt ttcactgtgt tagccaggat ggtctcgatc tccaggatgg 6480tctcgatctc caggatggtc tcgatctcct gacgtcgtga tccacccgcc tcggcctccc 6540aaaatgctgg gattacaggt gtgagccact gtgcccggcc aaaagaacag aaattatttt 6600atcctgaagt aagctgttta tatttgggat tatactgaac ctatttgtcc aataacctga 6660gttttcaaat aattttagtt ctataagtac tataattata taaatattaa tgaattcaga 6720ttagctgaaa ggaaaaaaag tagaagcctg actacttggt gctaactact aaagattttg 6780gcagaatcaa tgttggattt ggctttcctg tcccttcccc atgccagccc cccagagtgt 6840tctgccttgt gctgcctccc ttcacctgga gtgccacacc cctctctctg ccagttcagc 6900tcttcattct tcaaggcctg accttgtctg acccttgtgc ctctaaaccc gtggccccac 6960ctctcttggt tcctatgtca ggtgatgttt gtgtttttgg ttatgcccat ctccatagcc 7020agaccaagca ctctggaagc cagggttggg tgcttattta tctgtttgcc atgcagaaaa 7080tatcttgcac aaaattacct ctgttaagga atctgaagct gaatttagtt tggctgagtc 7140agggttgggt tttttttaag gggctgtggg gtgaaatgtt gactggaagc cacccacaaa 7200cacacacctg ctggttagga acccggctgt gggtggttct gagctgtttg gcttcagttg 7260acagtttctg attgccctga gcaccaggtc tcatcttgca tctcatcctg gcctggagaa 7320cattcagttt ccttccaacc cttcccacct ttcccccact cccttggagg aactgaagtt 7380ggggttgagg agagccagat ggctggagtg ggtatttgaa ggtctttctg tcacctgttc 7440agtgtggtct gccccacccc tgctgaccaa gactgactga aatgtaaaat aatacagacc 7500atctcacact cagaaagctg gcacattttt gaaagcccaa gtgtgggtaa gtgcgtggaa 7560caacgataat tcacactgct ttatgagtag aaattgtgag aaatattgtg ccaggcaatt 7620tgcaaaatct tggaaggttg

tgtgcactta accacccagc aactactcct ggatgcatcc 7680tagagaagtg ccatgtgaac agagaatgat tttaagactt cactgaagta ttgtttaggt 7740agcaagattg ggaaaagcct gcatttcatc agcagaagaa tggataaata aatgggttgt 7800ttttggtcct tggaaagtga atatgaaaga gttacgtctc aacacagata gatgaaaaat 7860tatgctgaga aagttggtga agctacatac aaggtaccct tagtgtaaag ttaagcatac 7920tgtgtacctg tgggcacgtt acttcaactt gtttttcact ttttctgtaa aatgggatag 7980tagtggcaat ctcacagggt gattgtgggt gggggggtgg tcaatgaagt aatgcatgta 8040aaatgcttag aatagtgtct agcatgtaag ccttgtggac atatagaaag tgttattgtt 8100ttgcacagta atctattttc tgtggattca aataatatga aatgagtata aaatcatgta 8160ttggaacgat gtgtgcaagt caccattctg ccttcctaag gcaggagacc tgatggattt 8220ggggagggta catggggcct tcagttgtgt tttctttgtt tttttctaaa aattgatgca 8280gaggcatcac aatgttaaga tttttacagg gtagtgtggt gggtactttt taactgtttg 8340cttaaagtgt ttcaaagtaa aaatatttct taagcatagt ctctgacatc ttatgacttt 8400atagaaatcc ctgccaggta catgatgtta cttagagtga gccacctaat ctttcaaggc 8460ttccatttcc tcacctgcaa aaattgggag tgtcagaggc gtttgaacca gagtgactcc 8520atcttgaata gggaactggg taaaataagg ctgagaccta ctgggctgca ttcccaggag 8580gttaggcatt ccaagtcatg ggatgagata ggaggtcagc acaagataca gtcataaaga 8640ccttgccaat aaaacagcat gtggtaaaga agccggtcaa aaccaagatg gcaatgaaag 8700tgacctctgg ttgtcctcac tgctgtaatt atactgtaat tataatgcat tagcatgcta 8760acagacactc ccaccagtgc catgacagtt tacaaacgcc atggcaggcc tggcacgatg 8820gctcatgcct gtaatcccag cactttggga ggccaaggcg ggcggattat gaggtcagga 8880gattgagacc agcctggcca acatggtgaa accctgtctc tactaaaaaa tacaaaaatt 8940agctgggcat ggtggcacgt gcctgtaatc ccagctactt gagaggctga ggcaggagaa 9000tctctagatc ccgggagttg gaggttgcag tgagccaaga tggtgccact gcactccagc 9060ctgggtgaca gagcaagact ctgtctcaaa aaaaaaaaaa aaaaatgcca tggcaacatc 9120aggaagtaac actatatggt ctaaaaagga gaggaaccct cagttctggt aattgctcac 9180acctttcccg ggaaacttat gaataatcta ccccttgttt agcatataat caataaataa 9240ccataaaaat aggcaaccag cagcccttgg ggctactctt cctatggagt agccattctg 9300tatttcttta ctttcttaat attaactact ttcactttgt ggactcgccc gaattctttt 9360cttgagcaag gtccaagaac tctctcttgg ggactggatc acgaccgctt tctggtaaca 9420ggagcaatac aaatggctat tagtaaacga acgcttaagt aactctagta catctatgat 9480gaaataccag gccacagttt ttttaaaaat gaggatgttc tctatatact gacgcacagt 9540caccaagaca tgcactttat aggaaattat aagctcctcc ttgcctaaat ctccttaatt 9600ttgcttaata aataacaagt gaagccagaa gctgtaaact cataatttta atgaagaaat 9660gggatccaga agatggagat gtttttcatt ctactttatt agatgaggaa aaggagagta 9720aatacttgta tgccagctac cacacctttt aaagttaatg ttaaccctgc taagaacagc 9780aaggagattt tatatcagcc aggttttagt tgccagaaac cacaccagtt attttagcaa 9840agaccttata aagaagtgta ctatatttaa gtagctaaaa aggcaagaag aaaatgccaa 9900ggtgtcttgg aggtagcaac tgctaagcag ctgccacccc aggactgggg gagctagaat 9960agagtctccc catgtttgac gtttgtctgc taatagcatt caagctctcc ccctcagttt 10020gcctggatct ggacaagctg ataagaaagc ccagccactt cctctttgcg tcagagggga 10080agttcaaact gtgcaaaccc tgactgtcaa atgcactgag ctctcataac cacaacaaaa 10140accagagcca cttgttcgtc ctttcactga agcccaacag actggcgtgg gtgcccgctt 10200tgctttccct agaaggcctc atgtgagtag taaacttttt cataacctca aaaaaaaaaa 10260aaaa 10264181330DNAHomo sapiens 18aaaaagaact gcttctcttt ctttccccct ccaagttcct agtggagggc tgagtccagc 60atcccagact cgtgtgacta tataggcaag catttgggga cctacttcac tttgataccc 120tagccttcag cagctcaagg tgttggcctt tggataggag gcttccaagt agtaaagctc 180cctgctctca gcaagcccaa caccatgggg aagggagatg tcttagaggc agcaccaacc 240accacagcct accattccct catggatgaa tatggttatg aggtgggcaa ggccattggc 300catggctcct atgggtcggt atatgaggct ttctacacaa agcagaaggt tatggtggca 360gtcaagatca tctcaaagaa gaaggcctct gatgactatc ttaacaagtt cctgccccgt 420gaaatacagg taatgaaagt cttgcggcac aagtacctca tcaacttcta tcgggccatt 480gagagcacat ctcgagtata catcattctg gaactggctc agggtggtga tgtccttgaa 540tggatccagc gctacggggc ctgctctgag ccccttgctg gcaagtggtt ctcccagctg 600accctgggca ttgcctacct gcacagcaag agcatcgtgc accgcctgat gcccagcctt 660tctgctgctg gtagggactt aaagttggag aacctgttgc tggacaagtg ggagaatgtg 720aagatatcag actttggctt tgccaagatg gtgccttcta accagcctgt gggttgtagc 780ccttcttacc gccaagtgaa ctgcttttcc cacctcagcc agacttactg tggcagcttt 840gcttacgctt gcccagagat cttacgaggc ttgccctaca accctttcct gtctgacacc 900tggagcatgg gcgtcatcct ttacactcta gtggtcgccc atctgccctt tgatgacacc 960aatctcaaaa agctgctaag agagactcag aaggaggtca ctttcccagc taaccatacc 1020atctcccagg agtgcaagaa cctgatcctc cagatgctac gccaagccac taagcgtgcc 1080accattctgg acatcatcaa ggattcctgg gtgctcaagt tccagcctga gcaacccacc 1140catgagatca ggctgcttga ggccatgtgc cagctccaca acaccactaa acagcaccaa 1200tccttgcaaa ttacgacctg aaaatggctg agggaggggg ctaagagagg agcaaagcag 1260gaggtcttgg gctaaaaatc ttttttacca aaaataaatc taagtctgat ttagtttcat 1320caaaaaaaaa 1330192779DNAHomo sapiens 19ccggggaccg tttgtagtta ggatccgctg tggcgtcctg agtggagttt gggaccccag 60ggagggaggg tgtgggcgtt cgggtccaga ggagctgttt agtatccaag atgaatgaca 120gcctgtttgt cagtttggac agacttttgc tagaatttgt cttccagtat gagcaagaca 180taagtactaa agaagagatg attcaaagaa ttaataaatg ctgtgaagat attaaggaaa 240acaaagtaac tatttgtagg atacacgaaa ctataaatgc aacagatgag gaaattgatc 300attactgtaa acatagtgag gagattaaag acaactgtag aaactggaag ccaacatgtg 360atgtttttcg taaacatgaa gattatatgc aggaccaatt tactgtttat caaggaactg 420ttgaaaaaga caaagaaatg tatcatgatt atatatgtca gtataaagaa gttttgaagc 480agtaccaact aaaatactca gaaacaccct tttcacgtga atattatgag aagaaaagag 540aacatgaaga aattcaaagc agagtgttgg catgtactga acaattaaaa atgaatgaaa 600caatttttat gaaatttcga gtgcctgctc cctttccatc acttactaaa tggactttaa 660acattgttaa tttgagatgt gaaacacaag atattcttaa acatgccagc aatcttacca 720aaagttcatc cgaattgaag aaagaagtag atgaaatgga aatagaaatt aattatttaa 780accagcagat atctaggcat aatgaaacta aggctctttc agaaactctg gaagaaaaga 840acaaaaatac agaaaacaga aaagaactga aagaaagaat ttttggaaaa gatgagcatg 900tacttacatt gaataaaact caaagcagtc aattatttct tccttatgaa tctcagaaat 960tagtaagacc aataaagatg cattcttcag aaccaagagt tgcagatata aaagaagaaa 1020gttctgcgaa gcagtcaaag cttgccaata ttgactttag acaaaaagaa aatgatacac 1080agatatttaa tgactctgct gtggataacc attcaaaatg ttcacatatt acgactatca 1140caagttcaca aaagtttatg caagtcagat tgttaacccc acagaaacaa tcaaattcca 1200atcagtggtc ggaaaaaggg gataaagatg ctgagtatgg agataaaggg acagtaagac 1260aagtaagaga atcaaaatgt acttcacaag ctatatatac tgaacatttt gggaagtcaa 1320tagaaaatga tagtgatgaa gtagaagaga gagctgagaa ttttccacga acgtctgaaa 1380ttcctatatt tttaggaact cccaaagctg tgaaagcacc tgagtcattg gagaaaataa 1440aattccctaa aacccccccg ttcgaaatta acagaaatag aaatgcagta cctgaagttc 1500aaacagaaaa ggaatcccct ggactttctt ttcttatgag ttatacttct agatcacctg 1560gattgaattt atttgattct tctgtatttg atacagaaat ctcatcagat cagtttaatg 1620aacattattc tgcaagaaat ctaaatcctc tgtcatcaga gcaagagatt ggaaacttac 1680ttgagaagcc agaaggagaa gatggcttta cattttcttt tccatcagac acttcaactc 1740atacatttgg agctggaaaa gatgatttta gttttccatt ttcatttgga cagggtcaaa 1800attcaatacc ttcttcttct ttaaaaggtt tttcatcttc ctcacaaaat acaacacagt 1860ttactttttt ttgagctagt cattaattcc ttaaattatt ttactgttct gtgttcatga 1920gggcataaat ttacattatt gcttaaaaca tgaagactgc tttcttttat tgattaaagc 1980agtaatgttt acattatttg attatattta ttgaaatatt gaaatactga atattttggg 2040ttttgtgtgt gctattaact aatcattatt tattttggtt ttgattttgc gagccgtggt 2100caggtagaac ttttattaat cttaatagaa tttgatgctt ttttcattac tctttattta 2160aatattaagc ctgcttctcc ttggaaccta aggttttttt ctggaagtat tgttggtact 2220ttgataagaa caagaactgc agtagtaact ccagagttag tgctgaagcg tactttagct 2280actaaaaatt tctattaaaa ttattgggtt tcacttctgc ttcactatgt agtatacaga 2340gtggtactgt aataataatt tcaaataatt tatgttaata acaaaatctg tgttattttc 2400ttctaatata acacatggta caattctaat tttatgagtt atgctaatgc tttcaatggc 2460taaaaattaa atgtaaaggg caagagtaat ttctgaaaat tggattgttg tatcagtggt 2520gatcctgtta atattctttt ttgcttaaat attttttgaa gaacatttac aattttgtct 2580ccttcaataa caaaaatttc ttctttatgt tttgtgttca gtatttgtca attaattata 2640tagcttaagt gaagatattt aagatttgat gaacttctgt aaacattttg ctcaatatca 2700ttgtattttg tgctttgtaa attagctgta ctgagttacc aagtaataaa gggtttgact 2760ccaaaaaaaa aaaaaaaaa 2779204144DNAHomo sapiens 20ggaccggaag tggaagtggt cttccaaggc ttttttgccg ctggtgtcag gagtattttc 60atattccaat accgataaat ctttgaggtt tctgggtgtc tctggggagc ccctgggcca 120gattttcctc tagactccag cccatctctt cagagcagct ctgcttgagt tcacagatga 180ctgccaagct tcagacaccc tacagaaaaa gggttgagac ccagtgtggc catgccagct 240aattggacct cacctcagaa atcctcagcc ctggctccag aggatcatgg cagctcctat 300gagtgttaac cttgatgcct gaaagaactg gaaattatga agatagattc agaagtcaaa 360tatgttaact aactgcattg aagagtagaa gaaaacaata gcctaggatc agtgtccttc 420agggatgtgg ctatcgattt cagcagagag gaatggcggc acctggaccc ttctcagaga 480aacctgtacc gggatgtgat gctggagacc tacagccacc tgctctcagt aggatatcaa 540gttcctgaag cagaggtggt catgttggag caaggaaagg aaccatgggc actgcagggt 600gagaggccac gtcagagctg cccaggagag aaattatggg accataatca atgtagaaaa 660atcctcagtt ataaacaagt atcctctcaa cctcaaaaaa tgtatcctgg ggagaaagct 720tatgaatgcg ccaaatttga aaagatattc acccagaagt cacagctcaa agtacacctg 780aaagttcttg caggagaaaa gctctatgta tgcattgaat gtgggaaggc ttttgtacag 840aagccagaat ttattataca ccagaaaacc catatgagag agaaaccctt taaatgcaat 900gaatgtggaa aatccttttt tcaagtgtcg tccctcttca ggcatcagag aattcatacc 960ggagagaaac tctatgaatg cagccagtgt gggaaaggct tctcttataa ctcagatctc 1020agtatacatg agaaaattca tactggagag agacaccatg aatgcactga ctgtggcaaa 1080gcattcacac aaaagtccac actcaagatg catcagaaaa tccatacagg cgagagatcc 1140tacatctgta ttgaatgcgg acaggccttc atccagaaga cccatttgat tgcacaccga 1200agaattcata ctggagaaaa accatatgag tgcagtaact gtggcaaatc cttcatttcc 1260aagtcacaac ttcaggtaca tcaacgtgtt cacacaagag tgaagcccta tatatgtacc 1320gaatatggga aggtcttcag caataattcc aacctcgtta cacataagaa agttcaaagt 1380agagagaaat cttccatatg tactgagtgt gggaaggcct ttacctacag gtcagagttg 1440attattcatc agagaattca cactggagag aaaccttatg aatgcagtga ctgtgggaaa 1500gccttcactc agaagtcagc actcacagtg catcagagaa ttcatacagg agaaaaatcg 1560tatatatgca tgaaatgtgg actggccttc attcagaagg cacacttgat tgcacatcaa 1620ataattcata ctggagagaa acctcataaa tgtggtcact gtgggaaatt gtttacctcc 1680aagtcgcaac tccatgttca taaacgaatt cacacaggag aaaagcccta tatgtgcaat 1740aaatgtggga aggcattcac caaccggtca aatctcatta cacatcagaa aactcataca 1800ggagagaaat cttatatatg ttccaaatgt ggaaaggcct tcacccagag gtcagacttg 1860attacacatc agagaatcca tactggggag aagccttatg aatgcaatac ttgtggaaaa 1920gccttcactc agaagtcaca cctcaatata catcagaaaa ttcacactgg agagagacag 1980tatgaatgcc acgaatgtgg gaaagccttc aaccagaaat caatactcat tgttcatcag 2040aaaattcata caggagagaa accctatgta tgcactgagt gtggaagagc tttcatccgc 2100aagtcaaact ttattactca tcaaagaatt catactggag agaagcctta tgaatgcagt 2160gactgtggga agtcctttac ctccaagtct cagctcctgg tgcatcagcc agttcacaca 2220ggagagaaac cctatgtgtg tgccgagtgc gggaaggcct ttagtggcag gtcaaatctc 2280agtaagcacc agaaaactca taccggagaa aagccctaca tttgttctga atgtgggaag 2340acctttcgac agaagtcaga gttgattaca catcacagaa ttcatactgg agagaaacct 2400tatgagtgca gtgactgtgg gaagtctttc actaaaaaat cacagctcca agtgcatcag 2460cgaattcaca ctggagagaa gccttacgtg tgtgctgagt gtgggaaggc ctttactgac 2520aggtccaatt tgaataaaca tcagacaaca cacactggag acaaacccta caagtgtggc 2580atctgtggga aaggcttcgt tcagaaatca gtgttcagcg tccatcagag cagccacgct 2640tgagagaaac agtgtgagaa aacccccctg agggttgggt ctgattgtac actgttgcac 2700gcatgcagca gaaaaatatg tatattattg taaatagaaa tgaccacagc agaatgtcac 2760acatggctgt tctggagagg gcctctgaga aggcactgaa tgaggcgagg gacccttcct 2820acattgtcac catccccagt aaaccttggg tcattattca tactgacaag gaaccgagtc 2880aatttggtga ataggaaaag ccttctcatg aaaactacaa tagaatactg ttaccaaatt 2940cttcctaaga aagatcgtat taagttaacg ataatcctgt ttactgtgga ttaggtatag 3000tgccaacaaa ttgaatgata aaacaacata atacgtagtt attttgatag tgatgaatcc 3060taagttatgt gagttgttcg ttgtggaaca cattgtgtaa cagactcctg ggtgtttttc 3120tttctcattc gaatctacca cagttggtca tatccaaccc tcattcagta tttctatcaa 3180gaaagagatg ctacaaaaaa aaaaagaaaa aacctttatg tatacagacg taaacctcag 3240aatgtatgtt gagtccccac tgtcatctac caagacttgc acccccctca ttatctacca 3300tgactgtctc tcagcctcac gggccctcag cactttgtgt tttgaccccc agcaccgtgt 3360cttgtgaact cccatcacct tcaagaaagc ttccgaggta agaattttat ggtcatctgg 3420gacaacttaa atctcccttc tgctgtcata gttcttccaa ctcagttgcc tttttttttt 3480tttcgtactc atcactgact tgaagcttag tatctggctt ccttaaggat gtaactttca 3540tgtaacagat taataactta tatgaaaacc aacacaacca tatgtttagg gctggaaagg 3600gccatgacgc ctggccattt ttcctgtttt accttactct tatgtgtgtc acacttcatc 3660aattccggaa acagtttctg gagatctcct cattacctct tttacaatca cctcactcca 3720gcatggtgtc tgttacctct tcccacttgt gacaatgtct agtaaggtcc actctccatt 3780ctgtgtgatg accacttatt acaaccctca gaatagggga cagtggtgtg ccccctgcaa 3840tacaatggtt tctatctcct gatactttta ttacacctct agcaggatgt cttgtgatcc 3900tccttattga tttttccctc acgatgatga acaattatct cccgttactc acctagcagt 3960atctaactgt ccctaacaca gcatgtggga atgccctcaa tacggtggat gctgttaact 4020ttcttccttc ccctcaggca atggcggtga cttacaatga accataatgt ccacatttcc 4080caactgtatt ttggagcctc ttctgtcccc ttctttctag gaccccagtt aaaaaaaaaa 4140aaaa 4144211642DNAHomo sapiens 21acagagtagc tttgcaactg gctttgggga cttccgaaag ctaccagcac tgcactgtga 60gactctcatc cctgagctga attcatctga ttcgacggca agctttggtg agaacataga 120tatatttctg aggaaaatgg actcagactt ctcacatgcc ttccagaagg aactcacctg 180cgtcatctgt ttgaactacc tggtagaccc tgtcaccatc tgctgtgggc acagcttctg 240taggccctgt ctctgccttt cgtgggagga agcccaaagt cctgcaaact gccctgcatg 300cagggaacca tcaccgaaaa tggacttcaa aaccaatatt cttctgaaga atttagtgac 360cattgccaga aaagccagtc tctggcaatt cctgagctct gagaaacaaa tatgtgggac 420ccataggcaa acaaagaaga tgttctgtga catggacaag agtctcctct gcttgctgtg 480ctccaactct caggagcacg gggctcacaa acaccatccc atcgaagagg cagctgagga 540acaccgggag aaactcttaa agcaaatgag gattttatgg aaaaagattc aagaaaatca 600gagaaatcta tatgaggagg gaagaacagc cttcctctgg aggggcaatg tggttttacg 660ggcacagatg atcaggaatg agtataggaa gctgcatccg gttctccata aggaagaaaa 720acaacattta gagagactga acaaggaata ccaagagatt tttcagcaac tccagagaag 780ttgggtcaaa atggatcaaa agagtaaaca cttgaaagaa atgtatcagg aactaatgga 840aatgtgtcat aaaccagatg tggagctgct ccaggatttg ggagacatcg tggcaaggag 900tgagtccgtg ctgctgcaca tgccccagcc tgtgaatcca gagctcactg caggacccat 960cactggactg gtgtacaggc tcaaccgctt ccgagtggaa atttccttcc attttgaagt 1020aaccaatcac aatatcaggc tctttgagga tgtgagaagt tggatgttta gacgtggacc 1080tttgaattct gacagatctg actattttgc tgcatgggga gccagggtct tctcctttgg 1140gaaacactac tgggagctgg atgtggacaa ctcttgtgac tgggctctgg gagtctgtaa 1200caactcctgg ataaggaaga atagcacaat ggttaactct gaggacatat ttcttctttt 1260gtgtctgaag gtggataatc atttcaatct cttgaccacc tccccagtgt ttcctcacta 1320catagagaaa cctctgggcc gggttggtgt gtttcttgat tttgaaagtg gaagtgtgag 1380ttttttgaat gtcaccaaga gttccctcat atggagttac ccagctggct ccttaacttt 1440tcctgtcagg cctttctttt acactggcca cagatgatca ggattaagaa aacttactgt 1500ttgggaactc catatacaag ggagcccttc actgttgata caaagaaatc atactgttca 1560ggcttttttg tactttagtg tcacttcatt ttattgctat taaataaaaa atttgtaaaa 1620ggcaaaaaaa aaaaaaaaaa aa 1642225030DNAHomo sapiens 22agtggagggg agggggtaag tgatagggta ggggaggccc tgggaaaggc aggacctcga 60ggcgcggccg cgcgaggtga ccggagtcac agttcccgca ggcggcgaca gcagagcgcc 120cactgcctcc agcaggtaac cggccgcggc cgcgcggcgc cggcccagcg cgggcgtggg 180acgtggagac ccggaggagg gcggcgggac ccgggcgggg aaaggcgcgg cgtggcttgg 240ctcaggtgcg cttctcccac ctggcagctc gctcagggct gtggggggcg cctgtgaggg 300cggccccgcc ttgcgggctg gagattaata ttaagattgg aagtttgtgt cttttgctgg 360atattggaaa ttgaatgtaa tggcaacaga atttataaag agttgctgtg gaggatgttt 420ctatggtgaa acagaaaaac acaacttttc tgtggaaaga gattttaaag cagcagtccc 480aaatagtcaa aatgctacta tctctgtacc tccattgact tctgtttctg taaagcctca 540gcttggctgt actgaggatt atttgctttc caaattacca tctgatggca aagaagtacc 600atttgtggtg cccaagttta agttatctta cattcaaccc aggacacaag aaactccttc 660acatctggaa gaacttgaag gatctgccag agcatctttt ggagatcgaa aggtagaact 720ttccagttca tcccagcacg gacctagcta tgatgtgtat aacccattct atatgtatca 780gcacatttca cctgatttga gtcgacgctt tcctccccgt tcagaagtga cgagactgta 840tggatcggtt tgtgatttaa ggacgaacaa acttcccggt tcccctgggc taagcaaatc 900tatgtttgat cttacaaact catctcagcg attcatccag agacatgatt cattgtccag 960tgtacccagt agttcttctt caaggaaaaa ttctcagggg agtaacagaa gcctggatac 1020aattactcta tcaggagatg aaagggactt tgggagactg aatgtgaaat tgttttataa 1080ttcttcagta gaacagatct ggatcacagt tttacagtgc agagatttaa gttggccctc 1140tagttatgga gacactccta ctgtttctat aaaaggaata cttacattgc ccaaaccagt 1200gcatttcaaa tcttcagcca aggaaggttc caacgctatt gaatttatgg aaacgtttgt 1260atttgctatt aaacttcaaa atctacaaac tgtaagactt gtatttaaga ttcaaaccca 1320gactcccagg aagaaaacca ttggagaatg ctcaatgtca ctcagaaccc ttagcacaca 1380ggaaatggat tactctttgg atataacacc accttcaaaa atttctgttt gccatgcaga 1440acttgaattg gggacttgtt ttcaagcagt aaatagcaga attcagttac aaattcttga 1500ggcacggtac cttccaagct catcaacacc tctgactttg agttttttcg tgaaggtggg 1560aatgtttagc tcgggagagt tgatttataa gaaaaagaca cgcttactga aggcctccaa 1620tggaagagtc aagtggggag agactatgat ttttccactt atacagagtg aaaaagaaat 1680tgtttttctc attaagcttt acagtcgaag ctctgtaaga agaaaacact ttgtgggcca 1740gatttggata agtgaagaca gtaataacat tgaagcagtg aaccagtgga aagagacagt 1800aataaatcca gaaaaggttg ttatcaggtg gcacaaatta aatccatctt gaagacttca 1860cacattaatt tggtgaagaa cttgacattc ttttagaaga cttatgattt caatttgcta 1920ccaatgagaa gaggcaaatc aacaaatttg tcaatttatg ggggctataa ttatggtata 1980taatgtatct gatagaaaat ttgataagaa aatgtaatga attttatcag atatccaaag 2040taaaggaaat gttttaaaac tgcaacaaga gacacagaca gtaaaatcaa agtattatta 2100ggatgactaa ataaattata aagtctgtga gaatatcaac catagatagt tctttctata 2160ttatgttttt gcttttgtat tttaagcttt acttagatat

tcaaaacctg gtatatcaag 2220tctctgttag tactattggc atttagaaga ctttaccatt atttcagtgc taggcattat 2280tgattaggtc ttggctccac tgtttacctc ttgctatgta ttttctcccg gtaaaaatga 2340attgaaccat ttcaactatt ttctatattt ggagaaagtt tgtgccctgt gttttataat 2400ttttttaccc ataagacatc acattatccc tttgtaagct acttatctcc aaaaaacttc 2460agaaatagaa aactacattt tggcaggaat aattgaaaac accagaaggt tgaagtttaa 2520ttggaaaccc agaatataca tactttgctg ttttcttccc tcaaatattt tactatttgt 2580tttatttgga gttaaaataa gagtatcatc catatggtcc atcctaattc acagaattaa 2640atgagcttaa atagaaaatt cagtatttta tgataatcac ttcgttttta gtttttaaaa 2700tttagattat tctataattt accgtgtttg agtattttct catttttttc ataaccatac 2760ctgattatac tgtgtaacaa atattttcta ttgcagtttt ctttccagta cttattagaa 2820ctcagtattt ggaaataatt tcagcttaat tgaccataag aactgtggcc aaaaagaaca 2880gttttttgga gaggcagatg acattatacc tgattttaga aaatctcact ttatttttgc 2940taataagtag actaagtgct ctgtgttctc agtcttccct ttttttctgc ccccattctt 3000actttgtccc aggcatgcag agaaagatgg tgatatttta ggccaggagt ataccttgct 3060ataacctaag catgccttct ttattccagc tcctatgttc tgtgtatatc attaacattt 3120tcccaaataa acacttaatt ctcttttccc taggtgccat ctcctcaagc tacaaaatgt 3180ccacatctta tatccccttt gcttctactg ctctgatttt gtggtaccag tactctctgc 3240cactgaacat tttgaaatat ttttgtttta gatttgcaaa aaatgacata taggtcagta 3300ctcacatgga tttttaagat aaatcacctg tgtgataata ttttgaatct gagacgaata 3360caacttttaa aaattgtttt taaaaataga cttttttttt tagagcagtt gtaggttaac 3420agaaaaattg agaggaagat agagatttcc tttctcccct gacaaagccc tcaacagcct 3480cccaggctat cagtatcctg caccacaatg gtacatttgt tacaatcaat gaacctactc 3540tgaaacatca ttatcatcca aagttcatgg tttacattag agtcccctct tggtgttata 3600catgctagag gacaaatata tgatgatatg tatgcatcat tataatatag tatagtttcg 3660ctgccctaaa catcctctgc aaatgcaact attttaatgg gtaccaaaga agtaaatgta 3720tttactggct tttagataat aaataacggg ctttattgtt tattttaaaa gctacaattt 3780gttttagctg gtttctctgt tctattaatg ctttgaattt ccaaatttaa tatatgtagt 3840catgcattta acttaatatt taattatttg atttatttaa ttttctatat tcttacaatg 3900tatgtatgat gtataattta agggaaagct atgacttctc agtttcttag aatcctaggt 3960aaataaaaca ataaaaagaa aacccttaca tttaaaagag ctttcaggta cagaagtatt 4020gatacaacta agatcctaaa tgttttaatt agtgtttact taagcctttt tcaggtgagg 4080aggtactaat gctggttatt tccttgaagc tttatgtgga cctataaata aaaatccaat 4140ctcctgctaa taggtatgca tattgtgaga aaaacgttag gagctggtag taaaaaatga 4200gattctatgc caaaataact tctcttcata tttgcctagg catttcttga cctttaccca 4260cttacgcaag gagaaggaaa tcataatgat gtcatgtgat caaaggaaac catggaaggg 4320ttcacgctga tagctgatag cttttacagt gctcattcct aacagtggat ttacttgtaa 4380gctttcagat caacacaaat agctgcagcc tgggttaaaa tataacatca ctatttggct 4440tttgttttgc atgattttta aaagcagtac tcctagggaa atggcctctg aagtatatca 4500gtttcatctc ttaccaagac tgttaagaag aaactagtgg gattttgaac aagttatata 4560attgtggtct gaaaagaccc taaactgaag ttctgtttaa atatagttac atgaatttct 4620ctgatactaa tgtactcaac agccaggtat aaactatatc tcctagtaac attttccatt 4680tttgtttaat caaatacttg cttatgaagg atttcagaaa tttgtaataa atgtcagctt 4740ttgatagcat agcagtaatt gacatttcaa aaatatatat ttctttctgt gtttggttgg 4800gtgtaatgag gaaaatacct gataaaatgt ctgaagacac tttctaatgt tatcttggtg 4860cataagctgt aatttttatt caaaattaaa tttcaaatgt ttgcagtttt ggctaaaaca 4920ttgagttgaa agaattatga aaagtgggcc catatgaagt accatgttca ttttgaaata 4980tagatttaag atttagaaat atattaaaag agttaatgga gcctcctaaa 5030232164DNAHomo sapiens 23acccacagca ctcattcctg gagctactgc ttggttccct gagaggtccc agaactctgc 60aaagtgagtc cagcgctgag atttttcttg cagatctatc aggatgagca tccaggcccc 120acccagactc ctggagctgg cagggcagag cctgctgaga gaccaggcct tgtccatctc 180tgccatggag gagctgccca gggtgctcta tctcccactc ttcatggagg ccttccgcag 240gagacacttc cagactctga cggtgatggt gcaggcctgg cccttcacct gcctccctct 300gggatcactg atgaagacgc ttcatttgga gaccttaaaa gcattgctgg aagggcttca 360tatgctgctt acacagaagg atcgccccag gaggtggaaa cttcaagtgc tggatttgcg 420ggatgttgat gagaatttct gggccagatg gcctggagcc tgggccctgt cctgcttccc 480agagaccatg agtaagaggc agacagcaga ggactgtcca aggatgggag agcaccagcc 540cttaaaggtg ttcatagaca tctgcctcaa ggaaataccc caggatgaat gcctgagata 600cctctttcag tgggtttacc aaaggagagg tttagtacac ctgtgctgta gtaagctggt 660caattatcta acgccgatta aacatctcag aaagtcattg aaaataatat acctgaatag 720tattcaacag ctggaaattc gcaacatgtc ctggccacgt ctgataagaa agcttcgttg 780ttacctgaag gagatgaaga atcttcgcaa actcgttttc tccaggtgcc atcattccat 840gtcagataat gaactcgaag gacggttagt caccaaattc agctctgtgt tcctcaggct 900ggaacacctc cagttgctta aaataaaatt gatcaccttc ttcagtgggc acctggaaca 960gctgatcagg tgcctccaga accccttgga gaacttggaa ttaacttatg gctacctatt 1020ggaagaagac atgaagtgtc tctcccagta cccaagcctc ggttacctaa agcatctgaa 1080tctcagctac gtgctgctgt tccgcatcag tcttgaaccc ctcggagctc tgctagagaa 1140aattgctgcc tctctcgaaa ccctcatctt ggagggctgt cagatccact actcccaact 1200cagtgccatc ctgcctggcc tgagccactg ctcccagctc accaccttct actttggcag 1260aaattgtatg tctatgggtg ccctgaagga cctgttgcgc cacaccagtg ggctgagcaa 1320gttaagcctg gagacgtatc ctgcccctga ggagagtttg aattccttgg ttcgtgtcga 1380ttgggagatc ttcgccctac ttcgggctga gctgatgtgt acactgaggg aagtcaggca 1440gcccaagagg atcttcattg gtcccacccc ctgcccttcc tgtggctcat caccgtctga 1500ggaactggag ctccatcttt gctgctaggg aaggcgtgcc tagcggggta gagaaatcca 1560aagttctctt ccaggcactg ggacactaaa atctactatg taggtgcaaa ctatttttct 1620cttttcttat ttatttcatt ttttaataat tccaaaattt ttattaaaga caatttgaga 1680cagggtttct ctgtgttgct ctgggatcct cctgcctcag ctgggcttat gggatcctcc 1740tgcctcagct tcctaaagtg ctgggattac tggcatgagt gactgtgtcc aggccacatg 1800caacttaaag gaagcacagg gaagtgctca gtgtgaggga aaaaaacata acagcagggg 1860gcaaggctgg aggaaaatgt tgaggtgaca tcaatgagaa cttcagggac ccgtgtccta 1920cagagtcgga aagagaagct aaagttctac agtgatgaga atgttatccc tgcaaggatg 1980gttaccaagg aatatcagaa ataaagagca cctgaatgaa aacttttaac ctgttgtagc 2040aatttatcca ccagaaatat ctagttattg agttactgat ggaaaaataa tgaaatacta 2100ctttgtctgt gattgagttt cagctgtaga acatcaaagc aaccaaatag aatttgatca 2160tttt 2164241642DNAHomo sapiens 24cctgaagcta ctggttggtt ccctgagagg tcccagaact ctgcaaagtg agtccagcgc 60tgagattttt cttgcagatc catcaggatg agcatccagg ccccaccgag actactggag 120ctggcggggc agagcctgct gagagaccag gccttgtcca tctctgccat ggaggagctg 180cccagggtgc tctatctccc actcttcagg gaggccttca gcaggagaca cttccagact 240ctgacggtga tggtgcaggc ctggcctttc acctgcctcc ctctggtatc gctgatgaag 300acgcttcatc tggagccatt gaaagcattg ctggaagggc ttcatatgct gcttacacag 360aaggatcgcc ccaggaggtg gaaacttcaa gtgctggatt tgcgggatgt tgatgagaat 420ttctgggcca gatggcctgg agcctgggcc ctgtcctgct tcccagaggc catgagtaag 480aggcagacag cagaggactg tccaaggacg ggagagcacc agcccttaaa ggtgttcata 540gacatctgcc tcaaggaaat accccaggat gaatgcctga gatacctctt ccagtgggtt 600taccaaagga gaggtttagt acacctgtgc tgtagtaagc tggtcaatta tctaacgcca 660attaaatatc tcagaaagtc attgaaaata atatacatta atagtattgg ggagctggaa 720attcacaaca cgtgctggcc acatctgata agaaagcttt attgttacct gaaggagatg 780aagactcttt gcaaactcgt tttctccagg tgccatcatt acacgtcaga taatgaactc 840gagggatggt tagtcaccag attcacctct gtgttcctca ggctggaaca cctccagttg 900cttaaaataa aattgatcac cttcttcagt gggcacctgg aacagctgat caggtgcctc 960cagaacccct tggagaactt ggaattaact tgtggcaacc tattagaaga ggacttgaag 1020tgtctctccc agttcccaag cctcggttac ctaaagcatc tgaatctcag ctacgtgctg 1080ctgttccgca tcagtcttga acccctagga gctctgctag agaaaattgc tgcctctctc 1140gagaccctcg tgttagaggg ctgtcagatc cactactccc aactcagtgc catcctgcct 1200ggcctgagct gctgctccca gctcaccacc ttctactttg gcagcaattg catgtctatt 1260gacgccctga aggacctgct gcgccacacc agtgggctga gcaagttaag cctggagacg 1320tatcctgccc ctgaggagag tttgaattcc ttggttcgtg tcaattggga gatcttcacc 1380ccacttcggg ctgagctgat gtgtacactg agggaattca ggcagcccaa gaggatcttc 1440attggcccca ccccctgccc ttcctgtggc tcatcaccgt ctgaggaact ggagctccat 1500ctttgctgct agggaaggcg tgcccagtgg ggtagagaaa tccaaagttc tcttccaggc 1560acttggacac taaaatctac tatgtaggtg caaactattt ttctcttttc ttatttattt 1620cattttttaa taattccaaa at 1642254167DNAHomo sapiens 25accttcgcca tatatacccg gggcgctgcg ctccacctgg ccgccgcctc cagcccagca 60cctgcggagg gagcgctgac catggctccc tggcctgaat tgggagatgc ccagcccaac 120cccgataagt acctcgaagg ggccgcaggt cagcagccca ctgcccctga taaaagcaaa 180gagaccaaca aaacagataa cactgaggca cctgtaacca agattgaact tctgccgtcc 240tactccacgg ctacactgat agatgagccc actgaggtgg atgacccctg gaacctaccc 300actcttcagg actcggggat caagtggtca gagagagaca ccaaagggaa gattctctgt 360ttcttccaag ggattgggag attgatttta cttctcggat ttctctactt tttcgtgtgc 420tccctggata ttcttagtag cgccttccag ctggttggag gaaaaatggc aggacagttc 480ttcagcaaca gctctattat gtccaaccct ttgttggggc tggtgatcgg ggtgctggtg 540accgtcttgg tgcagagctc cagcacctca acgtccatcg ttgtcagcat ggtgtcctct 600tcattgctca ctgttcgggc tgccatcccc attatcatgg gggccaacat tggaacgtca 660atcaccaaca ctattgttgc gctcatgcag gtgggagatc ggagtgagtt cagaagagct 720tttgcaggag ccactgtcca tgacttcttc aactggctgt ccgtgttggt gctcttgccc 780gtggaggtgg ccacccatta cctcgagatc ataacccagc ttatagtgga gagcttccac 840ttcaagaatg gagaagatgc cccagatctt ctgaaagtca tcactaagcc cttcacaaag 900ctcattgtcc agctggataa aaaagttatc agccaaattg caatgaacga tgaaaaagcg 960aaaaacaaga gtcttgtcaa gatttggtgc aaaactttta ccaacaagac ccagattaac 1020gtcactgttc cctcgactgc taactgcacc tccccttccc tctgttggac ggatggcatc 1080caaaactgga ccatgaagaa tgtgacctac aaggagaaca tcgccaaatg ccagcatatc 1140tttgtgaatt tccacctccc ggatcttgct gtgggcacca tcttgctcat actctccctg 1200ctggtcctct gtggttgcct gatcatgatt gtcaagatcc tgggctctgt gctcaagggg 1260caggtcgcca ctgtcatcaa gaagaccatc aacactgatt tcccctttcc ctttgcatgg 1320ttgactggct acctggccat cctcgtcggg gcaggcatga ccttcatcgt acagagcagc 1380tctgtgttca cgtcggcctt gacccccctg attggaatcg gcgtgataac cattgagagg 1440gcttatccac tcacgctggg ctccaacatc ggcaccacca ccaccgccat cctggccgcc 1500ttagccagcc ctggcaatgc attgaggagt tcactccaga tcgccctgtg ccactttttc 1560ttcaacatct ccggcatctt gctgtggtac ccgatcccgt tcactcgcct gcccatccgc 1620atggccaagg ggctgggcaa catctctgcc aagtatcgct ggttcgccgt cttctacctg 1680atcatcttct tcttcctgat cccgctgacg gtgtttggcc tctcgctggc cggctggcgg 1740gtgctggttg gtgtcggggt tcccgtcgtc ttcatcatca tcctggtact gtgcctccga 1800ctcctgcagt ctcgctgccc acgcgtcctg ccgaagaaac tccagaactg gaacttcctg 1860ccgctgtgga tgcgctcgct gaagccctgg gatgccgtcg tctccaagtt caccggctgc 1920ttccagatgc gctgctgctg ctgctgccgc gtgtgctgcc gcgcgtgctg cttgctgtgt 1980ggctgcccca agtgctgccg ctgcagcaag tgctgcgagg acttggagga ggcgcaggag 2040gggcaggatg tccctgtcaa ggctcctgag acctttgata acataaccat tagcagagag 2100gctcagggtg aggtccctgc ctcggactca aagaccgaat gcacggcctt gtaggggacg 2160ccccagattg tcagggatgg ggggatggtc cttgagtttt gcatgctctc ctccctccca 2220cttctgcacc ctttcaccac ctcgaggaga tttgctcccc attagcgaat gaaattgatg 2280cagtcctacc taactcgatt ccctttggct tggtggtagg cctgcagggc acttttattc 2340caacccctgg tcactcagta atcttttact ccaggaaggc acaggatggt acctaaagag 2400aattagagaa tgaacctggc gggacggatg tctaatcctg cgcctagctg ggttggtcag 2460tagaacctat tttcagactc aaaaaccatc ttcagaaaga aaaggcccag ggaaggaatg 2520tatgagaggc tctcccagat gaggaagtgt actctctatg actatcaagc tcaggcctct 2580cccttttttt aaaccaaagt ctggcaacca agagcagcag ctccatggcc tccttgcccc 2640agatcagcct gggtcagggg acatagtgtc attgtttgga aactgcagac cacaaggtgt 2700gggtctatcc cacttcctag tgctccccac attccccatc agggcttcct cacgtggaca 2760ggtgtgctag tccaggcagt tcacttgcag tttccttgtc ctcatgcttc ggggatggga 2820gccacgcctg aactagagtt caggctggat acatgtgctc acctgctgct cttgtcttcc 2880taagagacag agagtggggc agatggagga gaagaaagtg aggaatgagt agcatagcat 2940tctgccaaaa gggccccaga ttcttaattt agcaaactaa gaagcccaat tcaaaagcat 3000tgtggctaaa gtctaacgct cctctcttgg tcagataaca aaagccctcc ctgttggatc 3060ttttgaaata aaacgtgcaa gttatccagg ctcgtagcct gcatgctgcc accttgaatc 3120ccagggagta tctgcacctg gaatagctct ccacccctct ctgcctcctt actttctgtg 3180caagatgact tcctgggtta acttccttct ttccatccac ccacccactg gaatctcttt 3240ccaaacattt ttccattttc ccacagatgg gctttgatta gctgtcctct ctccatgcct 3300gcaaagctcc agatttttgg ggaaagctgt acccaactgg actgcccagt gaactgggat 3360cattaagtac agtcgagcac acgtgtgtgc atgggtcaaa ggggtgtgtt ccttctcatc 3420ctagatgcct tctctgtgcc ttccacagcc tcctgcctga ttacaccact gcccccgccc 3480caccctcagc catcccaatt cttcctggcc agtgcgctcc agccttatct aggaaaggag 3540gagtgggtgt agccgtgcag caagattggg gcctccccca tcccagcttc tccaccatcc 3600cagcaagtca ggatatcaga cagtcctccc ctgaccctcc cccttgtaga tatcaattcc 3660caaacagagc caaatactct atatctatag tcacagccct gtacagcatt tttcataagt 3720tatatagtaa atggtctgca tgatttgtgc ttctagtgct ctcatttgga aatgaggcag 3780gcttcttcta tgaaatgtaa agaaagaaac cactttgtat attttgtaat accacctctg 3840tggccatgcc tgccccgccc actctgtata tatgtaagtt aaacccgggc aggggctgtg 3900gccgtctttg tactctggtg atttttaaaa attgaatctt tgtacttgca ttgattgtat 3960aataattttg agaccaggtc tcgctgtgtt gctcaggctg gtctcaaact cctgagatca 4020agcaatccgc ccacctcagc ctcccaaagt gctgagatca caggcgtgag ccaccaccag 4080gcctgattgt aatttttttt tttttttttt tactggttat gggaagggag aaataaaatc 4140atcaaaccca aaaaaaaaaa aaaaaaa 4167262664DNAHomo sapiens 26agagccagcc cggaggagct agaaccttcc ccgcatttct ttcagcagcc tgagtcagag 60gcgggctggc ctggcgtagc cgcccagcct cgcggctcat gccccgatct gcccgaacct 120tctcccgggg tcagcgccgc gccgcgccac ccggctgagt cagcccgggc gggcgagagg 180ctctcaactg ggcgggaagg tgcgggaagg tgcggaaagg ttcgcgaaag ttcgcggcgg 240cgggggtcgg gtgaggcgca aaaggataaa aagcccgtgg aagcggagct gagcagatcc 300gagccgggct ggctgcagag aaaccgcagg gagagcctca ctgctgagcg cccctcgacg 360gcggagcggc agcagcctcc gtggcctcca gcatccgaca agaagcttca gccatgcagg 420ccccacggga gctcgcggtg ggcatcgacc tgggcaccac ctactcgtgc gtgggcgtgt 480ttcagcaggg ccgcgtggag atcctggcca acgaccaggg caaccgcacc acgcccagct 540acgtggcctt caccgacacc gagcggctgg tcggggacgc ggccaagagc caggcggccc 600tgaaccccca caacaccgtg ttcgatgcca agcggctgat cgggcgcaag ttcgcggaca 660ccacggtgca gtcggacatg aagcactggc ccttccgggt ggtgagcgag ggcggcaagc 720ccaaggtgcg cgtatgctac cgcggggagg acaagacgtt ctaccccgag gagatctcgt 780ccatggtgct gagcaagatg aaggagacgg ccgaggcgta cctgggccag cccgtgaagc 840acgcagtgat caccgtgccc gcctatttca atgactcgca gcgccaggcc accaaggacg 900cgggggccat cgcggggctc aacgtgttgc ggatcatcaa tgagcccacg gcagctgcca 960tcgcctatgg gctggaccgg cggggcgcgg gagagcgcaa cgtgctcatt tttgacctgg 1020gtgggggcac cttcgatgtg tcggttctct ccattgacgc tggtgtcttt gaggtgaaag 1080ccactgctgg agatacccac ctgggaggag aggacttcga caaccggctc gtgaaccact 1140tcatggaaga attccggcgg aagcatggga aggacctgag cgggaacaag cgtgccctgc 1200gcaggctgcg cacagcctgt gagcgcgcca agcgcaccct gtcctccagc acccaggcca 1260ccctggagat agactccctg ttcgagggcg tggacttcta cacgtccatc actcgtgccc 1320gctttgagga actgtgctca gacctcttcc gcagcaccct ggagccggtg gagaaggccc 1380tgcgggatgc caagctggac aaggcccaga ttcatgacgt cgtcctggtg gggggctcca 1440ctcgcatccc caaggtgcag aagttgctgc aggacttctt caacggcaag gagctgaaca 1500agagcatcaa ccctgatgag gctgtggcct atggggctgc tgtgcaggcg gccgtgttga 1560tgggggacaa atgtgagaaa gtgcaggatc tcctgctgct ggatgtggct cccctgtctc 1620tggggctgga gacagcaggt ggggtgatga ccacgctgat ccagaggaac gccactatcc 1680ccaccaagca gacccagact ttcaccacct actcggacaa ccagcctggg gtcttcatcc 1740aggtgtatga gggtgagagg gccatgacca aggacaacaa cctgctgggg cgttttgaac 1800tcagtggcat ccctcctgcc ccacgtggag tcccccagat agaggtgacc tttgacattg 1860atgctaatgg catcctgagc gtgacagcca ctgacaggag cacaggtaag gctaacaaga 1920tcaccatcac caatgacaag ggccggctga gcaaggagga ggtggagagg atggttcatg 1980aagccgagca gtacaaggct gaggatgagg cccagaggga cagagtggct gccaaaaact 2040cgctggaggc ccatgtcttc catgtgaaag gttctttgca agaggaaagc cttagggaca 2100agattcccga agaggacagg cgcaaaatgc aagacaagtg tcgggaagtc cttgcctggc 2160tggagcacaa ccagctggca gagaaggagg agtatgagca tcagaagagg gagctggagc 2220aaatctgtcg ccccatcttc tccaggctct atggggggcc tggtgtccct gggggcagca 2280gttgtggcac tcaagcccgc cagggggacc ccagcaccgg ccccatcatt gaggaggttg 2340attgaatggc ccttcgtgat aagtcagctg tgactgtcag ggctatgcta tgggccttct 2400agactgtctt ctatgatcct gcccttcaga gatgaacttt ccctccaaag ctagaacttt 2460cttcccagga taactgaagt cttttgactt tttgggggga gggcggttca tcctcttctg 2520cttcaaataa aaagtcatta atttattaaa acttgtgtgg cactttaaca ttgctttcac 2580ctatattttg tgtactttgt tacttgcatg tatgaatttt gttatgtaaa atatagttat 2640agacctaaat aaaaaaaaaa aaaa 2664272163DNAHomo sapiens 27ctaatcttcc agttgccccc tattgacttt aaaccaaagc tttgattcat gaccactggg 60atccagccag cgggggattt cttttcctga tggactgtac ccaggatctc acatttgctt 120aagcagctcc cacaattggg ttcttataaa aggacacgga agagacaagc tcagttttct 180ctgaaggaga aggactgcac ttagaactgc attttggcga cctctgaaat tcagtactgc 240agtgaatgag cttctgatct tgaggtgaac ttaacaaaat tatttttgga aaaatcgttg 300tgggaaccat taaaagaact ccaggaaaca tgaattctgg aatcttacag gtctttcagg 360gggaactcat ctgccccctg tgcatgaact acttcataga cccggtcacc atagactgtg 420ggcacagctt ttgcaggcct tgtttctacc tcaactggca agacatccca tttcttgtcc 480agtgctctga atgcacaaag tcaaccgagc agataaacct caaaaccaac attcatttga 540agaagatggc ttctcttgcc agaaaagtca gtctctggct attcctgagc tctgaggagc 600aaatgtgtgg cactcacagg gagacaaaga agatattctg tgaagtggac aggagcctgc 660tctgtttgct gtgctccagc tctcaggagc accggtatca cagacaccgt cccattgagt 720gggctgctga ggaacaccgg gagaagcttt tacagaaaat gcagtctttg tgggaaaaag 780cttgtgaaaa tcacagaaac ctgaatgtgg aaaccaccag aaccagatgc tggaaggatt 840atgtgaattt aaggctagaa gcaattagag ctgagtatca gaagatgcct gcatttcatc 900atgaagaaga aaaacataat ttggagatgc tgaaaaagaa ggggaaagaa atttttcatc 960gacttcattt aagtaaagcc aaaatggctc ataggatgga gattttaaga ggaatgtatg 1020aggagctgaa cgaaatgtgc cataaaccag atgtggagct acttcaggct tttggagaca 1080tattacacag gagtgagtcc gtgctgctgc acatgcccca gcctctgaat ccagagctca 1140gtgcagggcc catcactgga ctgagggaca ggctcaacca attccgagtg catattactc 1200tgcatcatga agaagccaac aatgatatct ttctgtatga aattttgaga agcatgtgta 1260ttggatgtga ccatcaagat gtaccctatt tcactgcaac acctagaagt tttcttgcat

1320ggggtgttca gactttcacc tcgggcaaat attactggga ggtccatgta ggggactcct 1380ggaattgggc ttttggtgtc tgtaatatgt atcggaaaga gaagaatcag aatgagaaga 1440tagatggaaa ggcgggactc tttcttcttg ggtgtgttaa gaatgacatt caatgcagtc 1500tctttaccac ctccccactt atgctgcaat atatcccaaa acctaccagc cgagtaggat 1560tattcctgga ttgtgaggct aagactgtga gctttgttga tgttaatcaa agctccctaa 1620tatacaccat ccctaattgc tctttctcac ctcctctcag gcctatcttt tgctgtattc 1680acttctgacc agagacaaat cagaaatgtg ttcacatgct gtgggaaccc ctttatccca 1740ggaagtcctc ttccttgtgc cttaacatac aggacaaata ggctctattt tatgtcttga 1800attgccttct aatgttatca aaactcattt attgtgttac tattaaatat gctgaaaacg 1860ctaaaagtat acgtattggt tctttattaa ataatttttg aaaaatcatt attcatgatc 1920atggcataca gtatattctc ttttttttct ttatttatga ctgtcactga gtgaaataat 1980agatgacaga catgtctgaa tgaagtaaaa atcaatggaa gacagtcggg atcttttgct 2040tcatgcaaaa aacttggagt gaagtctcaa tgataactgg gaaatgtttt tcttcctctt 2100tatctaacta tattacactt atccatcagg tttcattgta ttaatctatc ctttgaggta 2160ata 2163282160DNAHomo sapiens 28acccacagca ctcattcctg aagctactgg ttggttccct gagaggtccc agaactctgc 60gaagtgagtc cagcgctgag attttccttg cagatctatc aggatgagca tccaggcccc 120acccagacta ctggagctgg cagggcagag cctgctgaga gaccaggcct tgtccatctc 180tgccatggag gagctgccca gggtgctcta tctcccactc ttcatggagg ccttcagcag 240gagacacttc cagactctga cggtgatggt tcaggcctgg cccttcacct gcctccctct 300gggatcactg atgaagacgc ttcatttgga gaccttaaaa gcattgctgg aagggcttca 360tatgctgctt acacagaagg atcgccccag gaggtggaaa cttcaagtgc tggatttgcg 420ggatgttgac gagaatttct gggccagatg gcctggagcc tgggccctgt cctgcttccc 480agagaccacg agtaagaggc agacagcaga ggactgtcca aggatgggag agcaccagcc 540cttaaaggtg ttcatagaca tctgcctcaa ggaaataccc caggatgaat gcctgagata 600cctcttccag tgggtttacc aaaggagagg tttagtacac ctgtgctgta gtaagctggt 660caattatcta acgccgatta aatatctcag aaagtcattg aaaataatat acctgaatag 720tattcaagag ctggaaattc gcaacatgtc ctggccacgt ctgataagaa agcttcgttg 780ttacctgaag gagatgaaga atcttcgcaa actcgttttc tccaggtgcc atcattacac 840gtcagataat gaactccaag gacggttagt tgccaaattc agctctgtgt tcctcaggct 900ggaacacctt cagttgctta aaataaaatt gatcaccttc ttcagtgggc acctggaaca 960gctgatcagg tgcctccaga accccttgga gaacttggaa ttaacttatg gctacctatt 1020ggaagaagac atgaagtgtc tctcccagta cccaagcctc ggttacctaa agcatctgaa 1080tctcagctac gtgctgctgt tccgcatcag tcttgaaccc ctcggagctc tgctggagaa 1140aattgctgcc tctctcaaaa ccctcatctt ggagggctgt cagatccact actcccaact 1200cagtgccatc ctgcctgccc tgagccggtg ctcccagctc accaccttct actttggcag 1260aaattgcatg tctattgacg ccctgaagga cctgctgcgc cacaccagtg ggctgagcaa 1320gttaagcctg gagacgtatc ctgcccctga ggagagtttg aattccttgg ttcgtgtcaa 1380ttgggagatc ttcaccccac ttcgggctga gctgatgtgt acactgaggg aagtcaggca 1440gcccaagagg atcttcattg gccccacccc ctgcccttcc tgtggctcat caccgtctga 1500ggaactggag ctccatcttt gctgctaggg aaggcgtgcc cagtggggta gagaaatcca 1560aagttctctt ccaggcactt ggacactaaa atctactatg tgggtgcaaa ctatttttct 1620cttttcttat ttatttcatt ttttaataat tccaaaattt ttattaaaga caatttgaga 1680cagggtttcg ctgtgttgct ccagctggtc tcaaactgct gggcttatgg gatcctcctg 1740cctcagcttc ctaaagtgct gggattactg gcatgagtga ctgtgtccag gccacatgca 1800acttaaagga agcacaggca agtgttcagt gtgagggaaa aaacataaca gcagggggca 1860aggttggagg aaaatgttga ggtgacatca gtgagaactt cagggacccg tgtcctagag 1920tcggaaagag aagctaaagt tctacagtga tgagactgtt atccctgcaa ggatggttac 1980caaggaatat cagcaataaa gagcacctga atgaaaactt ttaacctgtt gtgcaattta 2040tccatcagaa atctctagtt atcgagttac ggatggaaaa ataacgaaat actaatttgt 2100ctgtgattga gtttcagttg tagaacatca aagcaaccaa ataaaaatta gatcattttg 2160295937DNAHomo sapiens 29tgcaggtaac aagggcaaca gcctgagcat ctcagagccc agaggcagag cgttagccga 60ttgcttccag catcatctgg ggcacagtgg ggtcttggtt cctcaatggg cctgagtgga 120tctaactctg cgaagttaga tcccaacagc catcacagtt tgcagacaat gtcattaaga 180ccatccagat aacttcctaa ctccagtttt gtgcccacca agcatccttc tgatttcaaa 240ttggcctcgc atgccatgtg caactgggag agagtgtgtg gacagaaatg gggccaattg 300actatttccc ttggctgtca tatttttcat taataaacta actctccagc cacaaataca 360cactcagaat gcctcttgct actccagatc ctccattcac tgtgaaggca atcatgggga 420ttatgaattc catctcccag gtgtggatta aactgcatgc caggggaggt ttctgtggtt 480ccaatctacc ccgcttagta catcagggct caacaggatc aggtcaaagc tggaaggatc 540ctgagagccc acagaaataa tgactcctgt gctgaggttc acaggagtag cactggggtc 600tgtgaattct tggcaaaaat tcagaaaacc taagggaatc catgcattag ctgataatga 660ggccatacag actaactaaa gcatcagcca cctcattaaa ctgggaagct taatactgtt 720tttattgcac aatcatttct aaatgtcttt tattaataaa attggggaaa tgaatttgtt 780attctttaat aagtgcagtg tgtttagctg acaaaatttt tacaaagatg gggatcaatg 840ggttgcaaga atactaaaag atgttcttgt tctgcagggt tggaagcccc taagccacca 900tgcactaccc atcattttac aaaagaagga gaaactgagg aacaaagaaa cacatatttt 960ccctcaagct tcagattccc tttaaactct taggatatcc cataaccccc tgtagcttat 1020ggcagctaga ttcatgacag acaatctctc tagagtcaat ttggtttttc tcttaactca 1080ctcaagcctc tgggaatgaa aggtctagcc cttgaaggct acttttggta gaagacgagg 1140ttcagtatta aaaaggagga cagaggatgg aaaagaacac aactacatca atagtttctc 1200cacattattt gatgttcaga acagtcccat gaagaagata taatattccc tttctacaga 1260ccaaaaaatt aatatttgga gaggtagaaa gaccacccaa gggaacacat atttagacgg 1320aaagcccagt tctgtctagt gtttaagtcc tggcccattc agctactctg ctctgtaact 1380atcacccatt tcagcaccgc ggacagaggc agagccctca gtcttccctg taggtgggat 1440ggaggcagag ggtggtaaga tgggtgctaa gtcccaggga agatatgtat ccaccaaagt 1500gcctgaatga tgagagggaa gtcagagcta aggaaggaca catcatggac atctctttac 1560atgtgtatca aattgcgttc tgtttagaac cattttctag cctcccacca aggacgtaaa 1620caggacaagc actgtcatct gtaaagtgcc actcccagac tgccacccag agttcataaa 1680aggctcagat gaatcaatag gtgggaaagt tatctggaat ttataaaaat tcactgttaa 1740ggagacgact atcacagaca aaccccaaaa tcagcggttt aacacaatag acattttttc 1800tgacttgtgt aaagccccag acagggatcg ctgcctgtca aaaggcctga ctggcacttg 1860tcttgaggca cactctggtg cccaggcccc ttgctccttg ttgctctgcc acctctgaag 1920tggcttctaa ggtcactgta tttatctgtg tcaagacaga ggaaaaaatc atgaataaat 1980aaacccaggg agatttgtat gagccaagcc tgaaattggc acacatcact ttggctcata 2040cgtggaggac tgggagtgca gaaagatgag gaaatgggtc ttactgaaca cagaaccaca 2100gaactctgtc tccctcctct ccaaagctga gaaattgcca caatcagaaa gtgtgattcc 2160catctgagag tttaagagca ggaatagatt aaagacaaat catgtaaaat accttgactc 2220ctagacttgc cgaagcattc agcctgagcc atctttacat gtggataatc ttggatttcc 2280caactgggct tcttgcacac tccatggtag aacgtcagag gaaatttttt tcaagcaaga 2340gctgttagat catgagattc cccagaaaga tacagataca ggtatatgtc atattactgg 2400agattctaat tcagctacct tcacaggcct gggaatgtgt ttttgacaca ggggcattag 2460gattgtttca gctgcaagtg acagaagtct agctcacacg gtcttaagca agaaaggaaa 2520tgtattgatt cctataagtg caccaggatc tagatacact gtcaggcaca gctggatgca 2580gactccaaca gtctcattgg gatacccacc ctctcctccc atctctgacc atcccaggtg 2640gctcccttca tgggtcaaag tggccaccag aagctccaga tacatctttt tatcaacagc 2700cccaggagaa cttctctttc tcagtaaaat cccacaaggt attttttggc actaattgtc 2760ttggcttggt cccattctta tccctgaacc aaccactgtg accaaaaacg tggtattctc 2820tgattatcca agcctgattc acgagcccac ccatgagtct gatatgaggt ccagtccata 2880caatctacac aagctaggtc tggggcatgg tggtgcccca aggacagctg gggtgccttt 2940cctagaagaa agggggcagg gaatgggtgc tggaccagca tcagcagcag aattcttagg 3000cactagactg tggggggctc agagaggcat gggagccctg aggtccccac aaggtgtggg 3060atagtctttc agaccttcag ggggggtctc tgctcactaa cctgctcaaa gcaccctggc 3120ccacaccgtg ggcagctgga aggtgccagt ccactgaaca tgtgtgattg gcatgaatct 3180cctgctttcc tttgtcagaa ggctaggagt ggatttgacc cgtttactca gaccctctca 3240gtggccctgc tcccacaggc tcaccccagc agggcccagt gcttggctgc caatgacgcc 3300aaggatatta gctgacagtg acttaaaaca ggggttcatg acctcagggt aaccgaggaa 3360cccctgaatc tgaattcatc agtctgtgta tatgaatgtg agtgcctctc ctttccccac 3420aggaaaaaat ctacaacttt catcaaatta ctcagggaga ctttattcta aagagactga 3480aaacgaccaa catcaatttt gactcctatg ggcatctgta aatagcttca aggttttaag 3540gtgaaatgtc atgtaccaaa atacacattc tggagaagca ggaagctaca gaccaacttg 3600agatgaaagt ctcatatcaa tgttttccca agtgtgctcc ttggaataca ggttgacatg 3660atatgatgcc cagcaaggga aacaaaacat attcatgttc aaattagttg gggaaatgct 3720ggactaaata aggtttgatg ggattctttt ttctgcagga cttctcagaa ggggctagag 3780taggcaaagt ttcccagatt tacctaataa aagcattgtt tctgtgggag tttcatttgt 3840tattacatgt ttcctgaatg cagattcata gactatcctt tggggaaccc tcgtcctcac 3900gggatgtatg ttcatggtgg tgtcttcgag tttgtgccct tgtgaagcat tctggcagca 3960agcgtctgaa cacttccaaa agggggcgat atttaggaga aatcgctcag cctgaattag 4020aacaaatgca gctgctggtg tctcttggtg cctgggagcc ctagagtgtc agagggagga 4080gcgtgcacac tggaaccagc agcctggtgc tgcgtctcag ctctgtcgct aactggctat 4140gcacctctgg gcatggcact taacccttct gagccccagc ccgccatctg taaaaagggc 4200ttgatgtgag gattgaatga gatcatgcag ggaacacaat gtctggcact tggaagcgtc 4260caccataaag agccaaggca gtagatggcc cagctgggtt tgttccaggc agagtttacc 4320ctctgccctg gaggctccag gaaatgctgc cacgtggctc ctattgcctt aaccacatcc 4380gacctgttcc tgacagctcc ccacatctcc agctcctttg ctggtgctcc aggcacctcc 4440aaacgtggcg agcccctctc ccctgccctc ttgtggcagc tgacctggca ggagtgggac 4500caagacatcc aaggcagctc ctttccacct gcatgggcac tttcctcagg acatccttgc 4560ccctggcacc accttgggcc agcaagccac atggaaatgg atgcagaggc accactgttt 4620gctgacaatt atacactgtc cttaaggtca cccttggcga tctgtcacca ggagcagaca 4680aacccacacc tcaaccatcc catcagagct tgtttctatc tgcatctgtc atcgctgatc 4740gcatttgaat gggtttagtc tctattttaa ataaaagatt tatgccttag ctgtcagagc 4800ctgcctttat ttgaaaattt aatcttgttt ctaggagtct agattaactt attagattta 4860ggcgtccctc gtgggtctct gagagaggag gagtagattc tcctccctgc attcggccct 4920gcacacccga cagtgagagc caagagctgg atgggcttct ccatccagca cgcccaggct 4980ggacagaggc ccccaactca ggcaactttg tcgggtaacc gtgtgtccag ggagtgcttt 5040cttgcacgct ccgtctccgg gccagcttcc aggacctgtg ctcactgcaa gggacaccca 5100tcgagccggc cctttctcag aggttttggg gaggcttcag gaaggacccc cagtgggggc 5160ccagcttgtc aacatgggct gtgccaagga gttctgagtt tccttcaggt ctgtattgta 5220tcttccaccc cctcagagct ccctccctac tgcttagacc acacgaagct gtggggctgt 5280gggcagccag ttcacttccc tgaccttgtc tgcaggtgga gacagtggca gtgcccctcc 5340cgggctgctg cgtcactcag tgtaaaagca gggaggcact ggagagctgc tgtctgcaag 5400tttgttgcta cttcaaaagg tgcaggtggg ccctcacctc ctttagaggt gaggatgagc 5460tacccaaagt gaaaaggagc ttctcagcgt cgcaatggag tcacggccag gctgcccaca 5520ccagccgtcc ggacctgcac cagtgccacg gggtctgccc catcttctct tccctctcct 5580tcccctctcc ctctctctcc ctgtctcttt ctcctctctc catgctattg actgaatgtt 5640tgaattccct gcaaatgcat tcctaacccc caatgtgact gtgtttggag acagggtctt 5700taggaggtaa ctgaggttaa atgaggttgt aaagatgggg ccctgaaccg atgggactgg 5760ggtccttatg agaagaggaa aaggggtccc tccccatgga gggacgacca cagcgaggca 5820gcagccgccc acacgccaga gaaggggact cagagggaag ccttgcttca ccggcacctt 5880gatcttgact cctagcctcc agaattgtga taaataaatt tctgttgctt aagcccc 5937302364DNAHomo sapiens 30ctgctgaaaa agcagaaaga gattaccagc cacagacggg tcatgagcgc ggtattactg 60ctggccctcc tggggttcat cctcccactg ccaggagtgc aggcgctgct ctgccagttt 120gggacagttc agcatgtgtg gaaggtgtcc gacctgcccc ggcaatggac ccctaagaac 180accagctgcg acagcggctt ggggtgccag gacacgttga tgctcattga gagcggaccc 240caagtgagcc tggtgctctc caagggctgc acggaggcca aggaccagga gccccgcgtc 300actgagcacc ggatgggccc cggcctctcc ctgatctcct acaccttcgt gtgccgccag 360gaggacttct gcaacaacct cgttaactcc ctcccgcttt gggccccaca gcccccagca 420gacccaggat ccttgaggtg cccagtctgc ttgtctatgg aaggctgtct ggaggggaca 480acagaagaga tctgccccaa ggggaccaca cactgttatg atggcctcct caggctcagg 540ggaggaggca tcttctccaa tctgagagtc cagggatgca tgccccagcc agtttgcaac 600ctgctcaatg ggacacagga aattgggccc gtgggtatga ctgagaactg cgatatgaaa 660gattttctga cctgtcatcg ggggaccacc attatgacac acggaaactt ggctcaagaa 720cccactgatt ggaccacatc gaataccgag atgtgcgagg tggggcaggt gtgtcaggag 780acgctgctgc tcctagatgt aggactcaca tcaaccctgg tggggacaaa aggctgcagc 840actgttgggg ctcaaaattc ccagaagacc accatccact cagcccctcc tggggtgctt 900gtggcctcct atacccactt ctgctcctcg gacctgtgca atagtgccag cagcagcagc 960gttctgctga actccctccc tcctcaagct gcccctgtcc caggagaccg gcagtgtcct 1020acctgtgtgc agccccttgg aacctgttca agtggctccc cccgaatgac ctgccccagg 1080ggcgccactc attgttatga tgggtacatt catctctcag gaggtgggct gtccaccaaa 1140atgagcattc agggctgcgt ggcccaacct tccagcttct tgttgaacca caccagacaa 1200atcgggatct tctctgcgcg tgagaagcgt gatgtgcagc ctcctgcctc tcagcatgag 1260ggaggtgggg ctgagggcct ggagtctctc acttgggggg tggggctggc actggcccca 1320gcgctgtggt ggggagtggt ttgcccttcc tgctaactct attaccccca cgattcttca 1380ccgctgctga ccacccacac tcaacctccc tctgacctca taacctaatg gccttggaca 1440ccagattctt tcccattctg tccatgaatc atcttcccca cacacaatca ttcatatcta 1500ctcacctaac agcaacactg gggagagcct ggagcatccg gacttgccct atgggagagg 1560ggacgctgga ggagtggctg catgtatctg ataatacaga ccctgtcctt tctcccagtg 1620ctgggatttc tccatgtgag ggggcagcag gacacccagg gatctagcgt gggggaggag 1680aggagcctaa tgagaaaatg accatctaaa gcctgccctt cattggtctg gttcacgtct 1740ccaaaccagc ttggatggta gcagagactt cagggtgctc cagccaaacg tatttgggca 1800tcaccatgac ctgggagggg aagatgcact gagacgtatg aggcttccag cctagcagcc 1860agggccctag cacaaacagg aggctcgccc catctgagca actgcaggag aggttagtac 1920agtcatgcat tgcttaacga cagggacgtg tcgttagaaa tgtgtcgtta ggtgatttta 1980tgaccatagg aacattgtag cgtgcactta caccaaccca gatggtacag cccaatacac 2040acccaggatg gacgctagag tcgactgctc ctaggctaca agcctgcagt gcatgttatg 2100gtgtgaatac tgcaggcaat cttaacacca cggcaagtat ttgtgcatct acacacatct 2160aaacatagaa aaggtacagc ataaatacac tattgtcatc tcagcagacc accgttctat 2220acgcaattcg tcgctgaccc aaacgttgct atgtagcatc tgcgtatcgt gggataattg 2280acatgagggc ttgagagaac tccagaaaaa aatgggttag cattttccca gagctgttat 2340cattgggtct ctcttaccac cata 2364311189DNAHomo sapiens 31atgcgcagac cgaggcaggg aggcgggggt gcgggaggct ccgcggctgc gcgggcgcgt 60gctggtggcc tcggcggagg ctcagtcccg gcccgcgccc gcggcgcccc cgctgcggcc 120cgagcggcct ggctgcggga tctgtgcgca cggatggcgc ggccgccccg gcagcacccg 180ggggtctggg cgtcgctgct cctgctgcta ctgacggggc ccgccgcctg cgcagccagc 240cccgcggacg acggtgcggg cccggggggc cggggacccc ggggacgcgc gcggggggac 300acgggcgccg acgaggcggt gccgcgccac gactcctcct acggcacctt cgcgggggag 360ttctacgacc tgcgctacct gtcggaggag ggttacccct tccctactgc tcctcctgtg 420gatccatttg ccaaaatcaa agtggacgac tgtggaaaaa ctaagggatg ctttagatat 480ggcaaaccag gctgtaatgc agagacctgt gactatttcc tcagctaccg gatgataggg 540gctgatgtag aatttgagct gagtgcagac acagatggtt gggtagcagt tggattctct 600tcagacaaga aaatgggtgg tgatgatgtc atggcctgcg tccatgatga caatggcagg 660gtccgcatac agcacttcta taatgtaggc cagtgggcaa aggagattca gagaaaccct 720gccagagatg aagaaggagt ttttgagaac aatcgcgtca cctgcagatt taaacgccct 780gtgaatgttc ccagagatga aacaattgtt gatctgcatt tgagttggta ttatctgttt 840gcttggggtc cagccattca gggctctatc actcgacatg atatagactc accgccggct 900tcagagcgtg ttgtcagtat ttacaagtat gaagacattt ttatgccatc agctgcctat 960caaaccttct catctccatt ttgtttgctt ctgattgttg ctctgacctt ctacctattg 1020atgggaaccc cctaaccaca gctgcagggc caacagatta catggattgg gaagtcttta 1080gtataaatat attttttaaa gaatatccag tataatttta gcttcaatta tttaagaaaa 1140aaaacctcat ataatttcag ctttttggaa gaaagaacaa gcttctttt 1189321272DNAHomo sapiens 32gcaaaaccgt gagctggatt tataatcgcc ctataaagct ccagaggcgg tcaggcacct 60gcagaggagc cccgccgctc cgccgactag ctgcccccgc gagcaacggc ctcgtgattt 120ccccgccgat ccggtccccg cctccccact ctgcccccgc ctaccccgga gccgtgcagc 180cgcctctccg aatctctctc ttctcctggc gctcgcgtgc gagagggaac tagcgagaac 240gaggaagcag ctggaggtga cgccgggcag attacgcctg tcagggccga gccgagcgga 300tcgctgggcg ctgtgcagag gaaaggcggg agtgcccggc tcgctgtcgc agagccgagg 360tggcctgttt ctgcgccgga ccagtcgagg actctggaca gtagaggccc cgggacgacc 420gagctgatgg cgtcttcgac cccatcttcg tccgcaacct cctcgaacgc gggagcggac 480cccaatacca ctaacctgcg ccccacaacg tacgatacct ggtgcggcgt ggcccatgga 540tgcaccagaa aactggggct caagatctgc ggcttcttgc aaaggaccaa cagcctggaa 600gagaagagtc gccttgtgag tgccttcaag gagaggcaat cctccaagaa cctgctttcc 660tgtgaaaaca gcgaccggga tgcccgcttc cggcgcacag agactgactt ctctaatctg 720tttgctagag atctgcttcc ggctaagaac ggtgaggagc aaaccgtgca attcctcctg 780gaagtggtgg acatactcct caactatgtc cgcaagacat ttgatcgctc caccaaggtg 840ctggactttc atcacccaca ccagttgctg gaaggcatgg agggcttcaa cttggagctc 900tctgaccacc ccgagtccct ggagcagatc ctggttgact gcagagacac cttgaagtat 960ggggttcgca caggtcatcc tcgatttttc aaccagctct ccactggatt ggatattatt 1020ggcctagctg gagaatggct gacatcaacg gccaatacca acatgccatc agacatgagg 1080gagtgttggt tgctacggtg atggggctca gagcagaacc aaagcatgat tgtgacctcc 1140agaggtgatg gtaactgcac acatggtttc caagggtctt cctcctaaat ttccaggggc 1200ctcccaagga aaatggacat attctttttg gaaataaaat acttctacca acataaaaaa 1260aaaaaaaaaa aa 1272332621DNAHomo sapiens 33ccttttttgg cctcgacggc ggcaacccag cctccctcct aacgccctcc gcctttggga 60ccaaccaggg gagctcaagt tagtagcagc caaggagagg cgctgccttg ccaagactaa 120aaagggaggg gagaagagag gaaaaaagca agaatccccc acccctctcc cgggcggagg 180gggcgggaag agcgcgtcct ggccaagccg agtagtgtct tccactcggt gcgtctctct 240aggagccgcg cgggaaggat gctggtccgc aggggcgcgc gcgcagggcc caggatgccg 300cggggctgga ccgcgctttg cttgctgagt ttgctgcctt ctgggttcat gagtcttgac 360aacaacggta ctgctacccc agagttacct acccagggaa cattttcaaa tgtttctaca 420aatgtatcct accaagaaac tacaacacct agtacccttg gaagtaccag cctgcaccct 480gtgtctcaac atggcaatga ggccacaaca aacatcacag aaacgacagt caaattcaca 540tctacctctg tgataacctc agtttatgga aacacaaact cttctgtcca gtcacagacc 600tctgtaatca gcacagtgtt caccacccca gccaacgttt caactccaga gacaaccttg 660aagcctagcc tgtcacctgg aaatgtttca gacctttcaa ccactagcac tagccttgca 720acatctccca ctaaacccta tacatcatct tctcctatcc taagtgacat caaggcagaa 780atcaaatgtt caggcatcag agaagtgaaa ttgactcagg gcatctgcct ggagcaaaat 840aagacctcca gctgtgcgga gtttaagaag gacaggggag agggcctggc ccgagtgctg 900tgtggggagg agcaggctga tgctgatgct ggggcccagg tatgctccct gctccttgcc 960cagtctgagg tgaggcctca gtgtctactg ctggtcttgg ccaacagaac agaaatttcc

1020agcaaactcc aacttatgaa aaagcaccaa tctgacctga aaaagctggg gatcctagat 1080ttcactgagc aagatgttgc aagccaccag agctattccc aaaagaccct gattgcactg 1140gtcacctcgg gagccctgct ggctgtcttg ggcatcactg gctatttcct gatgaatcgc 1200cgcagctgga gccccacagg agaaaggctg ggcgaagacc cttattacac ggaaaacggt 1260ggaggccagg gctatagctc aggacctggg acctcccctg aggctcaggg aaaggccagt 1320gtgaaccgag gggctcagga aaacgggacc ggccaggcca cctccagaaa cggccattca 1380gcaagacaac acgtggtggc tgataccgaa ttgtgactcg gctaggtggg gcaaggctgg 1440gcagtgtccg agagagcacc cctctctgca tctgaccacg tgctaccccc atgctggagg 1500tgacatctct tacgcccaac ccttccccac tgcacacacc tcagaggctg ttcttggggc 1560cctacacctt gaggaggggc aggtaaactc ctgtccttta cacattcggc tccctggagc 1620cagactctgg tcttctttgg gtaaacgtgt gacgggggaa agccaaggtc tggagaagct 1680cccaggaaca atcgatggcc ttgcagcact cacacaggac ccccttcccc taccccctcc 1740tctctgccgc aatacaggaa cccccagggg aaagatgagc ttttctaggc tacaattttc 1800tcccaggaag ctttgatttt taccgtttct tccctgtatt ttctttctct actttgagga 1860aaccaaagta accttttgca cctgctctct tgtaatgata tagccagaaa aacgtgttgc 1920cttgaaccac ttccctcatc tctcctccaa gacactgtgg acttggtcac cagctcctcc 1980cttgttctct aagttccact gagctccatg tgccccctct accatttgca gagtcctgca 2040cagttttctg gctggagcct agaacaggcc tcccaagttt taggacaaac agctcagttc 2100tagtctctct ggggccacac agaaactctt tttgggctcc tttttctccc tctggatcaa 2160agtaggcagg accatgggac caggtcttgg agctgagcct ctcacctgta ctcttccgaa 2220aaatcctctt cctctgaggc tggatcctag ccttatcctc tgatctccat ggcttcctcc 2280tccctcctgc cgactcctgg gttgagctgt tgcctcagtc ccccaacaga tgcttttctg 2340tctctgcctc cctcaccctg agccccttcc ttgctctgca cccccatatg gtcatagccc 2400agatcagctc ctaaccctta tcaccagctg cctcttctgt gggtgaccca ggtccttgtt 2460tgctgttgat ttctttccag aggggttgag cagggatcct ggtttcaatg acggttggaa 2520atagaaattt ccagagaaga gagtattggg tagatatttt ttctgaatac aaagtgatgt 2580gtttaaatac tgcaattaaa gtgatactga aacacaaaaa a 2621341816DNAHomo sapiens 34ggcgaccacg gtgtcttcaa aagccccgtc agggttggct tcctggggcc ggaccgactg 60tgggtcagtt tgcaccagcg ctctggaatc gagttacgcg cgaaagggca gagtttctgg 120aggaaaccgc agcctctcaa ccgctgaccg ggtctcagaa ggcccccggc agggccgctt 180ggcgggaact gaccacgcgc cagtcaggct ctccagggac ctgcgcaggc gcgtgtgggc 240ggagtcgtgc gcagggggcg gggcttcggg aaggagccac agagagggcg gggcgtagga 300cctgcgcttc gggggtggag tcggagcggc gcggcggcgg tcatgcggga cgcggatgca 360gacgcaggcg gaggcgctga cggcggggat ggccggggtg gccacagctg ccgcgggggc 420gtggacacag ccgcagctcc ggccggtgga gctcccccag cgcacgcgcc aggtccgggc 480agagacgccg cgtctgcggc cagggggtca cgaatgcggc cgcacatatt caccctcagc 540gtgcctttcc cgaccccctt ggaggcggaa atcgcccatg ggtccctggc accagatgcc 600gagccccacc aaagggtggt tgggaaggat ctcacagtga gtggcaggat cctggtcgtc 660cgctggaaag ctgaagactg tcgcctgctc cgaatttccg tcatcaactt tcttgaccag 720ctttccctgg tggtgcggac catgcagcgc tttgggcccc ccgtttcccg ctaagcctgg 780cctgggcaaa tggagcgagg tcccactttg cgtctccttg taggcagtgc gtccatcctt 840ccctagggca ggaattccca cagttgctac tttcctggga gggcctcatg ttttatctgg 900ttcttaaatg tttgttacta cagaaaataa aactgcgcta ctattccaag tctgagttta 960tttgcagctg gggcacctcc caatattctt gttgtgcttg ggttgctggg ggggggttct 1020agaattcaga tattcaagga gtacaaggaa attgaagaca atttaggaaa tggaagaaaa 1080tgaaaatcaa ttgggttctg tcattcagga ttaactactg tcaacatttt ggaatacttc 1140ctcagtttta cagttgcact tacatagtaa atgtgtaact gtaatataca ccacataata 1200tttgcaagtt tagtgttaaa tttttttcct gatttttaaa tctaacatga gcttttttcc 1260tctaacgatc agtgaagaaa gtgctggggc aattgactag tgtctggggc aaggagttgg 1320ctccctggaa aatacagtgt ctccagcctt agggctcttt tatagattct atcagatttt 1380ctgagagtga aaaggaagag gtacaactgc ttttattctc agaaaacaag gaaatggttt 1440gatccttttg agtcttgctt tgaagatgtg ctgtgtggga ccagagcagc tcttaactgt 1500aggcttgttt ccctctatgg aggcaacaaa caccattctg ggcaccctgg ccagtgctgc 1560ctaggtgaac atgagcttct ctatcctggt gggtggggac agctgctagt ccctgtcctg 1620cttgcacact ggagttaccg ttcatcctct cctgctgggg tgatggcctt ccctggtctt 1680gggtagcttc ctcacacgcc tgtgctcacc agtagtcgta gtccgctgca cactggaacg 1740ggagcctctg tggatatcca gggttctttc cctgtgcagc tctcttctct ctggttctcc 1800gccctgcaaa ctccag 181635773DNAHomo sapiens 35gtggggtcag caagagaaac tctacggcta tgggagagcc tgcgttcacc tcttttccga 60gcccacctgt tctggggaag ctcaaaagaa acatgatgcc ctgggcttta cagaagaaac 120gagaaatcca catggccaag gcccatcgga gacgagctgc gaggtctgct ctccccatga 180gactcaccag ctgcatcttc cggaggccgg tgacaaggat caggtctcat cctgacaacc 240aggtcagacg cagaaaaggg gacgagcacc tggagaagcc gcagcaactc tgcgcctacc 300ggagactgca ggccctgcag ccctgcagca gccaaggaga aggttcaagt ccactgcatt 360tggagagcgt cttaagtatc cttgcaccgg ggacggccgg tgaatctctg gacagagctg 420gtgctgagcg tgtgcgcatc ccgcttgagc ccacccctgg gcggtttcca gctgtggcag 480gggggccaac cccaggaatg ggttgtcagc tcccaccgcc cctctctggc caattggtga 540ctcctgcaga tatccggaga caggccagga gggtgaagaa agccagggag agactggcca 600aggccttgca ggcagacagg ctggccaggc aggcagaaat gctgacaggt gggtgaagct 660cagtcctggg ctttcggtcc ctttctttta atgcccatcc tcattcctac tctgaattgt 720cacacttttc ccttccccac cagttcttta ataaaagtat ttgaaaggca aca 77336773DNAHomo sapiens 36gtggggtcag caagagaaac tctacggcta tgggagagcc tgcgttcacc tcttttccga 60gcccacctgt tctggggaag ctcaaaagaa acatgatgcc ctgggcttta cagaagaaac 120gagaaatcca catggccaag gcccatcgga gacgagctgc gaggtctgct ctccccatga 180gactcaccag ctgcatcttc cggaggccgg tgacaaggat caggtctcat cctgacaacc 240aggtcagacg cagaaaaggg gacgagcacc tggagaagcc gcagcaactc tgcgcctacc 300ggagactgca ggccctgcag ccctgcagca gccaaggaga aggttcaagt ccactgcatt 360tggagagcgt cttaagtatc cttgcaccgg ggacggccgg tgaatctctg gacagggctg 420gtgctgagcg tgtgcgcagc ccgcttgagc ccacccctgg gcggtttcca gctgtggcag 480gggggccaac cccaggaatg ggttgtcagc tcccaccgcc cctctctggc caattggtga 540ctcctgcaga tatccggaga caggccagga gggtgaagaa agccagggag agactggcca 600aggccttgca ggcagacagg ctggccaggc aggcagaaat gctgacatgt agatgaagcg 660cagtcctggg ctttcggtcc ctttctttta atgcccatcc tcattcctac tctgaattgt 720cacacttttc ccttccccac cagttcttta ataaaagtat ttgaaaggca aca 773371583DNAHomo sapiens 37ctcctgggcc tctcaaagtc tgagccccgc tccgctgatg cctgtctgca gaatccgcac 60caaccagcac catgcccatg actctggggt actgggacat ccgtgggctg gcccacgcca 120tccgcttgct cctggaatac acagactcaa gctatgtgga aaagaagtac acgctggggg 180acgctcctga ctatgacaga agccagtggc tgaatgaaaa attcaagctg ggcctggact 240ttcccaatct gccctacttg attgatgggg ctcacaagat cacccagagc aatgccatcc 300tgcgctacat tgcccgcaag cacaacctgt gtggggagac agaagaggag aagattcgtg 360tggacatttt ggagaaccag gttatggata accacatgga gctggtcaga ctgtgctatg 420acccagattt tgagaaactg aagccaaaat acttggagga actccctgaa aagctaaagc 480tctactcaga gtttctgggg aagcggccat ggtttgcagg agacaagatc acctttgtgg 540atttccttgc ctatgatgtc cttgacatga agcgtatatt tgagcccaag tgcttggacg 600ccttcctaaa cttgaaggac ttcatctccc gctttgaggg tttgaagaag atctctgcct 660acatgaagtc cagccaattc ctccgaggtc ttttgtttgg aaagtcagct acatggaaca 720gcaaataggg cccagtgatg ccagaagatg ggagggagga gccaaccttg ctgcctgcga 780ccctggagga cagcctgact ccctggacct gccttcttcc tttttccttc tttctactct 840cttctcttcc ccaaggcctc attggcttcc tttcttctaa catcatccct ccccgcatcg 900aggctcttta aagcttcagc tccccactgt cctccatcaa agtccccctc ctaacgtctt 960cctttccctg cactaacgcc aacctgactg cttttcctgt cagtgctttt ctcttctttg 1020agaagccaga ctgatctctg agctccctag cactgtcctc aaagaccatc tgtatgccct 1080gctccctttg ctgggtccct accccagctc cgtgtgatgc ccagtaaagc ctgaaccatg 1140cctgccatgt cttgtcttat tccctgaggc tcccttgact caggactgtg ctcgaattgt 1200gggtggtttt ttgtcttctg ttgtccacag ccagagctta gtggatgggt gtgtgtgtgt 1260gtgtgttggg ggtggtgatc aggcaggttc ataaatttcc ttggtcattt ctgccctcta 1320gccacatccc tctgttcctc actgtgggga ttactacaga aaggtgctct gtgccaagtt 1380cctcactcat tcgcgctcct gtaggccgtc tagaactggc atggttcaaa gaggggctag 1440gctgatgggg aagggggctg agcagctccc aggcagactg ccttctttca ccctgtcctg 1500atagacttcc ctgatctaga tatccttcgt catgacactt ctcaataaaa cgtatcccac 1560cgtattgtaa aaaaaaaaaa aaa 1583381862DNAHomo sapiens 38acccaaagtc ttcaagcctg gagttcctgc ttggttcttc ctgaggtctg agcaccttct 60agactacatc cagatctgtt ttccctgcag attcatgaag atgagcatcc ggactccacc 120cagactcctg gagcttgcag ggcggagcct gctgagggac caagctttgg ccatgtccac 180cctggaggag ctgcccacag aacttttccc cccactgttc atggaggcct tcagcaggag 240acgctgtgag gccctgaagc tgatggtgca ggcctggccc ttccgccgcc tccctctgag 300gcctctgata aagatgcctt gtctggaggc cttccaagct gtgctcgatg ggcttgatgc 360actgcttacc caaggggttc gtcccaggag gtggaaactc caagtgctgg atttacagga 420tgtctgtgag aacttctgga tggtttggtc tgaagctatg gcccatgggt gcttcctcaa 480tgccaagagg aacaaaaaac cagtgcagga ctgtccaagg atgagaggac ggcagccctt 540gactgtgttc gtagaacttt ggctcaagaa caggactctg gatgaatacc tcacctacct 600ccttctatgg gtcaagcaga ggaaagattt actacacctg tgctgtaaga agctgaaaat 660tttgggaatg cccttccgca atatcagaag catcctgaaa atggtgaacc tagactgtat 720ccaggaggtg gaagtgaatt gcaagtgggt actgcccatc ctgacacagt ttaccccata 780cctgggccac atgaggaatc ttcagaagct cgttctctcc cacatggatg tctctcgcta 840cgtttcccca gagcagaaga aggagattgt tacccagttc accactcagt tcctcaagct 900gcgctgcctc caaaagcttt atatgaactc tgtttctttc ctcgaaggcc acctggacca 960gctgctcagc tgtctgaaga cctcgttaaa agtcctcaca ataactaact gtgtgctttt 1020ggaatcagac ttgaagcatc tatcccagtg cccgagtatc agtcaactaa agaccctgga 1080cctgagtggc atcagactga ccaattatag tcttgtgcct ctccaaattc tcctagaaaa 1140agttgcagcc acccttgagt acctggattt agatgactgt ggcatcatag actcccaagt 1200caacgccatc ctgcctgccc tgagccgctg ctttgagctc aacaccttca gcttctgtgg 1260aaatcccatc tgcatggcca ccctggagaa cctgctgagc cacacaatca tactcaaaaa 1320cttatgtgtg gagctgtatc ctgccccccg agagagttat ggtgctgatg gtactctctg 1380ctggagcaga tttgctcaaa ttagggctga gctgatgaac agagtgaggg acttaaggca 1440ccccaagagg atcttgttct gtactgacta ctgccctgac tgtggcaaca ggtcatttta 1500tgacctggag gcagatcaat actgctgttg aatgcctgcc tatttggatg ggtatgtcaa 1560acgctttctt ctggacactt ggaaactaaa acctaggtct taggtacatc ctaaagggag 1620cacagaaccc atcatttcac acataggctc tgaaagtggg aaaggaaagc tgatcaagca 1680ggggccggac ttgggggaaa tgttgccatg gattcgatgg gactttgggg acctgtgtcc 1740tgtagattcg aaaatgggaa tctgaatgtc tagagtggaa ttcaggcttg agaatacatg 1800agggagttac tcttgcatgg atggttgtaa agaaacaatc agaaataaag gaaaactgag 1860ca 1862391855DNAHomo sapiens 39acccaaagtc ttcaagcctg gagttcctgc ttggttcttc ctgaggactg agcaccttct 60agactacatc cagatctgtt ttccctgcag attcgtgaag atgagcatcc ggactccacc 120cagactcctg gagcttgcag ggcggagcct gctgagggac caagccttgg ccatgtccac 180cctggaggag ctgcccacag aacttttccc cccactgttc atggaggcct tcagcaggag 240acgctgtgag gccctgaagc tgatggtgca ggcctggccc ttccgccgcc tccctctgag 300gcctctgata aagatgcctt gtctggaggc cttccaagct gtgctcgatg ggctggatgc 360actgcttacc caaggggttc atcccaggag gtggaaactt caagtgctgg atttacagga 420tgtctgtgag aacttctgga tggtttggtc tgaagctatg gcccatgggt gcttcctcaa 480tgccaagagg aacaaaaaac cagtgcagga ctgtccaagg atgagaggac agcagccctt 540gactgtgttc gtagaacttt ggctcaagaa caggactctg gatgaatacc tcacctgcct 600ccttctatgg gtcaagcaga ggaaagattt actacacctg tgctgtaaga agctgaaaat 660tttgggaatg cccttccgca atatcagaag catcctgaaa atggtgaacc tagactgtat 720ccaggaggtg gaagtgaatt gcaagtgggt actgcccatc ctgacacagt ttaccccata 780cctgggccac atgaggaatc ttcagaagct cgttctctcc cacatggatg tctctcgcta 840cgtttcccca gagcagaaga aggagattgt tacccagttc accactcagt tcctcaagct 900gtgctgcctc caaaagcttt ctatgaactc tgtttctttc ctcgaaggcc acctggacca 960gctgctcagc tgtctgaaga cctcgttaaa ggtcctcaca ataactaact gtgtgctttt 1020ggaatcagac ttgaagcatc tatcccagtg cccgagtatc agtcaactaa agaccctgga 1080cctgagtggc atcagactga ccaattacag tcttgtgcct ctccaaattc tcctagaaaa 1140agttgcagcc acccttgagt acctggattt agatgactgt ggcatcatag actcccaagt 1200caacgccatc ctgcctgccc tgagccgctg ctttgagctc aacaccttca gcttctgtgg 1260aaatcccatc tccatggcca ccctggagaa cctgctgagc cacacaatca tactcaaaaa 1320cttatgcgtg gagctgtatc ctgccccccg ggagagttat gatgctgatg gtactctctg 1380ctggagcaga tttgctcaaa ttagggctga gctgatgaag agagtgaggg acttaaggca 1440ccccaagagg atcttgttct gtactgactg ctgccctgac tgtggcaaca ggtcatttta 1500tgacctggag gcagatcaat gctgctgttg aatgcctgcc tatttgggtg gatatgtcaa 1560acgctttctt ctggacactt ggaaactaaa acctaggtct taggtacatc ctatagggag 1620cacagaaccc atcatttcac acatgggctc tgaaagtggg aaaggaaagg tgatcaagca 1680ggggcaggac ttgggggaag tgttgccatg gattcgatgg gactttgggg acctgtgtcc 1740tgtagagtgg aaaatgggaa tttgaatgtc tagagtggag gcttgagaat acttgaggga 1800gttactcttg gatgcatggt tgtaaagaaa caatcagaaa taaaggaaaa ctgag 1855401648DNAHomo sapiens 40gcagggaatg agctcctgat cttggggagt acttaaaaga attttttctt ggaagaatta 60ctgcaggaaa cattcataga accttgggaa acatgaattc tggaatcttg caagtcttcc 120agagggcact cacctgtccc atctgcatga actacttcct agacccagtc accatagact 180gtgggcacag cttttgccgg ccctgtttgt acctcaactg gcaagacacg gcagttcttg 240ctcagtgctc tgaatgcaag aagacaacgc ggcagagaaa cctcaacact gacatttgtt 300tgaagaacat ggctttcatt gccagaaaag ccagcctccg gcaattcctt agctctgagg 360agcaaatatg tgggatgcac agagagacaa agaagatgtt ctgtgaagtg gacaagagcc 420tgctctgttt gccgtgctcc aactctcagg agcaccggaa tcacatacac tgtcccattg 480agtgggctgc tgaggaacgc cgggaggagc tcctaaaaaa aatgcagtct ttatgggaaa 540aagcttgtga aaatctcaga aatctgaaca tggaaaccac aagaaccaga tgctggaagg 600attatgtgag tttaaggata gaagcaatca gagctgaata tcagaagatg cctgcatttc 660tccatgaaga agagcaacat cacttggaaa ggctgcgaaa ggagggcgag gacatttttc 720agcaactcaa tgaaagcaaa gccagaatgg aacattccag ggagctttta agaggaatgt 780atgaggatct gaagcaaatg tgccataaag cagatgtgga gctactccag gcttttggag 840acatattaca caggtatgag tctctgctgc tgcaagtgtc tgagcctgtg aatccagagc 900tcagtgcagg gcccatcact ggactgctgg acagcctcag tggattcaga gttgatttta 960ctctgcagcc tgaaagagcc aatagtcata tcttcctgtg tggagatttg agaagcatga 1020atgttggatg tgaccctcaa gatgatcccg atatcactgg aaaatctgaa tgttttcttg 1080tatggggggc tcaggctttc acatctggca aatattattg ggaggttcat atgggggact 1140cttggaattg ggcttttggt gtctgtaaca attattggaa agagaagaga cagaatgaca 1200agatagatgg agaggaggga ctctttcttc ttggatgtgt taaggaggac actcactgca 1260gtctctttac cacctcccca cttgtggtgc aatatgttcc aagacctacc agcacagtag 1320gattattcct ggattgtgaa ggtagaaccg tgagctttgt tgatgttgat caaagttccc 1380tgatatacac catccccaat tgctccttct cacctcctct caggcctatc ttttgctgta 1440gtcacttctg accagagaaa agtcagaaat gtgcctgtat gctctgggaa cctgtttatc 1500ccagaaagcc ctctttttcg cacctcatca aacagaacaa ataagttata tttaatgtct 1560ttagttgcat tctaatgtca tcaaaactca tttatagtgt ttctattaaa tatggtgaaa 1620acattaaaaa aaaaaaaaaa aaaaaaaa 1648411617DNAHomo sapiens 41ccaccccccc cccccaccac caccaccacc accaccccgc cggccggccc caggcctcga 60cgccctgggt cccttccggg gtggggcggg ctgtcccagg ggggctcacc gccattcatg 120aaggggtgga gcctgcctgc ctgtgggcct ttacaagggc ggctggctgg ctggctggct 180gtccgggcag gcctcctggc tgcacctgcc gcagtgcaca gtccggctga ggtgcacggg 240agcccgccgg cctctctctg cccgcgtccg tccgtgaaat tccggccggg gctcaccgcg 300atggccctcc cgacaccctc ggacagcacc ctccccgcgg aagcccgggg acgaggacgg 360cgacggagac tcgtttggac cccgagccaa agcgaggccc tgcgagcctg ctttgagcgg 420aacccgtacc cgggcatcgc caccagagaa cggctggccc aggccatcgg cattccggag 480cccagggtcc agatttggtt tcagaatgag aggtcacgcc agctgaggca gcaccggcgg 540gaatctcggc cctggcccgg gagacgcggc ccgccagaag gccggcgaaa gcggaccgcc 600gtcaccggat cccagaccgc cctgctcctc cgagcctttg agaaggatcg ctttccaggc 660atcgccgccc gggaggagct ggccagagag acgggcctcc cggagtccag gattcagatc 720tggtttcaga atcgaagggc caggcacccg ggacagggtg gcagggcgcc cgcgcaggca 780ggcggcctgt gcagcgcggc ccccggcggg ggtcaccctg ctccctcgtg ggtcgccttc 840gcccacaccg gcgcgtgggg aacggggctt cccgcacccc acgtgccctg cgcgcctggg 900gctctcccac agggggcttt cgtgagccag gcagcgaggg ccgcccccgc gctgcagccc 960agccaggccg cgccggcaga gggggtctcc caacctgccc cggcgcgcgg ggatttcgcc 1020tacgccgccc cggctcctcc ggacggggcg ctctcccacc ctcaggctcc tcggtggcct 1080ccgcacccgg gcaaaagccg ggaggaccgg gacccgcagc gcgacggcct gccgggcccc 1140tgcgcggtgg cacagcctgg gcccgctcaa gcggggccgc agggccaagg ggtgcttgcg 1200ccacccacgt cccaggggag tccgtggtgg ggctggggcc ggggtcccca ggtcgccggg 1260gcggcgtggg aaccccaagc cggggcagct ccacctcccc agcccgcgcc cccggacgcc 1320tccgcctccg cgcggcaggg gcagatgcaa ggcatcccgg cgccctccca ggcgctccag 1380gagccggcgc cctggtctgc actcccctgc ggcctgctgc tggatgagct cctggcgagc 1440ccggagtttc tgcagcaggc gcaacctctc ctagaaacgg aggccccggg ggagctggag 1500gcctcggaag aggccgcctc gctggaagca cccctcagcg aggaagaata ccgggctctg 1560ctggaggagc tttaggacgc ggggttggga cggggtcggg tggttcgggg cagggcg 1617

Claims

1.-21. (canceled)

22. A method of treating Facioscapulohumeral muscular dystrophy (FSHD) in a subject, the method comprising administering to the subject one or more inhibitory nucleic acids targeting one or more of SLC34A2, TRIM49, TRIM43, CD177, NAAA, HSPA6, TC2N, or CD34.

23. A method of treating FSHD in a subject, the method comprising administering to the subject two or more inhibitory nucleic acids targeting two or more of SLC34A2, TRIM49, TRIM43, PRAMEF1, CD177, NAAA, HSPA6, TC2N, or CD34.

24. The method of claim 22 or 23, wherein the inhibitory nucleic acid is a double-stranded RNA, siRNA, shRNA, or antisense oligonucleotide.

25. The method of claim 24, wherein the antisense oligonucleotide is a morpholino oligonucleotide.

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