WO2017079551A1 - Methods for diagnosis and prognosis of venous thrombosis - Google Patents

Abstract

The present invention discloses novel variant alleles and polymorphisms in the myosin genes and genes in the myosin interactive network that are associated with or linked to venous thrombosis. The invention provides diagnosis or prognosis methods for detecting the increased risk of developing venous thrombosis in a subject. The invention also provides kits for carrying out the diagnosis and prognosis methods disclosed herein.

Description

Methods for Diagnosis and Prognosis of Venous Thrombosis

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Number 62/251,280 (filed November 5, 2015). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made in part with the U.S. government support by the National Institutes of Health Grant No. ROl HL021544. The U.S. Government therefore has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Venous thrombosis (VT) is the formation of a blood clot in the veins. A number of genetic and non-genetic factors are linked to thrombosis. These include, e.g., medical conditions such as surgery and trauma, prolonged immobilization, cancer, myeloproliferative disorders, and pregnancy; mutations in proteins involved in blood clotting or anticoagulation (e.g., factor V, prothrombin, and methylenetetrahydrofolate reductase); and increased expression levels of factors VIII, IX or XI, or fibrinogen.

[0004] Current genetic and environmental factors do not adequately explain the majority of clinical venous thrombosis events (VTEs), and there is a major unmet need for further basic research which can be translated to the clinic. Since acute VTE represents a failure to regulate thrombin generation, discovery and characterization of VT genetic or protein biomarkers and risk factors are current major challenges with clinical implications.

[0005] There is a strong need for better means for diagnosing or prognosing venous thrombosis. The present invention addresses this and other unmet needs in the art.

SUMMARY OF THE INVENTION

[0006] In one aspect, the present invention provides methods of prognosing an increased risk of developing a venous thrombosis in a subject. The methods entail (a) obtaining a nucleic acid sample from the subject, and (b) determining in the nucleic acid sample one or more single nucleotide polymorphisms (SNPs) shown in Tables 1-5. The detection of the SNPs enables diagnosis of an increased possibility of the risk of developing a venous thrombosis in the subject. In some embodiments, the employed nucleic acid sample contains genomic DNA of the subject. In some of these embodiments, the genomic DNA is extracted from a blood tissue sample of the subject. In some other embodiments, the employed nucleic acid sample contains cDNA or mRNA of the subject. In some of these methods, the cDNA or mRNA is prepared from a skeletal muscle sample of the subject. In some preferred embodiments, the subject to be tested is a human. In some methods, the SNPs are detected with a nucleic acid array.

(0007] Some methods of the invention require the detection of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more SNPs shown in Tables 1-5. Some methods require detection of one or more SNPs in myosin genes shown in Tables 1-3. In some of these methods, at least 2, 3, 4, or more SNPs present in one myosin gene shown in Tables 1-3 are detected. In some other methods, at least one SNP present in each of 2, 3, or 4 different myosin genes shown in Tables 1-3 are detected. Some other methods require detection of one or more SNPs in genes of myosin interactive network shown in Tables 4 and 5. In some of these methods, at least 2, 3, 4, or more SNPs present in one gene shown in Tables 4 and S are detected. In some methods, at least one SNP present in each of 2, 3, 4 or 5 different genes shown in Tables 4 and 5 are detected. Some methods of the invention require detection of a combination of (a) at least one SNP present in myosin genes as shown in Tables 1-3, and (b) at least one SNP present in genes of myosin interactive network as shown in Tables 4 and 5. Some of these methods require detection of a combination of (a) at least 2, 3, 4, 5, 6 or more SNPs present in myosin genes shown in Tables 1-3; and (b) at least 2, 3, 4, 5, 6 or more SNPs present in genes of myosin interactive network shown in Tables 4 and 5. Some other methods require the detection of a combination of (a) at least one SNP in each of 2, 3, or 4 different myosin genes shown in Tables 1-3; and (b) at least one SNP present in each of 2, 3, 4 or 5 different genes shown in Tables 4 and S.

[0008] Some methods of the invention can further include detecting in the nucleic acid sample at least one additional SNP or variant allele that is known to be associated with or linked to venous thrombosis. Examples of such known SNPs include rs6025, rs 1799963, rs8176719, rs2066865, rs2036914, rs2069951, rs2289252, rs4149755, rs2069952, rs2227589, rs!69713, rs3136516, rsl800595, rsl 799809, rs867186, rsl063856, rsl613662, rs3136520, «1800291, rsl039084, rs2001490, rs6003, rs670659, rs6048, rs4524, rs5985, 1208 indel, rs8176592, rs3822057, rsl523127, «3742264, rs514659, rsl799810 and rs710446.

[0009] In another aspect, the invention provides methods of identifying patients that may be in need of prophylactic antithrombotic therapy. These methods involve (a) obtaining a nucleic acid sample from each of a population of candidate subjects, and (b) determining in the nucleic acid sample one or more single nucleotide polymorphisms (SNPs) shown in Tables 1-5. Detection of these SNPs allows identification from the candidate subjects ones who may be in need of prophylactic antithrombotic therapy. In another aspect, the invention provides kits for diagnosing or prognosing an increased possibility of having venous thrombosis in a subject The kits contain (a) at least one pair of allele-specific

oligonucleotides that are capable of detecting the single nucleotide polymorphisms (SNPs) shown in Tables 1 -5, and (b) an instruction sheet containing description of using the of allele- specific oligonucleotides for diagnosing or prognosing venous thrombosis.

[0010] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows prothrombotic activity of skeletal muscle myosin.

[0012] Figure 2 illustrates myosin interactive network and myosin related genes.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

[0013] The present invention is predicated in part on the discoveries by the present inventors that some specific variants in myosin genes have predictive value in the diagnosis of the risk of venous thrombosis. As detailed herein, the inventors found that there is a high prevalence of the myosin heavy chain polymorphisms and polymorphisms in genes belonging to myosin interactive network among individuals with VTE. These inherited markers indicate an increased susceptibility for VTE, and provide a basis for employing more accurate diagnostic, prognostic, preventative and therapeutic regimes in individuals heterozygous or homozygous for the polymorphism of these genes.

[0014] In accordance with these discoveries, the invention provides diagnostic methods and kits for detecting a single nucleotide polymorphism (SNP) in chromosome 17 myosin clustering region and/or other myosin genes and/or genes belonging to myosin interactive network. The methods or kits can be used to evaluate the risk of thrombosis in a subject. The present invention also provides methods for predicting an increased risk or probability of developing thrombosis in a patient based upon variants in myosin heavy chain gene and other causative loci. The presence of the SNP(s) indicates an increased risk of developing venous thrombosis in the patient. The invention also provides methods to evaluate the genetic thrombosis risk using combination of multiple polymorphisms in myosin genes and/or genes belonging to myosin interactive network to increase the sensitivity. The detection of a combination of clustering SNPs in these genes could increase the sensitivity for identifying the subjects under VTE risk dramatically.

[0015] Unless otherwise stated, the present invention can be performed using standard procedures, as described, for example in Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos. 4,965,343, and 5,849,954;

Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998). The following sections provide additional guidance for practicing the compositions and methods of the present invention.

II. Definitions

[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (eds.), Oxford University Press (revised ed., 2000); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3PrdP ed., 2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4PthP ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0017] The term "biological sample", as used herein, refers to any sample from a biological source and includes, without limitation, cell cultures or extracts thereof, biopsied material obtained from a mammal or extracts thereof, and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

[0018] As used herein, the terms "diagnose," "diagnosis," and "diagnostics" include, but are not limited to any of the following: detection of VT (or VTE) that an individual may presently have, predisposition/susceptibility screening (i.e., determining the increased risk of an individual in developing VT in the future, or determining whether an individual has a decreased risk of developing VT in the future), determining a particular type or subclass of VT in a subject known to have or to have had VT, and confirming or reinforcing a previously made diagnosis of risk for VT. The terms also encompass evaluation of an individual to determine which therapeutic or prophylactic strategy that individual is most likely to positively respond to, prediction whether a patient is likely to respond to a particular treatment, and evaluation the future prognosis of an individual having VT. Such diagnostic uses are based on detection of one or more of the SNPs disclosed herein.

[0019] Diagnosis does not require ability to determine the presence or absence of a particular condition or disease with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, a positive diagnosis indicates an increased probability that a certain disease or condition is present in the subject compared to control subjects (e.g., subjects in the general population or subjects with a negative diagnosis). Similarly, a prognosis signals an increased probability that a given course or outcome will occur in a patient relative to the probability for the control subjects.

[0020] The term "genotype" as used herein broadly refers to the genetic composition of an organism, including, for example, whether a diploid organism is heterozygous or homozygous for one or more alleles of interest.

[0021] Hybridizations are usually performed under stringent conditions that allow for specific binding between an oligonucleotide and a target DNA containing one of the polymorphisms shown in Tables 1-5. A stringent condition is defined as any suitable buffer concentrations and temperatures that allow specific hybridization of the oligonucleotide to highly homologous sequence spanning at least one of the polymorphic sites shown in Tables 1-5 and any washing conditions that remove non-specific binding of the oligonucleotide. For example, conditions of 5X SSPE (730 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C are suitable for allele-specific probe hybridizations. The washing conditions usually range from room temperature to 60°C.

[0022] Hybridization probes are capable of binding in a base-specific manner to a complementary strand of nucleic acid. Such probes include nucleic acids and peptide nucleic acids (e.g., as described in Nielsen et al., Science 254, 1497-1500, 1991).

{0023] Linkage describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome, and can be measured by percent recombination between the two genes, alleles, loci or genetic markers that are physically-linked on the same chromosome. Loci occurring within 50 centimorgan of each other are linked. Some linked markers occur within the same gene or gene cluster.

[0024] Linkage disequilibrium (LD) refers to the co-inheritance of alleles (e.g., alternative nucleotides) at two or more different SNP sites at frequencies greater than would be expected from the separate frequencies of occurrence of each allele in a given population. The expected frequency of co-occurrence of two alleles that are inherited independently is the frequency of the first allele multiplied by the frequency of the second allele. Alleles that co- occur at expected frequencies are said to be in "linkage equilibrium." In contrast, LD refers to any non-random genetic association between allele(s) at two or more different SNP sites, which is generally due to the physical proximity of the two loci along a chromosome. LD can occur when two or more SNPs sites are in close physical proximity to each other on a given chromosome and therefore alleles at these SNP sites will tend to remain unseparated for multiple generations with the consequence that a particular nucleotide (allele) at one SNP site will show a non-random association with a particular nucleotide (allele) at a different SNP site located nearby. Hence, genotyping one of the SNP sites will give almost the same information as genotyping the other SNP site that is in LD.

[0025] Various degrees of LD can be encountered between two or more SNPs with the result being mat some SNPs are more closely associated (i.e., in stronger LD) than others. Furthermore, the physical distance over which LD extends along a chromosome differs between different regions of the genome, and therefore the degree of physical separation between two or more SNP sites necessary for LD to occur can differ between different regions of the genome.

[0026] A marker in linkage disequilibrium with disease predisposing variants can be particularly useful in detecting susceptibility to disease (or association with sub-clinical phenotypes) notwithstanding that the marker does not cause the disease. For example, a marker (X) that is not itself a causative element of a disease, but which is in linkage disequilibrium with a gene (including regulatory sequences) (Y) that is a causative element of a phenotype, can be used detected to indicate susceptibility to the disease in circumstances in which the gene Y may not have been identified or may not be readily detectable. Younger alleles (i.e., those arising from mutation relatively late in evolution) are expected to have a larger genomic segment in linkage disequilibrium. The age of an allele can be determined from whether the allele is shared among different human ethnic groups and/or between humans and related species.

[0027] Polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A single nucleotide polymorphism (SNP) occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations). A single nucleotide polymorphism usually arises due to substitution of one nucleotide for another at the polymorphic site. Single nucleotide polymorphisms according to the present application may fall within coding sequences of genes, non-coding regions of genes or the intronic regions between genes. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine or vice versa. Single nucleotide polymorphisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele. Unless otherwise noted, the terms "polymorphism" and "single nucleotide polymorphism" (SNP) are used herein interchangeably. As used herein, a set of polymorphisms or a set of SNPs means at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the SNPs shown in Tables 1-5.

[0028] Myosin is a highly conserved, ubiquitous family of protein found in all eukaryotic cells, where it provides the motor function for diverse movements such as cytokinesis, phagocytosis, and muscle contraction. All myosins contain an amino-terminal motor/head domain and a carboxy-terminal tail domain. Due to the extensive number of different molecules identified to date, myosins have been divided into seven distinct classes based on the properties of the head domain. One such class, class Π myosins, consists of the conventional two-headed myosins that form filaments and are composed of two myosin heavy chain (MYH) subunits and four myosin light chain subunits. The MYH subunit contains the ATPase activity providing energy that is the driving force for contractile processes mentioned above, and numerous MYH isoforms exist in vertebrates to carry out this function. [0029] Myosin-II isoforms are the major contractile proteins in muscle and also play several crucial roles in non-muscle contractility. Myosin-II molecules contain two motor domains and assemble into bipolar filaments. A number of genes encoding the myosin heavy chain isoforms (MYH cluster genes) have been identified. These include MYH1 (skeletal muscle, adult), MYH2 (skeletal muscle, adult), MYH3 (skeletal muscle, embryonic), MYH4 (skeletal muscle), MYH6 (cardiac muscle), MYH7 (cardiac muscle), MYH7B (cardiac muscle), MYH8 (skeletal muscle, perinatal), ΜΥΉ9 (non-muscle), MYH 10 (non-muscle), MYHI 1 (smooth muscle), MYH 13 (skeletal muscle), MYH 14 (non-muscle), MYH 15 and MYH 16. In humans and mice, skeletal myosin heavy chain (MYH) genes are clustered on a single chromosome (17p in humans and 11 in mice).

[0030] The term subject or patient can include human or non-human animals. Thus, the methods and described herein are applicable to both human and veterinary disease and animal models. Preferred subjects are "patients," i.e., living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology.

[0031] The phrase "substantially identical," in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 73%, preferably at least 85%, more preferably at least 90%, 95% or higher nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm such as those described below for example, or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 40-50 residues in length, preferably over a longer region than 50 amino acids, more preferably at least about 90-100 residues, and most preferably the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide for example.

[0032] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences) relative to the reference sequence, based on the designated program parameters.

[0033] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection. Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm Jiih.govA. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence. The phrase

"hybridizing specifically to", refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[0034] Venous thrombosis (VT) or venous thrombosis event (VTE) refers to the formation of a blood clot in the veins. VT may result from a genetic mutation alone or in concert with environmental factors, such as smoking. VTEs occur for the first time in about 100 per 100,000 people each year in the United States. About one-third of patients with symptomatic VT manifest pulmonary embolism (PE), whereas two-thirds manifest deep vein thrombosis (DVT) without PE. DVT is an acute VT in a deep vein, usually in the thigh, legs, or pelvis, and it is a serious and potentially fatal disorder that can arise as a complication for hospital patients, but may also affect otherwise healthy people. Large blood clots in VTE may interfere with blood circulation and impede normal blood flow. In some instances, blood clots may break off and travel to distant major organs such as the brain, heart or lungs as in PE and result in fatality.

ΠΙ. Prothrombotic myosins and SNPs in myosin genes associated with venous thrombosis

[0035] The present inventors discovered that skeletal muscle myosin is prothrombotic. Specifically, studies were initiated to define procoagulant or anticoagulant properties of skeletal muscle myosins. These proteins are not previously known to function in blood clotting. Remarkably, it was discovered purified skeletal muscle myosin acts as procoagulant in platelet rich plasma, plasma and purified protein system composed of factor Xa, factor Va and prothrombin. It was observed that, even under blood flow in a model using whole blood, skeletal muscle myosin acts as a prothrombotic (Fig.1). This indicates that skeletal muscle myosins represent a new family of proteins which are involved in the coagulation/thrombosis development.

[0036] In addition, a number of myosin heavy chain polymorphisms and polymorphisms in genes belonging to myosin interactive network were identified with high prevalence in subjects with clinical venous thrombosis events. The novel VTE associated polymorphisms identified herein, including the involved genes, are listed in Tables 1-5. Specifically, the Exome genotyping database of Scripps Venous Thrombosis registry subjects was analyzed for the association with VTE and pulmonary embolism (PE) risk. The database was created by Affymetrix Axiome Exome array using N=214 controls and N=107 VTE Caucasian cases. When the "collapsing method" was applied for rare variant (MAF<0.01) analysis for some regions, association with PE (a subset of VTE) risk was detected at ten MYH rare variants at chromosome 17p 13.1 (Table 1). This is a highly conserved region with clustered MYH genes (e.g., ΜΥΉ1, 2, 4, and 8). Among the 10 MYH rare variants, rsl 11567318 was significantly linked to VTE (p=6.5xl0-6, and after FDR correction, p<0.05). VTE associated rare variants (MAF<0.01 for both control and general population) were collected based on the analysis of total VTE and subsets of VTE (recurrent VTE, PE and idiopathic VTE). For the chromosome 17 MYH rare variant cluster, 16/107 VTE cases (15%) had a rare variant vs. 1/212 (0.5%) controls (OR=17, p=2.0 x 10-8) (Table 2 and Table 3).

[0037] Other than the MYH gene cluster, genes belonging to skeletal muscle myosin interactive network (Fig. 2) were also analyzed. It was found that some SNPs in these genes, including OBSCN (Table 4), TTN (Table 5), NEB (Table 5), MYBPC3 (Table 5), and TNNI3IK (Table 4), were also associated with the risk of VTE. Prevalences of these variants in VTE subjects and controls are summarized in Table 6. For example, for the OBSCN variant cluster, 19.6% VTE cases had a rare variant vs. 1.4% controls (OR=37, p=1.4 x 10-7) (Table 3). For the TTN and NEB variant clusters, 21.5% and 9.5% VTE cases had a rare variant vs 0.5 % and 0.5% controls, respectively (OR=58 and 19, p=3.8xl0-l 1 and 3.0 x 10- 4, respectively) (Table 6).

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

[0038] The SNPs disclosed herein are all known and characterized in the art. Detailed structural and technical information of these variants, including location and identity of the specific nucleotide residue substituted in each variant, can be readily obtained from die Entrez databases (e.g., the dbSNP collection) with the corresponding "rs" numbers of the SNPs listed in Tables 1-5. As detailed below, identification of these thrombosis-associated polymorphisms can have many clinical implications. For example, detection of one SNP or a set of polymorphisms described herein can allow one to design and perform diagnostic assays for evaluation of genetic risks for thrombosis and other related conditions. Detection and analysis of these polymorphisms can also be useful in designing prophylactic and therapeutic regimes customized to the underlying abnormalities (e.g., VTEs). Thus, some embodiments of the invention are directed to selecting or identifying subjects that may be in need of antithrombotic therapy or prophylactic antithrombotic therapy. Detection of the

polymorphisms described herein can also be employed in clinical trials of drugs for treatment of these diseases and the underlying biological abnormalities. rv. Detecting polymorphisms in myosin genes and related genes

[0039] The presence of polymorphisms in myosin and the related genes described herein indicate an increased susceptibility for VTE. This provides a basis for employing more accurate diagnostic, prognostic, preventative and therapeutic regimes in individuals heterozygous or homozygous for the polymorphism of these genes. The invention accordingly provides methods for predicting the risk of thrombosis (e.g., VTE) in subjects by detecting one or more of the SNPs described herein. Through detection of a combination of SNPs in these genes, the invention also provides diagnostic methods with an increased sensitivity for identifying the subjects under VTE risk. In some methods, one or more SNPs shown in Tables 1-5 are detected. In some methods, a set of polymorphisms or several SNPs in one MYH gene (e.g., MYH8, MYH2, MYH1, or MYH7B) or in one gene in myosin interactive network (e.g., 1 1 N, NEB or OBSCN) are detected. In some methods, a combination of at least one SNP in each of two or more MYH genes (e.g., MYH1, ΜΥΉ8, and ΜΥΉ7Β) are detected. In some other methods, a combination of at least one SNP in one MYH gene and at least one SNP in one gene in myosin interactive network are detected. In still some other embodiments, a combination of at least one SNP in each of two or more genes in the myosin interactive network (e.g., TTN, NEB and OBSCN) are detected.

[0040] In practicing the diagnostic or prognostic methods of the invention, the presence of polymorphisms described herein in the genome of a subject can be detected with many methods known in the art. Detection of the SNPs can be performed by analyzing a target nucleic acid (e.g., genomic DNA or cDNA) sample from the subject (e.g., a human patient). The target nucleic acid sample is typically isolated or generated with a biological sample (e.g., cells or tissues) obtained from the subject For assay of genomic DNA, virtually any biological sample (other than pure red blood cells) is suitable. For example, convenient tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal epithelium, skin and hair. For assay of cDNA or mRNA, the tissue sample must be obtained from an organ in which the target nucleic acid is expressed, e.g., the skeleton muscle.

Methods of preparing nucleic acids from biological samples are well known in the art and can be readily adapted to obtain a nucleic acid sample that is compatible with the detection methods of the invention. Automated sample preparation systems for extracting nucleic acids from a biological sample are commercially available, and examples are Qiagen's BioRobot 9600, Applied Biosystems' PRISM™ 6700 sample preparation system, and Roche Molecular Systems' COBAS AmpliPrep System.

[0041] Analysis of the target nucleic acid sample can be performed with many methods routinely practiced in the art. Typically, the methods employ agents that can specifically detect SNPs disclosed herein, e.g., via binding to and recognize sequence fragments in the target nucleic acid that correspond to the SNP. The agents preferably can differentiate between different alternative nucleotides at a target SNP position, thereby allowing the identity of the nucleotide present at the target SNP position to be determined. In various embodiments, the agents can hybridize to a target SNP-containing nucleic acid sequence by complementary base-pairing in a sequence specific manner, and discriminates the target variant sequence from other nucleic acid sequences. Examples of the SNP-detection agents suitable for the invention include a probe that hybridizes to a target nucleic acid containing one or more of the SNPs referred to in Tables 1-5. In a preferred embodiment, the probe can differentiate between nucleic acids having a particular nucleotide (allele) at a target SNP position from other nucleic acids that have a different nucleotide at the same target SNP position. Another example of the agents is a primer that acts as an initiation point of nucleotide extension along a complementary strand of a target polynucleotide. The SNP sequence information provided herein is also useful for designing primers, e.g., allele-specific primers, to amplify (e.g., using PCR) any SNP of the present invention. In some other embodiments, the employed SNP detection agent is an isolated or synthetic DNA or RNA polynucleotide probe or primer or PNA oligomer, or a combination of DNA, RNA and/or PNA, that hybridizes to a segment of a target nucleic acid molecule containing a SNP identified in Tables 1-5. A detection agent in the form of a polynucleotide may optionally contain modified base analogs, intercalators or minor groove binders. Multiple detection agents such as probes may be, for example, affixed to a solid support (e.g., arrays or beads) or supplied in solution (e.g., probe/primer sets for enzymatic reactions such as PCR, RT-PCR, TaqMan assays, or primer-extension reactions) in the diagnostic kits of the invention.

[0042] The primers or probes for detecting SNPs of the invention are typically at least about 8 nucleotides in length. In some embodiments, the primer or probe is at least about 10 nucleotides in length. In some embodiments, the primer or probe is at least about 12 nucleotides in length, or at least about 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. While the maximal length of a probe can be as long as the target sequence to be detected, depending on the type of assay in which it is employed, it is typically less than about SO, 60, 65, or 70 nucleotides in length. In the case of a primer, it is typically less than about 30 nucleotides in length. In some embodiments of the invention, the primer or probe is within the length of about 18 and about 28 nucleotides. However, in other embodiments, such as nucleic acid arrays and other embodiments in which probes are affixed to a substrate, the probes can be longer, such as on the order of 30-70, 75, 80, 90, 100, or more nucleotides in length.

[0043] For analyzing SNPs, it may be appropriate to use oligonucleotides specific for alternative SNP alleles. Such oligonucleotides that detect single nucleotide variations in target sequences may be referred to by such terms as "allele-specific oligonucleotides", "allele-specific probes", or "allele-specific primers". The design and use of allele-specific probes for analyzing polymorphisms is described in, e.g., Cotton et al. (ed.), Mutation Detection A Practical Approach, Oxford University Press, 1998; Saiki et al., Nature 324, 163-166 (1986); Dattagupta, EP235,726; and Saiki, WO 89/11548. The design of each allele-specific primer or probe depends on variables such as the precise composition of the nucleotide sequences flanking a SNP position in a target nucleic acid molecule, and the length of the primer or probe, and the stringency of the condition under which the hybridization between the probe or primer and the target sequence is performed. Higher stringency conditions utilize buffers with lower ionic strength and/or a higher reaction temperature, and tend to require a more perfect match between probe/primer and a target sequence in order to form a stable duplex. If the stringency is too high, however,

hybridization may not occur at all. In contrast, lower stringency conditions utilize buffers with higher ionic strength and/or a lower reaction temperature, and permit the formation of stable duplexes with more mismatched bases between a probe/primer and a target sequence. Optimal stringency of hybridization conditions for a given detection assay can be readily determined in accordance with protocols routinely practiced in the art.

[0044] Detection of specific SNPs or SNP genotyping can be accomplished with many methods well known in the art. These include, e.g., TaqMan assays, molecular beacon assays, nucleic acid arrays, allele-specific primer extension, allele-specific PCR, arrayed primer extension, homogeneous primer extension assays, primer extension with detection by mass spectrometry, pyrosequencing, multiplex primer extension sorted on genetic arrays, ligation with rolling circle amplification, homogeneous ligation, OLA (U.S. Pat. No.

4,988,167), multiplex ligation reaction sorted on genetic arrays, restriction-fragment length polymorphism, single base extension-tag assays, and the Invader assay. See, e.g., Chen et al., Pharmacogenomics J. 2003; 3:77-96; Kwok et al., Curr. Issues Mol. Biol. 2003; 5:43-60; Shi, Am J. Pharmacogenomics. 2002; 2:197-205; Kwok, Annu. Rev. Genomics Hum. Genet 2001; 2:235-58; and Marnellos, Curr. Opin. Drug Discov. Devel. 2003; 6:317-21. These methods may be used in combination with other routinely practiced detection mechanisms, e.g., luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, and electrical detection.

[0045] Many of the methods described herein for SNP detection require amplification of DNA from target samples. This can be accomplished by, e.g., PCR. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al, Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Patent 4,683,202 (each of which is incorporated by reference for all purposes). Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.

[0046] The identity of bases occupying the SNP positions (polymorphic sites) in the SNPs shown in Tables 1-5 can be determined by many routinely practiced methods. One method is single base extension methods. Single base extension methods are described in, e.g., US 5,846,710, US 6,004,744, US 5,888,819 and US 5,856,092. In brief, the methods work by hybridizing a primer that is complementary to a target sequence such that the 3' end of the primer is immediately adjacent to but does not span a site of potential variation in the target sequence. That is, the primer comprises a subsequence from the complement of a target polynucleotide terminating at the base that is immediately adjacent and 5' to the polymorphic site. The hybridization is performed in the presence of one or more labelled nucleotides complementary to base(s) that may occupy the site of potential variation. For example, for a biallelic polymorphisms two differentially labelled nucleotides can be used. For a tetraallelic polymorphisms four differentially labelled nucleotides can be used. In some methods, particularly methods employing multiple differentially labelled nucleotides, the nucleotides are dideoxynucleotides. Hybridization is performed under conditions permitting primer extension if a nucleotide complementary to a base occupying the site of variation in the target sequence is present Extension incorporates a labelled nucleotide thereby generating a labelled extended primer. If multiple differentially labelled nucleotides are used and the target is heterozygous then multiple differentially labelled extended primers can be obtained. Extended primers are detected providing an indication of which bass occupy the site of variation in the target polynucleotide.

[0047] Another method for determining identity of residues at the SNP position is via the use of allele-specific probes. The design and use of allele-specific probes for analyzing polymorphisms is described by e.g., Saiki et al., Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11S48. Allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymorphisms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15 mer at the 7 position; in a 16 mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms.

[0048] Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence. The polymorphisms can also be identified by hybridization to nucleic acid arrays, some examples of which are described by WO 95/11995 (incorporated by reference in its entirety for all purposes). In some embodiments, the polymorphisms in myosin genes or myosin interactive network genes described herein can be detected via commercially available micro arrays that are specifically designed for detecting SNPs. For example, human SNPs may be detected with Affymetrix Genome-Wide Human SNP Nsp/Sty Assay Kit and the Affymetrix MDA genotyping array (Yang et al., Nat. Methods 6: 663-666, 2009). [0049] Allele-specific amplification methods can also be used in the practice of the invention. With these methods, an allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarily. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer that hybridizes at a distal site. Amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarily to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. In some methods, the mismatch is included in the 3'-most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer. See, e.g., WO 93/22456.

[0050] In some embodiments, direct-sequencing can be employed in determining identity of the nucleotide residue at the polymorphic sites of the variant alleles shown in Tables 1-5. The direct analysis of the sequence of polymorphisms of the present invention can be accomplished using either the dideoxy- chain termination method or the Maxam-Gilbert method (see Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)). In some other embodiments, denaturing gradient gel electrophoresis may be used. Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Brlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992), Chapter 7. In still some other embodiments, single-strand conformation polymorphism analysis is suitable for practicing the methods of the invention. Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures that are partially dependent on the base sequence. The different electrophoretic mobilities of single- stranded amplification products can be related to base-sequence difference between alleles of target sequences.

V. Diagnosing and prognosing increased risk of thrombosis and related applications

[0051] The present invention provides methods of SNP genotyping for uses in various clinical or therapeutic applications, e.g., screening for venous thrombosis or determining predisposition thereto. SNP genotyping is a process to determine which specific nucleotide (i.e., allele) is present at each of one or more SNP positions, such as the SNPs listed in Tables 1-5. As described above, nucleic acid samples can be genotyped to determine which allele(s) is/are present at any given genetic region (e.g., SNP position) of interest by methods well known in the art. Detection of the SNPs or variant alleles in myosin genes or genes in the myosin interactive network as disclosed herein can have a number of clinical applications. In some embodiments, detection of the polymorphisms described herein is useful in diagnosing or confirming diagnosis of risk for thrombosis based on symptoms or susceptibility thereto in the subject. In some embodiments, detection of the variant alleles allow one to assess the risk of developing venous thrombosis (VTB) or specific sub-phenotypes including recurrent VTE, PE and idiopathic VTE. In some embodiments, detection of one or more SNPs in the myosin genes or related genes as described herein can enable selection or identification of subjects who may be in need of aggressive antithrombotic treatment or in need of prophylactic antithrombotic therapy. Another clinical application is to apply the information of detected SNPs in assessing the need for taking preventive measures against developing these conditions and likely response to drugs used to treat these conditions.

[0052] In general, a genetic association between one or more specific SNPs and a particular phenotypic trait of interest (e.g., VT or a subset thereof) needs to be established for using SNP genotyping in the various clinical applications described herein. The genetic association study can be carried out in line with methods well known in the art. See, e.g., Modern Epidemiology, Lippincott Williams & Wilkins (1998), 609-622; Genetic Data Analysis, Weir B., Sinauer (1990); Applied Logistic Regression, Hosmer and Lemeshow, Wiley (2000); Daly et al, Nature Genetics, 29, 232-235, 2001 ; Schaid et al, Am. J. Hum. Genet., 70, 425-434, 2002; Multiple comparisons and multiple tests, Westfall et al, SAS Institute (1999); Benjamini and Hochberg, Journal of the Royal Statistical Society, Series B 57, 1289-1300, 1995; Resampling-based Multiple Testing, Westfall and Young, Wiley (1993); Ewens and Spielman, Am. J. Hum. Genet 62, 450-458, 1995; Pritchard et al. Am. J. Hum. Gen. 1999, 65:220-228; Devlin et al. Biometrics 1999, 55:997-1004; and Devlin et al. Genet. Epidem.20001, 21:273-284. As exemplification, correlation of detected polymorphisms with VTE or subtypes thereof is performed for a population of individuals who have been tested for the presence or absence of thrombosis (or an intermediate phenotype) and for one or more the SNPs. As exemplified herein in Table 1 and Table 6, the presence or absence of one or a set of the SNPs is determined for a set of the individuals, some of whom exhibit a particular trait, and some of which exhibit lack of the trait. The alleles of each polymorphism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the trait of interest. Correlation can be performed by standard statistical methods including chi -squared test, Analysis of Variance, parametric linkage analysis, non-parametric linkage analysis, and statistically significant correlations between polymorphism and phenotypic characteristics are noted. For example, it may be found that (he presence of variant A (e.g., rs 111 S67318 in MYH8 gene) correlates with VTE. As a further example, it might be found that the combined presence of variant A and variant B at another allele (e.g., rs200843338 in TTN gene) correlates with venous thrombosis or a sub-phenotype.

[0053] An established association/correlation between genotypes and disease-related phenotypes can be useful in many settings. For example, in the case of a highly statistically significant association between one or more SNPs with predisposition to a disease for which treatment is available, detection of such a genotype pattern in a subject may justify immediate administration of treatment, or at least the institution of regular monitoring of the subject. Detection of the susceptibility alleles associated with serious disease in a couple

contemplating having children may also be valuable to the couple in their reproductive decisions. In the case of a weaker but still statistically significant association between a SNP and a human disease, immediate therapeutic intervention or monitoring may not be justified after detecting the susceptibility allele or SNP. Nevertheless, the subject can be motivated to begin simple life-style changes (e.g., diet, exercise) that can be accomplished at little or no cost to the individual but would confer potential benefits in reducing the risk of developing conditions for which that individual may have an increased risk by virtue of having the risk allele(s).

[0054] The methods of the present invention in general are not intended to provide an absolute identification of subjects who have or are at risk of developing VT or related conditions. Rather, a positive diagnosis provides an indication (hat there is a higher degree or likelihood that the subject has VT, or is under an increased risk of developing VT, as compared to control subjects or general population. Also, while detection of one SNP disclosed herein may indicates or suggest a positive diagnosis, combined detection of several or a set of such polymorphisms typically increases the probability of an accurate diagnosis. For example, the presence of a single polymorphic form known to correlate with thrombosis might indicate a probability of 20% that an individual is susceptible to thrombosis, whereas detection of five polymorphisms, each of which correlates with less than 20% probability, might indicate a probability up to 80% that an individual has or is susceptible to thrombosis. In some embodiments, detection of multiple SNPs in several genes shown in Tables 1-5 may also enhance accuracy of the diagnostic or prognostic methods of the invention.

[0055] Some methods of the invention are directed to detection of a plurality of SNPs in linkage disequilibrium (LD). For diagnostic purposes and similar uses described herein, if a particular SNP site is found to be useful for diagnosing of risk for VT (e.g., has a significant statistical association with the condition and/or is recognized as a causative polymorphism for the condition), then the skilled artisan would recognize that other SNP sites which are in LD with this SNP site would also be useful for diagnosing the condition. Thus, polymorphisms (e.g., SNPs and/or haplotypes) that are not the actual disease-causing (causative)

polymorphisms, but are in LD with such causative polymorphisms, are also useful. In such instances, the genotype of the polymorphism(s) that is/are in LD with the causative polymorphism is predictive of the genotype of the causative polymorphism and,

consequently, predictive of the phenotype (e.g., VT) that is influenced by the causative SNP(s). Therefore, polymorphic markers that are in LD with causative polymorphisms are useful as diagnostic markers, and are particularly useful when the actual causative

polymorphism^) is/are unknown.

(0056] The diagnosis or prognosis methods of the invention rely on the detection of one or more SNPs shown in Tables 1-5. Some methods of the invention require the detection of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the SNPs. In some methods, the detected plurality of SNPs are all present in one of the genes shown in Tables 1-5 (e.g., one MYH gene or one gene in the myosin interactive network). In some methods, the detected plurality of SNPs of detected are present in multiple genes shown in Tables 1-5. For example, the methods can require the detection of one or more SNPs present in each of 2 or more myosin genes as shown in Tables 1-3. The methods can also require the detection of one or more SNPs present in each of 2 or more genes in the myosin interactive network as shown in Tables 4 and 5. In some other embodiments, the diagnostic or prognostic methods of the invention require the detection of a combination of (1) one or more SNPs that are present in each of at least one myosin gene as shown in Tables 1-3 and (2) one or more SNPs that are present in each of at least one gene in the myosin interactive network as shown in Tables 4 and 5.

[0057] In some embodiments, detection of the polymorphisms shown in Tables 1-5 can be combined with analysis of polymorphisms in other genes or other known risk factors of venous thrombosis such as family history. In some embodiments, in addition to detecting one or more SNPs described in Tables 1-5, the methods of the invention can additionally detect other SNPs or variant alleles known in the art that are associated with the existence of or implicated in the development of venous thrombosis. One of such well known venous thrombosis related SNPs is listed in Table 6, rs6025, which is present in Factor V (F5 gene). Many other SNPs in various genes that are known to be associated with or linked to venous thrombosis are also described in the art. See, e.g., de Hann et al., Blood 120, 656-663, 2012; van Hylckama Vlieg et al., Circ. Cardiovasc. Genet. 7, 806-813, 2014; and Bruzelius et al., J. Thromb. Haemost. 13, 219-227, 2015. Examples include, e.g., SNPs rsl799963, rs8176719, rs2066865, rs2036914, rs2069951, rs2289252, rs4149755, rs2069952, rs2227589, rsl69713, rs3136516, rsl800595, rsl799809, rs867186, rsl063856, rsl613662, rs3136520, rsl800291, rsl039084, rs2001490, rs6003, rs670659, rs6048, rs4524, rs5985, 1208 indel, rs8176592, rs3822057, rsl523127, rs3742264, rs514659, rsl799810 and rs710446. The detection of one or more of these known thrombosis related polymorphism sites along with the SNPs in die myosin gene clustering region or genes in myosin interactive network described herein can further aid the diagnosis or prognosis of venous thrombosis.

[0058] In some embodiments, detection of the SNPs disclosed herein can also be useful for conducting clinical trials of drug candidates for treating or preventing thrombosis. Such trials are performed on treated or control populations having similar or identical polymorphic profiles at a defined collection of polymorphic sites. Use of genetically matched populations eliminates or reduces variation in treatment outcome due to genetic factors, leading to a more accurate assessment of the efficacy of a potential drug. Furthermore, the polymorphisms of the invention may be used after the completion of a clinical trial to elucidate differences in response to a given treatment. For example, the set of polymorphisms may be used to stratify the enrolled patients into disease sub-types or classes. It may further be possible to use the polymorphisms to identify subsets of patients with similar polymorphic profiles who have unusual (high or low) response to treatment or who do not respond at all (non-re sponders). In this way, information about the underlying genetic factors influencing response to treatment can be used in many aspects of the development of treatment (these range from the identification of new targets, through the design of new trials to product labeling and patient targeting). Additionally, the polymorphisms may be used to identify the genetic factors involved in adverse response to treatment (adverse events). For example, patients who show adverse response may have more similar polymorphic profiles than would be expected by chance. This would allow the early identification and exclusion of such individuals from treatment. It would also provide information that might be used to understand the biological causes of adverse events and to modify the treatment to avoid such outcomes.

VI. Diagnostic kits and systems

[0059] The invention further provides diagnostic kite or systems for carrying out the various diagnostic or prognostic methods of the invention. The diagnostic kits or systems typically contain agents that are capable of detecting one or more of the specific SNPs disclosed herein (e.g., allele-specific oligonucleotide probe pairs). In addition to the SNP- detection agents described above, the kits can also contain other components (e.g., a buffer, enzymes such as DNA polymerases or ligases, chain extension nucleotides such as deoxynucleotide triphosphates, and in the case of Sanger-type DNA sequencing reactions, chain terminating nucleotides, positive control sequences, negative control sequences, and the like) necessary to carry out an assay or reactions required for performing the diagnostic methods of the invention, such as amplification and/or detection of a SNP-containing nucleic acid molecule. The kits may further contain means for determining the amount of a target nucleic acid, and means for comparing the amount with a standard, and can comprise instructions for using the kit to detect the SNP-containing nucleic acid molecule of interest. For example, the kits can contain an instruction sheet which contains description of using the kits for diagnosing or prognosing venous thrombosis in human subjects. In some

embodiment of the present invention, kits are provided which contain the necessary reagents to carry out one or more assays to detect one or more SNPs disclosed herein. In some embodiments, the diagnostic kits/systems contain nucleic acid arrays, or compartmentalized kits, including m icrofluidic/lab-on-a-ch ip systems.

[0060] In some embodiments, the diagnostic kits or systems of the invention encompass combinations of multiple SNP detection reagents, or one or more SNP detection reagents in combination with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packages such as packaging intended for commercial sale, substrates to which SNP detection reagents are attached, electronic hardware components, etc.). In some embodiments, the diagnostic kits and systems, including but not limited to, packaged probe and primer sets (e.g., TaqMan probe/primer sets), arrays/m icroarrays of nucleic acid molecules, and beads that contain one or more probes, primers, or other detection reagents for detecting one or more SNPs of the present invention. The kits/systems can optionally include various electronic hardware components; for example, arrays ("DNA chips") and microfluidic systems ("lab-on-a-chip" systems) provided by various

manufacturers typically comprise hardware components. Other kits/systems (e.g., probe/primer sets) may not include electronic hardware components, but may be comprised of, for example, one or more SNP detection reagents (along with, optionally, other biochemical reagents) packaged in one or more containers. In some embodiments, the diagnostic kits of the present invention can also include materials or reagents that are needed to prepare the target nucleic acid samples from subjects in need of the diagnostic test, which are used in the subsequent amplification and/or detection of a SNP-containing nucleic acid molecule.

[0061] In various embodiments, the diagnostic kits/systems may contain, for example, one or more probes, or pairs of probes, that hybridize to a nucleic acid molecule at or near each target SNP position. Multiple pairs of allele-specific probes may be included in the kit/system to simultaneously assay large numbers of SNPs, at least one of which is a SNP disclosed herein (Tables 1-5). In some kits/systems, the allele-specific probes are

immobilized to a substrate such as an array or bead. For example, the same substrate can comprise allele-specific probes for detecting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or any number of or all of the SNPs shown in Tables 1-5. The kits can also additionally include detection agents (e.g., probes) for detecting other SNPs or variant alleles known to be linked to venous thrombosis.

[0062] The arrays ("microarrays" or "DNA chips") in the kits of the invention can be any array of distinct polynucleotides or oligonucleotides affixed to a substrate, such as glass, plastic, paper, nylon or other type of membrane, filter, chip, or any other suitable solid support. The oligonucleotides or polynucleotides can be synthesized directly on the substrate, or synthesized separate from the substrate and then affixed to the substrate. They can be prepared in accordance with methods routinely practiced in the art. See, e.g., U.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680), Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), Brown et al. (U.S. Pat. No. 5,807,522), Zammatteo et al. ("New chips for molecular biology and diagnostics", Biotechnol. Annu. Rev. 2002; 8:85-101), Sosnowski et al. ("Active microelectronic array system for DNA hybridization, genotyping and

pharmacogenomic applications", Psychiatr Genet. 2002, 12:181-92), Heller ("DNA microarray technology: devices, systems, and applications", Annu Rev Biomed Eng. 2002; 4: 129-53. Epub 2002 Mar. 22), Kolchinsky et al. ("Analysis of SNPs and other genomic variations using gel-based chips", Hum. Mutat. 2002, 19:343-60), and McGall et al. ("High- density genechip oligonucleotide probe arrays", Adv. Biochem. Eng. Biotechnol. 2002; 77:21-42).

[0063] Some diagnostic kits of the invention are compartmentalized kits. These kits include any kit in which reagents are contained in separate containers. Such containers include, for example, small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the test samples and reagents are not cross-contaminated, or from one container to another vessel not included in the kit, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another or to another vessel. Such containers may include, for example, one or more containers which will accept the test sample, one or more containers which contain at least one probe or other SNP detection reagent for detecting one or more

SNPs of the present invention, one or more containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and one or more containers which contain the reagents used to reveal the presence of the bound probe or other SNP detection reagents. The kit can optionally further comprise compartments and/or reagents for, for example, nucleic acid amplification or other enzymatic reactions such as primer extension reactions, hybridization, ligation, electrophoresis (preferably capillary electrophoresis), mass spectrometry, and/or laser-induced fluorescent detection. The kit may also include instructions for using the kit. Exemplary compartmentalized kits include microfluidic devices known in the art (see, e.g., Weigl et al., "Lab-on-a-chip for drug development", Adv.

Drug Deliv. Rev. 2003 Feb. 24; 55(3):349-77). In such microfluidic devices, the containers may be referred to as, for example, microfluidic "compartments", "chambers", or "channels"

***

[0064] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. [0065] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing or prognosing an increased risk of developing a venous thrombosis in a subject, comprising (a) obtaining a nucleic acid sample from the subject, and (b) determining in the nucleic acid sample one or more single nucleotide polymorphisms (SNPs) shown in Tables 1-5; thereby diagnosing the existence of an increased risk of developing a venous thrombosis in the subject.

2. The method of claim 1, wherein the nucleic acid sample comprises genomic DNA of the subject.

3. The method of claim 1, wherein the genomic DNA is extracted from a blood tissue sample of the subject.

4. The method of claim 1 , wherein the nucleic acid sample comprises cDNA or mRNA of the subject.

5. The method of claim 1 , wherein the cDNA or mRNA is prepared from a skeletal muscle sample of the subject.

6. The method of claim 1 , wherein the subject is a human.

7. The method of claim 1, wherein the SNPs are detected with a DNA array.

8. The method of claim 1, wherein the detected SNPs comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more SNPs shown in Tables 1-5.

9. The method of claim 1, wherein the detected SNPs comprise SNPs in the myosin gene cluster shown in Tables 1-3.

10. The method of claim 9, wherein the detected SNPs comprise at least 2, 3, 4, or more SNPs present in one myosin gene shown in Tables 1-3.

11. The method of claim 9, wherein the detected SNPs comprise at least one SNP present in each of 2, 3, or 4 different myosin genes shown in Tables 1-3.

12. The method of claim 1, wherein the detected SNPs comprise SNPs in genes of myosin interactive network shown in Tables 4 and 5.

13. The method of claim 12, wherein the detected SNPs comprise at least 2, 3, 4, or more SNPs present in one gene shown in Tables 4 and 5.

14. The method of claim 12, wherein the detected SNPs comprise at least one SNP present in each of 2, 3, 4 or 5 different genes shown in Tables 4 and 5.

15. The method of claim 1, wherein the detected SNPs comprise a combination of (a) at least one SNP present in myosin genes as shown in Tables 1-3, and (b) at least one SNP present in genes of myosin interactive network as shown in Tables 4 and 5.

16. The method of claim IS, wherein the detected SNPs comprise a combination of (a) at least 2, 3, 4, 5, 6 or more SNPs present in myosin genes shown in Tables 1-3; and (b) at least 2, 3, 4, 5, 6 or more SNPs present in genes of myosin interactive network shown in Tables 4 and 5.

17. The method of claim 15, wherein the detected SNPs comprise a combination of (a) at least one SNP in each of 2, 3, or 4 different myosin genes shown in Tables 1-3 and (b) at least one SNP present in each of 2, 3, 4 or 5 different genes shown in Tables 4 and S.

18. The method of claim 1, further comprising detecting in the nucleic acid sample at least one additional SNP or variant allele that is known to be associated with or linked to venous thrombosis.

19. The method of claim 18, wherein the at least one additional SNP or variant allele is selected from the groups consisting of SNPs rs6025, rsl799963, rs8176719, rs2066865, rs2036914, rs2069951, rs2289252, rs4149755, «2069952, rs2227589, rsl69713, rs3136516, rsl800595, rsl799809, rs867186, rsl063856, rsl613662, rs3136520, rsl800291, rsl039084, rs2001490, rs6003, rs670659, rs6048, rs4524, rs5985, 1208 indel, rs8176592, rs3822057, rsl523127, rs3742264, rs514659, rsl799810 and rs710446.

20. A method of identifying patients that may be in need of antithrombotic therapy or prophylactic antithrombotic therapy, comprising (a) obtaining a nucleic acid sample from each of a population of candidate subjects, and (b) detennining in the nucleic acid sample one or more single nucleotide polymorphisms (SNPs) shown in Tables 1-5, thereby identifying patients that may be in need of antithrombotic therapy or prophylactic antithrombotic therapy.

21. A kit, comprising (a) at least one pair of allele-specific oligonucleotides that are capable of detecting the single nucleotide polymorphisms (SNPs) shown in Tables 1-5, and (b) an instruction sheet containing description of using the of allele-specific oligonucleotides for diagnosing or prognosing venous thrombosis.