EP1240354A2

EP1240354A2 - Single nucleotide polymorphisms in genes

Info

Publication number: EP1240354A2
Application number: EP00959964A
Authority: EP
Inventors: Eric S. Lander; Michele Gargill; James S. Ireland; Stacey Bolk; George Q. Daley; Jeanette J. Mccarthy
Original assignee: Whitehead Institute for Biomedical Research; Millennium Pharmaceuticals Inc
Current assignee: Whitehead Institute for Biomedical Research; Millennium Pharmaceuticals Inc
Priority date: 1999-09-10
Filing date: 2000-09-07
Publication date: 2002-09-18
Also published as: AU7119400A; WO2001018250A2; WO2001018250A3

Abstract

The invention provides nucleic acid segments of the human genome, particularly nucleic acid segments from a gene, including polymorphic sites. Allele-specific primers and probes hybridizing to regions flanking or containing these sites are also provided. The nucleic acids, primers and probes are used in applications such as phenotype correlations, forensics, paternity testing, medicine and genetic analysis. A role for the thrombospondin gene(s) in vascular disease is also disclosed. Use of single nucleotide polymorphisms in the thrombospondin gene(s) for diagnosis, prediction of clinical course and treatment response, development of therapeutics and development of cell-culture-based and animal models for research and treatment are disclosed.

Description

SINGLE NUCLEOTIDE POLYMORPHISMS IN GENES

BACKGROUND OF THE INVENTION

The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution, generating variant forms of progenitor nucleic acid sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). The variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form, or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymoφhisms.

Several different types of polymoφhism have been reported. A restriction fragment length polymoφhism (RFLP) is a variation in DNA sequence that alters the length of a restriction fragment (Botstein et al., Am. J. Hum. Genet. 32, 314-331 (1980)). The restriction fragment length polymoφhism may create or delete a restriction site, thus changing the length of the restriction fragment. RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; W090/11369; Donis-Keller, Cell 51, 319-337 (1987); Lander et al., Genetics 121, 85-99 (1989)). When a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the animal will also exhibit the trait.

Other polymoφhisms take the form of short tandem repeats (STRs) that include tandem di-, tri- and tetra-nucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymoφhisms. VNTRs have been used in identity and paternity analysis (US 5,075,217; Armour et al, FEBS Lett. 307, 113-115 (1992); Horn et al., WO 91/14003; Jeffreys, EP 370,719), and in a large number of genetic mapping studies.

Other polymoφhisms take the form of single nucleotide variations between individuals of the same species. Such polymoφhisms are far more frequent than RFLPs, STRs and VNTRs. Some single nucleotide polymoφhisms (SNP) occur in protein-coding nucleic acid sequences (coding sequence SNP (cSNP)), in which case, one of the polymoφhic forms may give rise to the expression of a defective or otherwise variant protein and, potentially, a genetic disease. Examples of genes in which polymoφhisms within coding sequences give rise to genetic disease include β-globin (sickle cell anemia), apoE4 (Alzheimer's Disease), Factor V Leiden (thrombosis), and CFTR (cystic fibrosis). cS Ps can alter the codon sequence of the gene and therefore specify an alternative amino acid. Such changes are called "missense" when another amino acid is substituted, and "nonsense" when the alternative codon specifies a stop signal in protein translation. When the cSNP does not alter the amino acid specified the cSNP is called "silent".

Other single nucleotide polymoφhisms occur in noncoding regions. Some of these polymoφhisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymoφhisms have no phenotypic effects. Single nucleotide polymoφhisms can be used in the same manner as

RFLPs and VNTRs, but offer several advantages. Single nucleotide polymoφhisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymoφhism. The greater frequency and uniformity of single nucleotide polymoφhisms means that there is a greater probability that such a polymoφhism will be found in close proximity to a genetic locus of interest than would be the case for other polymoφhisms. The different forms of characterized single nucleotide polymoφhisms are often easier to distinguish than other types of polymoφhism (e.g., by use of assays employing allele-specific hybridization probes or primers). Only a small percentage of the total repository of polymoφhisms in humans and other organisms has been identified. The limited number of polymoφhisms identified to date is due to the large amount of work required for their detection by conventional methods. For example, a conventional approach to identifying polymoφhisms might be to sequence the same stretch of DNA in a population of individuals by dideoxy sequencing. In this type of approach, the amount of work increases in proportion to both the length of sequence and the number of individuals in a population and becomes impractical for large stretches of DNA or large numbers of persons.

SUMMARY OF THE INVENTION

Work described herein pertains to the identification of polymoφhisms which can predispose individuals to disease, by resequencing large numbers of genes in a large number of individuals. Various genes from a number of individuals have been resequenced as described herein, and SNPs in these genes have been discovered (see the Table and Fig. 3). Some of these SNPs are cSNPs which specify a different amino acid sequence, some of the SNPs are silent cSNPs and some of these cSNPs specify a stop signal in protein translation. Some of the identified SNPs were located in non-coding regions.

The invention relates to a gene which comprises a single nucleotide polymoφhism at a specific location. In a particular embodiment the invention relates to the variant allele of a gene having a single nucleotide polymoφhism, which variant allele differs from a reference allele by one nucleotide at the site(s) identified in the Table and Fig. 3. Complements of these nucleic acid sequences are also included. The nucleic acid molecules can be DNA or RNA, and can be double- or single-stranded. Nucleic acid molecules can be, for example, 5-10, 5-15, 10-20, 5-25, 10-30, 10-50 or 10-100 bases long.

The invention further provides allele-specific oligonucleotides that hybridize to the reference or variant allele of a gene comprising a single nucleotide polymoφhism or to the complement thereof. These oligonucleotides can be probes or primers.

The invention further provides a method of analyzing a nucleic acid from an individual. The method determines which base is present at any one of the polymoφhic sites shown in the Table and/or Fig. 3. Optionally, a set of bases occupying a set of the polymoφhic sites shown in the Table and /or Fig. 3 is determined. This type of analysis can be performed on a number of individuals, who are tested for the presence of a disease phenotype. The presence or absence of disease phenotype is then correlated with a base or set of bases present at the polymoφhic site or sites in the individuals tested. Thus, the invention further relates to a method of predicting the presence, absence, likelihood of the presence or absence, or severity of a particular phenotype or disorder associated with a particular genotype. The method comprises obtaining a nucleic acid sample from an individual and determining the identity of one or more bases (nucleotides) at polymoφhic sites of genes described herein, wherein the presence of a particular base is correlated with a specified phenotype or disorder, thereby predicting the presence, absence, likelihood of the presence or absence, or severity of the phenotype or disorder in the individual.

The thrombospondins are a family of extracellular matrix (ECM) glycoproteins that modulate many cell behaviors including adhesion, migration, and proliferation. Thrombospondins (also known as thrombin sensitive proteins or TSPs) are large molecular weight glycoproteins composed of three identical disulfide-linked polypeptide chains. The results described herein also reveal an important association between alterations, particularly SNPs, in TSP genes, particularly TSP-1 and TSP-4, and vascular disease. In particular, SNPs in these genes which are associated with premature coronary artery disease (CAD)(or coronary heart disease) and myocardial infarction (MI) have been identified and represent a potentially vital marker of upstream biology influencing the complex process of atherosclerotic plaque generation and vulnerability.

Thus, the invention relates to the TSP gene SNPs identified as described herein, both singly and in combination, as well as to the use of these SNPs, and others in TSP genes, particularly those nearby in linkage disequilibrium with these SNPs, for diagnosis, prediction of clinical course and treatment response for vascular disease, development of new treatments for vascular disease based upon comparison of the variant and normal versions of the gene or gene product, and development of cell-culture based and animal models for research and treatment of vascular disease. The invention further relates to novel compounds and pharmaceutical compositions for use in the diagnosis and treatment of such disorders. In preferred embodiments, the vascular disease is CAD or MI.

The invention relates to isolated nucleic acid molecules comprising all or a portion of the variant allele of TSP-1 (e.g., as exemplified by SEQ ID NO: 1), and to isolated nucleic acid molecules comprising all or a portion of the variant allele of TSP-4 (e.g., as exemplified by SEQ ID NO: 3). Preferred portions are at least 10 contiguous nucleotides and comprise the polymoφhic site, e.g., a portion of SEQ ID NO: 1 which is at least 10 contiguous nucleotides and comprises the "G" at position 2210, or a portion of SEQ ID NO: 3 which is at least 10 contiguous nucleotides and comprises the "C" at position 1186. The invention further relates to isolated gene products, e.g., polypeptides or proteins, which are encoded by a nucleic acid molecule comprising all or a portion of the variant allele of TSP-1 or TSP-4 (e.g., SEQ ID NO: 1 or SEQ ID NO: 3, respectively). The invention also relates to nucleic acid molecules which hybridize to and/or share identity with the variant alleles identified herein (or their complements) and which also comprise the variant nucleotide at the SNP site.

The invention further relates to isolated proteins or polypeptides comprising all or a portion of the variant amino acid sequence of TSP-1 (e.g., as exemplified by SEQ ID NO: 2), and to isolated proteins or polypeptides comprising all or a portion of the variant amino acid sequence of TSP-4 (e.g., as exemplified by SEQ ID NO: 4). Preferred polypeptides are at least 10 contiguous amino acids and comprise the polymoφhic amino acid, e.g., a portion of SEQ ID NO: 2 which is at least 10 contiguous amino acids and comprises the serine at residue 700, or a portion of SEQ ID NO: 4 which is at least 10 contiguous amino acids and comprises the proline at residue 387. The invention further relates to isolated nucleic acid molecules encoding such proteins and polypeptides, as well as to antibodies which bind, e.g., specifically, to such proteins and polypeptides.

The invention further relates to a method of diagnosing or aiding in the diagnosis of a disorder associated with the presence of one or more of (a) a G at nucleotide position 2210 of SEQ ID NO: 1 ; or (b) a C at nucleotide position 1186 of SEQ ID NO: 3 in an individual. The method comprises obtaining a nucleic acid sample from the individual and determining the nucleotide present at one or more of the indicated nucleotide positions, wherein presence of one or more of (a) a G at nucleotide position 2210 of SEQ ID NO: 1 ; or (b) a C at nucleotide position 1186 of SEQ ID NO: 3 is indicative of increased likelihood of said disorder in the individual as compared with an appropriate control, e.g., an individual having the reference nucleotide at one or more of said positions. In a particular embodiment the disorder is a vascular disease selected from the group consisting of atherosclerosis, coronary heart or artery disease, MI, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism. In a preferred embodiment, the vascular disease is selected from the group consisting of CAD and MI. The invention further relates to a method of diagnosing or aiding in the diagnosis of a disorder associated with one or more of (a) a G at nucleotide position 2210 of SEQ ID NO: 1; or (b) a C at nucleotide position 1186 of SEQ ID NO: 3 in an individual. The method comprises obtaining a nucleic acid sample from the individual and determining the nucleotide present at one or more of the indicated nucleotide positions, wherein presence of one or more of (a) an A at nucleotide position 2210 of SEQ ID NO: 1; or (b) a G at nucleotide position 1186 of SEQ ID NO: 3 is indicative of decreased likelihood of said disorder in the individual as compared with an appropriate control, e.g., an individual having the variant nucleotide at said position. In a particular embodiment the disorder is a vascular disease selected from the group consisting of atherosclerosis, coronary heart or artery disease, MI, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism. In a preferred embodiment, the vascular disease is selected from the group consisting of CAD and MI.

In one embodiment, the invention relates to a method for predicting the likelihood that an individual will have a vascular disease (or aiding in the diagnosis of a vascular disease), comprising the steps of obtaining a DNA sample from an individual to be assessed and determining the nucleotide present at one or more of nucleotide positions 2210 of SEQ ID NO: 1 or 1186 of SEQ ID NO: 3. The presence of the reference nucleotide at one or more of these positions indicates that the individual has a lower likelihood of having a vascular disease than an individual having the variant nucleotide at one or more of these positions, or a lower likelihood of having severe symptomology. In a particular embodiment, the individual is an individual at risk for development of a vascular disease.

The invention further relates to a method of diagnosing or aiding in the diagnosis of a disorder associated with the presence of one or more of (a) a serine at amino acid position 700 of SEQ ID NO: 2; or (b) a proline at amino acid position 387 of SEQ ID NO: 4 in an individual. The method comprises obtaining a biological sample containing the TSP-1 and/or TSP-4 protein or relevant portion thereof from the individual and determining the amino acid present at one or more of the indicated amino acid positions, wherein presence of one or more of (a) a serine at amino acid position 700 of SEQ ID NO: 2; or (b) a proline at amino acid position 387 of SEQ ID NO: 4 is indicative of increased likelihood of said disorder in the individual as compared with an appropriate control, e.g., an individual having the reference amino acid at one or more of said positions.

The invention further relates to a method of diagnosing or aiding in the diagnosis of a disorder associated with one or more of (a) a serine at amino acid position 700 of SEQ ID NO: 2; or (b) a proline at amino acid position 387 of SEQ ID NO: 4 in an individual. The method comprises obtaining a biological sample containing the TSP-1 and/or TSP-4 protein or relevant portion thereof from the individual and determining the amino acid present at one or more of the indicated amino acid positions, wherein presence of one or more of (a) an asparagine at amino acid position 700 of SEQ ID NO: 2; or (b) an alanine at amino acid position 387 of SEQ ID NO: 4 is indicative of decreased likelihood of said disorder in the individual as compared with an appropriate control, e.g., an individual having the variant amino acid at one or more of said positions. In one embodiment, the invention relates to a method for predicting the likelihood that an individual will have a vascular disease (or aiding in the diagnosis of a vascular disease), comprising the steps of obtaining a biological sample comprising the TSP-1 and/or TSP-4 protein or relevant portion thereof from an individual to be assessed and determining the amino acid present at one or more of amino acid positions 700 of SEQ ID NO: 2 or 387 of SEQ ID NO: 4. The presence of the reference amino acid at one or more of these positions indicates that the individual has a lower likelihood of having a vascular disease than an individual having the variant amino acid at one or more of these positions, or a lower likelihood of having severe symptomology. In a particular embodiment, the individual is an individual at risk for development of a vascular disease.

In another embodiment, the invention relates to pharmaceutical compositions comprising a reference TSP-1 and/or TSP-4 gene or gene product, or active portion thereof, for use in the treatment of vascular diseases. The invention further relates to the use of agonists and antagonists of TSP-1 and TSP-4 activity for use in the treatment of vascular diseases. In a particular embodiment the vascular disease is selected from the group consisting of atherosclerosis, coronary heart or artery disease, MI, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism. In a preferred embodiment, the vascular disease is selected from the group consisting of CAD and MI.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-1D show the reference nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences for TSP-1.

Figs. 2A-2C show the reference nucleotide (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4) sequences for TSP-4.

Fig. 3 shows a table providing detailed information about the SNPs identified herein. Column one shows the internal polymoφhism identifier. Column two shows the accession number for the reference sequence in the TIGR database

(http://www.tigr.org/tdb/hgi/searching/hgi_reports.html). Column three shows the nucleotide position for the SNP iste. Column four shows the gene in which the polymoφhism was identified. Column five shows the polymoφhic site and additional flanking sequence on each side of the polymoφhism. Column six shows the type of mutation produced by the polymoφhism. Columns seven and eight show the reference and alternate (variant) nucleotides, respectively, for the SNP. Columns nine and ten show the reference and alternate (variant) amino acids, respectively, encoded by the alleles of the gene. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a gene which comprises a single nucleotide polymoφhism (SNP) at a specific location. The gene which includes the SNP has at least two alleles, referred to herein as the reference allele and the variant allele. The reference allele (prototypical or wild type allele) has been designated arbitrarily and typically corresponds to the nucleotide sequence of the gene which has been deposited with GenBank or TIGR under a given Accession number. The variant allele differs from the reference allele by one nucleotide at the site(s) identified in the Table. The present invention also relates to variant alleles of the described genes and to complements of the variant alleles. The invention also relates to nucleic acid molecules which hybridize to and/or share identity with the variant alleles identified herein (or their complements) and which also comprise the variant nucleotide at the SNP site.

The invention further relates to portions of the variant alleles and portions of complements of the variant alleles which comprise (encompass) the site of the SNP and are at least 5 nucleotides in length. Portions can be, for example, 5-10, 5-15, 10- 20, 5-25, 10-30, 10-50 or 10-100 bases long. For example, a portion of a variant allele which is 21 nucleotides in length includes the single nucleotide polymoφhism (the nucleotide which differs from the reference allele at that site) and twenty additional nucleotides which flank the site in the variant allele. These nucleotides can be on one or both sides of the polymoφhism. Polymoφhisms which are the subject of this invention are defined in the Table with respect to the reference sequence deposited in GenBank or TIGR under the Accession number indicated. For example, the invention relates to a portion of a gene (e.g., AT3) having a nucleotide sequence as deposited in GenBank (e.g., UI 1270) comprising a single nucleotide polymoφhism at a specific position (e.g., nucleotide 11918). The reference nucleotide for AT3 is shown in column 8, and the variant nucleotide is shown in column 9 of the Table. The nucleotide sequences of the invention can be double- or single-stranded. The invention further provides allele-specific oligonucleotides that hybridize to the reference or variant allele of a gene comprising a single nucleotide polymoφhism or to the complement thereof. These oligonucleotides can be probes or primers.

The invention further provides a method of analyzing a nucleic acid from an individual. The method determines which base is present at any one of the polymoφhic sites shown in the Table and/or Fig. 3. Optionally, a set of bases occupying a set of the polymoφhic sites shown in the Table and/or Fig. 3 is determined. This type of analysis can be performed on a number of individuals, who are tested for the presence of a disease phenotype. The presence or absence of disease phenotype is then correlated with a base or set of bases present at the polymoφhic site or sites in the individuals tested.

Thus, the invention further relates to a method of predicting the presence, absence, likelihood of the presence or absence, or severity of a particular phenotype or disorder associated with a particular genotype. The method comprises obtaining a nucleic acid sample from an individual and determining the identity of one or more bases (nucleotides) at polymoφhic sites of genes described herein, wherein the presence of a particular base is correlated with a specified phenotype or disorder, thereby predicting the presence, absence, likelihood of the presence or absence, or severity of the phenotype or disorder in the individual.

DEFINITIONS A nucleic acid molecule or oligonucleotide can be DNA or RNA, and single- or double-stranded. Nucleic acid molecules and oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means. Preferred nucleic acid molecules and oligonucleotides of the invention include segments of DNA, or their complements, which include any one of the polymoφhic sites shown in the Table. The segments can be between 5 and 250 bases, and, in specific embodiments, are between 5-10, 5-20, 10-20, 10-50, 20-50 or 10-100 bases. For example, the segment can be 21 bases. The polymoφhic site can occur within any position of the segment. The segments can be from any of the allelic forms of DNA shown in the Table. As used herein, the terms "nucleotide", "base" and "nucleic acid" are intended to be equivalent. The terms "nucleotide sequence", "nucleic acid sequence", "nucleic acid molecule" and "segment" are intended to be equivalent.

Hybridization probes are oligonucleotides which bind in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al., Science 254, 1497-1500 (1991). Probes can be any length suitable for specific hybridization to the target nucleic acid sequence. The most appropriate length of the probe may vary depending upon the hybridization method in which it is being used; for example, particular lengths may be more appropriate for use in micro fabricated arrays, while other lengths may be more suitable for use in classical hybridization methods. Such optimizations are known to the skilled artisan. Suitable probes and primers can range from about 5 nucleotides to about 30 nucleotides in length. For example, probes and primers can be 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 nucleotides in length. The probe or primer preferably overlaps at least one polymoφhic site occupied by any of the possible variant nucleotides. The nucleotide sequence can correspond to the coding sequence of the allele or to the complement of the coding sequence of the allele.

As used herein, the term "primer" refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template. The term primer site refers to the area of the target DNA to which a primer hybridizes. The term primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the 3' end of the sequence to be amplified. As used herein, 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. It can be measured by percent recombination between the two genes, alleles, loci or genetic markers. As used herein, polymoφhism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymoφhic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population. A polymoφhic locus may be as small as one base pair. Polymoφhic markers include restriction fragment length polymoφhisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu. The first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms. A diallelic or biallelic polymoφhism has two forms. A triallelic polymoφhism has three forms. Work described herein pertains to the resequencing of large numbers of genes in a large number of individuals to identify polymoφhisms which can predispose individuals to disease. For example, polymoφhisms in genes which are expressed in liver may predispose individuals to disorders of the liver. By altering amino acid sequence, SNPs may alter the function of the encoded proteins. The discovery of the SNP facilitates biochemical analysis of the variants and the development of assays to characterize the variants and to screen for pharmaceutical that would interact directly with one or another form of the protein. SNPs (including silent SNPs) also enable the development of specific DNA, RNA, or protein-based diagnostics that detect the presence or absence of the polymoφhism in particular conditions.

A single nucleotide polymoφhism occurs at a polymoφhic 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 polymoφhism usually arises due to substitution of one nucleotide for another at the polymoφhic site. 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 polymoφhisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele. Typically the polymoφhic site is occupied by a base other than the reference base. For example, where the reference allele contains the base "T" at the polymoφhic site, the altered allele can contain a "C", "G" or "A" at the polymoφhic site.

The invention also relates to nucleic acid molecules which hybridize to the variant alleles identified herein (or their complements) and which also comprise the variant nucleotide at the SNP site. Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C, or equivalent conditions, are suitable for allele-specific probe hybridizations. Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleotide sequence and the primer or probe used.

The invention also relates to nucleic acid molecules which share substantial sequence identity to the variant alleles identified herein (or their complements) and which also comprise the variant nucleotide at the SNP site. Particularly preferred are nucleic acid molecules and fragments which have at least about 60%, preferably at least about 70, 80 or 85%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98% identity with nucleic acid molecules described herein. The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison puφoses (e.g., gaps can be introduced in the sequence of a first sequence). The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100). In certain embodiments, the length of a sequence aligned for comparison puφoses is at least 30%, preferably at least 40%, more preferably at least 60%, and even more preferably at least 70%, 80% or 90% of the length of the reference sequence. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non- limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incoφorated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al., Nucleic Acids Res., 25:389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. In one embodiment, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied (e.g., W=5 or W=20).

The term "isolated" is used herein to indicate that the material in question exists in a physical milieu distinct from that in which it occurs in nature. For example, an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstance, the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC. Preferably, an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present.

I. Novel Polymoφhisms of the Invention

Some of the novel polymoφhisms of the invention are shown in the Table. Columns one and two show designations for the indicated polymoφhism. Column three shows the Genbank or TIGR Accession number for the wild type (or reference) allele. Column four shows the location of the polymoφhic site in the nucleic acid sequence with reference to the Genbank or TIGR sequence shown in column three. Column five shows common names for the gene in which the polymoφhism is located. Column six shows the polymoφhism and a portion of the 3' and 5' flanking sequence of the gene. Column seven shows the type of mutation; N, non-sense, S, silent, M, missense. Columns eight and nine show the reference and alternate nucleotides, respectively, at the polymoφhic site. Columns ten and eleven show the reference and alternate amino acids, respectively, encoded by the reference and variant, respectively, alleles. Other novel polymoφhisms of the invention are shown in Fig. 3.

II. Analysis of Polymoφhisms A. Preparation of Samples

Polymoφhisms are detected in a target nucleic acid from an individual being analyzed. 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, 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. For example, if the target nucleic acid is a cytochrome P450, the liver is a suitable source.

Many of the methods described below 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.

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.

B. Detection of Polymoφhisms in Target DNA

There are two distinct types of analysis of target DNA for detecting polymoφhisms. The first type of analysis, sometimes referred to as de novo characterization, is carried out to identify polymoφhic sites not previously characterized (i.e., to identify new polymoφhisms). This analysis compares target sequences in different individuals to identify points of variation, i.e., polymoφhic sites. By analyzing groups of individuals representing the greatest ethnic diversity among humans and greatest breed and species variety in plants and animals, patterns characteristic of the most common alleles/haplotypes of the locus can be identified, and the frequencies of such alleles/haplotypes in the population can be determined. Additional allelic frequencies can be determined for subpopulations characterized by criteria such as geography, race, or gender. The de novo identification of polymoφhisms of the invention is described in the Examples section. The second type of analysis determines which form(s) of a characterized (known) polymoφhism are present in individuals under test. There are a variety of suitable procedures, which are discussed in turn.

1. Allele-Specific Probes

The design and use of allele-specific probes for analyzing polymoφhisms is described by e.g., Saiki et al, Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the coπesponding segment from another individual due to the presence of different polymoφhic forms 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 polymoφhic 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. 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 polymoφhisms within the same target sequence.

2. Tiling Arrays

The polymoφhisms can also be identified by hybridization to nucleic acid arrays, some examples of which are described in WO 95/11995. One form of such arrays is described in the Examples section in connection with de novo identification of polymoφhisms. The same array or a different array can be used for analysis of characterized polymoφhisms. WO 95/11995 also describes subarrays that are optimized for detection of a variant form of a precharacterized polymoφhism. Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence. The second group of probes is designed by the same principles as described in the Examples, except that the probes exhibit complementarity to the second reference sequence. The inclusion of a second group (or further groups) can be particularly useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (e.g., two or more mutations within 9 to 21 bases).

3. Allele-Specific Primers

An allele-specific primer hybridizes to a site on target DNA overlapping a polymoφhism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates 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 polymoφhic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3'-most position of the oligonucleotide aligned with the polymoφhism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).

4. Direct-Sequencing The direct analysis of the sequence of polymoφhisms 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)).

5. Denaturing Gradient Gel Electrophoresis

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. Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992), Chapter 7.

6. Single-Strand Conformation Polymoφhism Analysis Alleles of target sequences can be differentiated using single-strand conformation polymoφhism 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 which are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.

7. Single-Base Extension

An alternative method for identifying and analyzing polymoφhisms is based on single-base extension (SBE) of a fluorescently-labeled primer coupled with fluorescence resonance energy transfer (FRET) between the label of the added base and the label of the primer. Typically, the method, such as that described by Chen et al, (PNAS 94: 10756-61 (1997), incoφorated herein by reference) uses a locus- specific oligonucleotide primer labeled on the 5' terminus with 5-carboxyfluorescein (FAM). This labeled primer is designed so that the 3' end is immediately adjacent to the polymoφhic site of interest. The labeled primer is hybridized to the locus, and single base extension of the labeled primer is performed with fluorescently labeled dideoxyribonucleotides (ddNTPs) in dye-terminator sequencing fashion, except that no deoxyribonucleotides are present. An increase in fluorescence of the added ddNTP in response to excitation at the wavelength of the labeled primer is used to infer the identity of the added nucleotide.

III. Methods of Use

After determining polymoφhic form(s) present in an individual at one or more polymoφhic sites, this information can be used in a number of methods.

A. Forensics

Determination of which polymoφhic forms occupy a set of polymoφhic sites in an individual identifies a set of polymoφhic forms that distinguishes the individual. See generally National Research Council, The Evaluation of Forensic DNA Evidence (Eds. Pollard et al, National Academy Press, DC, 1996). The more sites that are analyzed, the lower the probability that the set of polymoφhic forms in one individual is the same as that in an unrelated individual. Preferably, if multiple sites are analyzed, the sites are unlinked. Thus, polymoφhisms of the invention are often used in conjunction with polymoφhisms in distal genes. Preferred polymoφhisms for use in forensics are biallelic because the population frequencies of two polymoφhic forms can usually be determined with greater accuracy than those of multiple polymoφhic forms at multi-allelic loci.

The capacity to identify a distinguishing or unique set of forensic markers in an individual is useful for forensic analysis. For example, one can determine whether a blood sample from a suspect matches a blood or other tissue sample from a crime scene by determining whether the set of polymoφhic forms occupying selected polymoφhic sites is the same in the suspect and the sample. If the set of polymoφhic markers does not match between a suspect and a sample, it can be concluded (barring experimental error) that the suspect was not the source of the sample. If the set of markers does match, one can conclude that the DNA from the suspect is consistent with that found at the crime scene. If frequencies of the polymoφhic forms at the loci tested have been determined (e.g., by analysis of a suitable population of individuals), one can perform a statistical analysis to determine the probability that a match of suspect and crime scene sample would occur by chance. p(ID) is the probability that two random individuals have the same polymoφhic or allelic form at a given polymoφhic site. In biallelic loci, four genotypes are possible: AA, AB, BA, and BB. If alleles A and B occur in a haploid genome of the organism with frequencies x and y, the probability of each genotype in a diploid organism is (see WO 95/12607): Homozygote: p(AA)= x² Homozygote: p(BB)= y² = (1-x)² Single Heterozygote: p(AB)= p(BA)= xy = x(l-x) Both Heterozygotes: p(AB+BA)= 2xy = 2x(l-x)

The probability of identity at one locus (i.e, the probability that two individuals, picked at random from a population will have identical polymoφhic forms at a given locus) is given by the equation: p(ID) = (x²)² + (2xy)² + (y²)².

These calculations can be extended for any number of polymoφhic forms at a given locus. For example, the probability of identity p(ID) for a 3-allele system where the alleles have the frequencies in the population of x, y and z, respectively, is equal to the sum of the squares of the genotype frequencies: p(ID) - x⁴ + (2xy)² + (2yz)² + (2xz)² + z⁴ + y⁴

In a locus of n alleles, the appropriate binomial expansion is used to calculate p(ID) and p(exc).

The cumulative probability of identity (cum p(ID)) for each of multiple unlinked loci is determined by multiplying the probabilities provided by each locus. cum p(ID) = p(IDl)p(ID2)p(ID3).... p(IDn)

The cumulative probability of non-identity for n loci (i.e. the probability that two random individuals will be different at 1 or more loci) is given by the equation: cum p(nonΙD) = 1-cum p(ID).

If several polymoφhic loci are tested, the cumulative probability of non- identity for random individuals becomes very high (e.g., one billion to one). Such probabilities can be taken into account together with other evidence in determining the guilt or innocence of the suspect.

B. Paternity Testing

The object of paternity testing is usually to determine whether a male is the father of a child. In most cases, the mother of the child is known and thus, the mother's contribution to the child's genotype can be traced. Paternity testing investigates whether the part of the child's genotype not attributable to the mother is consistent with that of the putative father. Paternity testing can be performed by analyzing sets of polymoφhisms in the putative father and the child.

If the set of polymoφhisms in the child attributable to the father does not match the set of polymoφhisms of the putative father, it can be concluded, barring experimental error, that the putative father is not the real father. If the set of polymoφhisms in the child attributable to the father does match the set of polymoφhisms of the putative father, a statistical calculation can be performed to determine the probability of coincidental match.

The probability of parentage exclusion (representing the probability that a random male will have a polymoφhic form at a given polymoφhic site that makes him incompatible as the father) is given by the equation (see WO 95/12607): p(exc) = xy(l-xy) where x and y are the population frequencies of alleles A and B of a biallelic polymoφhic site.

(At a triallelic site p(exc) = xy(l-xy) + yz(l- yz) + xz(l-xz)+ 3xyz(l-xyz))), where x, y and z and the respective population frequencies of alleles A, B and C).

The probability of non-exclusion is p(non-exc) = l-p(exc)

The cumulative probability of non-exclusion (representing the value obtained when n loci are used) is thus: cum p(non-exc) = p(non-excl)p(non-exc2)p(non-exc3).... p(non-excn)

The cumulative probability of exclusion for n loci (representing the probability that a random male will be excluded) cum p(exc) = 1 - cum p(non-exc).

If several polymoφhic loci are included in the analysis, the cumulative probability of exclusion of a random male is very high. This probability can be taken into account in assessing the liability of a putative father whose polymoφhic marker set matches the child's polymoφhic marker set attributable to his/her father.

C. Correlation of Polymoφhisms with Phenotypic Traits

The polymoφhisms of the invention may contribute to the phenotype of an organism in different ways. Some polymoφhisms occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect may be neutral, beneficial or detrimental, or both beneficial and detrimental, depending on the circumstances. For example, a heterozygous sickle cell mutation confers resistance to malaria, but a homozygous sickle cell mutation is usually lethal. Other polymoφhisms occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single polymoφhism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymoφhisms in different genes. Further, some polymoφhisms predispose an individual to a distinct mutation that is causally related to a certain phenotype. Phenotypic traits include diseases that have known but hitherto unmapped genetic components (e.g., agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, polycystic kidney disease, hereditary spherocytosis, von Willebrand's disease, tuberous sclerosis, hereditary hemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome, osteogenesis imperfecta, and acute intermittent poφhyria). Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is or may be genetic, such as autoimmune diseases, inflammation, cancer, diseases of the nervous system, and infection by pathogenic microorganisms. Some examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependent and non-independent), systemic lupus erythematosus and Graves disease. Some examples of cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary, pancreas, prostate, skin, stomach and uterus. Phenotypic traits also include characteristics such as longevity, appearance (e.g., baldness, obesity), strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments.

The correlation of one or more polymoφhisms with phenotypic traits can be facilitated by knowledge of the gene product of the wild type (reference) gene. The genes in which cSNPs of the present invention have been identified are genes which have been previously sequenced and characterized in one of their allelic forms.

Correlation is performed for a population of individuals who have been tested for the presence or absence of a phenotypic trait of interest and for polymoφhic markers sets. To perform such analysis, the presence or absence of a set of polymoφhisms (i.e. a polymoφhic set) 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 polymoφhism 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 such as a K-squared test and statistically significant correlations between polymoφhic form(s) and phenotypic characteristics are noted. For example, it might be found that the presence of allele Al at polymoφhism A correlates with heart disease. As a further example, it might be found that the combined presence of allele Al at polymoφhism A and allele Bl at polymoφhism B correlates with increased milk production of a farm animal.

Such correlations can be exploited in several ways. In the case of a strong correlation between a set of one or more polymoφhic forms and a disease for which treatment is available, detection of the polymoφhic form set in a human or animal patient may justify immediate administration of treatment, or at least the institution of regular monitoring of the patient. Detection of a polymoφhic form correlated with serious disease in a couple contemplating a family may also be valuable to the couple in their reproductive decisions. For example, the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymoφhism from her husband to her offspring. In the case of a weaker, but still statistically significant correlation between a polymoφhic set and human disease, immediate therapeutic intervention or monitoring may not be justified. Nevertheless, the patient can be motivated to begin simple life-style changes (e.g., diet, exercise) that can be accomplished at little cost to the patient but confer potential benefits in reducing the risk of conditions to which the patient may have increased susceptibility by virtue of variant alleles. Identification of a polymoφhic set in a patient correlated with enhanced receptiveness to one of several treatment regimes for a disease indicates that this treatment regime should be followed.

For animals and plants, coπelations between characteristics and phenotype are useful for breeding for desired characteristics. For example, Beitz et al, US 5,292,639 discuss use of bovine mitochondrial polymoφhisms in a breeding program to improve milk production in cows. To evaluate the effect of mtDNA D- loop sequence polymoφhism on milk production, each cow was assigned a value of 1 if variant or 0 if wildtype with respect to a prototypical mitochondrial DNA sequence at each of 17 locations considered. Each production trait was analyzed individually with the following animal model:

Y,_jkpn= μ + YS, + P. + X_k + β, + ... β₁₇ + PE_n + a„ +e_p where Y_ljknp is the milk, fat, fat percentage, SNF, SNF percentage, energy concentration, or lactation energy record; μ is an overall mean; YS, is the effect common to all cows calving in year-season; X_k is the effect common to cows in either the high or average selection line; β, to β,₇ are the binomial regressions of production record on mtDNA D-loop sequence polymoφhisms; PE_n is permanent environmental effect common to all records of cow n; a„ is effect of animal n and is composed of the additive genetic contribution of sire and dam breeding values and a Mendelian sampling effect; and e_p is a random residual. It was found that eleven of seventeen polymoφhisms tested influenced at least one production trait. Bovines having the best polymoφhic forms for milk production at these eleven loci are used as parents for breeding the next generation of the herd.

D. Genetic Mapping of Phenotypic Traits The previous section concerns identifying correlations between phenotypic traits and polymoφhisms that directly or indirectly contribute to those traits. The present section describes identification of a physical linkage between a genetic locus associated with a trait of interest and polymoφhic markers that are not associated with the trait, but are in physical proximity with the genetic locus responsible for the trait and co-segregate with it. Such analysis is useful for mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene(s) responsible for the trait. See Lander et al, Proc. Natl. Acad. Sci. (USA) 83, 7353-7357 (1986); Lander et al, Proc. Natl. Acad. Sci. (USA) 84, 2363-2367 (1987); Donis-Keller et al, Cell 51, 319-337 (1987); Lander et al, Genetics 121, 185-199 (1989)). Genes localized by linkage can be cloned by a process known as directional cloning. See Wainwright, Med. J. Australia 159, 170-174 (1993); Collins, Nature Genetics 1, 3-6 (1992).

Linkage studies are typically performed on members of a family. Available members of the family are characterized for the presence or absence of a phenotypic trait and for a set of polymoφhic markers. The distribution of polymoφhic markers in an informative meiosis is then analyzed to determine which polymoφhic markers co-segregate with a phenotypic trait. See, e.g., Kerem et al, Science 245, 1073-1080 (1989); Monaco et al, Nature 316, 842 (1985); Yamoka et al, Neurology 40, 222- 226 (1990); Rossiter et al, FASEB Journal 5, 21-27 (1991). Linkage is analyzed by calculation of LOD (log of the odds) values. A lod value is the relative likelihood of obtaining observed segregation data for a marker and a genetic locus when the two are located at a recombination fraction θ, versus the situation in which the two are not linked, and thus segregating independently (Thompson & Thompson, Genetics in Medicine (5th ed, W.B. Saunders Company, Philadelphia, 1991); Strachan, "Mapping the human genome" in The Human Genome (BIOS Scientific Publishers Ltd, Oxford), Chapter 4). A series of likelihood ratios are calculated at various recombination fractions (θ), ranging from θ = 0.0 (coincident loci) to θ = 0.50 (unlinked). Thus, the likelihood at a given value of θ is: probability of data if loci linked at θ to probability of data if loci unlinked. The computed likelihoods are usually expressed as the log₁₀ of this ratio (i.e., a lod score). For example, a lod score of 3 indicates 1000:1 odds against an apparent observed linkage being a coincidence. The use of logarithms allows data collected from different families to be combined by simple addition. Computer programs are available for the calculation of lod scores for differing values of θ (e.g., LIPED, MLINK (Lathrop, Proc. Nat. Acad. Sci. (USA) 81, 3443-3446 (1984)). For any particular lod score, a recombination fraction may be determined from mathematical tables. See Smith et al, Mathematical tables for research workers in human genetics (Churchill, London, 1961); Smith, Ann. Hum. Genet. 32, 127-150 (1968). The value of θ at which the lod score is the highest is considered to be the best estimate of the recombination fraction. Positive lod score values suggest that the two loci are linked, whereas negative values suggest that linkage is less likely (at that value of θ) than the possibility that the two loci are unlinked. By convention, a combined lod score of +3 or greater (equivalent to greater than 1000:1 odds in favor of linkage) is considered definitive evidence that two loci are linked. Similarly, by convention, a negative lod score of - 2 or less is taken as definitive evidence against linkage of the two loci being compared. Negative linkage data are useful in excluding a chromosome or a segment thereof from consideration. The search focuses on the remaining non- excluded chromosomal locations.

IV. Modified Polypeptides and Gene Sequences The invention further provides variant forms of nucleic acids and corresponding proteins. The nucleic acids comprise one of the sequences described in the Table, column 5, in which the polymoφhic position is occupied by one of the alternative bases for that position. Some nucleic acids encode full-length variant forms of proteins. Similarly, variant proteins have the prototypical amino acid sequences encoded by nucleic acid sequences shown in the Table, column 5, (read so as to be in- frame with the full-length coding sequence of which it is a component) except at an amino acid encoded by a codon including one of the polymoφhic positions shown in the Table. That position is occupied by the amino acid coded by the corresponding codon in any of the alternative forms shown in the Table.

Variant genes can be expressed in an expression vector in which a variant gene is operably linked to a native or other promoter. Usually, the promoter is a eukaryotic promoter for expression in a mammalian cell. The transcription regulation sequences typically include a heterologous promoter and optionally an enhancer which is recognized by the host. The selection of an appropriate promoter, for example tφ, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected. Commercially available expression vectors can be used. Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.

The means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra. A wide variety of host cells can be employed for expression of the variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide. Processing includes glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, and the like. As used herein, "gene product" includes mRNA, peptide and protein products.

The protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, Methods in Enzymology Volume 104, Academic Press, New York (1984); Scopes, Protein Purification, Principles and Practice, 2nd Edition, Springer- Verlag, New York (1987); and Deutscher (ed), Guide to Protein Purification, Methods in Enzymology, Vol. 182 (1990). If the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. If not secreted, the protein can be isolated from a lysate of the host cells.

The invention further provides transgenic nonhuman animals capable of expressing an exogenous variant gene and/or having one or both alleles of an endogenous variant gene inactivated. Expression of an exogenous variant gene is usually achieved by operably linking the gene to a promoter and optionally an enhancer, and microinjecting the construct into a zygote. See Hogan et al, "Manipulating the Mouse Embryo, A Laboratory Manual," Cold Spring Harbor Laboratory. Inactivation of endogenous variant genes can be achieved by forming a transgene in which a cloned variant gene is inactivated by insertion of a positive selection marker. See Capecchi, Science 244, 1288-1292 (1989). The transgene is then introduced into an embryonic stem cell, where it undergoes homologous recombination with an endogenous variant gene. Mice and other rodents are preferred animals. Such animals provide useful drug screening systems.

In addition to substantially full-length polypeptides expressed by variant genes, the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides. Biologically active fragments include any portion of the full-length polypeptide which confers a biological function on the variant gene product, including ligand binding, and antibody binding. Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.

Polyclonal and/or monoclonal antibodies that specifically bind to variant gene products but not to corresponding prototypical gene products are also provided. Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof. Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.

V. Kits

The invention further provides kits comprising at least one allele-specific oligonucleotide as described herein. Often, the kits contain one or more pairs of allele-specific oligonucleotides hybridizing to different forms of a polymoφhism. In some kits, the allele-specific oligonucleotides are provided immobilized to a substrate. For example, the same substrate can comprise allele-specific oligonucleotide probes for detecting at least 10, 100 or all of the polymoφhisms shown in the Table. Optional additional components of the kit include, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions. Usually, the kit also contains instructions for carrying out the methods. The thrombospondins are a family of extracellular matrix (ECM) glycoproteins that modulate many cell behaviors including adhesion, migration, and proliferation. Thrombospondins (also known as thrombin sensitive proteins or TSPs) are large molecular weight glycoproteins composed of three identical disulfide-linked polypeptide chains. TSPs are stored in the alpha-granules of platelets and secreted by a variety of mesenchymal and epithelial cells (Majack et al, Cell Membrane 3:51-11 (1987)). Platelets secrete TSPs when activated in the blood by such physiological agonists such as thrombin. TSPs have lectin properties and a broad function in the regulation of fibrinolysis and as a component of the ECM, and are one of a group of ECM proteins which have adhesive properties. TSPs bind to fibronectin and fibrinogen (Lahav et al, Eur J Biochem 145:151-6 (1984)), and these proteins are known to be involved in platelet adhesion to substratum and platelet aggregation (Leung, J Clin Invest 74: 1164-1112 (1986)). Recent work has implicated TSPs in response of cells to growth factors. Submitogenic doses of PDGF induce a rapid but transitory, increase in TSP synthesis and secretion by rat aortic smooth muscle cells (Majack et al, J Biol Chem 101: 1059-10 (1985)). PDGF responsiveness to TSP synthesis in glial cells has also been shown (Asch et al, Proc Natl Acad Sci 83:2904-8 (1986)). TSP mRNA levels rise rapidly in response to PDGF (Majack et al, JBiol Chem 262:8821-5 (1987)). TSPs act synergistically with epidermal growth factor to increase DNA synthesis in smooth muscle cells (Majack et al, Proc Natl Acad Sci S3 :9050-4 (1986)), and monoclonal antibodies to TSPs inhibit smooth muscle cell proliferation (Majack et al, JBiol Chem 106:415-22 (1988)). TSPs modulate local adhesions in endothelial cells, and TSPs, particularly TSP-1 primarily derived from platelet granules, are known to be an important activator of transforming growth factor beta-1 (TGFB-1) (Crawford et al, Cell 93:1159 (1998)) and appear to be a potential link between platelet-thrombosis and development of atherosclerosis. To determine pivotal genes associated with premature coronary artery disease, we analyzed DNA from 347 patients with MI or coronary revascularization before age 40 (men) or 45 (women) and 422 general population controls. Cases were drawn (one per family) from a retrospective collection of sibling pairs with premature CAD. Controls were ascertained through random-digit dialing. Both cases and controls were Caucasian. A complete database of phenotypic and laboratory variables for the affected patients afforded logistic regression to control for age, diabetes, body mass index, gender.

Thrombospondin (TSP) 4 and 1 emerged as important SNPs associated with premature CAD and MI. For CAD, 148 of 347 patients carried at least one copy of the TSP-4 variant compared with 142 of 422 control subjects; adjusted odds ratio 1.47, p=0.01. For premature MI, the association was even stronger: 91 of 187 cases vs. 142 of 422 controls had the variant; adjusted odds ratio 2.08, p=0.0003. The TSP-1 SNP was rare. Nonetheless, homozygosity for the variant allele gave an adjusted odds ratio of 9.5, p=.04. Specific reference nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO:

2) sequences for TSP-1 are shown in Figs. 1 A- ID. Specific reference nucleotide (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4) sequences for TSP-4 are shown in Figs. 2A-2C. It is understood that the invention is not limited by these exemplified reference sequences, as variants of these sequences which differ at locations other than the SNP sites identified herein can also be utilized. The skilled artisan can readily determine the SNP sites in these other reference sequences which correspond to the SNP sites identified herein by aligning the sequence of interest with the reference sequences specifically disclosed herein, and programs for performing such alignments are commercially available. For example, the ALIGN program in the GCG software package can be used, utilizing a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4, for example. Two SNPs have been specifically studied as described herein. The first

(G334u4) is a change from A (reference nucleotide) to G (alternate or variant nucleotide) at nucleotide position 2210 of the nucleic acid sequence of TSP-1 (Figs. 1A-1D), resulting in a missense amino acid mutation from asparagine (reference) to serine (alternate) at amino acid 700. The second SNP (G355u2) is a change from G (reference) to C (alternate) at nucleotide position 1186 of the nucleic acid sequence of TSP-4 (Figs. 2A-2C), resulting in a missense amino acid alteration from alanine (reference) to proline (alternate) at amino acid 387. With respect to the G355u2 SNP, individuals with CAD carried at least one copy of the variant "C" allele more frequently than control individuals (43% as compared with 34%). With respect to the G355u2 SNP, individuals with MI carried at least one copy of the variant "C" allele more frequently than control individuals (49%> as compared with 34%). With respect to the G334u4 SNP, individuals with CAD carried two copies of the variant "G" allele more frequently than control individuals (1.7% as compared with 0.2%>). With respect to the G334u4 SNP, individuals with MI carried two copies of the variant "G" allele more frequently than control individuals (2% as compared with 0.2%).

As used herein, the term "polymoφhism" refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymoφhic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population. A polymoφhic locus may be as small as one base pair, in which case it is referred to as a single nucleotide polymoφhism (SNP).

Thus, the invention relates to a method for predicting the likelihood that an individual will have a vascular disease, or for aiding in the diagnosis of a vascular disease, or predicting the likelihood of having altered symptomology associated with a vascular disease, comprising the steps of obtaining a DNA sample from an individual to be assessed and determining the nucleotide present at one or more of nucleotide positions 2210 of the TSP-1 gene or 1186 of the TSP-4 gene. In a preferred embodiment, the nucleotides present at both of these nucleotide positions are determined. In one embodiment the TSP-1 gene has the nucleotide sequence of SEQ ID NO: 1 and the TSP-4 gene has the nucleotide sequence of SEQ ID NO: 3. The presence of one or more of a G (the variant nucleotide) at position 2210 of SEQ ID NO: 1 or a C (the variant nucleotide) at position 1186 of SEQ ID NO: 1186 indicates that the individual has a greater likelihood of having a vascular disease, or a greater likelihood of having severe symptomology associated with a vascular disease, than if that individual had the reference nucleotide at one or more of these positions. Conversely, the presence of one or more of an A (the reference nucleotide) at position 2210 of SEQ ID NO: 1 or a G (the reference nucleotide) at position 1186 of SEQ ID NO: 3 indicates that the individual has a reduced likelihood of having a vascular disease or a likelihood of having reduced symptomology associated with a vascular disease than if that individual had the variant nucleotide at one or more of these positions.

In a particular embodiment, the individual is an individual at risk for development of a vascular disease. In another embodiment the individual exhibits clinical symptomology associated with a vascular disease. In one embodiment, the individual has been clinically diagnosed as having a vascular disease. Vascular diseases include, but are not limited to, atherosclerosis, coronary heart disease, myocardial infarction (MI), stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism. In preferred embodiments, the vascular disease is CAD or MI.

The genetic material to be assessed can be obtained from any nucleated cell from the individual. 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, skin and hair. For assay of cDNA or mRNA, the tissue sample must be obtained from a tissue or organ in which the target nucleic acid is expressed. Many of the methods described herein 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.

The nucleotide which occupies the polymoφhic site of interest (e.g., nucleotide position 2210 in TSP-1 and or nucleotide position 1186 in TSP-4) can be identified by a variety of methods, such as Southern analysis of genomic DNA; direct mutation analysis by restriction enzyme digestion; Northern analysis of RNA; denaturing high pressure liquid chromatography (DHPLC); gene isolation and sequencing; hybridization of an allele-specific oligonucleotide with amplified gene products; single base extension (SBE). In a prefeoed embodiment, determination of the allelic form of TSP is carried out using SBE-FRET methods as described herein, or using chip-based oligonucleotide arrays as described herein.

The invention also relates to a method for predicting the likelihood that an individual will have a vascular disease, or for aiding in the diagnosis of a vascular disease, or predicting the likelihood of having altered symptomology associated with a vascular disease, comprising the steps of obtaining a biological sample comprising TSP-1 and/or TSP-4 protein or relevant portion thereof from an individual to be assessed and determining the amino acid present at one or more of amino acid positions 700 of the TSP-1 gene product (e.g., as exemplified by SEQ ID NO: 2) or 387 of the TSP-4 gene product (e.g., as exemplified by SEQ ID NO: 4). In a preferred embodiment, the amino acids present at both of these amino acid positions are determined. As used herein, the term "relevant portion" of the TSP-1 and TSP-4 proteins is intended to encompass any portion of the protein which comprises the polymoφhic amino acid positions. The presence of one or more of a serine (the variant amino acid) at position 700 of SEQ ID NO: 2, or a proline (the variant amino acid) at position 387 of SEQ ID NO: 4 indicates that the individual has a greater likelihood of having a vascular disease, or a greater likelihood of having severe symptomology associated with a vascular disease, than if that individual had the reference amino acid at one or more of these positions. Conversely, the presence of one or more of an asparagine (the reference amino acid) at position 700 of SEQ ID NO: 2, or an alanine (the reference amino acid) at position 387 of SEQ I D NO: 4 indicates that the individual has a reduced likelihood of having a vascular disease or a likelihood of having reduced symptomology associated with a vascular disease, than if that individual had the varaint amino acid at one or more of these positions. In a particular embodiment, the individual is an individual at risk for development of a vascular disease. In another embodiment the individual exhibits clinical symptomology associated with a vascular disease. In one embodiment, the individual has been clinically diagnosed as having a vascular disease. In this embodiment of the invention, the biological sample contains protein molecules from the test subject. In vitro techniques for detection of protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. Furthermore, in vivo techniques for detection of protein include introducing into a subject a labeled anti-protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Polyclonal and/or monoclonal antibodies that specifically bind to variant gene products but not to corresponding reference gene products, and vice versa, are also provided. Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof comprising the variant portion. Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.

The polymoφhisms of the invention may be associated with vascular disease in different ways. The polymoφhisms may exert phenotypic effects indirectly via influence on replication, transcription, and translation. Additionally, the described polymoφhisms may predispose an individual to a distinct mutation that is causally related to a certain phenotype, such as susceptibility or resistance to vascular disease and related disorders. The discovery of the polymoφhisms and their correlation with CAD and MI facilitates biochemical analysis of the variant and reference forms and the development of assays to characterize the variant and reference forms and to screen for pharmaceutical agents that interact directly with one or another form of the protein.

Alternatively, these particular polymoφhisms may belong to a group of two or more polymoφhisms in the TSP gene(s) which contributes to the presence, absence or severity of vascular disease. An assessment of other polymoφhisms within the TSP gene(s) can be undertaken, and the separate and combined effects of these polymoφhisms, as well as alternations in other, distinct genes, on the vascular disease phenotype can be assessed.

Correlation between a particular phenotype, e.g., the CAD or MI phenotype, and the presence or absence of a particular allele is performed for a population of individuals who have been tested for the presence or absence of the phenotype.

Correlation can be performed by standard statistical methods such as a Chi-squared test and statistically significant correlations between polymoφhic form(s) and phenotypic characteristics are noted. This correlation can be exploited in several ways. In the case of a strong correlation between a particular polymoφhic form, e.g., the variant allele for TSP-1 and/or TSP-4, and a disease for which treatment is available, detection of the polymoφhic form in an individual may justify immediate administration of treatment, or at least the institution of regular monitoring of the individual. Detection of a polymoφhic form correlated with a disorder in a couple contemplating a family may also be valuable to the couple in their reproductive decisions. For example, the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymoφhism from her husband to her offspring. In the case of a weaker, but still statistically significant correlation between a polymoφhic form and a particular disorder, immediate therapeutic intervention or monitoring may not be justified. Nevertheless, the individual can be motivated to begin simple life-style changes (e.g., diet modification, therapy or counseling) that can be accomplished at little cost to the individual but confer potential benefits in reducing the risk of conditions to which the individual may have increased susceptibility by virtue of the particular allele. Furthermore, identification of a polymoφhic form correlated with enhanced receptiveness to one of several treatment regimes for a disorder indicates that this treatment regimen should be followed for the individual in question. Furthermore, it may be possible to identify a physical linkage between a genetic locus associated with a trait of interest (e.g., CAD or MI) and polymoφhic markers that are or are not associated with the trait, but are in physical proximity with the genetic locus responsible for the trait and co-segregate with it. Such analysis is useful for mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene(s) responsible for the trait. See Lander et al, Proc. Natl. Acad. Sci. (USA) 83, 7353-7357 (1986); Lander et al, Proc. Natl. Acad. Sci. (USA) 84, 2363-2367 (1987); Donis-Keller et al, Cell 51, 319-337 (1987); Lander βt al, Genetics 121, 185-199 (1989)). Genes localized by linkage can be cloned by a process known as directional cloning. See Wainwright, Med. J. Australia 159, 170-174 (1993); Collins, Nature Genetics 1, 3-6 (1992). Linkage studies are discussed in more detail above. In another embodiment, the invention relates to pharmaceutical compositions comprising a reference TSP-1 and/or TSP-4 gene or gene product for use in the treatment of vascular disease, e.g., CAD and MI. As used herein, a reference TSP gene product is intended to mean gene products which are encoded by the reference allele of the TSP gene. In addition to substantially full-length polypeptides expressed by the genes, the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides. Biologically active fragments include any portion of the full-length polypeptide which confers a biological function on the variant gene product, including ligand binding, and antibody binding. Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.

For instance, the polypeptide or protein, or fragment thereof, of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition. The particular physiological medium may include, but is not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists, and will depend on the ultimate pharmaceutical formulation desired. Methods of introduction of exogenous peptides at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal. Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents and treatment regimens.

The invention further pertains to compositions, e.g., vectors, comprising a nucleotide sequence encoding reference or variant TSP-1 and/or TSP-4 gene products. For example, reference genes can be expressed in an expression vector in which a reference gene is operably linked to a native or other promoter. Usually, the promoter is a eukaryotic promoter for expression in a mammalian cell. The transcription regulation sequences typically include a heterologous promoter and optionally an enhancer which is recognized by the host. The selection of an appropriate promoter, for example t p, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected. Commercially available expression vectors can be used. Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.

The means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra. A wide variety of host cells can be employed for expression of the variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide. Processing includes glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, and the like.

It is also contemplated that cells can be engineered to express the reference allele of the invention by gene therapy methods. For example, DNA encoding the reference TSP gene product, or an active fragment or derivative thereof, can be introduced into an expression vector, such as a viral vector, and the vector can be introduced into appropriate cells in an animal. In such a method, the cell population can be engineered to inducibly or constitutively express active reference TSP gene product. In a preferred embodiment, the vector is delivered to the bone marrow, for example as described in Corey et al. (Science 244: 1215-1281 (1989)).

The invention further relates to the use of compositions (i.e., agonists) which enhance or increase the activity of the reference (or variant) TSP (e.g., TSP-1 or TSP-4) gene product, or a functional portion thereof, for use in the treatment of vascular disease. The invention also relates to the use of compositions (i.e., antagonists) which reduce or decrease the activity of the variant (or reference) TSP (e.g., TSP-1 or TSP-4) gene product, or a functional portion thereof, for use in the treatment of vascular disease. The invention also relates to constructs which comprise a vector into which a sequence of the invention has been inserted in a sense or antisense orientation. For example, a vector comprising a nucleotide sequence which is antisense to the variant TSP-1 or TSP-4 allele may be used as an antagonist of the activity of the TSP-1 or TSP-4 variant allele. Alternatively, a vector comprising a nucleotide sequence of the TSP-1 or TSP-4 reference allele may be used therapeutically to treat vascular diseases. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.

Preferred recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein . The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a nucleic acid of the invention can be expressed in bacterial cells (e.g., E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention. Accordingly, the invention further provides methods for producing a polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.

The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a nucleic acid of the invention has been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into their genome or homologous recombinant animals in which endogenous nucleotide sequences have been altered. Such animals are useful for studying the function and/or activity of the nucleotide sequence and polypeptide encoded by the sequence and for identifying and/or evaluating modulators of their activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, an "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing a nucleic acid of the invention into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The sequence can be introduced as a transgene into the genome of a non-human animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of a polypeptide in particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191 and in Hogan, Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and or expression of mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding the transgene can further be bred to other transgenic animals carrying other transgenes.

The invention also relates to the use of the variant and reference gene products to guide efforts to identify the causative mutation for vascular diseases or to identify or synthesize agents useful in the treatment of vascular diseases, e.g., CAD and MI. Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al, Science, 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro, or in vitro activity. Sites that are critical for polypeptide activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol, 224:899-904 (1992); de Vos et al. Science, 255:306-312 (1992)).

Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of proteins of the invention in clinical trials. An exemplary method for detecting the presence or absence of proteins or nucleic acids of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the protein, or nucleic acid (e.g., mRNA, genomic DNA) that encodes the protein, such that the presence of the protein or nucleic acid is detected in the biological sample. A preferred agent for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA sequences described herein, preferably in an allele-specific manner. The nucleic acid probe can be, for example, a full-length nucleic acid, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

The invention also encompasses kits for detecting the presence of proteins or nucleic acid molecules of the invention in a biological sample. For example, the kit can comprise a labeled compound or agent (e.g., nucleic acid probe) capable of detecting protein or mRNA in a biological sample; means for determining the amount of protein or mRNA in the sample; and means for comparing the amount of protein or mRNA in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect protein or nucleic acid.

The following Examples are offered for the puφose of illustrating the present invention and are not to be construed to limit the scope of this invention. The teachings of all references cited herein are hereby incoφorated herein by reference. EXAMPLES Identification of Single Nucleotide Polymoφhisms

The polymoφhisms shown in the Table were identified by resequencing of target sequences from individuals of diverse ethnic and geographic backgrounds by hybridization to probes immobilized to microfabricated arrays. The strategy and principles for design and use of such arrays are generally described in WO 95/11995.

A typical probe aoay used in this analysis has two groups of four sets of probes that respectively tile both strands of a reference sequence. A first probe set comprises a plurality of probes exhibiting perfect complementarily with one of the reference sequences. Each probe in the first probe set has an interrogation position that corresponds to a nucleotide in the reference sequence. That is, the interrogation position is aligned with the corresponding nucleotide in the reference sequence, when the probe and reference sequence are aligned to maximize complementarily between the two. For each probe in the first set, there are three corresponding probes from three additional probe sets. Thus, there are four probes corresponding to each nucleotide in the reference sequence. The probes from the three additional probe sets are identical to the corresponding probe from the first probe set except at the interrogation position, which occurs in the same position in each of the four corresponding probes from the four probe sets, and is occupied by a different nucleotide in the four probe sets. In the present analysis, probes were 25 nucleotides long. Arrays tiled for multiple different references sequences were included on the same substrate.

Publicly available sequences for a given gene were assembled into Gap4 (http://www.biozentrum.unibas.ch/~biocomp/staden Overview.html). PCR primers covering each exon were designed using Primer 3 (http://www- genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). Primers were not designed in regions where there were sequence discrepancies between reads. Genomic DNA was amplified in at least 50 individuals using 2.5 pmol each primer, 1.5 mM MgCl₂, 100 μM dNTPs, 0.75 μM AmpliTaq GOLD polymerase, and 19 ng DNA in a 15 μl reaction. Reactions were assembled using a PACKARD MultiPROBE robotic pipetting station and then put in MJ 96-well tetrad thermocyclers (96°C for 10 minutes, followed by 35 cycles of 96°C for 30 seconds, 59°C for 2 minutes, and 72°C for 2 minutes). A subset of the PCR assays for each individual were run on 3% NuSieve gels in 0.5X TBE to confirm that the reaction worked.

For a given DNA, 5 μl (about 50 ng) of each PCR or RT-PCR product were pooled (Final volume = 150-200 μl). The products were purified using QiaQuick PCR purification from Qiagen. The samples were eluted once in 35 μl sterile water and 4 μl 10X One-Phor-All buffer (Pharmacia). The pooled samples were digested with 0.2 μ DNasel (Promega)for 10 minutes at 37°C and then labeled with 0.5 nmols biotin-N6-ddATP and 15 μ Terminal Transferase (GibcoBRL Life Technology) for 60 minutes at 37°C. Both fragmentation and labeling reactions were terminated by incubating the pooled sample for 15 minutes at 100°C.

Low-density DNA chips (Affymetrix,CA) were hybridized following the manufacturer's instructions. Briefly, the hybridization cocktail consisted of 3M TMAC1, 10 mM Tris pH 7.8, 0.01% Triton X-100, 100 mg/ml herring sperm DNA (Gibco BRL), 200 pM control biotin-labeled oligo. The processed PCR products were denatured for 7 minutes at 100°C and then added to prewarmed (37°C) hybridization solution. The chips were hybridized overnight at 44°C. Chips were washed in IX SSPET and 6X SSPET followed by staining with 2 μg/ml SARPE and 0.5 mg/ml acetylated BSA in 200 μl of 6X SSPET for 8 minutes at room temperature. Chips were scanned using a Molecular Dynamics scanner.

Chip image files were analyzed using Ulysses (Affymetrix, CA) which uses four algorithms to identify potential polymoφhisms. Candidate polymoφhisms were visually inspected and assigned a confidence value: high confidence candidates displayed all three genotypes, while likely candidates showed only two genotypes (homozygous for reference sequence and heterozygous for reference and variant). Some of the candidate polymoφhisms were confirmed by ABI sequencing. Identified polymoφhisms were compared to several databases to determine if they were novel. Results are shown in the Table. Association of Thrombospondin Gene Polymoφhisms with Vascular Disease To determine pivotal genes associated with premature coronary artery disease, we analyzed DNA from 347 patients with MI or coronary revascularization before age 40 (men) or 45 (women) and 422 general population controls. Cases were drawn (one per family) from a retrospective collection of sibling pairs with premature CAD. Controls were ascertained through random-digit dialing. Both cases and controls were Caucasian. A complete database of phenotypic and laboratory variables for the affected patients afforded logistic regression to control for age, diabetes, body mass index, gender.

Thrombospondin (TSP) 4 and 1 emerged as important SNPs associated with premature CAD and MI. For CAD, 148 of 347 patients carried at least one copy of the TSP-4 variant compared with 142 of 422 control subjects; adjusted odds ratio 1.47, p=0.01. For premature MI, the association was even stronger: 91 of 187 cases vs. 142 of 422 controls had the variant; adjusted odds ratio 2.08, p=0.0003. The TSP-1 SNP was rare. Nonetheless, homozygosity for the variant allele gave an adjusted odds ratio of 9.5, p=.04.

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COL10A1, collagen, type X, alpha 1 (Schmid metaphyseal

G704U3 WIAF-14071 X60382 1824 chondrodysplasia) CATACCACGT [G/C] CATGTGAAAG S G C V V

COL10A1, collagen, type X, alpha 1 (Schmid metaphyseal

G704U4 WIAF-14072 X60382 1582 chondrodysplasia) AGTCATGCCT [G/C] AGGGTTTTAT M G C E Q

COL11A1, collagen, type XI, alpha

G705al WIAF-13228 J04177 686 1 AGAAGAAAAC [T/A] GTGACAATGA S T A T T

COL11A1, collagen, type XI, alpha

G705a2 WIAF-13229 J04177 698 1 TGACAATGAT [T/A] GTTGATTGTA S T A I I

COL11A1, collagen, type XI, alpha

G705a3 WIAF-13230 J04177 888 1 TAGTCCAGAC [T/A] GTGACTCTTC M T A c s

COL11A1, collagen. type XI, alpha

G705a4 WIAF-13231 J04177 894 1 AGACTGTGAC [T/A] CTTCAGCACC M T A s T

COL11A1, collagen, type XI, alpha

G705a5 WIAF-13232 J04177 651 1 TGACGGGAAG [T/A] GGCATCGGGT M T A w R

COL11A1, collagen, type XI, alpha 00

G705a6 WIAF-13233 J04177 661 1 TGGCATCGGG [T/A] AGCAATCAGC M T A V E 4^

COL11A1, collagen. type XI, alpha

G705a7 WIAF-13234 J04177 1597 1 CGTCCTGGCT [T/C] ACCAGGGGCT M T C L s

COL11A1, collagen, type XI, alpha

G705a8 WIAF-13235 J04177 2745 1 TGGGTTTCCA [G/A] GTGCCAATGG M G A G S

COL11A1, collagen, type XI, alpha

G705a9 WIAF-13236 J04177 4385 1 GTCCAGAAGG [T/A] CTTCGGGGCA S T A G G

COL11A1, collagen, type XI, alpha

G705al0 WIAF-13237 J04177 4576 1 GAAAAAGGTG [A/T] CCGAGGGCTC M A T D V

COL11A1, collagen, type XI, alpha

G705all WIAF-13238 J04177 4306 1 GCTAAGGGGG [A/C] AGCAGGTGCA M A C E A

COL11A1, collagen, type XI, alpha

G705al2 WIAF-13239 J04177 4837 1 AGACATACTG [A/G] AGGCATGCAA M A G E G

COL11A1, collagen, type XI, alpha

G705al3 WIAF-13240 J04177 4931 1 AACAAGACAT [C/T] GAGCATATGA S C T I I

COL11A1, collagen, type XI, alpha

G705al4 WIAF-13346 J04177 299 1 AAGCACTAGA [T/G] TTTCACAATT M T G D E

COL11A1, collagen. type XI, alpha

G705al5 WIAF-13347 J04177 22251 GGGAGCCTGG [G/C] CCTCCAGGTC S G C G G

CO

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C0L4A4 , collagen, type IV, alpha

G724a4 WIAF-12123 X81053 18384 ACCAGGAAAG [C/A] ATGGTGCCTC M C A H N

COL4A4 , collagen, type IV, alpha

G724u5 WIAF-12364 X81053 3764 CTGTTTGCCA [C/T] TGTGTTCCTG S C T H H

COL4A4 , collagen. type IV, alpha

G724u6 WIAF-1236S X81053 20184 TCCAGGGGAT [C/G] ATGAAGATGC M C G H D

COL4A4 , collagen. type IV, alpha

G724u7 WIAF-12366 X81053 47564 GCCTTCCCGT [A/G] TTTAGCACGC S A G V V

COL4A4 , collagen, type IV, alpha

G724U8 WIAF-12377 X81053 35954 CTGGACCACC [A/G] GGGTGCCCAG S A G P P

COL4A4 , collagen, type IV, alpha

G724U9 WIAF-12378 X81053 35164 GGAGCATCCG [G/C] AGAGCAGGGC M G C G A

COL4A4 , collagen, type IV, alpha

G724U10 WIAF-12379 X81053 42884 CTGGTCTTCC [A/G] GGTCCCAGAG S A G P P

COL4A4 , collagen, type IV, alpha

G724U11 WIAF-12380 X81053 51404 GCCACTTTTT [C/A] GCAAATAAGT M C A F L

COL4A4 , collagen, type IV, alpha

G724U12 WIAF-12387 X81053 2074 GACTTGCCTG [C/T] GATGTGGTCT C T oo

G727ul WIAF-12362 D90279 5135 COL5A1 , collagen, type V, alpha 1 TTCAAGGTTT [A/T] CTGCAACTTC M A T Y F CO

G727u2 WIAF-12369 D90279 4686 COL5A1, collagen, type V, alpha 1 AACAGGGTAT [C/T] ACTGGTCCTT S C T I I

G727u3 WIAF-12370 D90279 4608 COL5A1 , collagen, type V, alpha 1 TCGGTCCTCC [G/C] GGTGAACAGG S G c P P

G727a4 WIAF-13300 D90279 2034 COL5A1 , collagen, type V, alpha 1 ACGGCCTGGC [T/A] GGGTTGCCAG S T A A A

G727a5 WIAF-13301 D90279 2073 COL5A1 , collagen, type V, alpha 1 GTGACCCTGG [T/C] CCTTCCGGCC S T c G G

G727a6 WIAF-13302 D90279 3763 COL5A1 , collagen, type V, alpha 1 CGGGCAGAAA [G/A] GTGATGAAGG M G A G S

COL7A1, collagen, type VII , alpha 1 (epidermolysis bullosa, dystrophic, dominant and

G729ul WIAF-11844 L02870 2345 recessive) ATGGACTGGA [G/A] CCAGATACTG S G A E E

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While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWE CLAIM:

1. A method of diagnosing or aiding in the diagnosis of a vascular disease in an individual comprising a) obtaining a nucleic acid sample from the individual; and b) determining the nucleotide present at nucleotide position 2210 of the thrombospondin- 1 gene, wherein presence of a G at nucleotide position 2210 is indicative of increased likelihood of a vascular disease in the individual as compared with an individual having an A at nucleotide position 2210.

2. The method of Claim 1, wherein the thrombospondin- 1 gene has the nucleotide sequence of SEQ ED NO: 1.

3. The method of Claim 1, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.

4. The method of Claim 3, wherein the vascular disease is myocardial infarction.

5. The method of Claim 3, wherein the vascular disease is coronary heart disease.

6. A method of diagnosing or aiding in the diagnosis of a vascular disease in an individual comprising a) obtaining a nucleic acid sample from the individual; and b) determining the nucleotide present at nucleotide position 2210 of the thrombospondin- 1 gene, wherein presence of an A at nucleotide position 2210 is indicative of decreased likelihood of a vascular disease in the individual as compared with an individual having a G at nucleotide position 2210.

7. The method according to Claim 6, wherein the thrombospondin- 1 gene has the nucleotide sequence of SEQ ID NO: 1.

8. The method according to Claim 6, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.

9. The method according to Claim 8, wherein the vascular disease is myocardial infarction.

10. The method according to Claim 8, wherein the vascular disease is coronary heart disease.

11. A method for predicting the likelihood that an individual will have a vascular disease, comprising the steps of: a) obtaining a DNA sample from an individual to be assessed; and b) determining the nucleotide present at nucleotide position 2210 of the thrombospondin- 1 gene, wherein presence of a G at nucleotide position 2210 is indicative of increased likelihood of a vascular disease in the individual as compared with an individual having an A at nucleotide position 2210.

12. The method according to Claim 11, wherein the thrombospondin- 1 gene has the nucleotide sequence of SEQ ID NO: 1.

13. The method according to Claim 11, wherein the individual is an individual at risk for development of a vascular disease.

14. The method according to Claim 11, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.

15. The method according to Claim 14, wherein the vascular disease is myocardial infarction.

16. The method according to Claim 14, wherein the vascular disease is coronary heart disease.

17. A nucleic acid molecule comprising all or a portion of the nucleic acid sequence of SEQ ID NO: 1 wherein said nucleic acid molecule is at least 10 nucleotides in length and wherein the nucleic acid sequence comprises a polymoφhic site at nucleotide position 2210 of SEQ ID NO: 1.

18. The nucleic acid molecule according to Claim 17, wherein the nucleotide at the polymorphic site is different from a nucleotide at the polymorphic site in a corresponding reference allele.

19. An allele-specific oligonucleotide that hybridizes to the nucleic acid molecule of Claim 17.

20. A peptide of SEQ ID NO: 2 which is at least ten contiguous amino acids, wherein the peptide comprises the serine at amino acid position 700 of SEQ ID NO: 2.

21. A method of diagnosing or aiding in the diagnosis of a vascular disease in an individual comprising a) obtaining a biological sample comprising thrombospondin- 1 protein or relevant portion thereof from the individual; and b) determining the amino acid present at amino acid position 700 of the thrombospondin- 1 protein, wherein presence of an asparagine at amino acid position 700 is indicative of increased likelihood of a vascular disease in the individual as compared with an individual having a serine at amino acid position 700.

22. The method of Claim 21, wherein the thrombospondin- 1 protein has the amino acid sequence of SEQ ID NO: 2.

23. The method of Claim 22, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.

24. The method of Claim 23, wherein the vascular disease is myocardial infarction.

25. The method of Claim 23 , wherein the vascular disease is coronary heart disease.

26. A method of diagnosing or aiding in the diagnosis of a vascular disease in an individual comprising a) obtaining a biological sample comprising thrombospondin- 1 protein or relevant portion thereof from the individual; and b) determining the amino acid present at amino acid position 700 of the thrombospondin- 1 protein, wherein presence of a serine at amino acid position 700 is indicative of reduced likelihood of a vascular disease in the individual as compared with an individual having an asparagine at amino acid position 700.

27. The method according to Claim 26, wherein the thrombospondin- 1 protein has the amino acid sequence of SEQ ID NO: 2.

28. The method according to Claim 26, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous ^' thromboembolism and pulmonary embolism.

29. The method of Claim 28, wherein the vascular disease is myocardial infarction.

30. The method of Claim 28, wherein the vascular disease is coronary heart disease.

31. A method of diagnosing or aiding in the diagnosis of a vascular disease in an individual comprising a) obtaining a nucleic acid sample from the individual; and b) determining the nucleotide present at nucleotide position 1186 of the thrombospondin-4 gene, wherein presence of a C at nucleotide position 1186 is indicative of increased likelihood of a vascular disease in the individual as compared with an individual having an G at nucleotide position 1186.

32. The method of Claim 31 , wherein the thrombospondin-4 gene has the nucleotide sequence of SEQ ID NO: 3.

33. The method of Claim 31 , wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.

34. The method of Claim 33, wherein the vascular disease is myocardial infarction.

35. The method of Claim 33, wherein the vascular disease is coronary heart disease.

36. A method of diagnosing or aiding in the diagnosis of a vascular disease in an individual comprising a) obtaining a nucleic acid sample from the individual; and b) determining the nucleotide present at nucleotide position 1186 of the thrombospondin-4 gene, wherein presence of a G at nucleotide position 1186 is indicative of decreased likelihood of a vascular disease in the individual as compared with an individual having a C at nucleotide position 1186.

37. The method according to Claim 36, wherein the thrombospondin-4 gene has the nucleotide sequence of SEQ ID NO: 3.

38. The method according to Claim 36, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.

39. The method according to Claim 38, wherein the vascular disease is myocardial infarction.

40. The method according to Claim 38, wherein the vascular disease is coronary heart disease.

41. A method for predicting the likelihood that an individual will have a vascular disease, comprising the steps of: a) obtaining a DNA sample from an individual to be assessed; and b) determining the nucleotide present at nucleotide position 1186 of the thrombospondin-4 gene, wherein presence of a C at nucleotide position 1186 is indicative of increased likelihood of a vascular disease in the individual as compared with an individual having a G at nucleotide position 1186.

42. The method according to Claim 41, wherein the thrombospondin-4 gene has the nucleotide sequence of SEQ ID NO: 3.

43. The method according to Claim 41, wherein the individual is an individual at risk for development of a vascular disease.

44. The method according to Claim 41, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.

45. The method according to Claim 44, wherein the vascular disease is myocardial infarction.

46. The method according to Claim 44, wherein the vascular disease is coronary heart disease.

47. A nucleic acid molecule comprising all or a portion of the nucleic acid sequence of SEQ ID NO: 3 wherein said nucleic acid molecule is at least 10 nucleotides in length and wherein the nucleic acid sequence comprises a polymoφhic site at nucleotide position 1186 of SEQ ID NO: 3.

48. The nucleic acid molecule according to Claim 47, wherein the nucleotide at the polymoφhic site is different from a nucleotide at the polymoφhic site in a corresponding reference allele.

49. An allele-specific oligonucleotide that hybridizes to the nucleic acid molecule of Claim 47.

50. A peptide of SEQ ID NO: 4 which is at least ten contiguous amino acids, wherein the peptide comprises the proline at amino acid position 387 of SEQ ID NO: 4.

51. A method of diagnosing or aiding in the diagnosis of a vascular disease in an individual comprising a) obtaining a biological sample comprising thrombospondin-4 protein or relevant portion thereof from the individual; and b) determining the amino acid present at amino acid position 387 of the thrombospondin-4 protein, wherein presence of an alanine at amino acid position 387 is indicative of increased likelihood of a vascular disease in the individual as compared with an individual having a proline at amino acid position 387.

52. The method of Claim 51 , wherein the thrombospondin-4 protein has the amino acid sequence of SEQ ID NO: 4.

53. The method of Claim 52, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.

54. The method of Claim 53, wherein the vascular disease is myocardial infarction.

55. The method of Claim 53, wherein the vascular disease is coronary heart disease.

56. A method of diagnosing or aiding in the diagnosis of a vascular disease in an individual comprising a) obtaining a biological sample comprising thrombospondin-4 protein or relevant portion thereof from the individual; and b) determining the amino acid present at amino acid position 387 of the thrombospondin-4 protein, wherein presence of a proline at amino acid position 387 is indicative of reduced likelihood of a vascular disease in the individual as compared with an individual having an alanine at amino acid position 387.

57. The method according to Claim 56, wherein the thrombospondin-4 protein has the amino acid sequence of SEQ ID NO: 4.

58. The method according to Claim 56, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.

59. The method of Claim 58, wherein the vascular disease is myocardial infarction.

60. The method of Claim 58, wherein the vascular disease is coronary heart disease.

61. A nucleic acid molecule selected from the group consisting of the genes listed in the Table, wherein said nucleic acid molecule is at least 10 nucleotides in length and comprises a polymoφhic site identified in the Table, wherein a nucleotide at the polymoφhic site is different from a nucleotide at the polymoφhic site in a corresponding reference allele.

62. A nucleic acid molecule according to Claim 61, wherein said nucleic acid molecule is at least 15 nucleotides in length.

63. A nucleic acid molecule according to Claim 61, wherein said nucleic acid molecule is at least 20 nucleotides in length.

64. A nucleic acid molecule according to Claim 61, wherein the nucleotide at the polymoφhic site is the variant nucleotide for the gene listed in the Table.

65. An allele-specific oligonucleotide that hybridizes to a portion of a gene selected from the group consisting of the genes listed in the Table, wherein said portion is at least 10 nucleotides in length and comprises a polymoφhic site identified in the Table, wherein a nucleotide at the polymoφhic site is different from a nucleotide at the polymoφhic site in a corresponding reference allele.

66. An allele-specific oligonucleotide according to Claim 65 that is a probe.

67. An allele-specific oligonucleotide according to Claim 65, wherein a central position of the probe aligns with the polymoφhic site of the portion.

68. An allele-specific oligonucleotide according to Claim 65 that is a primer.

69. An allele-specific oligonucleotide according to Claim 68, wherein the 3' end of the primer aligns with the polymoφhic site of the portion.

70. An isolated gene product encoded by a nucleic acid molecule according to Claim 61.

71. A method of analyzing a nucleic acid sample, comprising obtaining the nucleic acid sample from an individual; and determining a base occupying any one of the polymoφhic sites shown in the Table.

72. A method according to Claim 71, wherein the nucleic acid sample is obtained from a plurality of individuals, and a base occupying one of the polymoφhic positions is determined in each of the individuals, and wherein the method further comprising testing each individual for the presence of a disease phenotype, and correlating the presence of the disease phenotype with the base.