WO2001064957A1 - Polymorphisms associated with insulin-signaling and glucose-transport pathways - Google Patents

Abstract

The invention discloses a collection of polymorphic sites in genes known or suspected to have a role in metabolic diseases including diabetes, obesity, cardiovascular disorders, hypertension and some forms of cancers that may share the same signaling pathways indicated in this application. The invention provides nucleic acids including such polymorphic sites. The nucleic acids can be used as probes or primers or for expressing variant proteins. The invention also provide methods of analyzing the polymorphic forms occupying the polymorphic sites.

Description

PATENT APPLICATION

POLYMORPHISMS ASSOCIATED WITH INSULIN-SIGNALING AND GLUCOSE -TRANSPORT PATHWAYS

CROSS-REFERENCE TO RELATED APPLICATION The present application derives priority from USSN 60/187,176, filed March 2, 2000, which is incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

The maintenance of glucose homeostasis in humans requires a dynamic balance among glucose absorption in the gut, glucose utilization by brain, muscle and adipose tissue, and glucose synthesis and storage by liver {for a recent review, see Shepherd and Kahn, NEJM 341, 248-257 (1999)} . Blood glucose levels are regulated by a complex interaction between circulating hormones (mainly insulin and glucagon) and cellular proteins involved in insulin signaling and glucose transport. Resistance to insulin-stimulated glucose uptake in insulin-responsive tissues (muscle and adipose tissue) is considered as the primary cause of type II diabetes afflicted by more than 200 millions individuals worldwide. In addition, insulin resistance (IR) represents a fundamental biochemical abnormality and has been strongly associated with a cluster of metabolic diseases, also called syndrome X, that include reduced levels of circulating high-density lipoproteins, hypertension, obesity and coronary heart disease. All of these are known to be the major contributors of mortality and morbidity in developed countries {Reaven, J Internal Medicine, 236:13-22 (1994)}. Although insulin was discovered more than 75 years ago, only until recently have we begun to understand the mechanism that regulates insulin-stimulation of glucose transport into cells. Based on genetic evidence it is clear that insulin resistance is due to genetic defects in a variety of genes in functionally-related pathways although many key genes in these pathways remain poorly understood {Pedersen O, Exp Clin Endocrinal Disbetes, 107:113-118 (1999)}. Intense research over the past two decades has led to the discovery of genes for insulin, insulin receptor, insulin receptor substrates. phosphatidylinositol-3(PI3)-kinase, glucose transporters, glycogen synthase, .and glucokinase. However, mutations in these genes are rare and genetic polymorphisms in these genes account for a small portion (<15%) of IR-related syndromes including diabetes. Clearly, novel genes and associated genetic variants downstream of insulin signaling and glucose transport pathways remain to be identified. In recent years, several candidate genes have been cloned in humans as well as in model organisms using various molecular and cell biology tools. These include: regulator of G-protein signaling (RGS2, and RGS5), synaptosomal-associated protein 23 (SNAP23), aldolase B (ALDOB), and protein phosphatase 1, catalytic subunit (PP1CB). This patent application concerns the identification of novel genetic variants in the 3 'untranslated region of these 5 genes and their potential utility as genetic markers for insulin resistance and associated metabolic syndrome.

SUMMARY OF THE INVENTION The invention provides nucleic acid sequence between 10 and 100 bases comprising at least 10 contiguous nucleotides including a polymorphic site or an immediately adjacent base from each nucleotide sequence shown in Table 1 or the complement thereof. The nucleic acids can be either DNA or R A and between 10 and 100 bases in length- The base occupying the polymorphic site in such nucleic acids can be either reference bases shown in Table 1 or an alternative base complementary to the bases shown in Table 1. In some nucleic acids, the polymorphic site is occupied by a base that correlates with nsulin resistance or susceptibility thereto. The invention further provides allele-specific oligonucleotides that hybridize to a nucleic acid segment shown in Table 1 or its complement, including the polymorphic site or an immediately adjacent base. Such oligonucleotides are useful as probes or primers.

The invention further provides methods of analyzing a nucleic acid sequence. Such methods entail obtaining the nucleic acid from an individual; and determining a base occupying any one of the polymorphic sites shown in Table 1 or other polymorphic sites in complete linkage disequilibrium therewith. Some methods determine a set of bases occupying a set of the polymorphic sites shown in Table 1. In some methods, the nucleic acid is obtained from a plurality of individuals, and a base occupying one of the polymorphic positions is determined in each of the individuals. Each individual is then tested for the presence of disease or sub-clinical phenotypes, and correlating the presence of the phenotypes with the base, particularly _insulin resistance and related metabolic diseases

In another aspect, the invention provides nucleic acids comprising an isolated nucleic acid sequence of Table 1, or the complement thereof, wherein the polymorphic site within the sequence or its complement is occupied by a base other than the reference base show in Table 1. Such nucleic acids are useful, for example, in regulating the stability of rnRNA encoding proteins. Further functional analysis of these variants can be performed by in vitro gene expression experiments or by production of transgenic animals. The invention further provides methods of diagnosing a phenotype. Such methods entail determining which polymorphic form(s) are present in a DNA sample from a subject at one or more polymorphic sites shown in Table 1, and diagnosing the presence of a phenotype correlated with the form(s) in the subject.

The invention also provides methods of screening polymorphic sites linked to polymorphic sites shown in Table 1 for suitability for diagnosing a phenotype. Such methods entail identifying a polymorphic site linked to a polymorphic site shown in Table 1, wherein a polymorphic form of the polymorphic site shown in Table 1 has been correlated with a phenotype. One then determines haplotypes in a population of individuals to indicate whether the linked polymorphic site has a polymorphic form in linkage disequlibrium with the polymoφhic form correlated with the phenotype.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 A depicts a block diagram of a computer system suitable for implementing the present invention. Fig. IB depicts a network of computer systems.

DEFINITIONS A nucleic acid can be DNA or RNA, .and single- or double-stranded. Oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means. Preferred nucliec acids of the invention include segments of DNA, or their complements including any one of the polymorphic sites shown in Table 1. The segments are usually between 5 and 100 contiguous bases, and often range from 5, 10, 12, 15, 20, or 25 nucleotides to 10, 15, 30, 25, 20, 50 or 100 nucleotides. Nucleic acids between 5-10, 5-20, 10-20, 12-30, 15-30, 10-50, 20-50 or 20-100 bases are common. The polymorphic site can occur within any position of the segment. The segments can be from any of the allelic forms of DNA shown in Table 1. For brevity in Table 1, the symbol T is used to represent both thymidine in DNA and uracil in RNA. Thus, in RNA oligonucleotides, the symbol T should be construed to indicate a uracil residue.

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

The term primer refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions (i.e., 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 40 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 means 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.

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

Polymorphism 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 fonn. Diploid organisms may be homozygous or heterozygous for allelic forms. A diallelic polymoφhism has two forms. A triallelic polymoφhism has three forms.

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.

A set of polymoφhisms means at least 2, and sometimes 5, or more of the polymoφhisms shown in Table 1.

Hybridizations are usually performed under stringent conditions that allow for specific binding between an ohgonucleotide and a target DNA containing one of the polymoφhic sites shown in Table 1. A stringent condition is defined as any suitable buffer concentrations and temperatures that allow specific hybridization of the ohgonucleotide to highly homologous sequence spanning at least one of the polymoφhic sites shown in Table 1 and any washing conditions that remove non-specific binding of the ohgonucleotide.. For example, conditions of _up to 1M NaCl or up to 3M TMAC (Tetømethylammonium chlorideand a temperature of up to 60C are suitable for allele- specific hybridizations of an ohgonucleotide up to 25 bases in length. The washing conditions usually range from room temperature to 60C.

An isolated nucleic acid means an object species invention that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). Preferably, an isolated nucleic acid comprises at least about 50- percent (on a molar basis) of all macromolecular species present. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods).

Linkage disequilibrium (LD) or allelic association means the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population. For example, if locus X has alleles a and b, which occur equally frequently, and linked locus Y has alleles c and d, which occur equally frequently, one would expect the haplotype ac to occur with a frequency of 0.25 in a population of individuals. If ac occurs more frequently, then alleles a and c are considered in linkage disequilibrium. Linkage disequilibrium may result from natural selection of certain combination of alleles or because an allele has been introduced into a population too recently to have reached equilibrium (random association) between linked alleles. A marker in linkage disequilibrium with disease predisposing variants can be particularly useful in detecting susceptibility to disease (or association with sub- clinical phenotypes) notwithstanding that the marker does not cause the disease. For example, a marker (X) that is not itself a causative element of a disease, but which is in linkage disequilibrium with a gene (including regulatory sequences) (Y) that is a causative element of a phenotype, can be used detected to indicate susceptibility to the disease in circumstances in which the gene Y may not have been identified or may not be readily detectable. Younger alleles (i.e., those arising from mutation relatively late in evolution) are expected to have a larger genomic segment in linkage disequilibrium. The age of an allele can be determined from whether the allele is shared among different human ethnic groups and/or between humans and related species.

DETAILED DESCRIPTION The invention provides a collection of novel polymoφhisms in five genes encoding products known or suspected to have roles in biochemical pathways relating to insulin signaling and glucose transport. Detection of polymoφhisms in such genes is useful in designing and performing diagnostic assays for evaluation of genetic risks for metabolic diseases including diabetes, obesity, cardiovascular disorders, hypertension and some forms of cancer. Analysis of polymoφhisms is also useful in designing prophylactic and therapeutic regimes customized to underlying abnormalities. Detection of polymoφhisms is also useful for conducting clinical trials of drugs for treatment of these diseases and the underlying biological abnormalities. As with other human polymoφhisms, the polymoφhisms of the invention also have more general applications, such as forensics, paternity testing, linkage analysis and positional cloning.

I. Novel Polymoφhisms of the Invention.

The present application provides 6 polymoφhisms in 5 genes that may play a significant role in insulin signaling and glucose transport. Two polymoφhisms (KVFP002 and KVFP003) are located 4 bp apart in the SNAP23 gene in the

3 'untranslated region. In a sample of 10 unrelated individuals (20 chromosomes) of European ancestry, these two polymoφhisms .are in complete linkage disequilibrium, exliibiting only two of the four possible haplotypes, C(+1546)-C(+1550) and T(+1546)- T(+1550). Table 1 shows the base occupying the polymoφhic sites, the location of these polymoφhisms with respect to the full-length cDNA, and the DNA sequence flanking the polymoφhic sites. Table 1 also provides information about the primers for amplification by PCR and the size of each amplified product. We identified these polymoφhisms by direct-sequencing of PCR products amplified from genomic DNA from 10 individuals of European Caucasian ancestry (Coriell Human Diversity Panel HD01). The allelic frequencies in 20 chromosomes from these 10 individuals are also given in Table 1. All 6 polymoφhisms are located in the 3' untranslated region (3'UTR) of the respective genes. The seventh column of the table shows two bases that can occupy each polymoφhic site. The first base is designated an being a wildtype or reference base and the second base as an alternative or variant base.

TABLE 1

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. 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 (each of which is incoφorated by reference for all puφoses).

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 isotheπnal 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

The identity of bases occupying the polymoφhic sites shown in Table 1 can be determined in an individual (e.g., a patient being analyzed) by several methods, which are described in turn. 1. Single Base Extension Methods

Single base extension methods are described by e.g., US 5,846,710, US 6,004,744, US 5,888,819 and US 5,856,092. In brief, the methods work by hybridizing a primer that is complementarity to a target sequence such that the 3' end of the primer is immediately adjacent to but does not span a site of potential variation in the target sequence. That is, the primer comprises a subsequence from the complement of a target polynucleotide terminating at the base that is immediately adjacent and 5' to the polymoφhic site. The hybridization is performed in the presence of one or more labelled nucleotides complementary to base(s)that may occupy the site of potential variation. For example, for a biallelic polymoφhisms two differentially labelled nucleotides can be used. For a tetraallelic polymoφhis four differentially labelled nucleotides can be used. In some methods, particularly methods employing multiple differentially labelled nucleotides, the nucleotides are dideoxynucleotides. Hybridization is performed under conditions permitting primer extension if a nucleotide complementarity to a base occupying the site of variation in the target sequence is present. Extension incoφorates a labelled nucleotide thereby generating a labelled extended primer. If multiple differentially labelled nucleotides are used and the target is heterozygous then multiple differentially labelled extended primers can be obtained. Extended primers are detected providing an indication of which bas(s) occupy the site of variation in the target polynucleotide.

2. 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. The polymoφhisms can also be identified by hybridization to nucleic acid arrays, some example of which are described by WO 95/11995 (incoφorated by reference in its entirety for all puφoses).

3. Allele-Specific Amplification Methods

An allele-specific primer hybridizes to a site on target DNA overlapping a polymoφhism and only primes amplification of an allelic foπn 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 leading to a detectable product signifying the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the 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. In some methods, the mismatch is included in the 3'-most position of the ohgonucleotide 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 confoπnation 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 fonn 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 difference between alleles of target sequences.

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. Association Studies with insulin resistance and related metabolic diseases

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. By analogy, 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.

The polymoφhism shown in Table 1 can be analyzed for a coπelation with insulin resistance, the metabolic processes that may lead to diabetes, obesity, hypertension, cardiovascular disease, and some forms of cancers and response to drugs used to treat these diseases. For puφoses of these studies, insulin resistance can be defined as _impaired insulin effect to stimulate glucose transport in insulin — responsive tissues. Some useful sub-phenotypes for association studies are _fasting circulating glucose and/or insulin levels, circulating glucose and/or insulin levels during oral glucose tolerance test (OGTT), acute insulin response test, and steady-state plasma glucose test (SSPG). To perform an OGTT, a patient is asked to fast overnight and is then given a beverage containing 75 grams of glucose. Blood samples are then drawn every half an hour for 2 to 3 hours. The levels of glucose and insulin in samples collected at different time points can be used as quantitative measures of whole body insulin response and insulin resistance. A quantitative test for acute insulin response is performed by an intravenous injection of glucose solution followed by frequent blood sampling (every 5- 10 minutes for 1 hour) to determine the amount of insulin output by pancreas after glucose injection. Acutre insulin response (AIR) is the mean insulin increment in the plasma insulin concentration above the basal in the first 8 minutes after glucose injection Correlation is performed for a population of individuals who have been tested for the presence or absence of metabolic diseases or an intermediate phenotype and for one or more polymoφhic markers. To perform such analysis, the presence or absence of a set of polymoφhic forms (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. Coπelation can be performed by standard statistical methods such as a K-squared test and statistically significant coπelations 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 _type II diabetes or a sub-phenotype as a dichotomous trait. 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 _type II diabetesor a sub-phenotype.

B. Diagnosis of metabolic diseases Polymoφhic forms that coπelate with metabolic diseases or intermediate phenotypes are useful in diagnosing metabolic diseases or susceptibility thereto. Combined detection of several such polymoφhic forms (for example, 2, 5, of the polymoφhisms listed in Table 1) typically increases the probability of an accurate diagnosis. For example, the presence of a single polymoφhic form known to coπelate with type II diabetes might indicate a probability of 20%) that an individual has or is susceptible to diabetes, whereas detection of five polymoφhic forms, each of which coπelates with less than 20%> probability, might indicate a probability up to 80%> that an individual has or is susceptible to diabetes. Analysis of the polymoφhisms of the invention can be combined with that of other polymoφhisms or other risk factors of diabetes, such as family history or obesity, as well as other known risk factors such as obesity and high blood pressure.

Patients diagnosed with diabetes can be treated with conventional therapies and/or can be counseled to undertake remedial life style changes, such as a low fat diet, or more exercise. Conventional therapies for diabetes include the use of sulphonylurea-class agents that stimulate insulin release by pancreatic beta cells, the use of acarbose-class agents that reduce glucose absoφtion in the guts, the use of biguanide and metformin to reduce hepatic glucose production.In recently years, a new class of therapeutic agents, thiazolidinediones, has been developed to specifcally traget insulin resistance by increasing insulin sensitivity in adipose tissues. These include Rezulin (Parke-Davis), Avandia (SB), and Actos (Lilly). Finally, insulin injection is often recommeded for those patients with beta-cell failure and is frequently used in combination of one of the above-listed drugs.

C. Drug Screening The polymoφhism(s) showing the strongest coπelation with insulin resistance within a given gene are likely either to have a causative role in the manifestation of the phenotype or to be in linkage disequilibrium with the causative variants. Such a role can be confirmed by in vitro gene expression of the variant gene or by producing a transgenic animal expressing a human gene bearing such a polymoφhism and determining whether the animal develops insulin resistance. Polymoφhisms in coding regions that result in amino acid changes usually cause insulin resistance by decreasing, increasing or otherwise altering the activity of the protein encoded by the gene in which the polymoφhism occurs. Polymoφhisms in coding regions that introduce stop codons usually cause insulin resistance by reducing (heterozygote) or eliminating (homozygote) functional protein produced by the gene. Occasionally, stop codons result in production of a truncated peptide with abeπant activities relative to the full-length protein. Polymoφhisms in regulatory regions typically cause insulin resistance by causing increased or decreased expression of the protein encoded by the gene in which the polymoφhism occurs. Polymoφhisms in intronic or untranslated sequences can cause insulin resistance either through the same mechanism as polymoφhisms in regulatory sequences or by causing altered spliced patterns resulting in an altered protein. The precise role of polymoφhisms in the five genes shoen in Table 1 can be elucidated by several means. Alterations in expression levels of a protein can be determined by measuring protein levels in samples groups of persons characterized as having or not having insulin resistance (or intermediate phenotypes). Alterations in enzyme activity can similarly be detected by assaying for enzyme activity in samples from the above groups of persons. Alterations in receptor transducing activity can be detected by comparing receptor ligand binding, either in vitro or in a cellular expression system.

Having identified certain polymoφhisms as having causative roles in insulin resistance, and having elucidated at least in general terms whether such polymoφhisms increase or decrease the activity or expression level of associated proteins, customized therapies can be devised for classes of patients with different genetic subtypes of metabolic diseases. For example, if a polymoφhism in a given protein causes insulin resistance by increasing the expression level or activity of the protein, the associated diseases associated with the polymoφhism can be treated by administering an antagonist of the protein. If a polymoφhism in a given protein causes insulin resistance by decreasing the expression level or activity of a protein, the form of metabolic diseases associated with the polymoφhism can be treated by administering the protein itself, a nucleic acid encoding the protein that can be expressed in a patient, or an analog or agonist of the protein. Agonists, antagonists can be obtained by producing and screening large combinatorial libraries. Combinatorial libraries can be produced for many types of compound that can be synthesized in a step by step fashion. Such compounds include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocychc compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates. Large combinatorial libraries of the compounds can be constructed by the encoded synthetic libraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO 93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which is incoφorated by reference for all puφoses). Peptide libraries can also be generated by phage display methods. See, e.g., Devlin, W0 91/18980. The libraries of compounds can be initially screened for specific binding to the protein for which agonists or antagonists are to be identified, or to its natural binding partner. Prefeπed agents bind with a Kd < μM. For example, for receptor ligand combinations, the assay can be performed using cloned receptor immobilized to a support such as a microtiter well and binding of compounds can be measured in competition with ligand to the receptor. Agonist or antagonist activity can then be assayed using a cellular reporter system or a transgenic animal model.

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

D. Other Diseases

In addition to diabetes, the polymoφhisms in Table 1 can also be tested for association with other diseases in wliich insulin resistance is a common feature but the underlying genetic defects are poorly understood. These include: .polysystic overian syndrome, obesity, hypertension, familial combined hyperlipidemia, hypertriglyceridemia, diabretic complications, and different forms of cancer in which abnormalities in cell-cyle control and apoptosis are commonly observed. The reported polymoφhisms may also be in LD with neraby genes (with

30 kb or greater) that are not related to insulin resistance but contribute to phenotypes 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.

Such correlations can be exploited in several ways. In the case of a strong coπelation 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 coπelated 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 coπelation 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 coπelated with enhanced receptiveness to one of several treatment regimes for a disease indicates that this treatment regime should be followed.

E. Forensics

Determination of which polymoφhic forms occupy a set of polymoφhic sites in an individual identifies a set of polymoφhic foπns 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. Prefeπed polymoφhisms for use in forensics are diallelic 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 eπor) 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 diallelic 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 are (see WO 95/12607):

Homozygote: p(AA)= x2 Homozygote: p(BB)= y2 = (l-x)2 Single Heterozygote: p(AB)= p(B A)= xy = x( 1 -x)

Both Heterozygotes: ρ(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(nonID) = 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.

F. 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 putative father, it can be concluded, barring experimental eπor, 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 diallelic 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.

G. Genetic Mapping of Phenotypic Traits The polymoφhisms shown in table 1 can also be used to establish 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) (each of which is incoφorated by reference in its entirety for all purposes).

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 loglO 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 coπesponding proteins. The nucleic acids comprise one of the sequences described in Table 1 in which the polymoφhic position is occupied by an alternative base for that position. Some nucleic acid encode full-length vari-ant forms of proteins. Similarly, variant proteins have the prototypical amino acid sequences of encoded by nucleic acid sequence shown in Table 1 (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 coπesponding codon in 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 frp, 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. Prefeπed 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.

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 cont.amin.ants, 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 prefeπed 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 coπesponding 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 coπesponding 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 ohgonucleotide as described above. 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 ohgonucleotide probes for detecting at least 10, 100 or all of the polymoφhisms shown in Table 1. 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. VI. Computer Systems For Storing Polvmoφhism Data

Fig. 1A depicts a block diagram of a computer system 10 suitable for implementing the present invention. Computer system 10 includes a bus 12 which interconnects major subsystems such as a central processor 14, a system memory 16

(typically RAM), an input/output (I/O) controller 18, an external device such as a display screen 24 via a display adapter 26, serial ports 28 and 30, a keyboard 32, a fixed disk drive 34 via a storage interface 35 and a floppy disk drive 36 operative to receive a floppy disk 38, and a CD-ROM (or DVD-ROM) device 40 operative to receive a CD-ROM 42. Many other devices can be connected such as a user pointing device, e.g., a mouse 44 connected via serial port 28 and a network interface 46 connected via serial port 30.

Many other devices or subsystems (not shown) may be connected in a similar manner. Also, it is not necessary for all of the devices shown in Fig. 1A to be present to practice the present invention, as discussed below. The devices and subsystems may be interconnected in different ways from that shown in Fig. 1A. The operation of a computer system such as that shown in Fig. 1A is well known. Databases storing polymoφhism information according to the present invention can be stored, e.g., in system memory 16 or on storage media such as fixed disk 34, floppy disk 38, or CD- ROM 42. An application program to access such databases can be operably disposed in system memory 16 or sorted on storage media such as fixed disk 34, floppy disk 38, or CD-ROM 42.

Fig. IB depicts the interconnection of computer system 10 to remote computers 48, 50, and 52. Fig. IB depicts a network 54 interconnecting remote servers 48, 50, and 52. Network interface 46 provides the connection from client computer system 10 to network 54. Network 54 can be, e.g., the Internet. Protocols for exchanging data via the Internet and other networks are well known. Information identifying the polymoφhisms described herein can be transmitted across network 54 embedded in signals capable of traversing the physical media employed by network 54.

Information identifying polymoφhisms shown in Table 1 is represented in records, which optionally, are subdivided into fields. Each record stores information relating to a different polymoφhisms in Table 1. Collectively, the records can store information relating to all of the polymoφhisms in Table 1, or any subset thereof, such as 5, 10, 50, or 100 polymoφhisms from Table 1. In some databases, the information identifies a base occupying a polymoφhic position and the location of the polymoφhic position. The base can be represented as a single letter code (i.e., A, C, G or T/U) present in a polymoφhic form other than that in the reference allele. Alternatively, the base occupying a polymoφhic site can be represented in IUPAC ambiguity code as shown in Table 1. The location of a polymoφhic site can be identified as its position within one of the sequences shown in Table 1. For example, in the first sequence shown in Table 1, the polymoφhic site occupies the Aor C base. The position can also be identified by reference to, for example, a chromosome, and distance from known markers within the chromosome. In other databases, information identifying a polymoφhism contains sequences of 10-100 bases shown in Table 1 or the complements thereof, including a polymoφhic site. Preferably, such information records at least 10, 15, 20, or 30 contiguous bases of sequences including a polymoφhic site.

From the foregoing, it is apparent that the invention includes a number of general uses that can be expressed concisely as follows. The invention provides for the use of any of the nucleic acid segments described above in the diagnosis or monitoring of diseases, particularly insulin resistance and related metabolic syndrome. The invention further provides for the use of any of the nucleic acid segments in the manufacture of a medicament for the treatment or prophylaxis of such diseases. The invention further provides for the use of any of the DNA segments as a pharmaceutical. All publications and patent applications cited above are incoφorated by reference in their entirety for all puφoses to the same extent as if each individual publication or patent application were specifically and individually indicated to be so incoφorated by reference. Although the present invention has been described in some detail by way of illustration and example for puφoses of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid of between 10 and 100 bases comprising at least 10 contiguous nucleotides including a polymoφhic site or an adjacent base from a sequence shown in Table 1 or the complement thereof.

2. The nucleic acid of claim 1 that is DNA.

3. The nucleic acid of claim 1 that is RNA.

4. The nucleic acid of claim 1 that is less than 50 bases.

5. The nucleic acid of claim 1 that is less than 20 bases.

6. The nucleic acid of claim 1, wherein the polymoφhic form occupying the polymoφhic site is a reference base shown in Table 1.

7. The nucleic acid of claim 1, wherein the polymoφhic form occupying the polymoφhic site is an alternative base shown in Table 1.

8. The nucleic acid of claim 7, wherein the alternative base coπelates with insulin resistance or susceptibility thereto.

9. The nucleic acids of claim 1, which are from genes encoding RGS5, SNAP23, ALDOB, RGS2, or PPP1CB.

10. An allele-specific ohgonucleotide that hybridizes to a sequence including a polymoφhic site or a base adjacent thereto shown in Table 1 or the complement thereof.

11. The allele-specific ohgonucleotide of claim 10 that is a probe.

12. An isolated nucleic acid comprising a sequence of Table 1 or the complement thereof, wherein the polymoφhic site within the sequence or its complement is occupied by a base other than the reference base show in Table 1.

13. A method of analyzing a nucleic acid, comprising: obtaining nucleic acid samples from a plurality of individual; and determining abase occupying any one of the polymoφhic sites shown in Table 1 or other polymoφhic sites in equilibrium dislinkage therewith in each of the individuals and testing each individual for the presence of a phenotype, and coπelating the^' presence of the disease phenotype with the base

14. The method of claim 13, wherein the determining comprises determining a set of bases occupying a set of the polymoφhic sites shown in Table 1.

15. A method of diagnosing a phenotype comprising: detennining which polymoφhic form(s) are present in a sample from a subject at one or more polymoφhic sites shown in Table 1; diagnosing the presence of a phenotype coπelated with the form(s) in the subject.

16. The method of claim 15, wherein the phenotype is insulin resistance or related sub-phenotypes.

17. A method of screening for a polymoφhic site suitable for diagnosing a phenotype, comprising: identifying a polymoφhic site linked to a polymoφhic site shown in Table 1, wherein a polymoφhic form of the polymoφhic site shown in Table 1 has been coπelated with a phenotype; and determining haplotypes in a population of individuals to indicate whether the linked polymoφhic site has a polymoφhic form in linkage disequlibriu with the polymoφhic form coπelated with the phenotype.

18. The method of claim 17, wherein the polymoφhic fonn of the polymoφhic site shown in Table 1 has been coπelated with _insulin resistance and/or associated metabolic syndrome.

19. The method of claim 17, wherein the linked polymoφhic site and the polymoφhic site shown in Table 1 are from the same gene.

20. A computer-readable storage medium for storing data for access by an application program being executed on a data processing system, comprising: a data structure stored in the computer-readable storage medium, the data structure including information resident in a database used by the application program and including:

a plurality of records, each record of the plurality comprising infonnation identifying a polymoφhisms shown in Table 1.

21. The computer-readable storage medium of claim 20, wherein each record has a field identifying a base occupying a polymoφhic site and a location of the polymoφhic site.

22. The computer-readable storage medium of claim 20, wherein each record identifies a nucleic acid segment of between 10 and 100 bases from a fragment shown in Table 1 including a polymoφhic site, or the complement of the segment.

23. The computer-readable storage medium of claim 20, comprising at least 10 records, each record comprising information identifying a different polymoφhism shown in Table 1.

24. A signal carrying data for access by an application program being executed on a data processing system, comprising: a data structure encoded in the signal, said data structure including information resident in a database used by the application program and including: a plurality of records, each record of the plurality comprising information identifying a polymoφhism shown in Table 1