WO2008121788A2 - Haplotypes of lipoprotein lipase, visceral adiposity and fasting insulin levels - Google Patents

Abstract

The invention provides various methods of diagnosing susceptibility to and/or protection against conditions associated with insulin resistance syndrome in an individual. In one embodiment, the present invention provides a method of determining protection against insulin resistance syndrome in an individual by determining the presence of haplotype (1) at the LPL locus. In another embodiment, the present invention provides a method of determining susceptibility to insulin resistance syndrome in an individual by determining the presence of haplotype (2) at the LPL locus and/or haplotype (4) at the LPL locus. In another embodiment, the individual is Hispanic.

Description

HAPLOTYPES OF LIPOPROTEIN LIPASE, VISCERAL ADIPOSITY AND FASTING

INSULIN LEVELS

GOVERNMENT RIGHTS This invention was made with U.S. Government support on behalf of the Insulin

Resistance Atherosclerosis Study ("IRAS") Family Study project, supported in part by National Institutes of Health Grants HL-60894, HL-60944, HL-60919, and HL-61019. Further support came from National Institutes of Health Program Project Grant HL- 28481. The U.S. Government may have certain rights in this invention.

FIELD OF THE INVENTION

The invention relates generally to the fields of metabolism and, more specifically, to genetic methods for diagnosing insulin resistance and/or sensitivity.

BACKGROUND

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Metabolic syndrome affects an estimated 50 million people in the United States alone. Those with metabolic syndrome, also called insulin resistance syndrome, often have an increased risk of diabetes, and diseases that are related to plaque build ups in artery walls such as coronary heart disease. The result of insulin resistance is an impaired metabolic response to the body's own insulin so that active muscle cells cannot take up glucose effectively. The blood insulin levels are chronically higher, which in turn inhibits fat cells from releasing energy stores. Although the specific causes of metabolic syndrome are not completely understood, primary risk factors include abdominal obesity, and insulin resistance where the body is unable to use insulin efficiently.

Elevated liver enzyme levels, likely a reflection of fatty liver, have also been associated with insulin resistance and metabolic syndrome. Family studies have shown that both the metabolic syndrome and liver enzymes are heritable, with heritability and co-heritability analyses indicating significant evidence for a genetic contribution to liver enzyme levels.

Lipoprotein lipase (LPL) has the ability to hydrolyze circulating triglycerides and thus allow uptake of free fatty acids in adipose tissue and muscle where lipid accumulation influences obesity and insulin-stimulated glucose uptake. LPL is also expressed in vascular wall macrophages where it influences atherosclerosis, and has been previously shown to be associated with insulin resistance. Thus, LPL is a possible candidate gene for insulin resistance and other metabolic traits.

Although there have been some associations found between risk factors and metabolic traits, the exact cause and contribution factors for many of the metabolic diseases are largely unknown. Thus, there is need in the art to determine genes, allelic variants, biological pathways, and other factors that contribute to metabolic traits, including but not limited to metabolic syndrome and insulin resistance.

SUMMARY OF THE INVENTION

Various embodiments provide methods for evaluating the likelihood of an individual to have or develop insulin resistance comprising obtaining a DNA sample from the individual, and analyzing the DNA sample for at least one haplotype of a human gene coding lipoprotein lipase ("LPL"), the at least one haplotype selected from the group consisting of haplotype 1 , haplotype 2 and haplotype 4, where the presence of haplotype 1 is predictive of increased insulin sensitivity, the presence of haplotype 2 is predictive of increased insulin resistance, and the presence of haplotype 4 is predictive of increased insulin resistance. In other embodiments, the individual is Hispanic. Other embodiments provide for methods of determining a low probability of developing insulin resistance syndrome in an individual, relative to a subject who has and maintains insulin resistance syndrome, comprising determining the presence or absence of haplotype 1 at the lipoprotein lipase ("LPL") locus in the individual, and diagnosing a low probability of developing insulin resistance syndrome in the individual, relative to a subject who has and maintains insulin resistance syndrome, based upon the presence of haplotype 1 at the LPL locus. In other embodiments, the haplotype 1 at the LPL locus comprises SEQ. ID. NO.: 1 , SEQ. ID. NO.: 2, SEQ. ID. NO.: 3, SEQ. ID. NO.: 4, SEQ. ID. NO.: 5, SEQ. ID. NO.: 6, SEQ. ID. NO.: 7, SEQ. ID. NO.: 8, SEQ. ID. NO.: 9, SEQ. ID. NO.: 10, SEQ. ID. NO.: 11 , and/or SEQ. ID. NO.: 12. In other embodiments, the individual is Hispanic.

Other embodiments provide for methods of diagnosing susceptibility to insulin resistance syndrome in an individual, relative to a healthy subject, comprising determining the presence or absence of at least one risk haplotype at the lipoprotein lipase ("LPL") locus selected from the group consisting of haplotype 4 and haplotype 2, and diagnosing susceptibility to insulin resistance syndrome in the individual, relative to a healthy subject, based upon the presence of at least one risk haplotype. In other embodiments, the risk haplotype at the LPL locus comprises SEQ. ID. NO.: 1 , SEQ. ID. NO.: 2, SEQ. ID. NO.: 3, SEQ. ID. NO.: 4, SEQ. ID. NO.: 5, SEQ. ID. NO.: 6, SEQ. ID. NO.: 7, SEQ. ID. NO.: 8, SEQ. ID. NO.: 9, SEQ. ID. NO.: 10, SEQ. ID. NO.: 11 , and/or SEQ. ID. NO.: 12. In other embodiments, the individual is Hispanic.

Various embodiments provide for methods of diagnosing insulin sensitivity in an individual, comprising determining the presence of haplotype 1 and haplotype 2 at the lipoprotein lipase ("LPL") locus, and diagnosing insulin sensitivity in the individual based upon the presence of haplotype 1 at the LPL locus and haplotype 2 at the LPL locus. In other embodiments, the individual is Hispanic.

Other embodiments provide for methods of diagnosing insulin resistance in an individual, comprising determining the presence of haplotype 4 at the lipoprotein lipase ("LPL") locus and haplotype 2 at the LPL locus, and diagnosing insulin resistance in the individual based upon the presence of haplotype 4 at the LPL locus and haplotype 2 at the LPL locus. In other embodiments, the individual is Hispanic.

Other embodiments provide methods of diagnosing insulin resistance in an individual, comprising determining the presence or absence of haplotype 4 at the lipoprotein lipase ("LPL") locus, haplotype 2 at the LPL locus, and an increase in visceral fat mass relative to a healthy individual, and diagnosing insulin resistance in the individual based upon the presence of haplotype 4 at the LPL locus, haplotype 2 at the LPL locus, and an increase in visceral fat mass relative to a healthy individual. In other embodiments, the individual is Hispanic.

Various embodiments provide for methods of treating insulin resistance syndrome, comprising determining the presence of one or more risk haplotypes at the lipoprotein lipase ("LPL") locus, and treating the insulin resistance syndrome. In other embodiments, one of the one or more risk haplotypes at the LPL locus is haplotype 2. In other embodiments, one of the one or more risk haplotypes at the LPL locus is haplotype 4. In other embodiments, the insulin resistance syndrome comprises diabetes, plaque build up in artery walls, and/or obesity.

Various embodiments also provide methods of treating insulin resistance syndrome in an individual, comprising determining the presence of a high level of lipoprotein lipase ("LPL") expression relative to a healthy subject, and treating the insulin resistance syndrome. In other embodiments, the visceral adipose tissue demonstrates a high level of LPL expression.

Various embodiments provide methods of diagnosing insulin resistance in an individual, comprising determining the presence or absence of haplotype 4 at the lipoprotein lipase ("LPL") locus and the presence or absence of high expression of LPL relative to a healthy subject, and diagnosing insulin resistance in the individual based upon the presence of haplotype 4 at the LPL locus and the presence of a high expression of LPL relative to a healthy subject. In other embodiments, haplotype 4 at the LPL locus comprises SEQ. ID. NO.: 1 , SEQ. ID. NO.: 2, SEQ. ID. NO.: 3, SEQ. ID. NO.: 4, SEQ. ID. NO.: 5, SEQ. ID. NO.: 6, SEQ. ID. NO.: 7, SEQ. ID. NO.: 8, SEQ. ID. NO.: 9, SEQ. ID. NO.: 10, SEQ. ID. NO.: 11 , and/or SEQ. ID. NO.: 12. In other embodiments, the adipose visceral tissue demonstrates a high level of LPL expression. In other embodiments, the individual is Hispanic.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawing, which illustrate, by way of example, various embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

Figure 1 depicts a haplotype block defined by 12 LPL variants spanning intron 7 to exon 10. The gene structure of the 3' end of LPL is shown at top. The locations of the genotyped SNPs relative to the exons are indicated. D' values are indicated (%) in the plot. The dark solid blocks indicate D' = 1 (100%) for the corresponding pair of variants. The lighter solid boxes also indicate D' = 1 , but with a low confidence score.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton ef a/., Dictionary of Microbiology and Molecular Biology 3^rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

"SNP" as used herein means single nucleotide polymorphism. "Haplotype" as used herein refers to a set of single nucleotide polymorphisms (SNPs) on a gene or chromatid that are statistically associated.

"Risk haplotype" as used herein refers to a haplotype whose presence is associated with an increase in susceptibility to a disease, including but not limited to insulin resistance.

"Protective haplotype" as used herein refer to a decrease in susceptibility to disease, including but not limited to insulin resistance.

"Insulin resistance syndrome" as used herein is also described as "metabolic syndrome," and may include the clustering of a number of key risk factors for cardiovascular disease, such as diabetes, hypertension and lipid disorders.

As used herein, an "increase in visceral fat mass" means a high level of visceral fat mass relative to levels ordinarily found in an healthy individual.

Measurements of adiposity may involve evaluating waist-to-hip ratios and other techniques readily available to one of skill in the art.

The identities of the LPL haplotypes and markers, their location on the gene and their nucleotide substitutions may be found in Figure 1 , as well as Table 2 and 3. As used herein, "LPL" means lipoprotein lipase.

Examples of rs312, rs319, rs320, rs327, rs328, rs330, rs4922115, rs3289, rs3200218, rs1059611 , rs15285, and rs3866471 are described herein as SEQ. ID. NOS: 1-12, respectively.

An example of an LPL gene is described herein as SEQ. ID. NO.: 13, and an example of LPL expressed as a peptide is described herein as SEQ. ID. NO.: 14.

As used herein, the term "IRAS Family Study" means the Insulin Resistance Atherosclerosis Study Family Study, a study designed to explore the genetics of insulin resistance and visceral adiposity. As used herein, the term "MACAD" means the Mexican-American Coronary Artery Disease project, a study aimed at identifying genes common to insulin resistance and atherosclerosis.

As used herein, the term "biological sample" means any biological material from which nucleic acid molecules can be prepared. As non-limiting examples, the term material encompasses whole blood, plasma, saliva, cheek swab, or other bodily fluid or tissue that contains nucleic acid.

The methods may include the steps of obtaining a biological sample containing nucleic acid from the individual and determining the presence or absence of a SNP and/or a haplotype in the biological sample.

As disclosed herein, the inventors demonstrated that LPL 3' end haplotypes are associated with indexes of insulin sensitivity/resistance. The inventors also evaluated haplotype association with other metabolic phenotypes measured in the IRAS Family Study. The inventors found that the same two LPL haplotypes (haplotypes 1 and 4) associated with insulin sensitivity and insulin resistance in the MACAD project were also associated with indexes of insulin sensitivity/resistance in the IRAS Family Study Hispanics. The inventors also provide a mechanism unifying the phenotypes associated with haplotype 4, that the increased LPL activity with haplotype 4 is mainly expressed in visceral adipose tissue, leading to increased visceral fat mass and a consequent increase in insulin resistance. Finally, the inventors discovered that the second most common haplotype in IRAS Family Studyg was associated with increased fasting insulin and adverse effects on lipid parameters, representing a new risk haplotype in LPL. The inventors uncovered a new risk haplotype, haplotype 2, which suggests a complex nature of LPL's effect on features of the insulin resistance syndrome.

In one embodiment, the present invention provides methods of diagnosing susceptibility to insulin resistance syndrome in an individual by determining the presence or absence of a risk haplotype at the LPL locus, wherein susceptibility to insulin resistance syndrome is determined by the presence of a risk haplotype at the LPL locus. In another embodiment, the risk haplotype at the LPL locus is haplotype 2 and/or haplotype 4. In another embodiment, the presence of haplotype 2 and/or haplotype 4 at the LPL locus is indicative of susceptibility to insulin resistance. In another embodiment, the presence of haplotype 2 and/or haplotype 4 is associated with an increase in visceral fat mass relative to a healthy individual. In another embodiment, the present invention provides methods of treatment of insulin resistance syndrome by determining the presence of haplotype 2 and/or haplotype 4 at the LPL locus and treating the insulin resistance syndrome. In another embodiment, the individual is Hispanic.

In one embodiment, the present invention provides methods of determining protection against insulin resistance syndrome by determining the presence or absence of a protective haplotype at the LPL locus, wherein the presence of the protective haplotype is indicative of a low probability of insulin resistance syndrome. In another embodiment, the protective haplotype at the LPL locus is haplotype 1. In another embodiment, haplotype 1 at the LPL locus is associated with insulin sensitivity. In another embodiment, the individual is Hispanic.

Variety of Methods and Materials

A variety of methods can be used to determine the presence or absence of a variant allele or haplotype. As an example, enzymatic amplification of nucleic acid from an individual may be used to obtain nucleic acid for subsequent analysis. The presence or absence of a variant allele or haplotype may also be determined directly from the individual's nucleic acid without enzymatic amplification.

Analysis of the nucleic acid from an individual, whether amplified or not, may be performed using any of various techniques. Useful techniques include, without limitation, polymerase chain reaction based analysis, sequence analysis and electrophoretic analysis. As used herein, the term "nucleic acid" means a polynucleotide such as a single or double-stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA. The term nucleic acid encompasses nucleic acid molecules of both natural and synthetic origin as well as molecules of linear, circular or branched configuration representing either the sense or antisense strand, or both, of a native nucleic acid molecule.

The presence or absence of a variant allele or haplotype may involve amplification of an individual's nucleic acid by the polymerase chain reaction. Use of the polymerase chain reaction for the amplification of nucleic acids is well known in the art (see, for example, Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994)).

A TaqmanB allelic discrimination assay available from Applied Biosystems may be useful for determining the presence or absence of a variant allele. In a TaqmanB allelic discrimination assay, a specific, fluorescent, dye-labeled probe for each allele is constructed. The probes contain different fluorescent reporter dyes such as FAM and VICTM to differentiate the amplification of each allele. In addition, each probe has a quencher dye at one end which quenches fluorescence by fluorescence resonant energy transfer (FRET). During PCR, each probe anneals specifically to complementary sequences in the nucleic acid from the individual. The 5' nuclease activity of Taq polymerase is used to cleave only probe that hybridize to the allele. Cleavage separates the reporter dye from the quencher dye, resulting in increased fluorescence by the reporter dye. Thus, the fluorescence signal generated by PCR amplification indicates which alleles are present in the sample. Mismatches between a probe and allele reduce the efficiency of both probe hybridization and cleavage by Taq polymerase, resulting in little to no fluorescent signal. Improved specificity in allelic discrimination assays can be achieved by conjugating a DNA minor grove binder (MGB) group to a DNA probe as described, for example, in Kutyavin et al., "3¹- minor groove binder-DNA probes increase sequence specificity at PCR extension temperature, "Nucleic Acids Research 28:655-661 (2000)). Minor grove binders include, but are not limited to, compounds such as dihydrocyclopyrroloindole tripeptide (DPI,).

Restriction fragment length polymorphism (RFLP) analysis may also be useful for determining the presence or absence of a particular allele (Jarcho et al. in Dracopoli et al., Current Protocols in Human Genetics pages 2.7.1-2.7.5, John Wiley & Sons, New York; lnnis et al.,(Ed.), PCR Protocols, San Diego: Academic Press, Inc. (1990)). As used herein, restriction fragment length polymorphism analysis is any method for distinguishing genetic polymorphisms using a restriction enzyme, which is an endonuclease that catalyzes the degradation of nucleic acid and recognizes a specific base sequence, generally a palindrome or inverted repeat. One skilled in the art understands that the use of RFLP analysis depends upon an enzyme that can differentiate two alleles at a polymorphic site.

Allele-specific oligonucleotide hybridization may also be used to detect a disease-predisposing allele. Allele-specific oligonucleotide hybridization is based on the use of a labeled oligonucleotide probe having a sequence perfectly complementary, for example, to the sequence encompassing a disease-predisposing allele. Under appropriate conditions, the allele-specific probe hybridizes to a nucleic acid containing the disease-predisposing allele but does not hybridize to the one or more other alleles, which have one or more nucleotide mismatches as compared to the probe. If desired, a second allele-specific oligonucleotide probe that matches an alternate allele also can be used. Similarly, the technique of allele-specific oligonucleotide amplification can be used to selectively amplify, for example, a disease-predisposing allele by using an allele-specific oligonucleotide primer that is perfectly complementary to the nucleotide sequence of the disease-predisposing allele but which has one or more mismatches as compared to other alleles (Mullis et al., supra, (1994)). One skilled in the art understands that the one or more nucleotide mismatches that distinguish between the disease-predisposing allele and one or more other alleles are preferably located in the center of an allele-specific oligonucleotide primer to be used in allele-specific oligonucleotide hybridization. In contrast, an allele- specific oligonucleotide primer to be used in PCR amplification preferably contains the one or more nucleotide mismatches that distinguish between the disease-associated and other alleles at the 3' end of the primer.

A heteroduplex mobility assay (HMA) is another well known assay that may be used to detect a SNP or a haplotype. HMA is useful for detecting the presence of a polymorphic sequence since a DNA duplex carrying a mismatch has reduced mobility in a polyacrylamide gel compared to the mobility of a perfectly base-paired duplex (Delwart et al., Science 262:1257-1261 (1993); White et al., Genomics 12:301-306 (1992)).

The technique of single strand conformational, polymorphism (SSCP) also may be used to detect the presence or absence of a SNP and/or a haplotype (see

Hayashi, K., Methods Applic. 1 :34-38 (1991)). This technique can be used to detect mutations based on differences in the secondary structure of single-strand DNA that produce an altered electrophoretic mobility upon non-denaturing gel electrophoresis. Polymorphic fragments are detected by comparison of the electrophoretic pattern of the test fragment to corresponding standard fragments containing known alleles.

Denaturing gradient gel electrophoresis (DGGE) also may be used to detect a SNP and/or a haplotype. In DGGE, double-stranded DNA is electrophoresed in a gel containing an increasing concentration of denaturant; double-stranded fragments made up of mismatched alleles have segments that melt more rapidly, causing such fragments to migrate differently as compared to perfectly complementary sequences (Sheffield et al., "Identifying DNA Polymorphisms by Denaturing Gradient Gel Electrophoresis" in lnnis et al., supra, 1990).

Other molecular methods useful for determining the presence or absence of a SNP and/or a haplotype are known in the art and useful in the methods of the invention. Other well-known approaches for determining the presence or absence of a SNP and/or a haplotype include automated sequencing and RNAase mismatch techniques (Winter et al., Proc. Natl. Acad. Sci. 82:7575-7579 (1985)). Furthermore, one skilled in the art understands that, where the presence or absence of multiple alleles or haplotype(s) is to be determined, individual alleles can be detected by any combination of molecular methods. See, in general, Birren et al. (Eds.) Genome Analysis: A Laboratory Manual Volume 1 (Analyzing DNA) New York, Cold Spring Harbor Laboratory Press (1997). In addition, one skilled in the art understands that multiple alleles can be detected in individual reactions or in a single reaction (a "multiplex" assay). In view of the above, one skilled in the art realizes that the methods of the present invention for diagnosing or predicting susceptibility to or protection against various genetic diseases in an individual may be practiced using one or any combination of the well known assays described above or another art- recognized genetic assay.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1 Replication of Prior Studies

In prior studies of Mexican Americans, an association was described of LPL gene haplotypes with insulin sensitivity/resistance and atherosclerosis. The most common haplotype (haplotype 1) was protective while the fourth most common haplotype (haplotype 4) conferred risk for insulin resistance and atherosclerosis; the latter haplotype was also associated with increased post-heparin plasma LPL activity. In the study of Hispanics in the IRAS Family Study, described herein, the inventors sought to replicate the LPL haplotype association with insulin sensitivity/resistance. The inventors genotyped 1424 subjects from 90 families at 12 LPL single nucleotide polymorphisms (SNPs) and reconstructed haplotypes spanning intron 7 to exon 10. The haplotype structure was identical to that observed in prior studies. Association analyses were conducted using the QTDT program, adjusting for age, sex, BMI. Haplotype 1 was associated with decreased fasting insulin (P=O.01); haplotype 4 was associated with increased fasting insulin (P=O.02) and increased visceral fat mass (P=O.002). The inventors discovered a novel association of the second most common haplotype (haplotype 2) with increased fasting insulin (P=O.01), increased triglycerides (P=O.02), increased triglyceride to HDL-C ratio (P=O.04), and increased apolipoprotein B (P=O.02). This study provides independent replication of prior results of LPL haplotypes 1 and 4 as associated with measures of insulin sensitivity and resistance, respectively. It also provides a mechanism unifying the phenotypes associated with haplotype 4, that the increased LPL activity with haplotype 4 is mainly expressed in visceral adipose tissue, leading to increased visceral fat mass and a consequent increase in insulin resistance. The inventors also uncovered a new risk haplotype, haplotype 2, which suggests a complex nature of LPL's effect on features of the insulin resistance syndrome.

Example 2 Investigation of LPL

LPL hydrolyzes triglycerides in circulating chylomicrons and VLDL particles, allowing uptake of free fatty acids and monoacylglycerol in target tissues. LPL is expressed in capillary beds of adipose tissue and muscle, where lipid uptake and accumulation impacts on obesity and insulin-stimulated glucose uptake. LPL is also expressed in vascular wall macrophages and smooth muscle cells, where its function may influence foam cell development and vascular reactivity/blood pressure, respectively (Mead JR, Cardiovasc Res 55:261-269, 2002). Given these diverse roles, the LPL gene has been examined as a candidate gene for insulin resistance and the multiple manifestations of the insulin resistance (metabolic) syndrome. The inventors' investigation of the LPL gene has focused on haplotypes in the 3' end of the gene, distal to a recombination hotspot in intron 6 (Templeton AR, Am J Hum Genet 66:69- 83, 2000). Six haplotype-tagging SNPs were identified and haplotypes based on these 6 SNPs, spanning intron 7 to intron 9, were associated with prevalent coronary artery disease (CAD) in Mexican Americans with a family history of CAD in the MACAD study (Goodarzi MO, Genet Med 5:322-327, 2003). In the same population, it was demonstrated that the same haplotypes were associated with insulin sensitivity/resistance (Goodarzi MO, Diabetes 53:214-220, 2004). A consistent pattern emerged, with the most common haplotype associated with protection against CAD and insulin resistance and the fourth most common haplotype predisposing to these conditions. The inventors believed that these effects were due to linkage disequilibrium of these haplotypes with functional variants in the 3' untranslated region (UTR) of LPL, encoded by exon 10; in rodents, LPL 3' UTR sequences influence translational regulation of LPL(Ranganathan G, J Biol Chem 275:40986-40991 , 2000; Ranganathan G, J Biol Chem 272:2515-2519, 1997). To test this, the inventors sequenced exon 10 in individuals with and without the risk haplotype; the additional polymorphisms discovered in exon 10 were combined with the original 6 SNPs and genotyped in the entire cohort, resulting in haplotypes based on 19 SNPs (Goodarzi MO, J Clin Endocrinol Metab 90:4816-4823, 2005). The extended fourth most common haplotype (designated 19-4) showed association with postheparin plasma LPL activity, showing the presence of functional variants; this haplotype was also associated with multiple phenotypes relevant to the metabolic syndrome, including body mass index, blood pressure, HDL-C and triglyceride levels, as well as insulin resistance (Goodarzi MO, J Clin Endocrinol Metab 90:4816-4823, 2005).

Example 3 Subjects

Individual participants were members of Hispanic families recruited for the IRAS Family Study from two clinical sites, San Antonio, TX; and the San Luis Valley, CO (Henkin L, The IRAS Family Study design and methods. Ann Epidemiol 13:211- 217, 2003.). Briefly, families were identified from probands of the original IRAS study (Wagenknecht LE, Ann Epidemiol 5:464-472, 1995), and were selected based on large family sizes. Ascertainment was also supplemented with non-IRAS participants (and their family members) who were recruited from the general population. Families were not selected based on any phenotypic criteria.

Example 4 Genotyping and Haplotype Determination

Whole blood obtained from each IRAS Family Study participant was frozen and stored at -70⁰C and then shipped in batches to the Molecular Genetics Laboratory at Wake Forest University School of Medicine. Genomic DNA was extracted from the blood samples and stored. The inventors genotyped 12 single nucleotide polymorphisms (SNPs) in the LPL gene. The inventors genotyped the original six LPL 3' end SNPs, rs312, rs319, rs320, rs327, rs328, rs330 (designated 7315, 8292, 8393, 8852, 9040, 9712, respectively in prior publications (Goodarzi MO, Nat Genet 19:233- 240, 1998). The inventors genotyped the following exon 10 variants: rs4922115, rs3289, rs3200218, rs1059611 , rs15285, rs3866471. These six variants were predicted to tag the common haplotypes in exon 10.

The 12 SNPs were genotyped at Cedars-Sinai Medical Center in 1424 subjects from 90 families using the 5'-exonuclease assay (TaqMan MGB). PCR primers and TaqMan MGB probes for these 12 SNPs were previously reported (Goodarzi MO, Genet Med 5:322-327, 2003; Goodarzi MO, J Clin Endocrinol Metab90:4816-4823, 2005).

The program Haploview was used to determine haplotype frequencies as well as delineate haplotype blocks (Barrett JC, Bioinformatics 21 :263-265, 2005). Haploview constructs haplotypes by using an accelerated expectation maximization algorithm similar to the partition/ligation method (Qin ZS, Am J Hum Genet 71 :1242- 1247, 2002), which creates highly accurate population founder frequency estimates of the phased haplotypes based on the maximum likelihood derived from the unphased input genotypes. Haploview was used to calculate linkage disequilibrium (LD, the D' statistic and r2) between each pairwise combination of all 12 SNPs used in haplotype block determination. To determine haplotype blocks, Haploview searches for regions of strong LD (D' > 0.8) running from one marker to another, wherein the first and last markers in a block are in strong LD with all intermediate markers.

Based on the pedigree structures and genotype data of all individuals in each pedigree, haplotypes were constructed as the most likely set (determined by the maximum likelihood method) of fully determined parental haplotypes of the marker loci for each individual in the pedigree, using the simulated annealing algorithm implemented in the program Simwalk2 (Sobel E, Am J Hum Genet 58:1323- 1337, 1996). Using this method, the inventors were able to assign a haplogenotype to 1262 of the 1424 genotyped subjects.

Example 5 Phenotyping

All participants in the IRAS Family Study were interviewed by project staff who were trained and monitored centrally. Subjects provided information concerning their medical history (current health status and clinical conditions, including type 2 diabetes and its complications, hypertension, and cardiovascular disease events and procedures), health behaviors, and demographic features. Blood was collected after a 12 hour fast in EDTA, immediately placed on ice. Plasma was separated by centrifugation at 4 ⁰C for a total of 30,000 G-minutes. Plasma samples were stored at -70 ⁰C prior to analysis.

Height and weight were measured to the nearest 0.5 cm and 0.1kg, respectively. BMI was calculated as weight/height2 (kg/m2). Girths (minimum waist, waist at the umbilicus and hips) were measured following a standardized protocol. Waist circumference, taken as the minimum circumference between the thorax and the hips, was measured to the nearest 0.5 cm using a steel tape. Systolic and diastolic blood pressures were measured three times; the average of the second and third measurements was used in analyses.

Indexes of glucose homeostasis were assessed by the frequently sampled intravenous glucose tolerance test (IVGTT) (Bergman RN, Endocr Rev 6:45-86, 1985), with minimal model (MINMOD) analyses (Pacini G, Comput Methods Programs Biomed 23:113-122, 1986). The IVGTT protocol, with two modifications (injection of insulin rather than tolbutamide and a reduced blood sampling protocol) has been previously described in detail (Bergman RN, Diabetes 52:2168-2174, 2003; Rich SS, Diabetes 53:1866-1875, 2004). Insulin sensitivity index (Sl) and glucose effectiveness (SG) were calculated using MINMOD software. The acute insulin response to glucose (AIRG) was the mean insulin increment in the plasma insulin concentration above the basal in the first 8 min after the administration of glucose. Disposition index (Dl) was calculated as Dl = AIRG x Sl. Plasma glucose and insulin values were obtained using standard methods and used to derive the HOMA index of insulin resistance (Matthews DR, Diabetologia 28:412-419, 1985). Of the genotyped subjects, 978 subjects from 86 families were haplotyped and had measures of insulin sensitivity.

Total cholesterol and triglyceride were measured using enzymatic methods. LDL-C was calculated using the Friedewald equation (Fhedewald WT, Clin Chemi 8:499-502, 1972) if triglyceride was less than 400 mg/dL or otherwise by ultracentrifugation. HDL-C was measured using the direct method (Sugiuchi H, Clin Chem 41 :717-723, 1995). Apo B was measured by immunoprecipitation. The protocol for computed tomographic evaluation of visceral and subcutaneous fat at the L2-L3 and L4-L5 levels has been previously described.

Example 6 Data Analysis

Log-transformed or square-root transformed trait values were used as appropriate to reduce skewness for all statistical analyses. Trait values between men and women were compared using generalized estimating equations, adjusting for familial relationships.

Association was evaluated by quantitative transmission disequilibrium testing for both individual polymorphisms and haplotypes using the QTDT program (Abecasis GR, Am J Hum Genet 66:279-292, 2000). The transmission disequilibrium test (TDT) was first developed for dichotomous traits in which alleles transmitted and not transmitted from the parents to affected offspring are compared to determine whether one allele is associated with the disease in question (Spielman RS, Am J Hum Genet 52:506-516, 1993). The TDT was later extended to quantitative traits (Allison DB, Am J Hum Genet 60:676-690, 1997). Abecasis et al. developed a general approach for scoring allelic transmission that accommodates families of any size and uses all available genotypic information (Abecasis GR, Am J Hum Genet 66:279-292, 2000). Family data allow the construction of an expected genotype for every non- founder, and orthogonal deviates from this expectation are a measure of allelic transmission. The QTDT program implements this general transmission disequilibrium testing using the orthogonal model of Abecasis (Abecasis GR, Eur J Hum Genet 8:545-551 , 2000).

Age, gender, and body mass index were specified as covariates in all analyses. Environmental variance, polygenic variance, and additive major locus were specified in the variance model. The within family component of association was evaluated, to eliminate any effects of population stratification. Trait values by haplotype are presented as the mean values in carriers of a particular haplotype versus non-carriers.

The primary phenotypes for association analysis were indexes of insulin sensitivity (fasting insulin, HOMA, and Sl), given the goal of replicating association of LPL haplotypes with insulin sensitivity/resistance. Overall P values as well as haplotype-specific P values were calculated for the primary traits. Secondary phenotypes analyzed included other traits given by the IVGTT (AIRG, Dl₁ SG), lipid traits, measures of adiposity (body mass index, waist to hip ratio, subcutaneous adipose tissue, and visceral adipose tissue) and blood pressure traits. Only the haplotypes showing association with the primary traits were analyzed for association with the secondary traits.

Example 7

Results - Clinical Characteristics The clinical characteristics of the 978 subjects (398 men, 580 women) who were haplotyped and phenotyped for insulin sensitivity/resistance are shown in Table 1. There were no significant differences between men or women in fasting insulin, HOMA, or Sl.

Example 8 Table 1 - Clinical Characteristics of the IRAS Family Subjects

Trait Men (n=398) Women (n=580)

Age (yr) 399 ±142 (373) 411 ± 133 (405)

Body mass index (kg/m²) 279 ± 51 (277) 286 ± 61 (274)

Fasting glucose (mmoI/L)* 529 ± 049 (522) 510 ±054 (500)

Fasting insulin (pmol/L) 1062 ±718(933) 1098 ± 847 (861)

HOMA 645 ± 488 (530) 643 ± 553 (483)

S₁ 216 ± 191 (172) 218 ±191 (168)

AIRG 7645 ± 6097 (5916) 7353 ± 6250 (5799)

DI 13485 ±12640(10285) 13170 ±12592(10096)

S₀ 00208 ± 00082 (00208) 00213 ±00095 (00207)

Total cholesterol (mmol/L)* 465 ± 097 (458) 449 ±091 (442)

LDL-C (mmol /L)* 294 ± 082 (284) 274 ± 076 (270)

HDL-C (mmol/L)* 103 ±031 (098) 119± 032 (116)

Triglycerides (mmol/L)* 149± 101 (124) 1230 ± 084 (100)

Tπglyceπde/HDL-C ratio* 388 ± 353 (273) 275 ± 276 (193)

Apolipoprotein B (g/L)* 011 ±006 (010) 015 ±007 (014)

SBP (mmHg)* 1191 ± 144(1160) 1136± 174(1100)

DBP (mmHg)* 789± 94 (790) 735 ± 93 (730)

Waist-to-hip ratio* 092 ± 006 (092) 080 ± 006 (080)

Subcutaneous adipose tissue (cm²)* 2630 ±1277 (2423) 3742 ± 1480 (3521)

Visceral adipose tissue (cm²)* 1203 ±607 (1113) 967 ± 537 (859)

Data are mean ± SD (median) *P<0001 between men and women

Example 9 Results - Frequencies of LPL SNPs The frequencies of the 12 LPL SNPs are shown in Table 2. The genotype frequencies for all markers were in Hardy-Weinberg equilibrium. Linkage disequilibrium among the twelve markers (D') ranged from 0.19 to 1.0 (average pairwise D' of 0.98; the majority of D' values were between 0.93 and 1.0). Expressed as r2, linkage disequilibrium ranged from 0.001 to 0.97(average r2 of 0.26). Given the high linkage disequilibrium among the SNPs, one haplotype block was identified, spanning all 12 SNPs from intron 7 through exon 10 (Figure 1). The common haplotypes in this block are displayed in Table 3. The haplotypes observed in this Hispanic population were also observed in prior studies of Hispanics ascertained via a proband with coronary artery disease (MACAD study), with modest differences in haplotype frequency. Table 3 displays IRAS haplotypes next to the corresponding MACAD haplotypes. Cladistic analysis suggested that the haplotype structure of the 3' end of the LPL gene is ancient (Templeton AR, Genetics 156:1259-1275, 2000); consistent with this, the inventors observed the same common LPL haplotypes in the IRAS Hispanics as previously observed in several other populations (Goodarzi MO, Genet Med 5:322-327, 2003).

Example 10

Table 2 - Frequency and Position Information on 12 LPL Variants

Variant Variation Location Position MAF

Designation rs312 (7315) G/C Intron 7 -4822 0.14 rs319 (8292) A/C Intron 8 -3845 0.19 rs320 (8393)* T/G Intron 8 -3744 0.29 rs327 (8852) T/G Intron 8 -3285 0.30 rs328 (9040)+ C/G Exon 9 -3097 0.088 rs330 (9712) G/A Intron 9 -2425 0.19 rs4922115 G/A Exon 10 - 3' UTR 10 0.17 rs3289 T/C Exon 10 - 3' UTR 372 0.025 rs3200218 A/G Exon 10 - 3' UTR 1251 0.18 rsl059611 T/C Exon 10 — S' UTR 1743 0.11 rs 15285 C/T Exon 10 — 3' UTR 1847 0.30 rs3866471 C/A Exon ID3' UTR 1849 0.17

MAF = minor allele frequency. Allele frequency data is from genotyping of 1424 subjects. Position is given to show relative distance of SNPs from one another; the numbering corresponds to the position relative to the first nucleotide of exon 10. Numbers in parentheses correspond to the naming of SNPs in prior studies. *rs320 is the Hindlll variant; -|τs328 is the Ser447stop variant.

Example 11 Table 3 - LPL Haplotypes Defined By Genotyping 12 Polymorphisms

Designation Haplotype based on 12 Corresponding variants in the Hispanic haplotype from the

American IRAS cohort MACAD study* -_

111111111111 (C 19 - - 1 (0.536)

2 121111112111 (0.176) 19 - - 2 (0.177) 3 212212211122 (0.122) 19 - - 3 (0.107) 4 112221111221 (0.096) 19 - - 4 (0.072) 5 112212211122 (0.027) 19 - - 6 (0.017) 6 111111111121 (0.025) 19 - - 5 (0.023) 7 111111121111 (0.025) 19 - - 7 (0.009)

Haplotype founder frequencies are shown in parentheses after each haplotype. The 12-variant based haplotypes were derived from 1262 haplogenotyped subjects. 1 indicates the major allele at each SNP, 2 the minor allele. *The Mexican-American Coronary Artery Disease (MACAD) study, a study of adult offspring of Mexican- American probands with coronary artery disease.

Example 12 Phenotype associated with haplotype

No LPL haplotype was significantly associated with the insulin sensitivity index from the IVGTT (overall P value for haplotypic association >0.1). However, LPL haplotypes were significantly associated with fasting insulin and the HOMA index (overall P values for haplotypic association 0.0011 and 0.0008, respectively). Significant individual haplotype associations were observed for these traits with the first, second, and fourth most common haplotypes. Haplotype 1 was associated with increased insulin sensitivity, as seen by the lower fasting insulin (P=O.010) in haplotype 1 carriers versus non-carriers (Table 4).

Conversely, haplotype 2 was associated with insulin resistance, i.e. higher fasting insulin (P=O.0096). Haplotype 4 was also associated with insulin resistance; carriers of haplotype 4 had higher fasting insulin (P=O.022). Association results for HOMA values tracked exactly as the fasting insulin association results. With the exception of an association of haplotype 4 with increased fasting glucose (P=O.026), the other indexes of insulin sensitivity/resistance, fasting glucose and Sl, were not statistically significantly associated with any of these three haplotypes; however, their mean values agreed with haplotype 1 as associated with insulin sensitivity and haplotypes 2 and 4 with insulin resistance (Table 4).

Secondary analyses investigating association of haplotypes 1 , 2, and 4 with lipid, adiposity, and blood pressure traits were next carried out. The inventors found no further associations of any traits with haplotype 1. Haplotype 4 was associated with increased visceral fat mass (P=O.0019) but not with any other of the traits in the secondary analyses. Haplotype 2 was associated with deleterious effects on lipid variables, including increased triglycerides (P=O.021), increased triglyceride/HDL ratio (P=O.041), and increased apolipoprotein B level (P=O.023). Example 13 Table 4 - Mean Phenotype Levels By Haplotype Carrier Status

Trait Haplotype Haplotype Haplotype Haplotype 1 2 3 4

Carrier Non-carrier Carrier Non- Carrier Non- Carrier Non-

(n=737) (n=241) (n=378) carrier (n=224) carrier (n=132) carrier

(n=600) (n= (n=

754) 846)

Primary Phenotypes

Fasting 5.17 =1= 5.21±0.53 5.22 ± 5.15±0.53 5.1 1±0.52 5.206 5.23 ± 5.17 glucose 0.53 0.53 ± 0.53 0.54§ ±0.53

(mmol/L

Fasting 106.2 ± 1 13.4 ± 79.6 114.1 ± 104.0 ± 1 14.1 ± 106.2 113.4±78. 106.9 insulin 79.6t 82.5+ 77.5 85.4 ± 78.2 9§ ± 79.6

(pmol/L)

Si 2.17 ± 2.17 ± 2.12 2.11 ± 2.21 ± 2.19±1.93 2.17 ± 2.10 ±2.11 2.18 ± 1.83 1.94 1.89 1.90 1.88

Secondary Phenotypes

VAT 1 12.6 1 16.7 ± 57.8 114.1 1 13.4 =1= 1 16.8 ± 112.7 1 19.1±64. 1 12.8 (cm²) ±62.9 ±60.0 62.7 60.2 ± 62.2 7* ±61.2

TG 1.38 1.47 ± 1.00 1.49 ± 1.35 ± 1.41±0.95 1.41 1.28 ± 1.43 ± (mmol/L) ±0.95 1.05* 0.90 ±0.97 0.85 0.98

TG/HDL 3.33±3.18 3.56 ± 3.43 3.62 ± 3.25 ± 3.21 ± 3.44±3 3.10 ± 3.44 ± 3.52 || 3.06 2.91 .35 2.85 3.31

Apo B 0.88 ± 0.91 ± 0.23 0.91 ± 0.88 ± 0.890 ± 0.891 0.872 ± 0.89 ±

(g/L) 0.239 0.22* 0.23 0.23 ± 0.23 0.21 0.23

Data are mean ± SD. *Signifϊcant association of phenotype with haplotype. P=0.002; +P=o.oi; ^P=O.02; §P=0.03; || P=0.04

Example 14 Conclusions In the study of Hispanic families of the IRAS Family Study, the inventors demonstrated association of LPL haplotype 1 with decreased fasting insulin and haplotype 4 with increased fasting insulin and with increased visceral fat mass. They also identified haplotype 2 as predisposing to both insulin resistance and dyslipidemic features.

Prior work demonstrated association of haplotype 1 with insulin sensitivity and haplotype 4 with insulin resistance in Hispanics with a family history of CAD in the MACAD study (Goodarzi MO, Diabetes 53:214-220, 2004). In the present study of Hispanic families, not selected based on any phenotype, the inventors have demonstrated association of haplotype 1 with decreased fasting insulin and haplotype 4 with increased fasting insulin. Variation in the 3' end of LPL appears to influence insulin sensitivity/resistance in Hispanics.

Example 15

Mechanism Unifying Previous Findings

In the prior MACAD study, insulin resistance was quantified by fasting measures and the hyperinsulinemic euglycemic clamp. While only the clamp derived measures achieved statistical significance for associations with LPL haplotypes 1 and 4, the mean trait values for the indexes based on fasting measures (fasting insulin and HOMA) were consistent (Goodarzi MO, Diabetes 53:214-220, 2004). In other words, carriers of haplotype 4 had decreased M value, increased fasting insulin, and increased HOMA; carriers of haplotype 1 had opposite trait values. In the present study, statistical significance for genetic association with LPL haplotypes was found with fasting insulin but not the physiologic insulin sensitivity index, Sl, from the IVGTT, although the mean SI values were consistent in terms of insulin sensitivity for haplotype 1 (higher Sl) versus insulin resistance for haplotype 4 (lower Sl). Initially this might seem surprising given the prior demonstration of higher heritability of SI compared to fasting insulin (Bergman RN, Diabetes 52:2168-2174, 2003). As fasting insulin is also influenced by insulin secretion and clearance, LPL haplotypes could show association with fasting insulin because they also influence these other aspects of insulin dynamics. As LPL is not expressed in the adult liver (the main organ of insulin clearance) but is expressed in pancreatic beta cells, an effect on insulin secretion is possible. The lack of association with AIRG suggests such an effect, if any, is on basal insulin secretion, not glucose-stimulated insulin secretion. However, given the results from previous work in the MACAD study and the lack of association with AIRG or Dl in the present study, the inventors believe that the association of LPL with fasting insulin/HOMA in IRAS is largely due to an effect on insulin sensitivity.

LPL is mainly expressed in adipose tissue and muscle. In adipose tissue, LPL promotes lipid storage. Overactivity of LPL may lead to excessive adipose accumulation. Excess adiposity may contribute to insulin resistance via altered secretion of adipocytokines such as adiponectin, leptin, tumor necrosis factor, and resistin. In muscle, LPL activity leads to fatty acid uptake, for use as fuel. If muscle oxidative capacity is exceeded, intramyocellular lipid deposition occurs, which is well known to inhibit insulin signaling and lead to impaired insulin-stimulated muscle glucose uptake (Boden G, Diabetes 50:1612-1617, 2001). In fact, transgenic mice with muscle-specific LPL overexpression exhibit whole body and muscle insulin resistance (Pulawa LK, Curr Opin Clin Nutr Metab Care 5:569-574, 2002). As it has been shown post-heparin plasma LPL activity is elevated in carriers of haplotype 4 (Goodarzi MO, J Clin Endocrinol Metab90:4816-4823, 2005), it is conceivable that such individuals have excess lipid accumulation in adipocytes and myocytes, both of which then contribute to insulin resistance by the mechanisms discussed above. Provocative support for this mechanism comes from the fact that haplotype 4 was associated with increased visceral fat mass. Increased visceral adiposity is thought to be a major risk factor for development of insulin resistance and its sequelae (Chan JC, Semin Vase Med 2:45-57, 2002; Lebovitz HE, Diabetes Care 28:2322-2325, 2005). Prior work identified haplotype 4 as predisposing to insulin resistance and increased LPL activity, albeit measured in post-heparin plasma, which reflects whole-body LPL activity. The present study describes a mechanism unifying these findings, that the increased LPL activity with haplotype 4 is mainly expressed in visceral adipose tissue, leading to increased visceral fat mass and the consequent increase in insulin resistance.

The association of haplotype 4 with body mass index in the MACAD cohort is clarified by the IRAS data, suggesting excess visceral adipose accumulation with haplotype 4. In the MACAD cohort, LPL haplotype 4 was also associated with adverse effects on blood pressure (Goodarzi MO, J Clin Endocrinol Metab90:4816- 4823, 2005), while no blood pressure associations were observed for this haplotype in IRAS. This could reflect that fact that the MACAD subjects were ascertained by a family history of CAD, whereas IRAS families were not selected based on a particular phenotype.

Haplotype 4 is a marker for an ancestral chromosome on which arose functional variant(s) that influence LPL activity and metabolic phenotypes. The minor alleles of six variants (rs328, rs11570891 , rs1803924, rs3735964, rs1059611 , rs10645926), the latter five of which were identified by sequencing exon 10, are found uniquely on haplotype 4 (Goodarzi MO, J Clin Endocrinol

Metab90:4816-4823, 2005). These 3' UTR variants are candidates for variants that alter the expression of LPL and thus increase LPL activity. Ser447Stop (rs328 or 9040), the rare allele of which is found only on haplotype 4, has in many studies been associated with increased LPL activity both in population genetic studies (Groenemeijer BE, REGRESS Study Group. Circulation 95:2628-2635, 1997) and in in vitro experimentation (Kozaki K, J Lipid Res 34:1765-1772, 1993; Zhang H, Biochim Biophys Acta 1302:159-166, 1996).

In the present study, haplotype 2 was associated with increased fasting insulin and adverse effects on lipids, with increased triglycerides, triglyceride to HDL-C ratio, and apolipoprotein B levels. Haplotype 2 thus emerges as predisposing to multiple facets of the metabolic syndrome. Indeed, the fact that Hispanics have the highest age-specific prevalence of the metabolic syndrome in the U.S. (Park YW, Arch Intern Med 163:427-436, 2003) may in part be explained by the high frequency (-18%) of haplotype 2 in this ethnic group.

While the description above refers to particular embodiments of the present invention, it should be readily apparent to people of ordinary skill in the art that a number of modifications may be made without departing from the spirit thereof. The presently disclosed embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. Furthermore, one of skill in the art would recognize that the invention can be applied to various metabolic conditions and disorders and diseases besides that of insulin resistance syndrome. It will also be readily apparent to one of skill in the art that the invention can be used in conjunction with a variety of phenotypes, such as additional genetic variants, biochemical markers, abnormally expressed biological pathways, and various clinical manifestations.

Claims

1. A method for evaluating the likelihood of an individual to have or develop insulin resistance comprising: obtaining a DNA sample from the individual; and analyzing the DNA sample for at least one haplotype of a human gene coding lipoprotein lipase ("LPL"), the at least one haplotype selected from the group consisting of haplotype 1 , haplotype 2 and haplotype 4, wherein the presence of haplotype 1 is predictive of increased insulin sensitivity, the presence of haplotype 2 is predictive of increased insulin resistance, and the presence of haplotype 4 is predictive of increased insulin resistance.

2. The method of claim 1 , wherein the individual is Hispanic.

3. A method of determining a low probability of developing insulin resistance syndrome in an individual, relative to a subject who has and maintains insulin resistance syndrome, comprising: determining the presence or absence of haplotype 1 at the lipoprotein lipase ("LPL") locus in the individual; and diagnosing a low probability of developing insulin resistance syndrome in the individual, relative to a subject who has and maintains insulin resistance syndrome, based upon the presence of haplotype 1 at the LPL locus.

4. The method of claim 3, wherein the haplotype 1 at the LPL locus comprises SEQ. ID. NO.: 1 , SEQ. ID. NO.: 2, SEQ. ID. NO.: 3, SEQ. ID. NO.: 4, SEQ. ID. NO.: 5, SEQ. ID. NO.: 6, SEQ. ID. NO.: 7, SEQ. ID. NO.: 8, SEQ. ID. NO.: 9, SEQ. ID. NO.: 10, SEQ. ID. NO.: 11 , and/or SEQ. ID. NO.: 12.

5. The method of claim 3, wherein the individual is Hispanic.

6. A method of diagnosing susceptibility to insulin resistance syndrome in an individual, relative to a healthy subject, comprising: determining the presence or absence of at least one risk haplotype at the lipoprotein lipase ("LPL") locus selected from the group consisting of haplotype 4 and haplotype 2; and diagnosing susceptibility to insulin resistance syndrome in the individual, relative to a healthy subject, based upon the presence of at least one risk haplotype.

7. The method of claim 6, wherein the risk haplotype at the LPL locus comprises SEQ. ID. NO.: 1 , SEQ. ID. NO.: 2, SEQ. ID. NO.: 3, SEQ. ID. NO.: 4, SEQ. ID. NO.: 5, SEQ. ID. NO.: 6, SEQ. ID. NO.: 7, SEQ. ID. NO.: 8, SEQ. ID. NO.: 9, SEQ. ID. NO.: 10, SEQ. ID. NO.: 11 , and/or SEQ. ID. NO.: 12.

8. The method of claim 6, wherein the individual is Hispanic.

9. A method of diagnosing insulin sensitivity in an individual, comprising: determining the presence of haplotype 1 and haplotype 2 at the lipoprotein lipase ("LPL") locus; and diagnosing insulin sensitivity in the individual based upon the presence of haplotype 1 at the LPL locus and haplotype 2 at the LPL locus.

10. The method of claim 9, wherein the individual is Hispanic.

11. A method of diagnosing insulin resistance in an individual, comprising: determining the presence of haplotype 4 at the lipoprotein lipase ("LPL") locus and haplotype 2 at the LPL locus; and diagnosing insulin resistance in the individual based upon the presence of haplotype 4 at the LPL locus and haplotype 2 at the LPL locus.

12. The method of claim 11 , wherein the individual is Hispanic.

13. A method of diagnosing insulin resistance in an individual, comprising: determining the presence or absence of haplotype 4 at the lipoprotein lipase

("LPL") locus, haplotype 2 at the LPL locus, and an increase in visceral fat mass relative to a healthy individual; and diagnosing insulin resistance in the individual based upon the presence of haplotype 4 at the LPL locus, haplotype 2 at the LPL locus, and an increase in visceral fat mass relative to a healthy individual.

14. The method of claim 13, wherein the individual is Hispanic.

15. A method of treating insulin resistance syndrome, comprising: determining the presence of one or more risk haplotypes at the lipoprotein lipase ("LPL") locus; and treating the insulin resistance syndrome.

16. The method of claim 15, wherein one of the one or more risk haplotypes at the LPL locus is haplotype 2.

17. The method of claim 15, wherein one of the one or more risk haplotypes at the LPL locus is haplotype 4.

18. The method of claim 15, wherein the insulin resistance syndrome comprises diabetes, plaque build up in artery walls, and/or obesity.

19. A method of treating insulin resistance syndrome in an individual, comprising: determining the presence of a high level of lipoprotein lipase ("LPL") expression relative to a healthy subject; and treating the insulin resistance syndrome.

20 The method of claim 19, wherein the visceral adipose tissue demonstrates a high level of LPL expression.

21. A method of diagnosing insulin resistance in an individual, comprising: determining the presence or absence of haplotype 4 at the lipoprotein lipase

("LPL") locus and the presence or absence of high expression of LPL relative to a healthy subject; and diagnosing insulin resistance in the individual based upon the presence of haplotype 4 at the LPL locus and the presence of a high expression of LPL relative to a healthy subject.

22. The method of claim 21 , wherein haplotype 4 at the LPL locus comprises SEQ. ID. NO.: 1 , SEQ. ID. NO.: 2, SEQ. ID. NO.: 3, SEQ. ID. NO.: 4, SEQ. ID. NO.: 5, SEQ. ID. NO.: 6, SEQ. ID. NO.: 7, SEQ. ID. NO.: 8, SEQ. ID. NO.: 9, SEQ. ID. NO.: 10, SEQ. ID. NO.: 11 , and/or SEQ. ID. NO.: 12.

23. The method of claim 21 , wherein the adipose visceral tissue demonstrates a high level of LPL expression.

24. The method of claim 21 , wherein the individual is Hispanic.