WO2000020436A1 - An isolated polynucleotide associated with type ii diabetes mellitus and methods of use thereof - Google Patents

Abstract

The present invention provides isolated polynucleotides comprising sequences from a region of human chromosome 20q between D20S119 and D20S195, wherein the isolated polynucleotides are useful to as probes in screening for Type II diabetes. The invention further provides vectors and isolated host cells comprising the isolated polynucleotides. The invention further provides methods of detecting polymorphisms on chromosome 20q between D20S119 and D20S195, and methods of detecting a propensity to develop Type II diabetes, using the isolated polynucleotides of the invention.

Description

AN ISOLATED POLYNUCLEOTIDE ASSOCIATED WITH TYPE II DIABETES MELLITUS AND METHODS OF USE THEREOF

TECHNICAL FIELD This invention relates to a polynucleotide associated with Type II diabetes, and to vectors and host cells comprising the polynucleotide. The invention further provides diagnostic methods using the polynucleotides of the invention.

BACKGROUND ART

Diabetes mellitus is a syndrome which results in disregulation of glucose homeostasis with multiple etiologic factors that generally involve absolute or relative insulin deficiency or insulin resistance or both. All causes of diabetes ultimately lead to hyperglycemia, which is the hallmark of this disease syndrome. Several clinical subclasses are recognized, including: Type I (insulin-dependent or IDDM), Type II (non-insulin-dependent diabetes mellitus), maturity-onset diabetes of the young (MODY) and gestational diabetes. Overall, in the United States the prevalence of diabetes is about 2 to 4 percent, with

IDDM comprising 7 to 10 percent of all cases. The prevalence of IDDM is probably more accurate than the estimates for Type II diabetes. This is due at least in part to the relative ease of ascertainment of IDDM, while many patients with Type II diabetes are asymptomatic and thus this form of the disease goes undiagnosed. Type II diabetes, the most common form of diabetes found in the United States, is characterized by a later age of onset, insulin resistance and impaired insulin secretion. Obesity and increased hepatic glucose output are also associated with Type II diabetes. Indeed, in the United States, 80 to 90 percent of Type II diabetes patients are obese. The precise role of obesity in the causes of Type II diabetes and the development of complications associated with diabetes remains equivocal.

Type II diabetes has been shown to have a strong familial transmission: 40% of monozygotic twin pairs with Type II diabetes also have one or several first degree relatives affected with the disease. Barnett et al. (1981) Diabetologia 20:87-93. In the Pima Indians, the relative risk of becoming diabetic is increased twofold for a child born to one parent who is diabetic, and sixfold when both parents are affected (Knowler, W. C, et al. (1988) Genetic Susceptibility to Environmental Factors. A Challenge for Public Intervention, Almquist & Wiksele International: Stockholm, p. 67-74). Concordance of monozygotic twins for Type II diabetes has been observed to be over 90%, compared with approximately 50% for monozygotic twins affected with Type I diabetes (Barnett, A.H., et al. (1981) Diabetologia 20(2):87-93). Non-diabetic twins of Type II diabetes patients were shown to have decreased insulin secretion and a decreased glucose tolerance after an oral glucose tolerance test (Barnett, AH., et al. (1981) Brit. Med. J. 282: 1656-1658).

Central fat, particularly intra-abdominal adipose tissue (IAAT), is associated with increased risk for Type II diabetes (Vague, J. (1996) Obesity Res. 4(2):201-3; Kissebah, AH., et al. (1982) J. of Clinical Endocrinology & Metabolism 54(2):254-60; Bjomtorp, P. (1992) Obesity 579-586). Diabetes is a complex syndrome affected not only by familial transmission but by environmental factors as well (Kahn, C.R. et al. (1996) Ann. Rev. of Med. 47:509-31; Aitman, T . and Todd, A.J. (1995) Baillieres Clin. Endocrinology & Metabolism 9(3):631-56). There is a high prevalence of the disease in world populations. Expression is strongly age- dependent and the etiology is heterogeneous. The combined effect of these factors makes mapping the genes responsible for Type II diabetes particularly challenging. For example, a major pitfall for using linkage analysis with a complex trait such as diabetes is the difficulty in establishing transmission models. The high prevalence of the disease in world populations, reduced penetrance, and the presence of phenocopies each contributes to reducing the power of linkage studies. Sib pair studies and the transmission disequilibrium test, non-parametric methods which do not require a model for mode of inheritance, are hampered by heterogeneity and the large number of phenocopies expected for such a complex common disease. A number of published findings suggest linkage of diabetes to chromosome 20q (Ji et al. (1997) Diabetes 46:876-81; Bowden, D.W., et al. (1997) Diabetes 46:882-86; Velho et al. (1997) Diabetes and Metabolism 23:34-37; and Zouali et al. (1997) HumanMolec. Genet. 6:1401-1408), but definition of a locus linked to susceptibility to Type II diabetes has thus far been unsuccessful.

Segregation analyses of Type II diabetes or related phenotypes have provided support for a major gene (Hanson, R.L., et al. (1995) Amer. J. of Human Genetics 57:(1): 160-70; Serjeantson, S.W. and Zimmet, P. (1991) Baillieres Clin. Endocrinology & Metabolism 5(3):477-93; Elston, R.C., et al. (1974) Amer. J. of Human Genetics 26(1): 13-34), though in some analysis models incorporating a major gene effect did not provide a significantly better fit than those with multifactorial inheritance, and more complex models were required to explain the data (Cook, J.T., et al. (1994) Diabetologia 37(12): 1231-40; McCarthy, M.I. et al. ( 1994) Diabetologia 37( 12) : 1221 -30). Segregation analysis of Type II diabetes is complicated by the fact that disease expression is strongly age dependent and, in certain populations, by the increase in recent years of the incidence of the disease. Since obesity is commonly associated with Type II diabetes, it can also influence the familial relationships. Mutations in hepatocyte nuclear factor-4α gene, which is located on chromosome 20, have been associated with maturity onset diabetes of the young (MODY), a form of Type II diabetes. Yamagata et al. (1996) Nature 384:458-460. However, genetic studies appear to have ruled out a role for the so-called MODY1 gene as a major late-onset Type II diabetes susceptibility gene. Velho and Froguel (1998) Eur. J. Endocrinol. 138:233-239. Ji et al. ((1997) Diabetes 46:876-881) tested whether a gene or genes in the MODY1 region of chromosome 20 contributes to the development of Type II diabetes. They reported a possible linkage between Type II diabetes and markers D20S119, D20S178, and D20S197. Bowden et al. ((1997) Diabetes 46:882-886) also examined the potential contribution of MODY genes to Type II diabetes susceptibility in African American and Caucasian Type II diabetes- affected sibling pairs with a history of adult-onset diabetic nephropathy. While a linkage was seen among Caucasian sib pairs between MODYl-linked marker D20S197 and Type II diabetes, no evidence for linkage of MODY 1 marker to Type II diabetes in Africa- American sib pairs was observed. Linkage disequilibrium (LD) analysis is a powerful tool for mapping disease genes and may be particularly useful for investigating complex traits. LD mapping is based on the following expectations: for any two members of a population, it is expected that recombination events occurring over several generations will have shuffled their genomes, so that they share little in common with their ancestors. However, if these individuals are affected with a disease inherited from a common ancestor, the gene responsible for the disease and the markers that immediately surround it will likely be inherited without change, i.e., will be identical by descent (IBD), from that ancestor. The size of the regions that remain shared, or IBD, are inversely proportional to the number of generations separating the affected individuals and their common ancestor. Thus, established populations are suitable for fine scale mapping and recently founded ones are appropriate for using LD to roughly localize disease genes. Because isolated populations typically have had a small number of founders, they are particularly suitable for LD approaches. LD analysis has been used in several positional cloning efforts. Kerem et al. (1989) Science 245:1073-1080; Hastbacka et al. (1992) Nat. Genet. 2:204-211; and Hastbacka et al. (1994) Cell 78:1073-1087. However, the initial localization had been achieved using conventional linkage methods. Positional cloning is the isolation of a gene solely on the basis of its chromosomal location, without regard to its biochemical function. It has been proposed that LD mapping could be used to screen the human genome for disease loci, without conventional linkage analysis. Lander and Botstein (1986) Proc. Natl. Acad. Sci. USA 83 :7353-7357. This approach was not practical until a set of mapped markers covering the genome became available. Weissenbach et al. (1992) Nature 359:794-801. These markers include microsatellites. Microsatellites are highly polymorphic markers based on variable numbers of short tandem repeats of 1 to 6 base pairs, whose abundance has been estimated at an average of one in every 6 kilobase of human genomic sequence. Thousands of microsatellites have been characterized. Since unique nucleotide sequences flanking microsatellites have been identified, and since each locus is small enough to be analyzed using polymerase chain reaction, microsatelhte analysis has emerged as a powerful tool for genetic analysis.

Even with the availability of mapped markers, mapping of complex traits has proven difficult. It has been suggested that mapping of complex traits, such as susceptibility to Type II diabetes, would require very large sample sizes and extremely dense marker maps, making whole genome population-based studies with relatively small sample sizes have been characterized unfeasible. Risch and Merikangas (1996) Science 273.1516-1517. Instead, it was suggested that very large sample sizes and extremely dense marker maps could be needed for whole genome association studies of complex traits, using standard association tests. However, an absence of LD around disease genes was assumed; this assumption is valid in large, heterogeneous study populations but not in genetically homogeneous ones. In homogeneous populations, LD may be maintained for distances of several centimorgans (cM) around disease genes due to the fact that affected individuals are IBD for the regions around disease genes. Additionally, in such populations one may test for association using methods that differentiate such IBD regions from background levels of haplotype sharing (Jorde, L.B. (1995) Amer. J. of Human Genetics 56(1): 11-14).

Identification of Type II diabetes gene(s) is of major interest, with enormous diagnostic and therapeutic potential. The foregoing discussion highlights the difficulties which have been encountered in attempts to identify genetic loci which contribute to Type II diabetes. Indeed, genome-wide scans by several groups have revealed that Type II diabetes is far more complex and heterogeneous than many had originally thought. Hanson (1997) Diabetes 46: S 1 :51 A; Mahtani et al. (1996) Nature Genetics 14:90-4; and Hanis et al. (1996) Nature Genetics 13:161-6. Because a genetic locus has not yet been identified which is unequivocally associated with Type II diabetes, methods for detecting susceptibility to this disease are lacking. In addition, methods for diagnosing the disease are currently insufficient.

DISCLOSURE OF THE INVENTION The present invention provides an isolated polynucleotide comprising sequences from a region on chromosome 20q between D20S 195 and D20S 119. In one embodiment, the invention provides an isolated polynucleotide comprising sequences from a region on chromosome 20q between D20S195 and D20S119 derived from an individual without Type II diabetes. The isolated polynucleotides of the invention are useful in methods of detecting a polymorphism in a region on chromosome 20q between D20S 195 and D20S 119, as well as in other diagnostic methods disclosed herein. The invention further provides vectors and host cells comprising the isolated polynucleotides.

The present invention further provides methods of detecting a polymorphism on chromosome 20q between D20S 195 and D20S 119. Any of a number of known methods can be used to detect a polymorphism in this region. In some embodiments, the methods involve contacting a polynucleotide sample, such as a DNA sample, derived from a human with a probe derived from a region on chromosome 20q between D20S195 and D20S119 under stringent hybridization conditions and determining whether specific hybridization has occurred. Depending on the probe, hybridization, or lack thereof, is indicative of a polymorphism. In one embodiment of the invention, the probe is derived from an individual in the subject Bahamian population who does not have Type II diabetes, i.e., a normal individual. Lack of hybridization with this probe is indicative of a polymorphism on chromosome 20q between D20S195 and D20S119.

The present invention also provides a method for detecting a propensity of an individual to develop Type II diabetes. The method generally involves analyzing a polynucleotide sample derived from an individual to be tested for the propensity for the presence of a DNA polymorphism in a region on chromosome 20q between D20S195 and D20S119, wherein the DNA polymorphism is associated with Type II diabetes. Any of a number of known methods can be used to detect the polymorphism in this region.

The invention further provides methods of confirming a phenotypic diagnosis of Type II diabetes. A polynucleotide sample derived from an individual is analyzed for the presence of a polymorphism which is associated with Type II diabetes and which is in a region on chromosome 20q between D20S195 and D20S119.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a pedigree chart showing families related to a single founder in the subject population. Individuals with diabetes are denoted by black symbols, deceased individuals by a diagonal slash. Squares indicate males; circles indicate females; diamond denote gender unknown. Arrows point to individuals whose genotype was determined. Boxed dots indicate consanguinity; numbers above boxed dots are kinship coefficients.

Figure 2 is a chart showing genotyping data on seven distantly related members of the subject population which have Type II diabetes.

Figures 3 A and 3B show chromosome map positions corresponding to the set of 400 primer pairs that define a 10 cM resolution linkage map.

Figure 4 shows the nucleotide sequence of microsatelhte marker D20S195 (GenBank Accession No. Z24371). The sequence is given 5' to 3'. Figure 5 shows the nucleotide sequence of microsatelhte marker D20S119 (GenBank

Accession No. Z17198). The sequence is given 5' to 3'.

Figure 6 shows the nucleotide sequence of microsatelhte marker D20S107 (GenBank Accession No. Z16656). The sequence is given 5' to 3'.

Figure 7 shows the nucleotide sequence of microsatelhte marker D20S170 (GenBank Accession No. Z23468). The sequence is given 5' to 3'.

MODES OF CARRYINGOUT THEINVENTION

We have localized a region associated with Type II diabetes to a small interval on chromosome 20q between D20S195 and D20S119. To achieve this localization, we have identified a population, a Bahamian island community, most of whose members are descended from a small number of founders. The diabetes in this population resembles Type II diabetes seen in the United States, i.e., it is characterized by adult age-of-onset and is associated with abdominal obesity. This population was analyzed by linkage disequilibrium using microsatelhte markers. Attempts to identify a chromosomal region associated with Type II diabetes have heretofor been unsuccessful.

Localization of this Type II diabetes-associated interval in this population provides for a polynucleotide probe(s) comprising sequences included within the interval on chromosome 20q between D20S195 and D20S119 from a normal individual in the population who does not have Type II diabetes. This polynucleotide can thus serve as a hybridization probe in methods for detecting a polymorphism on chromosome 20q between D20S195 and D20S119 in the DNA of an individual. Accordingly, localization of this Type II diabetes- associated interval allows the development of methods for detecting a polymorphism on chromosome 20q between D20S 195 and D20S 119 in the DNA of an individual. Such methods make possible the identification of polymorphisms associated with Type II diabetes. Identification of this interval further allows identification and characterization of a gene(s) associated with Type II diabetes. The localization further allows development of methods for detecting a propensity in an individual to develop Type II diabetes, and methods for confirming a phenotypic diagnosis of Type II diabetes.

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as "Current Protocols in Molecular Biology", eds. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987) and periodic updates. Techniques relating to linkage disequilibrium can be found in numerous publications, including, for example, Terwilliger (1995) Am. J Hum. Genet. 56:777. The transmission disequilibrium test (TDT) technique has been described in, for example, Spielman et al. (1993) Am. J. Hum. Genet. 52:506. Techniques relating to detection of mutations can be found in various publications, including for example, "Laboratory Methods for Detection of Mutations and Polymorphisms in DNA" (1997) CRC Press, G.R. Taylor, ed.; and "Laboratory Protocols for Mutation Detection" (1996) Oxford University Press, U. Landegrun, ed. Definitions

Hybridization reactions can be performed under conditions of different "stringency".

Conditions that increase stringency of a hybridization reaction of widely known and published in the art. See, for example, Sambrook et al. (1989). Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25°C, 37°C, 50°C and 68°C; buffer concentrations of 10 X SSC, 6 X SSC, 1 X SSC, 0.1 X SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6 X SSC, 1 X SSC, 0.1 X SSC, or deionized water.

"T_m" is the temperature in degrees Celcius at which 50% of a polynucleotide duplex made of complementary strands hydrogen bonded in anti-parallel direction by Watson-Crick base pairing dissociates into single strands under conditions of the experiment. T_m may be predicted according to a standard formula, such as:

T_m = 81.5 + 16.6 logpf] + 0.41 (%G/C) - 0.61 (%F) - 600/L where [Xf] is the cation concentration (usually sodium ion, Na⁺) in mol/L; (%G/C) is the number of G and C residues as a percentage of total residues in the duplex; (%F) is the percent formamide in solution (wt/vol); and L is the number of nucleotides in each strand of the duplex.

Stringent conditions for both DNA/DNA and DNA/RNA hybridization are as described by Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, herein incorporated by reference. For example, see page 7.52 of Sambrook et al.

A polynucleotide or polynucleotide region has a certain percentage (for example, 80%), 85%>, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases are the same in comparing the two sequences. This ahgnment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters for a given alignment program are used. The term "a propensity to develop Type II diabetes", as used herein, intends a statistically significant increase in the probability of developing measurable symptoms of Type II diabetes in an individual having a particular genetic lesion(s) or polymorphism(s) compared with the probability in an individual lacking the genetic lesion or polymorphism. "Polymorphism", as used herein, refers to a difference in the nucleotide sequence of a given region as compared to a nucleotide sequence in a homologous region of another individual, in particular, a difference in the nucleotide sequence of a given region which differs between individuals of the same species. Polymorphisms include single nucleotide differences, differences in sequence of more than one nucleotide, insertions, inversions and deletions.

The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single-, double-stranded and triple helical molecules. "Oligonucleotide" generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art.

The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art.

A "substantially isolated" or "isolated" polynucleotide is one that is substantially free of the sequences with which it is associated in nature. By substantially free is meant at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of the materials with which it is associated in nature. As used herein, an "isolated" polynucleotide also refers to recombinant polynucleotides, which, by virtue of origin or manipulation: (1) are not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) are linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.

The term "vector" refers to a DNA molecule that can carry inserted DNA and be perpetuated in a host cell. Vectors are also known as cloning vectors, cloning vehicles or vehicles. The term includes vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication vectors that function primarily for the replication of nucleic acid, and expression vectors that function for transcription and/or translation of the DNA or R A. Also included are vectors that provide more than one of the above functions. A "host cell" includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the polynucleotides of the present invention. An "isolated host cell" is one which is not associated with, i.e., has been physically dissociated with, the organism from which it was derived.

A "individual " is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

As used herein, a "normal individual" is a member of the subject Bahamian population who is a member of the pedigree family shown in Figure 1, who does not have Type II diabetes and who does not carry a chromosome 20 allele for Type II diabetes. The "subject Bahamian population" is that described in Example 1, and includes members of the pedigree family shown in Figure 1, as well as distantly related individuals whose DNA was examined in order to identify shared alleles. An isolated polynucleotide of the invention which is derived from a normal individual and which is contained within an isolated host cell is being deposited with Coriell Cell Repository. As used herein, an "affected individual" is one who has symptoms of Type II diabetes. An isolated polynucleotide of the invention which is derived from an affected individual from the subject Bahamian population and which is contained within an isolated host cell is being deposited with Coriell Cell Repository. An isolated polynucleotide comprising sequences from a region on chromosome 20q between D20S119 and D20S195 which serves as a "normal" control is derived from 1) a "normal" individual, as described above; and/or 2) the normal homolog of the region on chromosome 20q between D20S119 and D20S195 from a heterozygous, affected individual from the subject Bahamian population.

A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples. "Transformation" or "transfection" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, lipofection, transduction, infection, electroporation, CaPO₄ precipitation, DEAE-dextran, particle bombardment, etc. The exogenous polynucleotide may be maintained as a non- integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.

Isolated polynucleotides comprising a region on chromosome 20q between D20S195 and D20S119

The present invention provides isolated polynucleotides comprising a region of chromosome 20q between D20S195 and D20S119. These polynucleotides comprise at least one polymorphism associated with Type II diabetes, and/or can be used to detect at least one polymorphism associated with Type II diabetes, and therefore have utility in a variety of diagnostic methods, as described herein.

Accordingly, the present invention encompasses isolated polynucleotides comprising a region of chromosome 20q between D20S195 and D20S119, vectors containing these polynucleotides, host cells containing these polynucleotides, and compositions comprising these polynucleotides. These polynucleotides are isolated and/or produced by chemical and/or recombinant methods, or a combination of these methods. Unless specifically stated otherwise, "polynucleotides" shall include all embodiments of the polynucleotide of this invention. These polynucleotides are useful as probes, primers, in expression systems, and in diagnostic methods as described herein.

An isolated polynucleotide of the present invention comprises a sequence contained within a region flanked by microsatelhte markers D20S 195 and D20119, (SEQ LD NO : 1 and SEQ LD NO:2, respectively; Figures 4 and 5, respectively). In one embodiment, the invention provides isolated polynucleotides comprising a region of chromosome 20q between D20S195 and D20S119 from a normal individual.

The isolated polynucleotide need not include the entire region of chromosome 20q between D20S 195 and D20S 119 as long as at least one polymorphism associated with Type II diabetes is included within the polynucleotide fragment, or as long as the polynucleotide fragment can detect at least one polymorphism associated with Type II diabetes. Using the oligonucleotide primers derived from SEQ D NO:l and SEQ LD NO: 2, polynucleotides of about 300 kb (kilo base pairs) to about 1000 kb can be identified and isolated. Oligonucleotide primers derived from SEQ LD NO: 1 and SEQ LD NO: 2 which are useful in amplifying microsatelhte markers D20S195 and D20S119, respectively, are those which flank the repeat sequence. For example, oligonucleotide primers which would amplify D20S119 include the following: 5' agctaactgacacagtttcag 3' (nucleotides 1-21 of SEQ LD NO:2); and 5' agtacattttctggcacttga 3' (complement of nucleotides 300 to 320 of SEQ LD NO:2).

Accordingly, a polynucleotide of the invention may be about 100 contiguous nucleotides, about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1200, 1500, 1700, 2000, 3000, 4000, 5000, 6000, 7000 contiguous nucleotides or larger of the sequence flanked by microsatelhte markers D20S195 and D20119, which can be amplified using oligonucleotide primers derived from SEQ LD NO: 1 and SEQ LD NO:2.

Also encompassed in the present invention are isolated polynucleotides comprising 150 contiguous kilobases having at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, even more preferably greater than 90% sequence identity to a sequence flanked by (i.e., comprised within) microsatelhte markers DS20S195 and DS20119, said polynucleotide comprising at least one polymorphism associated with Type II diabetes, and/or capable of detecting at least one polymorphism associated with Type II diabetes.

Also within the invention is an isolated polynucleotide at least about 15 nucleotides in length (preferably at least about 30, more preferably at least 100, more preferably at least about 150, even more preferably at least about 200, even more preferably at least about 250, even more preferably at least about 300, even more preferably at least about 400, and most preferably at least 450), including (a) a strand which hybridizes under stringent conditions to a DNA sequence flanked by (i.e., comprised within) microsatelhte markers D20S195 and D20119 from an individual with Type II diabetes or a normal individual, (b) the complement thereof, or (c) a double-stranded DNA including both (a) and (b). Multiple copies of this isolated DNA (useful, for example, as a hybridization probe or PCR primer) can be produced by recombinant means, by transfecting a cell with a vector containing this DNA.

Microsatelhte markers D20S107 and D20S170 are located between D20S195 and D20S119. As described in Example 2, Terwilliger analysis of D20S107 and D20S170 showed a maximum LOD score between these two markers. Accordingly, the invention encompasses isolated an isolated polynucleotide comprising the interval between D20S107 and D20S170 (SEQ ID NO:3 and SEQ ID NO:4, respectively; Figures 6 and 7, respectively). The invention further encompasses an isolated polynucleotide comprising sequences flanked by D20S195 and D20S107. In addition to D20S107 and D20S170, over 100 markers which lie between D20S195 and D20S119 are known, and the sequences are available. The sequences of these markers are available through linkages at the Web site http://cedar.genetics.soton.ac.uk/pub/chrom20/map.html, updated as of September 14, 1998. The nucleotide sequences of D20S195, D20S119, D20S107, D20S170, as well as these additional markers can be used to design oligonucleotide primers to prime PCR reactions to amplify polynucleotides between the markers, as described above. The amplified polynucleotides can be isolated by conventional means and, if desired, cloned into cloning and/or expression vectors. The amplified polynucleotides can be further tested for the presence of a sequences and/or polymorphisms associated with Type II diabetes. These isolated polynucleotides are encompassed by the present invention. Identification of a region on chromosome 20q associated with Type II diabetes

To identify a region of chromosome 20q between D20S195 and D20S119 as being associated with Type II diabetes, an iterative approach combining genome screening and localization techniques based on the findings from the genome screening can be followed. The following steps can be performed.

Step 1. Complete genotyping can be carried out, using a full set of genome screening markers on the subjects and their relatives, using markers shown in Figures 3 A and 3B.

Step 2. The genome screening results can be analyzed using various methods for detecting association between diabetes and marker loci, including but not limited to, transmission disequilibrium tests (TDT, Spielman et al. (1992) Nature Genetics 1 :82-3), the linkage disequilibrium analysis of Terwilliger (Terwilliger (1995) Amer. J. of Human Genetics 56(3):777-787), and the ancestral haplotype reconstruction method (Service et al. (1997) J. of Human Genetics 159:A236).

Based on computer simulations, we have a high probability of detecting diabetes loci with the Bahamian study population, even if diabetes is highly etiologically heterogeneous in the population. Subjects are generally analyzed in blocks of 20. Steps 1 and 2 are performed each time that a block of 20 new subjects is assembled. However, steps 1 and 2 can be performed with progressively larger samples until the data from the entire genome screening set have been analyzed. The analyses of the genome screening data are used to identify genome regions that should be investigated more intensively, rather than to test for statistically significant associations. For this reason, we used an inclusive threshold for identifying these regions, namely a p-value (in any of the three tests) of 0.05.

Step 3. Additional genotyping experiments can be performed in the regions identified in Step 2. For regions identified via the TDT or linkage disequilibrium of Terwilliger additional markers are typed that flank the original markers of interest at distances of about lcM.

For regions identified via the above-mentioned tests, one can type additional markers lying between the markers that form the initially detected haplotype, and the others flanking the original markers at distances of about 1 cM. This plan is feasible given the current density of polymorphic markers and the placement of such markers on the genetic and physical maps.

We analyzed the genotyping data in these highlighted regions to test for statistically significant associations. For determining the degree of significance we used conservative corrections for multiple testing. For association tests, guidelines for statistical significance have not been formally specified, as they have been for linkage tests; however, we followed an approach that is stringent.

The complete set of genotyping data (using the genome screening set of markers) can be re-analyzed each time the screen for a new block of subjects has been completed. In this case, it is possible that genome regions that initially met the thresholds for follow-up investigations will fail to meet these thresholds when more subjects are added. Such regions are then no longer targeted for intensive investigations. This strategy should minimize bias in the selection of regions to be targeted in the final round of analyses.

Genetic linkage analysis was performed using a set of highly polymorphic DNA markers, specifically microsatelhte markers. The microsatelhte markers which can be used for whole genome screening are described in the microsatellites Genethon map and are shown in Figures 3 A and 3B. We initially chose to focus on chromosome 20, based on indications in the literature that one or more regions on chromosome 20 are potentially involved in MODY. Microsatelhte markers specific for chromosome 20 are shown in Table 2.

Weissenbach et al. (1992) Nature 359:794-801; and Weber et al. (1993) Am. J. Hum. Gen. 53 : 1079-1095. Oligonucleotide sequences which serve as primers for extending a polynucleotide sequence and which are specific for each microsatelhte, are available in the Genome Data Bank. Using the set of markers shown in Table 2, we analyzed DNA samples from subjects with diabetes who are descendants of the founder population (pedigree shown in Figure 1). As described more fully in Example 2, we identified a segment less than 6 cM in length that localizes to a region on chromosome 20q between D20S195 and D20S119 and which is associated with Type II diabetes. Using different sets of microsatelhte markers, other regions of the genome can be analyzed for a linkage to susceptibility to Type II diabetes. In addition, using microsatelhte markers within the interval flanked by D20S 119 and D20S195, one can further narrow the interval, using the methods described herein.

Preparation of polynucleotides of the invention

The polynucleotides of this invention can be obtained using any known method, including, but not limited to, chemical synthesis, recombinant methods, and a PCR. PCR allows reproduction of DNA sequences. PCR technology is well known in the art and is described in U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston (1994). Other methods for amplifying a template polynucleotide are known to those skilled in the art and can be used to prepare the polynucleotides of the present invention.

As described in Example 2, the isolated polynucleotides of the present invention can be identified using oligonucleotide primer pairs derived from the sequences given in SEQ D NO: 1 and SEQ LD NO:2 (Figures 4 and 5, respectively) to identify a region of chromosome 20q between D20S195 and D20S119. Alternatively, an isolated polynucleotide can be identified using oligonucleotide primer pairs derived from the sequences given in SEQ ID NO: 3 and SEQ ID NO:4 (Figures 6 and 7, respectively), corresponding to D20S107 and D20S170, respectively. In addition, oligonucleotide primers derived from sequences of additional microsatelhte markers within the region flanked by D20S 195 and D20S 119 can be used to identify an isolated polynucleotide of the present invention. Amplification can be achieved by any known method, including, but not limited to, a polymerase chain reaction. Polynucleotides which serve as templates for amplification can be obtained from an individual having Type II diabetes, or a normal individual. One of skill in the art will recognize that a variety of oligonucleotide primer pairs can be used to identify polynucleotides comprising a region of chromosome 20q between D20S195 and D20S119. For example, by selecting oligonucleotide primers which hybridize to sequences proximal to but 5' or 3' of oligonucleotide primer sequences derived from SEQ LD NO:l and SEQ LD NO:2, one can amplify a polynucleotide comprising a region of chromosome 20q between D20S 195 and D20S 119. Alternative primer pairs can be overlapping or non-overlapping with oligonucleotide sequences derived from SEQ ID NO:l and SEQ ID NO:2.

Oligonucleotide primers derived from SEQ ID NO: 1 and SEQ LD NO: 2 can also be used as primers to determine a nucleotide sequence of a region of chromosome 20q between D20S 195 and D20S 119, using well known techniques of determining a nucleotide sequence, including, but not limited to, the dideoxy chain termination method. For example, an oligonucleotide having the sequence 5' gcacacatacacccctgaaaa 3 ' (nucleotides 331 to 351 of SEQ LD NO: 1) can be used to prime synthesis for sequencing from D20S195 in the direction of D20S119; and an oligonucleotide having the sequence 5' tgaaactgtgtcagttagct 3 ' (complementary to nucleotides 1-20 of SEQ ID NO:2) can be used to prime synthesis for sequencing from D20S119 in the direction of D20S195. Similarly, oligonucleotide primers having sequences derived from SEQ ID NO:3 and SEQ ID NO:4 can be used to prime synthesis for sequencing between D20S107 and D20S170, between D20S195 and D20S107, and between D20S119 and D20S170. Using the sequence data thus obtained using this technique, also called "primer walking", further oligonucleotide primers can be designed and additional nucleotide sequence information obtained. The sequence date thus obtained can be used to design additional primers for amplifying sequences comprised within D20S195 and D20S119. Using this method, smaller isolated polynucleotides comprised within D20S195 and D20S119 can be obtained.

In addition to D20S107 and D20S170, several microsatelhte markers which lie between D20S195 and D20S119 are known and the sequences are available. The nucleotide sequences of D20S195, D20S119, D20S107, D20S170, as well as these additional markers can be used to design oligonucleotide primers to prime PCR reactions to amplify polynucleotides between the microsatelhte markers, as described above. In this way, a "PCR contig library", i.e., a library of adjacent PCR amplification products, can be generated, covering the entire region between D20S195 and D20S119. The amplified polynucleotides can be isolated by conventional means and, if desired, inserted into cloning and/or expression vectors. The amplified polynucleotides can be further tested for the presence of sequences and/or polymorphisms associated with Type II diabetes. Ascertainment of whether an isolated polynucleotide is associate with Type II diabetes can be performed using the methods described in Example 2. The polynucleotides can also be generated using methods known in the art, such as chemical synthesis, site-directed mutagenesis, and/or recombinant methods.

Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence. For preparing the polynucleotides of the invention using recombinant methods, a polynucleotide comprising a sequence comprised within D20S195 and D20S119 can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification. Identification of the isolated polynucleotide as being comprised within D20S195 and D20S119 can be achieved as described above, using PCR primer pairs of known sequence, such as those derived from SEQ ID NO: 1 and SEQ ID

NO:2, as described herein, or using primer pairs identified by "primer walking", as described above. Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, f-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known within the art. See, e.g., Sambrook et al. (1989).

Cloning and expression vectors comprising an isolated polynucleotide of the invention The present invention further includes a variety of vectors (i.e., cloning and expression vectors) having cloned therein a polynucleotide(s) comprising a region of chromosome 20q between D20S195 and D20S119, as described above. These vectors can be used for expression of recombinant polypeptides as well as a source of polynucleotides comprising a region of chromosome 20q between D20S 195 and D20S 119. Cloning vectors can be used to obtain replicate copies of the polynucleotides of the invention that they contain, or as a means of storing the polynucleotides in a depository for future recovery. Expression vectors (and host cells containing these expression vectors) can be used to obtain polypeptides produced from the polynucleotides they contain. They may also be used where it is desirable to express polypeptides, encoded by an operably linked polynucleotide, in an individual, such as for eliciting an immune response via the polypeptide(s) encoded in the expression vector(s). Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are known in the art and need not be described in detail herein. For example, see Gacesa and Ramji, Vectors, John Wiley & Sons (1994).

Various methods for cloning DNA fragments are known in the art and can be used. These methods include, for example, cloning into mammalian artificial chromosomes (MAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), bacteriophage vectors such as PI, bacterial vectors, and the like. "Current Protocols in Molecular Biology", eds. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987) and periodic updates; Ikeno et al. (1998) Nature Biotech. 16:431-439; Harrington et al. (1997) Nat. Genet. 15:345-355; and Burke et al. (1987) Science 236:806- 812. A vector comprising a polynucleotide of the invention can be introduced into a host cell and/or a target cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent, such as vaccinia virus). The choice of means of introducing vectors or polynucleotides of the invention will often depend on the host cell.

Isolated host cells comprising a region on chromosome 20q between D20S195 andD20S119 The invention further provides isolated host cells transfected or transformed with (i.e., comprising) the above-described isolated polynucleotides, or above-described expression or cloning vectors of this invention. These cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The cells which are suitable for use in the methods of the present invention with respect to expression, transcriptional control, or for purposes of cloning and propagating a polynucleotide of the present invention can be prokaryotic or eukaryotic.

Host systems are known in the art and need not be described in detail herein. Prokaryotic hosts include bacterial cells, for example E. coli, B. subtilis, and mycobacteria. Among eukaryotic hosts are yeast, insect, avian, plant, C. elegans (or nematode) and mammalian cells. Examples of mammalian cells are COS cells, mouse L cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, human adipocyte cell lines, and African green monkey cells. Xenopus laevis oocytes, or other cells of amphibian origin, may also be used. Hybrid cells can also be used and include, but are not limited to, human cell-hamster somatic cell hybrids. Also suitable as host cells are mammalian cells lacking an endogenous region on chromosome 20q between D20S195 and D20S119. Somatic cell hybrids formed from the fusion of human cells with those from a different species are readily available, for example from Coriell Cell Repositories, and methods for their production are known in the art. Useful somatic cell hybrids between human cells and non-human cells are those which contain the portion of human chromosome 20 comprising sequences between D20S195 and D20S119, but lacking the homologous chromosomal region of the non-human species. Such hybrid cells can be analyzed for the presence of a region on chromosome 20q between D20S195 and D20S119. This can be achieved, for example, by amplifying DNA from the hybrid cell using oligonucleotide primers, as described above, or hybridization assays using a polynucleotide of the invention, as described above. Functional assays can also be used and include glucose uptake assays. In these assays, uptake of glucose is measured in the presence of insulin to stimulate glucose uptake. If a hybrid cell contains a region on chromosome 20q between D20S195 and D20S119, derived from a normal individual, that is involved with glucose metabolism, then glucose uptake in the presence of added insulin will be significantly above background for the cell. If a hybrid cell contains a region on chromosome 20q between D20S195 and

D20S119, derived from an individual with Type II diabetes, and the region is involved with glucose metabolism, then glucose uptake will be at background levels for the cell, or will be at a significantly lower level than for the normal cell.

An example of a glucose uptake assay is as follows. Adipocytes (1-2 x 10⁵ cells/ml) are suspended in Krebs-Ringer phosphate buffer with 3% (0.45 M) bovine serum albumin and 1.5 mM pyruvate and incubated in plastic tubes with or without insulin (1 nM final concentration) at 37°C for 15 minutes. Cells are incubated with 6.0 μCi of l-[³H]-2-deoxy- D-glucose (final concentration is 34 μM) added to the cell mixture and incubated for 3 minutes at 37°C. At the end of the 3 minute incubation, the cells are separated from the liquid by centrifugation through dinonyl pthalate oil. The oil layer is then removed and the 1- [ H]-2-deoxy-D-glucose associated with the oil layer is quantitated by liquid scintillation counting. Nonspecific glucose transport is determined in the presence of 50 μM cytochalasin B.

Methods using the polynucleotides of the present invention The above-described isolated polynucleotides can be used for a wide variety of purposes, which will vary with the desired or intended result. Accordingly, the present invention includes methods using the isolated polynucleotides of the invention, which have been described above.

Methods of detecting a polymorphism on chromosome 20q between D20S195 andDS20S119 The present invention provides methods for detecting a polymorphism on chromosome 20q between D20S195 and D20S119. Any of a number of known methods can be used to detect a polymorphism in this region. As one example, a polynucleotide sample derived from an individual is analyzed for specific hybridization to a probe, under stringent hybridization conditions, wherein said probe comprises a polynucleotide comprising a sequence which is contained within in a region flanked by microsatelhte markers D20S 195 and D20S119. If the probe is derived using template polynucleotide from an individual with

Type II diabetes, then specific hybridization is indicative of a polymorphism in this regions. If the probe is derived from a normal individual, then lack of hybridization is indicative of a polymorphism in this region.

A polynucleotide sample can be derived from an individual using established methods. Depending on the method used for analyzing the polynucleotide sample, it may be desirable to extract the polynucleotide from the biological sample. This can be accomplished by any known means, for example, digesting a cell sample with proteinase K then extracting the polynucleotide.

Any of a number of techniques known to those skilled in the art can be used to detect a polymorphism in a region on chromosome 20q between D20S195 and D20S119, using an isolated polynucleotide of the invention. These include, but are not limited to, direct sequencing of the interval from affected individuals (Chadwick et al. (1996) Biotechniques 20:676-683); and hybridization with one or more probes derived from a region on chromosome 20q between D20S195 and D20S119, including allele-specific oligonucleotide hybridization (Wong and Senadheera (1997) Clin. Chem. 43: 1857-1861). The region being detected can optionally be amplified by known techniques, including, but not limited to, a polymerase chain reaction. Other analytical techniques include, but are not limited to, single- strand conformation analysis; restriction length polymorphism (RFLP) analysis; enzymatic mismatch cleavage techniques such as glycosylase mediated polymorphism detection (Naughan and McCarthy (1998) Nucl. Acids Res. 26:810-815); heteroduplex PCR (Deuter and Muller (1998) Hum. Mutat. 11 :84-89); and fiberoptic DΝA sensor array techniques (Healey et al. (1997) Anal. Biochem. 251 :270-279). Automated methods of detecting polymorphisms have been developed and can be used in the methods of the present invention. See, for example, Marshall and Hodgson (1998) Nature Biotechnol. 16:27-31. Other methods include, for example, PCR-RFLP. Hani et al. (1998) J. Clin. Invest. 101 :521-526. In one embodiment, the method comprises hybridization of selected oligonucleotide primers, such as those derived from SEQ ID NO: 1 and SEQ ID NO:2, to a DNA sample derived from the individual to be tested, followed by amplification by any known technique, for example, a polymerase chain reaction (PCR). The polymorphic amplified fragments are then separated acccording to their size by electrophoresis on acrylamide denaturing gels, transferred onto a membrane, such as a nylon membrane, and, after treating the membrane to reduce non-specific binding, hybridized with microsatelhte probes comprising a detectable label. Alternatively, after separating the samples on gels, the sizes of the amplification products are compared visually for differences indicative of the presence of one or more polymorphisms associated with Type II diabetes. Alternatively, automated genotyping (utilizing fluorescent dyes and including size standards in each run) can be used to detect size differences between amplified sequences. Whether a given polymorphism is associated with Type II diabetes can be determined by analyzing a polynucleotide from a normal individual, a distantly related affected individual and an affected individual, all from the subject Bahamian population. A polymorphism found in a region on chromosome 20q between D20S195 and D20S119 in the normal individual would not be expected to be associated with Type II diabetes. However, a polymorphism in this region which is found in an affected individual from the pedigree family and in a distantly related affected individual, but which is not found in the normal individual would be expected to be associated with Type II diabetes. A polymorphism detected in this region in an individual who is not a member of the subject Bahamian population, would be expected to be associated with Type II diabetes if that polymorphism were absent from the homologous region in a normal individual.

Once it has been established that a given polymorphism is associated with Type II diabetes, an isolated polynucleotide comprising the polymorphism can be used to screen individuals for Type II diabetes.

Methods of detecting a propensity of an individual to develop Type II diabetes The present invention provides methods for detecting a propensity of an individual to develop Type II diabetes. The methods generally involve analyzing a polynucleotide sample derived from an individual for the presence of a DNA polymorphism on chromosome 20q between D20S195 and D20S119, wherein the polymorphism is associated with Type II diabetes. Methods of analysis are described above. Any known method can be used. Once a polymorphism associated with Type II diabetes has been detected in the DNA of an individual, the individual can then be monitored closely for the occurrence of symptoms associated with Type II diabetes. Symptoms may include polyuria and polydipsia. In addition to monitoring the individual, other measures may be taken, for example, modification of the individual's diet may be indicated.

Methods of confirming a phenotypic diagnosis of Type II diabetes

The present invention provides methods for confirming a phenotypic diagnosis of

Type II diabetes. The methods generally involve analyzing a polynucleotide sample derived from an individual diagnosed as having Type II diabetes for the presence of a polymorphism on chromosome 20q between D20S195 and D20S119, wherein the polymorphism is associated with Type II diabetes. Methods of detecting a polymorphism on chromosome 20q between D20S195 and D20S119 have been described above.

Primer walking in a region on chromosome 20q between D20S195 andD20S119

Oligonucleotide primers having sequences derived from SEQ ID NO:l, SEQ ID

NO:2, SEQ LD NO:3, and SEQ LD NO:4 can also be used as primers to determine a nucleotide sequence of a region of chromosome 20q between D20S195 and D20S119, using well known techniques of determining a nucleotide sequence, including, but not limited to, the dideoxy chain termination method. Using the sequence data thus obtained, further oligonucleotide primers can be designed and additional nucleotide sequence information obtained. Using this technique, also called "primer walking", information regarding polymorphisms in the region can be obtained, and coding sequences can be identified.

Identification of coding sequences in a region on chromosome 20q between D20S195 and D20S119

The Type II diabetes-associated interval on chromosome 20q between D20S195 and D20S119 can be used to identify polynucleotide coding sequences which encode one or more polypeptides. The sequences of these polynucleotides can be determined, and conceptual translations made. The nucleotide sequence of such polynucleotides from individuals displaying symptoms of Type II diabetes can be determined and compared with sequences from individuals not displaying these symptoms.

Direct cDNA selection can be used to isolate segments of expressed DNA from a region on chromosome 20q between D20S195 and D20S119, using primer walking, as described above, or any other method. M. Lovett, J. Kere, L. M. Hinton, Proc. Natl. Acad. Sci. USA 88 9628-9632 (1991); and Jou et al. Genomics 24:410-413 (1994). By using bacterial artificial chromosomes (BAC) (e.g., commercially available from Research Genetics Inc., Huntsville, Alabama), a group of cDNAs can be identified, and hybridization and PCR- amplification (or other techniques for amplifying a polynucleotide) experiments can be used to determine if these cDNA segments are derived from the interval. The cDNAs can then be used to determine whether specific sequences are differentially expressed in affected individuals compared to non-carrier individuals. For this purpose, cell lines can be generated from lymphoid cells isolated from an individual. Measurement of mRNA levels in lymphoblastoid cell lines can be used as an initial screen. The cell lines can be prepared by drawing blood from individuals, transforming the lymphoblasts with EBN and growing the immortalized cells in culture, using known techniques. Total RΝA and DΝA are extracted from the cultured human lymphoblastoid cell lines. Northern blot hybridization can be used to determine reduced levels of a specific sequence compared to levels from an unaffected, non-carrier individual as a result of mutations in the Type II diabetes gene on the chromosomes from these affected individuals which results in decreased levels of mature mRNA and play a primary role in Type II diabetes. Thus, alterations in gene sequences in affected individuals can be determined. Any known technique can be used to amplify the polynucleotide comprising the gene(s) associated with Type II diabetes. These include polymerase chain reaction techniques. A polymerase chain reaction (PCR) can be used to amplify the gene and to determine its sequence from affected individuals. Sequence comparison with unaffected, non-carrier individuals can be carried out to identify polymorphisms in the gene sequence(s) that are associated with Type II diabetes.

The identification of the biochemical defect(s) that causes Type II diabetes could provide a basis for treatments for this disease. In addition, knowledge that certain mutations in the gene(s) are responsible for the disease allows mutation detection tests to be used as a definitive diagnosis for Type II diabetes. Thus, the present invention provides an isolated polynucleotide that can be used in the identification of the presence (or absence) of a polymorphism in a Type II diabetes gene in a human and thus can be used in the diagnosis of Type II diabetes or in the genetic counseling of individuals, for example, those with a family history of Type II diabetes (although the general population can be screened as well). In particular, it should be noted that any mutation in a Type II diabetes gene away from the normal gene sequence is an indication of a potential genetic flaw; even so-called "silent" mutations that do not encode a different amino acid at the location of the mutation are potential disease mutations, since such mutations can introduce into (or remove from) the gene an untranslated genetic signal that interferes with the transcription and/or translation of the gene and/or processing of the mRNA. Thus, advice can be given to a patient concerning the potential for transmission of Type II diabetes if any mutation is present. While an offspring with the mutation in question may or may not have symptoms of Type II diabetes, patient care and monitoring can be selected that will be appropriate for the potential presence of the disease; such additional care and/or monitoring can be eliminated (along with the concurrent costs) if there are no differences from the normal gene sequence. As additional information (if any) becomes available (e.g., that a given silent mutation or conservative replacement mutation does or does not result in Type II diabetes), the advice given for a particular mutation may change. However, the change in advice given does not alter the initial determination of the presence or absence of mutations in the gene causing Type II diabetes.

Generally, mutations are identified in the human gene(s) for use in a method of detecting the presence of a genetic defect that causes or may cause Type II diabetes, or that can or may transmit Type II diabetes to an offspring of the human. Initially, the practitioner will be looking simply for differences from the sequence identified as being normal and not associated with disease, since any deviation from this sequence has the potential of causing disease, which is a sufficient basis for initial diagnosis, particularly if the different (but still unconfirmed) gene is found in a person with a family history of Type II diabetes. As specific mutations are identified as being positively correlated with Type II diabetes (or its absence), practitioners will in some cases focus on identifying one or more specific mutations of the gene that changes the sequence of a protein product of the gene or that results in the gene not being transcribed or translated. However, simple identification of the presence or absence of any mutation in the gene of a patient will continue to be a viable part of genetic analysis for diagnosis, therapy and counseling.

The actual technique used to identify the gene or gene mutant is not itself part of the practice of the invention. Any of the many techniques to identify gene mutations, such as direct sequencing of the gene from affected individuals, hybridization with specific probes, which includes the technique known as allele-specific oligonucleotide hybridization, either without amplification or after amplification of the region being detected, such as by PCR. Other analysis techniques include single-strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP), enzymatic mismatch cleavage techniques and transcription/translation analysis. All of these techniques are described in a number of patents and other publications, including, for example, "Laboratory Methods for Detection of Mutations and Polymorphisms in DNA" (1997) CRC Press, G.R. Taylor, ed.

Depending on the patient being tested, different identification techniques can be selected to achieve particularly advantageous results. For example, for a group of patients known to be associated with particular mutations of the gene, oligonucleotide ligation assays, "mini- sequencing" or allele-specific oligonucleotide (ASO) hybridization can be used. For screening of individuals who are not known to be associated with a particular mutation, single- strand conformation polymorphism, total sequencing of genetic and/or cDNA and comparison with standard sequences are preferred.

In many identification techniques, some amplification of the host genomic DNA (or of messenger RNA) will take place to provide for greater sensitivity of analysis. In such cases it is not necessary to amplify the entire gene, merely the part of the gene or the specific location within the gene that is being detected. Thus, the method of the invention generally comprises amplification (such as via PCR) of at least a segment of the gene, with the segment being selected for the particular analysis being conducted by the diagnostician.

Portions of the interval can be cloned into vectors, for example phage or plasmid vectors. Such vectors can be used to identify candidate cDNAs for screening for mutations in the DNA of Type II diabetes patients. The candidate cDNAs can be subsequently screened for mutations in DNA from Type

II diabetes patients. From the minimal candidate region defined by genetic mapping experiments a segment is left that is sufficiently large to contain multiple different genes.

Coding sequences from the surrounding DNA can be identified, and these sequences can be screened until a probable candidate cDNA are found. Candidates may also be identified by scanning databases consisting of partially sequenced cDNAs, known as expressed sequence tags, or ESTs. The database can be used to identify all cDNAs that map to the minimal candidate region for Type II diabetes. These cDNAs can then be used as probes to hybridize to the PI contig, and new microsatellites are isolated, which are used to genotype the "LD" sample. Maximal linkage disequilibrium in the vicinity of one or two cDNAs is identified.

Coding sequences can also be identified by exon amplification. Exon amplification targets exons in genomic DNA by identifying the consensus splice sequences that flank exon- intron boundaries. Briefly, exons are trapped in the process of cloning genomic DNA into an expression vector. These clones are transfected into COS cells, RT-PCR is performed on total or cytoplasmic RNA isolated from the COS cells using primers that are complementary to the splicing vector. Exon amplification can be performed using any known method. Another widely used approach is direct selection, which involves screening cDNAs using large insert clone contigs, with several steps to maximize the efficiency of hybridization and recovery of the appropriate hybrid.

Once cDNAs are identified, the most plausible candidates can be screened by direct sequencing, SSCP or using chemical cleavage assays. Genetic and physical data can be used to map a Type II diabetes gene to a less than 6 cM region of chromosome 20q between D20S195 and D20S119. New markers from this region can be tested in order to locate a Type II diabetes gene in a region small enough to provide higher quality genetic tests for Type II diabetes, and to find the mutated gene(s). Narrowing down the region in which the gene is located will lead to sequencing of a Type II diabetes gene as well as cloning thereof. Further genetic analysis employing, for example, new polymorphisms flanking D20S195 and D20S119 as well as the use of cosmids, yeast artificial chromosome (YAC) clones, or mixtures thereof, are employed in the narrowing down process. The next step in narrowing down the candidate region can include cloning of the chromosomal region 20q including proximal and distal markers in a contig formed by overlapping cosmids and YACS. Subsequent subcloning in cosmids, BACs, YACs, plasmids, phages, or other vectors can generate additional probes for more detailed mapping.

Functional assays can also be applied to determine whether a given polynucleotide comprises a Type II diabetes gene. For example, a polynucleotide of the invention and/or a vector of the invention can be introduced into a mammalian cell which lacks a region on chromosome 20q between D20S 195 and D20S 119. A vector of the invention which comprises a polynucleotide comprising a region on chromosome 20q between D20S195 and D20S 119 can be introduced into the cell by any known method. Thereafter, a functional assay such as the above-described glucose uptake assay can be used to determine whether the polynucleotide comprises a gene encoding a polypeptide involved with glucose uptake. In this case, a polynucleotide of the invention would be derived from a normal individual. If a the transformed cell contains a region on chromosome 20q between D20S195 and D20S119, derived from a normal individual, that is involved with glucose metabolism, then insulin- stimulated glucose uptake will be significantly above background for the cell. Background levels are established by performing a glucose uptake test on the untransformed cell which lacks a region on chromosome 20q between D20S 195 and D20S 119. Nectors comprising portions of the region on chromosome 20q between D20S195 and D20S119 from a normal individual can be generated and these vectors tested in the manner described above until the smallest fragment of the region is identified which results in the transformed cell having insulin-stimulated glucose uptake levels significantly above background levels. "Significantly above background levels" indicates that glucose uptake is at least about 20%, more preferably at least about 30%, more preferably at least about 40%, even more preferably at least about 50% or more, over glucose uptake levels of the untransformed cell, i.e., the cell which lacks a region on chromosome 20q between D20S195 and D20S119.

The following examples are provided to illustrate but not limit the invention.

EXAMPLES

EXAMPLE 1

Pedigree Analyses

The study population that we have identified for mapping Type II diabetes genes, a founder population in the Bahamas, is ideal for population-based disease gene mapping. We have adopted several theoretical principles in carrying out the studies described herein, including those previously described. Freimer et al. (1996) Amer. J. of Med. Genetics 67:254-63; and PCT Publication No. WO 98/07887.

We have identified a model population with a diabetogenic pattern of body fat distribution and increased prevalence of adult onset diabetes (Type II). The study population is Caucasian and is genetically and environmentally homogeneous. We first explored the utility of such a population for a diabetes study because we identified several extremely large and genetically informative diabetes pedigrees. Figure 1 shows a pedigree analysis of families belonging to this population. We soon discovered that this population is ideally suited for genetic mapping because this population is genetically homogeneous, having expanded rapidly from a small number of founders. Geographically, the study population is particularly isolated. Scheduled ferry service to the islands did not exist two decades ago and is now only available on a limited basis. Such limited access and the lack of hotels and restaurants to support tourism have contributed to a continued history of genetic isolation for the population. The founder population was derived primarily from 70 founders of English ancestry whose ship wrecked on the uninhabited islands in the 17th century, and the introduction a century later of a small number of Tory Loyalists, also of English ancestry. In the latter part of the 18th Century, a descendant of the original founding population and the daughter of a Tory Loyalist founded the principal island population to be used for our study. They had thirteen children and the population expanded rapidly due to a continued history of large sibships. Because of the "isolationist" nature of this community, the population has remained genetically isolated. We located a 700-member pedigree for the population on the primary study island. The current estimate of the number of descendants still remaining in the more isolated sections of the Bahamas is on the order of 4,000 people. Pedigree analysis is shown in Figure 1.

The power to detect disease genes can be investigated by simulating linkage using any known method, including, but not limited to, TDT and Terwilliger. Specifications involved in disease transmission are not necessary for these methods. Based on our preliminary findings, our population exhibits a similar expansion to that of the Costa Rican founder population previously described (WO 98/07887). It is assumed that the affected individuals in the Costa Rican population are on average 12 generations removed from a common ancestor who transmitted the disease allele/s. A second population in which affected individuals are approximately 15 generations removed from a common ancestor who transmitted the disease allele/s was included in the analysis. We estimate, based on our preliminary findings, that the Bahamian population will fall near this threshold. It was estimated that the Bahamian population has a similar degree of relatedness. These tests were compared under several scenarios, varying the genetic model for disease transmission and the size of the study sample.

Genealogical screening was conducted to establish the place of origin of each subject's family, back to the great-grandparental generation. This protocol was based on the assumption that those individuals whose great-grandparents' surnames match the original 70 founders or Tory Loyalist families, are likely to be descended from the founder population established in the 17th and 18th Centuries. There has been little new immigration into these remote communities/islands. A subject was included in the study if at least five of his/her eight great-grandparents were born in the islands. Of the subjects who have been interviewed, we have completed the genealogical assessment for 50 community residents. Of these, 20 subjects have been diagnosed with diabetes. Clinical presentation of subject population

The first study subjects to be recruited were identified during a diabetes health fair which our study group hosted on the island in 1996. Anthropometric measurements were assessed and estimates were made of central fat levels in this genetically isolated population. Intra-abdominal adipose tissue (IAAT) levels were significantly greater in the Bahamian founder population than for Caucasian, age-matched controls measured in Birmingham (p=0.0001). A series of anthropometric and skinfold measurements were assessed and used to estimate the levels of IAAT. Prediction equations for estimating the levels of IAAT have been described. Kekes-Szabo, T., et al. (1996) Intl. J. of Obesity & Related Metabolic Disorders 20(8):753-8; and Kekes-Szabo, T. (1994) Obesity Res. 2(5):450-457. Kekes- Szabo, T., et al. (1996) Intl. J. of Obesity & Related Metabolic Disorders 20(8):753-8; and Kekes-Szabo, T. (1994) Obesity Res. 2(5):450-457. The prediction equation for estimating the levels of IAAT used in these studies was as follows:

(2.57 x umbilicus circumference + 0.92 x age + 0.69 x suprailiac skinfold) - 188.61 The diabetes in our study population is characteristic of Type II diabetes seen in other Caucasian populations: central obesity-associated diabetes mellitus with an adult age of onset and no initial requirement for insulin. Clinical characteristics of individuals examined are shown in Table 1. Values are given as mean ± standard deviation.

Table 1.

Clinical characteristic Non-diabetic Diabetic

N 8 8

% male 25 25

Current age (years) 61 ±11 62 ±11 Current BMI (kg/m²) 23 ±4 37 ±12

Fasting Blood Glucose (mg/dL) 97 ±10 197 ±66 Blood Pressure (mmHg) 131/80 ±12/10 147/87 ±21/5 Cholesterol (mg/dL) 226 ±37 260 ±56 Triglycerides (mg/dL) 143 ±88 222 ±100

Average age at diagnosis (years) - 53 ±10

The diabetes in the individuals studied resembles neither MODY nor Type I diabetes.

In concordance with our observations of elevated levels of IAAT, many of the Bahamian subjects who participated in the health fair reported a strong family history of diabetes. Subjects have been assessed for diabetes either through medical records (fasting glucose or oral glucose tolerance test (OGTT)) or assessed at one of our health fairs (fasting glucose), in accordance with the published guidelines of the American Diabetes Association.

EXAMPLE 2

Linkage disequilibrium analysis The microsatelhte marker set that is used for the random, whole genome screen is the

ABI Prism Linkage Mapping Set Version 2 (Applied Biosystems, Inc./Perkin Elmer, Foster

City, CA). This Mapping Set consists of 400 fluorescent-labeled PCR primer pairs that define a 10-cM resolution linkage map, as shown in Figures 3 A and 3B.

The primers are selected to amplify microsatelhte loci selected from the Genethon human linkage map. Weissenbach et al. (1992); Weber et al. (1993). The set is divided into 28 panels, each containing 10 to 20 PCR primer pairs. Each of the primer pairs has been optimized for accuracy and is labeled with one of three dyes. During each run, a CEPH (Centre d' etude du polymorphisme humain; a collaborative genetic mapping of the human genome) standard control DNA sample is included as a size reference for each marker tested. For each marker tested, CEPH individuals 1331-01, 1331-02, and 1347-02 are used as size standards. CEPH DNAs are obtained from the NIGMS Human Genetic Mutant Cell Repository Coriell Institute for Medical Research (Camden, NJ).

Each sample is co-electrophoresed with an internal size standard. The Genescan 400HD ROXL size standards are an improved size standard for the ABI instrument. The size standard includes 21 evenly spaced fragments labeled with ROX. Because it is labeled with a different color dye than the samples, the size standard can be included with each sample. Any sample which did not meet stringent criteria for size calling (for example, peak heights must be greater than 100 to avoid possible miscalling of a homozygote) is repeated.

One advantage of automated genotyping (utilizing fluorescent dyes and including size standards in each run) compared to manual genotyping (utilizing isotopic labeling and size standards run in adjacent lanes), is that genome screens can proceed before the entire sample is collected. The ABI Prism Linkage Mapping Set Version 2 is designed to maximize the efficiency of typing multiple markers in a single run; once the markers are labeled with fluorescent dye they can be used at any time over the course of the project. Once the samples are amplified, they can be stored over time and run at a later date. Thus, it is straightforward to continually screen the genome, as new samples are completed. Individuals are genotyped using highly polymorphic microsatelhte markers spaced across the genome with lOcM resolution, followed by a screen using more densely spaced markers (i.e., about 800-1000 markers resulting in a map with 3-5cM resolution). Denser maps of markers can be used, as they become available, to localize more accurately a locus (loci) associated with Type II diabetes.

Genome screening results can be analyzed using any known method for detecting association between diabetes and marker loci, including, but not limited to, transmission disequilibrium tests: 1) TDT, Spielman, R.S. et al. (1992) Nature Genetics l(2):82-3); 2) and the linkage disequilibrium analysis of Terwilliger (Terwilliger, J. (1995) Amer. J. of Human Genetics 56(3):777-787). Based on computer simulations, we had a high probability of detecting diabetes loci with these samples, even if diabetes is highly etiologically heterogeneous in the population. We used an inclusive threshold for identifying regions associated with Type II diabetes, namely a p-value (in any of the three tests) of 0.05.

Analysis of chromosome 20 markers The Type II diabetes Linkage Analysis Consortium has provided a framework of 10 markers on chromosome 20 for initial analysis. The markers which were used to type DNAs from individuals in our Bahamian population are shown in Table 2.

Table 2. Marker GenBank Accession No.

D20S186 Z23375

D20S112 Z16842

D20S195 Z24371

D20S107 Z16656

D20S119 Z17198

D20S178 Z23757

D20S100 Z16487

D20S171 Z23313

Due to the growing consensus for linkage to diabetes on chromosome 20q (Ji, L., et al. (1997) Diabetes 46(5):876-81; Bowden, D.W., et al. (1997) Diabetes 46(5):882-86; Velho, G., et al. (1997) Diabetes and Metabolism 23(Supρl. 2):34-37; www.sph.umich.edu/group/statgen/consortium), we used the chromosome 20 markers shown in Table 2 to test DNA samples from affected individuals from our pedigree family (Figure 1) as well as from seven distantly-related, affected individuals. "Affected" individuals are those diagnosed with Type II diabetes. The set of markers shown in Table 2 covers a region of 69.3 cM on chromosome 20.

Blood samples were obtained from each individual tested, and DNA was isolated from the samples using standard techniques. DNA samples from affected individuals in the pedigree, as well as distantly related, affected individuals, were tested by PCR.

PCR reactions were conducted using the following protocol. The reaction mixture contained 1.0 μl primer mix (5 μM each primer), 1.2 μl DNA template (50 ng/μl), 9.0 μl True Allele PCR Premix, and 3.8 μl sterile deionized water. True Allele PCR Premix (Perkin Elmer/ ABI) contains PCR Buffer II, GeneAmp deoxynucleotide mix, AmpliTaq Gold DNA polymerase and MgCl₂. For each microsatelhte marker-specific primer pair, one member of each pair was labelled with a fluorescent dye. In addition to the primer pairs specific to each chromosome 20 marker tested, size standard primer pairs and corresponding template were included. The Genescan 400HD ROXI size standards are an improved size standard for the ABI instrument used in our studies. The size standard includes 21 evenly spaced fragments labeled with ROX, a red fluorescent dye. Each sample was subjected to capillary electrophoresis using an ABI310 Genetic Analyzer (Perkin Elmer/ ABI, Foster City, CA) automated sequencing instrument. Since PCR reactions for each microsatelhte marker included size standard control PCR reactions, each sample is co-electrophoresed with an internal size standard. The instrument then calculates, based on the size standard, the size of the fragments amplified with microsatelhte marker-specific PCR primers. "Shared alleles" are identified as microsatelhte marker-specific PCR fragments which are identical in size between and/or among individuals tested.

Six of these seven subjects shared a common allele with an affected individual in the pedigree for an anonymous marker located at chromosome 20ql2-13; four of these share an ancestral haplotype which extends greater than lOcM, as shown in Figure 2. The one subject who does not share alleles at this lOcM region is assumed to be a phenocopy.

The marker D20S107 demonstrated a higher than expected allele sharing among the affecteds. In order to assign a set of alleles to each copy of chromosome 20, an additional marker set, containing markers close to D20S 107, were used to test the DNAs from the same panel of pedigree members. The additional markers used were D20S477, D20S478, D20S170, and D20S481 (GenBank Accession Numbers G08047, G08048, Z23468, and G08051, respectively). Using these markers, a haplotype was generated.

Once the haplotyping was complete, closely spaced markers that surround D20S107 were used for Terwilliger linkage disequilibrium analysis. These markers are shown in Table 3.

Table 3.

Marker Sequence ID

D20S195 SEQ LD NO:l

D20S107 SEQ LD NO:3

D20S170 SEQ K> NO:4

D20S119 SEQ ID NO:2

These markers cover an 11.4 cM region on chromosome 20. The maximum LOD score was found between D20S107 and D20S170. The interval between D20S107 and D20S170 is 1.2 cM. Thus, it appears that a locus associated with Type II diabetes is in the vicinity of D20 SI 07.

Using this method, we localized the interval to a small region between D20S195 and D20S119, most likely close to D20S107. We did not detect the HNF-4α V3931 mutation.

All publications and patent applications mentioned in this specification are herein 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. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method for detecting a propensity of an individual to develop Type II diabetes, comprising analyzing a polynucleotide sample derived from the individual for the presence of a polymorphism on chromosome 20q between D20S195 and D20S119, wherein said polymorphism is associated with Type II diabetes.

2. The method of claim 1 , wherein the polymorphism is between D20S 195 and D20S107.

3. The method of claim 1 , wherein the polymorphism is between D20S 107 and D20S170.

4. The method of claim 1, wherein the analysis of the polynucleotide sample is performed by a method selected from the group consisting of detection of specific hybridization, measurement of allele size, restriction fragment length polymorphism analysis, and allele-specific hybridization analysis.

5. The method of claim 1, wherein the presence of the polymorphism is determined by amplifying a segment of DNA within D20S195 and D20S119 that spans the polymorphism.

6. The method of claim 5, further comprising the step of determining the size of the amplified segment.

7. The method of claim 1, wherein the presence or absence of the polymorphism is determined by contacting a polynucleotide from the individual with a polynucleotide probe capable of hybridizing to the polymorphism under stringent conditions; and determining whether hybridization has occurred, thereby indicating the presence of the polymorphism.

8. A method of detecting a polymorphism of chromosome 20q between microsatelhte markers D20S195 and D20S119, comprising the steps of: a) contacting a polynucleotide sample derived from an individual with at least one probe for a sufficient amount of time to allow for specific hybridization of the sample polynucleotide to the probe under stringent hybridization conditions, wherein the probe comprises a sequence from a region between D20S195 and D20S119 derived from a normal individual; and b) determining the presence of specific hybridization between the sample polynucleotide and the probe, wherein lack of specific hybridization of the probe and the sample polynucleotide provides for the detection of a polymorphism on chromosome 20q between D20S195 and D20S119.

9. A method of confirming a phenotypic diagnosis of Type II diabetes in an individual, comprising analyzing a sample of a polynucleotide derived from an individual diagnosed as having Type II diabetes for the presence of a polymorphism on chromosome 20q between D20S 195 and D20S 119, wherein said DNA polymorphism is associated with Type II diabetes.

10. A method of confirming a phenotypic diagnosis of Type II diabetes in an individual, comprising the steps of: a) introducing into a cell which lacks a region on chromosome 20q between D20S195 and D20S119 a polynucleotide from an individual to be tested, said polynucleotide comprising a region on chromosome 20q between D20S195 and D20S119; and b) measuring glucose uptake by said transformed cell in comparison to glucose uptake by a normal cell, wherein decreased glucose uptake is indicative of Type II diabetes.

11. An isolated polynucleotide comprising at least about 15 contiguous nucleotides of a sequence within a region of chromosome 20q, said region flanked by D20S195 and D20S119, wherein said sequence is associated with Type II diabetes and the isolated polynucleotide is useful as a probe in screening for Type II diabetes.

12. The polynucleotide of claim 11, wherein the sequences are comprised within a region flanked by D20S195 and D20S107.

13. The polynucleotide of claim 11, wherein the sequences are comprised within a region flanked by D20S107 and D20S170.

14. The polynucleotide of claim 11, wherein the isolated polynucleotide is from a normal individual.

15. The polynucleotide of claim 11, wherein the isolated polynucleotide is from an individual with Type II diabetes.

16. The polynucleotide of claim 11 wherein said polynucleotide is about 700 kb in length.

17. The polynucleotide of claim 11 wherein said polynucleotide is about 500 kb in length.

18. The polynucleotide of claim 11 wherein said polynucleotide is about 300 kb in length.

19. The polynucleotide of claim 11 wherein said polynucleotide is about 100 kb in length.

20. The polynucleotide of claim 11 wherein said polynucleotide is about 50 kb in length.

21. A polynucleotide vector comprising the polynucleotide of claim 11.

22. An isolated host cell comprising the polynucleotide of claim 11.

23. An isolated host cell comprising the vector of claim 21.

24. An isolated polynucleotide comprising at least about 25 contiguous nucleotides of a sequence within a region of chromosome 20q, said region flanked by D20S195 and D20S119, wherein said sequence is associated with Type II diabetes and the isolated polynucleotide is useful as a control in a screening assay for Type II diabetes.

25. A method of making a polynucleotide vector comprising a polynucleotide of claim 1 comprising inserting the polynucleotide of claim 1 into a polynucleotide vector.

26. An isolated polynucleotide comprising a region of chromosome 20q between D20S 195 and D20S 119, wherein said polynucleotide selectively hybridizes with a polynucleotide sample derived from an individual phenotypically diagnosed with Type II diabetes, and wherein said polynucleotide does not hybridize with a polynucleotide sample from an individual not affected by Type II diabetes.