NZ577804A - Genetic susceptibility variants of type 2 diabetes mellitus - Google Patents
Genetic susceptibility variants of type 2 diabetes mellitusInfo
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- NZ577804A NZ577804A NZ577804A NZ57780407A NZ577804A NZ 577804 A NZ577804 A NZ 577804A NZ 577804 A NZ577804 A NZ 577804A NZ 57780407 A NZ57780407 A NZ 57780407A NZ 577804 A NZ577804 A NZ 577804A
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Abstract
Provided is a method of determining a susceptibility to Type 2 diabetes in a human individual, comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, or in a genotype dataset derived from the individual, wherein the at least one polymorphic marker is selected from rs7756992, and markers in linkage disequilibrium therewith, and wherein determination of the presence or absence of the at least one allele is indicative of a susceptibility to Type 2 diabetes.
Description
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GENETIC SUSCEPTIBILITY VARIANTS OF TYPE 2 DIABETES MELLITUS
BACKGROUND OF THE INVENTION
t
Diabetes mellitus, a metabolic disease wherein carbohydrate utilization is reduced and lipid and protein utilization is enhanced, is caused by an absolute or relative deficiency of insulin. In the more severe cases, diabetes is characterized by chronic hyperglycemia, glycosuria, water and electrolyte loss, ketoacidosis and coma. Long term complications include development of neuropathy, retinopathy, nephropathy, generalized 10 degenerative changes in large and small blood vessels and increased susceptibility to infection. The most common form of diabetes is Type II, non-insulin-dependent diabetes that is characterized by hyperglycemia due to impaired insulin secretion and insulin resistance in target tissues. Both genetic and environmental factors contribute to the disease. For example, obesity plays a major role in the development of the disease. 15 Type 2 diabetes is often a mild form of diabetes mellitus of gradual onset.
The health implications of Type 2 diabetes are enormous. In 1995, there were 135 million adults with diabetes worldwide. It is estimated that close to 300 million will have diabetes in the year 2025. (King, H., et al., Diabetes Care, 21(9): 1414-1431
(1998)). The prevalence of Type 2 diabetes in the adult population in Iceland is 2.5% 20 (Vilbergsson, S., etal., Diabet. Med., 14(6): 491-498 (1997)), which comprises approximately 5,000 people over the age of 34 who have the disease.
Type 2 diabetes is characterized by hyperglycemia, which can occur through mechanisms such as impaired insulin secretion, insulin resistance in peripheral tissues and increased glucose output by the liver. Most Type 2 diabetes patients suffer serious 25 complications of chronic hyperglycemia including nephropathy, neuropathy, retinopathy and accelerated development of cardiovascular disease. The prevalence of Type 2 diabetes worldwide is currently 6% but is projected to rise over the next decade (Amos, A. F., McCarty, D. J., Zimmet, P., Diabet Med 14 Suppl 5, SI (1997)). This increase in prevalence of Type 2 diabetes is attributed to increasing age of the population and rise in 30 obesity.
There is evidence for a genetic component to the risk of Type 2 diabetes, including prevalence differences between various racial groups (Zimmet, P. eta/., Am J Epidemiol 118, 673 (1983), Knowler, W.C., Pettitt, D.J., Saad, M.F., Bennett, P.H., Diabetes Metab Rev 6, 1 (1990)), higher concordance rates among monozygotic than dizygotic twins
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(Newman, B. etal., Diabetologia 30, 763 (1987), Barnett, A.H., Eff, C., Leslie, R.D., Pyke, D.A., Diabetologia 20, 87 (1981)) and a sibling relative risk (As) for Type 2 diabetes in European populations of approximately 3.5 (Gloyn, A.L., Ageing Res Rev 2, 111 (2003)).
Two approaches have thus far been used to search for genes associated with Type 5 2 diabetes. Single nucleotide polymorphisms (SNPs) within candidate genes have been tested for association and have, in general, not been replicated or confer only a modest risk of Type 2 diabetes - the most widely reported being a protective Prol2Ala polymorphism in the peroxisome proliferator activated receptor gamma gene (PPARG2) (Altshuler, D. eta/., Nat Genet 26, 76 (2000)) and an at risk polymorphism in the 10 potassium inwardly-rectifying channel, subfamily J, member 11 gene (KIR6.2) (Gloyn A.L. etal., Diabetes 52, 568 (2003)).
Genome-wide linkage scans in families with the common form of Type 2 diabetes have yielded several loci, and the primary focus of international research consortia has been on loci on chromosomes 1, 12 and 20 observed in many populations (Gloyn, A.L., 15 Ageing Res Rev 2, 111 (2003)). The genes in these loci have yet to be uncovered.
However, in Mexican Americans, the calpain 10 (CAPN10) gene was isolated out of a locus on chromosome 2q (Horikawa, Y. eta/., Nat Genet 26, 163 (2000)). The rare Mendelian forms of Type 2 diabetes, namely maturity-onset diabetes of the young (MODY), have yielded six genes by positional cloning (Gloyn, A.L., Ageing Res Rev 2, 111 (2003)).
Genome-wide significant linkage to chromosome 5q for Type 2 diabetes mellitus in the Icelandic population has been reported (Reynisdottir, I. et a/., Am J Hum Genet 73, 323 (2003)); in the same study, suggestive evidence of linkage to lOq and 12q was also reported. Linkage to the lOq region has also been observed in Mexican Americans (Duggirala, R. et a/., Am J Hum Genet 64, 1127 (1999)).
The transcription factor 7-like 2 gene (TCF7L2; formerly TCF4) has been associated with Type 2 diabetes (P = 2.1 x 10(-9)) (Grant, S.F. eta/., Nat Genet 38, 320 (2006)). The original finding in an Icelandic cohort of association of the microsatellite marker DG10S478 within intron 3 of the gene (P = 2.1 x 10(-9)) was replicated in a Danish cohort (P = 4.8 x 10(-3)) and in a US cohort (P = 3.3 x 10(-9)). Compared with 30 non-carriers, heterozygous and homozygous carriers of the at-risk alleles (38% and 7% of the population, respectively) have relative risks of 1.45 and 2.41. This corresponds to a population attributable risk of 21%. %. Association of the TCF7L2 variant has now been replicated in 10 independent studies with similar relative risk found in the different populations studied. The TCF7L2 gene product is a high mobility group box-containing 35 transcription factor previously implicated in blood glucose homeostasis. It is thought to act through regulation of proglucagon gene expression in enteroendocrine cells via the Wnt signaling pathway.
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Despite the advances in unravelling the genetics of Type 2 diabetes, the high prevalence of the disease and increasing population affected shows an unmet medical need to define additional genetic factors involved in Type 2 diabetes to more precisely define the associated risk factors. People with impaired fasting glucose or impaired 5 glucose tolerance are asymptomatic but are at a high risk of developing Type 2 diabetes. Currently there is very little information to distinguish those within this high risk group, where lifestyle intervention would be the best choice for disease prevention, from those individuals for whom preventive medication would be more appropriate. Identification of susceptibility genes will allow a better understanding of the pathophysiology of the 10 disease and as a direct benefit for the patient it will facilitate better approaches for diagnosis and treatment. Also needed are therapeutic agents for prevention of Type 2 diabetes.
SUMMARY OF THE INVENTION
The present invention relates to methods of diagnosing an increased susceptibility to Type 2 diabetes, as well as methods of diagnosing a decreased susceptibility to Type 2 diabetes or diagnosing a protection against Type 2 diabetes, by evaluating certain markers or haplotypes that have been found to be associated with increased or decreased susceptibility of Type 2 diabetes.
In a first aspect, the present invention relates to a method of determining a susceptibility to Type 2 diabetes in a human individual, comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from the markers set forth in Tables 10-12, and markers in linkage
disequilibrium therewith, and wherein determination of the presence or absence of the at least one allele is indicative of a susceptibility to Type 2 diabetes. I one embodiment, the at least one polymorphic marker is selected from the markers set forth in Tables 10-12 and 14. In an alternative aspect the method of determining a susceptibility to Type 2 diabetes is a method of diagnosing a susceptibility to Type 2 diabetes.
In one embodiment, the at least one polymorphic marker is present within SEQ ID
NO:l, SEQ ID NO:2 or SEQ ID NO:3. In another embodiment, the at least one polymorphic marker comprises at least one marker selected from rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID
NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID
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NO: 10), rsl981647 (SEQ ID NO: 11), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic marker comprises at least one marker in strong linkage disequilibrium, as defined by numeric values for |D'| of greater than 0.8 5 and/or r2 of greater than 0.2, with one or more markers selected from the group consisting of the markers set forth in Table 22, Table 23 and Table 24. In one preferred embodiment, the at least one polymorphic marker is selected from markers rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 10 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), and markers in linkage disequilibrium therewith. In another preferred embodiment, the at least one polymorphic marker is selected from markers rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), and rs6931514 (SEQ ID NO:37). In one 15 embodiment, the at least one marker is selected from marker rs7756992 (SEQ ID NO: 21), and markers in linkage disequilibrium therewith. In another embodiment, the at least one markers is selected from the markers set forth in Table 22. In another embodiment, the at least one marker is selected from marker rsl0882091 (SEQ ID NO: 4), and markers in linkage disequilibrium therewith. In another embodiment, the at least 20 one markers is selected from the markers set forth in Table 23. In yet another embodiment, the at least one marker is selected from marker rs2191113 (SEQ ID NO: 13), and markers in linkage disequilibrium therewith. In another embodiment, the at least one markers is selected from the markers set forth in Table 24.
In one embodiment, the method of determining a susceptibility, or diagnosing a 25 susceptibility, of Type 2 diabetes, further comprises assessing the frequency of at least one haplotype in the individual. In one such embodiment, the at least one haplotype is selected from the haplotypes that comprise at least one polymorphic marker as set forth in Tables 1-6, and polymorphic markers in linkage disequilibrium therewith. In another embodiment, the at least one haplotype is selected from the haplotypes that comprise at 30 least one polymorphic marker selected from at least one marker selected from rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO:15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 35 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO: 11), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium therewith. In another embodiment, the at least one haplotype is selected from the haplotypes set forth in Tables 1-6 and 14.
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In a second aspect, the invention relates to a method of determining a susceptibility to Type 2 diabetes in a human individual, comprising determining whether at least one at-risk allele in at least one polymorphic marker is present in a genotype dataset derived from the individual, wherein the at least one polymorphic marker is 5 selected from the markers set forth in Tables 10-12, and markers in linkage disequilibrium therewith, and wherein determination of the presence of the at least one at-risk allele is indicative of increased susceptibility to Type 2 diabetes in the individual. The genotype dataset comprises in one embodiment information about marker identity, and the allelic status of the individual, i.e. information about the identity of the two alleles 10 carried by the individual for the marker. The genotype dataset may comprise allelic information about one or more marker, including two or more markers, three or more markers, five or more markers, one hundred or more markers, etc. In some embodiments, the genotype dataset comprises genotype information from a whole-genome assessment of the individual including hundreds of thousands of markers, or even 15 one million or more markers.
In one embodiment, the at least one polymorphic marker is present within SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3. In another embodiment, the at least one polymorphic marker comprises at least one marker selected from rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID 20 NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO:19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO: 11), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID 25 NO: 13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic marker comprises at least one marker in strong linkage disequilibrium, as defined by numeric values for |D'| of greater than 0.8 and/or r2 of greater than 0.2, with one or more markers selected from the group consisting of the markers set forth in Table 22, Table 23 and Table 24. In one preferred 30 embodiment, the at least one polymorphic marker is selected from markers rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), and markers in linkage disequilibrium therewith. In another preferred embodiment, the at least one polymorphic marker is 35 selected from markers rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), and rs6931514 (SEQ ID NO:37). In one embodiment, the at least one marker is selected from marker rs7756992 (SEQ ID NO: 21), and markers in linkage disequilibrium therewith. In another embodiment, the at 40 least one markers is selected from the markers set forth in Table 22. In another
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embodiment, the at least one marker is selected from marker rsl0882091 (SEQ ID NO: 4), and markers in linkage disequilibrium therewith. In another embodiment, the at least one markers is selected from the markers set forth in Table 23. In yet another embodiment, the at least one marker is selected from marker rs2191113 (SEQ ID NO: 5 13), and markers in linkage disequilibrium therewith. In another embodiment, the at least one markers is selected from the markers set forth in Table 24. In yet another embodiment, the at least one marker is selected from markers in linkage disequilibrium with the SLC30A gene on chromosome 8, between position 118,032,398 and 118,258,134 (NCBI Build 36 of the Human genome assembly). In one such embodiment, the at least 10 one marker is located within the SLC30A gene.
In one embodiment, the method of determining a susceptibility, or diagnosing a susceptibility, of Type 2 diabetes, further comprises assessing the frequency of at least one haplotype in the individual. In one such embodiment, the at least one haplotype is selected from the haplotypes that comprise at least one polymorphic marker as set forth 15 in Tables 1-6, and polymorphic markers in linkage disequilibrium therewith. In another embodiment, the at least one haplotype is selected from the haplotypes that comprise at least one polymorphic marker selected from at least one marker selected from rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 20 (SEQ ID NO:19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO: 11), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium 25 therewith. In another embodiment, the at least one haplotype is selected from the haplotypes set forth in Tables 1-6 and 14.
In certain embodiments of the invention, determination of the presence of at least one at-risk allele of at least one polymorphic marker in a nucleic acid sample from the individual is indicative of an increased susceptibility to Type 2 diabetes. In one 30 embodiment, the increased susceptibility is characterized by a relative risk (RR) or odds ratio (OR) of at least 1.15. In another embodiment, the increased susceptibility is characterized by a relative risk (RR) or odds ratio (OR) of at least 1.20.
In some embodiments, the presence of rs2497304 allele A, rs947591 allele A, rsl0882091 allele C rs7914814 allele T, rs6583830 allele A, rs2421943 allele G, 35 rs6583826 allele G, rs7752906 allele A, rsl569699 allele C, rs7756992 allele G,
rs9350271 allele A, rs9356744 allele C, rs9368222 allele A, rsl0440833 allele A, rs6931514 allele G, rsl860316 allele A, rsl981647 allele C, rsl843622 allele T,
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rs2191113 allele A, and/or rs9890889 allele A is indicative of increased susceptibility of Type 2 diabetes.
In particular embodiments, the presence of at least one protective allele in a nucleic acid sample from the individual is indicative of a decreased susceptibility of Type 2 5 diabetes. In another embodiment, the absence of at least one at-risk allele in a nucleic acid sample from the individual is indicative of a decreased susceptibility of Type 2 diabetes.
Particular embodiments of the methods of the invention relate to the at least one marker or haplotype being further associated with insulin response and/or impaired 10 glucose tolerance in an individual.
In other embodiments, the presence of, or the determination of, at least one allele or haplotype in an at-risk marker is indicative of an increased susceptibility to Type 2 diabetes, and wherein the at least one allele or haplotype is further indicative of decreased insulin response and/or impaired glucose tolerance.
In certain embodiments of the invention, linkage disequilibrium is characterized by numeric values for |D'| of greater than 0.8 and/or r2 of greater than 0.2. However, other values for the r2and |D'| measures are also possible in other embodiments, and such embodiments are also within the scope of the claimed invention, as described in further detail herein.
Another aspect of the invention relates to a method of assessing a susceptibility to
Type 2 diabetes in a human individual, comprising screening a nucleic acid from the individual for at least one polymorphic marker or haplotype in SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3, that correlates with increased occurrence of Type 2 diabetes in a human population, wherein the presence of an at-risk marker allele in the at least one 25 polymorphism or an at-risk haplotype in the nucleic acid identifies the individual as having elevated susceptibility to diabetes, and wherein the absence of the at least one at-risk marker allele or at-risk haplotype in the nucleic acid identifies the individual as not having the elevated susceptibility.
In one embodiment, the polymorphism or haplotype is selected from rs2497304 30 (SEQ ID NO:16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 35 (SEQ ID NO: 10), rsl981647 (SEQ ID NO: 11), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium
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therewith, as characterized by numeric values for |D'| of greater than 0.8 and/or r2 of greater than 0.2.
Certain embodiments of the invention further comprise a step of screening the nucleic acid for the presence of at least one at-risk genetic variant for Type 2 diabetes not 5 associated with LD Block C06 (SEQ ID NO:l), LD Block C10 (SEQ ID NO:2) and LD Block C17 (SEQ ID NO:3). Such additional genetic variants can in specific embodiments include any variant that has been identified as a susceptibility or risk variant for Type 2 diabetes, including other variants described herein. In one embodiment, the step comprises screening the nucleic acid for the presence or absence of at least one at-risk allele of at 10 least one at-risk variant for Type 2 diabetes in the TCF7L2 gene, wherein determination of the presence of the at least one at-risk allele is indicative of increased susceptibility of Type 2 diabetes. In another embodiment, the at least one at-risk variant in the TCF7L2 gene is selected from marker DG10S478, rsl2255372, rs7895340, rslll96205, rs7901695, rs7903146, rsl2243326 and rs4506565, and markers in linkage 15 disequilibrium therewith.
In another aspect of the present invention, the presence of the marker or haplotype found to be associated with Type 2 diabetes, and as such useful for determining a susceptibility to Type 2 diabetes, is indicative of a different response rate of the subject to a particular treatment modality for Type 2 diabetes.
In another aspect, the invention relates to a method of identification of a marker for use in assessing susceptibility to Type 2 diabetes in human individuals, the method comprising:
identifying at least one polymorphic marker within SEQ ID NO:l, SEQ ID NO:2 or
SEQ ID NO:3, or at least one polymorphic marker in linkage disequilibrium 25 therewith;
determining the genotype status of a sample of individuals diagnosed with, or having a susceptibility to, Type 2 diabetes; and determining the genotype status of a sample of control individuals;
wherein a significant difference in frequency of at least one allele in at least one 30 polymorphism in individuals diagnosed with, or having a susceptibility to, Type 2 diabetes, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing susceptibility to Type 2 diabetes.
In one embodiment, "significant" is determined by statistical means, e.g. the 35 difference is statistically significant. In one such embodiment, statistical significance is
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characterized by a P-vaiue of less than 0.05. In other embodiments, the statistical significance is characterized a P-value of less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001, less than 0.0000001, less than 0.0000000001, or less than 0.00000001.
In one embodiment, the at least one polymorphic marker is in linkage disequilibrium, as characterized by numerical values of r2 of greater than 0.2 and/or |D'| of greater than 0.8 with at least one marker selected from marker rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), 10 rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21),
rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO:ll), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO:13),
rs9890889 (SEQ ID NO:31).
In one embodiment, an increase in frequency of the at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, Type 2 diabetes, as compared with the frequency of the at least one allele in the control sample, is indicative of the at least one polymorphism being useful for assessing increased susceptibility to Type 2 diabetes. In another embodiment, a decrease in frequency of the 20 at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, Type 2 diabetes, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing decreased susceptibility to, or protection against, Type 2 diabetes.
Another aspect of the invention relates to a method of genotyping a nucleic acid 25 sample obtained from a human individual, comprising determining the presence or absence of at least one allele of at least one polymorphic marker in the sample, wherein the at least one marker is selected rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), 30 rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33),
rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID N0:10), rsl981647 (SEQ ID NO:ll), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium therewith, and wherein determination of the presence or 35 absence of the at least one allele of the at least one polymorphic marker is predictive of a susceptibility of Type 2 diabetes.
In one embodiment, genotyping comprises amplifying a segment of a nucleic acid that comprises the at least one polymorphic marker by Polymerase Chain Reaction (PCR),
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using a nucleotide primer pair flanking the at least one polymorphic marker. In another embodiment, genotyping is performed using a process selected from allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, nucleic acid sequencing, 5'-exonuclease digestion, molecular beacon assay, oligonucleotide ligation 5 assay, size analysis, and single-stranded conformation analysis. In one particular embodiment, the process comprises allele-specific probe hybridization. In another embodiment, the process comprises DNA sequencing. In a preferred embodiment, the method comprises:
1) contacting copies of the nucleic acid with a detection oligonucleotide probe and 10 an enhancer oligonucleotide probe under conditions for specific hybridization of the oligonucleotide probe with the nucleic acid;
wherein a) the detection oligonucleotide probe is from 5-100 nucleotides in length and specifically hybridizes to a first segment of the nucleic acid whose
nucleotide sequence is given by SEQ ID NO:l, SEQ ID NO:2 or SEQ ID
NO:3 that comprises at least one polymorphic site;
b) the detection oligonucleotide probe comprises a detectable label at its 3' terminus and a quenching moiety at its 5' terminus;
c) the enhancer oligonucleotide is from 5-100 nucleotides in length and is 20 complementary to a second segment of the nucleotide sequence that is 5'
relative to the oligonucleotide probe, such that the enhancer oligonucleotide is located 3' relative to the detection oligonucleotide probe when both oligonucleotides are hybridized to the nucleic acid; and d) a single base gap exists between the first segment and the second 25 segment, such that when the oligonucleotide probe and the enhancer oligonucleotide probe are both hybridized to the nucleic acid, a single base gap exists between the oligonucleotides;
2) treating the nucleic acid with an endonuclease that will cleave the detectable label from the 3' terminus of the detection probe to release free detectable
label when the detection probe is hybridized to the nucleic acid; and
3) measuring free detectable label, wherein the presence of the free detectable label indicates that the detection probe specifically hybridizes to the first segment of the nucleic acid, and indicates the sequence of the polymorphic site as the complement of the detection probe.
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In a particular embodiment, the copies of the nucleic acid are provided by amplification by Polymerase Chain Reaction (PCR). In another embodiment, the susceptibility determined is increased susceptibility. In another embodiment, the susceptibility determined is decreased susceptibility.
Another aspect of the invention relates to a method of assessing an individual for probability of response to a therapeutic agent for preventing and/or ameliorating symptoms associated with Type 2 diabetes, comprising: determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected 10 from rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), 15 rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO: 11), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium therewith, wherein determination of the presence of the at least one allele of the at least one marker is indicative of a probability of a positive response to the Type 2 diabetes therapeutic agent. In one embodiment, the Type 2 diabetes therapeutic agent 20 is selected from the agents set forth in Agent Table 1 and Agent Table 2.
Yet another aspect of the invention relates to a method of predicting prognosis of an individual diagnosed with, Type 2 diabetes, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is 25 selected from the group consisting of rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID
N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rs 1569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), 30 rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID N0:10), rsl981647 (SEQ ID NO:ll),
rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium therewith, wherein determination of the presence of the at least one allele is indicative of a worse prognosis of the Type 2 diabetes in the individual.
A further aspect of the invention relates to a method of monitoring progress of a treatment of an individual undergoing treatment for Type 2 diabetes, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from the group consisting of rs2497304 (SEQ ID
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NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO:ll), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO:13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium therewith, wherein determination of the presence of the at least one allele is indicative of the treatment outcome of the individual.
In one embodiment, the method further comprises assessing at least one biomarker in a sample from the individual. In another embodiment, the method further comprises analyzing non-genetic information to make risk assessment, diagnosis, or prognosis of the individual. The non-genetic information is in one embodiment selected from age, gender, ethnicity, socioeconomic status, previous disease diagnosis, medical history of subject, family history of Type 2 diabetes, biochemical measurements, and clinical measurements. In a particular preferred embodiment, a further step comprising calculating overall risk is employed.
The invention also relates to a kit for assessing susceptibility to Type 2 diabetes in a human individual, the kit comprising reagents for selectively detecting the presence or absence of at least one allele of at least one polymorphic marker in the genome of the individual, wherein the polymorphic marker is selected from the group consisting of polymorphic markers within the nucleic acid segments whose sequences are set forth in SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3, and markers in linkage disequilibrium therewith, and wherein the presence of the at least one allele is indicative of a susceptibility to Type 2 diabetes.
In one embodiment, the at least one polymorphic marker is selected from the group of markers set forth in Tables 10 - 12, and markers in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic marker is selected from the group of markers set forth in Tables 10 - 12 and Table 14, and markers in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic markers is selected from rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID N0:10), rsl981647 (SEQ ID NO:ll), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO:13), rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium therewith. In another embodiment, the at least one polymorphic markers is selected from rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID
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NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), 5 rsl860316 (SEQ ID N0:10), rsl981647 (SEQ ID N0:11), rsl843622 (SEQ ID N0:9), rs2191113 (SEQ ID NO: 13), and rs9890889 (SEQ ID NO:31).
In one embodiment, the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising the at least one polymorphic marker, a buffer and a detectable label. In one embodiment, the reagents 10 comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic nucleic acid segment obtained from the subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes one polymorphic marker, and wherein the fragment is at least 30 base pairs in size. In a particular embodiment the at least one oligonucleotide is completely 15 complementary to the genome of the individual. In another embodiment, the at least one oligonucleotide can comprise at least one mismatch to the genome of the individual. In one embodiment, the oligonucleotide is about 18 to about 50 nucleotides in length. In another embodiment, the oligonucleotide is 20-30 nucleotides in length.
In one preferred embodiment, the kit comprises:
a detection oligonucleotide probe that is from 5-100 nucleotides in length; an enhancer oligonucleotide probe that is from 5-100 nucleotides in length; and an endonuclease enzyme;
wherein the detection oligonucleotide probe specifically hybridizes to a first segment of the nucleic acid whose nucleotide sequence is given by SEQ ID NO:l, 25 SEQ ID NO:2 or SEQ ID NO:3 that comprises at least one polymorphic site; and wherein the detection oligonucleotide probe comprises a detectable label at its 3' terminus and a quenching moiety at its 5' terminus; wherein the enhancer oligonucleotide is from 5-100 nucleotides in length and is complementary to a second segment of the nucleotide sequence that is 5' relative to the 30 oligonucleotide probe, such that the enhancer oligonucleotide is located 3' relative to the detection oligonucleotide probe when both oligonucleotides are hybridized to the nucleic acid; wherein a single base gap exists between the first segment and the second segment, such that when the oligonucleotide probe and the enhancer oligonucleotide probe are both hybridized to the nucleic acid, a single 35 base gap exists between the oligonucleotides; and wherein treating the nucleic acid with the endonuclease will cleave the detectable label from the 3' terminus of the detection probe to release free detectable label when the detection probe is hybridized to the nucleic acid.
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A further aspect of the invention relates to the use of an oligonucleotide probe in the manufacture of a diagnostic reagent for diagnosing and/or assessing susceptibility to Type 2 diabetes in a human individual, wherein the probe hybridizes to a segment of a nucleic acid whose nucleotide sequence is given by SEQ ID NO: 1, SEQ ID NO:2 or SEQ 5 ID NO:3 that comprises at least one polymorphic site, wherein the fragment is 15-500 nucleotides in length. In one embodiment, the polymorphic site is selected from the polymorphic markers rs2497304 (SEQ ID NO:16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID N0:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), 10 rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO: 11), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID NO:31), and polymorphisms in linkage disequilibrium therewith.
Yet another aspect of the invention relates to a computer-readable medium on which is stored: an identifier for at least one polymorphic marker; an indicator of the frequency of at least one allele of said at least one polymorphic marker in a plurality of individuals diagnosed with Type 2 diabetes; and an indicator of the frequency of the least one allele of said at least one polymorphic markers in a plurality of reference individuals; 20 wherein the at least one polymorphic marker is selected from the polymorphic markers rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID NO:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), 25 rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO: 11), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID NO:31), and polymorphisms in linkage disequilibrium therewith. In one embodiment, linkage disequilibrium is defined as defined by numerical values of r2 of at least 0.2 and/or values of |D'| of at least 0.8.
In one embodiment, information about the ancestry of the plurality of individuals is included. In another embodiment, the plurality of individuals diagnosed with Type 2 diabetes and the plurality of reference individuals is of a specific ancestry.
Another aspect relates to an apparatus for determining a genetic indicator for Type 2 diabetes in a human individual, comprising: a computer readable memory; and a 35 routine stored on the computer readable memory; wherein the routine is adapted to be executed on a processor to analyze marker and/or haplotype information for at least one human individual with respect to at least one polymorphic marker selected from the markers rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4),
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rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), 5 rsl860316 (SEQ ID N0:10), rsl981647 (SEQ ID NO:ll), rsl843622 (SEQ ID NO:9),
rs2191113 (SEQ ID NO: 13), rs9890889 (SEQ ID N0:31), and markers in linkage disequilibrium therewith, as defined by numerical values of r2 of at least 0.2 and/or values of | D'| of at least 0.8, and generate an output based on the marker or haplotype information, wherein the output comprises a risk measure of the at least one marker or 10 haplotype as a genetic indicator of Type 2 diabetes for the human individual.
In one embodiment, the routine further comprises an indicator of the frequency of at least one allele of at least one polymorphic marker or at least one haplotype in a plurality of individuals diagnosed with Type 2 diabetes, and an indicator of the frequency of at the least one allele of at least one polymorphic marker or at least one haplotype in a 15 plurality of reference individuals, and wherein a risk measure is based on a comparison of the at least one marker and/or haplotype status for the human individual to the indicator of the frequency of the at least one marker and/or haplotype information for the plurality of individuals diagnosed with Type 2 diabetes.
In certain embodiments of the methods, uses, apparatus or kits of the invention, 20 linkage disequilibrium is characterized by numeric values for |D'| of greater than 0.8
and/or r2 of greater than 0.2. However, other values for the r2and |D'| measures are also possible in other embodiments and such embodiments are also within the scope of the claimed invention, as described in further detail herein.
In certain other embodiments of the methods, uses, apparatus or kits of the 25 invention, the individual is of a specific human ancestry. In one embodiment, the ancestry is selected from black African ancestry, Caucasian ancestry and Chinese ancestry. In another embodiment, the ancestry is black African ancestry. In another embodiment, the ancestry is European ancestry. In another embodiment, the ancestry is Caucasian ancestry. The ancestry is in certain embodiment self-reported by the individual 30 who undergoes genetic analysis or genotyping. In other embodiments, the ancestry is determined by genetic determination comprising detecting at least one allele of at least one polymorphic marker in a nucleic acid sample from the individual, wherein the presence or absence of the allele is indicative of the ancestry of the individual.
In particular other embodiments of the methods, uses, apparatus or kits of the 35 invention, the individual is obese. In other embodiments, the individual is non-obese.
Obesity is in one embodiment determined by values of BMI (Body Mass Index) of greater than 25. In another embodiment, obesity is defined by values of BMI greater than 30. Other cutoff integer or fractional values of BMI are also possible and within scope of the
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invention, including, but not limited to BMI of greater than 23, 24, 25.5, 26, 26.5, 27, 27.5 and so on. Non-obese individuals are in one embodiment defined as all those individuals who do not fulfill the criteria of obesity by BMI. In other embodiments, non-obese individuals are those with a particular cutoff of BMI, such as BMI less than 25, less 5 than 24, less than 23, less than 22, less than 21 or less than 20. Non-integer cutoff values of BMI values are also useful for defining non-obese individuals. In general, the obese and non-obese groups do not overlap in terms of their BMI values. In certain embodiments, the cutoff employed to define the groups is the same, e.g., greater than or smaller than BMI of 25. In other embodiments, a different cutoff is used, e.g., greater 10 than 27 for obese individuals and smaller than 23 for non-obese individuals. All relevant ranges of BMI that are suitable for defining obese and non-obese individuals are also possible and within scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention.
FIG 1 shows a plot linkage disequilibrium pattern in the region of chromosome 6p22.3 containing markers associated with Type 2 diabetes, (a) The X-axis shows positions with 20 respect to NCBI Build 35 genome assembly (identical to Build 36), and the Y-axis shows a measure of linkage disequilibrium in the region. The span of the CDKAL1 gene is indicated by the arrows, and the locations of exons by black bars perpendicular to the diagonal line. The SNP markers are plotted equidistantly rather than according to their physical positions. The figure shows the r2 measure of linkage disequilibrium, wherein the 25 shading is proportional to pair-wise values of r2 between markers, (b) A close-up of the 5' end of the CDKAL gene, showing the LD Block C06 region (SEQ ID NO: 1) within which several markers have been found to be associated with Type 2 diabetes. The location of several of the associated SNP markers is indicated on the figure.
FIG 2 shows linkage disequilibrium in the region of chromosome 10q23.33 containing 30 markers associated with Type 2 diabetes. The X-axis shows positions with respect to NCBI Build 35 genome assembly, and the Y-axis shows a measure of linkage disequilibrium in the region. The location of four associated SNP markers rs2497304, rs947591, rsl0882091 and rs7914814 is indicated as well as the span and exons of the three genes within the LD block, IDE, KIF11 and HHEX. The figure shows the r2 measure 35 of linkage disequilibrium, wherein the shading is proportional to pair-wise values of r2 between markers.
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FIG 3 shows linkage disequilibrium in the region of chromosome 17q24.3 containing markers associated with diabetes in non-obese and all patients. The location of five SNP markers, rsl860316, rsl981647, rsl843622, rs2191113 and rs9890889, is indicated. The figure shows the r2 measure of linkage disequilibrium, wherein the shading is 5 proportional to pair-wise values of r2 between markers.
FIG 4 shows a Q-Q plot of the 653,025 adjusted Chi2-statistics (circles) from the analysis of single SNPs and two marker haplotypes. The equiangular line (black line) is included in the plot for reference purpose. The dashed horizontal line indicates the threshold for genome-wide significance assuming a Bonferroni correction for the 653,025 SNPs / 10 haplotypes and three phenotypes tested.
FIG 5 presents a schematic view of the association of T2D to 6p22.3. a) The pair-wise correlation structure in a 1 Mb interval (20.5 - 21.5 Mb, NCBI Build34) on chromosome 6. The upper plot includes pair-wise D' for 1047 common SNPs (with MAF > 5%) from the HapMap release 19 for the CEU population, while the lower plot includes pair-wise r2 15 values for the same set of SNPs. b) Location of recombination hot-spots in this interval based on the HapMap dataset (Nature 437, 1299-1320 (27 October 2005))). c) Location of exons (vertical bars) of the two genes, E2F3 and CDKAL1, that map to the interval, d) Schematic view of the genome-wide association results in the interval for all T2D cases (black dots), non-obese T2D cases (open circles) and obese T2D cases (open triangles), 20 respectively. Plotted is -log P, where P is the adjusted P value, against the chromosomal location of the markers. All four panels use the same horizontal Mb scale indicated at the bottom of panel d).
FIG 6 shows CDKAL1 cDNA from INS-1 cells. Lanes 1 and 2 contain CDKAL1 cDNA amplified from exons 2 to 8 and exons 7 to 13, giving a band size of 596bp and 738bp, 25 respectively, p-actin (837bp) serves as a positive control in lane 3 and lane 4 is a negative control reaction without primers. Size standard is given on the left.
FIG 7 shows the association of rs7756992 and rsl3266634 to insulin secretion. Mean log-transformed insulin secretion levels, estimated by corrected insulin response (see Methods), for the three different genotypes of the two SNPs, rs7756992 and rsl3266634. 30 Results are shown for 3982 individuals (231 T2D cases and 3751 controls) from the
Danish Inter99 study that had an oral glucose tolerance test. The number of individuals is included under each column, and the standard error (s.e.m.) is indicated as horizontal bars. The included P values are from regression of the log-transformed insulin secretion levels on genotype status, adjusting for age, sex and affection status, assuming either an 35 additive model (Padd) or a recessive model (Prec)-
FIG 8 presents further analysis of association of rs7756992 and rsl3266634 with insulin secretion, a) Mean log-transformed insulin secretion levels, estimated by corrected
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insulin response (CIR) for the three different genotypes for the SNP rs7756992. The insulin secretion levels are estimated for a group of 3938 individuals from the Danish Inter99 cohort (223 T2D cases and 3715 controls) that had an OGTT. Results are shown for all individuals (leftmost bars) and males (middle bars) and females (rightmost bars) 5 separately. The number of individuals behind each estimate is indicated in parenthesis below the columns together with the corresponding genotype. The standard error of the mean is indicated with a bar on top of each column, b) Corresponding estimates for the different genotypes of the SNP rsl3266634 for 3926 individuals (228 T2D cases and 3698 controls).
DETAILED DESCRIPTION OF THE INVENTION
A description of preferred embodiments of the invention follows.
The present invention discloses polymorphic markers and haplotypes that have been found to be associated with Type 2 diabetes. Particular alleles at certain polymorphic SNP 15 markers and haplotypes comprising such alleles have been found to be associated with Type 2 diabetes. Such markers and haplotypes are useful for assessing susceptibility to Type 2 diabetes, as described in further detail herein. Further applications of the present invention include methods for assessing response to Type 2 diabetes therapeutic agents utilizing the polymorphic markers of the invention, as well as kits for assessing 20 susceptibility of an individual to Type 2 diabetes.
Definitions
The following terms shall, in the present context, have the meaning as indicated:
A "polymorphic marker", sometime referred to as a "marker", as described herein, 25 refers to a genomic polymorphic site. Each polymorphic marker has at least two sequence variations characteristic of particular alleles at the polymorphic site. Thus, genetic association to a polymorphic marker implies that there is association to at least one specific allele of that particular polymorphic marker. The marker can comprise any allele of any variant type found in the genome, including SNPs, microsatellites, insertions, 30 deletions, duplications and translocations.
An "allele" refers to the nucleotide sequence of a given locus (position) on a chromosome. A polymorphic marker allele thus refers to the composition (i.e., sequence) of the marker on a chromosome. Genomic DNA from an individual contains two alleles for any given polymorphic marker, representative of each copy of the marker on each
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chromosome. Sequence codes for nucleotides used herein are: A = 1, C = 2, G = 3, T = 4.
Sequence conucleotide ambiguity as described herein is as proposed by IUPAC-IUB. These codes are compatible with the codes used by the EMBL, GenBank, and PIR 5 databases.
IUB
Meaning
A
Adenosine
C
Cytidine
G
Guanine
T
Thymidine
R
G or A
Y
TorC
K
GorT
M
A or C
S
G or C
W
A orT
B
CGorT
D
AGorT
H
ACorT
V
A C or G
N
A C G or T (Any base)
A nucleotide position at which more than one sequence is possible in a population (either a natural population or a synthetic population, e.g., a library of synthetic molecules) is referred to herein as a "polymorphic site".
A "Single Nucleotide Polymorphism" or "SNP" is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the 15 same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides). The SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI).
A "variant", as described herein, refers to a segment of DNA that differs from the 20 reference DNA. A "marker" or a "polymorphic marker", as defined herein, is a variant. Alleles that differ from the reference are referred to as "variant" alleles.
A "microsatellite" is a polymorphic marker that has multiple small repeats of bases that are 2-8 nucleotides in length (such as CA repeats) at a particular site, in which the number of repeat lengths varies in the general population. An "indel" is a common form
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of polymorphism comprising a small insertion or deletion that is typically only a few nucleotides long.
A "haplotype," as described herein, refers to a segment of genomic DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid 5 organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus . In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles. Haplotypes are described herein in the context of the marker name and the allele of the marker in that haplotype, e.g., "3 rs7758851" refers to the 3 allele of marker rs7758851 10 being in the haplotype, and is equivalent to "rs7758851 allele 3". Furthermore, allelic codes in haplotypes are as for individual markers, i.e. 1 = A, 2 = C, 3 = G and 4 = T.
The term "susceptibility", as described herein, encompasses both increased susceptibility and decreased susceptibility. Thus, particular alleles at polymorphic markers and/or haplotypes of the invention may be characteristic of increased 15 susceptibility (i.e., increased risk) of Type 2 diabetes, as characterized by a relative risk (RR) or odds ratio (OR) of greater than one for the particular allele or haplotype. Alternatively, the markers and/or haplotypes of the invention are characteristic of decreased susceptibility (i.e., decreased risk) of Type 2 diabetes, as characterized by a relative risk of less than one.
A "nucleic acid sample" is a sample obtained from an individuals that contains nucleic acid. In certain embodiments, i.e. the detection of specific polymorphic markers and/or haplotypes, the nucleic acid sample comprises genomic DNA. Such a nucleic acid sample can be obtained from any source that contains genomic DNA, including as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from 25 skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
The term "Type 2 diabetes therapeutic agent" refers to an agent that can be used to ameliorate or prevent symptoms associated with Type 2 diabetes.
The term "Type 2 diabetes-associated nucleic acid", as described herein, refers to 30 a nucleic acid that has been found to be associated to Type 2 diabetes. This includes, but is not limited to, the markers and haplotypes described herein and markers and haplotypes in strong linkage disequilibrium (LD) therewith. In one embodiment, a Type 2 diabetes-associated nucleic acid refers to an LD-block found to be associated with Type 2 diabetes through at least one polymorphic marker located within the LD block.
The term "non-obese" refers, as described herein, to an individual with calculated
Body Mass Index (BMI) below a pre-determined threshold, such as a threshold of 30 or
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lower. Other thresholds useful for defining the term are also possible, as described in more detail herein. The formula for calculating BMI is given by [body weight (in kg)]/[height (in m)]2. The term "obese" refers to an individual with BMI above a certain pre-determined threshold, such as a threshold of 30.
The term "LD Block C06", as described herein, refers to the Linkage Disequilibrium
(LD) block on Chromosome 6 between markers rs4429936 and rs6908425, corresponding to position 20,634,996 - 20,836,710 of NCBI (National Center for Biotechnology Information) Build 35 (SEQ ID NO:l).
The term "LD Block C10", as described herein, refers to the Linkage Disequilibrium 10 (LD) block on Chromosome 10 between markers rs2798253 and rslll87152, corresponding to position 94,192,885 - 94,490,091 of NCBI (National Center for Biotechnology Information) Build 35 (SEQ ID NO:2).
The term "LD Block C17", as described herein, refers to the Linkage Disequilibrium (LD) block on Chromosome 17 between markers rsl 1077501 and rs4793497, 15 corresponding to position 66,037,656 - 66,163,076 of NCBI (National Center for Biotechnology Information) Build 35 (SEQ ID NO:3).
The term "CDKAL1", as described herein, refers to the CDK5 regulatory subunit associated protein 1-like 1 gene, which spans locations 20,642,736 - 21,340,611 in NCBI Build 35 of the human genome.
The term "SLC30A8", as described herein, refers to the Solute Carrier Family 30,
member 8, gene. This gene is located on chromosome 8, its longest isoform spanning as much as 225kb between positions 118,032,398 and 118,258,134 in NCBI Build 36 of the human genome assembly, corresponding to position 117,919,805 and 118,145,541, respectively in NCBI Build 34. In both these builds, the gene spans 225,736 bp of 25 genomic sequence.
Through genotyping of Icelandic Type 2 diabetes patients and population control individuals using the Illumina 330K chip that can be used to measure over 300,000 SNPs in the genome simultaneously, a number of variants associated with Type 2 diabetes have 30 been identified by the present invention. Association analysis using single SNPs, two marker haplotypes and extended haplotypes within areas of extensive linkage disequilibrium (LD blocks) was performed across the genome. After correcting the p-value for relatedness, 49 single markers and two marker haplotypes were initially identified at 21 loci (i.e. genetic susceptibility locations in the genome) that had a p-value 35 less than 5xl0"5 (Table 1). In addition, 10 extended haplotypes at 8 additional loci were
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selected by the same criteria (Table 2). Within the patient group, 700 individuals were non-obese (BMI<30) and those were tested separately for association. After correcting the p-value for relatedness, 36 single markers and two marker haplotypes at 20 loci had a p-value less than 5xl0"5 (Table 3). Three of those loci were also identified when the total 5 group was analyzed. In addition, 6 extended haplotypes at 4 additional loci were selected by the same criteria (Table 4). The obese group of 531 patients (BMI >30) was also analyzed separately for association. After correcting the p-value for relatedness, 38 single markers and two marker haplotypes at 16 loci had a p-value less than 5xl0"5 (Table 5). One of those loci was also identified when the total group was analyzed but no 10 overlap was found between the non-obese and obese groups using this criteria. In addition 10 extended haplotypes at 7 additional loci had a p-value less than 5xl0"5 in association analysis of obese diabetics (Table 6).
These single-marker association and two-marker and extended haplotype association results represent evidence for multiple susceptibility variants for Type 2 15 diabetes. It should be noted that for single-marker SNP analysis as presented herein, susceptibility variants can be represented by increased risk, wherein one allele is overrepresented in the patient group compared with controls. Alternatively, the susceptibility variants can be represented by the other allele of the SNP in question - for that allele, under-representation in patients compared with controls is expected. This is a 20 natural consequence of association analysis to genetic elements comprising two alleles. For multi-marker haplotypes or for polymorphic markers comprising more than one marker, at-risk association may be observed to one (or more) at-risk allele or haplotype. Protective variants in form of association (with RR-values less than unity) to one (or more) protective variants or haplotypes may also be observed, depending on the genetic 25 composition and haplotype structure in the genetic region in question.
One of the most significant association signals was identified by two single markers (rsl569699 and rs7756992) and three 2 marker haplotypes mapping to chromosome 6p22.3 (3 rs7758851 2 rsl569699, 1 rs4712527 3 rs7756992, 1 rs7756992 3 rs9295478 ; see Table 3). These markers are located within an area of extensive LD 30 (LD block) between position 20634996 and 20836710 on chromosome 6 (NCBI Build 35; SEQ ID NO:l) between markers rs4429936 and rs6908425 (Figure 1). This region contains the 5' end including exons 1-5 of the gene CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1) (NM_017774). The association of these markers was verified in two additional Type 2 diabetes cohorts (see Table 7).
Follow up studies of the association of rs7756992 allele G with increased risk of
Type 2 diabetes have established association of the marker to Type 2 diabetes in individuals of European ancestry (allele specific odds ratio (OR) = 1.16; P = 3.9x10 10), in individuals from Hong Kong of Han Chinese ancestry (OR = 1.25; P = 0.00018) (see
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Tables 14, 15 and 17). Additional variants within LD block C06 (SEQ ID NO:l) in LD with rs7756992 that have also been shown to be associated with Type 2 diabetes in European and Chinese populations include rsl569699, rs7752906, rs9350271, rs9356744, rs9368222, rsl0440833 and rs6931514 (Table 18). The genotype odds, ratio of the 5 rs77566992 allele G variant supports a nearly recessive mode of inheritance (Table 20). In particular, the OR for the homozygote is 1.45 and 1.55 in the European and Hong Kong groups, respectively. The rs77566992 allele G at-risk variant has been found to be correlated with decreased insulin response in carriers (Table 21, Figures 7 and 8). Homozygous carriers of the variant have been found to have an estimated 24% less 10 insulin response than heterozygotes or non-carriers suggesting that this variant confers risk of T2D through reduced insulin secretion. The rs7756992 marker, and markers in linkage disequilibrium therewith (including, but not limited to, rsl569699, rs7752906, rs9350271, rs9356744, rs9368222, rsl0440833 and rs6931514) can therefore be used to assess increased susceptibility to Type 2 diabetes in an individual.
The function of the gene product of CDKAL1 is not known. However, as implied in the gene name the protein product is similar to another protein, CDK5 regulatory subunit associated protein 1 (CDK5RAP1). CDK5RAP1 is expressed in neuronal tissues where it inhibits cyclin dependent kinase 5 (CDK5) activity by binding to the CDK5 regulatory subunit p35 (Ching, Y.P., Pang, A.S., Lam, W.H., Qi, R.Z. & Wang, J.H. J Biol Chem 277, 20 15237-40 (2002)). In pancreatic beta cells, CDK5 has been shown to play a role in the loss of beta cell function under glucotoxic conditions (Wei, F.Y. et al. Nat Med 11, 1104-8 (2005). Furthermore, inhibition of the CDK5/p35 complex prevents decrease of insulin gene expression that results from glucotoxicity (Ubeda, M., Rukstalis, J.M. & Habener, J.F. J Biol Chem 281, 28858-64 (2006)). CDKAL1 might play a role in the inhibition of 25 CDK5/p35 in pancreatic beta cells similar to that of CDK5RAP1 in neuronal tissue.
Reduced expression of CDKAL1 or reduced inhibitory function thus could lead to an impaired response to glucotoxicity. The present data shows that CDKAL1 is expressed in the rat pancreatic beta cell line INS-1 (Figure 6).
Based on the predicted function of CDKAL1 and known function of SLC30A8 we 30 would expect both rs7756992 and rsl3266634 to affect insulin secretion. To evaluate the effects of the two SNPs on insulin secretion we analyzed the effect of genotype status on corrected insulin response (CIR) in a set of individuals from the Inter99 study (part of Denmark B) that had undergone an oral glucose tolerance test (OGTT). For rs7756992, we demonstrated that the homozygote carriers of the risk allele had an estimated 24% 35 less CIR than the heterozygote carriers or non-carriers (P < 0.00001, Fig. 7). This observation is consistent with the variant's nearly recessive mode of inheritance with respect to disease risk. Furthermore, the effect observed on CIR is present in both males and females (Figure 8) and in T2D patients as well as controls, and adjusting for BMI status did not affect the results (Table 21). The effect of rsl3266634 on insulin response
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was smaller but significant and for this risk variant the reduction in CIR was consistent with an additive effect. No effect on insulin sensitivity was observed for either variant (Table 21).
The identification of CDKAL1 as a susceptibility gene forT2D adds a new piece to 5 the puzzle of how genetic factors may predispose to T2D. Although the function of this gene remains to be elucidated we have shown that it is expressed in pancreatic beta cells and that a variant within the gene is correlated with insulin secretion. The similarity to CDK5RAP1 further indicates that CDKAL1 may facilitate insulin production under glucotoxic conditions through interaction with CDK5. In conclusion, we have identified a 10 variant in the CDKAL1 gene that in a nearly recessive manner blunts the insulin response and predisposes to T2D.
The present invention has identified seven single markers and seven two marker haplotypes in a region on chromosome 10q23.33 to be associated with Type 2 diabetes (Table 1). Most of those markers are also associated to diabetes with elevated RR values 15 when obese patients are analyzed separately (Table 5). These markers are located within one LD block between positions 94192885and 94490091 (NCBI Build 35), corresponding to the genomic segment bridged by markers rs2798253 and rslll87152 (Figure 2). This LD block contains three genes, Insulin-degrading enzyme (IDE) (NM_004969), Kinesin family member 11 (KIF11) (NM_004523) and Homeobox, hematopoietically expressed 20 (HHEX) (NM_002729).
IDE may belong to a protease family responsible for intercellular peptide signaling. Though its role in the cellular processing of insulin has not yet been defined, insulin-degrading enzyme is thought to be involved in the termination of the insulin response (Fakhrai-Rad et al, Human Molecular Genetics 9:2149-2158, 2000). Genetic analysis of 25 the diabetic GK rat has revealed 2 amino acid substitutions in the IDE gene (H18R and A890V) in the GK allele which reduced insulin-degrading activity by 31% in transfected cells. However, when the H18R and A890V variants were studied separately, no effects were observed, suggesting a synergistic effect of the 2 variants on insulin degradation. No effect on insulin degradation was observed in cell lysates, suggesting that the effect may 30 be coupled to receptor-mediated internalization of insulin. Congenic rats with the IDE GK allele displayed postprandial hyperglycemia, reduced lipogenesis in fat cells, blunted insulin-stimulated glucose transmembrane uptake, and reduced insulin degradation in isolated muscle. Analysis of additional rat strains demonstrated that the dysfunctional IDE allele was unique to GK rats. The authors concluded that IDE plays an important role in 35 the diabetic phenotype in GK rats. IDE has been studied as a candidate gene for Type 2 diabetes in humans with inconsistent results. Two large studies have recently analyzed the association of IDE to Type 2 diabetes by mutation screening and haplotype analysis using tagging SNPs over the gene (Groves et al, Diabetes 52:1300-1305, 2003; Florez et
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al, Diabetes 55:128-135, 2006). Both studies conclude that common variants in IDE are unlikely to confer significant risk of Type 2 diabetes. These studies did however, not include the whole LD block as defined in figure 2 and at least some of the markers identified in our study as associated with Type 2 diabetes are outside the regions analyzed 5 in those previous studies. Based on the results reported here, markers in LD with IDE are associated with Type 2 diabetes, providing genetic evidence for the role of IDE in the etiology of Type 2 diabetes.
KIF11 encodes a motor protein that belongs to the kinesin-like protein family. Members of this protein family are known to be involved in various kinds of spindle 10 dynamics. The function of this gene product includes chromosome positioning,
centrosome separation and establishing a bipolar spindle during cell mitosis. This gene is not a good functional candidate for diabetes but has to be considered as a positional candidate due to its location within the associated LD block.
HHEX encodes a member of the homeobox family of transcription factors, many of 15 which are involved in developmental processes. Expression in specific hematopoietic lineages suggests that this protein may play a role in hematopoietic differentiation. HHEX is essential for pancreatic development; in HHEX negative mouse embryos there is a complete failure in ventral pancreatic specification (Bort et al, Development 131, 797-806, 2004). Other transcription factors involved in pancreatic development include the 20 MODY genes as well as other factors that have been implicated in late onset diabetes. HHEX is also an essential effector of Wnt antagonist for heart induction (Foley and Mercola, GENES & DEVELOPMENT 19:387-396, 2005). This puts HHEX in the same pathway as the recently established Type 2 diabetes gene TCF7L2 and together these data make HHEX a functional as well as positional candidate for Type 2 diabetes.
The association of rs2497304, rs947591, rsl0882091 and rs7914814 to Type 2
diabetes was verified in a Danish Type 2 diabetes case - control cohort and also in a US Caucasian cohort Type 2 diabetes cohort from the PENN CATH study (Table 8). When the two cohorts are combined the association of rs947591 reaches significance at the 0.05 level, with a risk of 1.1 in the combined cohort. When all the cohorts are combined the 30 risk is 1.15 for the rs947591 marker. These results indicate that variants within the LD block on Chromosome 10 that includes IDE and HHEX are susceptibility variants for Type 2 diabetes.
Five single markers and two marker haplotypes in a region of chromosome 17q24.3 were furthermore found to be associated with Type 2 diabetes in non-obese 35 patients (Table 3). Some of these markers show the strongest association reported in
Table 3 and association to this region was also observed when all diabetics were analyzed (Table 1). These markers are located within two adjacent LD blocks located between
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positions 66037656 and 66163076 (NCBI Build 35) on chromosome 17, between markers rsll077501 and rs4793497 (Figure 3). The association is significant at the genome-wide level. No known genes are located within these LD blocks. However, it is possible that variants in this region affect genes in neighboring regions including KCNJ2 and KCNJ16.
Alternatively these variants may affect unknown genes within these LD blocks.
Further evidence for the association of rs7756992, and correlated markers within the LD block C06 that contains the 5' end including exons 1-5 of the CDKALlgene (NM_017774) on chromosome 6p22.3, with Type 2 diabetes has come from additional association studies. Two equivalent markers, rs7754840 and rsl0946398, highly 10 correlated with rs7756992 (r2 0,68; D' 0,95) were shown to be significantly associated with Type II diabetes in three large studies (Saxena, R et al. Science 2007;316:1331-6; Zeggini, E et al. Science 2007;316:1336-41; Scott, LJ et al. Science 2007;316:1341-5). These studies thus further support the involvement of the CDKAL gene in Type 2 diabetes.
Association of rsl0882091 and correlated markers on chromosome 10q23.33 with 15 Type II diabetes is also supported by recent publications. A highly correlated marker,
rsllll875 (r2 0,51; D' = 1) was found to be significantly associated with Type II diabetes in four large studies (Sladek, R et al. Nature. 2007;445:828-30; Saxena, R et al. Science 2007;316:1331-6; Zeggini, E et al. Science 2007;316:1336-41; Scott, LJ et al. Science 2007;316:1341-5). Thus, recent studies provide additional support to the discoveries by 20 the present inventors that markers in the LD Block C10 region as described herein are risk factors for Type 2 diabetes.
The genomic sequence within populations is not identical when individuals are compared. Rather, the genome exhibits sequence variability between individuals at 25 many locations in the genome. Such variations in sequence are commonly referred to as polymorphisms, and there are many such sites within each genome For example, the human genome exhibits sequence variations which occur on average every 500 base pairs. The most common sequence variant consists of base variations at a single base position in the genome, and such sequence variants, or polymorphisms, are commonly 30 called Single Nucleotide Polymorphisms ("SNPs"). These SNPs are believed to have occurred in a single mutational event, and therefore there are usually two possible alleles possible at each SNP site; the original allele and the mutated allele. Due to natural genetic drift and possibly also selective pressure, the original mutation has resulted in a polymorphism characterized by a particular frequency of its alleles in any given 35 population. Many other types of sequence variants are found in the human genome,
including microsatellites, insertions, deletions, inversions and copy number variations. A polymorphic microsatellite has multiple small repeats of bases (such as CA repeats, TG on
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the complimentary strand) at a particular site in which the number of repeat lengths varies in the general population. In general terms, each version of the sequence with respect to the polymorphic site represents a specific allele of the polymorphic site. These sequence variants can all be referred to as polymorphisms, occurring at specific 5 polymorphic sites characteristic of the sequence variant in question. In general terms, polymorphisms can comprise any number of specific alleles. Thus in one embodiment of the invention, the polymorphism is characterized by the presence of two or more alleles in any given population. In another embodiment, the polymorphism is characterized by the presence of three or more alleles. In other embodiments, the polymorphism is 10 characterized by four or more alleles, five or more alleles, six or more alleles, seven or more alleles, nine or more alleles, or ten or more alleles. All such polymorphisms can be utilized in the methods and kits of the present invention, and are thus within the scope of the invention.
In some instances, reference is made to different alleles at a polymorphic site 15 without choosing a reference allele. Alternatively, a reference sequence can be referred to for a particular polymorphic site. The reference allele is sometimes referred to as the "wild-type" allele and it usually is chosen as either the first sequenced allele or as the allele from a "non-affected" individual (e.g., an individual that does not display a trait or disease phenotype).
Alleles for SNP markers as referred to herein refer to the bases A, C, G or T as they occur at the polymorphic site in the SNP assay employed. The allele codes for SNPs used herein are as follows: 1 = A, 2 =C, 3 =G, 4 =T. The person skilled in the art will however realise that by assaying or reading the opposite DNA strand, the complementary allele can in each case be measured. Thus, for a polymorphic site (polymorphic marker) 25 characterized by an A/G polymorphism, the assay employed may be designed to specifically detect the presence of one or both of the two bases possible, i.e. A and G. Alternatively, by designing an assay that is designed to detect the opposite strand on the DNA template, the presence of the complementary bases T and C can be measured. Quantitatively (for example, in terms of relative risk), identical results would be obtained 30 from measurement of either DNA strand (+ strand or - strand).
Typically, a reference sequence is referred to for a particular sequence. Alleles that differ from the reference are sometimes referred to as "variant" alleles. A variant sequence, as used herein, refers to a sequence that differs from the reference sequence but is otherwise substantially similar. Alleles at the polymorphic genetic markers 35 described herein are variants. Additional variants can include changes that affect a polypeptide. Sequence differences, when compared to a reference nucleotide sequence, can include the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift; the change of at least one nucleotide, resulting in a
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change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an 5 interruption of the coding sequence of a reading frame; duplication of all or a part of a sequence; transposition; or a rearrangement of a nucleotide sequence,. Such sequence changes can alter the polypeptide encoded by the nucleic acid. For example, if the change in the nucleic acid sequence causes a frame shift, the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature 10 stop codon, causing generation of a truncated polypeptide. Alternatively, a polymorphism associated with a disease or trait can be a synonymous change in one or more nucleotides {i.e., a change that does not result in a change in the amino acid sequence). Such a polymorphism can, for example, alter splice sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of an encoded polypeptide. It 15 can also alter DNA to increase the possibility that structural changes, such as amplifications or deletions, occur at the somatic level. The polypeptide encoded by the reference nucleotide sequence is the "reference" polypeptide with a particular reference amino acid sequence, and polypeptides encoded by variant alleles are referred to as "variant" polypeptides with variant amino acid sequences.
A haplotype refers to a segment of DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus . In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles, each allele 25 corresponding to a specific polymorphic marker along the segment. Haplotypes can comprise a combination of various polymorphic markers, e.g., SNPs and microsatellites, having particular alleles at the polymorphic sites. The haplotypes thus comprise a combination of alleles at various genetic markers.
Detecting specific polymorphic markers and/or haplotypes can be accomplished by 30 methods known in the art for detecting sequences at polymorphic sites. For example, standard techniques for genotyping for the presence of SNPs and/or microsatellite markers can be used, such as fluorescence-based techniques (Chen, X. eta/., Genome Res. 9(5)\ 492-98 (1999)), utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. Specific methodologies available for SNP genotyping include, 35 but are not limited to, TaqMan genotyping assays and SNPlex platforms (Applied Biosystems), mass spectrometry (e.g., MassARRAY system from Sequenom), mini-sequencing methods, real-time PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), Molecular Inversion Probe array technology (e.g., Affymetrix GeneChip), BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays) and
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Centaurus assay (Nanogen). By these or other methods available to the person skilled in the art, one or more alleles at polymorphic markers, including microsatellites, SNPs or other types of polymorphic markers, can be identified.
In certain methods described herein, an individual who is at an increased.
susceptibility (i.e., increased risk) for Type 2 diabetes, is an individual in whom at least one specific allele at one or more polymorphic marker or haplotype conferring increased susceptibility for Type 2 diabetes is identified (i.e., at-risk marker alleles or haplotypes). In one aspect, the at-risk marker or haplotype is one that confers a significant increased risk (or susceptibility) of Type 2 diabetes. In one embodiment, significance associated 10 with a marker or haplotype is measured by a relative risk (RR). In another embodiment, significance associated with a marker or haplotype is measured by an odds ratio (OR). In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.2, including but not limited to: at least 1.2, at least 1.3, at least 1.4, at 15 least 1.5, at least 1.6, at least 1.7, 1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, at least 4.0, and at least 5.0. In a particular embodiment, a risk (relative risk and/or odds ratio) of at least 1.2 is significant. In another particular embodiment, a risk of at least 1.3 is significant. In yet another embodiment, a risk of at least 1.4 is significant. In a further embodiment, a relative risk of at least about 1.5 is significant. In another 20 further embodiment, a significant increase in risk is at least about 1.7 is significant.
However, other cutoffs are also contemplated, e.g. at least 1.15, 1.25, 1.35, and so on, and such cutoffs are also within scope of the present invention. In other embodiments, a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 25 100%, 150%, 200%, 300%, and 500%. In one particular embodiment, a significant increase in risk is at least 20%. In other embodiments, a significant increase in risk is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and at least 100%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also 30 within scope of the present invention.
An at-risk polymorphic marker or haplotype of the present invention is one where at least one allele of at least one marker or haplotype is more frequently present in an individual at risk for the disease or trait (affected), compared to the frequency of its presence in a comparison group (control), and wherein the presence of the marker or 35 haplotype is indicative of susceptibility to the disease or trait. The control group may in one embodiment be a population sample, i.e. a random sample from the general population. In another embodiment, the control group is represented by a group of individuals who are disease-free. Such disease-free control may in one embodiment be characterized by the absence of one or more specific disease-associated symptoms. In
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another embodiment, the disease-free control group is characterized by the absence of one or more disease-specific risk factors. Such risk factors are in one embodiment at least one environmental risk factor. Representative environmental factors are natural products, minerals or other chemicals which are known to affect, or contemplated to 5 affect, the risk of developing the specific disease or trait. Other environmental risk factors are risk factors related to lifestyle, including but not limited to food and drink habits, geographical location of main habitat, and occupational risk factors. In another embodiment, the risk factors are at least one genetic risk factor.
As an example of a simple test for correlation would be a Fisher-exact test on a 10 two by two table. Given a cohort of chromosomes, the two by two table is constructed out of the number of chromosomes that include both of the markers or haplotypes, one of the markers or haplotypes but not the other and neither of the markers or haplotypes.
In other embodiments of the invention, an individual who is at a decreased susceptibility (i.e., at a decreased risk) for Type 2 diabetes is an individual in whom at least 15 one specific allele at one or more polymorphic marker or haplotype conferring decreased susceptibility for Type 2 diabetes is identified. The marker alleles and/or haplotypes conferring decreased risk are also said to be protective. In one aspect, the protective marker or haplotype is one that confers a significant decreased risk (or susceptibility) of the disease or trait. In another embodiment, the absence of an at-risk allele in a nucleic acid sample 20 from the individual is also indicative of a protection against disease, by virtue of the absence of at-risk alleles. In one embodiment, significant decreased risk is measured as a relative risk of less than 0.9, including but not limited to less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 and less than 0.1. In one particular embodiment, significant decreased risk is less than 0.7. In another 25 embodiment, significant decreased risk is less than 0.5. In yet another embodiment,
significant decreased risk is less than 0.3. In another embodiment, the decrease in risk (or susceptibility) is at least 20%, including but not limited to at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and 30 at least 98%. In one particular embodiment, a significant decrease in risk is at least about 30%. In another embodiment, a significant decrease in risk is at least about 50%. In another embodiment, the decrease in risk is at least about 70%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention.
The person skilled in the art will appreciate that for markers with two alleles present in the population being studied (such as SNPs), and wherein one allele is found in increased frequency in a group of individuals with a trait or disease in the population, compared with controls, the other allele of the marker will be found in decreased frequency in the group of
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individuals with the trait or disease, compared with controls. In such a case, one allele of the marker (the one found in increased frequency in individuals with the trait or disease) will be the at-risk allele, while the other allele will be a protective allele.
Linkage Disequilibrium
The natural phenomenon of recombination, which occurs on average once for each chromosomal pair during each meiotic event, represents one way in which nature provides variations in sequence (and biological function by consequence). It has been discovered that recombination does not occur randombly in the genome; rather, there are 10 large variations in the frequency of recombination rates, resulting in small regions of high recombination frequency (also called recombination hotspots) and larger regions of low recombination frequency, which are commonly referred to as Linkage Disequilibrium (LD) blocks (Myers, S. et al., Biochem Soc Trans 34:526-530 (2006); Jeffreys, A.J., et al.,Nature Genet 29:217-222 (2001); May, C.A., etal., Nature Genet 31:272-275(2002)).
Linkage Disequilibrium (LD) refers to a non-random assortment of two genetic elements. For example, if a particular genetic element (e.g., an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.50 (50%) and another element occurs at a frequency of 0.50 (50%), then the predicted occurrance of a person's having both elements is 0.25 (25%), assuming a random distribution of the elements. 20 However, if it is discovered that the two elements occur together at a frequency higher than 0.25, then the elements are said to be in linkage disequilibrium, since they tend to be inherited together at a higher rate than what their independent frequencies of occurrence (e.g., allele or haplotype frequencies) would predict. Roughly speaking, LD is generally correlated with the frequency of recombination events between the two 25 elements. Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurence of each allele or haplotype in the population. For populations of diploids, e.g., human populations, individuals will typically have two alleles for each genetic element (e.g., a marker, haplotype or gene).
Many different measures have been proposed for assessing the strength of linkage disequilibrium (LD). Most capture the strength of association between pairs of biallelic sites. Two important pairwise measures of LD are r2 (sometimes denoted A2) and |D'|. Both measures range from 0 (no disequilibrium) to 1 ('complete' disequilibrium), but their interpretation is slightly different. |D'| is defined in such a way that it is equal to 1 if just 35 two or three of the possible haplotypes are present, and it is <1 if all four possible haplotypes are present. Therefore, a value of |D'| that is <1 indicates that historical recombination may have occurred between two sites (recurrent mutation can also cause
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|D'| to be <1, but for single nucleotide polymorphisms (SNPs) this is usually regarded as being less likely than recombination). The measure r2 represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are present.
The r2 measure is arguably the most relevant measure for association mapping, 5 because there is a simple inverse relationship between r2 and the sample size required to detect association between susceptibility loci and SNPs. These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or 10 whether there is more or less LD in a region than predicted under a particular model). Measuring LD across a region is not straightforward, but one approach is to use the measure r, which was developed in population genetics. Roughly speaking, r measures how much recombination would be required under a particular population model to generate the LD that is seen in the data. This type of method can potentially also provide 15 a statistically rigorous approach to the problem of determining whether LD data provide evidence for the presence of recombination hotspots. For the methods, kits,procedures, media and apparati described herein, a significant r2 value can be at least 0.05, such as at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1.0. In one preferred 20 embodiment, the significant r2 value can be at least 0.2. Alternatively, linkage disequilibrium as described herein, refers to linkage disequilibrium characterized by values of | D'| of at least 0.2, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99. Thus, linkage disequilibrium represents a correlation between alleles of distinct markers. It is measured by correlation coefficient or |D'| (r2 up to 1.0 and |D'| up 25 to 1.0). In certain embodiments, linkage disequilibrium is defined in terms of values for both the r2 and |D'| measures. In one such embodiment, a significant linkage disequilibrium is defined as r2 > 0.1 and |D'| >0.8. In another embodiment, a significant linkage disequilibrium is defined as r2 > 0.2 and |D'| >0.9. Other combinations and permutations of values of r2and |D'|for determining linkage disequilibrium are also 30 possible, and within the scope of the invention. Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be determined in a collection of samples comprising individuals from more than one human population. In one embodiment of the invention, LD is determined in a sample from one or more of the HapMap populations (caucasian, african, japanese, Chinese), as defined 35 (http://www.hapmap.org). In one such embodiment, LD is determined in the CEU
population of the HapMap samples. In another embodiment, LD is determined in the YRI population. In yet another embodiment, LD is determined in samples from the Icelandic population.
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If all polymorphisms in the genome were identical at the population level, then every single one of them would need to be investigated in association studies. However, due to linkage disequilibrium between polymorphisms, tightly linked polymorphisms are strongly correlated, which reduces the number of polymorphisms that need to be 5 investigated in an association study to observe a significant association. Another consequence of LD is that many polymorphisms may give an association signal due to the fact that these polymorphisms are strongly correlated.
Genomic LD maps have been generated across the genome, and such LD maps have been proposed to serve as framework for mapping disease-genes (Risch, N. & 10 Merkiangas, K, Science 273:1516-1517 (1996); Maniatis, N., etal., Proc Natl Acad Sci USA 99:2228-2233 (2002); Reich, DE etal, Nature 411:199-204 (2001)).
It is now established that many portions of the human genome can be broken into series of discrete haplotype blocks containing a few common haplotypes; for these blocks, linkage disequilibrium data provides little evidence indicating recombination (see, e.g., 15 Wall., J.D. and Pritchard, J.K., Nature Reviews Genetics 4:587-597 (2003); Daly, M. et al., Nature Genet. 29:229-232 (2001); Gabriel, S.B. etal., Science 296:2225-2229
(2002); Patil, N. etal., Science 294:1719-1723 (2001); Dawson, E. etal., Nature 418:544-548 (2002); Phillips, M.S. etal., Nature Genet. 33:382-387 (2003)).
There are two main methods for defining these haplotype blocks: blocks can be 20 defined as regions of DNA that have limited haplotype diversity (see, e.g., Daly, M. et al., Nature Genet. 29:229-232 (2001); Patil, N. etal., Science 294:1719-1723 (2001); Dawson, E. et al., Nature 418:544-548 (2002); Zhang, K. et al., Proc. Natl. Acad. Sci. USA 99:7335-7339 (2002)), or as regions between transition zones having extensive historical recombination, identified using linkage disequilibrium (see, e.g., Gabriel, S.B. et 25 al., Science 296:2225-2229 (2002); Phillips, M.S. etal., Nature Genet. 33:382-387
(2003); Wang, N. etal., Am. J. Hum. Genet. 71:1227-1234 (2002); Stumpf, M.P., and Goldstein, D.B., Curr. Biol. 13:1-8 (2003)). More recently, a fine-scale map of recombination rates and corresponding hotspots across the human genome has been generated (Myers, S., etal., Science 310:321-32324 (2005); Myers, S. et al., Biochem
Soc Trans 34:526530 (2006)). The map reveals the enormous variation in recombination across the genome, with recombination rates as high as 10-60 cM/Mb in hotspots, while closer to 0 in intervening regions, which thus represent regions of limited haplotype diversity and high LD. The map can therefore be used to define haplotype blocks/LD blocks as regions flanked by recombination hotspots. As used herein, the terms 35 "haplotype block" or "LD block" includes blocks defined by any of the above described characteristics, or other alternative methods used by the person skilled in the art to define such regions.
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Haplotype blocks can be used to map associations between phenotype and haplotype status, using single markers or haplotypes comprising a plurality of markers. The main haplotypes can be identified in each haplotype block, and then a set of "tagging" SNPs or markers (the smallest set of SNPs or markers needed to distinguish among the 5 haplotypes) can then be identified. These tagging SNPs or markers can then be used in assessment of samples from groups of individuals, in order to identify association between phenotype and haplotype. If desired, neighboring haplotype blocks can be assessed concurrently, as there may also exist linkage disequilibrium among the haplotype blocks.
It has thus become apparent that for any given observed association to a 10 polymorphic marker in the genome, it is likely that additional markers in the genome also show association. This is a natural consequence of the uneven distribution of LD across the genome, as observed by the large variation in recombination rates. The markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait, and as such 15 are useful for use in the methods and kits of the present invention. One or more causative (functional) variants or mutations may reside within the region found to be associating to the disease or trait. Such variants may confer a higher relative risk (RR) or odds ratio (OR) than observed for the tagging markers used to detect the association. The present invention thus refers to the markers used for detecting association to the 20 disease, as described herein, as well as markers in linkage disequilibrium with the markers. Thus, in certain embodiments of the invention, markers that are in LD with the markers and/or haplotypes of the invention, as described herein, may be used as surrogate markers. The surrogate markers have in one embodiment relative risk (RR) and/or odds ratio (OR) values smaller than for the markers or haplotypes initially found to 25 be associating with the disease, as described herein. In other embodiments, the surrogate markers have RR or OR values greater than those initially determined for the markers initially found to be associating with the disease, as described herein. An example of such an embodiment would be a rare, or relatively rare (< 10% allelic population frequency) variant in LD with a more common variant (> 10% population 30 frequency) initially found to be associating with the disease, such as the variants described herein. Identifying and using such markers for detecting the association discovered by the inventors as described herein can be performed by routine methods well known to the person skilled in the art, and are therefore within the scope of the present invention.
It is possible that certain polymorphic markers in linkage disequilibrium witht the markers shown herein to be associated with Type 2 diabetes are located outside the physical boundaries of the LD block as defined. This is a consequence of the historical recombination rates in the region in question, which may have led to a region of strong LD (the LD block), with residual markers outside the block in LD with markers within the
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block. Such markers are also within scope of the present invention, as they are equally useful for practicing the invention by virtue of their genetic relationship with the markers shown herein to be associated with Type 2 diabetes. Examples are shown in Table 22 (rsl7234378; SEQ ID NO:44), Table 23 (rs7086285; SEQ ID NO:43) and Table 24 5 (rs9890889; SEQ ID NO:31; rs2009802; SEQ ID NO:38; rsl7718938; SEQ ID NO:39; , rs2109050; SEQ ID NO:41; rsl962801; SEQ ID NO:42.
Determination of haplotype frequency
The frequencies of haplotypes in patient and control groups can be estimated 10 using an expectation-maximization algorithm (Dempster A. et at., J. R. Stat. Soc. B, 39:1-38 (1977)). An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase can be used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies. Using a likelihood approach, an alternative hypothesis is tested, where a candidate at-risk-haplotype, which can include 15 the markers described herein, is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups. Likelihoods are maximized separately under both hypotheses and a corresponding 1-df likelihood ratio statistic is used to evaluate the statistical significance.
To look for at-risk and protective markers and haplotypes within a region of 20 interest, for example, association of all possible combinations of genotyped markers is studied, provided those markers span a practical region. The combined patient and control groups can be randomly divided into two sets, equal in size to the original group of patients and controls. The marker and haplotype analysis is then repeated and the most significant p-value registered is determined. This randomization scheme can be repeated, 25 for example, over 100 times to construct an empirical distribution of p-values. In a preferred embodiment, a p-value of <0.05 is indicative of a significant marker and/or haplotype association.
Haplotype Analysis
One general approach to haplotype analysis involves using likelihood-based inference applied to NEsted MOdels (Gretarsdottir S., eta/., Nat. Genet. 35:131-38 (2003)). The method is implemented in the program NEMO, which allows for many polymorphic markers, SNPs and microsatellites. The method and software are specifically designed for case-control studies where the purpose is to identify haplotype groups that 35 confer different risks. It is also a tool for studying LD structures. In NEMO, maximum
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likelihood estimates, likelihood ratios and p-values are calculated directly, with the aid of the EM algorithm, for the observed data treating it as a missing-data problem.
Even though likelihood ratio tests based on likelihoods computed directly for the observed data, which have captured the information loss due to uncertainty in phase and 5 missing genotypes, can be relied on to give valid p-values, it would still be of interest to know how much information had been lost due to the information being incomplete. The information measure for haplotype analysis is described in Nicolae and Kong (Technical Report 537, Department of Statistics, University of Statistics, University of Chicago; Biometrics, 60(2):368-75 (2004)) as a natural extension of information measures defined 10 for linkage analysis, and is implemented in NEMO.
For single marker association to a disease or trait (e.g., Type 2 diabetes), the Fisher exact test can be used to calculate two-sided p-values for each individual allele. Usually, all p-values are presented unadjusted for multiple comparisons unless specifically indicated. The presented frequencies (for microsatellites, SNPs and haplotypes) are allelic frequencies as opposed to carrier frequencies. To minimize any bias due the relatedness of the patients who were recruited as families for the linkage analysis, first and second-degree relatives can be eliminated from the patient list. Furthermore, the test can be repeated for association correcting for any remaining relatedness among the patients, by extending a variance adjustment procedure described in Risch, N. &Teng, J. (Genome Res., 8:1273-1288 (1998)), DNA pooling (ibid) for sibships so that it can be applied to general familial relationships, and present both adjusted and unadjusted p-values for comparison. The differences are in general very small as expected. To assess the significance of single-marker association corrected for multiple testing we can carry out a randomization test using the same genotype data. Cohorts of patients and controls can be randomized and the association analysis redone multiple times (e.g., up to 500,000 times) and the p-value is the fraction of replications that produced a p-value for some marker allele that is lower than or equal to the p-value we observed using the original patient and control cohorts.
For both single-marker and haplotype analyses, relative risk (RR) and the 30 population attributable risk (PAR) can be calculated assuming a multiplicative model
(haplotype relative risk model) (Terwilliger, J.D. & Ott, J., Hum. Hered. 42:337-46 (1992) and Falk, C.T. & Rubinstein, P, Ann. Hum. Genet. 51 (Pt 3):227-33 (1987)), i.e., that the risks of the two alleles/haplotypes a person carries multiply. For example, if RR is the risk of A relative to a, then the risk of a person homozygote AA will be RR times that of a 35 heterozygote Aa and RR2 times that of a homozygote aa. The multiplicative model has a nice property that simplifies analysis and computations— haplotypes are independent, i.e., in Hardy-Weinberg equilibrium, within the affected population as well as within the control population. As a consequence, haplotype counts of the affecteds and controls
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each have multinomial distributions, but with different haplotype frequencies under the alternative hypothesis. Specifically, for two haplotypes, h, and hjt risk(/7,)/risk(/j;) = (fi/Pi)/(fj/Pj), where fand p denote, respectively, frequencies in the affected population and in the control population. While there is some power loss if the true model is not 5 multiplicative, the loss tends to be mild except for extreme cases. Most importantly, p-values are always valid since they are computed with respect to null hypothesis.
Risk assessment and Diagnostics
As described herein, certain polymorphic markers and haplotypes comprising such 10 markers are found to be useful for risk assessment of Type 2 diabetes. Risk assessment can involve the use of the markers for diagnosing a susceptibility to Type 2 diabetes. Particular alleles of polymorphic markers are found more frequently in individuals with Type 2 diabetes, than in individuals without diagnosis of Type 2 diabetes. Therefore, these marker alleles have predictive value for detecting Type 2 diabetes, or a 15 susceptibility to Type 2 diabetes, in an individual. Tagging markers within haplotype blocks or LD blocks comprising at-risk markers, such as the markers of the present invention, can be used as surrogates for other markers and/or haplotypes within the haplotype block or LD block. Markers with values of r2 equal to 1 are perfect surrogates for the at-risk variants, i.e. genotypes for one marker perfectly predicts genotypes for the 20 other. Markers with smaller values of r2 than 1 can also be surrogates for the at-risk variant, or alternatively represent variants with relative risk values as high as or possibly even higher than the at-risk variant.
The at-risk variant identified may not be the functional variant itself, but is in this instance in linkage disequilibrium with the true functional variant. The present invention 25 encompasses the assessment of such surrogate markers for the markers as disclosed herein. Such markers are annotated, mapped and listed in public databases (e.g., dbSNP), as well known to the skilled person, or can alternatively be readily identified by sequencing the region or a part of the region identified by the markers of the present invention in a group of individuals, and identify polymorphisms in the resulting group of 30 sequences. As a consequence, the person skilled in the art can readily and without undue experimentation genotype surrogate markers in linkage disequilibrium with the markers and/or haplotypes as described herein. The tagging or surrogate markers in LD with the at-risk variants detected, also have predictive value for detecting association to Type 2 diabetes, or a susceptibility to Type 2 diabetes, in an individual.
The markers and haplotypes as described herein, e.g., the markers presented in
Tables 1 - 24, may be useful for risk assessment and diagnostic purposes for, either alone or in combination. The markers and haplotypes can also be combined with other markers
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conferring increased risk for Type 2 diabetes. Even in cases where the increase in risk by individual markers is relatively modest, i.e. on the order of 10-30%, the association may have significant implications. Thus, relatively common variants may have significant contribution to the overall risk (Population Attributable Risk is high), or combination of 5 markers can be used to define groups of individual who, based on the combined risk of the markers, is at significant combined risk of developing the disease. The markers described herein to be associated with Type 2 diabetes can therefore be combined with other polymorphic markers or haplotypes reported or found to be associated with Type 2 diabetes, so as to obtain an overall risk of the disease based on a plurality of genetic 10 markers.
In one such embodiment, the polymorphic markers or haplotypes described herein are assessed together with information about markers within the TCF7L2 gene.
Association of variants within this gene is well established (Grant S.F., et al., Nat Genet. -38:320-3 (2006)) and has been replicated in a large number of populations (Florez, J.C., 15 Curr Opin Clin Nutr Metabol Care 10:391-396 (2007). The marker rs7903146 within the TCF7L2 gene, or other markers in LD with the marker (e.g., rsl2255372) can be used to determine the genetic risk conferred by the at-risk variant in the gene (OR about 1.44).
Markers in other genes have recently been implicated in the etiology of Type 2 diabetes as risk factors, including PPARG (rsl801282), KCNJ11 (rs5215), TCF2 20 (rs4430796), WFS1 (rsl0010131), CDKN2A-2B (rsl081161), IGF2BP2 (rs4402960) and FTO (rs805136) (Frayling, T.M. Nature Reviews Genetics 8:657-662 (2007).. These markers, or markers in linkage disequilibrium therewith can likewise also be used in methods combining determination of the presence or absence of at-risk variants for Type 2 diabetes with the variants reported herein, so as to obtain an overall risk assessment of 25 Type 2 diabetes.
Thus, in one embodiment of the invention, a plurality of variants (genetic markers and/or biomarkers and/or haplotypes) is used for overall risk assessment. These variants are in one embodiment selected from the variants as disclosed herein. Other embodiments include the use of the variants of the present invention in combination with 30 other variants known to be useful for diagnosing a susceptibility to Type 2 diabetes. In such embodiments, the genotype status of a plurality of markers and/or haplotypes is determined in an individual, and the status of the individual compared with the population frequency of the associated variants, or the frequency of the variants in clinically healthy subjects, such as age-matched and sex-matched subjects. Methods known in the art, 35 such as multivariate analyses or joint risk analyses, may subsequently be used to determine the overall risk conferred based on the genotype status at the multiple loci. Assessment of risk based on such analysis may subsequently be used in the methods and kits of the invention, as described herein.
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As described in the above, the haplotype block structure of the human genome has the effect that a large number of variants (markers and/or haplotypes) in linkage disequilibrium with the variant originally associated with a disease or trait may be used as surrogate markers for assessing association to the disease or trait. The number of such 5 surrogate markers will depend on factors such as the historical recombination rate in the region, the mutational frequency in the region (i.e., the number of polymorphic sites or markers in the region), and the extent of LD (size of the LD block) in the region. These markers are usually located within the physical boundaries of the LD block or haplotype block in question as defined using the methods described herein, or by other methods 10 known to the person skilled in the art. However, sometimes marker and haplotype association is found to extend beyond the physical boundaries of the haplotype block as defined. Such markers and/or haplotypes may in those cases be also used as surrogate markers and/or haplotypes for the markers and/or haplotypes physically residing within the haplotype block as defined. As a consequence, markers and haplotypes in LD 15 (typically characterized by r2 greater than 0.1, such as r2 greater than 0.2, including r2 greater than 0.3, also including r2 greater than 0.4) with the markers and haplotypes of the present invention are also within the scope of the invention, even if they are physically located beyond the boundaries of the haplotype block as defined. This includes markers that are described herein (e.g., markers listed in Tables 22, 23 and 24), but may 20 also include other markers that are in linkage disequilibrium (e.g., characterized by r2 greater than 0.2 and/or |D'| > 0.8) with one or more of the markers listed in Tables 22, 23 and 24.
For the SNP markers described herein, the opposite allele to the allele found to be in excess in patients (at-risk allele) is found in decreased frequency in Type 2 diabetes. 25 These markers and haplotypes in LD and/or comprising such markers, are thus protective for Type 2 diabetes, i.e. they confer a decreased risk or susceptibility of individuals carrying these markers and/or haplotypes developing Type 2 diabetes. Alternatively speaking, the absence of at-risk alleles of at-risk variants implies the presence of the alternate allele for biallelic markers such as SNPs. Thus, the absence of at-risk variants 30 as described herein is indicative of a protection against Type 2 diabetes.
As described herein, haplotypes comprising a combination of genetic markers, e.g., SNPs and microsatellites, can be useful for risk assessment. Detecting haplotypes can be accomplished by methods known in the art and/or described herein for detecting sequences at polymorphic sites. Furthermore, correlation between certain haplotypes or 35 sets of markers and disease phenotype can be verified using standard techniques. A
representative example of a simple test for correlation would be a Fisher-exact test on a two by two table.
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In specific embodiments, a marker or haplotype found to be associated with Type 2 diabetes, is one in which a marker or haplotype is more frequently present in an individual at risk for Type 2 diabetes (e.g., an affected person), compared to the frequency of its presence in a healthy individual (control) or in a randomly selected 5 individual from the population (population control), wherein the presence of the marker allele or haplotype is indicative of Type 2 diabetes or a susceptibility to Type 2 diabetes. In other embodiments, at-risk markers in linkage disequilibrium with one or more markers found to be associated with Type 2 diabetes are tagging markers that are more frequently present in an individual at risk for Type 2 diabetes (e.g., affected individuals), 10 compared to the frequency of their presence in controls, wherein the presence of the tagging markers is indicative of increased susceptibility to Type 2 diabetes. In a further embodiment, at-risk markers alleles (i.e. conferring increased susceptibility) in linkage disequilibrium with one or more markers found to be associated with Type 2 diabetes are markers comprising one or more allele that is more frequently present in an individual at 15 risk for Type 2 diabetes, compared to the frequency of their presence in controls, wherein the presence of the markers is indicative of increased susceptibility to Type 2 diabetes.
Study population
In a general sense, the methods and kits of the invention can be utilized from 20 samples containing genomic DNA from any source, i.e. any individual. In preferred embodiments, the individual is a human individual. The individual can be an adult, child, or fetus. The present invention also provides for assessing markers and/or haplotypes in individuals who are members of a target population. Such a target population is in one embodiment a population or group of individuals at risk of developing the disease, based 25 on other genetic factors, biomarkers, biophysical parameters (e.g., weight, BMD, blood pressure), or general health and/or lifestyle parameters (e.g., history of disease or related diseases, previous diagnosis of disease, family history of disease).
The invention provides for embodiments that include individuals from specific age subgroups, such as those over the age of 40, over age of 45, or over age of 50, 55, 60, 30 65, 70, 75, 80, or 85. Other embodiments of the invention pertain to other age groups, such as individuals aged less than 85, such as less than age 80, less than age 75, or less than age 70, 65, 60, 55, 50, 45, 40, 35, or age 30. Other embodiments relate to individuals with age at onset of the disease in any of the age ranges described in the above. It is also contemplated that a range of ages may be relevant in certain 35 embodiments, such as age at onset at more than age 45 but less than age 60. Other age ranges are however also contemplated, including all age ranges bracketed by the age
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values listed in the above. The invention furthermore relates to individuals of either gender, males or females.
The Icelandic population is a Caucasian population of Northern European ancestry. A large number of studies reporting results of genetic linkage and association in the 5 Icelandic population have been published in the last few years. Many of those studies show replication of variants, originally identified in the Icelandic population as being associating with a particular disease, in other populations (Stacey, S.N., et al., Nat Genet. May 27 2007 (Epub ahead of print; Helgadottir, A., et al., Science 316:1491-93 (2007); Steinthorsdottir, V., etal., Nat Genet. 39:770-75 (2007); Gudmundsson, J., etal., Nat 10 Genet. 39:631-37 (2007); Amundadottir, L.T., etal., Nat Genet. 38:652-58 (2006);
Grant, S.F., et al., Nat Genet. 38:320-23 (2006)). Thus, genetic findings in the Icelandic population have in general been replicated in other populations, including populations from Africa and Asia. The variants described herein to be associated to the CDKAL gene, in particular the LD Block C06 (SEQ ID NO:l) have been replicated in several populations 15 of European, American, and Chinese (Hong Kong) origin. This supports the belief that these variants (rs7756992 and markers in linkage disequilibrium therewith) are at-risk variants for Type 2 diabetes in most populations.
Particular embodiments comprising individual human populations are thus also contemplated and within the scope of the present invention. Such embodiments relate to human subjects that are from one or more human population including, but not limited to, Caucasian populations, European populations, American populations, Eurasian populations, Asian populations, Central/South Asian populations, East Asian populations, Middle Eastern populations, African populations, Hispanic populations, and Oceanian populations. European populations include, but are not limited to, Swedish, Norwegian, Finnish, Russian, Danish, Icelandic, Irish, Kelt, English, Scottish, Dutch, Belgian, French, German, Spanish, Portuguese, Italian, Polish, Bulgarian, Slavic, Serbian, Bosnian, Czech, Greek and Turkish populations. The invention furthermore in other embodiments can be practiced in specific human populations that include Bantu, Mandenk, Yoruba, San, Mbuti Pygmy, Orcadian, Adygei, Russian, Sardinian, Tuscan, Mozabite, Bedouin, Druze, Palestinian, Balochi, Brahui, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash, Han, Dai, Daur, Hezhen, Lahu, Miao, Oroqen, She, Tujia, Tu, Xibo, Yi, Mongolan, Naxi, Cambodian, Japanese, Yakut, Melanesian, Papuan, Karitianan, Surui, Columbian, Maya and Pima.
In one preferred embodiment, the invention relates to populations that include 35 black African ancestry such as populations comprising persons of African descent or lineage. Black African ancestry may be determined by self reporting as African-Americans, Afro-Americans, Black Americans, being a member of the black race or being a member of the negro race. For example, African Americans or Black Americans are
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those persons living in North America and having origins in any of the black racial groups of Africa. In another example, self-reported persons of black African ancestry may have at least one parent of black African ancestry or at least one grandparent of black African ancestry.
The racial contribution in individual subjects may also be determined by genetic analysis. Genetic analysis of ancestry may be carried out using unlinked microsatellite markers such as those set out in Smith et al. (Am J Hum Genet 74, 1001-13 (2004)).
In certain embodiments, the invention relates to markers and/or haplotypes identified in specific populations, as described in the above. The person skilled in the art 10 will appreciate that measures of linkage disequilibrium (LD) may give different results when applied to different populations. This is due to different population history of different human populations as well as differential selective pressures that may have led to differences in LD in specific genomic regions. It is also well known to the person skilled in the art that certain markers, e.g. SNP markers, have different population frequency in 15 different populations, or are polymorphic in one population but not in another. The person skilled in the art will however apply the methods available and as thought herein to practice the present invention in any given human population. This may include assessment of polymorphic markers in the LD region of the present invention, so as to identify those markers that give strongest association within the specific population. 20 Thus, the at-risk variants of the present invention may reside on different haplotype background and in different frequencies in various human populations. However, utilizing methods known in the art and the markers of the present invention, the invention can be practiced in any given human population.
Utility of Genetic Testing
The knowledge about a genetic variant that confers a risk of developing Type 2 diabetes offers the opportunity to apply a genetic test to distinguish between individuals with increased risk of developing the disease (i.e. carriers of the at-risk variant) and those with decreased risk of developing the disease (i.e. carriers of the protective variant). The 30 core values of genetic testing, for individuals belonging to both of the above mentioned groups, are the possibilities of being able to diagnose the disease at an early stage and provide information to the clinician about prognosis/aggressiveness of the disease in order to be able to apply the most appropriate treatment.
For example, the application of a genetic test for Type 2 diabetes can identify high risk individuals among people with impaired fasting glucose (IFG) or impaired glucose tolerance (IGT). It is well established that while around a third of people who are found to
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have IFG/IGT develop Type 2 diabetes, glucose levels return to normal for an equal proportion of individuals. Identification of individuals within this group that are carriers of genetic risk variants will allow targeting of those individuals by preventive measures. For example, these individuals may benefit from a closer monitoring of blood glucose levels to 5 aid in early diagnosis. They may also need more stringent lifestyle intervention advice since individuals with certain genetic risk factors develop Type 2 diabetes at lower BMI levels than those without those factors.
Individuals with a family history of Type 2 diabetes and carriers of at-risk variants may benefit from genetic testing since the knowledge of the presence of a genetic risk 10 factor, or evidence for increased risk of being a carrier of one or more risk factors, may provide increased incentive for implementing a healthier lifestyle. Furthermore, closer monitoring of glucose levels should be advised for such individuals, facilitating early diagnosis and/or preventative treatment.
Genetic testing of Type 2 diabetes patients may furthermore give valuable 15 information about the primary cause of the disease and can aid the clinician in selecting the best treatment options and medication for each individual. For instance, patients with genetic risk factors for reduced insulin secretion may be likely to benefit from medication increasing insulin secretion while increasing insulin sensitivity in those individuals may be less effective.
METHODS OF THE INVENTION
Methods for risk assessment of Type 2 diabetes are described herein and are encompassed by the invention. The invention also encompasses methods of assessing an individual for probability of response to a therapeutic agent for Type 2 diabetes, as well as 25 methods for predicting the effectiveness of a therapeutic agent for Type 2 diabetes. Kits for assaying a sample from a subject to detect susceptibility to Type 2 diabetes are also encompassed by the invention.
DIAGNOSTIC AND SCREENING ASSAYS OF THE INVENTION
In certain embodiments, the present invention pertains to methods of assessing risk or diagnosing, or aiding in risk assessment or diagnosis of, Type 2 diabetes or a susceptibility to Type 2 diabetes, by detecting particular alleles at genetic markers that appear more frequently in Type 2 diabetes subjects or subjects who are susceptible to Type 2 diabetes. In a particular embodiment, the invention is a method of assessing 35 susceptibility to Type 2 diabetes by detecting at least one allele of at least one
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polymorphic marker (e.g., the markers described herein). The present invention describes methods whereby detection of particular alleles of particular markers or haplotypes is indicative of a susceptibility to Type 2 diabetes. Such prognostic or predictive assays can also be used to determine prophylactic treatment of a subject prior 5 to the onset of symptoms of Type 2 diabetes.
The present invention pertains in some embodiments to methods of clinical applications of diagnosis, e.g., diagnosis performed by a medical professional, which may include an assessment or determination of genetic risk variants. In other embodiments, the invention pertains to methods of risk assessment (or diagnosis) performed by a 10 layman. Recent technological advances in genotyping technologies, including high-throughput genotyping of SNP markers, such as Molecular Inversion Probe array technology (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays) have made it possible for individuals to have their own genome assessed for up to one million SNPs. The resulting genotype information, made 15 available to the individual can be compared to information from the public literature about disease or trait risk associated with various SNPs. The diagnostic application of disease-associated alleles as described herein, can thus be performed either by a health professional based on results of a clinical test or by a layman, including an individual providing service for performing an whole-genome assessment of SNPs. In other words, 20 the diagnosis or assessment of a susceptibility based on genetic risk can be made by health professionals, genetic counselors, genotype services providers or by the layman, based on information about his/her genotype and publications on various risk factors. In the present context, the term "diagnosing", and "diagnose a susceptibility", is meant to refer to any available diagnostic method, including those mentioned above.
In addition, in certain other embodiments, the present invention pertains to methods of diagnosing, or aiding in the diagnosis of, a decreased susceptibility to Type 2 diabetes, by detecting particular genetic marker alleles or haplotypes that appear less frequently in Type 2 diabetes patients than in individual not diagnosed with Type 2 diabetes or in the general population.
As described and exemplified herein, particular marker alleles or haplotypes (e.g.
the markers and haplotypes as listed in Tables 1-24, e.g., the markers and haplotypes as listed in Tables 1-6 and Tables 9-12, and markers in linkage disequilibrium therewith) are associated with Type 2 diabetes. In one embodiment, the marker allele or haplotype is one that confers a significant risk or susceptibility to Type 2 diabetes. In another 35 embodiment, the invention relates to a method of diagnosing a susceptibility to Type 2 diabetes in a human individual, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected
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from the group consisting of the polymorphic markers listed in Table 9, Table 10, Table 11, and Table 12, and markers in linkage disequilibrium (defined as r2 > 0.2) therewith. In another embodiment, the invention pertains to methods of diagnosing or assessing a susceptibility to Type 2 diabetes in a human individual, by screening for at least one 5 marker allele or haplotype as listed in Tables 1-6 and 9 - 12, or markers in linkage disequilibrium therewith. In another embodiment, the marker allele or haplotype is more frequently present in a subject having, or who is susceptible to, Type 2 diabetes (affected), as compared to the frequency of its presence in a healthy subject (control,
such as population controls). In certain embodiments, the significance of association of 10 the at least one marker allele or haplotype is characterized by a p value < 0.05. In other embodiments, the significance of association is characterized by smaller p-values, such as < 0.01, <0.001, <0.0001, <0.00001, <0.000001, <0.0000001, <0.00000001 or <0.000000001.
In these embodiments, the presence of the at least one marker allele or 15 haplotype is indicative of a susceptibility to Type 2 diabetes. These diagnostic methods involve detecting the presence or absence of at least one marker allele or haplotype that is associated with Type 2 diabetes. The haplotypes described herein include combinations of alleles at various genetic markers (e.g., SNPs, microsatellites). The detection of the particular genetic marker alleles that make up the particular haplotypes can be performed 20 by a variety of methods described herein and/or known in the art. For example, genetic markers can be detected at the nucleic acid level (e.g., by direct nucleotide sequencing or by other means known to the skilled in the art) or at the amino acid level if the genetic marker affects the coding sequence of a protein encoded by a Type 2 diabetes -associated nucleic acid (e.g., by protein sequencing or by immunoassays using antibodies that 25 recognize such a protein). The marker alleles or haplotypes of the present invention correspond to fragments of a genomic DNA sequence associated with Type 2 diabetes. Such fragments encompass the DNA sequence of the polymorphic marker or haplotype in question, but may also include DNA segments in strong LD (linkage disequilibrium) with the marker or haplotype (e.g., as determined by a value of ^greater than 0.2 and/or |D'| 30 > 0.8).
In one embodiment, diagnosis or assessment of a susceptibility to Type 2 diabetes can be accomplished using hybridization methods, such as Southern analysis, Northern analysis, and/or in situ hybridizations (see Current Protocols in Molecular Biology,
Ausubel, F. et ai., eds., John Wiley & Sons, including all supplements). The presence of a 35 specific marker allele can be indicated by sequence-specific hybridization of a nucleic acid probe specific for the particular allele. The presence of more than specific marker allele or a specific haplotype can be indicated by using several sequence-specific nucleic acid probes, each being specific for a particular allele. In one embodiment, a haplotype can be indicated by a single nucleic acid probe that is specific for the specific haplotype (i.e.,
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hybridizes specifically to a DNA strand comprising the specific marker alleles characteristic of the haplotype). A sequence-specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA. A "nucleic acid probe", as used-tierein, can be a DNA probe or an RNA probe that hybridizes to a complementary sequence. One of skill in the art would 5 know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample.
To diagnose a susceptibility to Type 2 diabetes, a hybridization sample is formed by contacting the test sample containing an Type 2 diabetes -associated nucleic acid, such as a genomic DNA sample, with at least one nucleic acid probe. A non-limiting example of 10 a probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe that is capable of hybridizing to mRNA or genomic DNA sequences described herein. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length that is sufficient to specifically hybridize under stringent conditions to appropriate mRNA or 15 genomic DNA. For example, the nucleic acid probe can comprise all or a portion of the nucleotide sequence of LD Block C06 (SEQ ID NO:l), LD Block C10 (SEQ ID NO:2) (e.g., the nucleotide sequence encoding the IDE, KIFll and/or the HHEX genes), LD Block C17 (SEQ ID NO:3) or the CDKAL1 gene, or the SLC30A8 gene, as described herein, optionally comprising at least one allele of a marker described herein, or at least one haplotype 20 described herein, or the probe can be the complementary sequence of such a sequence. In a particular embodiment, the nucleic acid probe is a portion of the nucleotide sequence of LD Block C06 (SEQ ID NO:l), LD Block C10 (SEQ ID NO:2) (e.g., the nucleotide sequence encoding the IDE, KIFll and/or the HHEX genes), LD Block C17 (SEQ ID NO:3) or the CDKAL1 gene, or the SLC30A8 gene as described herein, optionally comprising at 25 least one allele of a marker described herein , or at least one allele contained in the haplotypes described herein, or the probe can be the complementary sequence of such a sequence. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization can be performed by methods well known to the person skilled in the art (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. eta/., eds., 30 John Wiley & Sons, including all supplements). In one embodiment, hybridization refers to specific hybridization, i.e., hybridization with no mismatches (exact hybridization). In one embodiment, the hybridization conditions for specific hybridization are high stringency.
Specific hybridization, if present, is detected using standard methods. If specific 35 hybridization occurs between the nucleic acid probe and the nucleic acid in the test sample, then the sample contains the allele that is complementary to the nucleotide that is present in the nucleic acid probe. The process can be repeated for any markers of the present invention, or markers that make up a haplotype of the present invention, or multiple probes can be used concurrently to detect more than one marker alleles at a
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time. It is also possible to design a single probe containing more than one marker alleles of a particular haplotype (e.g., a probe containing alleles complementary to 2, 3, 4, 5 or all of the markers that make up a particular haplotype). Detection of the particular markers of the haplotype in the sample is indicative that the source of the sample has the 5 particular haplotype (e.g., a haplotype) and therefore is susceptible to DISEASE.
In one preferred embodiment, a method utilizing a detection oligonucleotide probe comprising a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is employed, as described by Kutyavin et at. (Nucleic Acid Res. 34:el28 (2006)). The fluorescent moiety can be Gig Harbor Green or 10 Yakima Yellow, or other suitable fluorescent moieties. The detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected. Preferably, the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are 15 designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template. The gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV. The enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch. Thus, by measuring the fluorescence of 20 the released fluorescent moiety, assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.
The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another 25 embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art. In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.
Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G. The use of modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions 35 containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C
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base in a double stranded DNA molecule. In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.
In another hybridization method, Northern analysis (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, supra) is used to identify the presence of a polymorphism associated with Type 2 diabetes. For Northern analysis, a test sample of RNA is obtained from the subject by appropriate means. As described herein, specific hybridization of a nucleic acid probe to RNA from the subject is indicative of a particular allele complementary to the probe. For representative examples of use of nucleic acid probes, see, for example, U.S. Patent Nos. 5,288,611 and 4,851,330.
Additionally, or alternatively, a peptide nucleic acid (PNA) probe can be used in addition to, or instead of, a nucleic acid probe in the hybridization methods described herein. A PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P., eta/., Bioconjug. Chem. 5:3-7 (1994)). The PNA probe can be designed to specifically hybridize to a molecule in a sample suspected of containing one or more of the marker alleles or haplotypes that are associated with Type 2 diabetes. Hybridization of the PNA probe is thus diagnostic for Type 2 diabetes or a susceptibility to Type 2 diabetes.
In one embodiment of the methods of the invention, diagnosis of Type 2 diabetes or a susceptibility to Type 2 diabetes is accomplished through enzymatic amplification of a nucleic acid from the subject. For example, a test sample containing genomic DNA can be obtained from the subject and the polymerase chain reaction (PCR) can be used to amplify a fragment comprising one ore more markers or haplotypes of the present invention found to be associated with Type 2 diabetes. As described herein, identification of a particular marker allele or haplotype associated with Type 2 diabetes can be accomplished using a variety of methods (e.g., sequence analysis, analysis by restriction digestion, specific hybridization, single stranded conformation polymorphism assays (SSCP), electrophoretic analysis, etc.). In another embodiment, diagnosis is accomplished by expression analysis using quantitative PCR (kinetic thermal cycling).
This technique can, for example, utilize commercially available technologies, such as TaqMan® (Applied Biosystems, Foster City, CA), to allow the identification of polymorphisms and haplotypes. The technique can assess the presence of an alteration in the expression or composition of a polypeptide or splicing variant(s) that is encoded by a Type 2 diabetes-associated nucleic acid. Further, the expression of the variant(s) can be quantified as physically or functionally different.
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In another embodiment of the methods of the invention, analysis by restriction digestion can be used to detect a particular allele if the allele results in the creation or elimination of a restriction site relative to a reference sequence. A test sample containing genomic DNA is obtained from the subject. PCR can be used to amplify particular regions 5 that are associated with Type 2 diabetes (e.g. the polymorphic markers and haplotypes of Tables 1-21, e.g., the polymorphic markers and haplotypes of Tables 1-6 and Tables 9-12, and markers in linkage disequilibrium therewith) nucleic acid in the test sample from the test subject. Restriction fragment length polymorphism (RFLP) analysis can be conducted, e.g., as described in Current Protocols in Molecular Biology, supra. The 10 digestion pattern of the relevant DNA fragment indicates the presence or absence of the particular allele in the sample.
Sequence analysis can also be used to detect specific alleles at polymorphic sites associated with Type 2 diabetes (e.g. the polymorphic markers and haplotypes of Tables 1-24, e.g., the polymorphic markers and haplotypes of Tables 1-6 and Tables 9-12, and 15 markers in linkage disequilibrium therewithe, e.g., the markers set forth in Tables 22, 23 and 24). Therefore, in one embodiment, determination of the presence or absence of a particular marker alleles or haplotypes comprises sequence analysis. For example, a test sample of DNA or RNA can be obtained from the test subject. PCR or other appropriate methods can be used to amplify a portion of a Type 2 diabetes-associated nucleic acid, 20 and the presence of a specific allele can then be detected directly by sequencing the polymorphic site (or multiple polymorphic sites) of the genomic DNA in the sample.
Allele-specific oligonucleotides can also be used to detect the presence of a particular allele at a Type 2 diabetes-associated nucleic acid (e.g. the polymorphic markers and haplotypes of Tables 1-21, e.g., the polymorphic markers and haplotypes of 25 Tables 1-6 and Tables 9-12, and markers in linkage disequilibrium therewith), through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, Saiki, R. etal., Nature, 324:163-166 (1986)). An "allele-specific oligonucleotide" (also referred to herein as an "allele-specific oligonucleotide probe") is an oligonucleotide of approximately 10-50 base pairs or 30 approximately 15-30 base pairs, that specifically hybridizes to a Type 2 diabetes-
associated nucleic acid, and which contains a specific allele at a polymorphic site (e.g., a polymorphism described herein). An allele-specific oligonucleotide probe that is specific for one or more particular a Type 2 diabetes-associated nucleic acid can be prepared using standard methods (see, e.g., Current Protocols in Molecular Biology, supra). PCR 35 can be used to amplify the desired region a Type 2 diabetes-associated nucleic acid. The DNA containing the amplified region can be dot-blotted using standard methods (see, e.g., Current Protocols in Molecular Biology, supra), and the blot can be contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified region can then be detected. Specific hybridization of an allele-specific
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oligonucleotide probe to DNA from the subject is indicative of a specific allele at a polymorphic site associated with Type 2 diabetes (see, e.g., Gibbs, R. et al., Nucleic Acids Res., 17:2437-2448 (1989) and WO 93/22456).
In another embodiment, arrays of oligonucleotide probes that are complementary 5 to target nucleic acid sequence segments from a subject, can be used to identify polymorphisms in a Type 2 diabetes-associated nucleic acid (e.g. the polymorphic markers and haplotypes of Tables 1-24, e.g. the polymorphic markers and haplotypes of Tables 1-6 and Tables 9-12, and markers in linkage disequilibrium therewith). For example, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise 10 a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also described as "Genechips™," have been generally described in the art (see, e.g., U.S. Patent No. 5,143,854, PCT Patent Publication Nos. WO 90/15070 and 92/10092). These arrays can generally be produced using mechanical synthesis methods or light directed synthesis 15 methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods (Fodor, S. eta/., Science, 251:767-773 (1991); Pirrung et al., U.S. Patent No. 5,143,854 (see also published PCT Application No. WO 90/15070); and Fodor. S. et al., published PCT Application No. WO 92/10092 and U.S. Patent No. 5,424,186, the entire teachings of each of which are incorporated by reference herein). 20 Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Patent No. 5,384,261; the entire teachings of which are incorporated by reference herein. In another example, linear arrays can be utilized.
Additional descriptions of use of oligonucleotide arrays for detection of polymorphisms can be found, for example, in U.S. Patent Nos. 5,858,659 and 5,837,832, 25 the entire teachings of both of which are incorporated by reference herein. Other methods of nucleic acid analysis can be used to detect a particular allele at a polymorphic site associated with Type 2 diabetes (e.g. the polymorphic markers and haplotypes of Tables 1-24, e.g. the polymorphic markers and haplotypes of Tables 1-6 and Tables 9-12, and markers in linkage disequilibrium therewith). Representative methods include, for 30 example, direct manual sequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA, 81: 1991-1995 (1988); Sanger, F., etal., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977); Beavis, et al., U.S. Patent No. 5,288,644); automated fluorescent sequencing; single-stranded conformation polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield, V., et 35 al., Proc. Natl. Acad. Sci. USA, 86:232-236 (1989)), mobility shift analysis (Orita, M., et al., Proc. Natl. Acad. Sci. USA, 86:2766-2770 (1989)), restriction enzyme analysis (Flavell, R., etal., Cell, 15:25-41 (1978); Geever, R., eta/., Proc. Natl. Acad. Sci. USA, 78:5081-5085 (1981)); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton, R., etal., Proc. Natl. Acad. Sci. USA, 85:4397-4401 (1985)); RNase protection
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assays (Myers, R., eta/., Science, 230:1242-1246 (1985); use of polypeptides that recognize nucleotide mismatches, such as E. coli mutS protein; and allele-specific PCR.
In another embodiment of the invention, diagnosis of Type 2 diabetes or a susceptibility to Type 2 diabetes can be made by examining expression and/or 5 composition of a polypeptide encoded by Type 2 diabetes-associated nucleic acid in those instances where the genetic marker(s) or haplotype(s) of the present invention result in a change in the composition or expression of the polypeptide. Thus, diagnosis of a susceptibility to Type 2 diabetes can be made by examining expression and/or composition of one of these polypeptides, or another polypeptide encoded by a Type 2 10 diabetes-associated nucleic acid, in those instances where the genetic marker or haplotype of the present invention results in a change in the composition or expression of the polypeptide. The haplotypes and markers of the present invention that show association to Type 2 diabetes may play a role through their effect on one or more of these nearby genes. Possible mechanisms affecting these genes include, e.g., effects on 15 transcription, effects on RNA splicing, alterations in relative amounts of alternative splice forms of mRNA, effects on RNA stability, effects on transport from the nucleus to cytoplasm, and effects on the efficiency and accuracy of translation.
A variety of methods can be used to make such a detection, including enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitation and 20 immunofluorescence. A test sample from a subject is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a Type 2 diabetes-associated nucleic acid. An alteration in expression of a polypeptide encoded by a Type 2 diabetes-associated nucleic acid can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide 25 produced). An alteration in the composition of a polypeptide encoded by a Type 2
diabetes-associated nucleic acid is an alteration in the qualitative polypeptide expression (e.g., expression of a mutant polypeptide or of a different splicing variant). In one embodiment, diagnosis of a susceptibility to Type 2 diabetes is made by detecting a particular splicing variant encoded by a Type 2 diabetes-associated nucleic acid, or a 30 particular pattern of splicing variants.
Both such alterations (quantitative and qualitative) can also be present. An "alteration" in the polypeptide expression or composition, as used herein, refers to an alteration in expression or composition in a test sample, as compared to the expression or composition of polypeptide encoded by a Type 2 diabetes-associated nucleic acid in a 35 control sample. A control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from a subject who is not affected by, and/or who does not have a susceptibility to, Type 2 diabetes (e.g., a subject that does not possess a marker allele or haplotype as described herein). Similarly, the presence of one or more
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different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample, can be indicative of a susceptibility to Type 2 diabetes. An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample, can be indicative of a specific allele in the instance where the allele alters a splice site relative to the reference in the control sample. Various means of examining expression or composition of a polypeptide encoded by a Type 2 diabetes-associated nucleic acid can be used, including spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and immunoassays (e.g., David et al., U.S. Pat. No. 4,376,110) such as immunoblotting (see, e.g., Current Protocols in Molecular Biology, particularly chapter 10, supra).
For example, in one embodiment, an antibody (e.g., an antibody with a detectable label) that is capable of binding to a polypeptide encoded by a Type 2 diabetes-associated nucleic acid can be used. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a labeled secondary antibody (e.g., a fluorescently-labeled secondary antibody) and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
In one embodiment of this method, the level or amount of polypeptide encoded by a Type 2 diabetes-associated nucleic acid in a test sample is compared with the level or 25 amount of the polypeptide encoded by a Type 2 diabetes-associated nucleic acid in a control sample. A level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of an alteration in the expression of the polypeptide encoded by the Type 2 diabetes-associated nucleic acid, and is diagnostic for 30 a particular allele or haplotype responsible for causing the difference in expression.
Alternatively, the composition of the polypeptide encoded by a Type 2 diabetes-associated nucleic acid in a test sample is compared with the composition of the polypeptide encoded by a Type 2 diabetes-associated nucleic acid in a control sample. In another embodiment, both the level or amount and the composition of the polypeptide can be 35 assessed in the test sample and in the control sample.
In another embodiment, the diagnosis of a susceptibility to Type 2 diabetes is made by detecting at least one Type 2 diabetes-associated marker allele or haplotype (e.g., associated alleles or haplotypes of the markers listed in Tables 1-21, such as Tables
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1-6 and Tables 9-12), in combination with an additional protein-based, RNA-based or DNA-based assay. The methods of the invention can also be used in combination with an analysis of a subject's family history and risk factors (e.g., environmental risk factors, lifestyle risk factors).
Kits
Kits useful in the methods of the invention comprise components useful in any of the methods described herein, including for example, primers for nucleic acid amplification, hybridization probes, restriction enzymes (e.g., for RFLP analysis), allele-10 specific oligonucleotides, antibodies that bind to an altered polypeptide encoded by a nucleic acid of the invention as described herein (e.g., a genomic segment comprising at least one polymorphic marker and/or haplotype of the present invention) or to a non-altered (native) polypeptide encoded by a nucleic acid of the invention as described herein, means for amplification of a nucleic acid associated with Type 2 diabetes, means 15 for analyzing the nucleic acid sequence of a nucleic acid associated with Type 2 diabetes, means for analyzing the amino acid sequence of a polypeptide encoded by a nucleic acid associated with Type 2 diabetes (e.g., the Type 2 diabetes protein encoded by the Type 2 diabetes gene), etc. The kits can for example include necessary buffers, nucleic acid primers for amplifying nucleic acids of the invention (e.g., a nucleic acid segment 20 comprising one or more of the polymorphic markers as described herein), and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase). Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with other Type 2 diabetes diagnostic assays.
In one embodiment, the invention is a kit for assaying a sample from a subject to detect the presence of Type 2 diabetes, symptoms associated with Type 2 diabetes, or a susceptibility to Type 2 diabetes in a subject, wherein the kit comprises reagents necessary for selectively detecting at least one allele of at least one polymorphism of the present invention in the genome of the individual. In a particular embodiment, the 30 reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising at least one polymorphism of the present invention. In another embodiment, the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a subject, wherein each oligonucleotide primer pair is designed to selectively amplify a 35 fragment of the genome of the individual that includes at least one polymorphism,
wherein the polymorphism is selected from the group consisting of the polymorphisms as listed in Tables 1-6 and 9-12, and polymorphic markers in linkage disequilibrium
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therewith (e.g., the markers set forth in Tables 22, 23 and 24). In yet another embodiment the fragment is at least 20 base pairs in size. Such oligonucleotides or nucleic acids (e.g., oligonucleotide primers) can be designed using portions of the nucleic acid sequence flanking polymorphisms (e.g., SNPs or microsatellites) that are indicative of 5 Type 2 diabetes. In another embodiment, the kit comprises one or more labeled nucleic acids capable of allele-specific detection of one or more specific polymorphic markers or haplotypes associated with Type 2 diabetes, and reagents for detection of the label. Suitable labels include, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.
In particular embodiments, the polymorphic marker or haplotype to be detected by the reagents of the kit comprises one or more markers, two or more markers, three or more markers, four or more markers or five or more markers selected from the group consisting of the markers set forth in Tables 9-12. In another embodiment, the marker or haplotype to be detected comprises the markers set forth in Tables 22-24. In another 15 embodiment, the marker or haplotype to be detected comprises markers rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID 20 NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO:ll), rsl843622 (SEQ ID NO:9), rs2191113 (SEQ ID NO: 13), and rs9890889 (SEQ ID NO:31), and markers in linkage disequilibrium therewith. In one such embodiment, linkage disequilibrium is defined by values of r2 greater than 0.2.
In one preferred embodiment, the kit for detecting the markers of the invention comprises a detection oligonucleotide probe, that hybridizes to a segment of template DNA containing a SNP polymorphisms to be detected, an enhancer oligonucleotide probe and an endonuclease. As explained in the above, the detection oligonucleotide probe comprises a fluorescent moiety or group at its 3' terminus and a quencher at its 5' 30 terminus, and an enhancer oligonucleotide, is employed, as described by Kutyavin eta/. (Nucleic Acid Res. 34:el28 (2006)). The fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties. The detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected. Preferably, the SNP is anywhere from the terminal residue to -6 residues from 35 the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template. The gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV.
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The enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch. Thus, by measuring the fluorescence of the released fluorescent moiety, assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.
The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.
In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection, and primers for such amplification are included in the reagent kit. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.
Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G. The use of modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule. In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.
In one such embodiments, the presence of the marker or haplotype is indicative of a susceptibility (increased susceptibility or decreased susceptibility) to Type 2 diabetes. In another embodiment, the presence of the marker or haplotype is indicative of response to a Type 2 diabetes therapeutic agent. In another embodiment, the presence of the marker or haplotype is indicative of prognosis of Type 2 diabetes. In yet another embodiment, the presence of the marker or haplotype is indicative of progress of treatment of Type 2 diabetes. Such treatment may include intervention by surgery, medication or by other means (e.g., lifestyle changes).
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Therapeutic agents for Type 2 diabetes
Currently available Type 2 diabetes medication (apart from insulin) falls into six main classes of drugs: sulfonylureas, meglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors and a new class of drugs called DPP-4 inhibitors. These classes of drugs work in different ways to lower blood glucose levels.
1. Sulfonylureas. Sulfonylureas stimulate the beta cells of the pancreas to release more insulin.
2. Meglitinides. Meglitinides are drugs that also stimulate the beta cells to release 10 insulin.
3. Biguanides. Biguanides lower blood glucose levels primarily by decreasing the amount of glucose produced by the liver. Metformin also helps to lower blood glucose levels by making muscle tissue more sensitive to insulin so glucose can be absorbed.
4. Thiazolidinediones. These drugs help insulin work better in the muscle and fat and 15 also reduce glucose production in the liver.
. Alpha-glucosidase inhibitors. These drugs help the body to lower blood glucose levels by blocking the breakdown of starches, such as bread, potatoes, and pasta in the intestine. They also slow the breakdown of some sugars, such as table sugar. Their action slows the rise in blood glucose levels after a meal. They should be taken with the first bite
of a meal.
6: DPP-4 Inhibitors. A new class of medications called DPP-4 inhibitors help improve AlC without causing hypoglycemia. They work by preventing the breakdown of a naturally occurring compound in the body, GLP-1. GLP-1 reduces blood glucose levels in the body, but is broken down very quickly so it does not work well when injected as a drug itself. 25 By interfering in the process that breaks down GLP-1, DPP-4 inhibitors allow it to remain active in the body longer, lowering blood glucose levels only when they are elevated.
Examples of available drugs in these classes are listed in Agent Table 1.
Agent Table 1.
Drug Class
Generic name
Brand name
Biguanides metformin
Glucophage, Glucophage XR, Glycon
metformin plus qlyburide
Glucovance
Thiazolidinediones pioglitazone
Actos
rosiglitazone
Avandia
Sulfonylureas acetohexamide
Dymelor
57
Drug Class
Generic name
Brand name
chlorpropamide
Diabinese
gliclazide Diamicron
Diamicron MR
glimepiride
Amaryl
glipizide
Glucotrol.Glucotrol XL
glyburide
Micronase, DiaBeta, Glynase PresTab
glyburide plus metformin
Glucovance
tolazamide
Tolinase
tolbutamide
Orinase, Tol-Tab
Meglitinides nateglinide
Starlix
repaglinide
Prandin, Gluconorm
Alpha-glucosidase inhibitors acarbose
Precose, Prandase
miglitol
Glyset
DPP-4 Inhibitors sitagliptin
Januvia
Additionally, a combination therapy comprising Biguanide and Sulphonylureas has bee used for treatment of Type 2 diabetes.
Additional Type 2 diabetes drugs are listed Agent Table 2.
Agent Table 2
Compound name(s)
Compound name (generated using Autonom, ISIS Draw version 2.5 from MDL Information Systems)
Company
Compound Reference
Indications
AR-0133418 (SN-4521)
1 -(4-Methoxy-benzyl)-3-(5-nitro-thiazol-2-yl)-urea
AstraZeneca
AD
AR-025028
NSD
AstraZeneca
CT-98023
N-[4-(2,4-Dichloro-phenyl)-5-(1 H-imidazol-2-yl)-pyrimidin-2-yl]-N'-(5-nitro-pyridin-2-yl)-ethane-1,2-diamine
Chiron Corp
non-insulin dependent diabetes
CT-20026
NSD
Chiron Corp
Wagman et al., Curr Pharm. Des 2004: 10(10) 1105-37
non-insulin dependent diabetes
CT-21022
NSD
Chiron Corp
non-insulin dependent diabetes
CT-20014
NSD
Chiron Corp
non-insulin dependent diabetes
CT-21018
NSD
Chiron Corp
non-insulin dependent diabetes
CHIR-98025
NSD
Chiron Corp
non-insulin dependent diabetes
CHIR-99021
NSD
Chiron Corp
Wagman et al., Curr Pharm. Des 2004: 10(10) 1105-37
non-insulin dependent diabetes
CG-100179
NSD
CrystalGenomics and Yuyu
WO-
2004065370
diabetes mellitus (Korea)
58
Compound name(s)
Compound name (generated using Autonom, ISIS Draw version 2.5 from MDL Information Systems)
Company
Compound Reference
Indications
4-[2-(4-Dimethylamino-3-nitro-phenylamino)-pyrimidin-4-yl]-3,5-dimethyl-1 H-pyrrole-2-carbonitrile
Cyclacel Ltd.
non-insulin dependent diabetes, among others.
NP-01139, NP-031112, NP-03112, NP-00361
4-Benzyl-2-methyl-[1,2,4]thiadiazolidine-3,5-dione
Neuropharma SA
CNS disorders, AD
3-[9-Fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-pyrrole-2,5-dione
Eli Lilly & Co
non-insulin dependent diabetes
GW-784752x, GW-784775, SB-216763, SB-415286
Cyclopentanecarboxylic acid
(6-pyridin-3-yl-furo[2,3-
d]pyrimidin-4-yl)-amide
GSK
W003024447 (compound referenced: 4-[2-(2-
bromophenyl)-4-(4-fluorophenyl)-1H-imidazol-5-yljpyridine non-insulin dependent diabetes, neurodegenerative disease
NNC-57-0511, NNC-57-0545, NNC-57-0588
1 -(4-Amino-furazan-3-yl)-5-piperidin-1-ylmethyl-1H-[1,2,3]triazole-4-carboxylic acid [1-pyridin-4-yl-meth-(E)-ylidene]-hydrazide
Novo Nordisk
non-insulin dependent diabetes,
CP-70949
NSD
Pfizer
Hypoglycemic agent
VX-608
NSD
Cerebrovascular ischemia, non-insulin dependent diabetes
KP-403 class
NSD
Kinetek
Nuclear factor kappa B modulator, Antiinflammatory, Cell cycle inhibitor, Glycogen synthase kinase-3 beta inhibitor
BYETTA (exenatide)
Exenatide: C184H282N50O60S -
Amino acid sequence:H-His-
Gly-Glu-Gly-Thr-Phe-Thr-
Ser-Asp-Leu-Ser-Lys-Gln-
Met-Glu-Glu-Glu-Ala-Val-
Arg-Leu-Phe-lle-Glu-Trp-
Leu-Lys-Asn-Gly-Gly-Pro-
Ser-Ser-Gly-Ala-Pro-Pro-
Pro-Ser-NH2
Amylin / Eli Lilly & Co
non-insulin dependent diabetes
Vildagliptin (LAF237)
NSD
Novartis
non-insulin dependent diabetes -DPP-4 inhibitor
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Therapeutic agents of the invention
Variants of the present invention {e.g., the markers and/or haplotypes as described herein) can be used to identify novel therapeutic targets for Type 2 diabetes.
For example, genes containing, or in linkage disequilibrium with, variants (markers and/or haplotypes) associated with Type 2 diabetes, or their products, as well as genes or their products that are directly or indirectly regulated by or interact with these variant genes or their products, can be targeted for the development of therapeutic agents to'treat Type 2 diabetes, or prevent or delay onset of symptoms associated with Type 2 diabetes. 10 Therapeutic agents may comprise one or more of, for example, small non-protein and non-nucleic acid molecules, proteins, peptides, protein fragments, nucleic acids (DNA, RNA), PNA (peptide nucleic acids), or their derivatives or mimetics which can modulate the function and/or levels of the target genes or their gene products.
The nucleic acids and/or variants of the invention, or nucleic acids comprising their 15 complementary sequence, may be used as antisense constructs to control gene expression in cells, tissues or organs. The methodology associated with antisense techniques is well known to the skilled artisan, and is described and reviewed in AntisenseDrug Technology: Principles, Strategies, and Applications, Crooke, ed., Marcel Dekker Inc., New York (2001). In general, antisense nucleic acid molecules are designed 20 to be complementary to a region of mRNA expressed by a gene, so that the antisense molecule hybridizes to the mRNA, thus blocking translation of the mRNA into protein. Several classes of antisense oligonucleotide are known to those skilled in the art,
including cleavers and blockers. The former bind to target RNA sites, activate intracellular nucleases (e.g., RnaseH or Rnase L), that cleave the target RNA. Blockers bind to target 25 RNA, inhibit protein translation by steric hindrance of the ribosomes. Examples of blockers include nucleic acids, morpholino compounds, locked nucleic acids and methylphosphonates (Thompson, Drug Discovery Today, 7:912-917 (2002)). Antisense oligonucleotides are useful directly as therapeutic agents, and are also useful for determining and validating gene function, for example by gene knock-out or gene knock-30 down experiments. Antisense technology is further described in Lavery et at., Curr. Opin. Drug Discov. Devel. 6:561-569 (2003), Stephens et al., Curr. Opin. Mol. Ther. 5:118-122 (2003), Kurreck, Eur. J. Biochem. 270:1628-44 (2003), Dias et al., Mol. Cancer Ter. 1:347-55 (2002), Chen, Methods Mol. Med. 75:621-636 (2003), Wang etal., Curr. Cancer Drug Targets 1:177-96 (2001), and Bennett, Antisense Nucleic Acid Drug.Dev. 12:215-24 35 (2002)
The variants described herein can be used for the selection and design of antisense reagents that are specific for particular variants. Using information about the variants described herein, antisense oligonucleotides or other antisense molecules that
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specifically target mRNA molecules that contain one or more variants of the invention can be designed. In this manner, expression of mRNA molecules that contain one or more variant of the present invention (markers and/or haplotypes) can be inhibited or blocked. In one embodiment, the antisense molecules are designed to specifically bind a particular 5 allelic form (i.e., one or several variants (alleles and/or haplotypes)) of the target nucleic acid, thereby inhibiting translation of a product originating from this specific allele or haplotype, but which do not bind other or alternate variants at the specific polymorphic sites of the target nucleic acid molecule.
As antisense molecules can be used to inactivate mRNA so as to inhibit gene 10 expression, and thus protein expression, the molecules can be used to treat a disease or disorder, such as Type 2 diabetes. The methodology can involve cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Such mRNA regions include, for example, protein-coding regions, in particular protein-coding regions 15 corresponding to catalytic activity, substrate and/or ligand binding sites, or other functional domains of a protein.
The phenomenon of RNA interference (RNAi) has been actively studied for the last decade, since its original discovery in C. elegans (Fire et al.,Nature 391:806-11 (1998)), and in recent years its potential use in treatment of human disease has been actively 20 pursued (reviewed in Kim & Rossi, Nature Rev. Genet. 8:173-204 (2007)). RNA
interference (RNAi), also called gene silencing, is based on using double-stranded RNA molecules (dsRNA) to turn off specific genes. In the cell, cytoplasmic double-stranded RNA molecules (dsRNA) are processed by cellular complexes into small interfering RNA (siRNA). The siRNA guide the targeting of a protein-RNA complex to specific sites on a 25 target mRNA, leading to cleavage of the mRNA (Thompson, Drug Discovery Today, 7:912-917 (2002)). The siRNA molecules are typically about 20, 21, 22 or 23 nucleotides in length. Thus, one aspect of the invention relates to isolated nucleic acid molecules, and the use of those molecules for RNA interference, i.e. as small interfering RNA molecules (siRNA). In one embodiment, the isolated nucleic acid molecules are 18-26 nucleotides in 30 length, preferably 19-25 nucleotides in,length, more preferably 20-24 nucleotides in length, and more preferably 21, 22 or 23 nucleotides in length.
Another pathway for RNAi-mediated gene silencing originates in endogenously encoded primary microRNA (pri-miRNA) transcripts, which are processed in the cell to generate precursor miRNA (pre-miRNA). These miRNA molecules are exported from the 35 nucleus to the cytoplasm, where they undergo processing to generate mature miRNA
molecules (miRNA), which direct translational inhibition by recognizing target sites in the 3' untranslated regions of mRNAs, and subsequent mRNA degradation by processing P-bodies (reviewed in Kim & Rossi, Nature Rev. Genet. 8:173-204 (2007)).
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Clinical applications of RNAi include the incorporation of synthetic siRNA duplexes, which preferably are approximately 20-23 nucleotides in size, and preferably have 3' overlaps of 2 nucleotides. Knockdown of gene expression is established by sequence-specific design for the target mRNA. Several commercial sites for optimal design and 5 synthesis of such molecules are known to those skilled in the art.
Other applications provide longer siRNA molecules (typically 25-30 nucleotides in length, preferably about 27 nucleotides), as well as small hairpin RNAs (shRNAs; typically about 29 nucleotides in length). The latter are naturally expressed, as described in Amarzguioui et al. (FEBS Lett. 579:5974-81 (2005)). Chemically synthetic siRNAs and 10 shRNAs are substrates for in vivo processing, and in some cases provide more potent gene-silencing than shorter designs (Kim et al., Nature Biotechnol. 23:222-226 (2005); Siolas et al., Nature Biotechnol. 23:227-231 (2005)). In general siRNAs provide for transient silencing of gene expression, because their intracellular concentration is diluted by subsequent cell divisions. By contrast, expressed shRNAs mediate long-term, stable 15 knockdown of target transcripts, for as long as transcription of the shRNA takes place (Marques et al., Nature Biotechnol. 23:559-565 (2006); Brummelkamp et al., Science 296: 550-553 (2002)).
Since RNAi molecules, including siRNA, miRNA and shRNA, act in a sequence-dependent manner, the variants of the present invention (e.g., the markers and 20 haplotypes as described herein) can be used to design RNAi reagents that recognize specific nucleic acid molecules comprising specific alleles and/or haplotypes (e.g., the alleles and/or haplotypes of the present invention), while not recognizing nucleic acid molecules comprising other alleles or haplotypes. These RNAi reagents can thus recognize and destroy the target nucleic acid molecules. As with antisense reagents, RNAi 25 reagents can be useful as therapeutic agents (i.e., for turning off disease-associated genes or disease-associated gene variants), but may also be useful for characterizing and validating gene function (e.g., by gene knock-out or gene knock-down experiments).
Delivery of RNAi may be performed by a range of methodologies known to those skilled in the art. Methods utilizing non-viral delivery include cholesterol, stable nucleic 30 acid-lipid particle (SNALP), heavy-chain antibody fragment (Fab), aptamers and nanoparticles. Viral delivery methods include use of lentivirus, adenovirus and adeno-associated virus. The siRNA molecules are in some embodiments chemically modified to increase their stability. This can include modifications at the 2' position of the ribose, including 2'-0-methylpurines and 2'-fluoropyrimidines, which provide resistance to Rnase 35 activity. Other chemical modifications are possible and known to those skilled in the art.
The following references provide a further summary of RNAi, and possibilities for targeting specific genes using RNAi: Kim & Rossi, Nat. Rev. Genet. 8:173-184 (2007),
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Chen & Rajewsky, Nat. Rev. Genet. 8: 93-103 (2007), Reynolds, eta/., Nat. Biotechnol. 22:326-330 (2004), Chi etal., Proc. Natl. Acad. Sci. USA 100:6343-6346 (2003), Vickers eta/., J. Biol. Chem. 278:7108-7118 (2003), Agami, Curr. Opin. Chem. Biol. 6:829-834 (2002), Lavery, eta/., Curr. Opin. Drug Discov. Devel. 6:561-569 (2003), Shi, Trends 5 Genet. 19:9-12 (2003), Shuey eta/., Drug Discov. Today 7:1040-46 (2002), McManus et al., Nat. Rev. Genet. 3:737-747 (2002), Xia etal., Nat. Biotechnol. 20:1006-10 (2002), Plasterk et al., curr. Opin. Genet. Dev. 10:562-7 (2000), Bosher et al., Nat. Cell Biol. 2:E31-6 (2000), and Hunter, Curr. Biol. 9:R440-442 (1999).
A genetic defect leading to increased predisposition or risk for development of a 10 disease, including Type 2 diabetes, or a defect causing the disease, may be corrected permanently by administering to a subject carrying the defect a nucleic acid fragment that incorporates a repair sequence that supplies the normal/wild-type nucleotide(s) at the site of the genetic defect. Such site-specific repair sequence may concompass an RNA/DNA oligonucleotide that operates to promote endogenous repair of a subject's genomic DNA. 15 The administration of the repair sequence may be performed by an appropriate vehicle, such as a complex with polyethelenimine, encapsulated in anionic liposomes, a viral vector such as an adenovirus vector, or other pharmaceutical compositions suitable for promoting intracellular uptake of the adminstered nucleic acid. The genetic defect may then be overcome, since the chimeric oligonucleotides induce the incorporation of the 20 normal sequence into the genome of the subject, leading to expression of the normal/wild-type gene product. The replacement is propagated, thus rendering a permanent repair and alleviation of the symptoms associated with the disease or condition.
The present invention provides methods for identifying compounds or agents that 25 can be used to treat Type 2 diabetes. Thus, the variants of the invention are useful as targets for the identification and/or development of therapeutic agents. Such methods may include assaying the ability of an agent or compound to modulate the activity and/or expression of a nucleic acid that includes at least one of the variants (markers and/or haplotypes) of the present invention, or the encoded product of the nucleic acid. This in 30 turn can be used to identify agents or compounds that inhibit or alter the undesired activity or expression of the encoded nucleic acid product. Assays for performing such experiments can be performed in cell-based systems or in cell-free systems, as known to the skilled person. Cell-based systems include cells naturally expressing the nucleic acid molecules of interest, or recombinant cells that have been genetically modified so as to 35 express a certain desired nucleic acid molecule.
Variant gene expression in a patient can be assessed by expression of a variant-containing nucleic acid sequence (for example, a gene containing at least one variant of the present invention, which can be transcribed into RNA containing the at least one
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variant, and in turn translated into protein), or by altered expression of a normal/wild-type nucleic acid sequence due to variants affecting the level or pattern of expression of the normal transcripts, for example variants in the regulatory or control region of the gene. Assays for gene expression include direct nucleic acid assays (mRNA), assays for 5 expressed protein levels, or assays of collateral compounds involved in a pathway, for example a signal pathway. Furthermore, the expression of genes that are up- or down-regulated in response to the signal pathway can also be assayed. One embodiment includes operably linking a reporter gene, such as luciferase, to the regulatory region of the gene(s) of interest.
Modulators of gene expression can in one embodiment be identified when a cell is contacted with a candidate compound or agent, and the expression of mRNA is determined. The expression level of mRNA in the presence of the candidate compound or agent is compared to the expression level in the absence of the compound or agent.
Based on this comparison, candidate compounds or agents for treating Type 2 diabetes 15 can be identified as those modulating the gene expression of the variant gene. When expression of mRNA or the encoded protein is statistically significantly greater in the presence of the candidate compound or agent than in its absence, then the candidate compound or agent is identified as a stimulator or up-regulator of expression of the nucleic acid. When nucleic acid expression or protein level is statistically significantly less 20 in the presence of the candidate compound or agent than in its absence, then the candidate compound is identified as an inhibitor or down-regulator of the nucleic acid expression.
The invention further provides methods of treatment using a compound identified through drug (compound and/or agent) screening as a gene modulator (i.e. stimulator 25 and/or inhibitor of gene expression).
In a further aspect of the present invention, a pharmaceutical pack (kit) is provided, the pack comprising a therapeutic agent and a set of instructions for administration of the therapeutic agent to humans diagnostically tested for one or more variants of the present invention, as disclosed herein. The therapeutic agent can be a 30 small molecule drug, an antibody, a peptide, an antisense or RNAi molecule, or other therapeutic molecules. In one embodiment, an individual identified as a carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent. In one such embodiment, an individual identified as a homozygous carrier of at least one variant of the present invention is instructed to take a prescribed 35 dose of the therapeutic agent. In another embodiment, an individual identified as a non-carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent.
64
Methods of assessing probability of response to therapeutic agents, methods of monitoring progress of treatment and methods of treatment
As is known in the art, individuals can have differential responses to a particular 5 therapy (e.g., a therapeutic agent or therapeutic method). Pharmacogenomics addresses the issue of how genetic variations (e.g., the variants (markers and/or haplotypes) of the present invention) affect drug response, due to altered drug disposition and/or abnormal or altered action of the drug . Thus, the basis of the differential response may be genetically determined in part. Clinical outcomes due to genetic variations affecting drug 10 response may result in toxicity of the drug in certain individuals (e.g., carriers or non-carriers of the genetic variants of the present invention), or therapeutic failure of the drug. Therefore, the variants of the present invention may determine the manner in which a therapeutic agent and/or method acts on the body, or the way in which the body metabolizes the therapeutic agent.
Accordingly, in one embodiment, the presence of a particular allele at a polymorphic site or haplotype is indicative of a different, e.g. a different response rate, to a particular treatment modality. This means that a patient diagnosed with Type 2 diabetes, and carrying a certain allele at a polymorphic or haplotype of the present invention (e.g., the at-risk and protective alleles and/or haplotypes of the invention) 20 would respond better to, or worse to, a specific therapeutic, drug and/or other therapy used to treat the disease. Therefore, the presence or absence of the marker allele or haplotype could aid in deciding what treatment should be used for a the patient. For example, for a newly diagnosed patient, the presence of a marker or haplotype of the present invention may be assessed (e.g., through testing DNA derived from a blood 25 sample, as described herein). If the patient is positive for a marker allele or haplotype at (that is, at least one specific allele of the marker, or haplotype, is present), then the physician recommends one particular therapy, while if the patient is negative for the at least one allele of a marker, or a haplotype, then a different course of therapy may be recommended (which may include recommending that no immediate therapy, other than 30 serial monitoring for progression of the disease, be performed). Thus, the patient's carrier status could be used to help determine whether a particular treatment modality should be administered. The value lies within the possibilities of being able to diagnose the disease at an early stage, to select the most appropriate treatment, and provide information to the clinician about prognosis/aggressiveness of the disease in order to be 35 able to apply the most appropriate treatment.
In some embodiments, the treatment modality comprises adminstering at least one of the therapeutic agents set forth in Agent Table 1 and Agent Table 2. In one
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embodiment, the therapeutic agent is selected from Biguanides, Thiazolidinediones, Sulfonylureas, Meglitinides, Alpha-glucosidase inhibitors and DPP-4 inhibitors. In one embodiment, the Biguanide is metformin or metformin plus glyburide. Other combination therapies comprising metformin, including combinations with thiazolidinediones, are also 5 contemplated and within the scope of the invention. In another embodiment, the Sulfunylurea is selected from acetohexamide, chlorpropamide, gliclazide Diamicron, glimepiride, glipizide, glyburide, tolazamide and tolbutamide. In another embodiment, the Thiazolidinedione is selected from pioglitazone, rosiglitazone and mitoglitazone or other thiazolidinedione derivatives. In another embodiment, the therapeutic agent is 10 selected from the agents set forth in Agent Table 2.
The present invention also relates to methods of monitoring progress or effectiveness of a treatment for Type 2 diabetes. This can be done based on the genotype and/or haplotype status of the markers and haplotypes of the present invention, i.e., by assessing the absence or presence of at least one allele of at least one polymorphic 15 marker as disclosed herein, or by monitoring expression of genes that are associated with the variants (markers and haplotypes) of the present invention. The risk gene mRNA or the encoded polypeptide can be measured in a tissue sample (e.g., a peripheral blood sample, or a biopsy sample). Expression levels and/or mRNA levels can thus be determined before and during treatment to monitor its effectiveness. Alternatively, or 20 concomitantly, the genotype and/or haplotype status of at least one risk variant for Type 2 diabetes presented herein is determined before and during treatment to monitor its effectiveness. Alternatively, biological networks or metabolic pathways related to the markers and haplotypes of the present invention can be monitored by determining mRNA and/or polypeptide levels. This can be done for example, by monitoring expression levels 25 or polypeptides for several genes belonging to the network and/or pathway, in samples taken before and during treatment. Alternatively, metabolites belonging to the biological network or metabolic pathway can be determined before and during treatment. Effectiveness of the treatment is determined by comparing observed changes in expression levels/metabolite levels during treatment to corresponding data from healthy 30 subjects.
The progress of therapy in individuals carrying at least one at-risk allele of at least one marker found to be associated with increased susceptibility or risk of Type 2 diabetes is thus monitored based on the genotype status of the individual. Individuals carrying at-risk variants as described herein may benefit from closer or more frequent monitoring of 35 progress of therapy than non-carriers, alternatively in combination with a particular treatment modality or therapeutic agent being adminstered, as described in the above.
In a further aspect, the markers of the present invention can be used to increase power and effectiveness of clinical trials. Thus, individuals who are carriers of at least one
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at-risk variant of the present invention, i.e. individuals who are carriers of at least one allele of at least one polymorphic marker conferring increased risk of developing Type 2 diabetes may be more likely to respond to a particular treatment modality. In one embodiment, individuals who carry at-risk variants for gene(s) in a pathway and/or 5 metabolic network for which a particular treatment (e.g., small molecule drug) is targeting, are more likely to be responders to the treatment. In another embodiment, individuals who carry at-risk variants for a gene, which expression and/or function is altered by the at-risk variant, are more likely to be responders to a treatment modality targeting that gene, its expression or its gene product. This application can improve the 10 safety of clinical trials, but can also enhance the chance that a clinical trial will demonstrate statistically significant efficacy, which may be limited to a certain sub-group of the population, e.g., individuals that are either carriers or non-carriers of the at-risk variants described herein. Thus, one possible outcome of such a trial is that carriers of certain genetic variants, e.g., the markers and haplotypes of the present invention, are 15 statistically significantly likely to show positive response to the therapeutic agent, i.e. experience alleviation of symptoms associated with Type 2 diabetes when taking the therapeutic agent or drug as prescribed.
In a further aspect, the markers and haplotypes of the present invention can be used for targeting the selection of pharmaceutical agents for specific individuals. 20 Personalized selection of treatment modalities, lifestyle changes or combination of the two, can be realized by the utilization of the at-risk variants of the present invention. Thus, the knowledge of an individual's status for particular markers of the present invention, can be useful for selection of treatment options that target genes or gene products affected by the at-risk variants of the invention. Certain combinations of 25 variants may be suitable for one selection of treatment options, while other gene variant combinations may target other treatment options. Such combination of variant may include one variant, two variants, three variants, or four or more variants, as needed to determine with clinically reliable accuracy the selection of treatment module.
In addition to the diagnostic and therapeutic uses of the variants of the present 30 invention, the variants (markers and haplotypes) can also be useful markers for human identification, and as such be useful in forensics, paternity testing and in biometrics. The specific use of SNPs for forensic purposes is reviewed by Gill {Int. J. Legal Med. 114:204-10 (2001)). Genetic variations in genomic DNA between individuals can be used as genetic markers to identify individuals and to associate a biological sample with an 35 individual. Genetic markers, including SNPs and microsatellites, can be useful to distinguish individuals. The more markers that are analyzed, the lower the probability that the allelic combination of the markers in any given individual is the same as in an unrelated individual (assuming that the markers are unrelated, i.e. that the markers are in perfect linkage equilibrium). Thus, the variants used for these purposes are preferably
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unrelated, i.e. they are inherited independently. Thus, preferred markers can be selected from available markers, such as the markers of the present invention, and the selected markers may comprise markers from different regions in the human genome, including markers on different chromosomes.
In certain applications, the SNPs useful for forensic testing are from degenerate codon positions (i.e., the third position in certain codons such that the variation of the SNP does not affect the amino acid encoded by the codon). In other applications, such for applications for predicting phenotypic characteristics including race, ancestry or physical characteristics, it may be more useful and desirable to utilize SNPs that affect the amino 10 acid sequence of the encoded protein. In other such embodiments, the variant (SNP or other polymorphic marker) affects the expression level of a nearby gene, thus leading to altered protein expression.
The present invention also relates to computer-implemented applications of the polymorphic markers and haplotypes described herein to be associated with Type 2 diabetes. Such applications can be useful for storing, manipulating or otherwise analyzing genotype data that is useful in the methods of the invention. One example pertains to storing genotype information derived from an individual on readable media, so as to be able to provide the genotype information to a third party (e.g., the individual), or for deriving information from the genotype data, e.g., by comparing the genotype data to information about genetic risk factors contributing to increased susceptibility to Type 2 diabetes, and reporting results based on such comparison.
One such aspect relates to computer-readable media. In general terms, such medium has capabilities of storing (i) identifer information for at least one polymorphic marker or a haplotye; (ii) an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in individuals with Type 2 diabetes; and an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in a reference population. The reference population can be a disease-free population of individuals. Alternatively, the reference population is a random sample from the general population, and is thus representativ of the population at large. The frequency indicator may be a calculated frequency, a count of alleles and/or haplotype copies, or normalized or otherwise manipulated values of the actual frequencies that are suitable for the particular medium.
Additional information about the individual can be stored on the medium, such as ancestry information, information about sex, physical attributes or charactersitics 35 (including height and weight), biochemical measurements (such as blood pressure, blood lipid levels, fasting glucose levels, insulin response measurements), or other useful
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information that is desirable to store or manipulate in the context of the genotype status of a particular individual.
The invention furthermore relates to an apparatus that is suitable for determination or manipulation of genetic data useful for determining a susceptibility to 5 Type 2 diabetes in a human individual. Such an apparatus can include a computer-readable memory, a routine for manipulating data stored on the computer-readable memory, and a routine for generating an output that includes a measure of the genetic data. Such measure can include values such as allelic or haplotype frequencies, genotype counts, sex, age, phenotype information, values for odds ratio (OR) or relative risk (RR), 10 population attributable risk (PAR), or other useful information that is either a direct statistic of the original genotype data or based on calculations based on the genetic data.
The above-described applications can all be practiced with the markers and haplotypes of the invention that have in more detail been described with respect to methods of assessing susceptibility to Type 2 diabetes. Thus, these applications can in 15 general be reduced to practice using markers listed in Tables 1-6, and markers in linkage disequilibrium therewith, e.g. the markers set forth in Tables 22, 23 and 24. In one embodiment, the markers or haplotypes are present within the genomic segments whose sequences are set forth in SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3. In another embodiment, the markers and haplotypes comprise at least one marker selected from 20 rs2497304 (SEQ ID NO: 16), rs947591 (SEQ ID N0:30), rsl0882091 (SEQ ID NO:4), rs7914814 (SEQ ID NO:24), rs6583830 (SEQ ID N0:20), rs2421943 (SEQ ID NO: 15), rs6583826 (SEQ ID NO: 19), rs7752906 (SEQ ID NO:32), rsl569699 (SEQ ID NO:6), rs7756992 (SEQ ID NO:21), rs9350271 (SEQ ID NO:33), rs9356744 (SEQ ID NO:34), rs9368222 (SEQ ID NO:35), rsl0440833 (SEQ ID NO:36), rs6931514 (SEQ ID NO:37), 25 rsl860316 (SEQ ID NO: 10), rsl981647 (SEQ ID NO: 11), rsl843622 (SEQ ID NO:9),
rs2191113 (SEQ ID NO: 13), and rs9890889 (SEQ ID NO:31), optionally including markers in linkage disequilibrium therewith, wherein linkage disequilibrium is defined by numerical values for r2 of greater than 0.2. In another embodiment, the marker or haplotype comprises at least one marker selected from rs2497304 allele A, rs947591 allele A, 30 rsl0882091 allele C rs7914814 allele T, rs6583830 allele A, rs2421943 allele G,
rs6583826 allele G, rs7752906 allele A, rsl569699 allele C, rs7756992 allele G,
rs9350271 allele A, rs9356744 allele C, rs9368222 allele A, rsl0440833 allele A, rs6931514 allele G, rsl860316 allele A, rsl981647 allele C, rsl843622 allele T,
rs2191113 allele A, and rs9890889 allele A. In yet another embodiment, the at least one 35 marker or haplotype comprises at least one marker selected from the markers set forth in Tables 22, 23 and 24.
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The nucleic acids and polypeptides described herein can be used in methods of diagnosis of a susceptibility to Type 2 diabetes, as well as in kits useful for such diagnosis.
An "isolated" nucleic acid molecule, as used herein, is one that is separated from 5 nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library). For example, an isolated nucleic acid of the invention can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or 10 chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC). An 15 isolated nucleic acid molecule of the invention can comprise at least about 50%, at least about 80% or at least about 90% (on a molar basis) of all macromolecular species present. With regard to genomic DNA, the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. For example, the isolated nucleic acid molecule can contain less than 20 about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
The nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant DNA contained in a vector is included 25 in the definition of "isolated" as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells or heterologous organisms, as well as partially or substantially purified DNA molecules in solution. "Isolated" nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention. An isolated nucleic acid molecule or nucleotide sequence can include a 30 nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means. Such isolated nucleotide sequences are useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human 35 tissue), such as by Northern blot analysis or other hybridization techniques.
The invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide
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sequence described herein (e.g., nucleic acid molecules that specifically hybridize to a nucleotide sequence containing a polymorphic site associated with a haplotype described herein). In one embodiment, the invention includes variants that hybridize under high stringency hybridization and wash conditions (e.g., for selective hybridization) to a 5 nucleotide sequence that comprises the nucleotide sequence of LD Block C06 (SEQ ID NO:l), LD Block CIO (SEQ ID NO:2; e.g., the nucleotide sequence encoding the IDE, KIFll and/or the HHEX genes) and LD Block C17 (SEQ ID NO:3), or the CDKALl gene or a fragment thereof (or a nucleotide sequence comprising the complement of the nucleotide sequence of LD Block C06 (SEQ ID NO:l), LD Block CIO (SEQ ID NO:2; e.g., 10 the nucleotide sequence encoding the IDE, KIFll and/or the HHEX genes) and LD Block C17 (SEQ ID NO:3), or the CDKALl gene or a fragment thereof), wherein the nucleotide sequence comprises at least one polymorphic allele contained in the haplotypes (e.g., haplotypes) described herein.
Such nucleic acid molecules can be detected and/or isolated by allele- or 15 . sequence-specific hybridization (e.g., under high stringency conditions). Stringency conditions and methods for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 and pages 6.3.1-6.3.6 in Current Protocols in Molecular Biology (Ausubel, F. et al., "Current Protocols in Molecular Biology", John Wiley & Sons, (1998)), and Kraus, M. and Aaronson, S., Methods Enzymol., 200:546-556 (1991), the entire teachings of which 20 are incorporated by reference herein.
The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a 25 function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence. The actual comparison of the two sequences can be 30 accomplished by well-known methods, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0), as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and 35 Gapped BLAST programs, the default parameters of the respective programs (e.g.,
NBLAST) can be used. See the website on the world wide web at ncbi.nlm.nih.gov. In one embodiment, parameters for sequence comparison can be set at score=100, wordlength = 12, or can be varied (e.g., W=5 or W=20).
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Other examples include the algorithm of Myers and Miller, CABIOS (1989), ADVANCE and ADAM as described in Torellis, A. and Robotti, C., Comput. Appl. Biosci. 10:3-5 (1994); and FASTA described in Pearson, W. and Lipman, D., Proc. Natl. Acad. Sci. USA, 85:2444-48 (1988). In another embodiment, the percent identity between two 5 amino acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, Cambridge, UK).
The present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid that comprises, or consists of, the nucleotide sequence of LD Block C06 (SEQ ID NO:l), LD 10 Block CIO (SEQ ID NO:2; e.g., the nucleotide sequence encoding the IDE, KIFll and/or the HHEX genes) and LD Block C17 (SEQ ID NO: 3), or the CDKALl gene or a fragment thereof (or a nucleotide sequence comprising, or consisting of, the complement of the nucleotide sequence of LD Block C06 (SEQ ID NO:l), LD Block CIO (SEQ ID NO:2; e.g., the nucleotide sequence encoding the IDE, KIFll and/or the HHEX genes)and LD Block 15 C17 (SEQ ID NO:3), or the CDKALl gene or a fragment thereof), wherein the nucleotide sequence comprises at least one polymorphic allele contained in the haplotypes (e.g., haplotypes) described herein. The nucleic acid fragments of the invention are at least about 15, at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500, 1000, 10,000 or more nucleotides in length.
The nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein. "Probes" or "primers" are oligonucleotides that hybridize in a base-specific manner to a complementary strand of a nucleic acid molecule.
In addition to DNA and RNA, such probes and primers include polypeptide nucleic acids
(PNA), as described in Nielsen, P. et al., Science 254:1497-1500 (1991). A probe or
primer comprises a region of nucleotide sequence that hybridizes to at least about 15,
typically about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule. In one embodiment, the probe or primer comprises at least one allele of at least one polymorphic marker or at least one haplotype described herein, or the complement thereof. In particular embodiments, a probe or
primer can comprise 100 or fewer nucleotides; for example, in certain embodiments from
6 to 50 nucleotides, or, for example, from 12 to 30 nucleotides. In other embodiments,
the probe or primer is at least 70% identical, at least 80% identical, at least 85%
identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. In another
embodiment, the probe or primer is capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.
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The nucleic acid molecules of the invention, such as those described above, can be identified and isolated using standard molecular biology techniques well known to the skilled person. The amplified DNA can be labeled (e.g., radiolabeled) and used as a probe for screening a cDNA library derived from human cells. The cDNA can be derived from 5 mRNA and contained in a suitable vector. Corresponding clones can be isolated, DNA can obtained following In vivo excision, and the cloned insert can be sequenced in either or both orientations by art-recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, 10 sequenced and further characterized.
In general, the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels, and as chromosome markers that are labeled to map related gene positions. The nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify Type 2 diabetes or a susceptibility 15 to Type 2 diabetes, and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample (e.g., subtractive hybridization). The nucleic acid sequences can further be used to derive primers for genetic fingerprinting, to raise anti-polypeptide antibodies using immunization techniques, and/or as an antigen to raise anti-DNA antibodies or elicit immune responses.
Antibodies
Polyclonal antibodies and/or monoclonal antibodies that specifically bind one form of the gene product but not to the other form of the gene product are also provided. Antibodies are also provided which bind a portion of either the variant or the reference 25 gene product that contains the polymorphic site or sites. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen-binding sites that specifically bind an antigen. A molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not 30 substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention. The term "monoclonal 35 antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. A monoclonal
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antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or a 5 fragment thereof. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain 10 the IgG fraction. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72 (1983)), the EBV-hybridoma 15 technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,1985, Inc., pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al., (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as 20 described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a monoclonal antibody 25 to a polypeptide of the invention (see, e.g., Current Protocols in Immunology, supra;
Galfre eta/., Nature 266:55052 (1977); R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); and Lemer, Yale J. Biol. Med. 54:387-402 (1981)). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods that also would be 30 useful.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind 35 the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene Su/fZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use
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in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 5 92/09690; PCT Publication No. WO 90/02809; Fuchs etal., Bio/Technology 9: 1370-1372 (1991); Hay etal., Hum. Antibod. Hybridomas 3:81-85 (1992); Huse et al., Science 246: 1275-1281 (1989); and Griffiths et al., EMBO J. 12:725-734 (1993).
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using 10 standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
In general, antibodies (e.g., a monoclonal antibody) can be used to isolate a polypeptide of the invention by standard techniques, such as affinity chromatography or 15 immunoprecipitation. A polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells. Moreover, an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies 20 can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. The antibody can be coupled to a detectable substance to facilitate its detection.
Examples of detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, biolumihescent materials, and radioactive 25 materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an 30 example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
Antibodies may also be useful in pharmacogenomic analysis. In such embodiments, antibodies against variant proteins encoded by nucleic acids as described 35 herein, such as variant proteins that are encoded by nucleic acids that contain at least one polymorpic marker of the invention, can be used to identify individuals that require modified treatment modalities.
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Antibodies can furthermore be useful for assessing expression of variant proteins in disease states, such as in active stages of Type 2 diabetes, or in an individual with a predisposition to Type 2 diabetes that is related to the function of the protein. Antibodies specific for a variant protein of the present invention that is encoded by a nucleic acid that 5 comprises at least one polymorphic marker or haplotype as described herein can be used to screen for the presence of the variant protein, for example to screen for a predisposition to Type 2 diabetes as indicated by the presence of the variant protein.
Antibodies can be used in other methods. Thus, antibodies are useful as diagnostic tools for evaluating proteins, such as variant proteins of the invention, in 10 conjunction with analysis by electrophoretic mobility, isoelectric point, tryptic or other protease digest, or for use in other physical assays known to those skilled in the art. Antibodies may also be used in tissue typing. In one such embodiment, a specific variant protein has been correlated with expression in a specific tissue type, and antibodies specific for the variant protein can then be used to identify the specific tissue type.
Subcellular localization of proteins, including variant proteins, can also be determined using antibodies, and can be applied to assess aberrant subcellular localization of the protein in cells in various tissues. Such use can be applied in genetic testing, but also in monitoring a particular treatment modality. In the case where treatment is aimed at correcting the expression level or presence of the variant protein or 20 aberrant tissue distribution or developmental expression of the variant protein, antibodies specific for the variant protein or fragments thereof can be used to monitor therapeutic efficacy.
Antibodies are further useful for inhibiting variant protein function, for example by blocking the binding of a variant protein to a binding molecule or partner. Such uses can 25 also be applied in a therapeutic context in which treatment involves inhibiting a variant protein's function. An antibody can be for example be used to block or competitively inhibit binding, thereby modulating (i.e., agonizing or antagonizing) the activity of the protein. Antibodies can be prepared against specific protein fragments containing sites required for specific function or against an intact protein that is associated with a cell or 30 cell membrane. For administration in vivo, an antibody may be linked with an additional therapeutic payload, such as radionuclide, an enzyme, an immunogenic epitope, or a cytotoxic agent, including bacterial toxins (diphtheria or plant toxins, such as ricin). The in vivo half-life of an antibody or a fragment thereof may be increased by pegylation through conjugation to polyethylene glycol.
The present invention will now be exemplified by the following non-limiting examples.
EXEMPLIFICATION
76
EXAMPLE 1
The following contains description of the identifiction of susceptibility factors found to be associated with Type 2 diabetes through single-point and haplotype analysis of SNP markers.
METHODS
Icelandic cohort
The Data Protection Authority of Iceland and the National Bioethics Committee of Iceland approved the study. All participants in the study gave informed consent. All personal identifiers associated with blood samples, medical information and genealogy were first encrypted by the Data Protection Authority, using a third-party encryption system. 15 For this study, 2400 Type 2 diabetes patients were identified who were diagnosed either through a long-term epidemiologic study done at the Icelandic Heart Association over the past 30 years or at one of two major hospitals in Reykjavik over the past 12 years. Two-thirds of these patients were alive, representing about half of the population of known Type 2 diabetes patients in Iceland today. The majority of these patients were contacted for this 20 study, and the cooperation rate exceeded 80%. All participants in the study visited the Icelandic Heart Association where they answered a questionnaire, had blood drawn and a fasting plasma glucose measurements taken. Questions about medication and age at diagnosis were included. The Type 2 diabetes patients in this study were diagnosed as described in our previously published linkage study (Reynisdottir et al., Am J Hum Genet 73, 25 323 (2003). In brief, the diagnosis of Type 2 diabetes was confirmed by study physicians through previous medical records, medication history, and/or new laboratory measurements. For previously diagnosed Type 2 diabetes patients, reporting of the use of oral glucose-lowering agent confirmed Type 2 diabetes. Individuals who were currently treated with insulin were classified as having Type 2 diabetes if they were also using or had previously used oral 30 glucose-lowering agents. In this cohort the majority of patients on medication take oral glucose-lowering agents and only a small portion (9%) require insulin. For hitherto undiagnosed individuals, the diagnosis of Type 2 diabetes and impaired fasting glucose (IFG) was based on the criteria set by the American Diabetes Association (Expert Committee on the Diagnosis and Classification of Diabetes Mellitus 1997). The average age of the Type 2 35 diabetes patients in this study was 69.7 years.
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Replication cohorts
The Danish study group was a set of Type 2 diabetes patients from the Steno Diabetes Center in Copenhagen (N = 1,018) and from the Inter99 population-based sample of 30-60 year old individuals living in the greater Copenhagen area and sampled at 5 Research Centre for Prevention and Health28 (N = 359). Diabetes and pre-diabetes categories were diagnosed according to the 1999 World Health Organization (WHO) criteria. An effectively random subset (N=2,400) of Danish controls with BMI measurements were obtained from the Inter99 collection. Informed written consent was obtained from all subjects before participation. The study was approved by the Ethical 10 Committee of Copenhagen County and was in accordance with the principles of the Helsinki Declaration.
The PENN CATH study in the US is a cross sectional study of the association of biochemical and genetic factors with coronary atherosclerosis in a consecutive cohort of patients undergoing cardiac catheterization at the University of Pennsylvania Medical 15 Center between July 1998 and March 2003. Type 2 diabetes was defined as history of fasting blood glucose >126mg/dl, 2-hour post-prandial glucose >200mg/dl, use of oral hypoglycemic agents, or insulin and oral hypoglycemic in a subject greater than age 40. The University of Pennsylvania Institutional Review Board approved the study protocol and all subjects gave written informed consent. Ethnicity was determined through self-20 report. A total of 468 Caucasian Type 2 diabetes cases were derived from this cohort. Additionally, 1024 unaffected (with respect to Type 2 diabetes) Caucasian controls were randomly drawn from the same study.
The DNA used for genotyping was the product of whole-genome amplification, by use of the GenomiPhi Amplification kit (Amersham), of DNA isolated from the peripheral 25 blood of the Danish and US Type 2 diabetes patients and controls.
Genotyping
A genome-wide scan of 1399 Icelandic diabetes patients was performed using Infinium HumanHap300 SNP chips from Illumina for assaying approximately 317,000 30 single nucleotide polymorphisms (SNPs) on a single chip (Illumina, San Diego, CA, USA). SNP genotyping for replication in other case-control cohorts was carried using the Centaurus platform (Nanogen).
Statistical Methods for Association Analysis
For single marker association to Type 2 diabetes, we used a likelihood ratio test to calculate a two-sided p-value for each allele. We calculated relative risk (RR) and population attributable risk (PAR) assuming a multiplicative model (C. T. Falk, P. Rubinstein, Ann Hum Genet 51 (Pt 3), 227 (1987); J. D. Terwilliger, J. Ott, Hum Hered 42, 337 (1992)). For the CEPH Caucasian HapMap data, we calculated LD between pairs 40 of SNPs using the standard definition of D' (R. C. Lewontin, Genetics 50, 757 (1964)) and
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R2 W. G. Hill, A. Robertson, Genetics 60, 615 (Nov, 1968). When plotting all SNP combinations to elucidate the LD structure in a particular region, we plotted D' in the upper left corner and p-values in the lower right corner. In the LD plots we present, the markers are plotted equidistantly rather than according to their physical positions.
RESULTS
Genome-wide association study
We successfully genotyped 1399 Icelandic Type 2 diabetes patients and 5275 10 population control individuals using the Illumina 330K chip. Association analysis was performed using single SNPs, two marker haplotypes and extended haplotypes within LD blocks. After correcting the p-value for relatedness we identified 49 single markers and two marker haplotypes at 21 loci (i.e. genetic susceptibility locations in the genome) that had a p-value less than 5xl0"5 (Table 1). In addition, 10 extended haplotypes at 8 15 additional loci were selected by the same criteria (Table 2). Within the patient group, 700 individuals were non-obese (BMI<30) and those were tested separately for association. After correcting the p-value for relatedness, 36 single markers and two marker haplotypes at 20 loci had a p-value less than 5xl0"5 (Table 3). Three of those loci were also identified when the total group was analysed. In addition 6 extended haplotypes at 4 additional loci 20 were selected by the same criteria (Table 4). The obese group of 531 patients (BMI >30) was also analysed separately for association. After correcting the p-value for relatedness 38 single markers and two marker haplotypes at 16 loci had a p-value less than 5xl0"5 (Table 5). One of those loci was also identified when the total group was analysed but no overlap was found between the non-obese and obese groups using this criteria. In 25 addition 10 extended haplotypes at 7 additional loci had a p-value less than 5xl0"5 in association analysis of obese diabetics (Table 6).
The single-marker association and two-marker and extended haplotype association analysis presented in Tables 1-6 thus represents evidence for multiple susceptibility variants for Type 2 diabetes. It should be noted that for single-marker SNP analysis as 30 presented herein, susceptibility variants can either be represented by increased risk, wherein one allele is overrepresented in the patient group compared with controls. Alternatively, the susceptibility variants can be represented by the other allele of the SNP in question - for that allele, under-representation in patients compared with controls is expected. This is a natural consequence of association analysis to genetic elements 35 comprising two alleles. For multi-marker haplotypes or for polymorphic markers comprising more than one marker, at-risk association may be observed to one (or more) at-risk allele or haplotype. Protective variants in form of association (with RR-values less than unity) to one (or more) protective variants or haplotypes may also be observed, depending on the genetic composition and haplotype structure in the genetic region in 40 question.
Table 1. Single markers and two marker haplotypes associated with Type 2 Diabetes. Associating alleles are indicated in front of each marker (Allelic code: A=l, C=2, G=3, T=4)
Chr
Pos
Punadj
Padj
Rrisk
Aff.frq
Ctrl.frq
Haplotype chrl
151511890
4.01 E-06
4.49E-05
1.223
0.407
0.360
3 rs3738028
chr2
40560735
2.41 E-06
3.06E-05
1.225
0.593
0.543
1 rs13414307
chr2
40560735
4.59E-07
8.27E-06
1.243
0.571
0.517
1 rs13414307 3 rs1990609
chr2
54969849
.53E-08
1.56E-06
1.287
0.335
0.281
3 rs930493 4 rs10173697
chr2
54977961
3.12E-06
3.75E-05
1.224
0.553
0.503
4 rs10173697
chr3
89323970
2.60E-06
3.25E-05
1.380
0.904
0.872
4 rs12486049
chr6
6965113
1.00E-06
1.53E-05
1.705
0.072
0.044
1 rs490213 3 rs814174
chr6
31556294
3.22 E-06
3.78E-05
1.232
0.372
0.325
2 rs2516424
chr6
31556294
1.93E-06
2.57E-05
1.240
0.368
0.320
2 rs2516424 2 rs4947324
chr6
132422361
3.10E-06
3.74E-05
1.262
0.278
0.234
3 rs9483377 2 rs997607
chr6
132422361
3.97E-06
4.54E-05
1.252
0.276
0.233
3 rs9483377 3 rs7745875
chr6
132422361
7.98E-07
1.25E-05
1.249
0.356
0.307
3 rs9483377
chr6
150460378
.01 E-07
8.86E-06
1.293
0.794
0.749
1 rs11155700
chr6
150461077
.15E-07
9.05E-06
1.292
0.794
0.749
2 rs12213837
chr6
164474219
3.07E-06
3.63E-05
0.813
0.479
0.531
4 rs206732 2 rs933251
chr7
87951463
4.36E-06
4.89E-05
1.273
0.753
0.705
1 rs2192319
chr8
124196776
1.21 E-06
1.78E-05
1.253
0.721
0.673
3 rs952656
chr8
124202699
.97E-07
9.96E-06
0.722
0.108
0.143
4 rs13252935 3 rs7824293
chr9
90164936
2.03E-06
2.62E-05
1.304
0.192
0.154
1 rs10993008
chr9
95493692
2.38E-06
3.03E-05
1.253
0.309
0.263
3 rs10990568 3 rs4743148
chr9
95510129
.85E-07
9.80E-06
1.252
0.365
0.315
3 rs4743148
chr10
53058229
1.39E-06
1.98E-05
1.240
0.377
0.328
4 rs7915186 4 rs3829170
chr10
53063104
1.37E-06
1.96E-05
1.239
0.386
0.336
4 rs3829170 3 rs7922112
chr10
94301795
2.54E-08
8.44E-07
1.276
0.614
0.555
3 rs2421943
chr10
94301795
2.11E-09
1.19E-07
1.297
0.585
0.521
3 rs2421943 2 rs7917359
chr10
94304784
1.49E-07
3.32E-06
0.797
0.443
0.499
3 rs7908111 3 rs2497304
chr10
94309972
6.60E-09
2.85E-07
0.779
0.455
0.517
3 rs1999763 4 rs10882091
chr10
94309972
6.60E-09
2.85E-07
0.779
0.455
0.517
. 3 rs1999763 3 rs6583830
chr10
94337810
1.36E-06
1.91E-05
1.228
0.518
0.467
3 rs6583826
chr10
94337810
7.18E-08
1.91 E-06
1.262
0.449
0.393
3 rs6583826 2 rs10882091
chr10
94364357
7.76E-08
2.04E-06
1.259
0.466
0.410
2 rs10882091 3 rs7923837
chr10
94364357
9.33E-08
2.30E-06
1.256
0.472
0.415
2 rs10882091
chr10
94372930
9.81 E-08
2.40E-06
1.256
0.472
0.415
4 rs7914814
chr10
94388098
9.33E-08
2.30E-06
1.256
0.472
0.415
1 rs6583830
chr10
94442410
8.41 E-08
2.17E-06
1.256
0.527
0.470
1 rs2275729 3 rs1111875
chr10
94482696
7.56E-08
1.95E-06
1.258
0.542
0.485
1 rs2497304
chr10
94485733
1.64E-06
2.21 E-05
1.225
0.526
0.475
1 rs947591
chr12
33373479
3.87E-06
4.37E-05
1.391
0.110
0.082
4 rs1905421
chr15
98156854
3.80E-06
4.30E-05
0.815
0.469
0.521
1 rs9920347 3 rs11635811
chr16
22705353
2.93E-06
3.57E-05
1.264
0.781
0.738
4 rs724466
chr16
72066252
4.23E-06
4.68E-05
0.625
0.038
0.059
2 rs1862773 4 rs825842
chr16
72086481
.86E-07
9.82E-06
0.612
0.043
0.069
4 rs2432543 3 rs4887826
chr17
66072384
7.34E-07
1.20E-05
1.236
" 0.564
0.511
3 rs17763769 1 rs1860316
chr17
66117911
1.18E-07
2.77E-06
0.781
0.282
0.335
3 rs1860316 2 rs17763811
chr17
66117911
6.79E-08
1.83E-06
1.281
0.707
0.653
1 rs1860316
chr17 66132788
chr17 66149102
chr17 66159416
chr20 36391335
1.80E-06 2.43E-05
1.39E-06 1.99E-05
7.32E-07 1.19E-05
2.09E-07 4.45E-06
1.226 0.563
1.239 0.665
1.266 0.744
1.250 0.550
0.513 2 rs1981647
0.615 4 rs1843622
0.696 1 rs2191113
0.495 3 rs4592915 2 rs2232580
Table 2. Multi - marker haplotypes associated with Type 2 Diabetes. Chr Pos Punadj Padj Rrisk Aff.frq Ctrl.frq Haplotype*
Chr2 19652497 2.00E-08 6.98E-07 2.492 0.027 0.011
3 rs1593746 3 rs4666491 3 re12710718 4 rs1579204 1 rs824506 2 rs1344652 1 rs4109456 3 rs1427547 2 rs1522490 1 rs6757410 4 rs1863776 Chr2 74747736 1.95E-06 2.59E-05 1.903 0.036 0.019
2 rs363674 2 rs759075 1 rs4853033 1 rs205651 4 rs363608 1 rs1063588 2 rs363612 1 rs150139 2 rs363617 4 rs1137 4 rs828902 1 rs205627 chr9 29300367 5.32E-07 9.29E-06 1.813 0.042 0.024
1 rs4879332 2 rs1928663 4 rs2183357 2 rs10813050 2 rs1928661 4 rs10491662 2 rs1169758 2 rs1169757 3 rs12378755 chr9 32290296 4.13E-06 4.68E-05 1.489 0.075 0.052
3 rs1537156 2 rs7024902 4 rs7037573 4 rs3928808 4 rs10970902 3 rs1331226 3 rs10758127 1 rs1331231 1 rs992710 2 rs1411866 3 rs10511901 3 rs2094703 1 rs7854942 4 rs2150637 Chr11 22912998 7.25E-07 1.19E-05 1.687 0.059 0.036
3 rs11026796 1 rs1019216 2 rs2302423 4 rs4923035 1 rs2429777 4 rs12575930 3 rs887567 2 rs733295 3 rs7113718 1 rs7934814 4 rs3909703 4 rs3862134 3 rs10833917 1 rs6483890 2 rs2433454 chr13 60726830 1.52E-06 2.12E-05 1.481 0.108 0.075
4 rs1411145 4 rs9539100 3 rs991666 3 rs1026924 3 rs4886330 3 rs1411568 3 rs1028965 1 rs9670441 chr16 72082296 1.71 E-06 2.29E-05 0.595 0.033 0.054
4 rs1424011 2 rs1862778 1 rs4888373 4 rs8053178 4 rs825842 4 rs2432543 2 rs6564272 3 rs4887826 3 rs825851 chr17 66118095 3.46E-08 1.05E-06 0.762 0.229 0.281
2 rs16913 2 rs10512540 3 rs17763769 1 rs2109051 3 rs1860316 3 rs9904090 4 rs1981647 2 rs1843622 2 rs4584866 3 rs17791650 3 rs9891997 3 rs2191113 Chr18 67477090 1.12E-06 1.64E-05 0.547 0.033 0.059
2 rs9956771 4 rs8088887 2 rs10514019 4 rs719328 4 rs1942399 2 rs1942396 4 rs948665 3 rs11151691 chrX 56884473 4.32E-06 4.85E-05 1.184 0.709 0.673
1 rs12858633 1 rs5960235 3 rs5914036 3 rs6612746
* Associating alleles are indicated in front of each marker (Allelic code: A=l, C=2, G=3, T=4)
82
Table 3. Single markers and two marker haplotypes associated with Type 2 Diabetes in non-obese patients
Chr
Pos
Punadj
Padj
Rrisk
Aff.frq
Ctrl.frq
Haplotype*
chrl
29759353
.23E-06
3.18E-05
0.661
0.104
0.149
4 rs4949283 2 rs502545
chr2
53360168
8.51 E-06
4.70E-05
1.411
0.855
0.807
1 rs1424963
chr5
87772535
1.95E-06
1.36E-05
1.394
0.244
0.188
3 rs10505855 2 rs12514611
chr6
6965113
.76E-06
3.39E-05
1.891
0.080
0.044
1 rs490213 3 rs814174
chr6
20650200
8.46E-06
4.68E-05
1.327
0.307
0.250
3 rs7758851 2 rs1569699
chr6
20771314
1.06E-06
8.14E-06
1.369
0.292
0.232
1 rs4712527 3 rs7756992
chr6
20787289
4.47E-06
2.79E-05
1.333
0.315
0.256
2 rs1569699
chr6
20787688
1.78E-06
1.28E-05
0.741
0.682
0.743
1 rs7756992 3 rs9295478
chr6
20787688
1.11 E-06
8.61 E-06
1.368
0.292
0.232
3 rs7756992
chr9
95447272
6.08E-06
3.61 E-05
0.764
0.469
0.536
2 rs10818991 2 rs10990303
chr11
23939149
3.05E-06
2.02E-05
1.525
0.128
0.088
4 rs1879230
chr11
130184827
9.00E-06
4.93E-05
1.303
0.416
0.353
4 rs11222327 1 rs1939905
chr13
26578564
2.15E-06
1.51 E-05
0.723
0.220
0.281
1 rs565707 1 rs6491198
chr13
26578564
8.29E-07
6.63E-06
1.381
0.763
0.700
2 rs565707
chr13
26635031
3.14E-06
2.03E-05
1.309
0.606
0.540
2 rs7984685
chr13
26637643
3.37E-06
2.15E-05
1.308
0.606
0.540
2 rs7998347
chr13
26801814
9.09E-06
4.97E-05
1.340
0.771
0.716
1 rs1333350
chr13
26801814
1.29E-06
9.76E-06
0.709
0.195
0.254
3 rs1333350 4 rs7987436
chr13
108034018
9.08E-06
4.97E-05
1.322
0.732
0.674
2 rs4771591
chr16
12697094
8.10E-06
4.59E-05
0.616
0.068
0.105
2 rs6498353 3 rs9941146
chr17
66072384
2.10E-07
2.09E-06
1.347
0.585
0.511
3 rs17763769 1 rs1860316
chr17
66117911
1.01E-09
2.42E-08
0.677
0.254
0.335
3 rs1860316 2 rs17763811
chr17
66117911
1.20E-09
2.73E-08
1.462
0.734
0.653
1 rs1860316
chr17
66132788
7.18E-07
.88E-06
1.329
0.583
0.513
2 rs1981647
chr17
66149102
4.33E-07
3.84E-06
1.355
0.684
0.615
4 rs1843622
chr17
66159416
4.49E-09
8.28E-08
1.467
0.771
0.696
1 rs2191113
chr17
66173475
4.75E-06
2.88E-05
1.472
0.885
0.839
1 rs9890889
chr18
41053807
4.27E-06
2.68E-05
1.389
0.218
0.167
3 rs10502860
chr18
63441694
8.25E-06
4.66E-05
0.687
0.121
0.167
4 rs764133 4 rs7237209
chr18
63465082
4.35E-06
2.67E-05
1.443
0.867
0.819
2 rs7237209
chr19
3316583
7.55E-06
4.33E-05
1.370
0.227
0.176
1 rs3810420
chr20
36391335
8.38E-06
4.65E-05
1.292
0.558
0.495
3 rs4592915 2 rs2232580
chr21
13769165
3.83E-06
2.40E-05
1.599
0.927
0.888
1 rs468601
chr21
33298252
1.17E-06
9.03E-06
1.358
0.311
0.249
3 rs2834061
chr21
39374906
4.04E-06
2.51 E-05
1.308
0.631
0.566
4 rs369906
chr22
29580921
8.60E-06
4.75E-05
1.347
0.265
0.212
3 rs8142410 3 rs5994353
*Associating alleles are indicated in front of each marker (Allelic code: A=l, C=2, G=3, T=4)
Table 4. Multi- marker haplotypes associated with Type 2 Diabetes in non-obese patients Chr Pos Punadj Padj Rrisk Aff.frq Ctrl.frq Haplotype*
chr2 19652497 3.14E-07 2.93E-06 2.859 0.031 0.011
3 rs1593746 3 rs4666491 3 rs12710718 4 rs1579204 1 rs824506 2 rs1344652 1 rs4109456 3 rs1427547 2 rs1522490 1 rs6757410 4 rs1863776
chr5 2458281 6.12E-06 3.62E-05 0.077 0.001 0.017
3 rs931283 1 rs160730 3 rs468085 4 rs464716 3 rs10052956 2 rs160729 3 rs315914 1 rs1039096
chr6 137323498 6.46E-06 3.73E-05 2.566 0.040 0.016
2 rs6570118 4 rs7743308 3 rs6928748 2 rs12214917 2 rs6936698 2 rs4896224 2 rs10872468
chr11 32116221 4.15E-06. 2.57E-05 1.362 . 0.266 0.211
1 rs224633 3 rs581573 4 rs223070 4 rs10488686 4 rs4922579 2 rs110688 4 rs1605271 3 rs4922901 3 rs7950374 1 rs1033584 1 rs12788147 3 rs11031625 2 rs880587 4 rs989570 2 rs10835861 chr17 66118095 7.82E-10 1.95E-08 0.660 0.205 0.281
2 rs16913 2 rs10512540 3 rs17763769 1 rs2109051 3 rs1860316 3 rs9904090 4 rs1981647 2 rs1843622 2 rs4584866 3 rs17791650 3 rs9891997 3 rs2191113 chr17 66204022 6.39E-06 3.76E-05 0.683 0.115 0.160
2 rs9890889 4 rs2367005 2 rs2109054 3 rs17792120 1 rs7221340 4 rs1486293 2 rs1486296 2 rs17763811 4 rs9807096 3 rs10512541 3 rs8065001 2 rs4793501 3 rs929474 3 rs9907514
*Associating alleles are indicated in front of each marker (Allelic code: A=l, C=2, G=3, T=4)
84
Table 5. Single markers and two marker haplotypes associated with Type 2 Diabetes in obese patients
Chr
Pos
Punadj
Padj
Rrisk
Aff.frq
Ctrl.frq
Haplotype*
chrl
104818519
.60E-06
2.85E-05
1.343
0.466
0.394
2
rs7553985
chrl
104824377
4.76E-06
2.48E-05
1.346
0.466
0.393
4
rs2166890
chrl
104825870
6.28E-06
3.14E-05
1.355
0.396
0.317
4
rs7552405
chr3
147025256
7.11 E-06
3.49E-05
1.696
0.097
0.059
3
rs7630694
chr3
197065940
2.81 E-06
1.58E-05
1.396
0.737
0.668
1
rs9858622
chr4
140287637
4.41 E-06
2.32E-05
1.431
0.804
0.741
1
rs13116075 1 rs6824182
chr4
140364285
1.05 E-05
4.86E-05
0.708
0.194
0.254
4
rs2292837 2 rs11725721
chr4
140397800
8.21 E-06
3.95E-05
0.704
0.194
0.254
3
rs3762864 2 rs11725721
chr5
76586085
9.46E-06
4.46E-05
0.750
0.438
0.510
1
rs832785 1 rs2859576
chr5
76586766
8.97E-06
4.26E-05
1.333
0.562
0.491
4
rS4704400
chrf>
9509965
7.50E-06
3.66E-05
1.335
0.495
0.424
4
rs214447
chr6
22837279
1.03E-05
4.80E-05
1.430
0.824
0.766
2
rs10498713 3 rs4426986
chr6
41191330
3.22E-06
1.77E-05
1.360
0.637
0.563
1
rs10456499
chr8
128358773
4.94E-06
2.56E-05
0.692
0.190
0.254
2
rs283710 2 rs412835
chr8
128362648
6.35E-07
4.42E-06
1.495
0.822
0.755
3
rs185852
chr8
128376264
1.57E-06
9.59E-06
0.680
0.189
0.255
2
rs283718 1 rs283720
chr9
126494483
2.67E-06
1.51 E-05
1.591
0.139
0.092
4
rs3814120
chr10
94301795
.53E-07
3.93E-06
1.393
0.602
0.521
3
rs2421943 2 rs7917359
chr10
94304784
8.39E-06
4.02E-05
0.747
0.427
0.499
3
rs7908111 3 rs2497304
chr10
94309972
3.74E-06
2.01 E-05
0.739
0.442
0.518
3
rs1999763 4 rs10882091
chr10
94309972
3.74E-06
2.01 E-05
0.739
0.442
0.518
3
rs1999763 3 rs6583830
chr10
94337810
1.89E-06
1.12E-05
1.364
0.469
0.393
3
rs6583826 2 rs10882091
chr10
94364357
1.76E-06
1.05E-05
1.363
0.486
0.410
2
rs10882091 3 rs7923837
chr10
94364357
2.58E-06
1.47E-05
1.355
0.491
0.415
2
rs10882091
chr10
94372930
2.66E-06
1.51 E-05
1.355
0.491
0.416
4
rs7914814
chr10
94388098
2.58E-06
1.47E-05
1.355
0.491
0.415
1
rs6583830
chr10
94482696
1.62E-06
9.85E-06
1.363
0.562
0.485
1
rs2497304
chr10
118562511
8.21 E-06
3.95E-05
1.384
0.302
0.238
4
rs1681748 4 rs2170862
chr10
118610986
9.43E-06
4.45E-05
1.367
0.320
0.256
4
rs2170862
chr10
118880683
3.29E-06
1.80E-05
1.379
0.347
0.278
3
rs10787760
chr11
106441899
8.79E-06
4.18E-05
1.533
0.142
0.097
4
rs1455593
chr12
30340321
4.54E-06
2.38E-05
0.723
0.296
0.368
1
rs1429622 3 rs1506382
chr14
81787150
3.94E-06
2.10E-05
1.363
0.439
0.365
1
rs799099 3 rs4899801
chr14
81843593
8.25E-06
3.97E-05
1.339
0.437
0.367
3
rs2066041
chr14
81899972
9.32E-06
4.40E-05
1.331
0.530
0.459
1
rs10483957
chr14
87823315
9.69E-07
6.35E-06
1.605
0.891
0.836
3
rs419028
chr16
24287484
6.15E-06
3.08E-05
1.388
0.372
0.300
1
rs11074618 2 rs985729
chr19
3065864
1.02 E-05
4.77E-05
1.433
0.825
0.767
3
rs3746069
*Associating alleles T=4)
are indicated in front of each marker (Allelic code:
A=l, C=2, G=3,
Table 6. Multi- marker haplotypes associated with Type 2 Diabetes in obese patients ®
o o
00
Chr Pos Punadj Padj Rrisk Aff.frq Ctrl.frq Haplotype* ®
•j\
chr2 2591675 4.35E-06 2.29E-05 0.654 0.126 0.181 S
4 rs7576292 4 rs6548079 4 rs1451199 1 rs2385306 2 rs1020530 1 rs12714359 2 rs7556672 3 rs1451198 chr4 112032007 7.13E-06 3.50E-05 1.699 0.097 0.060
2 rsl 6997168 4 rs2723316 1 rs6419178 3 rs1448817 3 rs2634073 2 rs2200733 2 rs2220427 2 rsl 3105878 3 rsl 0033464 Chr8 128361033 7.34E-07 5.01E-06 0.671 0.178 0.244
3 rs283709 2 rs283710 2 rs4871780 1 rsl 85852 2 rs412835
Chr10 68829632 4.50E-06 2.36E-05 2.428 0.039 0.017
4 rs7094426 1 rs1904614 3 rs10823028 3 rs2620924 1 rs12359451 2 rs11815372 3 rs7083570 3 rs2394375 2 rs1875151 4 rs10823057 4 rs6480272 3 rs1911356 chr11 106076550 9.88E-06 4.63E-05 0.655 0.114 0.164
3 rs1791587 3 rsl 793083 2 rs1791597 4 rs7104111 2 rsl 793064 1 rs4523664 2 rs623018 4 rs631214 3 rs602159 2 rsl 0890568 2 rs4553343 4 rsl 487906 3 rs4.121676 1 rs4121677 4 rs6588924 Chr13 94045239 4.93E-06 2.55E-05 0.058 0.001 0.012
1 rs726298 2 rs7339106 1 rs9556403 2 rs9590039 2 rs6492722 1 rs1572935 3 rs6492725 ,J' chr14 81810554 9.82E-07 6.42E-06 1.408 0.341 0.269
4 rs9323719 2 rs7143860 3 rs709900 2 rs10135954 1 rs799103 1 rs799099 3 rs8018202 4 rs709915 3 rs709918 3 rs2066041 1 rs1457990 3 rs4899801 1 rs10483957 chr15 63410029 6.68E-06 3.31 E-05 2.395 0.047 0.020
4 rs2019185 2 rs920688 1 rs894494 3 rs665287 1 rs626163 2 rs639812 2 rs894491 1 rs581427 4 rs603439 1 rs678113 2 rs602192 3 rs7182756 1 rs2280345 3 rs11071841 1 rs2277582 chr15 95944049 4.24E-06 2.25E-05 0.593 0.079 0.127
2 rs8029926 4 rs649034 4 rs2036348
chr18 38114511 4.94E-06 2.56E-05 0.555 0.055 0.094 4 rs9304267 3 rs3763494 1 rs882291 2 rs898785 3 rsl 1082268 4 rs8088748 2 rsl 0502781 3 rs717127 chr20 45233401 3.10E-06 1.71 E-05 1.397 0.322 0.254
1 rs6063073 4 rs6066209 3 rs2018876 2 rs3092781 4 rs6122563 3 rs8126262 1 rs6063083 3 rs6018337 4 rs7262634 pS
*Associating alleles are indicated in front of each marker (Allelic code: A=l, C=2, G=3, T=4) H
in o
o ©
o o o
In) O
86
Chromosome 6d22.3 locus
One of the most significant association signals for non-obese diabetic patients was identified by two single markers (rsl569699 and rs7756992) and two 2 marker haplotypes mapping to chromosome 6p22.3 (Table 3). These markers are located within one LD block at position 20634996-20836710 bases (NCBI Build 35) between markers rs4429936 and rs6908425 (SEQ ID NO:l; Figure 1). This LD block contains the 5' end including exons 1-5 of the gene CDK5 regulatory subunit associated protein 1-like 1 (CDKALl) (NM_017774). The CDKALl protein has catalytic activity as well as iron ion binding activity but the in vivo function in unknown. It is widely expressed including expression in pancreas.
To verify the association of rsl569699 and rs7756992 to Type 2 diabetes the two markers were genotyped in a Danish Type 2 diabetes case - control cohort and also in a US Caucasian cohort Type 2 diabetes cohort from the PENN CATH study (Table 7). The results show that the two markers are significantly associated with Type 2 diabetes in the Danish cohort and that it confers a similar risk in the US UPenn. cohort although the results do not reach statistical significance. When the two replication cohorts are combined the results are significant with a risk of around 1.2. When all the cohorts are combined the risk for each marker is over 1.2 comparing a group of nearly 3000 Type 2 diabetes patients (not accounting for BMI) and over 8000 controls. These results are genome wide significant.
WO 2008/065682 PCT/IS2007/000020
87
Table 7. Association of rsl569699 and rs7756992 to Type 2 diabetes
Iceland rs-Name Allele Chr Pos (B35) Aff.n Aff.frq Ctrl.n Ctrl.frq Rrisk Padj rsl 569699
2
chr6
20787289
1397
0.297
5264
0.256
1.224
0.000158
rs7756992
3
chr6
20787688
1398
0.270
5271
0.232
1.228
0.000204
Denmark (Steno)
rs-Name
Allele
Chr
Pos (B35)
Aff.n
Aff.frq
Ctrl.n
Ctrl.frq
Rrisk
P
rsl 569699
2
chr6
20787289
1108
0.361
2346
0.321
1.200
0.00079
rs7756992
3
chr6
20787688
1131
0.320
2361
0.274
1.247
0.000078
Upenn
rs-Name
Allele
Chr
Pos (B35)
Aff.n
Aff.frq
Ctrl.n
Ctrl.frq
Rrisk
P
rsl 569699
2
chr6
20787289
360
0.346
522
0.308
1.185
0.09944
rs7756992
3
chr6
20787688
392
0.293
690
0.261
1.176
0.103824
Combined replication cohorts
rs-Name
Allele
Chr
Pos (B35)
Aff.n
Aff.frq
Ctrl.n
Ctrl.frq
Rrisk
Pmh rsl 569699
2
chrf>
20787289
1468
1.195
0.00002
rs7756992
. 3
chr6
20787688
1523
3051
-
1.221
2.8E-06
Combined all cohorts
rs-Name
Allele
Chr
Pos (B35)
Aff.n
Aff.frq
Ctrl.n
Ctrl.frq
Rrisk
Pmh rsl 569699
2
chrf>
20787289
2865
1.207
1.1E-07
rs7756992
3
chr6
20787688
2921
-
8322
.
1.224
1.9E-09
These results show significant association to the 20634996-20836710 bp region (Build 34) on chromosome 6, between markers rs4429936 and rs6908425, in Type 2 diabetes. Values for relative risk (RR) are comparable in all three cohorts; the lack of significant association at the 0.05-level in the UPenn cohort is mainly due to lack of power compared with the other cohorts, although the RR value is slightly lower in this cohort as compared with Iceland (RR of 1.185 compared with 1.224 for rsl569699). Furthermore, RR-values for non-obese Type 2 diabetes patients in Iceland are even higher (RR = 1.33 for rsl569699).
Chromosome 10a23.33 locus
Seven single markers and seven two marker haplotypes in a region on chromosome 10q23.33 were found to be associated with Type 2 diabetes (Table 1). Most of those markers are also associated to diabetes with elevated RR values when obese patients are analysed separately (Table 5). These markers are located within one LD block between positions 94192885and 94490091 (NCBI Build 35), corresponding to the genomic segment bridged by markers rs2798253 and rslll87152 (Figure 2). This LD block contains three genes, Insulin-degrading enzyme (IDE)
88
(NM_004969), Kinesin family member 11 (KIFll) (NM_004523) and Homeobox, hematopoietically expressed (HHEX) (NM_002729).
IDE may belong to a protease family responsible for intercellular peptide signalling. Though its role in the cellular processing of insulin has not yet been defined, insulin-degrading enzyme is thought to be involved in the termination of the insulin response (Fakhrai-Rad et al, Human Molecular Genetics 9:2149-2158, 2000). Genetic analysis of the diabetic GK rat has revealed 2 amino acid substitutions in the IDE gene (H18R and A890V) in the GK allele which reduced insulin-degrading activity by 31% in transfected cells. However, when the H18R and A890V variants were studied separately, no effects were observed, suggesting a synergistic effect of the 2 variants on insulin degradation. No effect on insulin degradation was observed in cell lysates, suggesting that the effect may be coupled to receptor-mediated internalization of insulin. Congenic rats with the IDE GK allele displayed postprandial hyperglycemia, reduced lipogenesis in fat cells, blunted insulin-stimulated glucose transmembrane uptake, and reduced insulin degradation in isolated muscle. Analysis of additional rat strains demonstrated that the dysfunctional IDE allele was unique to GK rats. The authors concluded that IDE plays an important role in the diabetic phenotype in GK rats. IDE has been studied as a candidate gene for Type 2 diabetes in humans with inconsistent results. Two large studies have recently analysed the association of IDE to Type 2 diabetes by mutation screening and haplotype analysis using tagging SNPs over the gene (Groves et al, Diabetes 52:1300-1305, 2003; Florez et al, Diabetes 55:128-135, 2006). Both studies conclude that common variants in IDE are unlikely to confer significant risk of Type 2 diabetes. These studies did however, not include the whole LD block as defined in figure 2 and at least some of the markers identified in our study as associated with Type 2 diabetes are outside the regions analysed in those previous studies. Based on the results reported here, markers in LD with IDE are associated with Type 2 diabetes, providing genetic evidence for the role of IDE in the etiology of Type 2 diabetes.
KIFll encodes a motor protein that belongs to the kinesin-like protein family. Members of this protein family are known to be involved in various kinds of spindle dynamics. The function of this gene product includes chromosome positioning, centrosome separation and establishing a bipolar spindle during cell mitosis. This gene is not a good functional candidate for diabetes but has to be considered as a positional candidate due to its location within the associated LD block.
HHEX encodes a member of the homeobox family of transcription factors, many of which are involved in developmental processes. Expression in specific hematopoietic lineages suggests that this protein may play a role in hematopoietic differentiation. HHEX is essential for pancreatic development; in HHEX negative mouse embryos there is a complete failure in ventral pancreatic specification (Bort et al, Development 131, 797-806, 2004). Other transcription factors involved in pancreatic development include the MODY genes as well as other factors that have been implicated in late onset diabetes. HHEX is also an essential effector of Wnt antagonist for heart induction (Foley and Mercola, GENES & DEVELOPMENT 19:387-396, 2005). This puts HHEX in the same pathway as the recently established Type 2 diabetes gene TCF7L2 and together these data make HHEX a functional as well as positional candidate for Type 2 diabetes.
89
To verify the association of rs2497304, rs947591, rsl0882091 and rs7914814 to Type 2 diabetes, the markers were genotyped in a Danish Type 2 diabetes case - control cohort and also in a US Caucasian cohort Type 2 diabetes cohort from the PENN CATH study (Table 8). The results show that the association is not replicated in either cohort independently. However, when the two cohorts are combined the association of rs947591 reaches significance at the 0.05 level, with a risk of 1.1 in the combined cohort. When all the cohorts are combined the risk is 1.15 for the rs947591 marker.
These results indicate that variants within the LD block on Chromosome 10 that includes IDE and HHEX are susceptibility variants for Type 2 diabetes.
90
Table 8. Association analysis of markers on Chromosome 10 to Type 2 diabetes in Iceland, Denmark, and the US.
Iceland rs-Name Allele Chr Pos (B35) Aff.n Aff.frq Ctrl.n Ctrl.frq Rrisk Padj rsl 0882091
2
chr10
94364357
1399
0.472
5275
0.415
1.257
0.0000023
rs7914814
4
chr10
94372930
1399
0.472
5275
0.416
1.256
0.0000024
rs2497304
1
chr10
94482696
1399
0.542
5275
0.485
1.257
0.0000019
rs947591
1
chr10
94485733
1399
0.526
5273
0.475
1.226
0.0000221
Denmark (Steno)
rs-Name
Allele
Chr
Pos (B35)
Aff.n
Aff.frq
Ctrl.n
Ctrl.frq
Rrisk
P
rsl 0882091
2
chr10
94364357
1115
0.431
2341
0.413
1.077
0.15
rs7914814
4
chr10
94372930
1141
0.430
2360
0.410
1.088
0.10
rs2497304
1
chr10
94482696
1145
0.528
2348
0.509
1.079
0.14
rs947591
1
chr10
94485733
1140
0.502
2361
0.478
1.103
0.055
Upenn
rs-Name
Allele
Chr
Pos(B35)
Aff.n
Aff.frq
Ctrl.n
Ctrl.frq
Rrisk
P
rsl 0882091
2
chr10
94364357
386
0.377
640
0.375
1.008
0.93
rs7914814
4
chr10
94372930
394
0.379
683
0.381
0.995
0.95
rs2497304
1
chr10
94482696
408
0.460
778
0.454
1.021
0.81
rs947591
1
chr10
94485733
393
0.480
687
0.459
1.089
0.34
Combined replication cohorts
rs-Name
Allele
Chr
Pos(B35)
Aff.n
Aff.frq
Ctrl.n
Ctrl.frq
Rrisk
Pmh rsl 0882091
2
chr10
94364357
1501
1.052
0.19
rs7914814
4
chr10
94372930
1535
1.053
0.16
rs2497304
1
chr10
94482696
1553
1.057
0.16
rs947591
1
chr10
94485733
1533
-
3048
■
1.098
0.032
Combined all cohorts
rs-Name
Allele
Chr
Pos(B35)
Aff.n
Aff.frq
Ctrl.n
Ctrl.frq
Rrisk
Pmh rsl 0882091
2
chr10
94364357
2900
1.136
0.000017
rs7914814
4
chr10
94372930
2934
1.137
0.000012
rs2497304
1
chr10
94482696
2952
1.139
0.000011
rs947591
1
chr10
94485733
2932
-
8321
.
1.152
9.7E-07
Chromosome 17a24.3 locus
Five single markers and two two marker haplotypes in a region of chromosome 17q24.3 were found to be associated with Type 2 diabetes in non-obese patients (Table 3). Some of these markers show the strongest association reported in Table 3 and association to this region was also observed when all diabetics were analysed (Table 1). These markers are located within two adjacent LD blocks located between positions 66037656 and 66163076 (NCBI Build 35) on
91
chromosome 17, between markers rsll077501 and rs4793497 (Figure 3). The association is significant after correction for the number of tests performed in the single marker association analysis; i.e., the association is significant at the genome-wide level. No known genes are located within these LD blocks. However, it is possible that variants in this region affect genes in neighboring regions including KCNJ2 and KCNJ16. Alternatively these variants may affect unknown genes within these LD block regions.
92
Table 9. SNPs located within the CDKALl gene (Located between position 20,642,736 and 21,340,611 bp on Chromosome 6 in NCBI Build 35 and NCBI Build 36)
Pos Build 35/36
Marker ID
20642787
rs41271303
20642953
rsl 1963450
20643397
rs981043
20643513
rs981042
20643675
rsl 6883895
20643753
rsl 7512225
20643840
rs35035071
20643949
rs6904566
20644073
rs6927356
20644093
rs35281412
20644313
rs35915788
20644314
rs34025398
20644319
rs34361235
20644335
rs6905138
20644499
rsl 3194858
20644717
rs2179551
20644727
rs2179550
20644787
rs9465794
20644787
rs9465795
20644848
rs7747962
20644858
rs6910725
20644918
rs965054
20644971
rs2143407
20645032
rsl 0619380
20645431
rs2328525
20645661
rsl 3199286
20645841
rs10611252
20645940
rs7753499
20646023
rs7753956
20646024
rs34811195
20646024
rs7753670
20646107
rs3060613
20646109
rsl 1277970
20646110
rsl 1280099
20646114
rs6149468
20646139
rsl 6883900
20646175
rs7774291
20646441
rs10612082
20646476
rs9368198
20646502
rsl 3203336
20646504
rsl 3203631
20646619
rs6456355
20646644
rsl 0484635
20647190
rsl 2204173
20647320
rsl 3207544
20647851
rsl 2198728
20647984
rs28396084
20648327
rsl 2199073
20648500
rs9465796
20648561
rsl 2212600
20648596
rsl 3212040
20648663
rs35291340
20648722
rsl 2199324
20649085
rsl 2200871
20649159
rs9348432
Pos Build 35/36
Marker ID
20649183
rsl 2200834
20649236
rs34860173
20649324
rsl 1754872
20649498
rs6456356
20649517
rs9368199
20649682
rs2143406
20650176
rsl 0484634
20650200
rs7758851
20650398
rs34677076
20651447
rs6928571
20651461
rsl 2192584
20651608
rs34856684
20652015
rs9350255
20652091
rs9368200
20652136
rs12214002
20652245
rs9465797
20652300
rs9465798
20652574
rs28699301
20652650
rsl 3215844
20652678
rs12214315
20652722
rsl 1759517
20652786
rs13218957
20652806
rsl 3218962
20653186
rsl 0543744
20653201
rsl 2216047
20653447
rs9366354
20653890
rs9358342
20654091
rs9368201
20654382
rs34206163
20654506
rs9465799
20654794
rs34187071
20654867
rs9465800
20654890
rs6908974
20654992
rsl 3197372
20655361
rs13214145
20655793
rsl 6883910
20655968
rsl 2194705
20656271
rs35080661
20656465
rs7753467
20656466
rs7773488
20656986
rs34182285
20657084
rs34242699
20657780
rs9348433
20657942
rs9460519
20658083
rsl 2198377
20658096
rs9465801
20658195
rs9465802
20658822
rs28458932
20658823
rs9465803
20658981
rs2103682
20659321
rs9465804
20659580
rs34611621
20660058
rsl 2055423
20660653
rs9465805
20660829
rsl 1365187
Pos
Build
Marker ID
/36
20660836
rsl 1320714
20660918
rs9350256
20661764
rs7756211
20662069
rs9460520
20662498
rs34245467
20662930
rs9350257
20663855
rsl 1964554
20663990
rs9465806
20664109
rsl 1964635
20664190
rsl 3199421
20664314
rs6932320
20664570
rsl 2200078
20664659
rsl 3437555
20664884
rs9350258
20665256
rsl 2176441
20665260
rsl 2183826
20665264
rs9356738
20665272
rs9348434
20665343
rs9465807
20665804
rs4458667
20665995
rs7739402
20667590
rsl 6883914
20667591
rsl 6883916
20667900
rs9654584
20667999
rs9465808
20668414
rsl 7584626
20668565
rs7751682
20669667
rsl 1361279
20669681
rs34634263
20670059
rsl 2214549
20670364
rs7753519
20670575
rs28567007
20670597
rs7772137
20670719
rsl 2208597
20670998
rs9368202
20671877
rs2328526
20672452
rs34823358
20673287
rs28639914
20673363
rs34233572
20673415
rs4712506
20673935
rsl 3203450
20674280
rs9350259
20674435
rs6918457
20674595
rs35210537
20674749
rsl 1329887
20675016
rs9348435
20675068
rs35366106
20675342
rsl 6901563
20675352
rsl 2333229
20675520
rs9460521
20676092
rsl 0589899
20676351
rs2876573
20676957
rs6935461
20676963
rs6935465
20676968
rsl 0603174
Pos Build 35/36
Marker ID
20677060
rsl 2333291
20677967
rs2064321
20677985
rs35546893
20678018
rs4291090
20678121
rs2064320
20678268
rs9465810
20678275
rs9465811
20678423
rs9358344
20678756
rsl 0946390
20679114
rs6905281
20679339
rsl 6883932
20679612
rs34904067
20679660
rs7744002
20679763
rs35142564
20680095
rs9465812
20680678
rs7759094
20680784 .
rs9460522
20681538
rs7764551
20681585
rsl 0541455
20682409
rsl 6883935
20682542
rs13215603
20682568
rs962576
20683235
rsl 474720
20683797
rsl 6883944
20684155
rs34538343
20684269
rs9350260
20684353
rsl 6883951
20684645
rs9358345
20684862
rs1012627
20684890
rs9368203
20684939
rs35894322
20684965
rs4710932
20684984
rs6909117
20685540
rs1012626
20685748
rs1012625
20685760
rs7752194
20685958
rs9465813
20686014
rsl 2207923
20686355
rsl 6883963
20686831
rsl 3205786
20686887
rs35205364
20687102
rsl 0456232
20687189
rs9465814
20687201
rs35571892
20687740
rs9465815
20687753
rs36119371
20687921
rs28621813
20687926
rs9350261
20687928
rs7341226
20688175
rs6927481
20688323
rs35313444
20688373
rs6928198
20688404
rs6907897
20688545
rs6928586
20688872
rs9368204
93
Pos
Build
Marker ID
/36
20689021
rs9358346
20689589
rsl 1967546
20689593
rs34134803
20689772
rsl 0456233
20689807
rs7744833
20690122
rs9460523
20690123
rs9465816
20690432
rs6908077
20690630
rs9465817
20691069
rsl 1967445
20691263
rs34022950
20691793
rs9460524
20691994
rs34020592
20692003
rsl 1448102
20692339
rs9465818
20692402
rs9350262
20692513
rsl 3205241
20693000
rsl 2153939
20693100
rs6925593
20693119
rs4712507
20693225
rsl 0558806
20693267
rs35982532
20693276
rsl 1385529
20693360
rs9348436
20693416
rs9368206
20693438
rsl 3209542
20693452
rs9368207
20693630
rsl 3209907
20693635
rs6926658
20694018
rs12213132
20694182
rs4357125
20694554
rs6932944
20694607
rs6932962
20695026
rs9348437
20695332
rsl 2201857
20695356
rs9465819
20695447
rs6938955
20695539
rs9460525
20695827
rs9465820
20695964
rsl 0946391
20695968
rs9368208
20696003
rs9465821
20696183
rs6923790
20696399
rsl 0558139
20697309
rs6907459
20697320
rs6907767
20697321
rs9465822
20697349
rs6930283
20697706
rs6908042
20697741
rs6935317
20697761
rs35370102
20698266
rs9368209
20698366
rsl 3216746
20698367
rsl 3216747
20699007
rs35485532
20699747
rsl 3216324
20699817
rs4336434
20700046
rs4509107
20700428
rs9465823
20700465
rs6936705
Pos
Build
Marker ID
/36
20700679
rs34023799
20700929
rs6942313
20701057
rs28869917
20701318
rs34982231
20701631
rs9358349
20701770
rs9460526
20701829
rs9366356
20702163
rs36120092
20702181
rs9465824
20702519
rs4712512
20702561
rs4712513
20702646
rs4710934
20702658
rs9348438
20702902
rs9460529
20703363
rsl 3199587
20703470
rsl 3199384
20703526
rsl 0223680
20703606
rs9350263
20703768
rs9465825
20703832
rsl 0223876
20704100
rs35702271
20704171
rs9358350
20704432
rsl 2208985
20704771
rsl 2210459
20704892
rs35431707
20705144
rs36039523
20705297
rsl 1758281
20705350
rs28893199
20705757
rs34256347
20706019
rsl 2192740
20706282
rs13212326
20706486
rs12199184
20706753
rsl 0456234
20707009
rs4712514
20707422
rs9465826
20707607
rs9366357
20707867
rs2294809
20708549
rs2294808
20708813
rs7762750.
20708976
rs4712515
20708998
rsl 0522824
20708999
rs35660518
20709002
rsl 0679950
20709003
rs34870864
20709022
rs4712516
20709145
rs4710935
20709386
rs9465827
20709388
rsl 2204865
20709672
rsl 0946393
20709764
rsl 2209806
20709894
rsl 0946394
20709921
rsl 997778
20709971
rs35878587
20710359
rsl 997777
20710378
rs2223622
20710776
rsl 1964057
20711246
rs9460530
20711344
rs9460531
20711376
rs34329159
20711640
rs7764558
Pos Build 35/36
Marker ID
20711804
rs4710936
20712056
rsl 2213940
20712228
rsl 3215038
20712739
rsl 0946395
20712832
rs6939917
20712975
rs9358351
20713800
rs6925097
20713924
rs9465828
20713955
rs9465829
20713961
rs6902661
20714057
rs34373680
20714281
rs35051096
20714508
rs932405
20714591
rs6926585
20714635
rs3938395
20715464
rsl 1964664
20715551
rs35964987
20715663
rsl 2206413
20715758
rs35990187
20715763
rs4991654
20715910
rs9460532
20715991
rsl 3328250
20716030
rsl 3328252
20716194
rs4712517
20716257
rs4712518
20717220
rs7758129
20717475
rsl 3206462
20717483
rsl 3192442
20717486
rsl 3206468
20717492
rsl 3192445
20717498
rsl 3192450
20717504
rsl 3206477
20717510
rsl 3206483
20717577
rs12179168
20717586
rsl 2180975
20717611
rs12179172
20717860
rsl 2179563
20718357
rsl 1355836
20718696
rs2328527
20718709
rsl 1452882
20718920
rs2876574
20719905
rs9465831
20720031
rs34877824
20720290
rs13212501
20720647
rs9358352
20720703
rs9358353
20720761
rs28756205
20720889
rs9350265
20720989
rs7750508
20721130
rs7771052
20721141
rs9460533
20721195
rsl 3200415
20721216
rsl 0550932
20721312
rs9465832
20721463
rs9368211
20721471
rs9350266
20721507
rsl 1752592
20721515
rs9350267
20721754
rs978988
20721898
rs978987
Pos Build 35/36
Marker ID
20721906
rs978986
20722036
rs5874773
20722196
rs5874774
20722500
rs7756788
20722659
rs9348439
20722661
rs9356739
20722693
rs9356740
20722738
rs7760894
20723021
rs2796913
20723046
rs2608613
20723121
rsl 0456710
20723139
rs9476286
20723140
rs28368538
20723140
rs9476287
20723141
rs28612622
20723142
rs9463660
20723146
rs9463661
20723162
rs9461022
20723193
rs9461021
20723235
rs34980442
20723239
rs34049080
20723258
rs35218684
20723260
rs12175876
20723270
rsl 0948323
20723287
rs34400313
20723292
rs36081550
20723304
rsl 2175878
20723305
rs34520184
20723305
rs34756989
20723322
rs4629736
20723322
rs9296917
20723324
rs28562027
20723333
rs9381823
20723346
rs34769771
20723346
rs34774640
20723346
rs36038896
20723365
rsl 3209195
20723369
rs12213541
20723369
rs34112320
20723371
rs34615869
20723379
rs35949519
20723381
rs9257498
20723383
rs34200576
20723393
rs4960519
20723396
rs9261905
20723402
rs9267103
20723404
rsl 7367677
20723416
rs9261906
20723421
rs9267104
20723424
rs9267105
20723434
rs28810763
20723438
rs12179121
20723438
rs4714959
20723439
rsl 0946636
20723439
rs28763327
20723439
rs28847950
20723439
rs3933247
20723449
rs28808723
20723451
rs9767082
20723452
rs35132675
94
Pos Build 35/36
Marker ID
20723452
rs35517166
20723452
rs4714297
20723456
rsl 2182737
20723456
rs36163804
20723460
rs35790973
20723464
rs35236694
20723469
rs35567559
20723469
rs9267106
20723471
rs9800557
20723484
rsl 2182463
20723489
rsl 0948322
20723489
rs34052284
20723489
rs9268999
20723490
rs9258377
20723495
rs9257499
20723498
rs9265816
20723500
rs28771402
20723501
rs28771401
20723502
rs12194731
20723502
rs4451188
20723502
rs9717323
20723502
rs9765920
20723503
rs9265815
20723504
rs28771400
20723506
rs12190813
20723506
rs12215416
20723507
rsl 2178527
20723510
rs35234761
20723510
rs35887156
20723512
rs28797321
20723515
rs28771399
20723522
rsl 3207682
20723522
rs36099432
20723531
rs28749543
20723535
rsl 3196506
20723535
rs35716308
20723536
rs28831180
20723536
rs34938144
20723537
rs36142967
20723541
rs35313792
20723542
rs9260904
20723543
rs34597832
20723545
rs9767740
20723546
rs28771398
20723550
rs34384951
20723554
rsl 2524128
20723554
rsl 2665124
20723554
rs34922643
20723555
rs9260903
20723557
rs28771397
20723557
rs6915279
20723559
rs9261907
20723559
rs9766798
20723559
rs9767242
20723560
rs6914835
20723560
rs9260902
20723560
rs9767101
20723562
rs9261908
20723563
rsl 2207064
20723563
rs12213193
Pos
Build
Marker ID
/36
20723563
rs35750154
20723566
rs9260901
20723570
rs9269000
20723579
rsl 2178368
20723579
rs13197714
20723579
rs28771396
20723579
rs34443697
20723579
rs9267107
20723582
rs35113301
20723582
rs3933248
20723583
rs28819830
20723586
rsl 0947899
20723586
rs9260900
20723588
rsl 3204671
20723588
rs34094007
20723588
rs34963756
20723588
rs4304158
20723589
rs4298351
20723590
rsl 2182307
20723590
rsl 2201487
20723590
rs34097573
20723595
rs13219021
20723596
rs34456153
20723596
rs9767597
20723597
rs9767102
20723598
rs9269001
20723601
rsl 2180097
20723603
rsl 2178465
20723603
rs35128115
20723607
rs4273681
20723609
rsl 2193754
20723617
rs813814
20723617
rs9689672
20723619
rs36173068
20723623
rsl 2202172
20723623
rs28862376
20723625
rsl 2178884
20723625
rsl 2203117
20723625
rs34250588
20723626
rs12182581
20723632
rs34147797
20723635
rs9688484
20723636
rs9269002
20723636
rs9767733
20723637
rsl 0948989
20723637
rs34732676
20723637
rs9260899
20723638
rs35686233
20723639
rsl 1753098
20723639
rs35156647
20723646
rs34719653
20723646
rs35575623
20723648
rsl 2192046
20723649
rsl 1759854
20723649
rs28771395
20723649
rs35563402
20723649
rs36016334
20723649
rs4555911
20723653
rsl 1965757
20723654
rsl 0948990
Pos Build 35/36
Marker ID
20723654
rs28808296
20723654
rs34995142
20723654
rs809919
20723657
rsl 1571978
20723659
rsl 2208488
20723662
rsl 1751374
20723663
rs13217613
20723667
rsl 2528735
20723668
rs35160656
20723668
rs35322569
20723668
rs9766221
20723670
rs35120225
20723670
rs511868
20723674
rs9269003
20723675
rs34290316
20723675
rs35735496
20723675
rs9689102
20723676
rsl 0948991
20723676
rs9269004
20723677
rsl 3220607
20723677
rs34700647
20723677
rs9260898
20723678
rs9260897
20723684
rs28771394
20723686
rs12213200
20723687
rs9717987
20723688
rs28771393
20723688
rs9717716
20723689
rs28771392
20723690
rsl 1758009
20723690
rs34159662
20723690
rs34683172
20723690
rs9717331
20723691
rs9717853
20723692
rs9765875
20723696
rs12178974
20723696
rs36003577
20723698
rs9767747
20723700
rs28771391
20723704
rs13216352
20723707
rsl 2200762
20723707
rsl 2206373
20723707
rs34570202
20723707
rs36197940
20723707
rs9265814
20723714
rs34131282
20723717
rs12175478
20723720
rs28772692
20723722
rs35516674
20723722
rs35704013
20723727
rs34691406
20723728
rs28771390
20723728
rs4374863
20723730
rs34826149
20723734
rsl 2207894
20723734
rsl 2662476
20723736
rs9260896
20723739
rs28771389
20723740
rs28771388
20723741
rs9382592
Pos Build 35/36
Marker ID
20723745
rsl 2173375
20723745
rs9268072
20723745
rs9382110
20723746
rs4715211
20723747
rs35961188
20723747
rs34128950
20723747
rs4620119
20723749
rs9767236
20723752
rs34717143
20723755
rsl 3201202
20723756
rsl 3201503
20723762
rsl 3197088
20723762
rs35347849
20723766
rs35650828
20723766
rs34663083
20723767
rsl 3199241
20723767
rs34205031
20723768
rs34469031
20723768
rs28749541
20723768
rs9279137
20723769
rs9260895
20723770
rs35446958
20723770
rs12212483
20723775
rs28380829
20723777
rsl 2525384
20723777
rs34576984
20723782
rs283541
20723785
rs28380828
20723788
rsl 0948698
20723790
rs9265813
20723791
rs9689173
20723792
rs28380827
20723795
rs35399169
20723799
rs9260894
20723800
rsl 2173388
20723800
rs9269005
20723802
rs28359816
20723803
rs28380826
20723807
rs280297
20723807
rs34507582
20723807
rs9261623
20723810
rs12174621
20723813
rs28380825
20723815
rs35197377
20723816
rsl 7362870
20723816
rs35071522
20723816
rs34562190
20723816
rs35179751
20723816
rs4458721
20723817
rsl 2180385
20723817
rsl 2206581
20723817
rsl 2333308
20723817
rs9269006
20723821
rs9688475
20723829
rs9269007
20723832
rs9269008
20723832
rs9269009
20723834
rs35899754
20723836
rs13211190
20723837
rsl 2216274
95
Pos
Build
Marker ID
/36
20723838
rs12191544
20723838
rs4337934
20723841
rs9279295
20723842
rsl 2178577
20723849
rsl 2215604
20723853
rs9395360
20723854
rs28749540
20723855
rs28895226
20723855
rs9260893
20723861
rs4365925
20723864
rs9261912
20723865
rs28895227
20723870
rs9261913
20723873
rsl 2183502
20723873
rs34332895
20723873
rs9472692
20723876
rs35640475
20723884
rs9269010
20723888
rs12192337
20723888
rs35349786
20723891
rs34978372
20723893
rs280296
20723894
rsl 1755576
20723903
rs35553517
20723903
rs28803616
20723904
rs4358654
20723910
rs9260892
20723914
rsl 3196941
20723914
rs9717238
20723915
rsl 2175553
20723915
rs12191408
20723915
rs4460210
20723915
rs9260891
20723918
rs9395361
20723920
rs9766917
20723926
rs35073779
20723931
rs9765884
20723931
rs9767229
20723932
rs35247846
20723932
rs9717328
20723936
rs28805674
20723936
rs9260890
20723938
rs34926348
20723938
rs9717233
20723945
rs35869128
20723946
rs12175941
20723951
rs9260889
20723952
rs9766686
20723956
rsl 0046203
20723957
rs9260888
20723958
rsl 2180540
20723961
rs9766115
20723968
rs6912019
20723969
rs9767458
20723976
rsl 3202161
20723989
rsl 3218048
20724000
rsl 3218040
20724001
rsl 3206557
20724002
rsl 3202152
20724013
rs12210049
Pos
Build
Marker ID
/36
20724013
rs34552032
20724018
rs28890881
20724018
rs34478537
20724025
rs35191657
20724038
rs34450517
20724043
rs9689655
20724064
rs12180172
20724068
rs28865015
20724071
rs9717839
20724072
rs9717836
20724074
rs9885593
20724082
rs28887572
20724083
rs13216113
20724083
rs35254115
20724084
rsl 0949190
20724085
rs6923503
20724087
rsl 2214411
20724091
rs28747986
20724096
rsl 087363
20724096
rs1091092
20724104
rsl 2180759
20724107
rsl 1758052
20724112
rs9261321
20724113
rs28861738
20724113
rs36109104
20724123
rsl 3208115
20724129
rs9472693
20724130
rsl 2208570
20724130
rs28832660
20724131
rsl 3200482
20724131
rs9260886
20724131
rs9472694
20724159
rs9260885
20724172
rs280295
20724172
rs28749538
20724174
rsl 3201041
20724184
rsl 087362
20724187
rsl 2183850
20724190
rsl 2202552
20724208
rs9260884
20724210
rs9474341
20724211
rs9260883
20724222
rs34669820
20724225
rsl 2189886
20724234
rs12182114
20724266
rs9688643
20724272
rsl 2195237
20724281
rs35293595
20724281
rs9381428
20724306
rs34665644
20724309
rsl 2189599
20724311
rsl 2208503
20724312
rs9800791
20724330
rsl 2173681
20724337
rsl 2182094
20724343
rs12182119
20724352
rs12182102
20724392
rsl 2174506
20724393
rs9767809
20724394
rsl 2173703
Pos
Build
Marker ID
/36
20724401
rsl 2202889
20724435
rs28840538
20724466
rs9473983
20724467
rs34220946
20724487
rs9261322
20724491
rs9261323
20724517
rs526092
20724564
rs9473982
20724566
rs34307764
20724626
rs34490559
20724665
rs4449621
20724685
rs4714818
20724698
rs4714817
20724722
rs682384
20724738
rs682051
20724753
rsl 2530056
20724756
rs35086683
20724888
rsl 2528468
20725092
rsl 0650195
20725092
rs35956126
20725109
rs6937578
20725159
rsl 2530107
20725173
rs720448
20725176
rs9368212
20725250
rs35481531
20725253
rs720449
20725262
rs10650196
20725495
rs2064
20725499
rs2065
20725638
rs9368213
20725732
rs41455744
20726077
rs34960654
20726154
rs6900954
20726168
rs6456359
20726176
rs7739405
20726601
rs7760508
20726684
rsl 0456007
20726987
rs9460534
20727168
rsl 1966749
20727353
rs9358354
20727809
rs9368214
20727982
rs9350268
20728095
rs2069013
20728160
rs2069014
20728290
rs2069015
20728292
rsl 1967475
20729096
rs10214549
20729217
rs2223621
20729281
rs9465835
20729984
rs35404829
20729995
rsl 0687080
20730233
rs6915936
20730331
rs9295472
20730732
rsl 569660
20730929
rs7455009
20730945
rsl 569659
20730946
rs5874775
20730948
rsl 0601252
20730983
rsl 569658
20731030
rs34142400
Pos
Build
Marker ID
/36
20731063
rsl 6883996
20731181
rs35073110
20731184
rs5028948
20731263
rs34201758
20731371
rs6456360
20731373
rs6456361
20731380
rs6456362
20731584
rs6922571
20731925
rs35145358
20731950
rs9465836
20732013
rs7763304
20732158
rs9465837
20732218
rs7743314
20732337
rs714831
20732361
rs714830
20732484
rs7743789
20732584
rs5874776
20732589
rs5874777
20732965
rsl 6884003
20733079
rs9356741
20733080
rs9366358
20733246
rs9356742
20733430
rsl 1756987
20733470
rs9465838
20733613
rs2206579
20733920
rs35111339
20734361
rs6917583
20734388
rs6917599
20734630
rsl 3437429
20735418
rs34307011
20735420
rs4515379
20735756
rs2223620
20735870
rs9465839
20735993
rsl 2664972
20736790
rs28402356
20736798
rs6934727
20736873
rs35493429
20736881
rsl 1369825
20737261
rs4523079
20737470
rs7771213
20737568
rs9465840
20737687
rs9465841
20737767
rs9465842
20737837
rs9460535
20737992
rs34835908
20738060
rs9465843
20738139
rsl 3220465
20738159
rsl 3220352
20738321
rs4710937
20738375
rs9460536
20738376
rsl 3190734
20738451
rs9465844
20738712
rs35976895
20738713
rsl 1327958
20738932
rsl 3194407
20738990
rsl 0484633
20739524
rs2328528
20739789
rs35958155
20739809
rs4421185
20739932
rs2328529
96
Pos
Build
Marker ID
/36
20740089
rs9460537
20740745
rsl 7224527
20740886
rs34166991
20741024
rsl 7823073
20741276
rsl 2183074
20741316
rs9465845
20741844
rs35148963
20741886
rs7768642
20742320
rs9465846
20742407
rs9465847
20742551
rsl 7823127
20742594
rsl 6884021
20742630
rs4533976
20742865
rs7755830
20743005
rs7756031
20743139
rs35650451
20743188
rs10561117
20743241
rs6940200
20743799
rs9465848
20743808
rs34351919
20745083
rs4712519
20745285
rs9368215
20745566
rsl 2206285
20745853
rs34049994
20745988
rs7751957
20746702
rs9465849
20746857
rs7341291
20746957
rs6921014
20747388
rs9465850
20747583
rs7758612
20747681
rs35602526
20748295
rs9465851
20748399
rs34257578
20748403
rsl 1339738
20748516
rs9460538
20748850
rs4712520
20748883
rs4710938
20749084
rs35889049
20749315
rs9348440
20749879
rs9465852
20750054
rsl 2196009
20750306
rsl 1968248
20750353
rs34797179
20750764
rs6925328
20750913
rs36073053
20751140
rs36224625
20751145
rs35462488
20751150
rs3060659
20751153
rs5874778
20751155
rsl 1267861
20751201
rs4235999
20751230
rsl 6884038
20751706
rs2328530
20751731
rs2328531
20751750
rs28360636
20752162
rs6932676
20752183
rs9460539
20752303
rs6932876
20752346
rs34929755
20752359
rs28733367
Pos Build 35/36
Marker ID
20752360
rsl 1348111
20752473
rs6933219
20752702
rs6933165
20752872
rs35255583
20752901
rs34332316
20752923
rs4710939
20753486
rsl 1970417
20753659
rsl 1963217
20753670
rsl 1965473
20753833
rs2876575
20754049
rs7739974
20754976
rs7745175 .
20755173
rs7765784
20755178
rs6907731
20755250
rs35057896
20755313
rs7746072
20755639
rsl 0484632
20755770
rs35444529
20755849
rsl 7823571
20755941
rsl 1965062
20755962
rs6936199
20755965
rs6913126
20756178
rs6913509
20756613
rs34638218
20756673
rs35746011
20756741
rs9460540
20757090
rs36045545
20757129
rs35392790
20757233
rs6456364
20757260
rs2179553
20757513
rs9350269
20757531
rs9465854
20757787
rs2179552
20757936
rs6925233
20758033
rs2328532
20758248
rs7743970
20758317
rsl 3209457
20758318
rs34641285
20758344
rsl 3209538
20758387
rs2876576
20758479
rsl 3209572
20758653
rs28783153
20758712
rs9969037
20758800
rs7766844
20758969
rs7767133
20759291
rs7749464
20760100
rs2050225
20760696
rs9295474
20760744
rs9295475
20761529
rs2328545
20761548
rs28846771
20761779
rs2876582
20761899
rsl 0223446
20762094
rsl 3219723
20762095
rsl 3203489
20762154
rsl 3203583
20762172
rsl 3203887
20762279
rs6456366
20762876
rs9358355
20763089
rs9368216
Pos
Build
Marker ID
/36
20763375
rs9465855
20763384
rs9465856
20763463
rsl 6884070
20763482
rsl 6884072
20763647
rsl 3208604
20764169
rs9465857
20764307
rs9368217
20764559
rs9460541
20764746
rs9460542
20764765
rsl 1969955
20764779
rs4712522
20764924
rsl 6884074
20765172
rs34489684
20765324
rs2328546
20765543
rs4712523
20765844
rs4712524
20765898
rs35397753
20765991
rs4710940
20766197
rsl 3190727
20766215
rs35136485
20766311
rs35260725
20766335
rs35939620
20766566
rsl 7823996
20766713
rsl 6884082
20767438
rs6906327
20767566
rs6456367
20767785
rs6456368
20768202
rs7749083
20768344
rs6456369
20768669
rsl 0946396
20768672
rsl 0946397
20768710
rsl 1759505
20769000
rsl 3203361
20769013
rsl 0946398
20769122
rs7774594
20769229
rs7754840
20769249
rs9460543
20769508
rs9460544
20769529
rs9460545
20769711
rs2206740
20769806
rs5874779
20769807
rs33970890
20769815
rs5874780
20769816
rs35014292
20769816
rs35363501
20770092
rs6456370
20770102
rs979614
20770196
rs35456723
20770571
rs9368218
20770945
rs4712525
20771014
rs4712526
20771314
rs4712527
20771442
rs35191644
20771443
rs34470647
20771611
rs9460546
20771938
rs9465859
20772079
rs9465860
20772291
rs736425
20772508
rs3060776
20772509
rs34941928
Pos Build 35/36
Marker ID
20772512
rs5874781
20772761
rs35778487
20773060
rs742642
20773305
rs35248697
20773436
rsl1967127
20773528
rs7748382
20773547
rs9688549
20773548
rs9689351
20773570
rs28665000
20773886
rs7752236
20773925
rs7772603
20774001
rs7752780
20774034
rs7752906
20774160
rs34184260
20774223
rs2206739
20774225
rs2206738
20774250
rs2206737
20774436
rsl 1970425
20774484
rs36034806
20774899
rs35042364
20775218
rs35540121
20775361
rs9358356
20775667
rs9356743
20775778
rs9350270
20776366
rs34929853
20778035
rs34971765
20778443
rsl 1970596
20779261
rsl 2527373
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rsl 3217082
97
Pos Build 35/36
Marker ID
20786470
rs13217085
20786481
rsl 3217090
20786483
rsl 3217091
20786523
rsl 3200946
20786772
rsl 1968032
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rsl 569699
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rsl 1364854
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rsl 6884103
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rsl 0440832
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rsl 0440833
20796237
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rsl 2200791
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rsl 0577753
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rsl 6884131
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rsl 0806921
20805104
rsl 6884133
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rsl 0946401
20806114
rsl 6884135
20806582
rs35711395
20807220
rsl 1969783
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rsl 6884137
20808600
rsl 1970626
Pos Build 35/36
Marker ID
20809092
rs12190713
20809106
rsl 1398905
20809415
rsl 1961445
20809470
rs35982077
20809486
rsl 1305935
20810952
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rsl 6884140
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rsl 1753081
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rsl 3196379
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rsl 7226450
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rsl 073247
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rsl 6884146
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rsl 040558
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rsl 1963770
20823805
rsl 0946402
20823840
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rsl 3215905
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rsl 1756271
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Pos Build 35/36
Marker ID
20827858
rsl 2110493
20827866
rs12193125
20828258
rs9356746
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rsl 3219444
20829342
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rsl 7825025
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rsl 2197544
20839053
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rsl 1753021
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rsl 1753041
20843666
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rsl 0484631
20844216
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Pos
Build
Marker ID
/36
20845619
rs9358362
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rs9368228
20845737
rsl 7839864
20845816
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rsl 0568019
20859187
rsl 0598071
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rs12199271
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98
Pos Build 35/36
Marker ID
20859674
rsl 1756862
20859995
rs4712539
20860175
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rsl 6901574
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rsl 0946406
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rsl 1415727
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rsl 2664021
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rs4712540
Pos
Build
Marker ID
/36
20871244
rs35450702
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rsl 0536083
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rsl 0946407
20887060
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20887829
rs4076137
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rsl 3207747
20888221
rsl 3208059
Pos
Build
Marker ID
/36
20888329
rs4074058
20888884
rs9350282
20888894
rs3193655
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rsl 1545101
20888919
rs3193654
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rsl 2199265
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rsl 1752139
20891373
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rsl 0690190
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rsl 0647996
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rsl 0946408
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rsl 1344905
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rsl 2210335
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rsl 0946409
20903353
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rs9465892
Pos
Build
Marker ID
/36
20905830
rsl 2211211
20906051
rsl 1968853
20906198
rsl 0456236
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99
Pos
Build
Marker ID
/36
20919599
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Pos
Build
Marker ID
/36
20934012
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rsl 2210496
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rsl 7234378
Pos Build 35/36
Marker ID
20952897
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rsl 2211628
20964682
rs4712556
20965069
rs4710955
20965780
rs34481273
20965904
rsl 6884229
20966215
rsl 1965417
20966239
rs4712558
20966335
rs4712559
20966543
rs34865786
20966637
rsl 7826361
20966708
rs9366362
Pos
Build
Marker ID
/36
20966831
rs9366363
20967583
rsl 1966194
20968463
rsl 2524318
20968532
rs28412953
20968698
rs9358374
20968821
rs9350291
20968927
rs9350292
20969034
rs9350293
20969131
rsl 6901585
20969205
rs5007237
20969579
rs4383807
20969839
rs4330531
20970113
rsl 2662176
20970590
rs9356757
20970850
rs34873202
20970884
rs9368245
20970889
rs34101377
20971175
rsl 2201353
20971267
rs12214755
20972435
rsl 1269210
20973576
rs9465922
20973608
rs12191898
20973841
rs35647500
20973851
rs5874790
20973852
rs35372258
20974027
rs7749397
20974223
rs7454011
20974715
rs35244703
20974836
rs9368246
20974986
rs34062103
20975000
rs35487666
20975596
rsl 6884250
20975795
rsl 0554344
20976043
rs4370355
20976276
rsl 0588127
20976276
rs34382383
20977459
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20977678
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20977795
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20978072
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rs9350295
20978719
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20978768
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20978882
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20980033
rs35505816
20980715
rs34113324
20981367
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20981827
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20982392
rsl 6884254
20982541
rsl 3197235
20983561
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20983706
rs9368247
20984381
rsl 2665789
20984489
rs4077403
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20984662
rs4077405
20984894
rs4529295
20984917
rs35499986
20985132
rs4618519
20985379
rs36002274
100
Pos
Build
Marker ID
/36
20985482
rs9368248
20985514
rs7742802
20985952
rs4236004
20986261
rs4712561
20986658
rs9358375
20986864
rsl 2194583
20986999
rs34923632
20987073
rs4537128
20987237
rs4626408
20987282
rs12194813
20987312
rs6915746
20988259
rs9350297
20988570
rs12210231
20988821
rsl 2198243
20989365
rsl 0946415
20989383
rs7763704
20989596
rsl 0589878
20990154
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20990238
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20990820
rs4612156
20991595
rsl 0946416
20992059
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20992198
rs9460569
20993260
rs35314952
20993493
rs4131667
20993700
rs4334975
20993732
rs4272215
20994113
rsl 1756312
20994209
rs6926335
20995305
rs9366367
20995933
rs6938256
20996432
rs6938755
20996580
rs9465928
20996674
rs7773564
20996730
rs28843472
20996731
rsl 2210522
20996732
rs13191608
20996755
rsl 0806929
20996858
rs9268994
20996859
rs9268995
20996890
rs28895225
20996905
rs9268996
20996911
rs9268997
20996937
rs35328517
20996973
rs9268998
20997216
rsl 2055384
20997384
rsl 2055630
20997540
rs4947247
20998096
rs9460571
20998693
rsl 0946417
20998950
rsl 1752477
20999169
rs6913323
20999294
rsl 2194053
20999407
rs36009649
20999752
rsl 0594050
20999763
rs5874791
20999764
rs35553046
20999770
rs4363020
Pos
Build
Marker ID
/36
21000241
rsl 0456008
21000447
rs35294796
21000702
rs9368251
21001068
rs6916036
21001199
rs34587331
21001226
rs6456379
21001338
rs9368252
21001683
rs4626415
21002414
rs5874792
21002415
rs33925173
21002601
rs5874793
21002602
rs34720816
21002625
rsl 2528945
21004281
rsl 2206985
21004330
rsl 1757050
21004330
rs34449480
21004372
rs9368253
21004532
rs9295486
21004540
rs9465930
21004836
rsl 2208719
21005635
rs6918643
21005875
rs4712562
21006091
rs6456380
21006119
rs7759877
21006228
rs9358376
21006412
rs35620702
21006800
rs28665148
21006801
rs28599540
21007273
rs35400949
21007751
rsl 2153898
21007819
rs12154181
21007863
rs12154184
21008099
rs9368254
21008207
rs9368255
21009058
rs35958016
21009211
rs6456381
21009246
rs7758467
21009290
rs6456382
21009421
rs12153811
21009654
rs7739013
21009711
rs6922768
21009716
rs13191241
21009909
rsl 0640864
21009910
rs34135721
21010295
rs34154507
21010566
rs35386545
21010568
rs9368256
21010964
rs7740094
21010972
rs7758155
21011220
rs9366368
21011950
rs9358377
21012344
rs6908429
21012629
rs4712563
21012910
rs4710957
21013210
rsl 2214079
21013433
rs9460572
21013812
rs2876583
21013840
rs9465931
21014102
rs34068097
21014657
rsl 0080699
Pos
Build
Marker ID
/36
21014657
rs9460573
21014665
rs9460574
21014666
rs6920918
21015860
rs4413596
21016247
rs6909682
21016278
rs6910032
21016580
rsl 498427
21016869
rs34713988
21017395
rs9368257
21017439
rs6939006
21017512
rsl 7236121
21017740
rsl 1753331
21018757
rsl 2198263
21018818
rs35916632
21018915
rs9350298
21018982
rs4710958
21019053
rs35886192
21019304
rs6456385
21019380
rs7763674
21019654
rs7746308
21020651
rsl 2190631
21020765
rsl 498425
21021671
rs13197106
21022074
rs6913538
21022178
rs9460575
21022308
rs6914232
21022483
rs9460576
21022485
rs6914586
21022873
rs9465932
21022913
rs2030082
21022932
rs9350299
21023379
rsl 0536841
21023692
rs9465933
21024540
rs34537557
21025636
rs9368258
21026230
rs6938184
21026422
rs6938415
21026611
rs35678799
21028069
rs28440883
21028102
rs4712564
21028251
rs4712565
21028711
rs9366369
21028958
rs9465934
21029390
rs6913658
21030247
rs7765765
21030707
rsl 2662218
21030842
rs9460577
21030852
rs4712566
21030926
rs4712567
21030962
rs6936002
21031254
rs9460578
21031561
rs7771782
21032098
rs2328550
21032169
rs9465935
21033024
rs9460579
21033247
rs2328551
21033384
rs34787947
21033907
rs35164658
21034157
rs34463604
21034296
rsl 6884302
Pos
Build
Marker ID
/36
21034654
rsl 3215945
21034664
rs13215949
21034748
rsl 3202822
21034750
rsl 3203127
21034760
rsl 3203130
21035097
rs35259543
21035108
rsl 0687937
21035135
rs9358378
21035148
rs9465936
21035989
rs6903176
21036896
rs34272344
21037020
rs9366370
21037483
rs7747830
21037954
rs35552655,
21038014
rsl 1970010
21038092
rsl 1970030
21038468
rs35341341
21038480
rs5874794
21038957
rs34094116
21039069
rs9366371
21039135
rs7775995
21040473
rs6916666
21041656
rsl 6884308
21042140
rsl 1395158
21042539
rs34100038
21042991
rs7768647
21043110
rs35030815
21043259
rs9350300
21044027
rsl 0946419
21044393
rsl 0946420
21044437
rs9465937
21044451
rs9368259
21044452
rs33911838
21044591
rs9358379
21044671
rs9460580
21044802
rs35523552
21044952
rs16884310
21046071
rs34741537
21046084
rsl 2526287
21046565
rs9465938
21046566
rs28734969
21046585
rs7747773
21046841
rs9465939
21046900
rs4144650
21047359
rs4712569
21047690
rs4712570
21047713
rs6939250
21047725
rs4710959
21047769
rs4710960
21047987
rs13191844
21048358
rsl 2196595
21048740
rs9350301
21049043
rs9350302
21049105
rsl 1964084
21049192
rs6927419
21049306
rs12198512
21049369
rsl 1450413
21049370
rsl 1409601
21049371
rs33942714
21050175
rs4307160
101
Pos
Build
Marker ID
/36
21050624
rs4712571
21051211
rs9465941
21051466
rsl 0946423
21051483
rs9465942
21051538
rs34759060
21051555
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21051560
rs5874795
21051561
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21051563
rs12174149
21051568
rs9350303
21051569
rs9358381
21052585
rsl 1279071
21052648
rs9465943
21053046
rs9465944
21053295
rsl 0690950
21053629
rs34879403
21053757
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21053935
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21054784
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21054804
rsl 1361870
21054805
rs35132815
21055236
rsl 2203876
21055710
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21055853
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21055870
rs9368260
21056065
rs9460581
21056280
rs6930879
21056288
rs6930887
21056329
rs6931047
21056472
rs9350305
21058200
rs9350306
21058970
rsl 2197923
21059112
rs9460582
21059179
rs5874796
21059866
rs6922187
21060142
rs9465945
21060180
rsl 0558500
21060186
rsl 1289942
21060212
rs9348451
21060247
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21060361
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21060639
rs35863080
21061204
rs4631270
21061590
rs2015426
21061898
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21062676
rs9350307
21062805
rs1004172
21063311
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21064970
rs12199174
21066160
rs9368261
21067118
rs7738201
21067314
rsl 7236645
21067986
rsl 6884319
21068463
rs9918311
21068617
rs34769285
21068702
rsl 2205321
21068911
rs6456388
21069040
rs4712572
21069650
rsl 2524678
Pos Build 35/36
Marker ID
21069850
rs13211056
21070260
rsl 7236694
21070934
rs35382761
21071276
rs28612346
21071587
rs6915209
21071832
rs35420176
21072294
rs6940952
21072419
rs35708433
21072662
rs6941808
21072828
rsl 1966306
21073181
rsl 6884325
21073468
rs7743030
21073553
rs28429494
21074417
rs4712573
21074865
rsl 1752712
21075695
rs9460583
21075964
rs9460584
21076612
rs34425275
21076615
rsl 112255
21076815
rsl 112256
21077529
rs9348452
21077563
rs34660637
21077716
rs7746754
21077794
rs9350308
21078333
rs34215872
21079005
rs2030081
21079247
rs6940649
21079554
rs6941043
21079964
rs6913817
21080948
rs201361
21081289
rs34479545
21081631
rsl 980458
21082363
rs13210151
21082946
rs34266759
21083025
rs34192233
21083039
rsl 1427521
21083189
rs35882882
21083211
rsl 1330322
21083279
rsl 997685
21083363
rsl 0498699
21083433
rs201360
21085426
rs201359
21085653
rs201358
21085834
rs34764914
21087560
rs201357
21088272
rs4712575
21088448
rs4712576
21088450
rs4712577
21088472
rs365001
21088891
rs35829025
21089414
rs383684
21089616
rs444005
21090258
rs35693086
21090584
rsl 1961135
21091067
rs201356
21091773
rs201355
21091799
rsl 2193382
21091799
rs34391804
21091914
rs35723307
21091926
rs35448639
Pos Build 35/36
Marker ID
21093023
rs35957348
21093317
rs201354
21093369
rs201353
21093439
rs9350309
21093650
rs201352
21094109
rs201351
21094143
rs35853352
21094861
rs5874797
21094872
rs5874798
21094931
rs201350
21095014
rsl 997684
21095425
rs4560627
21095482
rsl 0699637
21095654
rs7453577
21096171
rsl 1300877
21096223
rs201349
21096419
rsl 0694429
21096428
rs3223730
21096922
rs201348
21097026
rs35010661
21097035
rs201347
21097047
rs10701919
21097265
rs201346
21098132
rs6456389
21099063
rsl 2528036
21099645
rs201345
21099698
rsl 3199754
21099916
rs35504365
21100226
rs35586782
21100247
rs28418444
21100248
rsl 3196975
21100263
rs35586503
21100320
rs9356759
21100418
rs9366372
21100871
rsl 0946424
21100949
rs201344
21101200
rsl 1964880
21101281
rsl 1967584
21101513
rs201343
21102229
rs201342
21102283
rs9350310
21102377
rs9368262
21102486
rs9368263
21103236
rs35996844
21103858
rs201341
21103935
rsl 2660707
21104054
rs201340
21104291
rs201339
21105034
rs396308
21105044
rs4712578
21105348
rs449233
21107366
rs9358383
21107874
rs36078234
21108856
rs9368264
21109330
rs9368265
21109358
rs9368266
21109523
rs9368267
21109553
rs6940871
21109878
rs35240009
21110331
rs6914677
Pos
Build
Marker ID
/36
21110432
rs9350311
21110750
rsl 2195827
21110788
rs9366373
21110864
rs34620040
21111239
rs9350312
21111513
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21111536
rs13191651
21111581
rs13191676
21111601
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21112269
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21112288
rs7744317
21112352
rs7762669
21113187
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21113252
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21113325
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21113687
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21115599
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21115719
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21115720
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rs2494728
21116011
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21116132
rsl 498426
21116718
rsl 2192642
21116822
rsl 3200036
21116851
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21116920
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21116972
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21117087
rs374654
21117572
rs6914615
21117867
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21118124
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rs416688
21118300
rsl 1382802
21118547
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21118567
rs201291
21119018
rs9465947
21119021
rsl 2197742
21119236
rs201292
21119698
rs35998561
21119973
rs201293
21120225
rs9358386
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rs35254358
21120883
rs6905258
21121364
rs201294
21121603
rs201295
21121962
rs7748932
21121994
rs2494729
21122535
rs3966676
21122822
rs34834774
21122975
rsl 6884376
21123408
rs201308
21123602
rs8180606
21123934
rs201309
21123964
rs201310
21124001
rs6456390
21124511
rs201311
21124822
rs5874799
21124823
rs34733906
21124830
rs400514
102
Pos Build 35/36
Marker ID
21124882
rs3061573
21125298
rsl 6884385
21125848
rs34894804
21125898
rs9460586
21126295
rsl 6884389
21126495
rs35678732
21126634
rsl 0946425
21127516
rs6899729
21127744
rsl 6884394
21128114
rs36115181
21128124
rs5874800
21128307
rs34639729
21128419
rs35260082
21128533
rs7744010
21128746
rs201290
21129343
rs201289
21129647
rs34802324
21129981
rs201288
21130208
rs201287
21131340
rs34891710
21131348
rsl 1438527
21131431
rs34878496
21131727
rsl 6884396
21132081
rs9465948
21132220
rsl 6884399
21133062
rsl 1297091
21133400
rsl 69293
21133737
rs201307
21133815
rsl 2206158
21133964
rs34317920
21134374
rs34305502
21134401
rs35570989
21135019
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21135740
rsl 2209877
21136038
rs6921781
21136172
rsl 6884406
21136940
rs201306
21136972
rs201305
21137253
rs201304
21137477
rs34243092
21137698
rs201303
21138072
rs201302
21138132
rs201301
21138504
rs35592283
21138790
rsl 0946426
21138980
rs6919322
21139058
rs4710961
21139068
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21139350
rs398913
21139414
rs4710962
21139480
rsl 2526927
21139855
rs9460587
21140661
rs415446
21141104
rs35761643
21141617
rsl 1961096
21141944
rs201300
21142185
rs35161624
21142199
rs35206549
21142635
rsl 3210277
21142726
rs12198160
Pos
Build
Marker ID
/36
21142744
rs7766970
21143715
rs201299
21143786
rs35061829
21144118
rs201298
21144658
rs201297
21145049
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21145593
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21146814
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rs9295489
21148244
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21148269
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21148356
rsl 7662372
21148684
rs201315
21148701
rsl 6884415
21150019
rsl 6884420
21150116
rsl 7730046
21150139
rs201316
21151059
rsl 6884425
21151118
rsl 555052
21151344
rsl 1753092
21151557
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rs201317
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rs9460589
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rs201319
21153050
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21153105
rs201321
21153294
rs201322
21153342
rs201323
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rs201325
21153907
rs9460590
21153987
rs201326
21154014
rs201327
21154069
rs201328
21154147
rs201329
21154297
rs201330
21154380
rsl 3217562
21154919
rs34472513
21154958
rs201331
21155233
rs201332
21155256
rs34312444
21155363
rs201333
21155639
rs201334
21155793
rs201335
21156103
rs201336
Pos
Build
Marker ID
/36
21156840
rs201337
21156885
rs201338
21157007
rs34847494
21157597
rs9942509
21157604
rs34987941
21158291
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21158841
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21160395
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21160650
rsl 1760181
21160654
rs2056951
21160683
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21160934
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21161031
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rs6456392
21162711
rsl 3215037
21162805
rs7752701
21163066
rs6456393
21163111
rs2328567
21163129
rs6456394
21163541
rsl 2661235
21163651
rs7754018
21163696
rs9350315
21163923
rs9918435
21165424
rs9465953
21165665
rs35560899
21165892
rs34142046
21166104
rs9465954
21166730
rsl 2202256
21166802
rs2206014
21167015
rsl 3219721
21167016
rsl 3205176
21167182
rsl 3219752
21167200
rsl 3205420
21167401
rs9350316
21167460
rs9368270
21167796
rs9465955
21169768
rs28532482
21170022
rsl 7237178
21170224
rsl 3209905
21170225
rsl 3209787
21170236
rsl 3210005
21170269
rsl 3210023
21170287
rsl 3210027
21170376
rs7744921
21170433
rs9465956
21170545
rs9465957
21170553
rs9465958
21170579
rsl 6884466
21170710
rsl 1969047
21170765
rsl 6884468
21170846
rsl 1969587
21171228
rs9465959
21171401
rs34908981
21172309
rs9460591
21173104
rs35603774
21173126
rs9465960
Pos
Build
Marker ID
/36
21173198
rs34559907
21173393
rs4710963
21173679
rs9465961
21173766
rs1001310
21173805
rs4712580
21174689
rs9460592
21174762
rs9460593
21176267
rsl 2209678
21177342
rs35738724
21177501
rs7762612
21177928
rs36174378
21177981
rsl 3199309
21178014
rs9295490
21178237
rsl 2660913
21178243
rsl 0946427
21178328
rsl 2199982
21178333
rs34600664
21178468
rs6932873
21178475
rs9356760
21178487
rs9465962
21178588
rs9356761
21178609
rsl 0650825
21179141
rs4382241
21179575
rs2013346
21179832
rsl 0498700
21180396
rs9350317
21180629
rsl 1757139
21180733
rsl 1757596
21181008
rsl 1757677
21181027
rs9465963
21181171
rsl 3203959
21181269
rs9366374
21181357
rsl 1968037
21181410
rs6918997
21181499
rs9368271
21181567
rs9460594
21181588
rs9460595
21182172
rs9295491
21182342
rs4283868
21182548
rs4413602
21183182
rsl 0541645
21183338
rsl 0541646
21183364
rsl 1407560
21183365
rs35953450
21183371
rs7766554
21183409
rs7766574
21183437
rsl 0541647
21183457
rs7766451
21183464
rs7766713
21183493
rs7766728
21183552
rs4280955
21183645
rs7767038
21183675
rs7766788
21183741
rs7770701
21184027
rsl 6884481
21184246
rs9295492
21184599
rs4367364
21184749
rs2068072
21184770
rs2068071
21185523
rsl 1963640
103
Pos Build 35/36
Marker ID
21185602
rsl 1961090
21185611
rsl 1963654
21185810
rs35119417
21186212
rs9358388
21186666
rs4599624
21187063
rsl 0080639
21187282
rs7749838
21187336
rsl 1967298
21187386
rs34700985
21187552
rs9366375
21187610
rs4310041
21187627
rs4496780
21187688
rs7750461
21187916
rs6917272
21188170
rs6917904
21188430
rs34700472
21188451
rs9350318
21189185
rs4323302
21189629
rs9366376
21189885
rsl 0456237
21189931
rsl 3219886
21190084
rs9368272
21190476
rs35189729
21191594
rs6904648
21191595
rsl 1751563
21191595
rs35898656
21192182
rs4593348
21192409
rs4421186
21192801
rs6915237
21193447
rs7773973
21193514
rsl 7834555
21193811
rs6901354
21194026
rs4130033
21194285
rs35287977
21194419
rsl 0806931
21194732
rsl 1964144
21194764
rsl 0946428
21196061
re12200487
21196232
rs4712581
21196650
rs6456395
21196835
rs6456396
21197051
rs9368273
21197394
rs4712582
21198212
rs34465715
21198601
rs6904348
21199015
rsl 1433815
21199016
rs36110847
21199138
rs9368274
21199756
rsl 2197841
21199815
rs12197853
21199817
rs12197854
21200342
rs7762660
21200361
rs7762670
21200554
rs7762964
21200605
rsl 3210302
21200944
rs34756370
21201333
rs7768086
21201341
rs13214341
21202104
rs33945169
21202717
rs6939622
Pos Build 35/36
Marker ID
21202933
rs4454125
21203121
rs4401656
21204432
rsl 1969472
21204602
rs9358389
21204788
rs4712583
21205444
rsl 1757261
21205844
rs34430280
21206360
rsl 1757901
21206756
rs34764667
21206833
rs35866346
21207116
rs9465965
21207331
rs7744319
21207645
rs9368275
21207687
rs9356762
21208023
rs9350319
21208328
rs9350320
21208606
rs9366377
21208909
rs5874801
21209654
rs9368276
21210536
rs4712584
21210553
rs34092761
21210726
rs9366378
21211436
rsl 1968036
21211664
rs9465966
21211803
rs4569951
21212138
rs4130302
21212414
rs35081613
21212547
rs9358390
21212715
rs6925464
21212715
rs9460596
21212847
rsl 7834987
21213142
rsl 2529531
21213851
rs36086759
21214087
rs6936205
21215067
rs4574622
21215086
rs9465967
21215088
rs9358391
21215150
rs35347692
21215159
rs5874802
21215207
rs4624863
21215258
rs9465968
21215359
rs4526186
21215371
rs34865903
21215437
rs7739050
21215448
rs4326226
21216093
rs4712585
21216145
rs9356763
21216170
rs4710964
21216871
rs9460597
21218688
rsl 1963150
21218907
rs6923546
21219519
rs6924221
21220306
rsl 0456238
21222458
rs34418857
21222459
rs34251532
21222460
rs34219362
21222644
rs35962170
21223003
rsl 6884514
21223188
rs7773318
21223279
rs4340995
Pos
Build
Marker ID
/36
21223295
rs9356764
21224572
rs35468559
21224879
rs3935207
21224965
rs35629967
21225019^
rs6927498
21225119
rsl 0613278
21225499
rs35372171
21225513
rsl 0554335
21225585
rs7764365
21225685
rs35234206
21225694
rsl 1418036
21225734
rs7768536
21226218
rs4389757
21227109
rs6456397
21227703
rs35183051
21228007
rs7741436
21228269
rs9356765
21228407
rs9366379
21228562
rs6926818
21228614
rsl 6884524
21228619
rs34425854
21228733
rs4074910
21228748
rs4076112
21229002
rs35885025
21229134
rs6909332
21229654
rs4438948
21229845
rs6905660
21230834
rs9358392
21231054
rs9368279
21231899
rs4315997
21231967
rs4479917
21232500
rsl 0946430
21232701
rs34821627
21232763
rs13191691
21232779
rsl 3207763
21232796
rsl 3207766
21232816
rs13191830
21233338
rs34857211
21234866
rs7750839
21235291
rs7751485
21235577
rsl 1757294
21235798
rs34183889
21236043
rsl 2055489
21236223
rs28421119
21237436
rs9295493
21237650
rsl 2055790
21237696
rs35692444
21237763
rs7766575
21237890
rs9356766
21237892
rs34802727
21237915
rs35927368
21238432
rsl 0708944
21238635
rs6932722
21238849
rs6937439
21238961
rs9986401
21239004
rs9465969
21239122
rs9986662
21239291
rs9295494
21239512
rs35030599
21240132
rs9460598
Pos
Build
Marker ID
/36
21240526
rs28403910
21241995
rs9465970
21242670
rs7760880
21242672
rs7761283
21242682
rsl 1463641
21242783
rs7765177
21242820
rs7765199
21242857
rs7764887
21243009
rs7765106
21243039
rs28610069
21243105
rs7765274
21243107
rs2446490
21243137
rs2493869
21243228
rs7765725
21243240
rs7765730
21243646
rsl 6884554
21243857
rs13216162
21244099
rs2446489
21244512
rs2446488
21244516
rs7771907
21245195
rs9350322
21245369
rs2446487
21246042
rs35201465
21246079
rs9358393
21246445
rs959712
21246447
rs35541643
21246451
rs34848377
21246456
rs5874803
21246457
rs33951051
21246458
rs5874804
21246459
rs35717786
21246466
rs5874805
21246467
rs35384149
21246474
rs959711
21246487
rs34589183
21246785
rs9688559
21246915
rs9689353
21246958
rs34154291
21246988
rs9688564
21246992
rs9688565
21247009
rs2446486
21247020
rs9688569
21247061
rs9688573
21247079
rs9295495
21247094
rs9465971
21247737
rs9460599
21247746
rs6916667
21247788
rs9465972
21248278
rs2446485
21248465
rs2446484
21249707
rs28665959
21249879
rs35583136
21249895
rs34896971
21249984
rs7746383
21250235
rs7746846
21250457
rs28360550
21250639
rs6932702
21251345
rs9348456
21251752
rs35724409
21251989
rs2328573
104
Pos Build 35/36
Marker ID
21252098
rsl 7835633
21252216
rs36008085
21252578
rsl 0498701
21252578
rs35938718
21252735
rs34957382
21253603
rsl 0708192
21254245
rsl 466340
21255061
rsl 0710231
21255726
rsl 466339
21255824
rs7740358
21256011
rsl 1405792
21256411
rs1471205
21256721
rs9368280
21257043
rs9295496
21257242
rsl 2206028
21257356
rs9368281
21258107
rs2168985
21258129
rsl 2207912
21258136
rs35882470
21258203
rs9368282
21258436
rs7752602
21258626
rs7752788
21258747
rs2446483
21258795
rs9356768
21258995
rs34495814
21259344
rs9350323
21259352
rs4530843
21259489
rs9460600
21259600
rs9460601
21260113
rsl 0946431
21260135
rs35088240
21260216
rsl 0946432
21260253
rsl 0946433
21260917
rsl 1961031
21261341
rs4144175
21261468
rsl 0604354
21261575
rsl 2203853
21261620
rs6901380
21261891
rs6906201
21262118
rs9460602
21262389
rs35396145
21262741
rs9350324
21262775
rs9295497
21262969
rs9366381
21263128
rs6456398
21263152
rs6456399
21263168
rs6456400
21263322
rs6456401
21263462
rsl 1456476
21263471
rs34116986
21263595
rs6913868
21263798
rs9366382
21263882
rs9348457
21264053
rs35931974
21264270
rs9366383
21264385
rs9366384
21264393
rs9356769
21264732
rs34306955
21264842
rs9350325
21264968
rs6924598
Pos Build 35/36
Marker ID
21265017
rs4712586
21265065
rs7761116
21265072
rs9358395
21265152
rs9368283
21265828
rs34689265
21266252
rs6931316
21267009
rs6916577
21267167
rs6937555
21267311
rsl 0645059
21267321
rsl 0652396
21267832
rsl 6884591
21268027
rsl 1442196
21268361
rs7739578
21268400
rs7739596
21268426
rs4527692
21268664
rs9295498
21268668
rs9295499
21268866
rsl 0080292
21268870
rs35915482
21268881
rs9465976
21268928
rs28581582
21268942
rs34844023
21269038
rs12179712
21269039
rs9465977
21269504
rs9465978
21271039
rs6929437
21271299
rs4995985
21271738
rs34031561
21271898
rs6914598
21272056
rs6935079
21272110
rs6935117
21272124
rs6935124
21272174
rs9368284
21272190
rs6915161
21272228
rs9356770
21272416
rs35558562
21272655
rs6916053
21272716
rs34191499
21273107
rs6941714
21273161
rs34326160
21273168
rs9460603
21273192
rs9348458
21273257
rs7776158
21273274
rsl 1965768
21274046
rs35674401
21274684
rs2125570
21275213
rs7768526
21275957
rs9368285
21276570
rs28360551
21277729
rs7763700
21277824
rs4425589
21277964
rs9348459
21278549
rsl 3197595
21278592
rs9460604
21278780
rs12180174
21278845
rs9465979
21279293
rs36058161
21279338
rsl 1969929
21279609
rsl 1965049
21279673
rs9295500
Pos Build 35/36
Marker ID
21279689
rs34012677
21279828
rs12178179
21279839
rs9465980
21280353
rs9358396
21281052
rsl 2194541
21281118
rs2061441
21281632
rs9460605
21281781
rsl 2525339
21282235
rs35462438
21282590
rs9465981
21282848
rs4637624
21282946
rs35969558
21283471
rsl 2525940
21283655
rs34603118
21283948
rsl 2528104
21284103
rsl 2526391
21284906
rs6939148
21284912
rs9460606
21285561
rsl 3219281
21285569
rs13219285
21285598
rs13219506
21285611
rs13219193
21285620
rs13219198
21285664
rsl 3219637
21285689
rsl 3205078
21285691
rsl 3205079
21285875
rsl 1308599
21286261
rs9465982
21288187
rsl 2214946
21288554
rs34495587
21289215
rsl 2523755
21289629
rs35642303
21290957
rs9295501
21291348
rs35815279
21291533
rsl 2527222
21291647
rs9465983
21291857
rs2493868
21291918
rs35979352
21292407
rs34248538
21292789
rsl 0946434
21292811
rs9465984
21293033
rs34599800
21293434
rs35442433
21293569
rs9465985
21294166
rs35712201
21294748
rs35539626
21294750
rs9465986
21294751
rs9465987
21294801
rsl 1961469
21295134
rs2446482
21295312
rs9465988
21295313
rs12191416
21295313
rs35985333
21295996
rs9465989
21296793
rs9350327
21297183
rs34750271
21297265
rs35013686
21297416
rsl 6884616
21297902
rs35898446
21297924
rsl 1751020
Pos
Build
Marker ID
/36
21297967
rsl 0452581
21298562
rsl 3192000
21298563
rs13191669
21298583
rs13192011
21298617
rsl 3192029
21298629
rs13192143
21298630
rsl 3207866
21298671
rs13191819
21298690
rs13192164
21298721
rs13192173
21298723
rs13191845
21299659
rs9465990
21299810
rs9460607
21299907
rs9465991
21299909
rs9366386
21299971
rs35944981
21300001
rs9366387
21300046
rs9366388
21300106
rs9368287
21300203
rsl 3193222
21300325
rsl 0080974
21300381
rs9295502
21300388
rsl 2528974
21300395
rs7759646
21300433
rs9465992
21300768
rsl 1964193
21301021
rs34456723
21301080
rs34094109
21301834
rsl 1759448
21302380
rsl 1962770
21303198
rs9366389
21303687
rsl 1753415
21304730
rs4712587
21304976
rs7748091
21305299
rs28469715
21305355
rs7748766
21305591
rs35164470
21305660
rs2125571
21305669
rs9465993
21306062
rs3793090
21307994
rs1531303
21308261
rs2305955
21308369
rsl 459047
21309244
rs35662535
21309281
rs9767650
21309286
rs9767186
21309387
rs9460608
21309472
rs9465994
21310133
rs9465995
21310563
rs36067162
21310749
rsl 1965158
21311344
rs9350328
21311426
rs5874806
21311451
rsl 0616274
21311452
rs5874807
21311454
rsl 1288843
21311471
rs9350329
21311502
rsl 824330
21311620
rs9717950
21311710
rs3898487
105
Pos Build 35/36
Marker ID
21311900
rs9350330
21311902
rs9350331
21312023
rs35603064
21312085
rs35615714
21312109
rs36017220
21312120
rsl 2196363
21312143
rs35881379
21312153
rs35710688
21312177
rs35017881
21312188
rs12196418
21312191
rs12196419
21312206
rs35883368
21312223
rsl 2196423
21312231
rs34046809
21312253
rs34108390
21312453
rs6921264
21312671
rs6921652
21312775
rs6926388
21312801
rsl 2527588
21313200
rsl 0456240
21313329
rsl 0456241
21313367
rsl 0456242
21313458
rsl 0456243
21313856
rs34046046
21313879
rsl 3213969
21313886
rs6932316
21313910
rs6932752
21313958
rsl 3214311
21313963
rs6932914
21313998
rs6932635
21314018
rs6912407
21314041
rs34849597
21314107
rs9366390
21314243
rsl 0223539
21314298
rsl 0223540
21314473
rs6913302
21315081
rs9366391
21315139
rs9356771
21315390
rsl 2530254
21315432
rs34085972
Pos Build 35/36
Marker ID
21315529
rs4291091
21315727
rs6940465
21315763
rs6901748
21316195
rs6902505
21316396
rs898167
21316398
rs898166
21316408
rs898165
21316820
rs34797264
21317102
rs9368288
21317206
rs9358397
21317611
rs2168984
21317978
rsl 563728
21318135
rs4712588
21318266
rsl 1267610
21318399
rs4712589
21318666
rs6915037
21318882
rsl 2664336
21319431
rs9465998
21319494
rs10214790
21319776
rs12201217
21320060
rs9350332
21320905
rs9358398
21321149
rs9358399
21321286
rs9358400
21321533
rsl 0214694
21321733
rs10214716
21322176
rs9460609
21322179
rs6929219
21322517
rsl 2527686
21322561
rsl 2527673
21322733
rs9350333
21323322
rsl 0946436
21323380
rs6913136
21323400
rsl 3200114
21323464
rsl 3200422
21323815
rs2328572
21323949
rs9350334
21324672
rs34913347
21324713
rsl 0946437
21324725
rsl 0946438
Pos
Build
Marker ID
/36
21325164
rs9358401
21325261
rs34055473
21325350
rs34921405
21325357
rs6904880
21325395
rs6456403
21325653
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21325832
rs9466000
21325853
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21326033
rs2100707
21326158
rs12111402
21326366
rs4712590
21326649
rs4710965
21327416
rs6937610
21327459
rsl 2110862
21327488
rs35624914
21327606
rsl 1349673
21327854
rsl 6884681
21327895
rs7738425
21328030
rsl 6884685
21328355
rsl 6884688
21328398
rs35663664
21328510
rsl 2203389
21328818
rs12191541
21328946
rs34618548
21330074
rsl 563726
21330730
rsl 6884693
21331119
rs2328574
21331209
rsl 6884699
21331264
rsl 6884705
21331267
rs6929141
21331293
rsl 6884709
21331392
rsl 6884713
21332034
rs9466002
21332081
rs9466003
21332103
rs9466004
21332139
rs9466005
21332272
rs9460610
21332409
rs7770316
21332488
rsl 1964983
21332496
rs7770752
Pos Build 35/36
Marker ID
21332625
rs7770637
21333229
rsl 870421
21333556
rs6942273
21333618
rs9466006
21333709
rs9466007
21334500
rs7763249
21335731
rs9368289
21335750
rs9368290
21335782
rsl 3202305
21335903
rs34362358
21335906
rsl 1415596
21336317
rs28484932
21336582
rs7754027
21336699
rs34022115
21336867
rs4710966
21337512
rsl 6884722
21338182
rs35571136
21338184
rs35739791
21338815
rs9460611
21338986
rs9460612
21339013
rsl 2200511
21339097
rs35791563
21339201
rs34084405
21339207
rs34158326
21339453
rsl 563727
21339524
rs3840416
21339530
rsl 1362523
21339688
rs7770664
21339861
rs35121088
21339935
rs4712591
21340199
rs35206923
21340202
rs28600127
21340213
rs4710967
21340214
rs4710968
21340218
rs13213171
21340219
rsl 3197226
21340225
rsl 2199601
21340594
rsl 137970
106
Table 10. SNPs within LD block C06 (SEQ ID NO:l) between positions 20,634,996 and 20,836,710 bp on Chromosome 6 in NCBI Build 35 and NCBI Build 36
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20634996
1
rs4429936
20635028
33
rs9465780
20635060
65
rs7743222
20635066
71
rs7743223
20635241
246
rs4516938
20635285
290
rs4628090
20635339
344
rs9465781
20635349
354
rs28450063
20635350
355
rs9465782
20635834
839
rs4712503
20635845
850
rs9465783
20635860
865
rs4712504
20636037
1042
rsl 0946388
20636813
1818
rs9460517
20636939
1944
rs34086777
20637089
2094
rs9465785
20637215
2220
rs7754223
20637279
2284
rs34173688
20637287
2292
rsl 1459684
20637288
2293
rs35781726
20637303
2308
rs9460518
20637450
2455
rsl 1362835
20637521
2526
rs7772956
20637824
2829
rsl 883641
20637875
2880
rsl 883640
20637944
2949
rsl 1402844
20638219
3224
rs35198704
20638372
3377
rs6923683
20638762
3767
rs12181295
20638829
3834
rs9465788
20638961
3966
rs34578766
20639509
4514
rs2206578
20639662
4667
rs35530523
20639708
4713
rs2206577
20639710
4715
rs34553771
20639718
4723
rs34581322
20639719
4724
rs5874771
20639909
4914
rs6902897
20640005
5010
rs34607984
20640118
5123
rs6923201
20640162
5167
rs6903415
20640249
5254
rs9465790
20640425
5430
rs6923750
20640859
5864
rsl 0717803
20641038
6043
rs9465791
20641141
6146
rs6909467
20641248
6253
rs34525680
20641293
6298
rs35457534
20641299
6304
rs35731703
20641303
6308
rsl 0554680
20641320
6325
rs35239102
20641362
6367
rs9368197
20641413
6418
rs9465792
20641494
6499
rs12212722
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20641581
6586
rs7765611
20641590
6595
rsl 0566792
20641598
6603
rsl 0566793
20641718
6723
rs34275610
20641718
6723
rsl 0566794
20641724
6729
rsl 1347538
20641733
6738
rs5874772
20642073
7078
rsl 6883887
20642385
7390
rs34088191
20642428
7433
rsl 0806920
20642440
7445
rsl 1370426
20642441
7446
rs33915274
20642442
7447
rsl 1459775
20642443
7448
rs34576540
20642494
7499
rsl 0708068
20642586
7591
rs4712505
20642787
7792
rs41271303
20642953
7958
rsl 1963450
20643397
8402
rs981043
20643513
8518
rs981042
20643675
8680
rsl 6883895
20643753
8758
rsl 7512225
20643840
8845
rs35035071
20643949
8954
rs6904566
20644073
9078
rs6927356
20644093
9098
rs35281412
20644313
9318
rs35915788
20644314
9319
rs34025398
20644320
9325
rs34361235
20644335
9340
rs6905138
20644499
9504
rsl 3194858
20644717
9722
rs2179551
20644727
9732
rs2179550
20644787
9792
rs9465794
20644787
9792
rs9465795
20644848
9853
rs7747962
20644858
9863
rs6910725
20644918
9923
rs965054
20644971
9976
rs2143407
20645032
10037
rsl 0619380
20645431
10436
rs2328525
20645661
10666
rsl 3199286
20645841
10846
rsl 0611252
20645940
10945
rs7753499
20646023
11028
rs7753956
20646024
11029
rs34811195
20646024
11029
rs7753670
20646107
11112
rs3060613
20646107
11112
rs6149468
20646109
11114
rsl 1277970
20646110
11115
rsl 1280099
20646139
11144
rsl 6883900
20646175
11180
rs7774291
20646443
11448
rsl 0612082
107
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20646476
11481
rs9368198
20646502
11507
rsl 3203336
20646504
11509
rsl 3203631
20646619
11624
rs6456355
20646644
11649
rsl 0484635
20647190
12195
rsl 2204173
20647320
12325
rsl 3207544
20647851
12856
rsl 2198728
20647984
12989
rs28396084
20648327
13332
rsl 2199073
20648500
13505
rs9465796
20648561
13566
rsl 2212600
20648596
13601
rs13212040
20648663
13668
rs35291340
20648722
13727
rsl 2199324
20649085
14090
rsl 2200871
20649159
14164
rs9348432
20649183
14188
rsl 2200834
20649236
14241
rs34860173
20649324
14329
rsl 1754872
20649498
14503
rs6456356
20649517
14522
rs9368199
20649682
14687
rs2143406
20650176
15181
rsl 0484634
20650200
15205
rs7758851
20650398
15403
rs34677076
20651447
16452
rs6928571
20651461
16466
rsl 2192584
20651608
16613
rs34856684
20652015
17020
rs9350255
20652091
17096
rs9368200
20652136
17141
rs12214002
20652245
17250
rs9465797
20652300
17305
rs9465798
20652574
17579
rs28699301
20652650
17655
rsl 3215844
20652678
17683
rs12214315
20652722
17727
rsl 1759517
20652786
17791
rs13218957
20652806
17811
rs13218962
20653188
18193
rsl 0543744
20653201
18206
rsl 2216047
20653447
18452
rs9366354
20653890
18895
rs9358342
20654091
19096
rs9368201
20654382
19387
rs34206163
20654506
19511
rs9465799
20654794
19799
rs34187071
20654867
19872
rs9465800
20654890
19895
rs6908974
20654992
19997
rsl 3197372
20655361
20366
rs13214145
20655793
20798
rsl 6883910
20655968
20973
rsl 2194705
20656271
21276
rs35080661
20656465
21470
rs7753467
20656466
21471
rs7773488
20656986
21991
rs34182285
20657084
22089
rs34242699
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20657780
22785
rs9348433
20657942
22947
rs9460519
20658083
23088
rsl 2198377
20658096
23101
rs9465801
20658195
23200
rs9465802
20658822
23827
rs28458932
20658823
23828
rs9465803
20658981
23986
rs2103682
20659321
24326
rs9465804
20659580
24585
rs34611621
20660058
25063
rsl 2055423
20660653
25658
rs9465805
20660829
25834
rsl 1365187
20660836
25841
rsl 1320714
20660918
25923
rs9350256
20661764
26769
rs7756211
20662069
27074
rs9460520
20662498
27503
rs34245467
20662930
27935
rs9350257
20663855
28860
rsl 1964554
20663990
28995
rs9465806
20664109
29114
rsl 1964635
20664190
29195
rsl 3199421
20664314
29319
rs6932320
20664570
29575
rsl 2200078
20664659
29664
rsl 3437555
20664884
29889
rs9350258
20665256
30261
rs12176441
20665260
30265
rsl 2183826
20665264
30269
rs9356738
20665272
30277
rs9348434
20665343
30348
rs9465807
20665804
30809
rs4458667
20665995
31000
rs7739402
20667590
32595
rs16883914
20667591
32596
rs16883916
20667900
32905
rs9654584
20667999
33004
rs9465808
20668414
33419
rsl 7584626
20668565
33570
rs7751682
20669667
34672
rsl 1361279
20669681
34686
rs34634263
20670059
35064
rsl 2214549
20670364
35369
rs7753519
20670575
35580
rs28567007
20670597
35602
rs7772137
20670719
35724
rsl 2208597
20670998
36003
rs9368202
20671877
36882
rs2328526
20672452
37457
rs34823358
20673287
38292
rs28639914
20673363
38368
rs34233572
20673415
38420
rs4712506
20673935
38940
rsl 3203450
20674280
39285
rs9350259
20674435
39440
rs6918457
20674595
39600
rs35210537
20674749
39754
rsl 1329887
20675016
40021
rs9348435
108
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20675068
40073
rs35366106
20675342
40347
rsl 6901563
20675352
40357
rsl 2333229
20675520
40525
rs9460521
20676094
41099
rsl 0589899
20676351
41356
rs2876573
20676957
41962
rs6935461
20676963
41968
rs6935465
20676968
41973
rsl 0603174
20677060
42065
rsl 2333291
20677967
42972
rs2064321
20677985
42990
rs35546893
20678018
43023
rs4291090
20678121
43126
rs2064320
20678268
43273
rs9465810
20678275
43280
rs9465811
20678423
43428
rs9358344
20678756
43761
rsl 0946390
20679114
44119
rs6905281
20679339
44344
rsl 6883932
20679612
44617
rs34904067
20679660
44665
rs7744002
20679763
44768
rs35142564
20680095
45100
rs9465812
20680678
45683
rs7759094
20680784
45789
rs9460522
20681538
46543
rs7764551
20681585
46590
rsl 0541455
20682409
47414
rsl 6883935
20682542
47547
rsl 3215603
20682568
47573
rs962576
20683235
48240
rsl 474720
20683797
48802
rsl 6883944
20684155
49160
rs34538343
20684269
49274
rs9350260
20684353
49358
rsl 6883951
20684645
49650
rs9358345
20684862
49867
rs1012627
20684890
49895
rs9368203
20684939
49944
rs35894322
20684965
49970
rs4710932
20684984
49989
rs6909117
20685540
50545
rs1012626
20685748
50753
rs1012625
20685760
50765
rs7752194
20685958
50963
rs9465813
20686014
51019
rsl 2207923
20686355
51360
rsl 6883963
20686831
51836
rsl 3205786
20686887
51892
rs35205364
20687102
52107
rsl 0456232
20687189
52194
rs9465814
20687201
52206
rs35571892
20687740
52745
rs9465815
20687753
52758
rs36119371
20687921
52926
rs28621813
20687926
52931
rs9350261
20687928
52933
rs7341226
20688175
53180
rs6927481
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20688323
53328
rs35313444
20688373
53378
rs6928198
20688404
53409
rs6907897
20688545
53550
rs6928586
20688872
53877
rs9368204
20689021
54026
rs9358346
20689589
54594
rsl 1967546
20689593
54598
rs34134803
20689772
54777
rsl 0456233
20689807
54812
rs7744833
20690123
55128
rs9460523
20690123
55128
rs9465816
20690432
55437
rs6908077
20690630
55635
rs9465817
20691069
56074
rsl 1967445
20691263
56268
rs34022950
20691793
56798
rs9460524
20691994
56999
rs34020592
20692003
57008
rsl 1448102
20692339
57344
rs9465818
20692402
57407
rs9350262
20692513
57518
rsl 3205241
20693000
58005
rsl 2153939
20693100
58105
rs6925593
20693119
58124
rs4712507
20693226
58231
rsl 0558806
20693267
58272
rs35982532
20693276
58281
rsl 1385529
20693360
58365
rs9348436
20693416
58421
rs9368206
20693438
58443
rsl 3209542
20693452
58457
rs9368207
20693630
58635
rsl 3209907
20693635
58640
rs6926658
20694018
59023
rs12213132
20694182
59187
rs4357125
20694554
59559
rs6932944
20694607
59612
rs6932962
20695026
60031
rs9348437
20695332
60337
rsl 2201857
20695356
60361
rs9465819
20695447
60452
rs6938955
20695539
60544
rs9460525
20695827
60832
rs9465820
20695964
60969
rsl 0946391
20695968
60973
rs9368208
20696003
61008
rs9465821
20696183
61188
rs6923790
20696401
61406
rsl 0558139
20697309
62314
rs6907459
20697320
62325
rs6907767
20697321
62326
rs9465822
20697349
62354
rs6930283
20697706
62711
rs6908042
20697741
62746
rs6935317
20697761
62766
rs35370102
20698266
63271
rs9368209
20698366
63371
rsl 3216746
20698367
63372
rsl 3216747
109
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20699007
64012
rs35485532
20699747
64752
rs13216324
20699817
64822
rs4336434
20700046
65051
rs4509107
20700428
65433
rs9465823
20700465
65470
rs6936705
20700679
65684
rs34023799
20700929
65934
rs6942313
20701057
66062
rs28869917
20701318
66323
rs34982231
20701631
66636
rs9358349
20701770
66775
rs9460526
20701829
66834
rs9366356
20702163
67168
rs36120092
20702181
67186
rs9465824
20702519
67524
rs4712512
20702561
67566
rs4712513
20702646
67651
rs4710934
20702658
67663
rs9348438
20702902
67907
rs9460529
20703363
68368
rsl 3199587
20703470
68475
rsl 3199384
20703526
68531
rsl 0223680
20703606
68611
rs9350263
20703768
68773
rs9465825
20703832
68837
rsl 0223876
20704100
69105
rs35702271
20704171
69176
rs9358350
20704432
69437
rsl 2208985
20704771
69776
rsl 2210459
20704892
69897
rs35431707
20705144
70149
rs36039523
20705297
70302
rsl 1758281
20705350
70355
rs28893199
20705757
70762
rs34256347
20706019
71024
rsl 2192740
20706282
71287
rsl 3212326
20706486
71491
rs12199184
20706753
71758
rsl 0456234
20707009
72014
rs4712514
20707422
72427
rs9465826
20707607
72612
rs9366357
20707867
72872
rs2294809
20708549
73554
rs2294808
20708813
73818
rs7762750
20708976
73981
rs4712515
20708998
74003
rsl 0522824
20708999
74004
rs35660518
20709002
74007
rsl 0679950
20709003
74008
rs34870864
20709022
74027
rs4712516
20709145
74150
rs4710935
20709386
74391
rs9465827
20709388
74393
rsl 2204865
20709672
74677
rsl 0946393
20709764
74769
rsl 2209806
20709894
74899
rsl 0946394
20709921
74926
rsl 997778
20709971
74976
rs35878587
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20710359
75364
rsl 997777
20710378
75383
rs2223622
20710776
75781
rsl 1964057
20711246
76251
rs9460530
20711344
76349
rs9460531
20711376
76381
rs34329159
20711640
76645
rs7764558
20711804
76809
rs4710936
20712056
77061 -
rs12213940
20712228
77233
rs13215038
20712739
77744
rsl 0946395
20712832
77837
rs6939917
20712975
77980
rs9358351
20713800
78805
rs6925097
20713924
78929
rs9465828
20713955
78960
rs9465829
20713961
78966
rs6902661
20714057
79062
rs34373680
20714281
79286
rs35051096
20714508
79513
rs932405
20714591
79596
rs6926585
20714635
79640
rs3938395
20715464
80469
rsl 1964664
20715551
80556
rs35964987
20715663
80668
rsl 2206413
20715758
80763
rs35990187
20715763
80768
rs4991654
20715910
80915
rs9460532
20715991
80996
rsl 3328250
20716030
81035
rsl 3328252
20716194
81199
rs4712517
20716257
81262
rs4712518
20717220
82225
rs7758129
20717475
82480
rsl 3206462
20717483
82488
rsl 3192442
20717486
82491
rsl 3206468
20717492
82497
rsl 3192445
20717498
82503
rsl 3192450
20717504
82509
rsl 3206477
20717510
82515
rsl 3206483
20717577
82582
rs12179168
20717586
82591
rsl 2180975
20717611
82616
rs12179172
20717860
82865
rsl 2179563
20718357
83362
rsl 1355836
20718696
83701
rs2328527
20718709
83714
rsl 1452882
20718920
83925
rs2876574
20719905
84910
rs9465831
20720031
85036
rs34877824
20720290
85295
rsl 3212501
20720647
85652
rs9358352
20720703
85708
rs9358353
20720761
85766
rs28756205
20720889
85894
rs9350265
20720989
85994
rs7750508
20721130
86135
rs7771052
20721141
86146
rs9460533
20721195
86200
rsl 3200415
110
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20721216
86221
rsl 0550932
20721312
86317
rs9465832
20721463
86468
rs9368211
20721471
86476
rs9350266
20721507
86512
rsl 1752592
20721515
86520
rs9350267
20721754
86759
rs978988
20721898
86903
rs978987
20721906
86911
rs978986
20722036
87041
rs5874773
20722196
87201
rs5874774
20722500
87505
rs7756788
20722659
87664
rs9348439
20722661
87666
rs9356739
20722693
87698
rs9356740
20722738
87743
rs7760894
20723021
88026
rs2796913
20723046
88051
rs2608613
20723121
88126
rsl 0456710
20723139
88144
rs9476286
20723140
88145
rs9476287
20723140
88145
rs28368538
20723141
88146
rs28612622
20723142
88147
rs9463660
20723146
88151
rs9463661
20723162
88167
rs9461022
20723193
88198
rs9461021
20723235
88240
rs34980442
20723239
88244
rs34049080
20723258
88263
rs35218684
20723260
88265
rsl 2175876
20723270
88275
rsl 0948323
20723287
88292
rs34400313
20723292
88297
rs36081550
20723304
88309
rs12175878
20723305
88310
rs34520184
20723305
88310
rs34756989
20723322
88327
rs4629736
20723322
88327
rs9296917
20723324
88329
rs28562027
20723333
88338
rs9381823
20723346
88351
rs34769771
20723346
88351
rs34774640
20723346
88351
rs36038896
20723365
88370
rsl 3209195
20723369
88374
rs12213541
20723369
88374
rs34112320
20723371
88376
rs34615869
20723379
88384
rs35949519
20723381
88386
rs9257498
20723383
88388
rs34200576
20723393
88398
rs4960519
20723396
88401
rs9261905
20723402
88407
rs9267103
20723404
88409
rsl 7367677
20723416
88421
rs9261906
20723421
88426
rs9267104
20723424
88429
rs9267105
20723434
88439
rs28810763
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20723438
88443
rsl 2179121
20723438 ■
88443
rs4714959
20723439
88444
rsl 0946636
20723439
88444
rs28763327
20723439
88444
rs28847950
20723439
88444
rs3933247
20723449
88454
rs28808723
20723451
88456
rs9767082
20723452
88457
rs35132675
20723452
88457
rs35517166
20723452
88457
rs4714297
20723456
88461
rsl 2182737
20723456
88461
rs36163804
20723460
88465
rs35790973
20723464
88469
rs35236694
20723469
88474
rs35567559
20723469
88474
rs9267106
20723471
88476
rs9800557
20723484
88489
rsl 2182463
20723489
88494
rsl 0948322
20723489
88494
rs34052284
20723489
88494
rs9268999
20723490
88495
rs9258377
20723495
88500
rs9257499
20723498
88503
rs9265816
20723500
88505
rs28771402
20723501
88506
rs28771401
20723502
88507
rsl 2194731
20723502
88507
rs4451188
20723502
88507
rs9717323
20723502
88507
rs9765920
20723503
88508
rs9265815
20723504
88509
rs28771400
20723506
88511
rs12190813
20723506
88511
rs12215416
20723507
88512
rsl 2178527
20723510
88515
rs35234761
20723510
88515
rs35887156
20723512
88517
rs28797321
20723515
88520
rs28771399
20723522
88527
rsl 3207682
20723522
88527
rs36099432
20723531
88536
rs28749543
20723535
88540
rsl 3196506
20723535
88540
rs35716308
20723536
88541
rs28831180
20723536
88541
rs34938144
20723537
88542
rs36142967
20723541
88546
rs35313792
20723542
88547
rs9260904
20723543
88548
rs34597832
20723545
88550
rs9767740
20723546
88551
rs28771398
20723550
88555
rs34384951
20723554
88559
rsl 2524128
20723554
88559
rsl 2665124
20723554
88559
rs34922643
20723555
88560
rs9260903
20723557
88562
rs28771397
111
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20723557
88562
rs6915279
20723559
88564
rs9261907
20723559
88564
rs9766798
20723559
88564
rs9767242
20723560
88565
rs6914835
20723560
88565
rs9260902
20723560
88565
rs9767101
20723562
88567
rs9261908
20723563
88568
rsl 2207064
20723563
88568
rs12213193
20723563
88568
rs35750154
20723566
88571
rs9260901
20723570
88575
rs9269000
20723579
88584
rs12178368
20723579
88584
rsl 3197714
20723579
88584
rs28771396
20723579
88584
rs34443697
20723579
88584
rs9267107
20723582
88587
rs35113301
20723582
88587
rs3933248
20723583
88588
rs28819830
20723586
88591
rsl 0947899
20723586
88591
rs9260900
20723588
88593
rsl 3204671
20723588
88593
rs34094007
20723588
88593
rs34963756
20723588
88593
rs4304158
20723589
88594
rs4298351
20723590
88595
rs12182307
20723590
88595
rsl 2201487
20723590
88595
rs34097573
20723595
88600
rs13219021
20723596
88601
rs34456153
20723596
88601
rs9767597
20723597
88602
rs9767102
20723598
88603
rs9269001
20723601
88606
rsl 2180097
20723603
88608
rsl 2178465
20723603
88608
rs35128115
20723607
88612
rs4273681
20723609
88614
rsl 2193754
20723617
88622
rs813814
20723617
88622
rs9689672
20723619
88624
rs36173068
20723623
88628
rsl 2202172
20723623
88628
rs28862376
20723625
88630
rsl 2178884
20723625
88630
rsl 2203117
20723625
88630
rs34250588
20723626
88631
rs12182581
20723632
88637
rs34147797
20723635
88640
rs9688484
20723636
88641
rs9269002
20723636
88641
rs9767733
20723637
88642
rsl 0948989
20723637
88642
rs34732676
20723637
88642
rs9260899
20723638
88643
rs35686233
20723639
88644
rsl 1753098
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20723639
88644
rs35156647
20723646
88651
rs35575623
20723646
88651
rs34719653
20723648
88653
rsl 2192046
20723649
88654
rsl 1759854
20723649
88654
rs28771395
20723649
88654
rs35563402
20723649
88654
rs36016334
20723649
88654
rs4555911
20723653
88658
rsl 1965757
20723654
88659
rsl 0948990
20723654
88659
rs28808296
20723654
88659
rs34995142
20723654
88659
rs809919
20723657
88662
rsl 1571978
20723659
88664
rsl 2208488
20723662
88667
rsl 1751374
20723663
88668
rs13217613
20723667
88672
rsl 2528735
20723668
88673
rs35160656
20723668
88673
rs35322569
20723668
88673
rs9766221
20723670
88675
rs35120225
20723670
88675
rs511868
20723674
88679
rs9269003
20723675
88680
rs34290316
20723675
88680
rs35735496
20723675
88680
rs9689102
20723676
88681
rsl 0948991
20723676
88681
rs9269004
20723677
88682
rsl 3220607
20723677
88682
rs34700647
20723677
88682
rs9260898
20723678
88683
rs9260897
20723684
88689
rs28771394
20723686
88691
rs12213200
20723687
88692
rs9717987
20723688
88693
rs28771393
20723688
88693
rs9717716
20723689
88694
rs28771392
20723690
88695
rsl 1758009
20723690
88695
rs34159662
20723690
88695
rs34683172
20723690
88695
rs9717331
20723691
88696
rs9717853
20723692
88697
rs9765875
20723696
88701
rs12178974
20723696
88701
rs36003577
20723698
88703
rs9767747
20723700
88705
rs28771391
20723704
88709
rsl 3216352
20723707
88712
rsl 2200762
20723707
88712
rsl 2206373
20723707
88712
rs34570202
20723707
88712
rs36197940
20723707
88712
rs9265814
20723714
88719
rs34131282
20723717
88722
rsl 2175478
20723720
88725
rs28772692
112
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20723722
88727
rs35516674
20723722
88727
rs35704013
20723727
88732
rs34691406
20723728
88733
rs28771390
20723728
88733
rs4374863
20723730
88735
rs34826149
20723734
88739
rsl 2207894
20723734
88739
rsl 2662476
20723736
88741
rs9260896
20723739
88744
rs28771389
20723740
88745
rs28771388
20723741
88746
rs9382592
20723745
88750
rs12173375
20723745
88750
rs9268072
20723745
88750
rs9382110
20723746
88751
rs4715211
20723747
88752
rs34128950
20723747
88752
rs35961188
20723747
88752
rs4620119
20723749
88754
rs9767236
20723752
88757
rs34717143
20723755
88760
rsl 3201202
20723756
88761
rsl 3201503
20723762
88767
rsl 3197088
20723762
88767
rs35347849
20723766
88771
rs34663083
20723766
88771
rs35650828
20723767
88772
rsl 3199241
20723767
88772
rs34205031
20723768
88773
rs28749541
20723768
88773
rs34469031
20723768
88773
rs9279137
20723769
88774
rs9260895
20723770
88775
rsl 2212483
20723770
88775
rs35446958
20723775
88780
rs28380829
20723777
88782
rsl 2525384
20723777
88782
rs34576984
20723782
88787
rs283541
20723785
88790
rs28380828
20723788
88793
rsl 0948698
20723790
88795
rs9265813
20723791
88796
rs9689173
20723792
88797
rs28380827
20723795
88800
rs35399169
20723799
88804
rs9260894
20723800
88805
rsl 2173388
20723800
88805
rs9269005
20723802
88807
rs28359816
20723803
88808
rs28380826
20723807
88812
rs280297
20723807
88812
rs34507582
20723807
88812
rs9261623
20723810
88815
rsl 2174621
20723813
88818
rs28380825
20723815
88820
rs35197377
20723816
88821
rsl 7362870
20723816
88821
rs34562190
20723816
88821
rs35071522
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20723816
88821
rs35179751
20723816
88821
rs4458721
20723817
88822
rsl 2180385
20723817
88822
rsl 2206581
20723817
88822
rsl 2333308
20723817
88822
rs9269006
20723821
88826
rs9688475
20723829
88834
rs9269007
20723832
88837
rs9269008
20723832
88837
rs9269009
20723834
88839
rs35899754
20723836
88841
rs13211190
20723837
88842
rsl 2216274
20723838
88843
rs12191544
20723838
88843
rs4337934
20723841
88846
rs9279295
20723842
88847
rsl 2178577
20723849
88854
rsl 2215604
20723853
88858
rs9395360
20723854
88859
rs28749540
20723855 _
88860
rs28895226
20723855
88860
rs9260893
20723861
88866
rs4365925
20723864
88869
rs9261912
20723865
88870
rs28895227
20723870
88875
rs9261913
20723873
88878
rsl 2183502
20723873
88878
rs34332895
20723873
88878
rs9472692
20723876
88881
rs35640475
20723884
88889
rs9269010
20723888
88893
rsl 2192337
20723888
88893
rs35349786
20723891
88896
rs34978372
20723893
88898
rs280296
20723894
88899
rsl 1755576
20723903
88908
rs28803616
20723903
88908
rs35553517
20723904
88909
rs4358654
20723910
88915
rs9260892
20723914
88919
rsl 3196941
20723914
88919
rs9717238
20723915
88920
rs12175553
20723915
88920
rs12191408
20723915
88920
rs4460210
20723915
88920
rs9260891
20723918
88923
rs9395361
20723920
88925
rs9766917
20723926
88931
rs35073779
20723931
88936
rs9765884
20723931
88936
rs9767229
20723932
88937
rs35247846
20723932
88937
rs9717328
20723936
88941
rs28805674
20723936
88941
rs9260890
20723938
88943 J
rs34926348
20723938
88943
rs9717233
20723945
88950
rs35869128
20723946
88951
rs12175941
113
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20723951
88956
rs9260889
20723952
88957
rs9766686
20723956
88961
rsl 0046203
20723957
88962
rs9260888
20723958
88963
rsl 2180540
20723961
88966
rs9766115
20723968
88973
rs6912019
20723969
88974
rs9767458
20723976
88981
rsl 3202161
20723989
88994
rsl 3218048
20724000
89005
rsl 3218040
20724001
89006
rsl 3206557
20724002
89007
rsl 3202152
20724013
89018
rsl 2210049
20724013
89018
rs34552032
20724018
89023
rs28890881
20724018
89023
rs34478537
20724025
89030
rs35191657
20724038
89043
rs34450517
20724043
89048
rs9689655
20724064
89069
rs12180172
20724068
89073
rs28865015
20724071
89076
rs9717839
20724072
89077
rs9717836
20724074
89079
rs9885593
20724082
89087
rs28887572
20724083
89088
rs13216113
20724083
89088
rs35254115
20724084
89089
rsl 0949190
20724085
89090
rs6923503
20724087
89092
rs12214411
20724091
89096
rs28747986
20724096
89101
rsl 087363
20724096
89101
rs1091092
20724104
89109
rsl 2180759
20724107
89112
rsl 1758052
20724112
89117
rs9261321
20724113
89118
rs28861738
20724113
89118
rs36109104
20724123
89128
rsl 3208115
20724129
89134
rs9472693
20724130
89135
rsl 2208570
20724130
89135
rs28832660
20724131
89136
rsl 3200482
20724131
89136
rs9260886
20724131
89136
rs9472694
20724159
89164
rs9260885
20724172
89177
rs280295
20724172
89177
rs28749538
20724174
89179
rsl 3201041
20724184
89189
rsl 087362
20724187
89192
rsl 2183850
20724190
89195
rsl 2202552
20724208
89213
rs9260884
20724210
89215
rs9474341
20724211
89216
rs9260883
20724222
89227
rs34669820
20724225
89230
rsl 2189886
20724234
89239
rs12182114
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20724266
89271
rs9688643
20724272
89277
rsl 2195237
20724281
89286
rs35293595
20724281
89286
rs9381428
20724306
89311
rs34665644
20724309
89314
rsl 2189599
20724311
89316
rsl 2208503
20724312
89317
rs9800791
20724330
89335
rsl 2173681
20724337
89342
rsl 2182094
20724343
89348
rs12182119
20724352
89357
rs12182102
20724392
89397
rsl 2174506
20724393
89398
rs9767809
20724394
89399
rs12173703
20724401
89406
rsl 2202889
20724435
89440
rs28840538
20724466
89471
rs9473983
20724467
89472
rs34220946
20724487
89492
rs9261322
20724491
89496
rs9261323
20724517
89522
rs526092
20724564
89569
rs9473982
20724566
89571
rs34307764
20724626
89631
rs34490559
20724665
89670
rs4449621
20724685
89690
rs4714818
20724698
89703
rs4714817
20724722
89727
rs682384
20724738
89743
rs682051
20724753
89758
rsl 2530056
20724756
89761
rs35086683
20724888
89893
rsl 2528468
20725092
90097
rs10650195
20725092
90097
rs35956126
20725109
90114
rs6937578
20725159
90164
rsl 2530107
20725173
90178
rs720448
20725176
90181
rs9368212
20725250
90255
rs35481531
20725253
90258
rs720449
20725262
90267
rsl 0650196
20725495
90500
rs2064
20725499
90504
rs2065
20725638
90643
rs9368213
20725732
90737
rs41455744
20726077
91082
rs34960654
20726154
91159
rs6900954
20726168
91173
rs6456359
20726176
91181
rs7739405
20726601
91606
rs7760508
20726684
91689
rsl 0456007
20726987
91992
rs9460534
20727168
92173
rsl 1966749
20727353
92358
rs9358354
20727809
92814
rs9368214
20727982
92987
rs9350268
20728095
93100
rs2069013
20728160
93165
rs2069014
114
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20728290
93295
rs2069015
20728292
93297
rsl 1967475
20729096
94101
rs10214549
20729217
94222
rs2223621
20729281
94286
rs9465835
20729984
94989
rs35404829
20729995
95000
rsl 0687080
20730233
95238
rs6915936
20730331
95336
rs9295472
20730732
95737
rsl 569660
20730929
95934
rs7455009
20730945
95950
rsl 569659
20730946
95951
rs5874775
20730948
95953
rsl 0601252
20730983
95988
rsl 569658
20731030
96035
rs34142400
20731063
96068
rsl 6883996
20731181
96186
rs35073110
20731184
96189
rs5028948
20731263
96268
rs34201758
20731371
96376
rs6456360
20731373
96378
rs6456361
20731380
96385
rs6456362
20731584
96589
rs6922571
20731925
96930
rs35145358
20731950
96955
rs9465836
20732013
97018
rs7763304
20732158
97163
rs9465837
20732218
97223
rs7743314
20732337
97342
rs714831
20732361
97366
rs714830
20732484
97489
rs7743789
20732584
97589
rs5874776
20732589
97594
rs5874777
20732965
97970
rsl 6884003
20733079
98084
rs9356741
20733080
98085
rs9366358
20733246
98251
rs9356742
20733430
98435
rsl 1756987
20733470
98475
rs9465838
20733613
98618
rs2206579
20733920
98925
rs35111339
20734361
99366
rs6917583
20734388
99393
rs6917599
20734630
99635
rsl 3437429
20735418
100423
rs34307011
20735420
100425
rs4515379
20735756
100761
rs2223620
20735870
100875
rs9465839
20735993
100998
rsl 2664972
20736790
101795
rs28402356
20736798
101803
rs6934727
20736873
101878
rs35493429
20736881
101886
rsl 1369825
20737261
102266
rs4523079
20737470
102475
rs7771213
20737568
102573
rs9465840
20737687
102692
rs9465841
20737767
102772
rs9465842
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20737837
102842
rs9460535
20737992
102997
rs34835908
20738060
103065
rs9465843
20738139
103144
rsl 3220465
20738159
103164
rsl 3220352
20738321
103326
rs4710937
20738375
103380
rs9460536
20738376
103381
rsl 3190734
20738451
103456
rs9465844
20738712
103717
rs35976895
20738713
103718
rsl 1327958
20738932
103937
rsl 3194407
20738990
103995
rsl 0484633
20739524
104529
rs2328528
20739789
104794
rs35958155
20739809
104814
rs4421185
20739932
104937
rs2328529
20740089
105094
rs9460537
20740745
105750
rsl 7224527
20740886
105891
rs34166991
20741024
106029
rsl 7823073
20741276
106281
rs12183074
20741316
106321
rs9465845
20741844
106849
rs35148963
20741886
106891
rs7768642
20742320
107325
rs9465846
20742407
107412
rs9465847
20742551
107556
rsl 7823127
20742594
107599
rsl 6884021
20742630
107635
rs4533976
20742865
107870
rs7755830
20743005
108010
rs7756031
20743139
108144
rs35650451
20743192
108197
rs10561117
20743241
108246
rs6940200
20743799
108804
rs9465848
20743808
108813
rs34351919
20745083
110088
rs4712519
20745285
110290
rs9368215
20745566
110571
rsl 2206285
20745853
110858
rs34049994
20745988
110993
rs7751957
20746702
111707
rs9465849
20746857
111862
rs7341291
20746957
111962
rs6921014
20747388
112393
rs9465850
20747583
112588
rs7758612
20747681
112686
rs35602526
20748295
113300
rs9465851
20748399
113404
rs34257578
20748403
113408
rsl 1339738
20748516
113521
rs9460538
20748850
113855
rs4712520
20748883
113888
rs4710938
20749084
114089
rs35889049
20749315
114320
rs9348440
20749879
114884
rs9465852
20750054
115059
rsl 2196009
20750306
115311
rsl 1968248
115
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20750353
115358
rs34797179
20750764
115769
rs6925328
20750913
115918
rs36073053
20751137
116142
rs35462488
20751140
116145
rs36224625
20751150
116155
rs3060659
20751153
116158
rs5874778
20751155
116160
rsl 1267861
20751201
116206
rs4235999
20751230
116235
rsl 6884038
20751706
116711
rs2328530
20751731
116736
rs2328531
20751750
116755
rs28360636
20752162
117167
rs6932676
20752183
117188
rs9460539
20752303
117308
rs6932876
20752346
117351
rs34929755
20752359
117364
rs28733367
20752360
117365
rsl 1348111
20752473
117478
rs6933219
20752702
117707
rs6933165
20752872
117877
rs35255583
20752901
117906
rs34332316
20752923
117928
rs4710939
20753486
118491
rsl 1970417
20753659
118664
rsl 1963217
20753670
118675
rsl 1965473
20753833
118838
rs2876575
20754049
119054
rs7739974
20754976
119981
rs7745175
20755173
120178
rs7765784
20755178
120183
rs6907731
20755250
120255
rs35057896
20755313
120318
rs7746072
20755639
120644
rsl 0484632
20755770
120775
rs35444529
20755849
120854
rsl 7823571
20755941
120946
rsl 1965062
20755962
120967
rs6936199
20755965
120970
rs6913126
20756178
121183
rs6913509
20756613
121618
rs34638218
20756673
121678
rs35746011
20756741
121746
rs9460540
20757090
122095
rs36045545
20757129
122134
rs35392790
20757233
122238
rs6456364
20757260
122265
rs2179553
20757513
122518
rs9350269
20757531
122536
rs9465854
20757787
122792
rs2179552
20757936
122941
rs6925233
20758033
123038
rs2328532
20758248
123253
rs7743970
20758317
123322
rsl 3209457
20758318
123323
rs34641285
20758344
123349
rsl 3209538
20758387
123392
rs2876576
20758479
123484
rsl 3209572
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20758653
123658
rs28783153
20758712
123717
rs9969037
20758800
123805
rs7766844
20758969
123974
rs7767133
20759291
124296
rs7749464
20760100
125105
rs2050225
20760696
125701
rs9295474
20760744
125749
rs9295475
20761529
126534
rs2328545
20761548
126553
rs28846771
20761779
126784
rs2876582
20761899
126904
rsl 0223446
20762094
127099
rsl 3219723
20762095
127100
rsl 3203489
20762154
127159
rsl 3203583
20762172
127177
rsl 3203887
20762279
127284
rs6456366
20762876
127881
rs9358355
20763089
128094
rs9368216
20763375
128380
rs9465855
20763384
128389
rs9465856
20763463
128468
rsl 6884070
20763482
128487
rsl 6884072
20763647
128652
rsl 3208604
20764169
129174
rs9465857
20764307
129312
rs9368217
20764559
129564
rs9460541
20764746
129751
rs9460542
20764765
129770
rsl 1969955
20764779
129784
rs4712522
20764924
129929
rsl 6884074
20765172
130177
rs34489684
20765324
130329
rs2328546
20765543
130548
rs4712523
20765844
130849
rs4712524
20765898
130903
rs35397753
20765991
130996
rs4710940
20766197
131202
rsl 3190727
20766215
131220
rs35136485
20766311
131316
rs35260725
20766335
131340
rs35939620
20766566
131571
rsl 7823996
20766713
131718
rsl 6884082
20767438
132443
rs6906327
20767566
132571
rs6456367
20767785
132790
rs6456368
20768202
133207
rs7749083
20768344
133349
rs6456369
20768669
133674
rsl 0946396
20768672
133677
rsl 0946397
20768710
133715
rsl 1759505
20769000
134005
rsl 3203361
20769013
134018
rsl 0946398
20769122
134127
rs7774594
20769229
134234
rs7754840
20769249
134254
rs9460543
20769508
134513
rs9460544
20769529
134534
rs9460545
20769711
134716
rs2206740
116
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20769806
134811
rs5874779
20769807
134812
rs33970890
20769815
134820
rs5874780
20769816
134821
rs35014292
20769816
134821
rs35363501
20770092
135097
rs6456370
20770102
135107
rs979614
20770196
135201
rs35456723
20770571
135576
rs9368218
20770945
135950
rs4712525
20771014
136019
rs4712526
20771314
136319
rs4712527
20771442
136447
rs35191644
20771442
136447
rs34470647
20771611
136616
rs9460546
20771938
136943
rs9465859
20772079
137084
rs9465860
20772291
137296
rs736425
20772508
137513
rs3060776
20772509
137514
rs34941928
20772512
137517
rs5874781
20772761
137766
rs35778487
20773060
138065
rs742642
20773305
138310
rs35248697
20773436
138441
rsl 1967127
20773528
138533
rs7748382
20773547
138552
rs9688549
20773548
138553
rs9689351
20773570
138575
rs28665000
20773886
138891
rs7752236
20773925
138930
rs7772603
20774001
139006
rs7752780
20774034
139039
rs7752906
20774160
139165
rs34184260
20774223
139228
rs2206739
20774225
139230
rs2206738
20774250
139255
rs2206737
20774436
139441
rsl 1970425
20774484
139489
rs36034806
20774899
139904
rs35042364
20775218
140223
rs35540121
20775361
140366
rs9358356
20775667
140672
rs9356743
20775778
140783
rs9350270
20776366
141371
rs34929853
20778035
143040
rs34971765
20778443
143448
rsl 1970596
20779261
144266
rsl 2527373
20779367
144372
rs35916847
20780262
145267
rsl 1968224
20780271
145276
rsl 1968225
20780276
145281
rs9465861
20780296
145301
rsl 1968264
20780406
145411
rsl 2189849
20780413
145418
rsl 2209627
20780432
145437
rsl 2189895
20780855
145860
rsl 1968848
20781135
146140
rsl 1963945
20781601
146606
rs35677128
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20781859
146864
rs7451008
20782670
147675
rs9368219
20782790
147795
rs1012636
20782945
147950
rs13217846
20783274
148279
rs1012635
20783700
148705
rs35665197
20783771
148776
rs35261542
20783828
148833
rs28823314
20783899
148904
rs28890810
20784051
149056
rs28871991
20784393
149398
rs34499031
20784650
149655
rsl 3208763
20784747
149752
rs28719685
20784789
149794
rs28856096
20785042
150047
rsl 1961863
20785211
150216
rsl 7824302
20785289
150294
rsl 2660618
20786302
151307
rsl 1371206
20786303
151308
rs34152621
20786409
151414
rs4712528
20786463
151468
rs13217082
20786470
151475
rsl 3217085
20786481
151486
rsl 3217090
20786483
151488
rsl 3217091
20786523
151528
rsl 3200946
20786772
151777
rsl 1968032
20786954
151959
rs9465863
20787289
152294
rsl 569699
20787386
152391
rs34168173
20787688
152693
rs7756992
20788045
153050
rs35312717
20788657
153662
rs9348441
20788843
153848
rs9368220
20788941
153946
rs6931254
20789327
154332
rs6911742
20790601
155606
rs35612982
20791039
156044
rs35816514
20791123
156128
rs34612860
20791143
156148
rs9350271
20791162
156167
rs35657899
20791179
156184
rsl 1364854
20791249
156254
rs9460547
20791646
156651
rsl 6884103
20791961
156966
rs2206736
20793465
158470
rs9356744
20794295
159300
rs34987372
20794427
159432
rs36005020
20794552
159557
rs7766070
20794975
159980
rs9368222
20795290
160295
rs35566695
20795781
160786
rsl 0440832
20796100
161105
rsl 0440833
20796237
161242
rs35747076
20796578
161583
rs6900217
20797104
162109
rs34433496
20797924
162929
rs7748720
20797928
162933
rs34175709
20798290
163295
rs6911357
20800493
165498
rsl 2200791
117
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20800955
165960
rs5874782
20800957
165962
rs36119385
20801341
166346
rsl 3219682
20802207
167212
rs4710941
20802270
167275
rs4620109
20802272
167277
rs28459626
20802273
167278
rs4712529
20802294
167299
rsl 0577753
20802504
167509
rs2223683
20802573
167578
rs2206735
20802863
167868
rs2206734
20802910
167915
rs34530846
20803458
168463
rsl 6884131
20804127
169132
rsl 0806921
20805104
170109
rsl 6884133
20805571
170576
rsl 7824500
20805652
170657
rsl 0946401
20806114
171119
rsl 6884135
20806582
171587
rs35711395
20807220
172225
rsl 1969783
20807364
172369
rsl 6884137
20808600
173605
rsl 1970626
20809092
174097
rs12190713
20809106
174111
rsl 1398905
20809415
174420
rsl 1961445
20809470
174475
rs35982077
20809486
174491
rsl 1305935
20810952
175957
rs9356745
20811700
176705
rs35043644
20811842
176847
rsl 6884140
20811931
176936
rs6931514
20812147
177152
rs35443650
20813281
178286
rs34671712
20813569
178574
rsl 1753081
20814081
179086
rs7739516
20814209
179214
rs6901559
20815176
180181
rsl 3196379
20815177
180182
rs13212234
20816204
181209
rs10536170
20817155
182160
rs9465869
20817688
182693
rs36070002
20818288
183293
rsl 7226450
20818905
183910
rsl 073247
20819131
184136
rs9465870
20819386
184391
rsl 7226492
20819433
184438
rs13213613
20819567
184572
rsl 6884146
20819958
184963
rs2206733
20820440
185445
rs3749925
20821121
186126
rs9460548
20821619
186624
rs9460549
20821685
186690
rsl 040558
20821893
186898
rs4712530
20822083
187088
rs35629277
20822362
187367
rs7451928
20822445
187450
rs6456371
20822589
187594
rsl 3220116
20822823
187828
rs2206732
20823169
188174
rs2179633
Position in Build 35/36
Position in SEQ ID NO:1
Marker ID
20823483
188488
rsl 1963770
20823805
188810
rsl 0946402
20823840
188845
rs4712531
20824098
189103
rs35738288
20824232
189237
rs9295478
20824549
189554
rs2328547
20824763
189768
rs3060781
20824764
189769
rs34686252
20824856
189861
rs13215905
20824884
189889
rs9368223
20824937
189942
rs2328548
20825025
190030
rsl 1427712
20825074
190079
rs6935599
20825100
190105
rs13216165
20825234
190239
rs9465871
20825383
190388
rsl 0946403
20826219
191224
rs2328549
20826449
191454
rsl 7226774
20827124
192129
rs9358357
20827211
192216
rs9368224
20827321
192326
rsl 1756271
20827372
192377
rs9358358
20827540
192545
rs9460550
20827858
192863
rsl 2110493
20827866
192871
rs12193125
20828258
193263
rs9356746
20828797
193802
rs9350272
20829322
194327
rs13219444
20829342
194347
rs12111216
-20829562
194567
rs9350273
20829700
194705
rs9368225
20830399
195404
rsl 7825025
20831036
196041
rs9368226
20832213
197218
rs6903175
20832229
197234
rs6903744
20832537
197542
rs12111351
20832754
197759
rs4712536
20832986
197991
rs9356747
20833076
198081
rs9356748
20833219
198224
rs7767391
20833402
198407
rs7747752
20833511
198516
rs9350274
20833853
198858
rs34170041
20833919
198924
rs6915155
20834014
199019
rs6914868
20834472
199477
rs4538697
20835549
200554
rs4712537
20836048
201053
rs34097377
20836492
201497
rs6928012
20836710
201715
rs6908425
118
Table 11. SNPs within LD block CIO (SEQ ID NO:2) between positions 94,192,885 and 94,490,091 bp on Chromosome 10 in NCBI Build 35 and NCBI Build 36
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94192885
1
rs2798253
94193597
713
rs36087110
94193803
919
rs35771118
94193950
1066
rsl 2359552
94193961
1077
rsl 1186999
94194166
1282
rs7916460
94194775
1891
rsl 0882065
94195841
2957
rsl 1187000
94196162
3278
rs4933231
94196306
3422
rsl 1187001
94196353
3469
rs4933725
94196465
3581
rsl 1187002
94196477
3593
rs4933726
94196509
3625
rs4933232
94196716
3832
rsl 1187003
94196844
3960
rs34115369
94197028
4144
rsl 0786047
94197152
4268
rsl 1814521
94197347
4463
rsl 1814555
94198457
5573
rs7476275
94198727
5843
rs3118967
94199011
6127
rsl 1187004
94199856
6972
rs7910977
94199919
7035
rs6583813
94199932
7048
rs511985
94200269
7385
rs7911558
94200789
7905
rsl 2415807
94201174
8290
rs35125831
94201284
8400
rs2251101
94201876
8992
rs7896688
94202516
9632
rs5786996
94202722
9838
rs913648
94203071
10187
rs5786997
94203072
10188
rs35771235
94203255
10371
rs34872659
94203768
10884
rs34266748
94204339
11455
rs4646958
94204560
11676
rsl 1187007
94205437
12553
rsl 1459510
94205449
12565
rs35832015
94206153
13269
rsl 2356364
94206407
13523
rsl 1593933
94206490
13606
rs3781241
94206524
13640
rs3781240
94206594
13710
rsl 0562725
94206599
13715
rsl 0617641
94206609
13725
rs28641489
94207018
14134
rsl 1187009
94207224
14340
rs36119168
94207391
14507
rsl 1594562
94207777
14893
rs3781239
94208177
15293
rs3824738
94208228
15344
rsl 2782629
94208261 ,
15377
rsl 2261501
94208278
15394
rsl 2781670
94208383
15499
rs568657
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94208423
15539
rs509954
94209484
16600
rs489517
94209509
16625
rs9420586
94209578
16694
rsl 1187010
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16713
rs2247348
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16864
rs307638
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17701
rs35118791
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17741
rs520711
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18218
rs7098739
94211382
18498
rs7081224
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18707
rs7093437
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19720
rs551266
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20812
rsl 042444
94213766
20882
rs7087334
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21261
rsl 887922
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21731
rs7898862
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21842
rsl 0882066
94214869
21985
rsl 1187011
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22048
rs7916011
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22113
rs7899603
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rs34934289
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rsl 2242504
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rs2275218
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rs538469
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rs35640611
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22939
rsl 1187012
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23256
rsl 1187013
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rs7893352
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rsl 1187014
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rs544537
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rsl 2243622
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26723
rsl 1187015
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rs7920976
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rs4646957
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rsl 1187016
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rs2250090
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rs35959170
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rs35551274
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rs7087153
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rsl 2762802
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rsl 2763971
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rsl 1187017
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rs2249960
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rsl 2262931
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rsl 1187018
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rsl 1323400
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rs7092468
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31851
rsl 2245118
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rs35223317
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rs35637537
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34352
rs35291821
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34506
rs7073248
94227405
34521
rs7091270
94227647
34763
rsl 2251346
94227782
34898
rs6583815
119
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94227902
35018
rsl 2411941
94227937
35053
rsl 7875326
94228149
35265
rs7077626
94228919
36035
rs35864975
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rs5030982
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36465
rs3831274
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36482
rs35611772
94229773
36889
rs7910605
94231074
38190
rsl 2356508
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38444
rs34093069
94231497
38613
rs35831196
94232484
39600
rs35250835
94232485
39601
rs5786998
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39602
rs35368064
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40302
rs12243214
94233203
40319
rs7917817
94233597
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rs2421940
94234183
41299
rs35120790
94234248
41364
rsl 0882067
94234880
41996
rs35436518
94234881
41997
rs34615998
94234883
41999
rsl 1595475
94235591
42707
rs35243007
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44088
rs35426658
94237227
44343
rs6583817
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44356
rs35863982
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rs35532620
94238290
45406
rsl 1187019
94238346
45462
rs12219139
94238396
45512
rs12219148
94238509
45625
rs34930778
94238512
45628
rs36015364
94238730
45846
rsl 1187020
94239054
46170
rs35650880
94239749
46865
rs7093418
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rsl 1596251
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47078
rs3737225
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rsl 1444132
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rs34841034
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rsl 1187021
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rs3837333
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rs34838821
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rs3781238
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rs35973022
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rs3781237
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rsl 0882068
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rs1855917
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rs1855916
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rsl 0882069
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rs9420151
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rsl 1187022
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rsl 0882070
94245384
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rs7075073
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53116
rsl 1187024
94246773
53889
rsl 1598525
94246972
54088
rs34822156
94247198
54314
rs7084090
94247956
55072
rsl 1187025
94247994
55110
rs6583818
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94249045
56161
rs34666358
94249117
56233
rsl 1187026
94249160
56276
rsl 1187027
94249226
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rs34459034
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rsl 1187028
94249316
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rs36049328
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56795
rs7097800
94249948
57064
rsl 0786048
94250085
57201
rsl 0882071
94250350
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rsl 2249976
94250507
57623
rs7068618
94250611
57727
rsl 1187029
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57808
rsl 0882072
94250983
58099
rsl 1187030
94251771
58887
rsl 1187031
94251786
58902
rsl 1187032
94252339
59455
rsl 1187033
94252515
59631
rsl 1187034
94252799
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rsl 1442945
94253137
60253
rsl 1187035
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60319
rsl 970244
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rsl 1187037
94253515
60631
rsl 970245
94253764
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rs5786999
94253765
60881
rs34057954
94253766
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rs10716816
94254606
61722
rs34708742
94254765
61881
rs35101389
94254975
62091
rsl 1187038
94255082
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rs34174850
94256325
63441
rs34053974
94256855
63971
rsl 1296200
94257747
64863
rsl 1460471
94258212
65328
rsl 1286004
94258296
65412
rs5787000
94258297
65413
rs33917554
94258314
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rs1832196
94258319
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rs34194084
94258381
65497
rs1832195
94258980
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rs35636429
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66284
rs4256898
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66462
rs34663898
94259587
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rs6583819
94259670
66786
rsl 1324773
94259792
66908
rsl 1187039
94260389
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rs34662862
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rs35891632
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rsl 0882073
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rsl 1498516
94261156
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rsl 7445028
94261438
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rs35831688
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rsl 1373926
94262304
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rs35405697
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rs35377675
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rs34457657
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69801
rs34774587
94262844
69960
rsl 1187040
94263091
70207
rs7086558
94263344
70460
rs7910569
120
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94263586
70702
rs34673600
94264572
71688
rs35270297
94264650
71766
rs4646956
94264789
71905
rsl 7875327
94265538
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rs9633693
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73244
rsl 2780132
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73622
rs7895832
94266635
73751
rs6583820
94267645
74761
rs7093773
94267750
74866
rsl 2257226
94267766
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rs7075851
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74962
rsl 0509645
94268591
75707
rs35693308
94271124
78240
rsl 1812558
94271625
78741
rsl 1187042
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rsl 0882074
94271861
78977
rsl 1187043
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79374
rsl 1187044
94272698
79814
rs7915971
94273091
80207
rs4933233
94273288
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rs35361515
94273349
80465
rsl 1187045
94273885
81001
rs35296767
94273981
81097
rsl 1187046
94274088
81204
rsl 1813097
94274094
81210
rsl 0882075
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81216
rsl 0882076
94274121
81237
rsl 1187047
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rsl 1187048
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81245
rsl 1187049
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81251
rsl 1187050
94274143
81259
rsl 1187051
94274150
81266
rsl 1187052
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81299
rsl 1818981
94274184
81300
rsl 1818982
94274213
81329
rsl 1187053
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81361
rsl 2355280
94274246
81362
rsl 2359894
94274253
81369
rsl 1187054
94274787
81903
rsl 2358677
94275109
82225
rs35688800
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rsl 2261046
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rs12261114
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rsl 2261174
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rs7894448
94275487
82603
rsl 2262694
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rs4641376
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rs35586301
94276174
83290
rsl 1187055
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rs7089987
94276400
83516
rs7073833
94276465
83581
rsl 0882077
94276595
83711
rsl 1459412
94276596
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rs34975586
94277360
84476
rs2421942
94280464
87580
rs7078413
94280662
87778
rs7079099
94280746
87862
rsl 2258487
94281644
88760
rs34747737
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94281681
88797
rs7901064
94282086
89202
rsl 7107709
94282197
89313
rs868057
94283137
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rs34880105
94283469
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rs35455474
94283592
90708
rsl 1819413
94283667
90783
rsl 1187056
94283823
90939
rs1855915
94283919
91035
rsl 2268712
94284271
91387
rs4646955
94285010
92126
rs7898114
94285220
92336
rsl 1450948
94285221
92337
rs35571064
94285296
92412
rs7898493
94285778
92894
rs7077418
94286057
93173
rsl 1187057
94286438
93554
rs34460166
94286967
94083
rs2275221
94288311
95427
rs1832197
94288480
95596
rsl 7107721
94288514
95630
rs5787001
94288515
95631
rs34593706
94288516
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rsl 1187059
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rsl 2249288
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rs12416180
94292030
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rsl 0882078
94292741
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rsl 1815736
94293623
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rs5004594
94293624
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rsl 970243
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100741
rs33928713
94293625
100741
rs5787002
94293956
101072
rsl 2218329
94294112
101228
rsl 1187060
94294428
101544
rs7915349
94295169
102285
rsl 7445328
94295389
102505
rsl 1187061
94295397
102513
rsl 7107734
94296406
103522
rsl 1187062
94296563
103679
rs10218994
94296625
103741
rsl 7445419
94296937
104053
rs12219325
94297315
104431
rsl 1286362
94297863
104979
rsl 0786049
94297879
104995
rs7900822
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105214
rsl 1187063
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105349
rsl 1187064
94298446
105562
rs10219017
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106121
rs34494546
94299843
106959
rs7909636
94300255
107371
rs34330550
94300414
107530
rsl 0882079
94300889
108005
rsl 2220493
94301795
108911
rs2421943
94301904
109020
rsl 1187065
94302165
109281
rs4406744
94302446
109562
rs35156639
94303116
110232
rs1987122
94303124
110240
rs9420144
94303675
110791
rs35707435
121
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94304293
111409
rsl 1418454
94304299
111415
rsl 1424864
94304548
111664
rs6583821
94304589
111705
rsl 0882080
94304784
111900
rs7908111
94305004
112120
rsl 0882081
94306623
113739
rs7902106
94306803
113919
rsl 2415874
94306808
113924
rs7917163
94307486
114602
rs4933728
94307610
114726
rsl 2412249
94307630
114746
rs3051565
94307851
114967
rsl 1187066
94307892
115008
rsl 0882082
94308049
115165
- rs7098744
94308378
115494
rsl 2777622
94308408
115524
rsl 2779093
94308409
115525
rsl 2777974
94309312
116428
rsl 1187067
94309594
116710
rsl 2765408
94309595
116711
rs34052181
94309972
117088
rsl 999763
94310119
117235
rsl 999764
94310400
117516
rsl 1187068
94310514
117630
rs7078418
94310644
117760
rs35429533
94310846
117962
rsl 1187069
94311213
118329
rs34630015
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118992
rsl 2264361
94311953
119069
rsl 0882083
94312407
119523
rsl 2264682
94312615
119731
rsl 1187070
94312726
119842
rsl 2266443
94312981
120097
rsl 2776190
94313002
120118
rs4933729
94313190
120306
rsl 2774925
94313202
120318
rsl 2774931
94314015
121131
rsl 1187071
94314384
121500
rs35009022
94314389
121505
rsl 1187072
94314566
121682
rsl 2241107
94314708
121824
rsl 1187073
94314816
121932
rsl 2763871
94314968
122084
rs35418143
94315124
122240
rsl 2411517
94315147
122263
rsl 1308616
94315157
122273
rs33935672
94315157
122273
rs5787003
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122607
rs7076966
94315748
122864
rs5787004
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122867
rs35925526
94315930
123046
rs5787005
94315944
123060
rs34378303
94316148
123264
rs2901598
94316150
123266
rs2421944
94316175
123291
rs34777726
94316176
123292
rs3051566
94316766
123882
rsl 2253172
94316799
123915
rsl 2253177
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94316926
124042
rsl 1187074
94317075
124191
rs7082686
94317136
124252
rs7100623
94317536
124652
rsl 2255048
94317963
125079
rsl 2355977
94318001
125117
rs35616984
94318580
125696
rs35826224
94318582
125698
rsl 0584127
94318582
125698
rs7915220
94318992
126108
rsl 1437287
94318993
126109
rs35875721
94319093
126209
rsl 0882084
94320200
127316
rs7097014
94321033
128149
rsl 2415246
94321119
128235
rs4304670
94321321
128437
rs2421945
94321612
128728
rsl 2252447
94321742
128858
rsl 0736067
94322696
129812
rs7909487
94323150
130266
rs34093946
94323222
130338
rsl 2783634
94323807
130923
rs4646954
94323935
131051
rs4646953
94323955
131071
rs35380063
94324493
131609
rsl 7875328
94324556
131672
rs34867369
94324758
131874
rs3758505
94325575
132691
rsl 1379220
94325779
132895
rs7099761
94326009
133125
rsl 1187075
94326342
133458
rs35628053
94326394
133510
rs3758504
94326684
133800
rs3758503
94326943
134059
rsl 1187076
94327161
134277
rs35376801
94327403
134519
rsl 2762388
94327431
134547
rsl 2762406
94327438
134554
rsl 2764060
94327683
134799
rs4933730
94327871
134987
rs7900584
94328020
135136
rs12414433
94328211
135327
rs7900991
94328632
135748
rs7921325
94328678
135794
rs7905037
94330087
137203
rsl 1187077
94330685
137801
rsl 1187078
94330689
137805
rsl 1273566
94331181
138297
rs6583822
94331572
138688
rsl 1187079
94331642
138758
rs7069538
94331770
138886
rs7087423
94332643
139759
rs2901597
94332963
140079
rs7092522
94332992
140108
rsl 2766927
94333545
140661
rsl 1187080
94334378
141494
rs34210106
94334395
141511
rs6583823
94334850
141966
rs7084215
94334897
142013
rs6583824
122
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94335207
142323
rs6583825
94335889
143005
rs2421941
94336041
143157
rsl 2262994
94336117
143233
rsl 2263054
94336859
143975
rsl 2573146
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144169
rsl 1187081
94337295
144411
rs34143873
94337314
144430
rsl 1455878
94337315
144431
rs35480775
94337549
144665
rs7921210
94337810
144926
rs6583826
94338024
145140
rsl 2776949
94338180
145296
rs7922041
94338205
145321
rs34073794
94338626
145742
rs10701812
94338815
145931
rs7911019
94339556
146672
rs7898478
94340196
147312
rs4611114
94340487
147603
rsl 889894
94340661
147777
rsl 2355158
94341882
148998
rsl 7875329
94341915
149031
rsl 1595187
94341951
149067
rsl 7875331
94342066
149182
rsl 7875332
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149251
rsl 1595260
94342136
149252
rsl 1599701
94342406
149522
rs7092778
94342485
149601
rsl 1187083
94342495
149611
rsl 7875333
94342598
149714
rsl 1187084
94342803
149919
rs2297743
94343099
150215
rs36015451
94343258
150374
rsl 2256435
94343418
150534
rsl 1818332
94343843
150959
rs3781236
94344062
151178
rs3824736
94344184
151300
rs3824735
94344608
151724
rsl 1429679
94344609
151725
rs34491012
94344692
151808
rsl 1187085
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152067
rsl 1187086
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152620
rsl 1817798
94345575
152691
rsl 2240557
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152899
rs35583124
94345930
153046
rsl 2262350
94346261
153377
rsl 1187087
94346544
153660
rs7099635
94346892
154008
rs4604791
94346932
154048
rsl 1556038
94347159
154275
rs7082683
94348003
155119
rs6583827
94349194
156310
rsl 1187089
94349700
156816
rsl 2218324
94349861
156977
rsl 1498685
94350652
157768
rsl 1187090
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157957
rsl 2411873
94351226
158342
rs7068118
94351364
158480
rs35624581
94351461
158577
rs35982149
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94352267
159383
rs35452156
94352383
159499
rs35807258
94352546
159662
rsl 0564653
94352561
159677
rs7095250
94352562
159678
rs7095369
94352579
159695
rsl 0564753
94352593
159709
rs3980475
94352908
160024
rs7096101
94353042
160158
rs9733604
94353107
160223
rsl 2266122
94353183
160299
rs7096520
94353519
160635
rs35612167
94353973
161089
rs4933731
94354724
161840
rsl 1187091
94354862
161978
rsl 0882085
94354869
161985
rsl 2763703
94355005
162121
rsl 0882086
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162537
rsl 1812310
94355638
162754
rsl 1187092
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162789
rsl 2242681
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162837
rsl 2257279
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163446
rsl 1393423
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163447
rs35558317
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163694
rsl 2244660
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163721
rsl 2244573
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163931
rsl 2244809
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163994
rsl 2259474
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164100
rsl 7875334
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164129
rsl 7107758
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164326
rsl 0786050
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164945
rsl 2246900
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164963
rsl 0882087
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165013
rs12261518
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rsl 1187093
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rsl 2247024
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165274
rsl 1187094
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165311
rsl 2261697
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165684
rsl 0882088
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165752
rsl 2248860
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166121
rs3835295
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rsl 0882089
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rs34446454
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rsl 1187095
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rs3835294
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rs12767141
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166612
rsl 2250905
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rsl 1187096
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167031
rsl 2356724
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167730
rsl 1599330
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rs35352564
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rs2275220
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170038
rs2275219
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170876
rsl 0882090
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171473
rsl 0882091
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171699
rsl 1187097
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171987
rsl 1187098
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172093
rsl 1187099
94365856
172972
rsl 2252642
94366308
173424
rsl 2252836
123
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94366409
173525
rsl 7875338
94366437
173553
rsl 7875337
94366604
173720
rs33921491
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173722
rsl 0550249
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173754
rs34075743
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173964
rs7069647
94367517
174633
rsl 1187100
94367754
174870
rsl 1187101
94367943
175059
rsl 2415792
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175145
rs3980476
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175299
rsl 2767059
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175946
rs6583828
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175973
rsl 1517020
94369044
176160
rs34515231
94369203
176319
rsl 0882092
94369352
176468
rs6583829
94369694
176810
rsl 2260688
94369814
176930
rsl 2781567
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177228
rs7084441
94371113
178229
rsl 7875339
94371489
178605
rs11187102
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178674
rsl 1187103
94371597
178713
rs34942989
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178745
rsl 1817621
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178757
rsl 1815573
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179114
rsl 1187104
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179136
rs7894183
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179266
rsl 2254289
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179521
rs36031409
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179548
rsl 2262063
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179558
rs7914248
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179561
rsl 2254474
94372736
179852
rs7898027
94372930
180046
rs7914814
94372946
180062
rs7898506
94373054
180170
rsl 7367408
94373182
180298
rsl 0082524
94373409
180525
rs7091887
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180540
rs7073868
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180777
rs7095585
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180954
rsl 2772554
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181086
rsl 2772956
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rsl 0882093
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181523
rsl 1187105
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181565
rsl 2258463
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181610
rs35518831
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181612
rs34197740
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181613
rs35008887
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181614
rs35083945
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182786
rsl 1187106
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183218
rs7071905
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183250
rs7087860
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183567
rs35368950
94376836
183952
rs7915915
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184034
rsl 2264372
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184430
rsl 573051
94377656
184772
rsl 0882094
94378087
185203
rsl 2244924
94378491
185607
rsl 7875340
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94379385
186501
rsl 1417254
94379401
186517
rs7068540
94379725
186841
rs28692618
94380110
187226
rs35297001
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187519
rs7073739
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187531
rsl 1595797
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187651
rsl 2243009
94380591
187707
rs7077137
94381028
188144
rs9731595
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188370
rs7078403
94381345
188461
rs7078179
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188793
rsl 1187107
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189383
rs2275217
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189555
rsl 7107764
94382499
189615
rsl 0639509
94382499
189615
rsl 7875342
94382500
189616
rs34767899
94382708
189824
rsl 1818705
94382761
189877
rsl 1818708
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189926
rsl 1818750
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189931
rs5787006
94383610
190726
rsl 1187108
94383671
190787
rs3051599
94383672
190788
rs35996949
94383672
190788
rs2421946
94383672
190788
rs5787007
94383674
190790
rs34710486
94383765
190881
rs28615450
94383941
191057
rsl 0736068
94383978
191094
rsl 1819282
94384075
191191
rsl 1819301
94384310
191426
rsl 1819351
94384382
191498
rsl 0882095
94384580
191696
rs7070990
94385075
192191
rs7089765
94385373
192489
rsl 0736069
94385475
192591
rsl 2257072
94385728
192844
rs7900689
94386335
193451
rsl 1812813
94386492
193608
rs4933732
94386500
193616
rsl 1594678
94386540
193656
rsl 1813238
94386568
193684
rsl 972360
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193710
rsl 1816962
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193819
rs4558084
94386890
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rs34948996
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194488
rs1547818
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194612
rs34497717
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rs7069680
94387964
195080
rs7085790
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195210
rs36015190
94388098
195214
rs6583830
94388569
195685
rs7090402
94388954
196070
rsl 1187109
94389669
196785
rs35137137
94389736
196852
rs35494689
94390313
197429
rs6583831
94390339
197455
rsl 1187110
94390489
197605
rsl 1187111
124
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94390602
197718
rs4933733
94390673
197789
rs34848251
94390842
197958
rs7922902
94391081
198197
rsl 1282830
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198198
rs35084253
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198468
rs6583832
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198482
rsl 0882096
94391873
198989
rs7079583
94391950
199066
rs7079602
94391966
199082
rsl 1187112
94392278
199394
rsl 2244429
94393212
200328
rs35312216
94393281
200397
rs28576319
94393578
200694
rs7088685
94394524
201640
rsl 7107776
94394677
201793
rs7902436
94394804
201920
rs7917359
94395324
202440
rs36070037
94395344
202460
rs34647366
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202822
rs33915127
94396217
203333
rsl 1187114
94396832
203948
rs7069619
94397097
204213
rs34629164
94397263
204379
rsl 1187115
94397647
204763
rs7073957
94397851
204967
rsl 0686721
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204975
rs34357393
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205097
rs36046902
94398388
205504
rsl 2264712
94398585
205701
rs34132402
94398837
205953
rsl 2358273
94398866
205982
rs35625726
94398869
205985
rsl 1285704
94399729
206845
rs7067733
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206896
rs6583833
94399806
206922
rs35230377
94400137
207253
rsl 0430651
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207922
rsl 2262781
94400870
207986
rsl 2359115
94401006
208122
rsl 2359131
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208271
rs7898318
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208296
rsl 2359158
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208352
rsl 2359159
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208356
rsl 2764793
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208361
rsl 2764800
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208362
rsl 2241432
94401250
208366
rsl 1383128
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rs7898430
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208824
rs35738968
94401710
208826
rsl 0537782
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208836
rs36000362
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208848
rs7893798
94402105
209221
rsl 1187116
94402138
209254
rsl 1187117
94402166
209282
rsl 1187118
94402579
209695
rsl 2411448
94402726
209842
rs35910196
94402828
209944
rs34963356
94403360
210476
rsl 7875344
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94403488
210604
rs34417963
94403643
210759
rs41290170
94403647
210763
rsl 7875345
94403675
210791
rs41290172
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210890
rs41290174
94403917
211033
rs1044146
94403990
211106
rsl 7875346
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211359
rs7078243
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211486
rsl 1187119
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211506
rs36107638
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211663
rs4933734
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211734
rsl 7875347
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212174
rs1044153
94405646
212762
rsl 1187120
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212929
rs35698103
94406209
213325
rs7071912
94406381
213497
rsl 1597699
94406501
213617
rsl 1598250
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213635
rs35135018
94406834
213950
rsl 2242617
94407185
214301
rsl 2264496
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214317
rsl 1814562
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214399
rs7916594
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214438
rs7093043
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214443
rs7092669
94407904
215020
rsl 2244738
94408110
215226
rsl 2573394
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215311
rs34223527
94408405
215521
rs7921040
94408480
215596
rs35583259
94408734
215850
rs2901599
94409046
216162
rsl 1187122
94409276
216392
rs6583834
94410546
217662
rs7076842
94411653
218769
rs9804194
94411998
219114
rs7914504
94412220
219336
rs35580348
94412694
219810
rs7079361
94412743
219859
rs7095377
94413406
220522
rs7096187
94413485
220601
rs6583835
94413764
220880
rs6583836
94413791
220907
rs7100344
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221169
rs2153827
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221294
rs6583837
94414402
221518
rs35978445
94414724
221840
rs2497321
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221874
rs2497320
94415097
222213
rsl 1187124
94415101
222217
rs7913315
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222232
rs2497319
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222263
rs7071919
94416183
223299
rs2488058
94416216
223332
rs2488057
94417125
224241
rs34940159
94417197
224313
rsl 2776166
94417669
224785
rsl 1187125
94418089
225205
rs7910187
94418694
225810
rs7914114
125
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94418703
225819
rsl 1554568
94418859
225975
rs7914143
94418888
226004
rsl 832887
94418890
226006
rs1418391
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226106
rs7914507
94419062
226178
rsl 7875348
94419074
226190
rs7899695
94419424
226540
rs12414381
94419447
226563
rs7918084
94419491
226607
rs7903302
94419506
226622
rsl 1187126
94419569
226685
rsl 1187127
94419688
226804
rsl 1187128
94419689
226805
rsl 2772782
94419887
227003
rsl 1187129
94420477
227593
rs2185756
94420505
227621
rs9419741
94420766
227882
rs34709928
94420766
227882
rs34848369
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227993
rs35250649
94421300
228416
rs9419742
94421540
228656
rsl 1594482
94421980
229096
rs2497318
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229424
rs2488055
94422330
229446
rs6583838
94422446
229562
rs2488054
94423353
230469
rs7909334
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230558
rs35288203
94423443
230559
rs7897560
94423460
230576
rs35413668
94423462
230578
rs7897565
94423473
230589
rsl 2761997
94423750
230866
rs34831256
94424074
231190
rs4421685
94425223
232339
rs28514404
94425223
232339
rs28591207
94425307
232423
rs35885794
94425468
232584
rs7098199
94425577
232693
rs34985734
94425653
232769
rs35906730
94425663
232779
rs4933735
94425739
232855
rs7098638
94426831
233947
rs7911264
94427225
234341
rsl 1187131
94427575
234691
rsl 1187132
94428318
235434
rsl 1448446
94428319
235435
rs34215006
94428322
235438
rsl 1439271
94428328
235444
rs35808040
94429339
236455
rs34911167
94429434
236550
rsl 1599074
94429447
236563
rsl 2774356
94429484
236600
rsl 2779070
94430250
237366
rs28757342
94430530
237646
rs35299744
94431405
238521
rs7089358
94431962
239078
rs35828108
94432143
239259
rs34615602
94432674
239790
rs7914280
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94432723
239839
rsl 7875349
94432994
240110
rs34669790
94434773
241889
rsl 0882098
94434827
241943
rsl 2778051
94434836
241952
rsl 1187133
94434881
241997
rsl 1187134
94435923
243039
rs2497317
94436021
243137
rs2488087
94436103
243219
rsl 2262390
94436615
243731
rsl 0786052
94437076
244192
rsl 0522178
94437120
244236
rsl 0531158
94437559
244675
rsl 2251379
94437891
245007
rs949579
94438736
245852
rsl 1187135
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245991
rs34593856
94439140
246256
rs12781513
94439449
246565
rs7475059
94439530
246646
rs4075563
94439906
247022
rs34266926
94439912
247028
rs2229328
94439992
247108
rs1418387
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247222
rs2901618
94440107
247223
rs2901619
94440209
247325
rs34372348
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247329
rs9420589
94441710
248826
rs41290176
94442254
249370
rsl 7851141
94442322
249438
rs2275730
94442410
249526
rs2275729
94443456
250572
rsl 1319879
94443457
250573
rs34616944
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251353
rs34433040
94445202
252318
rs35423905
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252973
rs34619410
94448868
255984
rs3076219
94449333
256449
rs34231544
94449598
256714
rs35657492
94450261
257377
rsl 1187138
94450630
257746
rsl 0882099
94450667
257783
rs10882100
94451033
258149
rs7100035
94451324
258440
rs7100357
94451790
258906
rsl 2263166
94451929
259045
rsl 2263147
94451954
259070
rsl 2218257
94451963
259079
rs2488085
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259081
rs2497316
94452407
259523
rs10882101
94452569
259685
rs5787008
94452862
259978
rs1111875
94453437
260553
rs7921459
94453671
260787
rs35245232
94453759
260875
rsl 977832
94453925
261041
rsl 977833
94453943
261059
rs35396842
94453943
261059
rsl 1309330
94453950
261066
rs34470242
94454287
261403
rsl 2778642
126
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94454556
261672
rs7911447
94454563
261679
rs7894796
94454564
261680
rs7911455
94454571
261687
rs7895123
94455183
262299
rs4933234
94455211
262327
rs34991559
94455539
262655
rs5015480
94456086
263202
rs2497315
94456407
263523
rsl 1187139
94456419
263535
rs12219514
94456475
263591
rsl 0882102
94456646
263762
rsl 2572190
94456890
264006
rsl 1187140
94456999
264115
rs7904159
94457006
264122
rs7904279
94457011
264127
rs7904508
94457018
264134
rs7904513
94457029
264145
rs7904292
94457125
264241
rsl 1187141
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264383
rs34744311
94458157
265273
rs9419743
94458159
265275
rsl 2254229
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265388
rsl 2246641
94458305
265421
rsl 2246541
94458432
265548
rsl 1813799
94458665
265781
rsl 1187142
94458914
266030
rsl 1187143
94459172
266288
rs28590736
94459519
266635
rs28428943
94459960
267076
rsl 1187144
94460495
267611
rsl 0882104
94460975
268091
rs34859807
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268095
rs2497314
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268691
rs4933736
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269301
rs34966020
94462949
270065
rs34290965
94463018
270134
rsl 1817277
94463116
270232
rs34600815
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270255
rs34793711
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270725
rs7087591
94464077
271193
rsl 0786053
94464091
271207
rs10882105
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271592
rsl 578672
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272200
rs35705157
94465091
272207
rs2901609
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rs34405337
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272756
rs4545448
94466041
273157
rs4504977
94466253
273369
rs4262638
94466275
273391
rsl 2777206
94466575
273691
rsl 2782663
94466582
273698
rsl 2782667
94466623
273739
rsl 2781544
94467199
274315
rsl 0748582
94467336
274452
rsl 1378649
94467337
274453
rs33926570
94467472
274588
rs2488082
94467519
274635
rsl 832886
94467850
274966
rs7898054
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94468084
275200
rsl 1187145
94468335
275451
rsl 1187146
94468644
275760
rs2488081
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275789
rs2488080
94469087
276203
rsl 112718
94469812
276928
rsl 2260097
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276973
rs34383024
94470058
277174
rs35816895
94470314
277430
rs10882106
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277487
rs2497313
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277598
rs7086841
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277782
rs2497312
94471062
278178
rs9420591
94471614
278730
rs4933235
94471897
279013
rs7923837
94471924
279040
rsl 0673051
94471925
279041
rs35275238
94471968
279084
rs35606816
94471975
279091
rs2497311
94472056
279172
rs7923866
94472115
279231
rsl 2780253
94472584
279700
rs35282244
94473140
280256
rs36026029
94473357
280473
rsl 1187147
94473463
280579
rs2488079
94473656
280772
rsl 0529320
94473689
280805
rs2497310
94473690
280806
rs2488078
94473695
280811
rs3221117
94473956
281072
rs2497309
94474172
281288
rs870786
94474324
281440
rs2497308
94474328
281444
rs2488077
94474418
281534
rs5787009
94474440
281556
rs5787010
94474442
281558
rs5787011
94474472
281588
rsl 1187148
94474486
281602
rs1418390
94474604
281720
rs2497307
94474750
281866
rs7081035
94474783
281899
rs7081294
94474989
282105
rs7081351
94475058
282174
rs7081745
94475060
282176
rsl 0568596
94475063
282179
rs7099393
94475082
282198
rsl 0665748
94475086
282202
rs34350311
94475191
282307
rs2497306
94475601
282717
rs2497305
94475656
282772
rs34372918
94475743
282859
rsl 1187149
94476016
283132
rsl 1597458
94476061
283177
rs2488076
94476146
283262
rsl 1593164
94476357
283473
rs34848929
94476358
283474
rsl 1597547
94476822
283938
rsl 1187150
94477781
284897
rs1544210
94478596
285712
rs35619602
127
Position in Build 35/36
Position in SEQ ID NO:2
Marker ID
94479180
286296
rsl 0630735
94479181
286297
rs35097519
94479192
286308
rs34237492
94479194
286310
rsl 1309242
94480154
287270
rs2488075
94480323
287439
rsl 2762754
94480347
287463
rsl 1593631
94480992
288108
rs9730884
94481595
288711
rsl 1379031
94481781
288897
rs10615317
94481897
289013
rs35598412
94482696
289812
rs2497304
94482730
289846
rs9419745
94482914
290030
rs35849687
94483045
290161
rs34009238
94483719
290835
rs2488074
94484440
291556
rsl 1187151
94484498
291614
rs35406218
94485046
292162
rs34249712
94485097
292213
rs2497303
Position in Build 35/36
Position in SEQ ID NO:2
Marker iD
94485259
292375
rs4933738
94485733
292849
rs947591
94485978
293094
rs7916355
94486361
293477
rs2051004
94488416
295532
rs4933236
94488811
295927
rsl 7107841
94488843
295959
rs33985961
94488845
295961
rsl 0578040
94488955
296071
rs2488073
94489325
296441
rs2488072
94489493
296609
rs34209030
94489557
296673
rs2488071
94489846
296962
rs7917254
94490010
297126
rsl 1318190
94490015
297131
rs34994435
94490015
297131
rsl 0588167
94490091
297207
rsl 1187152
128
Table 12. SNPs within LD block C17 between positions 66,037,656 and 66,163,076 bp on Chromosome 17 in NCBI build 35 and NCBI Build 36.)
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66037656
1
rsl 1077501
66038245
590
rsl 0445229
66038446
791
rs8067115
66038456
801
rsl 0445230
66038691
1036
rsl 0445231
•66039757
2102
rs28569992
66039800
2145
rs4606755
66039816
2161
rs4435300
66039936
2281
rs35154837
66039942
2287
rs9630701
66040960
3305
rsl 2165045
66040982
3327
rs7359539
66041088
3433
rs7359543
66042479
4824
rs365813
66043002
5347
rs4261590
66043301
5646
rs7223187
66043481
5826
rs6146132
66043562
5907
rs721249
66043745
6090
rs5821786
66043746
6091
rs33957619
66043759
6104
rs5821787
66043760
6105
rs33961999
66043764
6109
rsl 2950870
66044207
6552
rs350605
66044302
6647
rsl 0559381
66044390
6735
rsl 1650835
66044411
6756
rs7209364
66044489
6834
rs350604
66044496
6841
rsl 1657696
66044614
6959
rsl 1651554
66044626
6971
rsl 1657734
66045245
7590
rs350603
66045317
7662
rs2307760
66045722
8067
rsl 1653245
66045948
8293
rsl 1653355
66047524
9869
rs34832542
66047547
9892
rs2567294
66047580
9925
rsl 1655558
66047597
9942
rsl 84783
66047621
9966
rs353452
66047646
9991
rs9897791
66047700
10045
rsl 1655611
66047739
10084
rsl 825672
66047807
10152
rs35941755
66047887
10232
rs9896649
66048278
10623
rs34984463
66048288
10633
rsl 1374691
66048300
10645
rsl 1868103
66048450
10795
rs7220610
66048799
11144
rs7216368
66048942
11287
rs16913
66049292
11637
rs34941209
66049692
12037
rs8069108
66049716
12061
rs420762
66050080
12425
rs2630640
66050452
12797
rs34793380
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66050707
13052
rs1817630
66050903
13248
rsl 1077502
66050915
13260
rs7218450
66051172
13517
rs411602
66051859
14204
rsl 6975882
66051914
14259
rs41450951
66052282
14627
rsl 7780198
66052347
14692
rsl 0512540
66052398
14743
rs2630639
66052474
14819
rs4793432
66052546
14891
rs34696190
66052699
15044
rsl 2952273
66053325
15670
rs350612
66053541
15886
rsl 284043
66053695
16040
rs1298182
66053988
16333
rsl 092528
66054007
16352
rs1091892
66054019
16364
rsl 092390
66054025
16370
rsl 092391
66054076
16421
rs276805
66054488
16833
rsl 64784
66055098
17443
rsl 64785
66056158
18503
rs350611
66057036
19381
rs9736449
66057065
19410
rsl 161565
66057065
19410
rs350610
66057184
19529
rs36160618
66057341
19686
rs28835946
66057721
20066
rs350609
66057907
20252
rs36143257
66058061
20406
rsl 64786
66058223
20568
rs4506943
66058544
20889
rsl 64787
66058598
20943
rs35063328
66058616
20961
rs35629111
66058724
21069
rs35654390
66058733
21078
rs350608
66058804
21149
rs589894
66059113
21458
rs512280
66059121
21466
rs512274
66059131
21476
rs512241
66059267
21612
rs35813361
66059431
21776
rs34292805
66060049
22394
rs671190
66060102
22447
rs671117
66060111
22456
rs35419562
66060172
22517
rs8080393
66060192
22537
rs509784
66060244
22589
rs509924
66060364
22709
rs510865
66060367
22712
rs669895
66060401
22746
rs8075249
66060403
22748
rs8080759
66060407
22752
rs511552
66060416
22761
rs511578
66060618
22963
rsl 1326414
129
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66061168
23513
rs350607
66061287
23632
rs8081864
66061435
23780
rs8066762
66063324
25669
rsl 0048191
66063794
26139
rs34162560
66063983
26328
rs350606
66064178
26523
rs8078924
66065291
27636
rs4793451
66065798
28143
rs350613
66066258
28603
rsl 0432003
66066436
28781
rs350614
66066465
28810
rs34703743
66066481
28826
rs35908278
66066608
28953
rsl 1654062
66067303
29648
rs350615
66067453
29798
rsl 6975891
66067482
29827
rsl 7823280
66067699
30044
rs350616
66068320
30665
rs350617
66068798
31143
rs1991680
66069274
31619
rs9896037
66069554
31899
rsl 6975893
66069880
32225
rs8081551
66070068
32413
rsl 1654475
66071319
33664
rsl 1651609
66071575
33920
rs34132957
66071603
33948
rsl 2603169
66071721
34066
rs8073324
66072276
34621
rs1431455
66072384
34729
rsl 7763769
66073012
35357
rs350618
66073300
35645
rs1991679
66073592
35937
rs34134043
66073862
36207
rs7208933
66074000
36345
rsl 1655478
66074367
36712
rs35062489
66074796
37141
rs9900305
66075575
37920
rsl 6975908
66076138
38483
rs7224554
66076400
38745
rs35795750
66076402
38747
rs5821788
66076579
38924
rs350619
66076797
39142
rs34028570
66076805
39150
rs5821789
66076806
39151
rs35251724
66077103
39448
rs7210525
66077477
39822
rs2567296
66077488
39833
rsl 843621
66077930
40275
rs34077265
66077984
40329
rs528669
66078111
40456
rs8067160
66078127
40472
rs350620
66078527
40872
rs191621
66079419
41764
rs350621
66079437
41782
rs350622
66079660
42005
rsl 2452538
66079935
42280
rsl 7176093
66079969
42314
rs350623
66079990
42335
rsl 1077503
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66080024
42369
rsl 1077504
66080067
42412
rs350624
66080672
43017
rs35072892
66080920
43265
rsl 1657749
66080984
43329
rsl 6975914
66081110
43455
rs34693986
66081370
43715
rs350625
66081556
43901
rs818765
66081568
43913
rs415298
66081576
43921
rs376750
66081612
43957
rsl 0652573
66081700
44045
rs350626
66082086
44431
rs28590672
66083526
45871
rsl 6975922
66083670
46015
rs481417
66083783
46128
rs191622
66083825
46170
rs482515
66083858
46203
rs367218
66083931
46276
rs402214
66084484
46829
rs483543
66084515
46860
rs484253
66084734
47079
rs486202
66084769
47114
rs610662
66084772
47117
rsl 1310950
66084781
47126
rsl 2936985
66084782
47127
rsl 2945927
66084808
47153
rs610730
66084932
47277
rsl 825669
66084935
47280
rsl 825670
66084954
47299
rsl 825671
66085342
47687
rs8077690
66085473
47818
rsl 2602288
66086152
48497
rsl 6975937
66086744
49089
rs28694321
66087301
49646
rs35353185
66087527
49872
rs41486747
66087994
50339
rs718950
66088026
50371
rs718951
66089255
51600
rsl 1654235
66089418
51763
rsl 1077506
66090535
52880
rs1431454
66090620
52965
rs5821790
66090782
53127
rsl 367748
66090958
53303
rsl 2603995
66091042
53387
rsl 6975939
66091117
53462
rsl 1434683
66091324
53669
rs8081186
66091594
53939
rsl 49309
66091687
54032
rsl 84806
66091693
54038
rsl 49380
66091704
54049
rs151727
66091719
54064
rsl 89541
66091741
54086
rsl 1651021
66091844
54189
rs416121
66091912
54257
rs9302918
66092080
54425
rs9302919
66092700
55045
rs35618929
66092813
55158
rs35870620
66092904
55249
rsl 1652089
130
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66093601
55946
rsl 2938026
66093669
56014
rsl 2948379
66094196
56541
rs9911671
66094376
56721
rsl 6975941
66094422
56767
rs7220885
66094832
57177
rs601297
66094858
57203
rs601615
66094862
57207
rs601617
66094892
57237
rs601656
66095313
57658
rs418402
66096181
58526
rs35417478
66097168
59513
rsl 6975944
66097631
59976
rs34913709
66097633
59978
rs9894781
66097634
59979
rs9914075
66097640
59985
rsl 1658937
66097733
60078
rs8078784
66097760
60105
rs9915992
66098070
60415
rsl 0634138
66098071
60416
rs34728014
66098073
60418
rs34864826
66098076
60421
rsl 0551730
66098084
60429
rs5821791
66098085
60430
rs34310496
66098092
60437
rs34563419
66098173
60518
rs7503632
66098597
60942
rs9902449
66098928
61273
rs9894881
66098930
61275
rs9894882
66098976
61321
rsl 1656877
66099163
61508
rs5821792
66099494
61839
rsl 6975946
66099600
61945
rsl 7779190
66099816
62161
rsl 1650015
66100055
62400
rsl 0607347
66100062
62407
rsl 1372958
66100081
62426
rs34073356
66100089
62434
rs36104345
66100401
62746
rs2109051
66100605
62950
rs2159312
66101242
63587
rs990043
66101267
63612
rs576754
66101396
63741
rs2035582
66101665
64010
rs9905624
66101895
64240
rs693914
66102064
64409
rs558507
66102168
64513
rs35142117
66102168
64513
rsl 0596869
66102213
64558
rs560206
66102221
64566
rsl 2949221
66102269
64614
rs560368
66102315
64660
rs1911969
66102441
64786
rs35550717
66102450
64795
rs34779818
66102555
64900
rs9892329
66102591
64936
rsl 1658215
66102896
65241
rs35815207
66103027
65372
rs9914225
66103236
65581
rs9894021
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66103495
65840
rs9891523
66103561
65906
rs720877
66103923
66268
rs720876
66103928
66273
rs35174251
66104116
66461
rs9892968
66104437
66782
rsl 7779357
66104493
66838
rs34287249
66105315
67660
rs3042758
66105827
68172
rsl 872599
66106415
68760
rs7218838
66106622
68967
rs7209535
66106911
69256
rs9896809
66107082
69427
rs28507887
66107150
69495
rs8067542
66107151
69496
rsl 0641487
66107152
69497
rs33989506
66107167
69512
rs8081487
66108291
70636
rs4793495
66108545
70890
rs8073162
66108565
70910
rs8072591
66108901
71246
rs9905537
66108905
71250
rs8073114
66108924
71269
rs8072003
66108980
71325
rs35155940
66108991
71336
rsl 1459300
66108997
71342
rs36029337
66109457
71802
rsl 1656223
66110309
72654
rs6501400
66110507
72852
rs8074266
66110586
72931
rs388304
66110881
73226
rs4544280
66111138
73483
rsl 2601471
66111335
73680
rsl 2603574
66111468
73813
rsl 1077507
66111545
73890
rsl 1077508
66111926
74271
rs28546453
66112148
74493
rs412877
66112202
74547
rs391223
66112205
74550
rs7224183
66112227
74572
rs173318
66112234
74579
rs192147
66112749
75094
rs34361437
66113023
75368
rsl 2449913
66114764
77109
rs28496807
66114858
77203
rs7220084
66114926
77271
rs7224857
66115366
77711
rs7221542
66115371
77716
rs7221545
66115377
77722
rs34466876
66115416
77761
rsl 0610236
66115835
78180
rsl 979538
66116621
78966
rs8067103
66116703
79048
rs7216053
66116880
79225
rsl 2949351
66117903
80248
rs9913650
66117911
80256
rs1860316
66118086
80431
rs9914115
66118200
80545
rs9908443
66118485
80830
rs8079029
131
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66118495
80840
rsl 2601922
66118737
81082
rsl 0545098
66118737
81082
rsl 2603987
66119589
81934
rs9897225
66119616
81961
rs35975623
66119642
81987
rs9895773
66119822
82167
rs9898518
66119823
82168
rs28422091
66119823
82168
rs36094553
66119863
82208
rs3220372
66119992
82337
rs41408048
66120631
82976
rs28373290
66120827
83172
rs41459950
66121181
83526
rs36013413
66121413
83758
rsl 0564191
66121468
83813
rs7221715
66121873
84218
rsl 2940023
66122077
84422
rs4019476
66122410
84755
rs7222670
66122508
84853
rs7211934
66122635
84980
rs7212243
66122801
85146
rsl 1655139
66123046
85391
rs4793317
66123283
85628
rsl 71384
66123342
85687
rs4793496
66123566
85911
rs507683
66123595
85940
rs507607
66123682
86027
rs41528454
66123714
86059
rs41381246
66123879
86224
rs192146
66123955
86300
rs34149626
66123959
86304
rs35260054
66125089
87434
rs9908077
66125154
87499
rsl 1077509
66125233
87578
rs35234488
66125360
87705
rsl 1871352
66125993
88338
rs7209850
66126471
88816
rs421333
66126699
89044
rs413073
66126766
89111
rs2035581
66126795
89140
rs392974
66126799
89144
rs28532132
66126840
89185
rs3931227
66126841
89186
rsl 6975961
66126875
89220
rs532348
66127256
89601
rsl 1867791
66127283
89628
rsl 1871014
66127312
89657
rs34866225
66127313
89658
rs35958830
66128191
90536
rs9904090
66128304
90649
rsl 6975968
66129127
91472
rs9911708
66129806
92151
rs34448828
66129814
92159
rsl 1398461
66130210
92555
rs9907685
66130284
92629
rs9914666
66130455
92800
rsl 7717654
66131911
94256
rsl 6975970
66131995
94340
rs1981646
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66132156
94501
rsl 1656723
66132225
94570
rsl 6975976
66132788
95133
rs1981647
66133032
95377
rsl 6975979
66133370
95715
rsl 6975981
66134231
96576
rs9909661
66134831
97176
rs9890554
66135283
97628
rsl 1077510
66135627
97972
rs9302920
66135758
98103
rs34975186
66136201
98546
rsl 1870545
66136484
98829
rs9906234
66136910
99255
rsl 0621796
66136920
99265
rsl 1328278
66136922
99267
rsl 1328279
66137329
99674
rs35402203
66137331
99676
rs6501401
66137347
99692
rsl 0596163
66137437
99782
rs35029611
66137784
100129
rs35985303
66137798
100143
rsl 0595957
66137965
100310
rsl 0221271
66138452
100797
rsl 0221225
66138713
101058
rs34005576
66139441
101786
rs9913463
66139476
101821
rs9915148
66139800
102145
rsl 1650683
66140108
102453
rs34335723
66140414 r
102759
rsl 1654495
66141319
103664
rs35607820
66141543
103888
rsl 2601304
66141933
104278
rs1486290
66142011
104356
rsl 2452862
66142106
104451
rs35317540
66142226
104571
rsl 1077511
66142325
104670
rs35278774
66142329
104674
rs7216457
66142581
104926
rsl 1077512
66142607
104952
rs35634443
66142648
104993
rsl 1454851
66142729
105074
rsl 1654670
66142794
105139
rs34926966
66143200
105545
rs8078302
66143897
106242
rs562472
66144028
106373
rs28420303
66144129
106474
rs28542473
66144170
106515
rs28526433
66144232
106577
rsl 2185220
66144972
107317
rs7350903
66145018
107363
rs7350904
66145022
107367
rs7350905
66145067
107412
rs35353467
66145660
108005
rsl 6975985
66145765
108110
rsl 6975987
66145912
108257
rs412981
66145914
108259
rs412980
66145925
108270
rs9907746
66146236
108581
rs432688
66146640
108985
rs540331
132
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66146722
109067
rs473792
66146816
109161
rs2630644
66146912
109257
rsl 2949591
66146954
109299
rs2109053
66147095
109440
rs35296857
66147167
109512
rsl 7791270
66147246
109591
rsl 6975989
66147343
109688
rsl 7791282
66147436
109781
rs17718124
66147660
110005
rsl 6975993
66147678
110023
rs35106633
66147754
110099
rs34429407
66147913
110258
rs2240749
66147960
110305
rsl 6975998
66148045
110390
rs3217050
66148046
110391
rs2240750
66148178
110523
rsl 6976000
66148512
110857
rsl 6976002
66148962
111307
rsl 89580
66149102
111447
rsl 843622
66149149
111494
rs543765
66149213
111558
rs434729
66149222
111567
rs375709
66149257
111602
rsl 1656782
66149348
111693
rsl 1653519
66149386
111731
rsl 90256
66149859
112204
rs4584866
66150283
112628
rsl 6976008
66150360
112705
rsl 6976009
66150511
112856
rsl 6976011
66150609
112954
rsl 7718380
66150898
113243
rsl 1652208
66150909
113254
rsl 1652209
66151294
113639
rsl 0491179
66151858
114203
rsl 6976019
66152747
115092
rs35499697
66152804
115149
rsl 7791650
66152863
115208
rsl 6976023
66152963
115308
rsl 6976024
66152998
115343 j rs9891997
66153185
115530
rsl 2942978
66153557
115902
rsl 6976027
66153631
115976
rsl 7718538
66154056
116401
rsl 6976031
66155045
117390
rs34736208
66155048
117393
rsl 2952540
66155070
117415
rs34389302
66155101
117446
rsl 89581
66155303
117648
rs9910837
66155784
118129
rsl 7718586
66156561
118906
rs544680
66156593
118938
rs8066818
66157022
119367
rs408448
66157073
119418
rsl 1650843
Position in Build 35/36
Position in SEQ ID NO:3
Marker ID
66157111
119456
rs367742
66157179
119524
rs550945
66157197
119542
rsl 83590
66157224
119569
rs551058
66157327
119672
rs34098284
66157893
120238
rsl 83591
66157917
120262
rsl 83059
66157976
120321
rs404774
66158027
120372
rsl 1657329
66158091
120436
rs405068
66158109
120454
rs35166389
66158247
120592
rsl 6976038
66158414
120759
rsl 83592
66159001
121346
rs34458687
66159262
121607
rs35760966
66159416
121761
rs2191113
66159464
121809
rs5821793
66159546
121891
rs5821794
66159547
121892
rs35222039
66159556
121901
rsl 0648023
66159562
121907
rs3048626
66159637
121982
rs8072436
66159764
122109
rs2215270
66159891
122236
rsl 6976043
66160076
122421
rs8074760
66160292
122637
rsl 1657599
66160331
122676
rs34281212
66160370
122715
rs36074213
66160438
122783
rs171385
66160451
122796
rs412353
66160480
122825
rs422923
66160492
122837
rsl 1654012
66160668
123013
rs35429609
66160721
123066
rs2367004
66161977
124322
rs35222003
66161994
124339
rsl 1867678
66162691
125036
rsl 2953137
66162852
125197
rsl 0642929
66162854
125199
rs34937331
66162869
125214
rsl 0585639
66163076
125421
rs4793497
66037656
1
rsl 1077501
66038245
590
rsl 0445229
66038446
791
rs8067115
66038456
801
rsl 0445230
66038691
1036
rsl 0445231
66039757
2102
rs28569992
66039800
2145
rs4606755
66039816
2161
rs4435300
66039936
2281
rs35154837
WO 2008/065682 PCT/IS2007/000020
133
Table 13. Key to Sequence listing provided herein.
SEQ ID NO
Name
1
LD block C06
2
LD block C10
3
LD block C17
4
rsl 0882091
rsl 111875
6
rsl 569699
7
rsl7763769
8
rsl7763811
9
rsl843622
rsl860316
11
rsl981647
12
rsl999763
13
rs2191113
14
rs2275729
rs2421943
16
rs2497304
17
rs3829170
18
rs4712527
19
rs6583826
rs6583830
21
rs7756992
22
rs7758851
SEQ ID NO
Name
23
rs7908111
24
rs7914814
rs7915186
26
rs7917359
27
rs7922112
28
rs7923837
29
rs9295478
rs947591
31
rs9890889
32
rs7752906
33
rs9350271
34
rs9356744
rs9368222
36
rsl0440833
37
rs6931514
38
rs2009802
39
rsl7718938
40
rsl7223216
41
rs2109050
42
rs 1962801
43
rs7086285
44
rsl7234378
134
EXAMPLE 2
VARIANTS IN THE CDKALl GENE INFLUENCE INSULIN RESPONSE AND THE RISK OF TYPE 2 DIABETES
We have recently described a variant in TCF7L2 associated to T2D (Grant, S.F. et al. Nat Genet 38, 320-3 (2006); Helgason, A. et al. Nat Genet (2007)). In the following, we describe a genome-wide association study on Icelandic T2D patients, using the Illumina Hap300 chip. We individually tested 313,179 SNPs for association to T2D in a sample of 1399 T2D patients and 5275 controls. We further tested 339,846 two-marker haplotypes identified as efficient surrogates (r2 > 0.8) for a set of SNPs which were not included on the Hap300 chip but were typed in the HapMap project (Pe'er, I. et al. Nat Genet 38, 663-7 (2006)). In addition to analyzing the entire group of T2D patients we separately tested 700 non-obese T2D patients and 531 obese T2D patients for association. Overall, a total of 1,959,075 (653,025 variants x 3 phenotypes) tests were performed. The results were adjusted for relatedness between individuals and potential population stratification by genomic control (Devlin, B. & Roeder, K. Biometrics 55, 997-1004 (1999)) (see Methods). Specifically, the (unadjusted) chi-square statistics were divided by 1.287, 1.204 and 1.184 respectively for the analyses of all, non-obese and obese T2D cases. A previously identified SNP rs7903146 in the TCF7L2 gene gave the most significant results with OR = 1.38 and P = 1.82xlO"10 in all T2D patients. Although no other SNP or haplotype was significant after adjustment for the number of tests performed, more borderline significant signals were observed than expected by chance alone (Fig. 4). Hence we decided to further pursue the top signals.
METHODS
Icelandic study population
The Icelandic T2D group has been described previously (Reynisdottir, I. et al. Am 3 Hum Genet 73, 323-35 (2003)). A total of 1500 T2D patients were recruited for this genome-wide association study, using the Infinium II assay method and the Sentrix HumanHap300 BeadChip (Illumina, San Diego, CA, USA). Thereof, 1399 were successfully genotyped according to our quality control criteria (see Supplementary Methods) and used in the present case control-analysis; 531 of the genotyped cases were obese (BMI > 30). The controls used in this study consisted of 599 controls randomly selected from the Icelandic genealogical database and 4676 individuals from other ongoing genome-wide association studies at deCODE. The study was approved by the Data Protection Commission of Iceland and the National Bioethics Committee of Iceland. Written informed consent was obtained from all cases and controls.
135
Other study populations
The Danish female study group of 282 cases and 629 controls, herein termed Denmark A, was selected from the Prospective Epidemiological Risk Factor (PERF) study in Denmark (Tanko, L.B., et al. Bone 32, 8-14 (2003)). This is a group of postmenopausal women who took part in various screening placebo-controlled clinical trials and epidemiological studies performed at the Center for Clinical and Basic Research. At a follow-up examination of 5847 women in 2000-2001 medical history including diabetes type I and type II, family history, and current or previous long-term use of drugs were gathered during personal interviews using a preformed questionnaire. If subject was diagnosed as diabetes of either type I or type II, the time of diagnosis or treatment was also collected. The study was approved by the Ethical Committee of Copenhagen County and was in accordance with the principles of the Helsinki Declaration.
The second Danish study population of 1359 T2D cases and 4858 control individuals with normal glucose tolerance was from the Steno Diabetes Center in Copenhagen and from the Inter99 population-based sample of 30- to 60-year-old individuals living in the greater Copenhagen area and sampled at Research Centre for Prevention and Health (Jorgensen, T. et al. Eur J Cardiovasc Prev Rehabil 10, 377-86 (2003)). This dataset is referred to in the text as Denmark B. Diabetes and pre-diabetes categories were diagnosed according to the 1999 World Health Organization (WHO) criteria. An oral glucose tolerance test was performed on participants in the Inter99 study as described (Jorgensen, T. et al. Eur J Cardiovasc Prev Rehabil 10, 377-86 (2003)). Informed written consent was obtained from all subjects before participation. The study was approved by the Ethical Committee of Copenhagen County and was in accordance with the principles of the Helsinki Declaration.
The Philadelphia study population consisted of 468 T2D cases and 1024 control individuals. The study population was selected from the PENN CATH study, a cross-sectional study of the association of biochemical and genetic factors to coronary atherosclerosis in a study population of consecutive individuals undergoing cardiac catheterization at the University of Pennsylvania Medical Center. T2D was defined as a history of fasting blood glucose > 126 mg dl"1, 2 h postprandial glucose > 200 mg dl"1, use of oral hypoglycemic agents, or use of insulin and oral hypoglycemic in a subject older than age 40. The University of Pennsylvania Institutional Review Board approved the study protocol, and all subjects gave written informed consent. All cases and controls were of European ancestry. Ethnicity was determined through self-report.
The Dutch Breda study population consisted of 370 T2D cases and 916 control individuals. The cases were recruited in 1998-1999 in collaboration with the Diabetes Service Breda and 80 general practitioners from the region around Breda. All patients are diagnosed according to WHO criteria (plasma glucose levels >11.1 mmol/l or a fasting plasma glucose level > 7.0 mmol/l), and undergo clinical and laboratory evaluations for their diabetes at regular 3-month intervals. The
136
Medical Ethics Committee of the University Medical Centre in Utrecht approved the study protocol. All probands filled out an informed consent and a questionnaire on clinical data, including their diabetes related medication, height and weight at present and at the age of 20 year. The controls are Dutch blood bank donors with an average age of 48.
The Scottish study population consisted of type 2 diabetic cases and non-diabetic controls from the Wellcome Trust UK T2D case-control collection (Go-DARTS2) which is a sub-study of Diabetes Audit and Research Tayside (DARTS) (Morris, A.D. eta/. BMJ 315, 524-8 (1997)). All T2D patients were physician-diagnosed T2D cases recruited at primary or secondary care diabetes clinics, or invited to participate from primary care registers and have not been characterized for GAD anti-bodies or MODY gene mutations. The controls were invited to participate through the primary care physicians or through their workplace occupational health departments. Controls did not have a previous diagnosis of diabetes, but the glucose tolerance status of the controls is unknown. All individuals in this ongoing study were recruited in Tayside between October 2004 and July 2006. This study was approved by the Tayside Medical Ethics Committee and informed consent was obtained from all subjects.
All subjects in the Hong Kong study population were of southern Han Chinese ancestry residing in Hong Kong. The cases consisted of 1500 individuals with T2D selected from the Prince of Wales Hospital Diabetes Registry. Of these, 682 patients had young-onset diabetes (age-at-diagnosis < 40 years) with positive family history. An additional 818 cases were randomly selected from the same registry. The controls consisted of 1000 subjects with normal glucose tolerance (fasting plasma glucose < 6.1 mmol/l). Of these, 617 were recruited from the general population participating in a community-based cardiovascular risk screening program as well as hospital staff. In addition, 383 subjects were recruited from a cardiovascular risk screening program for adolescents. Informed consent was obtained for each participating subject. This study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.
The African study population comes from the Africa America Diabetes Mellitus study, which was originally designed as an affected sibling pair study with enrollment of available spouses as controls. It has since been expanded to include other family members of the affected pairs and population controls. Recruitment strategies and eligibility criteria for the families enrolled in this report have been described previously (Rotimi, C.N. etal. Ann Epidemiol 11, 51-8 (2001)). This West African case-control series consisted of individuals from the Yoruba (233 affected individuals, 432 controls) and Igbo (237 affected individuals, 276 controls) groups from Nigeria and the Akan (257 affected individuals, 248 controls), Ewe (22 affected individuals, 30 controls) and Gaa-Adangbe (123 affected individuals, 141 controls) groups from Ghana.
137
With the exception of the Scottish Go-DARTS study population the DNA used for genotyping in all replication study populations was the product of whole-genome amplification (GenomiPhi Amplification kit, Amersham) of DNA isolated from the peripheral blood.
Statistical analysis
Illumina Genome-Wide Genotyping. All Icelandic case- and control-samples were assayed with the Infinium HumanHap300 SNP chips (Illumina, SanDiego, CA, USA), containing 317,503 haplotype tagging SNPs derived from phase I of the International HapMap project. Of the SNPs assayed on the chip, 4,324 SNPs were excluded as the had (a) yield lower than 95% in cases or controls; (b) minor allele frequency less than 1% in the population; or (c) showed significant distortion from Hardy-Weinberg equilibrium in the controls (P-value < 0.001). Any samples with a call rate below 98% were excluded from the analysis. Thus, the final analyses presented in the text utilizes 313,179 SNPs.
Single SNP genotyping. Single SNP genotyping for all population studied, except for the Scottish Go-DARTS population, was carried out at deCODE Genetics in Reykjavik, Iceland by the Centaurus (Nanogen) platform (Kutyavin, I.V. et al. Nucleic Acids Res 34, el28 (2006)). The quality of each Centaurus SNP assay was evaluated by genotyping each assay in the CEU and/or YRI HapMap samples and comparing the results with the HapMap data. Assays with >1.5% mismatch rate were not used and a linkage disequilibrium (LD) test was used for markers known to be in LD. Single SNP genotyping for the Scottish population was carried out at the Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, Scotland, by the TaqMan® method.
Association analysis. For association analysis we utilized a standard likelihood ratio statistics, implemented in the NEMO software (Gretarsdottir, S. et al. Nat Genet 35, 131-8 (2003)) to calculate two-sided p-values and allele specific OR for each individual allele, assuming a multiplicative model for risk, i.e., that the risks of the two alleles a person carries multiply. Allelic frequencies, rather than carrier frequencies are presented for the markers, and p-values are given after adjustment for the relatedness of the subjects. When estimating genotype specific OR (Table 19) genotype frequencies in the population were estimated assuming HWE.
In general, allele/haplotype frequencies are estimated by maximum likelihood and tests of differences between cases and controls are performed using a generalized likelihood ratio test (Rice, J.A. Mathematical Statistics and Data Analysis, (Wadsworth Inc., Belmont, CA, 1995)). This method is particularly useful in situations where there are some missing genotypes for the marker of interest and genotypes of another marker, which is in strong LD with the marker of interest, are used to provide some partial information. This was used in the association tests presented in Table
138
17 to ensure that the comparison of the highly correlated markers was done using the same number of individuals. To handle uncertainties with phase and missing genotypes, maximum likelihood estimates, likelihood ratios and p-values are computed directly for the observed data, and hence the loss of information due to uncertainty in phase and missing genotypes is automatically captured by the likelihood ratios.
Results from multiple case-control groups were combined using a Mantel-Haenszel model (Mantel, N. & Haenszel, W. J Natl Cancer Inst 22, 719-48 (1959)) in which the groups were allowed to have different population frequencies for alleles, and genotypes but were assumed to have common relative risks.
Correction for relatedness of the subjects and Genomic Control. Some of the individuals in both the Icelandic patient and control groups are related to each other, causing the chi-square test statistic to have a mean >1 and median >0.6752. We estimated the inflation factor by calculating the average of the 653,025 chi-square statistics, which was a method of genomic control4 to adjust for both relatedness and potential population stratification. The inflation factor was estimated as 1.287, 1.204 and 1.184, for the analysis of all, non-obese and obese T2D cases, respectively. The results presented are based on adjusting the chi-square statistics by dividing each of them by the corresponding inflation factor.
Quantitative analysis. Data from oral glucose tolerance test on individuals from the Danish Inter99 study were used to calculate insulin secretion as corrected insulin response (CIR) using the following equation: (100 x insulin at 30 minutes) 4- [glucose at 30 minutes x(glucose at 30 minutes - 3.89 mmol)]. Insulin sensitivity was estimated as the reciprocal of the insulin resistance according to the homeostasis model assessment (HOMA): 22.5 / [fasting insulin x fasting glucose] (Matthews, D.R. eta/. Diabetologia 28, 412-9 (1985)). The association between CIR (HOMA) and genotype status was tested using a multiple regression where the log-transformed CIR (HOMA) where taken as the response variable and the explanatory variable was either the number of copies of risk allele an individual carries (an additive model) or an indicator variable for homozygous carriers of the risk allele (a recessive model). Adjustment for sex, age and affection status was done by including the appropriate terms as explanatory variables. For comparison insulin secretion was also calculated as (insulin at 30 minutes - insulin at 0 minutes) -f- (glucose at 30 minutes -glucose at 0 minutes), yielding comparable results.
139
Cell lines. The INS1 cells were provided by Hoffmann-LaRoche. They were grown in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 50 pg/ml penicillin-streptomycin (Invitrogen), 50 nM 2-mercaptoethanol (SIGMA), 1 mM MEM sodium pyruvate (Invitrogen) and 10 mM Hepes buffer solution (Invitrogen). They were split 1:2 twice per week by washing once in IX Hanks Balanced Salt Solution (Invitrogen) and then trypsinized (trypsin-EDTA; Invitrogen).
Preparation of RNA and cDNA amplification. INS1 cells were incubated for 48h in normal growth medium containing 10 mM glucose. At the time of harvest there were 2xl07 cells, which were used for the preparation of total RNA. RNA was extracted using RNeasy Midi Kit (Quiagen). cDNA was prepared using High-Capacity cDNA Archive Kit (Applied Biosystems). CDKALl cDNA was amplified using two different primer pairs between exons 2 and 8 (forward: 5'-
GGGGCTGCTCACATAATAATTCA-3'; reverse: 5'-TGTGCCAATGTCTCTGCCATA-3') and between exons 7 and 13 (forward: 5'-ACCTGGCCAGCTATCCCATT-3'; reverse: 5 '-CC ATTTTT CCC AT G AAT G C AG - 3'). Primers from beta-actin served as positive controls (forward 5'-ATCTGGCACCACACCTCCTACAATGAGCTGC-3'; reverse: 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3').
RESULTS AND DISCUSSION
For each phenotype tested we selected all single SNPs and two marker haplotypes with P < 0.00005 for replication in a case-control sample from Denmark (Denmark B). After eliminating redundant markers a total of 46 SNPs were taken further for the attempt at replication (Table 14). In addition, we included the five most significant non-synonymous SNPs present on the Illumina Hap300 chip. Out of those 51 SNPs, 47 were successfully genotyped in 1110 Danish T2D cases and 2272 controls. In the Danish group SNPs rs7756992 and rsl3266634 stood out and were significantly replicated with P= 0.00013 and OR = 1.24 and P = 0.0012 and OR = 1.20, respectively, in the Danish group of all T2D patients (Table 15). This is compared to P = 0.00021 and OR = 1.23 and P = 0.000061 and OR = 1.19, respectively in the initial Icelandic study. All of the other SNPs genotyped had P > 0.01 in the Danish group and were not pursued further. The first SNP, rs7756992, is located in intron 5 of the CDK5 regulatory subunit associated protein 1-like 1 (CDKALl) gene on 6p22.3. It resides in a large LD block of 201.7 kb that includes exons 1-5 of the CDKALl gene as well as the minimal promoter region but no other known genes (Figure 5). The second SNP, rsl3266634, is a non-synonymous SNP causing an arginine 325 to tryptophan change in the last exon of the solute carrier family 30 (zinc transporter), member 8 (SLC30A8)
gene on 8q24. The gene product of SLC30A8 is specific to the pancreas and it is expressed in beta
140
cells where it facilitates the accumulation of zinc from the cytoplasm into intracellular vesicles (Chimienti, F., eta/. Diabetes 53, 2330-7 (2004)). The risk allele of rsl3266634 on 8q24 has recently been found to confer risk of T2D in a genome wide association study of French T2D cases and controls (Sladek, R. eta/. Nature 445, 881-5 (2007)). Of other significantly associated SNPs in that study, we also replicated, in the initial Icelandic samples, association to two SNPs close to the HHEX gene (Table 16). However, we did not replicate the reported association to markers in the LOC387761 and EXT2 genes also described in that study.
We typed the SNPs rs7756992 and rsl3266634 in four other T2D case-control groups of European ancestry from Denmark (Denmark A), Scotland, the Netherlands and Philadelphia, US as well as case-control groups from Hong Kong and West Africa. Furthermore, the size of the Denmark B study group was expanded mostly by increasing the number of genotyped controls. The association of the G allele of rs7756992 was replicated with significance in the Scottish (OR = 1.11; P = 0.0042) and the Hong Kong (OR = 1.25; P = 0.00018) case-control groups (Table 17). Association in other study groups was not individually significant, but all were in the same direction. The observed association from combining all eight case-control groups gave an OR of 1.15 with a corresponding P of 9.0x10 12 (Table 17). Given that approximately 2 million tests were performed in the initial genome-scan, this association remained highly significant with Bonferonni adjustment Cadj = 1-8 xlO"5) (Skol, A.D., et ai. Nat Genet 38, 209-13 (2006)). Attempts at refining the association observed with rs7756992 by genotyping additional markers that correlate with the original signal in the HapMap CEPH (CEU) dataset, did not yield more significant results (Table 18). As could be expected the linkage disequilibrium observed for the West African population was considerably less than that seen for the Icelandic and Hong Kong groups (Table 19). Further work is needed to determine if an associated variant with a higher OR than observed for rs7756992 can be identified in the West African group. Likewise, for allele C of the non-synonymous SNP rsl3266634 the association to T2D was replicated with significance in three of the six additional groups (from Scotland, Philadelphia and Hong Kong) (Table 17). Even though the OR for Denmark B decreased with the larger sample size and the estimated effect was in the opposite direction (only slightly and non-significant) for Denmark A, the combined results from all study group yielded a genome-wide significant P of 2.5x10'" and an OR of 1.16 (Table 17).
In the Icelandic study the association to rs7756992 was more significant in non-obese T2D patients (OR = 1.37; P = 9.0xi0"6) than in the group of all patients (OR = 1.23; P = 0.00021) (Table 14 and Table 17). A higher OR in non-obese than in obese T2D patients was also observed for this variant in the other populations studied. For the combined populations of European origin the OR was 1.19; P = 7.29xi0"9 for the non-obese T2D patients compared to OR = 1.12; P = 0.00017 for the obese group. An even stronger effect was seen in the Hong Kong non-obese T2D group (OR = 1.36; P = 7.48xl0"6), compared to the obese group (OR = 1.13; P = 0.094), where obesity was defined as BMI > 25. When the results for all groups were combined, relative to
141
controls, OR = 1.19; P = 1.93xl0"n and OR = 1.13; P = 2.68xl0"5 was obtained for the non-obese and obese T2D patient groups, respectively. These results indicate that this variant does not confer increased risk of T2D through increased BMI.
Genotype odds ratio was estimated for each of the two loci (Table 20). Based on the results for the combined Caucasian study populations rs7756992 deviates significantly from the multiplicative model with OR for the heterozygote = 1.09 compared to OR = 1.45 for the homozygote, supporting a nearly recessive mode of inheritance. The same trend, although nonsignificant, was seen for the Hong Kong samples with heterozygote OR = 1.13 and OR = 1.55 for the homozygote. Conversely, a multiplicative model for the genotype relative risk provided an adequate fit for rsl3266634.
The function of the gene product of CDKALl is not known. However, as implied in the gene name the protein product is similar to another protein, CDK5 regulatory subunit associated protein 1 (CDK5RAP1). CDK5RAP1 is expressed in neuronal tissues where it inhibits cyclin dependent kinase 5 (CDK5) activity by binding to the CDK5 regulatory subunit p35 (Ching, Y.P., et al. J Biol Chem 277, 15237-40 (2002)). In pancreatic beta cells, CDK5 has been shown to play a role in the loss of beta cell function under glucotoxic conditions (Wei, F.Y. et al. Nat Med 11, 1104-8 (2005)). Furthermore, inhibition of the CDK5/p35 complex prevents decrease of insulin gene expression that results from glucotoxicity (Ubeda, M., et al. J Biol Chem 281, 28858-64 (2006)). It is tempting to speculate that CDKALl might play a role in the inhibition of CDK5/p35 in pancreatic beta cells similar to that of CDK5RAP1 in neuronal tissue. Reduced expression of CDKALl or reduced inhibitory function thus could lead to an impaired response to glucotoxicity. In this study we showed that CDKALl is expressed in the rat pancreatic beta cell line INS-1 (Figure 6). Further studies are needed to determine if the effect of CDKALl on increasing the risk of T2D is exerted through this pathway.
Based on the predicted function of CDKALl and known function of SLC30A8 we would expect both rs7756992 and rsl3266634 to affect insulin secretion. To evaluate the effects of the two SNPs on insulin secretion we analyzed the effect of genotype status on corrected insulin response (CIR) in a set of individuals from the Inter99 study (part of Denmark B) that had undergone an oral glucose tolerance test (OGTT). For rs7756992, we demonstrated that the homozygote carriers of the risk allele had an estimated 24% less CIR than the heterozygote carriers or non-carriers (P < 0.00001, Fig. 7). This observation is consistent with the variant's nearly recessive mode of inheritance with respect to disease risk. Furthermore, the effect observed on CIR is present in both males and females (Figure 8) and in T2D patients as well as controls, and adjusting for BMI status did not affect the results (Table 21). The effect of rsl3266634 on insulin response was smaller but significant and for this risk variant the reduction in CIR was consistent with an additive effect. No effect on insulin sensitivity was observed for either variant (Table 21).
142
The identification of CDKALl as a susceptibility gene for T2D adds a new piece to the puzzle of how genetic factors may predispose to T2D. Although the function of this gene remains to be elucidated we have shown that it is expressed in pancreatic beta cells and that a variant within the gene is correlated with insulin secretion. The similarity to CDK5RAP1 further indicates that CDKALl may facilitate insulin production under glucotoxic conditions through interaction with CDK5. In conclusion, we have identified a variant in the CDKALl gene that in a nearly recessive manner blunts the insulin response and predisposes to T2D.
Table 14. Association to T2D in the Icelandic discovery group.
The upper table includes association results for all SNPs or two-marker haplotypes that have an adjusted P value less than 10"5 for either all T2D cases, non-obese T2D cases or obese T2D cases. Included in the table is the chromosome, the position of the markers (or the midpoint for two-marker haplotypes) in NCBI Build 34, the markers and alleles tested, the corresponding surrogate SNP for two-markers haplotypes selected for replication, the frequency in controls and the frequency in cases, the odds ratio (OR) and adjusted P-value for the three case groups tested. The number of T2D cases in each of the three groups is included in parenthesis and the same set of 5275 controls is used in all tests. Note that information on BMI is missing for 168 of the cases. The lower table includes the corresponding values for the five most significant non-synonymous SNPs selected for replication. Included in column five are the corresponding genes and the codon changes. In both tables markers selected for further testing in the first replication group (Denmark B) are indicated with bold typesetting. Other markers / haplotypes were excluded from the replication study as they were a) highly correlated with another marker selected for replication, or b) belong to the TCF7L2 locus that has been studied previously.
All T2D cases (1399) NonObese T2D cases (700) Obese T2D cases (531)
Chr
Position
Markers
Allele
Surrogate3 (/*)
Con.frq
Case.frq
OR
P"
Case.frq
OR
P"
Case.frq
OR
P"
C01
29602516
rs4949283 rs502545
TC
rsl 0798895 G (1)
0.149
0.117
0.76
0.00016
0.104
0.66
0.000033
0.133
0.88
0.21
C01
104461151
rs7553985
C
-
0.394
0.430
1.16
0.0023
0.419
1.11
0.11
0.466
1.34
0.000027
C01
104467009
rs2166890
T
-
0.393
0.430
1.16
0.0018
0.419
1.11
0.091
0.466
1.35
0.000024
C01
104468502
rs7552405
T
-
0.317
0.355
1.19
0.00078
0.346
1.14
0.047
0.386
1.35
0.000030
C01
151915609
rs3738028
G
-
0.360
0.407
1.22
0.000046
0.417
1.27
0.00016
0.409
1.23
0.0038
C02
40632580
rsl 3414307 rsl 990609
AG
-
0.517
0.571
1.24
0.0000089
0.568
1.23
0.0011
0.582
1.30
0.00026
C02
40623619
rsl3414307
A
-
0.543
0.593
1.22
0.000033
0.589
1.21
0.0028
0.603
1.28
0.00056
. C02
55036788
rs930493 rs10173697
GT
-
0.281
0.335
1.29
0.0000017
0.325
1.23
0.0024
0.333
1.27
0.0016
C02
55040844
rsl 0173697
T
-
0.503
0.553
1.22
0.000040
0.545
1.18
0.0086
0.560
1.25
0.0014
C03
89162181
rsl2486049
T
-
0.872
0.904
1.38
0.000035
0.907
1.43
0.00043
0.901
1.34
0.0095
C03
146863467
rs7630694
G
-
0.060
0.070
1.20
0.065
0.056
0.93
0.60
0.097
1.70
. 0.000033
C03
196904151
rs9858622
A
-
0.668
0.701
1.17
0.0028
0.682
1.07
0.34
0.737
1.40
0.000016
C04
140508134
rsl 3116075 rs6824182
AA
rs10033117 C (1)
0.741
0.763
1.13
0.036
0.734
0.96
0.60
0.804
1.43
0.000024
C04
140604420
rs2292837rs11725721
TC
-
0.254
0.232
0.89
0.038
0.259
1.03
0.69
0.194
0.71
0.000047
C04
140621178
rs3762864rs11725721
GC
-
0.254
0.233
0.89
0.042
0.262
1.04
0.60
0.194
0.70
0.000038
C05
76637396
rs832785 rs2859576
AA
-
0.510
0.470
C05
76635083
rs4704400
T
-
0.490
0.530
C05 C06
87882885 6967990
rsl 0505855 rsl 2514611 rs490213 rs814174
GC AG
rsl 0452479 G (0.94) rs12201780 A (1)
0.188 0.044
0.224 0.072
C06
9509965
rs214447
T
-
0.424
0.449
C06
20779501
rs4712527 rs7756992
AG
-
0.232
0.270
C06
20805960
rs7756992 rs9295478
AG
-
0.743
0.701
C06
20787688
rs7756992
G
-
0.232
0.270
C06
31552682
rs2516424
C
-
0.325
0.372
C06
31592562
rs2516424 rs4947324
CC
-
0.320
0.368
C06
41130207
rsl0456499
A
-
0.563
0.597
C06
132387934
rs9483377 rs997607
GC
-
0.234
0.278
C06
132379686
rs9483377 rs7745875
GG
-
0.233
0.276
C06
132361238
rs9483377
G
-
0.307
0.356
C06
150399255
rsl1155700
A
-
0.749
0.794
C06
150399954
rs12213837
C
-
0.749
0.794
C06
164421443
rs206732 rs933251
TC
rsl 0085202 A (1)
0.531
0.479
C08
124084183
rs952656
G
-
0.673
0.721
C08
124092339
rsl 3252935 rs7824293
TG
-
0.143
0.108
C08
128249239
rs283710 rs412835
CC
-
0.254
0.222
C08
128250055
rsl85852
G
-
0.755
0.791
C08
128265112
rs283718 rs283720
CA
-
0.255
0.223
C09
88426790
rsl0993008
A
-
0.154
0.192
C09 C09
93768899 93802193
rs10818991 rs10990303 rs10990568 rs4743148
CC GG
rsl0985640 A
(0.85)
0.537 0.263
0.490 0.309
C09
93810412
rs4743148
G
-
0.315
0.365
C09
124790974
rs3814120
T
-
0.093
0.113
C10
52735263
rs7915186 rs3829170
TT
-
0.328
0.377
C10
52746400
rs3829170 rs7922112
TG
rs12247188T (0.9)
0.336
0.386
C10
93976392
rs2421943
G
-
0.555
0.614
C10
94022896
rs2421943 rs7917359
GC
-
0.521
0.585
C10
94068337
rs7908111 rs2497304
GG
-
0.499
0.443
0.85
0.00082
0.489
0.92
0.18
0.438
0.75
0.000043
1.18
0.0008
0.511
1.09
0.18
0.562
1.33
0.000043
1.25
0.00023
0.244
1.39
0.000015
0.200
1.08
0.38
1.71
0.000016
0.080
1.89
0.000037
0.063
1.48
0.033
1.11
0.034
0.416
0.97
0.61
0.495
1.34
0.000035
1.23
0.00021
0.292
1.37
0.0000090
0.250
1.11
0.21
0.81
0.000089
0.682
0.74
0.000013
0.718
0.88
0.11
1.23
0.00021
0.292
1.37
0.0000090
0.250
1.11
0.20
1.23
0.000039
0.375
1.25
0.00080
0.376
1.25
0.0020
1.24
0.000027
0.370
1.25
0.00074
0.373
1.26
0.0016
1.15
0.0040
0.575
1.05
0.43
0.637
1.36
0.000018
1.26
0.000040
0.272
1.22
0.0067
0.276
1.25
0.0065
1.25
0.000048
0.271
1.22
0.0052
0.273
1.23
0.0087
1.25
0.000013
0.348
1.20
0.0052
0.354
1.24
0.0040
1.29
0.0000095
0.786
1.23
0.0049
0.801
1.35
0.00039
1.29
0.0000097
0.786
1.23
0.0049
0.801
1.35
0.00040
0.81
0.000037
0.469
0.78
0.00015
0.497
0.87
0.058
1.25
0.000019
0.706
1.17
0.021
0.725
1.28
0.0012
0.72
0.000010
0.116
0.78
0.0099
0.104
0.69
0.00067
0.84
0.0024
0.245
0.95
0.51
0.190
0.69
0.000025
1.22
0.00050
0.764
1.05
0.49
0.822
1.49
0.0000046
0.84
0.0026
0.256
1.01
0.94
0.189
0.68
0.0000092
1.30
0.000027
0.181
1.21
0.019
0.194
1.32
0.0020
0.83
0.00019
0.469
0.76
0.000037
0.513
0.91
0.18
1.25
0.000032
0.314
1.28
0.00038
0.306
1.23
0.0076
1.25
0.000010
0.371
1.28
0.00013
0.358
1.21
0.0092
1.25
0.0046
0.094
1.01
0.91
0.140
1.59
0.000014
1.24
0.000021
0.374
1.22
0.0021
0.375
1.23
0.0049
1.24
0.000021
0.381
1.22
0.0027
0.387
1.24
0.0027
1.28
9.1 *10"7
0.600
1.20
0.0043
0.621
1.31
0.00017
1.30
1.3x10"®
0.565
1.19
0.0052
0.602
1.39
0.0000041
0.80
0.0000034
0.456
0.84
0.0072
0.427
0.75
0.000039
C10
94011761
rsl 999763 rsl 0882091
GT
-
0.517
0.455
C10
94023632
rsl 999763 rs6583830
GG
-
0.517
0.455
C10
94012407
rs6583826
G
-
0.467
0.518
C10
94025680
rs6583826 rsl 0882091
GC
-
0.393
0.449
C10
94092724
rs10882091 rs7923837
CG
-
0.410
0.466
C10
94038954
rsl 0882091
C
-
0.415
0.472
C10
94047527
rs7914814
T
-
0.416
0.472
C10
94062695
rs6583830
A
-
0.415
0.472
C10
94122233
rs2275729 rsl 111875
AG
-
0.470
0.527
C10
94157293
rs2497304
A
-
0.530
0.473
C10
94160330
rs947591
A
-
0.475
0.526
C10
114441018
rs7895307 rsl 2255372
GT
-
0.257
0.308
C10
114422936
rs7903146
T
-
0.300
0.372
C10
114434905
rs7903146 rsl 1196192
TT
-
0.220
0.282
C10
114438514
rs7904519
G
-
0.480
0.522
C10
114455586
rs7904519 rsl 0885409
GC
-
0.474
0.516
C10
114455586
rs7904519 rs10885409
AT
-
0.510
0.471
C10
114472659
rsl 0885409
C
-
0.484
0.523
C10
114473489
rs 12255372
T
-
0.294
0.351
C10
118261345
rsl681748 rs2170862
TT
-
0.238
0.265
C10
118285583
rs2170862
T
-
0.256
0.281
C10
118555280
rsl0787760
G
-
0.278
0.300
C11
23946882
rsl879230
T
-
0.088
0.111
C11
106474406
rsl455593
T
-
0.097
0.114
C12
30390375
rsl429622 rsl506382
AG
rs794598 C (0.9)
0.368
0.321
C12
33373479
rsl 905421
T
-
0.082
0.110
C13
25558690
rs565707 rs6491198
AA
-
0.281
0.249
C13
25478564
rs565707
C
-
0.700
0.734
C13
25535031
rs7984685
C
-
0.540
0.582
C13
25537643
rs7998347
C
-
0.540
0.582
C13
25715179
rs 1333350 rs7987436
GT
-
0.254
0.216
C14
80759910
rs799099 rs4899801
AG
-
0.365
0.390
0.78
2.9x10'7
0.472
0.83
0.0038
0.442
0.74
0.000019
0.78
2.9x10"'
0.472
0.83
0.0038
0.442
0.74
0.000019
1.23
0.000020
0.508
1.18
0.0080
0.527
1.28
0.00048
1.26
0.0000021
0.435
1.19
0.0062
0.469
1.36
0.000012
1.26
0.0000022
0.452
1.19
0.0063
0.486
1.36
0.000011
1.26
0.0000024
0.456
1.18
0.0079
0.491
1.36
0.000014
1.26
0.0000025
0.456
1.18
0.0081
0.491
1.35
0.000014
1.26
0.0000024
0.456
1.18
0.0079
0.491
1.36
0.000014
1.26
0.0000023
0.519
1.22
0.0018
0.534
1.29
0.00025
0.80
0.0000
0.481
0.82
0.00
0.466
0.77
0.000251
1.23
0.000023
0.521
1.21
0.0028
0.545
1.33
0.000053
1.29
0.0000049
0.330
1.42
4.5x10"'
0.269
1.06 .
0.45
1.38
1.9x10"10
0.396
1.53
2.4x10"11
0.342
1.21
0.010
1.39
3.4x10"9
0.298
1.51
9.4x10"9
0.263
1.27
0.0042
1.18
0.00045
0.553
1.34
0.0000026
0.483
1.01
0.84
1.18
0.00055
0.549
1.35
0.0000018
0.476
1.01
0.90
0.86
0.0013
0.441
0.76
0.000011
0.510
1.00
0.99
1.17
0.0014
0.555
1.33
0.0000060
0.483
0.99
0.94
1.29
4.9x10"7
0.371
1.41
1.6x10"'
0.317
1.11
0.15
1.15
0.013
0.245
1.04
0.59
0.302
1.38
0.000041
1.13
0.020
0.259
1.02
0.82
0.320
1.37
0.000043
1.12
0.037
0.269
0.96
0.53
0.347
1.38
0.000017
1.30
0.00097
0.128
1.53
0.000021
0.093
1.07
0.57
1.20
0.021
0.087
0.89
0.29
0.142
1.54
0.000040
0.82
0.000083
0.341
0.89
0.092
0.296
0.72
0.000023
1.39
0.000044
0.116
1.47
0.00020
0.107
1.35
0.011
0.85
0.0039
0.220
0.72
0.000016
0.274
0.97
0.69
1.19
0.0016
0.763
1.38
0.0000073
0.710
1.05
0.53
1.19
0.00043
0.606
1.31
0.000022
0.568
1.12
0.11
1.19
0.00046
0.606
1.31
0.000024
0.568
1.12
0.11
0.81
0.00030
0.195
0.71
0.000010
0.251
0.98
0.82
1.11
0.037
0.359
0.97
0.64
0.439
1.36
0.000022
C14
80763881
rs2066041
G
-
0.367
0.394
C14
80820260
rsl 0483957
A
-
0.459
0.493
C15
98094991
rs9920347 rsl 1635811
AG
rs2045107 C (0.9)
0.521
0.469
C16
12811478
rs6498353 rs9941146
CG
-
0.105
0.080
C16
22764405
rs724466
T
-
0.738
0.781
C16
24353768
rsl 1074618 rs985729
AC
rsl 1644596 G (1)
0.299
0.342
C16
73296557
rs1862773 rs825842
CT
-
0.059
0.038
C16
73311680
rs2432543 rs4887826
TG
-
0.069
0.043
C17
69180675
rsl 7763769 rsl 860316
GA
-
0.511
0.564
C17
69203439
rs1860316
A
-
0.653
0.707
C17
69242752
rs1860316 rs17763811
GC
-
0.335
0.282
C17
69218316
rs1981647
C
-
0.513
0.563
C17
69234630
rs1843622
T
-
0.615
0.665
C17
69244944
rs2191113
A
-
0.696
0.744
C17
69259003
rs9890889
A
-
0.839
0.869
C18
41051796
rsl0502860
G
-
0.167
0.194
C18
63451377
rs764133 rs7237209
TT
-
0.167
0.132
C18
63463071
rs7237209
C
-
0.819
0.852
C19
3316583
rs3810420
A
-
0.176
0.189
C20
37651862
rs4592915 rs2232580
GC
rs6127771 C (1)
0.495
0.550
C21
13769165
rs468601
A
-
0.888
0.908
C21
33296778
rs2834061
G
-
0.249
0.291
C21
39373432
rs369906
T
-
0.566
0.613
Gene
C03
69453958
rsl0510980
A
ENST00000343145 (K211R)
0.808
0.840
C08
118141371
rsl3266634
C
SLC30A8 (R325W)
0.646
0.685
C10
124472418
rs2495774
G
LOC390009 (Q27H)
0.547
0.594
C11
3624302
rs2271586
T
ART5 (T284K)
0.176
0.208
C19
8669900
rsl0410943
G
MGC33407 (A51V)
0.674
0.714
aA surrogate of the corresponding two marker haplotype with a correlation coefficient control (see Methods).
1.12
0.021
0.368
1.01
0.92
0.437
1.34
0.000038
1.15
0.0042
0.476
1.07
0.28
0.530
1.33
0.000042
0.81
0.000044
0.475
0.84
0.0056
0.468
0.81
0.0041
0.74
0.00054
0.068
0.62
0.000047
0.082
0.75
0.026
1.26
0.000038
0.781
1.27
0.0012
0.783
1.29
0.0025
1.21
0.00044
0.332
1.16
0.040
0.372
1.39
0.000032
0.63
0.000048
0.041
0.67
0.0075
0.039
0.64
0.0072
0.61
0.000010
0.042
0.60
0.00046
0.049
0.69
0.019
1.24
0.000013
0.585
1.35
0.0000023
0.543
1.14
0.069
1.28
0.0000020
0.734
1.46
3.2x10"®
0.687
1.17
0.039
0.78
0.0000028
0.254
0.68
2.6x10"®
0.301
0.86
0.039
1.23
0.000026
0.583
1.33
0.0000065
0.544
1.14
0.071
1.24
0.000021
0.684
1.35
0.0000043
0.640
1.11
0.14
1.27
0.000013
0.771
1.47
9.5x10"®
0.713
1.08
0.30
1.27
0.00053
0.885
1.47
0.000032
0.857
1.14
0.17
1.20
0.0035
0.218
1.39
0.000028
0.174
1.05
0.61
0.76
0.00010
0.121
0.69
0.000048
0.135
0.78
0.014
1.27
0.00028
0.867
1.44
0.000029
0.847
1.22
0.037
1.09
0.16
0.227
1.37
0.000045
0.146
0.80
0.021
1.25
0.0000048
0.558
1.29
0.000051
0.543
1.21
0.0060
1.25
0.0054
0.927
1.60
0.000026
0.895
1.08
0.48
1.24
0.000076
0.311
1.36
0.0000094
0.271
1.12
0.15
1.21
0.00010
0.631
1.31
0.000028
0.587
1.09
0.24
1.25
0.00065
0.836
1.22
0.019
0.845
1.30
0.0061
1.19
0.00060
0.678
1.16
0.030
0.697
1.26
0.0020
1.21
0.00011
0.592
1.20
0.0039
0.597
1.22
0.0043
1.23
0.00059
0.212
1.26
0.0033
0.203
1.20
0.042
1.20
0.00043
0.713
1.20
0.0076
0.708
1.17
0.035
°P values adjusted for relatedness and population stratification using genomic
Table 15. Association to T2D in the primary replication group (Denmark B).
Association results for the 47 SNPs tested in the primary replication cohort (Denmark B), consisting of 1110 T2D cases and 2272 controls. Included in the table is the chromosome, the position of the SNPs in NCBI Build 34, the marker and allele tested, frequency in controls and the frequency in cases, odds ratio (OR) and P value in all T2D cases, non-obese T2D cases and obese T2D cases, respectively. For all three groups of cases, the same group of controls is used and the number of cases is included in the parentheses. The two SNPs selected for replication in additional T2D case-control groups are highlighted with bold typesetting.
All T2D cases (1110) NonObese T2D cases (640) Obese T2D cases (470)
Chr
Position
Marker
Allele
Con.frq
Case.frq
OR
P
Case.frq
OR
P
Case.frq
OR
P
C01
29589307
rsl 0798895
A
0.832
0.828
0.97
0.68
0.831
0.99
0.94
0.824
0.94
0.55
C01
104461151
rs7553985
C
0.367
0.379
1.05
0.34
0.375
1.03
0.62
0.385
1.08
0.30
C01
151915609
rs3738028
G
0.385
0.410
1.11
0.050
0.419
1.15
0.029
0.397
1.05
0.47
C02
40623619
rsl 3414307
A
0.537
0.540
1.01
0.84
0.544
1.03
0.67
0.534
0.99
0.86
C03
69453958
rsl 0510980
A
0.826
0.833
1.05
0.50
0.835
1.06
0.50
0.831
1.03
0.74
C03
89162181
rsl 2486049
T
0.878
0.872
0.94
0.47
0.871
0.93
0.49
0.873
0.96
0.70
C03
146863467
rs7630694
G
0.053
0.054
1.02
0.85
0.051
0.95
0.72
0.059
1.12
0.46
C03
196904151
rs9858622
A
0.656
0.667
1.05
0.39
0.662
1.02
0.73
0.674
1.08
0.29
C04
140660180
rsl 0033117
C
0.740
0.746
1.03
0.65
0.747
1.04
0.65
0.744
1.02
0.81
C05
76635083
rs4704400
T
0.472
0.456
0.94
0.23
0.452
0.92
0.22
0.461
0.96
0.55
C05
87825021
rs10452479
G
0.229
0.238
1.05
0.43
0.240
1.06
0.43
0.235
1.04
0.68
C06
6971276
rs12201780
A
0.043
0.048
1.12
0.36
0.049
1.16
0.32
0.045
1.07
0.71
C06
9509965
rs214447
T
0.418
0.427
1.03
0.52
0.432
1.06
0.39
0.419
1.00
0.95
C06
20787688
rs7756992
G
0.276
0.322
1.24
0.00013
0.321
1.24
0.0021
0.323
1.25
0.0044
C06
31552682
rs2516424
C
0.363
0.380
1.07
0.19
0.374
1.05
0.48
0.387
1.11
0.18
C06
41130207
rsl 0456499
A
0.581
0.579
0.99
0.92
0.576
0.98
0.78
0.583
1.01
0.87
C06
132361238
rs9483377
G
0.306
0.331
1.12
0.039
0.334
1.14
0.061
0.327
1.10
0.20
C06
150399255
rsl 1155700
A
0.758
0.734
0.88
0.043
0.737
0.90
0.14
0.731
0.87
0.089
C06
164425224
rsl 0085202
G
0.430
0.426
0.99
0.78
0.424
■ 0.98
0.73
0.428
0.99
0.94
C08
118141371
rsl3266634
C
0.664
0.704
1.20
0.0012
0.701
1.19
0.013
0.707
1.22
0.012
C08
124084183
rs952656
G
0.672
0.672
1.00
0.98
0.680
1.04
0.56
0.660
0.95
0.51
C08
128250055
rs185852
G
0.796
0.797
1.01
0.92
0.794
0.99
0.88
0.801
1.03
0.72
C09
88426790
rsl 0993008
A
0.146
0.150
1.03
0.66
C09
93745181
rsl 0985640
G
0.430
0.434
1.01
0.78
C09
93810412
rs4743148
G
0.382
0.381
1.00
0.94
C09
124790974
rs3814120
T
0.089
0.090
1.02
0.84
C10
52758344
rsl 2247188
T
0.331
0.315
0.93
0.19
C10
94047527
rs7914814
T
0.413
0.432
1.08
0.14
C10
118555280
rs 10787760
G
0.294
0.276
0.91
0.15
C10
124472418
rs2495774
G
0.524
0.540
1.07
0.22
C11
23946882
rsl 879230
T
0.127
0.115
0.89
0.13
C11
3624302
rs2271586
T
0.190
0.201
1.07
0.28
C11
106474406
rs 1455593
T
0.081
0.080
0.98
0.81
C12
30434349
rs794598
T
0.623
0.600
0.91
0.063
C12
33373479
rs1905421
T
0.099
0.097
0.98
0.79
C14
80763881
rs2066041
G
0.427
0.415
0.95
0.35
C15
98060278
rs2045107
G
0.524
0.527
1.01
0.78
C16
12756032
rs6498353
C
0.136
0.134
0.98
0.80
C16
22764405
rs724466
T
0.695
0.715
1.10
0.085
C16
24356412
rsl 1644596
G
0.324
0.323
1.00
0.94
C16
73314817
rs4887826
G
0.064
0.052
0.82
0.068
C17
69203439
rs 1860316
A
0.679
0.682
1.01
0.82
C18
41051796
rs 10502860
G
0.222
0.197
0.86
0.044
C18
63463071
rs7237209
C
0.861
0.852
0.92
0.29
C19
3316583
rs3810420
A
0.181
0.191
1.07
0.30
C20
37645161
rs6127771
C
0.447
0.451
1.02
0.77
C21
33296778
rs2834061
G
0.250
0.255
1.03
0.66
3 o
K)
0.151
1.04
0.64
0.149
1.02
0.84
0.421
0.96
0.57
0.451
1.09
0.25
© o
00
0.370
0.95
0.41
0.398
1.07
0.39
o
0.076
0.85
0.16
0.109
1.27
0.052
•Jl
00
K)
0.312
0.92
0.22
0.318
0.94
0.45
0.434
1.09
0.18
0.429
1.07
0.35
0.268
0.88
0.080
0.288
0.97
0.73
0.542
1.07
0.27
0.538
1.06
0.46
0.118
0.91
0.36
0.110
0.85
0.14
0.194
1.02
0.77
0.211
1.14
0.13
0.081
0.99
0.92
0.078
0.96
0.77
0.594
0.88
0.058
0.608
0.94
0.37
0.086
0.85
0.17
0.113
1.16
0.24
0.427
1.00
1.00
0.398
0.89
0.11
0.522
0.99
0.92
0.534
1.04
0.55
148
0.140
1.04
0.68
0.124
0.90
0.35
0.719
1.12
0.10
0.710
1.08
0.34
0.336
1.06
0.43
0.305
0.92
0.27
0.054
0.84
0.21
0.050
0.78
0.11
0.684
1.02
0.74
0.679
1.00
1.00
0.198
0.87
0.12
0.196
0.86
0.13
0.848
0.89
0.22
0.857
0.97
0.74
0.188
1.05
0.54
0.195
1.10
0.30
0.442
0.98
0.77
0.462
1.06
0.39
0.267
1.09
0.23
0.239
0.94
0.48
^5
n
<z>
In)
o o ©
o o o
In)
o
Table 16. Association results for SNPs with reported association to T2D in Sladek et al.
Shown are association results for T2D in the Icelandic study group for the eight SNPs identified by Sladek et al (Nature 445, 881-5 (2007)) to associate with T2D. For the Icelandic group the table includes the frequency in cases and controls, odds ratio (OR) and adjusted P value for five of the eight SNP's. Corresponding values are shown for the replication cohort used in Sladek et al. Three of the markers, rslll3132, rsll037909 and rs3740878, are not on the Illumina 300K chip; however, a surrogate SNP rs729287 which has a correlation r2 = 1 to rsll037909 and rs3740878 (based on HapMap CEU data) has been typed in the Icelandic study group and results for this marker are included in the table.
Icelandic study group Sladek et al
Chr
Position
Marker
Allele
Controls
Cases
OR
P
Controls
Cases
ORa
P"
Nearest gene
C08
118141371
rsl 3266634
C
0.646
0.685
1.19
0.00060
0.699
0.746
1.26
.0*10"7
' SLC30A8
C10
94127459
rs1111875
G
0.550
0.588
1.17
0.0014
0.598
0.642
1.21
9.1x10"®
HHEX
C10
94146494
rs7923837
G
0.583
0.624
1.19
0.00058
0.623
0.665
1.20
2.2X10"5
HHEX
C10
114422936
rs7903146
T
0.300
0.372
1.38
1.9x10"10
0.293
0.406
1.65
<1.0*10~7
TCF7L2
C11
42211027
rs7480010
G
0.273
0.271
0.95
0.33
0.301
0.336
1.18
2.9*10^
LOC387761
C11
44207712
rs1113132
C
-
-
-
-
0.733
0.763
1.17
8.1x10^
EXT2
C11
44219923
rsl 1037909
T
-
-
-
-
0.729
0.760
1.18
4.5X10"4
EXT2
C11
44222111
rs3740878
A
-
- '
-
-
0.728
0.760
1.18
2.8x10^
EXT2
C11
44244399
rs729287
C
0.748
0.759
1.06
0.33
-
-
-
-
EXT2
a Allelic OR calculated from frequency information provided in Table 1 of Sladek et al. b P value (based on permutation) for Stage 2 in Table 1 in Sladek et al.
150
Table 17. Association results for the SNPs rs7756992 and rsl3266634 in six Caucasian T2D case-control groups and in case-control groups from Hong Kong and from West-Africa.
Study population (n//n) Frequency
Variant (allele)
Controls
Cases
OR (95% CI)
P value
Iceland (1399/5275)
rs7756992 (G)
0.232
0.270
1.23(1.10-1.37)
0.00021
rsl 3266634 (C)
0.646
0.685
1.19(1.08-1.31)
0.0006
Denmark A (263/597)
rs7756992 (G)
0.297
0.331
1.17(0.93-1.47)
0.18
rsl 3266634 (C)
0.686
0.672
0.94(0.75-1.17)
0.58
Denmark B (1359/4825)
rs7756992 (G)
0.279
0.320
1.21 (1.10-1.33)
0.000054
rsl 3266634 (C)
0.673
0.692
1.09 (0.99-1.19)
0.073
Philadelphia (447/950)
rs7756992 (G)
0.262
0.295
1.18(0.98-1.42)
0.073
rsl 3266634 (C)
0.678
0.760
1.51 (1.25-1.81)
1,5*10"5
Scotland (3742/3718)
rs7756992 (G)
0.267
0.288
1.11 (1.03-1.19)
0.0042
rsl 3266634 (C)
0.682
0.710
1.14(1.06-1.22)
0.00025
The Netherlands (368/915)
rs7756992 (G)
0.270
0.280
1.05 (0.86-1.27)
0.64
rsl 3266634 (C)
0.717
0.736
1.10(0.91-1.33)
0.33
Caucasian combined3 (7578/16280)
rs7756992 (G)
0.264
0.293
1.16(1.09-1.22)
3.9x10"10
rsl3266634 (C)
0.675
0.700
1.15(1.10-1.20)
3.3x10"9
Hong Kong(1457/986)
rs7756992 (G)
0.462
0.517
1.25(1.11-1.40)
0.00018
rsl3266634 (C)
0.523
0.566
1.19(1.06-1.33)
0.0035
West Africa3 (865/1106)
rs7756992 (G)
0.612
0.625
1.02 (0.92-1.14)
0.72
rsl 3266634 (C)
0.962
0.971
1.26 (0.88-1.81)
0.21
All groups combined (9900/18372)
rs7756992 (G)
1.15(1.11-1.20)
9..0*10"12
rsl 3266634 (C)
1.16(1.11-1.21)
2.5x10"11
Shown are the number of T2D cases and controls (nlm), the allelic frequency in the affected and control individuals, the allelic odds-ratio (OR) with 95 confidence intervals (CI 95%) and two-sided P values based on the multiplicative model. ''When combining results for the Caucasian groups and for the five West-African groups, OR's and P values are combined using a Mantel-Haenzsel model, while the frequency in cases and controls is estimated as a weighted average over the different study groups.
Table 18. Association of eight SNP's in CDKALl to T2D in Iceland, Hong Kong and West-Africa.
Association to T2D for eight SNP's in the CDKALl gene for three of the eight study groups; from Iceland, Hong Kong and West-Africa. The seven additional SNP's are all highly correlated to rs7756992.
Combined3 Iceland Hong Kong West-Africa0
SNP
Allele
Positionb
OR (95% CI)
P
Con.frq
Case.frq
OR
P
Con.frq
Case.frq
OR
P
Con.frq
Case.frq
OR
P
rs7752906
A
20774034
1.19(1.11-1.28)
6.5*10'7
0.296
0.338
1.22
0.00076
0.362
0.422
1.29
3.2x10"5
0.654
0.674
1.06
0.43
rsl 569699
C
20787289
1.19 (1.12-1.27)
1.4x10"7
0.257
0.297
1.22
0.00018
0.463
0.519
1.25
0.00019
0.627
0.656
1.10
0.17
rs7756992
G
20787688
1.17 (1.09-1.25)
3.1x10"®
0.232
0.270
1.23
0.00023
0.462
0.517
1.25
0.00018
0.612
0.625
1.02
0.72
rs9350271.
A
20791143
1.18(1.11-1.26)
9.6x10"7
0.257
0.298
1.23
0.00016
0.356
0.406
1.23
0.00055
0.695
0.712
1.07
0.38
rs9356744
C
20793465
1.18 (1.11-1.26)
7.9x10"7
0.256
0.297
1.23
0.00014
0.357
0.407
1.24
0.00045
0.696
0.713
1.06
0.39
rs9368222
A
20794975
1.20(1.12-1.28)
4.8x10"7
0.231
0.269
1.22
0.00029
0.355
0.405
1.24
0.00041
0.184
0.203
1.10
0.27
rsl0440833
A
20796100
1.18(1.11-1.27)
1.4X10"6
0.233
0.269
1.22
0.00046
0.354
0.407
1.25
0.00024
0.213
0.226
1.06
0.48
rs6931514
G
20811931
1.19 (1.11-1.27)
7.8x10 7
0.231
0.267
1.22
0.00047
0.464
0.520
1.25
0.00015
0.231
0.249
1.07
0.41
aResults for the three groups were combined using a Mantel-Haenszel model. "Basepair position in NCBI Build 34. cResults for the five West-African groups were combined using Mantel-Haenszel model and the allele frequencies shown are a weighted average of the frequency for the five groups.
152
Table 19. Pair-wise correlation for SNP's typed in CDKALl.
Pair-wise correlation, D' (lower left corner) and r2 (upper right corner), for the eight SNP's in CDKALl that were tested for association to T2D. The correlation is estimated for control individuals from the Icelandic, Hong Kong and West-African study groups, respectively.
rs7752906
rsi 569699
CM O)
h*
J
CM CM
CO o CO
rs6931514
D'\
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■n t-. £
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CO
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CO
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o
o e
Iceland
!
rs7752906
-
0.55
0.66
0.56
0.56
0.67
0.66
0.65
rsl 569699
0.83
-
0.87
0.99
0.98
0.85
0.83
0.83
rs7756992
0.98
1.00
-
0.86
0.86
0.99
0.97
0.96
rs9350271
0.84
1.00
1.00
-
1.00
0.86
0.85
0.84
rs9356744
0.84
1.00
1.00
1.00
-
0.87
0.86
0.85
rs9368222
0.99
1.00
1.00
1.00
1.00
-
0.98
0.97
rsl 0440833
0.96
0.97
1.00
0.98
0.99
1.00
-
0.99
rs6931514
0.96
0.97
0.99
0.98
0.99
0.99
1.00
-
Hong Kong
rs7752906
-
0.45
0.46
0.77
0.76
0.77
0.77
0.46
rsl 569699
0.84
-
0.99
0.63
0.63
0.62
0.62
0.98
rs7756992
0.84
1.00
-
0.63
0.62
0.64
0.64
0.99
rs9350271
0.89
1.00
0.99
-
1.00
0.99
0.99
0.62
rs9356744
0.88
0.99
0.99
1.00
-
0.99
0.99
0.62
rs9368222
0.89
0.99
1.00
1.00
1.00
-
1.00
0.63
rsl 0440833
0.89
1.00
1.00
1.00
1.00
1.00
-
0.63
rs6931514
0.84
0.99
1.00
0.99
0.99
1.00
1.00
-
West-Africa
rs7752906
-
0.16
0.32
0.13
0.14
0.12
0.07
0.08
rsl 569699
0.42
-
0.61
0.72
0.72
0.12
0.07
0.09
rs7756992
0.62
0.84
-
0.67
0.67
0.14
0.08
0.10
rs9350271
0.40
0.96
0.99
-
0.99
0.10
0.04
0.05
rs9356744
0.41
0.96
1.00
1.00
-
0.10
0.04
0.06
rs9368222
1.00
0.96
0.95
1.00
1.00
-
0.86
0.76
rsl 0440833
0.68
0.68
0.68
0.59
0.60 .
1.00
-
0.87
rs6931514
0.73
0.72
0.73
0.63
0.65
0.99
1.00
-
153
Table 20. Genotype specific odds ratio for rs7756992 and rsl3266634.
Study population Allelic Genotype odds ratio3
Variant (allele) OR (95% CI) 00 OX (95% CI) XX (95% CI) P"
Caucasian
rs7756992 (G)
1.16(1.09-1.22) 1
1.09(1.03-1.16)
1.45(1.31-1.61)
0.00052
rsl 3266634 (C)
1.15(1.11-1.20) 1
1.12(1.03-1.23)
1.30(1.18-1.43)
0.63
Hong Kong
rs7756992 (G)
1.25(1.11-1.40) 1
1.13(0.97-1.31)
1.55(1.23-1.95)
0.071
rsl 3266634 (C)
1.19(1.06-1.33) 1
1.13(0.96-1.34)
1.40(1.11-1.76)
0.43
aGenotype odds ratio for heterozygous (OX) and homozygous carrier (XX) compared with non-carriers (00). Test of the multiplicative model (the null hypotheses) versus the full model, one degree of freedom.
Table 21. Association to insulin secretion and insulin sensitivity.
Association of the risk variants rs7756992 (G) and rsl3266634 (C) to insulin secretion, estimated by corrected insulin response (CIR), and insulin sensitivity estimated the reciprocal of HOMA (homeostasis model assessment). The table includes number of T2D cases (n) and controls (m) used, the estimated effect and standard error and the P value obtained by regressing the log-transformed trait values on age, sex and either the number of risk alleles an individual carries (additive model) or an indicator variable for homozygous carriers of the risk allele (recessive model). When controls and T2D cases are analysed together an indicator variable for the affection status is included in the analysis. Also shown, for the combined group, is the corresponding P value obtained by adjusting for BMI status of the individuals in the analysis.
Analysis
Combined group
Controls
T2D
Trait
Group (n/m)
Effect (se)
P
P*
Effect (se)
P
Effect (se)
P
rs7756992 (add)
All (3715/223)
-0.083 (0.018)
4.0E-06
9.1 E-06
-0.080 (0.018)
1.3E-05
-0.142 (0.095)
0.14
t? O a>
(0 c
Males (1742/139)
-0.056 (0.025)
0.025
0.042
-0.058 (0.025)
0.021
-0.028 (0.119)
0.82
Females (1973/84)
-0.100 (0.025)
6.8E-05
0.00012
-0.088 (0.025)
0.00049
-0.342 (0.144)
0.02
rs7756992 (rec)
o
Q.
All (3715/223)
-0.243(0.041)
3.3E-09
4.9E-09
-0.230 (0.042)
3.7E-08
-0.417(0.199)
0.037
(A ©
Males (1742/139)
-0.225 (0.055)
4.9E-05
0.00014
-0.222 (0.056)
7.5E-05
-0.250 (0.250)
0.32
0£
{2
Females (1973/84)
-0.232 (0.059)
7.5E-05
7.6E-05
-0.204 (0.060)
0.00063
-0.696 (0.301)
0,023
"3
rsl 3266634 (add)
to c
All (3698/228)
-0.061 (0.017)
0.0005
0.00056
-0.059 (0.018)
0.00075
-0.083 (0.094)
0.38
Males (1736/143)
-0.079 (0.024)
0.0011
0.00091
-0.062 (0.024)
0.011
-0.262 (0.109)
0.017
Females (1962/85)
-0.048 (0.024)
0.047
0.052
-0.058 (0.024)
0.016
0.233 (0.166)
0.16
< 2
rs7756992 (add)
All (4430/1164)
-0.013(0.013)
0.33
0.7
0.002 (0.013)
0.85
-0.065 (0.038)
0.082
O x
Males (2062/691)
-0.002 (0.019)
0.94
0.51
0.022 (0.020)
0.26
-0.070 (0.049)
0.15
Females (2368/473)
-0.026 (0.018)
0.14
0.22
-0.018(0.018)
0.31
-0.061 (0.059)
0.3
rsl 3266634 (add)
All (4411/1166) -0.015(0.013) 0.24 0.19 -0.013(0.013) 0.31 -0.024(0.039) 0.55
Males (2058/697) -0.003(0.019) 0.88 0.81 -0.010(0.019) 0.61 0.019(0.050) 0.7
Females (2353/469) -0.028(0.017) 0.11 0.087 -0.016(0.017) 0.34 -0.092(0.063) 0.14 ' P value after adjusting for BMI by including a log(BMI) term among the explanatory variables.
156
Table 22. Surrogate markers for marker rs7756992 on chromosome 6. The table shows markers with values for r2 of greater than 0.2 in the HapMap Caucasian CEPH samples. The search was performed over a 2Mb region flanking rs77566992 (1Mb upstream and 5 1Mb downstream).
Surrogates for rs7756992 on chromosome 6
SNP
D*
R2
Pos B36
Pos SEQ ID NO:l rs9460517
0.82
0.30
20636813
1818
rs7772956
0.72
0.29
20637521
2526
rs6904566
0.73
0.32
20643949
8954
rs6927356
0.73
0.32
20644073
9078
rs6905138
0.73
0.32
20644335
9340
rsl3194858
0.73
0.32
20644499
9504
rs6456356
1.00
0.22
20649498
14503
rs9366354
0.84
0.40
20653447
18452
rs9368201
0.84
0.41
20654091
19096
rs9348433
0.84
0.40
20657780
22785
rsl3203450
0.73
0.32
20673935
38940
rsl012626
0.82
0.39
20685540
50545
rs9460523
0.55
0.23
20690122
55127
rs9350262
0.55
0.23
20692402
57407
rs4712507
0.56
0.24
20693119
58124
rs9366357
0.56
0.23
20707607
72612
rsl997777
1.00
0.22
20710359
75364
rsl1964057
0.56
0.23
20710776
75781
rsl2206413
1.00
0.22
20715663
80668
rs4515379
0.66
0.20
20735420
100425
rs9465841
0.66
0.20
20737687
102692
rsl3190734
0.62
0.31
20738376
103381
rs2328528
0.67
0.21
20739524
104529
rs2328529
0.67
0.21
20739932
104937
rs7768642
0.67
0.21
20741886
106891
rs9465846
0.67
0.21
20742320
107325
rs9465847
0.67
0.21
20742407
107412
rs7755830
1.00
0.32
20742865
107870
rs6940200
0.67
0.21
20743241
108246
rs9465850
0.67
0.22
20747388
112393
rs4710938
1.00
0.34
20748883
113888
rs9348440
0.79
0.23
20749315
114320
rs4235999
1.00
0.33
20751201
116206
rs4710939
1.00
0.35
20752923
117928
rsl 1965062
1.00
0.33
20755941
120946
rs9460540
1.00
0.33
20756741
121746
rs6456364
0.79
0.23
20757233
122238
rs9295474
0.95
0.68
20760696
125701
rs2328545
0.79.
0.23
20761529
126534
rs9368216
0.79
0.23
20763089
128094
rsl6884072
0.66
0.33
20763482
128487
rs9460541
0.66
0.33
20764559
129564
rs9460542
0.66
0.33
20764746
129751
rs4712522
0.95
0.68
20764779
129784
rsl6884074
0.66
0.32
20764924
129929
157
rs4712523
0.95
0.68
20765543
130548
rs4710940
0.95
0.52
20765991
130996
rsl3190727
0.66
0.33
20766197
131202
rs6906327
0.95
0.52
20767438
132443
rs6456367
0.95
0.68
20767566
132571
rs6456368
0.95
0.67
20767785
132790
rs7749083
0.66
0.33
20768202
133207
rs6456369
0.95
0.52
20768344
133349
rsl3203361
0.66
0.33
20769000
134005
rsl0946398
0.95
0.68
20769013
134018
rs7774594
0.95
0.67
20769122
134127
rs7754840
0.95
0.68
20769229
134234
rs9460544
0.95
0.68
20769508
134513
rs9460545
0.95
0.68
20769529
134534
rs979614
1.00
0.34
20770102
135107
rs4712525
0.95
0.68
20770945
135950
rs4712526
0.95
0.68
20771014
136019
rs9460546
0.95
0.68
20771611
136616
rs736425
0.66
0.33
20772291
137296
rs742642
0.79
0.23
20773060
138065
rs7748382
0.95
0.68
20773528
138533
rs7772603
0.95
0.68
20773925
138930
rs7752780
0.95
0.68
20774001
139006
rs7752906
0.95
0.70
20774034
139039
rsl 1970425
0.66
0.33
20774436
139441
rs9358356
0.95
0.67
20775361
140366
rs9356743
0.79
0.23
20775667
140672
rs9368219
1.00
0.53
20782670
147675
rsl012635
1.00
0.42
20783274
148279
rsl 569699
1.00
0.72
20787289
152294
rs9350271
1.00
0.78
20791143
156148
rs9356744
1.00
0.75
20793465
158470
rs7766070
1.00
1.00
20794552
159557
rs9368222
1.00
1.00
20794975
159980
rsl0440833
1.00
1.00
20796100
161105
rs2206734
1.00
0.53
20802863
167868
rs6931514
1.00
1.00
20811931
176936
rsll753081
1.00
0.53
20813569
178574
rsl040558
1.00
0.53
20821685
186690
rs9295478
0.62
0.30
20824232
189237
rs2328548
1.00
0.53
20824937
189942
rs6935599
1.00
0.53
20825074
190079
rs9465871
1.00
0.53
20825234
190239
rsl0946403
1.00
0.53
20825383
190388
rs2328549
1.00
0.30
20826219
191224
rs9358357
1.00
0.53
20827124
192129
rs9368224
1.00
0.53
20827211
192216
rs9358358
1.00
0.30
20827372
192377
rs9460550
1.00
0.53
20827540
192545
rs9356746
1.00
0.30
20828258
193263
rs9368226
1.00
0.50
20831036
196041
rsl2111351
0.61
0.29
20832537
197542
rs9356747
0.60
0.29
20832986
197991
rs9356748
1.00
0.30
20833076
198081
rs7767391
1.00
0.50
20833219
198224
rs7747752
0.62
0.30
20833402
198407
rsl7234378
0.80
0.24
20952720
158
Table 23. Surrogate markers for marker rsl0882091 on chromosome 10. The table shows markers with values for r2 of greater than 0.2 in the HapMap Caucasian CEPH samples. The search was performed over a 2Mb region flanking rsl0882091 (1Mb upstream and 1Mb downstream).
Surrogates for rsl0882091 on chromosome 10
SNP
D'
R2
Pos B36
Pos SEQ ID NO:2
rs7086285
0.71
0.23
94166068
rs2798253
0.93
0.32
94192885
1
rs6583813
1.00
0.33
94199919
7035
rsl1187007
1.00
0.35
94204560
11676
rs2149632
1.00
0.35
94222227
29343
rslll87025
0.95
0.48
94247956
55072
rslll87033
1.00
0.35
94252339
59455
rsl0509645
1.00
0.35
94267846
74962
rs7078413
0.49
0.23
94280464
87580
rs4646955
0.75
0.37
94284271
91387
rsl7445328
0.68
0.32
94295169
102285
rsl1187064
0.68
0.31
94298233
105349
rs2421943
1.00
0.45
94301795
108911
rsl1187065
0.95
0.48
94301904
109020
rsl1187078
1.00
0.35
94330685
137801
rs6583823
1.00
0.52
94334395
141511
rs2421941
0.96
0.93
94335889
143005
rs6583826
0.95
0.57
94337810
144926
rs3824735
1.00
0.36
94344184
151300
rsl0786050
1.00
1.00
94357210
164326
rsl1187094
1.00
0.21
94358158
165274
rsl1187096
1.00
0.35
94359568
166684
rs7914814
1.00
1.00
94372930
180046
rsl2772554
1.00
0.23
94373838
180954
rsl0882094
1.00
1.00
94377656
184772
rsl0882095
1.00
0.37
94384382
191498
rsl0736069
1.00
1.00
94385373
192489
rs7900689
1.00
1.00
94385728
192844
rs6583830
1.00
1.00
94388098
195214
rsl0882096
1.00
0.35
94391366
198482
rsl 1187114
1.00
0.36
94396217
203333
rs6583833
1.00
0.76
94399780
206896
rs7078243
1.00
0.78
94404243
211359
rs4933734
1.00
1.00
94404547
211663
rs7911264
1.00
0.73
94426831
233947
rs2488087
1.00
0.74
94436021
243137
rsl0882100
1.00
0.74
94450667
257783
rsl 111875
1.00
0.51
94452862
259978
rsl2778642
1.00
0.55
94454287
261403
rs5015480
1.00
0.51
94455539
262655
rsl0882102
1.00
0.52
94456475
263591
rsl 1187144
1.00
0.40
94459960
267076
rs7087591
1.00
0.39
94463609
270725
rsl0748582
1.00
0.39
94467199
274315
rs7923837
1.00
0.39
94471897
279013
rs7923866
1.00
0.39
94472056
279172
rs2497306
1.00
0.58
94475191
282307
rs2488075
1.00
0.60
94480154
287270
rs2497304
0.96
0.63
94482696
289812
159
rs947591
0.81
0.57
94485733
292849
rs2488071
0.62
0.24
94489557
296673
Table 24. Surrogate markers for marker rs2191113 on chromosome 17. The table shows markers with values for r2 of greater than 0.2 in the HapMap Caucasian CEPH 5 samples. The search was performed over a 2Mb region flanking rs2191113 (1Mb upstream and 1Mb downstream).
Surrogates for rs2191113 on chromosome 17
SNP
D'
R2
Pos B36
POS SEQ ID NO:3
rs350605
0.82
0.54
66044207
6552
rs350603
0.80
0.22
66045245
7590
rs420762
0.80
0.24
66049716
12061
rs350615
0.86
0.58
66067303
29648
rs350616
0.81
0.25
66067699
30044
rs350621
0.86
0.58
66079419
41764
rs350624
0.86
0.58
66080067
42412
rsl2602288
1.00
0.36
66085473
47818
rsl431454
0.82
0.26
66090535
52880
rs9302918
1.00
0.23
66091912
54257
rs9302919
0.81
0.26
66092080
54425
rs9911671
0.86
0.61
66094196
56541
rsl911969
0.86
0.60
66102315
64660
rs9894021
1.00
0.21
66103236
65581
rs720877
1.00
0.23
66103561
65906
rs720876
1.00
0.23
66103923
66268
rs7218838
0.86
0.61
66106415
68760
rs9896809
1.00
0.21
66106911
69256
rs7220084
0.82
0.26
66114858
77203
rsl860316
0.86
0.61
66117911
80256
rs8079029
0.90
0.62
66118485
80830
rs4019476
0.87
0.63
66122077
84422
rsl981647
0.82
0.26
66132788
95133
rs9890554
0.80
0.21
66134831
97176
rsl0221225
0.80
0.22
66138452
100797
rsl1650683
0.84
0.22
66139800
102145
rsl486290
0.82
0.27
66141933
104278
rs8078302
0.85
0.23
66143200
105545
rsl2949591
1.00
0.20
66146912
109257
rsl843622
1.00
0.61
66149102
111447
rs9891997
1.00
0.28
66152998
115343
rs9910837
1.00
0.28
66155303
117648
rs4793497
0.94
0.58
66163076
125421
rs9890889
0.89
0.24
66173475
rs2009802
0.71
0.23
66178475
rsl7718938
1.00
0.28
66184700
rsl7223216
0.89
0.24
66207685
rs2109050
0.89
0.24
66228633
rsl962801
1.00
0.31
66236090
160
Claims (27)
1. A method of determining a susceptibility to Type 2 diabetes in a human individual, comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, or in a genotype dataset derived from the individual, wherein the at least one polymorphic marker is selected from rs7756992, and markers in linkage disequilibrium therewith, and wherein determination of the presence or absence of the at least one allele is indicative of a susceptibility to Type 2 diabetes.
2. The method of Claim 1, wherein the at least one polymorphic marker is present within SEQ ID NO: 1.
3. The method of any of the preceding Claims, wherein said markers in linkage disequilibrium are defined by numeric values for |D'| of greater than 0.8 and/or r2 of greater than 0.2.
4. The method of any of claim 1 or 2, wherein the presence of at least one at-risk allele of at least one polymorphic marker is indicative of an increased susceptibility to Type 2 diabetes, and wherein the absence of the at least one at-risk allele is indicative of a decreased susceptibility of Type 2 diabetes.
5. The method of claim 4, wherein the increased susceptibility is characterized by a relative risk (RR) or odds ratio (OR) of at least 1.15.
6. The method of Claim 4 or 5, wherein the increased susceptibility is characterized by a relative risk (RR) or odds ratio (OR) of at least 1.20.
7. The method of Claim 4, wherein the at least one at-risk allele is rs7756992 allele G.
8. The method of any of the claims 1-3, wherein the presence of at least one protective allele in a nucleic acid sample from the individual is indicative of a decreased susceptibility of Type 2 diabetes.
9. The method of any of the claims 1-3, wherein the absence of at least one at-risk allele in a nucleic acid sample from the individual is indicative of a decreased susceptibility of Type 2 diabetes. 161
10. The method of any of Claims 1 to 9, further, comprising screening the nucleic acid or the genotype dataset for the presence or absence of at least one at-risk allele of at least one at-risk marker for Type 2 diabetes in the TCF7L2 gene, wherein determination of the presence of the at least one at-risk allele is indicative of increased susceptibility of Type 2 diabetes.
11. The method of Claim 10, wherein the at least one at-risk marker in the TCF7L2 gene is selected from marker DG10S478, rsl2255372, rs7895340, rslll96205, rs7901695, rs7903146, rsl2243326 and rs4506565, and markers in linkage disequilibrium therewith.
12. A method of identification of a marker for use in assessing susceptibility to Type 2 diabetes in human individuals, the method comprising a. identifying at least one polymorphic marker within SEQ ID NO: 1, or at least one polymorphic marker in linkage disequilibrium therewith; b. determining the genotype status of a sample of individuals diagnosed with, or having a susceptibility to, Type 2 diabetes; and c. determining the genotype status of a sample of control individuals; wherein a significant difference in frequency of at least one allele in at least one marker in individuals diagnosed with, or having a susceptibility to, Type 2 diabetes, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one marker being useful for assessing susceptibility to Type 2 diabetes.
13. The method of Claim 12, wherein linkage disequilibrium is characterized by numerical values of r2 of greater than 0.2 and/or |D'| of greater than 0.8.
14. The method of Claim 13, wherein an increase in frequency of the at least one allele in the at least one marker in individuals diagnosed with, or having a susceptibility to, Type 2 diabetes, as compared with the frequency of the at least one allele in the control sample, is indicative of the at least one marker being useful for assessing increased susceptibility to Type 2 diabetes, and wherein a decrease in frequency of the at least one allele in the at least one marker in individuals diagnosed with, or having a susceptibility to, Type 2 diabetes, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one marker being useful for assessing decreased susceptibility to, or protection against, Type 2 diabetes. 162
15. A method of assessing an individual for probability of response to a therapeutic agent for preventing and/or ameliorating symptoms associated with Type 2 diabetes, comprising: determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from rs7756992 (SEQ ID NO:21), and markers in linkage disequilibrium therewith, wherein determination of the presence of the at least one allele of the at least one marker is indicative of a probability of a positive response to the Type 2 diabetes therapeutic agent.
16. The method of Claim 15, wherein the Type 2 diabetes therapeutic agent is selected from the agents set forth in Agent Table 1 and Agent Table 2.
17. A method of predicting prognosis of an individual diagnosed with, Type 2 diabetes, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from the group consisting of rs7756992 (SEQ ID NO:21), and markers in linkage disequilibrium therewith, wherein determination of the presence of the at least one allele is indicative of a worse prognosis of the Type 2 diabetes in the individual.
18. A method of monitoring progress of a treatment of an individual undergoing treatment for Type 2 diabetes, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from the group consisting of rs7756992 (SEQ ID NO: 21), and markers in linkage disequilibrium therewith, wherein determination of the presence of the at least one allele is indicative of the treatment outcome of the individual.
19. The method of any of the Claims-17-18, wherein linkage disequilibrium is defined by numerical values of r2 of at least 0.2 and/or values of |D'| of at least 0.8
20. The method of any of the preceding Claims, further comprising assessing at least one biomarker in a sample from the individual.
21. The method of any of the preceding Claims, further comprising analyzing non-genetic information to make risk assessment, diagnosis, or prognosis of the individual.
22. The method of Claim 21, wherein the non-genetic information is selected from age, gender, ethnicity, socioeconomic status, previous disease diagnosis, medical history of received 17 may 2012. 163 subject, family history of Type 2 diabetes, biochemical measurements, and clinical measurements.
23. The method of any of the Claims -20-22, further comprising calculating overall risk.
24. Use of an oligonucleotide probe in the manufacture of a diagnostic reagent for diagnosing and/or assessing susceptibility to Type 2 diabetes in a human individual, wherein the probe hybridizes to a segment of a nucleic acid whose nucleotide sequence is given by SEQ ID NO: 1 that comprises at least one polymorphic site, wherein the fragment is 15-500 nucleotides in length.
25. The use according to Claim 24, wherein the polymorphic site is selected from the polymorphic markers rs7756992 (SEQ ID NO:21), and polymorphisms in linkage disequilibrium therewith.
26. A computer program when used to determine a genetic indicator for Type 2 diabetes in a human individual: wherein the computer program is adapted to be executed on a processor to analyze marker and/or haplotype information for at least one human individual with respect to at least one polymorphic marker selected from the markers rs7756992 (SEQ ID NO:21), and markers in linkage disequilibrium therewith, as defined by numerical values of r2 of at least 0.2 and/or values of |D'| of at least 0.8, and generate an output based on the marker or haplotype information, wherein the output comprises a risk measure of the at least one marker or haplotype as a genetic indicator of Type 2 diabetes for the human individual.
27. The method of any of the Claims 1-6 and 10-23, wherein the at least one polymorphic marker is selected from the markers set forth in Table 22. 28 A method according to any one of claims 1, 12 or 15-18, substantially as herein described with reference to any one of the accompanying Examples and Figures thereof. 29 Use according to claim 24, substantially as herein described with reference to any one of the accompanying Examples and Figures thereof. 30 A computer program according to claim 26, substantially as herein described with reference to any one of the accompanying Examples and Figures thereof.
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IS68572 | 2006-11-30 | ||
IS78630 | 2007-04-04 | ||
PCT/IS2007/000020 WO2008065682A2 (en) | 2006-11-30 | 2007-11-30 | Genetic susceptibility variants of type 2 diabetes mellitus |
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