Variants in Hepatocyte Nuclear Factor 4 Are Modestly Associated With Type 2 Diabetes in Pima Indians
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糖尿病学杂志 2005年第10期
1 Phoenix Epidemiology and Clinical Research Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Phoenix, Arizona
2 Washington University School of Medicine, St. Louis, Missouri
ABSTRACT
Single nucleotide polymorphisms (SNPs) within the hepatocyte nuclear factor 4 (HNF4) gene are associated with type 2 diabetes in Finns and Ashkenazi Jews. Previous studies in both populations have reported linkage to type 2 diabetes near the HNF4 locus on chromosome 20q12-13. To investigate whether HNF4 is a diabetes susceptibility gene in Pima Indians, a population with the highest reported prevalence of type 2 diabetes but with no evidence for linkage of the disease on chromosome 20q, 19 SNPs across the promoter and coding region of HNF4 were genotyped for association analysis. In a group of 1,037 Pima Indians (573 diabetic and 464 nondiabetic subjects), three SNPs in HNF4 (rs3212183 and rs2071197 located in introns 3 and 1, respectively, and rs6031558, an extremely rare SNP located in the P2 promoter region) were modestly associated with type 2 diabetes (rs3212183 odds ratio [OR] 1.34 [95% CI 1.07eC1.67], P = 0.009; rs2071197 1.34 [1.07eC1.66], P = 0.008; and rs6031558 3.18 [1.03eC9.84], P = 0.04, adjusted for age, sex, birth year, heritage, and family membership). We conclude that variants in HNF4 do not appear to be major determinants for type 2 diabetes in Pima Indians; however, HNF4 may have a minor role in type 2 diabetes susceptibility within this Native American population.
Hepatocyte nuclear factor 4 (HNF4) is a transcription factor expressed in many tissues including liver and pancreas (1). Its expression is controlled by two alternative promoters, P1 and P2, which produce at least nine transcripts. Transcripts from the P2 promoter are the predominant isoforms in pancreatic -cells (2). HNF4 has been suggested to have a role in metabolic pathways involved in glucose metabolism and insulin secretion (3,4). Mutations in both the coding and regulatory regions of HNF4 have been associated with maturity-onset diabetes of young (MODY type I), a dominantly inherited, early-onset form of type 2 diabetes (5).
Several genome-wide scans for type 2 diabetes susceptibility loci have identified linkage on chromosome 20q12-13 in a region that encompasses the HNF4 locus (6eC10). Recent fine-mapping follow-up studies independently identified multiple variants within the HNF4 gene that are associated with type 2 diabetes in the Finnish-U.S. Investigation of Type 2 Diabetes (FUSION) study population and in the Ashkenazi Jewish population (11,12). In both studies, the strongest association was observed with four SNPs (rs4810424, rs1884613, rs1882614, and rs2144908) from the P2 promoter region (11,12). However, several SNPs in the P1 promoter region are also modestly associated with type 2 diabetes in the FUSION study population but not in the Ashkenazi Jewish cohort. To investigate whether variants in HNF4 are associated with type 2 diabetes in Pima Indians who have a high prevalence of type 2 diabetes but show no evidence for linkage to type 2 diabetes on chromosome 20q13, a total of 19 SNPs across a 79-kb region encompassing both the P2 and P1 promoters and the coding region of HNF4 were genotyped in this population.
RESEARCH DESIGN AND METHODS
A total of 96 noneCfirst-degreeeCrelated Pima Indians who were diagnosed with type 2 diabetes at 25 years of age (mean age of onset 16.0 ± 0.5 years and mean BMI 35.0 ± 0.5 kg/m2) were selected for sequencing of the HNF4 coding region. Variants were genotyped for a family-based association study in 1,037 Pima Indians (from 332 nuclear families) who are participants of our ongoing longitudinal study of the etiology of type 2 diabetes among the Gila River Indian Community in Arizona (13,14). Among these 1,037 subjects, 573 had type 2 diabetes (64.4% women, mean BMI 34.4 ± 8.5 kg/m2, mean study age 44.7 ± 13.0 years, and mean age at diagnosis 33.8 ± 10.8 years) and 464 were nondiabetic (43.5% women, mean BMI 33.5 ± 8.4 kg/m2, and mean study age 34.0 ± 11.0 years). Diabetes status was determined according to the criteria of the World Health Organization (15).
Two variants were additionally genotyped in an independent case/control group of 170 full-heritage, noneCfirst-degreeeCrelated Pima Indians for analysis of early-onset diabetes. The case group consisted of the same 96 subjects used for the initial sequencing of the HNF4 gene, as described above. The control group consisted of 74 subjects with normal glucose tolerance (mean age 54.0 ± 0.4 years at last medical exam and mean BMI 36.0 ± 1 kg/m2).
Metabolic measurements of risk factors for type 2 diabetes in normal glucose-tolerant subjects.
Among the 1,037 subjects who were genotyped for the type 2 diabetes association analysis, 179 subjects were normal glucose-tolerant, full-heritage Pima Indian subjects who had undergone detailed metabolic testing as inpatients in our clinical research center. Glucose tolerance was determined by a 75-g oral glucose tolerance test with measurements of fasting 30-, 60-, 120-, and 180-min plasma glucose and insulin concentrations (15). To measure the acute insulin response, blood samples were collected before a 25-g glucose bolus infusion and at 3, 4, 5, 6, 8, and 10 min after infusion. The acute insulin response was calculated as the mean increment in plasma insulin concentrations from 3 to 5 min (16). Insulin sensitivity was assessed using the hyperinsulinemic-euglycemic clamp technique as described previously (16,17). Body composition was estimated by underwater weighing until January 1996 and by dual-energy X-ray absorptiometry (DPX-1; Lunar Radiation) thereafter (18).
Sequencing of HNF4 and genotyping of SNPs.
Sequencing was done using Big Dye terminator (Applied Biosystems) on an automated DNA capillary sequencer (model 3730; Applied Biosystems). Variants identified by either sequencing or databases were genotyped using the TaqMan Allelic Discrimination (AD) Assay (Applied BioSystems) on an ABI Prism 7700 (Applied BioSystems).
Statistical analysis.
Associations were assessed using the statistical analysis system of the SAS institute (Cary, NC). The relationship between trait and marker was generally assessed via an ordered categorical variable representing genotype (i.e., an additive model). For continuous variables, the general estimating equation procedure was used to adjust for covariates. These analyses account for the correlation among family members (i.e., siblings). In the family-based study, the association with diabetes was assessed by logistic regression with control for age, sex, heritage, and year of birth. These analyses were also performed with the general estimating equation to account for the correlation among siblings. In the case-control study of early-onset diabetes, the association was assessed by logistic regression with control for sex and heritage. Haplotype frequencies for pairs of SNPs were calculated in all 1,037 individuals with the Estimating Haplotypes (EH) program (19). D' was calculated as a measure of allelic association and 2 (r2) as a measure of concordance. Association between traits and individual haplotypes were examined with a modification of the zero-recombinant haplotyping procedure, as described previously (20).
RESULTS AND DISCUSSION
Four variants were identified by sequencing the coding region and intron-exon boundaries of HNF4 in Pima Indians (Fig. 1). Two of these variants were novel (Met86Arg and IVS9 + 378T-C) and two were present in databases (Thr130Ile = rs1800961 and C/T in the 3' untranslated region = rs6130615). In addition, 10 SNPs (rs1884614, rs2144908, rs2425637, rs2425640, rs6031552, rs6031558, rs3212183, rs1885088, rs1028583, and rs3818247) were selected for genotypic analysis in Pima Indians based on results from the FUSION and Ashkenazi Jewish studies of HNF4 (11,12). Two of these variants, rs1884614 and rs2144908, were associated with type 2 diabetes in both the FUSION and Ashkenazi Jewish studies, whereas the remaining eight variants were associated with type 2 diabetes in only one of the studies. These SNPs span a 79-kb region of HNF4 including the P2 and P1 promoters and the entire coding region of HNF4. To approximate an evenly spaced dense SNP map across the HNF4 locus, with an average inter-SNP distance of 3eC4 kb, seven additional database SNPs (hcv11183312, rs6017335, rs4364072, rs4812831, rs3212198, rs6103731, and rs1028583) were also selected, based on their relative positions, for genotyping in Pima Indians (Fig. 1).
Preliminary genotyping of these 21 SNPs in 90 Pima subjects showed that SNPs rs1884614 and rs2144908 were in complete genotypic concordance, as were SNPs rs6031552 and rs6031558. Therefore, rs2144908 and rs6031552 were excluded from further genotyping. The results of genotyping the remaining 19 HNF4 variants in 1,037 Pima subjects are given in Table 1. All SNPs were in Hardy-Weinberg equilibrium. Three SNPs, rs3212183, rs2071197, and rs6031558, were modestly associated with type 2 diabetes. SNP rs3212183, positioned in intron 3, had a minor allele (C) frequency of 0.43 in diabetic subjects versus 0.37 in nondiabetic subjects (OR 1.34 [95% CI 1.07eC1.67], P = 0.009). This variant was also modestly associated with type 2 diabetes in the FUSION study, where the C allele was also the risk allele. SNP rs2071197, which was not reported in either the FUSION or Ashkenazi Jewish study, was also associated with type 2 diabetes, with an allele (G) frequency of 0.51 in diabetic subjects versus 0.45 in nondiabetic subjects (1.34 [1.07eC1.66], P = 0.008). These two SNPs (rs3212183 and rs2071197) were in very high linkage disequalibrium (D' = 0.99) in Pima Indians and were also in high, but not complete, genotypic concordance (2 = 0.69). SNP rs6031558, positioned in the P2 promoter region, had a minor allele (C) frequency of 0.02 in diabetic subjects versus 0.01 in nondiabetic subjects. Although this SNP had a significant association with type 2 diabetes (3.18 [1.03eC9.84], P = 0.04), the rare frequency of this variant makes this analysis unreliable.
SNPs rs3212183 and rs2071197 were additionally genotyped in a separate case/control group of 170 full-heritage Pima Indians selected for early-onset (or "MODY-like") diabetes (age of onset 25 years). Neither SNP was associated with early-onset diabetes in this case/control study (P = 0.24, OR 0.75 [95% CI 0.47eC0.121], and P = 0.56, 0.87 [0.56eC1.37] for rs3212183 and rs2071197, respectively).
We further investigated whether rs3212183 and rs2071197, which were in extremely high linkage disequilibrium and associated with type 2 diabetes, were associated with metabolic risk factors that predict type 2 diabetes in 179 normal glucose-tolerant, full-heritage Pima Indians who had undergone detailed metabolic testing. The various tests assessed insulin action, insulin secretion, and rates of glucose turnover. Neither SNP was associated with various measures of insulin action (data only shown for rs3212183 in Table 2). Although HNF4 is known to have a primary role in insulin secretion, there was no association between these SNPs and insulin secretion, as measured by the acute insulin response following an intravenous bolus of glucose. It is possible that we lacked the power to detect a subtle association due to the relatively small sample number (n = 179). In support of this, subjects homozygous for the type 2 diabetes risk allele did have a trend toward lower insulin secretion compared with subjects heterozygous or homozygous for the nonrisk allele (Table 2).
We also analyzed whether any of the remaining 17 variants that were not associated with type 2 diabetes in Pima Indians were associated with these metabolic risk factors in the 179 genotyped subjects. All of these associations were also negative, with the exception of SNP rs1884614 from the P2 haplotype block. This variant, which is associated with type 2 diabetes in both the Finns and Ashkenazi Jews, was associated with insulin resistance, as assessed by a hyperinsulinemic-euglycemic clamp in Pima Indians. During both physiologic and maximally stimulating insulin concentrations, subjects with the C/C+C/T genotypes for rs1884614 had a lower glucose disposal (decreased insulin sensitivity) than subjects with the T/T genotypes (3.4 ± 0.1 vs. 3.7 ± 0.1 mg · kg EMBSeC1 · mineC1, P = 0.01 for physiologic insulin dose and 7.8 ± 0.2 vs. 8.4 ± 0.1 mg · kg EMBSeC1 · mineC1, P = 0.02 for maximally stimulating insulin dose, adjusted for age, sex, percent body fat, and nuclear family membership). However, since this variant was not associated with type 2 diabetes in Pima Indians and we cannot provide a known physiologic mechanism whereby a variant in HNF4a would affect glucose uptake in the muscle, this association may be spurious rather than confirmatory.
The pattern of linkage disequilibrium across the HNF4 locus was evaluated in Pima Indians by a pair-wise analysis of genotypes from the 19 variants in the 1,037 subjects (Fig. 2). Similar to that observed in the Finnish and Ashkenazi Jewish populations, SNPs in the P2 promoter region were in extremely high linkage disequilibrium. Linkage disequilibrium did not decay until 33 kb from the beginning of the P2 promoter, with the exception of a rare SNP rs6031558 that is positioned in the middle of this block and breaks the block due to low linkage disequilibrium (<0.2) with rs6017335 and rs4364072. SNPs in exons 4eC11 were also in high linkage disequilibrium. However, SNPs across the P1 promoter and exons 1eC3 tended to have lower linkage disequilibrium among themselves but still had D' values of >0.5, with some SNPs in the P2 region. When SNPs containing common variants (minor allele frequency >0.05) were analyzed, there were five haplotype "blocks" among these SNPs, as defined by contiguous SNPs with D' >0.9. Analyses of the individual haplotypes for SNPs within these blocks showed that the haplotype consisting of "G" at rs2071197 and "C" at rs3212183 had a higher prevalence of diabetes compared with the other combined haplotypes (OR 1.37 [95% CI 1.10eC1.72], P = 0.02), a finding that largely reflects the single marker associations. There were no statistically significant associations with any of the other haplotypes (data not shown).
The allele frequencies for some variants in HNF4 are consistent between different ethnic groups, while others are quite different. For example, the allele (C) for rs3212183 had a frequency of 0.43 in both diabetic Pima Indians and diabetic Finns and a frequency of 0.37 and 0.38 in nondiabetic Pima Indians and Finns, respectively. In contrast, for rs1884614 the risk allele (T) in the Finns and Ashkenazi Jews had a frequency of 0.20 and 0.27, respectively, but the T allele in Pima Indians had a frequency of 0.83. This suggests that different populations have different HNF4 haplotypes. A difference in allele frequencies of HNF4 variants between ethnic groups may also reflect the various population-attributed risks to type 2 diabetes due to these variants.
The strongest associations with type 2 diabetes in both the FUSION and the Ashkenazi Jews studies were observed with SNPs in the P2 promoter of HNF4. Recent studies (21,22) in the Amish and U.K. populations have also replicated associations of variants in HNF4 with type 2 diabetes. In the Amish study (21), the strongest association was with rs2425640 located in the P1 promoter. The U.K. study reported significant associations with two SNPs (rs4810424 and rs2144908) that tag the P2 promoter (22). In Pima Indians, the two common SNPs associated with type 2 diabetes were in introns 1 and 3. The fact that different SNPs in the HNF4 region are associated with diabetes in different populations suggests that none of these alleles themselves are causative functional variants but that they may be in linkage disequilibrium with a nearby functional allele. Alternatively, some of these alleles may be causative, but allelic heterogeneity across populations may make their identification difficult. Given the previous evidence implicating HNF4 variants in diabetes, we considered that it is desirable to preserve statistical power. Therefore no corrections of multiple comparisons were performed. Nonetheless, one cannot exclude chance as a potential explanation for the associations observed in the present study.
Although the sample size of the present study is fairly large in comparison with many other studies, it may nonetheless have low statistical power to detect variants with modest effects. We estimate that the power of the study is >70% to detect a significant (P < 0.05) association with a causal variant with minor allele frequency >0.05 accounting for 1% of the variance in liability to diabetes. Given an allele frequency of 0.2, this corresponds to an OR of 1.32, approximately the effect observed in the FUSION study. It is noteworthy that the ORs in the FUSION and Ashkenazi studies are likely an overestimation of the true effect due to testing of multiple variants within this region, and the power to detect a true, smaller, underlying effect will be lower. Examination of the 95% CIs for the ORs in Table 1 provides an evaluation of the extent of effect sizes most compatible with the data.
Although our genome-wide linkage scan in Pima Indians did not detect evidence for linkage to type 2 diabetes on chromosome 20q12-13, this association study showed that variants in introns 1 and 3 of HNF4 are modestly associated with type 2 diabetes. These results suggest that the HNF4 locus, or another gene near this locus, may be a common genetic determinant of type 2 diabetes in multiple populations, but its relative contribution may differ between populations.
ACKNOWLEDGMENTS
We thank Drs. Alan Permutt, Michael Boehnke, and Karen Mohlke for sharing their prepublication data.
FUSION, Finnish-U.S. Investigation of Type 2 Diabetes; HNF4, hepatocyte nuclear factor 4; SNP, single nucleotide polymorphism
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2 Washington University School of Medicine, St. Louis, Missouri
ABSTRACT
Single nucleotide polymorphisms (SNPs) within the hepatocyte nuclear factor 4 (HNF4) gene are associated with type 2 diabetes in Finns and Ashkenazi Jews. Previous studies in both populations have reported linkage to type 2 diabetes near the HNF4 locus on chromosome 20q12-13. To investigate whether HNF4 is a diabetes susceptibility gene in Pima Indians, a population with the highest reported prevalence of type 2 diabetes but with no evidence for linkage of the disease on chromosome 20q, 19 SNPs across the promoter and coding region of HNF4 were genotyped for association analysis. In a group of 1,037 Pima Indians (573 diabetic and 464 nondiabetic subjects), three SNPs in HNF4 (rs3212183 and rs2071197 located in introns 3 and 1, respectively, and rs6031558, an extremely rare SNP located in the P2 promoter region) were modestly associated with type 2 diabetes (rs3212183 odds ratio [OR] 1.34 [95% CI 1.07eC1.67], P = 0.009; rs2071197 1.34 [1.07eC1.66], P = 0.008; and rs6031558 3.18 [1.03eC9.84], P = 0.04, adjusted for age, sex, birth year, heritage, and family membership). We conclude that variants in HNF4 do not appear to be major determinants for type 2 diabetes in Pima Indians; however, HNF4 may have a minor role in type 2 diabetes susceptibility within this Native American population.
Hepatocyte nuclear factor 4 (HNF4) is a transcription factor expressed in many tissues including liver and pancreas (1). Its expression is controlled by two alternative promoters, P1 and P2, which produce at least nine transcripts. Transcripts from the P2 promoter are the predominant isoforms in pancreatic -cells (2). HNF4 has been suggested to have a role in metabolic pathways involved in glucose metabolism and insulin secretion (3,4). Mutations in both the coding and regulatory regions of HNF4 have been associated with maturity-onset diabetes of young (MODY type I), a dominantly inherited, early-onset form of type 2 diabetes (5).
Several genome-wide scans for type 2 diabetes susceptibility loci have identified linkage on chromosome 20q12-13 in a region that encompasses the HNF4 locus (6eC10). Recent fine-mapping follow-up studies independently identified multiple variants within the HNF4 gene that are associated with type 2 diabetes in the Finnish-U.S. Investigation of Type 2 Diabetes (FUSION) study population and in the Ashkenazi Jewish population (11,12). In both studies, the strongest association was observed with four SNPs (rs4810424, rs1884613, rs1882614, and rs2144908) from the P2 promoter region (11,12). However, several SNPs in the P1 promoter region are also modestly associated with type 2 diabetes in the FUSION study population but not in the Ashkenazi Jewish cohort. To investigate whether variants in HNF4 are associated with type 2 diabetes in Pima Indians who have a high prevalence of type 2 diabetes but show no evidence for linkage to type 2 diabetes on chromosome 20q13, a total of 19 SNPs across a 79-kb region encompassing both the P2 and P1 promoters and the coding region of HNF4 were genotyped in this population.
RESEARCH DESIGN AND METHODS
A total of 96 noneCfirst-degreeeCrelated Pima Indians who were diagnosed with type 2 diabetes at 25 years of age (mean age of onset 16.0 ± 0.5 years and mean BMI 35.0 ± 0.5 kg/m2) were selected for sequencing of the HNF4 coding region. Variants were genotyped for a family-based association study in 1,037 Pima Indians (from 332 nuclear families) who are participants of our ongoing longitudinal study of the etiology of type 2 diabetes among the Gila River Indian Community in Arizona (13,14). Among these 1,037 subjects, 573 had type 2 diabetes (64.4% women, mean BMI 34.4 ± 8.5 kg/m2, mean study age 44.7 ± 13.0 years, and mean age at diagnosis 33.8 ± 10.8 years) and 464 were nondiabetic (43.5% women, mean BMI 33.5 ± 8.4 kg/m2, and mean study age 34.0 ± 11.0 years). Diabetes status was determined according to the criteria of the World Health Organization (15).
Two variants were additionally genotyped in an independent case/control group of 170 full-heritage, noneCfirst-degreeeCrelated Pima Indians for analysis of early-onset diabetes. The case group consisted of the same 96 subjects used for the initial sequencing of the HNF4 gene, as described above. The control group consisted of 74 subjects with normal glucose tolerance (mean age 54.0 ± 0.4 years at last medical exam and mean BMI 36.0 ± 1 kg/m2).
Metabolic measurements of risk factors for type 2 diabetes in normal glucose-tolerant subjects.
Among the 1,037 subjects who were genotyped for the type 2 diabetes association analysis, 179 subjects were normal glucose-tolerant, full-heritage Pima Indian subjects who had undergone detailed metabolic testing as inpatients in our clinical research center. Glucose tolerance was determined by a 75-g oral glucose tolerance test with measurements of fasting 30-, 60-, 120-, and 180-min plasma glucose and insulin concentrations (15). To measure the acute insulin response, blood samples were collected before a 25-g glucose bolus infusion and at 3, 4, 5, 6, 8, and 10 min after infusion. The acute insulin response was calculated as the mean increment in plasma insulin concentrations from 3 to 5 min (16). Insulin sensitivity was assessed using the hyperinsulinemic-euglycemic clamp technique as described previously (16,17). Body composition was estimated by underwater weighing until January 1996 and by dual-energy X-ray absorptiometry (DPX-1; Lunar Radiation) thereafter (18).
Sequencing of HNF4 and genotyping of SNPs.
Sequencing was done using Big Dye terminator (Applied Biosystems) on an automated DNA capillary sequencer (model 3730; Applied Biosystems). Variants identified by either sequencing or databases were genotyped using the TaqMan Allelic Discrimination (AD) Assay (Applied BioSystems) on an ABI Prism 7700 (Applied BioSystems).
Statistical analysis.
Associations were assessed using the statistical analysis system of the SAS institute (Cary, NC). The relationship between trait and marker was generally assessed via an ordered categorical variable representing genotype (i.e., an additive model). For continuous variables, the general estimating equation procedure was used to adjust for covariates. These analyses account for the correlation among family members (i.e., siblings). In the family-based study, the association with diabetes was assessed by logistic regression with control for age, sex, heritage, and year of birth. These analyses were also performed with the general estimating equation to account for the correlation among siblings. In the case-control study of early-onset diabetes, the association was assessed by logistic regression with control for sex and heritage. Haplotype frequencies for pairs of SNPs were calculated in all 1,037 individuals with the Estimating Haplotypes (EH) program (19). D' was calculated as a measure of allelic association and 2 (r2) as a measure of concordance. Association between traits and individual haplotypes were examined with a modification of the zero-recombinant haplotyping procedure, as described previously (20).
RESULTS AND DISCUSSION
Four variants were identified by sequencing the coding region and intron-exon boundaries of HNF4 in Pima Indians (Fig. 1). Two of these variants were novel (Met86Arg and IVS9 + 378T-C) and two were present in databases (Thr130Ile = rs1800961 and C/T in the 3' untranslated region = rs6130615). In addition, 10 SNPs (rs1884614, rs2144908, rs2425637, rs2425640, rs6031552, rs6031558, rs3212183, rs1885088, rs1028583, and rs3818247) were selected for genotypic analysis in Pima Indians based on results from the FUSION and Ashkenazi Jewish studies of HNF4 (11,12). Two of these variants, rs1884614 and rs2144908, were associated with type 2 diabetes in both the FUSION and Ashkenazi Jewish studies, whereas the remaining eight variants were associated with type 2 diabetes in only one of the studies. These SNPs span a 79-kb region of HNF4 including the P2 and P1 promoters and the entire coding region of HNF4. To approximate an evenly spaced dense SNP map across the HNF4 locus, with an average inter-SNP distance of 3eC4 kb, seven additional database SNPs (hcv11183312, rs6017335, rs4364072, rs4812831, rs3212198, rs6103731, and rs1028583) were also selected, based on their relative positions, for genotyping in Pima Indians (Fig. 1).
Preliminary genotyping of these 21 SNPs in 90 Pima subjects showed that SNPs rs1884614 and rs2144908 were in complete genotypic concordance, as were SNPs rs6031552 and rs6031558. Therefore, rs2144908 and rs6031552 were excluded from further genotyping. The results of genotyping the remaining 19 HNF4 variants in 1,037 Pima subjects are given in Table 1. All SNPs were in Hardy-Weinberg equilibrium. Three SNPs, rs3212183, rs2071197, and rs6031558, were modestly associated with type 2 diabetes. SNP rs3212183, positioned in intron 3, had a minor allele (C) frequency of 0.43 in diabetic subjects versus 0.37 in nondiabetic subjects (OR 1.34 [95% CI 1.07eC1.67], P = 0.009). This variant was also modestly associated with type 2 diabetes in the FUSION study, where the C allele was also the risk allele. SNP rs2071197, which was not reported in either the FUSION or Ashkenazi Jewish study, was also associated with type 2 diabetes, with an allele (G) frequency of 0.51 in diabetic subjects versus 0.45 in nondiabetic subjects (1.34 [1.07eC1.66], P = 0.008). These two SNPs (rs3212183 and rs2071197) were in very high linkage disequalibrium (D' = 0.99) in Pima Indians and were also in high, but not complete, genotypic concordance (2 = 0.69). SNP rs6031558, positioned in the P2 promoter region, had a minor allele (C) frequency of 0.02 in diabetic subjects versus 0.01 in nondiabetic subjects. Although this SNP had a significant association with type 2 diabetes (3.18 [1.03eC9.84], P = 0.04), the rare frequency of this variant makes this analysis unreliable.
SNPs rs3212183 and rs2071197 were additionally genotyped in a separate case/control group of 170 full-heritage Pima Indians selected for early-onset (or "MODY-like") diabetes (age of onset 25 years). Neither SNP was associated with early-onset diabetes in this case/control study (P = 0.24, OR 0.75 [95% CI 0.47eC0.121], and P = 0.56, 0.87 [0.56eC1.37] for rs3212183 and rs2071197, respectively).
We further investigated whether rs3212183 and rs2071197, which were in extremely high linkage disequilibrium and associated with type 2 diabetes, were associated with metabolic risk factors that predict type 2 diabetes in 179 normal glucose-tolerant, full-heritage Pima Indians who had undergone detailed metabolic testing. The various tests assessed insulin action, insulin secretion, and rates of glucose turnover. Neither SNP was associated with various measures of insulin action (data only shown for rs3212183 in Table 2). Although HNF4 is known to have a primary role in insulin secretion, there was no association between these SNPs and insulin secretion, as measured by the acute insulin response following an intravenous bolus of glucose. It is possible that we lacked the power to detect a subtle association due to the relatively small sample number (n = 179). In support of this, subjects homozygous for the type 2 diabetes risk allele did have a trend toward lower insulin secretion compared with subjects heterozygous or homozygous for the nonrisk allele (Table 2).
We also analyzed whether any of the remaining 17 variants that were not associated with type 2 diabetes in Pima Indians were associated with these metabolic risk factors in the 179 genotyped subjects. All of these associations were also negative, with the exception of SNP rs1884614 from the P2 haplotype block. This variant, which is associated with type 2 diabetes in both the Finns and Ashkenazi Jews, was associated with insulin resistance, as assessed by a hyperinsulinemic-euglycemic clamp in Pima Indians. During both physiologic and maximally stimulating insulin concentrations, subjects with the C/C+C/T genotypes for rs1884614 had a lower glucose disposal (decreased insulin sensitivity) than subjects with the T/T genotypes (3.4 ± 0.1 vs. 3.7 ± 0.1 mg · kg EMBSeC1 · mineC1, P = 0.01 for physiologic insulin dose and 7.8 ± 0.2 vs. 8.4 ± 0.1 mg · kg EMBSeC1 · mineC1, P = 0.02 for maximally stimulating insulin dose, adjusted for age, sex, percent body fat, and nuclear family membership). However, since this variant was not associated with type 2 diabetes in Pima Indians and we cannot provide a known physiologic mechanism whereby a variant in HNF4a would affect glucose uptake in the muscle, this association may be spurious rather than confirmatory.
The pattern of linkage disequilibrium across the HNF4 locus was evaluated in Pima Indians by a pair-wise analysis of genotypes from the 19 variants in the 1,037 subjects (Fig. 2). Similar to that observed in the Finnish and Ashkenazi Jewish populations, SNPs in the P2 promoter region were in extremely high linkage disequilibrium. Linkage disequilibrium did not decay until 33 kb from the beginning of the P2 promoter, with the exception of a rare SNP rs6031558 that is positioned in the middle of this block and breaks the block due to low linkage disequilibrium (<0.2) with rs6017335 and rs4364072. SNPs in exons 4eC11 were also in high linkage disequilibrium. However, SNPs across the P1 promoter and exons 1eC3 tended to have lower linkage disequilibrium among themselves but still had D' values of >0.5, with some SNPs in the P2 region. When SNPs containing common variants (minor allele frequency >0.05) were analyzed, there were five haplotype "blocks" among these SNPs, as defined by contiguous SNPs with D' >0.9. Analyses of the individual haplotypes for SNPs within these blocks showed that the haplotype consisting of "G" at rs2071197 and "C" at rs3212183 had a higher prevalence of diabetes compared with the other combined haplotypes (OR 1.37 [95% CI 1.10eC1.72], P = 0.02), a finding that largely reflects the single marker associations. There were no statistically significant associations with any of the other haplotypes (data not shown).
The allele frequencies for some variants in HNF4 are consistent between different ethnic groups, while others are quite different. For example, the allele (C) for rs3212183 had a frequency of 0.43 in both diabetic Pima Indians and diabetic Finns and a frequency of 0.37 and 0.38 in nondiabetic Pima Indians and Finns, respectively. In contrast, for rs1884614 the risk allele (T) in the Finns and Ashkenazi Jews had a frequency of 0.20 and 0.27, respectively, but the T allele in Pima Indians had a frequency of 0.83. This suggests that different populations have different HNF4 haplotypes. A difference in allele frequencies of HNF4 variants between ethnic groups may also reflect the various population-attributed risks to type 2 diabetes due to these variants.
The strongest associations with type 2 diabetes in both the FUSION and the Ashkenazi Jews studies were observed with SNPs in the P2 promoter of HNF4. Recent studies (21,22) in the Amish and U.K. populations have also replicated associations of variants in HNF4 with type 2 diabetes. In the Amish study (21), the strongest association was with rs2425640 located in the P1 promoter. The U.K. study reported significant associations with two SNPs (rs4810424 and rs2144908) that tag the P2 promoter (22). In Pima Indians, the two common SNPs associated with type 2 diabetes were in introns 1 and 3. The fact that different SNPs in the HNF4 region are associated with diabetes in different populations suggests that none of these alleles themselves are causative functional variants but that they may be in linkage disequilibrium with a nearby functional allele. Alternatively, some of these alleles may be causative, but allelic heterogeneity across populations may make their identification difficult. Given the previous evidence implicating HNF4 variants in diabetes, we considered that it is desirable to preserve statistical power. Therefore no corrections of multiple comparisons were performed. Nonetheless, one cannot exclude chance as a potential explanation for the associations observed in the present study.
Although the sample size of the present study is fairly large in comparison with many other studies, it may nonetheless have low statistical power to detect variants with modest effects. We estimate that the power of the study is >70% to detect a significant (P < 0.05) association with a causal variant with minor allele frequency >0.05 accounting for 1% of the variance in liability to diabetes. Given an allele frequency of 0.2, this corresponds to an OR of 1.32, approximately the effect observed in the FUSION study. It is noteworthy that the ORs in the FUSION and Ashkenazi studies are likely an overestimation of the true effect due to testing of multiple variants within this region, and the power to detect a true, smaller, underlying effect will be lower. Examination of the 95% CIs for the ORs in Table 1 provides an evaluation of the extent of effect sizes most compatible with the data.
Although our genome-wide linkage scan in Pima Indians did not detect evidence for linkage to type 2 diabetes on chromosome 20q12-13, this association study showed that variants in introns 1 and 3 of HNF4 are modestly associated with type 2 diabetes. These results suggest that the HNF4 locus, or another gene near this locus, may be a common genetic determinant of type 2 diabetes in multiple populations, but its relative contribution may differ between populations.
ACKNOWLEDGMENTS
We thank Drs. Alan Permutt, Michael Boehnke, and Karen Mohlke for sharing their prepublication data.
FUSION, Finnish-U.S. Investigation of Type 2 Diabetes; HNF4, hepatocyte nuclear factor 4; SNP, single nucleotide polymorphism
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