Evidence that Rho guanine nucleotide exchange factor 11 (ARHGEF11) on 1q21 is a type 2 diabetes susceptibility gene in the Old Order Amish

Rho guanine nucleotide exchange factor 11 (ARHGEF11), located on chromosome 1q21, is involved in G protein signaling and is a pathway known to play a role in both insulin secretion and action. We genotyped 52 single nucleotide polymorphims (SNPs) in ARHGEF11 and compared the genotype frequencies of subjects with type 2 diabetes (n = 145) or type 2 diabetes/impaired glucose tolerance (IGT) (n = 293) with those of control subjects with normal glucose tolerance (NGT) (n = 358). Thirty SNPs, spanning the entire gene, were significantly associated with type 2 diabetes or type 2 diabetes/IGT. The most significantly associated SNP was rs6427340 (intron 2), in which the less common allele was the risk allele (odds ratio [OR] 1.82 [95% CI 1.20-2.70], P = 0.005 for type 2 diabetes vs. NGT and 1.79 [1.27-2.50], P = 0.0008 for type 2 diabetes/IGT vs. NGT). In an expanded set of nondiabetic subjects (n = 754), most of the type 2 diabetes-and IGT-associated SNPs were significantly associated with glucose levels during an oral glucose tolerance test, with the same SNP (rs6427340) showing the most significant associations (P = 0.007). All type 2 diabetes-and IGT-associated SNPs were in high linkage disequilibrium and constitute a single 133-kb haplotype block. These results, coupled with similar findings in Pima Indians, suggest that sequence variation in ARHGEF11 may influence risk of type 2 diabetes.     

Variants in hepatocyte nuclear factor 4alpha are modestly associated with type 2 diabetes in Pima Indians

Single nucleotide polymorphisms (SNPs) within the hepatocyte nuclear factor 4alpha (HNF4alpha) 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 HNF4alpha locus on chromosome 20q12-13. To investigate whether HNF4alpha 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 HNF4alpha were genotyped for association analysis. In a group of 1,037 Pima Indians (573 diabetic and 464 nondiabetic subjects), three SNPs in HNF4alpha (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 1.07-1.67], P = 0.009; rs2071197 1.34 [1.07-1.66], P = 0.008; and rs6031558 3.18 [1.03-9.84], P = 0.04, adjusted for age, sex, birth year, heritage, and family membership). We conclude that variants in HNF4alpha do not appear to be major determinants for type 2 diabetes in Pima Indians; however, HNF4alpha may have a minor role in type 2 diabetes susceptibility within this Native American population.     

Polymorphisms in the SLC2A2 (GLUT2) gene are associated with the conversion from impaired glucose tolerance to type 2 diabetes: the Finnish Diabetes Prevention Study

Impaired insulin secretion is a fundamental defect in type 2 diabetes. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the genes regulating insulin secretion (SLC2A2 [encoding GLUT2], GCK, TCF1 [encoding HNF-1alpha], HNF4A, GIP, and GLP1R) are associated with the conversion from impaired glucose tolerance (IGT) to type 2 diabetes in participants of the Finnish Diabetes Prevention Study. With the exception of SLC2A2, other genes were not associated with the risk of type 2 diabetes. All four SNPs of SLC2A2 predicted the conversion to diabetes, and rs5393 (AA genotype) increased the risk of type 2 diabetes in the entire study population by threefold (odds ratio 3.04, 95% CI 1.34-6.88, P = 0.008). The risk for type 2 diabetes in the AA genotype carriers was increased in the control group (5.56 [1.78-17.39], P = 0.003) but not in the intervention group. We conclude that the SNPs of SLC2A2 predict the conversion to diabetes in obese subjects with IGT.     

Single nucleotide polymorphisms in the proximal promoter region of the adiponectin (APM1) gene are associated with type 2 diabetes in Swedish caucasians

Adiponectin (APM1) is an adipocyte-derived peptide. The APM1 gene is located on chromosome 3q27 and linked to type 2 diabetes. In patients with type 2 diabetes, the adiponectin level in plasma is decreased in comparison to healthy subjects. To identify genetic defects of the APM1 gene that contribute to the development of type 2 diabetes, we genotyped 13 single nucleotide polymorphisms (SNPs) in 106 patients with type 2 diabetes, 325 patients with impaired glucose tolerance (IGT), and 497 nondiabetic control subjects in Swedish Caucasians by using dynamic allele-specific hybridization (DASH). We found that SNPs -11426(A/G) and -11377(G/C) in the proximal promoter region had significant differences of allele frequencies between type 2 diabetic patients and nondiabetic control subjects (P = 0.02 and P = 0.04, respectively). SNP-11426(A/G) was significantly associated with fasting plasma glucose in type 2 diabetic patients (P = 0.02) and in IGT subjects (P = 0.04), while the patients carrying CC and CG genotypes for SNP-11377(G/C) had a higher BMI than the patients with the GG genotype (P = 0.03). Haplotype analysis of 13 SNPs in the APM1 gene showed that estimates of haplotype frequencies in Swedish Caucasians are similar to those estimated in French Caucasians. However, no significant association of haplotypes with type 2 diabetes and IGT was detected in our study. The present study provides additional evidence that SNPs in the proximal promoter region of the APM1 gene contribute to the development of type 2 diabetes.     

Genetic variation in adiponectin receptor 1 and adiponectin receptor 2 is associated with type 2 diabetes in the Old Order Amish

Adiponectin receptor 1 (ADIPOR1) and adiponectin receptor 2 (ADIPOR2) are newly identified receptors for adiponectin, an adipocytokine with anti-inflammatory and insulin-sensitizing properties. We screened for polymorphisms by performing sequence analysis on all eight exons, splice junctions, and approximately 2 kb of the 5' flanking regions of each receptor. We detected 5 single nucleotide polymorphisms (SNPs) in ADIPOR1 and 16 SNPs in ADIPOR2. We genotyped these SNPs in Amish subjects with type 2 diabetes (n = 137), impaired glucose tolerance (IGT) (n = 139), and normal glucose tolerance (n = 342) to test for association with type 2 diabetes. Three intronic SNPs in ADIPOR1 were significantly associated with type 2 diabetes (P = 0.014-0.007; odds ratio [OR] 1.61-1.65) and in high linkage disequilibrium (r2 = 0.97-1.0). In ADIPOR2, we found that five SNPs delineated one large haplotype block (r2= 0.9-1.0) spanning >98 kb of the gene and promoter region, which was strongly associated with the combined type 2 diabetes/IGT trait (P < or = 0.001; OR 1.64-1.71). To our knowledge, these data provide the first evidence for association between variation in the adiponectin receptors and type 2 diabetes.     

Polymorphism in the calsequestrin 1 (CASQ1) gene on chromosome 1q21 is associated with type 2 diabetes in the old order Amish

Calsequestrin (CASQ)1 is involved in intracellular storage and release of calcium, a process that has been shown to mediate glucose transport in muscle. Its gene, CASQ1, is encoded on chromosome 1q21, a region that has been linked to type 2 diabetes in the Amish and several other populations. We screened all 11 exons, exon-intron junctions, and the proximal regulatory region of CASQ1 for mutations. We detected four novel single nucleotide polymorphisms (SNPs) (-1470C-->T, -1456delG, -1366insG, and 593C-->T). Ten informative SNPs within CASQ1 were genotyped in Amish subjects with type 2 diabetes (n = 145), impaired glucose tolerance (n = 148), and normal glucose tolerance (n = 358). Rs2275703 and rs617698 in introns 4 and 2 were significantly associated with type 2 diabetes (P = 0.008 and 0.04, respectively); three other SNPs showed borderline evidence for association to type 2 diabetes (P = 0.076-0.093). Furthermore, in nondiabetic subjects (n = 754), both rs2275703 and rs617698 were significantly associated with glucose area under the curve during an oral glucose tolerance test (P = 0.035 and 0.013, respectively). Haplotype analysis suggested that no haplotype could explain these associations better than rs2275703. These findings, coupled with similar findings in Utah Caucasians, suggest that sequence variation in CASQ1 may influence risk of type 2 diabetes.     

Single nucleotide polymorphisms of PPARD in combination with the Gly482Ser substitution of PGC-1A and the Pro12Ala substitution of PPARG2 predict the conversion from impaired glucose tolerance to type 2 diabetes: the STOP-NIDDM trial

Peroxisome proliferator-activated receptor (PPAR)-delta regulates fatty acid oxidation and improves insulin sensitivity. We screened six single nucleotide polymorphisms (SNPs) of the PPAR-delta gene (PPARD) for an association with the conversion from impaired glucose tolerance (IGT) to type 2 diabetes in 769 subjects participating in the STOP-NIDDM trial. A 2.7-fold increase in the risk of diabetes was observed in female carriers of the C allele of rs6902123 (95% CI 1.44-5.30; adjusted P = 0.002). In the placebo group, subjects possessing both the 482Ser allele of the PPAR-gamma coactivator-1alpha gene (PGC-1A) and the rare allele of two SNPs of PPARD (rs6902123 and rs3734254) had up to 2.5-fold increased risk for diabetes. Furthermore, women carrying the C allele of rs6902123 of PPARD and the Pro12Pro genotype of the PPAR-gamma2 gene (PPARG2) had a 3.9-fold (95% CI 1.79-8.63; P = 0.001)-higher risk for diabetes than women with protective genotypes. Expression levels of PPAR-delta in subcutaneous adipose tissue of 87 offspring of Finnish patients with type 2 diabetes did not differ among the genotype groups of SNPs of PPARD. We conclude that SNPs in PPARD modify the conversion from IGT to type 2 diabetes, particularly in combination with the SNPs of PGC-1A and PPARG2.     

Genetic analysis of HNF4A polymorphisms in Caucasian-American type 2 diabetes

Hepatocyte nuclear factor 4alpha (HNF4A), the gene for the maturity-onset diabetes of the young type 1 monogenic form of type 2 diabetes, is within the type 2 diabetes-linked region on chromosome 20q12-q13.1 and, consequently, is a positional candidate gene for type 2 diabetes in the general population. Previous studies have identified only a few rare coding mutations. However, recent studies suggest that single nucleotide polymorphisms (SNPs) located near the P2 (beta-cell) promoter of HNF4A are associated with diabetes susceptibility. In this study, we evaluated 23 SNPs spanning 111 kb including the HNF4A gene for association with type 2 diabetes in a collection of Caucasian type 2 diabetic patients with end-stage renal disease (n = 300) and control subjects (n = 310). None of the individual SNPs were associated with type 2 diabetes in this collection of case subjects (P values ranging from 0.06 to 0.99). However, haplotype analysis identifies significant differences between haplotype frequencies in type 2 diabetic case and control subjects (P = 0.013 to P < 0.001), with two uncommon "risk" haplotypes (2.4 and 2.2% of chromosomes) and two uncommon "protective" haplotypes (7.1 and 5.0% of chromosomes) accounting for the evidence of association. Our results suggest that type 2 diabetes linked to 20q12-13 is a heterogeneous disease in which different populations may have different type 2 diabetes susceptibility loci.     

Screening of 134 single nucleotide polymorphisms (SNPs) previously associated with type 2 diabetes replicates association with 12 SNPs in nine genes

More than 120 published reports have described associations between single nucleotide polymorphisms (SNPs) and type 2 diabetes. However, multiple studies of the same variant have often been discordant. From a literature search, we identified previously reported type 2 diabetes-associated SNPs. We initially genotyped 134 SNPs on 786 index case subjects from type 2 diabetes families and 617 control subjects with normal glucose tolerance from Finland and excluded from analysis 20 SNPs in strong linkage disequilibrium (r(2) > 0.8) with another typed SNP. Of the 114 SNPs examined, we followed up the 20 most significant SNPs (P < 0.10) on an additional 384 case subjects and 366 control subjects from a population-based study in Finland. In the combined data, we replicated association (P < 0.05) for 12 SNPs: PPARG Pro12Ala and His447, KCNJ11 Glu23Lys and rs5210, TNF -857, SLC2A2 Ile110Thr, HNF1A/TCF1 rs2701175 and GE117881_360, PCK1 -232, NEUROD1 Thr45Ala, IL6 -598, and ENPP1 Lys121Gln. The replication of 12 SNPs of 114 tested was significantly greater than expected by chance under the null hypothesis of no association (P = 0.012). We observed that SNPs from genes that had three or more previous reports of association were significantly more likely to be replicated in our sample (P = 0.03), although we also replicated 4 of 58 SNPs from genes that had only one previous report of association.     

Association testing of variants in the hepatocyte nuclear factor 4alpha gene with risk of type 2 diabetes in 7,883 people

Two recent publications reported association of common polymorphisms in the P2 promoter of hepatocyte nuclear factor 4alpha (HNF4alpha) (the MODY1 gene) with risk for type 2 diabetes. We attempted to reproduce this putative association by genotyping 11 single nucleotide polymorphism (SNPs) spanning the HNF4alpha coding region and the P2 promoter in >3,400 patients and control subjects from Sweden, Finland, and Canada. One SNP that was consistently associated in the two previous reports (rs1884613, in the P2 promoter region) also trended in the same direction in our sample, albeit with a lower estimated odds ratio (OR) of 1.11 (P = 0.05, one-tailed). We genotyped this SNP (rs1884613) in an additional 4,400 subjects from North America and Poland. In this sample, the association was not confirmed and trended in the opposite direction (OR 0.88). Meta-analysis of our combined sample of 7,883 people (three times larger than the two initial reports combined) yielded an OR of 0.97 (P = 0.27). Finally, we provide an updated analysis of haplotype structure in the region to guide any further investigation of common variation in HNF4alpha. Although our combined results fail to replicate the previously reported association of common variants in HNF4alpha with risk for type 2 diabetes, we cannot exclude an effect smaller than that originally proposed, heterogeneity among samples, variation in as-yet-unmeasured genotypic or environmental modifiers, or true association secondary to linkage disequilibrium (LD) with as-yet-undiscovered variant(s) in the region.     

Genetic variation near the hepatocyte nuclear factor-4 alpha gene predicts susceptibility to type 2 diabetes

The Finland-United States Investigation Of NIDDM Genetics (FUSION) study aims to identify genetic variants that predispose to type 2 diabetes by studying affected sibling pair families from Finland. Chromosome 20 showed our strongest initial evidence for linkage. It currently has a maximum logarithm of odds (LOD) score of 2.48 at 70 cM in a set of 495 families. In this study, we searched for diabetes susceptibility variant(s) at 20q13 by genotyping single nucleotide polymorphism (SNP) markers in case and control DNA pools. Of 291 SNPs successfully typed in a 7.5-Mb interval, the strongest association confirmed by individual genotyping was with SNP rs2144908, located 1.3 kb downstream of the primary beta-cell promoter P2 of hepatocyte nuclear factor-4 alpha (HNF4A). This SNP showed association with diabetes disease status (odds ratio [OR] 1.33, 95% CI 1.06-1.65, P = 0.011) and with several diabetes-related traits. Most of the evidence for linkage at 20q13 could be attributed to the families carrying the risk allele. We subsequently found nine additional associated SNPs spanning a 64-kb region, including the P2 and P1 promoters and exons 1-3. Our results and the independent observation of association of SNPs near the P2 promoter with diabetes in a separate study population of Ashkenazi Jewish origin suggests that variant(s) located near or within HNF4A increases susceptibility to type 2 diabetes.     

Promoter polymorphisms of the TNF-alpha (G-308A) and IL-6 (C-174G) genes predict the conversion from impaired glucose tolerance to type 2 diabetes: the Finnish Diabetes Prevention Study

High levels of cytokines are risk factors for type 2 diabetes. Therefore, we investigated whether the promoter polymorphisms of the tumor necrosis factor-alpha (TNF-alpha; G-308A) and interleukin 6 (IL-6; C-174G) genes predict the conversion from impaired glucose tolerance (IGT) to type 2 diabetes in the Finnish Diabetes Prevention Study. Altogether, 490 overweight subjects with IGT whose DNA was available were randomly divided into one of the two treatment assignments: the control group and the intensive, individualized diet and exercise intervention group. The -308A allele of the TNF-alpha gene was associated with an approximate twofold higher risk for type 2 diabetes compared with the G-308G genotype (odds ratio 1.80, 95% CI 1.05-3.09; P = 0.034). Subjects with both the A allele of the TNF-alpha gene and the C-174C genotype of the IL-6 gene had a 2.2-fold (CI 1.02-4.85, P = 0.045) higher risk of developing type 2 diabetes than subjects without the risk genotypes. We conclude that the -308A allele of the promoter polymorphism (G-308A) of the TNF-alpha gene is a predictor for the conversion from IGT to type 2 diabetes. Furthermore, this polymorphism seems to have a gene-gene interaction with the C-174C genotype of the IL-6 gene.     

EIF4A2 is a positional candidate gene at the 3q27 locus linked to type 2 diabetes in French families

One of the most replicated loci influencing type 2 diabetes-related quantitative traits (quantitative trait loci [QTL]) is on chromosome 3q27 and modulates both type 2 diabetes-and metabolic syndrome-associated phenotypes. A QTL for type 2 diabetes age of onset (logarithm of odds [LOD] score = 3.01 at D3S3686, P = 0.0001) was identified in a set of French families. To assess genetic variation underlying both age-of-onset QTL and our previous type 2 diabetes linkage in a 3.87-Mb interval, we explored 36 single nucleotide polymorphisms (SNPs) in two biologically relevant candidate genes for glucose homeostasis, kininogen (KNG1), and eukaryotic translation initiation factor 4alpha2 (EIF4A2). Analysis of 148 families showed significant association of a frequent SNP, rs266714, located 2.47 kb upstream of EIF4A2, with familial type 2 diabetes (family-based association test, P = 0.0008) and early age of onset (P = 0.0008). This SNP also contributes to both age-of-onset QTL (1.13 LOD score decrease P = 0.02) and type 2 diabetes linkage (genotype identical-by-descent sharing test, P = 0.02). However, no association was observed in three independent European diabetic cohorts. EIF4A2 controls specific mRNA translation and protein synthesis rate in pancreatic beta-cells, and our data indicates that EIF4A2 is downregulated by high glucose in rat beta-INS832/13 cells. The potential role of EIF4A2 in glucose homeostasis and its putative contribution to type 2 diabetes in the presence of metabolic stress will require further investigation.     

Endoneurial capillary abnormalities presage deterioration of glucose tolerance and accompany peripheral neuropathy in man

To explore whether microangiopathy is associated with disturbed glucose tolerance and peripheral neuropathy, we assessed endoneurial capillary morphology in sural nerve biopsies from men with diabetes, impaired glucose tolerance (IGT), and normal glucose tolerance (NGT). Baseline morphology was related to glucose tolerance and neuropathy at baseline and at follow-up 6 years later. Capillary density (in number per millimeters squared) at baseline was higher in subjects with diabetes (n = 10) compared with those with NGT (n = 5) at follow-up (median [interquartile range]) (86.0 [24.3] vs. 54.9 [17.1]; P = 0.0200) and in those progressing from IGT to diabetes (n = 4) compared with those with persistent IGT (n = 4) (86.7 [25.2] vs. 54.1 [14.6]; P = 0.0433). The capillary luminal area (in micrometers squared) was lower in subjects with NGT progressing to IGT (n = 2) or subjects with IGT progressing to diabetes (n = 3) compared with subjects with constant NGT (n = 6) or constant IGT (n = 4) (11.9 [2.4] vs. 20.8 [7.8]; P = 0.0201). The capillary basement membrane area (in micrometers squared) was increased in patients with peripheral neuropathy (n = 10) compared with those without (n = 7) (114.6 [68.8] vs. 75.3 [28.7]; P = 0.0084). In conclusion, increased capillary density was associated with current or future diabetes, decreased capillary luminal area with future deterioration in glucose tolerance, and increased basement membrane area with peripheral neuropathy.     

Association of common variation in the HNF1alpha gene region with risk of type 2 diabetes

It is currently unclear how often genes that are mutated to cause rare, early-onset monogenic forms of disease also harbor common variants that contribute to the more typical polygenic form of each disease. The gene for MODY3 diabetes, HNF1alpha, lies in a region that has shown linkage to late-onset type 2 diabetes (12q24, NIDDM2), and previous association studies have suggested a weak trend toward association for common missense variants in HNF1alpha with glucose-related traits. Based on genotyping of 79 common SNPs in the 118 kb spanning HNF1alpha, we selected 21 haplotype tag single nucleotide polymorphisms (SNPs) and genotyped them in >4,000 diabetic patients and control subjects from Sweden, Finland, and Canada. Several SNPs from the coding region and 5' of the gene demonstrated nominal association with type 2 diabetes, with the most significant marker (rs1920792) having an odds ratio of 1.17 and a P value of 0.002. We then genotyped three SNPs with the strongest evidence for association to type 2 diabetes (rs1920792, I27L, and A98V) in an additional 4,400 type 2 diabetic and control subjects from North America and Poland and compared our results with those of the original sample and of Weedon et al. None of the results were consistently observed across all samples, with the possible exception of a modest association of the rare (3-5%) A98V variant. These results indicate that common variants in HNF1alpha either play no role in type 2 diabetes, a very small role, or a role that cannot be consistently observed without consideration of as yet unmeasured genetic or environmental modifiers.