PCLO variants are nominally associated with early-onset type 2 diabetes and insulin resistance in Pima Indians

OBJECTIVE—A prior genome-wide association (GWA) study in Pima Indians identified variants within PCLO that were associated with early-onset type 2 diabetes. PCLO encodes a presynaptic cytomatrix protein that functions as a Ca²⁺ sensor that may be involved in insulin secretion and/or insulin action. Therefore, PCLO was analyzed as a candidate gene for type 2 diabetes.RESEARCH DESIGN AND METHODS—Sequencing of PCLO identified four nonsynonymous variants and a 10–amino acid insertion. These variants, together with 100 additional variants identified by sequencing or chosen from databases, were genotyped for association analysis in the same 895 subjects analyzed in the prior GWA study (300 case subjects with diabetes onset at aged <25 years, 334 nondiabetic control subjects aged >45 years, and 261 discordant siblings of the case or control subjects for within-family analyses), as well as 415 nondiabetic Pima Indians who had been metabolically phenotyped for predictors of diabetes. Selected variants were further genotyped in a population-based sample of 3,501 Pima Indians.RESULTS—Four variants were modestly associated with early-onset type 2 diabetes in both general and within-family analyses (P = 0.004–0.04, recessive model), where the diabetes risk allele was also nominally associated with a lower insulin-mediated glucose disposal rate (P = 0.009–0.14, recessive model) in nondiabetic Pima Indians. However, their association with diabetes in the population-based sample was weaker (P = 0.02–0.20, recessive model).CONCLUSIONS—Variation within PCLO may have a modest effect on early-onset type 2 diabetes, possibly as a result of reduced insulin action, but has minimal, if any, impact on population-based risk for type 2 diabetes.The Pima Indians of Arizona have an extremely high prevalence of type 2 diabetes (1). Their diabetes is characterized by obesity, dysfunction of insulin secretion, insulin resistance (decreased insulin-mediated glucose disposal), and increased rate of endogenous glucose output (2). Studies have shown that type 2 diabetes, insulin action, acute insulin response to glucose, and obesity are highly heritable in this population (3–5). To identify genes that underlie the development of type 2 diabetes in Pima Indians, we recently completed a genome-wide association (GWA) study using the Affymetrix 100K SNP genotyping array (6). Two single nucleotide polymorphisms (SNPs), rs10487656 and rs10487657, that ranked among the top 1% for a general association with early-onset type 2 diabetes (defined as age of onset <25 years) mapped within an intron of the PCLO gene. PCLO is located on chromosome 7q21 and encodes for a presynaptic cytomatrix protein that functions as a Ca²⁺ sensor that could potentially have a role in insulin secretion and/or insulin action (7–10); therefore, PCLO was analyzed as a positional and physiological candidate gene for 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.     

Molecular analysis of KCNJ10 on 1q as a candidate gene for Type 2 diabetes in Pima Indians

The KCNJ10 gene is located within a region on chromosome 1q linked to type 2 diabetes in the Pima Indians and six other populations. We therefore investigated this gene as a potential type 2 diabetes candidate gene in Pima Indians. KCNJ10 consists of two exons, spans approximately 33 kb, and we identified eight single-nucleotide polymorphisms (SNPs), including one (SNP2) in the coding region leading to a Glu359Lys substitution. Association studies were carried out in a case-control group composed of 149 affected and 150 unaffected Pimas, and the linkage analysis was performed in a linkage set of 1,338 Pimas. SNP1 in the promoter and SNP2 in the intron, which were in a complete linkage disequilibrium, and SNP5 in the 3' untranslated region showed association with diabetes in the case-control group (P = 0.02 and P = 0.01, respectively). When genotyped in the linkage set, only the KCNJ10-SNP1 variant showed a modest association with type 2 diabetes (P = 0.01). KCNJ10-SNP1 is in a strong linkage disquilibrium with SNP14 of the adjacent KCNJ9 locus, which we previously found to be associated with type 2 diabetes. After adjustment for KCNJ10-SNP1, the original linkage score at this locus was marginally reduced from 3.1 to 2.9. We conclude that these variants in KCNJ10 are unlikely to be the cause of linkage of type 2 diabetes with 1q in Pima Indians.     

Calsquestrin 1 (CASQ1) gene polymorphisms under chromosome 1q21 linkage peak are associated with type 2 diabetes in Northern European Caucasians

Genome-wide scans in multiple populations have identified chromosome 1q21-q24 as one susceptibility region for type 2 diabetes. To map the susceptibility genes, we first placed a dense single nucleotide polymorphism (SNP) map across the linked region. We identified two SNPs that showed strong associations, and both mapped to within intron 2 of the calsequestrin 1 (CASQ1) gene. We tested the hypothesis that sequence variation in or near CASQ1 contributed to type 2 diabetes susceptibility in Northern European Caucasians by identifying additional SNPs from the public database and by screening the CASQ1 gene for additional variation. In addition to 15 known SNPs in this region, we found 8 new SNPs, 3 of which were in exons. A single rare nonsynonymous SNP in exon 11 (A348V) was not associated with type 2 diabetes. The associated SNPs were localized to the region between -1,404 in the 5' flanking region and 2,949 in intron 2 (P = 0.002 to P = 0.034). No SNP 3' to intron 2, including the adjacent gene PEA15, showed an association. The strongest associations were restricted to individuals of Northern European ancestry ascertained in Utah. A six-marker haplotype was also associated with type 2 diabetes (P = 0.008), but neither transmission disequilibrium test nor family-based association studies were significant for the most strongly associated SNP in intron 2 (SNP CASQ2312). An independent association of SNPs in introns 2 and 4 with type 2 diabetes is reported in Amish families with linkage to chromosome 1q21-q24. Our findings suggest that noncoding SNPs in CASQ1 alter diabetes susceptibility, either by a direct effect on CASQ1 gene expression or perhaps by regulating a nearby gene such as PEA15.     

The role of insulin receptor substrate-1 gene (IRS1) in type 2 diabetes in Pima Indians

The insulin receptor substrate-1 (IRS1) is a critical element in insulin-signaling pathways, and mutations in the IRS1 gene have been reported to have a role in determining susceptibility to traits related to type 2 diabetes. In gene expression studies of tissue biopsies from nondiabetic Pima Indians, IRS1 mRNA levels were reduced in adipocytes from obese subjects compared with lean subjects, and IRS1 mRNA levels were also reduced in skeletal muscle from insulin-resistant subjects compared with insulin-sensitive subjects (all P < 0.05). Based on these expression differences and the known physiologic role of IRS1, this gene was investigated as a candidate gene for susceptibility to type 2 diabetes in Pima Indians, a population with an extremely high incidence and prevalence of type 2 diabetes. Thirteen variants were identified, and among these variants, several were in complete linkage disequilibrium. Four genotypically unique variants were further genotyped in 937 DNA samples from full-heritage Pima Indians. Three of the variants were modestly associated with type 2 diabetes (P < 0.05), one of which was additionally associated with 2-h plasma insulin and glucose as well as insulin action at physiologic and maximally stimulating insulin concentrations (all P < 0.05). The association of variants in IRS1 with type 2 diabetes and type 2 diabetes-related phenotypes and the differential expression of IRS1 in adipocytes and skeletal muscle suggest a role of this gene in the pathogenesis of type 2 diabetes in Pima Indians.     

Polymorphisms in the oxygen-regulated protein 150 gene (ORP150) are associated with insulin resistance in Pima Indians

The ORP150 gene that encodes the human oxygen-regulated protein (150 kDa) maps to chromosome 11q23, a region previously reported to be linked to type 2 diabetes and obesity in Pima Indians. This gene was also found to be differentially expressed in global gene expression studies comparing muscle mRNA from insulin-resistant versus insulin-sensitive subjects. Therefore, ORP150 was analyzed as a candidate gene for susceptibility to diabetes. Twelve variants were identified, and three unique representative polymorphisms were genotyped in 1,338 Pima Indians. None of these polymorphisms were associated with diabetes, but two polymorphisms were significantly associated with measures of insulin resistance. These data indicate that ORP150 has a role in insulin action but does not have a major role in determining susceptibility to type 2 diabetes in Pima Indians.     

Polymorphisms in the glucokinase-associated, dual-specificity phosphatase 12 (DUSP12) gene under chromosome 1q21 linkage peak are associated with type 2 diabetes

Linkage of type 2 diabetes to chromosome 1q21-q23 is well replicated across populations. In an initial 50-kb marker map (580 markers) across the linked region, one of the two strongest associations observed in Utah Caucasians was at marker rs1503814 (P < 0.00001 in pools, P < 0.004 in individuals). Based on this association, we typed additional markers and screened for sequence variation in the nearby DUSP12 gene. The strongest associations mapped to a highly conserved nongenic sequence just telomeric to rs1503814 and extended 10 kb telomeric through the DUSP12 gene and into the 5' end of the adjacent ATF6 gene. No coding variant could explain the association in the DUSP12 gene. An extended haplotype encompassing markers from -8,379 to +10,309 bp relative to the ATG start was more common in Caucasian case (0.381) than control subjects (0.285, P = 0.005) and was uniquely tagged by a 194-bp allele at either of two simple tandem repeat variants or by the T allele at marker +7,580. Markers -8,379 and +7,580 were nominally associated with type 2 diabetes in African-American subjects (P < 0.05), but with different alleles. Marker rs1503814 was strongly associated with postchallenge insulin levels among family members (P = 0.000002), but sequence variation in this region was not associated with type 2 diabetes in three other populations of European ancestry. Our data suggest that sequences in or upstream of DUSP12 may contribute to type 2 diabetes susceptibility, but the lack of replication suggests a small effect size.     

Variants within the calpain-10 gene on chromosome 2q37 (NIDDM1) and relationships to type 2 diabetes,insulin resistance,and impaired acute insulin secretion among Scandinavian Caucasians

Variations in the calpain-10 gene (CAPN10) have been identified among Mexican-Americans, and an at-risk haplotype combination (112/121) defined by three polymorphisms, UCSNP-43, -19, and -63, confers increased risk of type 2 diabetes. Here we examine the three polymorphisms in 1,594 Scandinavian subjects, including 409 type 2 diabetic patients, 200 glucose-tolerant control subjects, 322 young healthy subjects, 206 glucose-tolerant offspring of diabetic patients, and 457 glucose-tolerant 70-year-old men. The frequency of the 112/121 combination was not significantly different in 409 type 2 diabetic subjects compared with 200 glucose-tolerant control subjects (0.06 vs. 0.05; odds ratio 1.32 [95% CI 0.58-3.30]). In glucose-tolerant subjects, neither the single-nucleotide polymorphisms individually nor the 112/121 combination were associated with alterations in plasma glucose, serum insulin, or serum C-peptide levels at fasting or during an oral glucose tolerance test, estimates of insulin sensitivity, or glucose-induced insulin secretion. In conclusion, the frequency of the 112/121 at-risk haplotype of CAPN10 is low among Scandinavians and we were unable to demonstrate significant associations between the CAPN10 variants and type 2 diabetes, insulin resistance, or impaired insulin secretion.     

Genetic susceptibility to nephropathy in Pima Indians with type 2 diabetes mellitus

Summary: In Pima Indians, the incidence of end-stage renal disease, nearly all of which is attributable to type 2 diabetes mellitus, is more than 20 times that in the general United States population. Studies in the Pimas indicate that factors other than diabetes per se enhance susceptibility to the development of diabetic nephropathy. Aggregation of renal disease in families, a relationship between parental blood pressure and diabetic nephropathy in the offspring, and an association between higher prediabetic blood pressure and the occurrence of renal disease after the onset of diabetes all point to individual differences in susceptibility. Although clustering of environmental exposures may be responsible for these findings, they may also represent genetic transmission of susceptibility to renal disease. Recently, linkage analyses were performed in 98 diabetic sib-pairs, both affected by diabetic nephropathy. Two adjacent markers on chromosome 7 met the criteria for suggestive linkage with two-point analysis. Positioned between these markers is the gene coding for aldose reductase. Polymorphisms of this locus are associated with diabetic microvascular complications in other populations. Linkage studies provide evidence that familial aggregation of diabetic renal disease reflects, in part, genetic transmission of susceptibility that appears to be independent of the transmission of diabetes.     

Adiponectin receptor 1 gene (ADIPOR1) as a candidate for type 2 diabetes and insulin resistance

Considerable data support adiponectin as an important adipose-derived insulin sensitizer that enhances fatty acid oxidation and alters hepatic gluconeogenesis. Adiponectin acts by way of two receptors, ADIPOR1 and ADIPOR2. ADIPOR1 is widely expressed in tissues, including muscle, liver, and pancreas, and binds the globular form of adiponectin with high affinity. To test the hypothesis that sequence variations in or near the ADIPOR1 gene contribute to the risk of developing type 2 diabetes and the metabolic syndrome, we screened the eight exons (including the untranslated exon 1) of the ADIPOR1 gene with flanking intronic sequences and the 5' and 3' flanking sequences. We identified 22 single nucleotide polymorphisms (SNPs) in Caucasian and African-American subjects, of which a single nonsynonymous SNP (N44K) in exon 2 was present only in African-American subjects. We typed 14 sequence variants that had minor allele frequencies >5%. No SNP was associated with type 2 diabetes in Caucasians or African Americans, and no SNP was a determinant of insulin sensitivity or insulin secretion among nondiabetic members of high-risk Caucasian families. However, the two alleles of a SNP in the 3' untranslated region were expressed unequally, and ADIPOR1 mRNA levels were significantly lower among transformed lymphocytes from diabetic African-American individuals than among control cell lines. This altered gene expression might suggest a role for ADIPOR1 in the metabolic syndrome.     

Association of adiponectin mutation with type 2 diabetes: a candidate gene for the insulin resistance syndrome

Adiponectin, also referred to as AdipoQ or ACRP30, is a plasma protein produced and secreted exclusively from adipose tissue. The protein contains a collagen-like domain and a C1q-like globular domain. A protease-generated globular segment enhances fatty acid oxidation in muscles, thereby modulating lipid and glucose metabolism. Plasma adiponectin levels are inversely correlated with the severity of insulin resistance. A recent genome-wide scan study mapped a susceptibility locus for type 2 diabetes and the metabolic syndrome to chromosome 3q27, where the adiponectin gene is located. Here, we screened Japanese patients with type 2 diabetes and age- and BMI-matched nondiabetic control subjects for mutations in adiponectin gene. We identified four missense mutations (R112C, I164T, R221S, and H241P) in the globular domain. Among these mutations, the frequency of I164T mutation was significantly higher in type 2 diabetic patients than in age- and BMI- matched control subjects (P < 0.01). Furthermore, plasma adiponectin concentrations of subjects carrying I164T mutation were lower than those of subjects without the mutation. All the subjects carrying I164T mutation showed some feature of metabolic syndrome, including hypertension, hyperlipidemia, diabetes, and atherosclerosis. Our findings suggest that I164T mutation is associated with low plasma adiponectin concentration and type 2 diabetes.     

Reduced early insulin secretion in the etiology of type 2 diabetes mellitus in Pima Indians

We report the results of cross-sectional, prospective, and longitudinal studies identifying etiologic metabolic factors in the susceptibility to type 2 diabetes mellitus of the Pima Indians of Arizona, whose prevalence and incidence rates of the disease are the highest in the world. Diabetic Pima Indians are metabolically prototypic, with obesity, insulin resistance, a reduced acute insulin response to glucose, and increased endogenous glucose production. Cross-sectional studies show that the acute insulin response is absent in diabetic subjects and lower in impaired than in normal glucose-tolerant subjects. Prospective studies using proportional hazards analyses indicate that insulin resistance and a relatively low acute insulin response predict diabetes independently of age, gender, and each other, with obesity increasing susceptibility by worsening one or both predictors. Longitudinal studies show that glucose tolerance deteriorates as the degree of obesity increases due to worsening insulin resistance and decreases in early insulin secretion. Furthermore, since the children of diabetic pregnancies are at much greater risk of developing diabetes at a young age than those of nondiabetic pregnancies, the diabetic uterine environment may induce insulin resistance and/or reduced insulin secretion: early evidence confirms that adult normal glucose-tolerant offspring show a substantially decreased acute insulin response--the clearest demonstration yet of an environmental condition increasing susceptibility to type 2 diabetes mellitus. However, the genetic determinants require elucidation: correlation of the acute insulin response with the age of parental diabetes onset in fathers as well as mothers indicates a mechanism independent of the diabetic uterine environment.     

Effect of glycemia on mortality in Pima Indians with type 2 diabetes

The effect of plasma glucose concentration on overall and cause-specific mortality was examined in 1,745 Pima Indians (725 men, 1,020 women) > or = 15 years old with type 2 diabetes. During a median follow-up of 10.6 years (range 0.1-24.8), 533 subjects (275 men, 258 women) died; 113 of the deaths were attributable to cardiovascular disease, 96 to diabetes-related diseases (diabetic nephropathy for 92 of these), 249 to other natural causes, and 75 to external causes. After adjusting for age, sex, duration of diabetes, and BMI in a generalized additive proportional hazards model, higher baseline 2-h postload plasma glucose concentration predicted deaths from cardiovascular disease (P = 0.007) and diabetes-related diseases (P = 0.003), but not from other natural causes (P = 0.73). An increment of 5.6 mmol/l (100 mg/dl) in the 2-h plasma glucose concentration was associated with 1.2 times (95% CI 1.1-1.4) the death rate from cardiovascular disease, 1.3 times (95% CI 1.1-1.5) the death rate from diabetes-related diseases, and almost no change in the death rate from other natural causes (rate ratio = 1.0; 95% CI 0.94-1.1). In Pima Indians with type 2 diabetes, higher plasma glucose concentration predicts deaths from cardiovascular and diabetes-related diseases but has little or no effect on deaths from other natural or external causes.     

Normal coding sequence of insulin gene in Pima Indians and Nauruans, two groups with highest prevalence of type II diabetes

The nucleotide sequence of the insulin gene was determined in American Pima Indians and Micronesian Nauruans, two populations in whom the prevalence of non-insulin-dependent (type II) diabetes mellitus is the highest in the world. The insulin gene was amplified by the polymerase chain reaction to generate single-stranded DNA suitable for direct sequencing. The nucleotide sequences of the coding and adjacent regions of the insulin gene in six Pima Indians and two Nauruans with type II diabetes were identical to previously published insulin gene sequences of nondiabetic subjects.     

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.     

Genome-wide linkage analysis assessing parent-of-origin effects in the inheritance of type 2 diabetes and BMI in Pima Indians.

We examined the hypothesis that imprinted genes may affect the propensity to type 2 diabetes and obesity in Pima Indians. Multipoint variance component methods were used to assess linkage of BMI (kg/m(2)) and age-adjusted diabetes to loci derived from either father (LOD(FA)) or mother (LOD(MO)) in a genome-wide scan. Tentative evidence of loci where imprinted genes might be acting was found for diabetes with maternally derived alleles on chromosomes 5 (LOD(MO) = 1.5) and 14 (LOD(MO) = 1.6). Evidence of linkage of BMI to maternally derived alleles was found on chromosome 5 (LOD(MO) = 1.7) and to paternally derived alleles on chromosome 10p (LOD(FA) = 1.7). Additional analyses of sibling pairs who were affected by diabetes and younger than 25 years of age showed an increase of sharing of maternally derived alleles on chromosome 6 (LOD(MO) = 3.0). We also examined sites of a priori interest where action of imprinted genes has been proposed in diabetes or obesity. We found no evidence of parent-specific linkage (of either diabetes or BMI) on chromosome 11p, a region that contains several imprinted genes, but observed weak evidence of linkage of diabetes to paternally derived alleles (LOD(FA) = 0.9) in the region of chromosome 6q, believed to contain an exclusively paternally expressed gene or genes that cause transient neonatal diabetes mellitus. In conclusion, we determined regions of interest on chromosomes 5, 6, and 10 where imprinted genes might be affecting the risk of type 2 diabetes or obesity in Pima Indians.     

Positional candidate gene analysis of Lim domain homeobox gene (Isl-1) on chromosome 5q11-q13 in a French morbidly obese population suggests indication for association with type 2 diabetes

The Lim domain homeobox gene (Isl-1) is a positional candidate gene for obesity that maps on chromosome 5q11-q13, a locus linked to BMI and leptin levels in French Caucasians. Isl-1 might be involved in body weight regulation and glucose homeostasis via the activation of proglucagon gene expression, which encodes for glucagon and glucagon-like peptides. By mutation screening of 72 obese subjects, we identified three single-nucleotide polymorphisms (SNPs) in the Isl1 gene. The allele frequencies in the morbidly obese group did not differ from that of the control group. In the obese group, the -47G allele was associated with a decreased risk of type 2 diabetes (odds ratio 0.41, P = 0.019). The AG bearers displayed a higher maximal BMI than the AA bearers in the whole obese group (P = 0.026) as well as in the type 2 diabetic obese subgroup (P = 0.014). In obese families, this allele was not preferentially transmitted from heterozygous parents to their obese siblings, indicating that Isl-1 does not contribute to the linkage with obesity on 5cen-q. However, in French Caucasian morbidly obese subjects, the Isl1-47A-->G SNP may modulate the risk for type 2 diabetes and may increase body weight in diabetic morbidly obese subjects.