CYP7A1 Variants Are Associated with Postabsorptive Endogenous Glucose Production: A Genome-Wide Association Study in Nondiabetic Pima Indians

Increased post-absorptive endogenous glucose production (PEGP) is a major contributor to fasting hyperglycemia in individuals with type 2 diabetes (T2D). As compared to individuals with fasting normoglycemia, PEGP is increased at higher fasting glucose concentrations, suggesting that elevated PEGP is a marker of hyperglycemia and may be a metabolic risk factor for T2D. The aim of this study was to identify genetic variants associated with PEGP in non-diabetic Pima Indians, an ethnic group with high prevalence and heritability of T2D. A genome-wide association study (GWAS) was performed in a cohort of 552 non-diabetic Pima Indians (323M/229F, mean±SD, age: 27±6 yr, body fat: 32±8%, fasting glucose=89±10 mg/dL) using an Affymetrix Axiom Custom Array including 489,397 single-nucleotide polymorphisms (SNPs) selected to tag common variation (minor allele frequency≥5%, r2≥0.85) detected in Pima sequence data. PEGP was measured after an overnight fast by infusion of 3-[3H] glucose for 120 minutes before a hyperinsulinemic-euglycemic glucose clamp, and expressed per kg of fat free mass (FFM) estimated by hydrodensitometry or DXA. No SNP achieved genome-wide statistical significance (p=5x10-8), with the smallest 10 p-values ranging from 7x10-7 to 2x10-5. The top GWAS signal for PEGP, rs2162459 (p=7x10-7, with genomic control, adjusted for age, gender, percent body fat, heritage), maps to an intron in CYP7A1, and the G allele (frequency=0.23) is associated with higher PEGP (b=+0.09 mg/kg FFM/min per allele copy). This variant accounts for 5% of PEGP variance. CYP7A1 encodes the cholesterol 7α-hydroxylase which is the key regulatory enzyme converting cholesterol into bile acids in the liver. Increased expression of hepatic CYP7A1 improves insulin resistance in transgenic mice on a high-fat diet. However, rs2162459 is not associated with whole-body insulin action in Pima Indians (p=0.98). Our results suggest that common variation in CYP7A1 may influence PEGP in non-diabetic Pima Indians

Low Serum Insulinlike Growth Factor II Levels Correlate with High BMI in American Indian Adults

OBJECTIVE: Insulinlike growth factor II (IGF-II) regulates metabolism and growth. In humans, both positive and negative relationships have been reported between serum IGF-II levels and obesity. This study assessed the relationship between serum IGF-II levels and BMI and determined whether IGF-II levels predict weight gain. METHODS: Serum samples were available from 911 American Indians with a recorded BMI. IGF-II was measured using enzyme-linked immunosorbent assay. RESULTS: Serum IGF-II levels were negatively correlated with BMI (r = -0.17, P = 4.4 × 10-7 , adjusted for age, sex, and storage time). The strongest correlation was in participants aged ≥ 30 years (r = -0.28, P = 3.4 × 10-8 , N = 349), a modest correlation was in participants aged 20 to 29 years (r = -0.15, P = 7.6 × 10-3 , N = 322), and participants aged 15 to 19 years had no correlation (r = 0.05, P = 0.48, N = 240). IGF-II levels did not predict weight gain. However, among individuals who had genotypes for 64 established obesity variants (age ≥ 20 years, N = 671), a genetic risk score for high BMI was associated with lower IGF-II (β = -0.08 SD of IGF-II per SD of the genetic risk score, P = 0.025). CONCLUSIONS: There is a negative relationship between IGF-II levels and BMI, in which the correlation is stronger at older ages. The association between genetic risk for BMI and IGF-II levels suggests that this correlation may be due to an effect of obesity on IGF-II

Generation of Isogenic hiPSCs with Targeted Edits at Multiple Intronic SNPs to Study the Effects of the Type 2 Diabetes Associated KCNQ1 Locus in American Indians

The top genetic association signal for type 2 diabetes (T2D) in Southwestern American Indians maps to intron 15 of KCNQ1, an imprinted gene. We aim to understand the biology whereby variation at this locus affects T2D specifically in this genomic background. To do so, we obtained human induced pluripotent stem cells (hiPSC) derived from American Indians. Using these iPSCs, we show that imprinting of KCNQ1 and CDKN1C during pancreatic islet-like cell generation from iPSCs is consistent with known imprinting patterns in fetal pancreas and adult islets and therefore is an ideal model system to study this locus. In this report, we detail the use of allele-specific guide RNAs and CRISPR to generate isogenic hiPSCs that differ only at multiple T2D associated intronic SNPs at this locus which can be used to elucidate their functional effects. Characterization of these isogenic hiPSCs identified a few aberrant cell lines; namely cell lines with large hemizygous deletions in the putative functional region of KCNQ1 and cell lines hypomethylated at the KCNQ1OT1 promoter. Comparison of an isogenic cell line with a hemizygous deletion to the parental cell line identified CDKN1C and H19 as differentially expressed during the endocrine progenitor stage of pancreatic-islet development

A Genome-Wide Association Study of Acute Insulin Secretory Response in Pima Indians

Many of the established genes for type 2 diabetes (T2D) identified via genome-wide association studies appear to increase risk via an effect on acute insulin secretory response to glucose (AIR). Although the AIR is highly heritable in Pima Indians, established T2D genes account for only a fraction of this heritability. We performed a genome-wide analysis to identify variants that associate consistently with 2 different measures of acute insulin secretory function and with T2D. Genotypic data on 491,288 SNPs tagging 92% of common variation identified in whole genome sequences of Pima Indians (minor allele frequency ≥0.05, r2 ≥0.85) were available on 3708 full heritage Pima Indians informative for T2D. Among these, 300 (193 men, age: 27±6 yr, body fat: 31±8%) also had a measure of AIR when they had normal glucose tolerance. AIR was measured after an overnight fast by infusion of 25 g dextrose for 3 minutes, and calculated as the average increment in plasma insulin concentration from the 3rd to the 5th min after the glucose bolus. The corrected insulin response (CIR) was calculated at 30’ during the OGTT. No SNP achieved genome-wide statistical significance (p=5x10-8) for AIR or CIR (451 and 438 SNPs, respectively, had p<10−3 after genomic control, adjusted for age, sex, body fat, insulin action assessed during a glucose clamp, the first 5 genetic principal components and sibship, with 19 SNPs associated with both traits with directional consistency). However, one intronic SNP in TMEM132D (rs4301843) was consistent for the 2 traits and T2D, where the C allele (frequency=0.72) associated with lower AIR (b=−20% per copy, p=10-4), lower CIR (b=−21%, p=10-4) and higher T2D prevalence (OR=1.32, 95% CI: 1.14-1.52, p=2×10-4). TMEM132D encodes the transmembrane protein 132D, which may have an indirect role in T2D onset by binding to PPP1CA and other established T2D genes such as KCNQ1. Our results suggest that common variation in TMEM132D may influence AIR and predispose Pima Indians to T2D