Analysis of SLC16A11 Variants in 12,811 American Indians: Genotype-Obesity Interaction for Type 2 Diabetes and an Association With RNASEK Expression

Genetic variants in SLC16A11 were recently reported to be associated with type 2 diabetes in Mexican and other Latin American populations. The diabetes risk haplotype had a frequency of 50% in Native Americans from Mexico but was rare in Europeans and Africans. In the current study, we analyzed SLC16A11 in 12,811 North American Indians and found that the diabetes risk haplotype, tagged by the rs75493593 A allele, was nominally associated with type 2 diabetes (P = 0.001, odds ratio 1.11). However, there was a strong interaction with BMI (P = 5.1 × 10(-7)) such that the diabetes association was stronger in leaner individuals. rs75493593 was also strongly associated with BMI in individuals with type 2 diabetes (P = 3.4 × 10(-15)) but not in individuals without diabetes (P = 0.77). Longitudinal analyses suggest that this is due, in part, to an association of the A allele with greater weight loss following diabetes onset (P = 0.02). Analyses of global gene expression data from adipose tissue, skeletal muscle, and whole blood provide evidence that rs75493593 is associated with expression of the nearby RNASEK gene, suggesting that RNASEK expression may mediate the effect of genotype on diabetes

Whole-Genome Sequence Identifies Potentially Functional Variants in Cytochrome B5 Type A (CYB5A) that Associate with Obesity in Southwest American Indians

Southwest American Indians (SAI) suffer from a high obesity prevalence. We previously reported that rs7238987, in cytochrome B5 type A (CYB5A), strongly associated with increased body mass index (BMI) in a population-based study of SAI. CYB5A plays a role in fatty acid oxidation (FAO) and reductions in FAO have been linked to weight gain. However, in vitro studies did not confirm a functional effect of this variant. Therefore, the goal of the current study was to assess variation across the entire CYB5A locus to identify the causal variant contributing to obesity in this population. Genotypic data from a custom Axiom genotyping array on 7,700 SAI was merged with whole-genome sequence data from 335 SAI to impute variation within and 10,000 bp surrounding the CYB5A gene. This region contained 316 variants with a minor allele frequency >0.01; 3 variants had a Combined Annotation Dependent Depletion (CADD) score >10 (top 10% most likely to be deleterious) and a BMI association P ≤ 0.001 (adjusted for age, sex, birth year and the first 5 genetic principle components). Among these potentially deleterious variants, the obesity-risk T-allele in rs548402150, also significantly associated (P = 0.00003) with decreased CYB5A expression in skeletal muscle from biopsy data (n=207). Therefore, rs548402150 was prioritized for functional studies. This C/T variation falls within a CTCF consensus site in the 5’-UTR, which could have the ability to effect gene expression levels. In vitro luciferase reporter assays were used to assess altered promoter activity in human skeletal muscle cells. A 30% decrease in luciferase expression was identified for the promoter carrying the obesity-risk T-allele compared to the non-risk C-allele (P = 0.003). In summary, the further exploration of CYB5A using imputed data has identified a potential casual variant for the observed decreased CYB5A expression and increased risk of obesity in this population

Functional variants in cytochrome b5 type A (CYB5A) are enriched in Southwest American Indian individuals and associate with obesity

Objective This study aimed to identify genetic variants enriched in Southwest American Indian (SWAI) individuals that associate with BMI. Methods Whole genome sequencing data (n = 296) were used to identify potentially functional variants that are common in SWAI individuals (minor allele frequency ≥10%) but rare in other ethnic groups (minor allele frequency < 0.1%). Enriched variants were tested for association with BMI in 5,870 SWAI individuals. One variant was studied using a luciferase reporter, and haplotypes that included this variant were analyzed for association with various measures of obesity (n = 917-5,870), 24-hour energy expenditure (24-h EE; n = 419), and skeletal muscle biopsy expression data (n = 207). Results A 5′ untranslated region variant in cytochrome b5 type A (CYB5A), rs548402150, met the enrichment criteria and associated with increased BMI (β = 2%, p = 0.004). Functionally, rs548402150 decreased luciferase expression by 30% (p = 0.003) and correlated with decreased skeletal muscle CYB5A expression (β = −0.5 SD, p = 0.0008). Combining rs548402150 with two splicing quantitative trait loci in CYB5A identified a haplotype carried almost exclusively in SWAI individuals that associated with increased BMI (β = 3%, p = 0.0003) and decreased CYB5A expression, whereas the most common haplotype in all ethnic groups associated with lower BMI and percentage of body fatness, increased 24-h EE, and increased CYB5A expression. Conclusions Further studies on the effects of CYB5A on 24-h EE and BMI may provide insights into obesity-related physiology

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

2116-P: A Pipeline to Explore Rare Variation Which Can Contribute to Extreme Obesity in American Indians

Identification of high-impact loss-of-function variants for common disease is challenging because these variants are typically rare and thus statistical power is low when sample sizes are limited. In this study, we utilized whole-exome variant detection, variant prediction programs, tissue expression data, and case-control analysis to identify potentially highly penetrant obesity-susceptibility variants for prioritization for functional testing. Whole-exome sequencing was performed on DNA samples collected from a longitudinal, population-based study of American Indians (N=5432; maximum BMI recorded at ≥18 years). Among the 1 million biallelic SNVs detected in this cohort, those that were exonic or splicing, have minor allele frequency (MAF) ≤0.05, predicted to be damaging (CADD score ≥15), and located in genes expressed in hypothalamus were selected for analysis. These selection criteria resulted in 94,444 SNVs, and odds ratios were calculated for them by comparing the group of individuals with BMI in the top 5th percentile for this population (N=271, BMI ≥53.13 kg/m2) vs. those below the median (N=2,717, BMI <35.92 kg/m2). The resulting 19,382 variants with positive OR values were then divided into 4 bins based on MAFs, and those with a OR in the top 1% of the distribution of each bin were prioritized. In the resulting list of 291variants in 266 different genes, a missense variant p.I330V (rs201597085; OR=2.66; CADD=20.3; MAF=0.02) in GNAS was the best candidate, since GNAS was the only gene linked to obesity in humans. GNAS is a highly complex imprinted locus, and the main transcript derived from this locus (Gs-alpha) encodes the alpha subunit of the stimulatory guanine nucleotide-binding protein. The XLas isoform, a large variant of Gs-alpha, is paternally expressed. An association analysis with accountance for parental origin of the alleles is ongoing. Future functional studies would entail examining the effects of this missense variant on G protein-coupled receptor signaling