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

Missense Variant in Ceramide Synthase 2 (CERS2) is Associated with Higher Overnight Energy Expenditure and Hepatic Insulin Resistance in Healthy Humans

Research aim Genetic determinants of inter-individual differences in energy expenditure (EE) remain largely unknown. Participating in cellular signaling pathways, ceramides were identified as putative EE regulators. Current study investigated whether genetic variants within enzymes involved in ceramide synthesis and degradation affect EE. Methods EE was assessed for 24h by whole-room indirect calorimetry in a large cohort of Indigenous Americans informative for genome-wide imputa-tion data, body composition, and glucose disposal rate during euglycemic-hyperinsulinemic clamp. Real time cellular metabolism was measured using a Seahorse Analyzer. Association analyses of imputed genotype dosages with metabolic measurements were performed via linear mixed effects. Differences in mitochondrial respiration were assessed via mixed model analyses of repeated measurements obtained during the Seahorse experiments. Results Compared to the A allele, heterozygosity for missense variant rs267738 (E115A; A>C) in exon 4 of ceramide synthase 2 (CERS2) was associated with substantially higher sleeping EE (average=+116 kcal/day independent from differences in body composition) and increased rates of endogenous glucose production during insulin-stimulated (mean=+43%) and fasting (mean=+5%) conditions, both being markers of increased hepatic insulin resistance. The C allele for rs267738 did not affect ceramide synthesis in human hepatoma HepG2 cells but led to decreased (mean=−30%) basal mitochondrial respiration in vitro. Conclusions These findings are in line with previous mice studies that implicate CerS2 in hepatic insulin resistance and impaired mitochondrial respiration. In summary, we provide evidence that rs267738 in CERS2 affect human metabolism and induce hepatic insulin resistance, presumably via impairment of insulin signaling and alteration of mitochondrial function

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

Identification and characterization of the long non-coding RNA NFIA-AS2 as a novel locus for body mass index in American Indians

Background: Genome-wide association studies have shown that body mass index (BMI), an estimate of obesity, is highly polygenic. Individual variants typically have small effect sizes, making it challenging to identify unique loci in under-represented ethnic groups which lack statistical power due to their small sample size. Yet obesity is a major health disparity and is particularly prevalent in southwestern American Indians. Here, we identify and characterize a new locus for BMI that was detected by analyzing moderate associations with BMI obtained in a population-based sample of southwestern American Indians together with the well-powered GIANT dataset. Methods: Genotypes for 10.5 million variants were tested for association with BMI in 5870 American Indians and 2600 variants that showed an association P < 10-3 in the American Indian sample were combined in a meta-analysis with the BMI data reported in GIANT (N = 240,608). The newly identified gene, NFIA-AS2 was functionally characterized, and the impact of its lead associated variant rs1777538 was studied both in-silico and in-vitro. Results: Rs1777538 (T/C; C allele frequency = 0.16 in American Indians and 0.04 in GIANT, meta-analysis P = 5.0 × 10-7) exhibited a large effect in American Indians (1 kg/m2 decrease in BMI per copy of C allele). NFIA-AS2 was found to be a nuclear localized long non-coding RNA expressed in tissues pertinent to human obesity. Analysis of this variant in human brown preadipocytes showed that NFIA-AS2 transcripts carrying the C allele had increased RNA degradation compared to the T allele transcripts (half-lives = 9 h, 13 h respectively). During brown adipogenesis, NFIA-AS2 featured a stage-specific regulation of nearby gene expression where rs1777538 demonstrated an allelic difference in regulation in the mature adipocytes (the strongest difference was observed for L1TD1, P = 0.007). Conclusion: Our findings support a role for NFIA-AS2 in regulating pathways that impact BMI

An E115A missense variant in CERS2 is associated with increased sleeping energy expenditure and hepatic insulin resistance in American Indians

Genetic determinants of interindividual differences in energy expenditure (EE) are largely unknown. Sphingolipids, such as ceramides, have been implicated in the regulation of human EE via mitochondrial uncoupling. In this study, we investigated whether genetic variants within enzymes involved in sphingolipid synthesis and degradation affect EE and insulin-related traits in a cohort of American Indians informative for 24-h EE and glucose disposal rates during a hyperinsulinemic-euglycemic clamp. Association analysis of 10,084 genetic variants within 28 genes involved in sphingolipid pathways identified a missense variant (rs267738, A>C, E115A) in exon 4 of CERS2 that was associated with higher sleeping EE (116 kcal/day) and increased rates of endogenous glucose production during basal (5%) and insulin-stimulated (43%) conditions, both indicators of hepatic insulin resistance. The rs267738 variant did not affect ceramide synthesis in HepG2 cells but resulted in a 30% decrease in basal mitochondrial respiration. In conclusion, we provide evidence that the CERS2 rs267738 missense variant may influence hepatic glucose production and postabsorptive sleeping metabolic rate

Pleiotropic effects of an eQTL in the CELSR2/PSRC1/SORT1 cluster that associates with LDL-C and resting metabolic rate

Context: The locus CELSR2-PSRC1-SORT1, a primary genetic signal for lipids, has recently been implicated in different metabolic processes. Our investigation identified its association with energy metabolism. Objective: To determine biological mechanisms that govern diverse functions of this locus. Methods: Genotypes for 491,265 variants in 7,000 clinically characterized American Indians were previously determined using a custom-designed array specific for this longitudinally studied American Indian population. Among the genotyped individuals, 5,205 had measures of fasting lipid levels and 509 had measures of resting metabolic rate (RMR) and substrate oxidation rate assessed through indirect calorimetry. A genome-wide association study (GWAS) for LDL-C levels identified a variant in CELSR2 and the molecular impact of this variant on gene expression was assessed in skeletal muscle biopsies from 207 participants, followed by functional validation in mouse myoblasts using a luciferase assay. Results: A GWAS in American Indians identified rs12740374 in CELSR2 as the top signal for LDL-C levels (P = 1 × 10-22); further analysis of this variant identified an unexpected correlation with reduced RMR (effect = -44.3 kcal/day/minor-allele) and carbohydrate oxidation rate (effect = -5.21 mg/hour/kg-EMBS). Tagged variants showed a distinct linkage disequilibrium architecture in American Indians, highlighting a potential functional variant, rs6670347 (minor-allele frequency = 0.20). Positioned in the glucocorticoid receptor's core binding motif, rs6670347 is part of a skeletal muscle-specific enhancer. Human skeletal muscle transcriptome analysis showed CELSR2 as the most differentially expressed gene (P = 1.9 × 10-7), with the RMR-lowering minor allele elevating gene expression. Experiments in mouse myoblasts confirmed enhancer-based regulation of CELSR2 expression, dependent on glucocorticoids. Rs6670347 also associated with increased oxidative phosphorylation gene expression; CELSR2 as a regulator of these genes, suggests potential influence on energy metabolism through muscle oxidative capacity. Conclusion: Variants in the CELSR2/PSRC1/SORT1 locus exhibit tissue-specific effects on metabolic traits, with an independent role in muscle metabolism through glucocorticoid signaling

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