Assessment of the potential role of natural selection in type 2 diabetes and related traits across human continental ancestry groups: comparison of phenotypic with genotypic divergence.

AIMS/HYPOTHESIS: Prevalence of type 2 diabetes differs among human ancestry groups, and many hypotheses invoke differential natural selection to account for these differences. We sought to assess the potential role of differential natural selection across major continental ancestry groups for diabetes and related traits, by comparison of genetic and phenotypic differences. METHODS: This was a cross-sectional comparison among 734 individuals from an urban sample (none of whom was more closely related to another than third-degree relatives), including 83 African Americans, 523 American Indians and 128 European Americans. Participants were not recruited based on diabetes status or other traits. BMI was calculated, and diabetes was diagnosed by a 75 g oral glucose tolerance test. In those with normal glucose tolerance (n = 434), fasting insulin and 30 min post-load insulin, adjusted for 30 min glucose, were taken as measures of insulin resistance and secretion, respectively. Whole exome sequencing was performed, resulting in 97,388 common (minor allele frequency ≥ 5%) variants; the coancestry coefficient (FST) was calculated across all markers as a measure of genetic divergence among ancestry groups. The phenotypic divergence index (PST) was also calculated from the phenotypic differences and heritability (which was estimated from genetic relatedness calculated empirically across all markers in 761 American Indian participants prior to the exclusion of close relatives). Under evolutionary neutrality, the expectation is PST = FST, while for traits under differential selection PST is expected to be significantly greater than FST. A bootstrap procedure was used to test the hypothesis PST = FST. RESULTS: With adjustment for age and sex, prevalence of type 2 diabetes was 34.0% in American Indians, 12.4% in African Americans and 10.4% in European Americans (p = 2.9 × 10-10 for difference among groups). Mean BMI was 36.3, 33.4 and 33.0 kg/m2, respectively (p = 1.9 × 10-7). Mean fasting insulin was 63.8, 48.4 and 45.2 pmol/l (p = 9.2 × 10-5), while mean 30 min insulin was 559.8, 553.5 and 358.8 pmol/l, respectively (p = 5.7 × 10-8). FST across all markers was 0.130, while PST for liability to diabetes, adjusted for age and sex, was 0.149 (p = 0.35 for difference with FST). PST was 0.094 for BMI (p = 0.54), 0.095 for fasting insulin (p = 0.54) and 0.216 (p = 0.18) for 30 min insulin. For type 2 diabetes and BMI, the maximum divergence between populations was observed between American Indians and European Americans (PST-MAX = 0.22, p = 0.37, and PST-MAX = 0.14, p = 0.61), which suggests that a relatively modest 22% or 14% of the genetic variance, respectively, can potentially be explained by differential selection (assuming the absence of neutral drift). CONCLUSIONS/INTERPRETATION: These analyses suggest that while type 2 diabetes and related traits differ significantly among continental ancestry groups, the differences are consistent with neutral expectations based on heritability and genetic distances. While these analyses do not exclude a modest role for natural selection, they do not support the hypothesis that differential natural selection is necessary to explain the phenotypic differences among these ancestry groups. Graphical abstract.

Exome Sequencing Identifies A Nonsense Variant in DAO Associated With Reduced Energy Expenditure in American Indians.

BACKGROUND: Obesity and energy expenditure (EE) are heritable and genetic variants influencing EE may contribute to the development of obesity. We sought to identify genetic variants that affect EE in American Indians, an ethnic group with high prevalence of obesity. METHODS: Whole-exome sequencing was performed in 373 healthy Pima Indians informative for 24-hour EE during energy balance. Genetic association analyses of all high-quality exonic variants (≥5 carriers) was performed, and those predicted to be damaging were prioritized. RESULTS: Rs752074397 introduces a premature stop codon (Cys264Ter) in DAO and demonstrated the strongest association for 24-hour EE, where the Ter allele associated with substantially lower 24-hour EE (mean lower by 268 kcal/d) and sleeping EE (by 135 kcal/d). The Ter allele has a frequency = 0.5% in Pima Indians, whereas is extremely rare in most other ethnic groups (frequency < 0.01%). In vitro functional analysis showed reduced protein levels for the truncated form of DAO consistent with increased protein degradation. DAO encodes D-amino acid oxidase, which is involved in dopamine synthesis which might explain its role in modulating EE. CONCLUSION: Our results indicate that a nonsense mutation in DAO may influence EE in American Indians. Identification of variants that influence energy metabolism may lead to new pathways to treat human obesity. CLINICAL TRIAL REGISTRATION NUMBER: NCT00340132.

A Genome-Wide Association Study Using a Custom Genotyping Array Identifies Variants in GPR158 Associated With Reduced Energy Expenditure in American Indians.

Pima Indians living in Arizona have a high prevalence of obesity, and we have previously shown that a relatively lower energy expenditure (EE) predicts weight and fat mass gain in this population. EE is a familial trait (heritability = 0.52); therefore, in the current study, we aimed to identify genetic variants that affect EE and thereby influence BMI and body fatness in Pima Indians. Genotypic data from 491,265 variants were analyzed for association with resting metabolic rate (RMR) and 24-h EE assessed in a whole-room calorimeter in 507 and 419 Pima Indians, respectively. Variants associated with both measures of EE were analyzed for association with maximum BMI and percent body fat (PFAT) in 5,870 and 912 Pima Indians, respectively. rs11014566 nominally associated with both measures of EE and both measures of adiposity in Pima Indians, where the G allele (frequency: Pima Indians = 0.60, Europeans <0.01) associated with lower 24-h EE (ß = -33 kcal/day per copy), lower RMR (ß = -31 kcal/day), higher BMI (ß = +0.6 kg/m2), and higher PFAT (ß = +0.9%). However, the association of rs11014566 with BMI did not directionally replicate when assessed in other ethnic groups. rs11014566 tags rs144895904, which affected promoter function in an in vitro luciferase assay. These variants map to GPR158, which is highly expressed in the brain and interacts with two other genes (RGS7 and CACNA1B) known to affect obesity in knockout mice. Our results suggest that common ethnic-specific variation in GPR158 may influence EE; however, its role in weight gain remains controversial, as it either had no association with BMI or associated with BMI but in the opposite direction in other ethnic groups.

Assessment of established HDL-C loci for association with HDL-C levels and type 2 diabetes in Pima Indians.

AIMS/HYPOTHESIS: Epidemiological studies in Pima Indians identified elevated levels of HDL-cholesterol (HDL-C) as a protective factor against type 2 diabetes risk in women. We assessed whether HDL-C-associated single-nucleotide polymorphisms (SNPs) also associate with type 2 diabetes in female Pima Indians. METHODS: Twenty-one SNPs in established HDL-C loci were initially analysed in 2,675 full-heritage Pima Indians. SNPs shown to associate with HDL-C (12 SNPs) were assessed for association with type 2 diabetes in 7,710 Pima Indians (55.6% female sex). The CETP locus provided the strongest evidence for association with HDL-C and was further interrogated by analysing tag SNPs. RESULTS: Twelve of the 21 SNPs analysed had a significant association with HDL-C in Pima Indians; five SNPs representing four loci (CETP, DOCK6, PPP1R3B and ABCA1) reached genome-wide significance. Three SNPs, at CETP, KLF14 and HNF4A, associated with type 2 diabetes only in female participants with the HDL-C-lowering allele increasing diabetes risk (p values: 3.2 × 10(-4) to 7.7 × 10(-5)); the association remained significant even after adjustment for HDL-C. Additional analysis across CETP identified rs6499863 as having the strongest association with type 2 diabetes in female participants (p = 5.0 × 10(-6)) and this association remained independent of the HDL-C association. CONCLUSIONS/INTERPRETATION: SNPs at the CETP, HNF4A and KLF14 locus are associated with HDL-C levels and type 2 diabetes (in female participants). However, since HNF4A and KLF14 are established loci for type 2 diabetes, it is unlikely that HDL-C solely mediates these associations.

Variants associated with type 2 diabetes identified by the transethnic meta-analysis study: assessment in American Indians and evidence for a new signal in LPP.

AIM/HYPOTHESIS: A recent genome-wide trans-ancestry meta-analysis identified seven new loci associated with type 2 diabetes. We assessed the replication of the seven lead single nucleotide polymorphisms (SNPs) and evaluated these loci for additional signals in American Indians. METHODS: Seven SNPs were genotyped in 7,710 individuals from a longitudinally studied American Indian population, and associations with type 2 diabetes, BMI and related phenotypes were assessed. Previous genome-wide association study (GWAS) data from these individuals were used to screen for additional type 2 diabetes signals at these loci. A variant independent of the trans-ancestry meta-analysis was identified within LPP, and its replication was assessed in an additional 3,106 urban American Indians. RESULTS: SNP rs6813195 near to TMEM154 was nominally associated with type 2 diabetes (p = 0.01, OR 1.12 [95% CI 1.03, 1.22]) and adiposity: the type 2 diabetes risk allele was associated with a lower percentage body fat (ß = -1.451%, p = 4.8 × 10(-4)). Another SNP, rs3130501 near to POU5F1-TCF19, was associated with BMI (ß = -0.012, p = 0.004), type 2 diabetes adjusted for BMI (p = 0.02, OR 1.11 [95% CI 1.02, 1.22]), 2 h glucose concentrations (ß = 0.080 mmol/l, p = 0.02) and insulin resistance estimated by homeostatic model (ß = 0.039, p = 0.009). The independent variant identified at the LPP locus in our American Indian GWAS for type 2 diabetes was replicated in the additional samples (all American Indian meta-analysis, p = 8.9 × 10(-6), OR 1.29 [95% CI 1.15, 1.45]). CONCLUSIONS/INTERPRETATION: For two of the seven newly identified variants, there was nominal evidence for association with type 2 diabetes and related traits in American Indians. Identification of an independent variant at the LPP locus suggests the existence of more than one type 2 diabetes signal at this locus.

Common genetic variation in the glucokinase gene (GCK) is associated with type 2 diabetes and rates of carbohydrate oxidation and energy expenditure.

AIMS/HYPOTHESIS: Glucokinase (GCK) plays a role in glucose metabolism and glucose-stimulated insulin secretion. Rare mutations in GCK cause MODY. We investigated whether common variation (minor allele frequency ≥0.01) in GCK is associated with metabolic traits and type 2 diabetes. METHODS: Four exonic single-nucleotide polymorphisms (SNPs) and three SNPs predicted to cause loss of promoter function were identified in whole-genome sequence data from 234 Pima Indians. These seven tag SNPs and rs4607517, a type 2 diabetes variant established in other studies, were analysed in 415 full-heritage non-diabetic Pima Indians characterised for metabolic traits, and 7,667 American Indians who had data on type 2 diabetes and BMI. RESULTS: A novel 3' untranslated region (3'UTR) SNP, chr7:44184184-G/A, was associated with the rate of carbohydrate oxidation post-absorptively (ß = 0.22 mg [kg estimated metabolic body size (EMBS)](-1) min(-1), p = 0.005) and during a hyperinsulinaemic-euglycaemic clamp (ß = 0.24 mg [kg EMBS](-1) min(-1), p = 0.0002), the rate of carbohydrate oxidation in a respiratory chamber (ß = 311 kJ/day, p = 0.03) and 24 h energy expenditure, which was attributable to the thermic effect of food (ß = 520 kJ/day, p = 3.39 × 10(-6)). This 3'UTR SNP was also associated with diabetes (OR 1.36, 95% CI 1.11, 1.65, p = 0.002), where the A allele (allele frequency 0.05) was associated with a lower rate of carbohydrate oxidation, lower 24 h energy expenditure and higher risk for diabetes. In a Cox proportional hazards model, a rate of insulin-stimulated carbohydrate oxidation lower than the mean rate at baseline predicted a higher risk for developing diabetes than for those above the mean (hazard rate ratio 2.2, 95% CI 1.3, 3.6, p = 0.002). CONCLUSIONS/INTERPRETATION: Common variation in GCK influences the rate of carbohydrate oxidation, 24 h energy expenditure and diabetes risk in Pima Indians.

Variants in the LEPR gene are nominally associated with higher BMI and lower 24-h energy expenditure in Pima Indians.

Genome-wide association studies (GWASs) have been used to search for susceptibility genes for type 2 diabetes and obesity in the Pima Indians, a population with a high prevalence of both diseases. In these studies, a variant (rs2025804) in the LEPR gene was nominally associated with BMI in 1,082 subjects (P = 0.03 adjusted for age, sex, birth year, and family membership). Therefore the LEPR and leptin overlapping transcript (LEPROT) genes were selected for further sequencing and genotyping in larger population-based samples for association analyses with obesity-related phenotypes. Selected variants (n = 80) spanning these genes were genotyped in a sample of full-heritage Pima Indians (n = 2,842) and several common variants including rs2025804 were nominally associated with BMI (P = 0.05-0.003 adjusted for age, sex, birth year, and family membership). Four common tag variants associated with BMI in the full-heritage Pima Indian sample were genotyped in a second sample of mixed-heritage Native Americans (n = 2,969) and three of the variants showed nominal replication (P = 0.03-0.006 adjusted as above and additionally for Indian heritage). Combining both samples provided the strongest evidence for association (adjusted P = 0.0003-0.0001). A subset of these individuals (n = 403) had been metabolically characterized for predictors of obesity and the BMI risk alleles for the variants tagged by rs2025804 were also associated with lower 24-h energy expenditure (24hEE) as assessed in a human respiratory chamber (P = 0.0007 adjusted for age, sex, fat mass, fat-free mass, activity, and family membership). We conclude that common noncoding variation in the LEPR gene is associated with higher BMI and lower energy expenditure in Native Americans.