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Aims/hypothesis There is limited information on how polygenic scores (PSs), based on variants from genome-wide association studies (GWASs) of type 2 diabetes, add to clinical variables in predicting type 2 diabetes incidence, particularly in non-European-ancestry populations. Methods For participants in a longitudinal study in an Indigenous population from the Southwestern USA with high type 2 diabetes prevalence, we analysed ten constructions of PS using publicly available GWAS summary statistics. Type 2 diabetes incidence was examined in three cohorts of individuals without diabetes at baseline. The adult cohort, 2333 participants followed from age ≥20 years, had 640 type 2 diabetes cases. The youth cohort included 2229 participants followed from age 5–19 years (228 cases). The birth cohort included 2894 participants followed from birth (438 cases). We assessed contributions of PSs and clinical variables in predicting type 2 diabetes incidence. Results Of the ten PS constructions, a PS using 293 genome-wide significant variants from a large type 2 diabetes GWAS meta-analysis in European-ancestry populations performed best. In the adult cohort, the AUC of the receiver operating characteristic curve for clinical variables for prediction of incident type 2 diabetes was 0.728; with the PS, 0.735. The PS’s HR was 1.27 per SD (p=1.6 × 10−8; 95% CI 1.17, 1.38). In youth, corresponding AUCs were 0.805 and 0.812, with HR 1.49 (p=4.3 × 10−8; 95% CI 1.29, 1.72). In the birth cohort, AUCs were 0.614 and 0.685, with HR 1.48 (p=2.8 × 10−16; 95% CI 1.35, 1.63). To further assess the potential impact of including PS for assessing individual risk, net reclassification improvement (NRI) was calculated: NRI for the PS was 0.270, 0.268 and 0.362 for adult, youth and birth cohorts, respectively. For comparison, NRI for HbA1c was 0.267 and 0.173 for adult and youth cohorts, respectively. In decision curve analyses across all cohorts, the net benefit of including the PS in addition to clinical variables was most pronounced at moderately stringent threshold probability values for instituting a preventive intervention. Conclusions/interpretation This study demonstrates that a European-derived PS contributes significantly to prediction of type 2 diabetes incidence in addition to information provided by clinical variables in this Indigenous study population. Discriminatory power of the PS was similar to that of other commonly measured clinical variables (e.g. HbA1c). Including type 2 diabetes PS in addition to clinical variables may be clinically beneficial for identifying individuals at higher risk for the disease, especially at younger ages. Graphical abstract

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 −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.

Insulin-like growth factor binding protein 4 (IGFBP4) is involved in adipogenesis, and IGFBP4 null mice have decreased body fat through decreased PPAR-γ expression. In the current study, we assessed whether variation in the IGFBP4 coding region influences body mass index (BMI) in American Indians who are disproportionately affected by obesity. Whole exome sequence data from a population-based sample of 6779 American Indians with longitudinal measures of BMI were used to identify variation in IGFBP4 that associated with BMI. A novel variant that predicts a p.Ser76Thr in IGFBP4 (Thr-allele frequency = 0.02) was identified which associated with the maximum BMI measured during adulthood (BMI 39.8 kg/m

This study aimed to identify genetic variants enriched in Southwest American Indian (SWAI) individuals that associate with BMI.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).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.Further studies on the effects of CYB5A on 24-h EE and BMI may provide insights into obesity-related physiology.

HypothesisThe prevalence of type 2 diabetes is higher in Latino populations compared with other major ancestry groups. Not only has the Latino population been systematically underrepresented in large-scale genetic analyses, but previous studies relied on the imputation of ungenotyped variants based on the 1000 Genomes (1000G) imputation reference panel, which results in suboptimal capture of low-frequency or Latino-enriched variants. The NHLBI Trans-Omics for Precision Medicine (TOPMed) reference panel represents a unique opportunity to analyze rare genetic variations in the Latino population.MethodsWe evaluate the TOPMed imputation performance using genotyping array and whole-exome sequence data in 6 Latino cohorts. To evaluate the ability of TOPMed imputation of increasing the identified loci, we performed a Latino type 2 diabetes GWAS meta-analysis in 8,150 type 2 diabetes cases and 10,735 controls and replicated the results in 6 additional cohorts including whole-genome sequence data from the All of Us cohort.ResultsWe show that, compared to imputation with 1000G, the TOPMed panel improves the identification of rare and low-frequency variants. We identified 26 distinct signals including a novel genome-wide significant variant (minor allele frequency 1.6%, OR=2.0, P=3.4×10−9) near ORC5. A Latino-tailored polygenic score constructed from our data and GWAS data from East Asian and European populations improves the prediction accuracy in a Latino target dataset, explaining up to 7.6% of the type 2 diabetes risk variance.ConclusionsOur results demonstrate the utility of TOPMed imputation for identifying low-frequency variation in understudied populations, leading to the discovery of novel disease associations and the improvement of polygenic scores.

While the samples and data from the Pima Indians of the Gila River Indian Community have been included in many international HLA workshops and conferences and have been the focus of numerous population reports and the source of novel alleles at the classical HLA loci, they have not been studied for the non-classical loci. In order to expand our HLA-disease association studies, we typed over 300 whole genome sequences from full Pima heritage members, controlled for first degree relationship, and employed recently developed computer algorithms to resolve HLA alleles. Both classical-HLA-A, -B, and -C- and non-classical- HLA-E, -F, -G, -J, -L, -W, -Y, -DPA2, -DPB2, -DMA, -DMB, -DOA, -DRB2, -DRB9, TAP1- loci were typed at the 4-field level of resolution. We present allele and selected haplotype frequencies, test the genotype distributions for population structure, discuss the issues that are created for tests of Hardy-Weinberg equilibrium over the four sample spaces of high resolution HLA typing, and address the implications for the evolution of non-classical pseudogenes that are no longer expressed in a phenotype subject to natural selection.

Clinical and biomarker phenotypic associations for carriers of protein function-altering variants may help to elucidate gene function and health effects in populations. We genotyped 1127 Strong Heart Family Study participants for protein function-altering single nucleotide variants (SNV) and indels selected from a low coverage whole exome sequencing of American Indians. We tested the association of each SNV/indel with 35 cardiometabolic traits. Among 1206 variants (average minor allele count = 20, range of 1 to 1064), ~ 43% were not present in publicly available repositories. We identified seven SNV-trait significant associations including a missense SNV at ABCA10 (rs779392624, p = 8 × 10–9) associated with fasting triglycerides, which gene product is involved in macrophage lipid homeostasis. Among non-diabetic individuals, missense SNVs at four genes were associated with fasting insulin adjusted for BMI (PHIL, chr6:79,650,711, p = 2.1 × 10–6; TRPM3, rs760461668, p = 5 × 10–8; SPTY2D1, rs756851199, p = 1.6 × 10–8; and TSPO, rs566547284, p = 2.4 × 10–6). PHIL encoded protein is involved in pancreatic β-cell proliferation and survival, and TRPM3 protein mediates calcium signaling in pancreatic β-cells in response to glucose. A genetic risk score combining increasing insulin risk alleles of these four genes was associated with 53% (95% confidence interval 1.09, 2.15) increased odds of incident diabetes and 83% (95% confidence interval 1.35, 2.48) increased odds of impaired fasting glucose at follow-up. Our study uncovered novel gene-trait associations through the study of protein-coding variants and demonstrates the advantages of association screenings targeting diverse and high-risk populations to study variants absent in publicly available repositories.

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 (F(ST)) was calculated across all markers as a measure of genetic divergence among ancestry groups. The phenotypic divergence index (P(ST)) 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 P(ST)=F(ST), while for traits under differential selection P(ST) is expected to be significantly greater than F(ST.) A bootstrap procedure was used to test the hypothesis P(ST)=F(ST.) 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/m(2), 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)). F(ST) across all markers was 0.130, while P(ST) for liability to diabetes, adjusted for age and sex, was 0.149 (p=0.35 for difference with F(ST)). P(ST) 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 (P(ST-MAX)=0.22, p=0.37, and P(ST-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.

Applying exome sequencing to populations with unique genetic architecture has the potential to reveal novel genes and variants associated with traits and diseases. We sequenced and analyzed the exomes of 6,716 individuals from a Southwestern American Indian (SWAI) population with well-characterized metabolic traits. We found that the SWAI population has distinct allelic architecture compared to populations of European and East Asian ancestry, and there were many predicted loss-of-function (pLOF) and nonsynonymous variants that were highly enriched or private in the SWAI population. We used pLOF and nonsynonymous variants in the SWAI population to evaluate gene-burden associations of candidate genes from European genome-wide association studies (GWASs) for type 2 diabetes, body mass index, and four major plasma lipids. We found 19 significant gene-burden associations for 11 genes, providing additional evidence for prioritizing candidate effector genes of GWAS signals. Interestingly, these associations were mainly driven by pLOF and nonsynonymous variants that are unique or highly enriched in the SWAI population. Particularly, we found four pLOF or nonsynonymous variants in APOB, APOE, PCSK9, and TM6SF2 that are private or enriched in the SWAI population and associated with low-density lipoprotein (LDL) cholesterol levels. Their large estimated effects on LDL cholesterol levels suggest strong impacts on protein function and potential clinical implications of these variants in cardiovascular health. In summary, our study illustrates the utility and potential of exome sequencing in genetically unique populations, such as the SWAI population, to prioritize candidate effector genes within GWAS loci and to find additional variants in known disease genes with potential clinical impact.

AIMS: To examine the associations of average weight and weight velocity in three growth periods from birth through adolescence with type 2 diabetes incidence. METHODS: Child participants representing three growth periods- pre-adolescence (birth to ~8 years), early adolescence (~8 to ~13 years), and late adolescence (~13 to ~18 years) -were selected from a 43-year longitudinal study of American Indians. Age-sex-height-standardized average weight z score and weight z score velocity (change/year) were computed in each period. Participants were followed for up to 25 years from the end of each growth period until they developed diabetes. Associations of average weight z score or weight z score velocity with diabetes incidence were determined with sex-, birth date- and maternal diabetes-adjusted Poisson regression models. RESULTS: Among 2,100 participants representing the pre-adolescence growth period, 1,558 representing the early adolescence period, and 1,418 representing the late adolescence period, there were 290, 315, and 380 incident diabetes cases, respectively. During the first 10 years of follow-up, diabetes incidence rate ratio was 1.72 (95% CI 1.40–2.11)/standard deviation (SD) of log(10)weight z score mean in pre-adolescence, 2.09 (1.68–2.60)/SD of log(10)weight z score mean in early adolescence, and 1.85 (1.58–2.17)/SD of log(10)weight z score mean in late adolescence. Diabetes incidence rate ratio was 1.79 (1.49–2.17)/SD of log(10)weight z score velocity pre-adolescence, 1.13 (0.91–1.41)/SD log(10)weight z score velocity in early adolescence, and 1.29 (1.09–1.51)/SD log(10)weight z score velocity in late adolescence. There were strong correlations in the average weight z scores and weak correlations in the weight z score velocities between successive periods. CONCLUSIONS: Higher weight and accelerated weight gain in all growth periods associates with increased type 2 diabetes risk. Importantly, higher weight and greater weight velocity pre-adolescence jointly associate with the highest type 2 diabetes risk.

The top genetic association signal for type 2 diabetes (T2D) in Southwestern American Indians maps to intron 15 of

The Pima Indians of the Gila River Indian Community in Arizona have participated in a long-range study of type 2 diabetes mellitus since 1965 and have been the subject of HLA typing and population studies since the early days of serological assays. These data have been in numerous HLA workshops and conferences and have been the source of at least five novel alleles at the classical HLA loci. In recent time nearly the entire study group was subject to next generation sequencing by whole genome or exome technologies, which has allowed us to HLA type over 3000 full heritage persons with recently developed computer algorithms. We present here the results for the classical HLA Loci: HLA-A, B, C, DRA, DRB1, DRB3, DRB4, DRB5, DPA1, DPB1, DQA1, and DQB1 to the third field of resolution for synonymous alleles and type the likely four field resolution alleles from the subset of whole genome sequences. Allele frequencies, and haplotype frequencies at up to five loci, are presented as well as measures of population structure and heterozygosity. We define a core set of HLA variation that approximates the distribution for the Paleo-Indians and impute nine-locus, 4-field haplotypes that are expected to be common in full heritage peoples.

There is considerable evidence that birth weight is associated with increased risk of type 2 diabetes (T2D) in adulthood. However, evidence from populations with high prevalence of T2D on the genetic relationships between birth weight and T2D is limited. Data were obtained from a genome-wide association study (GWAS) in an Indigenous population from the Southwest US with high prevalence of T2D in which the relationship of T2D with birth weight is “U-shaped." Weighted polygenic scores (PSs) were constructed using imputed genotypes for genome-wide significant single-nucleotide polymorphisms (SNPs) from GWAS meta-analyses for birth weight (EGG Consortium, 126 SNPs) and T2D (DIAGRAM Consortium, 293 SNPs). Associations of birth weight PS and T2D PS with T2D and birth weight were calculated, accounting for participants’ pairwise relationships, using linear mixed models. Birth weight data (n=3700) were normalized separately by sex and analyzed for associations, adjusted for birth year, gestational age and the first 5 genetic principal components (PCs). T2D (n=7659) data were analyzed for associations, adjusted for age, sex, birth year and the first 5 genetic PCs. Birth weight PS had a significant, positive association with birth weight (β=0.134 SD birth weight per SD PS (95% CI 0.101, 0.168); p=4.1×10-15). The T2D PS was likewise significantly, positively associated with T2D (OR=1.48 per SD (1.38, 1.58); p=8.1×10-31). The birth weight PS was significantly, inversely associated with T2D (OR=0.91 (0.85, 0.97); p=0.0043); we found no evidence for a non-linear relationship between birth weight PS and T2D (p=0.80). The T2D PS was not significantly associated with birth weight (β=-0.005 (-0.040, 0.030); p=0.80). Results indicate that genetic variants that associate with birth weight and T2D from larger European GWAS largely also influence these traits in this Indigenous population. Current findings also support the notion that many variants conferring susceptibility to low birth weight also confer susceptibility to T2D. Disclosure L. E. Wedekind: None. W. Hsueh: None. M. Olaiya: None. S. Kobes: None. L. Baier: None. W. C. Knowler: None. A. Mahajan: Employee; Self; Genentech, Inc. M. Mccarthy: Employee; Self; Genentech, Inc. R. L. Hanson: None.

Background: There is limited information on how polygenic scores (PSs) — based on variants from genome-wide association studies (GWAS) of T2D — add to clinical variables in predicting T2D incidence. Methods: For participants in a longitudinal study in an Indigenous population from the Southwest United States (US) with high T2D prevalence, we analyzed 10 constructions of PS using publicly available GWAS summary statistics. T2D incidence was examined in three cohorts of individuals without T2D at baseline. The adult cohort, comprised of 2333 participants followed from age≥20y, had 640 T2D cases. The youth cohort was comprised of 2229 participants followed from age 5-19y (228 cases). The birth cohort was comprised of 2894 participants followed from birth (438 cases). We assessed contributions of the PS and clinical variables in predicting T2D incidence. Results: Of the 10 PS constructions, a PS using 293 genome-wide significant variants from a large T2D GWAS meta-analysis in European-ancestry populations performed best. In the adult cohort, area under the receiver operating characteristic curve (AUC) for clinical variables for prediction of T2D incidence was 0·728; with the PS, 0·735. The PS’s hazard ratio (HR) was 1.27 per SD (p=1·6×10-8; 95% CI 1·17-1·38). In the youth cohort, the analogous model’s AUC was 0·805; with the PS, 0·812. The PS’s HR was 1·49 (p=4·3×10-8; 95% CI 1·29-1·72). In the birth cohort, AUC for the clinical variables was 0·614; with the PS, 0·685. The PS’s HR was 1·48 (p=2·8×10-16; 95% CI 1·35-1·63). To further assess the potential impact of including PS for assessing individual T2D risk, the net reclassification improvement (NRI) was calculated: NRI for the PS was 0·264, 0·249, and 0·309 for adult, youth, and birth cohorts, respectively. For comparison, NRI for HbA1c was 0·292 and 0·150 for adult and youth cohorts, respectively. Across all cohorts, as estimated using decision curve analyses, the net benefit of including the PS in addition to clinical variables was most pronounced at moderately stringent threshold probability (pt) values for instituting a preventive intervention. Discussion: This study demonstrates that the PS contributes significantly to prediction of T2D incidence in addition to the information provided by clinical factors; however, overall improvement of the prediction model was modest. Discriminatory power of the PS for predicting incident T2D was similar to that of other commonly measured clinical factors (e.g. HbA1c). Including T2D PS in addition to clinical factors may be clinically beneficial for identifying individuals at higher risk for T2D, especially at younger ages. Funding Statement: This study used computational resources of the Biowulf system at the National Institutes of Health (NIH), Bethesda, MD. The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Intramural Research Program provided support for NIDDK-based coauthors. The NIH Oxford-Cambridge Scholars Program provided funding support for L.E.W.’s doctoral programme. M.I.M. was a Wellcome Senior Investigator and an NIHR Senior Investigator. This work was funded in Oxford by the Wellcome Trust (098381, 106130, 203141), NIH (U01- DK105535; U01-DK085545) and National Institute for Health Research (NIHR) (NF-SI-0617- 10090) and Oxford Biomedical Research Centre (BRC). Declaration of Interests: The views expressed in this article are those of the author(s) and not necessarily those of NIH, NHS, NIHR, or UK Department of Health. M.I.M. has served on advisory panels for Pfizer, Novo Nordisk, and Zoe Global; has received honoraria from Merck, Pfizer, Novo Nordisk, and Eli Lilly; and research funding from Abbvie, Astra Zeneca, Boehringer Ingelheim, Eli Lilly, Janssen, Merck, NovoNordisk, Pfizer, Roche, Sanofi Aventis, Servier, and Takeda. As of June 2019, M.I.M. and A.M. are employees of Genentech and holders of Roche stock. Ethics Approval Statement: Protocols were approved by the institutional review board of the National Institute of Diabetes and Digestive and Kidney Diseases.

Objective In an ongoing effort to identify the genetic variation that contributes to obesity in American Indians, known Bardet–Biedl syndrome (BBS) genes were analyzed for an effect on BMI and leptin signaling. Methods Potentially deleterious variants (Combined Annotation Dependent Depletion score > 20) in BBS genes were identified in whole‐exome sequence data from 6,851 American Indians informative for BMI. Common variants (detected in ≥ 10 individuals) were analyzed for association with BMI; rare variants (detected in 40 kg/m2), four were in BBS9. In vitro analysis of BBS9 found the Ile allele at Thr549Ile had a 20% increase in STAT3 activity compared with the Thr allele (P = 0.01). Western blot analysis showed the Ile allele had a 15% increase in STAT3 phosphorylation (P = 0.006). Comparable functional results were observed with Ser545Gly and Val209Leu but not Leu665Phe and Lys810Glu. Conclusions Potentially functional variants in BBS genes in American Indians are reported. However, functional evidence supporting a causal role for BBS9 in obesity is inconclusive.

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. Disclosure S.E. Day: None. M. Traurig: None. P. Piaggi: None. P. Kumar: None. S. Kobes: None. R.L. Hanson: None. C. Bogardus: None. L. Baier: None.

Our recent genome-wide association study (GWAS) identified variants strongly associated with type 2 diabetes (T2D) on chromosome 11p15 in American Indians at KCNQ1 (index variant rs2237895, P=2.5×10-8) and TH (rs11564707, P=3.8×10-8). To assess whether these variants influence epigenetic variation we analyzed associations with DNA methylation. Participants included 7659 American Indians (33% with T2D) who had participated in the GWAS, 842 of whom had DNA methylation measured in peripheral blood. Methylation was measured with the Infinium 450k array (Illumina, San Diego, CA). To identify local CpG sites associated with index variants, we analyzed associations for sites within 1 Mb of each gene (2162 sites for KCNQ1, 2325 for TH). Strong associations were identified: the diabetes risk allele at rs2237895 associated with decreased methylation at cg14637411 (β= 1.35 SD per copy, P=2.6×10-169), and the risk allele at rs11564707 associated with increased methylation at cg19878200 (β=0.46 SD, P=1.1×10-21). We further conducted Bayesian functional fine-mapping association of T2D and statistical colocalization of association of methylation at these sites with T2D using CAVIAR- including 1943 variants for KCNQ1 and 2954 variants for TH. Potentially causal variants for T2D were identified for each gene (30 for KCNQ1 and 33 for TH with posterior probability of causality >0.01); all were in strong linkage disequilibrium with the index variant. Strong evidence for colocalization of the association of T2D with methylation at cg14637411 (colocalization posterior probability >0.01, suggesting both traits are influenced by the same causal variants) was seen with 4 KCNQ1 variants: rs60808706, rs2299620, rs2237896 and rs2237897. Evidence for colocalization was weak for cg19878200 and T2D at TH variants. These results support the notion that KCNQ1 variants may influence T2D risk via an epigenetic effect, and they identify candidate functional variants. Disclosure R.L. Hanson: None. S. Kobes: None. W. Hsueh: None. Y.L. Muller: None. W.C. Knowler: None. L. Baier: None. Funding American Diabetes Association (1-12-DNP-01 to R.L.H.)

To identify variants that potentially impact human body fat via a role in adipogenesis, we analyzed whole-exome sequence data from a population-based sample of 6809 American Indians with longitudinal measures of BMI. A total of 623,236 exonic variants were detected and those that met the following criteria were prioritized for functional analysis: 1) non-synonymous, stop-gain or frameshift that occurred in ≥5 individuals; 2) a Combined Annotation Dependent Depletion (CADD) score ≥10 (top 10% of deleterious variants); 3) evidence for association with maximum BMI in adulthood (n=6002) or maximum BMI z-score in childhood (n=4882) (P In conclusion, whole-exome sequencing followed by in vitro analysis of lipid accumulation in OP9 cells has identified coding variants that may impact BMI via a role in adipogenesis. Disclosure Y.L. Muller: None. S.E. Day: None. C. Koroglu: None. S. Kobes: None. R.L. Hanson: None. W.C. Knowler: None. H. Kim: Employee; Self; Regeneron Pharmaceuticals. C. Van Hout: Employee; Self; Regeneron Genetics Center. N. Gosalia: None. A.R. Shuldiner: Employee; Self; Regeneron Genetics Center. Stock/Shareholder; Self; Regeneron Pharmaceuticals. C. Bogardus: None. L. Baier: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

Applying whole exome sequencing (WES) to populations with unique genetic architecture has the potential to reveal novel genes and variants associated with traits and diseases. We sequenced and analyzed the exomes of 6,716 individuals from an American Indian population in Southwest US (Southwestern American Indian, or SWAI) with well-characterized metabolic traits. We found that individuals of SWAI have distinct allelic architecture compared to individuals with European and East Asian ancestry, with many predicted loss-of-function (pLOF) and nonsynonymous variants that were highly enriched or private in SWAI. We evaluated gene-level associations with metabolic traits using pLOF and nonsynonymous variants in SWAI. Many of the candidate genes from previous GWAS studies for body mass index, type 2 diabetes, and plasma lipid levels were associated with respective traits in SWAI. Notably, these associations were mainly driven by pLOF and nonsynonymous variants that are unique or highly enriched in American Indians, many of which have not been observed in other populations or functionally characterized. Our study illustrates the utility and potential of WES in American Indians to prioritize candidate effector genes within GWAS loci and to find novel variants in known diseases genes with potential clinical impact.

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.

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.

Nighttime eating (NE) behavior has a genetic component and predicts weight gain. We hypothesized that some genetic variants, which affect NE would also show an effect on body mass index (BMI). We aimed to determine which known BMI variants associate with NE in Southwestern American Indians (SWAIs), who are at elevated risk for obesity. Known BMI variants from the GIANT-UK Biobank meta-analysis (N = 700,000) were analysed in SWAIs characterized for NE during an inpatient 3-day protocol. Variants were analysed for association with NE using whole-genome sequence data from 50 SWAIs (23 cases and 27 controls) and selected variants were genotyped in an additional 32 SWAIs (13 NE cases and 19 controls). Variants associated with NE in a meta-analysis of the two SWAI samples were further analysed for association with nightly caloric intake and functionality in hypothalamus, pituitary, and adrenal tissues. Variants were identified where the allele that associated with increased BMI in the GIANT-UK Biobank meta-analysis (P ≤ 1 × 10−8) also had a P

Prevalence of diabetes and obesity in Mexican Pima Indians is low, while prevalence is high in Pima Indians in the United States (US); although lifestyle likely accounts for much of the difference, the role of genetic factors is not well-explored. To examine this, we genotyped 359 single nucleotide polymorphisms, including established type 2 diabetes and obesity variants from genome-wide association studies (GWAS) and 96 random markers, in 342 Mexican Pimas. A multimarker risk score of obesity variants was associated with body mass index (BMI; β = 0.81 kg/m(2) per SD, P = 0.0066). The mean value of the score was lower in Mexican Pimas than in US Pimas (P = 4.3×10(−11)), and differences in allele frequencies at established loci could account for ~7% of the population difference in BMI; however, the difference in risk scores was consistent with evolutionary neutrality given genetic distance. To identify loci potentially under recent natural selection, allele frequencies at 283 variants were compared between US and Mexican Pimas, accounting for genetic distance. The largest differences were seen at HLA markers (e.g., rs9271720, difference = 0.75, P = 8.7×10(−9)); genetic distances at HLA were greater than at random markers (P = 1.6×10(−46)). Analyses of GWAS data in 937 US Pimas also showed sharing of alleles identical by descent at HLA that exceeds its genomic expectation (P = 7.0×10(−10)). These results suggest that, in addition to the widely-recognized balancing selection at HLA, recent directional selection may also occur, resulting in marked allelic differentiation between closely related populations.

Type 2 diabetes (T2D) affects more than 415 million people worldwide, and its costs to the health care system continue to rise. To identify common or rare genetic variation with potential therapeutic implications for T2D, we analyzed and replicated genome-wide protein coding variation in a total of 8,227 individuals with T2D and 12,966 individuals without T2D of Latino descent. We identified a novel genetic variant in the IGF2 gene associated with ∼20% reduced risk for T2D. This variant, which has an allele frequency of 17% in the Mexican population but is rare in Europe, prevents splicing between IGF2 exons 1 and 2. We show in vitro and in human liver and adipose tissue that the variant is associated with a specific, allele-dosage–dependent reduction in the expression of IGF2 isoform 2. In individuals who do not carry the protective allele, expression of IGF2 isoform 2 in adipose is positively correlated with both incidence of T2D and increased plasma glycated hemoglobin in individuals without T2D, providing support that the protective effects are mediated by reductions in IGF2 isoform 2. Broad phenotypic examination of carriers of the protective variant revealed no association with other disease states or impaired reproductive health. These findings suggest that reducing IGF2 isoform 2 expression in relevant tissues has potential as a new therapeutic strategy for T2D, even beyond the Latin American population, with no major adverse effects on health or reproduction.

Increased urine albumin excretion is highly prevalent in Hispanics/Latinos. Previous studies have found an association between urine albumin excretion and Amerindian ancestry in Hispanic/Latino populations. Admixture between racial/ethnic groups creates long-range linkage disequilibrium between variants with different allelic frequencies in the founding populations and it can be used to localize genes. Hispanic/Latino genomes are an admixture of European, African, and Amerindian ancestries. We leveraged this admixture to identify associations between urine albumin excretion (urine albumin-to-creatinine ratio [UACR]) and genomic regions harboring variants with highly differentiated allele frequencies among the ancestral populations. Admixture mapping analysis of 12,212 Hispanic Community Health Study/Study of Latinos participants, using a linear mixed model, identified three novel genome-wide significant signals on chromosomes 2, 11, and 16. The admixture mapping signal identified on chromosome 2, spanning q11.2–14.1 and not previously reported for UACR, is driven by a difference between Amerindian ancestry and the other two ancestries (P

AIMS/HYPOTHESIS: Individuals exposed to maternal diabetes in utero are more likely to develop metabolic and cardiovascular diseases later in life. This may be partially attributable to epigenetic regulation of gene expression. We performed an epigenome-wide association study to examine whether differential DNA methylation, a major source of epigenetic regulation, can be observed in offspring of mothers with type 2 diabetes during the pregnancy (OMD) compared with offspring of mothers with no diabetes during the pregnancy (OMND). METHODS: DNA methylation was measured in peripheral blood using the Illumina HumanMethylation450K BeadChip. A total of 423,311 CpG sites were analysed in 388 Pima Indian individuals, mean age at examination was 13.0 years, 187 of whom were OMD and 201 were OMND. Differences in methylation between OMD and OMND were assessed. RESULTS: Forty-eight differentially methylated CpG sites (with an empirical false discovery rate ≤0.05), mapping to 29 genes and ten intergenic regions, were identified. The gene with the strongest evidence was LHX3, in which six CpG sites were hypermethylated in OMD compared with OMND (p ≤ 1.1 × 10(−5)). Similarly, a CpG near PRDM16 was hypermethylated in OMD (1.1% higher, p = 5.6 × 10(−7)), where hypermethylation also predicted future diabetes risk (HR 2.12 per SD methylation increase, p = 9.7 × 10(−5)). Hypermethylation near AK3 and hypomethylation at PCDHGA4 and STC1 were associated with exposure to diabetes in utero (AK3: 2.5% higher, p = 7.8 × 10(−6); PCDHGA4: 2.8% lower, p = 3.0 × 10(−5); STC1: 2.9% lower, p =1.6 × 10(−5)) and decreased insulin secretory function among offspring with normal glucose tolerance (AK3: 0.088 SD lower per SD of methylation increase, p = 0.02; PCDHGA4: 0.08 lower SD per SD of methylation decrease, p = 0.03; STC1: 0.072 SD lower per SD of methylation decrease, p = 0.05). Seventeen CpG sites were also associated with BMI (p ≤ 0.05). Pathway analysis of the genes with at least one differentially methylated CpG (p < 0.005) showed enrichment for three relevant biological pathways. CONCLUSIONS/INTERPRETATION: Intrauterine exposure to diabetes can affect methylation at multiple genomic sites. Methylation status at some of these sites can impair insulin secretion, increase body weight and increase risk of type 2 diabetes.

Context In individuals with obesity, adipocyte endocrine function is affected by altered autophagy. Genetic variants in autophagy-related gene 7 (ATG7) correlated with serum chemerin (RARRES2) concentrations. Objectives To investigate a functional interplay between chemerin and ATG7, how it may relate to autophagy-mediated adipocyte dysfunction in obesity, and the relevance of genetic ATG7 variants in chemerin physiology. Design Adipose ATG7 mRNA expression and adiposity measures were available in two human study cohorts. The effect of a high-calorie diet on adipose Rarres2 and Atg7 expression was investigated in mice. In 3T3-L1 adipocytes, the effect of Atg7 knockdown on chemerin expression and secretion was studied. The influence of single nucleotide polymorphisms on ATG7 transcription and chemerin physiology was investigated using a luciferase assay. Setting Mouse model, clinical trials, in vitro studies. Participants Native American (n = 83) and white (n = 100) cohorts. Main outcome measure Adipocyte chemerin expression and secretion. Results In mice fed a high-calorie diet, adipose Atg7 mRNA expression did not parallel an increase in Rarres2 mRNA expression. ATG7 mRNA expression in human subcutaneous adipose tissue correlated with body mass index, fat mass (r > 0.27; P < 0.01), and adipocyte cell size (r > 0.24; P < 0.02). Atg7 knockdown in 3T3-L1 adipocytes decreased chemerin secretion by 22% (P < 0.04). Rs2606729 in ATG7 was predicted to alter ATG7 transcription and induced higher luciferase activity in vitro (P < 0.0001). Conclusions Human adipose ATG7 mRNA expression relates to measures of adiposity. Atg7 knockdown reduces chemerin secretion from adipocytes in vitro, supportive of a functional interplay between ATG7 and chemerin in autophagy-mediated adipocyte dysfunction.

A prior genome-wide association study in 7701 Pima Indians utilized a custom Axiom array (Affymetrix) which contained 474,196 SNPs. These SNPs tagged 92% of common variants (R2≥0.85, minor allele frequency ≥0.05) identified in the Pima Indian genome. SNP rs79788804, which tags 38 additional SNPs in intron 15 of KCNQ1, achieved genome-wide significance in its association with type 2 diabetes (T2D; OR=1.31, p=2 x10-8). A 3 SNP haplotype (rs79788804, rs2237892 and rs2299620) with >1 maternally-derived alleles strongly associated with T2D (ORM=2.05, p=2x10-12). ENCODE and Islet regulome databases mapped putative strong enhancers to 4 regions in intron 15 with multiple epigenetic marks and transcription factor binding sites. To validate these regulatory regions, we tested gene activation from the putative enhancers using CRISPR-CAS9-based acetyltransferase (dCas9p300) in human pancreatic beta cells (EndoC-βH3). Guide RNA specific acetylation by dCas9p300 at a 341 bp region spanning rs2299620, rs2237896, rs2237897 and rs74046911 resulted in a 1.5 fold gene activation for KCNQ1 and nearby gene TRPM5 (both p=0.001). A chromatin immunoprecipitation-qPCR assay confirmed that dCas9P300 acetylated chromatin at this targeted region. We further assessed the effects of 5 transcription factors (NKX2.2, MAFB, NKX6.1, FOXA2, PDX1) on transcriptional activity under control of these 4 enhancer regions. Mouse pancreatic β-cells (NIT1) were co-transfected with a vector expressing each transcription factor and a luciferase reporter construct containing the KCNQ1 SNPs. NKX2.2 had a 5.5 fold activation for the construct containing the same 341 bp KCNQ1 region, as compared to a 4.1 fold activation for the construct containing KCNQ1 promoter alone (p=0.02). Our data suggested that a 341 bp region containing rs2299620, rs2237896, rs2237897 and rs74046911 may harbor functional variant(s) which affect expression of KCNQ1 and TRPM5. Disclosure Y.L. Muller: None. S.E. Day: None. S. Kobes: None. W.C. Knowler: None. R.L. Hanson: None. C. Bogardus: None. L.J. Baier: None. Funding National Institutes of Health

Epidemiologic studies in many populations have shown that birth weight is associated with risk of type 2 diabetes (T2D) in adulthood. In a Southwestern American Indian population, lower and higher birth weight groups are at higher risk for T2D than those with normal birth weight. We performed a genome-wide association study (GWAS) of birth weight in 3700 Southwestern American Indians (2037 female; 1663 male). 496,190 SNPs (allele frequency>1%) were directly genotyped using an Axiom array designed to capture common variation in this community (Affymetrix; Santa Clara, CA). Birth weight was ascertained from Arizona state and medical records. T2D was determined according to American Diabetes Association criteria at a research exam or during clinical care. Birth weight data were normalized separately by sex and analyzed for genetic associations using a mixed model (SOLAR-Eclipse; Catonsville, MD) accounting for genetic relationships (based on genetic markers among all pairs of individuals) and adjusted for birth year and the 1st 5 genetic principal components. We identified 41 variants suggestively associated (p 5 of the 41 variants are also nominally associated with T2D (p Disclosure L.E. Wedekind: None. W. Hsueh: None. S. Kobes: None. M.T. Olaiya: None. W.C. Knowler: None. L.J. Baier: None. R.L. Hanson: None.

Although hundreds of genome-wide association studies-implicated loci have been reported for adult obesity-related traits, less is known about the genetics specific for early-onset obesity and with only a few studies conducted in non-European populations to date. Searching for additional genetic variants associated with childhood obesity, we performed a trans-ancestral meta-analysis of 30 studies consisting of up to 13 005 cases (≥95th percentile of body mass index (BMI) achieved 2–18 years old) and 15 599 controls (consistently

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 Disclosure C. Koroglu Altok: None. P. Piaggi: None. M. Traurig: None. R.L. Hanson: None. W.C. Knowler: None. H. Kim: Employee; Self; Regeneron Pharmaceuticals. N. Gosalia: Employee; Self; Regeneron Pharmaceuticals. C.V. Van Hout: Employee; Self; Regeneron Pharmaceuticals. B. Ye: Employee; Self; Regeneron Pharmaceuticals. A.R. Shuldiner: Employee; Self; Regeneron Pharmaceuticals. Stock/Shareholder; Self; Rhythm Pharmaceuticals, Inc. C. Bogardus: None. L.J. Baier: None. Funding National Institutes of Health

Genome-wide association studies (GWAS) have identified many variants associated with type 2 diabetes (T2D), but there is little information on their effects in high risk populations such as American Indians. We analyzed 211 primary variants, associated with T2D (P Disclosure R.L. Hanson: None. L.E. Wedekind: None. W. Hsueh: None. S. Kobes: None. L.J. Baier: None. C. Bogardus: None. W.C. Knowler: None.

AIMS/HYPOTHESIS: The aim of this work was to estimate the impact of birthweight on early-onset (age

MicroRNAs (miRNAs) play an important role in posttranscriptional regulation by binding to target sites in the 3′UTR of protein-coding genes. Genetic variation within target sites may potentially disrupt the binding activity of miRNAs, thereby impacting this regulation. In the current study, we investigated whether any established BMI-associated genetic variants potentially function by altering a miRNA target site. The genomic positions of all predicted miRNA target site seed regions were identified, and these positions were queried in the T2D Knowledge Portal for variants that associated with BMI in the GIANT UK Biobank. This in silico analysis identified ten target site variants that associated with BMI with a P value ≤ 5 × 10−8. These ten variants mapped to nine genes, FAIM2, CCDC171, ADPGK, ZNF654, MLXIP, NT5C2, SHISA4, SLC25A22, and CTNNB1. In vitro functional analyses showed that five of these target site variants, rs7132908 (FAIM2), rs4963153 (SLC25A22), rs9460 (ADPGK), rs11191548 (NT5C2), and rs3008747 (CCDC171), disrupted the binding activity of miRNAs to their target in an allele-specific manner. In conclusion, our study suggests that some established variants for BMI may function by altering miRNA binding to a 3′UTR target site.

BackgroundGenetic factors that influence kidney traits have been understudied for low frequency and ancestry-specific variants.MethodsWe combined whole genome sequencing (WGS) data from 23,732 participants from 10 NHLBI Trans-Omics for Precision Medicine (TOPMed) Program multi-ethnic studies to identify novel loci for estimated glomerular filtration rate (eGFR). Participants included European, African, East Asian, and Hispanic ancestries. We applied linear mixed models using a genetic relationship matrix estimated from the WGS data and adjusted for age, sex, study, and ethnicity.FindingsWhen testing single variants, we identified three novel loci driven by low frequency variants more commonly observed in non-European ancestry (PRKAA2, rs180996919, minor allele frequency [MAF] 0.04%, P=6.1×10-11; METTL8, rs116951054, MAF 0.09%, P=4.5×10-9; and MATK, rs539182790, MAF 0.05%, P=3.4×10-9). We also replicated two known loci for common variants (rs2461702, MAF=0.49, P=1.2×10-9, nearest gene GATM, and rs71147340, MAF=0.34, P=3.3×10-9, CDK12). Testing aggregated variants within a gene identified the MAF gene. A statistical approach based on local ancestry helped to identify replication samples for ancestry-specific variants.InterpretationThis study highlights challenges in studying variants influencing kidney traits that are low frequency in populations and more common in non-European ancestry.

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.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.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.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.

OBJECTIVE Meta-analyses of genome-wide association studies in Europeans have identified > 98 loci for BMI. Transferability of these established associations in Pima Indians was analyzed. METHODS Among 98 lead single nucleotide polymorphisms (SNPs), 82 had minor allele frequency ≥ 0.01 in Pima Indians and were analyzed for association with the maximum BMI in adulthood (n = 3,491) and BMI z score in childhood (n = 1,958). Common tag SNPs across 98 loci were also analyzed for additional signals. RESULTS Among the lead SNPs, 13 (TMEM18, TCF7L2, MRPS33P4, PRKD1, ZFP64, FTO, TAL1, CALCR, GNPDA2, CREB1, LMX1B, ADCY9, NLRC3) were associated with BMI (P ≤ 0.05) in Pima adults. A multi-allelic genetic risk score (GRS), which summed the risk alleles for 82 lead SNPs, showed a significant trend for a positive relationship between GRS and BMI in adulthood (beta = 0.48% per risk allele; P = 1.6 × 10-9 ) and BMI z score in childhood (beta = 0.024 SD; P = 1.7 × 10-7 ). GRS was significantly associated with BMI across all age groups ≥ 5 years, except for those ≥ 50 years. The strongest association was seen in adolescence (age 14-16 years; P = 1.84 × 10-9 ). CONCLUSIONS In aggregate, European-derived lead SNPs had a notable effect on BMI in Pima Indians. Polygenic obesity in this population manifests strongly in childhood and adolescence and persists throughout much of adult life.

We recently completed a genome wide association study for type 2 diabetes (T2D) in 7659 American Indians who are predominantly of Pima Indian heritage using a custom designed Axiom array. A novel intronic variant (hg19 chr10:72276664 G>C) in PALD1 was identified that associated with T2D (risk allele frequency (RAF)=0.01, OR=2.19[1.49-3.22] per risk allele [C], P=6.6×10-5 adjusted for age, sex, birthyear, PCs 1-5 and accounting for genetic relatedness). Further analysis in a dataset of Urban American Indians (N=3029) representing several tribal groups living in the Phoenix area, identified a directionally consistent association (OR=2.32, P=0.12), but this variant was rare among the entire Urban American Indian sample (RAF=0.003). A meta-analysis of both datasets resulted in a summary OR of 2.15[1.49-3.13], P=4.9×10-5. In Pima Indians, this variant is highly concordant (r2=0.92) with a novel missense variant (hg19 chr10:72298980 C>T, p.[P568S]) in PALD1. The missense variant also had a significant association with T2D in Pima Indians (RAF=0.02, OR=1.80[1.25-2.60], P=0.002) and in Urban American Indians (RAF=0.008, OR=2.29[1.03-5.12], P=0.04). However, after conditional analysis, only the intronic variant remains significant in the Pima Indian dataset. PALD1 is a predicted phosphatase and acts as a negative regulator of insulin signaling. Overexpression of PALD1 leads to lower insulin stimulated AKT phosphorylation and insulin receptor abundance. For preliminary functional studies, a short fragment (100bp) containing each allele of the PALD1 intronic variant (C or G) was cloned upstream of a SV40 promoter luciferase vector and transfected into C2C12 myoblasts. The T2D risk allele (C) fragment had significantly (P=0.004) higher SV40 promoter mediated luciferase activity as compared to the non-risk (G) allele. This result is consistent with the possible role of PALD1 in T2D pathogenesis. Further mechanistic studies are currently ongoing. Disclosure A.K. Nair: None. J.R. Sutherland: None. G.R. Elson: None. P. Piaggi: None. S. Kobes: None. R.L. Hanson: None. C. Bogardus: None. L. Baier: None.

Inflammatory processes may contribute to type 2 diabetes (T2D), but there is limited information about genetic associations with circulating levels of inflammatory cytokines. We performed a genome-wide association study (GWAS) of cytokines in 1061 nondiabetic Southwestern Native Americans (500 women; 561 men). Fasting serum concentrations of interleukin-6 (IL-6), plasminogen activator inhibitor-1 (PAI-1) and tumor necrosis factor-α (TNF-α) were measured using EMD Millipore assays. Genotypic data on 496,190 single-nucleotide polymorphisms (SNPs) with minor allele frequency >1% were derived from an Affymetrix Axiom array designed to capture common variation in Pima Indians. Data were normalized and analyzed for associations using a mixed model accounting for familial relationships, adjusted for age, sex and genetic principal components 1-5. The strongest associations were on chromosome 3 near A4GNT with IL-6 levels (rs2724711 p=4.2×10-6), and on chromosome 7 with PAI-1 levels (rs1608488 (p=8.3×10-7). An association of genome-wide statistical significance (rs1862840; p=1.2×10-8) was observed on chromosome 16 near WWOX with TNF-α levels. Each copy of the G allele (frequency=0.12) confers a 0.41-SD increase in TNF-α levels. In a larger cohort of Southwestern Native Americans, rs1862840 was not associated with T2D (N=7,659; p=0.17). Among 3,983 nondiabetic subjects it did associate with 2 hour corrected insulin response (β=0.06-SD decrease per G allele copy, p=0.011). Among 300 normal glucose tolerant subjects it associated with the acute insulin response to intravenous glucose (β=20% decrease per G allele copy, p=0.045). This study identified a novel association for TNF-α levels at WWOX (a gene previously implicated in T2D and insulin secretion in Han Chinese study populations) and suggests potential genetic links between inflammation and insulin secretion. Disclosure L.E. Wedekind: None. M. Walter: None. S. Kobes: None. P. Chen: None. W. Hsueh: None. R. Nelson: None. L. Baier: None. W.C. Knowler: None. R.L. Hanson: None.

A recent genome-wide association study (GWAS) in Samoans (a Polynesian population) identified strong associations of CREBRF variants with BMI. The G allele at rs12513649 (frequency=25.8%) was associated with ∼1.3 kg/m2 higher BMI per copy, as was the Gln allele at rs373863828 (which codes for an ArgGln substitution). The Gln allele at rs373863828 has frequency of 25.9% in Samoans and is rare in most global populations, although it has been observed at low frequency in some other Oceanic populations. The alleles associated with higher BMI were suggestively associated with lower risk of type 2 diabetes [T2D, odds ratio (OR)=0.69 per copy, P=4.7×10-7]. We investigated the associations of rs12513649 and rs373863828 with BMI and T2D in 2029 Pacific Islanders from Guam and Saipan, who participated in a GWAS. Frequencies of the G allele at rs12513649 and the Gln allele at rs373863828 were 8.1 and 5.4% respectively in full-heritage Chamorros (N=928), 4.1 and 0.2% in Filipinos (N=249), 6.1 and 3.7% in Chuukis (N=153), 2.2 and 1.1% in Carolinians (N=43), and 20.7 and 2.2% in Palauans (n=45). Using a genomic control procedure with adjustment for age, sex, end-stage renal disease and the first four genetic principal components, the G allele at rs12513649 and the Gln allele at rs373863828 were associated with higher BMI (β=1.60 kg/m2, P=0.002 and β=1.36 kg/m2, P=0.058 respectively). They were also modestly associated with taller height (β=0.8 cm, P=0.11, and β=1.5 cm, P=0.019). The alleles conferring higher BMI were associated with lower prevalence of T2D (OR=0.62, P=0.0and OR=0.45, P=0.001). When combined with previously published results for Samoans via meta-analysis, the association of rs373863828 with T2D achieved genome-wide statistical significance (OR=0.67, P=2.9×10-8). These results suggest that CREBRF variants may be important contributors to obesity in Oceanic populations, and they confirm the associations of the allele conferring higher BMI with lower risk of T2D. Disclosure R.L. Hanson: None. S. Safabakhsh: None. J. Curtis: None. D.K. Mahkee: None. L. Baier: None. W.C. Knowler: None. R. Nelson: None. W. Hsueh: None.

Strong associations between HLA alleles and infectious and autoimmune diseases are well established. Although obesity is also associated with these diseases, the relationship between HLA and obesity has not been systematically investigated in a large cohort. In the current study, we analyzed the association of HLA alleles with BMI using data from 1.3 million healthy adult donors from the Chinese Marrow Donor Program (CMDP). We found 23 HLA alleles, including 12 low-resolution and 11 high-resolution alleles, were significantly associated with BMI after correction for multiple testing. Alleles associated with high BMI were enriched in haplotypes that were common in both Chinese and European populations, whereas the alleles associated with low BMI were enriched in haplotypes common only in Asians. Alleles B*07, DRB1*07, DRB1*12, and C*03:02 provided the strongest associations with BMI (P = 6.89 × 10−10, 1.32 × 10−9, 1.52 × 10−9, and 4.45 × 10−8, respectively), where B*07 and DRB1*07 also had evidence for sex-specific effects (Pheterogeneity = 0.0067 and 0.00058, respectively). These results, which identify associations between alleles of HLA-B, DRB1, and C with BMI in Chinese young adults, implicate a novel biological connection between HLA alleles and obesity.

The genomic era, highlighted by large scale, genome-wide association studies (GWAS) for both common and rare diseases, have identified hundreds of disease-associated variants. However, most of these variants are not disease causing, but instead only provide information about a potential proximal functional variant through linkage disequilibrium. It is critical that these functional variants be identified, so that their role in disease risk can be ascertained. Luciferase assays are an invaluable tool for identifying and characterizing functional variants, allowing investigations of gene expression, intracellular signaling, transcription factors, receptor activity, and protein folding. In this chapter, we provide an overview of the different ways that luciferase assays can be used to validate functionality of a variant.