Whole Genome and Whole Exome Sequencing to Identify Genes for Type 2 Diabetes and Obesity

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

American Indians have extremely high rates of T2D, diabetic nephropathy and obesity, yet large-scale sequencing efforts to identify disease loci have not included individuals from this ethnic group. Therefore, variants that are unique or enriched for in American Indians, which may identify new therapeutic targets for these diseases, remain largely unknown. To identify common variation that increases susceptibility to type 2 diabetes (T2D) and/or obesity, whole genome sequence data was generated on 335 Pima Indians. Sequencing was performed by Illumina (N=301) and Complete Genomics, Inc (N=34). 13 million variants were found, including 11 million SNPs, 1.6 million Indels and 255,802 substitutions. Among all SNPs, 2.7 million were novel. To obtain information on rare variants which could potentially have a large effect size on disease risk, we recently obtained whole exome sequence data on 8500 American Indians informative for type 2 diabetes, obesity, diabetic nephropathy and lipid levels . For the whole exome sequence data, approximately 1.7 million variants were detected in 8500 American Indians. Among these variants, 95% were single nucleotide polymorphisms and 5% were short insertions or deletions. Approximately 493,0000 of these variants occurred in at least 5 subjects, and these underwent single variant analysis for type 2 diabetes and BMI. In addition, gene based models(Burden and SKAT methods) were also employed. The exome sequencing showed that Pima Indians have a distinct allelic architecture compared to populations of European and East Asian ancestry. The Pima Indian exomes had many predicted loss-of-function (pLOF) and nonsynonymous variants that were highly enriched or private. We evaluated gene-burden associations of candidate genes for type 2 diabetes, BMI, and four major plasma lipids and found 19 significant gene-burden associations for 11 genes, providing additional evidence for prioritizing candidate effector genes of GWAS signals. We also analyzed whole-exome sequence data in 373 healthy Pima Indians informative for 24-hour energy expenditure (24-h EE) since the identification of variants that influence energy metabolism may lead to new pathways to treat human obesity. We identified a variant which introduces a premature stop codon (Cys264Ter) in the DAO gene. This variant demonstrated the strongest association for 24-h EE, where the Ter allele associated with substantially lower 24-h EE (mean lower by 268 kcal/day) and sleeping EE (by 135 kcal/day). The Ter allele has a frequency=0.5% in Pima Indians, while 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. Insulin-like growth factor (IGF) is an important regulator of adipose tissue development. It is well established that the activity of IGF is regulated by IGF-binding proteins (IGFBPs). Recent studies of IGFBP4, which is highly expressed in adipose tissue, have shown that IGFBP4 is required for adipogenesis and influences the distribution of adipose depots in mice. Therefore, we sought to determine whether coding variation in IGFBP4 also contributes to obesity in 6809 American Indians. A novel variant predicting a Serine to a Threonine at codon 76 (Ser76Thr) was identified that had with a minor allele frequency (mAF) of 0.02 had a Combined Annotation Dependent Depletion (CADD) score >9.7 (a CADD of 10 means the top 10% most likely to be deleterious). This variant associated with BMI in both American Indian children and adults. An in vitro FOXO-reporter assay detecting PI3K/AKT mediated transcriptional activity was used to assess the effect of the Ser76Thr on IGF-I signaling. We found that COS7 cells expressing the BMI risk Thr-allele had a 55% decrease compared to the non-risk Ser-allele in FOXO1-induced transcriptional activity, due to increased FOXO1 nuclear exclusion that was mediated through increased activation of the PI3K/AKT by IGF-I signaling.