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

Indigenous people living in the United States have disproportionate 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 in specific Indigenous groups, 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 Indigenous Americans. 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 Indigenous American 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 Indigenous Americans. 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 Indigenous Americans living in Arizona have a distinct allelic architecture compared to populations of European and East Asian ancestry. The Indigenous 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 Indigenous Americans 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 the American Indian sample, 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 Indigenous Americans. 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. Lipid metabolism is an important element in regulating whole body insulin sensitivity and energy balance, and both excess and deficiency of lipid storage in white adipose tissue is linked to insulin resistance and risk for type 2 diabetes. A key enzyme in lipid metabolism is the short isoform of hormone sensitive lipase (HSL), encoded by the LIPE gene, which is involved in the hydrolysis of intracellular triglycerides and the release of fatty acids for use as energy substrate in adipose tissues. In animal studies, HSL null mice had increased circulating triglyceride and glucose levels and increased fat cell size; when cultured in media, fat cells from HSL null mice exhibited a decreased release of glycerol and free fatty acids. In humans, homozygous/bi-allelic pathogenic variants in LIPE are the cause of familial partial lipodystrophy type 6. A novel 19-bp frameshift deletion in LIPE has been reported in the Old Order Amish living in Lancaster, PA, and a novel nonsense variant, p.Ala507fsTer563 was identified in two siblings of European ancestry. Carriers of these loss-of-function mutations manifested clinical phenotypes including dyslipidemia, hepatic steatosis, insulin resistance, and T2D. Analysis of LIPE in whole-exome sequence data from Indigenous Americans identified an Arg611Cys variant (Cys-allele frequency = 0.087) significantly associated with T2D. Among these individuals who did not have T2D, those with the Cys-allele had increased insulin levels during an oral glucose tolerance test and a mixed meal test), and had increased lipid oxidation rates post-absorptively and during insulin infusion compared to individuals with the Arg-allele. In vitro functional studies showed that cells expressing the Cys-allele had a 17.2% decrease in lipolysis under isoproterenol stimulation and a 21.3% decrease in lipase enzyme activity measured by using p-nitrophenyl butyrate as a substrate compared to the Arg-allele. These data support that the Arg611Cys variant causes a modest impairment in lipolysis, thereby affecting glucose homoeostasis and risk of T2D in human populations. There is a collective push to diversify human genetic studies by including underrepresented populations. However, analyzing DNA sequence reads involves the initial step of aligning the reads to the GRCh38/hg38 reference genome which is inadequate for non-European ancestries. Therefore we conducted a study, using long-read sequencing technology, to construct de novo genome assemblies from two indigenous Americans from Arizona (IAZ). Each assembly included ∼17 Mb of DNA sequence not present [nonreference sequence (NRS)] in hg38, which consists mostly of repeat elements. Forty NRSs totaling 240 kb were uniquely anchored to the hg38 primary assembly generating a modified hg38-NRS reference genome. DNA sequence alignment and variant calling were then conducted with whole-genome sequencing (WGS) sequencing data from 387 IAZ using both the hg38 and modified hg38-NRS reference maps. Variant calling with the hg38-NRS map identified ∼50,000 single-nucleotide variants present in at least 5% of the WGS samples which were not detected with the hg38 reference map. We also directly assessed the NRSs positioned within genes. Seventeen NRSs anchored to regions including an identical 187 bp NRS found in both de novo assemblies. The NRS is located in HCN2 79 bp downstream of Exon 3 and contains several putative transcriptional regulatory elements. Genotyping of the HCN2-NRS revealed that the insertion is enriched in IAZ (minor allele frequency = 0.45) compared to other reference populations tested. This documented that inclusion of population-specific NRSs can dramatically change the variant profile in an underrepresented ethnic groups and thereby lead to the discovery of previously missed common variations.