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

美洲印第安人的T2 D、糖尿病肾病和肥胖症发病率极高，但大规模的测序工作，以确定疾病位点没有包括来自这个种族群体的个人。因此，在美洲印第安人中独特或丰富的变异，这可能会发现这些疾病的新治疗靶点，在很大程度上仍然未知。 为了确定增加2型糖尿病（T2 D）和/或肥胖症易感性的常见变异，在335名皮马印第安人身上生成了全基因组序列数据。测序由Illumina（N=301）和Complete Genomics，Inc（N=34）进行。发现了1300万个变异，包括1100万个SNP，160万个Indel和255，802个替换。在所有SNP中，有270万个是新的。为了获得可能对疾病风险具有较大影响的罕见变异的信息，我们最近获得了8500名美国印第安人的全外显子序列数据，这些数据为2型糖尿病，肥胖，糖尿病肾病和血脂水平提供了信息。 对于整个外显子组序列数据，在8500名美洲印第安人中检测到约170万个变体。在这些变异中，95%是单核苷酸多态性，5%是短插入或缺失。这些变异中约有493，0000个发生在至少5名受试者中，这些受试者接受了2型糖尿病和BMI的单变异分析。此外，还采用了基于基因的模型（Burden和SKAT方法）。 外显子组测序表明，皮马印第安人有一个独特的等位基因结构相比，欧洲和东亚血统的人口。皮马印第安人外显子组有许多预测的功能丧失（pLOF）和高度富集或私有的非同义变体。我们评估了2型糖尿病、BMI和四种主要血脂的候选基因的基因负担相关性，发现11个基因有19个显著的基因负担相关性，为优先考虑GWAS信号的候选效应基因提供了额外的证据。 我们还分析了373名健康的皮马印第安人的全外显子组序列数据，这些数据为24小时能量消耗（24小时EE）提供了信息，因为识别影响能量代谢的变异可能会导致治疗人类肥胖的新途径。我们发现了一个变体，它在DAO基因中引入了一个提前终止密码子（Cys 264 Ter）。该变体与24小时EE的相关性最强，其中Ter等位基因与24小时EE（平均降低268 kcal/天）和睡眠EE（降低135 kcal/天）大幅降低相关。Ter等位基因在皮马印第安人中的频率为0.5%，而在大多数其他种族中极为罕见（频率>0.01%）。体外功能分析显示，截短形式的DAO的蛋白质水平降低，与蛋白质降解增加一致。DAO编码D-氨基酸氧化酶，其参与多巴胺的合成，这可能解释其在调节EE中的作用。

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.