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

全基因组和全外显子组测序鉴定 2 型糖尿病和肥胖基因

基本信息

批准号：
10700678
负责人：
Leslie J Baier
金额：
$ 101.66万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10700678
关键词：
Adipocytes Adipose tissue Adult Affect Alleles American Indians Amish Animals Architecture Asian ancestry Biological Assay Body mass index Butyrates Candidate Disease Gene Cell Size Cells Child Code Codon Nucleotides Complex D-Amino Acid Dehydrogenase Data Development Diabetic Nephropathy Disease Dopamine Dyslipidemias East Asian Elements Energy Metabolism Enzymes Ethnic group European Exclusion Exhibits FOXO1A gene Familial partial lipodystrophy Fatty Acids Fatty Liver Frequencies Gene Frequency Genes Genetic Transcription Genomics Genotype Glucose Glycerol Heritability Hour Human Hydrolysis IGFBP4 gene Impairment In Vitro Individual Infusion procedures Insulin Insulin Resistance Insulin-Like Growth Factor I Insulin-Like Growth-Factor-Binding Proteins Isoproterenol Knockout Mice Large-Scale Sequencing Lead Link Lipase Lipids Lipolysis Measures Mediating Methods Minor Modeling Mus Mutation Non-Insulin-Dependent Diabetes Mellitus Nonesterified Fatty Acids Nonsense Codon Nuclear OGTT Obesity PI3K/AKT Pathogenicity Pathway interactions Phase Phenotype Pima Indian Plasma Population Prediabetes syndrome Predisposition Privatization Protein Isoforms Proteins Reporter Reporting Research Personnel Risk Role Serine Siblings Signal Transduction Single Nucleotide Polymorphism Sleep Somatomedins Techniques Testing Threonine Triglycerides Variant base clinical phenotype diabetes mellitus genetics diabetes risk disorder risk energy balance enzyme activity exome exome sequencing genome sequencing genome wide association study genome-wide insertion/deletion mutation insulin sensitivity lipid biosynthesis lipid metabolism loss of function loss of function mutation new therapeutic target novel oxidation protein degradation rare variant sterol esterase trait whole genome

项目摘要

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. 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 6782 American Indians identified an Arg611Cys variant (Cys-allele frequency = 0.087) significantly associated with T2D. Among these American Indians 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.

美洲印第安人患 T2D、糖尿病肾病和肥胖的比例极高，但用于识别疾病位点的大规模测序工作并未包括来自该族群的个体。因此，美洲印第安人中独特或丰富的变异可能为这些疾病确定新的治疗靶点，但在很大程度上仍然未知。为了确定增加 2 型糖尿病 (T2D) 和/或肥胖易感性的常见变异，我们生成了 335 名皮马印第安人的全基因组序列数据。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 万个插入缺失和 255,802 个替换。在所有 SNP 中，有 270 万个是新颖的。为了获得可能对疾病风险产生巨大影响的罕见变异的信息，我们最近获得了 8500 名美洲印第安人的全外显子序列数据，这些数据提供了 2 型糖尿病、肥胖、糖尿病肾病和血脂水平的信息。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 万个变异。在这些变异中，95%是单核苷酸多态性，5%是短插入或缺失。其中大约 493,0000 个变异发生在至少 5 名受试者中，并且这些变异接受了 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，与欧洲和东亚血统的人群相比，皮马印第安人具有独特的等位基因结构。皮马印第安人的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异。我们评估了 2 型糖尿病、BMI 和四种主要血浆脂质的候选基因的基因负荷关联，发现 11 个基因有 19 个显着的基因负荷关联，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 名健康皮马印第安人的全外显子序列数据，这些数据提供了 24 小时能量消耗 (24-h EE) 的信息，因为影响能量代谢的变异的识别可能会导致治疗人类肥胖的新途径。我们发现了一个在 DAO 基因中引入提前终止密码子 (Cys264Ter) 的变体。该变体表现出与 24 小时 EE 的最强关联，其中 Ter 等位基因与显着降低的 24 小时 EE（平均降低 268 kcal/天）和睡眠 EE（平均降低 135 kcal/天）相关。 Ter等位基因在皮马印第安人中的频率=0.5%，而在大多数其他种族群体中极为罕见（频率>0.01%）。体外功能分析表明，截短形式的 DAO 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，该酶参与多巴胺合成，这可能解释了它在调节 EE 中的作用。胰岛素样生长因子（IGF）是脂肪组织发育的重要调节因子。众所周知，IGF 的活性受 IGF 结合蛋白 (IGFBP) 的调节。最近对在脂肪组织中高表达的 IGFBP4 的研究表明，IGFBP4 是脂肪生成所必需的，并影响小鼠脂肪库的分布。因此，我们试图确定 IGFBP4 的编码变异是否也会导致 6809 名美洲印第安人的肥胖。鉴定出一种预测密码子 76 (Ser76Thr) 处丝氨酸变为苏氨酸的新变体，其次要等位基因频率 (mAF) 为 0.02，组合注释依赖性缺失 (CADD) 评分 >9.7（CADD 为 10 表示前 10 名） %最有可能是有害的）。这种变异与美洲印第安人儿童和成人的体重指数相关。使用检测 PI3K/AKT 介导的转录活性的体外 FOXO 报告基因测定来评估 Ser76Thr 对 IGF-I 信号传导的影响。我们发现，与非风险 Ser 等位基因相比，表达 BMI 风险 Thr 等位基因的 COS7 细胞在 FOXO1 诱导的转录活性中降低了 55%，这是由于 PI3K/AKT 激活增加介导的 FOXO1 核排斥增加。通过 IGF-I 信号传导。脂质代谢是调节全身胰岛素敏感性和能量平衡的重要因素，白色脂肪组织中脂质储存的过量和不足都与胰岛素抵抗和 2 型糖尿病的风险有关。脂质代谢中的关键酶是激素敏感脂肪酶 (HSL) 的短亚型，由 LIPE 基因编码，参与细胞内甘油三酯的水解和脂肪酸的释放，以用作脂肪组织中的能量底物。在动物研究中，HSL 缺失小鼠的循环甘油三酯和葡萄糖水平增加，脂肪细胞大小增加；当在培养基中培养时，HSL 缺失小鼠的脂肪细胞表现出甘油和游离脂肪酸的释放减少。在人类中，LIPE 中的纯合/双等位基因致病性变异是导致 6 型家族性部分脂肪营养不良的原因。生活在宾夕法尼亚州兰开斯特的旧秩序阿米什人中报道了 LIPE 中的一种新的 19-bp 移码缺失，以及一种新的无义变异, p.Ala507fsTer563 在欧洲血统的两个兄弟姐妹中被发现。这些功能丧失突变的携带者表现出临床表型，包括血脂异常、肝脂肪变性、胰岛素抵抗和 T2D。对 6782 名美洲印第安人的全外显子组序列数据进行 LIPE 分析，发现 Arg611Cys 变异（Cys 等位基因频率 = 0.087）与 T2D 显着相关。在这些没有患 T2D 的美洲印第安人中，与患有 Cys 等位基因的人相比，那些携带 Cys 等位基因的人在口服葡萄糖耐量试验和混合膳食试验中胰岛素水平升高，并且吸收后和胰岛素输注期间脂质氧化率升高。 Arg等位基因。体外功能研究表明，与 Arg 等位基因相比，表达 Cys 等位基因的细胞在异丙肾上腺素刺激下，脂肪分解作用降低了 17.2%，使用丁酸对硝基苯酯作为底物测得的脂肪酶活性降低了 21.3%。这些数据支持 Arg611Cys 变异会导致脂肪分解适度受损，从而影响人群中的葡萄糖稳态和 T2D 风险。