Follow-Up Studies of a Genome-Wide Association Analysis in Pima Indians

皮马印第安人全基因组关联分析的后续研究

• 批准号: 9549918
• 负责人: Leslie J Baier
• 金额: $ 105.77万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 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/9549918
• 关键词: 6-Phosphofructo-2-kinase; Acute; Adipose tissue; Adult; Affect; Age; Age of Onset; Albumins; Alleles; American Indians; Arizona; Basal metabolic rate; Biopsy; Body fat; Body mass index; Body measure procedure; Caucasians; Cell Communication; Cells; Childhood; Code; Communities; Creatinine; Custom; DNA; Data; Diabetes Mellitus; Diabetic Nephropathy; Disease; EGF gene; Eating; End stage renal failure; Energy Metabolism; Ethnic Origin; Ethnic group; Euglycemic Clamping; Extracellular Matrix; FOXO1A gene; Fatty acid glycerol esters; Feeding behaviors; Follow-Up Studies; Frequencies; Fructose; Gene Expression; Gene Expression Regulation; Gene Frequency; Genes; Genetic; Genetic Variation; Genome; Genotype; Glucose; High Prevalence; Hypothalamic structure; In Vitro; Individual; Inflammation; Insulin; Intravenous Bolus; Kidney; Kidney Glomerulus; Knowledge; Lead; Measures; Mediating; Meta-Analysis; Metabolic; Minor; Mouse Cell Line; Native Americans; Non-Insulin-Dependent Diabetes Mellitus; Notch Signaling Pathway; Obesity; Odds Ratio; Pathway interactions; Physiological; Pima Indian; Plasma; Play; Positioning Attribute; Predisposition; Protein Isoforms; Proteins; Publishing; RNA Splicing; Reporting; Reproducibility; Reverse Transcriptase Polymerase Chain Reaction; Risk; Rivers; Role; Sampling; Siblings; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism Map; Site; Small Interfering RNA; Structure of beta Cell of islet; Techniques; Time; Tissues; Transforming Growth Factors; Untranslated RNA; Up-Regulation; VEGFA gene; Variant; base; case control; cell growth; cohort; cytokine; design; diabetic; early onset; energy balance; falls; follow-up; forkhead protein; genetic variant; genome wide association study; genome-wide; genomic variation; glucose metabolism; insulin secretion; islet; kidney cell; knock-down; lipid biosynthesis; non-diabetic; notch protein; overexpression; pancreas development; phenotypic data; population based; protein function; response; risk variant; trait; urban Native American; whole genome

Analysis of our genotypic data from a 1 M SNP GWAS identified a SNP (rs1861612) in DNER that associated with T2D at genome-wide significance (odds ratio = 1.29 per copy of the T allele, P = 6.6 x 10-8). DNER (delta/notch-like EGF repeat containing) is expressed in islets and mediates notch signaling via cell-cell interaction. Notch signaling is critical for pancreatic development. We assessed the physiologic role of DNER in a mouse -cell line in which DNER was both over-expressed and knocked down by siRNA targeting. Notch pathway specific genes, Notch1, Hes1, and Neurog3 were significantly regulated by DNER (P <0.001), suggesting that alterations in DNER mediate an effect on T2D susceptibility through the notch signaling pathway. Although DNER has not been previously reported in GWASs for T2D, NOTCH2 is a highly reproducible T2D gene in other ethnicities. Analysis of the genotypic data for associaiton with BMI identified MAP2K3 as a new gene for obesity. This gene had not been reported as being among the top signals in published GWASs from other ethnic groups, however, we requested that the GIANT study of BMI in Caucasians look at specific SNPs in their GWAS data and several SNPs did have significant associations with BMI (P = 2 x 10-4). The effect of these variants was larger in American Indians as compared to Caucasians. Combining our American Indian data with the Caucasian data provided strong associations (P = 4 x 10-9). Functional studies on MAP2K3 showed that this gene has a role in adipogenesis, which is consistent with what is currently known about MAP signaling pathways. However, we also show that constitutive expression of MAP2K3 in the hypothalamus, a key tissue for modulating food intake, is associated with an up-regulation of genes involved in inflammation. This is an intriguing finding because several recent reports have proposed a causal role of hypothalamic inflammation in high fat induced obesity, as well as cytokines eliciting effects on feeding behavior. Follow-up of additional signals from our GWAS identified variation within the FOXO1A gene that modestly associated with early-onset (onset age <25years) T2D. FOXO1A encodes the forkhead transcription factor involved in pancreatic beta-cell growth and hypothalamic energy balance; therefore, variation across the FOXO1A locus was thoroughly interogated by genotyping all tag SNPs in a population-based sample of 7710 American Indians. An intronic SNP rs2297627 associated with early-onset T2D as well as T2D onset at any age, where the T2D risk allele also associated with lower acute insulin secretion. Another intronic SNP (rs1334241, D=0.99, r2=0.49 with rs2297627) associated with maximum adulthood BMI, maximum childhood BMI and % body fat. Therefore, we propose that common variation in FOXO1A modestly affects risk for T2D and obesity in American Indians. Our GWAS in Pima Indians also identified a variant within PFKFB2 (rs17258746) that associated with BMI. PFKFB2 encodes 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase isoform 2, which plays a role in glucose metabolism. To follow-up on the GWAS, tag SNPs across PFKFB2 were genotyped in American Indians who had longitudinal data on BMI (n=6839), T2D (n=7710), diabetic nephropathy (DN; n=2452), % body fat (n=555) and insulin secretion (n=298). Two SNPs were further genotyped in urban American Indians to assess replication for DN (n=864). PFKFB2 expression was measured in 201 adipose biopsies using real-time RT-PCR and 61 kidney biopsies using the Affymetrix U133 array. Two SNPs (rs17258746 and rs11120137), which capture the same signal, were associated with maximum BMI in adulthood, maximum BMI z-score in childhood and % body fat in adulthood. The adiposity-increasing allele correlated with lower PFKFB2 adipose expression. Lower expression of PFKFB2 further correlated with higher % body fat and BMI. This allele was also associated with increased risk for DN in both cohorts of American Indians and similarly correlated with lower PFKFB2 expression in kidney glomeruli. The same allele was also associated with lower insulin secretion assessed by acute insulin response and 30-min plasma insulin concentrations. Therefore, we propose that variation in PFKFB2 appears to reduce PFKFB2 expression in adipose and kidney tissues, and thereby increase risk for adiposity and DN. To identify additional genetic variations that increase risk for DN, we performed a genome-wide association study (GWAS) in 2388 subjects (1579 full heritage and the 809 non-full heritage Pima Indians) which represented 519 DN cases (albumin-to-creatinine ratio ACR300mg/g or ESRD) and 1869 controls (ACR<300mg/g), where all cases and controls had T2D. Genotyping utilized a custom Axiom array with 494K tag SNPs which capture 92% of common coding and non-coding SNPs (minor allele frequency of 0.05) and 56% of lower frequency coding-only SNPs (0.01-0.05) identified in the Pima genome. The power to detect an association with an effect size OR=1.20 was 0.54 for a risk allele with frequency of 0.50. As anticipated by the power calculations, no single SNP achieved genome-wide statistical significance (5x10-8). Therefore, we focused on those SNPs with reproducible evidence for association in full and non-full Pima Indian samples analyzed separately, which were also predicted to have a deleterious impact on protein function. These SNPs were further genotyped in an independent sample of 902 American Indians living in urban Phoenix (157 DN cases and 745 controls). SNP rs3732666 which predicts a S361G in the FBLN2 gene showed a consistent association with DN in full heritage (OR=1.33 per copy of G allele, frequency =0.28, p=7.1x10-3) and non-full heritage Pima Indians (OR=1.55, p=5.6x10-3) from the Gila River Indian Community, and also associated with DN in American Indians from urban Phoenix (OR=1.38, p=0.048). A meta-analysis of all 3 cohorts demonstrated the strongest association with DN OR=1.39, p=2.3x10-5. FBLN2 encodes the extracellular matrix remodeling protein fibulin2, which is a modulator of transforming growth factor b1 (TGFb1) and vascular endothelial growth factor a (VEGFA) pathways. In vitro studies of this missense variant in FBLN2 in cultured kidney cells are ongoing to establish whether the S361G variant affects TGFb1 and/or VEGFA signaling. Knowledge of the genetic basis of DN may lead to a better understanding of why some individuals with T2D develop DN, while others do not. Recently, we also completed analysis of a genome-wide association study for energy expenditure (EE) which represents the first ever genome-wide interrogation for variation that affects this predictor of obesity. We identified a variant in GPR158 that associates with two different measures of EE (24-hr EE measured in a metabolic chamber and resting metabolic rate) which is inversely related to body fatness and BMI in Pima Indians. The risk allele for this variant has a frequency of 0.60 in Pima Indians and 0.004 in Caucasians. We have also performed functional studies and have shown that this variant affects gene expression levels.

从1 M SNP GWA的基因型数据分析DNER中的SNP（rs1861612）与T2D在基因组显着性下相关的DNER（t2d = 1.29 c = 6.6 x 10-8）。 DNER（Delta/Notch样EGF重复重复）在胰岛中表达，并通过细胞 - 细胞相互作用介导Notch信号传导。 Notch信号对于胰腺发育至关重要。我们评估了DNER在小鼠细胞系中的生理作用，在小鼠细胞系中，DNER既过表达又被siRNA靶向击倒。 Notch途径特异性基因Notch1，Hes1和Neurog3受DNER显着调节（P <0.001），这表明DNER的变化通过Notch信号传导途径介导了对T2D易感性的影响。尽管DNER以前尚未在T2D的GWASS中报道，但Notch2是其他种族中高度可重现的T2D基因。 与BMI的协会的基因型数据分析将MAP2K3识别为肥胖的新基因。据报道，该基因在其他种族中发表的GWASS中的主要信号之一之一，但是，我们要求对高加索人的BMI研究在其GWAS数据中查看特定的SNP，而几个SNP确实与BMI有着显着的关联（P = 2 x 10-4）。与高加索人相比，这些变体在美洲印第安人中的影响更大。将我们的美洲印第安人数据与高加索数据相结合，提供了牢固的关联（p = 4 x 10-9）。对MAP2K3的功能研究表明，该基因在脂肪形成中起作用，这与当前有关MAP信号通路的知识一致。但是，我们还表明，在下丘脑中，MAP2K3的组成型表达是一种调节食物摄入的关键组织，与涉及炎症的基因的上调有关。这是一个有趣的发现，因为最近的几份报告提出了下丘脑炎症在高脂肪诱导的肥胖症中的因果作用，以及细胞因子对喂养行为的影响。 来自我们GWAS的其他信号的随访确定了与早发（发作年龄<25岁）T2D相关的FOXO1A基因内的变化。 FOXO1A编码涉及胰腺β细胞生长和下丘脑能量平衡的叉子转录因子；因此，通过在基于人群的7710美国印第安人样本中对所有TAG SNP进行基因分型，可以完全相互交互。与早发T2D以及T2D发作相关的内含子SNP RS2297627，T2D等位基因也与较低的急性胰岛素分泌有关。另一个内含子SNP（rs1334241，d = 0.99，r2 = 0.49，rs2297627）与最大成年BMI，最大儿童BMI和％体内脂肪有关。因此，我们建议FOXO1A的共同变化会适度影响美国印第安人T2D和肥胖的风险。 我们在皮马印第安人的GWAS还确定了与BMI相关的PFKFB2（RS17258746）中的一种变体。 PFKFB2编码6-磷酸2-激酶/果糖2,6-双磷酸酶同工型2，在葡萄糖代谢中起作用。为了对GWAS进行跟进，对PFKFB2的TAG SNP进行了基因分型，这些美国印第安人在BMI（n = 6839），T2D（n = 7710），糖尿病性肾病（DN; n = 2452），％体内脂肪（n = 55555）和胰岛素（n = 555）和胰岛素（n = 298）上的纵向数据（n = 6839）（n = 7710）（n = 7710）。在美洲印第安人中进一步对两个SNP进行了基因分型，以评估DN的复制（n = 864）。使用Affymetrix U133阵列，使用实时RT-PCR和61个肾脏活检在201脂肪活检中测量PFKFB2表达。捕获相同信号的两个SNP（RS17258746和RS11120137）与成年后的最大BMI，儿童时期的最大BMI Z评分和成年时体内脂肪有关。脂肪增强等位基因与较低的PFKFB2脂肪表达相关。 PFKFB2的较低表达进一步与较高的体内脂肪和BMI相关。该等位基因也与两位美国印第安人同类群中DN的风险增加有关，并且与肾肾小球中PFKFB2较低的表达相似。同样的等位基因也与通过急性胰岛素反应和30分钟血浆胰岛素浓度评估的胰岛素分泌较低有关。因此，我们建议PFKFB2的变化似乎会降低脂肪和肾脏组织中的PFKFB2表达，从而增加脂肪和DN的风险。 为了确定增加DN风险的其他遗传变异，我们在2388名受试者中进行了全基因组的关联研究（GWAS）（1579名全遗产和809个非满足遗产PIMA印第安人），代表了519个DN病例（白蛋白与creatinine to creatinine to creatinine Gatio Acr300mg/g或eSrd）和1869和<369的crip <369和<369的crip <300mg T2D。基因分型利用了一个具有494K标签SNP的自定义公理阵列，该阵列捕获了92％的常见编码和非编码SNP（次要等位基因频率为0.05），而PIMA基因组中鉴定出的较低频率仅编码仅SNP（0.01-0.05）的较低频率仅SNP（0.01-0.05）。检测与效应大小或= 1.20的关联的功率为0.54，对于频率为0.50的风险等位基因。正如功率计算所预期的那样，没有单个SNP获得全基因组统计学意义（5x10-8）。因此，我们专注于那些具有可再现的证据的SNP，以分别分析的全部和非满足PIMA印度样品中的关联证据，这些SNP对蛋白质功能产生了有害影响。在居住在城市凤凰城的902个美洲印第安人的独立样本中，将这些SNP进一步基因分型（157例DN病例和745个对照）。 SNP RS3732666预测FBLN2基因中的S361G与DN在完整遗产中表现出一致的关联（OR = 1.33 G erele，G等Urban Phoenix（OR = 1.38，P = 0.048）。对所有3个队列的荟萃分析表明，DN OR = 1.39，p = 2.3x10-5最强。 FBLN2编码细胞外基质重塑蛋白纤维蛋白2，它是转化生长因子B1（TGFB1）和血管内皮生长因子A（VEGFA）途径的调节剂。正在进行对培养肾细胞中FBLN2中这种错义变体的体外研究，以确定S361G变体是否影响TGFB1和/或VEGFA信号传导。了解DN的遗传基础可能会更好地理解为什么有些患有T2D的人会发展DN，而另一些人则没有。 最近，我们还完成了对全基因组能量消耗研究（EE）的分析，该研究代表了影响肥胖症预测指标的有史以来首次全基因组的质疑。我们确定了GPR158中的一种变体，该变体与两种不同的EE度量（在代谢室中测得的24小时EE和静息代谢率）与PIMA印第安人中的身体脂肪和BMI成反比。该变体的风险等位基因在PIMA印第安人的频率为0.60，高加索人的频率为0.004。我们还进行了功能研究，并表明这种变体会影响基因表达水平。