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.

对 1 M SNP GWAS 的基因型数据进行分析，发现 DNER 中的一个 SNP (rs1861612) 在全基因组范围内与 T2D 相关（比值比 = 每个 T 等位基因拷贝 1.29，P = 6.6 x 10-8）。 DNER（含有 δ/Notch 样 EGF 重复序列）在胰岛中表达，并通过细胞间相互作用介导 Notch 信号传导。 Notch 信号传导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，其中 DNER 过表达并被 siRNA 靶向敲低。 Notch 通路特异性基因 Notch1、Hes1 和 Neurog3 受 DNER 显着调节（P <0.001），表明 DNER 的改变通过 Notch 信号通路介导对 T2D 易感性的影响。尽管 DNER 此前尚未在 T2D 的 GWAS 中报道过，但 NOTCH2 是其他种族中高度可重复的 T2D 基因。 对与 BMI 相关的基因型数据的分析确定 MAP2K3 是一个新的肥胖基因。在已发表的其他种族群体的 GWAS 中，该基因尚未被报道为最重要的信号之一，但是，我们要求白种人 BMI 的 GIANT 研究查看其 GWAS 数据中的特定 SNP，并且一些 SNP 确实与 BMI 存在显着关联（ P = 2 x 10-4)。与白种人相比，这些变异对美洲印第安人的影响更大。将我们的美洲印第安人数据与高加索人数据相结合，得出了很强的关联性 (P = 4 x 10-9)。对 MAP2K3 的功能研究表明，该基因在脂肪生成中发挥作用，这与目前已知的 MAP 信号通路一致。然而，我们还表明，下丘脑（调节食物摄入的关键组织）中 MAP2K3 的组成型表达与炎症相关基因的上调有关。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中的因果作用，以及细胞因子对进食行为的影响。 对来自 GWAS 的其他信号的跟踪发现，FOXO1A 基因内的变异与早发（发病年龄 <25 岁）T2D 有一定关联。 FOXO1A 编码参与胰腺 β 细胞生长和下丘脑能量平衡的叉头转录因子；因此，通过对 7710 名美洲印第安人的人群样本中的所有标签 SNP 进行基因分型，彻底探究了 FOXO1A 基因座的变异。内含子 SNP rs2297627 与早发性 T2D 以及任何年龄的 T2D 发病相关，其中 T2D 风险等位基因还与较低的急性胰岛素分泌相关。另一个内含子 SNP（rs1334241，D=0.99，r2=0.49 与 rs2297627）与最大成年期 BMI、最大儿童期 BMI 和体脂百分比相关。因此，我们认为 FOXO1A 的常见变异对美洲印第安人患 T2D 和肥胖的风险影响不大。 我们在皮马印第安人中的 GWAS 还发现了 PFKFB2 (rs17258746) 中与 BMI 相关的变异。 PFKFB2 编码 6-磷酸果糖-2-激酶/果糖 2,6-双磷酸酶亚型 2，在葡萄糖代谢中发挥作用。为了跟踪 GWAS，我们对美洲印第安人的 PFKFB2 标签 SNP 进行了基因分型，这些印第安人拥有 BMI (n=6839)、T2D (n=7710)、糖尿病肾病 (DN；n=2452)、体脂百分比 (%) 的纵向数据（ n=555）和胰岛素分泌（n=298）。在美国城市印第安人中进一步对两个 SNP 进行基因分型，以评估 DN 的复制情况 (n=864)。使用实时 RT-PCR 测量 201 个脂肪活检组织中的 PFKFB2 表达，使用 Affymetrix U133 阵列测量 61 个肾脏活检组织中的 PFKFB2 表达。捕获相同信号的两个 SNP（rs17258746 和 rs11120137）与成年期最大 BMI、儿童期最大 BMI z 分数和成年期体脂百分比相关。肥胖增加的等位基因与较低的 PFKFB2 脂肪表达相关。 PFKFB2 的较低表达进一步与较高的体脂百分比和 BMI 相关。该等位基因还与两个美洲印第安人队列中 DN 风险增加相关，并且与肾小球中 PFKFB2 表达较低相关。通过急性胰岛素反应和 30 分钟血浆胰岛素浓度评估，相同的等位基因还与较低的胰岛素分泌相关。因此，我们认为 PFKFB2 的变异似乎会降低脂肪和肾组织中 PFKFB2 的表达，从而增加肥胖和 DN 的风险。 为了确定增加 DN 风险的其他遗传变异，我们对 2388 名受试者（1579 名纯血统和 809 名非血统皮马印第安人）进行了全基因组关联研究 (GWAS)，该研究代表了 519 名 DN 病例（白蛋白肌酐比值）比率 ACR300mg/g 或 ESRD）和 1869 名对照（ACR<300mg/g），其中所有病例和对照均患有 T2D。基因分型使用具有 494K 标签 SNP 的定制 Axiom 阵列，可捕获 Pima 基因组中识别的 92% 的常见编码和非编码 SNP（次要等位基因频率为 0.05）和 56% 的较低频率仅编码 SNP（0.01-0.05）。对于频率为 0.50 的风险等位基因，检测与效应大小 OR=1.20 关联的功效为 0.54。正如功效计算所预期的那样，没有任何一个 SNP 达到全基因组统计显着性 (5x10-8)。因此，我们重点关注那些在单独分析的完整和非完整皮马印第安样本中具有可重现证据的关联的 SNP，这些 SNP 也被预测会对蛋白质功能产生有害影响。这些 SNP 在居住在凤凰城城市的 902 名美国印第安人的独立样本（157 名 DN 病例和 745 名对照）中进一步进行了基因分型。预测 FBLN2 基因中 S361G 的 SNP rs3732666 显示出与完整遗传（每个 G 等位基因拷贝 OR = 1.33，频率 = 0.28，p = 7.1x10-3）和非完整遗传皮马印第安人（OR = 1.55，p=5.6x10-3)，来自吉拉河印第安人社区，也与美洲印第安人的 DN 相关凤凰城城市（OR=1.38，p=0.048）。对所有 3 个队列的荟萃分析表明，与 DN OR=1.39 的关联最强，p=2.3x10-5。 FBLN2 编码细胞外基质重塑蛋白 fibulin2，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。对培养肾细胞中 FBLN2 错义变异的体外研究正在进行中，以确定 S361G 变异是否影响 TGFb1 和/或 VEGFA 信号传导。了解 DN 的遗传基础可能有助于更好地理解为什么一些 T2D 患者会患上 DN，而其他人则不会。 最近，我们还完成了对能量消耗（EE）的全基因组关联研究的分析，这是有史以来第一次对影响肥胖预测因子的变异进行全基因组询问。我们发现了 GPR158 中的一个变体，该变体与两种不同的 EE 测量值（在代谢室中测量的 24 小时 EE 值和静息代谢率）相关，这与皮马印第安人的身体脂肪和 BMI 呈负相关。该变异的风险等位基因在皮马印第安人中的频率为 0.60，在白种人中的频率为 0.004。我们还进行了功能研究，并表明该变异会影响基因表达水平。