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

我们对来自1 M SNP GWAS的基因型数据进行分析，发现DNER中的SNP（rs 1861612）与T2 D在全基因组显著性上相关（比值比= 1.29/T等位基因拷贝，P = 6.6 x 10-8）。DNER（含有delta/notch-like EGF重复序列）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信号传导对胰腺发育至关重要。我们评估了DNER在小鼠细胞系中的生理作用，其中DNER既过表达又被siRNA靶向敲低。Notch通路特异性基因Notch 1、Hes 1和Neurog 3受DNER显著调节（P <0.001），表明DNER的改变通过notch信号通路介导对T2 D易感性的影响。虽然DNER以前没有在T2 D的GWAS中报道，但NOTCH 2在其他种族中是高度可重复的T2 D基因。 对与BMI相关的基因型数据的分析确定MAP 2K 3为肥胖的新基因。该基因尚未被报道为来自其他种族的已发表GWAS中的最高信号之一，然而，我们要求高加索人BMI的GIANT研究在其GWAS数据中查看特定SNP，并且一些SNP确实与BMI有显著关联（P = 2 x 10-4）。与高加索人相比，这些变异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人数据相结合，提供了强相关性（P = 4 x 10-9）。对MAP 2K 3的功能研究表明，该基因在脂肪形成中起作用，这与目前已知的MAP信号通路一致。然而，我们也表明，MAP 2K 3在下丘脑（调节食物摄入的关键组织）中的组成性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，以及细胞因子对摄食行为的影响。 对来自我们GWAS的额外信号的随访发现FOXO 1A基因内的变异与早发性（发病年龄<25岁）T2 D适度相关。FOXO 1A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名美国印第安人的基于人群的样本中所有标签SNP进行基因分型，对FOXO 1A基因座的变异进行了彻底的研究。内含子SNP rs 2297627与早发性T2 D以及任何年龄的T2 D发作相关，其中T2 D风险等位基因也与较低的急性胰岛素分泌相关。另一个内含子SNP（rs 1334241，D=0.99，r2=0.49与rs 2297627）与最大成年BMI、最大儿童BMI和%体脂相关。因此，我们认为FOXO 1A的常见变异对美国印第安人T2 D和肥胖的风险有一定影响。 我们在皮马印第安人中的GWAS还确定了PFKFB 2（rs 17258746）中与BMI相关的变体。PFKFB 2编码6-磷酸果糖-2-激酶/果糖2，6-二磷酸酶亚型2，其在葡萄糖代谢中起作用。为了对GWAS进行随访，在具有BMI（n=6839）、T2 D（n=7710）、糖尿病肾病（DN; n=2452）、%体脂（n=555）和胰岛素分泌（n=298）纵向数据的美国印第安人中对PFKFB 2的标签SNP进行基因分型。两个SNPs进一步基因分型在城市美国印第安人，以评估复制DN（n=864）。使用实时RT-PCR在201个脂肪活检组织中测量PFKFB 2表达，使用Affytron U133阵列在61个肾活检组织中测量PFKFB 2表达。两个SNPs（rs 17258746和rs 11120137）捕获了相同的信号，与成年期的最大BMI、儿童期的最大BMI z评分和成年期的%体脂相关。肥胖增加的等位基因与较低的PFKFB 2脂肪表达相关。PFKFB 2的低表达进一步与较高的体脂百分比和BMI相关。该等位基因也与两组美国印第安人DN风险增加相关，并且与肾小球中PFKFB 2表达降低相关。相同的等位基因也与急性胰岛素反应和30分钟血浆胰岛素浓度评估的胰岛素分泌降低相关。因此，我们认为PFKFB 2的变异似乎降低了脂肪和肾脏组织中PFKFB 2的表达，从而增加了肥胖和DN的风险。

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.