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

在我们的1MGWAs中，BMI的最高GWA值信号中有3个变异体定位于溶血磷脂酰甘油酰基转移酶1(LPGAT1)基因。LPGAT1属于酰基转移酶大家族，参与多种生物学过程，包括调节能量、动态平衡和体重的途径。因此，LPGAT1基因被分析为PIMA印第安人肥胖的候选基因。对生活在吉拉河印第安人社区的3391名全血统皮马印第安人的群体样本进行了基因分型，发现了位于LPGAT1内和邻近LPGAT1的变异(n=26)，包括通过测序确定的新的27bp缺失。在来自同一社区的3327名混血美洲原住民的第二个样本中，对选定的变体的复制进行了评估。在两个独立样本(由rs112662024和rs12058008标记)中，与体重指数(BMI)有名义关联的变异在组合样本(n=6718)中的关联为P=1-4x10-5。一个单倍型包括一个新的27个碱基缺失，这在高加索人中不会发生，在全血统的皮马印第安人中显示出与BMI的最强关联。体外功能分析提供了提示证据，即5-UTR中的27bp缺失可能影响转录或转录后调控。对人前脂肪细胞的LPGAT1基因进行分析，发现了一个额外的外显子，该外显子的序列可能是一个线粒体靶向肽。 在我们针对2型糖尿病的100万个SNP GWA中，我们最有力的发现是DNER中的一个SNP(Rs1861612)与整个基因组意义上的2型糖尿病相关(优势比=1.29每T等位基因副本，P=6.6x10-8)。DNER(Delta/Noch-like EGF Repeat Containing)在胰岛细胞中表达，通过细胞间相互作用介导Noch信号传导。Notch信号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，在该小鼠细胞系中，DNER过度表达并被siRNA靶向击倒。Notch通路特异性基因Notch1、Hes1和Neurog3受到DNER的显著调控(P&lt；0.001)，表明DNER的改变通过Notch信号通路介导了对T2D易感性的影响。虽然DNER以前没有在GWAS中被报道为2型糖尿病，但NOTCH2在其他种族中是一种高度可重复性的2型糖尿病基因。 在我们针对BMI的100万个SNP GWA中，我们最有力的发现之一是发现MAP2K3是一种新的肥胖基因。该基因在已发表的其他种族的GWASs中没有被报道为最重要的信号之一，然而，我们要求这项关于高加索人BMI的大型研究在他们的GWASs数据中查看特定的SNP，并且几个SNP确实与BMI有显著的关联(P=2×10-4)。与高加索人相比，这些变异在美洲印第安人中的影响更大。结合我们的美洲印第安人数据和高加索人数据提供了很强的关联性(P=4x10-9)。对MAP2K3的功能研究表明，该基因在脂肪形成中具有作用，这与目前已知的MAP信号通路是一致的。然而，我们也表明，MAP2K3在下丘脑的结构性表达与参与炎症的基因上调有关，下丘脑是调节食物摄入的关键组织。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。

In our 1 M GWAS, among the top GWAS signals for BMI were 3 variants that mapped within the lysophosphatidylglycerol acyltransferase 1 (LPGAT1) gene. LPGAT1 belongs to a large family of acyltransferases which are involved in a variety of biological processes including pathways that regulate energy homeostasis and body weight. Therefore LPGAT1 was analyzed as a candidate gene for obesity in Pima Indians. Variants (n = 26) located within and adjacent to LPGAT1 including a novel 27 bp deletion identified by sequencing were genotyped in a population-based sample of 3391 full-heritage Pima Indians living in the Gila River Indian Community. Replication of selected variants was assessed in a second sample of 3327 mixed-heritage Native Americans from the same community. Variants with nominal associations with body mass index (BMI) in each of the two independent samples (tagged by rs112662024 and rs12058008) had associations of P = 1-4 x 10-5 in the combined sample (n = 6718). A haplotype that included a novel 27 bp deletion, which does not occur in Caucasians, showed the strongest association with BMI in full heritage Pima Indians. In vitro functional analysis provided suggestive evidence that this 27 bp deletion in the 5-UTR may affect transcriptional or post-transcriptional regulation. Analysis of LPGAT1 cDNA from human preadipocytes identified an additional exon whose sequence could potentially serve as a mitochondrial targeting peptide. In our 1 million SNP GWAS for type 2 diabetes, our strongest finding was with a SNP (rs1861612) in DNER that was associated with type 2 diabetes 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 type 2 diabetes, NOTCH2 is a highly reproducible type 2 diabetes gene in other ethnicities. In our 1 million SNP GWAS for BMI, one of our strongest findings was identifying 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.