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

在我们的1 M GWAS中，BMI的顶级GWAS信号中有3个变异位于溶血磷脂酰甘油酰基转移酶1 （LPGAT1）基因内。LPGAT1属于酰基转移酶大家族，参与多种生物过程，包括调节能量稳态和体重的途径。因此，我们分析了LPGAT1作为皮马印第安人肥胖的候选基因。对生活在吉拉河印第安社区的3391名全遗传皮马印第安人的群体样本进行了基因分型，研究发现，位于LPGAT1内部和邻近的变异（n = 26）包括一个新的27 bp缺失。在来自同一社区的3327名混血印第安人的第二个样本中评估了所选变异的复制。在两个独立样本（rs112662024和rs12058008标记）中，与体重指数（BMI）有名义关联的变异在联合样本中具有P = 1-4 x 10-5的相关性（n = 6718）。一个单倍型包含一个新的27 bp缺失，这在白种人中没有发生，显示出与全遗传皮马印第安人的BMI最强的关联。体外功能分析提供了提示性证据，表明5-UTR中27 bp的缺失可能影响转录或转录后调控。对人前脂肪细胞LPGAT1 cDNA的分析发现了一个额外的外显子，其序列可能作为线粒体靶向肽。

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.