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

皮马印第安人的遗传研究一直受到阻碍，因为缺乏商业上可用的基因分型阵列，这些基因分型阵列包含丰富的或独特的该群体的变异，并且缺乏公开可用的基因组数据集，用于捕获该群体的连锁不平衡。为了克服这些问题，我们获得了335名皮马印第安人的全基因组序列数据，并与Affymetrix合作设计和制造了定制的皮马印第安人基因型阵列。然后使用该阵列对来自吉拉河印第安人社区的7,701名皮马印第安人进行基因分型，包括T2D、BMI和包括糖尿病肾病（DN）在内的T2D并发症的纵向数据，其中550名受试者具有来自CRC非糖尿病住院患者研究的相关代谢特征的额外数据。随后，使用该阵列对3209名城市印第安人（均为任何部落的50%美洲印第安人）进行了T2D和DN横断面数据的基因分型。利用基因型数据作为支撑，我们对7,701名皮马印第安人进行了7,996,530个信息得分为>.5的变异估算。这些输入的变量使我们能够对这些皮马样本进行全面分析，同时也使我们能够为大型协作荟萃分析提供皮马印第安人数据。我们的T2D和胰岛素分泌功能GWAS检测到KCNQ1中3个独立信号和TH中一个新发现的信号。KCNQ1 snp标记在KCNQ1的内含子15中有39个snp。利用Crispr/Cas9基因编辑技术在KCNQ1中靶向缺失内含子15 (48kb)， RT-PCR结果显示，这些细胞中KCNQ1及其邻近基因KCNQ-OT1和CDKN1C的RNA表达水平降低。我们通过系统地构建包含较少变体的结构，并通过引入荧光素酶测定评估它们对KCNQ1、KCNQ1- ot1和CDKN1C启动子活性的影响，来精确定位该内含子中的实际功能snp。此外，我们正在使用Crispr/Cas9基因编辑方法在人类β细胞系Endo-BHC1中产生敲除，以更好地评估这些变体在更具生物学性的细胞类型中的功能。TH中的另一个编码酪氨酸羟化酶的GWAS信号也在研究中。这种变异在其与T2D和胰岛素分泌功能的关联中具有性别特异性作用。我们的GWAS SNP与TH启动子中的2个SNP处于完美的连锁不平衡状态，这些SNP似乎是功能性的。其中一个功能性snp破坏了Y染色体上表达的SRY因子的结合位点。我们认为SRY的性别特异性表达是这些变异与T2D的性别特异性关联的基础。

Genetic studies in Pima Indians have been hampered by the lack of commercially available genotyping arrays which contain variation enriched or unique to this population, and the absence of publicly available genomic datasets that capture linkage disequilibrium for imputation in this population. To overcome these issues, we obtained whole-genome sequence data on 335 Pima Indians and in collaboration with Affymetrix designed and manufactured a custom Pima Indian genotypic array. This array was then used to genotype 7,701 Pima Indians from the Gila River Indian Community with longitudinal data on T2D, BMI and T2D complications including diabetic nephropathy (DN), of which 550 subjects had additional data on related metabolic traits from non-diabetic inpatient studies in our CRC. Subsequently, 3,209 Urban Indians (all are 50% American Indian of any tribe) with cross-sectional data on T2D and DN have been genotyped with this array. Using genotypic data as a scaffold, we imputed 7,996,530 variants with an info score >0.5 for the 7,701 Pima Indians. These imputed variants allow us to perform comprehensive analyses in these Pima samples, but also enable us to contribute our Pima Indian data for large collaborative meta-analyses. Our GWAS for T2D and insulin secretory function detected 3 independent signals in KCNQ1 and a newly identified signal in TH. The KCNQ1 SNPs tag 39 SNPs in intron 15 of KCNQ1. Crispr/Cas9 gene-editing was used to create a targeted deletion of intron 15 (48kb) in KCNQ1, and RT-PCR showed reduced RNA expression levels of KCNQ1 and neighboring genes KCNQ-OT1 and CDKN1C in these cells. We are pinpointing the actual functional SNPs in this intron by systematically making constructs containing fewer variants and assessing their impact on KCNQ1, KCNQ1-OT1 and CDKN1C promoter activity via in intro luciferase assays. In addition, we are using the Crispr/Cas9 gene-editing approach to generate knockouts in the human beta cell line Endo-BHC1 to better assess the function of these variants in a more biologic cell type. The other GWAS signal in TH, which encodes tyrosine hydroxylase, is also being studied. This variant had a gender specific effect in its association with T2D and insulin secretory function. Our GWAS SNP is in perfect linkage disequilibrium with 2 SNPs in the promoter of TH which appear to be functional. One of these functional SNPs disrupts the binding site for SRY, a factor expressed on the Y chromosome. We propose that gender-specific expression of SRY underlies the gender specific association of these variants with T2D. 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. 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.