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

皮马印第安人的遗传学研究一直受到阻碍，原因是缺乏商业上可用的包含丰富或独特于该群体的变异的基因分型阵列，以及缺乏公开可用的基因组数据集来捕捉该群体中的连锁不平衡。为了克服这些问题，我们获得了335名PIMA印第安人的全基因组序列数据，并与Affymetrix合作设计和制造了一个定制的PIMA印第安人基因分型阵列。该阵列随后被用于吉拉河印第安人社区的7,701名PIMA印第安人的T2D、BMI和T2D并发症(包括糖尿病肾病)的纵向数据，其中550名受试者在我们的CRC非糖尿病住院研究中获得了相关代谢特征的额外数据。随后，有3,209名城市印第安人(都是任何部落中50%的美国印第安人)在T2D和DN上有横断面数据，用这个阵列进行了基因分型。使用基因分型数据作为支架，我们为7,701名皮马印第安人计算了7,996,530个变种，信息得分为0.5。这些推测的变异使我们能够对这些PIMA样本进行全面分析，但也使我们能够为大型协作性荟萃分析贡献我们的PIMA印度数据。我们的T2D和胰岛素分泌功能的GWA在KCNQ1中检测到3个独立的信号，在TH中检测到一个新的信号。KCNQ1基因内含子15上的SNPs标记为39个SNPs。利用CRISPR/Cas9基因编辑技术获得KCNQ1内含子15(48kb)的靶向缺失，RT-PCR显示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异的构建体，并通过内含体荧光素酶分析来评估它们对KCNQ1、KCNQ1-OT1和CDKN1C启动子活性的影响，从而精确定位该内含子中的实际功能SNPs。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中产生敲除基因，以更好地评估这些变体在更具生物学意义的细胞类型中的功能。TH中的另一个编码酪氨酸羟基酶的GWAS信号也在研究中。该变异在与T2D和胰岛素分泌功能的关联中具有性别特异性效应。我们的GWASSNP与TH启动子中的两个SNP处于完美的连锁不平衡状态，这两个SNP似乎具有功能。其中一个功能SNPs破坏了SRY的结合位点，SRY是一种表达在Y染色体上的因子。我们认为SRY的性别特异性表达是这些变体与T2D的性别特异性关联的基础。 来自我们GWAS的其他信号的跟踪发现FOXO1A基因中的变异与早发性(发病年龄和25岁)T2D有一定的关联。FOXO1A编码与胰腺β细胞生长和下丘脑能量平衡有关的叉头转录因子；因此，通过对7710名美洲印第安人样本中的所有标签SNP进行基因分型，FOXO1A基因座上的变异被彻底消除。内含子SNP rs2297627与早发性T2D以及任何年龄起病的T2D相关，其中T2D风险等位基因还与急性胰岛素分泌减少相关。另一个内含子SNP(rs1334241，D=0.99，R2=0.49，rs2297627)与成年最大BMI、儿童最大BMI和体脂百分比相关。因此，我们认为FOXO1A基因的共同变异对美国印第安人患T2D和肥胖的风险有一定影响。 我们在皮马印第安人中的GWAs也在PFKFB2(Rs17258746)中发现了一个与BMI相关的变异。PFKFB2编码6-磷酸果糖-2-激酶/果糖2，6-二磷酸酶亚型2，在葡萄糖代谢中起作用。为了对GWAS进行随访，对有BMI(n=6839)、T2D(n=7710)、糖尿病肾病(DN；n=2452)、体脂百分比(n=555)和胰岛素分泌(n=298)纵向数据的美国印第安人进行了PFKFB2的Tag SNPs基因分型。对美国城市印第安人中的两个SNPs进行了进一步的基因分型，以评估糖尿病肾病的重复性(n=864)。采用实时RT-PCR技术检测了201例脂肪活检组织和61例肾活检组织中PFKFB2的表达。捕获相同信号的两个SNPs(rs17258746和rs11120137)与成年时的最大BMI、儿童时期的最大BMI z得分和成年时的体脂百分比相关。肥胖增加的等位基因与PFKFB2脂肪表达降低相关。PFKFB2的低表达进一步与较高的体脂百分比和BMI相关。在两组美洲印第安人中，该等位基因也与糖尿病肾病的风险增加相关，与肾小球中PFKFB2的低表达类似。通过急性胰岛素反应和30分钟血浆胰岛素浓度评估，同样的等位基因也与胰岛素分泌减少相关。因此，我们认为，PFKFB2基因的变异似乎降低了PFKFB2在脂肪和肾脏组织中的表达，从而增加了肥胖和糖尿病肾病的风险。 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全基因组范围内询问影响肥胖预测因素的变异。我们发现了GPR158中的一个变异，它与两种不同的EE(在代谢室中测量的24小时EE和静息代谢率)相关，后者与皮马印第安人的身体肥胖和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0.6，在高加索人中的频率为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。

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.