Molecular Analysis Of A Region On 1q Linked With Type 2 Diabetes

在先前的Pima印第安人2型糖尿病（T2 DM）易感基因的全基因组连锁扫描中，我们获得了与染色体1 q21-q23上标记连锁的最有力证据。随后，T2 DM的1 q连锁已在几个不同的人群中复制。我们寻找潜在的糖尿病易感基因的策略基于两种互补的方法：1）对密集分布的单核苷酸多态性（SNP）进行系统分析; 2）对变异/突变相关区域内的候选基因进行研究。测试信息性SNP与糖尿病的关联，并评估其对连锁的影响。对于单核苷酸多态性的系统分析，通过联合检测1 q上T2 DM连锁的几个小组，建立了一个国际合作的1号染色体联盟，目的是促进寻找潜在的糖尿病基因。这项工作涉及对来自5个群体的5000多名受试者（包括约1000名Pimas）进行分析，到目前为止，这项工作已经导致在大多数群体中跨越连锁峰的21 Mb的初始间隔内对超过4900个SNP进行基因分型。迄今为止，已经通过测序在糖尿病和非糖尿病Pimas的子集中分析了超过150个候选基因。此外，我们在NIDDK的实验室已经研究了150多个候选基因。CACNA 1 E作为该连锁的位置候选基因进行测序，并在已在连锁研究中分析的同一组皮马印第安人中对30个SNP进行基因分型。位于3UTR的一种新的G/A变异体与初发T2 DM相关（优势比=2.09/G等位基因拷贝; 95%CI =1.31-3.33，在加性和隐性模型下分别校正p=0.001和p=0.0006），并对染色体1 q21 -25的连锁证据有显著影响（p=0.004）。 此外，在372名接受代谢测试的非糖尿病Pima受试者中，该变异与胰岛素作用降低的多项指标相关，包括空腹时间增加，30，口服葡萄糖耐量试验期间60和120分钟血糖浓度和空腹血浆胰岛素升高（所有调整的p<0.01）以及在生理和最大刺激的胰岛素浓度下胰岛素刺激的葡萄糖处置速率降低（均p<0.002）。该3UTR变体的功能分析显示，与风险等位基因相比，非风险等位基因具有高2.3倍的表达。我们的数据表明，CACNA 1 E中的3UTR变异通过影响全身胰岛素作用而导致皮马印第安人对T2 DM的易感性。此外，ARHGEF 11，它编码的Rho鸟嘌呤核苷酸交换因子11，被分析作为一个位置候选基因的这种联系，因为这种蛋白质可能会刺激Rho依赖的信号，如胰岛素信号级联。ARHGEF 11基因，以及两个相邻的基因NTRK 1和INSRR，在24个皮马印第安人谁不是一级亲属测序。对这些基因的编码区、5个和3个非编码区以及启动子区进行测序，发现ARHGEF 11有28个变异体，NTRK 1有11个变异体，INSSR有8个变异体。这47个变异体，以及这些基因内/之间的84个额外的公共数据库变异体，在参与连锁研究的同一组皮马印第安人中进行基因分型以进行关联分析。ARHGEF 11中的一个R1467 H和几个与该变异体高度连锁不平衡的其他非编码变异体，在调整性别、家族成员和Pima遗传后，与初发T2 DM名义上相关（P=0.01，OR=3.39）。危险等位基因H的频率为0.10。在一个由262名糖尿病患者组成的亚组中，他们接受了详细的代谢测试，在调整了年龄、性别、核心家庭成员和体脂百分比后，风险等位基因H也与较低的平均胰岛素介导的葡萄糖处置率和较低的平均非氧化葡萄糖储存率相关（P0.01）。这些发现表明，ARHGEF 11内的变异名义上会增加T2 DM的风险，可能是由于胰岛素抵抗增加。 1号染色体联盟最近报道了对所有7个人群的SNP基因分型的荟萃分析。由于没有发现共同的SNP来解释所有这些人群中的连锁，联盟一直专注于对连锁的替代解释。一种假设是，这些连锁信号的基础是多个罕见的变异。因此，联合体已经对1号染色体上整个连锁区域的选定基因的所有外显子进行了深度重新测序。此外，正在对所有这些人群的样本进行全基因组拷贝数变异研究。

In a previous genome-wide linkage scan for genes predisposing to type 2 diabetes mellitus (T2DM) in the Pima Indians, we obtained the strongest evidence for linkage with markers on chromosome 1q21-q23. Subsequently, the 1q linkage of T2DM has been replicated in several, diverse populations. Our strategy to search for the underlying diabetes susceptibility gene(s) has been based on two complementary approaches: 1) systematic analysis of densely spaced single nucleotide polymorphisms (SNPs); and 2) investigation of candidate genes within the linked region for variants/mutations. Informative SNPs are tested for association with diabetes, and their effect on the linkage is also evaluated. For the systematic analysis of single nucleotide polymorphisms, an international collaborative Chromosome 1 Consortium has been established by uniting several of the groups which detected T2DM linkage on 1q, with the goal to facilitate search for the underlying diabetes gene(s). This startegy involves analysis of over 5000 subjects from five populations (including about 1000 Pimas), and so far this effort has led to genotyping of over 4900 SNPs within an initial interval of 21 Mb spanning the linkage peak in most populations. To date, more than 150 candidate genes have been analyzed by sequencing in a subset of diabetic and non-diabetic Pimas. In addition, our lab at NIDDK has investiagted more than 150 candidiate genes. CACNA1E was sequenced as a positional candidate gene for this linkage and 30 SNPs were genotyped in the same group of Pima Indians who had been analyzed in the linkage study. A novel G/A variant located in the 3UTR was associated with young-onset T2DM (odds ratio=2.09 per copy of the G allele; 95% CI=1.31-3.33, adjusted p=0.001 and p=0.0006 under an additive and recessive model, respectively) and had a significant effect upon the evidence for linkage at chromosome 1q21-25 (p=0.004). In addition, among 372 non-diabetic Pima subjects who had undergone metabolic testing, this variant was associated with multiple measures of reduced insulin action that included increased fasting, 30, 60 and 120 minute plasma glucose concentrations and increased fasting plasma insulin during an oral glucose tolerance test (all adjusted p<0.01) as well as a decreased rate of insulin-stimulated glucose disposal at both physiologically and maximally stimulated insulin concentrations (both p<0.002). Functional analysis of this 3UTR variant showed that the non-risk allele had a 2.3 fold higher expression as compared to the risk allele. Our data suggest that this 3UTR variant in CACNA1E contributes to T2DM susceptibility among the Pima Indians by effecting whole-body insulin action. In addition, ARHGEF11, which encodes the Rho guanine nucleotide exchange factor 11, was analyzed as a positional candidate gene for this linkage because this protein may stimulate Rho-dependent signals, such as the insulin signaling cascade. The ARHGEF11 gene, and two adjacent genes NTRK1 and INSRR, were sequenced in 24 Pima Indians who were not first-degree relatives. Sequencing of the coding regions, 5and 3 UTRs and putative promoter regions of these genes, identified 28 variants in ARHGEF11, 11 variants in NTRK1 and 8 variants in INSSR. These 47 variants, as well as 84 additional public database variants within/between these genes, were genotyped for association analysis in the same group of Pima Indians who had participated in the linkage study. An R1467H in ARHGEF11, and several additional non-coding variants that were in high linkage disequilibrium with this variant, were nominally associated with young-onset T2DM (P=0.01, OR=3.39) after adjusting for sex, family membership and Pima heritage. The risk allele H had a frequency of 0.10. In a subgroup of 262 on-diabetic full-heritage Pima Indians who had undergone detailed metabolic testing, the risk allele H was also associated with a lower mean insulin-mediated glucose disposal rate and a lower mean non-oxidative glucose storage rate after adjusting for age, sex, nuclear family membership, and percentage of body fat (P0.01). These findings suggest that variation within ARHGEF11 nominally increases risk of T2DM, possibly as a result of increased insulin resistance. The chromosome 1 consortium has recently reported on a meta-analysis of SNPs genotyped across all 7 populations. Because no common SNP was found that explained the linkage in all of these populations, the consortia has been focusing on alterantive explanations for the linkage. One hypothesis is that multiple rare variants underlie these linkage signals. Therefore the consortia has undertaken deep re-sequencing of all exons from selected genes across the region of linkage on chromosome 1. In addition, studies on genome-wide copy number variation are underway on samples from all of these populations.