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

在之前对皮马印第安人2型糖尿病（T2DM）易感性基因的全基因组连锁扫描中，我们获得了与染色体1q21-q23标记相关的最有力证据。随后，T2DM的1q连锁在几个不同的人群中得到了重复。我们寻找潜在糖尿病易感基因的策略基于两种互补的方法：1)密集间隔单核苷酸多态性（SNPs）的系统分析；2)变异/突变连锁区域内候选基因的研究。信息性snp与糖尿病的相关性进行了测试，并评估了它们对连锁的影响。为了对单核苷酸多态性进行系统分析，一个国际合作的1号染色体联盟已经成立，联合了几个在1q上检测到T2DM连锁的小组，目的是促进寻找潜在的糖尿病基因。该策略涉及对来自5个种群的5000多名受试者（包括约1000名Pimas）进行分析，到目前为止，这项工作已在大多数种群的连锁高峰的21 Mb初始间隔内导致超过4900个snp的基因分型。迄今为止，通过对糖尿病和非糖尿病Pimas亚群的测序，已经分析了150多个候选基因。此外，我们在NIDDK的实验室已经研究了150多个候选基因。CACNA1E作为该连锁的位置候选基因进行了测序，并在该连锁研究中分析的同一组皮马印第安人中对30个snp进行了基因分型。位于3UTR的一个新的G/A变异与年轻发病的T2DM相关（G等位基因的比值比为2.09 /拷贝；95% CI=1.31-3.33，分别在加性和隐性模型下调整p=0.001和p=0.0006），并且对染色体1q21-25的连锁证据有显著影响（p=0.004）。此外，在372名接受了代谢测试的非糖尿病皮马受试者中，这种变异与胰岛素作用降低的多项措施有关，包括禁食增加、在口服葡萄糖耐量试验中，60分钟和120分钟血浆葡萄糖浓度和空腹血浆胰岛素增加（均校正p<0.01），以及在生理和最大刺激胰岛素浓度下胰岛素刺激葡萄糖处置率降低（均p<0.002）。对该3UTR变异的功能分析表明，非风险等位基因的表达量比风险等位基因高2.3倍。我们的数据表明，CACNA1E的这种3UTR变异通过影响全身胰岛素作用，有助于皮马印第安人对2型糖尿病的易感性。此外，编码Rho鸟嘌呤核苷酸交换因子11的ARHGEF11被分析为该连锁的位置候选基因，因为该蛋白可能刺激Rho依赖信号，如胰岛素信号级联。对24名非一级亲属的皮马印第安人的ARHGEF11基因和两个相邻基因NTRK1和INSRR进行了测序。对这些基因的编码区、5和3个utr以及推测的启动子区进行测序，鉴定出ARHGEF11的28个变异，NTRK1的11个变异和INSSR的8个变异。这47个变体，以及这些基因内部或之间的84个公共数据库变体，在参与连锁研究的同一组皮马印第安人中进行了基因分型以进行关联分析。ARHGEF11中的R1467H，以及与该变异高度连锁不平衡的其他几个非编码变异，在调整性别、家族成员和皮马遗传后，名义上与年轻发病的T2DM相关（P=0.01， OR=3.39）。风险等位基因H的频率为0.10。在262名接受了详细代谢测试的非糖尿病全遗传皮马印第安人亚组中，在调整年龄、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性葡萄糖储存率相关（P0.01）。这些发现表明，ARHGEF11的变异名义上增加了2型糖尿病的风险，可能是胰岛素抵抗增加的结果。

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.