Positional Cloning Of A Diabetes Gene On Chromosome 11

先前在皮马印第安人中的基因组连锁扫描表明染色体11 q23 -24（LOD=3.6）上存在肥胖易感性位点。还有证据表明，相同的基因组区域包含2型糖尿病（T2 DM）的易感基因座（LOD=1.7）。双变量连锁分析的组合表型糖尿病的疾病位点（LOD= 5.2）提供了最有力的证据。连接区域跨越24 Mb。为了缩小这一区域，单核苷酸多态性（SNP）在整个24 Mb上以10 kb密度进行基因分型，用于连锁不平衡（LD）作图。迄今为止，我的实验室已经在我们的连锁研究中分析的相同受试者（N=1300）中对染色体11 q23 -25上的3400个SNP进行了基因分型。在FUSION芬兰人研究中，对我们最感兴趣的几个SNP进行了基因分型，FUSION芬兰人研究是来自Botnia、赫尔辛基和马尔莫的T2 DM或肥胖的独立病例/对照样本，来自丹麦的大量代谢表型白人，来自Leipziger学童研究的白人儿童，来自英国（牛津）的白人，来自VAGES研究的墨西哥裔美国人和来自斯塔尔县的墨西哥裔美国人。来自染色体11 q23 -25上我们的连锁区域中的基因的这些复制组的结果表明，在五项研究中的三项中，11号染色体上靠近ETS-1的变异体已经复制。7项研究中有6项显示11号染色体上的TREH变异具有显著相关性，但不同种族之间的风险等位基因不一致，因此该变异是否复制仍存在争议。在1个墨西哥裔美国人组中检测了CD 3E的一种变体，该变体显示T2 DM风险的非显著趋势与皮马印第安人相同。 我的实验室通过直接测序染色体11 q23 -25上连锁峰下的74个位置候选基因，进一步跟踪了LD定位鉴定的显著关联。这项工作仍在进行中，但我们在11号染色体上的基因测序工作表明，IGSF 4，CD 3E，TREH，ETS-1，SNX-19和OPCML中的变异与BMI和/或T2 DM最有说服力。我们最近完成了这些基因之一，TREH，编码海藻糖酶的变体的功能分析。我们测量了570名非糖尿病受试者的血清海藻糖酶活性，发现海藻糖酶活性与非同义Arg 486 Trp变体和启动子变体在-100 bp时高度相关（分别为p=10-12和p=10-15，经年龄、性别、遗传和家庭成员身份调整）。单倍型分析显示，对于3种变体（Trp 486、Ala 389和C，在-100 bp.海藻糖酶活性最低，T2 DM患病率最低。然而，在一项前瞻性研究中，我们无法证明低血浆海藻糖酶活性（与基因型无关）可预测T2 DM的发生。因此，我们相信我们已经揭示了海藻糖酶活性双峰外观的遗传基础（14），尽管这些变体与T2 DM相关，但它们可能不是T2 DM的病因。 我们还对ETS-1远端高度保守的基因间区域中的变体进行了功能研究。这些变量与皮马印第安人的BMI相关，在有证据表明与chrom相关的家庭中，这种关联变得显著更大。11q23。我的实验室一直在进行体外功能研究，以确定该区域是否含有增强子活性，我与NIDDK的小鼠代谢实验室合作，该实验室表明，与野生型同窝小鼠相比，雌性ETS-1半合子小鼠的体脂显著较高。

A prior genomic linkage scan in Pima Indians indicated an obesity susceptibility locus on chromosome 11q23-24 (LOD=3.6). There was also evidence that the same genomic region contained a susceptibility locus for type 2 diabetes mellitus (T2DM)(LOD=1.7). Bivariate linkage analysis for the combined phenotype diabesity gave the strongest evidence for a disease locus (LOD= 5.2). The region of linkage spans 24 Mb. To narrow this region, single nucleotide polymorphisms (SNPs) are being genotyped at a 10 kb density, across the entire 24 Mb, for linkage disequilibrium (LD) mapping. To date, my lab has genotyped 3400 SNPs across chromosome 11q23-25 in the same subjects that were analyzed in our linkage study (N=1300). Several of our most interesting SNPs have been genotyped in the FUSION study of Finns, independent case/control samples for either T2DM or obesity from Botnia, Helsinki, and Malmo, a large collection of metabolically phenotyped Caucasians from Denmark, Caucasian children from the Leipziger School Children study, Caucasians from the UK (Oxford), Mexican Americans from the VAGES study, and Mexican Americans from Starr County. Results from these replication groups for genes in our region of linkage on chromosome 11q23-25 have shown that variants near ETS-1 on chromosome 11 have replicated in three out of five studies. A variant in TREH on chromosome 11 showed significant associations in 6 out of seven studies, but the risk allele was inconsistent among the various ethnic groups, so it remains debatable whether this variant replicated. A variant in CD3E was tested in 1 Mexican American group where it showed a non-significant trend for risk of T2DM in the same direction as Pima Indians. My lab has further followed up on significant associations identified by LD mapping by directly sequencing 74 positional candidate genes under the peak of linkage on chromosome 11q23-25. This work is still in progress, but our gene sequencing efforts on chromosome 11 suggest that variants within IGSF4, CD3E, TREH, ETS-1, SNX-19, and OPCML are most convincingly associated with BMI and/or T2DM. We have recently completed functional analysis of variants in one of these genes, TREH, which encodes trehalase. We measured serum trehalase activity in 570 non-diabetic subjects and found that trehalase activity was highly associated with a non-synonymous Arg486Trp variant and a promoter variant at -100 b.p. (p=10-12 and p=10-15, respectively, adjusted for age, sex, heritage and family membership). Haplotype analysis showed that subjects who were homozygous for 3 variants (Trp486, Ala389, and C at -100 b.p.) had the lowest trehalase activity and had the lowest prevalence of T2DM. However, in a prospective study, we were unable to demonstrate that a low plasma trehalase activity (independent of genotype) predicted the development of T2DM. Therefore, we believe we have uncovered the genetic basis for the bimodal appearance of trehalase activity (14), and although these variants are associated with T2DM, they may not be causative for T2DM. We have also performed funtional studies for variants in a highly conserved, intergenic region distal to ETS-1. These varaints are associated with BMI in Pima Indians, where the association becomes significantly greater among the families with evidence for linkage on chrom. 11q23. My lab has been doing in vitro functional studies to determine whether this region contains enhancer activity, and I have collaborated with the Mouse Metabolism lab of NIDDK that has shown that female ETS-1 hemizygous mice have significantly higher body fat as compared to their wild type littermates.