Positional Cloning Of A Diabetes Gene On Chromosome 11

先前的皮马印第安人基因组连锁扫描显示，肥胖易感位点位于染色体11q23-24上（LOD=3.6）。也有证据表明，同一基因组区域包含2型糖尿病（T2DM）的易感性位点（LOD=1.7）。联合表型糖尿病的双变量连锁分析给出了疾病位点的最强证据（LOD= 5.2）。连锁区域跨越24 Mb。为了缩小这一区域，单核苷酸多态性（snp）以10 kb的密度在整个24 Mb中进行基因分型，以进行连锁不平衡（LD）定位。到目前为止，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11q23-25染色体上的3400个snp进行了基因分型。我们的几个最有趣的snp已经在芬兰人的FUSION研究中进行了基因分型，来自Botnia，赫尔辛基和马尔默的独立T2DM或肥胖病例/对照样本，来自丹麦的大量代谢表型高加索人，来自莱比锡学校儿童研究的高加索儿童，来自英国（牛津）的高加索人，来自VAGES研究的墨西哥裔美国人，以及来自斯塔尔县的墨西哥裔美国人。这些复制组对11q23-25染色体连锁区域的基因的结果表明，在5个研究中，有3个在11号染色体上靠近ETS-1的变异被复制。11号染色体上TREH的变异在7项研究中有6项显示出显著的相关性，但风险等位基因在不同的种族群体中并不一致，因此这种变异是否可以复制仍然存在争议。在一个墨西哥裔美国人群体中检测了CD3E的一种变异，结果显示，该变异与皮马印第安人患2型糖尿病的方向相同，没有显著的趋势。

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.