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）。为了缩小这一区域，我们以5kb的密度对1300名受试者的单核苷酸多态性（snp）进行基因分型，在整个24mb中进行连锁不平衡（LD）定位。几年前，我们的许多最有趣的snp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。对11q23-25染色体连锁区域基因的复制组结果显示，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。

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) were genotyped in 1300 subjects at a 5 kb density, across the entire 24 Mb, for linkage disequilibrium (LD) mapping. Several years ago, many of our most interesting SNPs were additionally 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 showed that variants near ETS-1 on chromosome 11 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. My lab 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. Variation within IGSF4 and ETS-1 were the most strongly associated with BMI and variation within TREH was the most strongly associated with T2DM. We completed functional analysis of variants within 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 also performed funtional studies for variants in a highly conserved, intergenic region distal to ETS-1. These variants are associated with BMI in Pima Indians, where the association becomes significantly greater among the families with evidence for linkage on chrom. 11q23. We collaborated with the Mouse Metabolism lab of NIDDK to show that female ETS-1 hemizygous mice have significantly higher body fat as compared to their wild type littermates. Within the past year we assembled two large population-based samples of Pima Indians from the Gila River Indian Community. The first sample consisted of 3501 full heritage Pima Indians (median age = 39 years with a prevalence of diagnosed diabetes = 45%). The second sample consisted of 3723 subjects who were predominately of mixed heritage (median age = 23 years with a prevalnce of diagnosed diabetes = 20%). We recently genotyped variation within TREH, IGSF4 and ETS-1 in these large samples of DNA. Variation within TREH was significantly associated with diabetes in both of these independent samples, signifying that this effect is widespread and highly reproducible. In contrast, variation within IGSF4 was very strongly associated with BMI among full heritage Pima Indians, but the effect was not as pronounced among individuals who were not full heritage Pima Indians. Finally, Ets-1 which provided the most significant associations with BMI among families which contributed to the initial linkage signal on Chromosome 11, does not appear to be a major gene for BMI in the general Pima population.