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）基因分型在1300名受试者在5 kb的密度，在整个24 Mb，连锁不平衡（LD）映射。几年前，我们许多最有趣的SNP在芬兰人的FUSION研究中进行了额外的基因分型，来自博特尼亚、赫尔辛基和马尔莫的T2 DM或肥胖症的独立病例/对照样本，大量来自丹麦的代谢表型高加索人，来自莱比锡学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，还有来自斯塔尔县的墨西哥裔美国人从这些复制组的基因在我们的区域的连锁在染色体11 q23 -25的结果表明，在11号染色体上的ETS-1附近的变异复制在三个研究。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。 我的实验室进一步跟进了LD定位确定的显着关联，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。IGSF 4和ETS-1内的变异与BMI的相关性最强，TREH内的变异与T2 DM的相关性最强。我们完成了编码海藻糖酶的TREH内变体的功能分析。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的变异体进行了功能研究。这些变异与皮马印第安人的BMI相关，在有证据表明与chrom相关的家庭中，这种相关性显着增加。11q23。我们与NIDDK的小鼠代谢实验室合作，表明雌性ETS-1半合子小鼠与野生型同窝小鼠相比具有显着更高的体脂。 在过去的一年里，我们收集了两个大的人口为基础的样本皮马印第安人从吉拉河印第安人社区。第一个样本包括3501名完整的皮马印第安人（中位年龄= 39岁，诊断糖尿病的患病率= 45%）。第二个样本包括3723例受试者，主要为混合遗传（中位年龄= 23岁，诊断糖尿病的患病率= 20%）。我们最近对这些大规模DNA样本中的TREH、IGSF 4和ETS-1变异进行了基因分型。在这两个独立的样本中，TREH内的变化与糖尿病显著相关，表明这种效应是广泛的和高度可重复的。相比之下，IGSF 4内的变化与完全继承皮马印第安人的BMI非常密切相关，但在非完全继承皮马印第安人的个体中，这种影响并不明显。最后，Ets-1提供了最显着的协会与BMI的家庭，有助于最初的连锁信号11号染色体上，似乎不是一个主要的基因BMI在一般皮马人口。

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.