Structural Analysis of Candidate Genes for type 2 Diabetes

在PIMAS中，一个基因被认为是2型糖尿病的候选基因，如果1)它与另一个人群的糖尿病表型有关，或2)它具有生理相关的功能，并位于与糖尿病相关的染色体区域。到目前为止，已经分析的候选基因包括那些与其他人群中的MODY相关的基因。我们在HNF1a、HNFlb和HNF3b基因中发现了几个多态性，但没有一个与PIMAS的早发性糖尿病相关。然而，HNFlb的一个多态性预测了蛋白质DNA结合区内的非保守氨基酸替代。因此，对HNF1B中的替换进行了功能变异研究。我们已将HNF1B的两种多态形式克隆到表达载体中。此外，我们还将受HNF1B调控的大鼠白蛋白启动子克隆到荧光素酶报告载体中。将含HNF1B的载体与荧光素酶报告基因共转染Cos-7细胞。我们已经发现这种不常见的氨基酸变体上调了大鼠白蛋白启动子的转录。我们还分析了Calain 10基因的一个多态性(UCSNP-43)，该基因被认为是导致墨西哥裔美国人糖尿病基因NIDDM1的原因。我们在1300名PIMA印第安人中对这种多态进行了基因分型，并发现UCSNP-43与非糖尿病PIMA患者的胰岛素抵抗和营养分配有关。在墨西哥裔美国人和两个欧洲人群体中，由3个多态组成的特定单倍型为糖尿病的发生提供了高危单倍型。因此，我们在1300例PIMA中对Calain 10基因另外两个多态进行了基因分型。然而，这种单倍型并不会增加皮马人群患糖尿病的风险。我们还对15号染色体上另一个不同的calain基因附近的另一个多态进行了基因分型，该基因被认为与墨西哥裔美国人的calain 10基因位点相互作用。我们没有发现与PIMAS患者15号染色体上的多态性有任何显著的关联。

A gene is considered a candidate gene for type 2 diabetes in Pimas if 1) it is associated with a diabetic phenotype in another population or 2) it has a physiologically relevant function and is positioned in a chromosomal region that is linked to diabetes. Candidate genes which have been analyzed to date include those genes which are associated with Mody in other populations. We identified several polymorphisms in the HNFla, HNFlb, and HNF3b genes, but none were associated with early onset diabetes in Pimas. One polymorphism in HNFlb, however, predicts a non-conservative amino acid substitution within the DNA binding region of the protein. Therefore the substitution in HNF1b was investigated for functional variability. We have cloned the two polymorphic forms of HNF1b into expression vectors. In addition, we have cloned the rat albumin promoter, which is regulated by HNF1b, into a luciferase reporter plasmid. Each of the vectors containing HNF1b were co-transfected with the luciferase reporter plasmid into Cos 7 cells. We have found that the uncommon amino acid variant upregulates transcription from the rat albumin promoter.We have also analyzed a polymorphism (UCSNP-43)in the calpain 10 gene which is thought to be responsible for the diabetic locus NIDDM1 in Mexican Americans. We have genotyped this polymophism in 1300 Pima Indians and have found an association between UCSNP-43 and insulin resistance and nutrient partitioning in non-diabetic Pimas. In Mexican Americans and two European populations, a specific haplotype consisting of 3 polymorphisms provided an at risk haplotype for the development of diabetes. Therefore we genotyped two additional polymorphisms in the calpain 10 gene in 1300 Pimas. This haplotype, however, did not confer an increased risk of diabetes in the Pima population. We have also genotyped another polymorphism near a different calpain gene on chromosome 15 which is thought to interact with the calpain 10 locus in Mexican Americans. We did not find any significant associations with the polymorphism on chromosome 15 in Pimas.