Detection of sequence variations in the human insulin-receptor gene by parallel denaturing gradient gel electrophoresis

We developed a parallel denaturing gradient gel electrophoresis (DGGE) protocol to maximize the detection of nucleotide variants in the DNA sequence coding for the mature insulin receptor and in splice site junctions. The melting behaviours of exons 2 to 22 and flanking intronic sequences were computer-simulated using two programs, MELT87 and SQHTX. The data obtained from computer analysis were used to select primers for amplification by polymerase chain reaction and optimal electrophoretic conditions. The ability of this protocol to detect nucleotide changes at the insulin-receptor locus was assessed by studying amplified DNA of a patient with leprechaunism whose insulin-receptor mutations were known and by screening the insulin-receptor gene for polymorphisms in a population of unrelated Caucasian individuals. Our results demonstrate that this DGGE protocol is sensitive since it detected (1) sequence variants reported to be undetectable by means of parallel DGGE, (2) previously characterized insulin-receptor nucleotide variants, and (3) unreported polymorphisms at the insulin-receptor locus of Caucasian individuals. It is also simple as perpendicular denaturing gradient gels are not required. Application of this protocol will facilitate the search for molecular defects underlying the pathogenesis of insulin resistance observed in genetic syndromes of severe insulin resistance as well as in other metabolic disorders. In addition, its ability to detect several regions of the insulin-receptor gene displaying a number of common polymorphic sites and being multiallelic will contribute to linkage studies in families with diabetic and/or insulin-resistant subjects. © 1993 Wiley-Liss, Inc.     

多囊卵巢综合征患者促排卵治疗后子宫内膜组织GLUT4的表达及意义

目的:观察多囊卵巢综合征(PCOS)患者经过胰岛素增敏剂和促排卵治疗后子宫内膜葡萄糖转运蛋白4(GLUT4)在基因水平和蛋白水平的表达,探讨改善胰岛素敏感性对PCOS子宫内膜的影响。方法:应用免疫组织化学法、反转录聚合酶链法(RT-PCR)比较PCOS组(20例PCOS患者)和对照组(15例月经规则的育龄妇女)子宫内膜GLUT4、胰岛素(INS)和胰岛素受体(INS-R)蛋白和基因水平的表达。结果:GLUT4、INS及INS-R的蛋白和mRNA在两组子宫内膜中均有表达,PCOS组GLUT4蛋白和mRNA表达强度均低于对照组(P<0.05、P<0.05),INS蛋白和mRNA表达水平均明显高于对照组(P<0.01、P<0.01),INS-R蛋白表达水平明显低于对照组(P<0.05),INS-R mRNA表达水平与对照组无显著性差异(P>0.05)。结论:PCOS患者经过胰岛素增敏剂和促排卵治疗后,子宫内膜GLUT4蛋白和基因水平的表达与对照组相比有差异,推测PCOS患者子宫内膜GLUT4的表达和转位受多因素影响。