A variant insulin promoter in non-insulin-dependent diabetes mellitus.

Abstract

To test the hypothesis that alterations in regulatory regions of the insulin gene occur in a subset of patients with non-insulindependent diabetes mellitus (NIDDM), the promoter region was studied by polymerase chain reaction (PCR) amplification directly from genomic DNA, followed by high-resolution polyacrylamide gel electrophoresis under nondenaturing conditions. By using this method a previously identified HincII polymorphism (GTIGAC to GTTGAG at position -56) in American Blacks was readily detected, indicating that single base changes could be observed. In the course of screening the insulin promoter from 40 American Black subjects with NIDDM, an apparent larger allele was found in two individuals. Both patients were shown to have in addition to a normal allele, a larger allele containing an 8-bp repeat, TGGTCTAA from positions -322 to -315 of the insulin promoter.To facilitate rapid screening for the 8-bp repeat, a high-resolution agarose gel electrophoretic analysis was adopted. DNA from American Black NIDDM subjects (a = 100) and nondiabetic subjects (a = 100) was PCR amplified and analyzed. The 8-bp repeat was present in five NIDDM subjects, and one nondiabetic subject. DNA from Mauritius Creoles, also of African ancestry, was analyzed, and the 8-bp repeat was present in 3 of 41 NIDDM subjects, and 0 of 41 nondiabetic subjects. Analysis of glucose metabolism in three presumed normal sibs of an NIDDM patient with an 8-bp repeat revealed that one sib had overt diabetes, and two sibs were glucose intolerant, but there was no consistent segregation of the insulin promoter variant with the diabetes phenotype. The variant promoter was not present in 35 Caucasian NIDDM patients or in 40 Pima Indians.To test the biological consequences of the 8-bp repeat sequence in the insulin promoter, a normal and variant promoter were subcloned into a luciferase plasmid, and reporter gene activity assessed by transient transfection into mouse insulinoma (ITC1) and hamster insulinoma (HIT) cells. The promoter activity of the variant allele was found to be reduced to 37.9 ± 10.3% of the activity of the normal promoter in HIT cells (P < 0.01, n = 4), and 49.1 ± 6.4% infTCl cells (P < 0.01, n = 6). These data thus suggest that a naturally occurring vari-

Glucokinase gene in gestational diabetes mellitus: population association study and molecular scanning

“…The fragment sizes in this study were larger than 150 base pair, however by using mutation detection enhancement gel (A. T. Biochem, Malvern, Pa., USA) and by examining under multiple electrophoretic conditions we are able to detect the known glucokinase mutations which are not found in this population [20]. The sensitivity of current PCR-SSCP analysis to detect the single base pair change has been assessed [20,37,38], it was not likely that the current study failed to detect existing mutations. However, the possibility of deletion, translocation, or a mutation which affects PCR in only one allele cannot be excluded with any known technique with 100% confidence and, in our previous study these possibilities have been excluded by Southern blot technique [20].…”

Genetic Determinants of Type 2 Diabetes

“…However, mutations in the promoter region could affect the regulation of the insulin gene, leading to a decrease of transcription and absolute or relative hypoinsulinemia. A variant allele of the promoter was observed in about 5 percent of African Americans with T2DM and shown to be associated with decreased transcriptional activity (Olansky et al, 1992). More recently, an association between T2DM and paternally transmitted class III alleles of the variable region upstream of the insulin gene (INS-VNTR) was observed in British families (Huxtable et al, 2000).…”

Massively parallel quantification of the regulatory effects of noncoding genetic variation in a human cohort

“…There are now several examples of noncoding genetic variants that alter the activity of regulatory elements and contribute substantially to complex traits and human diseases (Olansky et al 1992;Nicolae et al 2010;Maurano et al 2012;Corradin et al 2014;Stadhouders et al 2014;Guo et al 2015). Such examples are likely representative of a larger trend that genetic variations in regulatory elements are a major contributor to complex phenotypes and disease (Maurano et al 2012;Gusev et al 2014).…”