ANALYSIS OF THE INSULIN-RECEPTOR GENE IN NONINSULIN-DEPENDENT DIABETES-MELLITUS BY DENATURING GRADIENT GEL BLOTS - A CLINICAL RESEARCH-CENTER STUDY

We have used a new technique of denaturing gradient gel blotting to de termine the prevalence of alterations in the intracellular domain of t he insulin receptor in normal individuals and subjects with noninsulin -dependent diabetes mellitus (NIDDM). This method detects DNA sequence differences as restriction fragment melting polymorphisms (RFMP) and is sensitive to changes in sequence at both restriction sites and with in the fragments themselves. Using restriction digests with AluI, HaeI II, HinfI, RsaI, Sau3A, and Sau96, 12 RFMPs were found to localize to the region of the beta-subunit of the insulin receptor gene. Using exo n-specific probes, these RFMPs could be localized to specific regions surrounding individual exons, including exons 14, 15, 17, 19, 20, and 22. In general, linkage disequilibrium between polymorphisms was inver sely related to their distance in the gene structure, although there w as a ''hot spot'' for recombination between exons 19 and 20. No differ ence in melting temperatures or allele frequency was observed between NIDDM patients and controls. These data indicate that the region of th e insulin receptor gene coding, for the intracellular portion of the b eta-subunit is highly polymorphic and that polymorphisms surrounding s pecific exons can be identified by denaturing gradient gel blotting, b ut there is no evidence that variation at this locus contributes to NI DDM susceptibility in most individuals.