Rapid Publications Detection of Mutations in Insulin-Receptor Gene in NIDDM Patients by Analysis of Single-Stranded Conformation Polymorphisms STEPHEN O'RAHILLY, WOONG H. CHOI, PUSHPA PATEL, ROBERT C. TURNER, JEFFREY S. FLIER, AND DAVID E. MOLLER We used the recently described technique of single- stranded conformation-polymorphism (SSCP) analysis to examine the insulin-receptor locus. First, the ability of the method to detect known mutations and polymorphisms in the insulin-receptor coding sequence was assessed. Regions of the insulin- receptor sequence containing 16 different nucleotide changes, 9 in patient genomic DNA and 7 as cloned cDNA in plasmids, were analyzed. All 9 patient genomic DNA mutants and 5 of 7 plasmid mutants exhibited variant SSCP patterns. To investigate the potential of the technique for screening many patients, the 5 exons that encode the tyrosine kinase domain of the insulin receptor were examined in 30 unrelated white subjects with non-insulin-dependent diabetes mellitus (NIDDM). Exons 17-21 were amplified from genomic DNA with polymerase chain reaction and subjected to SSCP analysis. Exons 19, 20, and 21 revealed no bands of aberrant migration, suggesting a high degree of conservation of these sequences. One diabetic subject had an SSCP variant in exon 18. Direct sequencing of this subject's genomic DNA revealed a heterozygous missense mutation (Lys 1068 -> Glu 1068 ). Five different SSCP patterns were detected in exon 17. Based on direct sequencing, these patterns were explained by combinations of three different nucleotide substitutions, two of which were common silent polymorphisms. One subject had a heterozygous missense mutation Val 985 —• Met 985 . Allele-specific oligonucleotide hybridization confirmed the presence of these mutations in the appropriate diabetic subjects and also detected the Val 985 mutation in heterozygous form in 1 of 13 nondiabetic white subjects. SSCP analysis is a sensitive rapid method for screening for mutations in the insulin-receptor gene. Using SSCP, From the Department of Medicine, Beth Israel Hospital, Boston, Massachu- setts; and the Diabetes Research Laboratories, Radcliffe Infirmary, Oxford, United Kingdom. Address correspondence and reprint requests to David E. Moller, MD, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215. Received for publication 14 January 1991 and accepted in revised form 12 February 1991. we detected two previously unreported amino acid substitutions in the highly conserved tyrosine kinase domain of the insulin receptor. These represent the first potentially significant mutations found by screening candidate genes in NIDDM. The detection of one of these variants in a normoglycemic subject suggests that it is unlikely to cause the diabetic state, but given the complex genetic basis for NIDDM, a possible contributory role of each of these mutations mandates further study. Diabetes 40:777-82,1991 M utations of the insulin-receptor gene have been reported, to date, in 14 patients with rare syn- dromes of severe insulin resistance (1-3). In the general population, insulin resistance contrib- utes to non-insulin-dependent diabetes mellitus (NIDDM; 4) and may be a central feature of a constellation of atherogenic metabolic variables sometimes referred to as syndrome X (5). The role of inherited defects of the insulin receptor in these conditions is unknown. Some but not all studies of insulin-receptor restriction-fragment-length polymorphisms have suggested an association of certain haplotypes with NIDDM (6,7). The fact that the genetically affected relatives of some subjects with leprechaunism (8) or type A syndrome (3) demonstrate modest insulin resistance in the absence of any striking clinical features suggests that at least some insulin resistance in the general population is caused by mutations of the insulin-receptor gene. The insulin-receptor gene is large, with 4.1 kilobases (kb) of coding sequence distributed over 22 exons. Sequencing of the entire coding sequence is therefore a laborious pro- cess, and only four subjects with NIDDM (3 Pima Indians and 1 white) have had their entire sequence determined. No differences from the previously published "normal" se- quence (9,10) were detected (11-13). Several so-called "molecular scanning" methods have been designed to more rapidly detect deviations from the normal nucleotide sequence in short fragments of DNA (14), DIABETES, VOL. 40, JULY 1991 777