ORGANIZATION OF THE HUMAN CARBOXYPEPTIDASE-E GENE AND MOLECULAR SCANNING FOR MUTATIONS IN JAPANESE SUBJECTS WITH NIDDM OR OBESITY

Insulin is synthesized in the pancreatic beta cell as a larger precurs or molecule proinsulin which is converted to insulin and C-peptide by the concerted action of probormone convertase 2 (PC2), prohormone conv ertase 3 (PC3) and carboxypeptidase E (CPE). One of the features of no n-insulin-dependent diabetes mellitus (NIDDM) is an elevation in the p roinsulin level and/or proinsulin/insulin molar ratio suggesting that mutations in these three proinsulin processing enzymes might contribut e to the development of NIDDM, The identification of a mutation in the CPE,gene of the fat/fat mouse which leads to marked hyperproinsulinae mia and late-onset obesity and diabetes is consistent with a possible role for mutations in CPE in the development of diabetes and obesity i n humans. In order to test this hypothesis, we have isolated and chara cterized the human CPE,gene and screened it for mutations in a group o f Japanese subjects with NIDDM and obesity. The human CPE gene consist s of 9 exons spanning more than 60 kb. Primer extension analysis ident ified the transcriptional start site at -141 bp from the translational start site. Single strand conformational polymorphism analysis and nu cleotide sequencing of the promoter and entire coding region of the CP E gene in 269 Japanese subjects with NIDDM, 28 nondiabetic obese subje cts and 104 nonobese and nondiabetic controls revealed three nucleotid e changes, a G-to-T substitution at nucleotide -53, a G-to-A substitut ion at nucleotide -144 (relative to start of transcription) in the pro moter region and a silent G-to-A substitution in codon 219. None of th e nucleotide substitutions were associated with NIDDM or obesity. Thus , genetic variation in the CPE gene does not appear to play a major ro le in the pathogenesis of NIDDM or obesity in Japanese subjects.