Analysis of the HNF4 alpha gene in Caucasian type II diabetic nephropathic patients.

Linkage and association studies in Caucasian patients with Type II (non-insulin-dependent) diabetes mellitus suggest that one or more diabetes susceptibility gene(s) reside within human chromosome 20q12-13.1. This region of chromosome 20 contains the maturity-onset diabetes of the young type 1 gene, HNF4 alpha. The purpose of this study was to assess the possible involvement of HNF4 alpha in Type II diabetes. Mutation analysis was done on the 12 exons and promoter regions of the HNF4 alpha gene in 182 Caucasian diabetic nephropathic patients and 100 Caucasian control subjects. The functional consequences of a novel promoter mutation were examined using a reporter system in the HepG2 liver cell line and electrophoretic mobility shift assays. We identified two novel mutations in the HNF4 alpha, an R323H missense mutation in exon 8, and a 7 bp deletion (delta 7) in the proximal promoter region resulting in deletion of a single putative Sp1 binding site. Using a reporter assay system, the delta 7 sequence was found to exhibit a 51.2% (standard error +/- 4.2%) reduction in promoter activity relative to the normal sequence. In electrophoretic mobility shift assays using specific and non-specific competitors, the delta 7 sequence had a 45.5% (range 40.4-46.6) reduction in binding compared with the normal sequence. The delta 7 allele occurs in a family with multiple cases of Type II diabetes in a pattern consistent with coinheritance of the delta 7 allele and diabetes. Analysis of the HNF4 alpha gene revealed two possible mutations in 182 diabetic patients which suggests that the HNF4 alpha gene does not make a large contribution to diabetes susceptibility in the general population of Caucasian diabetic nephropathic patients. Functional analysis of the delta 7 promoter deletion suggests, however, that promoter mutations in otherwise normal genes could contribute to diabetes susceptibility.