Identification of a novel beta-cell glucokinase (GCK) promoter mutation (-71G>C) that modulates GCK gene expression through loss of allele-specific Sp1 binding causing mild fasting hyperglycemia in humans.

OBJECTIVE-Inactivating mutations in glucokinase (GCK) cause mild fasting hyperglycemia. Identification of a GCK mutation has implications for treatment and prognosis; therefore, it is important to identify these individuals. A significant: number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of GCK. We hypothesized that the GCK beta-cell promoter region, which currently is not. routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands. RESEARCH DESIGN AND METHODS-The p-cell GCK promoter was sequenced in patient DNA. The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system. Electrophoretic mobility shift assays (EMSAs) were used to determine the impact of the mutation on Sp1 binding. RESULTS-A novel -71G>C mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands. Family testing established cosegregation with fasting hyperglycemia (>= 5.5 mmol/l) in 39 affected individuals. Haplotype analysis in the U.K. family and four of the Slovakian families demonstrated that, the mutation had arisen independently. The mutation maps to a potential transcriptional activator binding site for Sp1. Reporter assays demonstrated that the mutation reduces promoter activity by up to fourfold. EMSAs demonstrated a dramatic reduction in Sp1 binding to the promoter sequence corresponding to the mutant allele. CONCLUSIONS-A novel P-cell GCK promoter mutation was identified that significantly reduces gene expression in vitro through loss of regulation by Sp1. To ensure correct diagnosis of potential GCK-MODY (maturity-onset diabetes of the young) cases, analysis of the beta-cell GCK promoter should be included. Diabetes 58:1929-1935, 2009