Abstract

Objective: Inactivating mutations in glucokinase (GCK) cause mild fasting hyperglycaemia. 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 β-cell promoter region, which is currently not routinely screened, could contain pathogenic mutations and we therefore sequenced this region in 60 such probands.

Research Design & Methods: The β-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 employed to determine the impact of the mutation on Sp1 binding.

Results: A novel −71G>C mutation was identified in a non-conserved region of the human promoter sequence in 6 apparently unrelated probands. Family testing established co-segregation with fasting hyperglycemia, (≥5.5mmol/L) in 39 affected individuals. Haplotype analysis in the UK family and 4 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 4 fold. EMSAs demonstrated a dramatic reduction in Sp1 binding to the promoter sequence corresponding to the mutant allele.

Conclusion: A novel β-cell GCK promoter mutation was identified which significantly reduces gene expression in vitro through loss of regulation by Sp1. To ensure correct diagnosis of potential GCK-MODY cases analysis of the β-cell GCK promoter should be included.