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

Daniela Gasperíková, Nicolas D Tribble, Juraj Staník, Miroslava Hucková, Nadezda Misovicová, Martijn van de Bunt, Lucia Valentínová, Beryl A Barrow, L'ubomir Barák, Radoslav Dobránsky, Eva Bereczková, Jozef Michálek, Kate Wicks, Kevin Colclough, Julian C Knight, Sian Ellard, Iwar Klimes, Anna L Gloyn

Research output: Contribution to journalArticlepeer-review

Abstract

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 beta-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 (> or = 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 beta-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.

Original languageEnglish
Pages (from-to)1929-35
Number of pages7
JournalDiabetes
Volume58
Issue number8
DOIs
Publication statusPublished - Aug 2009

Keywords

  • DNA Primers
  • Gene Expression Regulation, Enzymologic
  • Glucokinase
  • Humans
  • Hyperglycemia
  • Insulin-Secreting Cells
  • Mutation
  • Polymorphism, Single Nucleotide
  • Promoter Regions, Genetic
  • Sp1 Transcription Factor
  • Transfection

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