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.


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.
Gasperíková
Daniela
D
DIABGENE and Diabetes Laboratory, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovak Republic.
Tribble
Nicolas D
ND
Staník
Juraj
J
Hucková
Miroslava
M
Misovicová
Nadezda
N
van de Bunt
Martijn
M
Valentínová
Lucia
L
Barrow
Beryl A
BA
Barák
L'ubomir
L
Dobránsky
Radoslav
R
Bereczková
Eva
E
Michálek
Jozef
J
Wicks
Kate
K
Colclough
Kevin
K
Knight
Julian C
JC
Ellard
Sian
S
Klimes
Iwar
I
Gloyn
Anna L
AL
D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000235:genetics*; D006801:Humans; D006943:Hyperglycemia / Q000235:genetics*; D050417:Insulin-Secreting Cells / Q000201:enzymology*; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D016329:Sp1 Transcription Factor; D005941:Glucokinase