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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.

Gasperíková D, Tribble ND, Staník J, Hucková M, Misovicová N, van de Bunt M, Valentínová L, Barrow BA, Barák L, Dobránsky R, Bereczková E, Michálek J, Wicks K, Colclough K, Knight JC, Ellard S, Klimes I, Gloyn AL - Diabetes (2009)

Bottom Line: The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system.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.

View Article: PubMed Central - PubMed

Affiliation: DIABGENE and Diabetes Laboratory, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovak Republic.

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.

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DNA mobility shift assay with recombinant human Sp1 nuclear protein. Sp1 binds both the wild-type and Sp1 oligonucleotides, whereas the −71G>C mutation causes a dramatic reduction in Sp1 binding. A: Labeled GCK promoter (−53 to −88) and Sp1 consensus oligonucleotides were incubated in the presence or absence of Sp1 nuclear protein. Competition experiments were conducted using 100-fold molar excess of unlabeled oligonucleotides. B: Specific Sp1 and nonspecific USF antibodies were used to confirm the specificity of Sp1 binding that was visualized as a supershifted band of reduced mobility.
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Figure 3: DNA mobility shift assay with recombinant human Sp1 nuclear protein. Sp1 binds both the wild-type and Sp1 oligonucleotides, whereas the −71G>C mutation causes a dramatic reduction in Sp1 binding. A: Labeled GCK promoter (−53 to −88) and Sp1 consensus oligonucleotides were incubated in the presence or absence of Sp1 nuclear protein. Competition experiments were conducted using 100-fold molar excess of unlabeled oligonucleotides. B: Specific Sp1 and nonspecific USF antibodies were used to confirm the specificity of Sp1 binding that was visualized as a supershifted band of reduced mobility.

Mentions: To demonstrate that Sp1 can indeed bind to the putative binding site located at the −71bp region of the wild-type promoter, gel shift experiments were performed. Using the wild-type oligonucleotide, two clear bands that were specific on competition with molar excess of unlabeled self or an unlabeled probe corresponding to a consensus Sp1 binding site were observed (Fig. 3A).


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.

Gasperíková D, Tribble ND, Staník J, Hucková M, Misovicová N, van de Bunt M, Valentínová L, Barrow BA, Barák L, Dobránsky R, Bereczková E, Michálek J, Wicks K, Colclough K, Knight JC, Ellard S, Klimes I, Gloyn AL - Diabetes (2009)

DNA mobility shift assay with recombinant human Sp1 nuclear protein. Sp1 binds both the wild-type and Sp1 oligonucleotides, whereas the −71G>C mutation causes a dramatic reduction in Sp1 binding. A: Labeled GCK promoter (−53 to −88) and Sp1 consensus oligonucleotides were incubated in the presence or absence of Sp1 nuclear protein. Competition experiments were conducted using 100-fold molar excess of unlabeled oligonucleotides. B: Specific Sp1 and nonspecific USF antibodies were used to confirm the specificity of Sp1 binding that was visualized as a supershifted band of reduced mobility.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712784&req=5

Figure 3: DNA mobility shift assay with recombinant human Sp1 nuclear protein. Sp1 binds both the wild-type and Sp1 oligonucleotides, whereas the −71G>C mutation causes a dramatic reduction in Sp1 binding. A: Labeled GCK promoter (−53 to −88) and Sp1 consensus oligonucleotides were incubated in the presence or absence of Sp1 nuclear protein. Competition experiments were conducted using 100-fold molar excess of unlabeled oligonucleotides. B: Specific Sp1 and nonspecific USF antibodies were used to confirm the specificity of Sp1 binding that was visualized as a supershifted band of reduced mobility.
Mentions: To demonstrate that Sp1 can indeed bind to the putative binding site located at the −71bp region of the wild-type promoter, gel shift experiments were performed. Using the wild-type oligonucleotide, two clear bands that were specific on competition with molar excess of unlabeled self or an unlabeled probe corresponding to a consensus Sp1 binding site were observed (Fig. 3A).

Bottom Line: The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system.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.

View Article: PubMed Central - PubMed

Affiliation: DIABGENE and Diabetes Laboratory, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovak Republic.

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.

Show MeSH
Related in: MedlinePlus