Identification of a Novel β-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 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

Screening for GJB2 and GJB6 mutations in the Slovak deaf population

Introduction: Mutations of connexin genes account for up to 50% of prelingual bilateral sensorineural hearing loss (SNHL). In Slovakia the deaf population is estimated to reach about 9000 individuals. However, reliable data on bilateral SNHL etiology in Slovakia are not available to date. The aim of presented study was to analyze the GJB2 and GJB6 genes and describe their mutation spectrum in patients with bilateral SNHL and thus to determine epidemiology of one of the most dominant causes of SNHL in Slovakia. Methods: Since 2010 we performed molecular-genetic testing for GJB2 and GJB6 mutations in >500 subjects suffering from bilateral SNHL, that included 375 unrelated individuals selected for epidemiology analyses. Patients were recruited at 2 ORL clinics in Bratislava and special schools for hearing impaired children throughout Slovakia. Inclusion criteria were bilateral SNHL and age below 60 years at the time of hearing loss diagnosis. Direct sequencing and MLPA was used for DNA analysis. Results: We identified 11 mutations and six polymorphisms in GJB2 gene. Homozygous mutations occurred in 22% and compound heterozygotes in 9% of subjects. Negative subjects, without any pathogenic allele found accounted for 61%. Mutations c.35delG and c.71G>A were recorded most frequently (60.4% and 15.8% respectively). The large GJB6 deletion (delD13S1830) was found in 1 family.Conclusions: DNA analysis of patients with SNHL revealed mutation spectrum of GJB2 and GJB6 genes in our cohort and also confirmed exact genetic cause of hearing loss in almost one third of investigated subjects. Our results represent fundamental data for genetic counseling, clinical prognosis, improvement of diagnostic tools for clinical practice and possible personalized treatment in future.Supported by: Grants APVV 0148-10 and VEGA 1/0465/11Der Erstautor gibt keinen Interessenkonflikt an

Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia.

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which (approx)65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H and I436N when compared to wild type (IC(50) 14.6+/-0.1 mM and 20.3+/-1.6 mM vs.13.3+/-0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55(degrees Celsius) 8.8+/-0.8% and 3.1+/-0.4% vs. 42.5+/-3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening

Melanocortin-4 Receptor Gene Mutations in Obese Slovak Children

Summary The most common etiology of non-syndromic monogenic obesit