MODY type 2 P59S GCK mutant: founder effect in South of Italy

Delvecchio M, Ludovico O, Bellacchio E, Stallone R, Palladino T, Mastroianno S, Zelante L, Sacco M, Trischitta V, Carella M. MODY type 2 P59S GCK mutant: founder effect in South of Italy. Mutations in the glucokinase (GCK) gene are the most frequent cause of maturity onset diabetes of the young (MODY) in Italy. We evaluated GCK mutations in 32 unrelated patients younger than 18 years who had been diagnosed with MODY. Eleven different GCK heterozygous mutations were identified in 22 (68.7%) of the 32 probands. Nine mutations were missense and two were nonsense. Three of these mutations (E17X, P59S and E372X) have not been described previously and were shown to be associated with hyperglycaemia. Several prediction methods suggested that the E17X and E372X mutations result in a premature truncated protein and that the P59S mutation is pathogenic. This idea was further supported by evidence suggesting that Proline 59 is a highly conserved amino acid residue and that the P59S mutation does not appear to be present in non‐diabetic controls and in sequence variant databases. Furthermore, this mutation was found in six (27.3%) of the patients from the same geographical area, Gargano, pointing to the existence of a founder effect, which was confirmed by microsatellite analysis.     

早发糖尿病家系葡萄糖激酶基因突变的筛查

目的 早发2型糖尿病家系中可能存在MODY2基因（GCK）的致病突变,了解MODY2基因在早发家族性2型糖尿病发病中作用。方法 收集100个2型糖尿病家系,先证者均在40岁前被诊断为糖尿病,且至少还有一个一级亲属在45岁之前被诊断糖尿病。提取先证者血DNA,用PCR产物扩增MODY2基因的所有外显子和外显子／内含子拼接区,将PCR产物直接进行测序。SPSS进行统计分析。结果 在MODY2基因的筛查中发现3个DNA变异,一个在外显子4（ccc—ccg,为杂合的脯氨酸同义突变Pr0145Pro）,一个在外显子7（gcc—gcg,为杂合的丙氨酸同义突变ala233ala）,一个在内含子9（IVS＋8C〉T）。这三个变异等位基因频率分别为0．5％、0．5％、36％。对内含子IVS9＋8C〉T多态性的群体关联研究发现等位基因T在糖尿病组频率显著低于非糖尿病对照组（36％V847％,P〈0．05）,而C等位基因高于对照组（64％V853％,P〈0．05）。结论 MODY2基因内或附近的基因变异不是早发2型糖尿病家系的主要致病原因。     

Permanent neonatal diabetes caused by a homozygous nonsense mutation in the glucokinase gene

Abstract:  Glucokinase deficiency is an unfrequent cause of permanent neonatal diabetes (PND), as only seven patients have been reported, either homozygous for a missense or frameshift mutation or compound heterozygous for both of them. We report here the first known case caused by a homozygous nonsense mutation (Y61X) in the glucokinase gene ( GCK ) that introduces a premature stop codon, generating a truncated protein that is predicted to be completely inactive as it lacks both the glucose- and the adenosine triphosphate-binding sites. The proband, born to consanguineous parents, was a full-term, intra-uterine growth-retarded male newborn who presented with a glycaemia of 129 mg/dL (7.16 mmol/L) on his second day of life, increasing thereafter up to 288 mg/dL (15.98 mmol/L) and 530 mg/dL (29.41 mmol/L) over the next 24 h, in the face of low serum insulin (<3 μIU/mL; <20.83 pmol/L). He was put on insulin on the third day of life. Insulin has never been discontinued since then. The patient was tested negative for anti-insulin and islet cell antibodies at age 5 months. His father had non-progressive, impaired fasting glucose for several years. The mother was found to be mildly hyperglycaemic only when her glucose was checked after the child was diagnosed. In conclusion, biallelic GCK loss should be considered as a potential cause of PND in children born to consanguineous parents, even if they are not known to be diabetic at the time of PND presentation.     

Genetic and bioinformatics analysis of four novel GCK missense variants detected in Caucasian families with GCK‐MODY phenotype

Heterozygous loss‐of‐function mutations in the glucokinase (GCK) gene cause maturity‐onset diabetes of the young (MODY) subtype GCK (GCK‐MODY/MODY2). GCK sequencing revealed 16 distinct mutations (13 missense, 1 nonsense, 1 splice site, and 1 frameshift‐deletion) co‐segregating with hyperglycaemia in 23 GCK‐MODY families. Four missense substitutions (c.718A>G/p.Asn240Asp, c.757G>T/p.Val253Phe, c.872A>C/p.Lys291Thr, and c.1151C>T/p.Ala384Val) were novel and a founder effect for the nonsense mutation (c.76C>T/p.Gln26*) was supposed. We tested whether an accurate bioinformatics approach could strengthen family‐genetic evidence for missense variant pathogenicity in routine diagnostics, where wet‐lab functional assays are generally unviable. In silico analyses of the novel missense variants, including orthologous sequence conservation, amino acid substitution (AAS)‐pathogenicity predictors, structural modeling and splicing predictors, suggested that the AASs and/or the underlying nucleotide changes are likely to be pathogenic. This study shows how a careful bioinformatics analysis could provide effective suggestions to help molecular‐genetic diagnosis in absence of wet‐lab validations.     

电针“足三里”“肾俞”穴对T2DM大鼠GLUT2、GCK的影响

目的:探讨电针不同穴位对2型糖尿病大鼠的疗效差异及其机制。方法:40只雄性SD大鼠随机选取8只作为空白组,其余大鼠予高糖高脂饲料喂养50 d后,按35 mg/kg质量比腹腔注射2%链脲佐菌素(Streptozotocin,STZ)溶液进行T2DM造模,并将造模成功的32只大鼠随机分为模型组、电针足三里组、电针肾俞组、格列美脲灌胃组,每组8只。各电针组分别取双侧"足三里""肾俞"穴进行电针治疗,电针参数为2 Hz,2 m A连续波20 min/次,1次/d,6 d/周,连续4周。于干预前及干预后,检测各组大鼠灌胃前空腹血糖(Fasting Blood Glucose,FBG)、体质量及OGTT实验。末次干预结束,处死大鼠取血,取胰腺组织,采用放免法测血清胰岛素(FINS),计算胰岛素分泌指数(HOMA-B),Real-time PCR检测胰腺中葡萄糖转运蛋白-2(Glucose Transporter-2,GLUT2)、葡萄糖激酶(Glucokinase,GCK)的mRNA表达水平,HE染色观察胰腺的形态变化。结果:干预4周后,除空白组体质量显著增加(P0.01),其余各组体质量均显著减轻(P0.01);模型组FBG显著高于干预前、空白组(P0.01),格列美脲灌胃组显著低于模型组(P0.01),两电针组比较模型组均有下降趋势(P0.05);葡萄糖耐量实验(Oralglucose Tolerance,OGTT)实验结果与FBG同步;电针足三里组、格列美脲灌胃组FINS显著低于空白组、模型组及电针肾俞组(P0.01);模型组HOMA-B显著低于空白组(P0.01),各干预组比较模型组均有上升趋势(P0.05);模型组GLUT2的mRNA表达水平显著低于空白组(P0.01),电针足三里组、电针肾俞组显著高于模型组(P0.05,P0.01);模型组GCK的mRNA表达水平显著低于空白组(P0.01),各干预组均显著高于模型组(P0.01,P0.05),且电针足三里组显著高于格列美脲灌胃组(P0.01)。HE染色:模型组胰岛边界不清,体积明显变小,胰岛细胞数量减少且排列杂乱,细胞肿胀、坏死,可见细胞空泡变性。3个干预组与模型组比较,有不同程度的改善,胰岛边界较模型组清晰,体积增大,胰岛内细胞数量增加,排列也较均匀,细胞空泡变性减少。结论:电针足三里、肾俞穴可能是通过上调GLUT2、GCK mRNA的表达水平而改善T2DM大鼠的胰岛B细胞的分泌功能、保护胰岛B细胞的形态、降低FINS水平,其中足三里穴的效果更优。     

The association of common genetic variants in the APOA5, LPL and GCK genes with longitudinal changes in metabolic and cardiovascular traits

Aims/hypothesis  Common genetic variants influence plasma triacylglycerol, HDL-cholesterol (HDL-C) and glucose levels in cross-sectional studies. However, the longitudinal effects of these established variants have not been studied. Our aim was to examine the longitudinal associations of four such variants in the apolipoprotein A-V (APOA5), lipoprotein lipase (LPL), and glucokinase (GCK) genes with fasting glucose or lipid levels. Methods  The individuals analysed were participants in the Busselton Health Survey (n  = 4,554). Cross-sectional analyses of family data used the total association test. Longitudinal association analyses of unrelated participant data (n  = 2,864) used linear mixed-effects models. Results  The findings of cross-sectional association analyses replicated those of previous studies. We observed associations of the G and C alleles at the APOA5 single nucleotide polymorphisms (SNPs) rs662799 and rs3135506 with raised triacylglycerol levels (p  = 0.0003 and p < 0.0001, respectively), the 447X allele at the LPL SNP rs328 with reduced triacylglycerol levels (p  = 0.0004) and raised HDL-C levels (p  = 0.0004), and the A allele of the GCK SNP rs1799884 with raised fasting glucose level (p  = 0.015). Longitudinal association analyses showed that most of these associations did not change in the same individuals over an average follow-up time of 17.4 years, though there was some evidence that the association of the 447X allele of rs328 with raised HDL-C level significantly increased with age (p  = 0.01), and that the association of the C allele of rs3135506 with raised triacylglycerol level significantly increased over time (p  = 0.0007). Conclusions/interpretation  The current study suggests that the effects of established gene variants on lipid and glucose traits do not tend to alter with age during adulthood or over time. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Association of GCKR rs780094, alone or in combination with GCK rs1799884, with type 2 diabetes and related traits in a Han Chinese population

Aims/hypothesis  The GCKR rs780094 and GCK rs1799884 polymorphisms have been reported to be associated with dyslipidaemia and type 2 diabetes in white Europeans. The aim of this study was to replicate these associations in Han Chinese individuals and to identify the potential mechanisms underlying these associations. Methods  The single nucleotide polymorphisms rs780094 and rs1799884 were genotyped in a population-based sample of Han Chinese individuals (n = 3,210) and tested for association with risk of type 2 diabetes and related phenotypes. Results  The GCKR rs780094 A allele was marginally associated with reduced risk of type 2 diabetes (OR 0.85, 95% CI 0.73–1.00, p value under an additive model [p _(add)] = 0.05) and significantly associated with reduced risk of impaired fasting glucose (IFG) or type 2 diabetes (OR 0.86, 95% CI 0.77–0.96, p _[add] = 0.0032). It was also significantly associated with decreased fasting glucose and increased HOMA of beta cell function (HOMA-B) and fasting triacylglycerol levels (p _[add] = 0.0169–5.3 × 10⁻⁶), but not with HOMA of insulin sensitivity (HOMA-S). The associations with type 2 diabetes and IFG remained significant after adjustment for BMI, while adjustment for HOMA-B abolished the associations. The GCKR rs780094 was also associated with obesity and BMI, independently of its association with type 2 diabetes. The GCK rs1799884 A allele was significantly associated with decreased HOMA-B (p _[add] = 0.0005), but not with type 2 diabetes or IFG. Individuals with increasing numbers of risk alleles for both variants had significantly lower HOMA-B (p _[add] = 5.8 × 10⁻⁵) in the combined analysis. Conclusions/interpretation  Consistent with observations in white Europeans, the GCKR rs780094 polymorphism contributes to the risk of type 2 diabetes and dyslipidaemia in Han Chinese individuals. In addition, we showed that the effect on type 2 diabetes is probably mediated through impaired beta cell function rather than through obesity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. Q. Qi and Y. Wu contributed equally to this study.     

Molecular Diagnosis of Monogenic Diabetes and Clinical/Laboratory Features in Turkish Children

Objective:Monogenic diabetes is a heterogeneous disease that causes functional problems in pancreatic beta cells and hyperglycemia. The aim of this study was to determine the clinical and laboratory features, the admission characteristics and distribution of monogenic form of diabetes in childhood in Turkey.Methods:Patients aged 0-18 years, who were molecularly diagnosed with monogenic diabetes, and consented to participate, were included in the study.Results:Seventy-seven (45.6%) female and 92 male cases with a mean age of 8.18±5.05 years at diagnosis were included. 52.7% of the cases were diagnosed with monogenic diabetes by random blood glucose measurement. The reason for genetic analysis in 95 (56.2%) of cases was having a family member diagnosed with diabetes under the age of 25. At the time of diagnosis, ketone was detected in urine in 16.6% of the cases. Mean hemoglobin A1c on admission, fasting blood glucose, fasting insulin, and c-peptide values were 7.3±2.1%, 184.9±128.9 mg/dL, 9.4±22.9 IU/L, 1.36±1.1 and ng/L respectively. GCK-MODY was found in 100 (59.2%), HNF1A-MODY in 31 (18.3%), and variants in ABCC8 in 6 (3.6%), KCNJ11 in 5 (3%), HNF4A in 2 (1.2%), and HNF1B in 2 (1.2%).Conclusion:Recent studies have indicated HNF1A-MODY is the most frequent of all the MODY-monogenic diabetes cases in the literature (50%), while GCK-MODY is the second most frequent (32%). In contrast to these reports, in our study, the most common form was GCKMODY while less than 20% of cases were diagnosed with HNF1A-MODY.     

A novel heterozygous mutation in the glucokinase gene conferring exercise-induced symptomatic hyperglycaemia responsive to sulfonylurea

AimTo describe the atypical phenotype and genotype of an adolescent girl with symptomatic exercise-induced hyperglycaemia, responsive to sulfonylurea treatment.MethodsChart review, gene sequencing, and blinded continuous glucose monitoring (Medtronic iPro2) were used to characterise the case.ResultsA novel heterozygous mutation p.Q219x (c.655C>T) in exon 6 of the glucokinase gene (NM_000162.3) was confirmed in the patient and father. Initiation of gliclazide 20 mg twice daily was associated with resolution of symptoms and normalization of haemoglobin A1C (5.6%). Blinded continuous glucose monitoring demonstrated significantly less time spent in the hyperglycaemic range (sensor glucose > 8.0 mmol/L) when on twice daily gliclazide versus intermittent or no gliclazide (mean minutes/day with sensor glucose > 8 mmol/L: 53.6 ± 90.0 vs. 307.9 ± 246.6; P = 0.04).ConclusionsThis novel mutation in the glucokinase gene led to atypical symptomatic exercise-induced hyperglycaemia that was responsive to low dose sulfonylurea with self-reported additional benefit after reduction of carbohydrate intake. We postulate that her atypical clinical presentation was related to the intense elite-level physical activity combined with carbohydrate loading before exercise.