Genetic Analysis And Functional Study Of 2 Cases Of Congenital Hyperinsulinemia Caused By GCK Gene Mutation

BackgroundCongenital hyperinsulinism (CHI), also known as persistent hyperinsulinemic hypoglycemia of infancy (PHHI), is a rare heterogeneous disease, which was first reported by Aynsley-Green in 1981, is the most common cause of persistent hypoglycemia of infancy. What causes this disease is not entirely clear, while the pathological basis is the inappropriate, continued secretion of insulin by the pancreatic islet b-cells. At present, molecular genetic studies have shown that at least 9 gene mutations are linked to CHI, among which the most common genetic abnormality involves the ABCC8 and KCNJ11 genes encoding proteins SUR1 and Kir6.2, which are subunits of the ATP-sensitive potassium channel (KATP channel). While, the mutation of GCK gene which encodes glucokinase (GCK) is a rare type, which was reported in 1998 at the first time, only 9 families and 9 sporadic cases till now, and no report in Asians at all.Study 1 The analysis of pathogenic genes in 2 patients with congenital hyperinsulinismObjectiveTo investigate the clinical characteristics and molecular genetic features of 2 cases of CHI.MethodsThe clinical data of 2 patients with CHI in our hospital were summarized. The genomic DNA samples of the concerned family members were extracted, amplified by PCR and PCR products were directly sequenced.ResultsTwo cases of CHI were caused by GCK gene mutations. The mutation sites of GCKwere c.589 A>G and c.269 A>G respectively, leading to protein changes of p. M197V and p. K90R.Conclusion GCK-CHI owing to GCK gene mutation might occur in Asian population.Study 2 Functional study of GCK gene mutationsObjectiveTo identify the molecular mechanism of CHI due to GCK mutations-K90R and M197V.MethodsGCK plasmids carrying GST label mutants and wild type were constructed respectively. Recombination protein of GST-GCK mutants and wild type were expressed and purified in vitro. Dynamic and thermal stability analyses were conducted through Enzyme-coupled analysis.Results1. Compared with wild type, K90R mutation decreased the production of protein and M197V mutation increased the production of protein.2. K90R and M197V mutations led to the decrease of glucose S0.5 and Hill coefficient (h), and the increase of ATP-Km, meanwhile, K90R mutation resulted in a mild decline of Kcat, total Ia values were 1.620 and 4.690 respectively.ConclusionThe abnormality in enzyme kinetics was the cause of CHI due to K90R and M197V mutation.