Glucokinase (GCK) Mutations and Their Characterization in MODY2 Children of Southern Italy

Type 2 Maturity Onset Diabetes of the Young (MODY2) is a monogenic autosomal disease characterized by a primary defect in insulin secretion and hyperglycemia. It results from GCK gene mutations that impair enzyme activity. Between 2006 and 2010, we investigated GCK mutations in 66 diabetic children from southern Italy with suspected MODY2. Denaturing High Performance Liquid Chromatography (DHPLC) and sequence analysis revealed 19 GCK mutations in 28 children, six of which were novel: p.Glu40Asp, p.Val154Leu, p.Arg447Glyfs, p.Lys458_Cys461del, p.Glu395_Arg397del and c.580-2A>T. We evaluated the effect of these 19 mutations using bioinformatic tools such as Polymorphism Phenotyping (Polyphen), Sorting Intolerant From Tolerant (SIFT) and in silico modelling. We also conducted a functional study to evaluate the pathogenic significance of seven mutations that are among the most severe mutations found in our population, and have never been characterized: p.Glu70Asp, p.His137Asp, p.Phe150Tyr, p.Val154Leu, p.Gly162Asp, p.Arg303Trp and p.Arg392Ser. These seven mutations, by altering one or more kinetic parameters, reduced enzyme catalytic activity by >40%. All mutations except p.Glu70Asp displayed thermal-instability, indeed >50% of enzyme activity was lost at 50°C/30 min. Thus, these seven mutations play a pathogenic role in MODY2 insurgence. In conclusion, this report revealed six novel GCK mutations and sheds some light on the structure-function relationship of human GCK mutations and MODY2

Counterregulation during spontaneous nocturnal hypoglycemia in prepubertal children with type 1 diabetes.

OBJECTIVE: To examine counterregulatory responses during spontaneous nocturnal hypoglycemia in prepubertal children with type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 29 prepubertal patients with type 1 diabetes underwent two overnight profiles. Data were analyzed from 16 children (median [range] 8.7 [5.9-12.9] years of age) with a night of hypoglycemia and a nonhypoglycemic night. Children hypoglycemic (< 3.5 mmol/l) on night 1 were given 25% extra carbohydrate as uncooked cornstarch with their usual evening snack on night 2 to avoid hypoglycemia. Glucose, growth hormone, and cortisol were measured every 15 min, catecholamines every 30 min, and glucagon, pancreatic polypeptide, insulin, and ketones every 60 min. A group of 15 healthy control subjects, aged 9.5 (5.6-12.1) years, underwent one overnight profile. RESULTS: Median duration of hypoglycemia was 225 (30-630) min, and glucose nadir was 2.0 (1.2-3.3) mmol/l. Insulin levels were not different on the two nights (P = 0.9, analysis of variance), but children with diabetes had higher insulin levels than normal control subjects between 2300 and 0300, maximal at 0200 (mean +/- SEM 57.4 +/- 5.7 vs. 31.6 +/- 5.0 pmol/l, P = 0.002). Peak epinephrine was higher on the night of hypoglycemia (0.98 [0.52-2.09] nmol/l) versus nonhypoglycemia (0.32 [0.21-0.62] nmol/l), P = 0.001, but norepinephrine (1.29 [1.07-2.64] vs. 1.26 [1.04-1.88] nmol/l, P = 0.5), glucagon (93 [64.2-125.6] vs. 100.5 [54.6-158] ng/l, P = 0.6), pancreatic polypeptide (410.2 [191-643.2] vs. 270.8 [158.2-777.8] ng/l, P = 0.5), and cortisol (513 [300-679] vs. 475 [235-739] nmol/l, P = 0.6) were not different. Glucose threshold for epinephrine release was very low, 1.9 +/- 0.2 mmol/l. There was a short-lived rise in growth hormone from 75-105 min after onset of hypoglycemia, maximal at 90 min (7.8 +/- 1.2 vs. 3.5 +/- 0.9 ng/ml, P = 0.02). CONCLUSIONS: The prolonged nature of nocturnal hypoglycemic episodes may be explained in part by defective counterregulation. The risk of nocturnal hypoglycemia needs to be reduced before intensification of insulin therapy can be contemplated in this age-group