Methods
A family with congenital hyperinsulinism (CHI) was examined for GCK gene variants by Sanger sequencing. A combined approach, involving kinetic analysis (also using GK activators and inhibitors), intracellular translocation assays, insulin secretion measurements and structural modelling, was used to investigate the novel variant compared with known variants.
Results
We report on the novel gain-of-function GCK variant p.Val455Leu (V455L), inherited as an autosomal dominant trait in a German family with CHI and concomitant obesity (fasting blood glucose 2.1 mmol/l, BMI 45.0 kg/m2, HOMA-IR 1.5 in an adult female family member); one male family member developed type 2 diabetes until age 35 years (with fasting glucose 2.8-3.7 mmol/l, BMI 38.9 kg/m2, HOMA-IR 4.6). Kinetic characterisation of the V455L variant revealed a significant increase in glucose affinity (glucose concentration at which reaction rate is half its maximum rate [S0.5]: mutant 2.4 ± 0.3 mmol/l vs wild-type 7.6 ± 1.0 mmol/l), accompanied by a distinct additive susceptibility to both the endogenous activator fructose 2,6-bisphosphatase and the synthetic allosteric activator RO-28-1675. The effect of RO-28-1675 was more pronounced when compared with the previously known GK variants V455M and V455E. Binding to the inhibitor glucokinase regulatory protein was unimpaired for V455L and V455E but was reduced for V455M, whereas mannoheptulose inhibited all GK variants and the wild-type enzyme. Structural analyses suggested a role for residue 455 in rearrangements between the inactive and active conformations of GK and also in allosteric activation. Comparison with V455M and V455E and an overview of activating GK variants provided a context for the novel sequence aberration in terms of altered GK enzyme characteristics caused by single amino acid changes.