Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability

Abstract

Mutations in glucokinase (GCK) cause a spectrum of glycemic disorders. Heterozygous loss-of-function mutations cause mild fasting hyperglycemia irrespective of mutation severity due to compensation from the unaffected allele. Conversely, homozygous loss-of-function mutations cause permanent neonatal diabetes requiring lifelong insulin treatment. This study aimed to determine the relationship between in vitro mutation severity and clinical phenotype in a large international case series of patients with homozygous GCK mutations. Clinical characteristics for 30 patients with diabetes due to homozygous GCK mutations (19 unique mutations, including 16 missense) were compiled and assigned a clinical severity grade (CSG) based on birth weight and age at diagnosis. The majority (28 of 30) of subjects were diagnosed before 9 months, with the remaining two at 9 and 15 years. These are the first two cases of a homozygous GCK mutation diagnosed outside infancy. Recombinant mutant GCK proteins were analyzed for kinetic and thermostability characteristics and assigned a relative activity index (RAI) or relative stability index (RSI) value. Six of 16 missense mutations exhibited severe kinetic defects (RAI ≤ 0.01). There was no correlation between CSG and RAI (r(2) = 0.05, P = 0.39), indicating that kinetics alone did not explain the phenotype. Eighty percent of the remaining mutations showed reduced thermostability, the exceptions being the two later-onset mutations which exhibited increased thermostability. Comparison of CSG with RSI detected a highly significant correlation (r(2) = 0.74, P = 0.002). We report the largest case series of homozygous GCK mutations to date and demonstrate that they can cause childhood-onset diabetes, with protein instability being the major determinant of mutation severity.

Figure 1.

Ribbon model of the closed (glucose-bound) form of human GCK illustrating each of the 16 missense mutations. Glucose is indicated in stick form in the center of the active site.

Figure 2.

Linear regression analysis of Clinical Severity Grade against (A) RAI or (B) RSI for GCK missense mutations. The 95% confidence intervals for the linear regression lines are shaded in gray.

Figure 3.

Assessment of thermostability for WT and mutant human GST-GCK proteins. Logistic regression modelling was used to fit an activity curve to each protein in (A). Data were normalized to the baseline level of activity for each protein at 40°C. Each point represents mean activity ± SEM (n = 3 experiments except for WT and p.T342P n = 10 and p.D160N n = 6). The raw residuals, defined as the difference between observed and predicted values, for all proteins are shown in (B).