Background: Dominant mutations in ABCC8 can cause congenital hyperinsulinism (CHI), which is characterised by unregulated insulin secretion.
Objective and hypotheses: To understand the molecular basis of medically unresponsive CHI due to dominant ABCC8 mutations.
Method: We investigated ten patients with diazoxide unresponsive CHI who required a near total pancreatectomy. DNA sequencing revealed seven dominant heterozygous missense mutations in ABCC8 (one novel and six previously reported but uncharacterised mutations). Mutant cDNA constructs were transfected into HEK293 cells for functional studies. 86Rb+ was used as a surrogate to measure the efflux of K+. Electrophysiological techniques were used to measure whole-cell and single channel currents. Confocal microscopy was used to determine the subcellular location of mutant KATP channels, by co-transfecting with pDs-Red2-ER (endoplasmic reticulum marker) and Kir6.2-GFP.
Results: D1506E is a SUR1 mutation located in nucleotide binding domain 2 (NBD2). Homologous expression of D1506E under whole-cell patch-clamp, displayed only −2.88±1 pA/pF of current in the presence of 100 μM diazoxide. Similarly single channel data showed a current response of 4.5±1.8% in the presence of 1 mM ADP. Heterozygous expression of D1506E suggested a strong dominant negative effect on WT SUR1 subunits. Mutations in the transmembrane domain (TMD) were more responsive to channel activators. The A113V (SUR1 TMD0) mutant showed some activation to MgADP (83.5±7.3%). Confocal analysis demonstrated that the NBD2 mutations were not retained in the ER, which is indicative of membrane expression. The TMD mutations are relatively retained in the ER suggestive of a trafficking defect.
Conclusion: We define two groups of mutations with different cellular mechanisms. In the first, channel complexes with mutations in the NBD2 traffic normally but are unable to be activated by MgADP. In the second with mutations in the TMD are retained in the ER and have variable functional impairment.
01 - 03 Oct 2015
European Society for Paediatric Endocrinology