Nessa, A; (2014) Understanding the molecular basis of Congenital Hyperinsulinism due to autosomal dominant ABCC8 and KCNJ11 mutations. Doctoral thesis , UCL (University College London).

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Abstract

Background: Congenital Hyperinsulinism (CHI) is a rare heterogeneous disease characterised by unregulated insulin secretion. A prominent feature of CHI is severe hypoglycaemia which presents during the neonatal period. Immediate medical attention is required to prevent permanent neurological damage. The ABCC8 and KCNJ11 genes encode for the proteins SUR1 and Kir6.2 respectively. Four SUR1 and four Kir6.2 subunits assemble into a single ATP-sensitive potassium channel (KATP), which plays a pivotal role in regulating insulin secretion from pancreatic β-cells. The most common cause of CHI is due to recessive inactivating mutations in ABCC8/KCNJ11, which can lead to defects in KATP channel biogenesis, assembly and regulation. Dominant mutations in ABCC8/KCNJ11 causing medically unresponsive CHI have been reported, but the molecular mechanisms are not clear. Aim: To understand the molecular basis of medically unresponsive CHI due to dominant ABCC8 and KCNJ11 mutations. Patients: We studied 11 patients with diazoxide unresponsive CHI who required a near total pancreatectomy and 2 patients with diazoxide responsive CHI. DNA sequencing revealed dominant inactivating heterozygous missense mutations (9 ABCC8 and 1 KCNJ11). This includes three novel, and seven previously reported mutations. Methods: The mutations were created in plasmid constructs containing the WT cDNA sequence for ABCC8/KCNJ11 using site directed mutagenesis. These constructs were individually transfected into HEK293 cells for a series of functional studies. 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. Radioactive Rubidium (86Rb+) was used to measure the efflux of Potassium (K+) under stimulated conditions in intact cells. Electrophysiological techniques were also applied to measure the whole-cell and single channel currents. KATP channel activators, inhibitors and metabolic inhibition were used in the functional experiments. Results: The confocal analysis has demonstrated that the NBD2 mutations are not retained in the endoplasmic reticulum (ER), which is indicative of membrane expression. The transmembrane domain (TMD) mutations however are relatively retained in the ER which suggests there is a trafficking defect. D1506E is the most severe SUR1 NBD2 mutation which has been studied extensively. Homologous expression of D1506E under whole-cell patch-clamp, has revealed that there is only -2.88 ± 1 pA/pF of current in the cell in the presence of 100µM diazoxide. The single channel data shows a current response of only 4.5 ± 1.8% in the presence of 1mM ADP. Heterozygous expression of D1506E is suggestive of a strong dominant negative effect on WT SUR1 subunits. Mutations in the TMD however appear to be more responsive to channel activators such as diazoxide and metabolic inhibition, although there is relatively lower expression at the membrane. The A113V is a SUR1 TMD0 mutant which also shows that 52 ± 5% of the channel is retained in the ER. However the channel shows some activation to MgADP (83.5 ± 7.3%). Conclusions: The mechanism underlying medically unresponsive CHI caused by dominant mutations in NBD2, appears to be due the KATP channels inability to respond to channel agonists such as diazoxide. Mutations in NBD2 are likely to abolish the channels sensitivity to MgADP. The TMD0 mutations studied are paternal mutations causing diffuse disease; however the functional data suggests that there may be other unknown mechanisms involved in causing the disease.

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