Arya, VB; (2015) Understanding the novel genetic mechanisms of congenital hyperinsulinaemic hypoglycaemia. Doctoral thesis , UCL (University College London).

Text Final PhD Thesis 26 05 2015.pdf Available under License : See the attached licence file. Download (16MB)

Abstract

Background: Hyperinsulinaemic hypoglycaemia (HH) is characterized by unregulated secretion of insulin in the presence of hypoglycaemia. Mutations in nine different genes (ABCC8, KCNJ11, GLUD1, HNF4A, HNF1A, GCK, HADH, SLC16A1 and UCP2) have so far been identified as a cause of HH. Mutations in ABCC8 and KCNJ11, which encode the sulfonylurea receptor 1 (SUR1) and potassium inward-rectifying 6.2 (Kir6.2) subunits of ATP-sensitive potassium channel (KATP channel), are the most common cause of HH. At least two (possibly more) well-described histological subtypes are associated with HH: a focal form and a diffuse form. Diffuse HH are most commonly due to recessive and dominant mutations in ABCC8 and KCNJ11. Focal HH results due to somatic loss of the maternal 11p allele (11p15.1 to 11p15.5) involving the ABCC8 and KCNJ11 region in patients with paternally inherited mutation in ABCC8 or KCNJ11. HH can be transient and resolve within few weeks. Transient HH is seen in association with intrauterine growth restriction, maternal diabetes mellitus, perinatal asphyxia, erythroblastosis fetalis, maternal administration of sulfonylureas, and intravenous glucose infusions during labour. In large series of HH patients, the underlying genetic cause was not identified in approximately 50% of the patients. Whole-exome sequencing is a powerful and cost-effective tool for identifying genetic basis of diseases. It involves sequencing the protein coding regions of the human genome. Aim: To identify novel genetic mechanisms of HH. To functionally characterize two novel KATP channel mutations associated with a unique clinical phenotype. Patients: Four patients had protein-sensitive HH with normal serum ammonia. Two families (one consanguineous and one non-consanguineous) had two affected siblings with HH. All these patients had negative molecular genetics for ABCC8, KCNJ11, GLUD1 and HADH. One patient had a unique phenotype of HH and cardiac arrhythmias. In addition, three patients had two novel KATP channel mutations associated with a unique clinical phenotype (transient HH and combined focal/diffuse HH). Methods: Whole-exome sequencing (WES) was performed on nine patients (from seven families), their parents and unaffected siblings to identify novel, deleterious gene variants. Based on the available biological information, two novel gene variants (p.F108del K2P17 and p.A422V Kv6.2) were considered potential disease causing. The mutations were created in plasmid constructs containing the WT cDNA sequence for KCNK17 (K2P17) and KCNG2 (Kv6.2) using site-directed mutagenesis. These constructs were transfected into HEK293 cells, which were studied by whole-cell patch clamping. Two novel KATP channel mutations (p.T1516M SUR1 and p.*391Rext*94 Kir6.2) were also studied. For studying the p.*391Rext*94 Kir6.2 mutation, the WT human cDNA and 3′UTR sequence of KCNJ11 was cloned into pcDNA 3.1 vector. The KATP channel mutations were created in plasmid construct containing the WT cDNA sequence for ABCC8/KCNJ11 using site-directed mutagenesis. These constructs were transfected into HEK293 cells, which were then studied by whole-cell patch clamping. The p.*391Rext*94 Kir6.2 is a non-stop mutation and the transcripts can undergo degradation by non-stop decay phenomenon. The presence of p. *391Rext*94 Kir6.2 mutant transcript in the pancreatic tissue of the affected patient was studied by cDNA sequencing and Western blotting on the patient’s pancreatic tissue extracted RNA and protein fraction respectively. Results: WES identified two potential disease-causing heterozygous gene variants (p.F108del K2P17 and p.A422V Kv6.2) in a family with a phenotype of HH and cardiac arrhythmia, which were confirmed by Sanger sequencing. Whole-cell patch clamping experiments on HEK293 cells proved both variants to be pathogenic under heterozygous expression. Whole-cell patch clamping experiments on the two novel KATP channel mutations (p.T1516M SUR1 and p.*391Rext*94 Kir6.2) was indicative of pathogenic nature of the mutation. Analysis of the pancreatic tissue, obtained at surgery from the patient with non-stop KCNJ11 mutation (p.*391Rext*94 Kir6.2), by cDNA sequencing and Western blotting established the presence of transcript and protein with non-stop mutation. Conclusions: Mutations in KCNK17 (K2P17) and KCNG2 (Kv6.2) are potential novel genetic mechanisms for HH and cardiac arrhythmias. More patients with similar phenotype need to be screened for mutations in these two genes. A novel mechanism for HH (combined focal and diffuse HH phenotype) and molecular basis for a transient type of HH was identified.

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