ESPE Abstracts (2015) 84 P-2-491

Functional Analysis of Novel ABCC8 Mutations Found in Czech Patients with Congenital Hyperinsulinism

Klara Rozenkovaa,b, Petra Dusatkovaa, Lenka Dusatkovaa, Azizun Nessab, Barbora Obermannovaa, Jitka Kytnarovad, Zdenek Sumnika, Jan Lebla, Khalid Hussainb,c & Stepanka Pruhovaa

aFaculty of Medicine, Department of Paediatrics, Charles University in Prague and University Hospital Motol, Prague, Czech Republic; bGenetics and Epigenetics in Health and Disease, Genetics and Genomic Medicine Programme, Institute of Child Health, University College London, London, UK; cDepartment of Paediatric Endocrinology, Great Ormond Street Hospital for Children NHS Trust, London, UK; dFaculty of Medicine, Department of Paediatrics, Charles University in Prague and the General University Hospital in Prague, Prague, Czech Republic

Background: Congenital Hyperinsulinism (CHI) is a heterogeneous genetically determined condition that is characterised by unregulated secretion of insulin from pancreatic β-cells. The most common and severe cases are caused by mutations in the ABCC8 gene encoding the SUR1 subunit of the KATP channel subunit. To assess the pathogenic effect of novel ABCC8 mutations we performed in-vitro functional studies.

Objective and hypotheses: The aim of our study was to identify Czech patients with CHI caused by ABCC8 mutations and to perform in-vitro functional study of novel ABCC8 mutations found in our cohort.

Method: The molecular genetic analysis of ABCC8 gene was performed on DNA samples of 42 Czech patients with CHI. Novel mutations were created by site directed mutagenesis and transfected into HEK293 cells for functional studies using radioactive Rubidium (86+Rb). Mutant and wild type (WT) channels were exposed to different drug conditions: control (DMSO), 100 μM diazoxide, 100 μM diazoxide and 10 μM glibenclamide, 2.5 mM NaCN and 20 mM 2-deoxy-D-glucose and 2.5 mM NaCN, 20 mM 2-deoxy-D-glucose and 10 μM glibenclamide. 86+Rb efflux was measured in a liquid scintillation counter using Cherenkov radiation.

Results: Mutations in ABCC8 were identified in 12 out of 42 patients (28.6%) – one homozygous and 11 heterozygous (five novel: F48del, G389R, A478del, R657Q, and K1373del). The 86+Rb efflux assay showed that in mutant channels the activation by diazoxide was decreased by 41–91.4% (median 82.2%) when compared to WT channels. The most severe effect on KATP channel function was observed in case of A478del (activity decreased by 91.4%). On the other hand in case of R657Q there was a residual activation by diazoxide in correspondence with the patient’s phenotype (activity decreased by 41%).

Conclusion: We report the biggest cohort of Czech patients with CHI published so far. The proportion of heterozygous mutations is much higher when compared to other published cohorts, most probably due to lack of consanguinity in the Czech population. Moreover, using in-vitro functional study, we have proved the pathogenic effect of 5 novel heterozygous ABCC8 mutations on the pancreatic KATP channel function.

Funding: This work was supported by the ESPE Short-term Research Fellowship for Dr Klára Roženková and the Grant Agency of Charles University, Prague, Czech Republic (GAUK 248 213).