Co-inheritance of dominant and recessive ABCC8 mutations in a case of familial congenital hyperinsulinism (CHI):-molecular and functional studies

Abstract

Objectives: A male with CHI was born to an unaffected mother. His father had CHI with incomplete diazoxide response in infancy, then maintained on diazoxide (ceased at 7 years). The neonate had intensive medical support, a more severe phenotype, less diazoxide response (ceased due to pulmonary hypertension) and required surgery (diffuse disease). Objective (i) Molecular analysis: ABCC8, KCNJ11; (ii) Functional studies of likely pathogenic variants Methods: (i) Targeted massively parallel sequencing with Sanger sequencing confirmation (ii) COSm6 cells (green monkey kidney) transfected with wild type (WT) SUR1 and WT Kir6.2, or mutant SUR1 and WT Kir6.2. Western blots to assess SUR1 protein expression with processing efficiency estimated by core-and complex-glycosylated bands. Inside-out patch-clamp recording assessed channel gating properties (MgADP stimulation). Results: (i) Two heterozygous ABCC8 variants were detected. A paternal ABCC8 missense variant (c.4532T>C, p. Ile1511Thr) was predicted to have potentially damaging functional effects (previously reported monoallelic variant in diazoxide-responsive diffuse CHI). A maternal ABCC8 nonsense variant (c.742C>T, p. Arg248∗) resulting in a premature stop codon was predicted to have loss of function (previously reported recessive variant in diffuse CHI). (ii) I1511T SUR1: reduced protein bands compared to WT SUR1. R248∗ SUR1: no protein bands. Paternal (I1511T SUR1 co-expressed with WT SUR1 at 1:1 molar ratio; with Kir6.2): SUR1 bands similar intensity to WT SUR1. Maternal (R248∗ SUR1 with WT SUR1): reduced SUR1 bands. Proband: (I1511T SUR1 with R248∗ SUR1): reduced SUR1 bands. I1511T channels: reduced MgADP response. R248∗ channels: no detectable channel activity. I1511T and R248∗ channels: little MgADP response. Conclusions: Paternal mutation reduces channel function (reducing expression and MgADP response), predicting a dominant-negative effect. Maternal mutation affects WT allele expression, but residual WT channels reaching the cell surface respond to MgADP, hence mother clinically unaffected. The proband likely had an additive effect: maternal truncation mutation does not make it into the channel; paternal allele, although expressed, has lower maturation efficiency and reduced MgADP response compared to WT allele.L6279774342019-06-11 <br />