Functional characterization of ABCC8 variants of unknown significance based on bioinformatics predictions, splicing assays, and protein analyses: Benefits for the accurate diagnosis of congenital hyperinsulinism

Abstract

ABCC8 encodes the SUR1 subunit of the β‐cell ATP‐sensitive potassium channel whose loss of function causes congenital hyperinsulinism (CHI). Molecular diagnosis is critical for optimal management of CHI patients. Unfortunately, assessing the impact of ABCC8 variants on RNA splicing remains very challenging as this gene is poorly expressed in leukocytes. Here, we performed bioinformatics analysis and cell‐based minigene assays to assess the impact on splicing of 13 ABCC8 variants identified in 20 CHI patients. Next, channel properties of SUR1 proteins expected to originate from minigene‐detected in‐frame splicing defects were analyzed after ectopic expression in COSm6 cells. Out of the analyzed variants, seven induced out‐of‐frame splicing defects and were therefore classified as recessive pathogenic, whereas two led to skipping of in‐frame exons. Channel functional analysis of the latter demonstrated their pathogenicity. Interestingly, the common rs757110 SNP increased exon skipping in our system suggesting that it may act as a disease modifier factor. Our strategy allowed determining the pathogenicity of all selected ABCC8 variants, and CHI‐inheritance pattern for 16 out of the 20 patients. This study highlights the value of combining RNA and protein functional approaches in variant interpretation and reveals the minigene splicing assay as a new tool for CHI molecular diagnostics.

Splicing Outcomes of 5′ Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays

“…RNA analysis using either patient blood cells or immortalized lymphoblastoid cells represents an alternative option, providing that the gene of interest is normally expressed in these cells (Wai et al, 2020). In case of the non-feasibility of both approaches, a cell culture-based minigene splicing assay has often been devised (for some most recent examples, see Damasio et al, 2021;Hao et al, 2021;Kim et al, 2021;Kortum et al, 2021;Le Tertre et al, 2021;Morbidoni et al, 2021;Qian et al, 2021;Saint-Martin et al, 2021;Torrado et al, 2021).…”

A homozygous exonic variant leading to exon skipping in ABCC8 as the cause of severe congenital hyperinsulinism

“…This variant has been reported in a heterozygous state in two patients diagnosed with CHI (Park et al, 2016;Saint-Martin et al, 2021) gest birth stress, however, perinatal stress induced hyperinsulinism is a potential nongenetic etiology, as it leads to diazoxide-responsive disease that often resolves within the first year of life, a disease course similar to the reported patient (Hoe et al, 2006). The patient reported by Saint-Martin et al, did not respond to diazoxide and was confirmed to have focal disease based on histology.…”

“…We present this case of a patient with severe diffuse CHI and a homozygous c.4608G>A variant in ABCC8 . This variant has been reported in a heterozygous state in two patients diagnosed with CHI (Park et al, 2016; Saint‐Martin et al, 2021). A Korean patient reported by Park et al responded well to treatment with diazoxide and octreotide, and after 3 months, the patient had conjectured spontaneous remission.…”

“…To date, about 180 variants of unknown significance have been reported in ABCC8 , and they represent about 15% of all clinically reported variants in CHI. Interestingly, half of the ABCC8 VUS encountered in CHI patients are either intronic or synonymous (Saint‐Martin et al, 2021), and these variants could be reclassified after examination of their impact on splicing. The variant c.4608G>A is predicted to result in the synonymous amino acid substitution p.Ala1536Ala that occurs at the last nucleotide of exon 38, and the G>A change is predicted to weaken the normal splicing donor site signal by prediction programs (Alamut v2.11).…”

“…Further functional study for variant pathogenicity is fundamental in making definitive molecular diagnosis, determining therapeutic options and prognosis, and providing precise genetic counseling. However, due to the lack of ABCC8 expression in blood leukocytes, functional studies for pathogenicity of VUS have been challenging (Saint‐Martin et al, 2021). We present a case of an infant with severe CHI for whom we successfully demonstrated the altered splicing effect of an exonic VUS in ABCC8 , using the patient's surgically resected pancreas tissue and supporting the molecular diagnosis.…”