Functional Analysis of the PCCA and PCCB Gene Variants Predicted to Affect Splicing.

Igor Bychkov,Mikhail Skoblov,Alexandra Filatova,Galina Baydakova,Marina Kurkina,Alexandra Ilyushkina,Andrey Nekrasov,Ekaterina Zakharova,Artur Galushkin

doi:10.3390/ijms22084154

Abstract

It is estimated that up to one-third of all variants causing inherited diseases affect splicing; however, their deleterious effects and roles in disease pathogenesis are often not fully characterized. Given their prevalence and the development of various antisense-based splice-modulating approaches, pathogenic splicing variants have become an important object of genomic medicine. To improve the accuracy of variant interpretation in public mutation repositories, we applied the minigene splicing assay to study the effects of 24 variants that were predicted to affect normal splicing in the genes associated with propionic acidemia (PA)—PCCA and PCCB. As a result, 13 variants (including one missense and two synonymous variants) demonstrated a significant alteration of splicing with the predicted deleterious effect at the protein level and were characterized as spliceogenic loss-of-function variants. The analysis of the available data for the studied variants and application of the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) guidelines allowed us to precisely classify five of the variants and change the pathogenic status of nine. Using the example of the PA genes, we demonstrated the utility of the minigene splicing assay in the fast and effective assessment of the spliceogenic effect for identified variants and highlight the necessity of their standardized classification.

Highlights

Pathogenic variants in the PCCA and PCCB genes are responsible for the rare autosomal recessive metabolic disease called propionic acidemia (PA) (OMIM#606054)
Using freely available splicing prediction tools, we analyzed all pathogenic, likely pathogenic and of-uncertain-significance variants of the PCCA and PCCB genes available in the HGMD and ClinVar databases, and those previously identified in our lab [19]
Our results of the minigene assay suggest that PCCA: c.2119-9A>G causes an 8 b.p. insertion in PCCA exon 24 and is the spliceogenic loss-of-function variant, which leads to synthesis of the truncated protein with altered biotinyl-binding domain

Summary

Introduction

Pathogenic variants in the PCCA and PCCB genes are responsible for the rare autosomal recessive metabolic disease called propionic acidemia (PA) (OMIM#606054). The products of these genes form the heterododecameric enzyme propionyl CoA carboxylase (PCC), which converts propionyl CoA to methymalonyl CoA in the mitochondrial matrix [1]. PA is clinically heterogeneous and has a variable age of onset ranging from severe neonatal forms to mild adult-onset forms [2]. The symptoms of PA manifest in the early neonatal period and without treatment quickly become life-threatening. The symptoms include seizures, poor feeding, vomiting, hypotonia, metabolic acidosis, ketonuria, hypoglycemia, hyperammonemia and cytopenia [3]. Fast and proper genetic diagnosis can play an important role in patient management [3,4,5]

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