Deep Sequencing in a Case Study: Beyond the Common c.848T>C Pathogenic Variant.

Very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is caused by biallelic pathogenic variants in (acyl-CoA dehydrogenase very-long-chain), leading to impaired fatty acid oxidation and the accumulation of long-chain acylcarnitine. We report a single case of a two-year-old girl, whose neonatal metabolic screening revealed an acylcarnitine profile suggestive of VLCADD, with residual enzymatic activity of 19.8%. We performed whole-gene sequencing. We then carried out an in silico analysis of the potential effects of the variants with dedicated tools, assessing splicing, RNA structure, RNA binding factors, and protein structure. We also conducted gene expression analysis. Genetic testing identified her as compound heterozygous for the pathogenic variant (NM_000018.3):c.848T>C, inherited from her mother, and for the two paternal variants, c.-64T>C in the basal promoter and c.957G>A, a synonymous substitution in exon 10. Gene expression analysis revealed reduced mRNA levels in the proband's blood cells but without abnormal isoform production. A decreased expression of the paternal allele carrying the 957A was also observed. Despite this significant reduction in mRNA levels, the underlying mechanism remains unclear. Although currently healthy, due to the VLCAD residual activity within the range associated with the mild form of the disease, the child might be at potential risk for metabolic decompensation or late-onset VLCADD. Our results indicated an allelic imbalance in mRNA expression and c.957G>A is identified as a hypomorphic allele. This suggests that deep sequencing is a valuable tool for correlating genetic variants with enzymatic activity levels.