mRNA Sequencing to Identify Aberrant Splicing in X-linked Alport Syndrome

Introduction: X-linked Alport syndrome (XLAS) is a well-known monogenetic kidney disease caused by pathogenic variants in the COL4A5 gene. Routine analysis of exons and direct flanking regions fails to identify a pathogenic variant in 10% to 20% of patients with XLAS. Methods: We evaluated 11 selected patients with clinical features of XLAS, in whom routine analysis failed to identify a pathogenic variant. In 2 patients a variant of unknown significance was detected in the intronic splice site regions. We used mRNA analysis from fibroblasts or urine-derived podocyte-lineage cells to establish a genetic diagnosis. Results: In 2 patients with a variant of unknown significance (VUS), mRNA analysis confirmed the pathogenicity. In 9 patients, mRNA analysis was used to evaluate aberrant splicing and guide genomic DNA sequencing. In 7 patients a novel pathogenic deep-intronic variant was found. Overall, aberrant splicing was complete in 5 patients and partial in 4, whereas kidney disease was less severe in the latter group. Conclusion: This report highlights the importance of mRNA analysis to confirm pathogenicity or facilitate the search for intronic variants to establish a genetic diagnosis in XLAS. This analysis can serve as a diagnostic tool in patients suspected for Alport syndrome (AS) when routine genetic analysis fails to identify a pathogenic variant.