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

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.

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.

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.

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.