Deep Intronic Single-Nucleotide Variant in COL4A5 Causes X-Linked Alport Syndrome by Aberrant Splicing into a Pseudoexon Containing Intron 6

Background: Alport Syndrome (AS) confers a significant risk for kidney failure due to pathogenic variants in COL4A3/4/5 genes, leading to misfolding of the collagen IV (α345) heterotrimer of the basement membrane. Most patients present with genetic variants in the coding regions, detectable by exome based sequencing. However, intronic variants appear to be disease causing in some families, where analysis and verification is more complex. We here report a family in which several males suffered from kidney failure and hearing impairment in early adulthood. The females demonstrate microhematuria and slight proteinuria, but preserved kidney function into old age. The two sons of the index patient show microhematuria and significant proteinuria already before the age of five. This prompted a genetic consultation, with the clinical suspicion of X-linked AS. Methods: Exome based and whole genome sequencing. Cultivation of human urinary primary tubular cells and skin fibroblasts, to analyse the protein and mRNA expression of COL4A5. Sanger sequencing of variant specific product. Results: Exome based analysis of a panel of genes containing the Alport spectrum yielded no result. Genome sequencing identified a single nucleotide variant (SNV) in intron six (c.385-707G>T, NM_000495), which is absent from gnomAD database and generates a novel splice site by in silico prediction. An immunoblot for collagen IV (α5) from primary cells showed the wild-type allele product in the mother, but not from the sons. RT-PCR with intron six flanking primers showed comparable expression of COL4A5 in the mother, but not the sons. Inhibition of nonsense mRNA decay (NMD) with emetine enabled detection of a COL4A5 PCR product approximately 150 bp longer than the wild-type product. Purification and sequencing of this PCR product verified 147 bp of intronic sequence, confirming the aberrant splicing to a pseudoexon containing c.385-764 to c.385-618. The pseudoexon causes a frameshift with the theoretical protein product p.Gly129Alafs38*, prematurely terminated and probably not expressed because of efficient NMD. Conclusion: A molecular diagnosis was achieved by genome sequencing and verification in primary cells. The knowledge of the intronic SNV may enable access to clinical studies for the sons of the index patient, perhaps even genomic repair in future. Funding: Government Support – Non-U.S.