Trimerization profile of type IV collagen COL4A5 exon deletion in X-linked Alport syndrome

Background Alport syndrome (AS) is a genetic kidney disease caused by a mutation in type IV collagen alpha 3, alpha 4, and alpha 5, which are normally secreted as heterotrimer alpha 345(IV). Nonsense mutation in these genes causes severe AS phenotype. We previously revealed that the exon-skipping approach to remove a nonsense mutation in alpha 5(IV) ameliorated the AS pathology. However, the effect of removing an exon on trimerization is unknown. Here, we assessed the impact of exon deletion on trimerization to evaluate their possible therapeutic applicability and to predict the severity of mutations associated with exon-skipping. Methods We produced exon deletion constructs (Delta Exon), nonsense, and missense mutants by mutagenesis and evaluated their trimer formation and secretion activities using a nanoluciferase-based assay that we previously developed. Results Exon-skipping had differential effects on the trimer secretion of alpha 345(IV). Some Delta Exons could form and secrete alpha 345(IV) trimers and had higher activity compared with nonsense mutants. Other Delta Exons had low secretion activity, especially for those with exon deletion near the C-terminal end although the intracellular trimerization was normal. No difference was noted in the secretion of missense mutants and their Delta Exon counterpart. Conclusion Exon skipping is advantageous for nonsense mutants in AS with severe phenotypes and early onset of renal failure but applications may be limited to Delta Exons capable of normal trimerization and secretion. This study provides information on alpha 5(IV) exon-skipping for possible therapeutic application and the prediction of the trimer behavior associated with exon-skipping in Alport syndrome.