Last nucleotide substitutions of COL4A5 exons cause aberrant splicing

Abstract Background COL4A5 is a causative gene of X-linked Alport syndrome (XLAS). Male XLAS patients with nonsense variants show the most severe phenotypes of early-onset end-stage kidney disease; splicing variants show middle phenotypes; and missense variants show the mildest phenotypes. Therefore, genotyping for male XLAS patients can be used to predict kidney prognosis. Single-base substitutions at the last nucleotide position in each exon are known to affect splicing patterns, and could be splicing variants. However, in XLAS, these variants are generally considered to be missense variants, without conducting a transcript analysis, which underestimates some patients as mild phenotypes. This study aimed to investigate whether single-base substitutions at the last nucleotide position of COL4A5 exons cause aberrant splicing. Methods In total, 20 variants were found in the Human Gene Mutation Database (n = 14) and our cohort (n = 6). We performed functional splicing assays using a hybrid minigene analysis and in vivo transcript analyses of patients’ samples when available. Then, we investigated genotype–phenotype correlations for patients with splicing variants detected in this study by comparing data from our previous studies. Results Among 20 variants, 17 (85%) caused aberrant splicing. Male patients with splicing variants showed more severe phenotypes when compared to missense variants. Findings from the in vivo analyses for three variants were identical to those from the minigene assay. Conclusion Our study revealed that most single-base substitutions at the last nucleotide position of COL4A5 exons result in splicing variants, rather than missense variants, thereby leading to more severe phenotypes.