Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing

Abstract

Introduction: COL4A5 is a causative gene of X-linked Alport syndrome (XLAS). Male patients with XLAS with nonsense variants have the most severe phenotypes of early onset end-stage kidney disease (ESKD); those with splicing variants have middle phenotypes and those with missense variants have the mildest phenotypes. Therefore, genotyping for male patients with XLAS 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. Nevertheless, in XLAS, these variants are generally considered to be missense variants, without conducting a transcript analysis, which underestimates some patients as having 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 the 20 variants, 17 (85%) caused aberrant splicing. Male patients with splicing variants had more severe phenotypes when compared with those with missense variants. Findings from the in vivo analyses for 3 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.

Keywords: COL4A5; genotype–phenotype correlation; last nucleotide position; missense variants; single-base substitutions; splicing.

Graphical abstract

Figure 1

Flow diagram of variant selection. In total, 578 missense variants in COL4A5 were identified from HGMD and 14 missense variants caused by single-base substitutions at the last nucleotide position in each exon were included. There were 6 novel variants that were included in our cohort. Finally, 20 variants were included in this study. HGMD, Human Gene Mutation Database.

Figure 2

Kidney survival rate in male patients with XLAS. The solid line represents splicing variants detected in this study, and the dotted, dashed, and dot-dashed lines represent missense variants, intronic splicing variants, and nonsense variants reported in our previous study, respectively. The Kaplan–Meier kidney survival analysis results revealed that the median age for developing end-stage kidney disease was significantly lower for patients with splicing variants in our present study compared with those with missense variants in our previous study (27 years of age, 95% CI: 22–29 vs. 40 years of age, 95% CI: 35–45; Wilcoxon: P < 0.01). Nevertheless, there was no significant difference in the median age for developing end-stage kidney disease between patients with splicing variants in this study and those in our previous study with intronic splicing variants (27 years of age, 95% CI: 22–29 vs. 28 years of age, 95% CI: 24–35; P = 0.72) or nonsense variants (27 years of age, 95% CI: 22–29 vs. 18 years of age, 95% CI: 16–27; P = 0.09).