Aberrant Splicing Caused by Intronic Variants Positioned at Third to Fifth Nucleotides in COL4A5

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Introduction: Alport syndrome is an inherited kidney disease caused by the variants of COL4A3, COL4A4, or COL4A5, which encode type IV collagen. Among them, approximately 80% of cases have X-chromosome-linked pathogenic variants of COL4A5. According to recent advances in genetic analysis, a number of novel variants have been detected in Alport syndrome. It has been already known that single nucleotide substitutions in the introns positioned at 1st and 2nd from the last nucleotide of exons always cause aberrant splicing in general. Case Description: Methods: We extracted intronic 11 variants positioned at 3rd, 4th, and 5th from the last nucleotide of exons in COL4A5, in our Alport syndrome cohort from January 2006 to July 2022. We performed minigene in-vitro splicing assays, and if available, we also performed messenger RNA (mRNA) sequences obtained from patients’ samples. Results: The variants’ positions are as follows; 3rd in three, 4th in two, and 5th in six patients. All 11 patients had hematuria, and at least 9 of them had also proteinuria. The minigene splicing assay revealed aberrant splicing, particularly upstream entire exon skipping in all variants. Moreover, the results of mRNA sequences in six cases were consistent with those of the minigene splicing assay results. Discussion: This study revealed that the intronic variants positioned at 3rd, 4th, and 5th from the last nucleotide of exons in COL4A5 are highly likely to cause aberrant splicing. It is also revealed that the hybrid minigene splicing assay may be useful, especially when patients’ samples were not available for mRNA sequences. Intronic variants close to the exons are likely to be pathogenic and when those intronic variants are detected pathogenicities need to be evaluated.

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