Abstract
INTRODUCTION: Alport syndrome (AS) is an inherited kidney disease caused by variants in the COL4A3, COL4A4, or COL4A5 genes, resulting in type IV collagen abnormalities. Although autosomal dominant variants in COL4A3 and COL4A4 are increasingly being diagnosed, X-linked AS (XLAS) caused by COL4A5 variants predominates. Single nucleotide substitutions in introns positioned at first and second from the last nucleotide (called a consensus sequence) of exons always cause aberrant splicing. However, whether intronic variants at the third to fifth positions from the last nucleotide of exons can cause aberrant splicing is unclear. METHODS: We identified 11 intronic variants positioned at the third, fourth, and fifth nucleotides from the exon 3' end in COL4A5 from our AS cohort (January 2006-July 2022). We conducted in vitro splicing assays using minigenes and in silico splicing analysis using commercial splicing prediction software and evaluated mRNA sequences obtained from patients' samples when available. RESULTS: All 11 patients showed aberrant splicing patterns in the minigene splicing assays. In vivo analysis of 6 patients corroborated these findings. The commercial splicing prediction software accurately predicted splicing changes in 10 variants. CONCLUSIONS: This study shows that 11 intronic variants at the third to fifth positions in COL4A5 introns cause aberrant splicing. This finding highlights the importance of evaluating such variants for the diagnosis and prognosis of XLAS. Further investigation is warranted to confirm the pathogenicity of these variants and their effect on the prognosis of the kidney in XLAS.