Aberrant splicing caused by exonic single nucleotide variants positioned 2nd or 3rd to the last nucleotide in the COL4A5 gene

Background and objectivesThe evident genotype–phenotype correlation shown by the X-linked Alport syndrome warrants the assessment of the impact of identified gene variants on aberrant splicing. We previously reported that single nucleotide variants (SNVs) in the last nucleotide of exons inCOL4A5cause aberrant splicing. It is known that the nucleotides located 2nd and 3rd to the last nucleotides of exons can also play an essential role in the first step of the splicing process. In this study, we aimed to investigate whether SNVs positioned 2nd or 3rd to the last nucleotide of exons inCOL4A5resulted in aberrant splicing.MethodsWe selected eight candidate variants: six from the Human Gene Variant Database Professional and two from our cohort. We performed anin-vitrosplicing assay and reverse transcription-polymerase chain reaction (RT-PCR) for messenger RNA obtained from patients, if available.ResultsThe candidate variants were initially classified into the following groups: three nonsense, two missense, and three synonymous variants. Splicing assays and RT-PCR for messenger RNA revealed that six of the eight variants caused aberrant splicing. Four variants, initially classified as non-truncating variants, were found to be truncating ones, which usually show relatively more severe phenotypes.ConclusionWe revealed that exonic SNVs positioned 2nd or 3rd to the last nucleotide of exons in theCOL4A5were responsible for aberrant splicing. The results of our study suggest that attention should be paid when interpreting the pathogenicity of exonic SNVs near the 5′ splice site.