AbstractBackgroundFabry disease is an X-linked lysosomal storage disorder caused by insufficient α-galactosidase A (GLA) activity resulting from variants in theGLAgene, which leads to glycosphingolipid accumulation and life-threatening, multi-organ complications. Approximately 50 variants have been reported that cause splicing abnormalities inGLA. Most were found within canonical splice sites, which are highly conserved GT and AG splice acceptor and donor dinucleotides, whereas one-third were located outside canonical splice sites, making it difficult to interpret their pathogenicity. In this study, we aimed to investigate the genetic pathogenicity of variants located in non-canonical splice sites within theGLAgene.Methods13 variants, including four deep intronic variants, were selected from the Human Gene Variant Database Professional. We performed anin vitrosplicing assay to identify splicing abnormalities in the variants.ResultsAll candidate non-canonical splice site variants inGLAcaused aberrant splicing. Additionally, all but one variant was protein-truncating. The four deep intronic variants generated abnormal transcripts, including a cryptic exon, as well as normal transcripts, with the proportion of each differing in a cell-specific manner.ConclusionsValidation of splicing effects using anin vitrosplicing assay is useful for confirming pathogenicity and determining associations with clinical phenotypes.