Case Report: a novel non-canonical splice site variant in COL4A5 in a patient with Alport syndrome

Alport syndrome (AS) is a genetically heterogeneous disorder caused by mutations in type IV collagen genes, clinically characterized by progressive renal function deterioration. Despite advances in genetic screening technologies, cases resulting from non-canonical splice site variants remain diagnostically challenging, frequently leading to missed diagnoses or delayed therapeutic intervention. Here, we report a pediatric patient with AS and his mother, both harboring a novel splicing variant in the COL4A5 gene. The proband presented with microscopic hematuria and was found to carry a hemizygous COL4A5 variant (NM_033380.3: c.3374-3T > G) through whole-exome sequencing. To determine the pathogenicity of this variant, in vitro functional validation was performed using a minigene splicing assay, which confirmed aberrant mRNA splicing leading to exon 38 skipping. These findings established this variant as the genetic etiology of AS in this family. Through the integration of whole-exome sequencing and minigene splicing analysis, this study identifies c.3374-3T > G as a novel pathogenic non-canonical splice site mutation in the COL4A5 gene. Our findings not only expand the mutational spectrum of Alport syndrome and provide critical insights for early diagnosis and clinical management of similar cases, but also underscore the necessity of functional validation for non-canonical splice site variants.