Reclassification of candidate splicing variants refines clinically conflicting interpretations inSLC26A4-associated hearing loss

Purpose Variants in the human SLC26A4 gene are a major cause of hereditary hearing loss. Many splice site variants have been identified, but their pathogenicity is not well understood. Methods In accordance with the guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, we analysed the spectrum of SLC26A4 gene variants. We performed in silico analysis and in vitro splicing assays to evaluate novel or known variants of uncertain significance that may contribute to aberrant alternative splicing. Results In a cohort of 178 patients carrying SLC26A4 variants, selected from 202 hearing loss patients with or without inner ear malformations who underwent SLC26A4 gene testing, we identified a total of 50 variants. Among these, 10 intronic variants potentially affecting splicing collectively accounted for 54.8% of the total allele frequency of all identified variant types and were prioritised for messenger RNA (mRNA) splicing analysis. Further investigation demonstrated that four variants led to distinct types of aberrant splicing outcomes. Overall, the clinical significance of seven splice site variants was reclassified, representing at least 4.34% (14/323) of the variants within our cohort. Conclusion By using the standard classification of SLC26A4 variants, our results were able to interpret novel or uncertain SLC26A4 gene variants in a pathogenic or benign variant direction. This approach facilitates more refined genetic counselling for patients carrying SLC26A4 gene variants.