Mapping the Non‐Canonical Splicing Variants: Decrypting the Hidden Genetic Architecture of Idiopathic Male Infertility

Canonical splicing variants (±2) contribute significantly to genetic disorders, yet the clinical significance of non-canonical splicing variants (NCSVs) that occur outside of canonical splicing sites remains unknown in male infertility. A comprehensive evaluation of reported studies on hereditary male infertility revealed that the 2,404 pathogenic variants contained 120 canonical splicing variants and 32 NCSVs. Among the remaining 2,252 variants, the splicing variant analytical strategy identified 17 novel NCSVs that disrupt normal mRNA splicing from previously classified missense variants. This expands the contribution of NCSVs by 53.13% (17/32), with NCSVs accounting for 28.99% (49/169) of all the splicing variants. Moreover, thirteen positively validated NCSVs are identified in 12 of 718 idiopathic male infertility patients with negative results by conventional genetic analysis. The first pathogenic variant in the TATA element modulatory factor 1 (TMF1: c.2859+4A>G) results in TMF1 exon 14 skipping and decreased progressive sperm motility and morphological abnormalities in a patient with male infertility. Tmf1 NCSV knock-in mice recapitulated human phenotype, showing significantly decreased sperm count, motility, ultrastructural head defects, and subfertility. This study provides the first comprehensive landscape of NCSVs in male infertility, suggesting that NCSVs may constitute a hidden etiological factor for male infertility.