Identification of a missense variant (c.877G>T) disrupting canonical splicing and contributing to female infertility.

Background: deficiency is a significant cause of female infertility. Although multiple missense variants have been reported in prior studies, a number of these variants remain classified as variants of uncertain significance (VUS).

Methods: We present a patient of primary infertility characterized by oocyte maturation disorders and fertilization failure. Comprehensive genetic analysis was conducted through whole-exome sequencing (WES) to identify pathogenic variants, followed by Sanger sequencing for familial co-segregation analysis. Reverse transcription (RT-PCR), cDNA sequencing and quantitative RT-PCR were performed to validate the effect of the variant on pre-mRNA splicing.

Results: We identified compound heterozygous variants in the gene by WES: a pathogenic splice-site splicing variant (c.223-14_223-2del) and a missense variant (c.877G>T) initially classified as a VUS. Sanger sequencing confirmed that the proband carried biallelic variants, whereas her sisters with either wild-type genotypes or a single heterozygous variant exhibited normal fertility, supporting the co-segregation of the identified variants. Critically, RNA assays demonstrated that the missense variant c.877G>T disrupts canonical splicing of , resulting in exon 12 skipping.

Conclusion: This study provides the first experimental evidence that a missense variant (c.877G>T) can exert its pathogenicity through aberrant splicing, supporting its pathogenic reclassification and elucidating a genotype-phenotype correlation for missense variants through functional assays.