Functional Validation of a CC2D2A Non-Canonical Splice-Site Variant Supports Prenatal Molecular Testing in Joubert...

Background: Joubert syndrome (JS) is a genetically heterogeneous ciliopathy wherein non-canonical splice-site (NCSS) variants are frequently reported as variants of uncertain significance (VUS), complicating prenatal counseling. We aimed to clarify the clinical significance of an NCSS in CC2D2A through functional validation. Methods: Trio-WES was performed on a 13-year-old female proband with clinically diagnosed JS. Candidate variants were validated by Sanger sequencing and familial segregation. An in vitro minigene assay and RT-PCR on patient peripheral blood RNA were used to assess the splicing effect of c.439-5T>G. Variant classification followed the ACMG/AMP guidelines using the ClinGen quantitative Bayesian framework. Results: Compound heterozygous CC2D2A variants were identified: c.439-5T>G (maternal) and c.4732G>A (p.(Val1578Met), paternal); both were initially classified as VUS. Minigene analysis showed that c.439-5T>G caused aberrant splicing, retaining the intronic nucleotides c.439-4_439-1 (ATAG) at the exon 6-7 junction (r.438_439insauag) and introducing a frameshift and premature termination (p.(Pro147IlefsTer14)). Patient RNA confirmed this pattern. c.439-5T>G was reclassified as likely pathogenic (PVS1_VeryStrong + PM2_Supporting). c.4732G>A remained a VUS (PM3 + PM2_Supporting + PP3) on an independent allele basis; its trans configuration with the likely pathogenic allele supported a family-specific biallelic explanation but did not establish the pathogenicity of the missense allele. The fetus in a subsequent pregnancy was confirmed to carry both variants, establishing the compound heterozygous state. Conclusions: Minigene-based validation provided evidence to reclassify a CC2D2A NCSS from VUS to likely pathogenic, enabling variant-directed prenatal testing and informed reproductive counseling in this JS family. The paternal missense variant c.4732G>A (p.(Val1578Met)) remains a VUS; its trans configuration with the likely pathogenic allele supports, but does not establish, a biallelic explanation. This study illustrates how functional assays can clarify splicing variants that in silico predictions alone cannot reliably interpret.