Introduction
In rare disease diagnostics, combining in silico prediction with in vitro validation is key to elucidating the pathogenic mechanism of novel variants. The RDDC RNA Splicing Prediction Model plays a crucial role in this process, especially in unmasking the hidden pathogenicity of variants classified as "benign" or "VUS." In a study on a family with Joubert Syndrome (JS), the RDDC tool successfully predicted that a CPLANE1 gene "missense" variant actually functions by disrupting splicing. This prediction was subsequently confirmed perfectly by functional experiments, leading to the reclassification of the VUS to "Likely Pathogenic" (LP) and providing core evidence for the family's prenatal diagnosis and genetic counseling.
Research Challenge: WES Identifies a "VUS" Missense Variant
The challenge in this study arose from a family with a history of three adverse pregnancies, all involving JS-related malformations (e.g., cerebellar vermis agenesis). Whole Exome Sequencing (WES) revealed that the proband (fetus) carried compound heterozygous variants in the CPLANE1 gene: one known nonsense mutation (c.8893C>T) and one missense mutation c.203C>T (p.Ala68Val). Because the c.203C>T variant lacked sufficient evidence for pathogenicity in public databases, it was classified by ACMG as a "Variant of Uncertain Significance" (VUS). To provide accurate genetic counseling (e.g., the 25% recurrence risk and PGT options) for this family, it was imperative to determine if this VUS was truly pathogenic.
RDDC's Precise Prediction: Unveiling the Splicing Anomaly Behind the Missense Variant
To assess the potential pathogenic mechanism of the c.203C>T VUS, researchers utilized the RDDC RNA Splicing Prediction Model. Despite being a missense (exonic) variant, RDDC's AI model analyzed its potential to disrupt exonic splicing enhancers (ESEs) or silencers (ESSs). The prediction provided a clear and specific molecular hypothesis: the variant would cause the skipping of exon 3. This, in turn, would lead to a frameshift and the creation of a Premature Termination Codon (PTC).
This prediction re-contextualized a "missense" variant, which seemingly only affected one amino acid, as a "splicing" variant capable of causing a complete loss of protein function.
Experimental Validation: Upgrading from VUS to "Likely Pathogenic" (LP)
Based on RDDC's precise prediction, the research team quickly performed functional experiments (including RT-PCR and NMD-inhibition assays). The experimental results robustly confirmed RDDC's forecast:
- In cells carrying the
c.203C>Tvariant, the mRNA indeed underwent skipping of exon 3. - The resulting PTC triggered the Nonsense-Mediated mRNA Decay (NMD) pathway, leading to significant degradation of the mutant allele's mRNA.
The perfect concordance between the in silico prediction and the in vitro experimental results provided the critical evidence of pathogenicity (fulfilling ACMG criterion PS3). As a result, the team successfully reclassified the c.203C>T variant from a VUS to "Likely Pathogenic" (LP).
Case Implications
This case powerfully demonstrates that RDDC RNA Splicer is a robust tool for resolving the pathogenicity of VUS in WES data, particularly for uncovering hidden splicing mechanisms within exonic variants (missense or synonymous). It provides precise, testable molecular predictions (like exon skipping) that effectively guide subsequent functional experiments, serving as a critical bridge between genotype and phenotype, and between basic research and clinical applications like Preimplantation Genetic Testing (PGT).
Content Source and Disclaimer
This article is a compilation and interpretation of the scientific study cited below, intended to highlight the application of RDDC bioinformatics tools. All research data and conclusions belong to the original authors and publication.
Original Article:
Hong Z, Xiang S, Chen Z, et al. Unveiling the Pathogenic Role of Novel CPLANE1 Compound Heterozygous Variants in Joubert Syndrome: Insights Into mRNA Stability and NMD Pathway. Journal of Cellular and Molecular Medicine. 2025.
Article Link: https://doi.org/10.1111/jcmm.70484
