Introduction
The RDDC RNA Splicing Prediction Model bioinformatics AI tool demonstrates critical value in deciphering complex genetic patterns, particularly pathogenic mechanisms involving non-canonical splice sites and variants acting in cis. In a study of a family experiencing recurrent prenatal arthrogryposis (linked to Nemaline Myopathy, NM), the tool successfully predicted the synergistic effect of two novel non-canonical splicing variants located in cis within the NEB gene. RDDC's prediction (a 17 bp deletion within an exon) was subsequently precisely validated by a minigene assay, not only clarifying the pathogenicity of these "Variants of Uncertain Significance" (VUS) but also providing core evidence for genetic counseling and prenatal diagnosis for the family.
Research Challenge: WES Identifies Rare in cis Splicing VUS
The challenge in this study stemmed from a family with a history of recurrent adverse pregnancy outcomes, terminating the first and third pregnancies due to severe fetal arthrogryposis. To determine the genetic cause, the research team performed trio-WES on the third affected fetus and the parents. The results revealed compound heterozygous variants in the fetal NEB gene: one known pathogenic frameshift variant c.19049_19050delCA inherited from the father, and a more complex situation from the mother – two novel variants located on the same chromosome (in cis): c.24871G>T (in exon 178) and c.24871-10C>G (in the upstream intron 177). Both in cis variants affected non-canonical splice regions, were absent from population databases, and were classified as VUS by ACMG. Understanding the pathogenesis required clarifying how these two in cis variants collectively impacted NEB mRNA splicing.
RDDC's Precise Prediction: Revealing Synergistic Activation of a Cryptic Splice Site
To evaluate the potential pathogenic mechanism of these in cis VUSs before functional experiments, the researchers utilized the RDDC RNA Splicing Prediction Model bioinformatics AI tool, along with HSF. RDDC's prediction unveiled a sophisticated molecular mechanism:
Variant Effects Analysis
1. c.24871G>T (Exonic Variant): Predicted to disrupt the authentic splice acceptor site at the end of exon 178 and activate a cryptic acceptor site 17 bp downstream.
2. c.24871-10C>G (Intronic Variant): Predicted to reduce the score of the original acceptor site (from 0.981 to 0.9011), suggesting disruption of splicing regulatory elements, and potentially acting synergistically with the c.24871G>T variant to promote the activation of the same downstream cryptic acceptor site.
Synergistic Effect Prediction
Taken together, RDDC predicted that the synergistic action of these two in cis variants would ultimately lead to a 17 bp deletion within exon 178 (r.25063_25079del).
Experimental Validation: Confirming RDDC's Accuracy
The research team promptly validated RDDC's prediction using an in vitro minigene assay. After transfecting HEK293T and HeLa cells with constructs containing both in cis mutations, RT-PCR and sequencing showed that, besides the full-length transcript, the major aberrant transcript produced precisely lacked 17 bp from exon 178.
Validation Results
This experimental result was in perfect agreement with the molecular consequence predicted by RDDC. This 17 bp deletion causes a frameshift and premature termination codon (p.(Val8291Phefs*35)), resulting in a truncated, non-functional protein. The perfect concordance between the in silico prediction and the in vitro validation successfully reclassified the two in cis VUSs as "Likely Pathogenic" (LP).
Case Implications
This case powerfully demonstrates that RDDC RNA Splicer is a robust tool for deciphering the pathogenic mechanisms of complex splicing variants, including those at non-canonical sites and those acting in cis. It accurately predicts the impact of variants on splice signals and regulatory elements, including their synergistic effects, providing clear targets for subsequent functional validation. This "WES + RDDC prediction + functional validation" approach is crucial for improving the diagnostic rate of genetic disorders like prenatal arthrogryposis and for providing accurate genetic counseling and reproductive options (like PGT) to affected families.
Clinical Significance
This case demonstrates the important value of RDDC in the following aspects:
- Accurate prediction of pathogenic mechanisms of non-canonical splice site variants
- Revealing synergistic effects of in cis variants
- Providing scientific evidence for VUS reclassification
- Guiding functional validation experiment design
- Supporting genetic counseling and prenatal diagnosis decisions
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
Liu Y, Xu J, Lv Q, et al. Case report: identification of one frameshift variant and two in cis non-canonical splice variants of NEB gene in prenatal arthrogryposis. Frontiers in Genetics. 2023 Sep 13;14:1255850.
Article Link: https://pubmed.ncbi.nlm.nih.gov/37745844/
