Introduction
The combination of in silico prediction and multi-omics experimental validation provides a powerful strategy for elucidating the pathogenic mechanisms of non-canonical splice site variants. In a study focused on familial homocystinemia, the RDDC RNA Splicer tool, through its precise predictive capabilities, successfully uncovered the functional consequences of a novel variant located at a non-canonical splice site in the MTHFR gene. RDDC's prediction of an exon skipping pattern was subsequently precisely validated by TA cloning sequencing and RNA-seq data, not only clarifying the variant's pathogenicity but also establishing a "prediction-multi-omics validation" paradigm for diagnosing this condition.
Research Challenge: WES Identifies a VUS at a Non-Canonical Splice Site
This study centered on a 12-year-old patient presenting with seizures and significantly elevated plasma homocysteine levels (155.2 μmol/L), leading to a diagnosis of MTHFR deficiency. Whole Exome Sequencing (WES) revealed compound heterozygous variants in the MTHFR gene: a known missense variant c.1316T>C and a novel variant c.781-6G>A located at a non-canonical splice site (-6 position) in intron 5. The c.781-6G>A variant was absent from population databases, classifying it as a "Variant of Uncertain Significance" (VUS). Although other tools (like MaxEntScan) indicated a reduced score for this site, its specific impact on splicing required more precise prediction and experimental confirmation.
RDDC's Precise Prediction: Unveiling Three Potential Splicing Patterns
To evaluate the potential impact of the c.781-6G>A variant before committing experimental resources, the research team utilized the RDDC RNA Splicer tool. Based on advanced algorithms, this model can finely predict splicing alterations triggered by variants at non-canonical sites. For this specific variant, RDDC predicted three main possibilities:
Predicted Splicing Patterns
- Wild-type splicing: Some normal transcript might still be produced.
- 4 bp insertion: Activation of a cryptic splice site could lead to a short insertion.
- Exon 6 skipping: The entire exon 6 could be omitted.
These predictions suggested the variant likely disrupted splice acceptor function, leading to complex splicing outcomes, with exon skipping being one of the most probable loss-of-function mechanisms.
Experimental Validation: Confirming RDDC's Exon Skipping Prediction
The research team then employed multiple experimental methods to validate RDDC's predictions:
TA Cloning Sequencing
In samples from the patient and his heterozygous mother, besides the wild-type transcript, three aberrant splice products were detected: a 21 bp deletion within exon 6, a 4 bp insertion, and complete skipping of exon 6.
RNA-seq Validation
Sequencing depth for exon 6 in the patient's sample was significantly lower compared to other exons, directly confirming that exon 6 skipping was a major aberrant splicing event.
Minigene Assay
Minigene assay further experiments confirmed that the variant indeed promoted the skipping of exon 6.
The experimental results (particularly the exon skipping confirmed by both TA cloning and RNA-seq) were highly consistent with the pattern predicted by RDDC. This confirmed that the c.781-6G>A variant, by interfering with mRNA splicing, leads to an abnormal MTHFR protein structure, which, combined with the other missense variant, causes the disease.
Case Implications
This case powerfully demonstrates that RDDC RNA Splicer is a robust tool for deciphering the pathogenicity of VUS at non-canonical splice sites. It provides precise, specific molecular mechanism predictions (like exon skipping or base insertion), effectively guiding subsequent multi-omics validation experiments (e.g., RNA-seq, Minigene). This "RDDC prediction + multi-omics validation" approach provides an invaluable paradigm for the precise diagnosis and mechanistic study of MTHFR deficiency and other rare genetic disorders, highlighting the critical value of AI prediction tools in analyzing complex splicing events.
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
Li W, Ma X, Sun Y, et al. RNA sequencing combined with whole-exome sequencing revealed familial homocystinemia due to MTHFR deficiency and its complex splicing events. Gene. 2025 Jan 15;927:148560. Epub 2024 Oct 21.
Article Link: https://pubmed.ncbi.nlm.nih.gov/39571660/
