Introduction
The convergence of in silico prediction and in vitro experimental validation is the gold standard for elucidating the pathogenic mechanisms of novel splicing variants. In a study on the rare mitochondrial disorder Combined Oxidative Phosphorylation Deficiency 23 (COXPD23), the RDDC RNA Splicing Prediction Model (RNA Splicer) AI tool demonstrated its precision by successfully guiding subsequent functional experiments. The tool's predictions were in perfect agreement with the validation results from a minigene assay, providing critical evidence for the precise diagnosis of the disease.
Research Challenge: WES Identifies a Novel Splice Site VUS
This study focused on a 5-month-old male infant who presented with seizures, severe lactic acidosis, and cardiac injury, all characteristic of a mitochondrial metabolic disorder. Whole Exome Sequencing (WES) revealed compound heterozygous mutations in the GTPBP3 gene: a known missense variant, c.689A>C (p.Q230P), and a novel complex variant, c.809-1_809delinsA, located at the exon 7/intron 6 junction. This delins variant, which disrupted the canonical splice site, was unreported in public databases and classified as a "Variant of Uncertain Significance" (VUS). Determining its precise impact on mRNA splicing was essential for confirming its pathogenicity.
RDDC's Precise Prediction: Unveiling Multiple Aberrant Splicing Patterns
To evaluate the potential impact of the c.809-1_809delinsA VUS before conducting functional experiments, the research team utilized the RDDC RNA Splicing Prediction Model bioinformatics AI tool. This advanced algorithm can accurately predict splicing alterations caused by complex variants like delins. For this specific variant, RDDC predicted it would induce multiple aberrant splicing patterns, including the generation of a new acceptor site, exon skipping, or intron retention. All these predictions clearly pointed to a common outcome: a frameshift and the creation of a premature termination codon (PTC), leading to a loss of GTPBP3 gene function.
Experimental Validation: Confirming RDDC's Accuracy
The research team proceeded to validate RDDC's predictions using an in vitro minigene assay. The experimental results robustly confirmed RDDC's predictions: the construct carrying the c.809-1_809delinsA mutation indeed triggered aberrant splicing, producing two abnormal transcripts: one with an 8 bp deletion at the 5' end of exon 7 (caused by the new acceptor site), and another with a complete skip of exon 7. Both events, as predicted, lead to truncated proteins and likely degradation via the NMD pathway, resulting in a loss of GTPBP3 function. The in silico prediction and in vitro validation were in perfect agreement, successfully elucidating the pathogenic mechanism of this novel variant.
Case Implications
This case strongly demonstrates that RDDC RNA Splicer is a powerful tool for researchers facing non-canonical splice site VUS, including complex delins variants. It provides precise, specific molecular mechanism predictions (such as exon skipping or new acceptor site activation) that effectively guide subsequent functional validation experiments, greatly enhancing diagnostic efficiency. This "RDDC prediction + functional experiment" pathway provides an invaluable paradigm for the precise diagnosis and mechanistic study of COXPD23 and other rare mitochondrial diseases.
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:
Wang Y, He J, Dong F, et al. A novel mutation in GTPBP3 causes combined oxidative phosphorylation deficiency 23 by affecting pre-mRNA splicing. Heliyon. 2024 Mar 15;10(6):e27606.
Article Link: https://pubmed.ncbi.nlm.nih.gov/38515655/
