Introduction
"Synonymous variants" are often dismissed as benign in Whole Exome Sequencing (WES) analysis because they don't alter the protein's amino acid sequence. However, these seemingly "silent" variants can sometimes cause severe disease by impacting mRNA splicing. A recent study on a neonate with combined immunodeficiency and intestinal disease (GIDID2-like) perfectly illustrates the critical role of the RDDC RNA Splicer tool in uncovering the pathogenicity of such variants. In this research, RDDC accurately predicted that a novel synonymous variant in the PI4KA gene would cause a 5 bp deletion, a prediction subsequently confirmed perfectly by RT-PCR and minigene assays.
Clinical Challenge
The challenge originated from a preterm infant girl presenting with persistent diarrhea, sepsis, and immune abnormalities. WES identified two novel compound heterozygous mutations in her PI4KA gene: a missense mutation (p.Leu1949Pro) inherited from her father, and a synonymous mutation c.3453C>T (p.Gly1151=) from her mother. This synonymous variant, located at the exon 30-intron 30 junction and unreported in population databases, had unknown functional consequences. Determining whether and how this "silent" variant contributed to the patient's condition was crucial.
RDDC Prediction Analysis
To investigate the function of the c.3453C>T synonymous variant, the research team utilized the RDDC RNA Splicer tool to predict its effect on splicing. RDDC's analysis provided key, specific mechanistic hypotheses, predicting two potential aberrant splicing patterns:
Prediction Results
- A 5 bp deletion (GTGAG) in the mRNA: This would cause a frameshift and a premature termination codon (p.Gly1151GlyfsTer17).
- Skipping of exon 30: Resulting in a 93 bp deletion.
Both predictions pointed towards a loss-of-function outcome, providing a clear theoretical basis for subsequent experiments.
Experimental Validation
The research team promptly proceeded with validation using RT-PCR and minigene splicing assays. The experimental results strongly confirmed RDDC's first predicted mode: the c.3453C>T mutation indeed caused a 5 bp deletion in the mRNA. Furthermore, the experiments revealed significantly reduced levels of the mutant transcript (only 1/4th of the normal transcript), suggesting involvement of nonsense-mediated mRNA decay (NMD).
Validation Significance
The perfect concordance between the in silico prediction and the in vitro experimental results not only established the pathogenicity of the "synonymous" c.3453C>T variant but also elucidated its molecular mechanism—disrupting mRNA splicing to cause a GIDID2-like disease.
Clinical Significance
This case powerfully demonstrates that RDDC RNA Splicer is a potent tool for dissecting the potential pathogenicity of "silent" variants. It can provide accurate functional predictions for seemingly benign synonymous mutations, guiding experimental validation, thereby preventing missed diagnoses and providing critical information for precise diagnosis and genetic counseling in rare diseases.
Future Applications
This successful case provides important insights for clinical geneticists and researchers: when analyzing WES results, the potential pathogenicity of synonymous variants should not be overlooked, especially those located near splicing sites. The application of bioinformatics tools like RDDC RNA Splicer will greatly improve the accuracy and efficiency of rare disease diagnosis.
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
Stapels DAC, Zappeij-Kannegieter L, Parry DA, et al. A synonymous mutation in PI4KA impacts the transcription and translation process of gene expression. Human Mutation. 2023 Jan;44(1):210-218. Epub 2022 Nov 3.
Article Link: https://pubmed.ncbi.nlm.nih.gov/36341355/
