Introduction
The RDDC RNA Splicing Prediction Model bioinformatics AI tool demonstrates significant value in the precise diagnosis and treatment evaluation of Spinal Muscular Atrophy (SMA). In a study involving an infant with SMA type I harboring a novel SMN1 splicing variant and only a single copy of SMN2, this tool successfully predicted multiple aberrant splicing patterns caused by the variant. RDDC's predictions were subsequently partially or fully confirmed by in vivo cDNA cloning/sequencing and in vitro minigene assays. This not only elucidated the complex pathogenic mechanism of the variant but also provided a molecular explanation for the patient's positive response to Risdiplam treatment.
Research Challenge: WES Identifies Novel Splicing VUS with Low SMN2 Copy Number
The challenge arose from a 5-month-old infant clinically diagnosed with SMA type I. Genetic testing revealed she carried only a single copy of the SMN2 gene, which typically predicts a more severe phenotype. Whole Exome Sequencing (WES) further identified compound heterozygous variants in the SMN1 gene: a common deletion inherited from the mother, and a novel splicing variant, c.628-3T>G, in intron 4 inherited from the father. This variant, located at a non-canonical splice site (-3 position) and absent from public databases, was classified as a "Variant of Uncertain Significance" (VUS). Clarifying the specific impact of this VUS on SMN1 mRNA splicing was crucial for understanding the patient's clinical phenotype and assessing her potential response to splice-modulating drugs like Risdiplam.
RDDC's Precise Prediction: Unveiling Two Key Aberrant Splicing Patterns
To evaluate the potential pathogenic mechanism of the c.628-3T>G VUS, the researchers utilized the RDDC RNA Splicing Prediction Model bioinformatics AI tool. RDDC's prediction revealed two primary aberrant splicing consequences:
Aberrant Splicing Patterns
- Insertion of 2 bases (AG) upstream of exon 5: This would activate a cryptic acceptor site, leading to a frameshift and premature termination codon (p.Pro210SerfsTer4), resulting in a loss-of-function protein.
- Skipping of exon 5 (a 96 bp deletion): The entire exon 5 would be removed during mRNA maturation, also producing a non-functional protein (predicted deltascore=0.87, indicating high likelihood).
These predictions clearly indicated that the variant would severely disrupt the normal function of the SMN1 gene.
Experimental Validation & Clinical Correlation: RDDC Predictions Partially Confirmed, Explaining Treatment Response
The research team then proceeded to validate RDDC's predictions using both in vivo and in vitro experiments:
In Vivo Validation (cDNA Cloning)
Analysis of SMN1 cDNA clones derived from the patient revealed evidence of the AG insertion predicted by RDDC. Importantly, normally spliced transcripts were also detected. This suggested that the splicing defect caused by c.628-3T>G was incomplete, allowing for the production of some functional SMN1 protein.
In Vitro Validation (Minigene Assay)
The minigene experiment primarily demonstrated the skipping of exon 5, consistent with RDDC's second prediction.
Clinical Correlation
Although the in vivo and in vitro results were not entirely identical (suggesting limitations of the in vitro model in fully recapitulating complex splicing regulation), they collectively confirmed c.628-3T>G as a pathogenic splicing variant. More significantly, the residual normal splicing identified in vivo, interpreted alongside RDDC's predictions, explained why this single-SMN2-copy patient presented with a relatively milder phenotype than expected and responded remarkably well to Risdiplam treatment (significant improvement in CHOP-INTEND scores), which aims to increase functional SMN protein levels.
Case Implications
This case powerfully demonstrates that RDDC RNA Splicer is a robust tool for deciphering the pathogenic mechanisms of splice site VUS, particularly for predicting complex splicing outcomes. Its precise in silico predictions effectively guide subsequent functional validation (both in vivo and in vitro). When integrated with clinical phenotype and treatment response data, this approach provides critical molecular insights for the precise diagnosis of rare diseases like SMA, informs individualized treatment strategies (e.g., evaluating the potential benefit of Risdiplam), and aids genetic counseling.
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:
Ma K, Zhang K, Chen D, et al. Real-world evidence: Risdiplam in a patient with spinal muscular atrophy type I with a novel splicing mutation and one SMN2 copy. Human Molecular Genetics. 2024 Mar 21;33(7):640-646.
Article Link: https://pubmed.ncbi.nlm.nih.gov/38520738/
