Accurately linking in silico predictions with in vitro experimental validation is the gold standard for confirming the pathogenicity of novel splicing variants, especially for "Variants of Uncertain Significance" (VUS) at non-canonical sites. In a study on hypodysfibrinogenemia, RDDC's bioinformatics tool RDDCSC provided a critical and specific prediction, which was subsequently perfectly confirmed by minigene assay, offering decisive evidence for prenatal diagnosis in Autosomal Recessive Congenital Ichthyosis (ARCI).
Research Challenge: Functional Assessment of a Novel Splice Site VUS
This study focused on a 22-year-old male diagnosed with hypodysfibrinogenemia due to abnormal coagulation function tests (significantly lower fibrinogen activity than antigen levels) discovered during pre-operative screening. Gene sequencing identified a novel heterozygous variant in his FGA gene: IVS2+1G>T (c.180+1G>T), located at the canonical donor splice site of intron 2. This variant was confirmed to be de novo (absent in parents) and was not reported in population databases, classifying it as a VUS. Determining how this variant affects FGA mRNA splicing and protein function was key to establishing its pathogenicity.
RDDC's Precise Prediction: Revealing Potential Splicing Aberrations
To evaluate the potential impact of the IVS2+1G>T variant before conducting functional experiments, the research team utilized RDDC's AI prediction tool, the RNA Splicing Prediction Model (RNA Splicer). Based on advanced algorithms, this model predicts potential alterations in splicing patterns caused by variants. For this specific variant, RDDC predicted two primary aberrant splicing consequences:
- Insertion of the 368 bp intron 2 sequence: This would lead to a frameshift and premature termination codon.
- Skipping of exon 2 (126 bp).
Both predictions strongly suggested that the variant would severely disrupt normal mRNA processing, leading to truncated or internally deleted fibrinogen Aα chain proteins, thereby impairing function.
Experimental Validation: Confirming RDDC's Exon Skipping Prediction
The research team then proceeded to validate RDDC's predictions through in vitro cell experiments (RT-PCR and qPCR associated with minigene assays). The experimental results robustly confirmed RDDC's second predicted mode:
- RT-PCR clearly showed that the variant led to the skipping of exon 2 (126 bp).
- qPCR further indicated that, compared to wild-type, the mRNA expression level in the mutant group was significantly decreased, suggesting the aberrant transcript might be unstable or subject to degradation.
The high concordance between the in silico prediction (pointing to exon skipping) and the in vitro experimental results, combined with subsequent functional assays (showing significantly reduced fibrinogen polymerization and clotting activity) and ACMG guideline assessment, ultimately classified this VUS as a pathogenic mutation.
Case Implications
This case highlights the powerful capability of the RDDC RNA Splicer in deciphering the pathogenicity of splice site VUS. It provides precise, specific predictions of molecular mechanisms (like exon skipping), effectively guiding subsequent functional validation experiments and greatly improving diagnostic efficiency. This "RDDC prediction + functional experiment" approach offers a valuable paradigm for the precise diagnosis and mechanistic study of Congenital Fibrinogen Deficiencies (CFDs) and other rare hereditary coagulation disorders.
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 H¹, Zheng S¹, Yu X², Wu K², Zhao M¹, Zhu L². Genetic Mutation Analysis of Hypodysfibrinogenemia Caused by a Heterozygous FGA IVS2+1G>T Mutation in One Case. Journal of Wenzhou Medical University. 2024. (Note: Journal and full citation details incomplete)
Affiliation: ¹Wenzhou People's Hospital; ²(Information pending).
