Introduction
The RDDC RNA Splicer tool demonstrates significant value in increasing the molecular diagnostic rate for genetically heterogeneous diseases like albinism, particularly when analyzing the function of non-coding variants. A study focusing on 122 albinism patients unresolved by standard exome sequencing showed that systematic screening for non-coding variants, coupled with splicing effect prediction using bioinformatics tools like RDDC and subsequent functional validation, boosted the diagnostic yield by nearly 10%. RDDC's accurate predictions for several deep intronic variants (DIVs) and synonymous variants provided crucial in silico evidence leading to definitive diagnoses.
Research Challenge: The "Diagnostic Gap" After WES and Non-coding Variants
Albinism involves at least 21 genes and has a complex genetic basis. Standard exome sequencing (ES) provides a molecular diagnosis for only about 70% of patients, leaving a ~30% "diagnostic gap." Approximately 15% of these unresolved cases carry only a single known heterozygous pathogenic variant, strongly suggesting that the second pathogenic hit might lie in non-coding regions (like introns) missed by ES, or be a type of variant (like synonymous variants) that ES struggles to interpret. This study focused precisely on these 122 heterozygous patients, aiming to uncover these "hidden" pathogenic variants using Whole Genome Sequencing (WGS) or NGS Panels covering intronic regions, combined with functional prediction and validation.
RDDC's Precise Prediction: Unveiling Splicing Effects of DIVs and Synonymous Variants
The researchers first used WGS or NGS Panels to identify 37 candidate non-coding, low-frequency (MAF ≤ 0.001) variants likely affecting splicing in the 122 patients. Subsequently, they employed the RDDC RNA Splicer tool, alongside other algorithms, to predict the splicing effects of these candidates. RDDC played a key role in this stage:
Predicting Cryptic Splice Site Activation by DIVs
For the OCA2 gene variant c.2433-22889T>A, RDDC predicted the activation of a cryptic splice site, leading to a 159 bp pseudoexon insertion. This prediction provided a clear direction for RT-PCR validation, which confirmed the aberrant splicing and the resulting premature termination codon, establishing the DIV's pathogenicity.
Guiding Mechanistic Analysis of Synonymous Variants
For the OCA2 gene synonymous variant c.1857C>T in patient 4, RDDC predicted an impact on splicing. Although subsequent experiments did not find exon skipping, the prediction guided further analysis, suggesting a potential link to a 77 bp pseudoexon insertion.
Predicting Effects of Non-Canonical Splice Site Variants
For the SLC45A2 gene variant c.1157-765C>G, RDDC predicted the creation of a new splice site, resulting in a 275 bp pseudoexon insertion. RT-PCR and sequencing validated this outcome and the generation of a premature termination codon, confirming its pathogenicity.
Experimental Validation and Increased Diagnostic Yield
Based on the predictions from RDDC and other tools, the team performed RT-PCR and/or minigene assays on 14 candidate variants located in trans to the first identified pathogenic variant. The results confirmed that 9 of these variants indeed caused exon skipping or pseudoexon insertion. These validated pathogenic non-coding variants ultimately provided a definitive molecular diagnosis for 11 (or 12, slight discrepancy in source text) patients, boosting the diagnostic yield in this cohort of heterozygous patients by 9.8% (12/122). Among the patients with trans variants undergoing experimental validation, the confirmation rate reached 75% (12/16).
Case Implications
This study strongly demonstrates that for WES-unresolved albinism cases, systematically testing non-coding regions combined with splicing effect prediction using tools like RDDC RNA Splicer is an effective strategy to increase diagnostic yield. RDDC's accurate predictions provide a reliable theoretical basis for subsequent functional validation. This approach not only helps uncover the molecular mechanisms of albinism involving DIVs and synonymous variants but also offers new targets and strategies for genetic counseling and potential future splice-modulating therapies (like antisense oligonucleotides).
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:
Diallo M, Courdier C, Mercier E, et al. Functional Characterization of Splice Variants in the Diagnosis of Albinism. International Journal of Molecular Sciences. 2024 Mar 17;25(6):3440.
Article Link:
https://pubmed.ncbi.nlm.nih.gov/39201349/
