Introduction
The RDDC RNA Splicing Prediction Model (RNA Splicer) AI tool plays a crucial role in the prenatal diagnosis of Congenital Heart Disease (CHD), particularly in deciphering the function of non-canonical splice site variants. In a study involving a fetus with severe cardiac malformations, this tool successfully predicted that a novel variant located deep within an intron of the PLD1 gene would cause aberrant splicing. This prediction was subsequently precisely validated by RT-PCR and TA cloning sequencing, not only elucidating the pathogenic mechanism of the variant but also providing decisive molecular evidence for the family's genetic counseling and subsequent Preimplantation Genetic Testing for Monogenic diseases (PGT-M).
Research Challenge: WES Identifies an Intronic Variant of Uncertain Significance
This study involved a 24-week fetus presenting with severe right heart developmental abnormalities (pulmonary atresia, tricuspid dysplasia) detected via ultrasound. The mother had a previous history of pregnancy termination due to fetal malformations. To determine the genetic cause of the current fetal CHD, the research team performed Whole Exome Sequencing (WES) on fetal amniotic fluid and parental peripheral blood.
The results revealed novel compound heterozygous variants in the fetal PLD1 gene: a missense mutation c.1937G>C (p.G646A) inherited from the father, and a deep intronic variant c.1062-59A>G inherited from the mother. Neither variant was reported in public databases and both were classified by ACMG as "Variants of Uncertain Significance" (VUS).
The functional impact of the intronic variant c.1062-59A>G was particularly critical for determining its pathogenicity and posed a challenge for prenatal diagnosis and genetic counseling.
RDDC's Precise Prediction: Revealing Two Disruptive Splicing Patterns
To evaluate the potential pathogenic mechanism of the c.1062-59A>G VUS, the researchers utilized the RDDC RNA Splicing Prediction Model AI tool. This advanced algorithm accurately predicts the effects of variants on mRNA splicing.
Prediction Results
For this specific variant, RDDC predicted two primary aberrant splicing consequences:
- Insertion of a 76 bp intronic sequence (intron retention): This would cause a frameshift and premature termination codon.
- Skipping of exon 11: The entire exon 11 would be removed during mRNA maturation, also leading to a frameshift and premature termination.
Both predicted outcomes clearly indicated that this intronic variant would disrupt normal RNA processing, leading to a loss-of-function (LoF) of the PLD1 protein, consistent with the known mechanism for Congenital Valvular Dysplasia type 1 (CVDP1).
Experimental Validation and Clinical Application: From Prediction to Precise Prevention
Based on RDDC's clear predictions, the research team analyzed mRNA from the fetus and mother using RT-PCR and TA cloning sequencing. The experimental results robustly confirmed RDDC's predictions: both exon 11 skipping and the 76 bp intron retention were detected as aberrant splice products in the fetal and maternal samples.
Clinical Outcome
The high concordance between the in silico prediction and the in vitro validation definitively established the pathogenicity of the c.1062-59A>G variant. This crucial molecular diagnosis empowered the couple to make an informed decision, ultimately leading to the successful birth of a healthy baby via Preimplantation Genetic Testing for Monogenic diseases (PGT-M).
Clinical Significance
This case highlights the powerful capability of RDDC RNA Splicer in interpreting the function of non-canonical splice site VUS. It provides rapid, reliable functional predictions for novel, rare intronic variants identified by WES, effectively assisting clinicians in pathogenicity assessment. This significantly improves the diagnostic yield for rare diseases like CHD and provides precise molecular evidence for genetic counseling and reproductive interventions such as PGT-M.
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
Zhu L¹,², Chen M¹, Shi Y¹, Huang X¹, Ding H¹. Prenatal detection of novel compound heterozygous variants of the PLD1 gene in a fetus with congenital heart disease. Frontiers in Genetics. 2024 Feb 7;15:1347883.
Affiliation: ¹Department of Obstetrics and Gynecology, The First Affiliated Hospital of Ningbo University, Ningbo, China; ²(Information pending).
Article Link: https://pubmed.ncbi.nlm.nih.gov/39553471/
