Introduction
Accurately assessing the functional consequences of novel splicing variants is crucial when investigating the etiology of rare genetic diseases. A recent study on male infertility combined with hydronephrosis highlights the pivotal role of the RDDC (Rare Disease Data Center) RNA Splicing Prediction Model Tool in this process. In this research, RDDC provided precise pathogenicity predictions for two novel ADCY10 gene splicing variants, which were subsequently robustly confirmed by RT-PCR and Western blot experiments, successfully linking these variants to a complex phenotype of asthenozoospermia and renal abnormalities.
The Research Challenge: Determining the Functional Impact of Novel Splicing Variants
The study focused on four infertile male patients from two unrelated Pakistani families, all presenting with low sperm motility (asthenozoospermia) and hydronephrosis accompanied by kidney stones. Using Whole Exome Sequencing (WES), researchers identified three novel ADCY10 variants in these patients: one nonsense and two splicing variants (c.4286+1G>T and c.436+2T>G).
These variants were absent from population databases and co-segregated with the disease phenotype within the families. However, determining whether these splicing variants were truly pathogenic required understanding their impact on mRNA splicing and final protein function.
RDDC's Precise Prediction: Unveiling the Loss-of-Function Mechanism
To evaluate the potential impact of these two splicing variants, the research team employed the RDDC RNA Splicing Prediction Model Tool. RDDC's analysis delivered specific molecular consequence predictions:
- For the
c.4286+1G>Tvariant, RDDC predicted either a 190 bp insertion or a 118 bp deletion, both leading to premature termination of mRNA translation. - For the
c.436+2T>Gvariant, RDDC predicted either an 82 bp deletion leading to premature termination or a 114 bp exon skipping event.
Taken together, the RDDC tool predicted that both splicing variants would cause aberrant processing of ADCY10 mRNA, ultimately resulting in non-functional truncated proteins or nonsense-mediated mRNA decay (NMD)—a clear loss-of-function outcome.
Experimental Validation: Confirming RDDC's Accuracy
Based on RDDC's predictions, the team proceeded with experimental validation. RT-PCR and Western blot analyses revealed that both ADCY10 mRNA and protein levels were undetectable in the patients' blood and sperm lysates. This outcome perfectly corroborated RDDC's prediction of loss-of-function.
Furthermore, electron microscopy revealed sperm ultrastructural defects (such as disorganized mitochondrial sheaths and excessive residual cytoplasm) consistent with the loss of ADCY10 function.
Case Implications
This case powerfully demonstrates that the RDDC RNA Splicing Prediction Model is a potent tool for deciphering the pathogenicity of novel splicing variants. It provides precise, verifiable molecular mechanism predictions (like specific insertions, deletions, or exon skipping events), effectively guiding subsequent functional experiments.
Through the application of the RDDC tool, this study successfully classified the two novel ADCY10 splicing variants as pathogenic, not only expanding the phenotypic spectrum associated with ADCY10 (linking it for the first time to sperm midpiece swelling and head abnormalities) but also providing new targets for the genetic diagnosis of asthenozoospermia and hydronephrosis.
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:
Guo T, Sha Y, Mei L, et al. Novel biallelic ADCY10 variants cause asthenozoospermia with excessive residual cytoplasm and hydronephrosis in humans. Human Genetics. 2024 Mar 2;143(3):355-367.
Article Link: https://pubmed.ncbi.nlm.nih.gov/39891992/
