Case Study: RDDC RNA Splicer Accurately Predicts Female Infertility-Related Splicing Variants

Introduction

The perfect synergy between in silico prediction and in vitro experimental validation is key to elucidating the pathogenic mechanisms of novel splicing variants. In a study focused on female infertility characterized by Early Embryonic Arrest (EDA), the RDDC RNA Splicing Prediction Model bioinformatics AI tool demonstrated its precision by successfully guiding subsequent functional experiments. The tool's predictions for novel splicing variants in the TLE6 and NLRP5 genes were in complete agreement with the validation results from minigene assays, providing core evidence for resolving the pathogenicity of these "Variants of Uncertain Significance" (VUS) and guiding clinical diagnosis and Preimplantation Genetic Testing (PGT).

Research Challenge: Splicing VUS in Unexplained Infertility Identified by WES

This study focused on two female infertility patients experiencing recurrent IVF/ICSI failure due to embryos arresting at early developmental stages. To investigate the genetic etiology, the research team performed Whole Exome Sequencing (WES) on the patients and their families. This revealed two distinct sets of compound heterozygous variants:

• Patient 1 carried c.541+2dupT (splicing variant) and c.1075G>A (missense variant) in the TLE6 gene.
• Patient 2 carried c.1249C>T (missense variant) and c.2957+4A>G (splicing variant) in the NLRP5 gene.

Both TLE6 c.541+2dupT and NLRP5 c.2957+4A>G were novel splice site variants absent from public databases and classified as VUS. Determining whether these variants were the cause of the patients' embryonic arrest required clarifying their specific impact on mRNA splicing.

RDDC's Precise Prediction: Revealing Loss-of-Function Splicing Aberrations

To evaluate the potential pathogenic mechanisms of these two VUSs, the researchers employed the RDDC RNA Splicing Prediction Model bioinformatics AI tool. This advanced algorithm accurately predicts potential splicing pattern alterations caused by various types of variants, including insertions/duplications and non-canonical site variants.

For the TLE6 c.541+2dupT variant

RDDC predicted two primary aberrant splicing outcomes: a 19 bp deletion from exon 7 or an 118 bp retention of intron 7. Both scenarios would lead to frameshifts and premature termination codons, resulting in truncated proteins.

For the NLRP5 c.2957+4A>G variant

RDDC predicted two primary aberrant splicing outcomes: a 171 bp deletion of exon 11 or a 242 bp deletion spanning exons 10-11. These would also produce truncated proteins.

RDDC's predictions clearly indicated that both splicing variants were pathogenic via a loss-of-function (LoF) mechanism. They would disrupt the normal function of TLE6 and NLRP5 proteins (key members of the subcortical maternal complex, SCMC), potentially interfering with maternal mRNA clearance and zygotic genome activation (EGA), ultimately leading to embryonic arrest.

Experimental Validation and Clinical Value: From Prediction to Precise Intervention

The research team promptly validated RDDC's predictions using in vitro minigene assays. The experimental results robustly confirmed RDDC's forecasts:

TLE6 Variant Validation Results

For TLE6 c.541+2dupT, the experiments detected the two predicted aberrant splice products, generating truncated proteins p.Ala175SerfsTer134 and p.Gln181ArgfsTer2, respectively.

NLRP5 Variant Validation Results

For NLRP5 c.2957+4A>G, the experiments validated the aberrant splicing leading to the truncated protein p.Val906AlafsTer21.

The complete agreement between the in silico predictions and in vitro results provided strong evidence for the pathogenicity of these two novel splicing VUSs. This research not only expanded the mutation spectrum for TLE6 and NLRP5, offering new diagnostic targets for EDA, but more importantly, it showcased the critical value of RDDC RNA Splicer in aiding the interpretation of VUS pathogenicity. This provides a solid molecular foundation for subsequent PGT to select healthy embryos and achieve successful pregnancies.

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:
Li R, Mei M, Zhou L, et al. Biallelic Recessive Mutations in TLE6 and NLRP5 Cause Female Infertility Characterized by Human Early Embryonic Arrest. Human Mutation. 2024 Jun;45(6):e26786.

Article Link:
https://pubmed.ncbi.nlm.nih.gov/40225929/