Whole Genome Sequence Identifies the Second Allele: An Intronic Variant in RYR1 Contributes to Early-Onset Fetal Akinesia Deformation Sequence

Introduction: Fetal akinesia deformation sequence (FADS), a severe prenatal phenotype associated with congenital myopathies (CMs), is linked to mutations in the RYR1 gene. Although whole exome sequencing (WES) is a standard diagnostic approach, certain pathogenic variants may remain undetected. In this study, we utilized whole genome sequencing (WGS) to investigate a recurrent case of FADS with negative WES results. Methods: A couple who experienced four consecutive pregnancy losses, including two fetuses affected by FADS, underwent trio-WGS following negative WES findings. Variants were annotated using public databases and filtered according to ACMG guidelines. Functional validation of an intronic variant was conducted using RT-PCR, TA cloning, and Sanger sequencing. Results: WGS identified two novel RYR1 compound heterozygous variants in the affected fetus: a paternally inherited in-frame mutation (c.13659+655_14172 +588delinsCTGGCGCCCCATCTCAT) located the C-terminal hotspot and a maternally inherited deep intronic variant (c.4934+25G>A). Both of them were initially classified as variant of uncertain significance. RNA studies demonstrated that this intronic variant caused a 26-bp intron retention and frameshift, leading to its reclassification as pathogenic. And according to this PM3 evidence, the in-frame mutation was reclassified as likely pathogenic. Both variants were absent from population databases and exhibited segregation with the phenotype. Conclusion: This study highlights the utility of WGS in diagnosing fetal anomalies with negative WES results by identifying noncoding and structural variants. The identification of novel RYR1 mutations broadens the genetic spectrum of recessive CMs and emphasizes the necessity for functional assays to accurately interpret intronic variants. WGS is recommended for cases of recurrent fetal anomalies when WES is inconclusive, although careful interpretation of variants is advised.