What is the clinical evidence from this study?

Study design: Other. Population: Early-onset Fetal Akinesia Deformation Sequence (FADS) linked to RYR1 mutations (n=2). Intervention: Whole genome sequencing (WGS) vs. Whole exome sequencing (WES). Primary outcome: Identification of pathogenic genetic variants causing FADS (diagnostic yield increase).

What question did this study set out to answer?

To explore the genetic basis of fetal akinesia deformation sequence (FADS) when whole exome sequencing (WES) yields negative results.

February 19, 2026Open Access

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

Key Result

Whole genome sequencing identified two novel compound heterozygous RYR1 mutations causing fetal akinesia deformation sequence in cases where whole exome sequencing was negative, enabling genetic diagnosis in 100% of affected fetuses studied.

Key Points

To explore the genetic basis of fetal akinesia deformation sequence (FADS) when whole exome sequencing (WES) yields negative results.
Utilized trio-whole genome sequencing (WGS) for a couple with recurrent FADS.
Annotated variants using public databases and filtered according to ACMG guidelines.
Conducted functional validation of an intronic variant using RT-PCR, TA cloning, and Sanger sequencing.
Identified two novel compound heterozygous variants in the RYR1 gene in the affected fetus.
Reclassified an intronic variant as pathogenic due to its role in causing intron retention and frameshift.
Both identified variants were absent in population databases and showed segregation with the phenotype.

Structured PICO

Does whole genome sequencing identify pathogenic variants in cases of fetal akinesia deformation sequence with negative whole exome sequencing?

Population

A couple who experienced four consecutive pregnancy losses, including two fetuses affected by fetal akinesia deformation sequence (FADS), with negative whole exome sequencing (WES) results.

Intervention

Trio-whole genome sequencing (WGS) followed by functional validation of an intronic variant using RT-PCR, TA cloning, and Sanger sequencing.

Comparator

Prior negative whole exome sequencing (WES).

Outcome

Identification of pathogenic variants responsible for FADS.

Whole genome sequencing is effective in diagnosing fetal anomalies like FADS by identifying non-coding and structural variants missed by whole exome sequencing.

Main Result

Effect estimate: diagnostic yield increase

Absolute Event Rate: 100% vs 0%

Limitations

Very small sample size of only two affected fetuses from one family.
RNA validation performed on peripheral blood of carriers due to poor fetal tissue quality, potentially limiting functional interpretation.
No broader population or multicenter data to confirm generalizability of findings.

Abstract

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 deletion (c. 13659+655₁4172+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 (VUS). 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 deletion 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 non-coding 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.

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