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Clinical Impact of RNA Sequencing on VUS Resolution in a Diverse Rare Disease Cohort of Over 100,000 Patients

Laboratory Genetics and Genomics
  • Primary Categories:
    • Clinical Genetics
  • Secondary Categories:
    • Clinical Genetics
Introduction:
Variants of uncertain significance (VUS) are a persistent challenge for clinicians and a source of uncertainty for patients. The use of in silico predictive algorithms and multi-omics technologies are being increasingly utilized to provide additional data to incorporate into the variant classification process in hopes of minimizing the likelihood of reporting a VUS. RNA sequencing (RNAseq) is one technology that can aid in the resolution of VUS that are predicted to impact normal splicing of a gene, whereby the absence or presence of abnormally spliced transcripts can be used as part of the evidence to downgrade or upgrade VUS, respectively. This study sought to determine the real-world impact of an RNAseq VUS resolution program on a clinical cohort of rare disease patients that received exome-based testing as part of their diagnostic workup.
 

 





Methods:
An RNAseq VUS resolution program was available for rare disease patients who received exome-based tests performed at GeneDx between May 2019 and June 2022. Cases with VUS that were predicted to impact splicing and had sufficient expression in blood were eligible for the RNAseq VUS resolution program. Previously reported cases were also considered for eligibility during variant review and reclassification processes. RNAseq was performed on eligible variants when appropriate samples and consent were received from patients.

Results:
During this timeframe, over 100,000 exome-based tests were reported and VUS that were likely to benefit from RNAseq were identified in 134 (0.1%) patients. Of these patients, 69/134 (51%) consented and sent samples for RNAseq. Forty-eight of these patients (70%) received reports with variant classification changes on account of RNAseq findings. Specifically, 38/69 (55%) received an upgrade from VUS to likely pathogenic/pathogenic and 10/69 (14.5%) received a variant downgrade, nine of which were VUS downgraded to likely benign, and one was likely pathogenic downgraded to VUS. The RNAseq program impacted a total of 48 out of over 100,000 exome-based cases or about 0.048% of patients during this timeframe.

Conclusion:
While RNAseq can be a useful tool to assist in clinically impactful variant classification for a small subset of patients, the relative impact across a large cohort of clinically diverse patients receiving exome-based testing for rare disease is quite low. For patients with undiagnosed rare disease, the majority of reported VUS are missense variants for which RNAseq has limited utility. Thus, providing detailed and accurate clinical information, ordering trio-based testing, and selecting a laboratory with an extensive clinical and genomic database that incorporates machine learning algorithms remain key factors in clinically impactful VUS resolution for most patients.

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