Lessons learned: Targeted RNA sequencing as a tool for resolving variants of uncertain significance
Presenter Status
Fellow
Abstract Type
Clinical Research
Primary Mentor or Principal Investigator
Isabelle Thiffault
Presentation Type
Poster
Start Date
20-5-2026 11:00 AM
End Date
20-5-2026 12:00 PM
Abstract Text
Background: Exome and genome sequencing (ES/GS) has become increasingly available for individuals with a suspected underlying monogenic disease, and depending on the indication for testing, can achieve a diagnostic rate of 10-50%. However, a challenge that remains is resolving variants of uncertain significance (VUS) that are detected, causing inconclusive test results in roughly 23% of cases across North America. Mechanisms to resolve VUS include reanalysis over time, research collaborations for functional interrogation, and reflexing to additional technologies that are variant and gene mechanism dependent. RNA sequencing (RNAseq) is a technology that can aid in the resolution of VUS that are predicted to impact splicing. The detection of an abnormally spliced transcript(s) or nonsense-mediated mRNA decay (NMD) can be used as additional evidence for variant reclassification. Thus, there is a need for clinical testing that can functionally validate VUS and resolve molecular diagnoses.
Objectives/Goal: This retrospective study sought to elevate the impact of RNA studies for VUS resolution.
Methods/Design: Our cohort included 22 pediatric patients with various clinical phenotypes and a compelling genotype that received ES/GS-based testing between January 2023 to September 2025. VUS were identified in all patients.
Results: Fifteen patients (68%) received reports with variant classification changes due to RNA findings. Specifically, 13 cases (59%), received an upgraded report from VUS to likely pathogenic/pathogenic. Two of these patients had synonymous variants (c.615C>T (p.Gly205=) in HARS1 and c.60C>T (p.Gly20=) in SLC52A2) which we found to cause exon skipping and an in-frame deletion, respectively. Two small deep intronic deletions (c.82-28_82-16del in SNRPE and c.3610+262_3610+397del in TSC2) were found to cause loss-of- function due to a truncated product. In one patient, a missense variant resulted in skipping of two exons in MAGED2. The remaining eight positive patients had intronic variants located at +/- 3 to 39 positions of the exon. Of these intronic variants, 75% were predicted to affect splicing by in silico tools, with an average splice prediction score of ~53% (range:16-91%). Two patients (9%) received a variant downgrade due to lack of aberrant splicing, and 2/22 (9%) remained a VUS as RNA data was inconclusive. Lastly, 5/22 (23%) failed due to insufficient expression in blood.
Conclusions: Our study demonstrated the utility of RNA studies in providing sufficient evidence to reinterpret VUS in a small cohort of clinically diverse pediatric patients. Fifteen patients (68%) received an updated clinical report, 13 of which obtained a molecular diagnosis. Moreover, ~47% of our positive variants were not located at splicing junctions, and therefore, would most likely not be flagged as a likely candidate in ES/GS analysis pipelines. Additionally, two positive variants had Transcripts Per kilobase Million (TPM) in whole blood less than 0.1, indicating that these values can help narrow down which variants could be reflexed to targeted RNAseq, but they, as well as in silico splicing predictions, have significant limitations. As the cost of sequencing continues to decrease, this study demonstrated the utility of performing targeted RNAseq concurrently with DNA sequencing, representing an important advancement in genetic testing by improving classification of variants, therefore, improving the diagnostic yield.
Lessons learned: Targeted RNA sequencing as a tool for resolving variants of uncertain significance
Background: Exome and genome sequencing (ES/GS) has become increasingly available for individuals with a suspected underlying monogenic disease, and depending on the indication for testing, can achieve a diagnostic rate of 10-50%. However, a challenge that remains is resolving variants of uncertain significance (VUS) that are detected, causing inconclusive test results in roughly 23% of cases across North America. Mechanisms to resolve VUS include reanalysis over time, research collaborations for functional interrogation, and reflexing to additional technologies that are variant and gene mechanism dependent. RNA sequencing (RNAseq) is a technology that can aid in the resolution of VUS that are predicted to impact splicing. The detection of an abnormally spliced transcript(s) or nonsense-mediated mRNA decay (NMD) can be used as additional evidence for variant reclassification. Thus, there is a need for clinical testing that can functionally validate VUS and resolve molecular diagnoses.
Objectives/Goal: This retrospective study sought to elevate the impact of RNA studies for VUS resolution.
Methods/Design: Our cohort included 22 pediatric patients with various clinical phenotypes and a compelling genotype that received ES/GS-based testing between January 2023 to September 2025. VUS were identified in all patients.
Results: Fifteen patients (68%) received reports with variant classification changes due to RNA findings. Specifically, 13 cases (59%), received an upgraded report from VUS to likely pathogenic/pathogenic. Two of these patients had synonymous variants (c.615C>T (p.Gly205=) in HARS1 and c.60C>T (p.Gly20=) in SLC52A2) which we found to cause exon skipping and an in-frame deletion, respectively. Two small deep intronic deletions (c.82-28_82-16del in SNRPE and c.3610+262_3610+397del in TSC2) were found to cause loss-of- function due to a truncated product. In one patient, a missense variant resulted in skipping of two exons in MAGED2. The remaining eight positive patients had intronic variants located at +/- 3 to 39 positions of the exon. Of these intronic variants, 75% were predicted to affect splicing by in silico tools, with an average splice prediction score of ~53% (range:16-91%). Two patients (9%) received a variant downgrade due to lack of aberrant splicing, and 2/22 (9%) remained a VUS as RNA data was inconclusive. Lastly, 5/22 (23%) failed due to insufficient expression in blood.
Conclusions: Our study demonstrated the utility of RNA studies in providing sufficient evidence to reinterpret VUS in a small cohort of clinically diverse pediatric patients. Fifteen patients (68%) received an updated clinical report, 13 of which obtained a molecular diagnosis. Moreover, ~47% of our positive variants were not located at splicing junctions, and therefore, would most likely not be flagged as a likely candidate in ES/GS analysis pipelines. Additionally, two positive variants had Transcripts Per kilobase Million (TPM) in whole blood less than 0.1, indicating that these values can help narrow down which variants could be reflexed to targeted RNAseq, but they, as well as in silico splicing predictions, have significant limitations. As the cost of sequencing continues to decrease, this study demonstrated the utility of performing targeted RNAseq concurrently with DNA sequencing, representing an important advancement in genetic testing by improving classification of variants, therefore, improving the diagnostic yield.
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Poster Board Number: 13