Purpose: Genetic testing commonly yields a plethora of variants of uncertain significance (VUS) that can lead to ongoing uncertainty for patients and their caregivers. While all VUS hold uncertainty, some VUS have more evidence in support of pathogenicity while others have more evidence of a benign role. Sharing these nuances can help guide the investment in follow-up clinical and research investigations and may, at times, influence medical decision-making despite appreciated uncertainty. Methods: Four clinical laboratories have been subclassifying VUS to help prioritize investigation and guide reporting decisions. Each laboratory developed a distinct approach for how these subclasses are used in their laboratories and, in some cases, displayed on reports. We examined the composition of each laboratory's VUS subclasses and the likelihood variants from each subclass were reclassified towards pathogenic or benign. Results: We found that variants in the lowest subclass of VUS were never reclassified as likely pathogenic (LP) or pathogenic (P), while those in the highest subclass were much more likely to be reclassified as P/LP. Conclusion: Given that forthcoming professional guidance in variant classification will advise the use of VUS subclasses, the experience of our laboratories in using VUS subclasses can inform future practices.

All authors are employed by laboratories that provide fee-for-service genetic testing.

This publication was supported in part by the National Human Genome Research Institute of the National Institutes of Health through grant U24HG006834 (Rehm). The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

NOT HUMAN SUBJECT RESEARCH DETERMINATION Date: November 13, 2024 Title of Project: Distinct rates of VUS reclassification are observed when subclassifying VUS by evidence level Project Lead Name: Heidi Rehm The above referenced project does not meet the criteria for human subject research as defined by Mass General Brigham Insitutional Review Board policies and Health and Human Services regulations set forth in 45 CFR 46. Based on the information provided this activity does not constitute human subjects research because it does not involve human subjects. This research project does not involve individually identifiable information. This project only includes the use of de-identified samples/data obtained from humans with no intent to re-identify them. The project does not require IRB approval. Sincerely, Andrea C. Klaver, Expedited Specialist II, Institutional Review Board (IRB) Mass General Brigham

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