Understanding a variant of uncertain significance – take a close look at the interpretation
Eija Seppälä · May 29, 2020

A Variant of Uncertain Significance (VUS) can be a challenging result for both the clinician and the laboratory geneticist, not to mention difficult to explain to the patient. An ongoing collaboration between the clinician and the laboratory provides the best chance for future reclassification of a variant.

A VUS is often the easiest and least time-consuming variant to report; typically, it has not been described in the literature or variant databases so there is little published information to review. However, it is the least helpful result from a clinical point of view. At the time of the initial report, I do as much as possible to find a diagnostic result for the patient but VUSs are an inevitable result in some cases.

As a molecular geneticist, my goal is to minimize the number of VUSs reported as primary findings and ensure that we clearly define those in the report that are the most suspicious and have the greatest potential to be clinically relevant. To this end, I cannot emphasize enough the benefits of providing as much detailed clinical information about your patient as possible. This is especially important when ordering large gene panels or whole exome sequencing because broader tests like these cover multiple phenotypes, which may or may not be relevant to your particular patient.

I have been interpreting NGS data, classifying variants and re-evaluating rare VUSs in every medical specialty. Sparse clinical information may result in more VUSs being reported. Without detailed information about the patient’s clinical features, it is challenging to exclude rare variants that are inconsistent with the patient’s phenotype, thereby increasing the chance for one or more VUSs.

Before we can talk about reclassification, it is first important to understand how variants are classified as having “uncertain significance”.

Variant interpretation: A step by step process

At Blueprint Genetics, we start by focusing on variants that are rare in the general population. Assessing the allele frequency (AF) of a potential disease-causing variant depends on the mode of inheritance and the prevalence of the disease. A good tip for defining the allele frequency limit for a likely benign vs a likely pathogenic variant is to check the minor allele frequency (MAF) of the most common known pathogenic variant in that particular gene (in gnomAD, for example). If the variant in question is more common than the most common known disease-causing variant in the gene, it should have already been described if it is truly disease-causing. If it has not, then it is most likely a benign polymorphism.

After MAF filtering, we compare the remaining variants to the available data; both in our own internal database as well as in public databases, such as ClinVar, HGMD, and other various disease- or phenotype-specific databases. Has the variant been described in scientific literature? If yes, what kind of information is reported about patients with this variant? Has the variant been shown to segregate with the disease in families?

When evaluating for rare and ultra-rare diseases, collecting the above information can be a challenging process. If the variant has not been previously published, other questions can still provide helpful insights: Is the variant located in an important region of the protein? Is the affected amino acid conserved in evolution? Does the variant have an effect on the transcripts that are expressed in the impacted organ or tissue? Are there other variants reported that affect the same amino acid? Does the variant have an effect on splicing? Is the variant predicted to damage the protein function? All are important questions when looking for further evidence to assist with classification.

If the variant is rare such that it cannot be classified as likely benign but there is insufficient evidence to support a likely pathogenic classification, then it will be classified as a VUS. Over time and with additional evidence (from the patient, the family, from published reports or databases) may make it possible to reclassify a VUS, either “up” to Likely Pathogenic, or “down” to Likely Benign.

The ACMG recommends that VUSs should not be used in clinical decision-making. They suggest that efforts to resolve the classification of the variant to “pathogenic” or “benign” should be undertaken and while this effort to reclassify the variant is underway, additional monitoring of the patient for the disorder in question may be prudent.1

As a geneticist, I want to provide a thorough report and clarity around what information was used as a basis for the original classification. Best practices to evaluate quality interpretation is to:

  • Follow a standardized variant classification scheme.
  • Review complex VUSs with colleagues and/or expert clinical consultants.
  • Include familial segregation recommendations for suspicious VUSs directly in the test report. Sometimes testing informative family members is all that is required for reclassification, and making this as clear as possible upfront helps the clinician and patient to know when this is a viable option.
  • Transparency in reporting. When the report clearly states the evidence used to arrive at the classification, the limitations of the test and whether additional testing may be useful.

Eija Seppälä is a senior geneticist at Blueprint Genetics.

You might also be interested in: What are the next steps you can take to assist with variant reclassification? Read more: VUS, the unwanted result? Collaboration Between Clinicians and Laboratories helps lay the foundation for future reclassification


  1. Deignan JL, Chung WK, Kearney HM, et al. Points to consider in the reevaluation and reanalysis of genomic test results: a statement of the American College of Medical Genetics and Genomics (ACMG). Genetics in Medicine. 2019;21:1267-1270.

Last modified: October 30, 2020