Understanding genetic variants of uncertain significance

WHY IS THIS IMPORTANT?

Paediatricians order many first-line genetic tests, including chromosomal microarrays and gene panels. These tests may identify variants of uncertain significance (VUSs). A basic understanding of this type of result is important for test interpretation and clinical management. VUSs can be easily misinterpreted by healthcare providers and families.

WHAT ARE VUSs?

A five-tier classification system is typically used to interpret variation at the DNA sequence or chromosome level (1,2). Variants with insufficient or conflicting evidence supporting disease association, such that they cannot be classified as ‘pathogenic/likely pathogenic’, nor as ‘benign/likely benign’, are VUSs. The presence of a VUS, even in a relevant gene or chromosome, does not confirm a genetic diagnosis.

PRACTICAL TIP #1: VUSs DIFFER GREATLY IN THEIR LIKELIHOOD OF BEING PATHOGENIC

The interpretation of a genetic variant is informed by several factors. These include inherent characteristics like the frequency at which the variant is found in the general population, the prior observation of the variant in other individuals with similar presentations, and the predicted impact of the variant. The methods used and the limitations of the test, along with the specific policies of the laboratory, will determine if a given VUS is reported back to the ordering provider. Clinically, the pre-test suspicion for the associated genetic condition, and the overlap between the possible genetic condition and the patient’s features, are important considerations.

VUSs are unevenly distributed across a subjectively defined spectrum of suspected pathogenicity (Figure 1). When reviewing a VUS, one can try to assign it to a temperature category using details contained in the test report as well as the clinical context (3). ‘Hot’ VUSs are the most suspicious and may become pathogenic if additional proof of pathogenicity can be obtained. Most VUSs, however, are ‘cool’, ‘cold’, or ‘ice cold’, with insufficient evidence to classify the variant as ‘benign/likely benign’ rather than any compelling evidence of pathogenicity.

Figure 1.

Variants of uncertain significance (VUSs) exist along a spectrum of suspected pathogenicity (3). ‘Hot’ VUSs have a high level of supporting evidence, and, if additional affirmation of pathogenicity can be obtained, may be reclassified as pathogenic. ‘Warm’ VUSs are supported by evidence that points towards a diagnosis, but it may be impossible to readily obtain further data to substantiate a re-classification. ‘Tepid’ VUSs have moderate and sometimes conflicting evidence for pathogenicity, and the clinical context along with follow-up testing in the parents may aid in interpretation. Most VUSs, however, are ‘cool’, ‘cold’, or ‘ice cold’, and unlikely to be upgraded in their classification over time.

PRACTICAL TIP #2: TESTING PARENTS MAY HELP WITH INTERPRETATION

If a VUS is found to be new (de novo) in the child, and not inherited from either parent, this information may be enough to upgrade the variant to ‘likely pathogenic’. If a VUS is found to be inherited from an unaffected parent, the variant is less likely to be pathogenic. If a child has two VUSs in a gene that typically cause disease in an autosomal recessive manner, testing parents will clarify if the VUSs are in the same or different copies of the gene. Even after parental testing, variants often remain VUSs.

PRACTICAL TIP #3: THE CLASSIFICATION OF A VUS CAN CHANGE OVER TIME

Data to support or refute the clinical significance of a variant can emerge over time. For example, contemporary interpretations rely heavily on variant frequency in putatively healthy controls. As population-scale databases expanded to become more representative of human genetic diversity, a considerable proportion of VUSs as well as putatively diagnostic variants were found to be present at frequencies incompatible with causing severe genetic disorders (4). Inconclusive results should be re-reviewed periodically in case the classification of a VUS has changed. Few will ultimately be considered diagnostic. One study found that of the 272,581 VUSs reported in a large public database, <1% were reclassified over a 3-year period and, of these, 75% were downgraded to ‘likely benign’ or ‘benign’ (5).

PRACTICAL TIP #4: VUSs SHOULD NOT BE USED TO INFORM CLINICAL MANAGEMENT OR REPRODUCTIVE DECISION-MAKING

The dangers of over-interpreting genetic variants can be substantial (6). In our experience, vague documentation, limited post-test counselling, and family recollections that ‘something was found’ often contribute to the false impression that a child with a VUS has a molecularly confirmed diagnosis. When accepting a new patient into one’s practice, it is important to ensure that copies of all prior genetic test results are available and reviewed.

A patient may have a ‘hot’ VUS where it is not currently possible to generate additional clinical-grade evidence to upgrade the classification. Clinical correlation and judgment are important adjuncts to the formal laboratory-based interpretation criteria. In general, the decision to consider a VUS as diagnostic should be made after consultation with a genetics professional and the rationale should be clearly documented in the chart.

Summary

All human genomes contain thousands of VUSs. These can be a source of confusion when they are reported back to the ordering provider because of a possible connection to the patient’s phenotype. An abbreviated or e-consult with a genetics professional can help paediatricians triage VUSs and appropriately counsel families.

Funding: 2021 Starbucks Clinics Genetics/Genomics Research Studentship Award.

Potential Conflicts of Interest: There are no disclosures to declare. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.