Defining variants of uncertain significance
Genetic variants arise through errors in DNA replication or inherited sequence differences. The clinical significance of any given variant depends on the available evidence linking it to phenotypic outcomes. While most genetic variations are harmless polymorphisms, some mutations are linked to illnesses like cancers. VUS, by definition, cannot be classified as either pathogenic or benign based on current evidence. Currently, these variations are classified on a spectrum, from "benign" to "likely pathogenic," depending on the gene. The prevalence of VUS can vary, with well-studied genes like those involved in Cystic Fibrosis development having fewer VUS. Others, especially those linked to rare diseases, may have more.
Encountering a VUS during genomic testing is not uncommon. For example, roughly 35% of individuals undergoing next-generation sequencing (NGS) for hereditary breast cancer-related genes encounter one or more of them. Under the American College of Medical Genetics and Genomics (ACMG) framework, VUS is one of five formal variant classification categories, sitting between likely benign and likely pathogenic. For researchers and clinical teams, VUS present an interpretive gap: they cannot be used to guide eligibility decisions or treatment protocols, and their reclassification often takes years. Although most VUS will eventually be reclassified as likely pathogenic or benign, this process can be time-consuming, taking years to reach a definitive conclusion.
Until then, clinical recommendations are based on personal and family medical histories. Researchers can also use databases like ClinVar, along with scientific literature, to gather information on VUS they find to ensure that information on different variations is constantly being updated.
How VUS affect trial design and patient stratification
In clinical trials, variants of uncertain significance have the potential to complicate patient stratification, treatment decisions, and overall trial outcomes, all of which depend on precise genetic data interpretation. When researchers are sequencing all or part of the genome in order to make assumptions about a certain condition, it can be difficult to learn from VUS.
In trials targeting specific pathogenic variants, patients carrying a VUS in a clinically relevant gene are often not eligible for associated biomarker-targeted therapies. This creates a difficult decision for trial teams: excluding these patients may unnecessarily narrow an already limited pool, while including them introduces uncertainty into the dataset.
The lack of significant data can lead to their omission from medical reports, which excludes important genetic information from the study and leaves patients with uncertainties. When VUS are excluded from reports or deprioritised in analysis, trial data becomes incomplete and downstream clinical decisions are made on a narrower evidence base than the study itself could support.
Strategies for addressing VUS
In clinical trials, the following strategies can be implemented to manage variants of uncertain significance:
- Collaboration with genetic experts: Integrating genetic counseling into the screening pathway—rather than treating it as a post-result add-on—directly affects how efficiently VUS are communicated, how participants respond, and whether ambiguous results translate into trial delays or manageable exceptions within the eligibility framework.
- Long-term monitoring: Recognizing that VUS can evolve over time, longitudinal patient monitoring can be a crucial tool for researchers to accumulate data on the behavior and clinical significance of these mutations.
- Functional studies: Functional studies can also help researchers to learn about the impact of VUS on genetic processes. Researchers can conduct these studies to gain a deeper understanding of the clinical relevance of VUS.
Beyond these strategies, diversity of genomic data is essential in addressing VUS effectively. Currently, there is far more genetic information available for individuals of European ancestry, leading to a higher prevalence of VUS in other population groups. Genomic databases remain disproportionately populated by individuals of European ancestry, which elevates VUS rates in studies with more diverse enrollment. For sponsors designing trials with global or multi-ethnic populations, this is not only a scientific consideration—it is a protocol design and feasibility issue that affects how VUS rates are anticipated, disclosed, and managed across different patient groups.
At the same time, it's vital that researchers take the time to look into VUS that come up, particularly if a number of their participants possess the same one. Sharing the information they learn is also critical; they can collate their findings with ClinVar or research the VUS on the ClinVar Miner website, which has information on over 670,000 variants. Emerging approaches such as high-throughput functional assays also offer a path to resolving VUS classification by directly testing the biological impact of specific variants, complementing the evidence accumulated through population-level databases.
Role of genetic counseling
Genetic counseling plays a direct role in helping researchers and participants who undergo genetic testing understand the phenotypic implications of their genotype. VUS is one of the many things that they can provide support on. They communicate the significance and limitations of these uncertain variants with clarity and expertise, and by combining insights from family history and available variant information, genetic counselors can also provide patients with a better understanding of the potential clinical significance of their VUS.
While many patients are reassured by a VUS finding, others experience uncertainty about the ambiguity it introduces. Genetic counselors can offer guidance to these individuals, proposing options like more targeted genetic testing to mitigate the chances of encountering a VUS. They can also provide context that the majority of variants of uncertain significance are ultimately reclassified as benign. Through structured counseling, they help both patients and researchers make informed decisions throughout the genetic testing and analysis process.
Regulatory and classification guidelines for VUS
The American College of Medical Genetics and Genomics (ACMG) has created guidance for interpreting sequence variants, particularly for clinical genetic testing. With the rapid evolution of sequencing technology, the ACMG assembled a workgroup of clinical laboratory directors and clinicians to revise standards and guidelines for interpreting sequence variants. These recommendations provide a structured framework for classifying variants into five categories: pathogenic, likely pathogenic, uncertain significance, likely benign, and benign. This framework makes up the spectrum mentioned earlier:
The ACMG also emphasises the importance of performing clinical molecular genetic testing in CLIA-approved laboratories and having results interpreted by qualified experts.
A recent legal case involving the misclassification of a genetic variant linked to epilepsy underscored the need for guidelines like these and those created by the UK Association of Clinical Genetic Science. While these guidelines are not legally mandatory, adherence remains critical. Sponsors and laboratories should maintain transparency with participants about the potential challenges that surround genetic testing, including the possibility of receiving a VUS result. Continuous monitoring and re-evaluation of VUS is also vital, as new information can emerge that alters variant classifications. Finally, laboratories must be willing to share their data with clinical databases like ClinVar to collectively accumulate evidence for informed decision-making in the future.
Conclusion
Variants of uncertain significance present a real and growing challenge in precision medicine trials. As more therapies target specific genetic variants, the volume of VUS encountered during screening will only increase.
For sponsors and clinical teams, this means VUS management cannot be treated as an isolated laboratory concern. It requires coordinated workflows across genetic testing, counseling, data sharing, and long-term participant engagement. Taking a proactive, structured approach to VUS is essential not only for advancing scientific understanding, but for maintaining the trust and retention of the patients at the center of these programs.