Each gene has a specific sequence of nucleotides that is considered the standard genetic code or a gene’s reference sequence. When a patient’s sample is sent to a laboratory, the laboratory compares the patient’s genetic code with the reference sequence and looks for differences. These differences are referred to as variants. The laboratory must then determine which variants have the potential to affect gene function and are relevant for patient care. This interpretation is what a clinician receives in a report.
Variant classification underlies the basis of genetic medicine. Without it, clinicians would simply receive a report from a laboratory with a list of sequence variations. Today, most laboratories in the United States use the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines for the interpretation of sequence variants.1,2 The report recommends that sequence variants that cause Mendelian disorders be classified into 1 of 5 categories using specific criteria as a framework for classification. The 5 recommended categories are: (1) pathogenic; (2) likely pathogenic; (3) uncertain significance; (4) likely benign; and (5) benign.
From a clinical perspective, the most straightforward result is one that states that either a variant affects gene function (ie, mutation) or that the alteration does not affect function (ie, benign). The results that typically cause the most confusion or elicit the most questions from providers and patients are those that fall into the variant of uncertain significance (VUS) category. Although clinicians are becoming more accustomed to seeing these results with increased testing and next-generation panels, a lack of clear-cut evidence continues to cause frustration and confusion. This article addresses some of the questions that are frequently asked by providers.
What Is a VUS?
VUS is a term used to describe a change in the DNA sequence that does not have enough supporting evidence to be classified as either deleterious or benign. Given the availability of larger panels and the regular addition of new genes, most patients undergoing multigene panel testing will have at least 1 VUS. A VUS may also be detected in single-gene testing. Single-site testing for 1 specific alteration is one of the few tests in which a VUS is rarely, if ever, detected. Most laboratories consider a VUS to be a holding place rather than a final classification. Over time, a portion of the VUSs will be reclassified as either benign or deleterious.
Is It True That Information on a Test Request Form Can Be Useful for Variant Classification?
All information on the test request form is used in some way by the laboratory. People often think it is simply used to double-check the sample label and for billing purposes; however, it may also play a role in variant classification. Information regarding the diagnosis and family history can help a laboratory identify phenotypic trends associated with a VUS. In some cases, the information can help pull a variant out of the VUS category. If the information provided on the test request form provides enough phenotype suspicion, the laboratory will sometimes reach out to the provider for additional clinical information. For example, a patient with an MSH6 VUS may require a call to the clinician about immunohistochemistry testing.
How Can I Find More Information If I Receive a Report with a VUS?
The information on the report is usually a good place to start. Typically, the report will have some basic information regarding the classification, including any cited literature. Most laboratories are also responsive to inquiries about a specific case or classification. In some cases, you may want to see how other laboratories have classified a particular variant. One option is to use a publically available database to see whether other laboratories have observed the same variant and how they classified it. One such database is ClinVar, which is a National Institutes of Health publicly accessible database of variant classifications that are deposited by laboratories, researchers, and clinicians.3
If the test was not performed within the past year, a provider would also want to contact the laboratory that performed the test to determine whether any reclassifications have occurred. Sometimes it may also be useful to contact other laboratories regarding specific variants, because not all information is included in ClinVar. Although most laboratories in the United States presently use standard nomenclature, it has evolved over time. Therefore, the same variant may be reported using slightly different nomenclature. For a laboratory to compare what it has in its database, ideally, a provider would have a copy of the test report, which includes the testing laboratory, date of test, the specific alteration, and the reference sequence or nomenclature used.
Why Do I Hear About Discrepancies in Classification If Most Laboratories Use the ACMG-AMP Guidelines?
There are various reasons for classification discrepancies.4,5 One reason is that the ACMG-AMP guidelines are used as a framework. Along with this framework, laboratories use their own internal data and defined parameters when classifying variants. The 5 categories used by laboratories fall along a gradient; therefore, 2 laboratories could weigh a piece of evidence differently, which could push the variant’s location on the gradient more or less toward pathogenic or benign, resulting in 2 different classifications.
Another reason for classification discrepancies is that 2 laboratories could have different internal information and may use this information differently in their criteria. For example, if 1 laboratory has seen a NF1 variant 10 times in individuals with neurofibromatosis, it may be more likely to classify the variant as a mutation than a laboratory that has seen this variant only 1 time. The nomenclature and isoform used may also differ from laboratory to laboratory; therefore, an entry that appears to be identical to your variant may actually be a completely different alteration. If it appears that an alteration nomenclature or isoform discrepancy exists, typically the testing laboratory will help sort this information for you. Although most laboratories attempt to keep entries current, out-of-date entries are another reason for discrepancies in public databases.
What Is a Variant Reclassification? How Long Will It Take a Variant to Be Reclassified and How Will the Patient Be Notified?
A reclassification is a change in the interpretation of a variant. This generally happens because new data become available through additional information from clinicians, literature, or internal data. In some cases, family testing or research studies may be offered by a laboratory in an effort to gain more information for reclassification. To help the process, it may be helpful to share any new information or diagnosis for the patient with the laboratory.
The amount of time it takes for reclassification ranges from months to years or longer, and will depend heavily on the amount of evidence supporting the new classification. Some laboratories have a standard procedure for updating providers about reclassifications, whereas others do not. It may be helpful for providers to know their preferred laboratory’s notification policy. In addition, providers should encourage patients to contact them on an annual basis. This serves as a mechanism to assess patients to determine whether updated testing is needed and whether a reclassification has been issued for previous testing.
If a Patient Diagnosed with Breast Cancer at Age 35 Has a BRCA1 VUS, Should I Test Her Unaffected Sister for the VUS?
Normally, in this situation, testing the unaffected sister would not be indicated because we do not know the meaning of the variant. Multiple guidelines state that clinical testing for a familial VUS should not be performed unless it is done in a research setting.6 Testing in a research setting is usually performed to provide additional information for classification purposes. If a provider has questions or feels that another relative should be considered for testing, he or she can contact the testing laboratory to discuss this further. In some cases, laboratories may even offer complimentary VUS testing to specific relatives; however, this is usually done for research purposes in hopes of obtaining additional information for variant classification. Laboratories have their own internal protocols for which genes and which individuals in a family they will consider for research testing.
Should I Include Information About the VUS If I Plan to Order a Large Panel for the Unaffected Sister Based on a Paternal Family History of Breast and Colon Cancer?
In these cases, because the gene is already being tested on the panel, many providers will request that the laboratory review the VUS detected in the sister. For this to happen, it is useful to include the affected sister’s test report, rather than only written information regarding the variant. This ensures that the laboratory is reviewing the correct gene and variant because, as previously noted, some laboratories use different nomenclatures and isoforms.
How Do I Care for a Patient Who Has a VUS?
A VUS is not considered to be a pathogenic mutation. Therefore, care for a patient with a VUS should be based on the personal and family history, rather than the VUS test result.1,6
What About a Somatic VUS?
A VUS may also be detected in tumor tissue. If germline and tumor testing are ordered, then a VUS in the germline will be followed for reclassification. A somatic VUS, however, may not be reclassified given that tumors acquire additional mutations over time. As we learn more about somatic testing and treatment, it is possible that the importance of a somatic VUS may change over time and guidelines will evolve.7,8
How Do I Decide Which Laboratory to Use for Variant Classifications of a VUS?
Many factors may influence your decision to choose one laboratory over another. With respect to variant classifications of a VUS, some laboratories and/or particular tests do not list them on a test report. You would want to know which classifications are reported and decide whether this meets the needs of your patient. Although a VUS may lead to confusion for some patients, it could have a significant impact on their care and health should it be reclassified.
You will also want to know the laboratory’s reclassification procedure. For example, will they contact you with updates, and how will they contact you? Because the burden of reclassification communication often falls on the provider, it is important to ensure that the laboratory has a standard and effective procedure for communicating any change in classification.
Most clinical laboratories with professionals skilled in variant interpretation will have similar VUS rates. If a laboratory boasts having the lowest VUS rate or its VUS differs significantly from other laboratories, this should raise a red flag for you to ask more questions. A VUS rate is a VUS rate. Some laboratories make slightly more conservative calls than others, whereas others make more aggressive calls. VUS rates also vary by gene and slight differences in VUS rates could reflect differences in internal guidelines or data.
In addition, VUS rates can vary depending on how much of a gene is analyzed and what portions of that gene are used for the calculation. Some laboratories have different ranges for reporting variants, and some will include more intronic alterations than others. For example, if one laboratory includes +/- 5 base pairs from the exon and another laboratory uses +/- 20 base pairs from the exon for a calculation, the laboratory with the +/- 5 threshold may appear to have a lower VUS rate, because the laboratory with more intronic base pairs may have more variants to report. For these reasons and others, a thorough classification and assessment of each variant usually outweighs the VUS rate.
In summary, variant assessment is a complex process that relies on collaboration between the patient, the provider, and the laboratory. VUS remains a difficult and complex area with respect to genetic testing. Although it is unlikely that VUSs will disappear completely, advances in technology, additional data, and sharing of data will likely, over time, lower VUS rates. Until then, many patients undergoing genetic testing will likely experience having a VUS.
- Richards S, Aziz N, Bale S, et al; for the ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405-424.
- Toland AE, Forman A, Couch FJ, et al; for the BIC Steering Committee. Clinical testing of BRCA1 and BRCA2: a worldwide snapshot of technological practices. NPJ Genom Med. 2018;3:7-14.
- National Center for Biotechnology Information. ClinVar. www.ncbi.nlm.nih.gov/clinvar. Accessed May 18, 2018.
- Amendola LM, Jarvik GP, Leo MC, et al. Performance of ACMH-AMP variant-interpretation guidelines among nine laboratories in the Clinical Sequencing Exploratory Research consortium. Am J Hum Genet. 2016;98:1067-1076. Erratum in: Am J Hum Genet. 2016;99:247.
- Nykamp K, Anderson M, Powers M, et al. Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria. Genet Med. 2017;19:1105-1117.
- Eccles DM, Mitchell G, Monteiro AN, et al; for the ENIGMA Clinical Working Group. BRCA1 and BRCA2 genetic testing-pitfalls and recommendations for managing variants of uncertain clinical significance. Ann Oncol. 2015;26:2057-2065.
- Madhavan S, Ritter D, Micheel C, et al; for the ClinGen Somatic Working Group. ClinGen Cancer Somatic Working Group - standardizing and democratizing access to cancer molecular diagnostic data to drive translational research. Pac Symp Biocomput. 2018;23:247-258.
- Li MM, Datto M, Duncavage EJ, et al. Standards and guidelines for the interpretation and reporting of sequence variants in cancer: a joint consensus recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017;19:4-23.