A genetic variant is a change in the normal DNA sequence present in less than 1% of the population. As next-generation sequencing became routine, the number of identified variants exploded, challenging laboratories attempting to interpret their meaning. Previously, laboratories used internal systems, leading to inconsistent results when the same variant was tested at different facilities. To standardize clinical reporting, the American College of Medical Genetics and Genomics (ACMG) established unified guidelines in 2015 for interpreting sequence variants in Mendelian diseases. This standardized framework provides a systematic, evidence-based approach to determine the clinical significance of a genetic change.
The Five Variant Classification Categories
The ACMG guidelines establish a five-tier system for classifying variants, moving along a continuum from certainty of disease causation to certainty of harmlessness. The most definitive categories are Pathogenic (P), known to cause disease, and Benign (B), known to be harmless. Pathogenic results confirm a diagnosis, while Benign results mean the variant can be safely disregarded.
Falling between these extremes are the “Likely” categories, assigned when there is a high degree of confidence in the finding. A Likely Pathogenic (LP) variant has a greater than 90% probability of causing disease, and a Likely Benign (LB) variant has a greater than 90% probability of being harmless. Clinically, LP results are often acted upon similarly to Pathogenic results, guiding medical management and surveillance.
The middle classification is the Variant of Uncertain Significance (VUS). This is assigned when existing evidence is too contradictory or insufficient to confidently place the variant into one of the other four categories. VUS results account for a significant portion of newly identified variants and present the greatest challenge for clinicians. Since these variants cannot be definitively linked to the patient’s condition, they are generally not used to make medical decisions.
Principles of Evidence Weighting
The classification process relies on collecting and weighing specific scientific evidence supporting either a pathogenic or benign interpretation. This evidence is drawn from multiple sources, including population data, computational predictions, functional studies, and family segregation analysis. Each piece is assigned a standardized weight: Very Strong, Strong, Moderate, or Supporting for pathogenicity, and Stand-Alone, Strong, or Supporting for benign status.
Population data provides strong evidence for a benign classification if a variant is found at a high frequency in large databases of healthy individuals. If the variant is commonly seen in people without the associated genetic disorder, it is unlikely to be the cause of a severe disease. Conversely, evidence from a functional study showing that a protein is non-functional due to the variant would be considered Very Strong evidence for pathogenicity.
Evidence types are standardized to ensure consistent application across different laboratories. For example, a DNA sequence change resulting in a premature stop codon in a gene known to cause disease through loss of function is automatically assigned Very Strong evidence. This systematic weighting prevents an analyst from over- or under-valuing any single piece of information.
How Variant Classification is Performed
The final classification is determined by an algorithmic process that combines the weighted evidence according to established rules. This combinatorial logic requires specific thresholds of weighted evidence to be met for placement into one of the five categories. For a variant to be classified as Pathogenic, it must meet one Very Strong piece of evidence combined with a Moderate or Supporting piece, or multiple Strong pieces of evidence pointing toward pathogenicity.
The “Likely” categories are reached when the evidence is substantial but does not meet the criteria for a definitive classification. For example, a Likely Pathogenic result can be achieved with one Strong and one Moderate piece of evidence, or with multiple Moderate criteria.
The benign categories are reached by combining evidence that refutes a pathogenic role. A variant can be classified as Benign if it meets one Stand-Alone criterion, such as being too frequent in the general population, which overrides all other evidence. If the evidence for pathogenicity and benign status is conflicting or insufficient to reach the required thresholds, the variant defaults to the intermediate VUS category.
Clinical Implications of Variant Results
The classification of a genetic variant has immediate and long-term implications for a patient’s medical care and their family. A Pathogenic or Likely Pathogenic result can confirm a diagnosis, guide treatment, and initiate cascade screening for at-risk relatives. Conversely, a Benign result can end a diagnostic odyssey and prevent unnecessary medical interventions or anxiety.
The process of variant classification is not static; it requires periodic review, especially for VUS results. As new scientific literature is published, population databases grow, and functional studies are performed, the evidence base for a variant can change significantly. This new information frequently drives reclassification, where a variant’s category is updated, sometimes moving a VUS to a definitive Pathogenic or Benign classification.
External resources like the ClinVar database play a significant role by allowing clinical laboratories to share their variant interpretations and supporting evidence. This collaborative data sharing helps reduce discordance between laboratories and accelerates the reclassification process, particularly for VUS results. Long-term patient follow-up and the periodic re-analysis of initial genetic testing reports are necessary due to the continuous evolution of variant knowledge.