Clinical Variant Analysis – Classification Criteria of Pathogenic Variants

The ACMG Guidelines are utilized for the interpretation of variants. They are primarily applied to diagnose suspected inherited (primarily Mendelian) disorders in a clinical diagnostic laboratory setting. While evaluating variants no matter what the origin, it is important to distinguish between variants that are pathogenic (i.e., causative) for a disease and a variant that may be predicted to be disruptive/damaging to the protein for which it codes, but is not necessarily implicated in a disease. Once a variant has been determined to be pathogenic in one case it should be considered pathogenic in all cases studied. A single conclusion must be reached using the entire body of evidence in aggregate for each variant.

The ACMG Guidelines provide two sets of criteria: one for classification of pathogenic or likely pathogenic variants (Chapter 2) and one for classification of benign or likely benign variants (Chapter 3). Each pathogenic criterion is weighted as very strong (PVS1), strong (PS1-4), moderate (PM1-6) or supporting (PP1-5) and each benign criterion is weighted as stand-alone (BA1), strong (BS1-4), or supporting (BP1-6). For a given variant, the clinician selects the criteria based on the evidence observed for the variant. Then, the criteria are combined according to the scoring rules to choose a final classification from the five-tier system. The system allows for flexibility in variant classification based on additional knowledge that the geneticist may have from their laboratory or other sources such as ClinVar. In addition, a piece of evidence may be downgraded to a lower level if the evidence for fulfilling a criterion is not strong enough or upgraded to a higher level if the evidence for fulling a criterion is stronger than expected. So, let’s focus on the criteria used to classify a variant as pathogenic or likely pathogenic.

PVS1 The presence of a loss of function variant is considered very strong evidence for pathogenicity. These include nonsense, frameshift, start loss, exon deletion, and splice site variants. When applying PVS1, it is important to ensure that Loss of Function (LoF) variants is a known mechanism of disease in the corresponding gene. Additionally, one must exercise caution if the variant occurs in the last exon or the last 50 base pairs of the penultimate exon. Such variants should only be considered if they are upstream from at least one pathogenic LoF variant from a reputable source such as ClinVar or an internal database. Nykamp et al. (5) state that null variants failing to meet these criteria can still be considered evidence toward pathogenicity, but the value of this evidence is lower.

PS1 Generally, when a missense variant is known to be pathogenic, a different mutation that results in the same amino acid change can also be assumed to be pathogenic. However, one must ensure that the mechanism of pathogenicity occurs through altered protein function, rather than splice site disruption. 

PS2 A de novo variant is considered strong evidence for pathogenicity provided that the patient has a family history of disease that is consistent with de novo inheritance and the patient’s phenotype matches the gene’s disease association. The original guidelines include a requirement to prove paternity/maternity before these criteria can be applied. If family relationships are assumed, but not confirmed, it is recommended that PM6 be used instead. However, other authors have chosen to remove this requirement because the likelihood of undisclosed nonmaternity is relatively low (14).

PS3 Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product is considered strong evidence of pathogenicity.  Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established and reliable assays.

PS4 A significant increase in a variant’s prevalence in affected individuals relative to the prevalence in controls is considered strong evidence for pathogenicity. In the case of rare variants for which case-control studies do not demonstrate statistical significance, the previous observation of the variant in multiple unrelated patients with the same phenotype, along with its absence in controls, can be considered moderate evidence of pathogenicity.

PM3 This criterion is applied to all variants observed in trans with a pathogenic variant, which is another variant in the individual on a different haplotype for the associated recessive gene. Nykamp et al. (5) recommend upgrading this evidence to ‘strong’ if there are multiple observations of the variant in trans with other pathogenic variants.

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