The Significance of Unknown Significance: A Teachable Moment

Jeanette J. McCarthy, MPH, PhD; Bryce A. Mendelsohn, MD, PhD


July 05, 2016

Our Take

Any physician who orders genetic testing must understand the process of variant interpretation. This process is central to this lawsuit.

As an illustration, we will take the variant from this case and the information that would have been available at the time of testing and apply the guidelines set forth in 2015 by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology for variant classification.[4] These classification guidelines are based on a set of 28 criteria (16 pathogenic criteria and 12 benign criteria), representing both objective and subjective pieces of evidence, not all of which will apply to a given variant. These criteria are considered together in a weighted fashion to place a variant into one of five categories: pathogenic, likely pathogenic, VUS, likely benign, and benign. Some degree of professional experience and judgment must be used.

For this variant, none of the 12 benign criteria were met. The assessment of the 16 pathogenic criteria is shown in the Table.

Table. ACMG Guideline Criteria Applied to SCN1A Tyr413Asn and Epileptic Encephalopathy

ACMG Code ACMG Criterion Criterion Met? Notes
PM2 Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium (ExAC Browser) Yes This specific variant is not reported in any of the three control databases used today.
PP2 Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease Yes Missense is a known mechanism of Dravet syndrome. Nearly one half of ClinVar pathogenic variants in SCN1A are missense. However, there are also 401 possibly benign missense variants in SCN1A in the ExAC database.
PVS1 Null variant (ie, nonsense, frameshift, splice sites, initiation codon, exon deletion) in a gene where loss of function is a known mechanism of disease No It is not a null variant.
PM4 Protein length changes as a result of in-frame insertion/deletion in a nonrepeat region or stop-loss variants No It is not a protein length changing variant.
PM1 Located in a mutational hot spot and/or critical and well-established functional domain without benign variation Yes Mutation is located in protein region S6, which is known to form part of the pore of the ion channel.[5] Mutations are spread throughout the entire protein, with some clustering occurring in S6. However, benign variations are also found in this region (11 missense variants in this exon in ExAC database).
PS1 Same amino acid change as a previously established pathogenic variant, but a different nucleotide change No No overlapping pathogenic variants.
PM5 Novel missense change at an amino acid residue where a different missense change that was determined to be pathogenic has been seen before No No overlapping pathogenic variants.
PP3 Multiple lines of computational evidence support a deleterious effect on the gene or gene product Yes MutationTaster, Polyphen-2, and PhyloP support a deleterious effect.
PP5 Reputable source recently reports variant as pathogenic, but evidence is not available to the laboratory to perform an independent evaluation No This variant was not previously classified in ClinVar.
PS4 For rare variants, prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls Unknown Two unrelated patients with possibly the same phenotype reported. Absent in controls. Does this constitute "multiple"?
PP1 Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease No No familial cases are described in the literature.
PS3 Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product No No experimental studies are described in the literature.
PM3 For recessive disorders, detected in trans with a pathogenic variant NA Not suspected to be recessive.
PM6 Assumed to be de novo, but without confirmation of paternity and maternity Unknown Parents were not tested.
PS2 De novo (both maternity and paternity confirmed) in a patient with the disease and no family history Unknown Parents were not tested.
PP4 Patient's phenotype or family history is highly specific for a disease with a single genetic etiology No Several genes underlie epileptic encephalopathy.

ACMG = American College of Medical Genetics and Genomics; NA = not applicable


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