Table 3.

Criteria for Classifying Pathogenic Variants

Very strong evidence of pathogenicity
PVS1	Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease
	Caveats: Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7) Use caution interpreting LOF variants at the extreme 3’ end of a gene Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact Use caution in the presence of multiple transcripts
Strong evidence of pathogenicity
PS1	Same amino acid change as a previously established pathogenic variant regardless of nucleotide change
	Example:	Val->Leu caused by either G>C or G>T in the same codon
	Caveat:	Beware of changes that impact splicing rather than at the amino acid/protein level
PS2	De novo (both maternity and paternity confirmed) in a patient with the disease and no family history
	Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non- maternity
PS3	Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product
	Note: 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
PS4	The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls
	Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance.
	Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.
Moderate evidence of pathogenicity
PM1	Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation
PM2	Absent from controls (or at extremely low frequency if recessive) (see Table 6) in Exome Sequencing Project, 1000 Genomes or ExAC
	Caveat: Population data for indels may be poorly called by next generation sequencing
PM3	For recessive disorders, detected in trans with a pathogenic variant
	Note: This requires testing of parents (or offspring) to determine phase
PM4	Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants
PM5	Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before
	Example: Arg156His is pathogenic; now you observe Arg156Cys
	Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level
PM6	Assumed de novo, but without confirmation of paternity and maternity
Supporting evidence of pathogenicity
PP1	Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease
	Note: May be used as stronger evidence with increasing segregation data
PP2	Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease
PP3	Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc)
	Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.
PP4	Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology
PP5	Reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation