ACMG/AMP 2015 Variant Classification — NGSDiag V1 Guide
This guide implements the ACMG/AMP 2015 guidelines (Richards et al., Genetics in Medicine, 2015) as adapted by the French NGSDiag network (V1). Variants are classified into 5 categories: Pathogenic (P), Likely Pathogenic (LP), Variant of Uncertain Significance (VUS), Likely Benign (LB), Benign (B), by combining weighted evidence criteria.
Very Strong Pathogenic Criterion
PVS1 — Loss-of-function variant (nonsense, frameshift, canonical ±1,2 splice site, initiation codon, exon deletion) in a gene where loss-of-function is a known disease mechanism. NMD must be predicted and the exon constitutive.
Strong Pathogenic Criteria
PS1 — Same amino acid change as a previously established pathogenic missense variant (confirmed in ClinVar or literature).
PS2 — De novo variant (confirmed by parental testing with biological parentage confirmed) in a patient with the disease and no family history.
PS3 — Well-established in vitro or in vivo functional studies showing a deleterious effect on the gene or gene product.
PS4 — Prevalence of the variant significantly increased in affected individuals compared to controls (OR > 3 in case-control study).
Moderate Pathogenic Criteria
PM1 — Located in a mutational hotspot or well-established functional domain (active site, binding domain) without benign variation.
PM2 — Absent from controls or at extremely low frequency in gnomAD and other large population databases.
PM3 — For recessive disorders: detected in trans with a pathogenic variant (phase confirmed by parental analysis).
PM4 — Protein length changes due to in-frame deletions/insertions or stop-loss variants that may affect protein function.
PM5 — Novel missense change at an amino acid position where a different pathogenic missense has been reported.
PM6 — Assumed de novo variant, but without confirmation of parentage (clinically de novo, full penetrance expected).
Supporting Pathogenic Criteria
PP1 — Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease (LOD ≥ 1).
PP2 — Missense variant in a gene where missense variants are a common cause of disease AND benign missense variation is rare.
PP3 — Multiple lines of in silico evidence support a deleterious effect (SIFT, PolyPhen-2, REVEL, CADD, AlphaMissense). Do not combine with PVS1.
PP4 — Patient phenotype or family history is highly specific for a disease with a single genetic etiology (gene-disease match).
PP5 — Reputable source recently classifies variant as pathogenic (use with caution).
Stand-Alone Benign Criterion
BA1 — Allele frequency >5% in large population databases (gnomAD). Alone sufficient for Benign classification.
Strong Benign Criteria
BS1 — Allele frequency greater than expected for the disorder (incompatible with disease prevalence).
BS2 — Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder with full penetrance.
BS3 — Well-established in vitro or in vivo functional studies show no deleterious effect on protein function or splicing.
BS4 — Lack of segregation in affected members of a family (variant absent in multiple affected relatives).
Supporting Benign Criteria
BP1 — Missense variant in a gene where only truncating variants (LOF) cause disease.
BP2 — Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or in cis with an established pathogenic variant.
BP3 — In-frame deletions/insertions in a repetitive region without a known function.
BP4 — Multiple lines of in silico evidence suggest no impact on gene or gene product (SIFT tolerated, PolyPhen benign, low REVEL). Incompatible with PP3.
BP5 — Variant found in a case with an alternate molecular basis for disease already identified.
BP6 — Reputable source recently classifies variant as benign (use with caution).
BP7 — A synonymous variant for which splicing prediction algorithms predict no impact and the nucleotide is not conserved.
Classification Rules
Pathogenic: 1 PVS + 1 PS | 1 PVS + 2 PM | 1 PVS + 1 PM + 1 PP | 2 PS | 1 PS + 3 PM | 1 PS + 2 PM + 2 PP | 1 PS + 1 PM + 4 PP
Likely Pathogenic: 1 PVS alone | 1 PVS + 1 PM | 1 PS + 1–2 PM | 1 PS + 2 PP | 3 PM | 2 PM + 2 PP | 1 PM + 4 PP
Benign: 1 BA1 | 2 BS
Likely Benign: 1 BS + 1 BP | 2 BP
VUS: when no combination above is met, or conflicting evidence.