Documentation / Classification / Criteria Reference
Criteria Reference
Complete reference for all 28 ACMG evidence criteria. 19 are evaluated automatically, 9 require manual curation, and 1 (PM5) is currently disabled.
Pathogenic Evidence (16 criteria)
Null variant in gene where loss-of-function is a known disease mechanism
Conditions
Impact = HIGH
Consequence: frameshift, stop_gained, splice_acceptor, or splice_donor (canonical +/-1,2 only)
Gene constraint: pLI > 0.9 OR LOEUF < 0.35 OR gene in curated AR LoF list (~150 genes)
Disease association gate: gene must have established disease association (ClinVar P/LP 2+ stars, Orphanet, ClinGen HI, AR_LOF_GENES, or VCEP)
Last-exon downgrade (v3.9.0): truncating variants in last exon receive PVS1_Strong (+4 pts) instead of PVS1 Very Strong (+8 pts)
Exclusions
NMD-escaping transcripts (NMD_escaping_variant). Note: NMD_transcript_variant is NOT excluded (v3.11.3)
Non-canonical splice: splice_donor_5th_base_variant, splice_donor_region_variant, splice_acceptor_5th_base_variant (v3.11.4)
Stop-retained and stop-lost variants
HLA gene family
Homozygous reference genotypes (hom_ref) from multi-allelic VCF sites
Databases: VEP (consequence, impact, exon_number), gnomAD Constraint (pLI, LOEUF), curated AR LoF gene list, Orphanet, ClinVar (gene-level), ClinGen (HI score), VCEP
Does not evaluate reading frame rescue or alternative transcript usage
VCEP gene-specific PVS1 applicability gate available for ~50-60 genes
Same amino acid change as an established pathogenic variant
Conditions
ClinVar: Pathogenic or Likely Pathogenic
ClinVar review stars >= 2
Databases: ClinVar
Matches exact variant, not amino acid position (PM5 matching is disabled)
De novo variant (confirmed paternity and maternity)
Requires trio sequencing data and confirmed parental relationships.
Functional studies show a deleterious effect
Requires curation of published functional assay data.
Prevalence significantly increased in affected individuals
Requires case-control study data or odds ratios.
Located in a functional domain
Conditions
Variant overlaps a curated critical functional domain from CRITICAL_PFAM_DOMAINS list (~60 domains with documented pathogenic variant clustering)
Databases: VEP (domains)
Only curated critical domains trigger PM1 (kinase catalytic, DNA-binding, ion channel pores, enzyme active sites, VCEP-documented domains)
Generic structural domains (Caveolin, coiled-coil, DUF) excluded
Absent from controls or at extremely low frequency
Conditions
gnomAD global AF < 0.0001 (0.01%) OR absent from gnomAD (NULL = never observed in ~800K individuals)
Databases: gnomAD v4.1
ClinGen SVI PM2_Supporting downgrade not implemented
VCEP gene-specific PM2 thresholds available when enabled
Detected in trans with a pathogenic variant for recessive disorders
Conditions
Compound heterozygote candidate flag = true
Inferred from genotype data without formal phasing
Protein length change in a non-repetitive region
Conditions
In-frame insertion or deletion
Located within a Pfam functional domain
Not in a repetitive or low-complexity region
Exclusions
HLA gene family
Databases: VEP (consequence, domains)
Novel missense at known pathogenic amino acid position
Pending standardized protein-level coordinate matching in ClinVar preprocessing.
Assumed de novo without confirmation
Requires family structure information not available in single-sample analysis.
Cosegregation with disease in multiple affected family members
Requires multi-generational pedigree data.
Missense in a gene with low rate of benign missense variation
Conditions
Missense variant
Gene constraint: pLI > 0.5 AND mis_z > 2.0 (missense constraint)
Databases: VEP (consequence), gnomAD Constraint (pLI, mis_z)
Computational evidence supports a deleterious effect
Conditions
Path A (Missense): BayesDel_noAF with ClinGen SVI calibrated thresholds -- PP3_Strong (>= 0.518, +4 pts), PP3_Moderate (0.290-0.517, +2 pts), PP3_Supporting (0.130-0.289, +1 pt)
PM1 + PP3 double-counting guard: when PM1 applies alongside PP3_Strong, PP3 is downgraded to PP3_Moderate (combined cap at Strong equivalent)
Path B (Splice): SpliceAI max_score >= 0.2 AND PVS1 does not apply (ClinGen SVI double-counting guard). Always Supporting strength.
Databases: dbNSFP 4.9c (BayesDel_noAF), SpliceAI (max_score)
BayesDel_noAF excludes allele frequency to avoid circular reasoning with PM2/BA1/BS1
Patient phenotype matches gene disease association
Conditions
>= 3 patient HPO terms match the gene HPO profile
OR >= 2 matches with highly specific gene (<= 5 total HPO associations)
Databases: HPO (gene-phenotype associations)
Requires patient HPO terms to be provided.
Reputable source reports variant as pathogenic
Conditions
ClinVar: Pathogenic or Likely Pathogenic
Review stars >= 1 AND < 2 (lower confidence than PS1)
Databases: ClinVar
ClinGen SVI recommended retiring PP5; retained for maximum sensitivity.
Benign Evidence (12 criteria)
Allele frequency above 5%
Conditions
gnomAD global AF > 0.05 (5%)
Databases: gnomAD v4.1
BA1 overrides ALL other evidence including ClinVar. VCEP gene-specific BA1 thresholds may be lower.
Allele frequency greater than expected for disorder
Conditions
8-level inheritance-aware cascade: VCEP > ClinGen HI=3 > Orphanet AD > Orphanet XLD > HPO AD > Orphanet AR > AR_LOF_GENES > Constraint-implied AD > Default AR
AD threshold: AF >= 0.1%
AR threshold: AF >= 5%
Upper bound: AF <= BA1 threshold
Databases: gnomAD v4.1, ClinGen (HI score), Orphanet (inheritance), HPO (HP:0000006), gnomAD Constraint (pLI, LOEUF), AR_LOF_GENES
VCEP gene-specific BS1 thresholds override generic logic when enabled
Observed in healthy adults for fully penetrant early-onset disorder
Conditions
gnomAD homozygote count > 15
Databases: gnomAD v4.1
Functional studies show no deleterious effect
Requires curation of published functional assay data.
Lack of segregation in affected family members
Requires family segregation data.
Missense in a gene where primarily truncating variants cause disease
Conditions
Missense variant
MODERATE impact
pLI < 0.1 (LoF-tolerant gene)
mis_z < 2.0 or absent (v3.6.8 missense constraint guard)
No ClinVar Pathogenic missense in same gene (v3.8.2 guard)
Databases: VEP, gnomAD Constraint (pLI, mis_z), ClinVar (session-local)
Observed in trans with pathogenic for fully penetrant dominant
Conditions
Compound heterozygote candidate
ClinGen haploinsufficiency score = 30 (dosage sensitivity unlikely)
Databases: ClinGen
In-frame indel in repetitive region without known function
Conditions
In-frame indel
Repetitive/low-complexity region OR not in any Pfam domain
Databases: VEP (consequence, domains)
Computational evidence suggests no impact
Conditions
BayesDel_noAF with ClinGen SVI calibrated thresholds -- BP4_Moderate (<= -0.361, -2 pts), BP4_Supporting (-0.360 to -0.181, -1 pt)
SpliceAI max_score must be < 0.1 (no predicted splice impact)
Databases: dbNSFP 4.9c (BayesDel_noAF), SpliceAI
Variant found in case with alternate molecular basis
Requires case-level information about alternative diagnoses.
Reputable source reports variant as benign
Conditions
ClinVar: Benign or Likely Benign
Review stars >= 1
Databases: ClinVar
ClinGen SVI recommended retiring BP6; retained for maximum sensitivity.
Synonymous variant with no predicted splice impact
Conditions
Synonymous variant
Not in splice region
SpliceAI max_score <= 0.1
Databases: VEP, SpliceAI
Conservation filter intentionally omitted per Walker et al. 2023 Table S13.