Prerequisites: Basic Linux CLI & Sequencing Formats
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Chapter 9: Step 7 — The ACMG/AMP 2015 Masterclass: All 28 Criteria Deconstructed
The 2015 American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) consensus guidelines established the global gold standard for constitutional sequence variant classification. In this masterclass chapter, we deconstruct all 28 individual criteria across their standardized strength levels.
The 28 Evidence Criteria Architecture
The ACMG/AMP framework categorizes evidence into Pathogenic (P) and Benign (B) categories, each sub-divided by weight:
PVS1: Null variant (nonsense, frameshift, canonical ±1,2 splice site, initiation codon, single/multi-exon deletion) in a gene where Loss-of-Function (LoF) is a definitively established disease mechanism. ClinGen Caveats: Must verify the variant triggers Nonsense-Mediated Decay (NMD) and is not located in the final 50 bp of the penultimate exon or within the final exon.
STRONG PATHOGENIC (PS1 – PS4)
PS1 (Same Amino Acid): Same amino acid change as a previously established pathogenic variant regardless of nucleotide change (e.g. c.1799T>A vs c.1799_1800delTGinsAA both producing BRAF p.Val600Glu).
PS2 (De Novo Confirmed): De novo mutation confirmed with both biological maternity and paternity established in a patient with no family history.
PS3 (Functional In Vitro/In Vivo Assay): Well-established in vitro or in vivo functional studies supportive of a damaging effect on protein function, validated against known pathogenic and benign controls.
PS4 (Prevalence in Cases vs Controls): The prevalence of the variant in affected individuals is statistically significantly increased compared to healthy population controls (odds ratio > 5.0, confidence interval excluding 1.0).
MODERATE PATHOGENIC (PM1 – PM6)
PM1 (Mutational Hotspot): Located in a well-characterized mutational hotspot and/or critical functional domain (e.g. catalytic active site, zinc finger) devoid of benign variation.
PM2 (Absent/Ultra-Rare in Controls): Absent from controls (or extremely low frequency $\le 0.001\%$ in gnomAD). Note: ClinGen updated PM2 to Supporting strength (PM2_Supporting) in 2020.
PM3 (In Trans with Pathogenic): For recessive disorders, detected in trans (on the opposite chromosome) with a confirmed pathogenic variant.
PM4 (In-Frame Indel): Protein length changes resulting from an in-frame insertion or deletion in a non-repeat region.
PM5 (Novel Missense at Known Pathogenic Site): Novel missense change at an amino acid residue where a different missense change has been established as pathogenic.
PM6 (Assumed De Novo): Assumed de novo mutation, but without genetic confirmation of biological paternity and maternity.
SUPPORTING PATHOGENIC (PP1 – PP5)
PP1 (Co-Segregation): Co-segregation with disease in multiple affected family members across a pedigree.
PP2 (Missense in Low-Tolerance Gene): Missense variant in a gene that has a low rate of benign missense variation and where missenses are a common mechanism of disease.
PP3 (In Silico Damaging): Multiple independent computational predictors (CADD, REVEL $\ge 0.75$, AlphaMissense) support a deleterious effect.
PP4 (Phenotype Specificity): Patient's clinical phenotype or family history is highly specific for a single genetic disorder.
PP5 (Reputable Source Pathogenic): Reputable laboratory reports variant as pathogenic, but raw primary evidence is unavailable for independent evaluation (ClinVar 2+ stars).
2. Benign Evidence Criteria Breakdown
STAND-ALONE & STRONG BENIGN (BA1 & BS1 – BS4)
BA1 (Stand-Alone Benign): Allele frequency $> 5\%$ in gnomAD across any major population. Automatically and immediately classifies the variant as BENIGN regardless of other criteria.
BS1 (Frequency Greater Than Expected): Allele frequency is greater than expected for the disease prevalence and penetrance.
BS2 (Observed in Healthy Adult): Observed in a healthy adult for a fully penetrant childhood-onset dominant condition.
BS3 (Functional Assay Non-Damaging): Well-established functional assays show no damaging effect on protein activity or splicing.
BS4 (Lack of Segregation): Non-segregation of the variant with the disease phenotype in affected family members.
SUPPORTING BENIGN (BP1 – BP7)
BP1: Missense variant in a gene where only Loss-of-Function truncations cause disease.
BP2: Observed in trans with a pathogenic variant in a dominant disorder, or in cis with another pathogenic variant.
BP3: In-frame insertion/deletion in a repetitive region without known functional role.
Java Heap Exhaustion (GATK / Picard): If processes terminate with java.lang.OutOfMemoryError, set your environment variable explicitly: export _JAVA_OPTIONS="-Xmx16g -Djava.io.tmpdir=/tmp".
Index Timestamp Mismatch: If tools complain that a BAM index is missing or out of date, regenerate with samtools index -@ 4 sample.bam.
Conda Channel Conflicts: Avoid ABI incompatibilities between bioconda and conda-forge by setting strict priority: conda config --set channel_priority strict.
Chromosome Naming Contamination: Always verify whether reference FASTA headers use chr1 or 1; divergent naming causes silent alignment drops.
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