Understanding ACMG/AMP Guidelines: A Practical Approach

The Pre-Standardization Era and the Imperative for Change

To appreciate current framework rigor, one must understand the chaotic landscape necessitating its creation. Prior to 2015, clinical molecular genetics laboratories operated with autonomy often bordering on idiosyncrasy. Terminology describing genetic alterations—"mutation," "polymorphism," "variant," and "variant of unknown significance"—lacked standardized definitions. A "mutation" in one laboratory might imply definitive pathogenicity, while another might use the term simply denoting a change from reference sequence, regardless of clinical impact.

This semantic ambiguity created precarious environments for clinicians, who frequently received conflicting reports from different reference laboratories regarding identical genetic variants. Such discordance led to confusion in patient management, inconsistent genetic counseling, and in worst cases, inappropriate medical interventions or missed surveillance opportunities. The technological explosion of Next-Generation Sequencing (NGS) exacerbated these vulnerabilities. As the field transitioned from single-gene Sanger sequencing to massive parallel sequencing of gene panels, whole exomes (WES), and whole genomes (WGS), variant volumes requiring interpretation increased exponentially. A typical exome analysis yields approximately 80,000 to 100,000 variants per individual. The manual "artisan" approach was no longer scalable.

The 2015 ACMG/AMP Consensus: A Framework for Mendelian Disorders

In response to critical challenges, ACMG, AMP, and the College of American Pathologists (CAP) convened a joint workgroup to revise variant interpretation standards. The resulting 2015 publication by Richards et al. established the modern five-tier classification system now serving as the global standard for Mendelian disorders.

The core innovation was introducing 28 structured evidence criteria. Unlike previous guidelines relying on gestalt impressions, the 2015 framework deconstructed interpretation into discrete, evaluable components. These criteria were categorized by evidence type—population data, computational predictions, functional studies, segregation analysis, de novo status, and allelic data—and weighted by strength: Stand-alone (A), Very Strong (VS), Strong (S), Moderate (M), and Supporting (P). This structured approach allowed for a "combinatorial" method of classification. Surveys following guideline release indicated 97% of laboratories reporting approaches consistent with the ACMG-AMP framework.

The Rise of ClinGen and the Era of Specification

Recognizing that "one size does not fit all" has limitations in genetics' nuanced world, the Clinical Genome Resource (ClinGen) was established to provide expert consensus on rule application. A critical development in this post-2015 era has been formation of Variant Curation Expert Panels (VCEPs). These disease-specific expert groups specify ACMG/AMP criteria for specific gene-disease pairs. VCEPs define gene-specific allele frequency thresholds, validated functional assays, and critical protein domains. Furthermore, the ClinGen Sequence Variant Interpretation (SVI) Working Group has released general recommendations updating and refining original 2015 criteria. These updates—covering PVS1 decision trees, PM2 downgrading, and computational predictor calibration—effectively supersede the original 2015 paper text.

The Five-Tier Classification System Definitions

Understanding precise tier definitions is essential for accurate reporting and clinical communication. These definitions carry statistical weight and clinical implications:

The Bayesian Transformation

While 2015 guidelines provided a logic grid (e.g., "1 Strong + 2 Moderate = Pathogenic"), this system had limitations. It was rigid, unable to easily handle diverse evidence combinations, and mathematically opaque. In 2018, Tavtigian et al., working with ClinGen SVI, proposed a Bayesian framework mapping ACMG criteria to a point-based system. This framework has become the industry standard for automated and semi-automated variant classification.

The core concept is that each evidence strength level corresponds to Odds of Pathogenicity (OddsP). By converting these odds to logarithmic scale, we can assign additive "points" to each criterion.

This point system offers profound advantages for clinical laboratories. First, it resolves "conflicting" criteria mathematically. If a variant has PVS1 (+8 points) but also benign criterion BS1 (-4 points), the sum is +4, falling into VUS range (0-5 points). This prevents "contradiction error" inherent in grid system and accurately reflects uncertainty. Second, it allows granular "sub-tiering" of VUSs. A VUS with 5 points ("VUS-High" or "Hot VUS") might be prioritized for segregation analysis or research, whereas a VUS with 0 points is a "Cold VUS". Third, it allows expert panels to assign intermediate weights.

BA1 & BS1 Evolution

BA1 (Stand-alone Benign): Originally defined as allele frequency >5%. However, for many rare Mendelian disorders, this threshold is too conservative. VCEPs often lower this; for example, the Hearing Loss VCEP sets BA1 at >0.5% for autosomal recessive hearing loss genes.

BS1 (Benign Strong): Applied when allele frequency is greater than expected for the disorder but below BA1. Calculating the "maximum credible population allele frequency" (maximum tolerated allele frequency) is crucial here, using the Whiffin et al. calculator which considers disease prevalence, penetrance, and genetic heterogeneity.

The PM2 Downgrade

PM2 (Absent/Rare in Population): In 2015, absence in population databases was Moderate evidence. However, as gnomAD grew (now >140,000 exomes/genomes), it became clear that many rare variants are benign. Consequently, the ClinGen SVI recommendation is to downgrade PM2 to Supporting strength (PM2_Supporting). Absence of evidence is not strong evidence of pathogenicity.

PS4 (Case-Control)

PS4 applies when the prevalence of the variant in affected individuals is significantly increased compared to controls. This requires rigorous statistical power (Fisher's exact test). Note that small case series often fail to reach statistical significance for PS4, though they may qualify for PS4_Supporting.

Establishing LOF Mechanism

First, one must confirm that Loss of Function (LOF) is a known mechanism of disease for the gene. For genes where gain-of-function or dominant-negative effects are the primary mechanism (e.g., certain KCNQ1 variants), PVS1 cannot be applied.

Nonsense & Frameshift

The critical question is Nonsense Mediated Decay (NMD).

• NMD Predicted: Premature termination codon (PTC) is >50-55 bp upstream of the last exon-exon junction. Generally PVS1 (Strong/Very Strong).
• NMD Not Predicted: PTC in the last exon or last 50bp of penultimate exon. PVS1 may be downgraded to Strong or Moderate depending on whether the truncated region is critical for function.

Splicing Variants

Canonical splice sites (±1, 2) are generally PVS1. However, if the skipped exon is in-frame and not critical, the protein might retain function. In silico tools (SpliceAI, MaxEntScan) are critical here.

Initiation Codon Variants

Variants affecting the start codon (ATG) typically result in PVS1_Moderate or PVS1_Supporting, as alternative start sites may rescue protein expression.

PP3/BP4 Calibration

Computational predictions (PP3/BP4) have evolved from simple voting (SIFT/PolyPhen) to ensemble meta-predictors like REVEL. The ClinGen SVI recommends using calibrated scores. For example, a REVEL score >0.75 might be PP3_Moderate, while >0.93 might be PP3_Strong (Pejaver et al. 2022). Conversely, low scores support BP4.

PM1 vs PM5 Hotspots

PM1: Mutational hotspot or critical functional domain. Requires defined boundaries.
PM5: Novel missense change at an amino acid residue where a different missense change has been determined to be pathogenic. This is strong evidence that the residue is critical.

Functional Studies (PS3/BS3)

Functional assays are powerful but variable. Brnich et al. (2019) provide a framework for validating assays. Assays must have defined controls (known pathogenic and benign variants) and statistical thresholds. A well-validated assay can provide PS3_Strong or even PS3_Very_Strong evidence.

Segregation Analysis (PP1/BS4)

Segregation in affected family members increases probability of pathogenicity. Jarvik et al. provide a calculation for segregation evidence strength based on the number of meioses.

De Novo Occurrences (PS2/PM6)

PS2: De novo (both maternity and paternity confirmed). Strong evidence.
PM6: De novo (paternity not confirmed). Moderate evidence.

CAP Checklist (MOL.36118)

CAP accreditation requires laboratories to have a documented policy for variant classification. Reports must clearly state the classification and the criteria used.

Clinical Interpretation

The report should not just list the tier (e.g., "Pathogenic") but explain why. "This variant is classified as Pathogenic based on PVS1, PM2, and PP3..."

Duty to Recontact

As knowledge evolves, classifications change. Laboratories face the ethical and legal question of recontacting physicians when a VUS is reclassified to Pathogenic or Benign.