Table 4.

Summary of the Main Lines of Evidence Used to Assess the Pathogenicity of Genetic Variants as Described by the ACMG/AMP Variant Interpretation Guidelines, Including Their Specific Application to Variants Detected in HCM Patients, Alongside Current Criteria‐Specific Issues and Ongoing/Future Developments for a More Refined Application

ACMG/AMP Rule/Evidence Class	Application to HCM	Issues and Future Developments
PVS1—null/truncating variant in gene with loss‐of‐function mechanism for disease.	Applied to truncating variants in MYBPC3, detected in ≈10% of HCM patients.	Some variants may not lead to nonsense‐mediated decay and haploinsufficiency.95 Noncoding variants that may lead to a truncated transcript (eg, splicing variants) can be difficult to detect and interpret.
PS1/PM5—same amino acid change/change at same residue as an established pathogenic variant	Numerous established pathogenic HCM variants and many examples of different variants affecting same residues.	Difficult to unambiguously define what is an established pathogenic variant. Curation of ClinVar entries is ongoing and will create a high confidence set of the most common pathogenic variants.
PS3—proven deleterious effect with functional studies.	Animal or cell‐based studies can be used to assess the phenotypic effect of a variant detected in a patient.	Uncertainty about the translatability of evidence from in vitro or in vivo models to the clinical setting. Currently impractical for regular application in a clinical genetic setting. High throughput assays with demonstrated translatability for known pathogenic and benign variants could produce a valuable database for all possible single nucleotide variants in sarcomeric genes.
PS4—variant is significantly enriched in cases compared with controls.	There are numerous founder and recurrent pathogenic HCM variants that are observed in multiple HCM probands/families. Comparison with control or population data sets can identify significantly enriched variants.	Guidelines for MYH7 variants suggest presence in distinct numbers of HCM probands for strong (≥15), moderate (≥6) or supporting (≥2) evidence.100 However, this does not demonstrate statistically significant enrichment in cases, for which large case and control/population data sets are required. The threshold for defining a significant enrichment and methods for dealing with multiple testing in cohort studies need to be addressed. Most currently available data sets are derived from European ancestry populations that are unlikely to include recurrent variants from other population groups.
PM1/PP2—relative frequency of variants in cases and controls for genes or gene regions.	All sarcomeric genes enriched for rare variants in HCM, with several mutation hotspots, eg, MYH7 head domain.	Current rules are ambiguously defined and not based on quantitative measures. Methods now developed based on case‐control analysis and definition of enriched clusters provide a quantitative approach, with evidence strength dependent on level of enrichment in cases.96
PM2—variant is rare enough in the population to be plausibly pathogenic (also BA1, BS1, BS2).	Population frequency data from gnomAD and disease‐specific threshold based on HCM characteristics provide stringent variant rarity threshold.97	Low penetrance variants or modifiers may be less rare in the population than standard pathogenic variants, requiring evidence from other rules to achieve (likely) pathogenic classification. Some population groups are still not well represented in databases like gnomAD (eg, North Africa and West Asia), variants from patients from these groups need to be analyzed with caution.
PP1—segregation of variant with disease in family pedigrees.	Segregation evidence is available for many HCM‐causing variants (in literature and ClinVar). Strength applied to evidence depends on the number of informative meioses.94, 100	As many HCM variants are private or detected in small family pedigrees, this evidence class will not be informative for a large proportion of variants. Incomplete penetrance in HCM means phenotype‐negative variant carriers are uninformative for pedigree analysis. Data for this evidence can be difficult to derive from literature and ClinVar.
PP3—computational evidence to support a deleterious effect.	As with most other Mendelian diseases, predictions from several different algorithms are used to provide supportive evidence for pathogenicity.	Algorithms lack specificity and therefore provide limited supportive evidence. Consensus findings from several orthogonal techniques should be used but there are numerous available algorithms and little agreement on the most appropriate subset of algorithms to use. Algorithms are generically applied to all genes but may not be equally effective.

ACMG indicates American College of Medical Genetics; AMP, Association of Molecular Pathologists; and HCM, indicates hypertrophic cardiomyopathy.