Table 2.
ACMG/AMP criteria used for polyadenylation signal hexamer variant pathogenicity classification (see Supplementary Information for full explanations).
| Criteria | Criteria Description | Modification |
|---|---|---|
| Pathogenic Criteria | ||
| PVS1 | Loss of function allele in a gene where loss of function is a known mechanism of disease. | Not Applicable |
| PS1 | Same amino acid change as a previously established pathogenic variant regardless of nucleotide change | Not Applicable |
| PS2/PM6 |
De novo occurrence in an individual with disease and no family history. Each proven de novo case, count for 2 points, each assumed de novo case, count for 1 point. Very Strong: ≥8 points Strong: 4–7 points Moderate: 2–3 points Supporting: 1 point |
Nonea |
| PS3 | Well-established functional studies supportive of a damaging effect on gene or gene product. Moderate: For genes where loss of function is a known mechanism of disease, decreased RNA or protein levels (<75% of WT) in three or more cell lines from unrelated individuals who harbor that variant Supporting: For genes where loss of function is a known mechanism of disease, decreased RNA or protein levels (<75% of WT) in cells from affected individual who harbors the variant |
Variant type Specific |
| PS4 | The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls. If specifications have been provided by a ClinGen expert panel, case counting should consider their recommendation for strength. Popmax MAF in gnomAD should be <0.0006 to use these guidelines. For odds ratio calculations gnomAD can be used as a control set. Strong: ≥7 unrelated cases with associated condition. For variants with ≥7 unrelated cases an odds ratio can be calculated to determine strength level, an odds ratio ≥18.3 allows for PS4 to be used at strong. Moderate: 2–6 unrelated cases with associated condition. For variants with 2–6 unrelated cases an odds ratio can be calculated to determine strength level, an odds ratio ≥4.8 allows for PS4 to be used at moderate. Supporting: One case with associated condition. |
Strengthb |
| PM1 | Located in a mutational hot spot and/or critical and well-established functional domain. Downgraded to avoid overcounting with PP3. Supporting: Use for variants in predominant hexamers. |
Variant type Specific |
| PM2 | Absent from controls (gnomAD). Incorporated into PS4, do not consider separately unless ClinGen expert panel specifications for PS4 are used and PM2 is incorporated into those specifications. |
Not Applicable |
| PM3 | Variants in trans with pathogenic variant for recessive disorders. An individual cannot be counted for both PM3 and PS4. Example weighting below, see ClinGen Sequence Variant Interpretation Recommendation for in trans Criterion for complete explanation. Moderate: Identified with pathogenic variant in trans, phase known OR identified in homozygous state in two unrelated affected individuals. Supporting: Identified homozygous state in an affected individual. |
Nonea |
| PM4 | Protein length change. | Not Applicable |
| PM5 | Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Consider novel changes in hexamer sequence. Supporting: Single nucleotide variant in a hexamer where a different single nucleotide variant was previously determined to be likely pathogenic. New hexamers must be in lower functional group as predicted by Sheets et alc Previously established likely pathogenic variant must reach a classification of pathogenicity without PM5. |
Variant type Specific |
| PP1 | Co-segregation with disease in multiple affected family members. Strong: Co-segregation with disease in ≥7 reported meioses Moderate: Co-segregation with disease in 5–6 reported meioses Supporting: Co-segregation with disease in 3–4 reported meioses |
Noned |
| PP2 | Missense variant in gene with low rate of benign missense variants | Not Applicable |
| PP3 | Computational evidence suggests impact on gene or gene product. Supporting: CADD score of ≥10. |
Variant type Specific |
| PP4 | Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology. | Nonee |
| Benign Criteria | ||
| BA1 | Allele frequency is >0.05 in any general continental population dataset of at least 2000 observed alleles and found in a gene without a gene- or variant-specific BA1 modification. If specifications have been provided by an expert panel BA1 should be determined as set by the expert panel for the gene. |
Nonee |
| BS1 | Popmax allele frequency greater than expected for the disorder. If specifications have been provided by an expert panel BS1 should be determined as set by the expert panel for the gene. |
Nonee |
| BS2 | Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder with full penetrance expected at an early age. | Nonee |
| BS3 | Well-established functional studies show no damaging effect on protein function Moderate: No reduction in RNA or protein level in three or more cell lines from unrelated individuals who harbor the variant. Supporting: No reduction in RNA or protein level in cells from an individual who harbors the variant. |
Variant type Specific |
| BS4 | Lack of segregation in family members. | Nonee |
| BP1 | Missense variant in a gene for which loss of function is known mechanism of disease. | Not Applicable |
| BP2 | Observed in cis with a pathogenic variant in any inheritance pattern. | Nonee |
| BP3 | In-frame deletions/insertions in a repetitive region without a known function. | Not Applicable |
| BP4 | Computational evidence suggests no impact on gene or gene product. Supporting: CADD score of <5.0. |
Variant type Specific |
| BP5 | Variant found in a case with an alternate molecular basis for disease. | Nonee |
| BP7 | A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved | Not Applicable |
Criteria that have been modified for polyA variants are noted in the modification column. For criteria included in the original ACMG/AMP framework but not used in these specified criteria, the row is shown in gray.
bJohnston et al. [34].
cSee Sheets et al. Table 3.
dKelly et al. [33].
eRichards et al. [18].