Table 1.

Summary of DICER1-specific specifications of the ACMG/AMP variant curation guidelines.

Original ACMG/AMP Evidence Codes		DICER1 Specifications
Criteria	Criteria Description
PVS1	Null variant in a gene where loss of function is a known mechanism of disease.	Follow SVI-approved decision tree (Figure S1) with DICER1 specific modifications: NMD cutoff: p.Pro1850 In-frame exon(s): 5,10, 15, 18, 22 Non-coding exon(s): 1 Final exon: 27 10% of protein = 193 amino acids No criteria applied for disruption of start codon p.M1
PS1	Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.	Other variant must be interpreted as pathogenic by the DICER1 VCEP. Likely pathogenic changes do not apply. Same amino acid change: must confirm there is no difference in splicing. Non-canonical intronic splicing variants at same nucleotide: should have equal or worse splicing impact. Caveat: do not apply PM1 (full strength) or PM5 is PS1 is applied.
PS2	De novo (proven or assumed) in a patient with disease and no family history.	Follow the point structure outlined in the manuscript and summarized in Table 3. PS2_Very Strong: ≥4 points PS2: ≥2 but less than 4 points PS2_Moderate: ≥1 but less than 2 points PS2_Supporting: ≥0.5 but less than 1 point
PS3	Well-established in vitro or in vivo functional studies supportive of a damaging effect.	PS3: RNA assay demonstrates splicing impact that is out-of-frame OR in-frame with ≥ 193 residues affected OR disrupting the RNase IIIb domain. (Downgrade to PS3_Moderate if PVS1_Strong is also met) PS3_Moderate: RNA assay demonstrates splicing impact that is in-frame and disrupts < 193 residues, leaving the RNase IIIb domain intact. PS3_Supporting: In vitro cleavage assay with positive and negative controls demonstrates severely reduced capacity to produce 5p and/or 3p miRNA from pre-miRNA. Caveat: Do not apply PS3 at any strength if PVS1 is applied at full strength.
PS4	The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls.	Follow the points structure outlined in the manuscript and summarized in Table 3. PS4: ≥4 points PS4_Moderate: 2–3.5 points PS4_Supporting: 1–1.5 points Caveats: Do not apply for variants that meet BA1 or BS1. Do not apply proband points for an individual who has another germline variant that could have reasonably contributed to the phenotype or whose tumor sequencing suggests sporadic tumorigenesis.
PM1	Located in a mutational hot spot and/or critical and well-established functional domain.	PM1: Putative missense variants at residues affecting RNase IIb domain metal ion-binding (p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, p.E1813). PM1_Supporting: Putative missense variants affecting other residues in the RNase IIIb domain (p.Y1682 – p.S1846). Caveat: The full strength rule cannot be applied with PS1 or PM5.
PM2	Absent/rare from controls in an ethnically-matched cohort population sample.	This rule code is only applicable at a supporting level. PM2_Supporting: Allele frequency <0.000005 across gnomAD (non-cancer) with no more than one allele in any subpopulation and at least 20x coverage.
PM3	For recessive disorders, detected in trans with a pathogenic variant.	N/A – DICER1 syndrome follows autosomal dominant inheritance
PM4	Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.	PM4: In-frame indels with a residue within the RNase IIIb domain (p.Y1682 – p.S1846). PM4_Supporting: In-frame indels outside of the RNase IIIb domain (p.Y1682 – p.S1846) and repeat regions (p.D606-p.D609; p.E1418-p.E1420; p.E1422-p.E1425).
PM5	Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.	Other variant must be interpreted as pathogenic by the DICER1 VCEP. Likely pathogenic changes do not apply. The variant under assessment should have an equal or worse Grantham score. MaxEntScan and SpliceAI should demonstrate no splicing impact. Caveat: Do not apply with PS1 or with full-strength PM1.
PM6	Assumed de novo, but without confirmation of paternity and maternity.	N/A – considered redundant after PS2 modifications
PP1	Co-segregation with disease in multiple affected family members.	Phenotype-positive individuals should have high, moderate, or low-specificity phenotypes (see Table 2). PP1_Strong: ≥7 meioses across ≥2 families PP1_Moderate: 5 or 6 meioses across ≥1 family PP1: 3 or 4 meioses across ≥1 family Caveats: Do not apply for variants that meet BA1 or BS1. Segregation with a single low-specificity phenotype across multiple individuals does not fulfill PP1.
PP2	Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.	N/A - While DICER1 does meet recommended cutoff for missense constraint z score of ≥3.09 established by the SVI (4.23 on gnomAD), the DICER1 VCEP recommends this rule not be used due to the presence of various missense variants throughout the gene that are clinically interpreted as benign (9) or likely benign (30) in ClinVar.
PP3	Multiple lines of computational evidence support a deleterious effect on the gene or gene product.	Missense variants: REVEL score ≥0.75 OR concordance of MaxEntScan and SpliceAI for prediction of splice impact. Splicing variants: Concordance of MaxEntScan and SpliceAI for prediction of splice impact. Caveat: Do not apply in combination with PVS1.
PP4	Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.	Tumor testing of a neoplasm within the DICER1 syndrome phenotypic spectrum in a proband with the germline variant under assessment reveals the following: A previously reported somatic second hit in a DICER1 hotspot codon (Table S1) AND Retention of the germline variant under assessment See Figure 1 for a PP4 application flowchart. Caveats: PP4 cannot be applied to germline curation of variants in the DICER1 hotspot codons (p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, or p.E1813). PP4 cannotbe applied if tumor testing reveals any additional DICER1 non-hotspot variant(s).
PP5	Reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation.	N/A per published SVI guidance
BA1	Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes, or ExAC.	Allele frequency >0.003 (0.3%) in gnomAD (non-cancer) subpopulations. Subpopulations must have >2,000 alleles tested and a minimum of 5 alleles present.
BS1	Allele frequency is greater than expected for disorder.	Allele frequency >0.0003 (0.03%) in gnomAD (non-cancer) subpopulations. Subpopulations must have >2,000 alleles tested and a minimum of 5 alleles present.
BS2	Observed in a healthy adult.	BS2: 40+ unrelated females from a single source have reached age 50 without a tumor diagnosis (ratio of BS2-eligible females to PS4-eligible probands must be ≥ 40:1) OR 2+ observations of homozygosity in healthy individuals OR 1+ observation(s) of homozygosity in a healthy individual with status confirmed by parental testing. BS2_Supporting: 10+ unrelated females from a single source have reached age 50 without a tumor diagnosis (ratio of BS2-eligible females to PS4-eligible probands must be ≥ 10:1) OR 2+ observations of homozygosity in individuals lacking clinical information.
BS3	Well-established in vitro or in vivo functional studies shows no damaging effect on protein function.	BS3: For intronic or synonymous variants, ≥2 observations of no splicing impact via RNA assay. BS3_Supporting: In vitro cleavage assay with positive and negative controls demonstrates retained ability to produce 5p and 3p miRNA from pre-miRNA.
BS4	Lack of segregation in affected members of a family.	Variant observed in at least one phenotype-positive (must be high- or moderate-specificity phenotype; see Table 2), genotype-negative 1st, 2nd, or 3rd degree relative(s) of the proband. This rule does not apply to phenotype-negative, genotype-positive family members.
BP1	Missense variant in gene where primarily truncating variants cause disease.	N/A – truncating variants account for only a portion of disease-causing variants
BP2	Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern.	Observed in trans with a pathogenic or likely pathogenic DICER1 variant (phase confirmed) in at least 1 individual OR observed in cis and/or phase unknown in at least 3 individuals, at least 2 of whom carry unique pathogenic/likely pathogenic DICER1 variants. This rule code can only be used to compare variants asserted as pathogenic by the ClinGen DICER1 VCEP. Homozygous cases are not relevant for BP2 and should instead contribute to BS2.
BP3	In-frame deletions/insertions in a repetitive region without a known function.	N/A
BP4	Multiple lines of computational evidence suggest no impact on gene or gene product.	Missense variants: REVEL score < 0.50 AND concordance of MaxEntScan and SpliceAI predicting no splice effects. Synonymous, intronic, and non-coding variants: Concordance of MaxEntScan and SpliceAI predicting no splice effects.
BP5	Variant found in a case with an alternate molecular basis for disease.	N/A - Given the broad spectrum of DICER1-related neoplasms and the general lack of evidence of other high-penetrance germline variants that could account for such neoplasms (except perhaps for some low-specificity phenotypes), this rule should not be used at this time.
BP6	Reputable source recently reports variant as benign but the evidence is not available to the laboratory to perform an independent evaluation.	N/A per published SVI guidance
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.	This rule applies to silent variants and intronic variants at or beyond +7 to −21 positions. For other intronic or non-coding variants, BP7 may be applied if the variant is the reference nucleotide in ≥1 primate and/or ≥4 mammalian species. Caveat: BP7 cannot be applied unless BP4 is also met.