RUNX1 c.560C>T, p.(Ala187Val)

Reasoning

The variant is a missense change (p.Ala187Val) located within the RUNX1 Runt Homology Domain (RHD), defined by the MM-VCEP as amino acids 89-204. Ala187 is NOT one of the 13 hotspot residues (R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204), so PM1_strong does not apply; per the MM-VCEP RUNX1 v3.1 rule 'PM1_Supporting: Variant affecting one of the other AA residues 89-204 within the RHD', PM1 is met at Supporting strength.

Rule

PM1_Supporting: Variant affecting one of the other AA residues 89-204 within the RHD. (PM1_strong applies only to R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204.)

Evidence considered

• CSPEC (MM-VCEP RUNX1 v3.1) PM1 rule: PM1_strong for the 13 hotspot residues within the RHD (R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204); PM1_Supporting for any other AA residue 89-204 within the RHD.
• Variant normalization (prefetch.json / Mutalyzer): NM_001754.4:c.560C>T -> NP_001745.2:p.(Ala187Val); residue 187 falls within RHD range 89-204 and is not among the 13 listed hotspot residues.
• Myeloid-Malignancy-VCEP-RUNX1-Pilot-Results.xlsx consulted per index guidance to verify code-combination usage for PM1_supporting in the RUNX1 RHD; no residue-187 entries are present in the 969-row pilot file.

Sources

Reasoning

The RUNX1 MM-VCEP v3.1 specification applies PM2 at supporting strength: minor allele frequency <= 0.00005 with at least 2,000 alleles tested around the position and 20x coverage at the position, evaluated using the GrpMax FAF when available in gnomAD v4.1.0. The variant is completely absent (MAF = 0) from gnomAD v4.1 (GrpMax FAF therefore 0), gnomAD v2.1, and gnomAD-Canada v1.0, satisfying the <= 0.00005 threshold in every subpopulation. Datasets queried (gnomAD v4.1 exomes >700,000 individuals, gnomAD v2.1, gnomAD-Canada) far exceed the 2,000-allele requirement, and the ClinGen MM-VCEP expert-panel submission to ClinVar explicitly states the variant is 'completely absent from all population databases with at least 20x coverage for runx1', corroborating the coverage condition. PM2_supporting is met.

Rule

PM2_Supporting: Minor allele frequency <= 0.00005 with at least 2000 alleles tested around and 20x coverage at the position; evaluate using the GrpMax FAF when available in gnomAD v4.1.0 (RUNX1 MM-VCEP v3.1).

Evidence considered

• RUNX1 MM-VCEP v3.1 (cspec) PM2_Supporting rule: minor allele frequency <= 0.00005 with at least 2000 alleles tested around and 20x coverage at the position; panel recommends evaluating using the GrpMax FAF when available in gnomAD v4.1.0, otherwise requiring all subpopulations to meet the threshold.
• gnomAD v4.1 query for chr21-34859527-G-A (GRCh38) returned 'absent' (MAF = 0); absence means the GrpMax FAF is also 0, satisfying the <= 0.00005 threshold by a large margin.
• gnomAD v2.1 query for 21-36231824-G-A (GRCh37) returned 'absent' (MAF = 0).
• gnomAD-Canada v1.0 (HostSeq genomes) API query returned 'absent' (MAF = 0).
• ClinVar VCV002850045 submission SCV004449577 (Labcorp) evidence text: 'this variant is not present in population databases (gnomad no frequency)' - corroborating direct gnomAD absence observations; the submission's VUS classification itself was not used as evidence.
• ClinVar VCV002850045 submission SCV005205650 (ClinGen MM-VCEP expert panel) evidence text: 'it is completely absent from all population databases with at least 20x coverage for runx1 (pm2_supporting)' - expert-panel confirmation that the 20x coverage condition of the panel's own PM2_Supporting rule is satisfied.
• Only the supporting strength tier is available for PM2 in the RUNX1 MM-VCEP v3.1 specification (single PM2_Supporting strength rule), so PM2 is applied at supporting strength, not moderate.

Still missing

• The gnomAD query payloads (source_data) were empty; absence was recorded as search_status only, without per-position allele counts or numeric coverage statistics. Explicit per-position allele count and >= 20x coverage confirmation at chr21:34859527 would strengthen the audit trail, though the MM-VCEP ClinVar submission text confirms the 20x coverage condition.

Sources

Reasoning

Missense variant NM_001754.4:c.560C>T (p.Ala187Val) in RUNX1. Per the governing ClinGen Myeloid Malignancy VCEP RUNX1 specification v3.1 (cspec), PP3 for missense variants is met when REVEL score >= 0.88 OR SpliceAI >= 0.38. REVEL score is 0.977 (local REVEL v1.3 lookup), well above the 0.88 VCEP threshold, so PP3 is met at supporting strength via the missense in-silico path. SpliceAI max delta score is 0.001, far below the 0.38 splice-impact threshold, so the splicing sub-path does not contribute, but the OR condition is satisfied by REVEL alone. The variant is not a canonical splice site variant (no exclusion applies) and no RNA analysis is available (RNA-proven caveat does not apply). BayesDel (0.539) was not used because no verified published threshold is available to this pipeline.

Rule

PP3 (missense): REVEL >= 0.88 OR SpliceAI >= 0.38 — MM-VCEP RUNX1 specification v3.1 (cspec)

Evidence considered

• MM-VCEP RUNX1 specification v3.1 (cspec): PP3 for missense variants is met when REVEL score >= 0.88 or SpliceAI >= 0.38, including creation of cryptic novel splice sites; do not use for canonical splice site variants or for variants whose splicing effect is proven by RNA analysis.
• REVEL score for NM_001754.4:c.560C>T (p.Ala187Val) is 0.977 (local REVEL v1.3 lookup), which is >= the VCEP threshold of 0.88; PP3 is met via the missense in-silico path.
• SpliceAI max delta score is 0.001 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.001, DS_DL 0.0), below the 0.38 PP3 threshold, so no splice-impact contribution to PP3.
• Variant is a missense substitution in exon 6 of NM_001754.4 and is not a canonical splice site variant (canonical_splice_consensus = false), so the PP3 canonical-splice-site exclusion does not apply.
• BayesDel score 0.539458 was not used because no verified published calibration threshold with a named publication is available to this pipeline; treated as insufficiently calibrated for PP3/BP4.

Still missing

• BayesDel score 0.539458 could not be used as corroborating evidence because no verified published threshold with a named publication is available to this pipeline; PP3 is fully met via REVEL alone, so this gap does not change the outcome.
• No RNA assay data is available to confirm or refute the in silico predictions (relevant only to potential PVS1_variable(RNA)/BP7_variable(RNA) strength adjustments, not to PP3 itself).

Sources

Reasoning

PS1 requires the same amino acid change (p.Ala187Val) produced by a different nucleotide change to have been previously established as pathogenic (PS1) or likely pathogenic (PS1_Moderate) by the MM-VCEP. No such alternate-nucleotide variant was identified: the only ClinVar record for p.Ala187Val (VCV002850045, NM_001754.5:c.560C>T) is classified Uncertain Significance by both a clinical laboratory (Labcorp) and the ClinGen Myeloid Malignancy VCEP, and the automated same-residue candidate harvest (pm5_candidates.json) returned 0 same-residue candidates. In addition, MM-VCEP RUNX1 v3.1 release notes state a caveat not to use PS1 at any level if PM1 was applied; PM1_supporting is applied for this variant (Ala187 lies within the RHD AA 89-204), which independently bars PS1.

Rule

PS1: Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. PS1_Moderate: Same amino acid change as a previously established likely pathogenic variant regardless of nucleotide change. Caveat (v3.1): do not use PS1 at any level if PM1 was applied.

Evidence considered

• CSPEC (MM-VCEP RUNX1 v3.1) PS1 rule: same amino acid change as a previously established pathogenic variant (PS1, Strong) or likely pathogenic variant (PS1_Moderate) regardless of nucleotide change; the previously established variant must have been reviewed by the MM-VCEP and asserted pathogenic/likely pathogenic.
• CSPEC (MM-VCEP RUNX1 v3.1) release notes: caveat added not to use PM5/PS1 at any level if PM1 was applied.
• ClinVar variation 2850045: the sole ClinVar record for p.Ala187Val (c.560C>T) is Uncertain Significance (1 clinical laboratory) and Uncertain Significance by the ClinGen Myeloid Malignancy VCEP (expert panel) - no pathogenic/likely pathogenic precedent for this amino acid change.
• pm5_candidates.json: ClinVar harvest for same-residue variants at residue 187 (queries RUNX1[GENE] AND A187[VARNAME], AND Ala187[VARNAME], AND p.Ala187[All Fields]) found 0 same-residue candidates; harvest failed softly due to HTTP 429 rate limiting.
• Sherloc paper (PMID:28492532, only paper with full text) was scanned for 'Ala187', 'A187', 'c.560', '560C', '187Val' - 0 matches; no variant-specific content for this codon.

Still missing

• No alternate-nucleotide (non-c.560C>T) variant producing p.Ala187Val with an MM-VCEP-reviewed pathogenic/likely pathogenic assertion was identified; none is known in the collected evidence.
• Per MM-VCEP RUNX1 v3.1 caveat, PS1 cannot be applied at any strength when PM1 is applied (PM1_supporting applied here).

Sources

Reasoning

PM5 (Moderate) requires a different missense change at the same residue (Ala187) that has been determined pathogenic (or likely pathogenic for PM5_Supporting) by the MM-VCEP. No such comparator was identified: pm5_candidates.json reports 0 same-residue candidates (the ClinVar harvest was rate-limited with HTTP 429 and completed softly), and the MM-VCEP pilot file contains no residue-187 variants. The splicing caveat is satisfied (SpliceAI max delta 0.001 <= 0.20). However, MM-VCEP RUNX1 v3.1 explicitly states 'PM5 cannot be used if PM1 was applied at any strength level', and PM1_supporting is applied for this variant; this caveat independently bars PM5.

Rule

PM5: Missense change at an AA residue where a different missense change which has been determined to be pathogenic before (after accounting for Grantham scores). PM5_Supporting: ...determined to be likely pathogenic before. Caveat: PM5 cannot be used if PM1 was applied at any strength level.

Evidence considered

• CSPEC (MM-VCEP RUNX1 v3.1) PM5 rule: missense change at an AA residue where a different missense change has been determined pathogenic before (PM5, Moderate; >=2 such changes for PM5_Strong; likely pathogenic for PM5_Supporting), after accounting for Grantham scores; variant must not impact splicing (RNA assay or SpliceAI <= 0.20).
• CSPEC (MM-VCEP RUNX1 v3.1) caveat: PM5 cannot be used if PM1 was applied at any strength level.
• pm5_candidates.json: 4 candidates seen, 0 same-residue candidates, 0 expert-panel PLP candidates; harvest failed softly due to ClinVar HTTP 429 rate limiting, so no residue-187 comparator was collected.
• SpliceAI: max delta score 0.001 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.001, DS_DL 0.0), satisfying the PM5 splicing caveat (<= 0.20).
• Myeloid-Malignancy-VCEP-RUNX1-Pilot-Results.xlsx scanned for '187'/'A187'/'Ala187' - no same-residue comparator variants present.

Still missing

• Same-residue comparator harvest from ClinVar was incomplete (HTTP 429 rate limiting); a definitive survey of other missense changes at residue 187 (e.g., p.Ala187Thr/Asp) with MM-VCEP-reviewed pathogenicity is lacking.
• Per MM-VCEP RUNX1 v3.1 caveat, PM5 cannot be applied at any strength when PM1 is applied (PM1_supporting applied here).

Sources

Reasoning

PP5 is not for use per the MM-VCEP (ClinGen SVI VCEP Review Committee recommendation). Under the global PP5 policy, PP5 may be applied at supporting strength only when the exact variant has a ClinVar expert-panel classification of Pathogenic or Likely pathogenic. For this exact variant (ClinVar variation 2850045), the ClinGen Myeloid Malignancy VCEP - the only expert-panel submitter, reviewed by expert panel (3 stars) - classified it Uncertain Significance, not Pathogenic/Likely pathogenic. The other submission (Labcorp, single submitter, VUS) is an ordinary laboratory assertion that must never trigger PP5. The variant is also absent from the local MM-VCEP pilot results file (970 rows reviewed). The PP5 trigger condition is therefore not satisfied.

Rule

PP5: supporting when the exact variant has a ClinVar expert-panel (>=3 star) classification of Pathogenic/Likely pathogenic; MM-VCEP otherwise marks PP5 not for use

Evidence considered

• ClinVar variation 2850045: expert-panel submission SCV005205650 (ClinGen Myeloid Malignancy Variant Curation Expert Panel, 'reviewed by expert panel', 3 stars) classified the exact variant NM_001754.5(RUNX1):c.560C>T (p.Ala187Val) as Uncertain Significance; this is an expert-panel classification but it is not Pathogenic/Likely pathogenic, so PP5's trigger is not met
• ClinVar submission SCV004449577 (Labcorp Genetics formerly Invitae, criteria provided/single submitter, VUS) is an ordinary laboratory submission and per policy must never trigger PP5
• MM-VCEP RUNX1 v3.1 (cspec) PP5 entry: 'Not Applicable for this VCEP' - 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee'; global policy overrides this only when an expert-panel P/LP classification exists, which is not the case here
• Local MM-VCEP pilot results file (Myeloid-Malignancy-VCEP-RUNX1-Pilot-Results.xlsx, 970 rows) contains no row for c.560C>T / p.Ala187Val, so no VCEP precedent P/LP classification exists locally

Sources

Reasoning

The governing framework is the ClinGen Myeloid Malignancy VCEP RUNX1 specification v3.1 (cspec), which defines BA1 as MAF >= 0.0015 (0.15%) in any general continental population dataset with >= 2,000 alleles tested and variant present in >= 5 alleles. The variant NM_001754.4:c.560C>T (p.Ala187Val) was directly queried and is absent from gnomAD v2.1 (GRCh37 21-36231824-G-A), gnomAD v4.1 (GRCh38 chr21-34859527-G-A), and gnomAD-Canada v1.0 (HostSeq genomes); all three queries returned search_status 'absent' (MAF = 0). MAF 0 is far below the 0.0015 BA1 threshold, so BA1 is definitively not met.

Rule

BA1: Minor allele frequency >= 0.0015 (0.15%) in any general continental population dataset with >= 2,000 alleles tested and variant present in >= 5 alleles (RUNX1 MM-VCEP v3.1).

Evidence considered

• RUNX1 MM-VCEP v3.1 (cspec doc 135637580) BA1 rule: minor allele frequency >= 0.0015 (0.15%) in any general continental population dataset with >= 2,000 alleles tested and variant present in >= 5 alleles; threshold derived from the Whiffin/Ware calculator with FPD/AML prevalence 1 in 40, penetrance 85%, allelic heterogeneity 100%, maximum genetic heterogeneity 10%, 95% CI.
• Direct gnomAD v4.1 (exome) query for chr21-34859527-G-A (GRCh38) returned 'absent' (search_status 'absent'; found false); no alleles observed, MAF = 0.
• Direct gnomAD v2.1 (exome) query for 21-36231824-G-A (GRCh37) returned 'absent'; no alleles observed, MAF = 0.
• Direct gnomAD-Canada v1.0 (HostSeq genomes) API query for 21-34859527-G-A returned 'absent'; no alleles observed, MAF = 0.
• Absence in all three general population datasets (including gnomAD v4.1 with >700,000 exomes) makes MAF >= 0.0015 in any continental dataset impossible; BA1 not met regardless of population subgroup.

Sources

Reasoning

The RUNX1 MM-VCEP v3.1 specification defines BS1 as MAF between 0.00015 (0.015%) and 0.0015 (0.15%) in any general continental population dataset with >= 2,000 alleles tested and variant present in >= 5 alleles. The variant is absent (MAF = 0) from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, which is below the lower bound of the BS1 band (0.00015). BS1 is therefore not met.

Rule

BS1: Minor allele frequency between 0.00015 (0.015%) and 0.0015 (0.15%) in any general continental population dataset with >= 2,000 alleles tested and variant present in >= 5 alleles (RUNX1 MM-VCEP v3.1).

Evidence considered

• RUNX1 MM-VCEP v3.1 (cspec) BS1 rule: minor allele frequency between 0.00015 (0.015%) and 0.0015 (0.15%) in any general continental population dataset with >= 2,000 alleles tested and variant present in >= 5 alleles; the panel notes BS1 alone can support likely benign only within this range and absent contradictory evidence.
• Variant absent (MAF = 0) in gnomAD v4.1, gnomAD v2.1, and gnomAD-Canada v1.0 direct queries; MAF 0 is below the 0.00015 lower bound of the BS1 range, so the BS1 band is not entered.
• No population dataset queried reports any allele for c.560C>T, so the >= 5 allele requirement and the frequency band both fail.

Sources

Reasoning

The MM-VCEP RUNX1 v3.1 specification keeps BP2 applicable (Benign Supporting): a variant observed in trans with a pathogenic variant for this fully penetrant dominant disorder, observed in cis with a pathogenic variant, or observed in the homozygous state in individuals without FPD/AML phenotype can be considered supporting benign evidence. No such observation exists for NM_001754.4:c.560C>T (p.Ala187Val): the variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada v1.0 (no homozygotes); the ClinVar record (VCV002850045, 2 germline submissions) provides no parental/phase testing and no report of a second RUNX1 variant in trans or cis; and no publication in the case bundle reports this variant (the only available full text, PMID:28492532/Sherloc, is a criteria-refinement methodology paper with no RUNX1 variant content). BP2 therefore is not met.

Rule

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.

Evidence considered

• MM-VCEP RUNX1 v3.1 cspec BP2 note: "Biallelic pathogenic variants in RUNX1 have never been reported in FPD/AML patients. A variant in trans with a known pathogenic variant or observation of the variant in the homozygous state in individuals without FPD/AML phenotype can be considered supporting benign evidence."
• MM-VCEP RUNX1 v3.1 BP2 rule (Supporting): "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."
• gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0 all report this variant as absent; no homozygous (or any) allele observations exist in population data.
• ClinVar VCV002850045 contains two germline submissions (Labcorp, VUS, criteria provided single submitter; ClinGen Myeloid Malignancy VCEP, VUS, reviewed by expert panel with codes PM1_supporting and PM2_supporting); neither submission reports parental/phase information or a second RUNX1 variant in trans or cis.
• Full text of PMID:28492532 (Sherloc) was searched for c.560C>T / p.Ala187Val / RUNX1 case data and contains no mention of this variant or any allelic-configuration observations; no BP2 evidence can be drawn from the literature bundle.

Still missing

• No parental or phase (trans/cis) testing data for this variant is available in the case bundle; establishing a trans configuration with a pathogenic RUNX1 variant would require such data.
• No reports of homozygous observations of this variant in individuals without FPD/AML phenotype exist (consistent with complete absence from population databases).

Sources

Reasoning

Per the MM-VCEP RUNX1 specification v3.1 (cspec), BP4 for missense variants requires REVEL score < 0.50 AND SpliceAI <= 0.20. REVEL score is 0.977, which fails the REVEL < 0.50 condition; although SpliceAI (max delta 0.001) is <= 0.20, the AND conjunction is not satisfied, so BP4 is not met. The same REVEL value that meets PP3 (>= 0.88) is what fails BP4, so the two criteria are applied consistently without double-counting.

Rule

BP4 (missense): REVEL < 0.50 AND SpliceAI <= 0.20 — MM-VCEP RUNX1 specification v3.1 (cspec)

Evidence considered

• MM-VCEP RUNX1 specification v3.1 (cspec): BP4 for missense variants requires REVEL score < 0.50 AND SpliceAI <= 0.20; BP4 for synonymous and intronic variants requires SpliceAI <= 0.20.
• REVEL score is 0.977, which is NOT < 0.50; the REVEL condition of the missense BP4 rule fails.
• SpliceAI max delta score is 0.001 (<= 0.20), satisfying the splice component, but BP4 requires both conditions for missense variants, so BP4 is not met.
• BayesDel score 0.539458 was not used because no verified published calibration threshold with a named publication is available to this pipeline.

Still missing

• BayesDel score 0.539458 could not be considered because no verified published threshold with a named publication is available to this pipeline; even under a conventional damaging/benign split the score would not rescue the REVEL < 0.50 requirement, so this gap does not change the outcome.

Sources

Reasoning

BP6 is not for use per the MM-VCEP (ClinGen SVI VCEP Review Committee recommendation). Under the global BP6 policy, BP6 may be applied at supporting strength only when the exact variant has a ClinVar expert-panel classification of Benign or Likely benign. For this exact variant (ClinVar variation 2850045), the ClinGen Myeloid Malignancy VCEP - the only expert-panel submitter, reviewed by expert panel (3 stars) - classified it Uncertain Significance, not Benign/Likely benign. Neither the laboratory submission (Labcorp, VUS) nor any other collected source provides a benign expert-panel assertion. The BP6 trigger condition is therefore not satisfied.

Rule

BP6: supporting when the exact variant has a ClinVar expert-panel (>=3 star) classification of Benign/Likely benign; MM-VCEP otherwise marks BP6 not for use

Evidence considered

• ClinVar variation 2850045: expert-panel submission SCV005205650 (ClinGen Myeloid Malignancy Variant Curation Expert Panel, 'reviewed by expert panel', 3 stars) classified the exact variant as Uncertain Significance; this expert-panel classification is not Benign/Likely benign, so BP6's trigger is not met
• ClinVar submission SCV004449577 (Labcorp, single submitter, VUS) is an ordinary laboratory submission and per policy must never trigger BP6
• MM-VCEP RUNX1 v3.1 (cspec) BP6 entry: 'Not Applicable for this VCEP' - 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee'; global policy overrides this only when an expert-panel B/LB classification exists, which is not the case here

Sources

Reasoning

The governing ClinGen Myeloid Malignancy VCEP RUNX1 v3.1 specification (cspec) makes PS2 applicable and defines PS2_Supporting as 1 proven de novo occurrence (both maternity AND paternity confirmed) in a patient with FPD/AML phenotype, and PS2_Moderate as >=2 such occurrences, with paternity-only confirmation explicitly insufficient. No evidence of a de novo occurrence, parental testing, or absence of family history is present anywhere in the case evidence bundle. The ClinVar record (VCV002850045, reviewed by expert panel) contains two germline submissions (Labcorp SCV004449577; ClinGen MM-VCEP SCV005205650) that applied only PM1_supporting/PM2_supporting and PM2 respectively and reported no de novo or parental testing data. The literature pass contains no variant-specific case report of c.560C>T. Evidence is therefore insufficient to apply PS2 at any strength.

Evidence considered

• MM-VCEP RUNX1 v3.1 (cspec) PS2 specification: PS2_Supporting = 1 proven de novo occurrence (both maternity and paternity confirmed) in a patient with FPD/AML phenotype; PS2_Moderate = >=2 proven de novo occurrences; confirmation of paternity only is insufficient; no family history is defined as absence of the variant and any RUNX1-phenotypic criteria in first- and second-degree relatives.
• ClinVar record VCV002850045 (reviewed by expert panel, 3 stars) contains two germline submissions: Labcorp (SCV004449577, 2023, Uncertain significance) and ClinGen Myeloid Malignancy VCEP (SCV005205650, 2024, Uncertain Significance); neither submission reports a de novo occurrence or parental testing, and the applied criteria codes were PM1_supporting/PM2_supporting (expert panel) and PM2 (Labcorp).
• No proband-level parental testing or de novo occurrence data for NM_001754.4:c.560C>T exists in the case evidence bundle (prefetch, evidence, case summary, or literature pass).

Still missing

• No parental testing (maternity and paternity confirmation) data for the proband is available in the case evidence.
• No proven de novo occurrence of c.560C>T (p.Ala187Val) in a patient with FPD/AML phenotype has been reported in ClinVar or the literature pass.
• Family history status (absence of variant and RUNX1-phenotypic criteria in first- and second-degree relatives) is not documented.

Sources

Reasoning

The governing framework is the ClinGen Myeloid Malignancy VCEP (MM-VCEP) RUNX1 specification v3.1 (cspec doc 135637580, framework_mode=vcep), which keeps PS3 Applicable and defines it through transactivation assay evidence: PS3_Strong requires transactivation assays demonstrating altered transactivation (<20% of wt and/or reduced to levels similar to well-established pathogenic variants such as R201Q or R166Q) AND data from a secondary assay demonstrating altered function; PS3_Moderate requires altered transactivation OR >=2 secondary assays demonstrating altered function; PS3_Supporting requires enhanced transactivation (>115% of wt). The MM-VCEP assay instructions require transactivation assays to include wt and known pathogenic controls plus co-expression with CBF-beta (promoters M-CSFR, PF4, C-FMS, GZMB containing consensus RUNX1 binding sites TGTGGT). No variant-specific functional study of NM_001754.4:c.560C>T (p.Ala187Val) exists in the case evidence bundle: OncoKB explicitly reports 'OncoKB did not identify variant-specific reviewed functional evidence for this variant' (not_reviewed=true, no citations); ClinVar VCV002850045 lists the variant as VUS with the MM-VCEP expert-panel submission applying only PM1_supporting and PM2_supporting (no PS3); the only full-text paper (PMID:28492532, Sherloc) contains no mention of this variant and no RUNX1 functional data; PMID:33661592 (GeneReviews) is image-only with the variant not mentioned; COSMIC records a single somatic occurrence (n=1), which is not a functional assay; SpliceAI (max delta 0.001) and REVEL (0.977) are in-silico predictions, not well-established functional studies. Because no transactivation or secondary assay data for this variant was identified, PS3 cannot be met at any strength and the evidence is insufficient to assess it; per instructions, not_assessed is returned with the gaps identified. PVS1 does not apply (missense variant, not a null variant), so the PS3 'not applicable if variant meets PVS1' caveat does not block assessment.

Rule

PS3: Transactivation assays demonstrating altered transactivation (<20% of wt, and/or reduced to levels similar to well established pathogenic variants such as R201Q or R166Q) AND data from a secondary assay demonstrating altered function. Not applicable if variant meets PVS1. If variant meets PVS1_strong, upgrade to PVS1. PS3_Moderate: Transactivation assays demonstrating altered transactivation (<20% of wt and/or reduced to levels similar to well established pathogenic variants such as R201Q or R166Q) OR >= 2 secondary assays demonstrating altered function. PS3_Supporting: Transactivation assays demonstrating enhanced transactivation (>115% of wt).

Evidence considered

• MM-VCEP RUNX1 v3.1 cspec PS3 rule: "PS3: Transactivation assays demonstrating altered transactivation (<20% of wt, and/or reduced to levels similar to well established pathogenic variants such as R201Q or R166Q) AND data from a secondary assay demonstrating altered function. Not applicable if variant meets PVS1. If variant meets PVS1_strong, upgrade to PVS1."
• MM-VCEP RUNX1 v3.1 cspec PS3_Moderate rule: "Transactivation assays demonstrating altered transactivation (<20% of wt and/or reduced to levels similar to well established pathogenic variants such as R201Q or R166Q) OR >= 2 secondary assays demonstrating altered function."
• MM-VCEP RUNX1 v3.1 cspec PS3_Supporting rule: "Transactivation assays demonstrating enhanced transactivation (>115% of wt)."
• MM-VCEP RUNX1 v3.1 cspec PS3 assay guidance: transactivation assays using M-CSFR, PF4, C-FMS and GZMB promoters containing consensus RUNX1 binding sites TGTGGT, and "must include wt and known pathogenic controls, as well as co-expression with CBF-beta"; secondary assays include EMSA/yeast-hybrid (DNA binding), co-immunoprecipitation/FRET/affinity (CBF-beta heterodimerization), immunofluorescence and cell-fractionation with Western Blot (localization), colony-forming potential of sorted HSPCs, and xenotransplantation.
• OncoKB RUNX1 A187V page: "OncoKB did not identify variant-specific reviewed functional evidence for this variant; gene-level curated context is available for reviewer follow-up." Classification 'Unknown Oncogenic Effect', not_reviewed=true, no citations.
• ClinVar VCV002850045 (variation 2850045): variant classified VUS by Labcorp (SCV004449577, criteria provided single submitter) and VUS by the ClinGen Myeloid Malignancy VCEP (SCV005205650, reviewed by expert panel, criteria PM1_supporting and PM2_supporting); no functional evidence or PS3 code appears in either submission.
• Full text of PMID:28492532 (Sherloc) was searched for Ala187/A187/c.560C>T/transactivation/functional; no matches — this criteria-refinement methodology paper contains no variant-specific functional data.
• COSMIC records one somatic occurrence (COSV55882830, n=1) of this variant; a somatic cancer observation is not a functional assay and cannot substitute for transactivation/secondary assay evidence.
• SpliceAI (max delta 0.001) and REVEL (0.977) are in-silico predictions; per MM-VCEP instructions they are not well-established functional studies and do not address the PS3 transactivation/secondary assay requirements.
• Variant is a missense change (p.Ala187Val) not classified as a null variant by the PVS1 assessment, so the PS3 caveat 'not applicable if variant meets PVS1' does not apply.

Still missing

• No validated transactivation assay data for p.Ala187Val (per MM-VCEP: assay must include wt and known pathogenic controls and co-expression with CBF-beta, using promoters with consensus RUNX1 binding sites TGTGGT); altered transactivation (<20% of wt, or reduced to levels similar to R201Q/R166Q, or enhanced >115% of wt) cannot be demonstrated.
• No secondary assay data for p.Ala187Val (e.g., EMSA/yeast-hybrid for DNA binding, co-IP/FRET/affinity for CBF-beta heterodimerization, immunofluorescence/cell-fractionation for localization, colony-forming potential, xenotransplantation).
• No published functional study of p.Ala187Val was identified in the case evidence bundle; OncoKB has no variant-specific functional review for this variant and the literature bundle contains no functional-theme papers.

Sources

Reasoning

PS4 is applicable under the governing MM-VCEP RUNX1 v3.1 specification (quasi-case-control: PS4_Supporting = 1 proband meeting at least one RUNX1-phenotypic criterion, OR 31.8, with the variant absent from gnomAD or present once). The control-side condition is satisfied: c.560C>T is absent from gnomAD v2.1 and v4.1. However, no proband-level observation of this exact variant meeting RUNX1-phenotypic criteria is documented in any collected source. The exact-variant ClinVar record (2850045) contains a 3-star expert-panel submission (ClinGen Myeloid Malignancy VCEP) classified Uncertain Significance with codes PM1+PM2 only - no PS4 was applied by the panel, and the panel's final classification cannot itself be used as proband/case evidence. The only variant occurrence reported anywhere is a single somatic COSMIC entry (COSV55882830, n=1), which is not germline FPD/AML evidence and was excluded. PMID 28492532 (Sherloc, full text reviewed) contains no mention of this variant and no case data; PMID 33661592 (RUNX1 FPD/AML GeneReviews) is a gene-level review with no verifiable variant-specific content. The MM-VCEP notes that no published RUNX1 case-control study exists. With zero countable probands, PS4 cannot be assessed and no PS4 point can be awarded.

Rule

MM-VCEP RUNX1 v3.1: PS4 >= 4 probands meeting >=1 RUNX1-phenotypic criterion (OR 127.1); PS4_Moderate 2-3 probands (OR 63.5-95.3); PS4_Supporting 1 proband (OR 31.8); variant absent from gnomAD or present once

Evidence considered

• MM-VCEP RUNX1 v3.1 (cspec) PS4 rule: 'PS4: >= 4 probands meeting at least one of the _RUNX1_-phenotypic criteria (OR 127.1); PS4_Moderate: 2-3 probands (OR 63.5-95.3); PS4_Supporting: 1 proband (OR 31.8)'; MM-VCEP notes that no published RUNX1 case-control study exists and the code requires the proband to meet RUNX1-phenotypic criteria with the variant absent from gnomAD or present once
• ClinVar variation 2850045: expert-panel submission SCV005205650 (ClinGen Myeloid Malignancy VCEP, reviewed by expert panel, 2024-09-12) classified this exact variant Uncertain Significance and applied only PM1+PM2; laboratory submission SCV004449577 (Labcorp, single submitter, 2023-04-19) classified VUS applying only PM2; neither submission documents probands with RUNX1 phenotype carrying c.560C>T, and the panel's VUS classification is not usable as case evidence
• gnomAD v2.1 and gnomAD v4.1 (and gnomAD-Canada v1.0) are all absent for this variant (21-36231824-G-A / chr21-34859527-G-A), satisfying the quasi-case-control control-side condition but contributing zero proband count
• COSMIC reports one somatic cancer occurrence (COSV55882830, n=1); somatic tumor data does not constitute a germline proband meeting RUNX1-phenotypic criteria and was excluded from PS4
• PMID 28492532 (Sherloc, full text reviewed): variant-classification framework methods paper; no mention of c.560C>T, p.Ala187Val, or RUNX1 anywhere in the full text; provides no case or case-control data for PS4
• PMID 33661592 (RUNX1 FPD/AML GeneReviews): gene-level clinical review; no abstract snippet or full text in this bundle and no variant-specific content verifiable, so it cannot supply proband evidence

Still missing

• No documented probands meeting at least one RUNX1-phenotypic criterion (thrombocytopenia with normal platelet size/volume; platelet ultrastructural/functional defects; hematologic malignancy - AML/MDS most commonly) carrying c.560C>T
• No published case-control or cohort study reporting this exact variant in FPD/AML patients
• A single proband observation would be needed even for PS4_Supporting (OR 31.8) under the MM-VCEP rule

Sources

Reasoning

The MM-VCEP RUNX1 v3.1 specification (cspec) defines PM6_Supporting as 2 or 3 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with FPD/AML phenotype and PM6 as >=4 such occurrences. No assumed de novo occurrences of c.560C>T are reported in any source in the case evidence bundle: neither ClinVar submission (SCV004449577, SCV005205650) reports de novo status, and the literature pass contains no variant-specific case report. Evidence is insufficient to apply PM6 at any strength.

Evidence considered

• MM-VCEP RUNX1 v3.1 (cspec) PM6 specification: PM6_Supporting = 2 or 3 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with FPD/AML phenotype; PM6 = >=4 assumed de novo occurrences; PS2 and PM6 combined are capped at a maximum of one moderate or two supporting rules (1 point).
• ClinVar submissions for VCV002850045 (Labcorp SCV004449577; ClinGen MM-VCEP SCV005205650) report no de novo status or parental testing for c.560C>T.
• No variant-specific case reports describing assumed de novo occurrence of c.560C>T were identified in the literature pass (PMID:28492532 and PMID:33661592 contain no variant-specific data).

Still missing

• No assumed de novo occurrences (without parental confirmation) of c.560C>T in patients with FPD/AML phenotype have been reported.
• No parental testing or proband origin-of-variant information is documented in ClinVar submissions or the literature pass.

Sources

Reasoning

The MM-VCEP RUNX1 v3.1 specification (cspec) makes PP1 applicable and defines PP1 (supporting) as 3 or 4 meioses, PP1_Moderate as 5 or 6 meioses, and PP1_Strong as >=7 meioses observed within one or across multiple families, counting only genotype- and phenotype-positive individuals and obligate carriers; the VCEP waived the requirement for segregation in more than one family. No pedigree, family segregation, or meiosis data for c.560C>T exists in the case evidence bundle, and the literature pass contains no variant-specific case reports (PMID:28492532 is a criteria methodology article and PMID:33661592 is a GeneReviews overview, neither mentions the variant). Evidence is insufficient to apply PP1 at any strength.

Evidence considered

• MM-VCEP RUNX1 v3.1 (cspec) PP1 specification: PP1 = 3 or 4 meioses, PP1_Moderate = 5 or 6 meioses, PP1_Strong = >=7 meioses observed within one or across multiple families; only genotype- and phenotype-positive individuals and obligate carriers are counted; the MM-VCEP waived the requirement of demonstrating co-segregation in more than one family.
• ClinVar record VCV002850045 contains no family/segregation information for c.560C>T; both submissions (SCV004449577, SCV005205650) applied only PM1/PM2 evidence.
• Literature pass for this case contains only PMID:28492532 (Sherloc criteria methodology paper) and PMID:33661592 (RUNX1 FPD/AML GeneReviews overview); full text of PMID:28492532 was checked and contains no mention of c.560C>T, p.Ala187Val, or RUNX1, and provides no segregation data.

Still missing

• No co-segregation or meiosis count data for c.560C>T in any family is available.
• No variant-specific case report or family study describing segregation of c.560C>T was identified in the literature pass.
• Affected relatives' genotype/phenotype status (RUNX1-phenotypic criteria per PS2) is undocumented.

Sources

Reasoning

Under the governing MM-VCEP RUNX1 v3.1 specification (cspec doc 135637580), BS3 is Applicable and requires positive functional evidence of normal function: BS3 (Strong) requires transactivation assays demonstrating normal transactivation (80-115% of wt) AND data from a secondary assay demonstrating normal function; BS3_Supporting requires transactivation assays demonstrating normal transactivation (80-115% of wt). No such data exists for NM_001754.4:c.560C>T (p.Ala187Val) in the case evidence bundle: no transactivation assay of this variant (with wt and pathogenic controls per MM-VCEP) was identified; no secondary assay demonstrating normal function was identified; OncoKB reports no variant-specific reviewed functional evidence; ClinVar lists the variant as VUS (MM-VCEP expert panel applied only PM1_supporting and PM2_supporting); the sole full-text paper (PMID:28492532) does not mention the variant; PMID:33661592 (GeneReviews) is image-only without variant mention. Absence of damaging evidence is not the same as demonstrated normal function, so BS3 cannot be met at any strength; because the evidence is insufficient, not_assessed is returned with the gaps identified.

Rule

BS3: Transactivation assays demonstrating normal transactivation (80-115% of wt) AND data from a secondary assay demonstrating normal function. BS3_Supporting: Transactivation assays demonstrating normal transactivation (80-115% of wt).

Evidence considered

• MM-VCEP RUNX1 v3.1 cspec BS3 rule: "BS3: Transactivation assays demonstrating normal transactivation (80-115% of wt) AND data from a secondary assay demonstrating normal function."
• MM-VCEP RUNX1 v3.1 cspec BS3_Supporting rule: "BS3_Supporting: Transactivation assays demonstrating normal transactivation (80-115% of wt)."
• MM-VCEP RUNX1 v3.1 cspec BS3 note: transactivation assays must include wt and known pathogenic controls and co-expression with CBF-beta; secondary assays include EMSA (DNA binding), co-IP/FRET (CBF-beta heterodimerization), immunofluorescence/cell-fractionation (localization), colony-forming potential, and xenotransplantation.
• OncoKB RUNX1 A187V page: "OncoKB did not identify variant-specific reviewed functional evidence for this variant" — no variant-specific functional study supporting normal function was found.
• ClinVar VCV002850045: VUS by Labcorp and by the ClinGen Myeloid Malignancy VCEP (expert panel applied PM1_supporting and PM2_supporting only); no submission reports any functional assay (normal or abnormal) for this variant.
• Full text of PMID:28492532 (Sherloc) was searched for Ala187/A187/c.560C>T/transactivation/functional; no matches — no evidence of normal function for this variant in the literature bundle.
• The variant is absent from gnomAD v2.1, v4.1 and gnomAD-Canada v1.0; population absence is not a functional assay and does not satisfy the BS3 transactivation requirement.
• SpliceAI (max delta 0.001) predicts no splice impact, but this is an in-silico prediction, not a well-established functional study, and does not satisfy the BS3 transactivation/secondary-assay requirement.

Still missing

• No transactivation assay demonstrating normal transactivation (80-115% of wt) for p.Ala187Val exists in the case evidence bundle; BS3_Supporting (single-assay threshold) therefore cannot be applied either.
• No secondary assay demonstrating normal function for p.Ala187Val exists (no EMSA/DNA-binding, CBF-beta heterodimerization, localization, or colony-forming data showing wild-type-like function).
• A validated transactivation assay of p.Ala187Val performed per MM-VCEP requirements (wt and known pathogenic controls, CBF-beta co-expression) is required before BS3 or BS3_Supporting can be assessed.

Sources

Reasoning

The MM-VCEP RUNX1 v3.1 specification (cspec) defines BS4 as applicable when the variant is observed in >=2 informative meioses, with the MM-VCEP note that the code should only be applied for genotype-negative, phenotype-positive family members. No genotype-negative, phenotype-positive family members, informative meiosis data, or non-segregation observations for c.560C>T are reported in the case evidence bundle or in the ClinVar record. Evidence is insufficient to apply BS4.

Evidence considered

• MM-VCEP RUNX1 v3.1 (cspec) BS4 specification: BS4 is applicable when observed in >=2 informative meioses; MM-VCEP note states the code should only be applied for genotype-negative, phenotype-positive family members.
• ClinVar record VCV002850045 contains no family member testing data for c.560C>T; neither submission reports non-segregation observations.
• No variant-specific family data for c.560C>T was found in the literature pass (PMID:28492532 full text checked and negative for the variant; PMID:33661592 is a gene-level review).

Still missing

• No genotype-negative, phenotype-positive family member (informative meiosis) data for c.560C>T is available.
• No family testing or non-segregation observations are reported in ClinVar submissions or the literature pass.

Sources