Criteria Specification Registry

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Criteria Specification

ClinGen Myeloid Malignancy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RUNX1 Version 3.0.0

Type: Tavtigian et.al., 2020 - Bayesian adaptation of Richards et.al., 2015 Description : MM-VCEP Specifications for RUNX1 Variant Curation

Version : 3.0.0 Release Notes :

(1) New gnomAD MAF threshold for PM2_supporting ≤ 0.00005 to account for larger population in gnomAD v4.
(2) Upgraded strength of PM1 to PM1_strong when used for missense variants at the following residues: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. Added a caveat to not use PM5/PS1 at any level if PM1 was applied.
(3) Upgraded strength of PM4 to PM4_strong when used for missense variants at the following residues: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. Added an allowance to use PM4 for stop-loss variants.
(4) Established PVS1_variable (RNA) and BP7_variable (RNA) to be used when RNA data is available for splicing variants. PS1 is now able to be used for splicing variants with the same predicted splicing event as a known pathogenic/likely pathogenic splicing variant.
(5) Conservation data is no longer considered when applying BP7. BP7 is limited to intronic variants, and synonymous variants which don’t occur in the last 3 nucleotides preceding a canonical donor splice site or the first nucleotide following a canonical acceptor splice site.

Myeloid Malignancy VCEP

Released

8/14/2025

20:12:47

Rules for RUNX1

Release notes: (1) New gnomAD MAF threshold for PM2_supporting ≤ 0.00005 to account for larger population in gnomAD v4. (2) Upgraded strength of PM1 to PM1_strong when used for missense variants at the following residues: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. Added a caveat to not use PM5/PS1 at any level if PM1 was applied. (3) Upgraded strength of PM4 to PM4_strong when used for missense variants at the following residues: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. Added an allowance to use PM4 for stop-loss variants. (4) Established PVS1_variable (RNA) and BP7_variable (RNA) to be used when RNA data is available for splicing variants. PS1 is now able to be used for splicing variants with the same predicted splicing event as a known pathogenic/likely pathogenic splicing variant. (5) Conservation data is no longer considered when applying BP7. BP7 is limited to intronic variants, and synonymous variants which don’t occur in the last 3 nucleotides preceding a canonical donor splice site or the first nucleotide following a canonical acceptor splice site.

Gene: RUNX1 (HGNC:10471) HGNC Name: RUNX family transcription factor 1 Transcripts: NM_001754.4 (RUNX1c) Disease: hereditary thrombocytopenia and hematologic cancer predisposition syndrome (MONDO:0011071) Mode of Inheritance: Autosomal dominant inheritance

Criteria & Strength Specifications
PVS1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Caveats: • Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). • Use caution interpreting LOF variants at the extreme 3’ end of a gene. • Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. • Use caution in the presence of multiple transcripts. VCEP Specifications: ClinGen Myeloid Malignancy Variant Curation Expert Panel (MM-VCEP) notes: (1) We recommend using RUNX1 isoform c as the default transcript (NM_001754.4), since this is the isoform used for annotation by most clinical laboratories. (2) Three major isoforms (a, b, c) are expressed by use of two promotors and alternative splicing. Expression of the short human RUNX1a isoform has been shown to favor expansion of the hematopoietic stem cell (HSC) pool, whereas expression of the full length RUNX1b and RUNX1c isoforms function to promote hematopoietic differentiation. RUNX1 LOF variants are a common mechanism of disease in familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). C-terminal truncating variants not predicted to undergo nonsense-mediated mRNA decay (NMD) are classified as PVS1_strong, deletions of exon 1-3, presumably only affecting RUNX1 isoform c, meet PVS1_moderate. (3) Most splicing effects are based on predictions. The rules can be modified in the future if new functional evidence3 becomes available. The rules can be modified in the future if RNA evidence becomes available using Walker et al., 2023, PMID: 37352859 ⁵ as a guide; modification of strength should be based on the quality of the RNA study where: Comparison to a control is necessary Patient RNA is better than minigene assays Primers are designed to capture the possibility of multi-exonic/multi-cassette events NMD inhibitors (e.g., puromycin, proprietary molecule found in PAXgene tubes), particularly when the predicted effect is nonsense-mediated decay, are used Quantification of the effect by SNP analysis is better than PSI (percent splicing index) analysis is better than capillary electrophoresis is better than estimation by gel band density Multiple studies are better than a single study Caution should be used in modifying strength when the effect of the splicing impact is incomplete ("leaky" splice site) or unclear RUNX1* Specification: Per modified RUNX1 PVS1 decision tree for single-nucleotide variants (SNVs) and CNVs and table of splicing effects. Strength-modified: PVS1, PVS1_Strong, PVS1_Moderate PVS1_Variable (RNA): When RNA/splicing data is available for a variant, apply PVS1 at the appropriate strength level based on the predicted effect of the aberrant mRNA on protein translation as it corresponds to the PVS1_Variable splicing table. Strength may also be modified based on the quality of the RNA analysis (as described above). For "leaky" splice sites, strength level should be decreased by one if a near-complete impact is demonstrated, but no code should be applied if an incomplete impact is demonstrated. Refer to Walker et al., 2023, PMID: 37352859 ⁵ for additional guidance. SNVs, Indels/Delins, Splicing Variants [RUNX1 PVS1 decision tree for SNVs] CNVs [RUNX1 PVS1 decision tree for CNVs] Canonical Splice Site Variants (see Supplemental Table) [Summary of splicing effects] Stand Alone Very Strong Per modified RUNX1 PVS1 decision tree for SNVs and CNVs and table of splicing effects. Default Point Value: 8 Modification Type: Gene-specific Strong Per modified RUNX1 PVS1 decision tree for SNVs and CNVs and table of splicing effects. Default Point Value: 4 Modification Type: Gene-specific,Strength Moderate Per modified RUNX1 PVS1 decision tree for SNVs and CNVs and table of splicing effects. Default Point Value: 2 Modification Type: Gene-specific,Strength Supporting Default Point Value: 1 Not Applicable
PS1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. VCEP Specifications: MM-VCEP Notes: (1) The previously established pathogenic variant must be reviewed by the MM-VCEP and asserted pathogenic/likely pathogenic before this rule can be applied. (2) For missense variants, RNA data or agreement in splicing predictor show no impact on splicing. (3) For splice site variants, do not apply this code except for variants in the canonical donor/acceptor (“dinucleotide”) sites, the U2 donor motif (last 3 bases of the exon and 6 nucleotides of the intron), or the U2 acceptor motif (20 nucleotides of the intron and 1st base of the exon). Splicing predictions for the variant being evaluated and the known pathogenic/likely pathogenic (as assessed by VCEP rules) should match before consideration of the criterion, with at least similar scores. Do not apply for +2G>C variants. RUNX1 Specification: For missense variants: 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. For splice site variants: PS1_Variable: Follow recommendations from the ClinGen SVI Splicing Subgroup (Walker et al., 2023, PMID: 37352859 ⁵) [PS1 splicing application] Stand Alone Very Strong Strong PS1: Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. For splice site variants: PS1_Variable: Follow recommendations from the ClinGen SVI Splicing Subgroup (Walker et al., 2023, PMID: 37352859 ⁵) Default Point Value: 4 Modification Type: General recommendation,Strength Moderate PS1_Moderate: Same amino acid change as a previously established likely pathogenic variant regardless of nucleotide change. For splice site variants: PS1_Variable: Follow recommendations from the ClinGen SVI Splicing Subgroup (Walker et al., 2023, PMID: 37352859 ⁵) Default Point Value: 2 Modification Type: General recommendation,Strength Supporting Default Point Value: 1 Not Applicable
PS2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. VCEP Specifications: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. MM-VCEP notes: (1) The FPD/AML phenotype is not highly specific and there is substantial genetic heterogeneity. We thus concluded that due to the lack of a highly specific phenotype and genetic heterogeneity, the maximum allowable value is 1 point contributing to the overall score. (2) The phenotype of a deleterious RUNX1 mutation encompasses at least one of the following three criteria: Mild to moderate thrombocytopenia with normal platelet size and volume in the absence of other causative factors such as autoimmune (e.g. antibodies against platelet surface antigens) or drug-related thrombocytopenia. Platelet ultrastructural and/or functional defects including platelet alpha or dense granule secretion defects or impaired platelet aggregation - particularly in response to collagen and epinephrine. Diagnosis of a hematologic malignancy, most commonly affecting the myeloid lineage causing acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), less frequently involving the lymphoid lineage manifesting as T-acute lymphoblastic leukemia (T-ALL). There are rare case-reports of patients with germline RUNX1 mutations and mixed myeloproliferative syndromes/MDS such as chronic myelomonocytic leukemia, as well as case-reports of patients with B-ALL, and hairy-cell leukemia. (3) No family history is defined as the absence of the variant and any of the _RUNX1_-phenotypic criteria in first and second-degree relatives. (4) The maximum allowable strength by combining PS2 and PM6 is to apply one moderate or two supporting rules (the maximum allowable value is still 1 point). RUNX1 Specification: Following the ClinGen Sequence Variant Interpretation (SVI) Working Group guidance, de novo RUNX1 variants will be scored at the third tier of the point-based system (“Phenotype consistent with gene but not highly specific and high genetic heterogeneity”) with maximum allowable value of 1 point contributing to overall score: PS2_Moderate: ≥ 2 proven de novo occurrences (both maternity and paternity confirmed) in patients with FPD/AML phenotype. PS2_Supporting: 1 proven de novo occurrence (both maternity and paternity confirmed) in a patient with FPD/AML phenotype. Stand Alone Very Strong Default Point Value: 8 Strong Default Point Value: 4 Moderate PS2_Moderate: ≥ 2 proven de novo occurrences (both maternity and paternity confirmed) in patients with FPD/AML phenotype. Default Point Value: 2 Modification Type: Disease-specific,Strength Supporting PS2_Supporting: 1 proven de novo occurrence (both maternity and paternity confirmed) in a patient with FPD/AML phenotype. Default Point Value: 1 Modification Type: Disease-specific,Strength Not Applicable
PS3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established. VCEP Specifications: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. MM-VCEP notes: Transactivation assays demonstrating altered transactivation compared to wildtype (wt) are often performed as functional studies to evaluate the pathogenicity of a RUNX1 variant. Promoter sequences of M-CSFR, PF4, C-FMS and GZMB, containing consensus RUNX1 binding sites TGTGGT, have been used for this purpose. The transactivation assay must include wt and known pathogenic controls, as well as co-expression with CBFâ. Data from secondary assays are frequently used to evaluate an altered function of mutant RUNX1. Electrophoretic mobility shift assays and yeast hybrid assays are performed to demonstrate decreased DNA binding affinity, and co-immunoprecipitation assays, fluorescence resonance energy transfer assays and affinity assays can demonstrate diminished heterodimerization ability of mutant RUNX1 with CBFâ. Abnormal cellular localization of mutant RUNX1 can be shown by immunofluorescence and cell-fractionation with Western Blot. Sorted primary hematopoietic stem and progenitor cells can be used for demonstration of reduced colony-forming potential and xenotransplantation experiments may reveal abnormal function of mutant RUNX1 in vivo. RUNX1 Specification: 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). Stand Alone Very Strong Default Point Value: 8 Strong 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. Default Point Value: 4 Modification Type: Gene-specific,Strength Moderate 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. Default Point Value: 2 Modification Type: Gene-specific,Strength Supporting PS3_Supporting: Transactivation assays demonstrating enhanced transactivation (>115% of wt). Default Point Value: 1 Modification Type: Gene-specific,Strength Not Applicable
PS4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance. Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence. VCEP Specifications: MM-VCEP notes: (1) There is currently no published RUNX1 case control study. The criteria of a case control study can be added into the rules, if such a study will be published in the future. The original ACMG/AMP criterion states that in the absence of a published case-control study, the observation of the variant in multiple unrelated patients with the same phenotype and its absence in controls, may be used. The MM-VCEP created a “quasi-case-control study” with the estimated number of probands worldwide and the overall gnomAD population as control cohort. In order to apply this code, the proband has to meet the _RUNX1_-phenotypic criteria (see PS2) and the variant has to be either absent from gnomAD or only present once. RUNX1 Specification: PS4: ≥ 4 probands meeting at least one of the _RUNX1_-phenotypic criteria (OR 127.1). PS4_Moderate: 2-3 probands meeting at least one of the _RUNX1_-phenotypic criteria (OR 63.5-95.3). PS4_Supporting: 1 proband meeting at least one of the _RUNX1_-phenotypic criteria (OR 31.8). Stand Alone Very Strong Default Point Value: 8 Strong PS4: ≥ 4 probands meeting at least one of the _RUNX1_-phenotypic criteria (OR 127.1). Default Point Value: 4 Modification Type: Disease-specific,Strength Moderate PS4_Moderate: 2-3 probands meeting at least one of the _RUNX1_-phenotypic criteria (OR 63.5-95.3). Default Point Value: 2 Modification Type: Disease-specific,Strength Supporting PS4_Supporting: 1 proband meeting at least one of the _RUNX1_-phenotypic criteria (OR 31.8). Default Point Value: 1 Modification Type: Disease-specific,Strength Not Applicable
PM1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation. VCEP Specifications: MM-VCEP notes: The Runt homology domain (RHD) has been established as highly conserved DNA binding domain without any benign variation in ClinVar. Thirteen somatic and/or germline mutational hotspots within the RHD have been identified: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. Variants in other parts of the RHD (amino acid (AA) 89-204) have been described as likely pathogenic/pathogenic before. There was additional evidence of germline pathogenic/likely pathogenic RUNX2 variants affecting AA 89 and 94 (PMID 17290219) [⁰⁰](#PMID_ 17290219), a gene with a Runt Homology Domain that has 90% sequence homology with RUNX1. AA 89 is also still part of the b-sheet of the CBF heterodimerization domain, which is functionally important. Thus, we prompt to establish PM1_supporting with reduced strength-level for these variants. No reported germline RUNX1 mutations in AA residues 77-88 of the RHD to date. If there is more evidence available, this region may be expanded in the future to other parts of the RHD or the protein. RUNX1 Specification: PM1_strong: Variant affecting one of the following 13 AA residues within the RHD: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. PM1_Supporting: Variant affecting one of the other AA residues 89-204 within the RHD. Stand Alone Very Strong Strong PM1_strong: Variant affecting one of the following 13 AA residues within the RHD: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. Default Point Value: 4 Modification Type: Gene-specific,Strength Moderate Default Point Value: 2 Supporting PM1_Supporting: Variant affecting one of the other AA residues 89-204 within the RHD. Default Point Value: 1 Modification Type: Gene-specific,Strength Not Applicable
PM2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. Caveat: Population data for indels may be poorly called by next generation sequencing. VCEP Specifications: MM-VCEP notes: New gnomAD MAF threshold for PM2_supporting ≤ 0.00005 to account for larger population in gnomAD v4.The mean coverage of RUNX1 in the population database used should be at least 20x. RUNX1 Specification: PM2_Supporting: Minor allele frequency ≤ 0.00005 with at least 2000 alleles tested around and 20x coverage at the position. Stand Alone Very Strong Strong Moderate Default Point Value: 2 Supporting PM2_Supporting: Minor allele frequency ≤ 0.00005 with at least 2000 alleles tested around and 20x coverage at the position. Default Point Value: 1 Modification Type: Gene-specific,Strength Not Applicable
PM3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase. Stand Alone Very Strong Default Point Value: 8 Strong Default Point Value: 4 Moderate Default Point Value: 2 Supporting Default Point Value: 1 Not Applicable Comments: MM-VCEP notes: FPD/AML is inherited in an autosomal dominant manner, thus PM3 is not applicable.
PM4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. VCEP Specifications: Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. MM-VCEP notes: The RHD has been established as highly conserved DNA binding domain without any benign variation in ClinVar. Thirteen somatic and/or germline mutational hotspots within the RHD have been identified: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. Variants in other parts of the RHD (AA 89-204) have been described as likely pathogenic/pathogenic before. There was additional evidence of germline pathogenic/likely pathogenic RUNX2 variants affecting AA 89 and 94 (PMID 17290219)⁶, a gene with a Runt Homology Domain that has 90% sequence homology with RUNX1. AA 89 is also still part of the b-sheet of the CBF heterodimerization domain, which is functionally important. Thus, we prompt to establish PM4_supporting with reduced strength-level for these variants. No reported germline RUNX1 mutations in AA residues 77-88 of the RHD to date. If there is more evidence available, this region may be expanded in the future to other parts of the RHD or the protein. RUNX1 Specification: For in-frame/indel variants: PM4_strong: In-frame deletion/insertion impacting at least one of the following AA residues within the RHD: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. PM4_Supporting: In-frame deletion/insertion impacting at least one of the other AA residues 89-204 within the RHD. For stop loss variants: PM4: Stop-loss variant causing a protein extension. Stand Alone Very Strong Strong For in-frame/indel variants: PM4_strong: In-frame deletion/insertion impacting at least one of the following AA residues within the RHD: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. For stop loss variants: PM4: Stop-loss variant causing a protein extension. Default Point Value: 4 Modification Type: Gene-specific,Strength Moderate In-frame deletion/insertion impacting at least one of the following amino acid residues within the RHD: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204. Default Point Value: 2 Modification Type: Gene-specific,Strength Supporting PM4_Supporting: In-frame deletion/insertion impacting at least one of the other AA residues 89-204 within the RHD. Default Point Value: 1 Modification Type: Gene-specific,Strength Not Applicable
PM5 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level. VCEP Specifications: Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. MM-VCEP notes: RNA data or SpliceAI ≤ 0.20 The previously established pathogenic variant must be reviewed by the MM-VCEP and asserted pathogenic/likely pathogenic before this rule can be applied. For missense variants, the Grantham score of the alternate residue of the new variant should be equal or higher to that of the alternate residue of the known pathogenic/likely pathogenic variant. [Grantham score table] (Grantham, 1974, PMID: 4843792⁷) 4. There are at least two nonsense/frameshift variants that were curated as pathogenic in each exon (exons 3-7) without applying PM5_Supporting. RUNX1 Specification: PM5_Strong: Missense change at an AA residue where ≥ 2 different missense changes which have been determined to be pathogenic before (after accounting for Grantham scores). 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: Missense change at an AA residue where a different missense change which has been determined to be likely pathogenic before (after accounting for Grantham scores). PM5_Supporting is also applied to nonsense/frameshift variants that are downstream of c.98 (in transcript NM_001754.4). Caveats: Of note, the variant must not impact splicing based on RNA assay or SpliceAI ≤ 0.20. The nonsense/frameshift variants before c.98 only affect one of the RUNX1 functional transcript. PVS1 is also not appliable in this region based on the RUNX1 PVS1 decision tree. PM5 cannot be used if PM1 was applied at any strength level. Stand Alone Very Strong Strong PM5_Strong:* Missense change at an AA residue where ≥ 2 different missense changes which have been determined to be pathogenic before (after accounting for Grantham scores). Caveats: Of note, the variant must not impact splicing based on RNA assay or SpliceAI ≤ 0.20. The nonsense/frameshift variants before c.98 only affect one of the RUNX1 functional transcript. PVS1 is also not appliable in this region based on the RUNX1 PVS1 decision tree. PM5 cannot be used if PM1 was applied at any strength level. Default Point Value: 4 Modification Type: Strength Moderate 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). Caveats: Of note, the variant must not impact splicing based on RNA assay or SpliceAI ≤ 0.20. The nonsense/frameshift variants before c.98 only affect one of the RUNX1 functional transcript. PVS1 is also not appliable in this region based on the RUNX1 PVS1 decision tree. PM5 cannot be used if PM1 was applied at any strength level. Default Point Value: 2 Modification Type: Strength Supporting PM5_Supporting: Missense change at an AA residue where a different missense change which has been determined to be likely pathogenic before (after accounting for Grantham scores). PM5_Supporting* is also applied to nonsense/frameshift variants that are downstream of c.98 (in transcript NM_001754.4). Caveats: Of note, the variant must not impact splicing based on RNA assay or SpliceAI ≤ 0.20. The nonsense/frameshift variants before c.98 only affect one of the RUNX1 functional transcript. PVS1 is also not appliable in this region based on the RUNX1 PVS1 decision tree. *PM5 cannot be used if PM1 was applied at any strength level. Default Point Value: 1 Modification Type: Strength Not Applicable
PM6 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Assumed de novo, but without confirmation of paternity and maternity. VCEP Specifications: MM-VCEP notes: (1) FPD/AML phenotype is not highly specific and there is substantial genetic heterogeneity. We thus concluded that due to the lack of a highly specific phenotype and genetic heterogeneity, the maximum allowable value is 1 point contributing to the overall score. (2) The phenotype of a deleterious RUNX1 mutation encompasses at least one of the three phenotypic criteria (see PS2). (3) No family history is defined as the absence of the variant and any of the RUNX1-phenotypic criteria in first and second-degree relatives. (4) The maximum allowable strength by combining PS2 and PM6 is to apply one moderate or two supporting rules (the maximum allowable value is still 1 point). RUNX1 Specification: Following the SVI guidance, assumed de novo RUNX1 variants will be scored at the third tier of the point-based system with maximum allowable value of 1 point contributing to overall score: PM6: ≥ 4 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with FPD/AML phenotype. PM6_Supporting: 2 or 3 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with FPD/AML phenotype. Stand Alone Very Strong Default Point Value: 8 Strong Default Point Value: 4 Moderate RUNX1 Specification: Following the SVI guidance, assumed de novo RUNX1 variants will be scored at the third tier of the point-based system with maximum allowable value of 1 point contributing to overall score: PM6: ≥ 4 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with FPD/AML phenotype. Default Point Value: 2 Modification Type: Disease-specific,Strength Supporting RUNX1 Specification: Following the SVI guidance, assumed de novo RUNX1 variants will be scored at the third tier of the point-based system with maximum allowable value of 1 point contributing to overall score: PM6_Supporting: 2 or 3 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with FPD/AML phenotype. Default Point Value: 1 Modification Type: Disease-specific,Strength Not Applicable
PP1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data. VCEP Specifications: Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. MM-VCEP notes: The MM-VCEP adopted the approach being taken by other ClinGen-EPs and supported by the SVI and other work with additional meioses supporting higher evidence levels based on calculated LOD scores of 0.9, 1.5 and 2.1, respectively, with three or four meioses for PP1, five or six meioses for PP1_moderate and seven or more meioses for PP1_strong. Affected individuals show at least one of the _RUNX1_-phenotypic criteria (see PS2). Only genotype and phenotype positive individuals and obligate carriers are counted. The MM-VCEP waived the ACMG/AMP recommendations for demonstrating co-segregation in more than one family given that many RUNX1 variants are unique to families and do not occur in other unrelated families. RUNX1 Specification: PP1_Strong: ≥ 7 meioses observed within one or across multiple families. PP1_Moderate: 5 or 6 meioses observed within one or across multiple families. PP1: 3 or 4 meioses observed within one or across multiple families. Stand Alone Very Strong Strong PP1_Strong: ≥ 7 meioses observed within one or across multiple families. Default Point Value: 4 Modification Type: Disease-specific,Strength Moderate PP1_Moderate: 5 or 6 meioses observed within one or across multiple families. Default Point Value: 2 Modification Type: Disease-specific,Strength Supporting PP1: 3 or 4 meioses observed within one or across multiple families. Default Point Value: 1 Modification Type: Disease-specific,Strength Not Applicable
PP2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease. Stand Alone Very Strong Strong Moderate Supporting Default Point Value: 1 Not Applicable Comments: MM-VCEP notes: The recommended cutoff for PP2 by the SVI is a missense constraint z score of ≥ 3.09 which was not met by RUNX1 (2.48 on ExAC and 2.08 on gnomAD). In addition, there are 9 benign/likely benign missense RUNX1 variants in ClinVar.
PP3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant. VCEP Specifications: MM-VCEP notes: For in-silico evaluation of missense variants, the MM-VCEP recommends using REVEL, a meta-predictor combining 13 individual tools with high sensitivity and specificity and that has recently demonstrated highest performance compared to any individual tool or other ensemble methods. The threshold of REVEL is based on the evaluation of 25 germline PATH/LPATH and 25 BEN/LBEN missense variants in RUNX1. With the comparison of 11 different ensemble in-silico predictors (BayesDel AF, CADD, Condel, DANN, Eigen, FATHMM-MKL, MetaLR, MetaSVM, REVEL, UMD predictor, VEST) and their respective AUC, REVEL was found to be among the highest performing tools (AUC=1) and the new threshold for PP3 and BP4 was based on the REVEL scores at 90% sensitivity and 90% specificity, respectively. We compared the performance of the new splice predictor SpliceAI and MES, which has been shown to be the highest performing tool pre-SpliceAI by using a test set of 202 variants in genes associated with inherited hematologic malignancies/AA/BMF or cytopenia. The thresholds for PP3 and BP4 were established based on the SpliceAI score at 90% sensitivity (PP3, ≥ 0.38) and 90% specificity (BP4, ≤ 0.20). For some variant types that REVEL or SpliceAI scores are not available, multiple other predictors in agreements can be used in PP3. PP3 cannot be applied for canonical splice site variants. RUNX1 Specification: For missense variants: PP3: REVEL score ≥ 0.88 or SpliceAI ≥ 0.38, including the creation of cryptic novel splice sites. For synonymous and intronic (intron 4-8) variants: PP3: SpliceAI ≥ 0.38, including the creation of cryptic novel splice sites. Caveats: Do not use for variants with a predicted splicing effect that is proven by RNA analysis. See PVS1_Variable (RNA). Do not use for for canonical splice site variants. Stand Alone Very Strong Strong Default Point Value: 4 Moderate Default Point Value: 2 Supporting For missense variants: PP3: REVEL score ≥ 0.88 or SpliceAI ≥ 0.38, including the creation of cryptic novel splice sites. For synonymous and intronic (intron 4-8) variants: PP3: SpliceAI ≥ 0.38, including the creation of cryptic novel splice sites. Caveats: Do not use for variants with a predicted splicing effect that is proven by RNA analysis. See PVS1_Variable (RNA). Do not use for for canonical splice site variants. Default Point Value: 1 Modification Type: Disease-specific,Gene-specific Not Applicable
PP4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology. Stand Alone Very Strong Strong Default Point Value: 4 Moderate Default Point Value: 2 Supporting Default Point Value: 1 Not Applicable Comments: MM-VCEP notes: The FPD/AML phenotype is rather unspecific and can be caused by a number of other inherited predisposition syndromes, somatic mutations or environmental factors that are insufficient to meet the original ACMG/AMP rule PP4.
PP5
Original ACMG Summary Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation. Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BA1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. VCEP Specifications: MM-VCEP notes: FPD/AML with germline RUNX1 mutation is a rare disorder. The phenotype of carriers of a germline RUNX1 mutation includes three criteria (mild to moderate thrombocytopenia, platelet ultrastructural and/or functional defects and diagnosis of a hematologic malignancy). Of these three criteria, thrombocytopenia is the most common feature. Most clinical laboratories establish their platelet count reference values by measuring samples from at least 120 healthy individuals and identifying the most outlying 5% of observed values. Most often, these outlying observations are split evenly between the ends of the test result distribution in the reference population, 2.5% at each end of the distribution, resulting in a two-sided reference interval. Using this approach, the prevalence of thrombocytopenia can be defined as 1 in 40 (lower 2.5%) in general population. The penetrance in families with RUNX1 germline mutation is high to near-complete. We identified a family with a penetrance of 85% among known carriers of the mutation as the pedigree with the lowest penetrance to date. So far, no founder mutations in RUNX1 have been reported, de novo variants are rare but have been described. The MM-VCEP modified BA1 using extremely conservative values to account for the unknown prevalence and disease attribution to RUNX1. In order to obtain a _RUNX1_-specific population allele frequency for BA1, we utilized the Whiffin/Ware calculator (http://cardiodb.org/allelefrequencyapp/) with a prevalence of 1 in 40, a conservative unascertained penetrance estimate of 85%, an allelic heterogeneity of 100% and a maximum genetic heterogeneity of 10%. A 95% confidence interval was used to develop the threshold. The threshold developed for application of BA1 as a stand-alone criterion is a minor allele frequency of equal to or higher than 0.0015 (0.15%). The MM-VCEP also adopted the SVI recommendation that the variant be present in any general continental population dataset with a minimum number of 2,000 alleles and variant present in ≥ 5 alleles. RUNX1 Specification: 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. Stand Alone RUNX1 Specification: 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. Default Point Value: Not Applicable Modification Type: Disease-specific Very Strong Strong Moderate Supporting Not Applicable
BS1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Allele frequency is greater than expected for disorder. Stand Alone Very Strong Strong MM-VCEP notes: Similarly, for the BS1 calculation, we utilized the Whiffin/Ware calculator (http://cardiodb.org/allelefrequencyapp/) with a prevalence of 1 in 40, a conservative unascertained penetrance estimate of 85%, an allelic heterogeneity of 100% and a maximum genetic heterogeneity of 1% (one magnitude lower than for BA1). A 95% confidence interval was used to develop the threshold. We developed a range for application of BS1 for variants with a minor allele frequency between 0.00015 (0.015%) and 0.0015 (0.15%). The MM-VCEP also adopted the SVI recommendation that the variant be present in any general continental population dataset with a minimum number of 2,000 alleles and variant present in ≥ 5 alleles. The variant can be classified as likely benign based on BS1 alone if there is no contradictory evidence supporting pathogenicity. RUNX1 Specification: 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. Default Point Value: -4 Modification Type: Disease-specific Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable
BS2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. Stand Alone Very Strong Strong Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: MM-VCEP notes: BS2 is not applicable since FPD/AML patients display incomplete penetrance and the average age of onset of hematologic malignancies is 33 years.
BS3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing. VCEP Specifications: MM-VCEP notes: Transactivation assays demonstrating altered transactivation compared to wt are often performed as functional studies to evaluate the pathogenicity of a RUNX1 variant. Promoter sequences of M-CSFR, PF4, C-FMS and GZMB, containing consensus RUNX1 binding sites TGTGGT, have been used for this purpose. The transactivation assay must include wt and known pathogenic controls, as well as co-expression with CBFâ. Data from secondary assays are frequently used to evaluate an altered function of mutant RUNX1. Electrophoretic mobility shift assays and yeast hybrid assays are performed to demonstrate decreased DNA binding affinity, and co-immunoprecipitation assays, fluorescence resonance energy transfer assays and affinity assays can demonstrate diminished heterodimerization ability of mutant RUNX1 with CBFâ. Abnormal cellular localization of mutant RUNX1 can be shown by immunofluorescence and cell-fractionation with Western Blot. Sorted primary hematopoietic stem and progenitor cells can be used for demonstration of reduced colony-forming potential and xenotransplantation experiments may reveal abnormal function of mutant RUNX1 in vivo. RUNX1 Specification: 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). Stand Alone Very Strong Strong BS3: Transactivation assays demonstrating normal transactivation (80-115% of wt) AND data from a secondary assay demonstrating normal function. Default Point Value: -4 Modification Type: Gene-specific,Strength Moderate Default Point Value: -2 Supporting BS3_Supporting: Transactivation assays demonstrating normal transactivation (80-115% of wt). Default Point Value: -1 Modification Type: Gene-specific,Strength Not Applicable
BS4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Lack of segregation in affected members of a family. Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation. VCEP Specifications: MM-VCEP notes: This code should only be applied for genotype-negative, phenotype-positive family members. RUNX1 Specification: BS4: Applicable when observed in ≥ 2 informative meioses. Stand Alone Very Strong Strong BS4: Applicable when observed in ≥ 2 informative meioses. Default Point Value: -4 Modification Type: General recommendation Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable
BP1 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Missense variant in a gene for which primarily truncating variants are known to cause disease. Stand Alone Very Strong Strong Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: MM-VCEP notes: BP1 is not applicable for RUNX1, because both truncating and missense variants cause FPD/AML.
BP2 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary 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. VCEP Specifications: MM-VCEP notes: BP2 is applicable per the original ACMG/AMP guidelines. In vivo, mice lacking Runx1 die during mid-embryonic development. 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. Stand Alone Very Strong Strong Default Point Value: -4 Moderate Default Point Value: -2 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. Default Point Value: -1 Modification Type: None Not Applicable
BP3 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary In frame-deletions/insertions in a repetitive region without a known function. Stand Alone Very Strong Strong Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: MM-VCEP notes: RUNX1 does not contain a repetitive region without known function. BP3 is therefore deemed not applicable.
BP4 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant. VCEP Specifications: MM-VCEP notes: For in-silico evaluation of missense variants, the MM-VCEP recommends using REVEL, a meta-predictor combining 13 individual tools with high sensitivity and specificity and that has recently demonstrated highest performance compared to any individual tool or other ensemble methods. The threshold of REVEL is based on the evaluation of 25 germline PATH/LPATH and 25 BEN/LBEN missense variants in RUNX1. With the comparison of 11 different ensemble in-silico predictors (BayesDel AF, CADD, Condel, DANN, Eigen, FATHMM-MKL, MetaLR, MetaSVM, REVEL, UMD predictor, VEST) and their respective AUC, REVEL was found to be among the highest performing tools (AUC=1) and the new threshold for PP3 and BP4 was based on the REVEL scores at 90% sensitivity and 90% specificity, respectively. We compared the performance of the new splice predictor SpliceAI and MES, which has been shown to be the highest performing tool pre-SpliceAI by using a test set of 202 variants in genes associated with inherited hematologic malignancies/AA/BMF or cytopenia. The thresholds for PP3 and BP4 were established based on the SpliceAI score at 90% sensitivity (PP3, ≥ 0.38) and 90% specificity (BP4, ≤ 0.20). RUNX1 Specification: For missense variants: BP4: REVEL score < 0.50 AND SpliceAI ≤ 0.20 For synonymous and Intronic variants: BP4: SpliceAI ≤ 0.20 Stand Alone Very Strong Strong Default Point Value: -4 Moderate Default Point Value: -2 Supporting For missense variants: BP4: REVEL score < 0.50 AND SpliceAI ≤ 0.20 For synonymous and Intronic variants: BP4: SpliceAI ≤ 0.20 Default Point Value: -1 Modification Type: Disease-specific,Gene-specific Not Applicable
BP5 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary Variant found in a case with an alternate molecular basis for disease. Stand Alone Very Strong Strong Default Point Value: -4 Moderate Default Point Value: -2 Supporting Default Point Value: -1 Not Applicable Comments: BP5 is not applicable. In rare circumstances, a patient can carry two pathogenic variants in genes predisposing to hematologic malignancies.
BP6
Original ACMG Summary Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation. Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BP7 PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 Files References
Original ACMG Summary A synonymous 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. VCEP Specifications: MM-VCEP notes: Conservation is no longer a requirement for BP7 based on its limited predicted power and recommendations from the ClinGen SVI Splicing Subgroup (Cheung et al., 2019, 30503770; Walker et al., 2023, PMID: 37352859⁹). Splicing effects are currently based solely on predictions. The rules can be modified in the future if RNA evidence becomes available using Walker et al., 2023, PMID: 37352859⁹ as a guide; modification of strength should be based on the quality of the RNA study where: Comparison to a control is necessary Patient RNA is better than minigene assays Primers are designed to capture the possibility of multi-exonic/multi-cassette events NMD inhibitors (e.g., puromycin, proprietary molecule found in PAXgene tubes), particularly when the predicted effect is nonsense-mediated decay, are used Quantification of the effect by SNP analysis is better than PSI (percent splicing index) analysis is better than capillary electrophoresis is better than estimation by gel band density Multiple studies are better than a single study * Caution should be used in modifying strength when the effect of the splicing impact is incomplete ("leaky" splice site) or unclear. RUNX1 Specification: BP7: Applicable for Synonymous variants – excluding those in the last 3 nucleotides preceding a canonical donor splice site or the first nucleotide following a canonical acceptor splice site – with SpliceAI ∆ scores ≤ 0.20. Intronic variants with SpliceAI ∆ scores ≤ 0.20. BP7_Variable (RNA): Applicable for variants with RNA data, with weighting based on the quality of the available RNA data. Stand Alone Very Strong Strong Default Point Value: -4 Moderate Default Point Value: -2 Supporting BP7: Applicable for Synonymous variants – excluding those in the last 3 nucleotides preceding a canonical donor splice site or the first nucleotide following a canonical acceptor splice site – with SpliceAI ∆ scores ≤ 0.20. Intronic variants with SpliceAI ∆ scores ≤ 0.20. BP7_Variable (RNA): Applicable for variants with RNA data, with weighting based on the quality of the available RNA data. Default Point Value: -1 Modification Type: Disease-specific,Gene-specific Not Applicable

Point Based Variant Classification Categories

Category	Point Ranges
Pathogenic	10
Likely Pathogenic	6 - 9
Uncertain Significance	0 - 5
Likely Benign	-6 - -1
Benign	-7

Additional Notes :

Files & Images

MM-VCEP RUNX1 Pilot Results: MM-VCEP RUNX1 Pilot Results

RUNX1 PVS1 decision tree for SNVs: RUNX1 PVS1 decision tree for SNVs

Summary of splicing effects: Summary of splicing effects

Grantham score table: Grantham score table

PS1 splicing application: PS1 splicing application

RUNX1 PVS1 decision tree for CNVs: RUNX1 PVS1 decision tree for CNVs

References

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2. Matheny CJ, Speck ME, Cushing PR, et al. Disease mutations in RUNX1 and RUNX2 create nonfunctional, dominant-negative, or hypomorphic alleles. EMBO J. 2007;26(4):1163-1175. doi:10.1038/sj.emboj.7601568

3. Grantham R. Amino acid difference formula to help explain protein evolution. Science. 1974;185(4154):862-864. doi:10.1126/science.185.4154.862

4. Tavtigian SV, Harrison SM, Boucher KM, Biesecker LG. Fitting a naturally scaled point system to the ACMG/AMP variant classification guidelines. Hum Mutat. 2020;41(10):1734-1737. doi:10.1002/humu.24088

5. Walker LC Hoya M et al. Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup. Am J Hum Genet (2023) 110 (7) p. 1046-1067. 10.1016/j.ajhg.2023.06.002 37352859

6. Matheny CJ Speck ME et al. Disease mutations in RUNX1 and RUNX2 create nonfunctional, dominant-negative, or hypomorphic alleles. EMBO J (2007) 26 (4) p. 1163-75. 10.1038/sj.emboj.7601568 17290219

7. Grantham R Amino acid difference formula to help explain protein evolution. Science (1974) 185 (4154) p. 862-4. 10.1126/science.185.4154.862 4843792

8. Walker LC Hoya M et al. Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup. Am J Hum Genet (2023) 110 (7) p. 1046-1067. 10.1016/j.ajhg.2023.06.002 37352859

9. Walker LC Hoya M et al. Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup. Am J Hum Genet (2023) 110 (7) p. 1046-1067. 10.1016/j.ajhg.2023.06.002 37352859