MM-VCEP ACMG/AMP specifications for RUNX1 variants
| ACMG/AMP criteria code . | Original ACMG/AMP rule summary . | Specification . | Stand-alone . | Very strong . | Strong . | Moderate . | Supporting . | Comments . |
|---|---|---|---|---|---|---|---|---|
| PVS1 | Null variant in a gene where LOF is a known mechanism of disease | Gene-specific, strength | NA | Per modified RUNX1 PVS1 decision tree for SNVs, indels, and CNVs and table of splicing effects | NA | RUNX1 LOF variants are a common mechanism of disease in FPD/AML. Three major isoforms (A, B, and C) are expressed by use of 2 promotors and alternative splicing. C-terminal variants not predicted to undergo NMD are classified as PVS1_strong, deletions of exons 2 and 3, presumably only affecting RUNX1 isoform 1C, meet PVS1_moderate | ||
| PS1 | Same AA change as a previously established PATH variant regardless of nucleotide change | Strength | NA | NA | Same AA change as a previously established PATH variant regardless of nucleotide change | Same AA change as a previously established, LPATH variant regardless of nucleotide change | NA | (1) RNA data or agreement in splicing predictors show no splicing effects (SSF and MES predict either increase in canonical splice site score or decrease in canonical splice score by no more than 10% and no putative splice sites are created). (2) The previously established variant must be asserted PATH/LPATH based on MM-VCEP rules for RUNX1 before this rule can be applied |
| PS2 | De novo (maternity and paternity confirmed) in a patient with the disease and no family history | Disease-specific, strength | NA | NA | NA | Two or more proven de novo occurrences (maternity and paternity confirmed) in patients with the RUNX1-phenotype | One proven de novo occurrence (maternity and paternity confirmed) in a patient with the RUNX1-phenotype | (1) No family history is defined as: absence of the variant and any of the RUNX1-phenotypic criteria in first- and/or second-degree relatives. (2) The proband must exhibit at least 1 phenotypic FPD/AML criterion. (3) The maximum allowable strength by combining PS2 and PM6 criteria is to apply 1 moderate or 2 supporting rules |
| PS3 | Well-established in vitro or in vivo functional studies supportive of a damaging effect | Gene-specific, strength | NA | NA | Transactivation assays exhibiting altered transactivation (<20% of wt, and/or reduced to levels similar to well-established PATH variants such as R201Q or R166Q) and data from a secondary assay showing altered function. PS3 cannot be applied if the variant meets PVS1. If the variant meets criteria for PVS1_strong and PS3, we recommend either applying PVS1_strong and PS3_moderate or upgrading PVS1_strong to PVS1 without applying PS3 | Transactivation assays exhibiting altered transactivation (<20% of wt, and/or reduced to levels similar to well-established PATH variants such as R201Q or R166Q) or ≥2 secondary assays exhibiting altered function | Transactivation assays exhibiting enhanced transactivation (>115% of wt) | (1) Transactivation assays should include wt and known PATH controls, as well as coexpression with CBFβ. Promoter sequences of CSF1R (M-CSF-R), PF4, C-FMS, and GZMB, containing consensus RUNX1-binding sites have been used for transactivation assays. (2) The following secondary assays have been performed: EMSA and yeast hybrid assays (decreased DNA-binding affinity), co-IP, FRET, and affinity assays (diminished heterodimerization ability with CBFβ), IF and WB with cell fractionation (abnormal cellular localization), colony-forming assays (reduced colony-forming potential), and xenotransplantation experiments (abnormal function of mutant RUNX1 in vivo). (3) PS3 can also be applied for evidence of very low or abnormal mRNA/protein expression of the variant allele as a functional consequence of a null variant or incorrect mRNA/protein products |
| PS4 | The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in control subjects | Disease-specific, strength | NA | NA | Four or more probands meeting RUNX1-phenotypic criteria | Two to 3 probands meeting RUNX1-phenotypic criteria | One proband meeting RUNX1-phenotypic criteria | The affected individual has to fit at least 1 of the RUNX1-phenotypic criteria and the variant has to be either absent from gnomAD (overall population) or only present once |
| PM1 | Located in a mutational hotspot and/or critical and well-established functional domain without BEN variation | Gene-specific, strength | NA | NA | NA | Variant affecting 1 of the following 13 hotspot residues: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204 | Variant affecting 1 of the other AA residues 105-204 within the RHD | The RHD (AA 77-204) has been established as a highly conserved DNA-binding domain without any BEN variation in ClinVar. No germline PATH variants have been reported in residues in the region (AA 77-104) to date. The AA range under PM1_supporting may be expanded in the future to other parts of the protein if more evidence emerges |
| PM2 | Absent from control subjects | General recommendation | NA | NA | NA | Per original ACMG/AMP guidelines | NA | Variant must be completely absent from all population databases. The mean coverage of RUNX1 in the population database used should be at least 20× |
| PM3 | For recessive disorders, detected in trans with a PATH variant | NA | FPD/AML is inherited in an autosomal dominant manner | |||||
| PM4 | Protein length changes due to in-frame deletions/insertions in a nonrepeat region or stop-loss variants | Gene-specific, strength | NA | NA | NA | In-frame deletion/insertion affecting at least 1 of the 13 hotspot residues (R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204) | Other in-frame deletion/insertion affecting residues 105-204 within the RHD | See PM1 |
| PM5 | Missense change at AA residue where a different missense change determined to be PATH has been seen before | Strength | NA | NA | Missense change at the same residue where ≥2 different missense changes have previously been determined to be PATH. PM5_strong cannot be applied together with PM1 | Missense change at the same residue where a different missense change has previously been determined to be PATH | Missense change at the same residue where a different missense change has previously been determined to be LPATH | See PS1 |
| PM6 | Assumed de novo (but without confirmation of maternity and paternity) in a patient with the disease and no family history | Disease-specific, strength | NA | NA | NA | Four or more assumed de novo occurrences (without confirmation of maternity and paternity) in patients with the RUNX1-phenotype | Two or 3 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with the RUNX1-phenotype | See PS2 |
| PP1 | Cosegregation with disease in multiple affected family members | Disease-specific, strength | NA | NA | Seven or more meioses observed within 1 family or across multiple families | Five or 6 meioses observed within 1 family or across multiple families | Three or 4 meioses observed within 1 family or across multiple families | (1) Affected individuals exhibit at least 1 of the RUNX1-specific phenotypic criteria. (2) Only genotype- and phenotype-positive individuals and obligate carriers are counted. (3) Demonstration of cosegregation in multiple families is not required because many RUNX1 variants are unique and only occur in 1 family |
| PP2 | Missense variant in a gene that has a low rate of BEN missense variation and where missense variants are a common mechanism of disease | NA | Missense constraint z score for RUNX1 is <3.09 | |||||
| PP3 | Multiple lines of computational evidence support a deleterious effect on the gene or gene product | General recommendation | NA | NA | NA | NA | Per original ACMG/AMP guidelines | (1) PP3 should be applied for missense variants with a REVEL score >0.75. (2) PP3 should be applied for missense or synonymous variants if the variant alters the last 3 bases of an exon preceding a donor splice site or the first 3 bases of an exon following a splice acceptor site and the predicted decrease in the score of the canonical splice site (measured by both MES and SSF) is at least 75% regardless of the predicted creation/presence of a putative cryptic splice site. (3) PP3 should also be applied for intronic variants (in introns 4-8) located in reference to exons at positions +3 to +5 for splice donor sites or −3 to −5 for splice acceptor sites for which the predicted decrease in the score is at least 75% (measured by both MES and SSF) regardless of the predicted creation/presence of a putative cryptic splice site. (4) PP3 cannot be applied for canonical splice site variants |
| PP4 | Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology | NA | FPD/AML does not exhibit a highly specific phenotype, and there is substantial genetic heterogeneity | |||||
| PP5 | Reputable source recently reports variant as PATH, but the evidence is not available to the laboratory to perform an independent analysis | NA | According to SVI recommendations | |||||
| BA1 | Allele frequency is >5% in ESP, 1000G, or ExAC | Disease-specific | MAF ≥0.0015 (0.15%) | NA | NA | NA | NA | The variant is present in any general continental population dataset with a minimum number of 2000 alleles and variant present in ≥5 alleles |
| BS1 | Allele frequency is greater than expected for disorder | Disease-specific | NA | NA | MAF between 0.00015 (0.015%) and 0.0015 (0.15%) | NA | NA | (1) The variant is present in any general continental population dataset with a minimum number of 2000 alleles and variant present in ≥5 alleles. (2) Variant can be classified as LBEN based on BS1 alone if there is no contradictory evidence supporting pathogenicity |
| BS2 | Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age | NA | Patients with FPD/AML display incomplete penetrance, and the average age of onset of hematologic malignancies is 33 y | |||||
| BS3 | Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing | Gene-specific, strength | NA | NA | (1) Transactivation assays exhibiting normal transactivation (80%-115% of wt); and (2) data from a secondary assay exhibiting normal function | NA | Transactivation assays exhibiting normal transactivation (80%-115% of wt) | See PS3 (1) and (2) |
| BS4 | Lack of segregation in affected members of a family | General recommendation | NA | NA | Applied when seen in ≥2 informative meioses | NA | NA | This code should only be applied for genotype-positive, phenotype-negative (with sufficient laboratory evidence) family members |
| BP1 | Missense variant in a gene for which primarily truncating variants are known to cause disease | NA | FDP/AML is caused by both PATH missense and truncating variants | |||||
| BP2 | Observed in trans with a PATH variant for a fully penetrant dominant gene/disorder or observed in cis with a PATH variant in any inheritance pattern | General recommendation | NA | NA | NA | NA | Per original ACMG/AMP guidelines | BP2 can also be applied if the variant is detected in a homozygous state |
| BP3 | In-frame deletions/insertions in a repetitive region without a known function | NA | RUNX1 does not contain a repetitive region without known function | |||||
| BP4 | Multiple lines of computational evidence suggest no impact on gene or gene product | General recommendation | NA | NA | NA | NA | Per original ACMG/AMP guidelines | BP4 should be applied for missense variants if all of the following apply: (1) REVEL score <0.15; (2) SSF and MES predict either an increase in the canonical splice site score or a decrease in the canonical splice site score by no more than 10%; and (3) no putative cryptic splice sites are created. BP4 should also be applied for synonymous, intronic, and noncoding variants for which SSF and MES predict either an increase in the canonical splice site score or a decrease in the canonical splice site score by no more than 10% and no putative cryptic splice sites are created |
| BP5 | Variant found in a case with an alternate molecular basis for disease | NA | In rare circumstances, a patient can carry 2 variants in genes predisposing to hematologic malignancies | |||||
| BP6 | Reputable source recently reports variants as BEN, but the evidence is not available to the laboratory to perform an independent evaluation | NA | According to SVI recommendations | |||||
| BP7 | 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 | General recommendation | NA | NA | NA | NA | Per original ACMG/AMP guidelines. BP7 cannot be applied in combination with PP3 | Also applicable to intronic/noncoding variants at or beyond positions +7/–21 for which (1) SSF and MES predict either an increase in the canonical splice site score or a decrease in the canonical splice site score by no more than 10% and no putative cryptic splice sites are created; and (2) evolutionary conservation prediction algorithms predict the site as not conserved (eg, PhyloP score <0.1 or the variant is the reference nucleotide in 1 primate and/or 3 mammal species) |
| ACMG/AMP criteria code . | Original ACMG/AMP rule summary . | Specification . | Stand-alone . | Very strong . | Strong . | Moderate . | Supporting . | Comments . |
|---|---|---|---|---|---|---|---|---|
| PVS1 | Null variant in a gene where LOF is a known mechanism of disease | Gene-specific, strength | NA | Per modified RUNX1 PVS1 decision tree for SNVs, indels, and CNVs and table of splicing effects | NA | RUNX1 LOF variants are a common mechanism of disease in FPD/AML. Three major isoforms (A, B, and C) are expressed by use of 2 promotors and alternative splicing. C-terminal variants not predicted to undergo NMD are classified as PVS1_strong, deletions of exons 2 and 3, presumably only affecting RUNX1 isoform 1C, meet PVS1_moderate | ||
| PS1 | Same AA change as a previously established PATH variant regardless of nucleotide change | Strength | NA | NA | Same AA change as a previously established PATH variant regardless of nucleotide change | Same AA change as a previously established, LPATH variant regardless of nucleotide change | NA | (1) RNA data or agreement in splicing predictors show no splicing effects (SSF and MES predict either increase in canonical splice site score or decrease in canonical splice score by no more than 10% and no putative splice sites are created). (2) The previously established variant must be asserted PATH/LPATH based on MM-VCEP rules for RUNX1 before this rule can be applied |
| PS2 | De novo (maternity and paternity confirmed) in a patient with the disease and no family history | Disease-specific, strength | NA | NA | NA | Two or more proven de novo occurrences (maternity and paternity confirmed) in patients with the RUNX1-phenotype | One proven de novo occurrence (maternity and paternity confirmed) in a patient with the RUNX1-phenotype | (1) No family history is defined as: absence of the variant and any of the RUNX1-phenotypic criteria in first- and/or second-degree relatives. (2) The proband must exhibit at least 1 phenotypic FPD/AML criterion. (3) The maximum allowable strength by combining PS2 and PM6 criteria is to apply 1 moderate or 2 supporting rules |
| PS3 | Well-established in vitro or in vivo functional studies supportive of a damaging effect | Gene-specific, strength | NA | NA | Transactivation assays exhibiting altered transactivation (<20% of wt, and/or reduced to levels similar to well-established PATH variants such as R201Q or R166Q) and data from a secondary assay showing altered function. PS3 cannot be applied if the variant meets PVS1. If the variant meets criteria for PVS1_strong and PS3, we recommend either applying PVS1_strong and PS3_moderate or upgrading PVS1_strong to PVS1 without applying PS3 | Transactivation assays exhibiting altered transactivation (<20% of wt, and/or reduced to levels similar to well-established PATH variants such as R201Q or R166Q) or ≥2 secondary assays exhibiting altered function | Transactivation assays exhibiting enhanced transactivation (>115% of wt) | (1) Transactivation assays should include wt and known PATH controls, as well as coexpression with CBFβ. Promoter sequences of CSF1R (M-CSF-R), PF4, C-FMS, and GZMB, containing consensus RUNX1-binding sites have been used for transactivation assays. (2) The following secondary assays have been performed: EMSA and yeast hybrid assays (decreased DNA-binding affinity), co-IP, FRET, and affinity assays (diminished heterodimerization ability with CBFβ), IF and WB with cell fractionation (abnormal cellular localization), colony-forming assays (reduced colony-forming potential), and xenotransplantation experiments (abnormal function of mutant RUNX1 in vivo). (3) PS3 can also be applied for evidence of very low or abnormal mRNA/protein expression of the variant allele as a functional consequence of a null variant or incorrect mRNA/protein products |
| PS4 | The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in control subjects | Disease-specific, strength | NA | NA | Four or more probands meeting RUNX1-phenotypic criteria | Two to 3 probands meeting RUNX1-phenotypic criteria | One proband meeting RUNX1-phenotypic criteria | The affected individual has to fit at least 1 of the RUNX1-phenotypic criteria and the variant has to be either absent from gnomAD (overall population) or only present once |
| PM1 | Located in a mutational hotspot and/or critical and well-established functional domain without BEN variation | Gene-specific, strength | NA | NA | NA | Variant affecting 1 of the following 13 hotspot residues: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204 | Variant affecting 1 of the other AA residues 105-204 within the RHD | The RHD (AA 77-204) has been established as a highly conserved DNA-binding domain without any BEN variation in ClinVar. No germline PATH variants have been reported in residues in the region (AA 77-104) to date. The AA range under PM1_supporting may be expanded in the future to other parts of the protein if more evidence emerges |
| PM2 | Absent from control subjects | General recommendation | NA | NA | NA | Per original ACMG/AMP guidelines | NA | Variant must be completely absent from all population databases. The mean coverage of RUNX1 in the population database used should be at least 20× |
| PM3 | For recessive disorders, detected in trans with a PATH variant | NA | FPD/AML is inherited in an autosomal dominant manner | |||||
| PM4 | Protein length changes due to in-frame deletions/insertions in a nonrepeat region or stop-loss variants | Gene-specific, strength | NA | NA | NA | In-frame deletion/insertion affecting at least 1 of the 13 hotspot residues (R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204) | Other in-frame deletion/insertion affecting residues 105-204 within the RHD | See PM1 |
| PM5 | Missense change at AA residue where a different missense change determined to be PATH has been seen before | Strength | NA | NA | Missense change at the same residue where ≥2 different missense changes have previously been determined to be PATH. PM5_strong cannot be applied together with PM1 | Missense change at the same residue where a different missense change has previously been determined to be PATH | Missense change at the same residue where a different missense change has previously been determined to be LPATH | See PS1 |
| PM6 | Assumed de novo (but without confirmation of maternity and paternity) in a patient with the disease and no family history | Disease-specific, strength | NA | NA | NA | Four or more assumed de novo occurrences (without confirmation of maternity and paternity) in patients with the RUNX1-phenotype | Two or 3 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with the RUNX1-phenotype | See PS2 |
| PP1 | Cosegregation with disease in multiple affected family members | Disease-specific, strength | NA | NA | Seven or more meioses observed within 1 family or across multiple families | Five or 6 meioses observed within 1 family or across multiple families | Three or 4 meioses observed within 1 family or across multiple families | (1) Affected individuals exhibit at least 1 of the RUNX1-specific phenotypic criteria. (2) Only genotype- and phenotype-positive individuals and obligate carriers are counted. (3) Demonstration of cosegregation in multiple families is not required because many RUNX1 variants are unique and only occur in 1 family |
| PP2 | Missense variant in a gene that has a low rate of BEN missense variation and where missense variants are a common mechanism of disease | NA | Missense constraint z score for RUNX1 is <3.09 | |||||
| PP3 | Multiple lines of computational evidence support a deleterious effect on the gene or gene product | General recommendation | NA | NA | NA | NA | Per original ACMG/AMP guidelines | (1) PP3 should be applied for missense variants with a REVEL score >0.75. (2) PP3 should be applied for missense or synonymous variants if the variant alters the last 3 bases of an exon preceding a donor splice site or the first 3 bases of an exon following a splice acceptor site and the predicted decrease in the score of the canonical splice site (measured by both MES and SSF) is at least 75% regardless of the predicted creation/presence of a putative cryptic splice site. (3) PP3 should also be applied for intronic variants (in introns 4-8) located in reference to exons at positions +3 to +5 for splice donor sites or −3 to −5 for splice acceptor sites for which the predicted decrease in the score is at least 75% (measured by both MES and SSF) regardless of the predicted creation/presence of a putative cryptic splice site. (4) PP3 cannot be applied for canonical splice site variants |
| PP4 | Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology | NA | FPD/AML does not exhibit a highly specific phenotype, and there is substantial genetic heterogeneity | |||||
| PP5 | Reputable source recently reports variant as PATH, but the evidence is not available to the laboratory to perform an independent analysis | NA | According to SVI recommendations | |||||
| BA1 | Allele frequency is >5% in ESP, 1000G, or ExAC | Disease-specific | MAF ≥0.0015 (0.15%) | NA | NA | NA | NA | The variant is present in any general continental population dataset with a minimum number of 2000 alleles and variant present in ≥5 alleles |
| BS1 | Allele frequency is greater than expected for disorder | Disease-specific | NA | NA | MAF between 0.00015 (0.015%) and 0.0015 (0.15%) | NA | NA | (1) The variant is present in any general continental population dataset with a minimum number of 2000 alleles and variant present in ≥5 alleles. (2) Variant can be classified as LBEN based on BS1 alone if there is no contradictory evidence supporting pathogenicity |
| BS2 | Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age | NA | Patients with FPD/AML display incomplete penetrance, and the average age of onset of hematologic malignancies is 33 y | |||||
| BS3 | Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing | Gene-specific, strength | NA | NA | (1) Transactivation assays exhibiting normal transactivation (80%-115% of wt); and (2) data from a secondary assay exhibiting normal function | NA | Transactivation assays exhibiting normal transactivation (80%-115% of wt) | See PS3 (1) and (2) |
| BS4 | Lack of segregation in affected members of a family | General recommendation | NA | NA | Applied when seen in ≥2 informative meioses | NA | NA | This code should only be applied for genotype-positive, phenotype-negative (with sufficient laboratory evidence) family members |
| BP1 | Missense variant in a gene for which primarily truncating variants are known to cause disease | NA | FDP/AML is caused by both PATH missense and truncating variants | |||||
| BP2 | Observed in trans with a PATH variant for a fully penetrant dominant gene/disorder or observed in cis with a PATH variant in any inheritance pattern | General recommendation | NA | NA | NA | NA | Per original ACMG/AMP guidelines | BP2 can also be applied if the variant is detected in a homozygous state |
| BP3 | In-frame deletions/insertions in a repetitive region without a known function | NA | RUNX1 does not contain a repetitive region without known function | |||||
| BP4 | Multiple lines of computational evidence suggest no impact on gene or gene product | General recommendation | NA | NA | NA | NA | Per original ACMG/AMP guidelines | BP4 should be applied for missense variants if all of the following apply: (1) REVEL score <0.15; (2) SSF and MES predict either an increase in the canonical splice site score or a decrease in the canonical splice site score by no more than 10%; and (3) no putative cryptic splice sites are created. BP4 should also be applied for synonymous, intronic, and noncoding variants for which SSF and MES predict either an increase in the canonical splice site score or a decrease in the canonical splice site score by no more than 10% and no putative cryptic splice sites are created |
| BP5 | Variant found in a case with an alternate molecular basis for disease | NA | In rare circumstances, a patient can carry 2 variants in genes predisposing to hematologic malignancies | |||||
| BP6 | Reputable source recently reports variants as BEN, but the evidence is not available to the laboratory to perform an independent evaluation | NA | According to SVI recommendations | |||||
| BP7 | 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 | General recommendation | NA | NA | NA | NA | Per original ACMG/AMP guidelines. BP7 cannot be applied in combination with PP3 | Also applicable to intronic/noncoding variants at or beyond positions +7/–21 for which (1) SSF and MES predict either an increase in the canonical splice site score or a decrease in the canonical splice site score by no more than 10% and no putative cryptic splice sites are created; and (2) evolutionary conservation prediction algorithms predict the site as not conserved (eg, PhyloP score <0.1 or the variant is the reference nucleotide in 1 primate and/or 3 mammal species) |
Evidence codes are noted in bold font.
AA, amino acid; co-IP, coimmunoprecipitation; EMSA, electrophoretic mobility shift assay; FRET, fluorescence resonance energy transfer; IF, immunofluorescence; LOF, loss-of-function; MES, MaxEntScan; NA, not applicable; NMD, nonsense-mediated decay; RHD, Runt homology domain; SSF, Splice Site Finder; WB, western blot; wt, wild type.