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Please note this is a beta version of the ClinGen Evidence Repository. This resource is intended to provide access to variant level evidence used and applied by ClinGen Variant Curation Expert Panels in the classification of variants. In this beta version, the evidence is limited to curation notes and referenced literature (PMIDs).

For general information about ClinGen Expert Panels and Variant Curation please visit: Clinical Domain Working Groups. For specific inquiries regarding a variant classification or evidence curation (e.g. population database queried, segregation counts or other evidence used) or to submit general comments about the evidence repo, please email us.

The resource is undergoing updates and tesing. Should you encounter any issues regarding the data displayed, lack of functionality or other problems, please let us know so we can rectify these accordingly. Your help in this regard is greatly appreciated.

MM-VCEP ACMG/AMP specifications for RUNX1

Benign
Pathogenic
Stand Alone
Strong
Supporting
Supporting
Moderate
Strong
Very Strong
Population Data


• Allele frequency is >5% in ESP, 1000G, or ExAC.
• Comments:
• • The variant is present in in any general continental population dataset with a minimum number of 2,000 alleles and variant present in ≥ 5 alleles. BA1


• Allele frequency is greater than expected for disorder.
• MAF between 0.00015 (0.015%) and 0.0015 (0.15%)
• Comments:
• • The variant is present in any general continental population dataset with a minimum number of 2,000 alleles and variant present in ≥ 5 alleles.
• • Variant can be classified as likely benign based on BS1 alone if there is no contradictory evidence supporting pathogenicity. BS1


• The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.
• 1 proband meeting RUNX1-phenotypic criteria.
• Comments:
• • The affected individual has to fit at least one of the RUNX1-phenotypic criteria AND variant has to be either absent from gnomAD (overall population) or only present once. PS4-Supporting


• The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.
• 2-3 probands meeting RUNX1-phenotypic criteria.
• Comments:
• • The affected individual has to fit at least one of the RUNX1-phenotypic criteria AND variant has to be either absent from gnomAD (overall population) or only present once. PS4-Moderate


• Absent from controls.
• Per original ACMG/AMP guidelines.
• Comments:
• • Variant must be completely absent from all population databases. The mean coverage of RUNX1 in the population database used should be at least 20x. PM2


• The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.
• ≥ 4 probands meeting RUNX1-phenotypic criteria.
• Comments:
• • The affected individual has to fit at least one of the RUNX1-phenotypic criteria AND variant has to be either absent from gnomAD (overall population) or only present once. PS4

Computational And Predictive Data


• Multiple lines of computational evidence suggest no impact on gene or gene product.
• Comments:
• • BP4 should be applied for missense variants with a REVEL score < 0.15 as well as synonymous, intronic and non-coding variants for which SSF and MES predict either an increase in the canonical splice site score or a decrease of the canonical splice site score by no more than 10% and no putative cryptic splice sites are created. BP4


• 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.
• Per original ACMG/AMP guidelines. BP7 can not be applied in combination with PP3.
• Comments:
• • Also applicable to intronic/non-coding 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 of 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 (e.g. PhyloP score < 0.1 or the variant is the reference nucleotide in one primate and/or three mammal species.). BP7


• Missense change at the same residue where a different missense change has previously been determined to be likely pathogenic.
• Comments:
• • see PS1 PM5-Supporting


• Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.
• Other in-frame deletion/insertion impacting residues 105-204 within the RHD.
• Comments:
• • See PM1 PM4-Supporting


• Multiple lines of computational evidence support a deleterious effect on the gene or gene product.
• Comments:
• • PP3 should be applied for missense variants with a REVEL score of >0.75.
• • PP3 should also be applied for missense or synonymous variants if the variant alters the last three bases of an exon preceding a splice donor site or the first three bases of an exon following an acceptor splice 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.
• • PP3 should also be applied for intronic variants (in introns 4-8) located in reference to exons at positions +3 to +5 for donor splice sites or -3 to -5 for acceptor splice sites and the predicted decrease in the score of the canonical splice site is at least 75% (measured by both MES and SSF) regardless of the predicted creation/presence of a putative cryptic splice site.
• • PP3 cannot be applied for canonical splice site variants. PP3


• Null variant in a gene where LOF is a known mechanism of disease.
• Per modified RUNX1 PVS1 decision tree for SNVs and CNVs and table of splicing effects.
• Comments:
• • RUNX1 LOF variants are a common mechanism of disease in FPD/AML. Three major isoforms (A, B, C) are expressed by use of two 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. PVS1-Moderate


• Same AA change as a previously established likely pathogenic variant regardless of nucleotide change.
• Comments:
• • RNA data or agreement in splicing predictors show no splicing effects (SSF and MES predict either increase in canonical splice site score or decrease of canonical splice score by no more than 10% and no putative splice site are created).
• • The previously established PATH/LPATH variant must be asserted pathogenic/likely pathogenic based on MM-VCEP rules for RUNX1 before this rule can be applied PS1-Moderate


• Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.
• In-frame deletion/insertion impacting at least one of the 13 hotspot residues R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201
• Comments:
• • See PM1 PM4


• Missense change at the same residue where a different missense change has previously been determined to be pathogenic.
• Comments:
• • see PS1 PM5


• Null variant in a gene where LOF is a known mechanism of disease.
• Per modified RUNX1 PVS1 decision tree for SNVs and CNVs and table of splicing effects.
• Comments:
• • RUNX1 LOF variants are a common mechanism of disease in FPD/AML. Three major isoforms (A, B, C) are expressed by use of two 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. PVS1-Strong


• Missense change at the same residue where ≥ 2 different missense changes have previoulsy been determined to be pathogenic. PM5_strong cannot be applied together with PM1.
• Comments:
• • see PS1 PM5-Strong


• Same AA change as a previously established pathogenic variant regardless of nucleotide change.
• Comments:
• • RNA data or agreement in splicing predictors show no splicing effects (SSF and MES predict either increase in canonical splice site score or decrease of canonical splice score by no more than 10% and no putative splice site are created).
• • The previously established PATH/LPATH variant must be asserted pathogenic/likely pathogenic based on MM-VCEP rules for RUNX1 before this rule can be applied PS1


• Null variant in a gene where LOF is a known mechanism of disease.
• Per modified RUNX1 PVS1 decision tree for SNVs and CNVs and table of splicing effects.
• Comments:
• • RUNX1 LOF variants are a common mechanism of disease in FPD/AML. Three major isoforms (A, B, C) are expressed by use of two 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. PVS1

Functional Data


• Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.
• Transactivation assays demonstrating normal transactivation (80-115% of wt) AND
• data from a secondary assay demonstrating normal function.
• Comments:
• • see PS3 (1) and (2) BS3


• Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.
• Transactivation assays demonstrating normal transactivation (80-115% of wt).
• Comments:
• • see PS3 (1) and (2) BS3-Supporting


• Transactivation assays demonstrating enhanced transactivation (>115% of wt).
• Comments:
• • Transactivation assays should include wt and known pathogenic controls, as well as co-expression 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.
• • 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), xenotransplantation experiments (abnormal function of mutant RUNX1 in vivo).
• • 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 PS3-Supporting


• Located in a mutational hot spot and/or critical and well-established functional domain without benign variation.
• Variant affecting one of the other AA residues 105-204 within the RHD.
• Comments:
• • The RHD (AA 77-204) has been established as highly conserved DNA-binding domain without any benign variation in ClinVar. Residues in the region (AA 77-104) are also in the RHD, but no germline variants have been reported to date. The AA range under PM1_supporting may be expanded in the future to other parts of the protein if more evidence emerges. PM1-Supporting


• 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.
• Comments:
• • Transactivation assays should include wt and known pathogenic controls, as well as co-expression 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.
• • 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), xenotransplantation experiments (abnormal function of mutant RUNX1 in vivo).
• • 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 PS3-Moderate


• Located in a mutational hot spot and/or critical and well-established functional domain without benign variation.
• Variant affecting one of the following 13 hotspot residues: R107, K110, A134, R162, R166, S167, R169, G170, K194, T196, D198, R201, R204.
• Comments:
• • The RHD (AA 77-204) has been established as highly conserved DNA-binding domain without any benign variation in ClinVar. Residues in the region (AA 77-104) are also in the RHD, but no germline variants have been reported to date. The AA range under PM1_supporting may be expanded in the future to other parts of the protein if more evidence emerges. PM1


• 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
• cannot be applied if the variant meets *PVS1*. If the variant meets criteria for *PVS1_strong
• and *PS3*, we recommend applying either *PS3_moderate
• or upgrading *PVS1_strong
• to *PVS1
• without applying *PS3*.
• Comments:
• • Transactivation assays should include wt and known pathogenic controls, as well as co-expression 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.
• • 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), xenotransplantation experiments (abnormal function of mutant RUNX1 in vivo).
• • 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 PS3

Segregation Data


• Lack of segregation in affected members of a family.
• Applied when seen in ≥ 2 informative meioses.
• Comments:
• • This code should only be applied for genotype-positive, phenotype-negative (with sufficient laboratory evidence) family members. BS4


• Co-segregation with disease in multiple affected family members.
• 3 or 4 meioses observed within one or across multiple families.
• Comments:
• • Affected individuals show at least one of the RUNX1-specific phenotypic criteria.
• • Only genotype and phenotype positive individuals and obligate carriers are counted.
• • Demonstration of co-segregation in multiple families is not required since many RUNX1 variants are unique and only occur in one family. PP1


• Co-segregation with disease in multiple affected family members.
• 5 or 6 meioses observed within one or across multiple families.
• Comments:
• • Affected individuals show at least one of the RUNX1-specific phenotypic criteria.
• • Only genotype and phenotype positive individuals and obligate carriers are counted.
• • Demonstration of co-segregation in multiple families is not required since many RUNX1 variants are unique and only occur in one family. PP1-Moderate


• Co-segregation with disease in multiple affected family members.
• ≥ 7 meioses observed within one or across multiple families.
• Comments:
• • Affected individuals show at least one of the RUNX1-specific phenotypic criteria.
• • Only genotype and phenotype positive individuals and obligate carriers are counted.
• • Demonstration of co-segregation in multiple families is not required since many RUNX1 variants are unique and only occur in one family. PP1-Strong

De novo Data


• Assumed de novo (but without confirmation of maternity and paternity) in a patient with the disease and no family history.
• 2 or 3 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with the RUNX1-phenotype.
• Comments:
• • see PS2 PM6-Supporting


• 1 proven de novo occurrence (maternity and paternity confirmed) in a patient with the RUNX1-phenotype.
• Comments:
• • No family history is defined as: absence of the variant and any of the RUNX1-phenotypic criteria in first- or second degree relatives.
• • The proband must exhibit at least one phenotypic FPD/AML criterion. (3) The maximum allowable strength by combining PS2 and PM6 criteria is to apply one moderate or two supporting rules. PS2-Supporting


• Assumed de novo (but without confirmation of maternity and paternity) in a patient with the disease and no family history.
• ≥ 4 assumed de novo occurrences (without confirmation of maternity and paternity) in patients with the RUNX1-phenotype.
• Comments:
• • see PS2 PM6


• ≥ 2 proven de novo occurrences (maternity and paternity confirmed) in patients with the RUNX1-phenotype.
• Comments:
• • No family history is defined as: absence of the variant and any of the RUNX1-phenotypic criteria in first- or second degree relatives.
• • The proband must exhibit at least one phenotypic FPD/AML criterion. (3) The maximum allowable strength by combining PS2 and PM6 criteria is to apply one moderate or two supporting rules. PS2-Moderate

Allelic Data


• 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.
• Per original ACMG/AMP guidelines.
• Comments:
• • BP2 can also be applied if the variant is detected in a homozygous state. BP2

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