Variant classification summary

NM_004456.5:c.1937A>T

EZH2 · NP_004447.2:p.(Tyr646Phe) · NM_004456.5

GRCh37: chr7:148508727 T>A · GRCh38: chr7:148811635 T>A

Gene: EZH2 Transcript: NM_004456.5

Final call

Likely Pathogenic

PS3 strong PM1 moderate PM2 supporting

Variant details

Gene

EZH2

Transcript

NM_004456.5

Protein

NP_004447.2:p.(Tyr646Phe)

gnomAD AF

ClinVar

OncoKB

Likely Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

1

The NM_004456.5:c.1937A>T (p.Tyr646Phe) variant in EZH2 is absent from large population databases including gnomAD v2.1 and v4.1, consistent with a rare pathogenic variant (PM2_Supporting).

2

The variant affects Tyr646 in the SET domain of EZH2, the critical catalytic domain responsible for histone H3K27 methyltransferase activity. This residue is a well-established mutational hotspot recurrently altered in B-cell lymphomas, and benign variation at this position is not observed (PM1).

3

Multiple independent functional studies from four publications demonstrate that the p.Tyr646Phe (also known as Y641F) alteration produces a gain-of-function effect: altered substrate specificity leading to increased H3K27 trimethylation in vitro and in vivo (Morin et al. 2010, Sneeringer et al. 2010, Yap et al. 2011), and resistance to Jak2/β-TrCP-mediated degradation resulting in enhanced protein stability (Sahraeian et al. 2014). These well-established functional assays consistently support a damaging effect on the EZH2 protein (PS3_Strong).

4

In silico predictions are discordant: REVEL predicts a pathogenic effect (0.853) while BayesDel predicts a benign effect (0.139). SpliceAI predicts no splicing impact (max delta 0.01). These mixed computational predictions do not meet the threshold for either PP3 or BP4.

5

Overall classification: Likely Pathogenic. Using generic ACMG/AMP 2015 combination rules (PMID:25741868), the evidence profile of 1 Strong (PS3) + 1 Moderate (PM1) + 1 Supporting (PM2) satisfies the Likely Pathogenic threshold (1 Strong AND 1-2 Moderate).

Final determination: Generic ACMG/AMP 2015 fallback rules support a Likely Pathogenic classification based on the observed combination of pathogenic criteria.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	N/A	This is a missense variant (p.Tyr646Phe) that does not fall into the generic PVS1 null-variant buckets of nonsense, frameshift, or canonical ±1,2 splice consensus variants.	pvs1_generic_framework
PS1	N/A	No previously classified pathogenic variant with the same amino acid change (p.Tyr646Phe) was identified in ClinVar or the curated literature to support PS1.	clinvar
PS2	Not assessed	No de novo occurrence data (with confirmed maternity/paternity) was available for this variant. The exploratory evidence search for de novo reports failed to return usable results.
PS3	Met	Well-established functional studies from four independent publications demonstrate that the p.Tyr646Phe (Y641F) alteration causes a gain-of-function effect: altered substrate specificity with increased H3K27 trimethylation activity (PMID:20081860, PMID:21078963, PMID:21190999) and resistance to Jak2/β-TrCP-mediated degradation with increased protein stability (PMID:24469040). Multiple orthogonal functional assays (in vitro methyltransferase assays, cell-based H3K27me3 quantification, protein stability/degradation assays) consistently support a damaging effect on the EZH2 protein.	PMID:20081860 PMID:21078963 PMID:21190999 PMID:24469040
PS4	Not assessed	No case-control or cohort data comparing variant prevalence in affected individuals versus controls for a germline phenotype is available. The variant has been reported 143 times in COSMIC (somatic cancer), but these are somatic observations not suitable for PS4 in a germline context.
PS5	N/A	This variant is not reported as a de novo occurrence (PS2 prerequisite not met), so PS5 cannot be applied.
PM1	Met	The p.Tyr646Phe variant is located in the SET domain of EZH2 (residues 613-727), a well-established critical functional domain responsible for histone methyltransferase activity. Position 646 is a known mutational hotspot in both somatic cancer (COSMIC, Cancer Hotspots) and is within a region where benign variation is not observed. Multiple publications confirm that this residue is recurrently altered in disease.	PMID:20081860 PMID:21190999
PM2	Met	The variant is absent from gnomAD v2.1 (exomes) and gnomAD v4.1 (exomes/genomes), indicating it is not observed in large population databases and supporting a pathogenic role.	gnomad_v2 gnomad_v4
PM5	N/A	No previously classified pathogenic missense variant at the same amino acid residue (Tyr646) with a different amino acid change was identified in ClinVar. PM5 candidate harvesting found zero same-residue comparators.	pm5_candidates
PM6	Not assessed	No confirmed de novo report for this specific variant was identified. The exploratory evidence search did not return usable de novo data.
PP1	Not assessed	No segregation data (co-segregation of the variant with disease in multiple affected family members) is available for this variant.
PP2	Not assessed	While EZH2 missense variants are a known mechanism of Weaver syndrome (a germline overgrowth disorder), gnomAD missense constraint metrics (Z-score, o/e ratio) were not retrieved to confirm a low rate of benign missense variation in this gene. Both conditions of PP2 must be met to apply this criterion.
PP3	Not met	In silico predictions are discordant: REVEL predicts a pathogenic effect (score 0.853, above the >0.5 threshold), but BayesDel predicts a benign effect (score 0.139, below the >0.27 pathogenic threshold). SpliceAI predicts no splicing impact (max delta 0.01). Multiple lines of computational evidence do not consistently support a deleterious effect.	revel bayesdel spliceai
PP4	Not assessed	No patient phenotype or family history information is available for this case to assess whether the clinical presentation is highly specific for EZH2-related disease.
PP5	N/A	No reputable source (e.g., clinical diagnostic laboratory, expert panel) has reported this variant as pathogenic without the evidence being available for independent evaluation. The variant is absent from ClinVar.
BA1	Not met	The variant is absent from gnomAD v2.1 and v4.1. BA1 requires an allele frequency >1% in any population, which is not met.	gnomad_v2 gnomad_v4
BS1	Not met	The variant is absent from gnomAD v2.1 and v4.1. BS1 requires an allele frequency >0.3%, which is not met.	gnomad_v2 gnomad_v4
BS2	Not assessed	No data are available regarding observation of this variant in a homozygous or hemizygous state in healthy adults with full penetrance expected at an early age.
BS3	Not met	Well-established functional studies demonstrate a gain-of-function damaging effect for p.Tyr646Phe (Y641F), including increased H3K27 trimethylation and enhanced protein stability. BS3 requires functional studies showing no damaging effect, which is contradicted by the available evidence.	PMID:20081860 PMID:21078963 PMID:21190999 PMID:24469040
BS4	Not assessed	No segregation data are available to evaluate lack of segregation of the variant with disease in affected family members.
BP1	Not met	BP1 applies to missense variants in genes where primarily truncating variants are known to cause disease. EZH2-related Weaver syndrome is primarily caused by missense variants; early truncating mutations are notably absent, with a proposed dominant-negative rather than haploinsufficiency mechanism. BP1 is not applicable to this gene.	pvs1_gene_context
BP2	Not assessed	No data are available regarding observation of this variant in trans with a pathogenic variant for a fully penetrant dominant disorder, or in cis with a pathogenic variant.
BP3	N/A	BP3 applies to in-frame deletions/insertions in repetitive regions; this is a missense substitution.
BP4	Not met	Multiple lines of computational evidence do not consistently suggest no impact. REVEL score is 0.853 (pathogenic), contradicting a benign interpretation. BayesDel score is 0.139 (benign-leaning) but does not override the strongly pathogenic REVEL prediction.	revel bayesdel spliceai
BP5	Not assessed	No data are available regarding observation of this variant in a case with an alternate molecular basis for disease.
BP6	Not assessed	No reputable source has reported this variant as benign. The variant is absent from ClinVar.
BP7	N/A	BP7 applies to synonymous variants with no predicted splice impact. This is a missense variant (c.1937A>T, p.Tyr646Phe), not a synonymous change.

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