EZHIP c.1379C>G, p.(Ser460Cys)

Reasoning

PM2 is not met because the variant is observed in population databases at a frequency exceeding the generic rare-variant threshold of <0.1% in at least one ancestry-defined dataset: gnomAD-Canada South Asian AF 0.002014 (0.2014%). The variant is rare in the larger gnomAD datasets, but PM2 requires consideration of the highest credible ancestry-specific frequency rather than overall frequency alone. The Canada South Asian result is based on only 2 alleles among 993 and may represent a sampling or ancestry-specific threshold artifact, so human review is appropriate.

Rule

{'framework': 'generic_acmg_fallback', 'threshold': '<0.1% allele frequency', 'interpretation': 'PM2 requires absence or sufficiently rare frequency in population databases; the highest ancestry-specific observed frequency exceeds the generic rare-variant threshold.'}

Evidence considered

• gnomAD v2.1: overall AF 2.1806e-05, maximum South Asian AF 0.0001572, no homozygotes.
• gnomAD v4.1: overall AF 8.7536e-06, maximum South Asian AF 8.8913e-05, no homozygotes.
• gnomAD-Canada v1.0: overall AF 0.0001381 (2/14484 alleles); South Asian AF 0.002014 (2/993 alleles), no homozygotes, variant-level filters empty and low-complexity/segmental-duplication flags false.
• Using the generic operational PM2 threshold of <0.1%, the Canada South Asian AF of 0.2014% prevents PM2; the discordant small-subset result is a threshold/sampling artifact requiring human review.

Still missing

• No EZHIP-specific VCEP framework or disease-specific maximum credible allele frequency was available.
• The ancestry-specific gnomAD-Canada observation is small (2/993 alleles) and requires review for cohort, ancestry assignment, and technical artifacts.

Sources

Reasoning

PP5 is restricted here to an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. The case-evidence bundle reports NM_203407.3:c.1379C>G as absent from ClinVar, with no variation ID, submission, review status, or expert-panel assertion. PP5 is therefore not met; ordinary or aggregate ClinVar evidence would not qualify, and none is present.

Rule

requires exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic

Evidence considered

• ClinVar search result for the exact EZHIP variant reports no result and states that the variant is absent from ClinVar; no expert-panel Pathogenic or Likely pathogenic assertion is available.

Sources

Reasoning

BA1 is not met. The highest reported allele frequency is 0.002014 (0.2014%) in the South Asian subset of gnomAD-Canada; gnomAD v2.1 and v4.1 report maximum population frequencies of 0.0001572 and 0.0000889, respectively. These values are far below the generic BA1 threshold of >1%.

Rule

{'framework': 'generic_acmg_fallback', 'threshold': '>1% allele frequency', 'interpretation': 'BA1 requires a population frequency sufficiently common for a benign classification; the observed maximum is below threshold.'}

Evidence considered

• gnomAD v2.1: 4/183436 alleles, overall AF 2.1806e-05, maximum South Asian AF 0.0001572, no homozygotes.
• gnomAD v4.1: 5/571195 alleles, overall AF 8.7536e-06, maximum South Asian AF 8.8913e-05, no homozygotes.
• gnomAD-Canada v1.0: 2/14484 alleles, overall AF 0.0001381; both alleles were in the South Asian subset (2/993, AF 0.002014), with no homozygotes.
• The observed frequencies do not approach the generic BA1 threshold of >1%; no applicable EZHIP-specific VCEP specification was retrieved.

Sources

Reasoning

BS1 is not met under the generic operational threshold of >0.3% allele frequency. The gnomAD-Canada South Asian subset has AF 0.002014 (0.2014%), which is below 0.3% and is based on only 2 alleles among 993; the larger gnomAD v2.1/v4.1 datasets show substantially lower frequencies. Disease prevalence, penetrance, and an EZHIP-specific maximum credible population frequency are unavailable, so the near-threshold ancestry-specific observation warrants review but does not satisfy BS1.

Rule

{'framework': 'generic_acmg_fallback', 'threshold': '>0.3% allele frequency', 'interpretation': 'BS1 requires an allele frequency greater than the maximum expected for the disorder; no disease-specific threshold was available, so the generic operational threshold was used.'}

Evidence considered

• gnomAD v2.1 reports overall AF 2.1806e-05 and grpmax FAF 4.258e-05; highest South Asian AF is 0.0001572 (3/19079 alleles).
• gnomAD v4.1 reports overall AF 8.7536e-06 and grpmax FAF 2.973e-05; highest South Asian AF is 8.8913e-05 (4/44988 alleles).
• gnomAD-Canada v1.0 reports overall AF 0.0001381 (2/14484 alleles), with South Asian AF 0.002014 (2/993 alleles), no homozygotes, and no reported region flags for the variant.
• The maximum observed AF of 0.2014% is below the generic BS1 threshold of >0.3%; no disease-specific threshold is available.

Still missing

• No EZHIP-specific disease prevalence, penetrance, or maximum credible allele-frequency threshold was retrieved.
• The elevated gnomAD-Canada South Asian frequency is based on only 2 alleles in that ancestry subset and should be reviewed for ancestry/cohort and technical threshold artifacts.

Sources

Reasoning

BS2 is not met. The available population datasets report zero homozygotes for this X-chromosome variant, and there is no observation of unaffected homozygous or hemizygous individuals with documented phenotype and age. Absence of a qualifying observation cannot support BS2.

Rule

{'framework': 'generic_acmg_fallback', 'requirement': 'Observed homozygotes or hemizygotes in apparently healthy individuals with adequate age/phenotype information', 'interpretation': 'No qualifying unaffected carrier observation is available.'}

Evidence considered

• gnomAD v2.1 reports 0 homozygotes overall and 0 in the South Asian subset.
• gnomAD v4.1 reports 0 homozygotes overall and 0 in the South Asian subset.
• gnomAD-Canada v1.0 reports 0 homozygotes overall and 0 in the South Asian subset; the dataset provides no phenotype-based evidence of unaffected hemizygous carriers.

Still missing

• No phenotype-annotated healthy homozygous or hemizygous carriers were identified.

Sources

Reasoning

BP6 is restricted here to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. The case-evidence bundle reports NM_203407.3:c.1379C>G as absent from ClinVar, with no variation ID, submission, review status, or expert-panel assertion. BP6 is therefore not met; ordinary laboratory submissions, aggregate labels, and non-expert assertions would not qualify.

Rule

requires exact-variant ClinVar expert-panel classification of Benign or Likely benign

Evidence considered

• ClinVar search result for the exact EZHIP variant reports no result and states that the variant is absent from ClinVar; no expert-panel Benign or Likely benign assertion is available.

Sources

Reasoning

PS1 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

No case-level evidence documents a de novo occurrence of NM_203407.3:c.1379C>G (NP_981952.1:p.(Ser460Cys)) in an affected proband with confirmed maternity and paternity. The evidence bundle contains no proband identifier, parental genotypes, trio testing results, or phenotype-linked de novo observation.

Rule

Generic ACMG/AMP PS2 requires the variant to be observed de novo in an affected individual with confirmed maternity and paternity; strength can be increased only when multiple rigorously documented de novo observations and phenotype consistency meet applicable calibrated guidance.

Evidence considered

• The case summary identifies the variant as NM_203407.3:c.1379C>G in EZHIP but provides no proband or parental testing information.
• The consolidated evidence bundle reports no literature PMIDs and no case-level de novo or family data relevant to PS2.

Still missing

• Affected proband status and phenotype consistency are not documented.
• Maternal and paternal identity and genotypes are not documented.
• Validated parental testing or trio sequencing results are not available.
• Maternity and paternity confirmation and possible mosaicism assessment are not documented.

Reasoning

No ClinGen VCEP specification is available for EZHIP (framework_mode: generic_acmg, final_classification_framework.found=false), so generic ACMG/AMP PS3 requirements apply. No variant-specific functional/biochemical assay data (e.g., HMTase activity, H3K27me3 deposition, cell-based rescue assay) for NM_203407.3:c.1379C>G (p.Ser460Cys) was identified in any source in the case bundle. OncoKB explicitly notes it did not identify variant-specific reviewed functional evidence for this variant, providing only gene-level context (EZHIP recurrently altered in endometrial stromal sarcoma and posterior fossa ependymoma). literature_pass.json found zero PMIDs for this case, and vcep_materials/fulltext_map contain no functional assay content tied to this specific substitution. PS3 cannot be scored without a validated assay demonstrating an abnormal functional effect for this exact variant.

Evidence considered

• OncoKB evidence_sentence: 'OncoKB did not identify variant-specific reviewed functional evidence for this variant; gene-level curated context is available for reviewer follow-up. EZHIP, a polycomb binding protein, is recurrently altered by rearrangement and mutation in endometrial stromal sarcomas and posterior fossa ependymoma.' This confirms absence of variant-specific functional data and provides only gene-level biological context, insufficient to support PS3.

Still missing

• No published or unpublished functional assay (in vitro or in vivo) has tested NM_203407.3:c.1379C>G / p.(Ser460Cys) specifically.
• No variant-specific data on EZHIP's PRC2-inhibitory function (H3K27me3 suppression) for this substitution.
• No ClinGen EZHIP-specific VCEP specification exists to define an approved functional assay or strength calibration for PS3.

Sources

Reasoning

PS4 requires statistically significant enrichment of the exact variant in affected individuals or a case-control comparison. The case-evidence bundle contains no affected-case series, control comparison, allele counts in affected individuals, odds ratio, or statistical test for NM_203407.3:c.1379C>G; therefore PS4 cannot be assessed.

Rule

generic ACMG/AMP PS4: requires statistically significant case-control enrichment or validated affected-case enrichment for the exact variant

Evidence considered

• The consolidated case-evidence bundle provides no case-control study, affected-case count, odds ratio, p-value, or other variant-specific enrichment evidence.

Still missing

• Variant-specific affected-case counts and an appropriate control cohort are needed, with a quantitative enrichment analysis or statistically significant case-control result.

Reasoning

PM1 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

No applicable EZHIP-specific VCEP specification was retrieved, so the generic ACMG/AMP framework applies. The case bundle contains no affected-proband observations, second pathogenic/likely pathogenic EZHIP variant, phase information, or inheritance data demonstrating that NM_203407.3:c.1379C>G (p.Ser460Cys) is in trans with a pathogenic variant in a recessive disorder. PM3 therefore cannot be assessed.

Evidence considered

• The governing materials report no EZHIP ClinGen CSPEC/VCEP specification and identify generic ACMG/AMP fallback as the framework.
• The consolidated case-evidence bundle reports no ClinVar record and no literature PMIDs, and contains no family, proband, phase, or second-variant observations relevant to PM3.
• Generic PM3 requires evidence that the variant is observed in trans with a pathogenic variant in an affected individual in the context of a recessive disorder; that evidence is absent here.

Still missing

• Affected-proband or family-level clinical observations involving this variant are not provided.
• No pathogenic or likely pathogenic variant in EZHIP for phase comparison is provided.
• No validated trans/cis phase or inheritance-mode information is provided.

Sources

Reasoning

PM5 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

No case-level or publication evidence documents a presumed de novo occurrence of NM_203407.3:c.1379C>G in an affected individual without confirmed maternity and paternity. There are no reported meioses, parental genotypes, or phenotype-linked observations from which PM6 can be evaluated.

Rule

Generic ACMG/AMP PM6 may be considered for a presumed de novo occurrence in an affected individual when maternity and paternity are not confirmed, but the observation and phenotype must still be documented; it must not be inferred from rarity or from another source's classification.

Evidence considered

• The evidence bundle contains no family, parental, proband, or presumed de novo observation for this variant.
• The literature pass contains zero PMIDs, so no publication can be used to establish presumed de novo evidence for PM6.

Still missing

• Affected individual and phenotype are not documented.
• Parental testing status and the basis for presuming de novo status are not documented.
• Maternity and paternity confirmation are not documented.
• Independent presumed de novo observations or meioses are not documented.

Reasoning

No segregation data are available for NM_203407.3:c.1379C>G. The case bundle does not identify affected or unaffected relatives, variant-positive or variant-negative relatives, informative meioses, pedigree structure, or phenotype concordance; therefore supporting segregation evidence cannot be assigned.

Rule

Generic ACMG/AMP PP1 requires co-segregation of the variant with disease in affected family members; strength depends on the number and informativeness of meioses and the quality of phenotype and genotype documentation.

Evidence considered

• The consolidated evidence bundle provides no family segregation observations for this variant.
• No publication with a family pedigree, affected-relative testing, or informative meiosis was identified in the case evidence.

Still missing

• Pedigree and number of informative meioses are not documented.
• Genotypes of affected relatives are not documented.
• Genotypes and phenotypes of unaffected relatives are not documented.
• Phenotype consistency and disease penetrance considerations are not documented.

Reasoning

PP2 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

NM_203407.3:c.1379C>G (p.(Ser460Cys)) is a missense substitution. No ClinGen VCEP CSPEC was found for EZHIP (cspec.found=false; vcep_materials.json final_classification_framework.found=false, source=generic_acmg_fallback), so the case falls back to this lab's generic ACMG in-silico calibration rules (generic_acmg_classification_rules.md). Under that framework, missense variants are assessed using REVEL only, and REVEL is explicitly excluded from being substituted with SpliceAI or BayesDel scores. The prefetch REVEL lookup failed to return a score for this variant (chrX:51408395, GRCh38) because no REVEL data file was found covering this position. BayesDel returned a score (-0.514238) but per this pipeline's calibration policy no verified published threshold/PMID exists for BayesDel, so it cannot be used for PP3/BP4 regardless of the number. SpliceAI (max delta 0.011) is available but is explicitly not applicable to a missense variant's protein-level effect assessment per the governing rule set (splice-path scores are reserved for intronic/synonymous/non-canonical-splice-position variants). With no usable calibrated computational predictor score for this missense variant, PP3 cannot be assessed.

Evidence considered

• generic_acmg_classification_rules.md specifies: missense variants use REVEL only for PP3/BP4 (REVEL>0.750 -> PP3 supporting; REVEL<0.250 -> BP4 supporting; 0.250-0.750 gray zone), and BayesDel has no verified published threshold usable under this pipeline's policy.
• prefetch.json revel field: {"score": null, "found": false}, with error 'revel: No REVEL ZIP found for chrX:51408395'.
• No ClinGen VCEP CSPEC found for EZHIP (case_summary.json cspec.found=false; vcep_materials.json final_classification_framework.found=false, source='generic_acmg_fallback').

Still missing

• REVEL score unavailable for chrX:51408395 (GRCh38) / NM_203407.3:c.1379C>G - no REVEL data file covered this position in prefetch
• BayesDel score (-0.514238) exists but has no verified published threshold/PMID usable by this pipeline for PP3/BP4 calibration
• SpliceAI score is not applicable to a missense variant under the governing generic ACMG in-silico calibration rules

Reasoning

PP4 requires the patient's phenotype or highly specific clinical findings to be highly specific for a disease associated with the gene. No patient phenotype, diagnostic features, or gene-disease-specific phenotype comparison is provided in the case-evidence bundle. Gene-level context from OncoKB is not variant-specific clinical phenotype evidence and cannot establish PP4.

Rule

generic ACMG/AMP PP4: requires a highly specific patient phenotype for a disease associated with the gene

Evidence considered

• The consolidated case-evidence bundle contains no patient phenotype or clinical diagnostic feature set for this variant.
• OncoKB provides gene-level cancer context, is explicitly not variant-specific reviewed evidence, and does not establish phenotype specificity for PP4.

Still missing

• A documented patient phenotype and a validated disease-specific phenotype comparison are needed.

Reasoning

No ClinGen VCEP specification is available for EZHIP; generic ACMG/AMP BS3 requirements apply. No functional assay evidence of any kind (normal or abnormal) for NM_203407.3:c.1379C>G / p.(Ser460Cys) was found in the case bundle. OncoKB, the only functional-context source present, explicitly states it did not identify variant-specific reviewed functional evidence. There is no basis to conclude the variant shows a normal/wild-type functional effect in a validated assay.

Evidence considered

• OncoKB evidence_sentence confirms absence of variant-specific functional evidence for this variant, precluding a BS3 (normal function) call.

Still missing

• No functional assay data (validated or otherwise) exists for this specific variant to evaluate a normal functional readout.
• No ClinGen EZHIP-specific VCEP specification exists to define an approved assay or BS3 strength calibration.

Sources

Reasoning

No non-segregation evidence is available for NM_203407.3:c.1379C>G. The case bundle does not document unaffected relatives who were tested and found to carry the variant, nor does it provide pedigree, phenotype, penetrance, or genotype-quality information needed to interpret such a finding.

Rule

Generic ACMG/AMP BS4 requires lack of segregation in an affected family context, typically demonstrated by the variant occurring in unaffected relatives when the phenotype, age, penetrance, and testing quality make non-segregation informative; population frequency alone is insufficient.

Evidence considered

• The consolidated evidence bundle contains no unaffected-relative testing or non-segregation observation for this variant.
• Population observations and absence from ClinVar do not constitute BS4 evidence and were not used to assess this criterion.

Still missing

• Tested unaffected relatives and their phenotypes are not documented.
• Variant genotypes and assay quality for unaffected relatives are not documented.
• Pedigree structure, age-related penetrance, and phenotype definition are not documented.
• No informative non-segregation event is available for review.

Reasoning

BP1 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

No applicable EZHIP-specific VCEP specification was retrieved, so the generic ACMG/AMP framework applies. The case bundle contains no phase-resolved observation showing NM_203407.3:c.1379C>G (p.Ser460Cys) in trans with a pathogenic variant in a dominant disorder or in cis with a pathogenic variant in a recessive disorder. BP2 therefore cannot be assessed.

Evidence considered

• The governing materials report no EZHIP ClinGen CSPEC/VCEP specification and identify generic ACMG/AMP fallback as the framework.
• The consolidated case-evidence bundle contains no family segregation, phase, cis/trans, or compound-genotype observations relevant to BP2.
• Generic BP2 requires an appropriate phase observation with a pathogenic variant—typically trans for a dominant disorder or cis for a recessive disorder—and no such observation is available.

Still missing

• No affected or unaffected individual-level genotype observations are provided for allelic comparison.
• No pathogenic or likely pathogenic allele for cis/trans comparison is provided.
• No inheritance mode or validated phase information is provided.

Sources

Reasoning

Same gap as PP3: this is a missense variant (NM_203407.3:c.1379C>G, p.(Ser460Cys)) with no EZHIP-specific VCEP CSPEC available, so the generic ACMG in-silico calibration rules apply and require a REVEL score for missense variants. REVEL is not available for this position (prefetch error: 'No REVEL ZIP found for chrX:51408395'). BayesDel (-0.514238) is present but has no verified published calibration threshold usable by this pipeline. SpliceAI (max delta 0.011, well below any splice-impact threshold) cannot substitute for REVEL under the governing rule because SpliceAI is reserved for intronic/synonymous/non-canonical-splice-position variants, not missense substitutions. No calibrated in-silico predictor evidence is therefore available to support or refute BP4.

Evidence considered

• generic_acmg_classification_rules.md specifies: missense variants use REVEL only for PP3/BP4 (REVEL>0.750 -> PP3 supporting; REVEL<0.250 -> BP4 supporting; 0.250-0.750 gray zone), and BayesDel has no verified published threshold usable under this pipeline's policy.
• prefetch.json revel field: {"score": null, "found": false}, with error 'revel: No REVEL ZIP found for chrX:51408395'.
• prefetch.json spliceai field: max_delta_score/max_delta = 0.011 (DS_AG 0.002, DS_AL 0.001, DS_DG 0.002, DS_DL 0.011) - noted but not applicable to this missense variant per the governing rule.

Still missing

• REVEL score unavailable for chrX:51408395 (GRCh38) / NM_203407.3:c.1379C>G - no REVEL data file covered this position in prefetch
• BayesDel score (-0.514238) exists but has no verified published threshold/PMID usable by this pipeline for PP3/BP4 calibration
• SpliceAI score is not applicable to a missense variant under the governing generic ACMG in-silico calibration rules

Reasoning

BP5 requires the variant to be found in a patient with a phenotype clearly attributable to an alternative molecular basis. The bundle provides no patient phenotype, alternate molecular diagnosis, or evidence linking this variant to a case with an established alternative cause; therefore BP5 cannot be assessed.

Rule

generic ACMG/AMP BP5: requires an alternative molecular basis explaining the patient's phenotype

Evidence considered

• The consolidated case-evidence bundle contains no patient-level phenotype, alternate molecular diagnosis, or variant-specific BP5 case observation.

Still missing

• A documented affected patient with an independently established alternative molecular cause of the phenotype is needed.