POLE c.1100T>G, p.(Phe367Cys)

Reasoning

PM2 is met at Supporting strength. NM_006231.4:c.1100T>G (POLE p.Phe367Cys) is absent from the default all-comers gnomAD v2.1 and gnomAD v4.1 datasets, satisfying the supplied generic PM2 threshold of <=0.0001. The variant is a missense substitution, so the indel calling caveat does not apply.

Rule

PM2: Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 all-comers: the variant was absent, corresponding to an observed allele frequency of 0.
• gnomAD v4.1 all-comers: the variant was absent, corresponding to an observed allele frequency of 0.
• The supplied generic PM2 calibration is <=0.0001 and specifies PM2 at Supporting strength under the ClinGen SVI recommendation (PMID:25741868).
• No VCEP-specific population-source rule was provided for PM2, so the default all-comers population sources were used.

Sources

Reasoning

The queried variant is p.Phe367Cys. PATH-250-323.pdf identifies p.F367S (c.1100T>C) as one of six recurrent non-hotspot POLE exonuclease-domain mutations considered pathogenic in the TCGA-based analysis, and PATH-250-323-s002.xlsx records p.F367(S^C) with COSMIC 2 and TCGA 2. p.F367S is a different missense change at the same amino-acid residue and meets the generic PM5 comparator requirement. This is somatic endometrial-carcinoma evidence rather than a germline-established pathogenic classification, so human review is retained.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• PATH-250-323.pdf, abstract: the six additional POLE mutations in the POLE-score >=4 group were considered pathogenic.
• PATH-250-323.pdf, results text: the six recurrent non-hotspot variants considered pathogenic include F367S.
• PATH-250-323-s002.xlsx, row p.F367(S^C): COSMIC 2, TCGA 2, total 4; this confirms a different missense substitution at the same residue 367.

Still missing

• The comparator evidence is from somatic endometrial-carcinoma cohorts; no germline pathogenic classification for p.F367S was identified in the case sources.

Sources

Reasoning

The variant is a missense substitution, NM_006231.4:c.1100T>G, p.Phe367Cys/p.F367C. The governing POLE rule applies PP3 at Supporting strength when the exact missense variant appears in Supplementary Table S2 or S3, its REVEL class is likely disease causing, and the number of benign in silico results is <=1. Supplementary Table S3 contains the exact F367C row: SIFT Deleterious, PANTHER probably damaging, SNAP2 Effect, PolyPhen2 probably damaging, PROVEAN Deleterious, REVEL likely disease causing, and 0 benign results. All stated conditions are satisfied. The SpliceAI result was not used because the governing missense PP3 path is satisfied by the pre-assigned table rule and the same prediction must not be double-counted across computational criteria.

VCEP rule

Apply PP3_Supporting when the exact missense variant appears in Supplementary Table S2 or S3, the REVEL class is 'likely disease causing' (including minor spelling variants), and the number of benign in silico results is <=1.

Evidence considered

• León-Castillo et al. 2020 Supplementary Table S3, row F367C: SIFT=Deleterious; PANTHER=probably damaging; SNAP2=Effect; PolyPhen2=probably damaging; PROVEAN=Deleterious; REVEL=likely disease causing; Number of benign results=0.
• The exact variant was searched as c.1100T>G, p.Phe367Cys, and p.F367C; the governing in-silico table match is the F367C row.
• The PP3 rule requires an exact missense-table match, REVEL likely disease causing, and <=1 benign result; this row meets all three conditions.

Sources

Reasoning

The governing POLE rule requires the exact missense variant to be recurrent in both COSMIC and TCGA endometrial carcinoma cohorts, have a combined EC count >=10, and belong to the established/pathogenic set. PATH-250-323-s002.xlsx Supplementary Table S1 lists p.F367C / c.1100T>G as COSMIC 1, TCGA 0, total 1; the variant therefore fails recurrence in both cohorts and the >=10 requirement and is not one of the established/pathogenic variants specified by the framework. PATH-250-323.pdf also presents the established hotspot set as P286R, V411L, S297F, A456P, and S459F, not F367C.

VCEP rule

Apply PS4_Supporting only when the exact POLE missense variant is recurrent in both the COSMIC and TCGA endometrial carcinoma cohorts in Supplementary Table S1, has a combined EC count >=10, and belongs to the established/pathogenic set highlighted by León-Castillo et al. 2020. Under the current package this supports p.P286R, p.V411L, p.A456P, and p.S297F. Do not upgrade above Supporting because the evidence source is somatic recurrence context rather than germline case-control evidence.

Evidence considered

• León-Castillo et al. 2020 Supplementary Table S1, row p.F367C / c.1100T>G: COSMIC=1, TCGA=0, total=1.
• León-Castillo et al. 2020 PATH-250-323.pdf: the established pathogenic exonuclease-domain hotspot set is P286R, V411L, S297F, A456P, and S459F; F367C is not in that set.
• The exact variant was searched as c.1100T>G, p.Phe367Cys, and p.F367C in the available governing full-text files; no matching p.Phe367Cys or additional p.F367C entry was found in PATH-250-323.pdf beyond the c.1100T>G table entry.

Still missing

• PATH-250-323-s001.tif was unavailable for text conversion, so its visual content could not be independently reviewed.

Sources

Reasoning

The governing León-Castillo POLE framework specifies PM1 only for exact listed substitutions: the five hotspot variants, six exact pathogenic non-hotspot variants including p.F367S, or four exact supporting-tier variants. p.F367C is not one of those exact PM1 substitutions. Although PATH-250-323-s002.xlsx records p.F367C as recurrent (COSMIC 1, TCGA 0; total 1), recurrence alone does not satisfy the framework's exact-variant PM1 rule. The vcep_materials.json domain_tables key is empty, so there is no additional authoritative domain-table entry to apply.

VCEP rule

Apply PM1_Moderate to the exact non-hotspot exonuclease-domain missense variants identified in the paper as recurrent and POLE-score >=4 / classified as pathogenic in the TCGA-based analysis: p.F367S, p.L424I, p.M295R, p.P436R, p.M444K, and p.D368Y. Use this only for these exact substitutions, not all substitutions at the same codon.

Evidence considered

• vcep_materials.json PM1 rule: PM1_Moderate is limited to the exact substitutions p.F367S, p.L424I, p.M295R, p.P436R, p.M444K, and p.D368Y; p.F367C is not listed.
• PATH-250-323-s002.xlsx, row p.F367C c.1100T>G: COSMIC 1, TCGA 0, total 1.
• PATH-250-323.pdf: the paper identifies recurrent pathogenic non-hotspot variants including F367S, but does not make F367C an exact pathogenic PM1 substitution.

Still missing

• PATH-250-323-s001.tif was unavailable for text conversion and could not be reviewed visually.
• No domain_tables entry was present for POLE in vcep_materials.json.

Sources

Reasoning

PP5 is not met because the exact variant has no ClinVar record and therefore no exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. OncoKB's Likely Oncogenic label is not an exact-variant ClinVar expert-panel classification and cannot trigger generic PP5.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-variant search for NM_006231.4:c.1100T>G returned no result, with no variation ID, classification, or expert-panel submission.
• OncoKB reported Likely Oncogenic, but this is not a ClinVar expert-panel classification and was not used for PP5.

Sources

Reasoning

BA1 is not met. NM_006231.4:c.1100T>G (POLE p.Phe367Cys) is absent from the default all-comers gnomAD v2.1 and gnomAD v4.1 datasets, so no observed allele frequency reaches the stand-alone benign threshold of >=0.05. The variant is a missense substitution, so the indel calling caveat does not apply.

Rule

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 all-comers: NM_006231.4:c.1100T>G / GRCh37 12:133252327 A>C was absent, corresponding to an observed allele frequency of 0.
• gnomAD v4.1 all-comers: NM_006231.4:c.1100T>G / GRCh38 12:132675741 A>C was absent, corresponding to an observed allele frequency of 0.
• No VCEP-specific population-source rule was provided for BA1, so the default all-comers population sources were used.

Sources

Reasoning

BS1 is not met. The variant is absent from the default all-comers gnomAD v2.1 and gnomAD v4.1 datasets, providing no observed population frequency above the supplied generic BS1 threshold of >=0.01. No disease-prevalence, penetrance, or gene-specific frequency calibration establishes a lower threshold for this interpretation.

Rule

BS1: Allele frequency is greater than expected for disorder.

Evidence considered

• gnomAD v2.1 all-comers: the variant was absent, corresponding to an observed allele frequency of 0.
• gnomAD v4.1 all-comers: the variant was absent, corresponding to an observed allele frequency of 0.
• The supplied generic BS1 calibration is >=0.01; it identifies Whiffin et al. 2017 as the generic SVI default and per-gene derivation reference (PMID:28518168).
• No disease-prevalence or penetrance estimate in the available evidence establishes a POLE-specific BS1 threshold that would alter the supplied generic default.

Sources

Reasoning

The variant is within the governing missense scope and the exact F367C row is present in Supplementary Table S3, but the BP4 conditions are not satisfied. The governing rule requires REVEL Likely benign or Likely-benign and at least 4 benign in silico results. The row instead reports REVEL likely disease causing and 0 benign results. The same computational row is used to distinguish BP4 from PP3, not to apply both criteria.

VCEP rule

Apply BP4_Supporting when the exact missense variant appears in Supplementary Table S2 or S3, the REVEL class is 'Likely benign' or 'Likely-benign', and the number of benign in silico results is >=4.

Evidence considered

• León-Castillo et al. 2020 Supplementary Table S3, row F367C: REVEL=likely disease causing and Number of benign results=0.
• The BP4 rule requires an exact missense-table match, REVEL Likely benign or Likely-benign, and >=4 benign results; the exact row fails both predictor conditions.

Sources

Reasoning

BP6 is not met because the exact variant has no ClinVar record and therefore no exact-variant ClinVar expert-panel classification of Benign or Likely benign. No eligible reputable-source benign classification was identified.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-variant search for NM_006231.4:c.1100T>G returned no result, with no variation ID, classification, or expert-panel submission.

Sources

Reasoning

The variant is p.Phe367Cys (p.F367C). The reviewed VCEP materials document p.F367S and p.F367C at residue 367, but do not document a previously established pathogenic Phe367Cys substitution caused by a different nucleotide change. The available evidence therefore does not establish the same-amino-acid-change requirement for PS1.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• PATH-250-323-s002.xlsx, rows p.F367(S^C) and p.F367C/c.1100T>G: the table records different substitutions at residue 367, not an alternate nucleotide producing the same Phe367Cys amino-acid change.
• PATH-250-323.pdf, Table 1 area: F367S is listed as c.1100T>C, whereas the queried variant is c.1100T>G; these produce different amino-acid substitutions.

Still missing

• No established pathogenic alternate-nucleotide Phe367Cys record was identified in PATH-250-323.pdf or PATH-250-323-s002.xlsx.
• PATH-250-323-s001.tif was unavailable for text conversion and could not be checked for a same-amino-acid Phe367Cys entry.

Sources

Reasoning

The available case evidence does not report a proband with the disease, family history, or confirmed maternity and paternity testing for NM_006231.4:c.1100T>G; therefore de novo status cannot be assessed under PS2.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• The case evidence identifies NM_006231.4:c.1100T>G (POLE p.(Phe367Cys)) but contains no family, parental-testing, maternity, paternity, or proband-phenotype data relevant to PS2.

Still missing

• Proband disease phenotype and diagnostic confirmation
• Family-history status
• Maternity confirmation
• Paternity confirmation
• Parental testing results for NM_006231.4:c.1100T>G

Reasoning

No validated in vitro or in vivo functional assay, assay-control result, or reproducible clinical-laboratory functional study for POLE NM_006231.4:c.1100T>G (p.Phe367Cys) was identified. OncoKB provides variant-specific biological-effect context describing likely loss of function, but it does not document a qualifying assay result or validation sufficient to apply PS3 or calibrate its strength.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• OncoKB variant-specific record for POLE p.F367C describes the biological-effect context as Likely Loss-of-function and the curated oncogenicity label as Likely Oncogenic, but reports no functional assay, controls, validation data, or assay metric.
• The case literature pass found no PMIDs and paper_extracts.json was empty; therefore no variant-specific functional publication could be evaluated for PS3.

Still missing

• A variant-specific validated in vitro or in vivo functional study with appropriate controls and reproducible results is needed to assess PS3.

Sources

Reasoning

PM3 cannot be assessed because the available evidence does not establish that the relevant POLE disorder is recessive and provides no affected-proband observations, pathogenic variant in the same individual, parental/offspring testing, or phase determination showing this variant in trans with a pathogenic variant.

Rule

For recessive disorders, detected in trans with a pathogenic variant ... Note: This requires testing of parents (or offspring) to determine phase.

Still missing

• Establish the relevant POLE disorder and its inheritance mode.
• Obtain affected-proband observations and a pathogenic second allele, if present.
• Obtain parental or offspring testing to determine whether the variants are in trans.

Reasoning

The available case evidence does not report an affected proband or an assumed de novo occurrence without maternity and paternity confirmation; therefore PM6 cannot be assessed.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• The case evidence contains no documented parental testing, parental origin, de novo assessment, or proband phenotype for NM_006231.4:c.1100T>G.

Still missing

• Proband disease phenotype and diagnostic confirmation
• Parental-origin testing results
• Evidence supporting a de novo occurrence

Reasoning

The available case evidence contains no affected relatives, family segregation results, or documented disease status for family members; co-segregation cannot therefore be assessed under PP1.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• The case evidence provides no pedigree, affected relatives, family-member genotypes, informative meioses, or segregation observations for NM_006231.4:c.1100T>G.

Still missing

• Affected-family-member phenotypes
• Genotypes of informative relatives
• Number of informative meioses
• Segregation results
• Confirmation that POLE definitively causes the relevant disease

Reasoning

The reviewed case materials do not provide the two gene-level facts required for PP2: a low rate of benign missense variation and evidence that missense variants are a common disease mechanism for POLE in the applicable disorder context. The somatic POLE paper and recurrence table do not establish those generic gene-level conditions.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Still missing

• A validated POLE-specific benign missense variation rate was not available in the reviewed evidence.
• A validated POLE-specific demonstration that missense variation is a common disease mechanism was not available in the reviewed evidence.

Reasoning

No patient phenotype or family-history information is available in the case evidence. The criterion cannot be assessed without determining whether the phenotype is highly specific for a disease with a single genetic etiology.

Still missing

• Patient phenotype and family-history details are not available.

Reasoning

BS2 cannot be assessed from the available evidence. The criterion requires observation of the variant in a healthy adult in the relevant zygosity state with full penetrance expected at an early age; the available population records report the variant as absent and provide no healthy-carrier, homozygote, hemizygote, disease-prevalence, or penetrance observation.

Rule

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.

Evidence considered

• gnomAD v2.1 and v4.1 report the variant as absent; absence is not a documented observation of a healthy adult carrying the variant and therefore does not satisfy BS2.
• No case-specific source documents a healthy adult heterozygote, homozygote, or hemizygote, and no source establishes the required full early-age penetrance context.

Still missing

• A documented healthy adult carrying the variant in the relevant zygosity state is required.
• The applicable POLE disease inheritance, age-related penetrance, and expected full-penetrance context are not established for this criterion.

Sources

Reasoning

No validated in vitro or in vivo functional assay, assay-control result, or reproducible clinical-laboratory functional study showing that POLE NM_006231.4:c.1100T>G (p.Phe367Cys) has no damaging effect on protein function or splicing was identified. The available OncoKB record instead gives only variant-specific likely-loss-of-function context and does not establish benign function.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• OncoKB variant-specific record for POLE p.F367C describes the biological-effect context as Likely Loss-of-function, but provides no validated functional assay demonstrating preserved protein function or normal splicing.
• The case literature pass found no PMIDs and paper_extracts.json was empty; therefore no variant-specific functional publication could be evaluated for BS3.

Still missing

• A variant-specific validated in vitro or in vivo study demonstrating no damaging effect on POLE protein function or splicing is needed to assess BS3.

Sources

Reasoning

The available case evidence does not identify affected family members, their genotypes, or whether the variant segregates with disease; lack of segregation cannot therefore be established under BS4.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The case evidence contains no pedigree or family testing data showing affected relatives who lack the variant, and no information allowing assessment of phenocopies or multiple pathogenic variants.

Still missing

• Affected-family-member phenotypes
• Family-member genotypes
• Variant segregation results
• Assessment of possible phenocopies or more than one pathogenic variant in the family

Reasoning

The reviewed case materials do not establish that POLE disease is caused primarily by truncating variants. The León-Castillo materials address somatic POLE missense mutations in endometrial carcinoma and cannot by themselves establish the gene-level disease mechanism required for BP1.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Still missing

• A gene-level disease-mechanism review demonstrating predominantly truncating POLE pathogenic variants was not available in the reviewed evidence.

Reasoning

BP2 cannot be assessed because the available evidence contains no affected-proband or family segregation data and does not identify a pathogenic variant with which NM_006231.4:c.1100T>G is in trans or cis. The inheritance pattern and penetrance required for the dominant trans pathway are also not established.

Rule

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.

Still missing

• Identify any pathogenic variant in the same individual or family.
• Obtain parental or offspring testing to determine cis/trans phase.
• Establish the disorder's inheritance pattern and whether the dominant condition is fully penetrant.

Reasoning

The case evidence does not document an alternate molecular basis for the patient's disease. Somatic recurrence in COSMIC and a cancer database label do not establish an alternate molecular diagnosis for a patient case.

Rule

Variant found in a case with an alternate molecular basis for disease.

Still missing

• No documented alternate pathogenic molecular diagnosis or corresponding patient-level case information is available.