POLE c.4477G>C, p.(Ala1493Pro)

Reasoning

PM2 is met at supporting strength. NM_006231.4:c.4477G>C was absent from gnomAD v2.1 and gnomAD v4.1, and the queried gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer datasets were also negative. This supports an observed population allele frequency of zero in the available datasets and satisfies the supplied generic PM2 supporting threshold of <=0.0001. No governing VCEP or disease-specific population-source rule was supplied for this criterion, so the all-comers gnomAD datasets were used as the default, with non-cancer results recorded as corroboration.

Rule

PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v2.1 (exome) reported the variant as absent.
• gnomAD v4.1 reported the variant as absent.
• The queried gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records also reported the variant as absent; no governing VCEP rule required these subsets instead of the all-comers default.
• Applied supplied generic PM2 supporting threshold: <=0.0001, based on the SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020; PMID:25741868).

Sources

Reasoning

The POLE custom BP4 rule applies only when the exact missense variant appears in PATH-250-323-s003 or PATH-250-323-s004 with a likely benign REVEL class and at least four benign in-silico results. Searches of PATH-250-323.pdf, PATH-250-323-s003.xlsx, and PATH-250-323-s004.xlsx found no c.4477G>C, p.Ala1493Pro, or p.A1493P entry, so the custom BP4 rule cannot be applied. Under the framework's stated fallback, the generic missense workflow uses REVEL only; the REVEL score is 0.147, below the BP4 threshold of <0.250, so BP4 is met at supporting strength. The SpliceAI max delta score of 0.38 is not used for this missense-specific generic path.

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. If the variant is absent from the supplementary tables, do not use this custom BP4 rule and fall back to the project's generic in-silico workflow.

Evidence considered

• PATH-250-323.pdf: no row or passage matching c.4477G>C, p.Ala1493Pro, or p.A1493P.
• PATH-250-323-s003.xlsx, sheet 'Suplementary Table S2': no matching variant row; the table contains missense in-silico results with REVEL class and benign-result count.
• PATH-250-323-s004.xlsx, sheet 'Supplementary Table S3': no matching variant row; the table contains non-hotspot missense in-silico results with REVEL class and benign-result count.
• Local REVEL lookup: score 0.147. Generic missense BP4 threshold: REVEL <0.250.

Still missing

• No exact variant entry was found in PATH-250-323.pdf for c.4477G>C, p.Ala1493Pro, or p.A1493P.
• No exact variant entry was found in PATH-250-323-s003.xlsx for c.4477G>C, p.Ala1493Pro, or p.A1493P.
• No exact variant entry was found in PATH-250-323-s004.xlsx for c.4477G>C, p.Ala1493Pro, or p.A1493P.

Sources

Reasoning

The governing León-Castillo et al. 2020 POLE framework restricts PM1 to the exact listed exonuclease-domain substitutions. p.A1493P is not one of the five strong hotspot substitutions, six moderate recurrent pathogenic substitutions, or four supporting recurrent uncertain substitutions. vcep_materials.json has no domain_tables entry for POLE, and the queried variant was absent from PATH-250-323.pdf and PATH-250-323-s002.xlsx; PATH-250-323-s001.tif had no searchable conversion.

VCEP rule

Apply PM1_Strong to the five established POLE exonuclease-domain hotspot missense variants emphasized by León-Castillo et al. 2020: p.P286R, p.V411L, p.S297F, p.A456P, and p.S459F. These are the exact hotspot substitutions repeatedly treated in the paper as pathogenic for the POLE-ultramutated phenotype in endometrial carcinoma. ... 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. ... Apply PM1_Supporting to exact recurrent exonuclease-domain missense variants that the paper places in the POLE-score 3 / uncertain tier rather than the pathogenic tier: p.A465V, p.L424V, p.T278M, and p.A428T. This captures hotspot/domain context without promoting them to the paper's pathogenic set.

Evidence considered

• vcep_materials.json PM1 rule: Strong applies only to p.P286R, p.V411L, p.S297F, p.A456P, and p.S459F; Moderate only to p.F367S, p.L424I, p.M295R, p.P436R, p.M444K, and p.D368Y; Supporting only to p.A465V, p.L424V, p.T278M, and p.A428T.
• vcep_materials.json domain_tables: empty for POLE, so no additional authoritative domain list applies.
• PATH-250-323.pdf and PATH-250-323-s002.xlsx: no c.4477G>C, p.Ala1493Pro, or p.A1493P entry.
• PATH-250-323-s001.tif: converted text unavailable; visual review was not possible.

Still missing

• PATH-250-323-s001.tif was unavailable as searchable text, so its visual content could not be checked for the variant.

Sources

Reasoning

The POLE custom PP3 rule applies only when the exact missense variant appears in PATH-250-323-s003 or PATH-250-323-s004 with a likely disease-causing REVEL class and no more than one benign in-silico result. Searches of PATH-250-323.pdf, PATH-250-323-s003.xlsx, and PATH-250-323-s004.xlsx found no c.4477G>C, p.Ala1493Pro, or p.A1493P entry, so the custom PP3 rule cannot be applied. Under the framework's stated fallback, the generic missense workflow uses REVEL only; the REVEL score is 0.147, below the PP3 threshold of >0.750, so PP3 is not met. The SpliceAI max delta score of 0.38 is not used for this missense-specific generic path.

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. If the variant is absent from the supplementary tables, do not use this custom PP3 rule and fall back to the project's generic in-silico workflow.

Evidence considered

• PATH-250-323.pdf: no row or passage matching c.4477G>C, p.Ala1493Pro, or p.A1493P.
• PATH-250-323-s003.xlsx, sheet 'Suplementary Table S2': no matching variant row; the table contains missense in-silico results with REVEL class and benign-result count.
• PATH-250-323-s004.xlsx, sheet 'Supplementary Table S3': no matching variant row; the table contains non-hotspot missense in-silico results with REVEL class and benign-result count.
• Local REVEL lookup: score 0.147. Generic missense PP3 threshold: REVEL >0.750.

Still missing

• No exact variant entry was found in PATH-250-323.pdf for c.4477G>C, p.Ala1493Pro, or p.A1493P.
• No exact variant entry was found in PATH-250-323-s003.xlsx for c.4477G>C, p.Ala1493Pro, or p.A1493P.
• No exact variant entry was found in PATH-250-323-s004.xlsx for c.4477G>C, p.Ala1493Pro, or p.A1493P.

Sources

Reasoning

BA1 is not met. NM_006231.4:c.4477G>C (POLE p.Ala1493Pro) was absent from gnomAD v2.1 and gnomAD v4.1; the available population evidence therefore does not approach the generic stand-alone benign threshold of allele frequency >=0.05. The queried gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer datasets also reported the variant as absent. No population observation supports an allele frequency above 5%.

Rule

BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• gnomAD v2.1 (exome) reported NM_006231.4:c.4477G>C as absent; no allele frequency, allele count, or homozygote count was reported.
• gnomAD v4.1 reported NM_006231.4:c.4477G>C as absent; no allele frequency, allele count, or homozygote count was reported.
• The queried gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records also reported the variant as absent.
• Applied generic BA1 stand-alone threshold: >=0.05, ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868).

Sources

Reasoning

BS1 is not met. The variant was absent from gnomAD v2.1 and gnomAD v4.1, with no observed population allele frequency; this is below the supplied generic BS1 threshold of >=0.01. The queried non-cancer subsets were also negative. Although disease prevalence and penetrance are not otherwise quantified in the case materials, the available frequency evidence does not support an allele frequency greater than expected for the disorder at the supplied calibration threshold.

Rule

BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v2.1 (exome) reported the variant as absent, so no population allele frequency was observed.
• gnomAD v4.1 reported the variant as absent, so no population allele frequency was observed.
• The queried gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records also reported the variant as absent.
• Applied supplied generic BS1 calibration threshold: >=0.01; per-gene derivation is preferred (Whiffin et al. 2017, PMID:28518168).

Sources

Reasoning

The variant is a missense substitution, so PS1 is applicable, but no previously established pathogenic variant producing the same Ala1493Pro amino-acid change was identified. The governing VCEP files PATH-250-323.pdf and PATH-250-323-s002.xlsx contain no c.4477G>C, p.Ala1493Pro, or p.A1493P entry; PATH-250-323-s001.tif has no available converted text. ClinVar variation 3225459 is classified as uncertain significance by a single submitter and does not establish a pathogenic comparator.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• PATH-250-323.pdf: no entry for c.4477G>C, p.Ala1493Pro, or p.A1493P; the paper's listed established pathogenic hotspot substitutions are P286R, V411L, S297F, A456P, and S459F.
• PATH-250-323-s002.xlsx Supplementary Table S1: no entry for c.4477G>C, p.Ala1493Pro, or p.A1493P.
• PATH-250-323-s001.tif: converted text unavailable; visual review was not possible.
• ClinVar variation 3225459: NM_006231.4:c.4477G>C (p.Ala1493Pro), uncertain significance, single submitter.

Still missing

• No same-amino-acid pathogenic comparator was available in the reviewed evidence.
• PATH-250-323-s001.tif was unavailable as searchable text, so its visual content could not be checked for the variant.

Sources

Reasoning

PS2 cannot be assessed because the available case evidence does not document a proband with a disease phenotype, absence of family history, or testing confirming both maternity and paternity. No parental genotypes, parentage records, or de novo laboratory result are provided; therefore neither confirmed de novo status nor the disease-context requirements can be established.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. ... Confirmation of paternity only is insufficient.

Evidence considered

• The available case evidence identifies NM_006231.4:c.4477G>C (POLE; p.Ala1493Pro) but contains no parental testing, parentage confirmation, de novo assessment, proband phenotype, or family-history data relevant to PS2.

Still missing

• Document the proband's disease phenotype and relevant clinical diagnosis.
• Document the family history and confirm that no family history is present.
• Provide variant testing results for both biological parents and evidence confirming maternity and paternity.

Reasoning

PS3 cannot be assessed because the available evidence contains no variant-specific in vitro or in vivo functional study of POLE p.Ala1493Pro. The only fetched publication was a general cancer-predisposition referral guideline that does not mention POLE, this variant, or functional assay results, and the case evidence provides no validated assay, appropriate controls, assay reproducibility, or result demonstrating a damaging effect on the gene or gene product.

Rule

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

Evidence considered

• The available evidence identifies NM_006231.4:c.4477G>C (POLE; p.Ala1493Pro) but contains no variant-specific functional assay, assay controls, reproducibility data, or functional result supporting a damaging effect.
• PMID:25394175 was reviewed through its extracted full text; it does not mention POLE, c.4477G>C, p.Ala1493Pro, or functional assay results.

Still missing

• Provide a variant-specific in vitro or in vivo functional study for POLE p.Ala1493Pro.
• Provide assay methodology, appropriate positive and negative controls, replicate or reproducibility data, and a validated interpretation of the variant's effect on POLE function or splicing.

Sources

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

PM3 cannot be assessed from the available case evidence. No affected-proband observation, pathogenic second allele, parental or offspring testing, phase result, or inheritance mode is documented for POLE NM_006231.4:c.4477G>C (p.Ala1493Pro). Therefore, it is not possible to determine whether this variant is in trans with a pathogenic variant in a recessive disorder.

Rule

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

Evidence considered

• The case evidence identifies NM_006231.4:c.4477G>C (p.Ala1493Pro) but contains no documented affected-proband, parental, offspring, phase, cis/trans, second-allele, or inheritance-mode observation.
• ClinVar contains one single-submitter germline classification as uncertain significance, with no usable criterion-lead submission or segregation evidence; this does not establish a pathogenic allele for PM3.

Still missing

• Documented affected-proband observation relevant to the disorder
• Pathogenic variant on the other allele
• Parental or offspring testing establishing trans phase
• Confirmed inheritance mode for the relevant POLE disorder

Reasoning

The variant is a missense substitution, so PM5 is applicable, but no different pathogenic missense substitution at Ala1493 was identified. The PM5 candidate artifact reports zero same-residue candidates, and the reviewed POLE VCEP files contain no c.4477G>C, p.Ala1493Pro, or p.A1493P entry; PATH-250-323-s001.tif has no available converted text.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• pm5_candidates.json: zero total candidates and zero same-residue candidates at residue 1493.
• PATH-250-323.pdf and PATH-250-323-s002.xlsx: no c.4477G>C, p.Ala1493Pro, or p.A1493P entry.
• PATH-250-323-s001.tif: converted text unavailable; visual review was not possible.

Still missing

• No pathogenic alternate missense comparator at Ala1493 was available in the reviewed evidence.
• PATH-250-323-s001.tif was unavailable as searchable text, so its visual content could not be checked for same-residue comparators.

Sources

Reasoning

PM6 cannot be assessed because the available case evidence does not establish that the variant is de novo in the proband. No parental genotypes or parentage information are reported, so there is no basis to classify the variant as assumed de novo without confirmation of maternity and paternity.

Rule

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

Evidence considered

• The available case evidence contains no parental testing, parentage information, or report of an assumed de novo occurrence for NM_006231.4:c.4477G>C.

Still missing

• Provide parental variant testing results or a documented de novo claim based on family testing.
• Clarify whether maternity and paternity were confirmed or remain unconfirmed.

Reasoning

PP1 cannot be assessed because no affected relatives, informative meioses, segregation results, or phenotype-matched family members are documented. The available evidence also does not establish the relevant familial disease relationship needed to evaluate co-segregation in POLE.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... May be used as stronger evidence with increasing segregation data.

Evidence considered

• The available case evidence contains no family pedigree, affected relatives, segregation testing, informative meioses, or family phenotype data for NM_006231.4:c.4477G>C.

Still missing

• Provide a pedigree identifying affected and unaffected relatives.
• Provide clinical phenotype information for each potentially affected relative.
• Provide variant genotypes and segregation results for informative family members.

Reasoning

The variant is missense and PP2 is applicable, but the reviewed evidence does not establish both required gene-level conditions: a low rate of benign missense variation and missense as a common disease mechanism. The available POLE material is focused on somatic exonuclease-domain hotspots and does not provide the required generic PP2 gene-level evidence.

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

• No source in the reviewed case evidence established the required POLE benign-missense constraint and common missense disease mechanism together.

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

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

BS2 cannot be assessed from the available evidence. The population records report absence of the variant but do not provide an observed healthy adult carrying the variant, zygosity, age, phenotype, or penetrance information. Therefore, there is no direct evidence that the variant was observed in a healthy adult in the zygosity required for the disease model.

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 reported the variant as absent; these records contain no healthy-carrier observation, zygosity, age, or phenotype data.
• The queried gnomAD non-cancer subset records were also absent and likewise supplied no healthy-adult carrier observation.

Still missing

• A documented healthy adult observation with the relevant zygosity is unavailable.
• Disease inheritance model, expected age of onset, and full-penetrance assumptions are not established in the available case evidence.

Sources

Reasoning

BS3 cannot be assessed because the available evidence contains no variant-specific in vitro or in vivo functional study of POLE p.Ala1493Pro. The only fetched publication was a general cancer-predisposition referral guideline that does not mention POLE, this variant, or functional assay results, and the case evidence provides no validated assay, appropriate controls, assay reproducibility, or result showing no damaging effect on protein function or splicing.

Rule

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

Evidence considered

• The available evidence identifies NM_006231.4:c.4477G>C (POLE; p.Ala1493Pro) but contains no variant-specific functional assay, assay controls, reproducibility data, or functional result supporting a benign effect.
• PMID:25394175 was reviewed through its extracted full text; it does not mention POLE, c.4477G>C, p.Ala1493Pro, or functional assay results.

Still missing

• Provide a variant-specific in vitro or in vivo functional study for POLE p.Ala1493Pro.
• Provide assay methodology, appropriate positive and negative controls, replicate or reproducibility data, and a validated interpretation showing no damaging effect on protein function or splicing.

Sources

Reasoning

BS4 cannot be assessed because no affected family members, their variant genotypes, or non-segregation observations are documented. The available evidence therefore does not show that an affected relative lacks the variant, and the phenocopy caveat cannot be evaluated.

Rule

Lack of segregation in affected members of a family. ... The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals.

Evidence considered

• The available case evidence contains no affected-family-member genotypes, phenotype assessments, or documented non-segregation observations for NM_006231.4:c.4477G>C.

Still missing

• Provide a pedigree and phenotype documentation for affected relatives.
• Provide variant testing results for affected relatives.
• Assess possible phenocopies or multiple pathogenic variants if non-segregation is observed.

Reasoning

The variant is missense and BP1 is applicable, but the reviewed evidence does not establish that POLE disease is caused primarily by truncating variants. The León-Castillo materials concern somatic missense POLE mutations and do not provide the gene-level disease-mechanism evidence required for BP1.

Rule

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

Still missing

• No source in the reviewed case evidence established a primarily truncating POLE disease mechanism.

Reasoning

BP2 cannot be assessed from the available case evidence. No pathogenic variant in trans or cis with NM_006231.4:c.4477G>C (p.Ala1493Pro) is documented, and the evidence does not establish a fully penetrant dominant POLE disorder for applying the trans pathway. The absence of a documented phase result prevents evaluation of either BP2 pathway.

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.

Evidence considered

• The case evidence contains no documented pathogenic variant in cis or trans with NM_006231.4:c.4477G>C (p.Ala1493Pro), and no molecular or familial phase result.
• The available ClinVar record is a single-submitter uncertain-significance classification and provides no phase or pathogenic-partner observation.

Still missing

• Documented pathogenic variant on the other allele or in cis
• Parental, family, or molecular phasing evidence
• Confirmed inheritance and penetrance context for the relevant disorder

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

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