POLE c.4275C>T, p.(Gly1425=)

Reasoning

The variant is extremely rare in population databases: gnomAD v4.1 total AF 1.85871e-06 (3/1,614,022 alleles), grpmax FAF 6.8e-07, and 0 homozygotes; gnomAD v2.1 total AF 3.98105e-06 (1/251,190 alleles), also with 0 homozygotes. It is absent from gnomAD-Canada v1.0. This meets the non-VCEP rare-frequency PM2 supporting threshold of <0.1%.

Rule

Generic/non-VCEP population fallback: PM2 Supporting for an absent or extremely rare variant in population controls, operationalized here as AF <0.1%, with ancestry-aware review and no homozygote signal.

Evidence considered

• gnomAD v4.1: 3/1,614,022 total alleles, AF 1.85871e-06 (0.000185871%), grpmax FAF 6.8e-07, and 0 homozygotes; the highest population AF is 2.54254e-06 in European non-Finnish individuals.
• gnomAD v2.1: 1/251,190 total alleles, AF 3.98105e-06 (0.000398105%), highest population AF 8.80483e-06 in European non-Finnish individuals, and 0 homozygotes.
• gnomAD-Canada v1.0: absent.
• Operational non-VCEP PM2 rule: allele frequency below 0.1% supports PM2 at Supporting strength when the variant is not common in population controls; the observed frequencies are far below this threshold.

Sources

Reasoning

Per this pipeline's generic PP3/BP4 in-silico calibration (applied because the variant is synonymous, not missense, and is absent from the custom POLE literature framework's Le\u00f3n-Castillo et al. 2020 supplementary in-silico tables so that gene-specific missense-only rule does not govern), SpliceAI is the designated predictor for synonymous/non-missense variants, with max delta < 0.1 triggering BP4 supporting. SpliceAI max delta score = 0.037 (DS_AG 0.01, DS_AL 0.013, DS_DG 0.037, DS_DL 0.008), clearly below the 0.1 threshold, so BP4_Supporting is met.

Rule

generic_acmg_pp3_bp4_synonymous_spliceai_path

Evidence considered

• SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.04)'; underlying scores DS_AG=0.01, DS_AL=0.013, DS_DG=0.037, DS_DL=0.008, max_delta=0.037.
• Generic fallback rule (generic_acmg_classification_rules.md, cited as generic_acmg_combination_rules) specifies: for intronic/synonymous/non-canonical-splice-position variants, use SpliceAI only, and SpliceAI max delta < 0.1 triggers BP4 (supporting).
• Custom POLE literature-derived PP3/BP4 rule (Le\u00f3n-Castillo et al. 2020 supplementary Tables S2/S3, REVEL-class based) was checked but does not apply because it is scoped to exact missense variants present in those tables, and this variant is synonymous and absent from them.

Sources

Reasoning

The applicable local POLE framework limits PS4 to Supporting and requires the exact missense variant to be recurrent in both COSMIC and TCGA endometrial-carcinoma cohorts, have a combined count of at least 10, and be in the established pathogenic set. NM_006231.4:c.4275C>T is synonymous (p.Gly1425=), is reported only three times in COSMIC, and no qualifying TCGA recurrence or case-control enrichment is supplied.

Rule

León-Castillo 2020 local POLE PS4 rule: Supporting only for an exact established pathogenic POLE missense variant recurrent in both COSMIC and TCGA endometrial-carcinoma cohorts with combined count >=10.

Evidence considered

• The León-Castillo 2020 local POLE framework customizes PS4 only for exact recurrent pathogenic exonuclease-domain missense variants and caps it at Supporting.
• COSMIC reports the exact variant three times, below the local framework's required combined endometrial-carcinoma count of 10; the bundle provides no qualifying TCGA count.

Still missing

• A variant-specific, germline case-control study or qualifying recurrence data in both required endometrial-carcinoma cohorts is not available in the bundle.

Sources

Reasoning

NM_006231.4:c.4275C>T is a synonymous variant (NP_006222.2:p.(Gly1425=)); it is not a missense substitution, so the REVEL-based missense in-silico path does not apply and REVEL is not available for this variant (score null/not found). Per this pipeline's generic PP3/BP4 in-silico calibration (used because no CSPEC was found and the custom POLE literature-derived framework's PP3/BP4 rule only covers exact missense variants listed in its Le\u00f3n-Castillo et al. 2020 supplementary in-silico tables, which this synonymous variant is absent from), synonymous/non-missense variants are assessed with SpliceAI only, requiring max delta score > 0.2 to trigger PP3. SpliceAI predicts max delta score = 0.037 (DS_AG 0.01, DS_AL 0.013, DS_DG 0.037, DS_DL 0.008), far below the 0.2 supporting-pathogenic threshold, so PP3 is not met.

Rule

generic_acmg_pp3_bp4_synonymous_spliceai_path

Evidence considered

• Variant NM_006231.4:c.4275C>T / NP_006222.2:p.(Gly1425=) is synonymous, not missense, so the REVEL-based missense computational path is inapplicable and REVEL score is unavailable (null/not found) in prefetch data.
• The custom POLE literature framework's PP3/BP4 rule (from Le\u00f3n-Castillo et al. 2020 supplementary Tables S2/S3) requires the exact variant to be an in-table missense substitution with a listed REVEL class; this synonymous variant is not present in those tables, so that custom rule does not apply and the pipeline's generic in-silico fallback governs instead.
• Generic fallback for non-missense (synonymous/intronic) variants uses SpliceAI only: max delta > 0.2 triggers PP3 supporting. SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.04)' with computed max_delta = 0.037, which is well below the 0.2 PP3 threshold.

Sources

Reasoning

The exact variant has two Likely benign ClinVar submissions, both from clinical laboratories with single-submitter review status; the submission audit identifies zero expert-panel submissions. No exact-variant Pathogenic or Likely pathogenic expert-panel classification is present.

Rule

PP5 is applied only for an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification.

Evidence considered

• ClinVar variation 1127492 exactly matches NM_006231.4:c.4275C>T (p.Gly1425=); both submissions are Likely benign single-submitter laboratory assertions, and the audit reports zero expert-panel submissions.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification would be required for PP5.

Sources

Reasoning

The variant is far below a stand-alone benign high-frequency threshold. gnomAD v4.1 reports AF 1.85871e-06 (3/1,614,022 alleles), highest population AF 2.54254e-06 in European non-Finnish individuals, and grpmax FAF 6.8e-07; gnomAD v2.1 reports AF 3.98105e-06. These frequencies do not meet BA1.

Rule

Generic ACMG/AMP population evidence fallback: BA1 requires a clearly benign, population-level frequency above the stand-alone high-frequency threshold; this variant is orders of magnitude below the operational 1% BA1 threshold.

Evidence considered

• gnomAD v4.1: 3 alternate alleles among 1,614,022 total alleles; total AF 1.85871e-06 (0.000185871%), European non-Finnish AF 2.54254e-06 (0.000254254%), and grpmax FAF 6.8e-07.
• gnomAD v2.1: 1 alternate allele among 251,190 alleles; total AF 3.98105e-06 (0.000398105%).
• The variant is absent from gnomAD-Canada v1.0; absence in an additional reference population does not raise its frequency to BA1 range.

Sources

Reasoning

The observed population frequency is not greater than expected for a POLE disease-associated variant under the operational BS1 threshold. The maximum reported frequency is 2.54254e-06 in gnomAD v4.1 European non-Finnish individuals, with grpmax FAF 6.8e-07, and 3 total alternate alleles.

Rule

Generic ACMG/AMP population evidence fallback: BS1 requires an allele frequency greater than expected for the relevant disorder; this operating framework uses >0.3% as the BS1 threshold, which is not approached here.

Evidence considered

• gnomAD v4.1 reports total AF 1.85871e-06, highest population AF 2.54254e-06 in European non-Finnish individuals, and grpmax FAF 6.8e-07; no ancestry shows an elevated frequency compatible with BS1.
• gnomAD v2.1 reports total AF 3.98105e-06 and highest population AF 8.80483e-06 in European non-Finnish individuals.
• Both datasets show only rare singleton/three-allele observations, not an excess frequency.

Sources

Reasoning

No homozygotes are reported in gnomAD v2.1 or v4.1, and no unaffected adult homozygote or hemizygote observation is available. Therefore the evidence does not satisfy BS2; absence of homozygotes is not positive BS2 evidence.

Rule

Generic ACMG/AMP BS2 requires observation of the variant in healthy adults, typically in homozygous or hemizygous state for a dominant disease or in an appropriate genotype for a recessive disease. Zero homozygotes is not BS2.

Evidence considered

• gnomAD v4.1 reports 0 homozygotes overall and 0 homozygotes in every listed population; the variant is observed in only 3 alleles.
• gnomAD v2.1 reports 0 homozygotes overall and 0 homozygotes in every listed population; the variant is observed in only 1 allele.
• gnomAD-Canada v1.0 reports the variant as absent, so it provides no healthy homozygote observation.

Sources

Reasoning

Although the exact variant has a ClinVar Likely benign aggregate label, its two underlying assertions are ordinary single-submitter clinical-laboratory submissions and the audit identifies zero expert-panel submissions. This does not meet the required exact-variant ClinVar expert-panel Benign or Likely benign assertion.

Rule

BP6 is applied only for an exact-variant ClinVar expert-panel Benign or Likely benign classification.

Evidence considered

• ClinVar variation 1127492 exactly matches NM_006231.4:c.4275C>T (p.Gly1425=), but its two Likely benign submissions are non-expert clinical-laboratory assertions; the audit reports zero expert-panel submissions.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely benign classification would be required for BP6.

Sources

Reasoning

No proband-level case description or validated parental testing is provided. The available evidence does not establish that NM_006231.4:c.4275C>T occurred de novo, nor does it document maternity/paternity confirmation or parental mosaicism assessment.

Rule

Generic ACMG/AMP PS2 requires a confirmed de novo occurrence in an affected proband, with maternity and paternity confirmed; strength may be increased only when the phenotype is highly specific and/or additional de novo observations support the finding.

Evidence considered

• The consolidated case-evidence bundle contains no proband-level de novo observation, trio-testing result, parental genotype, maternity/paternity confirmation, or parental mosaicism result for this variant.

Still missing

• Proband phenotype and case-level clinical report
• Genotype results for both biological parents
• Validated maternity and paternity
• Assessment for parental mosaicism

Sources

Reasoning

No functional/biochemical assay data (in vitro polymerase activity, splicing minigene, RNA studies, or other validated functional assay) for NM_006231.4:c.4275C>T (NP_006222.2:p.(G1425=)) were found anywhere in the case evidence bundle. The POLE custom gene framework (based on Leon-Castillo et al. 2020) defines rules only for PM1, PS4, PP3, and BP4 (exonuclease-domain hotspot/recurrence and in silico context for somatic endometrial carcinoma variants) and does not address PS3/BS3 or provide any functional assay calibration. The only literature retrieved (PMID:25394175, PMID:28492532) does not mention this variant or any POLE functional assay; PMID:25394175 was explicitly checked in paper_extracts.json and confirmed to have no relevance. No functional study source is present in source_registry.json.

Still missing

• No validated functional assay (biochemical, splicing, or minigene) evidence located for this variant or gene in the current evidence bundle.
• No POLE-specific PS3/BS3 rule specification available in the custom gene framework provided for this case.

Reasoning

No affected proband or family-level observation was provided showing NM_006231.4:c.4275C>T in trans with a pathogenic or likely pathogenic variant, and no phase-resolved biallelic genotype or inheritance evidence is available. The variant is synonymous (NP_006222.2:p.(Gly1425=)); the available ClinVar, population, somatic, and general literature records do not establish PM3.

Rule

Generic PM3: pathogenic or likely pathogenic evidence for the variant in affected individuals with a recessive disorder, with the strength determined by the number and independence of confirmed affected observations and by phase evidence showing the variant in trans with another pathogenic or likely pathogenic allele. No applicable POLE-specific PM3 specification was provided.

Evidence considered

• The POLE case bundle identifies NM_006231.4:c.4275C>T as a synonymous variant, NP_006222.2:p.(Gly1425=), but contains no affected-proband, compound-heterozygous, biallelic, phase, or inheritance observation relevant to PM3.
• The local POLE framework specifies PM1, PS4, PP3, and BP4 only; PM3 therefore requires generic ACMG/AMP assessment, for which the required trans-phase affected-case evidence is absent.
• The only extracted paper finding states that PMID:25394175 does not mention POLE or this variant and provides no variant observation, segregation, or inheritance evidence.

Still missing

• Affected-proband observations with a second pathogenic or likely pathogenic variant are not available.
• Phase information establishing the two variants in trans is not available.
• Inheritance and segregation data for a relevant recessive disease model are not available.

Sources

Reasoning

No case-level report identifies this variant as presumed de novo, and no parental testing, maternity/paternity information, or phenotype-specific de novo observation is available. The variant's presence in population databases does not establish or refute a presumed de novo event in a particular proband.

Rule

Generic ACMG/AMP PM6 may be used for a presumed de novo variant when parental testing is unavailable, but the de novo inference must be supported by a documented affected proband and appropriate phenotype; it is weaker than PS2 because parentage and/or parental genotype is not confirmed.

Evidence considered

• The consolidated case-evidence bundle provides no presumed-de-novo case observation or parental testing record for NM_006231.4:c.4275C>T.

Still missing

• A published or clinical case identifying the proband
• Parental testing or documented presumed-de-novo basis
• Maternity and paternity confirmation
• Phenotype consistency with a POLE-related disorder

Sources

Reasoning

No affected relatives carrying the variant, unaffected relatives lacking the variant, pedigree, informative meioses, or segregation likelihood data are provided. Therefore, cosegregation with disease cannot be assessed.

Rule

Generic ACMG/AMP PP1 requires cosegregation of the variant with disease in affected family members; strength can be increased only with multiple informative meioses or an applicable validated quantitative segregation framework.

Evidence considered

• The consolidated case-evidence bundle contains no family pedigree, affected-relative genotype, unaffected-relative genotype, informative meiosis count, or cosegregation calculation for this variant.

Still missing

• Pedigree and disease status of relatives
• Genotypes of affected relatives
• Genotypes of informative unaffected relatives
• Number of informative meioses and segregation analysis

Sources

Reasoning

No proband phenotype, disease diagnosis, tumor characteristics, or phenotype-specific testing information is provided. Therefore phenotype specificity for a POLE-associated condition cannot be assessed.

Rule

Generic ACMG/AMP PP4: the patient's phenotype or family history must be highly specific for a disease with a single genetic etiology.

Evidence considered

• The bundle contains variant normalization and database evidence but no case-level phenotype or diagnostic workup.

Still missing

• Proband-level phenotype and diagnostic testing information sufficient to determine whether the phenotype is highly specific for a POLE-associated disorder.

Reasoning

No functional/biochemical assay data showing normal/wild-type function for NM_006231.4:c.4275C>T were found anywhere in the case evidence bundle. As with PS3, the custom POLE gene framework in this case does not define BS3 rules, and no functional-study source is present in source_registry.json or paper_extracts.json.

Still missing

• No validated functional assay evidence (showing normal function) located for this variant or gene in the current evidence bundle.
• No POLE-specific BS3 rule specification available in the custom gene framework provided for this case.

Reasoning

No informative unaffected relatives are documented as carrying or lacking the variant, and no phenotype or pedigree information is available to determine whether the variant fails to segregate with disease. Population frequency and ClinVar classification are not BS4 evidence.

Rule

Generic ACMG/AMP BS4 requires lack of segregation with disease in affected family members or presence of the variant in appropriately informative unaffected relatives; the relatives must be sufficiently phenotyped and informative for the disease mechanism.

Evidence considered

• The consolidated case-evidence bundle contains no documented unaffected relatives tested for this variant and no non-segregation analysis.

Still missing

• Pedigree with reliable disease-status ascertainment
• Genotypes of clinically unaffected relatives
• Age and phenotype information sufficient to interpret unaffected status
• Informative non-segregation observations

Sources

Reasoning

No phase-resolved observation was provided showing NM_006231.4:c.4275C>T in trans with a pathogenic or likely pathogenic variant in a dominant disorder, or in cis with a pathogenic variant in a recessive disorder. The available records do not establish the allelic relationship required for BP2.

Rule

Generic BP2: the variant is observed in trans with a pathogenic variant for a dominant disorder, or in cis with a pathogenic variant for a recessive disorder, with appropriate confirmation of phase and disease context. No applicable POLE-specific BP2 specification was provided.

Evidence considered

• The case bundle contains no phase-resolved cis/trans observation involving NM_006231.4:c.4275C>T and a pathogenic or likely pathogenic variant.
• The local POLE framework specifies PM1, PS4, PP3, and BP4 only; BP2 therefore requires generic ACMG/AMP assessment, but the required allelic phase and inheritance evidence is absent.
• The only extracted paper finding states that PMID:25394175 does not mention POLE or this variant and provides no variant observation, segregation, or inheritance evidence.

Still missing

• No family or proband genotype data establish cis or trans phase with a pathogenic or likely pathogenic variant.
• No inheritance model and no affected/unaffected status information are available for an informative individual or family.

Sources

Reasoning

No proband diagnosis, phenotype, or molecular workup is supplied, and no alternate molecular cause is documented. BP5 cannot be assessed.

Rule

Generic ACMG/AMP BP5: a variant is found in a case with an alternate molecular basis for disease.

Evidence considered

• No co-occurrence or alternate molecular diagnosis information is present in the bundle.

Still missing

• Proband-level phenotype and molecular testing results establishing an alternate molecular basis for the condition.