POLE c.1347G>A, p.(Thr449=)

Reasoning

BS1 is met at supporting strength under the laboratory's generic non-VCEP threshold. gnomAD v4.1 shows the variant at 0.38850% in Finnish individuals (243/62,548 alleles), exceeding 0.3%; gnomAD v2.1 independently shows 0.35565% in the Finnish population (85/23,900 alleles). The variant is also present at 0.06821% overall in gnomAD v4.1 and 0.10560% overall in gnomAD v2.1. Human review is warranted because the excess is driven by Finnish ancestry and the disease-specific maximum tolerated frequency/penetrance was not provided.

Rule

Generic non-VCEP BS1: apply at supporting strength when the allele frequency is >0.3% in an appropriate population dataset and is greater than expected for the relevant disease; account for ancestry, disease prevalence, penetrance, and technical artifacts.

Evidence considered

• The POLE local framework has no population-specific BS1 rule; it directs criteria outside its explicit customizations to generic ACMG/AMP fallback handling.
• gnomAD v4.1 reports a maximum observed Finnish allele frequency of 0.00388502 (243/62,548 alleles; 1 homozygote), while the overall frequency is 0.00068211 (1,100/1,612,642 alleles).
• gnomAD v2.1 reports a maximum observed Finnish allele frequency of 0.00355649 (85/23,900 alleles; no homozygotes), while the overall frequency is 0.00105597 (297/281,258 alleles; 1 homozygote).
• gnomAD-Canada v1.0 reports 7/18,418 alleles (0.03801%) and no homozygotes, providing an independent population dataset without a comparable elevated frequency.
• The laboratory's generic non-VCEP operating threshold for BS1 is >0.3%; the Finnish-specific frequencies exceed this threshold, but the ancestry concentration and absence of a POLE-specific prevalence/penetrance model limit confidence.

Still missing

• No POLE-specific disease prevalence, penetrance estimate, or maximum credible allele-frequency calculation was provided for this synonymous variant.
• The Finnish-enriched frequency should be checked against the relevant disease ancestry distribution and any known founder effect before treating BS1 as decisive.

Sources

Reasoning

BS2 is met at supporting strength based on homozygotes observed in the gnomAD reference population: 3 homozygotes in gnomAD v4.1 and 1 homozygote in gnomAD v2.1. This is compatible with a benign or low-penetrance allele for a predominantly autosomal-dominant POLE cancer-predisposition phenotype, but gnomAD does not provide phenotype-confirmed healthy-adult status or a disease-specific penetrance assessment; therefore the evidence should receive human review.

Rule

Generic BS2: observed homozygotes in a reference population may support benignity for a fully penetrant dominant disorder, but require confirmation that the reference individuals are appropriately healthy/aged and that the disease model is applicable.

Evidence considered

• gnomAD v4.1 reports 3 total homozygotes, including 1 Finnish homozygote and 2 European non-Finnish homozygotes; the overall allele count is 1,100/1,612,642.
• gnomAD v2.1 reports 1 total homozygote; the Finnish subpopulation has no homozygotes.
• gnomAD-Canada v1.0 reports no homozygotes among 18,418 alleles.
• Because reference-population homozygote observations are not equivalent to phenotype-confirmed healthy adults, the BS2 inference is supportive rather than high confidence.

Still missing

• The gnomAD records do not establish that each homozygote is an unaffected, adequately aged adult without the relevant POLE-associated phenotype.
• Disease-specific penetrance and the expected phenotype in POLE homozygotes were not provided.

Sources

Reasoning

The variant is synonymous (NP_006222.2:p.(Thr449=)). The POLE local BP4 rule applies only to an exact missense variant listed in Supplementary Table S2 or S3 and therefore does not apply. Under the generic non-missense splice-prediction pathway, SpliceAI maximum delta 0.07 is below the BP4 threshold (<0.1), supporting no predicted splice impact and BP4_Supporting.

Rule

Generic ACMG/AMP non-missense computational pathway: SpliceAI maximum delta <0.1 supports BP4; this variant's maximum delta is 0.07.

Evidence considered

• SpliceAI for NM_006231.4:c.1347G>A reports a maximum delta score of 0.07 (DS_DL 0.070; all other delta scores <=0.006).
• The local POLE BP4 specification is limited to exact missense variants in Supplementary Table S2 or S3; the queried synonymous variant is absent from both tables.
• The generic non-missense workflow uses SpliceAI maximum delta <0.1 for BP4_Supporting.

Sources

Reasoning

NM_006231.4:c.1347G>A is synonymous, NP_006222.2:p.(Thr449=), rather than a predicted null variant. It is not a nonsense, frameshift, or canonical +/-1,2 splice-site variant, and no significant splice effect is predicted (maximum SpliceAI delta 0.07). Therefore it does not meet the generic PVS1 null-variant decision framework.

Rule

Generic ClinGen SVI PVS1 fallback: PVS1 applies only to variants predicted to cause loss of function through an eligible null/splice consequence after transcript and NMD assessment; this synonymous, non-canonical-splice variant is outside those entry criteria.

Evidence considered

• The POLE local package is not an official ClinGen CSPEC/VCEP ACMG specification and does not provide a PVS1-specific rule; generic ClinGen SVI PVS1 guidance is therefore the applicable fallback.
• The variant-level assessment identifies the consequence as synonymous and states that c.1347G>A is outside the default PVS1 buckets of nonsense, frameshift, and canonical +/-1,2 splice variants.
• SpliceAI returned DS_AG 0.000, DS_AL 0.001, DS_DG 0.006, and DS_DL 0.070 (maximum 0.07), reported as no significant predicted splice impact.

Sources

Reasoning

The applicable local POLE rule permits PS4_Supporting only for an exact missense variant recurrent in both COSMIC and TCGA endometrial-carcinoma cohorts with combined count >=10 and belonging to the specified pathogenic set. NM_006231.4:c.1347G>A is synonymous (p.Thr449=), not one of the qualifying missense variants, and no germline case-control enrichment data were supplied.

Rule

Local POLE PS4 rule: exact qualifying recurrent missense variant in COSMIC and TCGA EC cohorts, combined EC count >=10, and member of the specified pathogenic set; otherwise generic ACMG/AMP PS4 requires statistically significant affected-versus-control enrichment.

Evidence considered

• The local POLE framework specifies PS4_Supporting only for exact recurrent pathogenic-set missense variants in both COSMIC and TCGA endometrial-carcinoma cohorts, capped at Supporting because this is somatic recurrence rather than germline case-control evidence.
• The normalized consequence is NP_006222.2:p.(Thr449=), a synonymous change, so the local missense-specific PS4 rule cannot be met.

Still missing

• No germline case-control study with exact-variant counts in affected individuals and ancestry-matched controls was supplied.

Sources

Reasoning

PM2 is not met because the variant is present at appreciable frequency in multiple population datasets, including gnomAD v4.1 (1,100/1,612,642 alleles; 3 homozygotes), gnomAD v2.1 (297/281,258 alleles; 1 homozygote), and gnomAD-Canada v1.0 (7/18,418 alleles; no homozygotes).

Rule

Generic PM2: apply only when the variant is absent or nearly absent from appropriately representative population databases after considering coverage and ancestry; presence in multiple datasets prevents application.

Evidence considered

• The POLE local framework has no population-specific PM2 rule, so generic population handling applies.
• gnomAD v4.1 reports the variant at AF 0.00068211 with 3 homozygotes; gnomAD v2.1 reports AF 0.00105597 with 1 homozygote; and gnomAD-Canada v1.0 reports AF 0.00038006 with no homozygotes.
• The variant is not absent from controls and therefore cannot satisfy an absence-from-population-databases PM2 rule.

Sources

Reasoning

The variant is a synonymous SNV, p.(Thr449=), with no change in amino-acid sequence or protein length. It is neither an in-frame insertion/deletion in a non-repeat region nor a stop-loss variant; PM4 is not met.

Rule

Generic ACMG/AMP fallback: PM4 requires a protein-length change from an in-frame insertion/deletion in a non-repeat region or a stop-loss variant; neither is present.

Evidence considered

• No POLE-specific PM4 specification was available in the local framework; generic ACMG/AMP PM4 is the applicable fallback.
• The normalized consequence is NP_006222.2:p.(Thr449=), confirming that the SNV leaves the encoded threonine and protein length unchanged.

Sources

Reasoning

The variant is synonymous (NP_006222.2:p.(Thr449=)). The POLE local PP3 rule applies only to an exact missense variant listed in Supplementary Table S2 or S3, so it is inapplicable here and the generic non-missense splice-prediction pathway governs. SpliceAI maximum delta is 0.07, below the PP3 threshold (>0.2); PP3 is therefore not met.

Rule

Generic ACMG/AMP non-missense computational pathway: SpliceAI maximum delta >0.2 supports PP3; this variant's maximum delta is 0.07.

Evidence considered

• SpliceAI for NM_006231.4:c.1347G>A reports DS_AG 0.000, DS_AL 0.001, DS_DG 0.006, DS_DL 0.070, and maximum delta 0.07.
• The local POLE PP3 specification is limited to exact missense variants in Supplementary Table S2 or S3; the queried synonymous variant is absent from both tables.
• The generic non-missense workflow uses SpliceAI maximum delta >0.2 for PP3_Supporting and <0.1 for BP4_Supporting.

Sources

Reasoning

No exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion is present. Therefore PP5 cannot be applied.

Rule

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

Evidence considered

• ClinVar variation 240388 has no expert-panel submissions and is reported as Benign/Likely benign by laboratory submitters; ordinary laboratory submissions and aggregate labels are excluded from PP5.

Sources

Reasoning

BA1 is not met. The highest observed population allele frequency is 0.38850% in gnomAD v4.1 Finnish individuals (243/62,548 alleles), below the laboratory's generic non-VCEP BA1 threshold of >1%. The overall gnomAD v4.1 frequency is 0.06821% (1,100/1,612,642 alleles).

Rule

Generic non-VCEP BA1: apply only when the allele frequency exceeds 1% in an appropriate population dataset; use the highest credible population frequency and consider ancestry-specific enrichment.

Evidence considered

• The POLE local framework customizes PM1, PS4, PP3, and BP4 only; it contains no population-specific BA1 rule, so generic population handling applies.
• gnomAD v4.1 reports an overall allele frequency of 0.00068211 (1,100/1,612,642 alleles) and a maximum observed subpopulation frequency of 0.00388502 in the Finnish population (243/62,548 alleles; 1 homozygote).
• gnomAD v2.1 reports an overall allele frequency of 0.00105597 (297/281,258 alleles) and a Finnish subpopulation frequency of 0.00355649 (85/23,900 alleles).
• The generic non-VCEP operating threshold used for BA1 is >1%; neither the overall nor maximum observed population frequency reaches that threshold.

Sources

Reasoning

This is a synonymous single-nucleotide substitution, not an in-frame insertion/deletion. Consequently, repeat-region context cannot trigger BP3, irrespective of whether residue 449 lies in a repeat region.

Rule

Generic ACMG/AMP fallback: BP3 requires an in-frame deletion/insertion in a repetitive region without a known function; the variant is an SNV and does not satisfy the variant-type prerequisite.

Evidence considered

• No POLE-specific BP3 specification was available in the local framework; generic ACMG/AMP BP3 is the applicable fallback.
• The normalized consequence is synonymous p.(Thr449=), so there is no in-frame protein deletion or insertion to evaluate against a repetitive-region context.

Sources

Reasoning

No exact-variant ClinVar expert-panel Benign or Likely benign assertion is present. Therefore BP6 cannot be applied.

Rule

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

Evidence considered

• ClinVar variation 240388 has no expert-panel submissions; its Benign/Likely benign labels are from laboratory submissions and are excluded from BP6.

Sources

Reasoning

No case-level evidence in the consolidated bundle documents a confirmed de novo occurrence of NM_006231.4:c.1347G>A in an affected proband with verified maternity/paternity and both parents tested negative. Proband phenotype consistency and parental-test results are not available, so PS2 cannot be applied.

Rule

Generic PS2 requires the variant to be confirmed de novo in an affected individual, with confirmed maternity and paternity; strength may depend on phenotype specificity and parental testing quality.

Evidence considered

• The consolidated case-evidence bundle contains no documented proband, parental testing, confirmed de novo result, meiosis count, or phenotype-segregation record for this variant.
• The applicable POLE custom framework does not specify PS2; generic ACMG/AMP assessment is therefore required for this criterion.
• Generic ACMG/AMP criterion framework reviewed in Richards et al. 2015 (PMID:25741868).

Still missing

• Affected proband phenotype and clinical diagnosis
• Confirmed maternity and paternity
• Parental genotypes showing both parents negative for the variant
• Testing method, coverage, and assessment of parental mosaicism

Sources

Reasoning

No variant-specific functional assay showing a damaging effect was identified for NM_006231.4:c.1347G>A (NP_006222.2:p.(Thr449=)). The applicable POLE local framework does not specify PS3/BS3, so the generic ACMG/AMP functional-evidence definition is used; the available case bundle contains no validated assay result, assay controls, or biologically relevant functional readout supporting PS3.

Rule

Generic ACMG/AMP PS3: apply only when well-established functional studies show a damaging effect on the gene or gene product; no strength is assigned without validated, disease-relevant assay evidence and adequate controls.

Evidence considered

• The case bundle identifies the variant as synonymous, NM_006231.4:c.1347G>A / NP_006222.2:p.(Thr449=), and provides no variant-specific functional assay result.
• The POLE framework retrieved for this case is a local literature-derived framework that customizes PM1, PS4, PP3, and BP4 only; it provides no PS3 rule, so generic ACMG/AMP guidance was used.
• Richards et al. (PMID:25741868) provides the general ACMG/AMP sequence-variant interpretation framework but does not report this POLE variant or a functional assay for it.

Still missing

• A validated variant-specific functional assay with appropriate positive and negative controls and a disease-relevant readout is not available.

Sources

Reasoning

No applicable POLE-specific specification for PM3 was retrieved; the local POLE framework customizes PM1, PS4, PP3, and BP4 only, so generic ACMG/AMP criteria apply. The available case bundle contains no affected-proband observation, second pathogenic or likely pathogenic allele, phase result, or evidence that NM_006231.4:c.1347G>A is in trans with such an allele. PM3 therefore cannot be assessed.

Rule

Generic PM3: apply only when the variant is observed in trans with a pathogenic or likely pathogenic variant in an affected individual, with strength determined by the number and quality of qualifying observations; phase and phenotype must be established.

Evidence considered

• The retrieved POLE framework is explicitly limited to PM1, PS4, PP3, and BP4 and provides no PM3 rule.
• Review of the consolidated case-evidence bundle found no affected-proband case series, compound-heterozygous or homozygous observation, pathogenic allele in trans, or validated phase result for NM_006231.4:c.1347G>A (NP_006222.2:p.(Thr449=)).
• Population observations and ClinVar classifications do not establish the phase-dependent affected-proband evidence required for PM3 and were not used to infer this criterion.

Still missing

• Affected-proband data for this variant
• A second pathogenic or likely pathogenic variant in the same gene in an affected individual
• Validated phase information demonstrating the variants are in trans
• Relevant inheritance-mode and family-segregation information

Sources

Reasoning

No case-level evidence in the consolidated bundle documents a presumed de novo occurrence of NM_006231.4:c.1347G>A when parental relationships or parental genotypes were not adequately established. Without a reported proband, phenotype, and supporting parental information, PM6 cannot be applied.

Rule

Generic PM6 is used for a presumed de novo occurrence in an affected individual when maternity and/or paternity are not confirmed, provided the clinical and case evidence supports the presumption.

Evidence considered

• The consolidated case-evidence bundle contains no documented presumed de novo case, parental genotypes, parentage assessment, or phenotype details for this variant.
• The applicable POLE custom framework does not specify PM6; generic ACMG/AMP assessment is therefore required for this criterion.
• Generic ACMG/AMP criterion framework reviewed in Richards et al. 2015 (PMID:25741868).

Still missing

• Affected proband phenotype and clinical diagnosis
• Evidence that the variant is absent from tested parents or presumed de novo
• Confirmation or limitation of maternity and paternity
• Testing methodology and parental mosaicism assessment

Sources

Reasoning

No affected relatives, informative meioses, phenotype-consistent segregation, or family-level genotype data are reported for NM_006231.4:c.1347G>A. Consequently, there is no evidence to support PP1 at any strength.

Rule

Generic PP1 requires co-segregation of the variant with disease in affected family members; strength depends on the number and informativeness of meioses and the specificity of the phenotype.

Evidence considered

• The consolidated case-evidence bundle contains no pedigree, affected-relative testing, segregation results, informative meioses, or phenotype-consistency data for this variant.
• The applicable POLE custom framework does not specify PP1; generic ACMG/AMP assessment is therefore required for this criterion.
• Generic ACMG/AMP criterion framework reviewed in Richards et al. 2015 (PMID:25741868).

Still missing

• Pedigree and affected-relative information
• Genotypes of affected relatives
• Number of informative meioses
• Phenotype specificity and segregation consistency
• Assessment of unaffected relatives and possible phenocopies

Sources

Reasoning

No proband phenotype, tumor phenotype, or phenotype-specificity assessment was supplied. ClinVar lists broad/heterogeneous condition labels, which do not establish that this individual has a phenotype highly specific for a POLE-related disorder.

Rule

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

Evidence considered

• ClinVar condition labels include carcinoma of colon, colorectal cancer susceptibility, hereditary cancer-predisposing syndrome, and unspecified/not-provided entries; these are not patient-level phenotype evidence and are not sufficiently specific to support PP4.

Still missing

• Structured proband phenotype and clinical history are unavailable.
• No evidence was supplied that the observed phenotype, if any, is highly specific for POLE-related disease and has appropriate differential diagnoses excluded.

Sources

Reasoning

No variant-specific functional assay showing a normal or non-damaging effect was identified for NM_006231.4:c.1347G>A (NP_006222.2:p.(Thr449=)). The synonymous consequence and low predicted splice impact are not functional assay evidence and cannot establish BS3. The applicable POLE local framework does not specify BS3, so generic ACMG/AMP functional-evidence guidance is used.

Rule

Generic ACMG/AMP BS3: apply only when well-established functional studies show no damaging effect on protein function or splicing; computational predictions and synonymous status alone are insufficient.

Evidence considered

• The case bundle identifies a synonymous variant and reports SpliceAI max delta 0.07, but this computational prediction is not a functional assay and does not establish BS3.
• The case bundle contains no validated variant-specific biochemical, cellular, protein, or organismal assay demonstrating normal function with adequate controls.
• Richards et al. (PMID:25741868) provides general ACMG/AMP guidance but does not report this POLE variant or a benign functional assay.
• A standardized multi-gene analysis (PMID:26467025) reports aggregate trends for synonymous variants and does not provide variant-specific POLE functional evidence; its findings cannot establish BS3 for this variant.

Still missing

• A validated variant-specific functional assay demonstrating preserved function, with appropriate controls and a disease-relevant readout, is not available.

Sources

Reasoning

No informative non-segregation evidence is documented for NM_006231.4:c.1347G>A. The bundle does not identify unaffected relatives who were appropriately evaluated and tested negative or other family members in whom the variant failed to track with the relevant phenotype, so BS4 cannot be applied.

Rule

Generic BS4 requires lack of segregation with disease in affected families, typically based on appropriately evaluated unaffected relatives or other informative non-segregation data.

Evidence considered

• The consolidated case-evidence bundle contains no unaffected-relative testing, non-segregation observation, pedigree, or phenotype-penetrance analysis for this variant.
• The applicable POLE custom framework does not specify BS4; generic ACMG/AMP assessment is therefore required for this criterion.
• Generic ACMG/AMP criterion framework reviewed in Richards et al. 2015 (PMID:25741868).

Still missing

• Pedigree with clinically evaluated unaffected relatives
• Genotypes of unaffected relatives
• Definition and age-related penetrance of the relevant phenotype
• Informative meioses and exclusion of phenocopies or reduced penetrance

Sources

Reasoning

No applicable POLE-specific specification for BP2 was retrieved; the local POLE framework customizes PM1, PS4, PP3, and BP4 only, so generic ACMG/AMP criteria apply. The available case bundle contains no validated observation of NM_006231.4:c.1347G>A in trans with a pathogenic variant for a dominant disorder or in cis with a pathogenic variant, and no phase or inheritance information. BP2 therefore cannot be assessed.

Rule

Generic BP2: apply when the variant is observed in trans with a pathogenic variant for a fully penetrant dominant disorder, or in cis with a pathogenic variant for any inheritance pattern; the allelic phase and relevant inheritance model must be established.

Evidence considered

• The retrieved POLE framework is explicitly limited to PM1, PS4, PP3, and BP4 and provides no BP2 rule.
• Review of the consolidated case-evidence bundle found no phase-resolved cis or trans observation involving NM_006231.4:c.1347G>A and another pathogenic or likely pathogenic allele, and no inheritance-mode evidence applicable to BP2.
• The available ClinVar and population records do not provide the required phase-dependent allelic observation and were not used to infer this criterion.

Still missing

• Phase-resolved genotype data involving this variant and another pathogenic or likely pathogenic variant
• Clarification of the relevant POLE disease inheritance model
• Affected-proband and family data supporting the allelic relationship

Sources

Reasoning

No patient-level evidence identifies an alternate molecular diagnosis that fully explains the clinical presentation. The available ClinVar condition labels and laboratory classifications do not constitute such evidence.

Rule

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

Evidence considered

• ClinVar contains condition labels and laboratory assertions for this variant, but no documented alternate molecular diagnosis in an affected individual.

Still missing

• Patient phenotype and comprehensive molecular testing results are unavailable.
• No independently established alternate molecular diagnosis explaining the phenotype was supplied.

Sources

Reasoning

The variant is synonymous and SpliceAI predicts no significant splice impact (maximum delta 0.07), satisfying the splice-prediction component of BP7. However, no nucleotide-conservation evidence is provided in the consolidated case evidence, so the full BP7 criterion cannot be assessed.

Rule

Generic ACMG/AMP BP7 requires a synonymous variant with no predicted splice impact and a nucleotide that is not highly conserved; conservation evidence is unavailable.

Evidence considered

• SpliceAI for NM_006231.4:c.1347G>A reports a maximum delta score of 0.07, consistent with no predicted splice impact.
• No conservation score or source assessing conservation at c.1347 is present in the consolidated case evidence.

Still missing

• Variant-position nucleotide-conservation evidence is required to assess the remaining BP7 requirement.

Sources