POLE c.547G>A, p.(Ala183Thr)

Reasoning

The applicable threshold is the SVI recommendation to apply PM2 at Supporting strength for a variant that is absent from, or at extremely low frequency in, population databases, using a frequency cut-off of <= 0.0001 (PMID:25741868, as supplied). The governing POLE framework customizes only PM1, PS4, PP3 and BP4, specifies no population-frequency rule and no non-cancer or exome-only source, so the generic threshold applies and the all-comers GNOMAD_V2_1 / GNOMAD_V4_1 entries are the default sources. Explicit substitution with this variant's numbers: gnomAD v4.1 total AF = 3.84184e-05 (62/1,613,808 alleles, homozygotes 0) with grpmax FAF 3.775e-05; gnomAD v2.1 total/exome AF = 7.9552e-06 (2/251,408 alleles, homozygotes 0) with grpmax FAF 2.92e-06. Is 3.84184e-05 <= 0.0001? Yes, by roughly 2.6-fold, and the v2.1 figure is roughly 12-fold below. The ancestry-specific maximum also stays below the cut-off: European (non-Finnish) 4.83167e-05 (57/1,179,716) in v4.1 and 1.75889e-05 (2/113,708) in v2.1. Component frequencies are likewise below 0.0001 (v4.1 exome 4.03695e-05; v4.1 genome 1.96969e-05). The non-cancer subsets were checked and agree: gnomAD v2.1 non-cancer exomes 8.44338e-06 (2/236,872) and gnomAD v3.1 non-cancer genomes 2.02782e-05 (3/147,942). The variant is also absent from gnomAD-Canada v1.0 (HostSeq genomes) and no homozygote has been observed in any cohort. Because the variant is not truly absent but is present at extreme rarity, PM2 is applied at Supporting strength only, never upgraded, consistent with the SVI downgrade; the original ACMG/AMP moderate framing (absence, or frequency below the expected carrier frequency, in a general population of more than 1000 individuals) is superseded here. A ClinVar submitter's own criterion-level PM2 assertion for this variant was noted as corroborating context, but the verdict rests entirely on the directly retrieved gnomAD allele frequencies above.

Rule

PM2 (Supporting, per SVI downgrade): allele frequency <= 0.0001 in population databases (PMID:25741868); no POLE-specific overriding rule exists; Supporting strength only, never upgraded.

Evidence considered

• gnomAD v4.1 (all-comers, GRCh38 chr12-132679528-C-T): total AF 3.84184e-05 (62/1,613,808 alleles, homozygotes 0), grpmax FAF 3.775e-05; exome 4.03695e-05 (59/1,461,500); genome 1.96969e-05 (3/152,308); highest ancestry European (non-Finnish) 4.83167e-05 (57/1,179,716). All values below 0.0001.
• gnomAD v2.1 (all-comers, GRCh37 12-133256114-C-T): total/exome AF 7.9552e-06 (2/251,408 alleles, homozygotes 0), grpmax FAF 2.92e-06; highest ancestry European (non-Finnish) 1.75889e-05 (2/113,708). Below 0.0001.
• Non-cancer sensitivity check: gnomAD v2.1 non-cancer exomes AF 8.44338e-06 (2/236,872 alleles, homozygotes 0); gnomAD v3.1 non-cancer genomes AF 2.02782e-05 (3/147,942 alleles, homozygotes 0). Both below 0.0001, so the source choice does not change the verdict.
• gnomAD-Canada v1.0 (HostSeq genomes): 'Absent from gnomAD-Canada v1.0'.
• Threshold basis: ClinGen SVI recommendation to apply PM2 at Supporting strength with a frequency cut-off of <= 0.0001 (PMID:25741868), as supplied; the original ACMG/AMP 2015 PM2 text requires absence, or frequency below the expected carrier frequency, in a general population of more than 1000 individuals.
• A ClinVar submitter criterion-lead signal records PM2 for this exact variant ('this variant is present in population databases (no rsid available, gnomad 0...'), noted as corroborating context only; the verdict is based on the directly retrieved gnomAD frequencies.
• Framework scope: no CSPEC is available for POLE (cspec.found = false) and the local custom POLE framework defines no population criterion, so the generic threshold and the all-comers gnomAD default sources were used.

Still missing

• The variant is present, not absent, in gnomAD v4.1 (62 alleles), so PM2 rests on extreme rarity rather than absence; the Supporting-strength cap was applied and PM2 was not upgraded.
• gnomAD-Canada v1.0 returned no record for this variant, so that cohort contributes an absence call only and no allele-frequency estimate.
• No POLE-specific PM2 rule or POLE-specific rarity threshold exists in the governing framework, so the generic 0.0001 Supporting threshold was used.

Sources

Reasoning

PP2 requires a missense variant in a gene in which missense variation is a common mechanism of disease and in which benign missense variation is rare. The mechanism limb is well supported for POLE: germline POLE disease (polymerase proofreading-associated polyposis / POLE-related colorectal cancer predisposition) is caused by heterozygous missense substitutions in the exonuclease/proofreading domain, as stated in the OncoKB gene-level curation carried in this case ('Germline heterozygous loss-of-function mutations in the exonuclease domain of POLE cause polyposis and predispose individuals to colorectal cancer', with supporting PMIDs 23263490, 23528559, 24501277, 26133394, 26493165), and the governing Leon-Castillo et al. 2020 package likewise treats missense exonuclease-domain substitutions as the pathogenic class (p.P286R, p.V411L, p.S297F, p.A456P, p.S459F). The assessed variant is a missense change, so this limb is satisfied. The second limb - a low rate of benign missense variation - is not directly verifiable from the material assembled for this case: no gene-level missense-constraint metric (gnomAD missense Z-score / MOI or equivalent) is present anywhere in the case evidence, and PP2 therefore rests on the reported gene-level rarity of benign POLE missense variants rather than a formal constraint statistic. PP2 is applied at supporting strength, the standard strength for this criterion, with the unverified constraint limb flagged for human review.

Rule

PP2 (ACMG/AMP 2015, PMID:25741868): missense variant in a gene with a low rate of benign missense variation in which missense variants are a common mechanism of disease; applied at supporting strength (the standard strength for this criterion). No POLE-specific PP2 specification exists in the governing local framework.

Evidence considered

• PP2 definition (PMID:25741868): missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease.
• OncoKB gene-level curation: 'Germline heterozygous loss-of-function mutations in the exonuclease domain of POLE cause polyposis and predispose individuals to colorectal cancer' - these germline alleles are missense proofreading-domain substitutions, establishing missense as a common disease mechanism for POLE.
• OncoKB background states that POLE 'contains an exonuclease "proofreading" domain' whose alteration results in accumulation of single-nucleotide variants and an ultramutated phenotype - a missense-driven mechanism.
• The governing Leon-Castillo et al. 2020 package treats missense exonuclease-domain substitutions (p.P286R, p.V411L, p.S297F, p.A456P, p.S459F) as the established pathogenic class in POLE, reinforcing that missense is the operative variant class for this gene.
• The assessed variant is a missense substitution (p.Ala183Thr), so the missense requirement of PP2 is satisfied.
• The constraint limb of PP2 could not be quantified: no missense Z-score, MOI or equivalent gene-level constraint statistic appears in evidence.json, gene_context.json or the case evidence bundle, and no publication providing a POLE constraint threshold is available in the VCEP package.

Still missing

• No gene-level missense-constraint metric (e.g. gnomAD missense Z-score / MOI) for POLE was present in the case evidence, so the 'low rate of benign missense variation' limb of PP2 could not be independently verified; PP2 therefore carries the needs_human_review flag.
• No POLE-specific PP2 rule exists in the governing local framework, so the generic ACMG/AMP PP2 definition was applied.

Sources

Reasoning

Scope: c.547G>A is a missense change, p.(Ala183Thr), so BP4 is in scope for the missense in-silico path. No ClinGen CSPEC exists for POLE, so the local POLE custom framework governs; its BP4 rule (variant present in Supplementary Table S2 or S3, REVEL class 'Likely benign', and >=4 benign in-silico results) cannot be applied because the exact variant is absent from both supplementary tables, and the framework directs fallback to the generic in-silico workflow in that situation. Under the generic ClinGen SVI REVEL calibration, the observed REVEL score of 0.037 sits below every benign threshold: <=0.29 for supporting, <=0.183 for moderate, and <=0.016 for strong (Pejaver et al. 2022, PMID:36413997). 0.037 does not reach the strong bin (it exceeds 0.016) but is comfortably inside the moderate bin (<=0.183), so BP4 is met at moderate strength. The independent direction of the only other predictor present is concordant: BayesDel is -0.676197, also benign-leaning, but it is given no weight because no generic ACMG-strength BayesDel calibration exists and the POLE materials specify no cutoff. SpliceAI returned no scores and, per the governing rule for missing SpliceAI data, that absence is not used to support BP4; BP4 here rests solely on the calibrated REVEL bin. This is the single computational criterion met for this variant.

Rule

Custom POLE framework BP4 (Supplementary Table S2/S3 REVEL class 'Likely benign' with >=4 benign in-silico results) is inapplicable because c.547G>A is absent from both tables; fall back to the generic in-silico workflow using the ClinGen SVI REVEL calibration: BP4 strong <=0.016, moderate <=0.183, supporting <=0.29 (PMID:36413997).

Evidence considered

• The local POLE custom framework's BP4 rule requires the exact missense variant to appear in Supplementary Table S2 or S3 with REVEL class 'Likely benign' and >=4 benign in-silico results, and explicitly directs fallback to the generic in-silico workflow when the variant is absent from those tables.
• Direct search of the converted Supplementary Table S2 (PATH-250-323-s003.xlsx.txt) and Supplementary Table S3 (PATH-250-323-s004.xlsx.txt) found no entry for p.A183T, p.Ala183Thr or c.547G>A in either table.
• REVEL score 0.037 from the local REVEL v1.3 lookup (GRCh38 chr12:132679528 C>T), compared against the supplied ClinGen SVI REVEL benign calibration: BP4 supporting <=0.29, moderate <=0.183, strong <=0.016 (Pejaver et al. 2022, PMID:36413997).
• The REVEL score does not reach the strong benign bin (0.037 > 0.016) but is inside the moderate benign bin (0.037 <= 0.183), so moderate strength was assigned.
• BayesDel score -0.676197 is directionally concordant with a benign computational profile but is excluded from the weight of evidence because no generic ACMG-strength calibration exists and no POLE-specific cutoff is provided.
• SpliceAI returned no scores for this variant; consistent with the case guidance, that missing data was not used to support BP4.
• León-Castillo et al. 2020 (PATH-250-323.pdf) uses in-silico tools only to triage endometrial carcinoma tumours for POLE-score/WES analysis and provides no pre-assigned germline ACMG code for this variant.

Still missing

• Searched PATH-250-323-s003.xlsx (Supplementary Table S2) in full: no entry for c.547G>A / p.Ala183Thr / p.A183T, so the POLE custom BP4 rule (which requires a 'Likely benign' REVEL class plus >=4 benign in-silico results from that table) could not be applied and the generic REVEL calibration was used instead.
• Searched PATH-250-323-s004.xlsx (Supplementary Table S3) in full: no entry for c.547G>A / p.Ala183Thr / p.A183T.
• No multi-tool benign consensus could be assembled (SIFT/PANTHER/SNAP2/PolyPhen2/PROVEAN are only available for the variants tabulated in Supplementary Tables S2 and S3, which do not include this variant); BP4 therefore rests on a single calibrated predictor, REVEL.
• BayesDel -0.676197 is directionally concordant but uncalibrated for ACMG strength, so it could not be added to the weight of evidence.

Sources

Reasoning

PS1 requires the variant to encode the same amino-acid change as a variant already established as pathogenic (ACMG/AMP 2015; PMID:25741868). NM_006231.4:c.547G>A produces p.(Ala183Thr). The governing local POLE framework derived from the Leon-Castillo et al. 2020 package treats only five substitutions as established pathogenic exonuclease-domain hotspots - p.P286R, p.V411L, p.S297F, p.A456P and p.S459F - and p.Ala183Thr is none of them; the framework's lower tiers (p.F367S, p.L424I, p.M295R, p.P436R, p.M444K, p.D368Y, p.A465V, p.L424V, p.T278M, p.A428T) are likewise unrelated to Ala183. The ClinVar record for this variant (ClinVarID 540667 / VCV000540667) aggregates four submissions, all uncertain significance (3 laboratories) or likely benign (1 laboratory), with no pathogenic submission, and contains no pathogenic POLE variant affecting codon 183. Supplementary Table S1 of the same package, which lists every recurrent POLE variant in the COSMIC and TCGA endometrial carcinoma cohorts, returns no entry for this variant when searched as 'p.A183T', 'c.547G>A', 'p.Ala183Thr' or 'A183'. No source examined reports a pathogenic variant encoding p.Ala183Thr (or any other pathogenic substitution at Ala183), so the same-amino-acid-change requirement of PS1 is not satisfied.

Rule

PS1 (ACMG/AMP 2015, Richards et al., PMID:25741868): apply when the variant produces the same amino-acid change as a previously established pathogenic variant. No POLE-specific PS1 rule is present in the governing local framework or in any available CSPEC/VCEP specification.

Evidence considered

• PMID:25741868 defines PS1 as the same amino-acid change as a previously established pathogenic variant, regardless of nucleotide change; no POLE-specific PS1 rule exists in the local framework, so the generic definition governs.
• The Leon-Castillo et al. 2020 package designates exactly five exonuclease-domain substitutions as established pathogenic hotspots in its framework: p.P286R, p.V411L, p.S297F, p.A456P and p.S459F.
• The assessed change p.(Ala183Thr) matches none of those established pathogenic amino-acid changes and none of the framework's secondary (POLE-score >=4 or POLE-score 3) variant tiers.
• ClinVar VCV000540667 for this variant carries 4 submissions: 3 x Uncertain significance and 1 x Likely benign (Ambry Genetics, 2024-12-23); no submitter classified it pathogenic, and no pathogenic POLE variant at codon 183 is reported.
• Supplementary Table S1 (PATH-250-323-s002) was searched for 'p.A183T', 'c.547G>A', 'p.Ala183Thr' and 'A183'; the table's only 183-containing entry is p.H1833N (c.5497C>A), a different residue (codon 1833), so no PS1 comparator is present there either.

Sources

Reasoning

The governing POLE framework is the local literature-derived Leon-Castillo et al. 2020 custom framework (framework_label 'Leon-Castillo et al. 2020 custom POLE framework', customized criteria PM1, PS4, PP3, BP4), whose PS4 specification is a somatic-recurrence adaptation explicitly capped at Supporting. The exact comparison operator, quoted literally from that framework's own PS4 rule text, is: the variant must be 'recurrent in both the COSMIC and TCGA endometrial carcinoma cohorts in Supplementary Table S1', have a 'combined EC count >=10', and 'belong to the established/pathogenic set highlighted by Leon-Castillo et al. 2020' (the framework names p.P286R, p.V411L, p.A456P and p.S297F). Explicit substitution with this variant's numbers: combined endometrial-carcinoma count (COSMIC + TCGA) for p.Ala183Thr = 0, so 0 >= 10? NO -> the custom PS4_Supporting rule does not fire. I searched the full 100-row Supplementary Table S1 (PATH-250-323-s002.xlsx.txt) for 'c.547G>A', 'p.A183T', 'p.Ala183Thr', '183' and '547' and found no row for this variant; its only match was the unrelated p.H1833N / c.5497C>A row. A183T is likewise absent from Supplementary Table S2 (TCGA in-silico table, 46 rows) and Supplementary Table S3 (non-TCGA non-hotspot table, 41 rows), and the main paper text (PATH-250-323.pdf.txt) never mentions c.547G>A, p.A183T or residue 183; the paper's recurrence counts are keyed to the five established hotspots P286R, V411L, A456P, S297F and S459F. Because the operator here is a '>=' (strengthens UPWARD with count), the correct direction is: higher recurrence count supports PS4, and A183T's count of 0 fails it. Independent live sources agree: COSMIC returned no result for this variant ('This variant has not previously been reported in somatic cancers (COSMIC)'), Cancer Hotspots reports 'This variant does not lie in a statistically significant hotspot', and OncoKB returned 'Unknown Oncogenic Effect' with is_variant_specific = false and no citations. Generic ACMG PS4 (increased prevalence in affected individuals versus controls) is also unsupported: no germline case-control data exist for this variant and no numeric enrichment value (odds ratio or prevalence ratio) is available anywhere in the consulted sources, so no >= threshold comparison can be performed for the generic criterion either. PS4 is therefore not met at any strength.

Rule

POLE custom PS4_Supporting (somatic-recurrence adaptation, Supporting cap): exact variant recurrent in both COSMIC and TCGA endometrial-carcinoma cohorts in Supplementary Table S1 AND combined EC count >=10 AND in the established/pathogenic set {p.P286R, p.V411L, p.A456P, p.S297F}.

Evidence considered

• Searched the full Supplementary Table S1 (PATH-250-323-s002.xlsx.txt, 100 variant rows) for 'c.547G>A', 'p.A183T', 'p.Ala183Thr', '183' and '547': no row for this variant; its combined COSMIC + TCGA endometrial-carcinoma count is therefore 0. The only '183' hit was the unrelated p.H1833N (c.5497C>A) row.
• Searched the main Leon-Castillo et al. 2020 text (PATH-250-323.pdf.txt) for c.547G>A, p.A183T, p.Ala183Thr and '183': no mention; the paper designates only P286R, V411L, S297F, A456P and S459F as established pathogenic hotspot mutations and states that >95% of POLE mutations outside the exonuclease domain are classified as non-pathogenic by POLE-score.
• Searched Supplementary Table S2 (TCGA in-silico table, PATH-250-323-s003.xlsx.txt) and Supplementary Table S3 (non-TCGA non-hotspot table, PATH-250-323-s004.xlsx.txt) in full: neither contains A183T, so the variant has no documented TCGA or other endometrial-carcinoma recurrence to feed the custom PS4 rule.
• Framework PS4 rule verbatim operator: 'recurrent in both the COSMIC and TCGA endometrial carcinoma cohorts in Supplementary Table S1', 'combined EC count >=10', 'established/pathogenic set'. Explicit inequality: 0 >= 10? no.
• COSMIC: 'This variant has not previously been reported in somatic cancers (COSMIC)' (no sample count available).
• Cancer Hotspots (POLE A183): no hotspot row found; 'This variant does not lie in a statistically significant hotspot'.
• OncoKB (POLE A183T): 'Unknown Oncogenic Effect', is_variant_specific = false, not_reviewed = true, zero citations - no variant-specific somatic or germline pathogenicity support.
• No germline case-control enrichment statistic for this variant was found in any consulted source, so the generic ACMG PS4 criterion has no numeric value to compare against a threshold.

Still missing

• No germline case-control enrichment data (odds ratio, or affected-versus-control prevalence) exist for POLE c.547G>A p.Ala183Thr in any consulted source, so the generic ACMG PS4 threshold comparison is unperformable for this variant.
• PATH-250-323-s001.tif (supplementary figure; declared for PM1/PS4) could not be converted to text and was not visually reviewed; it is a somatic-tumour summary figure not expected to carry germline case-control enrichment data.

Sources

Reasoning

PM1 requires the variant to lie in a mutational hotspot or in a critical, well-established functional domain without benign variation. The authoritative domain list for this gene was checked first: vcep_materials.json contains an empty domain_tables object with no POLE entry, so no approved POLE critical-domain table exists and the governing material is the Leon-Castillo et al. 2020 POLE package. Every substitution that package treats as pathogenic or plausibly pathogenic lies in the C-terminal proofreading/exonuclease region: the five pathogenic hotspots p.P286R, p.V411L, p.S297F, p.A456P, p.S459F; the POLE-score >=4 non-hotspot tier p.F367S, p.L424I, p.M295R, p.P436R, p.M444K, p.D368Y; and the POLE-score 3 / uncertain tier p.A465V, p.L424V, p.T278M, p.A428T. p.Ala183Thr sits more than 90 residues N-terminal to the most N-terminal of these and outside the exonuclease (proofreading) domain, which is the only well-established critical functional domain for POLE pathogenicity. The paper states that 'a large majority of mutations outside the exonuclease domain are not pathogenic' and that POLE mutations outside the exonuclease domain 'appear to be passengers secondary to the hypermutator phenotype', and its own POLE-score analysis found that 19/23 (83%) of non-exonuclease-domain mutations scored <=2. The variant is also not a recurrent somatic hotspot (CancerHotspots query 'POLE A183' returned no statistically significant hotspot row), is absent from COSMIC, is not among the recurrent variants in Supplementary Table S1, and is absent from Supplementary Table S1 when searched under all three notations. PM1 is not met.

Rule

PM1 (ACMG/AMP 2015, PMID:25741868) as implemented by the governing local POLE framework: apply only to the five established exonuclease-domain hotspots (PM1_Strong: p.P286R, p.V411L, p.S297F, p.A456P, p.S459F), to the listed POLE-score >=4 non-hotspot exonuclease-domain substitutions (PM1_Moderate), or to the listed POLE-score 3 exonuclease-domain substitutions (PM1_Supporting); all require exonuclease-domain location. Variants outside the exonuclease domain do not meet PM1.

Evidence considered

• vcep_materials.json 'domain_tables' is an empty object - there is no POLE-specific approved critical-domain list for this gene, so PM1 was adjudicated against the governing Leon-Castillo et al. 2020 package rather than an approved domain table.
• PM1 definition (PMID:25741868): located in a mutational hotspot and/or critical and well-established functional domain without benign variation.
• In the governing paper, 59/82 TCGA endometrial carcinomas with somatic POLE mutations were in the exonuclease domain; the five most common (p.P286R, p.V411L, p.S297F, p.A456P, p.S459F) were classified as pathogenic hotspots.
• The paper's abstract states: 'Only 1/23 (4%) tumours with non-EDM showed these genomic alterations, indicating that a large majority of mutations outside the exonuclease domain are not pathogenic.'
• The paper's discussion states that cases with 'POLE mutations outside the exonuclease domain: these appear to be passengers secondary to the hypermutator phenotype'.
• POLE-score analysis: '19/23 tumours with POLE mutations outside the exonuclease domain had scores <=2' - i.e. the non-domain region behaves as non-pathogenic in the paper's own functional readout.
• OncoKB gene-level curation (oncokb, and the same text reproduced in gene_context.json / pvs1_gene_context) places the critical functional domain of POLE at the exonuclease 'proofreading' domain, mutation of which causes the ultramutated phenotype.
• CancerHotspots query 'POLE A183' returned no result ('This variant does not lie in a statistically significant hotspot'), and COSMIC returned no record for c.547G>A; neither source registry key exists for these two lookups, so they are reported here as narrative.
• Supplementary Table S1 was searched for 'p.A183T', 'c.547G>A', 'p.Ala183Thr' and 'A183' with no match, so this variant is not among the recurrent exonuclease-domain variants tabulated by the framework.
• Because the governing material defines pathogenicity by exonuclease-domain location plus hotspot recurrence, and p.Ala183 (residue 183) is N-terminal to every variant it treats as exonuclease-domain (most N-terminal: p.T278M / p.D275V region), residue 183 does not qualify under PM1.

Still missing

• vcep_materials.json contains no POLE entry under domain_tables, so the exonuclease-domain boundary was inferred from the set of variants the governing Leon-Castillo et al. 2020 package itself classifies as exonuclease-domain rather than from an approved VCEP domain table.
• PATH-250-323-s001.tif (supplementary figure) could not be converted to text and was not reviewed visually.

Sources

Reasoning

PM5 requires a missense change at an amino-acid residue where a different missense change has been established as pathogenic. No such comparator exists for POLE codon 183. The dedicated same-residue comparator collection for this case (pm5_candidates.json) returned found=false with zero candidates seen, zero same-residue candidates and zero expert-panel or majority pathogenic candidates, and explicitly recorded that classic same-residue PM5 semantics could not be confirmed and that automatic harvesting was skipped. The ClinVar record for this variant (VCV000540667) aggregates four submissions, none pathogenic, and reports no pathogenic or likely pathogenic POLE variant at codon 183 that could serve as a PM5 comparator. The governing Leon-Castillo et al. 2020 package contains no variant at residue 183 in its recurrent-variant table, its only 183-containing entry being p.H1833N (codon 1833), and none of the framework's listed pathogenic or plausibly pathogenic substitutions (p.P286R, p.V411L, p.S297F, p.A456P, p.S459F, p.F367S, p.L424I, p.M295R, p.P436R, p.M444K, p.D368Y, p.A465V, p.L424V, p.T278M, p.A428T) is at Ala183. PM5 is not met.

Rule

PM5 (ACMG/AMP 2015, PMID:25741868): apply when a different missense change at the same amino-acid residue has been established as pathogenic. No POLE-specific PM5 rule is present in the governing local framework.

Evidence considered

• PM5 definition (PMID:25741868): missense change at an amino-acid residue where a different missense change determined to be pathogenic has been seen before.
• pm5_candidates.json: found=false, pm5_mode='unknown', 0 candidates seen, 0 same-residue candidates; recommendation level 'not_applicable' with the instruction to review manually rather than apply PM5 from that artifact.
• ClinVar VCV000540667: 4 submissions (3 x Uncertain significance, 1 x Likely benign); no pathogenic or likely pathogenic POLE missense variant at codon 183 is listed as a comparator.
• Supplementary Table S1 (PATH-250-323-s002) was searched for 'A183', 'p.A183T' and 'c.547G>A'; no entry at residue 183 exists, so the framework provides no same-residue pathogenic comparator either.

Still missing

• pm5_candidates.json reported pm5_mode 'unknown' and harvested no same-residue comparators, so the PM5 conclusion rests on the ClinVar record and the VCEP tables rather than on an automated codon-183 comparator search.

Sources

Reasoning

Scope: NM_006231.4:c.547G>A is NP_006222.2:p.(Ala183Thr), a missense substitution, so PP3 is in scope through both the missense in-silico path and the splice-impact path. No ClinGen CSPEC exists for POLE (cspec found: false), so the local POLE custom framework governs, and it states that its custom PP3 rule (REVEL class 'likely disease causing' with <=1 benign in-silico results in Supplementary Table S2 or S3) applies only when the exact variant appears in one of those tables, otherwise the generic in-silico workflow is used. The exact change is absent from both tables: Supplementary Table S2 lists 44 variants (a minimum of A1010T through Y1889C; the closest residue entries are M1754V, W1824C, V2025M, Y1889C) and Supplementary Table S3 lists 38 non-hotspot variants; p.Ala183Thr, p.A183T and c.547G>A appear in neither, confirmed by direct search of both converted tables. The generic workflow therefore applies. REVEL is available and is 0.037, which is below the PP3 supporting threshold of 0.644 (Pejaver et al. 2022, PMID:36413997) by a wide margin, so the missense path supplies no pathogenic computational support. BayesDel is 0.676197 on the benign side but no generic ACMG-strength calibration exists for BayesDel and the POLE materials specify no cutoff, so it is not_available and cannot be counted. The splice path is also non-contributory: SpliceAI returned no scores for this variant (max delta null, available: false), which is missing data rather than evidence of absent splice impact, and Pangolin (SG 0.001, SL -0.005) is not calibrated for PP3 here. With no predictor meeting a pathogenic threshold, PP3 is not met.

Rule

Custom POLE framework PP3 (Supplementary Table S2/S3 REVEL class 'likely disease causing' with <=1 benign in-silico result) is inapplicable because c.547G>A is absent from both tables; fall back to the generic in-silico workflow, where PP3 requires REVEL >=0.644 (supporting), >=0.773 (moderate) or >=0.932 (strong) per the ClinGen SVI REVEL calibration (PMID:36413997).

Evidence considered

• The local POLE custom framework's PP3 rule requires that the exact missense variant appear in Supplementary Table S2 or S3 with REVEL class 'likely disease causing' and <=1 benign in-silico results, and explicitly directs fallback to the generic in-silico workflow when the variant is absent from those tables.
• Direct search of the converted Supplementary Table S2 (PATH-250-323-s003.xlsx.txt) found no entry for p.A183T, p.Ala183Thr or c.547G>A; the table tabulates 44 TCGA EC POLE variants keyed by short protein notation.
• Direct search of the converted Supplementary Table S3 (PATH-250-323-s004.xlsx.txt) found no entry for p.A183T, p.Ala183Thr or c.547G>A; the table tabulates 38 non-TCGA EC cohort POLE variants keyed by short protein notation.
• REVEL score 0.037 from the local REVEL v1.3 lookup, compared against the supplied ClinGen SVI REVEL calibration (PP3 supporting >=0.644; Pejaver et al. 2022, PMID:36413997).
• BayesDel score -0.676197 was present but is treated as not_available because no generic ACMG-strength calibration is established and the POLE materials specify no BayesDel cutoff.
• SpliceAI returned no scores (max_delta null, spliceai_available false) and Pangolin SG 0.001 / SL -0.005 were recorded; neither provides calibrated PP3 support here.
• León-Castillo et al. 2020 (PATH-250-323.pdf) frames in-silico tools as a tumour-level triage step (>=4 tools predicting pathogenic or >1 predicting non-pathogenic) for endometrial carcinoma POLE-score classification, not as a germline ACMG/AMP PP3 rule, so it supplies context only and no pre-assigned PP3 code for this variant.

Still missing

• Searched PATH-250-323-s003.xlsx (Supplementary Table S2, in silico results for POLE mutations in TCGA EC) in full: no entry for c.547G>A / p.Ala183Thr / p.A183T. Its 44 listed variants include no residue-183 change.
• Searched PATH-250-323-s004.xlsx (Supplementary Table S3, in silico results for non-hotspot POLE mutations in non-TCGA EC cohorts) in full: no entry for c.547G>A / p.Ala183Thr / p.A183T.
• SpliceAI returned no scores for this variant (max delta null rather than 0), so the splice-impact arm of PP3 could not be evaluated; this is missing data and was not treated as evidence of absent splice impact.
• aGVGD, VarSeak, CADD and other missense predictors were not available in the evidence for this variant; REVEL was the only calibrated missense predictor present.

Sources

Reasoning

Per this group's binding rule, PP5 may only be applied for an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic; ordinary laboratory submissions, aggregate ClinVar labels and non-expert assertions must never trigger it. ClinVar VCV000540667 for NM_006231.4:c.547G>A (p.Ala183Thr) (variation ID 540667, review stars 1) has expert_panels empty and the submission audit records expert_panel_submissions = 0, with is_expert_panel = false for all four submissions (Labcorp Genetics/Invitae, GeneDx, Ambry Genetics, Department of Pathology and Laboratory Medicine, Sinai Health System). The aggregate record is 'Conflicting classifications of pathogenicity' / 'criteria provided, conflicting classifications' at 1 star, comprising 3 clinical laboratories reporting Uncertain significance and 1 clinical laboratory reporting Likely benign. There is no ClinGen VCEP or other expert-panel assertion of Pathogenic or Likely pathogenic for this exact variant, and the conflicting single-submitter VUS calls are precisely the class of ordinary submissions the rule excludes. OncoKB likewise returned 'Unknown Oncogenic Effect' with is_variant_specific = false, so no reputable-source pathogenic assertion exists. No numeric threshold or comparison operator applies to PP5; the criterion is qualitative and its trigger condition is absent, so PP5 is not met.

Rule

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

Evidence considered

• ClinVar VCV000540667 (variation ID 540667) for NM_006231.4:c.547G>A (p.Ala183Thr): expert_panels empty, expert_panel_submissions = 0, is_expert_panel = false for all 4 submissions (SCV000772614 Labcorp/Invitae, SCV003930996 GeneDx, SCV005928741 Ambry Genetics, SCV006055420 Sinai Health System).
• Aggregate ClinVar classification 'Conflicting classifications of pathogenicity', review status 'criteria provided, conflicting classifications', 1 star; 3 laboratories Uncertain significance and 1 laboratory Likely benign.
• No exact-variant expert-panel (ClinGen VCEP) classification of Pathogenic/Likely pathogenic exists, so the PP5 trigger condition is absent; ordinary single-submitter assertions are explicitly excluded by the governing rule.
• OncoKB (POLE A183T) reported 'Unknown Oncogenic Effect' with is_variant_specific = false and no citations, providing no reputable-source pathogenic assertion.

Sources

Reasoning

Framework first: no ClinGen CSPEC/VCEP specification exists for POLE in this case (case_summary cspec.found = false), and the governing framework is the local literature-derived Leon-Castillo et al. 2020 custom POLE framework, which customizes only PM1, PS4, PP3 and BP4 and explicitly states that outside those paper-derived rules one falls back to generic ACMG/AMP. No part of that package specifies a population-frequency criterion, a non-cancer cohort source, or an exome-only source, so the generic ACMG/AMP 2015 stand-alone benign threshold applies and the all-comers GNOMAD_V2_1 / GNOMAD_V4_1 entries are the default sources. The published operator is an allele frequency >= 0.05 for stand-alone benign support (ACMG/AMP 2015, PMID:25741868: 'if over 5% then it is considered as stand-alone support (BA1)'). Explicit substitution with this variant's numbers: gnomAD v4.1 (GRCh38 chr12-132679528-C-T) total AF = 3.84184e-05 (62/1,613,808 alleles, homozygotes 0), exome component 4.03695e-05 (59/1,461,500) and genome component 1.96969e-05 (3/152,308); gnomAD v2.1 (GRCh37 12-133256114-C-T) total/exome AF = 7.9552e-06 (2/251,408 alleles, homozygotes 0). Is 3.84184e-05 >= 0.05? No - the highest dataset-level frequency is approximately 1,300-fold below the threshold. Ancestry does not rescue it: the highest observed ancestry-specific frequency is European (non-Finnish) 4.83167e-05 (57/1,179,716), still ~1,000-fold below 0.05, and gnomAD v4.1 grpmax FAF is 3.775e-05. The non-cancer subsets were checked as a sensitivity analysis and are if anything lower: gnomAD v2.1 non-cancer exomes 8.44338e-06 (2/236,872) and gnomAD v3.1 non-cancer genomes 2.02782e-05 (3/147,942). The variant is absent from gnomAD-Canada v1.0 (HostSeq genomes). Homozygote count is 0 in every cohort in which the variant is seen. No consulted source reports a frequency anywhere near 5% in any population, so BA1 cannot be met at any strength.

Rule

BA1 stand-alone benign: allele frequency >= 0.05 in a general population database (ACMG/AMP 2015, PMID:25741868); no POLE-specific overriding rule exists.

Evidence considered

• gnomAD v4.1 (all-comers, GRCh38 chr12-132679528-C-T): total AF 3.84184e-05 (62/1,613,808 alleles, homozygotes 0); exome 4.03695e-05 (59/1,461,500); genome 1.96969e-05 (3/152,308); highest ancestry European (non-Finnish) 4.83167e-05 (57/1,179,716); grpmax FAF 3.775e-05.
• gnomAD v2.1 (all-comers, GRCh37 12-133256114-C-T): total/exome AF 7.9552e-06 (2/251,408 alleles, homozygotes 0); highest ancestry European (non-Finnish) 1.75889e-05 (2/113,708); grpmax FAF 2.92e-06.
• Non-cancer sensitivity check: gnomAD v2.1 non-cancer exomes AF 8.44338e-06 (2/236,872 alleles, homozygotes 0; NFE 1.94757e-05, 2/102,692); gnomAD v3.1 non-cancer genomes AF 2.02782e-05 (3/147,942 alleles, homozygotes 0). Neither approaches 5%.
• gnomAD-Canada v1.0 (HostSeq genomes): 'Absent from gnomAD-Canada v1.0' (no record returned).
• Threshold basis: ACMG/AMP 2015 stand-alone benign criterion, allele frequency over 5% (PMID:25741868, section 'PS4 PM2 BA1 BS1 BS2 Variant Frequency and Use of Control Populations').
• Framework scope: no CSPEC was available for POLE (cspec.found = false); the local custom POLE framework customizes only PM1, PS4, PP3 and BP4 and directs generic ACMG/AMP fallback elsewhere, so no gene-specific population source or threshold overrides the all-comers gnomAD default for BA1.

Still missing

• No POLE-specific BA1 threshold is derivable: the governing framework specifies no population-frequency rule and no disease prevalence or penetrance figures are present in the case files, so the generic ACMG/AMP 2015 5% stand-alone threshold was applied.
• gnomAD-Canada v1.0 (HostSeq) returned no record for this variant, so that cohort contributes an absence call only and no allele-frequency estimate.

Sources

Reasoning

The applicable threshold is the generic ACMG/AMP strong benign allele-frequency default of >= 0.01 (per-gene derivation preferred: Whiffin et al. 2017, PMID:28518168; supplied as the generic SVI default). A POLE-specific BS1 threshold could not be derived because neither the governing framework nor the case files contain POLE disease prevalence or penetrance figures, and the governing POLE framework customizes only PM1, PS4, PP3 and BP4 - it defines no population criterion and no non-cancer or exome-only source requirement, so the all-comers GNOMAD_V2_1 / GNOMAD_V4_1 entries remain the default and the generic 0.01 threshold applies. Explicit substitution with this variant's numbers: gnomAD v4.1 total AF 3.84184e-05 (62/1,613,808) and v2.1 total AF 7.9552e-06 (2/251,408); the highest ancestry-specific frequency is European (non-Finnish) 4.83167e-05 (57/1,179,716), and gnomAD v4.1 grpmax FAF is 3.775e-05. Is 4.83167e-05 >= 0.01? No - the highest ancestry frequency is roughly 200-fold below the BS1 threshold, and the cohort-level frequencies are ~250-1,250-fold below it. The non-cancer sensitivity check is likewise far below: v2.1 non-cancer exomes 8.44338e-06 (2/236,872) and v3.1 non-cancer genomes 2.02782e-05 (3/147,942). One datapoint nominally crosses the threshold and is the reason this criterion is flagged for human review: gnomAD v3.1 non-cancer genomes reports HGDP:HAN AF 0.0119048 (1/84 alleles). That estimate rests on a single carrier allele in an 84-allele ancestry bin whose grpmax FAF is 0.0, giving an extremely wide confidence interval; it is not an adequately powered frequency estimate, it is not reproduced in the same ancestry group in the larger all-comers datasets, and the same variant's European (non-Finnish) frequency is 4.83167e-05 (about 250-fold lower). Treating a 1-of-84 observation as population-frequency evidence would be a small-cohort artifact, not a valid threshold crossing, so BS1 is not met; the artifact is documented for reviewer confirmation rather than silently discarded.

Rule

BS1 strong benign: allele frequency >= 0.01 in a general population database (generic SVI default, Whiffin et al. 2017, PMID:28518168); no POLE-specific overriding derivation available.

Evidence considered

• gnomAD v4.1 (all-comers): total AF 3.84184e-05 (62/1,613,808 alleles, homozygotes 0); exome 4.03695e-05 (59/1,461,500); genome 1.96969e-05 (3/152,308); highest ancestry European (non-Finnish) 4.83167e-05 (57/1,179,716); grpmax FAF 3.775e-05.
• gnomAD v2.1 (all-comers): total/exome AF 7.9552e-06 (2/251,408 alleles, homozygotes 0); highest ancestry European (non-Finnish) 1.75889e-05 (2/113,708); grpmax FAF 2.92e-06.
• Non-cancer sensitivity check: gnomAD v2.1 non-cancer exomes AF 8.44338e-06 (2/236,872 alleles, homozygotes 0; NFE 1.94757e-05, 2/102,692); gnomAD v3.1 non-cancer genomes AF 2.02782e-05 (3/147,942 alleles, homozygotes 0), with HGDP:HAN 0.0119048 (1/84 alleles) and grpmax FAF 0.0 - one allele in an 84-allele bin, not an adequately powered estimate.
• gnomAD-Canada v1.0 (HostSeq genomes): absent.
• Threshold basis: generic strong benign allele-frequency default of 0.01, attributed to Whiffin et al. 2017 (PMID:28518168); ACMG/AMP 2015 (PMID:25741868) defines BS1 as an allele frequency greater than expected for the disorder, and no POLE-specific expected frequency is available.
• Framework scope: the governing POLE framework customizes only PM1, PS4, PP3 and BP4 and specifies no population source or threshold, so the all-comers gnomAD entries were used as the default source and the generic BS1 threshold applied.

Still missing

• No POLE-specific BS1 threshold could be derived (no prevalence or penetrance data for POLE-associated disease exist in the case files; the governing framework defines no population rule), so the generic 0.01 SVI default (Whiffin et al. 2017, PMID:28518168) was used.
• gnomAD v3.1 non-cancer reports HGDP:HAN AF 0.0119048 (1/84 alleles), which nominally exceeds 0.01; it is a single-allele observation in an 84-allele bin with grpmax FAF 0.0 and is treated as a small-cohort artifact - reviewer confirmation is requested.
• gnomAD-Canada v1.0 returned no record, so no independent Canadian population frequency is available to cross-check the European-dominant signal.

Sources

Reasoning

BS2 requires observation of the variant in a well-documented healthy adult individual for a condition that is fully penetrant at an early age - homozygous for a recessive condition, heterozygous for a dominant condition, or hemizygous for an X-linked condition (ACMG/AMP 2015, PMID:25741868: 'if the disease under investigation is fully penetrant at an early age and the variant is observed in a well-documented healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) condition then this is considered strong evidence for a benign interpretation'). Two independent conditions fail here. First, no homozygous observation exists in any dataset: gnomAD v4.1 homozygotes 0 (62/1,613,808 alleles), gnomAD v2.1 homozygotes 0 (2/251,408 alleles), gnomAD v2.1 non-cancer exomes homozygotes 0 (2/236,872), gnomAD v3.1 non-cancer genomes homozygotes 0 (3/147,942), and the variant is absent from gnomAD-Canada v1.0, so the homozygous (recessive) arm has no supporting observation at all. Second, the heterozygous (dominant) arm requires a fully penetrant early-onset phenotype, and the POLE-associated phenotype is a dominant, adult-onset, incompletely penetrant cancer-predisposition state (polymerase proofreading-associated polyposis / POLE-associated cancer predisposition); healthy adult gnomAD participants carrying this allele therefore do not satisfy the criterion's premise, and the 62 heterozygous alleles seen in gnomAD v4.1 adult participants and 2 in gnomAD v2.1 carry no BS2 weight. The governing POLE framework customizes only PM1, PS4, PP3 and BP4 and defines no BS2 rule, so the generic ACMG/AMP definition above applies unchanged. No numeric threshold applies to this criterion and no evidence in the consulted sources supports it. The single ClinVar Likely benign submission for this variant was deliberately not used as BS2 evidence, since a criterion may never be inferred from another source's final classification.

Rule

BS2 strong benign: variant observed in a well-documented healthy adult individual for a recessive (homozygous), dominant (heterozygous) or X-linked (hemizygous) condition that is fully penetrant at an early age (ACMG/AMP 2015, PMID:25741868); no POLE-specific overriding rule exists.

Evidence considered

• gnomAD v4.1 homozygote count 0 for total (62/1,613,808 alleles), exome (59/1,461,500) and genome (3/152,308) components; gnomAD v2.1 homozygote count 0 (2/251,408 alleles); gnomAD v2.1 non-cancer exomes homozygotes 0 (2/236,872); gnomAD v3.1 non-cancer genomes homozygotes 0 (3/147,942); gnomAD-Canada v1.0 absent. No homozygous observation in any dataset.
• The variant is present as heterozygotes in adult population cohorts (62 alleles in gnomAD v4.1), but the POLE-associated phenotype is a dominant, adult-onset, incompletely penetrant cancer predisposition, so the 'fully penetrant at an early age' premise required for the heterozygous arm of BS2 is not satisfied.
• Threshold basis: ACMG/AMP 2015 BS2 definition (PMID:25741868) - observation in a well-documented healthy adult for a recessive (homozygous), dominant (heterozygous) or X-linked (hemizygous) condition that is fully penetrant at an early age; no numeric threshold applies.
• Framework scope: the governing POLE custom framework defines no BS2 rule and no penetrance specification (customized criteria are PM1, PS4, PP3 and BP4), so the generic ACMG/AMP definition was applied.

Still missing

• No homozygous carrier of POLE c.547G>A (p.Ala183Thr) was found in any consulted population dataset, so the recessive arm of BS2 has no observation; the variant is nonetheless rare enough (62 alleles in 1,613,808 in gnomAD v4.1) that absence of homozygotes is expected and is not itself informative.
• No POLE-specific BS2 rule or penetrance specification exists in the governing framework, and no penetrance estimate for POLE-associated disease was present in the case files, so BS2 was assessed against the generic ACMG/AMP 2015 definition only.

Sources

Reasoning

BP1 applies only to a missense variant in a gene in which primarily truncating variants are known to cause disease. POLE is not such a gene. The germline POLE disease mechanism is missense: OncoKB gene-level curation in this case records that heterozygous loss-of-function changes in the exonuclease/proofreading domain cause polyposis and colorectal cancer predisposition, and those alleles are missense substitutions rather than truncating ones. The governing Leon-Castillo et al. 2020 package likewise defines the pathogenic class by missense exonuclease-domain substitutions (p.P286R, p.V411L, p.S297F, p.A456P, p.S459F; POLE-score >=4 non-hotspot substitutions such as p.F367S, p.L424I, p.M295R, p.P436R, p.M444K, p.D368Y) rather than by truncation. Because missense variation is an established mechanism of POLE disease, the gene-level precondition for BP1 fails and no benign direction can be taken from this criterion.

Rule

BP1 (ACMG/AMP 2015, PMID:25741868): missense variant in a gene for which primarily truncating variants are known to cause disease. No POLE-specific BP1 rule exists in the governing local framework.

Evidence considered

• BP1 definition (PMID:25741868): missense variant in a gene for which primarily truncating variants are known to cause disease; BP1 is used as benign supporting evidence.
• OncoKB gene-level curation lists the known germline disease-causing POLE alleles as missense exonuclease-domain changes (polyposis / colorectal cancer predisposition), not truncating variants.
• The governing Leon-Castillo et al. 2020 package's pathogenic POLE class is entirely missense (five hotspots plus the POLE-score >=4 non-hotspot substitutions), so missense - not truncation - is the established POLE disease mechanism.
• The assessed variant is a missense substitution (p.Ala183Thr), but the gene-level premise of BP1 is false for POLE, so BP1 does not apply.

Sources

Reasoning

BP6 (a reputable source reports the variant as benign) is, per this group's binding rule, applicable only for an exact-variant ClinVar expert-panel classification of Benign or Likely benign; ordinary laboratory submissions and aggregate labels must never trigger it. ClinVar VCV000540667 for NM_006231.4:c.547G>A (p.Ala183Thr) has expert_panels empty and expert_panel_submissions = 0, with is_expert_panel = false for all four submissions. The single Likely benign call (SCV005928741, Ambry Genetics, 'criteria provided, single submitter', germline clinical testing, no PMIDs) is an ordinary single-submitter laboratory assertion, not an expert-panel classification, and the aggregate record is 'Conflicting classifications of pathogenicity' at 1 star, comprising 3 laboratories reporting Uncertain significance and only 1 reporting Likely benign. No exact-variant expert-panel benign classification exists, so the BP6 trigger condition is absent and BP6 is not met; this is a qualitative trigger test with no numeric threshold or comparison operator.

Rule

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

Evidence considered

• ClinVar VCV000540667 (variation ID 540667): expert_panels empty, expert_panel_submissions = 0, is_expert_panel = false for all 4 submissions.
• The single Likely benign call (SCV005928741, Ambry Genetics, single submitter, germline clinical testing, no PMID trail) is an ordinary laboratory assertion explicitly excluded from triggering BP6 by the governing rule.
• Aggregate ClinVar status 'Conflicting classifications of pathogenicity', 1 star, with 3 laboratories Uncertain significance and 1 laboratory Likely benign; no exact-variant expert-panel benign classification exists.

Sources

Reasoning

PS2 requires a de novo occurrence of POLE NM_006231.4:c.547G>A (p.Ala183Thr) confirmed by testing of both parental samples with parentage established by identity testing. No proband-level genotype, no parental testing result and no de novo report for this variant exists in any source available for this case: the ClinVar record (variation 540667) comprises four germline submissions with no assertion of a de novo occurrence, and the two full-text publications retrieved for this case do not mention the variant. The local POLE framework (Leon-Castillo et al. 2020 custom package) specifies only PM1, PS4, PP3 and BP4, so PS2 was evaluated against generic ACMG/AMP 2015 definitions. With no confirmed de novo observation, PS2 cannot be met, and the criterion is not assessable rather than refuted.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom framework) customises only PM1, PS4, PP3 and BP4, so PS2 was evaluated under generic ACMG/AMP 2015 definitions. The de novo criterion requires that both parental samples were tested and shown by identity testing to be the biological parents, that the family history is consistent with de novo inheritance, and that the phenotype matches the gene-disease association with reasonable specificity (PMID:25741868, section 'PS2 PM6 De novo variants'; the same text notes that PM6 applies when parentage is assumed rather than confirmed).
• ClinVar variation 540667 for NM_006231.4(POLE):c.547G>A (p.Ala183Thr) holds four germline submissions - Invitae/Labcorp, GeneDx and Sinai Health System as Uncertain significance and Ambry Genetics as Likely benign - all single-submitter, with no expert-panel submission. None of them asserts a de novo occurrence, and the criterion signals extracted from the submission texts are limited to PM2 and PP3; no parental genotypes accompany any submission.
• No proband, trio or de novo observation for c.547G>A was located in any source in this case: the two retrieved full texts (PMID:25394175, an ACMG/NSGC cancer-referral practice guideline, and PMID:25741868, the ACMG/AMP classification guideline) do not mention POLE or the variant, and neither carries case-level segregation or de novo data.

Still missing

• No proband-level genotype, indication for testing or clinical phenotype is available for the individual in whom c.547G>A was observed.
• No parental testing (trio) data: neither parental genotypes nor identity/parentage confirmation have been reported.
• No de novo occurrence of c.547G>A is reported in the ClinVar record (variation 540667, 4 germline submissions), in PMID:25394175, in PMID:25741868, or in any other retrieved source.

Sources

Reasoning

PS3 requires well-established functional studies demonstrating a damaging effect on the gene or gene product. No such study exists in the retrieved evidence for POLE NM_006231.4:c.547G>A (p.Ala183Thr). OncoKB returned 'Unknown Oncogenic Effect' for this exact substitution with is_variant_specific = false and no variant-level functional evidence or citations, so it contributes nothing to PS3. The governing specification for this case is a local, literature-derived POLE framework based on Leon-Castillo et al. 2020 (framework_mode custom_gene_framework; official_clingen = false; precedence_rank 2); that framework customizes only PM1, PS4, PP3 and BP4 and contains no PS3 specification or POLE-specific PS3 strength calibration, so no gene-specific rule can be applied either. Leon-Castillo et al. 2020 itself contains no laboratory functional assay: its stated approach to evaluating the 'functional status' of POLE variants is six in-silico tools (SIFT, PROVEAN, PolyPhen-2, PANTHER, SNAP2, REVEL), and p.Ala183Thr is not named anywhere in the paper nor listed in its recurrent-variant (Supplementary Table S1) or in-silico (Supplementary Tables S2/S3) tables. The two full-text papers retrieved for this case (Richards et al. 2015 ACMG/AMP guideline; ACMG/NSGC cancer-referral practice guideline) are methodological documents that neither mention POLE nor report any assay. The only quantitative variant-level biology available is computational predictor output (REVEL 0.037; BayesDel -0.676197), and computational prediction is by definition PP3/BP4 evidence, not the experimentally established functional evidence PS3 requires; REVEL 0.037 in particular is a benign-leaning score and cannot be repurposed as damaging functional evidence. ClinVar submissions for this variant contain no PS3: the Labcorp submission cites only the Sherloc methodology paper, the GeneDx submission cites in-silico reasoning, the Ambry 'Likely benign' submission cites no PMID at all, and there are no expert-panel submissions. Absence of the variant from COSMIC is a somatic-recurrence observation, not functional data. PS3 is therefore not assessed because the evidence does not exist in this case, not because an available assay was negative.

Evidence considered

• OncoKB entry for POLE A183T: classification 'Unknown Oncogenic Effect', is_variant_specific = false, not_reviewed = true, citations_pmids = [] - OncoKB explicitly states it did not identify variant-specific reviewed functional evidence, so there is no curated assay-based damaging-effect claim for this substitution.
• Governing specification: local custom POLE framework derived from Leon-Castillo et al. 2020 (framework_label 'Leon-Castillo et al. 2020 custom POLE framework', official_clingen = false, precedence_rank 2, scoring_model ACMG/AMP 2015 with custom gene-specific criterion specifications). Its customized_criteria list is exactly [PM1, PS4, PP3, BP4]; no PS3 trigger or strength rule is defined, and the framework notes state that outside the explicit paper-derived rules it falls back to generic ACMG/AMP.
• León-Castillo et al. 2020 (J Pathol 250:323-335, PATH-250-323.pdf): full text contains no laboratory assay - the methods state 'To evaluate the functional status of somatic POLE mutations, we used six widely-used in silico tools: SIFT, PROVEAN, PolyPhen-2, PANTHER, SNAP2, and the meta predictor REVEL.' No p.Ala183Thr / c.547G>A mention anywhere (0 text matches for 'A183' or '183'), and the paper's framework is explicitly somatic endometrial-carcinoma hotspot context rather than a germline ACMG/AMP assay framework.
• PATH-250-323-s002.xlsx (Supplementary Table S1, recurrent POLE variants in COSMIC and TCGA EC cohorts): p.Ala183Thr is absent from the table (the only '183' match is the unrelated p.H1833N / c.5497C>A), so this variant is not among the recurrent/pathogenic exonuclease-domain variants curated there.
• PATH-250-323-s003.xlsx (Supplementary Table S2) and PATH-250-323-s004.xlsx (Supplementary Table S3): p.Ala183Thr is absent from both in-silico result tables, so even the paper's computational variant-level data do not cover this substitution.
• PMID:25741868 (Richards et al. 2015, ACMG/AMP): defines PS3 as 'well-established functional studies show a damaging effect' but reports no variant-level or POLE data and does not mention c.547G>A; it is the definitional source for PS3 only.
• PMID:25394175 (ACMG/NSGC cancer-predisposition referral practice guideline): contains no functional assay data at all and does not mention POLE or p.Ala183Thr.
• ClinVar record 540667 (4 submissions, no expert panel): Labcorp/Invitae VUS cites only 28492532 (Sherloc methodology refinement, no variant-level data); GeneDx VUS cites in-silico reasoning only; Ambry Likely benign cites no PMID; Sinai Health System VUS cites only 25741868. No submission asserts PS3 or reports an assay for this variant.
• In-silico predictors available for this variant: REVEL 0.037 and BayesDel -0.676197. These are computational predictions; under ACMG/AMP they are PP3/BP4 evidence and cannot be converted into PS3, which requires experimentally established functional evidence. BayesDel additionally has no verified published ACMG-strength calibration available to this case.
• Somatic-recurrence data: the variant was not found in COSMIC and does not lie in a statistically significant cancer hotspot (CancerHotspots query 'POLE A183' returned no row). Absence of somatic recurrence and lack of hotspot significance are not functional assay results and were not used as PS3 evidence.

Still missing

• No laboratory functional assay of POLE p.Ala183Thr (e.g. polymerase epsilon proofreading fidelity, mutation-rate/ultramutation reporter, yeast or human-cell complementation) was identified in any retrieved source, so PS3 cannot be evaluated.
• The governing POLE framework (Leon-Castillo et al. 2020-derived, non-official local framework) defines no PS3 rule and provides no POLE-specific PS3 strength calibration to substitute for one.
• No retrieved publication reports this exact variant, so no assay-linked variant-level functional report could be traced through the ClinVar citation trail (only 28492532 and 25741868 were validated, both methodology papers).

Sources

Reasoning

PM3 requires that POLE NM_006231.4:c.547G>A (p.Ala183Thr) be detected in trans with a pathogenic variant, i.e. it is a recessive-modality criterion: the two hits must be shown on opposite alleles in the same individual. POLE has both dominant and recessive disease contexts, and both appear among the conditions attached to the ClinVar record for this variant (Colorectal cancer, susceptibility to, 12 and Hereditary cancer-predisposing syndrome on the dominant side; Facial dysmorphism-immunodeficiency-livedo-short stature syndrome and the IMAGe-like 'intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, genital anomalies, and immunodeficiency' condition on the recessive side), so a trans observation would be admissible if one existed. No source available for this case provides one. The ClinVar record (variation 540667, VCV000540667) comprises a single variant with four germline submissions, all single-submitter and none expert-panel (three Uncertain significance - Invitae/Labcorp, GeneDx, Sinai Health System - and one Likely benign - Ambry Genetics); it reports no second POLE variant, no zygosity, no phase and no affected relative. gnomAD records the variant at 2/251,408 alleles (AF 7.9552e-06) in v2.1 and 62/1,613,808 alleles (AF 3.84184e-05) in v4.1, with zero homozygotes in every dataset, so no individual is reported in whom a second allele could be examined. The two full texts held for this variant (PMID:25741868, the ACMG/AMP classification framework, and PMID:25394175, an ACMG/NSGC cancer-referral guideline) do not name POLE or p.(Ala183Thr) and contain no case-, phase- or family-level data. The governing local POLE package (Leon-Castillo et al. 2020, somatic endometrial-carcinoma hotspot context) specifies only PM1, PS4, PP3 and BP4 and gives no PM3 rule, so the generic ACMG/AMP 2015 definition applies. With no proband genotype, no candidate trans partner and no phasing data, the configuration PM3 requires is untested rather than disproven, so the criterion is not assessable; no other source's classification was used as a proxy for it.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom framework) customises only PM1, PS4, PP3 and BP4, so PM3 was evaluated under generic ACMG/AMP 2015, where PM3 is moderate pathogenic evidence requiring detection of the variant in trans with a pathogenic variant (PMID:25741868). No official ClinGen CSPEC/VCEP was available for POLE.
• ClinVar variation 540667 (VCV000540667) for NM_006231.4(POLE):c.547G>A (p.Ala183Thr): four germline submissions, all single-submitter, 1-star 'criteria provided, conflicting classifications', no expert-panel submission - Uncertain significance x3 (Invitae/Labcorp, GeneDx, Sinai Health System) and Likely benign x1 (Ambry Genetics). The submission texts yield no second POLE variant, no zygosity and no phase information; the submitted classifications themselves were not used to infer PM3, since a criterion may not be derived from another source's final classification.
• Inheritance context for the gene: ClinVar attaches both dominant conditions (Colorectal cancer, susceptibility to, 12; Hereditary cancer-predisposing syndrome) and biallelic/recessive conditions (Facial dysmorphism-immunodeficiency-livedo-short stature syndrome; the IMAGe-like intrauterine growth retardation / metaphyseal dysplasia / adrenal hypoplasia / genital anomalies / immunodeficiency condition) to this variant, and the case's gene-level PVS1 eligibility context records no inheritance-mode entry for POLE, so neither a dominant-only nor a recessive-only reading of this variant is documented and the trans configuration PM3 asks about remains open.
• Population data: gnomAD v2.1 2/251,408 alleles (AF 7.9552e-06; highest subpopulation European non-Finnish 2/113,708, AF 1.75889e-05; homozygotes = 0), gnomAD v4.1 62/1,613,808 alleles (AF 3.84184e-05; homozygotes = 0), gnomAD v2.1 non-cancer exomes 2/236,872 (AF 8.44338e-06), gnomAD v3.1 non-cancer 3/147,942 (AF 2.02782e-05), and absent from gnomAD-Canada v1.0. No dataset reports a homozygote, so there is no population-level homozygous observation from which any allelic configuration could be inferred.
• Case-level literature: the two full texts fetched for this variant (PMID:25741868 and PMID:25394175) were searched for 'POLE', 'c.547G>A', 'p.Ala183Thr'/'A183T' and for case, phase, trans/cis and family terms; neither names POLE or the variant. PMID:25394175 is an ACMG/NSGC referral-indications guideline with no case, segregation or frequency data, so it contributes no affected-proband observation.
• The local POLE package (PATH-250-323 PDF and supplementary tables S1-S3) covers somatic endometrial-carcinoma recurrence, hotspots and in-silico outputs only; it contains no germline allelic-phase material and no PM3 rule, so it cannot supply the in-trans observation PM3 requires.
• No numeric threshold applies to PM3 in the supplied calibration data or in the governing framework; the criterion turns on an observation (variant in trans with a pathogenic variant) that no available source provides.

Still missing

• No proband-level genotype, zygosity or indication for testing is documented for c.547G>A, so the allelic phase of the variant cannot be established.
• No second POLE variant (pathogenic or otherwise) is reported alongside c.547G>A in any consulted source, so an in-trans partner cannot be identified or excluded.
• No parental or family genotypes are available from which the phase of c.547G>A could be inferred.
• No homozygous carrier and no tumour genotype co-occurring with this variant is reported (gnomAD homozygotes = 0 in every dataset; no COSMIC record), so no indirect cis/trans signal exists either.

Sources

Reasoning

PM6 requires a de novo occurrence of POLE NM_006231.4:c.547G>A (p.Ala183Thr) presumed on clinical grounds, without confirmation that both parental samples are the biological parents. Neither requirement can be reached here: no report of a de novo occurrence for this variant exists in the ClinVar record or in any publication retrieved for this case, and no parental genotypes (confirmed or assumed) are available. The local POLE framework does not specify PM6, so generic ACMG/AMP 2015 definitions apply; because a de novo event cannot be established at any level of confidence, the criterion is not assessable rather than refuted.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom framework) customises only PM1, PS4, PP3 and BP4; PM6 is not specified, so generic ACMG/AMP 2015 definitions apply. Per PMID:25741868 ('PS2 PM6 De novo variants'), PM6 applies when a de novo occurrence is assumed but parentage is not confirmed, and still requires a family history consistent with de novo inheritance and reasonable phenotype specificity.
• ClinVar variation 540667 records four germline submissions for c.547G>A (three Uncertain significance, one Likely benign; no expert panel). No submission reports a de novo event, trio testing, or an affected carrier whose phenotype could be matched to POLE-associated disease; the only criterion signals extracted from the submissions are PM2 and PP3.
• Neither retrieved full text (PMID:25394175, PMID:25741868) mentions POLE c.547G>A, and the somatic endometrial-carcinoma POLE package held for this gene addresses hotspot/recurrence context rather than germline de novo events, so no de novo observation for this variant is available anywhere in the case.

Still missing

• No de novo occurrence of c.547G>A has been reported in any source, so even the unconfirmed de novo premise required by PM6 cannot be established.
• Parental genotypes are unavailable, so neither confirmed parentage (PS2) nor assumed de novo status (PM6) can be evaluated.
• No clinical phenotype or indication for testing is documented for the variant carrier, which is required to judge whether the phenotype matches the POLE disease association.

Sources

Reasoning

PP1 requires co-segregation of POLE c.547G>A (p.Ala183Thr) with disease across multiple affected members of a family, ideally with the number of informative meioses stated. No pedigree, no affected relative genotypes and no meioses are reported for this variant in the ClinVar record (variation 540667, four germline submissions, none of which cites segregation data) or in any publication retrieved for this case. The local POLE framework does not specify PP1, and no segregation data exist, so the criterion is not assessable rather than refuted.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom framework) customises only PM1, PS4, PP3 and BP4; PP1 is not specified, so generic ACMG/AMP 2015 applies, where PP1 covers co-segregation with disease in multiple affected family members as supporting pathogenic evidence (PMID:25741868, Table 3/Table 4 criterion framework; PP1 is weighted supporting).
• ClinVar variation 540667 for c.547G>A: four germline submissions (Invitae/Labcorp, GeneDx, Sinai Health System as Uncertain significance; Ambry Genetics as Likely benign), all single-submitter and none expert-panel. No submission reports a pedigree, co-segregation, or the number of meioses; the submission texts yield only PM2 and PP3 signals, so the ClinVar record contributes no segregation evidence.
• Neither retrieved full text (PMID:25394175 - ACMG/NSGC cancer-referral guideline; PMID:25741868 - ACMG/AMP classification guideline) mentions POLE c.547G>A or provides family data for it; the somatic endometrial-carcinoma POLE package held for this gene contains hotspot/recurrence material only, with no germline family study. No numeric segregation threshold could be applied because no segregation data exist for this variant.

Still missing

• No family pedigree or number of informative meioses is documented for c.547G>A.
• No genotype data for affected or unaffected relatives are available, so co-segregation cannot be scored at any strength.
• No publication retrieved for this case reports this variant in a family, and none provides segregation data.

Sources

Reasoning

PP4 requires that the patient's phenotype or family history be highly specific for a disease with a single genetic etiology. No proband-level phenotype, HPO term list, family history, clinical indication or referring phenotype was captured anywhere in this case: case_summary.json, evidence.json, prefetch.json and the phenotype-prevalence evidence bundle contain variant-level annotations only (normalization, population frequencies, ClinVar submissions, in-silico scores). The only phenotype-like information is the set of ClinVar condition labels attached to VCV000540667 ('Colorectal cancer, susceptibility to, 12', 'Facial dysmorphism-immunodeficiency-livedo-short stature syndrome', 'Hereditary cancer-predisposing syndrome', and further labels), which are broad, non-specific cancer-predisposition and syndromic differentials attached to a conflicting 1-star record rather than a documented proband phenotype; these reflect different submitters' differentials and cannot be treated as a phenotype highly specific for a single genetic etiology. The governing POLE custom framework does not define a PP4 rule (customized criteria are PM1, PS4, PP3 and BP4), so no gene-specific PP4 specificity specification exists and the generic ACMG/AMP definition applies; without any documented phenotype there is no specificity to weigh. This is a missing-data gap rather than a negative finding, and it is closable if the proband's clinical phenotype or HPO terms are supplied. No numeric threshold or comparison operator is defined for PP4 in the governing framework or in any consulted VCEP file, so no >=/<= inequality applies.

Evidence considered

• Reviewed the case bundle and prefetch.json for proband phenotype, clinical indication, HPO terms or family history: none present; the case carries variant-level annotations only.
• ClinVar VCV000540667 condition labels are broad, non-specific cancer-predisposition/syndromic terms ('Colorectal cancer, susceptibility to, 12', 'Facial dysmorphism-immunodeficiency-livedo-short stature syndrome', 'Hereditary cancer-predisposing syndrome') from conflicting single submitters, not a phenotype highly specific for a single genetic etiology.
• The governing POLE custom framework customizes only PM1, PS4, PP3 and BP4, so PP4 has no gene-specific specification and no numeric threshold; the generic ACMG/AMP PP4 definition was applied and could not be evaluated for lack of phenotype data.

Still missing

• No proband phenotype, HPO terms, family history or clinical indication was captured in this case (case_summary.json / evidence.json / prefetch.json), so PP4 phenotype specificity is unassessable.
• No POLE-specific PP4 rule or numeric specificity threshold exists in the governing framework; PP4 was therefore tested only against the generic ACMG/AMP definition.
• PATH-250-323-s001.tif (supplementary figure) could not be converted to text and was not visually reviewed; it is a somatic-tumour figure and is not expected to carry proband phenotype data.

Sources

Reasoning

BS3 requires well-established functional studies demonstrating no damaging effect on protein function. No functional assay of POLE p.Ala183Thr was identified in any retrieved source, so there is no experimental result to support a benign functional claim. The benign-leaning signals present in this case are computational and therefore belong to BP4, not BS3: REVEL 0.037 and BayesDel -0.676197 are predictor outputs, and predictor scores cannot be substituted for assay evidence (BayesDel additionally has no verified published ACMG-strength calibration available here). OncoKB's 'Unknown Oncogenic Effect' is explicitly not a variant-specific functional determination, and its gene-level background describes POLE exonuclease-domain biology rather than this substitution. Leon-Castillo et al. 2020, the basis of the local POLE framework, reports that the large majority of mutations outside the exonuclease domain are not pathogenic, but that conclusion rests on tumour genomic signatures (ultramutated phenotype, C>A/T>G/C>G substitution classes, TMB) in somatic endometrial carcinoma cohorts, not on a functional assay of this variant, and the framework itself specifies no BS3 rule and is not an official ClinGen CSPEC; p.Ala183Thr is also absent from its Supplementary Tables S1-S3. SpliceAI returned no scores for this variant, and missing splice data must not be used to support a benign criterion. No ClinVar submission for this variant asserts BS3 or reports a non-damaging functional assay, and the single 'Likely benign' submission cites no PMIDs, so it cannot be used as functional evidence. BS3 is therefore not assessed for want of assay evidence rather than because an assay showed normal function.

Evidence considered

• No functional assay evidence for POLE c.547G>A (p.Ala183Thr) exists in any retrieved source: the only two full-text papers fetched for this case are PMID:25741868 (ACMG/AMP guideline) and PMID:25394175 (ACMG/NSGC referral guideline), neither of which mentions POLE or this variant or reports assay data.
• REVEL score 0.037 (local REVEL v1.3 lookup, source_registry 'revel'): a computational ensemble prediction, benign-leaning. Computational predictor output is the domain of PP3/BP4 and is not a functional study, so it cannot satisfy BS3; the BP4 call itself is not made here.
• BayesDel score -0.676197 (local BayesDel lookup, source_registry 'bayesdel'): computational, benign-leaning, and with no verified published ACMG-strength calibration available to this case, so it is not usable for any benign criterion here and certainly not as BS3 assay evidence.
• OncoKB for POLE A183T: 'Unknown Oncogenic Effect', is_variant_specific = false, no citations - OncoKB does not curate this variant as functionally neutral or damaging; its gene-level text concerns exonuclease-domain proofreading hotspots generally, not p.Ala183Thr.
• León-Castillo et al. 2020 (PATH-250-323.pdf): abstract reports 'Only 1/23 (4%) tumours with non-EDM showed these genomic alterations, indicating that a large majority of mutations outside the exonuclease domain are not pathogenic' - this is a somatic tumour genomic-signature observation (ultramutated phenotype, POLE-score), not a functional assay of p.Ala183Thr, and the paper neither names nor tabulates this variant (absent from Supplementary Tables S1-S3).
• León-Castillo et al. 2020 methods evaluate variant 'functional status' only via six in-silico tools (SIFT, PROVEAN, PolyPhen-2, PANTHER, SNAP2, REVEL); the local framework built on this paper defines rules for PM1, PS4, PP3 and BP4 only, with no BS3 specification, and is not an official ClinGen CSPEC.
• PMID:25741868 defines BS3 as 'well-established functional studies show no damaging effect' but supplies no variant-level or POLE data; it is the definitional source for BS3 only.
• ClinVar record 540667: no submitter reports BS3 or a functional assay. The single Likely benign submission (Ambry Genetics) cites no PMIDs and cannot be used as functional evidence; the three VUS submissions cite in-silico reasoning (GeneDx), Sherloc methodology (Labcorp), or the ACMG guideline (Sinai Health System). No expert-panel submissions are present.
• Not used for BS3: SpliceAI returned no scores for this variant; that missing data must not be treated as evidence of no splice impact. Absence from COSMIC and lack of a statistically significant CancerHotspots residue row are somatic-recurrence observations, not functional data supporting a non-damaging effect.

Still missing

• No functional assay of POLE p.Ala183Thr demonstrating preserved protein function (proofreading fidelity, mutation-rate reporter, or complementation assay) was identified in any retrieved source, so BS3 cannot be evaluated.
• The governing local POLE framework defines no BS3 rule and is not an official ClinGen CSPEC, so no gene-specific BS3 strength specification is available.
• The only benign-leaning variant-level evidence is computational (REVEL 0.037; BayesDel -0.676197); it is PP3/BP4 material and must not be promoted to BS3.

Sources

Reasoning

BS4 requires evidence that POLE c.547G>A (p.Ala183Thr) does not segregate with disease in a family with multiple affected members - typically an affected relative who carries the phenotype but not the variant. No family study, pedigree or relative genotype data exist for this variant: the ClinVar record (variation 540667) reports four germline submissions without any segregation or non-segregation data, and no retrieved publication reports the variant at all. The local POLE framework does not specify BS4, so generic ACMG/AMP 2015 definitions apply, and with no family data the criterion is not assessable rather than met or refuted.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom framework) customises only PM1, PS4, PP3 and BP4; BS4 is not specified, so generic ACMG/AMP 2015 applies, where BS4 is supporting benign evidence requiring lack of segregation in affected family members (PMID:25741868, who weighted the benign criteria as stand-alone BA1, strong BS1-4, supporting BP1-6).
• ClinVar variation 540667 for c.547G>A: four germline submissions, none expert-panel, with no reported family structure, pedigree, or variant status of affected relatives; the submissions assert no non-segregation observation, so no BS4 evidence is present in the ClinVar record.
• The single Likely benign submission (Ambry Genetics, SCV005928741) is a classification without accompanying segregation evidence; it was not used to infer BS4, since a criterion may not be derived from another source's final classification. Neither retrieved full text (PMID:25394175, PMID:25741868) mentions POLE c.547G>A, and the somatic endometrial-carcinoma POLE package contains no germline family data, so no non-segregation evidence exists for this variant.

Still missing

• No family with multiple affected members is documented for c.547G>A, which BS4 requires.
• No genotypes for affected relatives are available, so non-segregation (variant-negative affected individual) cannot be demonstrated.
• The Likely benign ClinVar submission (Ambry Genetics) provides no stated segregation or non-segregation data and cannot substitute for family evidence.

Sources

Reasoning

BP2 is supporting benign evidence that requires a specific allelic observation: the variant is seen in trans with a pathogenic variant of a fully penetrant dominant disorder, or in cis with a pathogenic variant under any inheritance pattern. It therefore depends on a co-occurring pathogenic variant whose phase relative to c.547G>A is known. Nothing of that kind exists for POLE c.547G>A (p.Ala183Thr). No proband genotype, phased pedigree or relative genotype is available anywhere in this case: the ClinVar record (variation 540667, VCV000540667) contains only this single variant with four single-submitter germline submissions (three Uncertain significance, one Likely benign) and no expert-panel review, and reports no co-occurring POLE variant, no zygosity and no phase. The population datasets contain no allele-configuration observation either: gnomAD reports zero homozygotes for this variant in v2.1 (2/251,408 alleles, AF 7.9552e-06), v4.1 (62/1,613,808 alleles, AF 3.84184e-05) and both non-cancer subsets, and the variant is absent from gnomAD-Canada; COSMIC returns no somatic record from which a co-occurring POLE alteration might be read. The two full texts held for this variant (PMID:25741868 and PMID:25394175) do not mention POLE or p.(Ala183Thr), and the local POLE package (Leon-Castillo et al. 2020) specifies only PM1, PS4, PP3 and BP4, so generic ACMG/AMP 2015 governs BP2 as well. With no phased observation in cis or trans available, and a benign assertion that cannot be borrowed from another submitter's classification, BP2 cannot be evaluated from the available evidence and is recorded as not assessed rather than met or refuted.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom framework) customises only PM1, PS4, PP3 and BP4, so BP2 was evaluated under generic ACMG/AMP 2015, where BP2 is supporting benign evidence requiring the variant to be observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or in cis with a pathogenic variant in any inheritance pattern (PMID:25741868).
• ClinVar variation 540667 for c.547G>A: one variant record, four germline submissions (Uncertain significance x3 - Invitae/Labcorp, GeneDx, Sinai Health System; Likely benign x1 - Ambry Genetics), all single-submitter and none expert-panel, with 1-star conflicting review status. The record states no second POLE variant, no phase and no zygosity. The single Likely benign submission is a classification without accompanying allelic evidence and was not used to infer BP2.
• Population data: gnomAD v2.1 2/251,408 alleles (AF 7.9552e-06, homozygotes = 0), gnomAD v4.1 62/1,613,808 alleles (AF 3.84184e-05, homozygotes = 0), gnomAD v2.1 non-cancer exomes 2/236,872 (AF 8.44338e-06, homozygotes = 0), gnomAD v3.1 non-cancer 3/147,942 (AF 2.02782e-05, homozygotes = 0), and absent from gnomAD-Canada v1.0. No homozygous individual is reported, so the datasets contain no allele-configuration observation relevant to BP2.
• No co-occurring pathogenic variant was identified in any searched source: the somatic search for the variant in COSMIC returned no record, the ACMG/NSGC referral guideline (PMID:25394175) does not cover POLE or polymerase proofreading-associated polyposis, and the ACMG/AMP framework paper (PMID:25741868) contains no variant-level data.
• Gene-level context: ClinVar attaches both dominant and recessive POLE conditions to this variant, and the case's gene-level PVS1 eligibility context records no inheritance-mode entry for POLE, so the modality in which a pathogenic partner allele would have to be observed (cis under any inheritance pattern, or trans for a fully penetrant dominant disorder) is not fixed by any available source.
• No numeric threshold applies to BP2 in the supplied calibration data or in the governing framework; the criterion turns on a phased co-occurrence observation that no available source provides.

Still missing

• No phased genotype or pedigree data exist for c.547G>A, so its cis/trans configuration relative to any poised pathogenic POLE variant is unknown.
• No co-occurring pathogenic POLE variant (germline or somatic) is reported with c.547G>A in the ClinVar record or in any other consulted source.
• No homozygous carrier of c.547G>A is reported (gnomAD homozygotes = 0 in v2.1, v4.1 and both non-cancer subsets), so no allele-configuration observation exists; in any case the population datasets carry no phenotype that could be attached to such an observation.

Sources

Reasoning

BP5 requires that the variant be observed in a case with an alternate molecular basis for disease (an affected individual carrying this variant who also has a separate, sufficient genetic explanation). No case-level genotype or phenotype data exist in this case: there is no proband record, no co-occurring pathogenic variant in another gene and no documented alternate molecular diagnosis anywhere in case_summary.json, evidence.json or prefetch.json. All four ClinVar submissions for VCV000540667 are germline single-submitter clinical-testing records; the submission audit records condition_labels empty for three of them and 'Not Provided' for GeneDx, with no co-occurring-variant or alternate-diagnosis information attached. The governing POLE custom framework does not customize BP5 (customized criteria are PM1, PS4, PP3, BP4), so no gene-specific BP5 operator or numeric likelihood-ratio threshold exists in the applicable framework; the generic ACMG/AMP BP5 definition applies and no evidence for it was collected. Critically, no likelihood-ratio-style or other numeric value is defined for BP5 in this framework or in any consulted VCEP file, so there is no threshold and no <=/>=-style inequality to evaluate. This is a missing-data gap rather than a negative finding and is closable if case-level genotype and co-occurring-variant data are supplied.

Evidence considered

• Reviewed the case bundle, prefetch.json and clinvar_submission_audit.json for any case with an alternate molecular basis for disease: none present; no co-occurring variant, second diagnosis or alternate molecular cause is recorded.
• ClinVar submissions for VCV000540667 carry no condition labels or co-occurring-variant information (condition_labels empty for SCV000772614, SCV005928741, SCV006055420; 'Not Provided' for SCV003930996).
• The governing POLE custom framework customizes only PM1, PS4, PP3 and BP4; BP5 is not gene-specified, has no numeric threshold, and generic ACMG BP5 requires case-level alternate-basis evidence that is absent.

Still missing

• No case-level clinical or genotype data (proband genotype, co-occurring pathogenic variant in another gene, or alternate molecular diagnosis) were captured in this case, so BP5 is unassessable.
• No POLE-specific BP5 rule exists in the governing framework (customized criteria are PM1, PS4, PP3, BP4 only) and no VCEP text supplies a numeric BP5 likelihood-ratio operator, so no threshold applies to this criterion.

Sources