POLE c.286-8C>G, p.?

Reasoning

The exact variant is absent (AF=0) from gnomAD v2.1 and gnomAD v4.1, two large population datasets spanning diverse ancestries, which is below the project's non-VCEP PM2 operating threshold of <0.1%. POLE-associated polyposis/CRC predisposition is a rare autosomal dominant disorder (FAP prevalence approximately 1/11,300-37,600; POLE subset rarer), so absence of this intronic allele in population cohorts is consistent with a rare disease-associated allele rather than a common polymorphism. PM2 is met and applied at supporting strength, per the ClinGen SVI convention (Whiffin et al. 2017, PMID 28518168) that absence/ultra-rareness in large population databases warrants PM2 at supporting rather than moderate strength.

Rule

PM2 (ACMG/AMP 2015, PMID 25741868): absent in controls or at extremely low frequency; project non-VCEP gnomAD operating threshold <0.1%. Observed AF = 0 -> met (supporting, per Whiffin et al. 2017, PMID 28518168).

Evidence considered

• Absent (AF=0) in gnomAD v2.1 (GRCh37) and gnomAD v4.1 (GRCh38); AF=0 < project non-VCEP PM2 threshold of 0.1% (user profile convention).
• Base rule: ACMG/AMP 2015 PM2 (PMID 25741868) - absent in controls or extremely low frequency.
• PM2 supporting-strength calibration: Whiffin N et al., 'Using high-resolution variant frequencies to empower clinical genome interpretation', Genet Med 2017 (PMID 28518168), recommending PM2 at supporting strength for absence/ultra-rareness in population databases.
• Disease rarity context: FAP prevalence 1/11,300-37,600 and birth incidence 1/8,300-10,000 (PMID 41551532, retrieved in pvs1_gene_context), with POLE mutations a minority subset; expected pathogenic allele frequency well below 0.1%.

Still missing

• gnomAD-Canada v1.0 query failed (HTTP 500); PM2 rests on absence in gnomAD v2.1 and v4.1 only.
• No ancestry-stratified allele counts are capturable because the variant is absent from both datasets.

Sources

Reasoning

Intronic variant evaluated with SpliceAI only per the governing generic in-silico workflow (the custom POLE BP4 rule applies only to exact missense variants present in Supplementary Tables S2/S3; this variant is intronic and absent, triggering the framework's generic fallback). SpliceAI max delta 0.08 is <0.1, the SVI-recommended BP4 threshold (Walker et al. 2023, ClinGen SVI Splicing Subgroup, PMID 37442131), with no predicted impact on the exon 4 acceptor (DS_AG 0.0) or the exon 3 donor (DS_DG 0.0); SpliceAI therefore predicts no significant splice impact, supporting BP4 at Supporting strength (default BP4 strength in the governing framework: benign supporting). The same prediction is not double-counted: it is applied to BP4 only, with PP3 not met and BP7 not applicable.

Rule

Generic fallback (custom POLE BP4 rule inapplicable - variant is not a missense variant present in Supplementary Tables S2/S3): SpliceAI max delta <0.1 -> BP4 supporting. Met at 0.08.

Evidence considered

• SpliceAI Lookup (spliceai): max delta 0.08 (DS_AG 0.0, DS_AL 0.08, DS_DG 0.0, DS_DL 0.04) - SpliceAI predicts no significant splice impact; below the BP4 threshold.
• Generic in-silico operating thresholds (generic_acmg_combination_rules, source PMID 25741868): for intronic/synonymous/non-canonical-splice-position variants evaluate SpliceAI only; SpliceAI max delta <0.1 -> BP4 supporting, per SVI-recommended thresholds (Walker et al. 2023, ClinGen SVI Splicing Subgroup report, PMID 37442131). Max delta 0.08 is <0.1, so BP4 supporting is met.
• Custom POLE BP4 rule (final_classification_framework; vcep_path_250_323_s003 / vcep_path_250_323_s004): applies only to exact missense variants listed in Supplementary Tables S2/S3; c.286-8C>G is intronic and absent, so the framework falls back to the generic in-silico workflow.

Still missing

• BP4 rests on a single in-silico predictor (SpliceAI) per the governing single-mechanism rule; no RNA/functional assay confirmation exists for this variant (BS3 territory, outside this group's scope).

Sources

Reasoning

Variant NM_006231.4:c.286-8C>G is an intronic single-nucleotide substitution located 8 bp upstream of exon 3 (VariantValidator exon annotation '3i'; protein consequence NP_006222.2:p.?). No official ClinGen CSPEC exists for POLE, and the governing local custom framework (final_classification_framework, León-Castillo et al. 2020) customizes only PM1/PS4/PP3/BP4 (all missense-oriented), so PVS1 was assessed under the generic ClinGen SVI PVS1 recommendations (pvs1_generic_framework, PMC6185798). The variant is not a nonsense, frameshift, or canonical +-1/2 splice-site variant: pvs1_variant_assessment places it in variant_bucket 'other' with canonical_splice_consensus false and apply_generic_pvs1_framework false. SpliceAI (spliceai) predicts no significant splice impact (max delta = 0.08, below the 0.1 threshold), so there is no evidence of a null allele, NMD-triggering transcript, or protein truncation. PVS1 is therefore not met. The gene-level LoF-mechanism gate (pvs1_gene_context) was eligible for POLE, but eligibility alone does not satisfy the variant-level null-variant requirement.

Rule

ClinGen SVI PVS1 recommendations (PMC6185798); generic ACMG/AMP 2015 (PMID:25741868)

Evidence considered

• pvs1_variant_assessment
• pvs1_generic_framework
• pvs1_gene_context
• spliceai
• generic_acmg_combination_rules
• final_classification_framework

Still missing

• No RNA-based splicing assay or minigene functional study is available to absolutely exclude a subtle cryptic-splice effect; SpliceAI max delta 0.08 argues against one but is not definitive.
• No transcript-level quantification (e.g., NMD assessment) was performed for this variant.

Sources

Reasoning

Variant is intronic (NM_006231.4:c.286-8C>G, intron 3, position -8; non-canonical splice position, VariantValidator exonic context '3i'). The governing local custom POLE framework (León-Castillo et al. 2020) customizes PP3 only for exact missense variants present in Supplementary Tables S2/S3 with REVEL class 'likely disease causing' and <=1 benign in-silico result; this variant is intronic and absent from those tables (direct inspection found only missense entries, e.g. p.P286R in S2, p.P286L/p.P286S in S3), so the framework's own fallback clause mandates the generic in-silico workflow. Under the generic rule, intronic/non-missense variants are evaluated with SpliceAI only (exactly one mechanism per variant): SpliceAI max delta 0.08 does not exceed the >0.2 PP3 threshold (SVI-recommended thresholds; Walker et al. 2023, ClinGen SVI Splicing Subgroup, PMID 37442131), so no splice-impact prediction supports PP3. Missense-path predictors are not applicable to an intronic variant: REVEL score not found (found=false in local REVEL v1.3 data) and BayesDel has no verified published threshold in this pipeline (treated as not usable). PP3 not met.

Rule

Generic fallback (custom POLE PP3 rule inapplicable - variant is not a missense variant present in Supplementary Tables S2/S3): SpliceAI max delta >0.2 -> PP3 supporting. Max delta 0.08 does not meet the threshold.

Evidence considered

• SpliceAI Lookup (spliceai): max delta 0.08 (DS_AG 0.0, DS_AL 0.08, DS_DG 0.0, DS_DL 0.04) - below the PP3 splice-impact threshold; no PP3-supporting splice prediction.
• Generic in-silico operating thresholds (generic_acmg_combination_rules, source PMID 25741868): for intronic/synonymous/non-canonical-splice-position variants evaluate SpliceAI only; SpliceAI max delta >0.2 -> PP3 supporting, per SVI-recommended thresholds (Walker et al. 2023, ClinGen SVI Splicing Subgroup report, PMID 37442131). Max delta 0.08 is not >0.2, so PP3 is not met.
• Custom POLE PP3 rule (final_classification_framework; vcep_path_250_323_s003 / vcep_path_250_323_s004): applies only to exact missense variants listed in Supplementary Tables S2/S3; c.286-8C>G is intronic and absent, so the framework falls back to the generic in-silico workflow.

Sources

Reasoning

PP5 requires an exact-variant ClinVar expert-panel (expert-panel) classification of Pathogenic/Likely pathogenic. The ClinVar record for NM_006231.4(POLE):c.286-8C>G (variation 2005051) contains exactly one submission (SCV003204076, Labcorp Genetics, formerly Invitae) classified Likely benign with review status 'criteria provided, single submitter' (1 star); expert_panel_submissions = 0. There is no expert-panel P/LP assertion, and per adjudication rules ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger PP5. Additionally, the existing classification is in the benign direction, not pathogenic.

Rule

PP5 restricted to an exact-variant ClinVar expert-panel classification of Pathogenic/Likely pathogenic (supporting); non-expert, single-submitter laboratory assertions are excluded by adjudication policy.

Evidence considered

• ClinVar (clinvar): variation 2005051 - classification 'Likely benign', review status 'criteria provided, single submitter', 1 submission (Labcorp), expert_panel_submissions = 0

Still missing

• No ClinVar expert-panel (3-star) Pathogenic/Likely pathogenic classification exists for this exact variant
• Only a single-submitter laboratory classification (1 star) is available, which is excluded from triggering PP5 by rule

Sources

Reasoning

The exact variant NM_006231.4(POLE):c.286-8C>G (NC_000012.11:g.133256816G>C / NC_000012.12:g.132680230G>C) is absent (allele frequency = 0) from both gnomAD v2.1 and gnomAD v4.1, with clean successful queries and no captured allele counts. This is far below the project's non-VCEP BA1 operating threshold of >1% and the original ACMG/AMP 2015 BA1 threshold of >5% in reference populations. There is no population-frequency evidence that this intronic variant is a common benign polymorphism, so BA1 is not met.

Rule

BA1 (ACMG/AMP 2015, PMID 25741868): allele frequency >5% in reference population; project non-VCEP gnomAD operating threshold >1%. Observed AF = 0 -> not met.

Evidence considered

• Absent (AF=0) in gnomAD v2.1 (GRCh37 12-133256816-G-C, direct variant lookup, no errors).
• Absent (AF=0) in gnomAD v4.1 (GRCh38 chr12-132680230-G-C, direct variant lookup, no errors).
• Project non-VCEP gnomAD operating threshold for BA1 is >1% (user profile convention); original ACMG/AMP 2015 BA1 threshold >5% (Richards et al. 2015, PMID 25741868, via generic_acmg_combination_rules). AF=0 does not approach either threshold.

Still missing

• gnomAD-Canada v1.0 could not be queried (HTTP 500); absence determination rests on gnomAD v2.1 and v4.1 only.

Sources

Reasoning

BS1 requires the allele frequency to be greater than expected for the disorder. POLE-associated polyposis/colorectal cancer predisposition is a rare autosomal dominant condition (FAP prevalence approximately 1/11,300-37,600, with the POLE subset rarer), so a pathogenic POLE allele would be expected at very low frequency. The variant is instead absent (AF=0) from gnomAD v2.1 and v4.1, below the project's non-VCEP BS1 operating threshold of >0.3%. The frequency is not elevated; BS1 is not met.

Rule

BS1 (ACMG/AMP 2015, PMID 25741868): allele frequency greater than expected for the disorder; project non-VCEP gnomAD operating threshold >0.3%. Observed AF = 0 -> not met.

Evidence considered

• Absent (AF=0) in gnomAD v2.1 and gnomAD v4.1 (direct variant lookups, no errors).
• Project non-VCEP gnomAD operating threshold for BS1 is >0.3% (user profile convention); base rule ACMG/AMP 2015 BS1 (PMID 25741868). AF=0 is far below the threshold.
• Disease rarity context: FAP birth incidence 1/8,300-10,000 and prevalence 1/11,300-37,600 (quoted in PMID 41551532, retrieved in pvs1_gene_context); POLE mutations account for a minority of FAP cases, supporting a very low expected pathogenic allele frequency.

Still missing

• No disease-expected carrier-frequency calculation was performed beyond the qualitative rarity of POLE-associated polyposis; AF=0 makes any threshold comparison moot.

Sources

Reasoning

BS2 requires the variant to be observed in healthy adult individuals (homozygous for a recessive disorder, or heterozygous for a dominant disorder with full penetrance expected at an early age). No carriers at all - neither heterozygotes nor homozygotes - are observed in gnomAD v2.1 or v4.1 (AF=0). Absence of the variant in healthy controls is not evidence that it is benign; BS2 requires positive observation of the variant in healthy individuals, which is not present. BS2 is not met.

Rule

BS2 (ACMG/AMP 2015, PMID 25741868): observed in a healthy adult (homozygous for recessive, or heterozygous for dominant fully penetrant early-onset disorder). No carriers observed (AF=0) -> not met.

Evidence considered

• Absent (AF=0) in gnomAD v2.1 and gnomAD v4.1; therefore zero healthy-adult carrier observations and zero homozygotes.
• Base rule ACMG/AMP 2015 BS2 (PMID 25741868, via generic_acmg_combination_rules).

Still missing

• No control-cohort carrier observations are available for this variant; only absence data exists.
• No population-genotype breakdown (heterozygote/homozygote counts) was captured because the variant is absent from both queried datasets.

Sources

Reasoning

BP6 requires an exact-variant ClinVar expert-panel classification of Benign/Likely benign. Although the ClinVar classification for variation 2005051 is 'Likely benign', it originates from a single clinical laboratory (SCV003204076, Labcorp Genetics) with review status 'criteria provided, single submitter' (1 star) and expert_panel_submissions = 0. Per adjudication rules, ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger BP6, so BP6 is not met despite the benign-direction label.

Rule

BP6 restricted to an exact-variant ClinVar expert-panel classification of Benign/Likely benign (supporting); non-expert, single-submitter laboratory assertions are excluded by adjudication policy.

Evidence considered

• ClinVar (clinvar): variation 2005051 - classification 'Likely benign', review status 'criteria provided, single submitter', 1 submission (Labcorp), expert_panel_submissions = 0

Still missing

• No ClinVar expert-panel (3-star) Benign/Likely benign classification exists for this exact variant
• The existing 'Likely benign' label is a single-submitter laboratory classification and is explicitly excluded from triggering BP6 by rule

Sources

Reasoning

PS2 (de novo occurrence, maternity and paternity confirmed) cannot be evaluated: the case bundle contains no proband case report, no phenotype/pedigree section, and no parental testing results for NM_006231.4:c.286-8C>G. No de novo occurrence of this variant is reported in ClinVar (SCV003204076 provides no criteria or case-level data), and the only retrieved publication (PMID:28492532, Sherloc) is a methodology paper that never mentions POLE or this variant. With no observation of a de novo event, PS2 is not_assessed; the specific gap is absence of proband-parent trio data with confirmed parentage.

Rule

Generic ACMG/AMP 2015 (Richards et al. 2015, PMID 25741868; source_registry key 'generic_acmg_combination_rules'): PS2 applies to a de novo variant with confirmed maternity and paternity. No gene-specific specification governs PS2 for POLE: the local custom framework (León-Castillo 2020) customizes only PM1/PS4/PP3/BP4, and no ClinGen CSPEC/VCEP for POLE exists (cspec found = false), so generic ACMG/AMP applies.

Evidence considered

• Case bundle (review_manifest.json, case_summary.json, evidence.json, literature_pass.json) contains no proband, pedigree, or parental-testing data
• ClinVar record VCV002005051 / SCV003204076 (1-star, single submitter) carries no criteria and no case-level segregation or de novo information; its final 'Likely benign' classification was not used to infer any criterion
• PMID:28492532 (Sherloc) reviewed in full text; framework discussion of de novo evidence only, no variant-specific data

Still missing

• No proband case report or phenotype data for the index case carrying c.286-8C>G
• No parental testing (maternity/paternity confirmed) demonstrating a de novo event
• No literature/ClinVar case-level source reporting this variant as de novo

Sources

Reasoning

PS3 (generic ACMG/AMP 2015, PMID 25741868) requires well-established in vitro or in vivo functional studies showing a damaging effect on protein function or splicing. For NM_006231.4:c.286-8C>G (intronic, 8 bp upstream of exon 3 in the acceptor region; p.?), the case bundle contains no functional assay evidence of any kind: literature_pass.json lists only PMID 28492532 (Sherloc, a classification-methods paper that does not mention this variant or POLE); evidence.json contains no RNA/minigene/protein assay results; and the local POLE framework source (León-Castillo et al. 2020) evaluates functional status only with six in silico tools and covers no splice-region variants. SpliceAI reports max delta 0.08 (DS_AL 0.08, DS_DL 0.04, DS_AG 0.0, DS_DG 0.0), but that is an in silico splice prediction, not well-established functional evidence, and cannot meet PS3. Evidence is therefore insufficient: PS3 is not assessed pending a functional splicing assay for the exon 3 acceptor region.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868) PS3: 'Well-established in vitro or in vivo functional studies show a damaging effect on protein function or splicing' (Strong). No POLE VCEP/CSPEC PS3 specification exists; the local custom POLE framework (León-Castillo 2020) customizes only PM1/PS4/PP3/BP4, so the generic rule governs.

Evidence considered

• Generic ACMG/AMP 2015 PS3 rule (Richards et al. 2015, PMID 25741868): well-established in vitro or in vivo functional studies show a damaging effect on protein function or splicing; no such studies are present for this variant
• No POLE VCEP/CSPEC exists and the local custom POLE framework (León-Castillo 2020) specifies rules only for PM1/PS4/PP3/BP4, so PS3 is governed by the generic ACMG/AMP rule
• SpliceAI lookup for NM_006231.4:c.286-8C>G: max delta score 0.08 (DS_AL 0.08, DS_DL 0.04, DS_AG 0.0, DS_DG 0.0) — in silico splice prediction only; not functional assay evidence; not used to meet PS3
• PMID:28492532 (Sherloc) full text reviewed: variant c.286-8C>G and POLE not mentioned; confirms PS3 requires well-established functional assays
• ClinVar VCV002005051 single-submitter Likely benign classification not used to infer PS3/BS3 (a criterion may not be inferred from another source's final classification)
• León-Castillo et al. 2020 (PATH-250-323.pdf) reviewed in full: evaluates functional status only via six in silico tools; contains no wet-lab functional assays and no mention of c.286-8C>G

Still missing

• No RNA-based or other functional assay (e.g., RT-PCR on carrier RNA or a minigene splicing assay spanning the exon 3 acceptor region) for NM_006231.4:c.286-8C>G
• No published functional study of this exact variant identified by the literature pass (only PMID 28492532, a methods paper, was retrieved)
• SpliceAI max delta 0.08 is in silico only and cannot substitute for functional assay evidence

Sources

Reasoning

No germline case-control or cohort enrichment data exist for POLE c.286-8C>G, so the generic ACMG/AMP PS4 rule (prevalence of the variant in affected individuals significantly increased versus controls) cannot be evaluated. The governing local custom POLE framework (León-Castillo et al. 2020, J Pathol 250:323-335; registry key vcep_path_250_323) defines PS4 as a somatic-recurrence adaptation capped at Supporting that applies ONLY to exact recurrent exonuclease-domain missense hotspots (p.P286R, p.V411L, p.A456P, p.S297F) with combined COSMIC+TCGA endometrial carcinoma count >=10; NM_006231.4:c.286-8C>G is an intronic (splice-region, exon 3i) variant, is not one of these missense hotspots, and is absent from COSMIC (no somatic cancer report) and from gnomAD. The only literature lead flagged for PS4 (PMID:28492532, Sherloc methods paper, cited by the Labcorp ClinVar submission) was reviewed in full text and contains no variant-specific case data. No proband cases, recurrence counts, or affected-vs-control statistics were identified in any source.

Rule

Generic ACMG/AMP PS4 (PMID 25741868): prevalence of the variant in affected individuals significantly increased compared with controls. Custom POLE framework PS4 rule (vcep_path_250_323) applies only to exact recurrent exonuclease-domain missense hotspots in COSMIC+TCGA EC cohorts (S1, combined count >=10) and is inapplicable to this intronic variant; fallback to generic ACMG/AMP per framework notes.

Evidence considered

• Custom POLE framework PS4 rule (León-Castillo et al. 2020, J Pathol 250:323-335, local framework vcep_path_250_323): PS4_Supporting only when the exact missense variant is recurrent in both COSMIC and TCGA EC cohorts in Supplementary Table S1 with combined EC count >=10 and belongs to the established/pathogenic hotspot set (p.P286R, p.V411L, p.A456P, p.S297F) - not applicable to this intronic variant
• Supplementary Table S1 (vcep_path_250_323_s002): recurrent POLE variants in COSMIC/TCGA EC cohorts - c.286-8C>G not listed
• ClinVar (clinvar): single germline laboratory submission SCV003204076 (Labcorp), Likely benign, no case-control data; conditions 'not provided'
• PMID:28492532 (Sherloc full text): methodology/refinement paper; full-text search for POLE, c.286-8C>G, endometrial, proofread returned zero matches - no case-control or variant-level data
• Context (not a formal citation): COSMIC search found no somatic report; gnomAD v2.1/v4.1 absent

Still missing

• No germline case-control or cohort enrichment study data for c.286-8C>G in POLE-associated phenotypes (e.g., polymerase proofreading-associated polyposis / cancer predisposition)
• Custom POLE framework PS4 rule is limited to exact recurrent exonuclease-domain missense hotspots; no generic case-control dataset (cases vs. controls) was available to apply the generic rule
• No proband case series or phenotypic cohort reporting this variant was found

Sources

Reasoning

PM3 (recessive disorders, variant detected in trans with a pathogenic variant) is not governed by the local custom POLE framework (León-Castillo et al. 2020), which customizes only PM1, PS4, PP3, and BP4; the generic ACMG/AMP 2015 rule (PMID 25741868) therefore applies. POLE is associated with autosomal recessive phenotypes (e.g., FILS syndrome, OMIM 615139), so the criterion is applicable in principle. However, the case bundle contains no affected-proband observation of NM_006231.4:c.286-8C>G, no second POLE variant, and no phase data establishing a trans configuration with any pathogenic POLE variant. The only report of this variant is a single-submitter ClinVar record (VCV002005051 / SCV003204076, Labcorp/Invitae) classified Likely benign with condition 'not provided' and no criteria supplied; it provides no allelic or phase information. The sole retrieved publication (PMID:28492532, Sherloc) is a criteria-methodology paper with no variant-specific, trans/cis, biallelic, or phase content. PM3 cannot be evaluated without proband-level allelic data, so it is recorded as not_assessed with the specific data gaps listed.

Rule

Generic ACMG/AMP 2015 (PMID 25741868): PM3 - for recessive disorders, detected in trans with a pathogenic variant. Not customized by the local León-Castillo POLE framework (customizes only PM1/PS4/PP3/BP4); generic fallback applies.

Evidence considered

• ClinVar record 2005051 (VCV002005051 / SCV003204076, Labcorp Genetics/Invitae): single-submitter Likely benign, criteria provided/single submitter, condition 'not provided'; contains no second-variant, phase, or proband information relevant to PM3.
• PMID:28492532 (Sherloc) full text reviewed: methodology-only paper; no matches for 'in trans', 'in cis', 'PM3', 'BP2', 'biallelic', 'compound heterozyg', or 'phase'; no variant-specific observations for c.286-8C>G.
• Generic ACMG/AMP 2015 rule basis (PMID 25741868): PM3 applies for recessive disorders when the variant is detected in trans with a pathogenic variant; no such observation exists here.

Still missing

• No affected-proband observation of NM_006231.4:c.286-8C>G with phenotype data for any POLE-associated recessive disorder (e.g., FILS syndrome) is present in the case bundle.
• No pathogenic/likely pathogenic POLE variant in trans with c.286-8C>G has been reported; no biallelic or compound-heterozygous configuration is documented.
• No phase data (parental testing, segregation, or linkage) is available to establish trans versus cis configuration.
• The single ClinVar submission (SCV003204076) provides no condition, no phase, and no criteria, and cannot be used to infer any allelic observation.

Sources

Reasoning

PM6 (de novo occurrence, parentage assumed but not confirmed) cannot be evaluated for the same reason as PS2: no de novo observation of c.286-8C>G exists in the case bundle, in the ClinVar submission (no criteria/case data provided), or in the retrieved literature. In the absence of any reported de novo event, PM6 is not_assessed; the gap is absence of any proband-parent trio observation, with or without confirmed parentage.

Rule

Generic ACMG/AMP 2015 (Richards et al. 2015, PMID 25741868; source_registry key 'generic_acmg_combination_rules'): PM6 applies to a de novo variant without confirmation of maternity and paternity. No POLE-specific specification exists for PM6 (custom framework customizes only PM1/PS4/PP3/BP4; no ClinGen CSPEC for POLE), so generic ACMG/AMP applies.

Evidence considered

• Case bundle contains no de novo observation for this variant
• ClinVar VCV002005051 / SCV003204076 provides no criteria or case-level data; final classification not used to infer criteria
• PMID:28492532 (Sherloc) reviewed in full text; de novo rules discussed conceptually only (e.g., 'The observation that a variant has arisen de novo in a relevant and established disease gene is counted as strong evidence'), with no variant-specific observations

Still missing

• No de novo occurrence of c.286-8C>G reported in any source in this case
• No parental testing data of any kind (confirmed or assumed parentage)
• No proband case report to anchor a de novo claim

Sources

Reasoning

PP1 (co-segregation of the variant with disease in affected family members) cannot be evaluated: no affected family members are tested or described anywhere in the case bundle, and no segregation data for c.286-8C>G appear in ClinVar or in the retrieved literature. The custom POLE framework does not specify PP1, and the Sherloc paper (PMID:28492532) discusses segregation evidence only as a general framework concept. PP1 is therefore not_assessed; the gap is a complete absence of family/pedigree segregation data.

Rule

Generic ACMG/AMP 2015 (Richards et al. 2015, PMID 25741868; source_registry key 'generic_acmg_combination_rules'): PP1 applies when the variant co-segregates with disease in affected family members. No POLE-specific specification exists for PP1 (custom framework customizes only PM1/PS4/PP3/BP4; no ClinGen CSPEC for POLE), so generic ACMG/AMP applies.

Evidence considered

• Case bundle contains no segregation/family data (no proband section, no pedigree, no family testing)
• ClinVar VCV002005051 / SCV003204076 provides no segregation information; final classification not used to infer criteria
• PMID:28492532 (Sherloc) reviewed in full text; PP1 cosegregation discussed as a framework rule only, no variant-specific segregation data

Still missing

• No affected family members tested for c.286-8C>G
• No segregation counts or LOD-style data for this variant
• No pedigree information for the index case in the case bundle

Sources

Reasoning

PP4 requires the patient's phenotype or family history to be highly specific for a disease caused by variants in the gene (single genetic etiology). No proband phenotype, clinical indication, or family-history information is available for this case: the sole ClinVar submission lists conditions as 'not provided', and no case report, cohort, or phenotype registry data were retrieved. Phenotype specificity therefore cannot be evaluated, and PP4 is not assessed.

Rule

Generic ACMG/AMP PP4 (PMID 25741868): phenotype or family history highly specific for a gene with a single genetic etiology. Not assessable in the absence of any phenotype data.

Evidence considered

• ClinVar (clinvar): variation 2005051, SCV003204076, api_conditions = ['not provided'] - no phenotype information supplied

Still missing

• No proband phenotype or clinical indication data (ClinVar condition 'not provided')
• No family-history or segregation-context phenotype information
• No POLE-associated phenotype documentation (e.g., polyposis, early-onset cancer history) for the tested individual

Sources

Reasoning

BS3 (generic ACMG/AMP 2015, PMID 25741868) requires well-established in vitro or in vivo functional studies showing NO damaging effect on protein function or splicing. No such functional study exists for NM_006231.4:c.286-8C>G in the case bundle. SpliceAI max delta 0.08 predicts no significant splice impact but is an in silico observation, not well-established functional evidence. The ClinVar record (VCV002005051) is a single-submitter (1-star, Labcorp/Invitae) Likely benign classification whose cited PMID is only the Sherloc methods paper; a laboratory's final classification cannot be used to infer BS3. Absence from gnomAD v2.1/v4.1 and COSMIC provides no functional evidence. Evidence is therefore insufficient: BS3 is not assessed pending a well-established functional assay (e.g., RNA splicing assay) demonstrating normal splicing.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868) BS3: 'Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing' (Benign Strong). No POLE VCEP/CSPEC BS3 specification exists; the local custom POLE framework (León-Castillo 2020) customizes only PM1/PS4/PP3/BP4, so the generic rule governs.

Evidence considered

• Generic ACMG/AMP 2015 BS3 rule (Richards et al. 2015, PMID 25741868): well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing; no such studies are present for this variant
• No POLE VCEP/CSPEC exists and the local custom POLE framework (León-Castillo 2020) specifies rules only for PM1/PS4/PP3/BP4, so BS3 is governed by the generic ACMG/AMP rule
• SpliceAI max delta 0.08 for NM_006231.4:c.286-8C>G: computational splice prediction; not well-established functional evidence of a benign effect; not used to meet BS3
• ClinVar VCV002005051 (SCV003204076, Labcorp Genetics/Invitae, Likely benign, criteria provided single submitter, evaluated 2022-06-12): single-submitter 1-star classification; explicitly not used to infer BS3
• Absence from gnomAD v2.1 and v4.1 and no COSMIC somatic report: population/somatic absence provides no functional evidence for BS3
• PMID:28492532 (Sherloc) full text reviewed: confirms BS3 requires a well-established assay showing no deleterious effect; no functional data for this variant exists in the ClinVar-cited literature

Still missing

• No well-established functional study (e.g., RNA splicing assay of the exon 3 acceptor region, minigene assay) demonstrating normal splicing for c.286-8C>G
• In silico splice prediction (SpliceAI max delta 0.08) alone cannot satisfy BS3
• ClinVar single-submitter Likely benign classification and gnomAD/COSMIC absence are not functional evidence and were not used for BS3

Sources

Reasoning

BS4 (lack of segregation in affected family members) cannot be evaluated because no affected family members have been tested for c.286-8C>G in any source in this case. BS4 requires an observed non-segregation event (an affected family member who does not carry the variant, or a carrier without disease in an appropriately powered pedigree); no such observation exists here. Population-frequency absence (gnomAD v2.1/v4.1/Canada: absent) is not BS4 evidence and was not used for this criterion. BS4 is therefore not_assessed; the gap is an absence of any family-testing data, whether segregating or non-segregating.

Rule

Generic ACMG/AMP 2015 (Richards et al. 2015, PMID 25741868; source_registry key 'generic_acmg_combination_rules'): BS4 applies when there is a lack of segregation of the variant with disease in affected family members. No POLE-specific specification exists for BS4 (custom framework customizes only PM1/PS4/PP3/BP4; no ClinGen CSPEC for POLE), so generic ACMG/AMP applies.

Evidence considered

• Case bundle contains no family-testing or non-segregation data
• ClinVar VCV002005051 / SCV003204076 provides no segregation information; final 'Likely benign' classification was explicitly NOT used to infer BS4 (never infer a criterion from another source's final classification)
• gnomAD v2.1/v4.1/Canada absence noted but does not bear on BS4 (population frequency is outside this group's scope)
• PMID:28492532 (Sherloc) reviewed in full text; BS4 (nonsegregation) discussed as a framework concept only, no variant-specific data

Still missing

• No affected family members tested (no non-segregation observation possible)
• No pedigree/family data for the index case
• No BS4-capable family study in the retrieved literature

Sources

Reasoning

BP2 (observed in trans with a pathogenic variant for a fully dominant disorder, or observed in cis with a pathogenic variant in any inheritance pattern) is not customized by the local custom POLE framework, so the generic ACMG/AMP 2015 rule (PMID 25741868) applies. POLE has autosomal dominant phenotypes (polymerase proofreading-associated polyposis / colorectal cancer predisposition), so BP2 is applicable in principle. However, no second POLE variant is observed anywhere in the case bundle, and no cis/trans configuration or phase information exists for c.286-8C>G. The variant is intronic (c.286-8, p.?) with no proband-level report; the single ClinVar submission is a one-star Likely benign record without condition or criteria, and the only retrieved paper (PMID:28492532, Sherloc) contains no allelic observations. Without any observation of a second variant in cis or trans, BP2 cannot be evaluated and is recorded as not_assessed.

Rule

Generic ACMG/AMP 2015 (PMID 25741868): BP2 - observed in trans with a pathogenic variant for a fully dominant disease/disorder, or observed in cis with a pathogenic variant in any inheritance pattern. Not customized by the local León-Castillo POLE framework; generic fallback applies.

Evidence considered

• ClinVar record 2005051 (VCV002005051 / SCV003204076): single-submitter Likely benign with no condition, no phase, and no second variant; does not provide any cis/trans observation for BP2.
• PMID:28492532 (Sherloc) full text reviewed: no trans/cis, biallelic, or phase content; methodology-only paper with no variant-specific observations.
• Generic ACMG/AMP 2015 rule basis (PMID 25741868): BP2 requires observation in trans with a pathogenic variant for a dominant disorder or in cis with a pathogenic variant; no such observation exists here.

Still missing

• No second POLE variant is reported in this case, so a cis or trans configuration with a pathogenic variant cannot be established.
• No familial segregation or parental phase testing data is available for any fully dominant POLE phenotype (e.g., polymerase proofreading-associated polyposis).
• No affected-proband observation with genotype data for c.286-8C>G exists in the case bundle; the ClinVar submission (SCV003204076) documents only a single-submitter Likely benign call with condition 'not provided'.

Sources

Reasoning

BP5 requires evidence that the variant was found in a case with an alternate molecular basis for disease. No proband-level data on an alternate pathogenic variant, alternate diagnosis, or alternative molecular explanation is available in any source for this case (no proband phenotype or co-occurrence information; ClinVar condition 'not provided'). BP5 therefore cannot be assessed.

Rule

Generic ACMG/AMP BP5 (PMID 25741868): variant found in a case with an alternate molecular basis for disease. Not assessable without proband-level data.

Evidence considered

• ClinVar (clinvar): no co-occurrence or alternate-cause information present in submission SCV003204076

Still missing

• No proband-level data on an alternate molecular basis of disease (no alternate diagnosis, no co-occurring causal variant information)

Sources