POLE c.6331-24C>T, p.?

Reasoning

Scope: c.6331-24C>T is an intronic variant in a splice region (24 nt from the adjacent exon boundary, outside the canonical ±1,2 dinucleotides), so the splice path via SpliceAI is the applicable computational benign-evidence path. SpliceAI Lookup returned a maximum delta score of 0.076 (DS_DG 0.076, DS_AL 0.028, DS_AG 0.004, DS_DL 0.001), which is below the <=0.1 BP4_supporting cutoff of the Jaganathan et al. 2019 Cell SpliceAI calibration (PMID:30661751), indicating no predicted gain or loss of a donor/acceptor site. The independent Pangolin scores in the same lookup (SG 0.044, SL -0.014) concur. Because the variant produces no amino-acid substitution (NP_006222.2:p.?), there is no REVEL or BayesDel score, and none should be used; the benign computational signal rests solely on the splice prediction and is counted once, as BP4_supporting. The governing POLE (León-Castillo et al. 2020) custom framework has its own BP4_Supporting rule, but it is restricted to exact missense variants present in Supplementary Table S2 or S3 with a 'Likely benign' REVEL class and >=4 benign tool results; this variant is in neither table (searched 'c.6331-24C>T', '6331', 'p.?' — no match), and the framework instructs fallback to the generic in-silico workflow, under which BP4_supporting is met. The strength remains supporting rather than stronger because a single predictor family with a null call, unconfirmed by RNA analysis, is the entire basis.

Rule

Generic ClinGen SVI SpliceAI calibration (Jaganathan et al. 2019, Cell; PMID:30661751): BP4_supporting if SpliceAI max delta <= 0.1. The gene-specific POLE (León-Castillo et al. 2020) BP4_Supporting rule is limited to exact missense variants present in Supplementary Table S2/S3 and is not applicable to this intronic variant; the framework directs use of the generic in-silico workflow in that case.

Evidence considered

• Variant consequence type settled first: c.6331-24C>T is intronic and non-canonical (outside ±1,2 splice consensus positions), so BP4 was evaluated only through the SpliceAI path and no missense predictor (REVEL) was used; the prefetch confirms REVEL 'found: false' and BayesDel 'found: false' for this variant.
• SpliceAI Lookup (hg37, distance 500, basic annotation, masked, source_data.spliceai_available true) returned DS_AG 0.004, DS_AL 0.028, DS_DG 0.076, DS_DL 0.001, max_delta_score 0.076, compared against the Jaganathan et al. 2019 Cell cutoff supplied for this task (BP4 supporting <= 0.1; PMID:30661751).
• Pangolin scores from the same lookup (SG 0.044, SL -0.014) independently show no predicted splice gain or loss.
• León-Castillo et al. 2020 framework notes and criteria.json: the custom BP4_Supporting rule requires the exact missense variant to be present in Supplementary Table S2 or S3 with a 'Likely benign'/'Likely-benign' REVEL class and >=4 benign in-silico results, and the framework states that variants absent from those tables are handled by the project's generic in-silico workflow.
• Supplied prefetch evidence sentence for the SpliceAI lookup states 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.08)'; the sub-scores in the underlying source_data give an exact maximum of 0.076, and both values sit below the 0.1 BP4 cutoff, so the discrepancy does not change the verdict.

Still missing

• The POLE governing framework's custom BP4 rule (León-Castillo et al. 2020) is missense-only and has no entry for this variant: PATH-250-323-s003.xlsx.txt (Supplementary Table S2) and PATH-250-323-s004.xlsx.txt (Supplementary Table S3) were searched for 'c.6331-24C>T', '6331' and 'p.?' with no matching row, so no pre-assigned BP4 code exists for this variant; the generic SpliceAI path was used instead.
• No RNA-based splice assay (RT-PCR/minigene) is available to confirm the negative SpliceAI call; BP4 rests on a single in-silico evidence line, so it is capped at supporting strength.

Sources

Reasoning

Not met. NM_006231.4:c.6331-24C>T is a single-nucleotide intronic substitution 24 bp upstream of the intron 45/exon 46 boundary (VariantValidator: NC_000012.11:g.133202927G>A / NC_000012.12:g.132626341G>A, variant_exonic_positions = '45i', MANE/RefSeq Select transcript NM_006231.4, CCDS9278.1, predicted protein NP_006222.2:p.?). It is therefore not one of the null-variant types to which PVS1 is restricted under the ClinGen SVI PVS1 recommendations (PMC6185798): nonsense, frameshift, canonical +-1/2 splice-consensus, initiation-codon, or single/multi-exon deletion. The applicable prediction path for an intronic/splice-region variant is splicing, and SpliceAI (basic, distance 500, hg37) is negative: DS_AG 0.004, DS_AL 0.028, DS_DG 0.076, DS_DL 0.001, max delta 0.076, with the largest delta being a donor-gain prediction located ~422 nt away; Pangolin SG 0.044 / SL -0.014. The max delta of 0.076 is below even the SpliceAI recommended supporting cutoff of 0.2, and far below the 0.5 high-precision cutoff (Jaganathan et al. 2019, Cell, PMID:30661751), so no gain, loss or strengthening of a splice site is predicted and there is no computational basis for an aberrant, NMD-triggering or protein-truncating transcript. Position 24 bp from the exon boundary also lies outside the standard splice-region window (within 1-3 bases of the exon or 3-8 bases of the intron) discussed in ACMG/AMP 2015 (PMID:25741868), and that guideline states that for splice variants a null effect is assumed, not established, and that confirmation of impact requires RNA or protein analysis; no RNA or protein data exist for this variant in this case, and REVEL is not found (and would be irrelevant for a non-missense variant). The gene-level gate PVS1 also relies on is not substantiated: the local POLE package (Leon-Castillo et al. 2020) is a somatic endometrial-carcinoma hotspot/missense resource that customizes only PM1, PS4, PP3 and BP4, no ClinGen CSPEC/VCEP exists for POLE (cspec.found = false), and the case's PVS1 gene-context file records lof_mechanism_supported = true on the strength of five PMIDs whose abstracts concern MAN2B1, FBXW7, SRSF1, ASXL3 and cutaneous melanoma/pigmentation genes, none of which mentions POLE. Because the criterion is not met, no strength tier is assigned: the SVI downgrade routes to 'strong', 'moderate' or 'supporting' are only reached after a null effect has been established, and no such downgrade was applied here.

Rule

ClinGen SVI PVS1 recommendations (source_registry key pvs1_generic_framework, PMC6185798) applied under generic ACMG/AMP 2015 fallback: PVS1 requires a predicted-null variant; this non-canonical intronic substitution (24 bp from the exon 46 acceptor) has no predicted splice effect (SpliceAI max delta 0.076 < 0.2) and no RNA/protein confirmation, so PVS1 is not met and no strength tier is assigned.

Evidence considered

• Variant normalization (prefetch/variant_validator): NM_006231.4:c.6331-24C>T, genomic NC_000012.11:g.133202927G>A and NC_000012.12:g.132626341G>A; variant_exonic_positions = '45i' for both assemblies (intron 45); MANE Select / RefSeq Select transcript NM_006231.4, CCDS9278.1; predicted protein consequence NP_006222.2:p.? with no amino-acid change; primary_assembly_loci confirm the substitution is a single G>A.
• SpliceAI Lookup (source_registry key 'spliceai'; basic, distance 500, hg37): DS_AG 0.004, DS_AL 0.028, DS_DG 0.076, DS_DL 0.001; max delta score 0.076; DP_AG -24, DP_AL -57, DP_DG -422, DP_DL -174; SpliceAI reports 'no significant splice impact'. Pangolin SG 0.044, SL -0.014. Compared with the SpliceAI recommended supporting cutoff 0.2 and high-precision cutoff 0.5 (Jaganathan et al. 2019, Cell, PMID:30661751), both far above this variant's delta.
• Variant-level PVS1 scaffold in the case data (source_registry key 'pvs1_variant_assessment') records canonical_splice_consensus = false, consequence_class 'unknown', variant_bucket 'other' and apply_generic_pvs1_framework = false, with the rationale that the variant does not fall into the generic null-variant buckets of nonsense, frameshift or canonical +-1,2 splice consensus.
• ClinGen SVI PVS1 recommendations (source_registry key 'pvs1_generic_framework', PMC6185798): PVS1 is restricted to variants predicted to be null (nonsense, frameshift, canonical +-1/2 splice, initiation codon, single- or multi-exon deletion), and for splice-region variants an aberrant transcript must be demonstrated rather than assumed before PVS1 can be applied.
• ACMG/AMP 2015 full text (PMID:25741868, PVS1 section): 'For splice site variants, the variant may lead to exon skipping, shortening or inclusion of intronic material, due to alternative donor/acceptor site usage or creation of new sites. Although splice site variants are predicted to lead to a null effect, confirmation of impact requires functional analysis by either RNA or protein analysis.' The same section also cautions that truncating variants in the last exon or last 50 bp of the penultimate exon escape NMD and that alternatively spliced exons can make an assumed null benign - no NMD-relevant aberrant transcript is predicted here in any case, since no splice change is predicted. No POLE or c.6331-24C>T-specific data appear in this guideline.
• Governing framework check: no ClinGen CSPEC found (prefetch cspec.found = false) and no VCEP specification for POLE; the local framework (source_registry keys 'final_classification_framework' and 'vcep_path_250_323', Leon-Castillo et al. 2020 J Pathol 250:323-335 plus Supplementary Tables S1-S3) is a somatic endometrial-carcinoma resource whose customized criteria are PM1, PS4, PP3 and BP4 only - all exonuclease-domain missense hotspot/substitution rules - and it contains no PVS1 or splice decision tree. Generic ACMG/AMP 2015 plus the ClinGen SVI PVS1 recommendations therefore govern PVS1.
• Gene-level LoF gate audit: output/pvs1_gene_context.json sets germline_disease_context_found = true, lof_mechanism_supported = true and pvs1_gene_gate = 'eligible' citing five PMIDs whose titles/abstracts concern MAN2B1 alpha-mannosidosis, FBXW7 neurodevelopmental syndrome, SRSF1 haploinsufficiency, ASXL3 Bainbridge-Ropers syndrome and cutaneous melanoma/pigmentation genes; none mentions POLE. This gate was not accepted as POLE loss-of-function mechanism evidence and PVS1 was not granted on its basis.

Still missing

• No RNA or protein-based splice assay exists for this intronic variant; a demonstrated aberrant (e.g. out-of-frame) transcript would be required to revisit PVS1, and no such data are present in this case.
• No gene-specific PVS1 decision tree is available: no ClinGen CSPEC/VCEP for POLE, and the local Leon-Castillo et al. 2020 package customizes only PM1/PS4/PP3/BP4.
• The gene-level LoF-mechanism justification present in the case data is not usable evidence (supporting PMIDs concern unrelated genes); a genuine POLE LoF-mechanism literature basis was not retrievable in this run.

Sources

Reasoning

PS4 requires that the prevalence of the variant in affected individuals is significantly increased compared with its prevalence in controls (ACMG/AMP 2015, PMID:25741868). No germline case-control dataset, cohort enrichment statistic, odds ratio, or p-value exists anywhere in this case for NM_006231.4:c.6331-24C>T; the only population data are unselected reference frequencies (gnomAD v2.1 AF 0.000237, 66/278,448 alleles; gnomAD v4.1 AF 0.000130, 209/1,611,370 alleles), which are control-side data and cannot establish enrichment. The governing gene-specific specification for this variant's gene is the local POLE framework derived from Leon-Castillo et al. 2020 (J Pathol 250:323-335) and its supplementary tables, which declares PS4. That framework's PS4 specification is explicit and narrow: PS4_Supporting applies only when the exact POLE missense variant is recurrent in BOTH the COSMIC and TCGA endometrial carcinoma cohorts in Supplementary Table S1, has a combined EC count >=10, and belongs to the paper's established/pathogenic set (P286R, V411L, A456P, S297F). The variant under review is intronic (NM_006231.4:c.6331-24C>T, exon 45i per VariantValidator, NP_006222.2:p.?), i.e. not a missense exonuclease-domain substitution at all, and I searched Supplementary Table S1 in full (all 108 rows, every notation: 'c.6331-24C>T', 'p.?', '6331', plus the genomic coordinates 133202927/132626341 and ClinVar ID 1697379) and found no entry for it in any notation. Every row of Table S1 is a protein-level/CDS missense or truncating notation; no intronic variant is listed. COSMIC likewise reports no somatic occurrence, so neither arm of the framework's dual-recurrence requirement can be satisfied. Because no numeric enrichment value was extracted, the directional operator check for this criterion is vacuous - there is no value to compare against a >= operator. PS4 is therefore not met, and the separate matter of the paper's somatic hotspot recurrence context for the five established exonuclease-domain missense mutations does not transfer to this intronic variant.

Rule

PS4_Supporting (local POLE framework, Leon-Castillo et al. 2020): apply only to an exact missense exonuclease-domain variant recurrent in both COSMIC and TCGA in Supplementary Table S1 with combined EC count >=10 and belonging to the established pathogenic set (p.P286R, p.V411L, p.A456P, p.S297F).

Evidence considered

• The local POLE framework governing this gene (Leon-Castillo et al. 2020) restricts PS4 to an exact-variant somatic-recurrence rule: the exact missense exonuclease-domain variant must be recurrent in both the COSMIC and TCGA endometrial carcinoma cohorts in Supplementary Table S1, with a combined EC count >=10, and must belong to the paper's established pathogenic set (p.P286R, p.V411L, p.A456P, p.S297F). PS4 is capped at Supporting in that framework.
• Searched PATH-250-323-s002.xlsx (Supplementary Table S1, complete 108-line table) for 'c.6331-24C>T', 'p.?', '6331', 'intron', 'splice': no match. The table lists only protein/CDS-level missense and truncating notations (e.g. p.P286R c.857C>G; p.V411L c.1231G>T), with no intronic entry.
• Searched PATH-250-323.pdf (full text, 2091 lines) for the variant in all supplied notations and for nomenclatures '6331', 'splice', 'intronic', 'germline', 'case-control', 'odds ratio': no match for this variant; the paper evaluates somatic exonuclease-domain mutations only.
• Searched the genomic coordinates g.133202927G>A / g.132626341G>A and ClinVar variation ID 1697379 across the entire POLE VCEP package: no match, confirming this intronic variant is outside the scope of the governing package.
• gnomAD reference frequencies (v2.1 AF 0.000237, 66/278,448 alleles; v4.1 AF 0.000130, 209/1,611,370 alleles; zero homozygotes in both) are control-population data and provide no affected-individual arm, so they cannot support PS4.
• ClinVar variation 1697379 supplies a Likely benign classification from a single clinical laboratory with no assertion of increased prevalence in affected individuals.

Still missing

• PATH-250-323-s001.tif is declared by this gene's VCEP index as governing PM1 and PS4 but could not be converted to text; it was therefore unavailable for the PS4 recurrence/hotspot search. It is a supplementary figure expected to mirror the data in Supplementary Table S1, which was searched in full and contains no entry for c.6331-24C>T.
• No germline case-control or cohort enrichment dataset (odds ratio, p-value) is available for POLE c.6331-24C>T; PS4 could not be tested against a numeric enrichment value.
• Searched PATH-250-323-s002.xlsx (Supplementary Table S1, full text) and PATH-250-323.pdf (full text) for c.6331-24C>T, p.?, 6331, 133202927, 132626341 and 1697379 - no entry for this variant in either file.

Sources

Reasoning

PM1 requires the variant to lie in a mutational hotspot and/or a critical, well-established functional domain without benign variation. The vcep_materials.json 'domain_tables' key is empty ({}), so no authoritative VCEP-approved POLE critical-domain list exists to check the variant against. The governing local POLE package specifies PM1 only for exact protein-level exonuclease-domain substitutions: Strong for the five established hotspots p.P286R, p.V411L, p.S297F, p.A456P and p.S459F; Moderate for the paper's pathogenic non-hotspot set (p.F367S, p.L424I, p.M295R, p.P436R, p.M444K, p.D368Y); and Supporting for the POLE-score-3 set (p.A465V, p.L424V, p.T278M, p.A428T). NM_006231.4:c.6331-24C>T is intronic (intron 45, 24 nt from the exon 46 acceptor) and produces p.? - it is none of those substitutions, has no residue, and therefore cannot fall within the coding exonuclease domain those rules describe. Supplementary Table S1 lists no intronic or splice-region variant at all, and SpliceAI max delta 0.076 for this substitution indicates no gain of a functional splice hotspot, so it cannot be treated as a splice-region hotspot either. PM1 is therefore not met.

Rule

PM1 not met: local POLE PM1 rules apply only to exact exonuclease-domain missense substitutions, and this intronic p.? variant lies outside the exonuclease domain and outside every listed hotspot.

Evidence considered

• vcep_materials.json contains an empty domain_tables object ({}), so there is no VCEP-derived critical functional domain list for POLE against which the variant's residue could be checked.
• The local POLE framework in final_classification_framework.json defines PM1_Strong for the exact hotspots p.P286R, p.V411L, p.S297F, p.A456P, p.S459F; PM1_Moderate for p.F367S, p.L424I, p.M295R, p.P436R, p.M444K, p.D368Y; and PM1_Supporting for p.A465V, p.L424V, p.T278M, p.A428T - all protein-level substitution rules with no provision for intronic variants.
• PATH-250-323.pdf was searched ('c.6331-24C>T', '6331', 'p.?', 'intron', 'intronic', 'splice'): the paper defines the five pathogenic exonuclease-domain hotspots in somatic endometrial carcinoma and states that a large majority of mutations outside the exonuclease domain are not pathogenic; it analyses no intronic or splice variant and never mentions this variant.
• PATH-250-323-s002.xlsx Supplementary Table S1 was read in full (101 variant rows): entries are exonic missense, nonsense and frameshift changes with COSMIC/TCGA recurrence counts; no row corresponds to c.6331-24C>T and no intronic variant is listed.
• SpliceAI annotation for this variant (DS_AG 0.004, DS_AL 0.028, DS_DG 0.076, DS_DL 0.001; max delta 0.076) shows no predicted splice-altering effect, so the substitution does not create or destroy a splice hotspot or a functional splice element.
• VariantValidator normalization places the variant in intron 45 (exon context '45i') with protein consequence NP_006222.2:p.?, i.e. it does not encode any residue of the POLE exonuclease domain.
• PMID:25741868 supplies the generic ACMG/AMP PM1 definition (mutational hotspot and/or critical and well-established functional domain without benign variation) applied here.

Still missing

• PATH-250-323.pdf and PATH-250-323-s002.xlsx (both declared by the POLE index as governing PM1) were searched for 'c.6331-24C>T', '6331' and 'p.?' and contain no entry for this variant; Supplementary Table S1 contains no intronic or splice-region variant whatsoever, so no pre-assigned PM1-style code for this variant exists in either file.
• PATH-250-323-s001.tif (declared as governing PM1) could not be converted to text and was therefore not searchable; it is a supplementary figure of the same Leon-Castillo dataset and remains visually unverified.
• Only a local custom POLE framework exists (index.txt states it is 'not an official ClinGen CSPEC/VCEP rule set'; cspec.found=false) and vcep_materials.json 'domain_tables' is empty, so no authoritative VCEP critical-domain boundary list could be applied; PM1 rests on the local framework's exact-substitution missense rules.

Sources

Reasoning

PM2 at supporting strength requires the variant to be absent from, or at a frequency at or below 0.0001 in, a large control/general population cohort (supplied generic calibration, ACMG/AMP 2015 PMID:25741868 as refined by the ClinGen SVI PM2-supporting recommendation). This variant is clearly not that rare, and it is not absent from controls: gnomAD v2.1 all-comers total AF 0.000237028 (66 carrier alleles / 278,448), popmax FAF 0.00044857, European (non-Finnish) AF 0.00048502 (61/125,768); gnomAD v4.1 all-comers total AF 0.000129703 (209 carrier alleles / 1,611,370), popmax FAF 0.00014455, European (non-Finnish) AF 0.000163774 (193/1,178,456). The non-cancer subsets are equivalent or higher: gnomAD v2.1 non-cancer exomes AF 0.000226672 (53/233,818), popmax FAF 0.00044857, NFE 0.000527727; gnomAD v3.1 non-cancer genomes AF 0.000169008 (25/147,922), popmax FAF 0.00019435, NFE 0.000309. Both the total AF and the popmax FAF exceed 0.0001 in all four datasets (8/8 dataset-metric comparisons), with the lowest single value (v4.1 all-comers total AF 0.000129703) still above the threshold. PM2 is therefore not met, and the conclusion is unchanged whether the all-comers gnomAD v2.1/v4.1 entries are used as the default source (as the governing POLE framework requires, since it specifies no alternative) or the non-cancer subsets are substituted.

Rule

PM2 (supporting pathogenic): absent from, or at an allele/filtering frequency <= 0.0001 in, a large control/general population cohort (ACMG/AMP 2015 with ClinGen SVI supporting-strength calibration, PMID:25741868).

Evidence considered

• PM2 threshold applied from the supplied published calibration: supporting pathogenic strength when the variant is absent or at allele/filtering frequency <= 0.0001 in a large control population (ACMG/AMP 2015, PMID:25741868; ClinGen SVI recommendation to downgrade PM2 to supporting).
• gnomAD v2.1 (all-comers totals; default source): total AF 0.000237028 (AC 66 / AN 278,448), homozygotes 0; popmax FAF 0.00044857; European (non-Finnish) AF 0.00048502 (61/125,768).
• gnomAD v4.1 (all-comers totals; default source): total AF 0.000129703 (AC 209 / AN 1,611,370), homozygotes 0; popmax FAF 0.00014455; European (non-Finnish) AF 0.000163774 (193/1,178,456).
• gnomAD v2.1 non-cancer exomes (GNOMAD_V2_1_NON_CANCER reported by its exome-only figures): AF 0.000226672 (53/233,818), homozygotes 0; popmax FAF 0.00044857; European (non-Finnish) AF 0.000527727 (61/115,590).
• gnomAD v3.1 non-cancer genomes (GNOMAD_V3_1_NON_CANCER; genomes only, v3 has no exome cohort): AF 0.000169008 (25/147,922), homozygotes 0; popmax FAF 0.00019435; European (non-Finnish) AF 0.000309 (20/64,800).
• The variant is definitively not absent from controls: it is carried on 66 alleles in gnomAD v2.1 and 209 alleles in gnomAD v4.1, with consistent presence across the non-cancer subsets; read-depth/absence checking is therefore not applicable.
• Source selection: the governing POLE framework is the local Leon-Castillo-derived custom framework (official_clingen = false), which specifies no non-cancer or exome-only population source for PM2, so the all-comers GNOMAD_V2_1/GNOMAD_V4_1 entries are the default; the v2.1 non-cancer exome and v3.1 non-cancer genome subsets were nevertheless evaluated and give the same 'not met' result.
• gnomAD-Canada v1.0 (HostSeq genomes) lookup for this variant failed with HTTP 500 (Internal Server Error); this is a retrieval failure, not a confirmed absence, and it was excluded from the PM2 assessment rather than counted as support.
• The single-submitter ClinVar classification (Likely benign, SCV002550037) was not used as evidence for PM2.

Sources

Reasoning

Scope: NM_006231.4:c.6331-24C>T (NC_000012.11:g.133202927G>A) is an intronic substitution 24 nucleotides upstream of an exon boundary, not a missense, truncating or synonymous change, so computational evidence must come from the splice path (SpliceAI) and not from REVEL/BayesDel. SpliceAI Lookup returned DS_AG 0.004, DS_AL 0.028, DS_DG 0.076, DS_DL 0.001 (variant consequence 'sequence_variant'), giving a maximum delta score of 0.076. That is far below the >=0.2 supporting and >=0.5 moderate cutoffs of the Jaganathan et al. 2019 Cell SpliceAI calibration (PMID:30661751), so no computational evidence of a deleterious splice effect exists and PP3 is not met. The governing POLE (León-Castillo et al. 2020) custom framework does provide its own PP3_Supporting rule, but that rule is explicitly restricted to an exact missense variant listed in Supplementary Table S2 or S3 with a 'likely disease causing' REVEL class; this intronic variant appears in neither table (searched 'c.6331-24C>T', '6331', 'p.?' — no match in either file), and the framework itself instructs that a variant absent from the supplementary computational tables falls back to the generic in-silico workflow. The generic path is therefore the governing rule here, and under it PP3 is not met. No RNA-based functional data or de novo splice-junction assay is available to suggest a splicing effect that the predictor missed, and the ACMG/AMP 2015 guidance that splice predictions are low-specificity, adjunct-only evidence (PMID:25741868) would in any case not convert a null prediction into PP3 support.

Rule

Generic ClinGen SVI SpliceAI calibration (Jaganathan et al. 2019, Cell; PMID:30661751): PP3_supporting if SpliceAI max delta >= 0.2; PP3_moderate if >= 0.5. The gene-specific POLE (León-Castillo et al. 2020) PP3_Supporting rule is limited to exact missense variants present in Supplementary Table S2/S3 and is not applicable to this intronic variant; the framework directs use of the generic in-silico workflow in that case.

Evidence considered

• Variant consequence type settled first: c.6331-24C>T is intronic (ClinVar record gives the HGVS as NC_000012.12(NM_006231.4):c.6331-24C>T under 'genome_context_intronic_sequence', protein consequence NP_006222.2:p.?; the generic PVS1 scaffold classes it as non-canonical, non-±1,2 and bucket 'other'), so only the SpliceAI path was evaluated and REVEL/BayesDel were deliberately not consulted for PP3.
• SpliceAI Lookup (hg37, distance 500, basic annotation, masked) returned DS_AG 0.004, DS_AL 0.028, DS_DG 0.076, DS_DL 0.001, max delta score 0.076; compared against the Jaganathan et al. 2019 Cell cutoffs supplied for this task (PP3 supporting >= 0.2, moderate >= 0.5; PMID:30661751).
• Pangolin scores from the same lookup (SG 0.044, SL -0.014) are concordant with no gain or loss of splice signal; Pangolin was used only as a consistency check because no ACMG-strength Pangolin calibration is published.
• León-Castillo et al. 2020 framework notes and criteria.json: the custom PP3_Supporting rule requires the exact missense variant to be present in Supplementary Table S2 or S3 with a 'likely disease causing' REVEL class and <=1 benign in-silico result, and states that when a variant is absent from those tables the project's generic in-silico workflow is used instead.
• Both supplementary in-silico tables were searched directly; Table S2 contains 44 protein-level missense entries (e.g. A456P, P286R, S297F, V411L) and Table S3 contains 37 protein-level missense/indel entries (e.g. A456V, S297Y, L698Cfs*94), none of which is this intronic variant.
• ACMG/AMP 2015 (PMID:25741868) note that splice-site prediction software has high sensitivity but lower specificity (~60-80%), that multiple computational tools should be combined and counted as a single piece of evidence, and that predictions should not be the sole source of evidence for a clinical assertion.

Still missing

• The POLE governing framework's own PP3 rule (León-Castillo et al. 2020, custom) is missense-only and its supporting tables contain no entry for c.6331-24C>T: PATH-250-323-s003.xlsx.txt (Supplementary Table S2) and PATH-250-323-s004.xlsx.txt (Supplementary Table S3) were searched for 'c.6331-24C>T', '6331' and 'p.?' and contain no matching row, so no pre-assigned PP3 code exists for this variant.
• No RNA/cDNA splice assay or minigene result is available to functionally confirm the SpliceAI negative prediction; such data are not required for PP3/BP4 here but would raise confidence.

Sources

Reasoning

PP5 requires that a reputable source reports the variant as pathogenic, with the evidence not available to the laboratory to evaluate independently. Per the governing rule applying to this criterion in this pipeline, PP5 may be applied only for an exact-variant ClinVar expert-panel (ClinGen VCEP/Expert Panel) classification of Pathogenic or Likely pathogenic; ordinary laboratory submissions and aggregate ClinVar labels must never trigger it. The exact variant NM_006231.4(POLE):c.6331-24C>T is present in ClinVar as variation ID 1697379, variant title NM_006231.4(POLE):c.6331-24C>T, and its classification is Likely benign - the opposite direction - with review status 'criteria provided, single submitter' (1 star). The submission audit explicitly records expert_panel_submissions = 0 and an empty expert-panels set; the single submission is SCV002550037 from the Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital, an ordinary clinical-testing laboratory submission (origin germline, method clinical testing, is_expert_panel false), and its only cited paper is PMID:25741868, the generic ACMG/AMP 2015 guideline, which names neither POLE nor this variant. There is no expert-panel pathogenic assertion anywhere in the record, so PP5 is not met.

Rule

PP5 applies only to an exact-variant ClinVar expert-panel (ClinGen VCEP/Expert Panel) Pathogenic or Likely pathogenic classification; ordinary laboratory submissions and aggregate ClinVar labels never trigger it (ACMG/AMP 2015 as implemented for this pipeline; PMID:25741868).

Evidence considered

• ClinVar variation 1697379 (exact transcript-HGVS match 'NM_006231.4(POLE):c.6331-24C>T') is classified Likely benign with review status 'criteria provided, single submitter' (1 star) - not a pathogenic expert-panel assertion.
• Submission audit for this variant records expert_panel_submissions = 0 and an empty expert-panels set; the sole submission, SCV002550037 (Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital), is flagged is_expert_panel false, trust_tier 'single_submitter'.
• The only PMID cited by that submission is 25741868 (ACMG/AMP 2015 consensus guideline), which does not mention POLE or this variant and therefore supplies no variant-level pathogenic assertion.
• No ClinGen VCEP/Expert Panel assertion for POLE c.6331-24C>T exists in the retrieved evidence.

Sources

Reasoning

POLE NM_006231.4:c.6331-24C>T (NC_000012.11:g.133202927G>A; deep intronic, exon 45i, 24 nt from the exon 45 acceptor) is present at low but clearly non-trivial frequency in every population dataset examined. gnomAD v2.1 all-comers: total AF 0.000237028 (66/278,448 alleles), 0 homozygotes, popmax FAF 0.00044857, highest ancestry European (non-Finnish) AF 0.00048502 (61/125,768). gnomAD v4.1 all-comers: total AF 0.000129703 (209/1,611,370 alleles), 0 homozygotes, popmax FAF 0.00014455, European (non-Finnish) AF 0.000163774 (193/1,178,456). The non-cancer subsets give the same picture (gnomAD v2.1 non-cancer exomes AF 0.000226672, 53/233,818, NFE 0.000527727, 61/115,590; gnomAD v3.1 non-cancer genomes AF 0.000169008, 25/147,922, popmax FAF 0.00019435, NFE 0.000309, 20/64,800). The BA1 stand-alone benign threshold requires an allele frequency >= 0.05 in a control/general population dataset (ACMG/AMP 2015, PMID:25741868). The single highest ancestry-specific frequency observed anywhere is 0.000528, roughly 95-fold below BA1, and no dataset or ancestry reaches even 0.001. BA1 is therefore not met, and the verdict is identical whether the all-comers gnomAD v2.1/v4.1 totals or the non-cancer subsets are used. The ClinVar record for this variant was not used as evidence for this criterion.

Rule

BA1 (stand-alone benign): allele frequency >= 0.05 in a control/general population dataset (ACMG/AMP 2015, PMID:25741868).

Evidence considered

• gnomAD v2.1 (all-comers totals; default source because the governing POLE framework specifies no population source): variant 12-133202927-G-A, total AF 0.000237028 (AC 66 / AN 278,448), homozygotes 0; popmax FAF 0.00044857; highest ancestry European (non-Finnish) AF 0.00048502 (AC 61 / AN 125,768), homozygotes 0; exome AF 0.000214523 (53/247,060), genome AF 0.000414171 (13/31,388).
• gnomAD v4.1 (all-comers totals; default source): variant chr12-132626341-G-A, total AF 0.000129703 (AC 209 / AN 1,611,370), homozygotes 0; popmax FAF 0.00014455; highest ancestry European (non-Finnish) AF 0.000163774 (AC 193 / AN 1,178,456), homozygotes 0; exome AF 0.000126107 (184/1,459,078), genome AF 0.000164158 (25/152,292).
• gnomAD v2.1 non-cancer exomes (sensitivity analysis; GNOMAD_V2_1_NON_CANCER reported by its exome-only figures): AF 0.000226672 (53/233,818), homozygotes 0; European (non-Finnish) AF 0.000527727 (61/115,590), which is the highest ancestry-specific value seen in any dataset in this case; popmax FAF 0.00044857.
• gnomAD v3.1 non-cancer genomes (sensitivity analysis; v3 has no exome cohort, so this source is genomes-only): AF 0.000169008 (25/147,922), homozygotes 0; popmax FAF 0.00019435; European (non-Finnish) AF 0.000309 (20/64,800).
• The 'highest population' annotation for gnomAD v3.1 non-cancer (1KG:ITU, AF 0.0049 / MAF 0.490%) rests on a single allele in a 204-allele sample and is treated as statistical noise, not as a frequency signal.
• BA1 threshold applied from the supplied published calibration: stand-alone benign at allele frequency >= 0.05, ACMG/AMP 2015 (PMID:25741868). Threshold comparison used the highest ancestry-specific frequency (0.000528) and the popmax FAF values (0.00014455-0.00044857); all are far below 0.05, so BA1 fails by roughly two orders of magnitude in every source.
• Ancestry pattern is not a private founder signal: the variant is most frequent in European (non-Finnish) samples in every dataset, is present at low frequency in other groups (e.g. Admixed American 1/35,310 in v2.1), and has 0 homozygotes everywhere, consistent with a low-level recurring variant rather than a single-population founder allele.
• A single-submitter ClinVar classification (Likely benign, SCV002550037) exists for this variant but was not used as evidence for BA1: a criterion is never inferred from another source's final classification.
• gnomAD-Canada v1.0 (HostSeq genomes) did not contribute to this criterion; its lookup failed with HTTP 500 and was therefore excluded rather than treated as a negative result.

Sources

Reasoning

BS1 (strong benign) requires an allele frequency greater than that expected for the disorder. No gene-specific population threshold exists for POLE: there is no ClinGen CSPEC for this gene, and the governing local POLE custom framework (leon-castillo-2020-custom-framework-v1, official_clingen = false) customizes only PM1, PS4, PP3 and BP4 and defines no BA1/BS1/BS2/PM2 thresholds, expressly falling back to generic ACMG/AMP outside its rules. The applicable threshold is therefore the generic SVI default of >= 0.01 (per-gene Whiffin-style derivation preferred, PMID:28518168). Against that threshold the variant falls well short: the highest frequency observed anywhere is 0.000528 (gnomAD v2.1 non-cancer exomes, European non-Finnish, 61/115,590), with all-comers v2.1 total AF 0.000237 and popmax FAF 0.00044857, all-comers v4.1 total AF 0.000130 and popmax FAF 0.00014455, v2.1 non-cancer exomes total AF 0.000227, and v3.1 non-cancer genomes total AF 0.000169 and popmax FAF 0.00019435. Every dataset and ancestry value is roughly 19-fold or more below 0.01, none reaches even 0.001, and the variant is present at a frequency consistent with a rare rather than a common polymorphism. BS1 is therefore not met under the applicable threshold, and the same conclusion holds whether all-comers totals or non-cancer subsets are used. The caveat is that a per-gene derived threshold could in principle sit below the generic default and was not derivable from the materials available.

Rule

BS1 (strong benign): allele frequency greater than expected for the disorder; generic SVI default >= 0.01, per-gene Whiffin-style derivation preferred but not derivable here (PMID:28518168, PMID:25741868).

Evidence considered

• BS1 threshold applied from the supplied published calibration: strong benign at allele frequency >= 0.01, generic SVI default with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168).
• Frequency values used for the comparison, all far below 0.01: gnomAD v2.1 all-comers total AF 0.000237028 (66/278,448) and popmax FAF 0.00044857; gnomAD v4.1 all-comers total AF 0.000129703 (209/1,611,370) and popmax FAF 0.00014455; gnomAD v2.1 non-cancer exomes total AF 0.000226672 (53/233,818) and popmax FAF 0.00044857; gnomAD v3.1 non-cancer genomes total AF 0.000169008 (25/147,922) and popmax FAF 0.00019435.
• Highest ancestry-specific frequency in any dataset: European (non-Finnish) 0.000527727 (61/115,590) in gnomAD v2.1 non-cancer exomes; the same ancestry in gnomAD v4.1 all-comers is 0.000163774 (193/1,178,456). Even this maximum is ~19-fold below the 0.01 generic BS1 threshold and ~95-fold below 0.05.
• No homozygotes are reported in any dataset (v2.1 all-comers 0; v4.1 all-comers 0; v2.1 non-cancer exomes 0; v3.1 non-cancer genomes 0), so the frequency signal rests entirely on heterozygote carrier alleles.
• Governing framework check: no ClinGen CSPEC exists for POLE (cspec found = false), and the local POLE framework derived from Leon-Castillo et al. 2020 customizes only PM1, PS4, PP3 and BP4, stating that outside its explicit rules the interpreter should fall back to generic ACMG/AMP; no gene-specific population threshold or population source was available to override the generic default.
• Gene-level disease context (adult-onset, incompletely penetrant colorectal cancer/polyposis predisposition) means the 'greater than expected for the disorder' comparison is inherently approximate for this gene; because no prevalence or penetrance figures were available in the case materials, the generic threshold was used and the derivation shortfall is recorded as a gap.

Still missing

• No per-gene (Whiffin-style) BS1 threshold could be derived for POLE: the governing local POLE framework specifies no population thresholds, and the case materials contain no POLE-associated disease prevalence, penetrance, or allelic-heterogeneity inputs required for that derivation. The supplied generic SVI default (>= 0.01) was applied; the ~19-fold margin makes the 'not met' verdict insensitive to modest downward adjustment of the threshold.

Sources

Reasoning

BP6 requires that a reputable source report the variant as benign, with the evidence not available to the laboratory to evaluate independently. Per the governing rule applying to this criterion in this pipeline, BP6 may be applied only for an exact-variant ClinVar expert-panel (ClinGen VCEP/Expert Panel) classification of Benign or Likely benign; ordinary laboratory submissions, aggregate ClinVar labels and non-expert assertions must never trigger it. The exact variant NM_006231.4(POLE):c.6331-24C>T does have a ClinVar record (variation ID 1697379, title NM_006231.4(POLE):c.6331-24C>T) whose classification is Likely benign - the correct direction for BP6 - but the supporting assertion does not meet the qualifying standard: review status is 'criteria provided, single submitter' (1 star), the submission audit records expert_panel_submissions = 0 with an empty expert-panels set, and the single submission SCV002550037 is an ordinary clinical-testing laboratory assertion (is_expert_panel false, trust_tier 'single_submitter'). A 1-star, single-laboratory Likely benign label is exactly the kind of ordinary submission that must not trigger BP6, so BP6 is not met. Note the directionality point for this group: BP6 is not a numeric-threshold criterion here (no likelihood-ratio or frequency cutoff is defined by the governing framework for BP6), so no operator comparison is required or performed.

Rule

BP6 applies only to an exact-variant ClinVar expert-panel (ClinGen VCEP/Expert Panel) Benign or Likely benign classification; ordinary laboratory submissions, aggregate ClinVar labels and non-expert assertions never trigger it (ACMG/AMP 2015 as implemented for this pipeline; PMID:25741868).

Evidence considered

• ClinVar variation 1697379 (exact match 'NM_006231.4(POLE):c.6331-24C>T') is classified Likely benign but with review status 'criteria provided, single submitter' (1 star).
• Submission audit records 1 total submission, 0 expert-panel submissions and an empty expert-panels set; SCV002550037 is a single clinical-testing laboratory (Center for Genomic Medicine, Rigshospitalet) with is_expert_panel false and trust_tier 'single_submitter'.
• No ClinGen VCEP/Expert Panel benign assertion for POLE c.6331-24C>T exists in the retrieved evidence, so the exact-variant expert-panel precondition for BP6 is not satisfied.
• The governing local POLE framework defines no BP6 rule, so the generic ACMG/AMP definition applies and its expert-panel-only qualifying standard is not met.

Sources

Reasoning

The governing POLE framework available for this case is the local León-Castillo et al. 2020 custom package, which customizes only PM1, PS4, PP3 and BP4 and contains no PS2 specification; generic ACMG/AMP 2015 (Richards et al.) therefore governs PS2. Generic PS2 requires a confirmed de novo occurrence of NM_006231.4:c.6331-24C>T (an intronic exon 45 variant, NP_006222.2:p.?) in a patient with the gene-associated phenotype, with the biological parents demonstrated not to carry the variant and with parentage confirmed (at least one of maternity/paternity established). The case materials contain no proband, no affected individual, no parental genotypes, no trio or Sanger follow-up, and no phenotype data, so a confirmed de novo occurrence cannot be established or excluded. The only external variant record, the ClinVar entry (single submitter, Likely benign, condition 'not specified'), reports no family or parental testing information and its classification cannot be used to infer any criterion. No published report of this variant exists among the retrieved literature; the only paper triaged for this case is the ACMG/AMP guideline itself, which contains no POLE or variant-specific data.

Rule

Generic ACMG/AMP 2015 PS2: confirmed de novo occurrence (both parents tested and shown not to carry the variant, with parentage confirmed) in an individual with the gene-associated phenotype.

Evidence considered

• The local POLE framework (León-Castillo et al. 2020, custom_internal, precedence rank 2) lists customized_criteria as only PM1, PS4, PP3 and BP4; PS2 is not specified, so the generic ACMG/AMP 2015 rule applies.
• Generic ACMG/AMP 2015 defines PS2 as a confirmed de novo (both parents tested and negative, parentage confirmed) occurrence in a patient with the relevant phenotype.
• The case carries no proband, family, trio, or phenotype record for NM_006231.4:c.6331-24C>T; the variant is intronic (c.6331-24C>T, exon 45i) with protein consequence NP_006222.2:p.?.
• The ClinVar record (variation 1697379, SCV002550037, Center for Genomic Medicine, Rigshospitalet, single submitter, germline origin, condition 'not specified') provides no parental testing or de novo observation, and a submitter's classification is not used to infer a criterion.

Still missing

• No proband or affected individual carrying c.6331-24C>T is documented in the case materials.
• No parental genotypes or trio testing (Sanger or NGS) demonstrating absence of the variant in both biological parents.
• No confirmation of parentage/maternity-paternity for a de novo claim.
• No phenotype or indication data to establish phenotype consistency with the POLE-associated disorder.

Sources

Reasoning

Not assessed. No ClinGen CSPEC/VCEP specification exists for POLE, and the governing local framework (Leon-Castillo et al. 2020, J Pathol 250:323-335) customizes only PM1, PS4, PP3 and BP4 and states explicitly that it is not an official ACMG rule set, so PS3 falls back to the generic ACMG/AMP 2015 definition: well-established functional studies showing a damaging effect on the gene or protein. NM_006231.4:c.6331-24C>T is an intronic variant (intron 45, 24 bp from the exon boundary; predicted protein consequence p.?, no amino-acid change), so the assay classes able to satisfy PS3 would be mRNA/splicing assays (direct RNA or cDNA analysis, in vitro minigene splicing assay) or protein-level assays. No such assay of this variant exists in any source consulted. The only publication retrieved for this case is the ACMG/AMP 2015 guideline itself, whose full text contains no mention of POLE or of c.6331-24C>T, no assay data, and whose only relevant content is generic (splice-region variant impact requires functional RNA or protein confirmation). The ClinVar record (VCV001697379; single submitter, Rigshospitalet; Likely benign) contains no assay data and its classification was not used to infer this criterion. OncoKB returned no classification and COSMIC no record. The local POLE package (paper plus Supplementary Tables S1-S3) contains no functional assay data, no PS3 specification and no mention of this intronic variant; its tables hold only missense recurrence counts and missense in-silico results. The only functional-adjacent datum available is a computational splice prediction (SpliceAI max delta 0.076), which is not an assay result and cannot establish a damaging effect. PS3 is therefore not assessable rather than negatively supported: the criterion's evidence base is absent, not contradicted.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868): PS3 = 'Well-established functional studies show a damaging effect on gene or protein function.' No POLE-specific PS3 specification exists in the governing local framework (Leon-Castillo et al. 2020 customizes only PM1, PS4, PP3, BP4), so generic ACMG applies.

Evidence considered

• Governing framework: no ClinGen CSPEC/VCEP is available for POLE; this case runs in custom_gene_framework mode on the local POLE package derived from Leon-Castillo et al. 2020 (J Pathol 250:323-335). That framework customizes only PM1, PS4, PP3 and BP4 and its own notes record that it is not an official ClinGen CSPEC/VCEP rule set, so PS3 has no applicable gene-specific specification and falls back to generic ACMG/AMP 2015.
• Variant class driving the applicable assay type: NM_006231.4:c.6331-24C>T is intronic, located 24 bp from the exon boundary within intron 45 (variant_exonic_positions start/end exon = 45i on both GRCh37 and GRCh38), with predicted protein consequence NP_006222.2:p.? and no amino-acid substitution. It is therefore not a missense change, and neither a REVEL score nor a protein-function missense assay is the relevant evidence class; the relevant classes are RNA/mRNA processing assays or protein-level analysis.
• PS3 requirement (generic ACMG/AMP 2015): 'Functional studies can be a powerful tool in support of pathogenicity; however, not all functional studies are effective in predicting an impact on a gene or protein function' and, importantly for this variant type, 'Although splice site variants are predicted to lead to a null effect, confirmation of impact requires functional analysis by either RNA or protein analysis.' No such RNA or protein analysis is available for c.6331-24C>T, and no assay validity/reproducibility information exists to judge.
• Retrieved publication inventory: this case retrieved exactly one publication, PMID:25741868 (the ACMG/AMP 2015 guideline), which surfaced through the ClinVar submission's cited PMID trail. Its full text was reviewed (paper_extracts entry plus raw text) and contains no mention of POLE, no mention of c.6331-24C>T, and no functional, cohort or assay data of any kind.
• Local POLE framework package (PATH-250-323.pdf and Supplementary Tables S1-S3) was searched for this variant and for functional-assay content: it is a somatic endometrial-carcinoma study of exonuclease-domain missense hotspots (p.P286R, p.V411L, p.S297F, p.A456P, p.S459F) based on POLE-score and mutational-signature analysis; the PDF text contains no occurrence of '6331', 'intron', 'splice', 'PS3', 'BS3', 'minigene' or 'functional assay', and the supplementary shared-string contents contain no occurrence of '6331' or 'intron'. The package supplies no PS3-satisfying evidence for this intronic variant.
• ClinVar VCV001697379 (SCV002550037, Center for Genomic Medicine, Rigshospitalet; criteria provided, single submitter; Likely benign; condition 'not specified') contains no functional assay data; its classification was consulted only to trace its cited literature (PMID:25741868) and was explicitly NOT used to infer PS3 or any other criterion.
• SpliceAI result for NM_006231.4:c.6331-24C>T: max delta score 0.076 (DS_DG 0.076 at DP_DG -422; DS_AG 0.004; DS_AL 0.028; DS_DL 0.001; Pangolin SG 0.044, SL -0.014). This is a computational prediction of splice impact, not a functional assay result, and cannot satisfy PS3; it is relevant only to in-silico splicing evidence.
• No REVEL score and no BayesDel score exist for this variant (both absent from the local resources), which is expected for a non-missense variant and in any case could not substitute for functional evidence under PS3.
• OncoKB returned no classification and COSMIC returned no record for this variant, so neither provides functional or assay evidence; the pre-computed theme triage for this case surfaced no paper containing functional assay data (literature_pass criterion_screened and criteria_index were empty).

Still missing

• No variant-specific functional assay of POLE NM_006231.4:c.6331-24C>T was identified in any source consulted: no RNA/cDNA (RT-PCR) analysis, no in vitro minigene splicing assay, and no POLE proofreading or polymerase-extension fidelity assay. Such an assay is the only route to PS3 for this intronic variant.
• Literature coverage for this variant was limited to the ACMG/AMP 2015 guideline, retrieved through the ClinVar submission's PMID trail; a dedicated search for published functional or RNA studies of POLE c.6331-24C>T was not performed, so the absence of a published assay is not conclusive.
• No assay controls, reproducibility or validation data of any kind exist for this variant, so PS3 strength calibration could not be considered.

Sources

Reasoning

PM3 is a recessive-modality criterion: it requires that POLE NM_006231.4:c.6331-24C>T (intron 45, c.6331-24, i.e. 24 bp upstream of the exon 46 acceptor) be shown to lie in trans with a pathogenic variant in the same individual. The governing specification for this case is a local, literature-derived POLE package (Leon-Castillo et al. 2020, somatic endometrial-carcinoma hotspot context) that customises only PM1, PS4, PP3 and BP4 and sets no PM3 rule, and no ClinGen CSPEC/VCEP exists for POLE (cspec found = false), so the generic ACMG/AMP 2015 definition applies (PMID:25741868). POLE has both dominant germline contexts (polyposis and colorectal-cancer predisposition) and biallelic recessive ones (facial dysmorphism-immunodeficiency-livedo-short stature syndrome), so a trans observation would be admissible if one existed; none is documented in any source available for this variant. The ClinVar record (variation 1697379, VCV001697379) contains a single germline submission (SCV002550037, Center for Genomic Medicine, Rigshospitalet Copenhagen University Hospital; Likely benign; review status 'criteria provided, single submitter'; no expert panel) whose condition is recorded only as 'not specified', and reports no second POLE variant, no zygosity, no phase and no affected relative. The population datasets report zero homozygotes (gnomAD v2.1 66/278,448 alleles, AF 0.000237, European non-Finnish 61/125,768, AF 0.000485, homozygotes 0; gnomAD v4.1 209/1,611,370 alleles, AF 0.000130, homozygotes 0; the gnomAD-Canada v1.0 query returned no usable result), so no individual is reported in whom a second allele could be examined. The only full text held for this variant (PMID:25741868) does not name POLE or the variant and carries no case-, phase- or family-level data. With no proband genotype, no candidate trans partner and no phasing data, the configuration PM3 requires is untested and the criterion is recorded as not assessed rather than met or refuted.

Rule

Generic ACMG/AMP PM3 (PMID:25741868): for recessive disorders, the variant is detected in trans with a pathogenic variant; applied generically because the governing local POLE package specifies only PM1, PS4, PP3 and BP4 and no ClinGen CSPEC/VCEP exists for POLE.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom framework, source 'local_custom_framework', official_clingen false, precedence_rank 2) customises only PM1, PS4, PP3 and BP4 for exonuclease-domain hotspot/recurrence and table-grounded in-silico evidence and states explicitly that outside those rules generic ACMG/AMP applies; it contains no PM3 rule. No ClinGen CSPEC/VCEP for POLE was retrieved (cspec found = false), so PM3 was evaluated under the generic ACMG/AMP 2015 definition: for recessive disorders, the variant detected in trans with a pathogenic variant (PMID:25741868).
• ClinVar variation 1697379 (VCV001697379) for NM_006231.4(POLE):c.6331-24C>T: one germline submission from a clinical laboratory (SCV002550037, Center for Genomic Medicine, Rigshospitalet Copenhagen University Hospital), classification Likely benign, review status 'criteria provided, single submitter' (1 star), no expert-panel submission, condition recorded only as 'not specified', cited PMID 25741868. The submission provides no second POLE variant, no zygosity, no phase and no relative genotype. The submitted classification itself was not used to infer PM3, since a criterion may not be derived from another source's final classification.
• Inheritance context for the gene: the gene-level summary available for this case describes germline POLE variants as causing polyposis and predisposing to colorectal cancer and as linked to a rare syndrome of facial dysmorphism, immunodeficiency, livedo and short stature, i.e. both dominant and biallelic-recessive contexts exist; the case's gene-context artifact nevertheless records no inheritance-mode entry for POLE and the ClinVar condition attached to this variant is 'not specified', so neither a dominant-only nor a recessive-only reading is fixed for this variant and the trans configuration PM3 asks about remains open.
• Population data: gnomAD v2.1 66/278,448 alleles (AF 0.000237028; highest subpopulation European non-Finnish 61/125,768, AF 0.00048502; grpmax FAF 0.00044857; homozygotes 0) and gnomAD v4.1 209/1,611,370 alleles (AF 0.000129703; European non-Finnish 193/1,178,456, AF 0.000163774; grpmax FAF 0.00014455; homozygotes 0). No dataset reports a homozygote, so there is no population-level observation of this allele on both chromosomes from which any allelic configuration could be inferred. The gnomAD-Canada v1.0 query returned no result (source error), so no absence of the variant can be inferred from that dataset and it contributes nothing to PM3.
• Case-level literature: the only full text held for this variant (PMID:25741868, the ACMG/AMP 2015 standards) was checked for 'POLE', the c.6331-24C>T HGVS forms and the trans/cis and PM3/BP2 terms; it names neither POLE nor the variant and contains no case, phase, segregation or family data. It supplies only the generic PM3 definition and the observation that PM3 may be upgraded to strong when the variant is detected in trans (on opposite chromosomes) with pathogenic variants in multiple observations.
• The local POLE package underlying the framework (PATH-250-323 PDF and supplementary tables S1-S3) covers somatic endometrial-carcinoma recurrence, hotspot identity 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.6331-24C>T, so the allelic phase of the variant cannot be established.
• No second POLE variant (pathogenic, likely pathogenic or otherwise) is reported alongside c.6331-24C>T in any consulted source, so an in-trans partner can be neither identified nor excluded.
• No parental, sibling or other family genotypes are available from which the phase of c.6331-24C>T could be inferred.
• No homozygote and no somatic/tumour genotype co-occurring with this variant is reported (gnomAD homozygotes = 0 in every dataset; the COSMIC search returned no record), so no indirect cis/trans signal exists either.
• The inheritance mode of the POLE-associated disorder being evaluated is not specified in this case (ClinVar condition 'not specified'; the gene-level PVS1 context carries no inheritance entry), so the recessive setting PM3 presupposes is not documented.

Sources

Reasoning

The local León-Castillo et al. 2020 POLE framework customizes only PM1, PS4, PP3 and BP4, so PM6 is governed by generic ACMG/AMP 2015 (Richards et al.). Generic PM6 requires an assumed de novo occurrence of NM_006231.4:c.6331-24C>T without confirmation of parental testing, i.e. at least a documented proband carrying the variant for whom parental samples are unavailable or unconfirmed, together with phenotype consistency. This case provides no proband and no affected individual carrying the variant, so no assumed de novo event can even be recorded, and there is consequently no phenotype to check for consistency. The single ClinVar submission (Likely benign, one laboratory, condition 'not specified') contains no family-level or de novo information; its classification is not used to infer a criterion. No paper reporting this variant was retrieved; the only available publication is the ACMG/AMP guideline, which contains no POLE or variant-specific data.

Rule

Generic ACMG/AMP 2015 PM6: assumed de novo occurrence without confirmation of parental testing, in a patient consistent with the gene-associated phenotype.

Evidence considered

• The governing local POLE framework customizes only PM1, PS4, PP3 and BP4; PM6 is unspecified and falls back to generic ACMG/AMP 2015.
• Generic ACMG/AMP 2015 defines PM6 as an assumed de novo occurrence without confirmation of parental testing (paternity and maternity not confirmed).
• No proband, affected relative, parental sample status, or clinical phenotype is recorded anywhere in the case materials for c.6331-24C>T.
• The ClinVar submission for this variant is a single non-expert laboratory entry (Likely benign, condition 'not specified') with no de novo or family information.

Still missing

• No proband or affected individual with c.6331-24C>T documented, so an assumed de novo occurrence cannot be recorded.
• No statement of whether parental samples were available, tested, or unconfirmed.
• No phenotype data to assess consistency of an assumed de novo occurrence with the POLE-associated disorder.

Sources

Reasoning

The local León-Castillo et al. 2020 POLE framework customizes only PM1, PS4, PP3 and BP4 and specifies no segregation rule, so generic ACMG/AMP 2015 (Richards et al.) governs PP1. Generic PP1 requires co-segregation of NM_006231.4:c.6331-24C>T with disease in multiple affected family members of a single pedigree, i.e. genotype and phenotype data across a family showing the variant tracking with the POLE-associated phenotype. This case contains no pedigree, no affected or unaffected relative genotypes, no meioses, and no phenotype information at all, so neither the presence nor the absence of co-segregation can be established and no PP1 strength can be assigned. The variant's only external record, the single-submitter ClinVar entry with condition 'not specified', contains no segregation data, and a submitter's classification is never used to infer a criterion. The available POLE literature package (somatic endometrial-carcinoma hotspot review) addresses none of these criteria, and the only triaged publication is the ACMG/AMP guideline itself.

Rule

Generic ACMG/AMP 2015 PP1: co-segregation of the variant with disease in multiple affected family members.

Evidence considered

• The governing local POLE framework customizes only PM1, PS4, PP3 and BP4; no PP1 specification exists in it, so generic ACMG/AMP 2015 applies.
• Generic ACMG/AMP 2015 defines PP1 as co-segregation with disease in multiple affected family members within a pedigree.
• No family, pedigree, meiosis, relative-genotype, or phenotype data for c.6331-24C>T exist in the case materials (case summary, evidence, literature pass, paper extracts).
• The provided POLE literature package (León-Castillo et al. 2020 and its supplementary tables) covers PM1/PS4/PP3/BP4 in a somatic endometrial carcinoma context and contains no segregation evidence.

Still missing

• No pedigree or family structure for the proband is provided.
• No genotype data for affected or unaffected relatives, and no meioses counted.
• No phenotype information linking relatives to the POLE-associated disorder, so segregation with disease cannot be assessed.
• No quantitative co-segregation (likelihood ratio) dataset is available.

Sources

Reasoning

PP4 requires that the patient's phenotype or family history be highly specific for a disease with a single genetic aetiology (ACMG/AMP 2015, PMID:25741868). No phenotype information exists for this case: the case record, evidence compilation and prefetch all contain no HPO terms, no clinical diagnosis, no tumour type, no age of onset and no family history, and the ClinVar record for this variant carries the condition label 'not specified'. The governing local POLE framework (Leon-Castillo et al. 2020 package) customizes only PM1, PS4, PP3 and BP4 and provides no PP4 specification, so the generic ACMG/AMP definition would apply - but there is no proband phenotype against which to test disease specificity, and no gene-level or variant-level phenotype-specificity literature was retrieved to substitute. PP4 cannot be judged met or not met on the available evidence and is therefore not assessed.

Evidence considered

• Searched the phenotype/clinical fields of the case record and prefetch data for phenotype, HPO, indication, condition, proband or patient descriptors: no proband phenotype or family history is present.
• ClinVar variation 1697379 (exact match, NM_006231.4(POLE):c.6331-24C>T) lists the condition as 'not specified', providing no disease-specificity information.
• The Leon-Castillo et al. 2020 POLE package customizes only PM1, PS4, PP3 and BP4; it contains no PP4 rule, so no gene-specific phenotype-specificity specification is available.

Still missing

• No clinical phenotype, HPO term set, disease diagnosis, tumour type, age of onset or family history is recorded anywhere in this case; PP4 requires the proband's phenotype to be assessed for specificity to a single-gene disorder.
• The ClinVar record for the exact variant only carries the condition label 'not specified'.
• No POLE-specific PP4 specification exists in the governing local framework, so the generic ACMG/AMP definition would have to be applied - but it cannot be operationalized without proband phenotype data.

Sources

Reasoning

Not assessed. As with PS3, no CSPEC/VCEP specification exists for POLE and the governing local framework (Leon-Castillo et al. 2020) customizes only PM1, PS4, PP3 and BP4, so BS3 falls back to the generic ACMG/AMP 2015 definition: well-established functional studies showing no damaging effect on protein function or splicing. NM_006231.4:c.6331-24C>T is an intronic variant (intron 45, 24 bp from the exon boundary; p.?), so a BS3-satisfying study would need to demonstrate preserved splicing or preserved POLE function directly, for example normal mRNA/cDNA products on RT-PCR or a minigene assay showing no aberrant transcript, or normal proofreading/polymerase activity. No functional study of this variant exists in any source consulted: the single retrieved publication (ACMG/AMP 2015 guideline) reports no data on POLE or on this variant, the ClinVar submission carries no assay data and its Likely benign classification was not used to infer this criterion, OncoKB returned no classification and COSMIC no record, and the local POLE package plus its supplementary tables contain only somatic hotspot recurrence and missense in-silico content with no mention of this intronic variant. The available SpliceAI result (max delta 0.076, below the published recommended thresholds) is a computational prediction, not a functional demonstration, and the absence of a REVEL score simply reflects a non-missense variant; neither can satisfy BS3. BS3 is therefore not assessable: the absence of functional evidence must not be converted into a benign functional call.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868): BS3 = 'Well-established functional studies show no damaging effect on protein function or splicing.' No POLE-specific BS3 specification exists in the governing local framework (Leon-Castillo et al. 2020 customizes only PM1, PS4, PP3, BP4), so generic ACMG applies.

Evidence considered

• Governing framework: no ClinGen CSPEC/VCEP for POLE; the local POLE framework derived from Leon-Castillo et al. 2020 customizes only PM1, PS4, PP3 and BP4, contains no BS3 specification, and its notes state it is not an official ClinGen CSPEC/VCEP rule set, so BS3 rests on generic ACMG/AMP 2015.
• Variant class: NM_006231.4:c.6331-24C>T lies in intron 45, 24 bp from the exon boundary (variant_exonic_positions = 45i), with predicted protein consequence NP_006222.2:p.? and no amino-acid change. The BS3-relevant evidence class is therefore a splicing/mRNA assay (RT-PCR/cDNA, minigene) or a protein-level POLE function assay showing a normal result; neither exists for this variant.
• BS3 requirement (generic ACMG/AMP 2015): the guideline's 'PS3 BS3 Functional studies' section defines BS3 as well-established functional studies showing no damaging effect and requires that an assay's biological relevance be judged (enzymatic/functional readout reflecting the biological environment, full function versus one component of function) together with validation, reproducibility and robustness data. No assay of any kind exists for this variant, so none of these conditions can be evaluated.
• Splice-region relevance (generic ACMG/AMP 2015): the guideline states that mRNA-level assays (direct RNA/cDNA analysis, in vitro minigene splicing assays) 'can be highly informative when evaluating the effects of variants at splice junctions and within coding sequences, untranslated regions, as well as deeper intronic regions', and that although splice-region variants are predicted to lead to a null effect, 'confirmation of impact requires functional analysis by either RNA or protein analysis'. The confirmatory assay has not been done for c.6331-24C>T.
• Local POLE framework package (PATH-250-323.pdf and Supplementary Tables S1-S3) was searched for this variant and for functional content: it is a somatic endometrial-carcinoma study of exonuclease-domain missense hotspots using POLE-score and ultramutation-signature analysis; the PDF text contains no occurrence of '6331', 'intron', 'splice', 'BS3', 'minigene' or 'functional assay', and the supplementary tables contain no occurrence of '6331' or 'intron'. It therefore supplies no BS3-supporting proofreading, splicing or protein-function data, and no assay performed on this variant.
• ClinVar VCV001697379 (SCV002550037, single submitter, Likely benign, condition 'not specified') provides no functional assay data; its classification was NOT used to infer BS3, per the rule that no criterion may be inferred from another source's final classification.
• SpliceAI for c.6331-24C>T: max delta 0.076 (DS_DG 0.076; DS_AG 0.004; DS_AL 0.028; DS_DL 0.001). This is an in-silico prediction and, even at a benign-leaning value, cannot be counted as BS3 functional evidence; BS3 requires an assay result showing no damaging effect. The absence of REVEL and BayesDel scores reflects the non-missense consequence type and likewise provides no functional evidence.
• OncoKB returned no classification and COSMIC no record; the functional-theme literature triage for this case yielded no paper with assay data and no candidate functional criteria, leaving no BS3-relevant study to weigh.

Still missing

• No functional or RNA-level study of POLE NM_006231.4:c.6331-24C>T was identified that could demonstrate absence of a damaging effect: no RT-PCR/cDNA transcript analysis, no minigene splicing assay, and no POLE proofreading or polymerase-activity assay, and therefore no assay controls or reproducibility data.
• Literature retrieval for this case returned only the ACMG/AMP 2015 guideline (PMID:25741868, via the ClinVar submission's PMID trail), so the absence of a published negative functional study cannot be treated as established and a targeted variant-specific literature search remains open.
• A benign-leaning in-silico splice prediction for this intronic variant, if one existed, would inform BP4 (in-silico evidence) rather than BS3, which requires assay data; no such assay data are available.

Sources

Reasoning

The local León-Castillo et al. 2020 POLE framework customizes only PM1, PS4, PP3 and BP4 and contains no non-segregation rule, so generic ACMG/AMP 2015 (Richards et al.) governs BS4. Generic BS4 requires demonstrated lack of segregation of NM_006231.4:c.6331-24C>T with disease, i.e. genotype and phenotype data showing affected family members who do not carry the variant (or equivalent non-segregation across informative meioses). This case provides no pedigree, no relative genotypes, no meioses and no phenotype data, so non-segregation is neither demonstrated nor suggested; absence of segregation data is not evidence of non-segregation and cannot support BS4. The variant's population frequency (gnomAD v2.1 total AF 0.000237, 66/278448 alleles, no homozygotes; v4.1 AF 0.00013) and the single-submitter ClinVar Likely-benign record are not segregation evidence, and a submitter's classification is never used to infer a criterion. No published segregation study of this variant was retrieved.

Rule

Generic ACMG/AMP 2015 BS4: lack of segregation of the variant with disease in affected family members.

Evidence considered

• The governing local POLE framework customizes only PM1, PS4, PP3 and BP4; BS4 is unspecified and falls back to generic ACMG/AMP 2015.
• Generic ACMG/AMP 2015 defines BS4 as lack of segregation in affected family members (a benign-supporting observation requiring family genotype data).
• No family genotype, pedigree, or meiosis data exist in the case materials; the absence of segregation data is not evidence of non-segregation.
• The only variant-level external data are population frequencies (gnomAD v2.1 total AF 0.000237028, 66/278448 alleles, 0 homozygotes; v4.1 AF 0.000129703, 209/1611370 alleles, 0 homozygotes) and one non-expert ClinVar submission (Likely benign, condition 'not specified', no family data) - none of which is segregation evidence.

Still missing

• No pedigree, no genotyped affected relatives, and no informative meioses for non-segregation analysis.
• No phenotype data for relatives, so segregation status cannot be evaluated either way.
• Absence of any segregation dataset must not be read as evidence of non-segregation.
• No quantitative likelihood-ratio dataset for non-segregation is available.

Sources

Reasoning

BP2 is supporting benign evidence for a specific allelic configuration: the variant observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant under any inheritance pattern. It therefore requires a pathogenic partner allele whose phase relative to POLE c.6331-24C>T is known. No such observation exists in any source available for this variant. The ClinVar record (variation 1697379, VCV001697379) holds a single germline submission (SCV002550037, Rigshospitalet; Likely benign; 1 star; no expert panel) with the condition recorded only as 'not specified', and states no co-occurring POLE variant, no zygosity and no phase. The trans limb presupposes a fully penetrant dominant disorder, and neither the case's ClinVar record (condition 'not specified') nor the gene-level information available for this case establishes full penetrance for POLE (germline variants are described as predisposing to colorectal cancer, a susceptibility rather than a fully penetrant model, while the recognised biallelic POLE syndrome is recessive). The cis limb requires the variant to be phased on the same allele as a pathogenic variant, and no proband genotype, family genotype or phasing result exists in this case, which was reviewed at the variant level only. The population datasets contain no allele-configuration observation either: gnomAD reports zero homozygotes for this variant in v2.1 (66/278,448 alleles, AF 0.000237) and v4.1 (209/1,611,370 alleles, AF 0.000130), the gnomAD-Canada v1.0 query returned no usable result, and no somatic record of the variant was retrieved from COSMIC. The only full text held for this variant (PMID:25741868) does not mention POLE or the variant beyond supplying the generic BP2 definition. Because neither limb can be tested from the available records, BP2 is recorded as not assessed rather than met or refuted.

Rule

Generic ACMG/AMP BP2 (PMID:25741868): observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant in any inheritance pattern; applied generically because the governing local POLE package specifies only PM1, PS4, PP3 and BP4 and no ClinGen CSPEC/VCEP exists for POLE.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom framework, official_clingen false) specifies only PM1, PS4, PP3 and BP4, and no ClinGen CSPEC/VCEP exists for POLE (cspec found = false), so BP2 was evaluated under the generic ACMG/AMP 2015 definition: observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant in any inheritance pattern (PMID:25741868).
• ClinVar variation 1697379 (VCV001697379) for NM_006231.4(POLE):c.6331-24C>T: one germline submission (SCV002550037, Center for Genomic Medicine, Rigshospitalet Copenhagen University Hospital), Likely benign, 1 star, no expert panel, condition 'not specified'. The record describes a single variant and provides no co-occurring POLE variant, no zygosity, no phase and no family genotype, so neither the cis limb nor the trans limb of BP2 has an observation to rest on. The submitted Likely benign classification was not used as criterion evidence.
• Penetrance and inheritance context: the trans limb of BP2 requires a fully penetrant dominant gene/disorder. Neither the case's ClinVar condition ('not specified') nor the gene-level summary available for this case establishes full penetrance - germline POLE variants are described as causing polyposis and predisposing to colorectal cancer (a susceptibility model), while the recognised biallelic POLE syndrome (facial dysmorphism, immunodeficiency, livedo, short stature) is recessive - and the case's gene-context artifact records no inheritance-mode entry for POLE.
• Population and somatic data: gnomAD v2.1 66/278,448 alleles (AF 0.000237028; European non-Finnish 61/125,768, AF 0.00048502; homozygotes 0) and gnomAD v4.1 209/1,611,370 alleles (AF 0.000129703; homozygotes 0); no homozygote is reported, so the datasets contain no observation of this allele paired with a second POLE allele. The gnomAD-Canada v1.0 query returned no result (source error), so it cannot be read as an absence, and the somatic (COSMIC) search returned no record of this variant from which any co-occurring alteration could be read.
• Case-level literature: the only full text held for this variant (PMID:25741868, the ACMG/AMP 2015 standards) was checked for 'POLE', the c.6331-24C>T HGVS forms and the trans/cis terms; it neither names POLE nor the variant and provides no co-occurrence, phase or segregation data, contributing only the generic BP2 definition.
• The local POLE package underlying the framework (PATH-250-323 PDF and supplementary tables S1-S3) addresses somatic endometrial-carcinoma recurrence, hotspot identity and in-silico output only and contains no BP2 rule and no germline phase material.
• 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.6331-24C>T, so its cis/trans configuration relative to any potential pathogenic POLE variant is unknown.
• No co-occurring pathogenic or likely pathogenic POLE variant (germline or somatic) is reported with c.6331-24C>T in the ClinVar record or in any other consulted source.
• No homozygote or compound-heterozygous carrier of c.6331-24C>T is reported (gnomAD homozygotes = 0 in v2.1 and v4.1; the gnomAD-Canada query returned no result), so no allele-configuration observation exists; population datasets also carry no phenotype that could be attached to such an observation.
• The inheritance mode and penetrance of the POLE-associated disorder being evaluated are not specified for this case, so it cannot be determined whether the trans limb of BP2 (which requires a fully penetrant dominant disorder) could apply.

Sources

Reasoning

BP5 requires that the variant be found in a case in which an alternate molecular basis (alternative genetic cause or a different pathogenic variant explaining the phenotype) has been identified (ACMG/AMP 2015, PMID:25741868). This is a case-level observation: it needs a described proband, the co-occurring alternate genetic finding, and a statement that the alternate finding accounts for the phenotype. No such case exists in the material available for this variant - there is no proband phenotype, no additional variant report, no tumour molecular profile and no co-occurring pathogenic finding recorded anywhere in the case record or prefetch data, and the governing local POLE framework (Leon-Castillo et al. 2020 package) defines no BP5 rule at all. BP5 therefore cannot be evaluated and is not assessed.

Evidence considered

• Searched the case record and prefetch data for case-level content (proband, co-occurring variant, alternate molecular basis, molecular diagnosis): none present.
• ClinVar variation 1697379 provides only a germline, single-submitter Likely benign classification for an unspecified condition, with no case-level alternate-basis information.
• The governing local POLE framework customizes only PM1, PS4, PP3 and BP4 and defines no BP5 rule.

Still missing

• No case-level data (proband, co-occurring variants, tumour molecular profile, or alternate genetic diagnosis) are recorded for this variant; BP5 is an inherently case-level criterion and cannot be assessed without them.
• The governing local POLE framework (Leon-Castillo et al. 2020) contains no BP5 rule, so only the generic ACMG/AMP definition would apply, and it has no case data to operate on.
• The ClinVar record for the exact variant does not report an alternate molecular basis for any case.

Sources