POLE c.4291-30_4291-27delinsAAT, p.?

Reasoning

POLE NM_006231.4:c.4291-30_4291-27delinsAAT is absent from gnomAD v2.1 (GRCh37 12-133220173-CAAG-ATT) and gnomAD v4.1 (GRCh38 chr12-132643587-CAAG-ATT), and also from the gnomAD v2.1 non-cancer (exomes-only) and gnomAD v3.1 non-cancer (genomes-only) subsets; the population allele frequency is 0, which is <= the PM2 supporting threshold of 0.0001 (ClinGen SVI 2020 recommendation to apply PM2 at Supporting strength, PMID:25741868). No official POLE CSPEC/VCEP exists, and the local León-Castillo et al. 2020 framework defines no PM2 rule and no gene-specific population source, so the generic supporting-strength PM2 is applied to the all-comers gnomAD totals. PM2 is therefore met at Supporting strength.

Rule

PM2 supporting: allele frequency <= 0.0001 (ClinGen SVI 2020 downgrade of PM2 to Supporting; PMID:25741868).

Evidence considered

• gnomAD v2.1 (GRCh37, 12-133220173-CAAG-ATT): absent (allele frequency 0).
• gnomAD v4.1 (GRCh38, chr12-132643587-CAAG-ATT): absent (allele frequency 0).
• gnomAD v2.1 non-cancer (exomes-only) and gnomAD v3.1 non-cancer (genomes-only) subsets: both absent.
• PM2 applied at Supporting strength per the ClinGen SVI 2020 recommendation (PMID:25741868); no POLE-specific PM2 rule or population source exists in the governing local León-Castillo et al. 2020 framework.

Still missing

• PM2 relies on observed absence from large population reference cohorts; absence is not proof of true rarity, and PM2 does not account for incomplete cohort coverage.
• gnomAD-Canada v1.0 (HostSeq) timed out; the variant is absent from gnomAD v2.1 and v4.1 regardless.
• No non-cancer/exome-only population source was mandated for PM2, so the all-comers gnomAD v2.1/v4.1 figures were used.

Sources

Reasoning

The only governing PS4 specification available for POLE is the local custom framework derived from Leon-Castillo et al. 2020 (J Pathol 250:323-335). That rule is deliberately 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 established/pathogenic hotspot set (P286R, V411L, A456P, S297F); it is capped at Supporting and is explicitly a somatic-recurrence adaptation, not germline case-control evidence. The queried variant NM_006231.4:c.4291-30_4291-27delinsAAT is an intronic variant (protein consequence p.?), not a missense substitution, so it cannot satisfy the rule's exonuclease-domain missense prerequisite at all. I searched Supplementary Table S1 (PATH-250-323-s002.xlsx) for 'c.4291-30_4291-27delinsAAT', 'delinsAAT', '4291' and 'p.?' and there is no entry for this variant; its combined EC recurrence count is therefore 0. Writing the required comparison explicitly against the governing threshold: combined EC count 0 >= 10? no. Independently, the generic ACMG/AMP PS4 concept (a statistically increased prevalence of the variant in affected probands versus controls) has no supporting data: the variant is absent from the COSMIC somatic cancer database and no germline case series or case-control study mentioning it was retrieved (literature pass returned 0 PMIDs; paper_extracts is empty). PS4 is not met.

Rule

PS4_Supporting: exact recurrent POLE missense variant recurrent in both COSMIC and TCGA EC cohorts (Supplementary Table S1), combined EC count >= 10, and within the established/hotspot set (P286R, V411L, A456P, S297F); capped at Supporting.

Evidence considered

• Searched PATH-250-323-s002.xlsx (Supplementary Table S1, Leon-Castillo et al. 2020) for 'c.4291-30_4291-27delinsAAT', 'delinsAAT', '4291' and 'p.?': no row for this variant. Table S1 is keyed by protein/CM variants such as p.P286R c.857C>G (181 EC), p.V411L c.1231G>T (114), p.A456P c.1366G>C (20), p.S297F c.890C>T (18); it contains no intronic/splice entry.
• The custom POLE framework's PS4 rule: 'Apply PS4_Supporting 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 established/pathogenic set highlighted by Leon-Castillo et al. 2020. Under the current package this supports p.P286R, p.V411L, p.A456P, and p.S297F.'
• Variant is an intronic 4-bp delins (c.4291-30_4291-27) with protein consequence p.?, per the normalization record; it is not a missense exonuclease-domain hotspot.
• Evidence pass: COSMIC search for POLE c.4291-30_4291-27delinsAAT returned no result; literature pass returned 0 PMIDs; no case-control enrichment evidence was available.

Still missing

• PATH-250-323-s001.tif, declared by the VCEP index for PS4, could not be converted to text (conversion unavailable); no extractable content, so it was not possible to confirm or exclude a variant entry there.
• No germline case-control or case-series enrichment data exist for this variant; no POLE case-control study of this intronic change was retrieved, so a generic ACMG/AMP PS4 could not be calculated even in principle.

Sources

Reasoning

NM_006231.4:c.4291-30_4291-27delinsAAT (NP_006222.2:p.?) is a 4-bp deletion-insertion in intron 33 (VariantValidator places both breakpoints at exon 33i; genomic NC_000012.12:g.132643587_132643590delinsATT), so it is an intronic/splice-region variant and PP3 is assessed on the SpliceAI path only; no REVEL or other missense predictor applies to it. SpliceAI reports a maximum delta score of 0.135 (DS_AL 0.135, DS_AG 0.011, DS_DG 0.016, DS_DL 0.000; Pangolin SG 0.047 / SL -0.019), which falls below the recommended PP3 supporting cutoff of >= 0.2 (Jaganathan et al. 2019, PMID:30661751), so no weighted splice-influence evidence is available. The gene's governing local framework (Leon-Castillo et al. 2020 custom POLE framework) provides a PP3 supporting rule only for an exact missense variant present in Supplementary Table S2 or S3 with a 'likely disease causing' REVEL class; this variant is a not a missense substitution and is absent from both tables, so that custom rule cannot be applied and assessment falls back to the generic SpliceAI path. PP3 is therefore not met.

Rule

PP3: computational evidence supports a deleterious effect on gene or gene product (splice-region path, SpliceAI). For intronic/splice-region variants, SpliceAI max delta >= 0.2 (supporting) or >= 0.5 (moderate) (Jaganathan et al. 2019, PMID:30661751).

Evidence considered

• VariantValidator/normalization annotated the input as NM_006231.4:c.4291-30_4291-27delinsAAT with predicted protein consequence NP_006222.2:p.? and both breakpoints within intron 33 ('start_exon': '33i', 'end_exon': '33i'), establishing an intronic/splice-region context rather than a missense coding change.
• SpliceAI Lookup returned a maximum delta score of 0.135 (DS_AG 0.011, DS_AL 0.135, DS_DG 0.016, DS_DL 0.000), below the published PP3 supporting cutoff of >= 0.2 (Jaganathan et al. 2019, Cell, PMID:30661751); DS_AL at the acceptor is the only sub-score above 0.1.
• Pangolin was also reported (splice gain 0.047, splice loss -0.019), corroborating the absence of a strong splice effect.
• The governing local POLE framework descends from Leon-Castillo et al. 2020 (PATH-250-323.pdf) and its supplementary tables; its PP3 rule is explicitly restricted to an exact missense variant present in Table S2/S3 with a 'likely disease causing' REVEL class, which this intronic variant is not.
• A full-text search of all converted POLE VCEP files (PATH-250-323.pdf, s003, s004) found no occurrence of 'c.4291-30_4291-27delinsAAT', 'delinsAAT', '4291', or any splice/intron keyword, confirming no pre-assigned code for this variant.

Still missing

• Searched the governing VCEP in-silico materials PATH-250-323-s003.xlsx (Table S2) and PATH-250-323-s004.xlsx (Table S3) and the main PATH-250-323.pdf text in full: no entry for c.4291-30_4291-27delinsAAT, NP_006222.2:p.?, or any intronic/splice-region notation was found; both tables list only missense substitutions, so no pre-assigned PP3 code exists for this variant.
• No MaxEntScan, SpliceSiteFinder, or other orthogonal splice predictor was available to corroborate or extend the SpliceAI call at this position.

Sources

Reasoning

PP5 applies only to an exact-variant ClinVar expert-panel (review status 'practice guideline' or 'reviewed by expert panel') Pathogenic/Likely pathogenic classification. The ClinVar search for this variant (POLE[GENE] AND c.4291-30_4291-27delinsAAT[VARNAME], plus transcript and 6 alternative search terms including the GRCh37 genomic description) returned no record at all: exact_match false, no variation ID, no classification, no review status, no expert panels and no submissions. With no ClinVar record there is no expert-panel assertion of any kind, so PP5 is not met.

Evidence considered

• ClinVar search (POLE[GENE] AND c.4291-30_4291-27delinsAAT[VARNAME]) plus six alternative terms returned no record; prefetch exact_match=false and evidence pass recorded 'This variant is absent from ClinVar.'
• No variation ID, classification, review status, expert-panel flag or submitter is available, so no expert-panel assertion can be applied.

Sources

Reasoning

POLE NM_006231.4:c.4291-30_4291-27delinsAAT (NC_000012.11:g.133220173_133220176delinsATT) is absent from gnomAD v2.1 (all-comers and, per its exomes-only figures, the non-cancer subset) and from gnomAD v4.1 (v4 all-comers; v3.1 genomes-only non-cancer also negative). Allele frequency is therefore 0. In the absence of an official ClinGen CSPEC/VCEP for POLE, the governing source is the local León-Castillo et al. 2020 custom framework, which customizes only PM1/PS4/PP3/BP4 and specifies no population-frequency criterion or a non-cancer/exome-only population source; generic ACMG/AMP population thresholds therefore apply. BA1 requires a stand-alone allele frequency >= 0.05 (ACMG/AMP 2015, PMID:25741868). An allele frequency of 0 is far below this, so BA1 is not met.

Rule

BA1 stand-alone benign: allele frequency >= 0.05 (ACMG/AMP 2015, PMID:25741868).

Evidence considered

• gnomAD v2.1 (GRCh37, 12-133220173-CAAG-ATT): search_status 'absent', found=false, no allele frequency reported.
• gnomAD v4.1 (GRCh38, chr12-132643587-CAAG-ATT): search_status 'absent', found=false, no allele frequency reported.
• gnomAD v2.1 non-cancer (exomes-only) subset: absent; gnomAD v3.1 non-cancer (genomes-only) subset: absent.
• BA1 stand-alone threshold >= 0.05 taken from ACMG/AMP 2015 (PMID:25741868).
• The governing source is the local León-Castillo et al. 2020 POLE custom framework (customized criteria PM1/PS4/PP3/BP4 only); it defines no population-frequency rule, so generic ACMG/AMP thresholds were used, and no non-cancer/exome-only population source was mandated.

Still missing

• gnomAD-Canada v1.0 (HostSeq) returned a timeout and could not be checked, but it is a small supplemental cohort and cannot plausibly carry this variant at the 0.05 BA1 threshold given its absence from gnomAD v2.1 and v4.1.

Sources

Reasoning

This POLE NM_006231.4:c.4291-30_4291-27delinsAAT variant is absent from all available population datasets: gnomAD v2.1 (GRCh37 12-133220173-CAAG-ATT), gnomAD v2.1 non-cancer exomes-only subset, gnomAD v4.1 (GRCh38 chr12-132643587-CAAG-ATT), and gnomAD v3.1 non-cancer genomes-only subset all report search_status 'absent'. The observed allele frequency is 0. No applicable ClinGen CSPEC/VCEP exists for POLE, and the local León-Castillo et al. 2020 framework does not specify BS1 or any gene-specific frequency source, so the generic BS1 strong-benign threshold of allele frequency >= 0.01 applies (per-gene derivation preferred; Whiffin et al. 2017, PMID:28518168). An allele frequency of 0 is far below 0.01, so BS1 is not met.

Rule

BS1 strong benign: allele frequency >= 0.01 (generic SVI default; Whiffin et al. 2017, PMID:28518168).

Evidence considered

• gnomAD v2.1 (GRCh37, 12-133220173-CAAG-ATT): absent, no frequency.
• gnomAD v4.1 (GRCh38, chr12-132643587-CAAG-ATT): absent, no frequency.
• gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets: both absent.
• BS1 threshold >= 0.01 used as the generic SVI default (per-gene derivation preferred; Whiffin et al. 2017, PMID:28518168), since the local León-Castillo POLE framework specifies no BS1 rule.

Still missing

• No gene-specific BS1 threshold could be derived because no POLE ClinGen CSPEC/VCEP with a calibrated frequency threshold is available; the generic 0.01 SVI default was used.
• gnomAD-Canada v1.0 (HostSeq) timed out and was not evaluable.

Sources

Reasoning

BS2 is met when the variant is observed in a healthy adult individual in a genotype context appropriate to the disease model (homozygous for a recessive, or heterozygous for a fully penetrant dominant, disorder) in a population cohort. Because the variant is absent from gnomAD v2.1 and v4.1 (and their non-cancer subsets), there are zero observed carriers of any genotype, including zero homozygotes. No homozygous observation exists to support BS2, so the criterion is not met. No POLE-specific CSPEC/VCEP modifies BS2; the local León-Castillo et al. 2020 framework does not address BS2, so generic BS2 semantics apply.

Rule

BS2 strong benign: observed in a healthy adult for a recessive (homozygous) or dominant (heterozygous) disorder; no such observation exists (allele frequency 0).

Evidence considered

• gnomAD v2.1 (GRCh37, 12-133220173-CAAG-ATT): absent, zero carriers/homozygotes.
• gnomAD v4.1 (GRCh38, chr12-132643587-CAAG-ATT): absent, zero carriers/homozygotes.
• gnomAD v2.1 non-cancer and v3.1 non-cancer subsets: both absent.
• No POLE gene-specific BS2 specification exists; the local León-Castillo et al. 2020 custom framework customizes only PM1/PS4/PP3/BP4, so generic BS2 semantics apply.

Still missing

• gnomAD cohorts are not formally phenotype-screened healthy-adult cohorts, but the complete absence of the variant means there is no carrier observation to which BS2 could attach regardless of cohort annotation.

Sources

Reasoning

NM_006231.4:c.4291-30_4291-27delinsAAT (NP_006222.2:p.?) is an intronic/splice-region variant in intron 33, so BP4 is assessed on the SpliceAI path only. BP4 supporting requires a SpliceAI maximum delta score <= 0.1 (Jaganathan et al. 2019, PMID:30661751); the observed maximum delta is 0.135, driven by the acceptor-loss sub-score (DS_AL 0.135), which exceeds the cutoff. The score therefore sits in the gray zone between the BP4 (<= 0.1) and PP3 (>= 0.2) cutoffs, meaning neither benign nor pathogenic computational evidence is met; a gray-zone result does not license any cross-path substitution and there is no missense/REVEL path for an intronic variant. The governing Leon-Castillo 2020 custom POLE framework supplies a BP4 supporting rule only for an exact missense variant present in Table S2/S3 with a 'Likely benign' REVEL class and >= 4 benign in-silico results; this variant is not missense and is absent from both tables. BP4 is therefore not met.

Rule

BP4: computational evidence suggests no impact on gene or gene product (splice-region path, SpliceAI). For intronic/splice-region variants, SpliceAI max delta <= 0.1 (supporting) (Jaganathan et al. 2019, PMID:30661751).

Evidence considered

• Normalization placed the variant in intron 33 (both breakpoints '33i'), so the SpliceAI path is the sole applicable PP3/BP4 path and the missense REVEL path does not apply.
• SpliceAI maximum delta score 0.135 (DS_AL 0.135) exceeds the BP4 supporting cutoff of <= 0.1 (Jaganathan et al. 2019, Cell, PMID:30661751), so BP4 is not met.
• Because 0.135 lies between the 0.1 BP4 and 0.2 PP3 cutoffs, the computational result is indeterminate in both directions and cannot support either criterion.
• The custom POLE framework's BP4 rule is limited to missense variants in Table S2/S3 with a 'Likely benign' REVEL class and >= 4 benign tool results; this intronic variant is not eligible for that rule.

Still missing

• Searched the governing VCEP in-silico materials PATH-250-323-s003.xlsx (Table S2) and PATH-250-323-s004.xlsx (Table S3) and PATH-250-323.pdf in full: no entry for c.4291-30_4291-27delinsAAT, NP_006222.2:p.?, or any intronic/splice-region notation; no pre-assigned BP4 code exists for this variant.
• No orthogonal splice predictor (e.g. MaxEntScan) was available to corroborate the borderline SpliceAI result at the 0.1 cutoff.

Sources

Reasoning

BP6 applies only to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. The ClinVar search for this variant returned no record (exact_match false, no variation ID, no classification, no expert-panel review status, no submissions). No expert-panel Benign/Likely-benign assertion exists, and ordinary laboratory submissions or aggregate labels would not qualify in any case. BP6 is not met.

Evidence considered

• ClinVar search returned no record for this variant (no variation ID, classification or review status), so no expert-panel Benign/Likely-benign assertion exists.

Sources

Reasoning

PS2 (de novo occurrence confirmed by parental testing) requires a proband with the POLE-associated phenotype in whom the variant is demonstrated absent in both biological parents with parentage confirmed. No proband record, phenotype description, pedigree, or parental genotype data are present in the available case materials, and no source reports a de novo occurrence for this variant. ClinVar carries no record for POLE c.4291-30_4291-27delinsAAT and no literature report of this variant was identified. Absent parental testing, PS2 cannot be met and cannot be assessed.

Rule

PS2: de novo (confirmed) variant in a patient with the disease and no family history of the disease; requires demonstration that the variant is absent in both biological parents with confirmed parentage.

Evidence considered

• Framework review: no ClinGen CSPEC/VCEP specification was retrieved for POLE (cspec found=false); the applied local Leon-Castillo et al. 2020 custom framework customizes only PM1, PS4, PP3 and BP4, so PS2 falls back to generic ACMG/AMP 2015.
• No case material supplies a proband, phenotype, pedigree, or parental-testing data; no consulted source reports this variant as de novo.
• ClinVar search for POLE c.4291-30_4291-27delinsAAT returned no record, and no literature report or full-text describing this variant was identified.

Still missing

• No proband phenotype/disease information for this POLE variant.
• No parental genotype testing results, so de novo status and parentage cannot be established.

Sources

Reasoning

PS3 requires well-established functional studies demonstrating a damaging effect on gene or protein function. NM_006231.4:c.4291-30_4291-27delinsAAT is an intronic variant in POLE (VariantValidator places both endpoints in intron 33, exonic position '33i'), lying 27-30 bp upstream of the exon beginning at c.4291 and therefore outside the canonical +/-1,2 splice consensus. For a non-coding/splice-region variant the relevant functional evidence would be an RNA-based or equivalent assay (minigene splicing assay, RT-PCR/cDNA sequencing of patient RNA, or a comparable functional readout). No such assay data exist anywhere in this case: literature_pass.json reports total_pmids 0 ('No PMIDs found across evidence sources'), paper_extracts.json and fulltext_map.json are empty, no full text was fetched, and there is no ClinVar record, no COSMIC somatic record, and no other functional characterization of this variant. The only splicing-related result available is a computational SpliceAI prediction (max delta 0.135, DS_AL 0.135), which is not a functional assay and at 0.135 is indeterminate, sitting between the generic PP3 supporting cutoff (>=0.2) and the BP4 supporting cutoff (<=0.1); computational predictions cannot satisfy PS3. No gene-specific PS3 specification exists: the local León-Castillo et al. 2020 POLE framework customizes only PM1, PS4, PP3 and BP4, so generic ACMG/AMP PS3 semantics apply. PS3 is therefore not assessed, and the gap is the absence of any validated functional or RNA assay of this variant.

Evidence considered

• NM_006231.4:c.4291-30_4291-27delinsAAT is intronic in POLE (VariantValidator exonic position '33i' for both endpoints), located 27-30 bp upstream of the exon beginning at c.4291, i.e. non-canonical and outside the +/-1,2 splice consensus.
• PS3 requires well-established functional studies showing a damaging effect; for a non-coding/splice-region variant the applicable evidence class is an RNA-based or equivalent functional assay (minigene, RT-PCR/cDNA sequencing, or comparable readout).
• No functional assay data for this variant were found in any evidence source: literature_pass.json reports total_pmids 0 ('No PMIDs found across evidence sources'), paper_extracts.json and fulltext_map.json are empty, ClinVar has no record, COSMIC has no record, and the only POLE-package material is contextual hotspot/somatic literature with no assay of this variant.
• SpliceAI is a computational predictor, not a functional assay; its max delta score of 0.135 (DS_AL 0.135) is indeterminate between the generic PP3 supporting cutoff (>=0.2) and the BP4 supporting cutoff (<=0.1), and computational scores cannot satisfy PS3.
• No gene-specific PS3 rule was available; the local León-Castillo et al. 2020 POLE framework customizes only PM1, PS4, PP3 and BP4, so generic ACMG/AMP PS3 semantics were applied.

Still missing

• No functional/RNA assay data (minigene splicing assay, RT-PCR/cDNA sequencing, or equivalent) exist for c.4291-30_4291-27delinsAAT.
• No publications were retrieved for this variant (literature_pass total_pmids = 0; paper_extracts.json and fulltext_map.json empty), so no published functional characterization could be evaluated.
• SpliceAI max delta 0.135 is a computational prediction only and is indeterminate; PS3 cannot be supported without an experimental assay.

Sources

Reasoning

PM3 (Pathogenic, Moderate) requires that the variant be detected in trans with a pathogenic variant in a recessive disorder. No ClinGen CSPEC/VCEP specification was retrieved for POLE; the applied local León-Castillo et al. 2020 custom POLE framework customizes only PM1, PS4, PP3 and BP4 and says nothing about PM3, so the generic ACMG/AMP 2015 PM3 definition applies. POLE has two germline disease contexts relevant to phase: autosomal dominant polymerase proofreading-associated polyposis (heterozygous proofreading-domain variants) and autosomal recessive IMAGE-I syndrome (biallelic POLE variants); PM3 is only usable when a recessive, biallelic setting is established, and that setting is not stated for this case. The case materials contain no proband record, no phenotype, no zygosity, no pedigree, no parental genotypes and no second POLE variant. The only variant-level sources available report the variant itself: ClinVar returns no record for POLE c.4291-30_4291-27delinsAAT (empty variation_ids, exact_match false) and gnomAD v2.1/v4.1 report it as absent, so no pathogenic partner variant in trans is reported by any source. Because the trans/cis configuration of this variant cannot be established from any available evidence, PM3 can be neither met nor excluded and is recorded as not_assessed.

Rule

PM3: For recessive disorders, detected in trans with a pathogenic variant (ACMG/AMP 2015); not applicable to dominant disorders.

Evidence considered

• Framework review: no ClinGen CSPEC/VCEP was retrieved for POLE (cspec found=false); the applied local León-Castillo et al. 2020 custom POLE framework customizes only PM1, PS4, PP3 and BP4 and does not specify PM3, so generic ACMG/AMP 2015 PM3 semantics apply.
• Inheritance-mode review: POLE germline disease is split between autosomal dominant polymerase proofreading-associated polyposis (germline heterozygous proofreading-domain variants) and autosomal recessive IMAGE-I syndrome (biallelic POLE variants), so PM3 is only applicable if a recessive biallelic setting is established; no disease context is stated for this case.
• No proband genotype, zygosity, pedigree, parental genotype, or second POLE variant is present in the case materials; no consulted source reports a partner pathogenic variant in trans with c.4291-30_4291-27delinsAAT.
• ClinVar holds no record for this variant (variation_ids empty, exact_match false), so no ClinVar-classified partner variant or phase information exists to evaluate trans configuration.
• The variant is a 4-bp intronic deletion-insertion 27-30 bp upstream of the affected exon (NM_006231.4:c.4291-30_4291-27delinsAAT; NC_000012.11:g.133220173_133220176delinsATT) with predicted protein consequence p.? (NP_006222.2:p.?), i.e. no protein-level allelic information.

Still missing

• No proband genotype or zygosity for POLE c.4291-30_4291-27delinsAAT.
• No phase information (trans vs cis) and no second POLE variant reported in any source, including ClinVar.
• Inheritance mode of the proband's disease is not specified; POLE has both autosomal dominant (polymerase proofreading-associated polyposis) and autosomal recessive (IMAGE-I syndrome) germline contexts.
• No family segregation or parental testing data.

Sources

Reasoning

PM6 (assumed de novo, parentage not confirmed) requires a proband with the POLE-associated phenotype and evidence suggesting the variant arose de novo when parental confirmation is unavailable. No proband, phenotype, family history, or parental data are present in the available case materials, so an assumed-de-novo occurrence cannot be established for this variant. PM6 is therefore not assessable.

Rule

PM6: assumed de novo variant (parentage not confirmed) in a patient with the disease and no family history of the disease.

Evidence considered

• Framework review: no ClinGen CSPEC/VCEP for POLE; the local Leon-Castillo et al. 2020 framework does not specify PM6, so generic ACMG/AMP 2015 applies.
• No case material supplies a proband, phenotype, or family/parental context from which an assumed de novo occurrence could be inferred.

Still missing

• No proband phenotype/disease information.
• No family history or parental data to support an assumed de novo occurrence.

Sources

Reasoning

PP1 (co-segregation of the variant with disease in multiple affected family members) requires a family pedigree with genotype-phenotype segregation data. The available case materials contain no pedigree, no list of affected or unaffected relatives, and no family genotypes; no consulted source describes this variant co-segregating in a family. Segregation therefore cannot be evaluated.

Rule

PP1: co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease (strength varies with number of meioses/affecteds).

Evidence considered

• Framework review: no ClinGen CSPEC/VCEP for POLE; the local Leon-Castillo et al. 2020 framework does not specify PP1, so generic ACMG/AMP 2015 applies.
• No case material provides a pedigree or family genotype data, and no consulted source reports co-segregation of this variant with disease.

Still missing

• No pedigree or list of affected/unaffected relatives.
• No segregation genotypes for family members.

Sources

Reasoning

PP4 (phenotype or family history highly specific for a disease with a single genetic etiology) requires the affected individual's clinical phenotype to be known and compared against the gene's disease spectrum. No phenotype information was provided for this case: the case summary and evidence bundle contain no HPO terms, no clinical diagnosis, no age of onset and no indication text. The pvs1 gene context lists a single spurious disease string ('Alpha-mannosidosis due to a novel MAN2B1 truncating mutation...'), which is a generic gene-level literature artifact for an unrelated gene and cannot be used as this variant's phenotype. Because the proband's phenotype is unknown, PP4 cannot be evaluated and is returned not_assessed rather than not_met.

Evidence considered

• The consolidated case evidence carries no HPO terms, phenotype description, indication for testing, or family history; only the gene-level summary is present.
• The gene context describes POLE-related disease broadly (colorectal cancer/polyposis and the facial dysmorphism, immunodeficiency, livedo, short stature syndrome) but no case-specific phenotype is documented.

Still missing

• No proband clinical phenotype (HPO terms, diagnosis, onset, family history) is present anywhere in the case files; PP4 cannot be assessed without it.

Reasoning

BS3 requires well-established functional studies demonstrating no damaging effect on gene or protein function. NM_006231.4:c.4291-30_4291-27delinsAAT is intronic in POLE (VariantValidator exonic position '33i'), 27-30 bp upstream of the exon beginning at c.4291, so the relevant assay class would again be an RNA-based functional study (minigene splicing assay, RT-PCR/cDNA sequencing of patient RNA, or equivalent). No functional assay data of any kind exist in this case: literature_pass.json reports total_pmids 0, paper_extracts.json and fulltext_map.json are empty, no full text was fetched, and there is no ClinVar or COSMIC record for this variant. Absence of a variant from databases and absence of publications is not evidence of a non-damaging functional effect and cannot satisfy BS3. The only splicing-related result is the computational SpliceAI prediction (max delta 0.135), which is not a functional assay; at 0.135 it sits between the generic PP3 supporting cutoff (>=0.2) and the BP4 supporting cutoff (<=0.1) and is indeterminate, and a computational score can never establish a benign functional effect for BS3. No gene-specific BS3 specification exists (the local León-Castillo et al. 2020 POLE framework covers only PM1, PS4, PP3, BP4), so generic ACMG/AMP semantics apply. BS3 is therefore not assessed, and the gap is the same absence of any validated functional or RNA assay.

Evidence considered

• BS3 requires well-established functional studies showing no damaging effect on gene or protein function; no such studies exist for this variant.
• NM_006231.4:c.4291-30_4291-27delinsAAT is intronic in POLE (VariantValidator exonic position '33i'), 27-30 bp upstream of the exon beginning at c.4291; the applicable evidence class for a benign functional claim would be an RNA-based assay (minigene, RT-PCR/cDNA sequencing, or equivalent).
• No functional assay data were identified: literature_pass.json reports total_pmids 0, paper_extracts.json and fulltext_map.json are empty, no full text was fetched, and no ClinVar or COSMIC record exists for this variant.
• Absence of the variant from ClinVar, COSMIC and gnomAD, and the lack of any published assay, is not evidence of a non-damaging functional effect and cannot satisfy BS3.
• SpliceAI is a computational predictor, not a functional assay; its max delta score of 0.135 is indeterminate between the generic PP3 supporting cutoff (>=0.2) and the BP4 supporting cutoff (<=0.1), and a computational score cannot satisfy BS3.
• No gene-specific BS3 rule was available; the local León-Castillo et al. 2020 POLE framework customizes only PM1, PS4, PP3 and BP4, so generic ACMG/AMP BS3 semantics were applied.

Still missing

• No functional/RNA assay data (minigene splicing assay, RT-PCR/cDNA sequencing, or equivalent) exist for c.4291-30_4291-27delinsAAT.
• No publications were retrieved for this variant (literature_pass total_pmids = 0; paper_extracts.json and fulltext_map.json empty), so no published functional characterization could be evaluated.
• Absence from ClinVar, COSMIC and gnomAD is not evidence of a non-damaging functional effect; BS3 cannot be supported without an experimental assay.

Sources

Reasoning

BS4 (lack of segregation) requires family genotype data demonstrating non-segregation, i.e. the variant absent from affected relatives or present in unaffected relatives. No family members or genotypes are available in the case materials, so non-segregation cannot be assessed and BS4 cannot be met.

Rule

BS4: lack of segregation in affected members of a family (variant absent in affected relatives / present in unaffected relatives), supporting a benign interpretation.

Evidence considered

• Framework review: no ClinGen CSPEC/VCEP for POLE; the local Leon-Castillo et al. 2020 framework does not specify BS4, so generic ACMG/AMP 2015 applies.
• No case material provides family genotype data; nothing reports non-segregation of this variant with disease.

Still missing

• No pedigree or family genotypes to test segregation.
• No data on whether the variant is absent from affected relatives or present in unaffected relatives.

Sources

Reasoning

BP2 (Benign, Supporting) requires observation of the variant either in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or in cis with a pathogenic variant in any inheritance pattern. No ClinGen CSPEC/VCEP specification was retrieved for POLE, and the applied local León-Castillo et al. 2020 custom POLE framework customizes only PM1, PS4, PP3 and BP4, so the generic ACMG/AMP 2015 BP2 definition applies. The criterion is entirely phase-dependent: it can only be triggered when a second, independently pathogenic variant is present and its cis/trans relationship to this variant is known. The case provides no proband genotype, no zygosity, no family study and no second variant, and ClinVar holds no record for POLE c.4291-30_4291-27delinsAAT (empty variation_ids, exact_match false); no source reports any co-occurring pathogenic variant in cis or in trans with this variant. The required cis/trans observation therefore cannot be made, so BP2 is recorded as not_assessed rather than not_met.

Rule

BP2: 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 (ACMG/AMP 2015).

Evidence considered

• Framework review: no ClinGen CSPEC/VCEP for POLE; the local León-Castillo et al. 2020 custom POLE framework does not specify BP2, so generic ACMG/AMP 2015 BP2 semantics apply.
• BP2 is phase-dependent: it requires a second pathogenic variant, either observed in trans for a fully penetrant dominant disorder or in cis with this variant in any inheritance pattern.
• No case material supplies a proband genotype, zygosity, family study, or second POLE variant, so no cis- or trans-relationship to a pathogenic variant can be established.
• ClinVar search for POLE c.4291-30_4291-27delinsAAT returned no record (variation_ids empty, exact_match false), so there is no co-occurring ClinVar-classified pathogenic variant to place in cis or in trans.

Still missing

• No co-occurring pathogenic variant (in cis or in trans) identified for this variant.
• No proband genotype or phase information to determine cis/trans configuration.
• No family genotypes or pedigree data.
• No second-variant testing or a second ClinVar/ClinVar-submitted variant reported.

Sources

Reasoning

BP5 (variant found in a case with an alternate molecular basis for the disease) requires a defined affected case or family in which a different pathogenic variant or established alternative cause explains the phenotype. No case-level genotype/phenotype data are available here: there is no second variant, no alternate diagnosis and no clinical narrative for this proband. Without an identified alternate molecular basis there is nothing to test the criterion against, so BP5 is returned not_assessed rather than not_met.

Evidence considered

• No second variant, alternate genetic diagnosis, or case narrative is present in the case summary or evidence bundle.

Still missing

• No case-level data (co-occurring pathogenic variant, co-segregating alternate cause, or clinical narrative) exist in the case files; BP5 cannot be assessed.