POLE c.941C>A, p.(Ser314Ter)

Reasoning

NM_006231.4:c.941C>A produces NP_006222.2:p.(Ser314Ter), a nonsense variant. No official ClinGen POLE CSPEC exists (cspec.found=false) and the local Leon-Castillo et al. 2020 custom framework only specifies rules for missense PM1/PS4/PP3/BP4, not PVS1, so the generic ClinGen SVI PVS1 decision tree (PMC6185798) is the governing framework, consistent with the pipeline's own pvs1_variant_assessment scaffold. Exon-level mapping places c.941 in exon 10 of 49 coding exons (variant_exonic_positions start_exon=end_exon='10'; CDS exon boundaries in prefetch selector_short.exon.c). The premature stop at codon 314 is far upstream of the final exon-exon junction (48 further exons remain, CDS spans 6861 nt / 2286 aa), so NMD is predicted rather than escape. The truncation removes >85% of the 2286-aa protein, including essentially the entire exonuclease proofreading domain and the C-terminal polymerase/zinc-finger domains that follow, so the truncated region is unambiguously biologically critical, not a dispensable distal segment. Under the generic PVS1 decision tree (nonsense variant, NMD predicted, critical region removed) this supports full PVS1_VeryStrong. needs_human_review is set because the germline gene-disease/loss-of-function mechanism support in pvs1_gene_context is weak and largely off-target: of the five 'supporting_sources' literature hits, only one (PMID:41551532, a Hungarian FAP cohort) even mentions POLE, and there it is reported only as a VUS, not as an established pathogenic LOF finding; the other four papers (FBXW7, SRSF1, melanoma germline panel, BRCA1-related HBOC case report) do not concern POLE at all and appear to be non-specific keyword matches. Established germline POLE disease is predominantly caused by heterozygous exonuclease-domain missense variants with a dominant-negative/altered-proofreading (gain-of-function) mechanism (polymerase proofreading-associated polyposis), while a recessive, partial loss-of-function mechanism is only documented for biallelic genotypes (FILS/IMAGe-like phenotype), not for isolated heterozygous truncating alleles. No zygosity or family/segregation information is provided in this case bundle to confirm which mechanism applies to this specific allele. PVS1 strength is therefore assigned per the standard truncation/NMD technical criteria but flagged for human review of the gene-disease mechanism/zygosity fit before it is treated as unconditionally applicable.

Rule

Generic ACMG/AMP PVS1 (ClinGen SVI PMC6185798 fallback) applied to a nonsense variant predicted to trigger NMD and remove the majority of the protein, including the exonuclease and polymerase domains; no POLE-specific CSPEC or local custom PVS1 rule exists.

Evidence considered

• prefetch.json normalize step: NM_006231.4:c.941C>A predicted protein NP_006222.2:p.(Ser314Ter); position_first=313, position_last_predicted=314 confirms an early premature stop.
• variant_validator variant_exonic_positions places the variant in exon 10 (start_exon=end_exon='10') of the transcript.
• prefetch.json selector_short.exon.c lists 49 total coding exons for NM_006231.4, confirming the premature stop at codon 314 is 39 exons upstream of the terminal exon, well beyond the 50-bp NMD-escape boundary.
• prefetch.json selector_short.cds.c gives CDS length c.1-6861 (2286 aa full-length protein), so the p.(Ser314Ter) truncation removes roughly 1972 of 2286 codons (>85% of the protein), including the exonuclease proofreading domain and downstream polymerase domains.
• pvs1_variant_assessment.json classifies this as consequence_class='nonsense', variant_bucket='nonsense', framework_source=PMC6185798 (ClinGen SVI PVS1 recommendations), apply_generic_pvs1_framework=true, suggested_default_strength='PVS1'.
• pvs1_gene_context.json flags germline_disease_context_found=true and lof_mechanism_supported=true, but its own supporting_sources are largely off-target: PMID:41551532 (Hungarian FAP cohort) lists POLE variants only as VUS (n=5), and the remaining four cited PMIDs (35395208 FBXW7, 37071997 SRSF1, 41673532 melanoma germline panel, 41947901 BRCA1 HBOC case report) do not discuss POLE at all, indicating a non-specific literature match rather than a confirmed POLE LOF disease mechanism.
• The local Leon-Castillo et al. 2020 custom POLE framework (final_classification_framework) explicitly customizes only PM1, PS4, PP3, and BP4 for somatic hotspot missense interpretation and does not provide a POLE-specific PVS1 rule, so generic ACMG/AMP PVS1 fallback via PMC6185798 is appropriate per case_summary.json final_classification_framework.rule_payload.notes.

Still missing

• The pvs1_gene_context literature search returned papers that are mostly unrelated to POLE (FBXW7, SRSF1, melanoma panel, HBOC case report); only one hit (PMID:41551532) mentions POLE and only as a VUS, so the automated germline LOF-mechanism support is weak and needs independent confirmation from POLE-specific germline disease literature (e.g., FILS/IMAGe-like syndrome or PPAP case series) before finalizing full-strength PVS1.
• No zygosity, family history, or clinical phenotype data are provided in this case bundle to determine whether this single heterozygous truncating allele fits the recognized POLE disease mechanism(s), which are predominantly dominant missense gain-of-function or recessive biallelic partial loss-of-function; this should be confirmed by a human reviewer.

Sources

Reasoning

PM2 is met at Moderate strength under the generic ACMG/AMP fallback: the exact variant is absent from the queried gnomAD v2.1 exome, gnomAD v4.1 exome, and gnomAD-Canada v1.0 population datasets. The available evidence contains no population frequency or ancestry-specific count suggesting a threshold artifact.

Rule

Generic ACMG/AMP PM2: absent from controls (or at extremely low frequency if recessive), applied at Moderate strength because no POLE-specific PM2 specification was retrieved.

Evidence considered

• gnomAD v2.1 reported GRCh37 12-133252759-G-T absent.
• gnomAD v4.1 reported GRCh38 chr12-132676173-G-T absent.
• gnomAD-Canada v1.0 reported GRCh38 12-132676173-G-T absent.
• No official ClinGen CSPEC was retrieved for POLE. The retrieved local POLE framework specifies only PM1, PS4, PP3, and BP4 and directs generic ACMG/AMP fallback outside those explicit rules.

Still missing

• No POLE-specific PM2 frequency specification was retrieved; the local POLE framework customizes PM1, PS4, PP3, and BP4 only, so PM2 follows generic ACMG/AMP fallback.

Sources

Reasoning

NM_006231.4:c.941C>A is a nonsense variant (NP_006222.2:p.(Ser314Ter)), not a missense substitution, so the POLE custom framework's PP3 rule (which is scoped explicitly to missense variants listed in Leon-Castillo et al. Supplementary Table S2/S3 with a REVEL class) does not apply, and this variant is not present in those tables. Falling back to the generic ACMG in-silico calibration for non-missense variants, which uses SpliceAI only: SpliceAI max delta score = 0.132 (DS_AG 0.056, DS_AL 0.132, DS_DG 0.011, DS_DL 0.02), which falls within the gray zone (0.1-0.2) defined by the generic SVI-based threshold set, so PP3 is not met on splice-impact evidence.

Rule

generic_acmg_classification_rules.md SpliceAI gray-zone rule (non-missense path)

Evidence considered

• SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.13).' with component scores DS_AG=0.056, DS_AL=0.132, DS_DG=0.011, DS_DL=0.02.
• Generic ACMG fallback rule (generic_acmg_classification_rules.md): for intronic/synonymous/non-canonical-splice-position (i.e. non-missense) variants, SpliceAI max delta > 0.2 triggers PP3, and max delta in [0.1, 0.2] is a gray zone where neither PP3 nor BP4 is met.
• POLE custom framework (case_summary.json cspec.criteria PP3 rule) restricts its PP3_Supporting rule to exact missense variants appearing in Supplementary Table S2 or S3 with an assigned REVEL class; c.941C>A is a nonsense variant absent from those tables, so this custom rule is inapplicable and the generic fallback governs.

Still missing

• No gene-specific (POLE) SpliceAI or nonsense-specific in-silico threshold framework was retrieved; generic SVI-based SpliceAI thresholds were used.
• REVEL score not available (found=false) so no missense-path evidence to consider even if it were applicable.

Sources

Reasoning

No exact-variant ClinVar record was found for NM_006231.4:c.941C>A (p.(Ser314Ter)); therefore there is no exact-variant Pathogenic/Likely pathogenic expert-panel assertion to support PP5.

Rule

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

Evidence considered

• ClinVar exact-variant searches in the case bundle returned no result and report that this variant is absent from ClinVar.

Sources

Reasoning

BA1 is not met: the variant is absent from the queried gnomAD v2.1 exome, gnomAD v4.1 exome, and gnomAD-Canada v1.0 datasets, so no allele frequency is available that could meet a stand-alone benign frequency threshold.

Rule

Generic ACMG/AMP population-frequency fallback: BA1 requires an allele frequency above the applicable stand-alone benign threshold.

Evidence considered

• gnomAD v2.1 queried GRCh37 12-133252759-G-T and reported the variant absent.
• gnomAD v4.1 queried GRCh38 chr12-132676173-G-T and reported the variant absent.
• gnomAD-Canada v1.0 queried GRCh38 12-132676173-G-T and reported the variant absent.

Still missing

• No POLE-specific BA1 threshold was retrieved; however, absence across the queried population datasets precludes BA1 under any frequency-based threshold.

Sources

Reasoning

BS1 is not met: the variant is absent from the queried gnomAD v2.1 exome, gnomAD v4.1 exome, and gnomAD-Canada v1.0 datasets, so there is no frequency greater than expected for POLE-associated disease.

Rule

Generic ACMG/AMP population-frequency fallback: BS1 requires an allele frequency greater than expected for the disorder.

Evidence considered

• The v2.1, v4.1, and Canada population queries each reported this exact normalized allele absent; no overall or ancestry-specific allele frequency is available to support BS1.

Still missing

• No POLE-specific BS1 threshold, disease prevalence, or penetrance calibration was retrieved; this does not affect the conclusion because the queried datasets report the variant absent.

Sources

Reasoning

BS2 is not met: no observation of this variant in multiple well-phenotyped unaffected adults was provided. Population-dataset absence cannot establish BS2.

Rule

Generic ACMG/AMP BS2 requires observation in healthy adults for a disorder expected to be fully penetrant at an early age.

Evidence considered

• The available population evidence consists of variant absence in gnomAD datasets, not unaffected-adult phenotype data.

Still missing

• Require observations in appropriately phenotyped unaffected adults, with an age and phenotype definition suitable for the relevant POLE-associated disease spectrum and penetrance.

Reasoning

Same evidence basis as PP3: c.941C>A is a nonsense variant, so the POLE custom BP4 rule (missense-only, scoped to Supplementary Table S2/S3 REVEL classes) does not apply and the variant is absent from those tables. Under the generic ACMG fallback for non-missense variants, SpliceAI max delta = 0.132 falls in the gray zone (0.1-0.2), which is below the >0.2 PP3 threshold but above the <0.1 BP4 threshold, so BP4 is not met.

Rule

generic_acmg_classification_rules.md SpliceAI gray-zone rule (non-missense path)

Evidence considered

• SpliceAI evidence_sentence and scores: max delta score = 0.132 (DS_AG=0.056, DS_AL=0.132, DS_DG=0.011, DS_DL=0.02), falling in the generic gray-zone band (0.1-0.2) where neither PP3 nor BP4 applies.
• Generic ACMG fallback rule (generic_acmg_classification_rules.md): SpliceAI max delta < 0.1 triggers BP4 for non-missense variants; 0.132 does not clear this threshold.
• BayesDel score of 0.655873 was retrieved from prefetch but is excluded from PP3/BP4 consideration because no verified published calibration threshold with a named PMID is available to this pipeline for BayesDel.
• POLE custom framework BP4 rule (case_summary.json cspec.criteria BP4 rule) is scoped to exact missense variants in Supplementary Table S2/S3 with REVEL class 'Likely benign'; c.941C>A is nonsense and absent from those tables, so this custom rule does not apply.

Still missing

• No gene-specific (POLE) SpliceAI-based BP4 threshold framework was retrieved; generic SVI-based SpliceAI thresholds were used.
• BayesDel score is available (0.655873) but per this pipeline's calibration policy no verified published threshold exists for BayesDel, so it cannot be used for BP4 regardless of the score value; treated as not_available for this criterion.

Sources

Reasoning

No exact-variant ClinVar record was found for NM_006231.4:c.941C>A (p.(Ser314Ter)); therefore there is no exact-variant Benign/Likely benign expert-panel assertion to support BP6.

Rule

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

Evidence considered

• ClinVar exact-variant searches in the case bundle returned no result and report that this variant is absent from ClinVar.

Sources

Reasoning

Not assessed: no proband phenotype, parental genotypes, parentage confirmation, or germline de novo testing result is provided for NM_006231.4:c.941C>A (p.Ser314Ter).

Rule

Generic ACMG/AMP fallback: PS2 requires a confirmed de novo occurrence in an affected individual with a phenotype consistent with the relevant POLE-associated disease, with maternity and paternity confirmed.

Evidence considered

• No official POLE ClinGen CSPEC/VCEP specification was retrieved. The available local POLE framework customizes only PM1, PS4, PP3, and BP4; PS2 therefore follows generic ACMG/AMP evidence semantics.
• The consolidated case-evidence bundle contains no proband-level phenotype, parental genotype, parentage, or de novo testing information.

Still missing

• Obtain proband phenotype and indication for testing.
• Obtain parental testing results from appropriate germline samples and confirmation of biological parentage.
• Document whether the parental testing was sufficiently sensitive to assess possible parental mosaicism.

Reasoning

No functional/well-established assay data (e.g., in vitro exonuclease proofreading activity assay, cDNA/minigene splicing assay, reporter assay) for NM_006231.4:c.941C>A (p.(Ser314Ter)) were found anywhere in the case bundle (evidence.json, literature_pass.json, vcep_materials.json, fulltext_map.json). The local custom POLE gene framework (Leon-Castillo et al. 2020) addresses only PM1, PS4, PP3, and BP4; it contains no PS3/BS3 functional-assay specification, and no official ClinGen POLE CSPEC/VCEP is present in this case. No generic-ACMG-eligible functional study data were located for this variant.

Still missing

• No validated functional assay (biochemical exonuclease activity, cell-based proofreading/mutator-phenotype assay, or splicing assay) reported for this specific variant.
• No POLE-specific PS3/BS3 VCEP specification available to define assay validation requirements or strength calibration.

Reasoning

No affected-proband observations with a pathogenic or likely pathogenic POLE variant established in trans with NM_006231.4:c.941C>A were provided. PM3 cannot be assessed.

Rule

Generic ACMG/AMP PM3 fallback: requires a recessive-disease observation of the variant in trans with a pathogenic/likely pathogenic variant in an affected individual.

Evidence considered

• No ClinGen CSPEC was retrieved for POLE. The available local POLE framework specifies PM1, PS4, PP3, and BP4 only, so PM3 is evaluated under generic ACMG/AMP fallback.
• The consolidated case-evidence bundle contains no affected-proband observations, no co-occurring POLE variant, and no phase or segregation result.

Still missing

• Affected-proband genotype data identifying a second POLE pathogenic/likely pathogenic variant.
• Phase evidence demonstrating that the two POLE variants are in trans.
• Disease-specific inheritance and phenotype information supporting a recessive POLE disease context for the proband.

Reasoning

Not assessed: the case bundle provides no parental testing result or reported de novo occurrence for NM_006231.4:c.941C>A (p.Ser314Ter).

Rule

Generic ACMG/AMP fallback: PM6 may support an assumed de novo occurrence in an affected individual with a phenotype consistent with the relevant disease when parentage is not confirmed; no such occurrence is documented here.

Evidence considered

• No official POLE ClinGen CSPEC/VCEP specification was retrieved. The available local POLE framework does not specify PM6; generic ACMG/AMP evidence semantics apply.
• The consolidated case-evidence bundle contains no parental genotype or de novo report.

Still missing

• Obtain parental germline testing results.
• If the variant is reported de novo, document the proband phenotype and whether biological parentage was confirmed.

Reasoning

Not assessed: no affected or unaffected relatives, family structure, genotypes, phenotypes, or informative meioses are reported for this variant.

Rule

Generic ACMG/AMP fallback: PP1 requires co-segregation of the variant with a phenotype consistent with the relevant disease in informative meioses; no segregation data are available.

Evidence considered

• No official POLE ClinGen CSPEC/VCEP specification was retrieved. The available local POLE framework does not specify PP1; generic ACMG/AMP evidence semantics apply.
• The consolidated case-evidence bundle contains no pedigree, relative-level genotype, phenotype, or meiosis data.

Still missing

• Obtain a pedigree with disease status, age/age at onset, and POLE genotype for informative relatives.
• Determine the relevant POLE-associated disease phenotype and penetrance before evaluating segregation.
• Document the number of informative meioses and any phenocopies or reduced penetrance.

Reasoning

No proband phenotype, tumor phenotype, or phenotype-specificity assessment is present in the consolidated case-evidence bundle. PP4 cannot be evaluated without a documented phenotype that is highly specific for a POLE-associated condition.

Rule

Generic ACMG/AMP PP4: phenotype must be highly specific for a disease with a single genetic etiology.

Evidence considered

• The consolidated bundle contains no case-level phenotype or clinical presentation for the individual carrying the variant.

Still missing

• Provide the proband's phenotype, relevant clinical diagnosis, age at onset, and testing context, with assessment of whether the phenotype is highly specific for POLE-associated disease.

Reasoning

No functional assay evidence showing a benign/no-impact result for NM_006231.4:c.941C>A (p.(Ser314Ter)) was found in the case bundle. As with PS3, no POLE-specific BS3 VCEP specification is present and no generic-ACMG-eligible functional study data were located for this variant.

Still missing

• No functional assay data of any kind (pathogenic or benign direction) available for this variant in the collected evidence.
• No POLE-specific BS3 VCEP specification available to define assay validation requirements or strength calibration.

Reasoning

Not assessed: no well-phenotyped affected relatives who lack NM_006231.4:c.941C>A (p.Ser314Ter), or other informative non-segregation observations, are reported.

Rule

Generic ACMG/AMP fallback: BS4 requires convincing lack of segregation in affected family members for a phenotype consistent with the relevant disease; no such data are available.

Evidence considered

• No official POLE ClinGen CSPEC/VCEP specification was retrieved. The available local POLE framework does not specify BS4; generic ACMG/AMP evidence semantics apply.
• The consolidated case-evidence bundle contains no family segregation or non-segregation observations.

Still missing

• Obtain genotype and phenotype data for affected relatives in an informative pedigree.
• Establish that any affected non-carrier has a phenotype sufficiently specific to the relevant POLE-associated disease and assess phenocopies.

Reasoning

No co-occurrence data identify NM_006231.4:c.941C>A in cis with a pathogenic variant or in trans with a pathogenic variant for a fully penetrant dominant condition. BP2 cannot be assessed.

Rule

Generic ACMG/AMP BP2 fallback: requires observation in cis with a pathogenic variant, or in trans with a pathogenic variant for a fully penetrant dominant disorder.

Evidence considered

• No ClinGen CSPEC was retrieved for POLE. The available local POLE framework specifies PM1, PS4, PP3, and BP4 only, so BP2 is evaluated under generic ACMG/AMP fallback.
• The consolidated case-evidence bundle contains no second variant, phase result, co-occurrence observation, or inheritance information that could satisfy BP2.

Still missing

• Co-occurrence data for NM_006231.4:c.941C>A and a pathogenic/likely pathogenic variant.
• Phase evidence establishing cis or trans configuration.
• For a trans observation, evidence that the relevant disorder is fully penetrant and dominantly inherited.

Reasoning

The bundle provides no proband phenotype and no evidence of an alternate molecular diagnosis that fully explains a documented phenotype. BP5 cannot be assessed.

Rule

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

Evidence considered

• No case-level phenotype or alternate molecular diagnosis is supplied in the consolidated bundle.

Still missing

• Provide the proband phenotype and diagnostic workup, including any alternate molecular diagnosis and evidence that it fully explains the phenotype.