POLE c.331-30G>A, p.?

Reasoning

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868) defines PM2 as moderate pathogenic evidence when a variant is absent from (or below the expected carrier frequency if recessive) a large general population or control cohort (>1000 individuals). NM_006231.4:c.331-30G>A is present in gnomAD at extremely low frequency: gnomAD v4.1 AF 3.37335e-05 (0.00337%; 54/1,600,784 alleles; exome 51/1,448,582; genome 3/152,202), gnomAD v2.1 AF 1.19612e-05 (0.00120%; 3/250,812 alleles), with 0 homozygotes in both datasets, grpmax FAF 3.621e-05, and absence from gnomAD-Canada v1.0. Zero homozygous carriers in ~1.6 million alleles and a population-maximum AF ~30-fold below any frequency at which population data would argue for benignity (BA1 >5%, BS1 'greater than expected'; PMID 25741868) are consistent with the 'extremely low frequency' reading of PM2. Caveat: the variant is not literally absent from gnomAD (54 alleles in v4.1), so under a strict absence requirement for autosomal dominant inheritance (PPAP) PM2 would not be met, and the recessive clause (below expected carrier frequency for FILS/IMAGE-I) could not be quantified for lack of disease prevalence data; PM2 is therefore applied at supporting strength under the extremely-low-frequency operationalization and flagged for human confirmation.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868): PM2 when variant is absent from (or below expected carrier frequency if recessive) a large general-population or control cohort; applied here at supporting strength under the extremely-low-frequency reading.

Evidence considered

• gnomAD v4.1 AF = 3.37335e-05 (0.00337%), 54/1,600,784 alleles, 0 homozygotes; grpmax FAF = 3.621e-05 (source: gnomad_v4)
• gnomAD v2.1 AF = 1.19612e-05 (0.00120%), 3/250,812 alleles, 0 homozygotes (source: gnomad_v2)
• Variant absent from gnomAD-Canada v1.0 (source: gnomad_canada)
• PM2 definition: variant absent from (or below expected carrier frequency if recessive) a large general population or control cohort (>1000 individuals) (Richards et al. 2015, PMID 25741868); applied under the extremely-low-frequency reading given 0 homozygotes in ~1.6 million alleles and population-maximum AF far below any benignity-relevant frequency (BA1 >5%, PMID 25741868)

Still missing

• Variant is present (not absent) in gnomAD v4.1 (54/1,600,784 alleles); PM2 applied under the 'extremely low frequency' reading rather than strict absence - if strict absence for autosomal dominant inheritance is required, PM2 would not be met
• No POLE-specific disease prevalence data were available to quantify the recessive 'below expected carrier frequency' clause (FILS / IMAGE-I)
• No disease-matched control cohort (e.g., unaffected relatives or population-matched colorectal cancer screening cohort) was available for a case-control frequency comparison

Sources

Reasoning

Generic in-silico fallback for intronic/non-canonical-splice variants uses SpliceAI only: BP4 (supporting) applies when SpliceAI max delta < 0.1 (SVI Splicing Subgroup thresholds, Walker et al. 2023, PMID 37352859). Observed max delta 0.06, with all four individual delta scores below 0.1 (DS_AG 0.06, DS_AL 0.02, DS_DG 0.02, DS_DL 0.0) and Pangolin scores near zero (SG 0.05, SL -0.01), predicting no impact on splicing. The custom POLE framework BP4 rule (exact missense variant in Tables S2/S3 with REVEL 'likely benign' and >=4 benign results) is inapplicable: the variant is intronic and absent from both tables. The same SpliceAI prediction is not double-counted: PP3 is not met and BP7 is not applicable, so the single splice-impact prediction supports BP4 alone at supporting strength.

Rule

Generic in-silico fallback (intronic/non-canonical-splice variant): SpliceAI max delta < 0.1 -> BP4 (supporting) per generic_acmg_combination_rules (SVI-recommended thresholds, Walker et al. 2023, PMID 37352859). Custom POLE framework BP4 rule inapplicable because the variant is intronic and absent from Supplementary Tables S2/S3.

Evidence considered

• SpliceAI max delta 0.06 < 0.1, the BP4 threshold for intronic variants (SVI Splicing Subgroup thresholds per Walker et al. 2023, PMID 37352859) -> BP4 (supporting)
• Generic in-silico calibration (generic_acmg_combination_rules): for intronic, synonymous, or non-canonical-splice-position variants use SpliceAI only; SpliceAI max delta < 0.1 -> BP4 (supporting); max delta 0.06 falls in this range, not the [0.1, 0.2] gray zone
• ACMG/AMP 2015 BP4 definition, PMID 25741868: 'Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing, etc.)' - governing definition; the generic calibration designates SpliceAI as the splice predictor for intronic variants
• Custom POLE framework BP4 rule (vcep_path_250_323_s003/s004): requires the exact missense variant in Supplementary Tables S2/S3 with REVEL 'likely benign' and >=4 benign results; variant is intronic and absent from both tables -> rule inapplicable, generic fallback governs

Sources

Reasoning

NM_006231.4:c.331-30G>A is an intronic single-nucleotide substitution (intron 4, 30 bp upstream of exon 5; VariantValidator exonic position '4i') with predicted protein consequence p.? and consequence class 'sequence_variant'. It falls into none of the generic PVS1 null-variant buckets (nonsense, frameshift, canonical +/-1/2 splice site, initiation codon, single/multi-exon deletion): canonical_splice_consensus = false and variant_bucket = 'other' per pvs1_variant_assessment.json, which explicitly sets apply_generic_pvs1_framework = false. SpliceAI lookup reports max delta score 0.06 (DS_AG 0.06, DS_AL 0.02, DS_DG 0.02, DS_DL 0.00), providing no evidence of splice-site disruption or of an NMD-competent aberrant transcript; no RNA/minigene functional data are present in the case bundle. Although the POLE gene-level PVS1 gate is eligible (germline LoF mechanism supported per pvs1_gene_context.json), the variant itself is not a null variant and no splice-impact evidence exists, so PVS1 is not met.

Rule

Generic ACMG/AMP 2015 PVS1 (PMID:25741868) as operationalized by the ClinGen SVI PVS1 framework (pvs1_generic_framework, PMC6185798; Tayoun et al. 2018): null-variant buckets (nonsense, frameshift, canonical +/-1/2 splice, initiation codon, single/multi-exon deletion) with a known LoF mechanism; non-bucket variant types (e.g., intronic SNVs) qualify only with demonstrated or high-confidence predicted NMD-competent splice disruption.

Evidence considered

• VariantValidator (prefetch.json): c.331-30G>A is intronic (exon annotation '4i'), 30 bp upstream of the exon beginning at c.331; protein consequence p.?; transcript NM_006231.4 is MANE Select for POLE.
• pvs1_variant_assessment.json: consequence_class 'unknown', canonical_splice_consensus false, variant_bucket 'other', apply_generic_pvs1_framework false; rationale: 'This variant does not fall into the default generic PVS1 null-variant buckets of nonsense, frameshift, or canonical ±1,2 splice consensus variants.'
• SpliceAI lookup (spliceai): max delta score 0.06 with DS_AG 0.06, DS_AL 0.02, DS_DG 0.02, DS_DL 0.0; 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.06).'
• pvs1_gene_context.json: POLE germline LoF mechanism supported (PVS1 gene gate 'eligible'); no official CSPEC/VCEP or local explicit PVS1 framework, so generic PVS1 fallback follows the ClinGen SVI PVS1 recommendations (PMC6185798).
• final_classification_framework.json: local León-Castillo custom POLE framework customizes only PM1, PS4, PP3, BP4 and notes 'Outside the explicit León-Castillo paper-derived rules, fall back to generic ACMG/AMP' - no POLE-specific PVS1 rule exists.

Still missing

• No RNA/minigene functional assay data; SpliceAI (max delta 0.06) alone cannot exclude a rare cryptic splice effect, but the variant meets no PVS1 null-variant bucket regardless, so the not_met call is not affected.

Sources

Reasoning

Variant is intronic (NM_006231.4:c.331-30G>A, 30 bp 5' of exon 4; NP_006222.2:p.?, no coding consequence; canonical splice consensus position: no), so the missense in-silico path is not applicable (REVEL found:false, score null; BayesDel found:false) and the splice-impact path (SpliceAI) governs. The custom POLE framework PP3 rule (exact missense variant in Supplementary Tables S2/S3 with REVEL class 'likely disease causing' and <=1 benign in-silico result) is inapplicable: the variant is intronic and absent from both tables (verified by direct inspection of PATH-250-323-s003.xlsx and s004.xlsx). Generic in-silico fallback for intronic/non-canonical-splice variants uses SpliceAI only: PP3 requires SpliceAI max delta > 0.2 (SVI Splicing Subgroup thresholds, Walker et al. 2023, PMID 37352859). Observed max delta 0.06 (DS_AG 0.06, DS_AL 0.02, DS_DG 0.02, DS_DL 0.0) is not > 0.2 and predicts no splice impact, so PP3 is not met. No other positive computational evidence for pathogenicity is available (COSMIC absent; ClinVar VUS with no criteria signals).

Rule

Generic in-silico fallback (intronic/non-canonical-splice variant): SpliceAI max delta > 0.2 -> PP3 (supporting) per generic_acmg_combination_rules (SVI-recommended thresholds, Walker et al. 2023, PMID 37352859). Custom POLE framework PP3 rule inapplicable because the variant is intronic and absent from Supplementary Tables S2/S3.

Evidence considered

• SpliceAI max delta 0.06 (DS_AG 0.06, DS_AL 0.02, DS_DG 0.02, DS_DL 0.0, Pangolin SG 0.05/SL -0.01) is not > 0.2, the PP3 threshold for intronic variants (SVI Splicing Subgroup thresholds per Walker et al. 2023, PMID 37352859; SpliceAI algorithm and 0.2 cutoff per Jaganathan et al. 2019, PMID 30661751) -> PP3 not met
• Custom POLE framework PP3 rule (vcep_path_250_323_s003/s004): requires the exact missense variant in Supplementary Tables S2/S3 with REVEL 'likely disease causing' and <=1 benign result; c.331-30G>A is intronic and absent from both tables (direct inspection, no '331' match in any data row) -> rule inapplicable, generic fallback governs
• REVEL/BayesDel not applicable: variant has no protein consequence (p.?); REVEL found:false and BayesDel found:false in prefetch (and BayesDel has no verified published threshold available to this pipeline in any case)

Sources

Reasoning

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868) defines BA1 as a stand-alone benign indicator when allele frequency in a large control population is over 5%. The highest observed frequency for NM_006231.4:c.331-30G>A is in European (non-Finnish) gnomAD v4.1 at 4.62231e-05 (0.00462%, 54/1,168,248 alleles); total gnomAD v4.1 AF is 3.37335e-05 (0.00337%, 54/1,600,784 alleles) and gnomAD v2.1 AF is 1.19612e-05 (0.00120%, 3/250,812 alleles). Even the population-maximum filtering allele frequency (grpmax FAF 3.621e-05) is roughly three orders of magnitude below the 5% BA1 threshold. No ancestry group or subpopulation approaches a benignity-relevant frequency, so BA1 is not met.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868): BA1 when allele frequency is >5% in a large, diverse control population (stand-alone benign).

Evidence considered

• gnomAD v4.1 total AF = 3.37335e-05 (0.00337%), 54/1,600,784 alleles, 0 homozygotes (source: gnomad_v4)
• gnomAD v2.1 total AF = 1.19612e-05 (0.00120%), 3/250,812 alleles, 0 homozygotes (source: gnomad_v2)
• Highest subpopulation AF = 4.62231e-05 (0.00462%, European non-Finnish, gnomAD v4.1); grpmax FAF = 3.621e-05
• BA1 stand-alone threshold = allele frequency over 5% in a large control population (Richards et al. 2015, PMID 25741868)

Still missing

• No separate 1000 Genomes / ESP queries were performed, but gnomAD v2.1 and v4.1 provide large, diverse population coverage sufficient for the BA1 assessment.

Sources

Reasoning

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868) defines BS1 as strong benign evidence when allele frequency in a control population is greater than expected for the disorder. POLE-associated germline disorders are rare (polymerase proofreading-associated polyposis, autosomal dominant; FILS and IMAGE-I syndromes, autosomal recessive). The observed allele frequency (max 0.00462% NFE in gnomAD v4.1; total 0.00337% v4.1; 0.00120% v2.1) is orders of magnitude below any allele-frequency range at which BS1 is applied for rare Mendelian disorders, and no POLE-specific expected allele frequency was available in the case materials to support a 'greater than expected' determination. BS1 is not met.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868): BS1 when allele frequency is greater than expected for the disorder (strong benign).

Evidence considered

• Observed maximum AF = 4.62231e-05 (0.00462%, European non-Finnish, gnomAD v4.1); total v4.1 AF = 3.37335e-05 (0.00337%) (source: gnomad_v4)
• gnomAD v2.1 AF = 1.19612e-05 (0.00120%), 3/250,812 alleles (source: gnomad_v2)
• BS1 requires allele frequency greater than expected for the disorder (Richards et al. 2015, PMID 25741868); observed AF is orders of magnitude below the range where BS1 applies for rare Mendelian disorders

Still missing

• No disease-prevalence-based expected allele frequency for POLE-associated disorders (PPAP, FILS, IMAGE-I) was available to quantify the BS1 'greater than expected' comparison; not needed for the call because the observed AF is far below any benignity-relevant range.

Sources

Reasoning

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868) defines BS2 as strong benign evidence when a variant is observed in a well-documented healthy adult individual for a disease fully penetrant at an early age (homozygous for recessive, heterozygous for dominant, hemizygous for X-linked). Zero homozygotes are observed in gnomAD v2.1 (0/250,812 alleles) and gnomAD v4.1 (0/1,600,784 alleles); the 54 heterozygous carriers in v4.1 come from a general-population cohort that is not phenotype-verified as unaffected for POLE-related disorders, and the dominant POLE phenotype (PPAP) is adult-onset with incomplete/age-dependent penetrance, so the 'fully penetrant at an early age' condition is not satisfied. BS2 is not met.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID 25741868): BS2 when variant is observed in a healthy adult for a fully penetrant early-onset disorder - homozygous (AR), heterozygous (AD), or hemizygous (XL) (strong benign).

Evidence considered

• 0 homozygotes in gnomAD v2.1 (250,812 alleles) and gnomAD v4.1 (1,600,784 alleles) (sources: gnomad_v2, gnomad_v4)
• 54 heterozygous carriers (54/1,600,784 alleles) in gnomAD v4.1; general-population carriers are not well-documented unaffected adults for POLE-related disorders
• BS2 requires observation in a well-documented healthy adult for a fully penetrant early-onset disorder (Richards et al. 2015, PMID 25741868); PPAP is adult-onset with incomplete penetrance

Sources

Reasoning

BP1 is not met on two independent grounds. (1) BP1 ('Missense variant in a gene for which primarily truncating variants are known to cause disease', Richards et al. 2015, PMID 25741868) requires a missense variant, and c.331-30G>A is intronic (p.?). (2) The gene-level premise of BP1 is false for POLE: the established disease mechanism is missense in the exonuclease domain — the governing local framework (León-Castillo et al. 2020) is built entirely on pathogenic missense hotspot substitutions (p.P286R, p.V411L, p.S297F, p.A456P, p.S459F) — so POLE is not a primarily-truncating gene and BP1 would not apply even to a missense variant in this gene.

Evidence considered

• Richards et al. 2015 (PMID 25741868): BP1 = 'Missense variant in a gene for which primarily truncating variants are known to cause disease.'
• Local custom POLE framework (León-Castillo et al. 2020, PATH-250-323.pdf): POLE disease mechanism is dominated by exonuclease-domain missense (five established pathogenic hotspot substitutions), contradicting the primarily-truncating premise of BP1; the queried variant is additionally intronic, not missense.

Sources

Reasoning

No de novo occurrence data exists for NM_006231.4:c.331-30G>A (POLE, intronic, c.331-30, protein p.?). The case bundle contains no proband phenotype report, no parental testing results, and no paternity/maternity identity testing for this variant. The sole ClinVar submission (SCV004024405, Center for Genomic Medicine, Rigshospitalet) is 'Uncertain significance' (1 star, single submitter, criteria provided but no criteria listed) with condition 'not specified', and the only retrieved publication (PMID:25741868) is the ACMG/AMP 2015 guideline itself, which contains no variant-specific case data. Governing rule: generic ACMG/AMP 2015 fallback (PS2 = de novo with parental identity confirmed, strong), because the local Leon-Castillo 2020 custom POLE framework (framework_mode custom_gene_framework) customizes only PM1/PS4/PP3/BP4 and explicitly falls back to generic ACMG/AMP outside those rules. With no de novo observation at all, PS2 cannot be evaluated; insufficient evidence.

Rule

Generic ACMG/AMP 2015 (Richards et al. 2015, PMID:25741868) via generic_acmg_combination_rules fallback; PS2 = 'De novo (paternity and maternity confirmed)' at strong. Not customized by the local Leon-Castillo 2020 POLE framework (customizes only PM1/PS4/PP3/BP4).

Still missing

• No parental testing data: no evidence of de novo occurrence and no paternity/maternity identity confirmation for this variant.
• No proband case report describing phenotype and family history for this variant exists in the case bundle.
• Actionable follow-up: obtain parental testing and identity-confirmation results from the submitting laboratory (SCV004024405) or clinical records.

Sources

Reasoning

PS3 requires well-established functional studies demonstrating a deleterious effect of the variant. For NM_006231.4:c.331-30G>A (POLE, intronic, 30 bp upstream of exon 5) no variant-specific functional assay evidence exists in the case bundle or retrieved literature: the only publication available (PMID:25741868) is the ACMG/AMP 2015 guidelines paper itself, which defines the criterion but contains no functional data on this variant; the sole ClinVar submission (SCV004024405, VUS, criteria provided/single submitter) cites only that framework paper; COSMIC has no record for this variant; and no RT-PCR/RNA-seq, minigene splicing assay, or polymerase/exonuclease activity assay is present in the bundle. Governing framework: no ClinGen CSPEC exists for POLE, and the local custom POLE framework (vcep_path_250_323, Leon-Castillo et al. 2020) specifies rules only for PM1/PS4/PP3/BP4, so generic ACMG/AMP 2015 (PMID:25741868) governs PS3/BS3. With no functional study of any kind, PS3 cannot be evaluated and is returned not_assessed.

Rule

PS3 (generic ACMG/AMP 2015, PMID:25741868): Well-established functional studies show a deleterious effect (Strong). Generic fallback applies because no ClinGen CSPEC is available for POLE and the local custom POLE framework (vcep_path_250_323) customizes only PM1/PS4/PP3/BP4, not functional criteria.

Evidence considered

• ACMG/AMP 2015 (PMID 25741868) defines PS3 as 'well-established functional studies show a deleterious effect' and specifies that assay validity depends on how closely the assay reflects the biological environment, validation/reproducibility data, and that mRNA-level assays (direct RNA/cDNA analysis, in vitro minigene splicing assays) are the informative approaches for variants at splice junctions and intronic regions.
• ClinVar record 2575150 / SCV004024405 was consulted: the single submission classifies the variant as Uncertain significance (criteria provided, single submitter) and its only citation is the ACMG/AMP 2015 framework paper; no functional assay is cited.
• COSMIC search returned no result for POLE c.331-30G>A, providing no somatic/functional context (absence of a record is not functional evidence).

Still missing

• No validated functional assay evidence for c.331-30G>A (no mRNA-level assays such as patient RNA RT-PCR/cDNA analysis or in vitro minigene splicing assays; no protein-level polymerase/exonuclease activity assays) identified in the case bundle or retrieved literature.
• ClinVar submission SCV004024405 (Uncertain significance, single submitter) cites only PMID 25741868 (ACMG/AMP framework); no variant-specific functional study is referenced.

Sources

Reasoning

PS4 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

PM3 (generic ACMG/AMP 2015, Richards et al. PMID 25741868) requires that the variant be detected in trans with a known pathogenic variant in the context of a recessive disorder, observed in an affected proband. No ClinGen CSPEC exists for POLE, and the local custom POLE framework (León-Castillo et al. 2020) specifies rules only for PM1/PS4/PP3/BP4, so PM3 falls back to the generic rule. The case bundle contains no proband or family genotype data for NM_006231.4:c.331-30G>A: there is no second POLE variant reported in any affected individual, no cis/trans (phase) determination, and no affected proband with a recessive POLE phenotype. The only clinical observation is a single-submitter ClinVar Uncertain significance record (SCV004024405, condition 'not specified'), which carries no allelic or phase information; gnomAD v4.1 shows a rare heterozygous population observation (AF 3.37e-05, 0 homozygotes) that is not proband evidence. SpliceAI max delta 0.06 and the deep-intronic location do not substitute for the required allelic observation. Because the decisive evidence (trans configuration with a pathogenic variant) is entirely absent rather than affirmatively refuted, PM3 is returned not_assessed pending phase/segregation data.

Rule

Generic ACMG/AMP 2015 PM3 (Richards et al., PMID 25741868): 'For recessive disorders, detected in trans with a pathogenic variant.' Applied because the local POLE custom framework (León-Castillo et al. 2020 custom POLE framework v1) customizes only PM1/PS4/PP3/BP4 and its notes direct fallback to generic ACMG/AMP outside its explicit rules.

Evidence considered

• ACMG/AMP 2015 PM3 rule definition (Richards et al., PMID 25741868, 'Cis/trans testing' section): for two heterozygous variants in a recessive-disorder gene, demonstrating the second variant is in trans with a known pathogenic variant is moderate evidence of pathogenicity for that variant, upgradable to strong with multiple trans observations; used as the governing rule because the POLE custom framework does not customize PM3
• ClinVar VariationID 2575150 (SCV004024405, Center for Genomic Medicine, Rigshospitalet): Uncertain significance, single submitter, condition 'not specified', germline origin; reviewed and found to contain no allele/phase/trans information
• gnomAD v4.1 (12-132680076-C-T): AF 3.37335e-05 (54/1,600,784 alleles), 0 homozygotes; population observation only, provides no proband or phase evidence

Still missing

• No phase (cis/trans) testing data: no proband or parental genotypes establishing that c.331-30G>A occurs in trans with a known pathogenic POLE variant
• No second pathogenic POLE variant reported in any affected proband (no compound-heterozygote observation)
• Recessive disease context unconfirmed: the only clinical record (ClinVar SCV004024405) is a single-submitter VUS with condition 'not specified'; no affected proband with a recessive POLE phenotype (e.g., biallelic FILS syndrome) is documented
• No literature or database report of this variant observed in trans with a pathogenic variant

Sources

Reasoning

No assumed-de-novo evidence exists for NM_006231.4:c.331-30G>A. There is no observation of the variant arising de novo with or without confirmed parental identity: no parental samples, no family-history statement consistent with de novo inheritance, and no proband phenotype matching a POLE-associated disease presentation are recorded anywhere in the bundle (ClinVar submission SCV004024405 has condition 'not specified' and no criteria; the only publication, PMID:25741868, is the ACMG/AMP 2015 methodology paper with no variant-specific data). Governing rule: generic ACMG/AMP 2015 fallback (PM6 = assumed de novo without confirmation of paternity and maternity, moderate), as the local POLE framework does not customize PM6. Insufficient evidence -> not_assessed.

Rule

Generic ACMG/AMP 2015 (Richards et al. 2015, PMID:25741868) via generic_acmg_combination_rules fallback; PM6 = 'Assumed de novo, but without confirmation of paternity and maternity' at moderate. Not customized by the local Leon-Castillo 2020 POLE framework.

Still missing

• No assumed de novo observation: no parental testing results at all (neither confirmed nor unconfirmed parentage) for this variant.
• No proband phenotype or family-history data to support a de novo inheritance pattern for a dominant disorder.
• Actionable follow-up: obtain parental testing results and identity-testing status from the submitting laboratory (SCV004024405).

Sources

Reasoning

No segregation data exists for NM_006231.4:c.331-30G>A. No affected family members have been genotyped for this variant, no meioses/segregation events are reported, and no family pedigree data are present in the case bundle. The ClinVar submission (SCV004024405) provides no familial information, and the only retrieved publication (PMID:25741868) is the ACMG/AMP 2015 guideline with no variant-specific data. gnomAD allele frequencies (v2.1 AF 1.20e-05, 3/250812; v4.1 AF 3.37e-05, 54/1600784; 0 homozygotes in both) address population-frequency criteria (BA1/BS1/PM2/BS2), not PP1 co-segregation, and were not used here. Governing rule: generic ACMG/AMP 2015 fallback (PP1 = co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease, supporting), as the local POLE framework does not customize PP1. Insufficient evidence -> not_assessed.

Rule

Generic ACMG/AMP 2015 (Richards et al. 2015, PMID:25741868) via generic_acmg_combination_rules fallback; PP1 = 'Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease' at supporting. Not customized by the local Leon-Castillo 2020 POLE framework.

Still missing

• No segregation analysis: no affected relatives genotyped, no meioses counted for this variant.
• No family pedigree data in the bundle; POLE germline disease context is literature-only (no official VCEP/CSPEC).
• Actionable follow-up: obtain family segregation data (genotyping of affected and unaffected relatives) from the submitting laboratory or referring clinician.

Sources

Reasoning

PP4 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

PP5 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

BS3 requires well-established functional studies showing no damaging effect on protein function or splicing. For this intronic POLE variant the only splice-related datum is SpliceAI (max delta score 0.06; DS_AG=0.06, DS_AL=0.02, DS_DG=0.02, DS_DL=0.0), which is an in silico prediction and explicitly not a well-established functional study; per ACMG/AMP 2015 the appropriate BS3-class functional evidence for an intronic/splice-region variant would be mRNA-level assays (patient RNA RT-PCR/cDNA analysis, minigene splicing assays), none of which are present in the case bundle. The ACMG/AMP 2015 paper (PMID:25741868) was reviewed for the rule definition only and contains no data on this variant. Because the required type of evidence is entirely absent, BS3 cannot be evaluated; the low SpliceAI score is not usable as functional evidence and is left to the in-silico group (BP4 pathway per the generic fallback). Returned not_assessed.

Rule

BS3 (generic ACMG/AMP 2015, PMID:25741868): Well-established functional studies show no damaging effect on protein function or splicing (Strong, benign). Generic fallback applies for the same framework reasons as PS3.

Evidence considered

• ACMG/AMP 2015 (PMID 25741868) defines BS3 as 'well-established functional studies show no damaging effect on protein function or splicing' and identifies direct analysis of RNA and/or cDNA derivatives and in vitro minigene splicing assays as the informative functional approaches for splice-junction/intronic variants.
• SpliceAI lookup (source key 'spliceai') returned max delta score 0.06 for NM_006231.4:c.331-30G>A, below the generic in-silico BP4 threshold (<0.1), but this is an in silico prediction, not a functional assay, and is insufficient to satisfy BS3.
• ClinVar record 2575150 / SCV004024405 was consulted: VUS submission with no functional study cited; absence of functional evidence does not support BS3.

Still missing

• No well-established functional studies demonstrating absence of a damaging effect on splicing or protein function for c.331-30G>A.
• Only in silico splice prediction (SpliceAI max delta 0.06) is available; a functional splicing assay (e.g., patient RNA RT-PCR/cDNA analysis or minigene assay) would be required before BS3 could be considered.

Sources

Reasoning

No non-segregation evidence exists for NM_006231.4:c.331-30G>A. BS4 requires documented lack of segregation in affected family members (affected individuals tested and found not to carry the variant); no family studies of any kind exist for this variant in the case bundle. Population allele frequencies (gnomAD v2.1 AF 1.20e-05, grpmax FAF 7.03e-06; v4.1 AF 3.37e-05, grpmax FAF 3.62e-05, 0 homozygotes) are evidence for population-frequency criteria (BA1/BS1/BS2/PM2, handled by other groups), not for BS4, and were therefore not applied here. Governing rule: generic ACMG/AMP 2015 fallback (BS4 = lack of segregation in affected family members, strong benign), as the local POLE framework does not customize BS4. Insufficient evidence -> not_assessed.

Rule

Generic ACMG/AMP 2015 (Richards et al. 2015, PMID:25741868) via generic_acmg_combination_rules fallback; BS4 = 'Lack of segregation in affected family members' at strong (benign). Not customized by the local Leon-Castillo 2020 POLE framework.

Still missing

• No family segregation data: no affected family members genotyped to demonstrate absence of the variant.
• No pedigree or familial testing documentation anywhere in the case bundle.
• Actionable follow-up: obtain testing results of affected relatives from the submitting laboratory (SCV004024405) to evaluate non-segregation.

Sources

Reasoning

BP2 (generic ACMG/AMP 2015, Richards et al. PMID 25741868) requires either (a) the variant observed in trans with a pathogenic variant in a fully dominant disorder, or (b) the variant observed in cis with a pathogenic variant in any inheritance pattern. No ClinGen CSPEC exists for POLE and the local custom POLE framework (León-Castillo et al. 2020) does not customize BP2, so the generic rule governs. The case bundle contains no cis/trans phase determination for NM_006231.4:c.331-30G>A with any pathogenic POLE variant: no parental or family testing results, no co-occurrence observations in cis or in trans, and no report of this intronic variant alongside a known pathogenic variant. The ClinVar record is a single-submitter VUS with condition 'not specified' and no allelic context; gnomAD population frequencies do not address phase. Because the required allelic observations are absent rather than affirmatively refuted, BP2 is returned not_assessed pending phase/co-occurrence data.

Rule

Generic ACMG/AMP 2015 BP2 (Richards et al., PMID 25741868): 'Observed in trans with a pathogenic variant for a fully dominant disease/disorder; OR observed in cis with a pathogenic variant in any inheritance pattern.' Applied because the local POLE custom framework (León-Castillo et al. 2020 custom POLE framework v1) customizes only PM1/PS4/PP3/BP4 and its notes direct fallback to generic ACMG/AMP outside its explicit rules.

Evidence considered

• ACMG/AMP 2015 BP2 rule definition (Richards et al., PMID 25741868, 'Cis/trans testing' section): finding a second variant in cis with a pathogenic variant is supporting (not definitive) evidence for a benign role, and in dominant disorders detection of a variant in trans with a pathogenic variant is supporting evidence for a benign impact; used as the governing rule because the POLE custom framework does not customize BP2
• ClinVar VariationID 2575150 (SCV004024405): Uncertain significance, single submitter, condition 'not specified'; reviewed and found to contain no cis/trans or co-occurrence data
• gnomAD v4.1 (12-132680076-C-T): AF 3.37335e-05, 0 homozygotes; population frequencies cannot establish phase with a pathogenic variant

Still missing

• No cis/trans phase data: no parental or family testing demonstrating whether c.331-30G>A occurs in cis or in trans with any pathogenic POLE variant
• No observation of the variant in trans with a pathogenic variant in a dominant disorder context
• No observation of the variant in cis with a pathogenic variant in any inheritance pattern
• ClinVar record (VCV002575150) provides no co-occurrence or phase information

Sources

Reasoning

BP5 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys

Reasoning

BP6 could not be assessed because its agent did not produce valid output.

Still missing

• attempt 1: evidence_refs must contain only source_registry keys
• attempt 2: evidence_refs must contain only source_registry keys
• attempt 3: evidence_refs must contain only source_registry keys