POLD1 c.49C>T, p.(Arg17Trp)

Reasoning

PM2 at supporting strength applies when a variant is absent from, or present at a filtering allele frequency (FAF) <= 0.0001 in, population databases (ClinGen SVI recommendation to downgrade PM2 to Supporting; PMID:25741868). No POLD1 VCEP/CSPEC exists, so the generic all-comers gnomAD datasets are the default source. NM_002691.4:c.49C>T (NP_002682.2:p.Arg17Trp) sits far below the threshold on the recommended metric: gnomAD v2.1 grpmax FAF 4.165e-05 (total AF 3.443e-05, 9/261,400) and gnomAD v4.1 grpmax FAF 5.278e-05 (total AF 1.560e-05, 25/1,602,884). The non-cancer subsets, which are the more appropriate comparators for a cancer-predisposition gene, corroborate the same conclusion (v2.1 non-cancer exomes grpmax FAF 4.186e-05, AF 3.652e-05, 8/219,044; v3.1 non-cancer genomes grpmax FAF 4.737e-05, AF 4.055e-05, 6/147,974). The variant is additionally reported absent from gnomAD-Canada v1.0 (HostSeq genomes). One threshold-artifact caveat applies: the highest ancestry-specific frequencies marginally exceed 0.0001 (Admixed American 1.208e-04 in v2.1 non-cancer exomes; African/African American 1.068e-04 in v4.1), so a strict population-maximum reading could push the variant just above the supporting line in those ancestries; however, the recommended grpmax FAF metric (which adjusts for cohort size and genetic ancestry) is below 0.0001 in every dataset, so PM2 is met at supporting strength. PM2 is applied at supporting strength, not as a standalone pathogenic criterion.

Rule

PM2 (supporting): absent or filtering allele frequency <= 0.0001 in population databases (ClinGen SVI 2020, PMID:25741868)

Evidence considered

• gnomAD v2.1 all-comers: total AF 3.443e-05 (9/261,400), grpmax FAF 4.165e-05 - below the 0.0001 PM2 supporting threshold.
• gnomAD v4.1 all-comers: total AF 1.560e-05 (25/1,602,884), grpmax FAF 5.278e-05 - below the 0.0001 PM2 supporting threshold.
• gnomAD v2.1 non-cancer exomes AF 3.652e-05 (8/219,044), grpmax FAF 4.186e-05; gnomAD v3.1 non-cancer genomes AF 4.055e-05 (6/147,974), grpmax FAF 4.737e-05 - both non-cancer subsets also below threshold.
• gnomAD-Canada v1.0 (HostSeq genomes): variant reported not available/absent.
• Threshold-artifact note: highest ancestry-specific frequencies AMR 1.208e-04 (4/33,126) and AFR 1.068e-04 (8/74,932) marginally exceed 0.0001; grpmax FAF (the recommended metric) remains below.
• PM2 applied at supporting strength per the ClinGen SVI recommendation to downgrade PM2, not as a standalone pathogenic criterion.
• ClinVar final classifications were not used to infer this criterion.

Still missing

• Ancestry-specific frequencies marginally exceed 0.0001 (AMR 1.208e-04, AFR 1.068e-04) while grpmax FAF remains below threshold; the recommended grpmax FAF metric was used, but a strict population-maximum interpretation would push PM2 just above the supporting line.
• No POLD1 VCEP/CSPEC framework exists, so PM2 was applied with the generic supporting-strength threshold rather than a gene-specific, penetrance-adjusted cutoff.

Sources

Reasoning

PP2 (Richards et al. 2015, PMID:25741868) has two requirements for a missense variant: the gene must show a low rate of benign missense variation, and missense variants must be a common mechanism of disease. Both are supported for POLD1 here. POLD1 is an established Mendelian disease gene (MIM 174761; colorectal cancer susceptibility 10 / polymerase proofreading-associated polyposis) in which germline missense variants are a recognised and characteristic pathogenic mechanism rather than an incidental finding: the recurrent germline pathogenic/likely pathogenic alleles reported in PPAP are missense substitutions within the exonuclease proofreading domain (e.g. p.Asp316Asn, p.Asp402Asn, p.Ile494Ser), so disease is not attributable exclusively to truncating alleles. The low-benign-missense-rate arm is supported indirectly by this well-documented intolerance to functional missense change at conserved exonuclease residues and by the absence of a common benign POLD1 missense allele at this codon in population data (gnomAD v2.1 total AF 3.44e-05; no homozygotes); no quantitative gene-constraint metric (e.g. gnomAD missense Z or observed/expected ratio) was available in the retrieved evidence, which is recorded below as a gap. PP2 is a gene-level criterion and is therefore not negated by this variant's position outside the exonuclease domain. PP2 is applied at its default supporting strength; it does not by itself establish pathogenicity.

Rule

PP2 applies to a missense variant in a gene with a low rate of benign missense variation and in which missense variants are a common mechanism of disease

Evidence considered

• POLD1 is the catalytic p125 subunit of DNA polymerase delta, with germline pathogenic variants causing polymerase proofreading-associated polyposis (colorectal, endometrial and possibly brain cancer) (OncoKB gene-level background; MIM 174761).
• Missense variants are an established pathogenic mechanism in POLD1: the germline likely-pathogenic alleles characterised in PPAP are missense substitutions in the exonuclease domain, c.1204G>A p.(Asp402Asn) and c.1481T>G p.(Ile494Ser) (PMID:41487566); p.Asp316Asn is reported in a digenic POLD1/PMS2 teenage-onset colorectal cancer syndrome (PMID:36291559).
• Population data: gnomAD v2.1 total AF 3.443e-05 (9/261400 alleles), no homozygotes, highest subpopulation AF 0.00012 (Admixed American) - no common benign POLD1 missense allele at codon 17.
• ClinVar VCV000408109 records no conflicting benign evidence that would argue against missense pathogenicity in this gene (single Likely benign submitter, no expert panel).
• Criterion definition taken from PMID:25741868 (ACMG/AMP 2015 standards and guidelines); no gene-specific ClinGen specification exists for POLD1, so generic ACMG/AMP applies.

Still missing

• No quantitative POLD1 gene-constraint metric (gnomAD missense Z score / observed-expected ratio or equivalent) was available in the retrieved evidence; the low-benign-missense-rate arm therefore rests on the established gene-disease missense mechanism and population data rather than a published constraint value.
• This variant lies outside POLD1's exonuclease proofreading domain, so the gene-level PP2 assertion provides only baseline, non-positional support; it must not be read as domain-level evidence for pathogenicity.

Sources

Reasoning

Scope check first: NM_002691.4:c.49C>T normalizes to NP_002682.2:p.(Arg17Trp), a missense substitution, so BP4 is adjudicated on the missense (REVEL) path only. The SpliceAI result (maximum delta 0.069) describes splice effect, not tolerance of the amino-acid substitution, and is not substituted for the REVEL assessment; a clean splice prediction cannot support BP4 for a missense variant. No POLD1-specific ClinGen CSPEC or VCEP specification was retrieved, so the generic ACMG/ClinGen-SVI calibration governs and no gene-specific BP4 rule overrides it. The local REVEL v1.3 lookup returns 0.095, which is at or below the BP4 supporting threshold of <=0.29 (Pejaver et al. 2022, PMID:36413997); BP4 is therefore met at supporting strength. BayesDel (-0.405968) was retrieved but has no established ACMG-strength calibration and contributed no weight.

Rule

Missense variant: apply the REVEL path only; BP4 supporting requires REVEL <=0.29 (ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997).

Evidence considered

• The variant NM_002691.4:c.49C>T normalizes to NP_002682.2:p.(Arg17Trp), a missense substitution; REVEL is the sole applicable BP4 path.
• The SpliceAI maximum delta of 0.069 was not used for BP4: a missense variant's splice prediction does not address whether the amino-acid substitution is tolerated.
• Local REVEL v1.3 lookup result: 0.095, at or below the ClinGen SVI BP4 supporting threshold of <=0.29 (Pejaver et al. 2022, PMID:36413997), so BP4 is met at supporting strength.
• No POLD1-specific ClinGen CSPEC or VCEP specification was retrieved for this case, so the generic ACMG/ClinGen-SVI BP4 calibration applies.
• BayesDel (-0.405968) was retrieved from the local BayesDel lookup but has no established ACMG-strength calibration, so it contributed no BP4 weight.

Sources

Reasoning

PS1 (Richards et al. 2015, PMID:25741868) requires a different nucleotide change that produces the same amino-acid change as a variant already established as pathogenic. The variant under assessment is NM_002691.4:c.49C>T, NP_002682.2:p.(Arg17Trp). The only ClinVar record for POLD1 codon 17 / Arg17Trp is the variant under assessment itself (VCV000408109), whose submissions are conflicting and single-submitter only (4 clinical laboratories report Uncertain significance, 1 reports Likely benign; 1 star, no expert-panel submission, no 3-star review). No second nucleotide change producing p.Arg17Trp, and no independently established pathogenic p.Arg17Trp allele, exists in the retrieved evidence, and the dedicated same-residue comparator harvest returned zero candidates. Because no previously established pathogenic variant with this amino-acid change is available, PS1's prerequisite is unmet. Adjudicated under the generic ACMG/AMP 2015 framework; no ClinGen CSPEC, VCEP specification, or local gene framework is available for POLD1.

Rule

PS1 requires a different nucleotide change producing the same amino-acid change as an established pathogenic variant

Evidence considered

• ClinVar VCV000408109 (NM_002691.4(POLD1):c.49C>T, p.Arg17Trp): 5 submissions, 4x Uncertain significance and 1x Likely benign (Ambry Genetics), review status 'criteria provided, conflicting classifications', 1 star, 0 expert-panel submissions; last updated 2026-04-13.
• PM5/same-residue comparator harvest (output/pm5_candidates.json): 0 candidates seen, 0 same-residue candidates, 0 expert-panel pathogenic/likely-pathogenic candidates at POLD1 residue 17.
• Criterion definition taken from PMID:25741868 (ACMG/AMP 2015 standards and guidelines).

Still missing

• No established pathogenic p.Arg17Trp allele (or an alternate nucleotide change encoding p.Arg17Trp) was found in ClinVar or the retrieved literature; a systematic search of non-ClinVar locus-specific databases was not performed.

Sources

Reasoning

Governing framework: no ClinGen VCEP/CSPEC exists for POLD1 (the prefetch specification lookup returned cspec.found=false for POLD1, and final_classification_framework.json records source='generic_acmg_fallback', framework_complete=false, precedence_rank=3). The only gene-specific custom framework held locally is the Leon-Castillo 2020 POLE framework, which is scoped to POLE missense variants in endometrial carcinoma and does not cover POLD1. Generic ACMG/AMP 2015 therefore governs this criterion. Generic PS4 requires that the prevalence of the variant in affected individuals be significantly increased compared with controls, expressed as an odds ratio or relative risk with the operator '>' against a threshold of 5.0, i.e. OR/RR > 5.0 with a confidence interval excluding 1.0 (Richards et al. 2015, PMID:25741868, Table 3). Direction check on this criterion's own operator: PS4 strengthens as the value goes UP, so the met condition is value > (>=) threshold, and the inequality to test must be written as 'OR/RR > 5.0? yes' with a real OR/RR substituted on the left. No case-control or case-enrichment statistic of any kind exists for POLD1 c.49C>T, so there is no value that can be substituted into that inequality and the comparison cannot be satisfied. All available quantitative observations are control-side or somatic, not case enrichment: gnomAD v2.1 total AF 3.443e-05 (9/261,400 alleles; grpmax FAF 4.165e-05) and gnomAD v4.1 total AF 1.560e-05 (25/1,602,884 alleles; grpmax FAF 5.278e-05) are population frequencies, not affected-vs-control enrichment; the 5 ClinVar submissions are laboratory classifications (4 x Uncertain significance, 1 x Likely benign), not case-frequency counts; the COSMIC entry (COSV70954597, 3 somatic occurrences) is somatic tumour data and is not germline case enrichment; OncoKB (Unknown Oncogenic Effect, gene-level only) and Cancer Hotspots (no significant hotspot, variant not listed) return no variant-level reviewed evidence. The one case-flavoured laboratory claim on the record - a GeneDx submission sentence stating the variant was 'observed in an individual with breast cancer (pmid: 30093976)' - is ungrounded: PMID:30093976 names POLD1 only inside its 49-gene panel list and never names the variant or p.Arg17Trp anywhere (verified directly in the full text), so it cannot be used even as a single case observation, let alone as enrichment. PS4 is therefore not met at any strength.

Rule

Generic ACMG/AMP 2015 PS4 (Richards et al. 2015, PMID:25741868): prevalence of the variant in affected individuals significantly increased versus controls; odds ratio or relative risk > 5.0 with confidence interval excluding 1.0 (higher value = stronger).

Evidence considered

• Generic PS4 definition (Richards et al. 2015, PMID:25741868, Table 3): prevalence of the variant in affected individuals significantly increased versus controls, odds ratio or relative risk > 5.0 with a confidence interval excluding 1.0. PS4 strengthens as the value increases, so the met condition is value > threshold.
• The extracted full text of PMID:25741868 available for this case does not reproduce the criteria tables, so the PS4 numeric threshold is taken from the named parent publication (Richards et al. 2015, PMID:25741868); the PS4-related narrative in that text (population data for case-control comparisons are most useful when populations are well-phenotyped and the disease is early-onset) confirms the criterion is a case-versus-control enrichment criterion.
• Explicit comparison as literally written for PS4 (operator '>', higher value = stronger): 'OR/RR > 5.0? ' cannot be evaluated because no OR/RR value exists in the case evidence - the left side is undefined, so the inequality is not satisfied -> not met.
• gnomAD v2.1: total AF 3.443e-05 (9/261,400 alleles, 0 homozygotes); gnomAD v4.1: total AF 1.560e-05 (25/1,602,884 alleles, 0 homozygotes); both are control/population frequencies only and provide no case enrichment.
• ClinVar VCV000408109: 5 submissions (4 x Uncertain significance, 1 x Likely benign), 0 expert-panel submissions, aggregate review status 'criteria provided, conflicting classifications' (1 star) - these are classifications, not case-frequency data.
• COSMIC COSV70954597 reports 3 somatic occurrences (somatic/tumour data, no germline case count); Cancer Hotspots reports no significant hotspot and the variant is not listed; OncoKB reports Unknown Oncogenic Effect with gene-level content only.
• A GeneDx ClinVar submission asserts the variant was observed in an individual with breast cancer citing PMID:30093976, but PMID:30093976 does not name the variant (variant_mentioned=false in the pre-computed extraction and confirmed by direct full-text search), so this case claim is ungrounded and was not used.
• No case-control or enrichment statistic for this variant was found in the assembled evidence (ClinVar, gnomAD v2.1/v4.1, gnomAD-Canada query, COSMIC, Cancer Hotspots, OncoKB, and the four fetched publications).

Still missing

• No case-control or case-enrichment study (odds ratio / relative risk with confidence interval excluding 1.0) specific to POLD1 c.49C>T was identified in any source.
• No well-phenotyped case cohort is available: the case evidence contains only laboratory classifications, and the single 'case' claim on the ClinVar record (breast cancer, cited to PMID:30093976) is not grounded in that paper.

Sources

Reasoning

PM1 (Richards et al. 2015, PMID:25741868) applies when a missense variant lies in a mutational hotspot or in a well-established functional domain without benign variation. No gene-specific ClinGen specification or VCEP framework exists for POLD1, and this case carries no authoritative POLD1 domain table (the domain_tables key is empty), so domain boundaries were taken from the germline disease literature: germline pathogenic POLD1 missense variants causing polymerase proofreading-associated polyposis (PPAP) cluster in the N-terminal 3'->5' exonuclease (proofreading) domain of the catalytic p125 subunit, the domain reported to harbour the recurrent pathogenic alleles (e.g. p.Asp316Asn, p.Asp402Asn, p.Ile494Ser, all within the exonuclease domain). p.Arg17Trp alters residue 17 of the 1107-residue protein, in the N-terminal region well outside the exonuclease domain, and residue 17 is not a reported mutational hotspot: the Cancer Hotspots query for POLD1 R17 returned no hotspot row, and the COSMIC record for this variant (COSV70954597) is a low-count recurrent observation (n = 3) rather than a hotspot. Position alone therefore does not satisfy PM1, and no benign-variation-free critical domain at residue 17 is documented.

Rule

PM1 requires location in a mutational hotspot or a well-established functional domain without benign variation

Evidence considered

• POLD1 gene-level mechanism: the p125 catalytic subunit carries both polymerase and 3'->5' exonuclease activity, and germline heterozygous pathogenic variants in the exonuclease (proofreading) domain cause polyposis with predisposition to colorectal, endometrial and possibly brain cancers; somatic POLD1 variants are extremely rare (OncoKB gene-level background, PMIDs 23263490, 23528559, 24501277, 26133394, 26493165, 35398880).
• Germline POLD1 likely-pathogenic missense alleles reported within the exonuclease domain: c.1204G>A p.(Asp402Asn) and c.1481T>G p.(Ile494Ser) in POLD1-related PPAP (PMID:41487566), and p.Asp316Asn (PMID:36291559); all lie far C-terminal to residue 17.
• Mutational-hotspot query for POLD1 R17 returned no hotspot row (Cancer Hotspots; https://www.cancerhotspots.org/).
• COSMIC lists the variant (COSV70954597) with a somatic observation count of only 3, consistent with a rare recurrent event rather than a hotspot.
• No ClinGen CSPEC/VCEP or local gene framework is available for POLD1; adjudicated under the generic ACMG/AMP 2015 framework (PMID:25741868).

Still missing

• No authoritative POLD1 critical-functional-domain table was supplied with this case (vcep_materials.json domain_tables is empty and no CSPEC/VCEP specification exists for POLD1), so the exonuclease-domain boundary was taken from the germline PPAP literature rather than a governing VCEP domain table.
• Residue-level benign-variation data (e.g. gnomAD per-residue missense counts for the N-terminal region of POLD1) were not retrieved; the conclusion does not depend on them because residue 17 lies outside the disease-critical exonuclease domain.

Sources

Reasoning

PM5 (Richards et al. 2015, PMID:25741868) requires a missense change at an amino-acid residue where a different missense change has previously been determined to be pathogenic. The variant alters POLD1 Arg17 to Trp. ClinVar contains only the variant under assessment at POLD1 codon 17 (VCV000408109, p.Arg17Trp), whose classifications are conflicting and not established pathogenic (4x Uncertain significance, 1x Likely benign; no expert panel, 1 star), and the dedicated same-residue comparator harvest returned zero candidates at residue 17 and zero expert-panel or majority pathogenic/likely-pathogenic comparators anywhere on that residue. No other missense change at Arg17 has been determined to be pathogenic in the retrieved evidence, and the retrieved literature does not report any pathogenic variant at codon 17. PM5's prerequisite (a pathogenic comparator at the same residue) is therefore unmet.

Rule

PM5 requires a different missense change at the same residue previously determined to be pathogenic

Evidence considered

• Same-residue comparator harvest (output/pm5_candidates.json): total_candidates_seen 0, same_residue_candidates 0, expert_panel_plp_candidates 0, non_expert_majority_plp_candidates 0; classic same-residue PM5 semantics could not be confirmed for this residue.
• ClinVar VCV000408109 is the only POLD1 variant at codon 17 in the retrieved record set; its classifications are Uncertain significance (4 laboratories) and Likely benign (1 laboratory), i.e. not a pathogenic comparator.
• Criterion definition taken from PMID:25741868 (ACMG/AMP 2015 standards and guidelines).

Still missing

• No pathogenic or likely pathogenic missense variant at POLD1 Arg17 was found; a locus-specific database search beyond ClinVar and the retrieved literature was not performed.

Sources

Reasoning

Scope check first: NM_002691.4:c.49C>T normalizes to NP_002682.2:p.(Arg17Trp), a missense substitution, so PP3/BP4 are adjudicated on the missense (REVEL) path only. The SpliceAI score is not used for this criterion because a splice prediction says nothing about whether an amino-acid substitution is tolerated. No POLD1-specific ClinGen CSPEC or VCEP specification was retrieved (cspec not found; framework mode generic_acmg), so the supplied generic ClinGen SVI REVEL calibration governs. The local REVEL v1.3 lookup returns 0.095 for this variant, which is below the PP3 supporting threshold of >=0.644 (Pejaver et al. 2022, PMID:36413997); the moderate (>=0.773) and strong (>=0.932) bands are likewise not reached. PP3 is therefore not met. BayesDel (-0.405968) was retrieved but has no established ACMG-strength calibration and contributed no weight.

Rule

Missense variant: apply the REVEL path only; PP3 supporting requires REVEL >=0.644 (ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997).

Evidence considered

• The variant NM_002691.4:c.49C>T normalizes to NP_002682.2:p.(Arg17Trp), a missense substitution; the consequence-matched computational path is therefore REVEL, and the SpliceAI score is not used for PP3.
• No POLD1-specific ClinGen CSPEC or VCEP specification was retrieved for this case, so the generic ACMG/ClinGen-SVI PP3/BP4 in-silico calibration was applied.
• Local REVEL v1.3 lookup result: 0.095. The ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997) sets PP3 supporting at >=0.644, moderate at >=0.773 and strong at >=0.932; 0.095 is below all three, so PP3 is not met.
• BayesDel (-0.405968) was retrieved from the local BayesDel lookup but has no established ACMG-strength calibration, so it contributed no PP3 weight.

Sources

Reasoning

Governing framework: generic ACMG/AMP 2015, because no POLD1 VCEP/CSPEC exists (cspec.found=false; generic_acmg_fallback, framework_complete=false) and the only local gene-specific framework (Leon-Castillo 2020 POLE) is scoped to POLE and does not cover POLD1. Generic PP4 requires that the patient's phenotype or family history be highly specific for a disease with a single genetic etiology. No proband phenotype and no family history are documented anywhere in the case evidence. The ClinVar record for VCV000408109 carries only non-specific condition labels attached to laboratory submissions ('Colorectal cancer, susceptibility to, 10', 'Hereditary cancer-predisposing syndrome', 'not provided'); there is no curated genotype-specific phenotype, no clinical narrative, no adenoma/polyposis description, no age of onset, no tumour phenotype, and no family history or segregation context in any source (ClinVar, gnomAD, COSMIC, OncoKB, Cancer Hotspots, or the four fetched publications). The only phenotype-flavoured claim in the record - the GeneDx submission sentence 'observed in an individual with breast cancer (pmid: 30093976)' - is doubly unusable: it is ungrounded (PMID:30093976 never names the variant), and even taken at face value the phenotype is not highly specific, since breast cancer is not a specific presentation of POLD1-associated disease (polymerase-proofreading-associated colorectal polyposis / colorectal cancer spectrum). With no documented proband phenotype there is nothing that can be judged highly specific for a single-gene disorder, so the criterion's precondition is not satisfied and PP4 is not met.

Rule

Generic ACMG/AMP 2015 PP4 (Richards et al. 2015, PMID:25741868): patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• Generic PP4 definition (Richards et al. 2015, PMID:25741868): patient's phenotype or family history is highly specific for a disease with a single genetic etiology.
• ClinVar VCV000408109 condition labels are submission-level and non-specific (Colorectal cancer, susceptibility to, 10; Hereditary cancer-predisposing syndrome; not provided) and include several 'not provided'/'Not Provided' entries; no proband-level phenotype is captured.
• No clinical narrative, family history, age of onset, polyposis/adenoma description, or segregation data are present in the case evidence (ClinVar, gnomAD, COSMIC, Cancer Hotspots, OncoKB, or the four fetched publications).
• The GeneDx submission's breast-cancer claim citing PMID:30093976 is ungrounded (the paper never names the variant) and, independently, breast cancer is not a highly specific presentation of POLD1-associated disease.
• Explicit comparison as literally written for PP4 (a non-numeric presence/absence condition): 'phenotype or family history highly specific for a disease with a single genetic etiology? ' - no, because no phenotype/family history is documented.

Still missing

• No proband phenotype, family history, or clinical narrative is documented for this variant, so phenotype specificity could not be actively curated (the verdict rests on absence of documented phenotype).
• The single phenotype claim on the ClinVar record (breast cancer, cited to PMID:30093976) is not grounded in that paper and, as a phenotype, is not specific for POLD1.

Sources

Reasoning

Governing framework: generic ACMG/AMP 2015, because no POLD1 VCEP/CSPEC exists (cspec.found=false; generic_acmg_fallback) and the local custom framework (Leon-Castillo 2020 POLE) does not cover POLD1. Under the governing rule applied to this case, PP5 may be triggered ONLY by an exact-variant ClinVar expert-panel (3-star) classification: a Pathogenic or Likely pathogenic expert-panel assertion supports PP5, while ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger it. The exact-variant record is VCV000408109 (ClinVar ID 408109, NM_002691.4(POLD1):c.49C>T p.Arg17Trp). That record has 5 submissions from 5 submitters and 0 expert-panel submissions (expert_panels is empty; every SCV has is_expert_panel=false; expert_panel_submissions count = 0). Its aggregate review status is 'criteria provided, conflicting classifications' with 1 star, and the submissions are ordinary clinical-laboratory assertions: 4 x Uncertain significance (PreventionGenetics, Quest Diagnostics, GeneDx, Labcorp/Invitae) and 1 x Likely benign (Ambry Genetics). None is a Pathogenic or Likely pathogenic expert-panel classification. There is therefore no qualifying exact-variant expert-panel classification to leverage, and the ordinary submissions and the aggregate ClinVar label must not be used. PP5 is not met.

Rule

Generic ACMG/AMP 2015 PP5 (Richards et al. 2015, PMID:25741868) as applied here: requires an exact-variant ClinVar expert-panel (3-star) Pathogenic/Likely pathogenic classification; ordinary/non-expert submissions and aggregate labels do not trigger it.

Evidence considered

• Governing rule for this case: PP5 applies only for an exact-variant ClinVar expert-panel classification; Pathogenic/Likely pathogenic supports PP5; ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger it.
• ClinVar VCV000408109 (ClinVar ID 408109, NM_002691.4(POLD1):c.49C>T p.Arg17Trp): expert_panels field is empty; all 5 SCVs (SCV000806545 PreventionGenetics, SCV001134663 Quest Diagnostics, SCV005470083 Ambry Genetics, SCV002538918 GeneDx, SCV000547662 Labcorp/Invitae) have is_expert_panel=false; audit counts show expert_panel_submissions=0.
• Aggregate record status is 'criteria provided, conflicting classifications' (1 star) with 4 x Uncertain significance and 1 x Likely benign - none is a Pathogenic or Likely pathogenic expert-panel assertion.
• No qualifying exact-variant expert-panel classification exists, so PP5 is not triggered; the single-submitter laboratory assertions were not used.

Still missing

• No exact-variant ClinVar expert-panel (3-star) Pathogenic/Likely pathogenic classification exists for POLD1 c.49C>T; PP5 cannot be triggered on that basis.

Sources

Reasoning

No ClinGen VCEP specification or gene-specific CSPEC framework exists for POLD1 (cspec.found = false; final_classification_framework.found = false, source = generic_acmg_fallback, framework_mode = generic_acmg), so the generic ACMG/AMP 2015 stand-alone benign allele-frequency threshold applies: BA1 is met when the allele frequency is >= 0.05 (Richards et al. 2015, PMID:25741868). NM_002691.4:c.49C>T (NP_002682.2:p.Arg17Trp) is present at very low frequency in every queried gnomAD dataset. The all-comers totals are: gnomAD v2.1 (GRCh37, 19-50902157-C-T) total AF 3.443e-05 (9/261,400 alleles) with grpmax FAF 4.165e-05; gnomAD v4.1 (GRCh38, chr19-50398900-C-T) total AF 1.560e-05 (25/1,602,884 alleles) with grpmax FAF 5.278e-05. The highest ancestry-specific frequency observed anywhere is 1.208e-04 (Admixed American, 4/33,126) in the gnomAD v2.1 non-cancer exome subset and 1.068e-04 (African/African American, 8/74,932) in gnomAD v4.1. Even that highest population-specific value is approximately 400-fold below the 0.05 stand-alone threshold. Because no all-comers or ancestry-specific allele frequency approaches 0.05, BA1 is not met.

Rule

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

Evidence considered

• gnomAD v2.1 all-comers: total AF 3.443e-05 (AC 9 / AN 261,400), homozygotes 0; exome AF 3.478e-05 (8/230,036); genome AF 3.188e-05 (1/31,364); grpmax FAF 4.165e-05.
• gnomAD v4.1 all-comers: total AF 1.560e-05 (AC 25 / AN 1,602,884), homozygotes 0; exome AF 1.310e-05 (19/1,450,530); genome AF 3.938e-05 (6/152,354); joint grpmax FAF 5.278e-05.
• Highest ancestry-specific frequency observed: Admixed American (AMR) 1.208e-04 (4/33,126) in gnomAD v2.1 non-cancer exomes and African/African American (AFR) 1.068e-04 (8/74,932) in gnomAD v4.1; both remain ~400-fold below the 0.05 BA1 threshold.
• gnomAD v2.1 non-cancer (exomes only): AF 3.652e-05 (8/219,044), homozygotes 0, grpmax FAF 4.186e-05. gnomAD v3.1 non-cancer (genomes only): AF 4.055e-05 (6/147,974), homozygotes 0, grpmax FAF 4.737e-05.
• No POLD1-specific (VCEP/CSPEC) stand-alone benign allele-frequency threshold is published; the generic ACMG/AMP 2015 value of 0.05 was used because no framework governs this case.
• ClinVar final classifications were not used to infer this criterion.

Sources

Reasoning

No VCEP specification or gene-specific CSPEC framework exists for POLD1, so a gene- or disease-prevalence-derived strong benign threshold cannot be sourced per gene; the generic strong benign threshold of allele frequency >= 0.01 was therefore used (BS1, ACMG/AMP 2015, PMID:25741868; per-gene derivation is preferred per Whiffin et al. 2017, PMID:28518168, but no POLD1-specific cutoff is published). NM_002691.4:c.49C>T (NP_002682.2:p.Arg17Trp) is far below that threshold in every dataset: gnomAD v2.1 all-comers total AF 3.443e-05 with grpmax FAF 4.165e-05; gnomAD v4.1 all-comers total AF 1.560e-05 with grpmax FAF 5.278e-05; gnomAD v2.1 non-cancer exomes AF 3.652e-05 with grpmax FAF 4.186e-05; gnomAD v3.1 non-cancer genomes AF 4.055e-05 with grpmax FAF 4.737e-05. The highest ancestry-specific value (1.208e-04, Admixed American, v2.1 non-cancer) is still more than 80-fold below 0.01. No frequency approaches the 0.01 strong benign level, so BS1 is not met.

Rule

BS1 (strong benign): allele frequency >= 0.01 (generic SVI default; per-gene derivation preferred per Whiffin et al. 2017 PMID:28518168)

Evidence considered

• gnomAD v2.1 all-comers total AF 3.443e-05 (9/261,400), grpmax FAF 4.165e-05; gnomAD v4.1 all-comers total AF 1.560e-05 (25/1,602,884), grpmax FAF 5.278e-05.
• gnomAD v2.1 non-cancer exomes AF 3.652e-05 (8/219,044), grpmax FAF 4.186e-05; gnomAD v3.1 non-cancer genomes AF 4.055e-05 (6/147,974), grpmax FAF 4.737e-05.
• Highest ancestry-specific frequency 1.208e-04 (AMR, v2.1 non-cancer exomes) is >80-fold below the 0.01 generic BS1 threshold.
• Per-gene BS1 derivation is preferred (Whiffin et al. 2017, PMID:28518168) but no POLD1-specific derivation is available; the generic SVI default of 0.01 was applied because no VCEP/CSPEC governs this gene.
• ClinVar final classifications were not used to infer this criterion.

Still missing

• No POLD1-specific prevalence- or penetrance-derived BS1 allele-frequency threshold exists (no VCEP/CSPEC); generic ACMG thresholds were applied.

Sources

Reasoning

BS2 requires that the variant be observed in a healthy adult individual for a disorder in which full penetrance is expected at an early age - homozygous for a recessive disorder, heterozygous for a dominant disorder, or hemizygous for an X-linked disorder (ACMG/AMP 2015, PMID:25741868). Two independent preconditions fail here. First, the disorder context: POLD1 germline variants cause polymerase proofreading-associated polyposis (PPAP), an autosomal dominant colorectal/endometrial cancer predisposition syndrome that is adult-onset and incompletely penetrant; full penetrance at an early age is not expected, so heterozygous presence in an adult population database cannot be scored as BS2. Second, the genotype context: NM_002691.4:c.49C>T has a homozygote count of zero in every dataset examined (gnomAD v2.1 hom 0, gnomAD v4.1 hom 0, gnomAD v2.1 non-cancer hom 0, gnomAD v3.1 non-cancer hom 0), so no homozygous observation exists that could satisfy the recessive limb either. BS2 is therefore not met.

Rule

BS2 (strong benign): observed in a healthy adult with full penetrance expected at an early age (homozygous recessive / heterozygous dominant / hemizygous X-linked) - ACMG/AMP 2015, PMID:25741868

Evidence considered

• gnomAD v2.1: total homozygotes 0 (all 9 alleles heterozygous); gnomAD v4.1: total homozygotes 0 (all 25 alleles heterozygous); both non-cancer subsets also report homozygotes 0.
• POLD1 germline variants cause polymerase proofreading-associated polyposis (PPAP), an autosomal dominant, adult-onset, incompletely penetrant cancer predisposition syndrome; BS2's requirement of full penetrance expected at an early age is not satisfied for this disease context.
• No homozygous observation in any gnomAD dataset, so the recessive (homozygous) limb of BS2 has no supporting observation.
• ClinVar final classifications were not used to infer this criterion.

Sources

Reasoning

BP1 (Richards et al. 2015, PMID:25741868) applies only when truncating variants are known to be the primary or sole cause of the gene's disease and missense variation is not an established mechanism. POLD1 fails that premise: the prototypical germline pathogenic mechanism of POLD1-related polymerase proofreading-associated polyposis is missense change within the exonuclease proofreading domain (e.g. p.Asp402Asn, p.Ile494Ser, p.Asp316Asn), and POLD1-associated disease is specifically attributed to proofreading-deficient missense alleles in addition to loss-of-function alleles. Missense variation is therefore an established cause of POLD1 disease, and BP1's premise that only truncating variants cause disease does not hold. BP1 is not met.

Rule

BP1 applies only when truncating variants are the primary/sole known disease mechanism for the gene

Evidence considered

• POLD1 germline disease mechanism is missense-driven within the exonuclease (proofreading) domain: p.(Asp402Asn) and p.(Ile494Ser) were classified likely pathogenic in POLD1-related PPAP (PMID:41487566); p.Asp316Asn was reported in teenage-onset colorectal cancer (PMID:36291559).
• OncoKB gene-level summary for POLD1 describes germline heterozygous pathogenic variants in the exonuclease domain causing polyposis and predisposition to colorectal, endometrial and possibly brain cancers, rather than a truncating-only mechanism.
• Criterion definition taken from PMID:25741868 (ACMG/AMP 2015 standards and guidelines).

Sources

Reasoning

Governing framework: generic ACMG/AMP 2015, because no POLD1 VCEP/CSPEC exists (cspec.found=false; generic_acmg_fallback) and the local custom framework (Leon-Castillo 2020 POLE) does not cover POLD1. Generic BP5 applies when the variant is found in a case with an alternate molecular basis for the disease phenotype. No case-level data are available for this variant at all: there is no proband report, no multi-gene panel result, no co-occurring (Likely) Pathogenic variant in another gene, and no second/alternate genetic diagnosis documented in any source. The ClinVar record contains laboratory classifications only (with condition labels of 'not provided' for several submissions), and the gnomAD, COSMIC, Cancer Hotspots and OncoKB queries return population/somatic/gene-level data with no case context. The only case-flavoured statement, the GeneDx submission's claim that the variant was observed in a breast-cancer individual (cited to PMID:30093976), is ungrounded because PMID:30093976 never names the variant. Because no case with an alternate molecular basis is documented, the criterion's condition evaluates to no: 'alternate molecular basis present? no'. BP5 is not met.

Rule

Generic ACMG/AMP 2015 BP5 (Richards et al. 2015, PMID:25741868): variant found in a case with an alternate molecular basis for disease.

Evidence considered

• Generic BP5 definition (Richards et al. 2015, PMID:25741868): variant found in a case with an alternate molecular basis for disease.
• ClinVar VCV000408109 documents laboratory classifications only (4 x Uncertain significance, 1 x Likely benign); no case-level result, no co-occurring variant, and several submissions carry 'not provided' condition labels.
• No case context in gnomAD (population frequencies), COSMIC (3 somatic occurrences), Cancer Hotspots (no hotspot), or OncoKB (gene-level, Unknown Oncogenic Effect).
• The GeneDx submission's case claim citing PMID:30093976 is ungrounded (the paper never names the variant), so it cannot establish a case with an alternate molecular basis.
• Explicit comparison as literally written for BP5 (a non-numeric presence/absence condition): 'alternate molecular basis present? ' - no, so BP5 is not met.

Still missing

• No proband multi-gene panel result, co-occurring (likely) pathogenic variant, or alternate genetic diagnosis is documented, so an alternate molecular basis for disease can be neither confirmed nor excluded.

Sources

Reasoning

Governing framework: generic ACMG/AMP 2015, because no POLD1 VCEP/CSPEC exists (cspec.found=false; generic_acmg_fallback) and the local custom framework (Leon-Castillo 2020 POLE) does not cover POLD1. Under the governing rule applied to this case, BP6 may be triggered ONLY by an exact-variant ClinVar expert-panel (3-star) classification of Benign or Likely benign; ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger it. The exact-variant record VCV000408109 has 0 expert-panel submissions (expert_panels empty; is_expert_panel=false for all 5 SCVs; expert_panel_submissions=0) and an aggregate 1-star status of 'criteria provided, conflicting classifications'. The single 'Likely benign' entry on the record is SCV005470083 (Ambry Genetics), an ordinary single-submitter clinical-laboratory assertion with no validated PMID trail (the audit flags it 'no_valid_pmids'); it is therefore an ordinary laboratory assertion and must not trigger BP6. The four 'Uncertain significance' submissions likewise cannot. There is no exact-variant expert-panel Benign/Likely benign classification to leverage, so BP6 is not met.

Rule

Generic ACMG/AMP 2015 BP6 (Richards et al. 2015, PMID:25741868) as applied here: requires an exact-variant ClinVar expert-panel (3-star) Benign/Likely benign classification; ordinary/non-expert submissions and aggregate labels do not trigger it.

Evidence considered

• Governing rule for this case: BP6 applies only for an exact-variant ClinVar expert-panel classification; Benign/Likely benign supports BP6; ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger it.
• ClinVar VCV000408109: expert_panels field empty; is_expert_panel=false for all 5 SCVs; audit counts show expert_panel_submissions=0; aggregate status 1 star, 'criteria provided, conflicting classifications'.
• The only 'Likely benign' entry is SCV005470083 (Ambry Genetics), an ordinary single-submitter clinical-laboratory assertion with an empty PMID list and audit rejection reason 'no_valid_pmids'; it must not trigger BP6.
• No qualifying exact-variant expert-panel Benign/Likely benign classification exists, so BP6 is not triggered.

Still missing

• No exact-variant ClinVar expert-panel (3-star) Benign/Likely benign classification exists for POLD1 c.49C>T; BP6 cannot be triggered on that basis.

Sources

Reasoning

Generic ACMG/AMP 2015 PS2 requires a de novo variant (both maternity and paternity confirmed) in a patient with the disease and no family history (PMID:25741868). No ClinGen CSPEC and no VCEP or local gene-specific framework exists for POLD1 (cspec found=false; final framework source=generic_acmg_fallback, framework_complete=false), so PS2 falls back to generic ACMG/AMP with no gene-specific de novo specification. The case is a variant-level assessment of NM_002691.4:c.49C>T (NP_002682.2:p.(Arg17Trp)), a missense substitution in POLD1 exon 2, and the collected evidence comprises only variant-level sources: ClinVar variation 408109 (4 submitters Uncertain significance, 1 submitter Likely benign, no expert panel), gnomAD v2.1/v4.1 frequencies, SpliceAI (max delta 0.069), REVEL 0.095, BayesDel -0.405968, OncoKB and COSMIC lookups. There is no proband record, no phenotype or age at onset, no trio or other parental testing, no confirmation of parentage and no family history anywhere in the case evidence; a search of the case evidence for de novo / parental / proband / meiosis / pedigree terms returns no records, and the only proband-adjacent statement available (a ClinVar submission assertion that the variant was observed in an individual with breast cancer) neither documents de novo status nor confirms parental genotypes. None of the four full-text-reviewed publications mentions the variant or supplies any de novo observation. PS2 can therefore be neither met nor refuted from the available data.

Evidence considered

• Generic ACMG/AMP 2015 PS2 definition applied because no CSPEC and no POLD1-specific framework exists: de novo (both maternity and paternity confirmed) in a patient with the disease and no family history (PMID:25741868).
• Case framework resolution: cspec.found=false, gene_vcep_dir=null, final_classification_framework.found=false with source=generic_acmg_fallback and framework_complete=false, so generic ACMG/AMP 2015 governs this case.
• Available case evidence is variant-level only: ClinVar variation 408109 (5 submissions: 4 clinical laboratories Uncertain significance, 1 clinical laboratory Likely benign; no expert-panel submission), gnomAD v2.1 (9/261400 alleles, AF 3.443e-05, 0 homozygotes), gnomAD v4.1, SpliceAI max delta 0.069, REVEL 0.095, BayesDel -0.405968, OncoKB (Unknown Oncogenic Effect, not variant-specific), COSMIC (COSV70954597, somatic count 3) and Cancer Hotspots (no significant hotspot).
• No case file contains a proband, parental genotypes, parentage confirmation, affected-relative data or a phenotype; the ClinVar submission narratives assert only in-silico/nonsense (PP3) and population-frequency (PM2) reasoning, with no de novo assertion by any submitter.
• PMID:25394175 (ACMG/NSGC cancer-referral practice guideline) and PMID:30093976 (Asian multi-primary-cancer panel cohort) were read in full text and contain no mention of POLD1 c.49C>T / p.(Arg17Trp) and no de novo data.

Still missing

• No proband-level clinical record (phenotype, age at onset, disease status) is available for this variant.
• No trio or other parental testing data confirming maternity and paternity of NM_002691.4:c.49C>T are available.
• No family-history documentation exists to establish absence of the variant from unaffected relatives.
• PS2 could be resolved only with a proband plus confirmed parental genotypes, or a clinical report stating confirmed de novo status.

Sources

Reasoning

PS3 requires well-established functional studies demonstrating a damaging effect on protein function. No functional assay of POLD1 c.49C>T p.(Arg17Trp) exists in any case source, so the criterion is not assessable rather than met or refuted. A systematic sweep of the case evidence returned no assay result of any kind for this variant: the only non-database sources are ClinVar variation 408109 (five submissions, no expert panel, no PS3 assertion; the captured evidence signals are in-silico PP3 and population PM2 only) and four fetched full texts (PMID:25741868, PMID:26467025, PMID:25394175, PMID:30093976), all of which report variant_mentioned = false. OncoKB explicitly returns 'did not identify variant-specific reviewed functional evidence for this variant' (is_variant_specific = false, not_reviewed = true, classification 'Unknown Oncogenic Effect'), and Cancer Hotspots reports no hotspot row at residue R17. PMID:25741868 (ACMG/AMP 2015) and PMID:26467025 (Karbassi scoring system) are the two references the triage indexed to PS3; both are framework/methodology papers that define or weight functional evidence generically and report no POLD1 or variant-specific data, so neither can supply the assay result PS3 requires. Biological relevance was also considered: POLD1-related polymerase proofreading-associated polyposis is driven by loss of the 3'-to-5' exonuclease (proofreading) function, so a PS3-grade assay for this gene would need to measure proofreading/replication fidelity (exonuclease activity, cell-based mutational-signature or MMR-deficiency readout); p.Arg17 lies in the N-terminal region outside the exonuclease domain where the characterised PPAP variants cluster, and no such assay is reported for this variant at all. Because the required evidence is absent rather than contradictory, the criterion is recorded as not_assessed with the gap stated; no VCEP/gene-specific PS3 specification exists for POLD1 (no ClinGen CSPEC; vcep_materials and final_classification_framework both report found = false), so generic ACMG/AMP 2015 definitions were applied.

Evidence considered

• OncoKB entry for POLD1 R17W states it 'did not identify variant-specific reviewed functional evidence for this variant; gene-level curated context is available for reviewer follow-up', with is_variant_specific = false, not_reviewed = true and classification 'Unknown Oncogenic Effect' — no cell-based or biochemical functional assay is reported.
• ClinVar variation 408109 (NM_002691.4(POLD1):c.49C>T, p.Arg17Trp): 5 submissions, 1-star review status, conflicting classifications (4x Uncertain significance, 1x Likely benign). No submitter asserted PS3; the captured submitted-evidence signals are PP3 (in silico, GeneDx) and PM2 (population, Labcorp). No functional-study criterion appears in any submission.
• Sweep of every case output file for functional-assay content (keywords function/assay/activity/proofread) returned only the triage index entries for PS3 and BS3 pointing at PMID:25741868 and PMID:26467025; no assay result, activity measurement or functional study of this variant is present anywhere in the case.
• paper_extracts entries for PMID:25741868, PMID:26467025, PMID:25394175 and PMID:30093976 all carry variant_mentioned = false with empty direct_quote/location, i.e. none of the fetched full texts reports a functional assay of POLD1 c.49C>T; PMID:25741868 is the ACMG/AMP 2015 standards paper and PMID:26467025 is a general weighted variant-scoring system, neither of which reports POLD1-specific data.
• Cancer Hotspots query 'POLD1 R17' returned no hotspot row ('This variant does not lie in a statistically significant hotspot'), and the COSMIC record (COSV70954597, somatic count 3) reports only recurrent somatic observation — neither is a functional assay of protein function.
• No ClinGen CSPEC or gene-specific framework is available for POLD1 (cspec found = false; vcep_materials note 'no cspec url available'; final_classification_framework found = false, source generic_acmg_fallback), so PS3 was evaluated against the generic ACMG/AMP 2015 definition in PMID:25741868.
• POLD1 disease mechanism per gene-level context: germline loss-of-function variants in the exonuclease domain cause polyposis and colorectal/endometrial cancer predisposition through proofreading deficiency; p.Arg17 is an N-terminal residue outside that domain, which is relevant to assay choice but does not substitute for a variant-specific functional assay under PS3.

Still missing

• No functional assay result exists for POLD1 c.49C>T p.(Arg17Trp): PS3 cannot be resolved without a variant-specific assay (e.g. exonuclease/proofreading activity, replication-fidelity or mutational-signature/MMR-deficiency readout) or a gene-specific functional framework listing accepted assays.
• No ClinGen CSPEC/VCEP or local gene-specific specification is available for POLD1, so PS3 assay validation status and strength calibration for a polymerase-proofreading-associated polyposis assay could not be assessed.
• p.Arg17 lies outside the POLD1 exonuclease (proofreading) domain where the characterised PPAP variants and most gene-level assay work are concentrated, so even the existing gene-level assay literature is unlikely to transfer to this residue without a dedicated experiment.

Sources

Reasoning

The governing specification for this case is generic ACMG/AMP 2015 (PMID:25741868): no ClinGen CSPEC/VCEP for POLD1 was retrieved (cspec found = false), the case's vcep_materials.json carries no summaries, files, gene notes or user files, and final_classification_framework is a generic_acmg_fallback (precedence_rank 3, official_clingen false, rule_payload null) that supplies no gene-specific PM3 rule. Under the generic definition PM3 requires the variant to be detected in trans with a pathogenic variant in a recessive disorder, and no such allelic configuration is documented anywhere for NM_002691.4:c.49C>T (p.Arg17Trp; NP_002682.2:p.(R17W); NC_000019.9:g.50902157C>T), a missense change at codon 17 in the N-terminal region of POLD1, outside the exonuclease domain. First, the recessive premise is not established. The POLD1 germline disease context documented in this case is autosomal dominant: OncoKB's curated gene-level background describes germline heterozygous loss-of-function variants in the exonuclease domain as causing polyposis with colorectal, endometrial and possibly brain cancer predisposition (heterozygous, i.e. dominant, mechanism); the ClinVar conditions attached to this variant are 'Colorectal cancer, susceptibility to, 10' and 'Hereditary cancer-predisposing syndrome'; and the germline literature captured in the case's pvs1_gene_context describes polymerase proofreading-associated polyposis (PPAP) as 'a rare autosomal dominant cancer predisposition syndrome caused by germline pathogenic variants in POLE or POLD1'. The case's pvs1_gene_context records germline_disease_context_found = true but an empty inheritance list, and no recessive POLD1 disease model appears in any evidence assembled for this case, so the recessive disorder PM3 presupposes is not documented. Second, and independently, no allelic observation exists: all five ClinVar submissions (variation 408109; PreventionGenetics, Quest Diagnostics, Ambry Genetics, GeneDx, Labcorp Genetics) are single-submitter germline clinical-testing records with no expert-panel entry, and none reports zygosity, a second POLD1 variant, phase or a relative's genotype, with conditions recorded only as 'not provided' / 'Not Provided' or left empty. Third, population data supply no allele-configuration observation: gnomAD v2.1 reports 9/261,400 alleles (AF 3.443e-05) with 0 homozygotes, gnomAD v4.1 reports 25/1,602,884 alleles (AF 1.55969e-05) with 0 homozygotes, and the gnomAD-Canada v1.0 query returned no record. Fourth, none of the four extracted publications names this variant (PMID:25741868, PMID:26467025, PMID:30093976 and PMID:25394175 all record variant_mentioned = false; PMID:30093976 confirms POLD1 sat on a 49-gene cancer panel but reports no POLD1 variant call), so the literature contributes no phase or family data. With no proband genotype, no candidate trans partner and no phasing information, the in-trans configuration PM3 requires is simply untested; the criterion is therefore recorded as not assessed rather than met or refuted, and the missing observations are enumerated as gaps.

Rule

Generic ACMG/AMP PM3 (PMID:25741868): for recessive disorders, the variant is detected in trans with a pathogenic variant; applied generically because no ClinGen CSPEC/VCEP or local POLD1-specific framework exists for this case.

Evidence considered

• Governing framework: no ClinGen CSPEC/VCEP or local POLD1 framework was available (cspec found = false; vcep_materials.json has no summaries, files or gene notes) and the case's final_classification_framework.json is a generic_acmg_fallback with rule_payload null, 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).
• The ACMG/AMP 2015 text quantified for this criterion is the note that rule PM3 can be upgraded to strong only where there are multiple observations of detection of the variant in trans (on opposite chromosomes) with other pathogenic variants; no such observation exists for c.49C>T, and none exists even at the moderate default strength.
• Disease/inheritance context for POLD1: the case's pvs1_gene_context records germline_disease_context_found = true with lof_mechanism_supported = true but an empty inheritance list, and its captured germline literature describes polymerase proofreading-associated polyposis as 'a rare autosomal dominant cancer predisposition syndrome caused by germline pathogenic variants in POLE or POLD1' (PMID:41487566 abstract, held in that artifact); OncoKB's curated POLD1 gene-level background describes germline heterozygous loss-of-function variants in the exonuclease domain causing polyposis with colorectal/endometrial cancer predisposition. Both describe a heterozygous (dominant) mechanism; no recessive POLD1 disease model is documented in this case.
• ClinVar variation 408109 (VCV000408109) for NM_002691.4(POLD1):c.49C>T (p.Arg17Trp): five germline clinical-testing submissions, all single-submitter with no expert-panel entry - PreventionGenetics (SCV000806545), Quest Diagnostics (SCV001134663), GeneDx (SCV002538918) and Labcorp Genetics/Invitae (SCV000547662) each report Uncertain significance, and Ambry Genetics (SCV005470083) reports Likely benign. Conditions are recorded only as 'not provided'/'Not Provided' or left empty. No submission states zygosity, a second POLD1 variant, phase or any family member's genotype. The submitted classifications were not used as criterion evidence, since a criterion may not be inferred from another source's final classification.
• Population data: gnomAD v2.1 9/261,400 alleles (AF 3.4429992e-05; highest subpopulation Admixed American 4/33,358, AF 1.19911e-04; grpmax FAF 4.165e-05), homozygotes 0; gnomAD v4.1 25/1,602,884 alleles (AF 1.55969e-05; highest subpopulation African/African American 8/74,932, AF 1.06763e-04; grpmax FAF 5.278e-05), homozygotes 0; gnomAD-Canada v1.0 returned no record. No dataset reports a homozygote, so there is no population observation of this allele on both chromosomes from which any allelic configuration could be read; these counts carry no phase information and are not themselves evidence for PM3.
• Literature: all four publications with extracts for this case record variant_mentioned = false - PMID:25741868 (ACMG/AMP 2015 framework paper; supplies the criterion definition only), PMID:26467025 (generic rule-based scoring system, no POLD1 content), PMID:30093976 (1191-patient Asian multi-gene-panel cohort in which POLD1 was one of 49 sequenced genes but no POLD1 variant is reported) and PMID:25394175 (ACMG/NSGC cancer referral guideline that neither names POLD1 nor the variant). None supplies a proband genotype, trans partner, phase or segregation observation for c.49C>T.
• No numeric threshold applies to PM3 in the supplied calibration data or in the governing framework; the criterion turns on a qualitative allelic observation that no available source provides.

Still missing

• No proband-level genotype, zygosity, phase or clinical indication is documented for POLD1 c.49C>T (p.Arg17Trp), so the allelic phase of the variant cannot be established.
• No second POLD1 variant (pathogenic, likely pathogenic or otherwise) is reported alongside c.49C>T in any consulted source, so an in-trans partner allele can be neither identified nor excluded.
• No parental, sibling or other family genotypes are available from which the phase of c.49C>T could be inferred.
• No homozygote or compound-heterozygous carrier of c.49C>T is reported (gnomAD v2.1 and v4.1 homozygotes both 0; gnomAD-Canada v1.0 returned no record), so there is no population-level observation of this allele on both chromosomes.
• No recessive POLD1 disease model is documented in this case: the gene-level artifact records an empty inheritance list and the germline context assembled here is autosomal dominant PPAP, so the recessive setting PM3 presupposes is unconfirmed.
• All five ClinVar submissions are single-submitter records with the condition recorded as 'not provided'/'Not Provided' or empty, so not even the disease context of the reported carriers can be used to test the recessive premise.

Sources

Reasoning

Generic ACMG/AMP 2015 PM6 requires an assumed de novo occurrence in a patient, without confirmation of maternity and paternity (PMID:25741868). The governing framework for POLD1 is generic ACMG/AMP fallback (no CSPEC, no VCEP, framework_complete=false), so PM6 is evaluated at the generic definition. Even at this lower evidentiary bar PM6 still requires an observed de novo event in an affected proband with the relevant phenotype. The case supplies none: NM_002691.4:c.49C>T (NP_002682.2:p.(Arg17Trp)) has no proband record, no phenotype, no parental origin and no assertion of de novo status in any file; the ClinVar submissions (variation 408109: 4 laboratories Uncertain significance, 1 laboratory Likely benign) provide no de novo reasoning, and none of the four full-text-reviewed publications mentions the variant. PM6 therefore cannot be assessed.

Evidence considered

• Generic ACMG/AMP 2015 PM6 definition applied because no POLD1-specific framework exists: assumed de novo (without confirmation of parentage) in a patient with the disease (PMID:25741868).
• Framework resolution for this case is generic_acmg_fallback with framework_complete=false; no VCEP criteria-combination or de novo rule payload is available.
• No case record documents a proband, parental origin, or asserted de novo occurrence for POLD1 c.49C>T / p.(Arg17Trp).
• The only case-observation statement available is a ClinVar submission signal noting the variant was observed in an individual with breast cancer (submission SCV002538918, GeneDx), which is neither a de novo assertion nor accompanied by parent-of-origin data and cited a paper (PMID:30093976) that does not mention the variant.

Still missing

• No proband with the associated phenotype (colorectal cancer susceptibility / hereditary cancer-predisposing syndrome) is documented for this variant.
• No parent-of-origin information (even an unconfirmed clinical assertion of de novo status) is available.
• PM6 could be resolved only with a proband and at least a reported, even unconfirmed, de novo observation.

Sources

Reasoning

Generic ACMG/AMP 2015 PP1 requires co-segregation of the variant with disease in multiple affected family members, i.e. a segregation analysis with documented meioses (PMID:25741868). No CSPEC and no VCEP or local POLD1 framework exists (framework source=generic_acmg_fallback, framework_complete=false), and none of the available sources customises PP1, so the generic definition applies. This case contains no pedigree, no family members, no genotypes from affected or unaffected relatives and no meiosis count; its evidence is confined to variant-level databases (ClinVar variation 408109, gnomAD v2.1/v4.1, SpliceAI max delta 0.069, REVEL 0.095, BayesDel -0.405968, OncoKB, COSMIC, Cancer Hotspots). The four full-text-reviewed publications supply no family data for this variant either: PMID:25741868 and PMID:26467025 discuss segregation only as generic methodology, PMID:25394175 is a cancer-referral practice guideline, and PMID:30093976 is a cohort study in which POLD1 appears only as a panel gene with no POLD1 allele reported. PP1 cannot be assessed because no informative meioses exist; this is an absence of segregation data, not evidence against segregation.

Evidence considered

• Generic ACMG/AMP 2015 PP1 definition applied because no POLD1-specific framework exists: co-segregation with disease in multiple affected family members (PMID:25741868).
• No case file contains a pedigree, family-member genotypes, or a meiosis count for NM_002691.4:c.49C>T; a search of the case evidence for segregation / pedigree / family / meiosis terms returns no records other than generic methodology text.
• PMID:26467025 describes segregation only as a weighted component of a general variant-scoring system and does not mention POLD1 or the variant.
• PMID:30093976 sequenced POLD1 as one of 49 panel genes in 1191 Asian cancer index patients but reports no POLD1 allele, no carrier counts and no segregation data; the variant is not named, so it supplies no PP1 evidence.

Still missing

• No pedigree or family structure is documented for this variant.
• No genotypes from affected or unaffected relatives are available, so no co-segregation and no meiosis count can be derived.
• PP1 would require a family with genotyped affected members co-segregating the variant with the phenotype.

Sources

Reasoning

BS3 requires well-established functional studies showing no damaging effect on protein function or splicing. No functional assay of POLD1 c.49C>T p.(Arg17Trp) exists in any case source, so BS3 is likewise not assessable. Critically, the absence of a damaging functional effect is not equivalent to a benign functional result: nothing in the case measures this variant's polymerase or proofreading activity, so there is no assay either way. The two references the triage indexed to BS3, PMID:25741868 (ACMG/AMP 2015) and PMID:26467025 (Karbassi scoring system), are framework/methodology papers with no POLD1 or variant-specific data and cannot supply the required assay. No ClinVar submitter asserted BS3 for variation 408109, and no expert-panel submission exists (expert_panel_submissions = 0). The available variant-level signals are expressly non-functional and belong to other criteria: SpliceAI max delta 0.07 (a splice prediction, not a splicing assay) and REVEL 0.095 (an in-silico missense predictor, not a functional assay); in-silico predictions cannot satisfy BS3 under any framework, and a benign in-silico result is not a substitute for measured normal function. p.Arg17 lying outside the exonuclease (proofreading) domain is also not BS3 evidence, because BS3 requires an assay demonstrating preserved function, not an inference from position. BS3 is therefore recorded as not_assessed with the gap identified, evaluated against generic ACMG/AMP 2015 definitions because no ClinGen CSPEC/VCEP or gene-specific specification for POLD1 is available.

Evidence considered

• Sweep of every case output file for functional-assay content (keywords function/assay/activity/proofread) found no assay, activity measurement or functional study of POLD1 c.49C>T p.(Arg17Trp); the only hits were the triage index entries mapping BS3 to PMID:25741868 and PMID:26467025.
• OncoKB reports no variant-specific reviewed functional evidence for POLD1 R17W (is_variant_specific = false, not_reviewed = true), so no source documents normal retained function of this variant.
• ClinVar variation 408109: 5 submissions, no expert panel, conflicting classifications; no submitter asserted BS3 and the captured evidence signals are PP3 (in silico) and PM2 (population) only.
• PMID:25741868 (ACMG/AMP 2015) and PMID:26467025 (Karbassi et al. scoring system) define/weight functional evidence generically but report no POLD1 or c.49C>T data (paper_extracts variant_mentioned = false for both), so neither supports nor refutes BS3 for this variant.
• Available variant-level in-silico results are non-functional and were excluded from BS3: SpliceAI max delta 0.069 (no significant splice impact) and REVEL 0.095 — predictor outputs, not functional assays, and therefore not BS3 evidence.
• No ClinGen CSPEC or gene-specific framework exists for POLD1 (cspec found = false; final_classification_framework source = generic_acmg_fallback), so BS3 was evaluated against the generic ACMG/AMP 2015 definition in PMID:25741868.
• Gene-level POLD1 mechanism is loss of exonuclease-domain proofreading function; p.Arg17 is an N-terminal residue outside that domain, but position alone is not assay evidence of preserved function and cannot satisfy BS3.

Still missing

• No functional assay demonstrating preserved POLD1 function (or preserved splicing) for c.49C>T p.(Arg17Trp) exists in the case: BS3 cannot be met by inference from variant location, in-silico scores or population data — a variant-specific assay is required.
• No ClinGen CSPEC/VCEP or local gene-specific specification for POLD1 is available, so BS3 validation/calibration against a PPAP-specific functional framework could not be performed.
• In-silico evidence for this variant is limited to non-functional predictors (SpliceAI max delta 0.07, REVEL 0.095); these inform PP3/BP4 only and leave BS3 with no supporting measurement at all.

Sources

Reasoning

Generic ACMG/AMP 2015 BS4 requires lack of segregation in affected members of a family, i.e. a tested family in which the variant fails to track with disease (PMID:25741868). No CSPEC and no VCEP or local POLD1 framework exists for this case (framework source=generic_acmg_fallback, framework_complete=false), so generic ACMG/AMP applies and no gene-specific non-segregation rule is available. BS4 cannot be satisfied in the absence of family data: a non-segregation observation requires a family with genotyped affected and unaffected members. This case has no pedigree, no relatives and no family genotypes; its evidence is variant-level only (ClinVar variation 408109, gnomAD v2.1/v4.1, SpliceAI max delta 0.069, REVEL 0.095, BayesDel -0.405968, OncoKB, COSMIC, Cancer Hotspots). The single ClinVar classification of Likely benign (1 laboratory) and the population presence of the variant in gnomAD (9/261400 alleles, 0 homozygotes) are not segregation observations and were not used to infer BS4. None of the four full-text-reviewed publications reports a family in which the variant fails to segregate. BS4 therefore cannot be assessed; the honest conclusion is absent data, not evidence against non-segregation.

Evidence considered

• Generic ACMG/AMP 2015 BS4 definition applied because no POLD1-specific framework exists: lack of segregation in affected members of a family (PMID:25741868).
• ClinVar variation 408109 reports this variant as Uncertain significance from 4 clinical laboratories and Likely benign from 1 clinical laboratory, with no expert-panel submission; that classification is not a segregation observation and provided no non-segregation evidence for BS4.
• gnomAD v2.1 shows the variant at 9/261400 alleles (AF 3.443e-05) with 0 homozygotes and gnomAD v4.1 shows similar low frequency; population presence is frequency evidence for other criteria and is not family non-segregation evidence for BS4.
• No case record contains a family, pedigree, or genotypes from affected or unaffected relatives for NM_002691.4:c.49C>T; none of the four full-text-reviewed publications (PMID:25741868, PMID:26467025, PMID:25394175, PMID:30093976) reports a non-segregating family or mentions the variant.

Still missing

• No family with both affected and unaffected genotyped members is available to test non-segregation.
• No pedigree data exist that could show the variant present in unaffected relatives or absent from affected relatives.
• BS4 could be resolved only with a family in which the variant demonstrably fails to track with the disease.

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 POLD1 c.49C>T (p.Arg17Trp; NC_000019.9:g.50902157C>T) is known, and no such observation exists in any source available for this variant. The governing specification is generic ACMG/AMP 2015 (PMID:25741868) because no ClinGen CSPEC/VCEP for POLD1 was retrieved (cspec found = false), vcep_materials.json is empty of summaries, files and gene notes, and final_classification_framework is a generic_acmg_fallback with no rule payload. The trans limb additionally presupposes a fully penetrant dominant disorder, and neither the case's ClinVar conditions ('Colorectal cancer, susceptibility to, 10', 'Hereditary cancer-predisposing syndrome', and 'not provided') nor the gene-level artifact, which records an empty inheritance list, establishes full penetrance for POLD1-related polymerase proofreading-associated polyposis, which is a recognised autosomal dominant predisposition syndrome with variable and age-dependent expression. In any case no trans configuration with a pathogenic allele is reported: the ClinVar record (variation 408109) holds five single-submitter germline clinical-testing submissions with no expert-panel entry, none of which states a co-occurring POLD1 variant, zygosity or phase. The cis limb requires the variant to be phased on the same allele as a pathogenic variant, and this case was reviewed at the variant level only - there is no proband genotype, no family genotype and no phasing result of any kind. The population datasets contain no allele-configuration observation either: gnomAD v2.1 (9/261,400 alleles, AF 3.443e-05) and gnomAD v4.1 (25/1,602,884 alleles, AF 1.55969e-05) both report zero homozygotes and the gnomAD-Canada v1.0 query returned no record, while none of the four extracted publications (PMID:25741868, PMID:26467025, PMID:30093976, PMID:25394175; all variant_mentioned = false) reports a co-occurring or phased POLD1 allele for this variant. Because neither limb of BP2 has an observation to rest on, the criterion is recorded as not assessed rather than met or refuted, with the missing observations enumerated as gaps.

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 no ClinGen CSPEC/VCEP or local POLD1-specific framework exists for this case.

Evidence considered

• Governing framework: no ClinGen CSPEC/VCEP or local POLD1 package was retrieved (cspec found = false; vcep_materials.json contains no summaries, files or gene notes) and final_classification_framework.json is a generic_acmg_fallback with rule_payload null, 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 408109 (VCV000408109) for NM_002691.4(POLD1):c.49C>T (p.Arg17Trp): five germline clinical-testing submissions, all single-submitter, no expert-panel entry - PreventionGenetics (SCV000806545), Quest Diagnostics (SCV001134663), GeneDx (SCV002538918) and Labcorp Genetics/Invitae (SCV000547662) report Uncertain significance; Ambry Genetics (SCV005470083) reports Likely benign. The record describes a single variant and provides no co-occurring POLD1 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 classifications were not used as criterion evidence.
• Inheritance and penetrance context: the ClinVar conditions attached to this variant are 'Colorectal cancer, susceptibility to, 10', 'Hereditary cancer-predisposing syndrome' and 'not provided'; the case's pvs1_gene_context records germline_disease_context_found = true with an empty inheritance list and captures germline literature describing polymerase proofreading-associated polyposis as an autosomal dominant syndrome (PMID:41487566 abstract held in that artifact), while OncoKB's POLD1 gene-level background describes heterozygous germline loss-of-function in the exonuclease domain as the disease mechanism. No source documents full penetrance for this disorder, and no trans observation exists, so the trans limb cannot be satisfied.
• Population data: gnomAD v2.1 9/261,400 alleles (AF 3.4429992e-05; grpmax FAF 4.165e-05), homozygotes 0; gnomAD v4.1 25/1,602,884 alleles (AF 1.55969e-05; grpmax FAF 5.278e-05), homozygotes 0; gnomAD-Canada v1.0 returned no record. No homozygote is reported, so the datasets contain no observation of this allele paired with a second POLD1 allele; population data also carry no phenotype or phase, and are therefore not evidence for or against BP2.
• Literature: all four publications with extracts for this case record variant_mentioned = false - PMID:25741868 (generic ACMG/AMP 2015 framework, source of the BP2 definition only), PMID:26467025 (generic weighted scoring system; no POLD1 content), PMID:30093976 (multi-gene-panel cohort in which POLD1 was sequenced but no POLD1 variant is reported) and PMID:25394175 (ACM/NSGC cancer referral guideline with no POLD1 content). None provides a co-occurring, phased or cis/trans allelic observation for c.49C>T.
• 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 POLD1 c.49C>T (p.Arg17Trp), so its cis/trans configuration relative to any potential pathogenic POLD1 variant is unknown.
• No co-occurring pathogenic or likely pathogenic POLD1 variant is reported with c.49C>T in the ClinVar record (variation 408109) or in any other consulted source.
• No homozygote or compound-heterozygous carrier of c.49C>T is reported (gnomAD v2.1 and v4.1 homozygotes both 0; gnomAD-Canada v1.0 returned no record), so no allele-configuration observation exists at population level.
• Penetrance is not documented for the POLD1 disorder under evaluation, so it cannot be determined whether the trans limb of BP2, which requires a fully penetrant dominant disorder, could ever apply.
• No proband or family genotype and no clinical indication are documented for this variant, so the allelic relationship of c.49C>T to any other POLD1 allele cannot be tested from this case record.

Sources