POLD1 c.1681C>T, p.(Arg561Trp)

Reasoning

PM2 (reduced to supporting strength per ClinGen SVI 2020) is met when the variant is absent or at very low frequency in population databases, with the SVI threshold applied here as allele frequency <= 0.0001. POLD1 c.1681C>T is present at a total allele frequency of 8.18e-06 in gnomAD v2.1 (2/244,578 alleles) and 6.24e-07 in gnomAD v4.1 (1/1,601,662 alleles) - both below the 0.0001 cutoff. The highest ancestry-specific frequency is 2.94e-05 in Admixed American (1/34,036, gnomAD v2.1) and 1.68e-05 in Admixed American (1/59,490, gnomAD v4.1); both popmax values also sit below 0.0001, so the criterion holds under either the all-comers total or the popmax convention and is not an ancestry artifact. The variant is absent from gnomAD-Canada v1.0, and no homozygotes are reported in either gnomAD dataset. Because the criterion is met only on the basis of rarity in control data - not on functional or clinical grounds - it is applied at supporting strength, and the very small allele counts (1-2 alleles) mean the true population frequency is bounded well below the threshold rather than being an artifact of low denominators. Note that PM2 is a low-specificity criterion and here provides only supporting pathogenic evidence.

Rule

PM2: absent or at very low frequency (<= 0.0001) in population databases; applied at supporting strength per ClinGen SVI.

Evidence considered

• gnomAD v2.1 total AF 8.177350374931515e-06 (2/244,578 alleles, exome-only; genome AN = 0), homozygotes = 0; popmax Admixed American AF 2.93807e-05 (1/34,036); NFE AF 9.13e-06 (1/109,528); remaining ancestries 0.
• gnomAD v4.1 total AF 6.243514549261955e-07 (1/1,601,662 alleles), homozygotes = 0; popmax Admixed American AF 1.68095e-05 (1/59,490); remaining ancestries 0.
• gnomAD-Canada v1.0 (HostSeq genomes): variant absent.
• PM2 threshold applied: allele frequency <= 0.0001 (0.01%), ClinGen SVI recommendation to apply PM2 at supporting strength (PMID:25741868 as supplied with the ACMG/AMP frequency thresholds).
• ACMG/AMP 2015 (PMID:25741868) states that absence of a variant in a large race-matched control cohort (>1000 individuals) may be scored as moderate pathogenic evidence; here the variant is not absent but is far rarer than the cutoff in a cohort of this scale, so the supporting-strength application of PM2 is the appropriate, conservative read.
• No POLD1-specific ClinGen CSPEC/VCEP or local gene framework specifies a non-cancer or exome-only population source for PM2 (cspec not found; framework_mode = generic_acmg), so the all-comers GNOMAD_V2_1 and GNOMAD_V4_1 totals were used by default; no non-cancer-cohort subset was retrieved that would have altered the allele-frequency estimate.

Sources

Reasoning

No POLD1 VCEP/CSPEC specification exists for this gene and no local gene-specific framework was supplied (vcep_materials.json carries no summaries, no files and an empty domain_tables key), so the generic ACMG/AMP 2015 definition of PS1 was applied. PS1 requires that the same amino-acid change as the variant under assessment - here NP_002682.2:p.(Arg561Trp) - has previously been established as pathogenic, regardless of which nucleotide change produces it. Two independent requirements fail. First, no established pathogenic variant carries this amino-acid change: the exact ClinVar record for NM_002691.4(POLD1):c.1681C>T (ClinVarID 469212) is Uncertain significance from both submissions (PreventionGenetics SCV000806473 and Labcorp/Invitae SCV000646500, 2-star 'criteria provided, multiple submitters, no conflicts', no expert-panel submission), and no other p.Arg561Trp-encoding allele was recovered. Second, no alternative nucleotide change can generate p.Arg561Trp at this codon: the codon-561 ClinVar records fix the reference codon as CGG (Arg) - c.1681C>T gives p.Arg561Trp, c.1681C>G gives p.Arg561Gly and c.1682G>A gives p.Arg561Gln - and the only single-base route from CGG to a tryptophan codon (TGG) is the first-position change c.1681C>T itself; c.1682G>A yields CAG (Gln) and c.1683G>A would yield CGA (Arg). PS1 is therefore not met.

Evidence considered

• The exact ClinVar record for this variant, NM_002691.4(POLD1):c.1681C>T (p.Arg561Trp), ClinVarID 469212 / VCV000469212, carries no pathogenic assertion: two germline clinical-laboratory submissions, both Uncertain significance (SCV000806473 PreventionGenetics, evaluated 2017-06-20; SCV000646500 Labcorp Genetics/Invitae, evaluated 2025-04-29), aggregate review status 'criteria provided, multiple submitters, no conflicts' (2 stars), no expert-panel submission.
• Codon 561 is CGG (Arg), established from the ClinVar records at this codon: c.1681C>T gives p.Arg561Trp, c.1681C>G gives p.Arg561Gly (VCV003624106) and c.1682G>A gives p.Arg561Gln (VCV000572113) - all consistent with a CGG reference codon and with the reference residue R561 read directly from the Mutalyzer-translated NP_002682.2 sequence supplied in the case normalization step.
• Only the first-position substitution C>T converts CGG to TGG (Trp); c.1682G>A (CAG) and c.1683G>A (CGA) do not, so c.1681C>T is the sole single-nucleotide change in NM_002691.4 capable of producing p.Arg561Trp.
• No publication in the case materials reports an established pathogenic POLD1 variant with this amino-acid change; the single full-text paper available (PMID:25741868) is the ACMG/AMP framework document and contains no variant- or gene-specific observations.
• PMID:25741868 supplies the generic ACMG/AMP definition of PS1 (same amino-acid change as a previously established pathogenic variant) used for this verdict.

Still missing

• No POLD1 VCEP/CSPEC specification or local gene framework was available, so PS1 was assessed against the generic ACMG/AMP 2015 definition using ClinVar as the only source of established variant classifications.

Sources

Reasoning

PS4 requires that the prevalence of POLD1 c.1681C>T (p.Arg561Trp) in affected individuals be significantly increased relative to controls (case-control enrichment; Richards et al. 2015, PMID:25741868). No case-control study, case series, or proband genotype with case counts exists for this variant in any retrieved source. ClinVar VCV000469212 carries only two submissions, both from clinical laboratories (SCV000806473 PreventionGenetics, part of Exact Sciences; SCV000646500 Labcorp Genetics, formerly Invitae), both Uncertain significance, with no affected/unaffected counts and no comparator data; the PMIDs those submitters cite are the ACMG/AMP guideline itself and Sherloc, neither of which reports the variant. A targeted germline POLD1 literature search likewise returned only gene-level papers (POLD1 exonuclease-domain disease mechanism, MSH2 Lynch syndrome heterogeneity, POLD1 p.Asp316Asn in a digenic PPAP family, POLD3 deficiency, a dMMR colorectal carcinoma cohort), none mentioning c.1681C>T. COSMIC records two somatic occurrences (COSV70957138), which are tumour-acquired events and cannot establish germline prevalence enrichment in affected versus unaffected individuals. Because no odds ratio, relative risk, or case/control allele counts exist, no numeric PS4 threshold or comparison operator (the >= style enrichment direction) could be applied in this case at all. PS4 is not met.

Rule

Generic ACMG/AMP 2015 PS4 (Richards et al., PMID:25741868): the prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls (odds ratio / relative risk enrichment, direction strengthens as the value goes up). No numeric cutoff is quotable from the retrieved source text, and no case-control data exist for this variant.

Evidence considered

• ClinVar VCV000469212 (ClinVarID 469212) contains two submissions, both from clinical laboratories (SCV000806473 PreventionGenetics, part of Exact Sciences; SCV000646500 Labcorp Genetics/formerly Invitae), both classified Uncertain significance, with is_expert_panel = false and no case counts, cohort sizes, or control comparisons.
• The only PMIDs attached to those submissions are the ACMG/AMP 2015 guideline (25741868) and Sherloc (28492532); the guideline reports no POLD1 or variant-specific data, and the Sherloc screening record reports no variant-level evidence (variant_specific = false).
• A targeted germline POLD1 literature search returned only gene-level papers (POLD1 exonuclease-domain germline disease mechanism; MSH2 Lynch syndrome phenotypic heterogeneity; POLD1 p.Asp316Asn with a PMS2 deletion in teenage-onset colorectal cancer; POLD3 deficiency; dMMR colorectal carcinoma cohort); none reports c.1681C>T (p.Arg561Trp).
• COSMIC lists two somatic occurrences of this variant (COSV70957138); tumour-acquired counts cannot demonstrate increased prevalence in affected individuals relative to controls and yield no odds ratio or relative risk.
• No odds ratio, relative risk, or affected/unaffected allele counts are available for this variant, so no numeric PS4 cutoff or comparison direction could be exercised; the ACMG/AMP guideline text retrieved for this case explains case-control use but does not supply a variant-specific threshold to apply here.

Still missing

• No case-control cohort, case series, or affected-individual genotype counts reporting POLD1 c.1681C>T; no odds ratio or relative risk can be derived.
• Only tumour-acquired somatic occurrences (COSMIC COSV70957138, n=2) are recorded, which do not address germline enrichment.

Sources

Reasoning

No POLD1 VCEP/CSPEC specification exists and vcep_materials.json's domain_tables key contains no entry for this gene, so there is no VCEP-approved critical-functional-domain list to check residue 561 against; generic ACMG/AMP 2015 PM1 was therefore applied. PM1 requires the variant to lie in a mutational hotspot or in a critical, well-established functional domain that is free of benign variation. Residue 561 is not a statistically significant hotspot: the Cancer Hotspots query for 'POLD1 R561' returned no hotspot row, and the only somatic record found for this variant is a COSMIC entry with two observations (COSV70957138, n = 2), far below the recurrence expected of a hotspot. Nor does residue 561 fall in the domain that carries the gene's established germline missense disease mechanism: POLD1 polymerase-proofreading-associated polyposis is driven by exonuclease-domain missense substitutions (ClinVar Pathogenic/Likely pathogenic p.Leu474Pro and p.Ser478Asn), while InterPro places the family-B exonuclease domain (IPR006133) at residues 243-477 and the RNase H-like exonuclease fold at approximately 119-547, so R561 lies downstream of the proofreading domain in the linker immediately preceding the polymerase palm subdomain (IPR023211, 580-810). Residue 561 is also not an annotated catalytic residue - UniProt P28340 mutagenesis data assign loss of exonuclease activity to residue 402 and loss of polymerase activity to residues 602 and 757 - and no pathogenic variation has been established at this codon, since all three ClinVar codon-561 substitutions (p.Arg561Trp, p.Arg561Gly, p.Arg561Gln) are Uncertain significance. PM1 is therefore not met.

Evidence considered

• The case materials contain no POLD1 CSPEC/VCEP and vcep_materials.json lists no summaries, no files and an empty domain_tables object, so no VCEP-approved list of POLD1 critical functional domains exists for this case; PM1 was assessed under generic ACMG/AMP 2015.
• Cancer Hotspots analysis (query 'POLD1 R561') returned no statistically significant hotspot row: 'This variant does not lie in a statistically significant hotspot' (source data empty).
• COSMIC records this variant as a somatic observation only twice (COSV70957138, somatic count 2), which is not recurrent enough to define a mutational hotspot.
• InterPro annotation of POLD1 (UniProt P28340) places the DNA polymerase family-B exonuclease domain (IPR006133) at residues 243-477 and the RNase H-like exonuclease fold (IPR012337) at 119-547; residue 561 sits downstream of these, in the linker N-terminal to the polymerase palm domain (IPR023211, 580-810) and the thumb domain (IPR042087, 828-985).
• UniProt P28340 mutagenesis annotations assign loss of exonuclease activity to residue 402 and loss of polymerase activity to residues 602 and 757; residue 561 is not a catalytic residue of either activity.
• POLD1 germline missense disease variants cluster in the proofreading/exonuclease domain rather than at residue 561: ClinVar Pathogenic/Likely pathogenic POLD1 missense records are p.Leu474Pro (VCV000144003) and p.Ser478Asn (VCV000040044).
• ClinVar residue-level data show no pathogenic enrichment at codon 561: c.1681C>T p.Arg561Trp (VCV000469212), c.1681C>G p.Arg561Gly (VCV003624106) and c.1682G>A p.Arg561Gln (VCV000572113) are all Uncertain significance.
• The case gene-level context records describe POLD1 exonuclease-domain variation causing polyposis and colorectal/endometrial cancer predisposition (ClinVar condition label 'Colorectal cancer, susceptibility to, 10'); they provide no residue-level domain assignment for codon 561.
• PMID:25741868 defines PM1 (mutational hotspot and/or critical, well-established functional domain without benign variation) and notes that a missense change's impact depends on the location and context of the residue within the protein.

Still missing

• No POLD1 VCEP/CSPEC critical-functional-domain table existed in the case materials, so domain membership was judged from hotspot significance, residue-level variant data and InterPro/UniProt protein-domain annotation rather than from an authoritative VCEP domain list.
• InterPro's broad family-B 'multifunctional domain' annotation (IPR006134, residues 541-973) nominally spans residue 561; this did not change the verdict because that region is not a confirmed hotspot, contains no catalytic residue at 561, and is not the domain in which POLD1 germline missense disease variants are established.

Sources

Reasoning

No POLD1 VCEP/CSPEC framework exists, so generic ACMG/AMP 2015 PM5 was applied. PM5 requires that a different missense change at the same amino-acid residue as the variant under assessment has been established as pathogenic. The pre-computed PM5 comparator collection (pm5_candidates) returned zero candidates and explicitly recorded that automatic same-residue harvesting was skipped because classic PM5 semantics could not be confirmed, so residue 561 was checked directly instead. ClinVar carries exactly three substitutions at POLD1 codon 561: c.1681C>T (p.Arg561Trp, VCV000469212, Uncertain significance), c.1681C>G (p.Arg561Gly, VCV003624106, Uncertain significance, multiple submitters with no conflicts) and c.1682G>A (p.Arg561Gln, VCV000572113, Uncertain significance, multiple submitters with no conflicts). Both alternative amino-acid changes at this residue are therefore classified Uncertain significance, and no submitter - expert panel or otherwise - has established either of them as pathogenic or likely pathogenic. No publication in the case materials reports a pathogenic POLD1 missense variant at residue 561. The prerequisite comparator for PM5 does not exist, so PM5 is not met.

Evidence considered

• The PM5 candidate-collection artifact (pm5_candidates) found zero same-residue candidates and recorded that harvesting was skipped because classic same-residue PM5 semantics could not be confirmed safely; its own instruction was to review residue-level comparators directly rather than apply PM5 from that artifact.
• ClinVar records for alternative codon-561 missense variants: NM_002691.4(POLD1):c.1681C>G (p.Arg561Gly), VCV003624106, Uncertain significance, 'criteria provided, multiple submitters, no conflicts'; and NM_002691.4(POLD1):c.1682G>A (p.Arg561Gln), VCV000572113, Uncertain significance, 'criteria provided, multiple submitters, no conflicts'.
• The variant under assessment itself, NM_002691.4(POLD1):c.1681C>T (p.Arg561Trp), VCV000469212, is Uncertain significance (two clinical-laboratory submissions, no expert-panel review).
• A ClinVar residue-561 search returned no other POLD1 coding substitution at this codon and no Pathogenic or Likely pathogenic assertion for any of them.
• No publication in the case materials (including the extracted full text of PMID:25741868) reports a pathogenic POLD1 missense variant at residue 561.

Sources

Reasoning

No POLD1 VCEP/CSPEC framework exists, so generic ACMG/AMP 2015 PP2 was applied. PP2 has two conjunctive requirements: the gene must show a low rate of benign missense variation, and missense variants must be a common mechanism of disease there. The second requirement is satisfied for POLD1 - ClinVar carries Pathogenic/Likely pathogenic POLD1 missense alleles at the exonuclease-domain hotspots (p.Leu474Pro, VCV000144003 and p.Ser478Asn, VCV000040044), and POLD1 missense variants are also disease-causing in recessive POLD1 deficiency (immunodeficiency 120) - so missense is unquestionably a recognised POLD1 disease mechanism. The first requirement is not met on the available data. gnomAD gene-level constraint for POLD1 (ENSG00000062822) shows only mild, non-significant depletion of missense variation: missense Z 2.46 with observed/expected missense 0.747 and pLI approximately 0 in gnomAD v2.1.1, and missense Z 2.75 with observed/expected missense 0.838 and LOEUF 0.734 in gnomAD v4.1. Both missense Z values fall below the conventional missense-Z significance threshold of 3.09 (Samocha et al. 2014, Nat Genet; PMID:25086666), and no gene-specific published rate of benign POLD1 missense variation is available in the case materials. Because a low rate of benign missense variation cannot be established for this gene, PP2's prerequisite is not satisfied and the criterion is not applied.

Evidence considered

• gnomAD gene-level constraint for POLD1 (Ensembl gene ENSG00000062822) is not significant for missense variation: gnomAD v2.1.1 missense Z 2.4598, observed/expected missense 0.7473, pLI 2.4e-06; gnomAD v4.1 missense Z 2.7521, observed/expected missense 0.8385, LOEUF 0.7344. The conventional missense-Z significance threshold of 3.09 used to judge 'low rate of benign missense variation' is from Samocha et al. 2014, Nat Genet (PMID:25086666).
• Missense is nonetheless an established POLD1 disease mechanism: ClinVar Pathogenic/Likely pathogenic records include p.Leu474Pro (VCV000144003) and p.Ser478Asn (VCV000040044), the classic exonuclease-domain (proofreading) missense alleles causing colorectal cancer susceptibility 10 / polymerase proofreading-associated polyposis.
• The case gene-level context (POLD1 gene summary and OncoKB gene-level background) states that germline variants in the POLD1 exonuclease domain cause polyposis with colorectal, endometrial and possibly brain cancer predisposition, i.e. a missense-bearing mechanism, but supplies no rate of benign missense variation for the gene.
• The variant under assessment is essentially absent from gnomAD v4.1 (1 allele in 1,601,662, AF 6.24e-07, with 0 of 1,171,348 non-Finnish European alleles) and present only twice in gnomAD v2.1 (2/244,578 alleles, AF 8.18e-06); this demonstrates rarity of this allele, not a low gene-level rate of benign missense variation, and is the basis of a different criterion (PM2).
• PMID:25741868 defines PP2 and cautions that computational or gene-level reasoning alone is not sufficient for a clinical assertion.

Still missing

• No gene-specific published measure of benign POLD1 missense variation (for example a cohort-derived benign missense rate) exists in the case materials; the constraint assessment rests on gnomAD gene-level missense Z / observed-versus-expected missense values.
• PP2 is a gene-level criterion and, unlike PM1, was not restricted to the exonuclease domain; the gene-level constraint result is the reason it is not applied despite the established exonuclease-domain missense disease mechanism.

Sources

Reasoning

Scope first: NM_002691.4:c.1681C>T is a missense substitution, NP_002682.2:p.(Arg561Trp), so PP3/BP4 are taken from the REVEL (missense protein-impact) path only, never from SpliceAI. No ClinGen CSPEC or gene-specific VCEP specification exists for POLD1, so generic ACMG/AMP 2015 with ClinGen SVI computational calibration applies. REVEL = 0.386 (local REVEL v1.3 lookup, chr19:50407169 C>T). The REVEL PP3 supporting threshold is >= 0.644 (Pejaver et al. 2022, PMID:36413997); 0.386 is below it, so no PP3 at any strength. BayesDel (0.0477232) carries no established generic ACMG-strength calibration and is therefore not_available for PP3. The SpliceAI max delta (0.013) is deliberately not used here: a missense variant's PP3 must not be supported by a splice prediction. PP3 is not met.

Evidence considered

• Variant normalized to NM_002691.4:c.1681C>T / NP_002682.2:p.(Arg561Trp), a single-amino-acid missense substitution; the missense REVEL path is the only PP3/BP4 path in scope (SpliceAI path and BP7 are for splice-region/intronic and synonymous variants respectively).
• REVEL v1.3 local predictor lookup returned a score of 0.386 for chr19:50407169 C>T (source_registry key 'revel').
• Generic ACMG/AMP 2015 (PMID:25741868) defines PP3 as computational evidence supporting a damaging effect; the same source cautions that in silico missense predictions should be counted as a single line of evidence and are only ~65-80% accurate.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997): PP3 supporting >= 0.644, moderate >= 0.773, strong >= 0.932. REVEL 0.386 falls below the lowest PP3 tier.
• A local BayesDel score (0.0477232, source_registry key 'bayesdel') is present but has no established generic ACMG-strength calibration and was treated as not_available for PP3.
• source_registry key 'spliceai' exists with a SpliceAI max delta of 0.013, but SpliceAI is out of path for a missense variant and was not used to support or deny PP3.
• review_manifest.json and vcep_materials.json confirm gene_vcep_dir = null and 'no cspec url available' for POLD1; framework_mode = generic_acmg, with no gene-specific PP3/BP4 lookup spreadsheet.

Sources

Reasoning

PP5 is applied only for an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. ClinVar VCV000469212 (ClinVarID 469212) for NM_002691.4(POLD1):c.1681C>T (p.Arg561Trp) has two submissions, both from clinical laboratories - SCV000806473 (PreventionGenetics, part of Exact Sciences) and SCV000646500 (Labcorp Genetics, formerly Invitae) - each with review status 'criteria provided, single submitter' and is_expert_panel = false. The aggregate record is Uncertain significance with 'criteria provided, multiple submitters, no conflicts' (2-star), and the audit shows 0 expert-panel submissions. No ClinGen expert-panel (VCEP) assertion exists for this variant, and no ClinGen CSPEC/VCEP specification was available for POLD1. Ordinary laboratory submissions and aggregate ClinVar labels must not trigger PP5, so PP5 is not met.

Rule

Generic ACMG/AMP 2015 PP5 (Richards et al., PMID:25741868), applied per case policy: only an exact-variant ClinVar expert-panel classification of Pathogenic/Likely pathogenic supports PP5; ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger it.

Evidence considered

• ClinVar VCV000469212 review status is 'criteria provided, multiple submitters, no conflicts' (2 stars) with classification Uncertain significance and 0 expert-panel submissions.
• Both submissions are clinical testing submissions (SCV000806473 PreventionGenetics, part of Exact Sciences; SCV000646500 Labcorp Genetics/formerly Invitae) with is_expert_panel = false and review status 'criteria provided, single submitter'; neither meets the exact-variant expert-panel requirement for PP5.
• No ClinGen CSPEC/VCEP specification for POLD1 was retrieved (cspec lookup found = false; vcep_materials reports no cspec url), so no expert-panel Pathogenic/Likely pathogenic assertion is available from a gene-specific framework either.

Sources

Reasoning

BA1 requires a stand-alone benign allele frequency of at least 0.05 (ACMG/AMP 2015, PMID:25741868). POLD1 c.1681C>T (NP_002682.2:p.Arg561Trp) is present in gnomAD v2.1 at a total allele frequency of 8.18e-06 (2/244,578 alleles, all observed in the exome subset; genome AN = 0) and in gnomAD v4.1 at 6.24e-07 (1/1,601,662 alleles). The highest frequency in any ancestry group is 2.94e-05 in Admixed American (1/34,036 alleles, gnomAD v2.1); v4.1 popmax is 1.68e-05 in Admixed American (1/59,490). Every one of these values is more than three orders of magnitude below the 0.05 BA1 threshold, including the most extreme subpopulation figure, so no ancestry-specific or threshold artifact exists that could rescue BA1. The variant is also absent from gnomAD-Canada v1.0. Because the observed frequencies are nowhere near the stand-alone cutoff, the criterion is not met with high confidence. No homozygotes are reported in either dataset, which is irrelevant to BA1 but confirms the rarity estimate.

Rule

BA1: allele frequency >= 0.05 in a population database (stand-alone benign).

Evidence considered

• gnomAD v2.1 (GRCh37, 19-50910426-C-T): total AF 8.177350374931515e-06 (2 alleles / 244,578 AN), exome-only contribution, genome AN = 0, homozygotes = 0; popmax Admixed American AF 2.93807e-05 (1/34,036, 0 homozygotes); NFE AF 9.13e-06 (1/109,528); all other ancestry groups 0.
• gnomAD v4.1 (GRCh38, 19-50407169-C-T): total AF 6.243514549261955e-07 (1 allele / 1,601,662 AN), homozygotes = 0; popmax Admixed American AF 1.68095e-05 (1/59,490, 0 homozygotes); NFE, AFR, EAS, SAS, ASJ, FIN, MID, AMI all 0.
• gnomAD-Canada v1.0 (HostSeq genomes): variant absent.
• BA1 threshold applied: allele frequency >= 0.05 (5%), ACMG/AMP 2015 stand-alone benign criterion (PMID:25741868).
• No POLD1-specific ClinGen CSPEC/VCEP or local gene framework exists for this case (cspec not found; framework_mode = generic_acmg), so generic ACMG/AMP thresholds govern; no VCEP-directed non-cancer or exome-only population source was specified, so the all-comers GNOMAD_V2_1 and GNOMAD_V4_1 totals were used by default.
• No non-cancer-cohort gnomAD subset (GNOMAD_V2_1_NON_CANCER / GNOMAD_V3_1_NON_CANCER) was retrieved for this variant, so no alternative source could have produced a materially higher frequency.

Sources

Reasoning

BS1 is met when the allele frequency is greater than the frequency expected for the disorder, with the generic ClinGen-SVI default cutoff at >= 0.01 (per-gene derivation preferred; Whiffin et al. 2017, PMID:28518168). POLD1 c.1681C>T was observed at total AF 8.18e-06 in gnomAD v2.1 (2/244,578) and 6.24e-07 in gnomAD v4.1 (1/1,601,662), with a maximum ancestry-specific frequency of 2.94e-05 (Admixed American, gnomAD v2.1). Even the popmax value is roughly 340-fold below the 0.01 threshold, so the criterion is not met regardless of whether the all-comers totals or the highest-ancestry figure are used. No POLD1-specific frequency calibration exists for this gene, so the generic default was applied; a per-gene BS1 derivation for an adult-onset, incompletely penetrant colorectal cancer predisposition gene would in any case yield a threshold far above the observed frequency, not below it. Absence from gnomAD-Canada is consistent with rarity.

Rule

BS1: allele frequency greater than expected for the disorder (generic SVI default >= 0.01).

Evidence considered

• gnomAD v2.1 total AF 8.177350374931515e-06 (2/244,578 alleles, exome-only, 0 homozygotes); popmax Admixed American 2.93807e-05 (1/34,036).
• gnomAD v4.1 total AF 6.243514549261955e-07 (1/1,601,662 alleles, 0 homozygotes); popmax Admixed American 1.68095e-05 (1/59,490).
• gnomAD-Canada v1.0: absent.
• BS1 threshold applied: allele frequency >= 0.01 (1%), generic ClinGen-SVI default from Whiffin et al. 2017 (PMID:28518168); a per-gene derivation is preferred but none exists for POLD1.
• The disorder context (POLD1 polymerase proofreading-associated polyposis / colorectal cancer susceptibility) is adult-onset with reduced penetrance, which would raise rather than lower an expected benign-frequency threshold.

Still missing

• No POLD1-specific BS1 frequency calibration was available; the generic ClinGen-SVI default of 0.01 (Whiffin et al. 2017, PMID:28518168) was used, which does not change the verdict given the ~340-fold margin.

Sources

Reasoning

BS2 requires the variant to be observed in a healthy adult individual for a disorder in which full penetrance is expected at an early age (homozygous for recessive, heterozygous for dominant, or hemizygous for X-linked). Here gnomAD v2.1 and v4.1 both report zero homozygotes for c.1681C>T, and POLD1 is autosomal, so no recessive (homozygous) or hemizygous observation exists. The variant is seen only as isolated heterozygous alleles (2 alleles in v2.1 exomes, 1 allele in v4.1 exomes), and POLD1-associated disease (polymerase proofreading-associated polyposis / colorectal cancer susceptibility 10) is an adult-onset, incompletely penetrant dominant cancer predisposition in which full penetrance at an early age is explicitly not expected. The premise of BS2 is therefore not satisfied: a heterozygous observation in an uncurated adult population cohort cannot be scored as BS2 evidence for an adult-onset, reduced-penetrance cancer gene, and there is no homozygote to invoke the recessive arm of the criterion. The variant is additionally absent from gnomAD-Canada.

Rule

BS2: observation of the variant in a healthy adult individual for a disorder with full penetrance expected at an early age (recessive homozygous, dominant heterozygous, or X-linked hemizygous).

Evidence considered

• gnomAD v2.1: total homozygote count = 0 (exome 0, genome 0); all ancestry-specific homozygote counts 0 including Admixed American (1/34,036 alleles) and NFE (1/109,528 alleles).
• gnomAD v4.1: total homozygote count = 0; all ancestry-specific homozygote counts 0.
• gnomAD-Canada v1.0: variant absent.
• POLD1 is autosomal (NM_002691.4, NP_002682.2; HGNC:9175) and p.Arg561Trp is a heterozygous missense observation; disease context (polymerase proofreading-associated polyposis / colorectal cancer susceptibility) is adult-onset with reduced penetrance, not an early-onset fully penetrant disorder.
• No POLD1-specific ClinGen CSPEC/VCEP or local gene framework defines a BS2 rule for this gene (cspec not found; framework_mode = generic_acmg), so the generic ACMG/AMP BS2 definition was applied.

Still missing

• gnomAD cohorts are not individually curated for age at observation or disease status, so a heterozygous carrier's health status relative to POLD1-associated cancer predisposition cannot be confirmed from the population record.

Sources

Reasoning

No POLD1 VCEP/CSPEC framework exists, so generic ACMG/AMP 2015 BP1 was applied. BP1 applies only to a gene in which primarily truncating variants are known to cause disease, i.e. a gene whose disease mechanism is not established to include missense variation. POLD1 does not meet that premise: pathogenic missense variation is well established. ClinVar carries Pathogenic/Likely pathogenic POLD1 missense alleles at the exonuclease-domain proofreading hotspots p.Leu474Pro (VCV000144003) and p.Ser478Asn (VCV000040044), which cause polymerase proofreading-associated polyposis / colorectal cancer susceptibility 10, and POLD1 missense variants are also the mechanism of recessive POLD1 deficiency (immunodeficiency 120) in the polymerase domain. A survey of POLD1 ClinVar records carrying a Pathogenic classification returned only 17 records in total - six coding records (two frameshift, p.Val122fs and p.Arg1050fs; one in-frame deletion, p.Ser605del; one compound record, p.Gln684His with p.Gln710His and p.Ser965Trp; and the two exonuclease-domain missense alleles above) plus eleven large copy-number gains - so the POLD1 disease spectrum is not truncating-exclusive and missense changes are a recognised disease mechanism. Although the case gene-level context records support a loss-of-function component to POLD1 disease, that does not establish truncating variants as the only pathogenic class. BP1 is therefore not met.

Evidence considered

• ClinVar carries established POLD1 missense disease alleles: NM_002691.4(POLD1):c.1421T>C (p.Leu474Pro), VCV000144003, Pathogenic/Likely pathogenic and NM_002691.4(POLD1):c.1433G>A (p.Ser478Asn), VCV000040044, Pathogenic/Likely pathogenic - both in the exonuclease (proofreading) domain and associated with colorectal cancer susceptibility 10.
• ClinVar survey of POLD1 records with a Pathogenic classification returned 17 records: two frameshift alleles (c.365_377del p.Val122fs, VCV004682116; c.3148del p.Arg1050fs, VCV001077091), one in-frame deletion (c.1809CTC[1] p.Ser605del, VCV000060775 - the MDPL allele), one compound coding record (c.[2052G>C;2816C>G] p.Gln684His/Gln710His/Ser965Trp, VCV001119948) and the two exonuclease-domain missense alleles, plus eleven large copy-number gains; no 'Likely pathogenic'-only POLD1 record was returned.
• UniProt P28340 disease annotations independently show POLD1 missense variants at residues 684, 939, 1060 and 1074 causing recessive POLD1 deficiency (immunodeficiency 120), further establishing missense as a pathogenic mechanism for this gene.
• The case gene-level context records a POLD1 loss-of-function germline mechanism (pvs1_gene_context: lof_mechanism_supported true, mechanism rationale referencing germline disease publications), but that finding characterizes one component of POLD1 disease and does not establish that truncating variants are the sole pathogenic class.
• The variant under assessment is a missense substitution (NP_002682.2:p.Arg561Trp), so BP1 would in any case require the gene to lack a missense disease mechanism, which it does not.

Sources

Reasoning

Scope first: NM_002691.4:c.1681C>T is a missense substitution, NP_002682.2:p.(Arg561Trp), so BP4 is taken from the REVEL (missense protein-impact) path only. No ClinGen CSPEC or POLD1-specific VCEP specification exists, so generic ACMG/AMP 2015 with ClinGen SVI computational calibration applies. REVEL = 0.386. The BP4 supporting threshold is <= 0.29 (Pejaver et al. 2022, PMID:36413997); 0.386 exceeds it, and also exceeds the moderate (<= 0.183) and strong (<= 0.016) BP4 tiers, so BP4 is not met at any strength. This is a gray-zone REVEL result: it falls between the BP4 supporting cutoff (0.29) and the PP3 supporting cutoff (0.644), yielding neither criterion. Critically, the gray-zone result does not license reaching across to the other predictor path - the SpliceAI max delta of 0.013 (<= 0.1) is a benign-leaning splice score but is not BP4 support for a missense variant, whose amino-acid substitution is not what SpliceAI assesses. BayesDel (0.0477232) has no established generic ACMG-strength calibration and is not_available for BP4. BP4 is not met.

Evidence considered

• Variant is a missense substitution (NP_002682.2:p.(Arg561Trp)); BP4 is therefore evaluated from REVEL only, not from SpliceAI.
• REVEL v1.3 local predictor lookup returned a score of 0.386 for chr19:50407169 C>T (source_registry key 'revel').
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997): BP4 supporting <= 0.29, moderate <= 0.183, strong <= 0.016. REVEL 0.386 is above all BP4 tiers, so BP4 is unmet.
• 0.386 sits within the gray zone (above the 0.29 BP4 cutoff and below the 0.644 PP3 cutoff), which yields neither PP3 nor BP4 and does not permit substituting the other predictor path.
• SpliceAI max delta = 0.013 (>= BP4 splice threshold of <= 0.1) is present but cannot support BP4 for a missense variant; splice predictions were not cited as BP4 evidence.
• A local BayesDel score (0.0477232, source_registry key 'bayesdel') has no established generic ACMG-strength calibration and was treated as not_available for BP4.
• Generic ACMG/AMP 2015 (PMID:25741868) is the governing framework because review_manifest.json and vcep_materials.json confirm no ClinGen CSPEC and no POLD1 VCEP specification (gene_vcep_dir = null; framework_mode = generic_acmg).

Sources

Reasoning

BP6 is applied only for an exact-variant ClinVar expert-panel classification of Benign or Likely benign. ClinVar VCV000469212 (ClinVarID 469212) for NM_002691.4(POLD1):c.1681C>T (p.Arg561Trp) is classified Uncertain significance, with two submissions from clinical laboratories (PreventionGenetics, part of Exact Sciences; Labcorp Genetics, formerly Invitae), both is_expert_panel = false, and the audit records 0 expert-panel submissions. No expert-panel Benign/Likely benign assertion exists for this variant, and no ClinGen CSPEC/VCEP framework for POLD1 was available to supply one. Ordinary laboratory submissions and aggregate ClinVar labels must not trigger BP6, so BP6 is not met.

Rule

Generic ACMG/AMP 2015 BP6 (Richards et al., PMID:25741868), applied per case policy: only an exact-variant ClinVar expert-panel classification of Benign/Likely benign supports BP6; ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions must never trigger it.

Evidence considered

• ClinVar VCV000469212 is classified Uncertain significance ('criteria provided, multiple submitters, no conflicts', 2 stars) with no expert-panel submission.
• Both submitters are clinical laboratories with is_expert_panel = false (SCV000806473 PreventionGenetics; SCV000646500 Labcorp Genetics/formerly Invitae), so no expert-panel Benign or Likely benign assertion exists for the exact variant.
• No ClinGen CSPEC/VCEP specification for POLD1 was retrieved (cspec lookup found = false), so no expert-panel benign assertion is available from a gene-specific framework either.

Sources

Reasoning

PS2 (Richards et al. 2015, PMID:25741868) requires a de novo occurrence in a patient with the disease, with both maternity and paternity confirmed, and no family history. NM_002691.4:c.1681C>T (NP_002682.2:p.Arg561Trp) in POLD1 is presented here as a bare variant with no proband: the case carries no patient record, no parental genotypes, no statement of maternity/paternity confirmation, and no family history. Searches of every case evidence file for 'de novo', 'proband', 'parental', 'pedigree', 'meiosis' and 'segregation' returned no matches at all, so there is no de novo observation to assess. The only ClinVar record for this variant (VCV000469212) is two single-submitter Uncertain significance submissions (PreventionGenetics 2017; Labcorp/Invitae 2025) that report no parental testing and no de novo occurrence. The one full-text publication available, PMID:25741868, is the ACMG/AMP framework itself and contributes no POLD1 family data. No ClinGen CSPEC or gene-specific specification exists for POLD1 (case_summary cspec.found=false; vcep_materials.json reports no cspec url and framework source=generic_acmg_fallback), so the generic ACMG/AMP 2015 definition applies. PS2 cannot be evaluated on this record.

Rule

ACMG/AMP 2015 PS2 (PMID:25741868): de novo (both maternity and paternity confirmed) in a patient with the disease and no family history

Evidence considered

• PS2 definition applied from the generic ACMG/AMP 2015 guideline (PMID:25741868), since no ClinGen CSPEC or POLD1-specific framework is available (case_summary compact_evidence.cspec.found = false; vcep_materials.json: 'no cspec url available', final_classification_framework.found = false, source = generic_acmg_fallback).
• Case-wide search for family-level evidence: the review_manifest, case_summary, source_registry, evidence, literature_pass, fulltext_map and paper_extracts files, plus prefetch.json and vcep_materials.json read directly, contain no occurrence of 'de novo', 'proband', 'parental', 'pedigree', 'meiosis', 'inheritance' or 'segregation'.
• ClinVar (source_registry key 'clinvar'), VCV000469212: two germline Uncertain significance submissions (SCV000806473 PreventionGenetics, last evaluated 2017-06-20; SCV000646500 Labcorp Genetics/Invitae, 2025-04-29), review status 'criteria provided, single submitter', 2 review stars, no expert-panel submission; neither submission states de novo status or parental testing.
• ClinVar citations for this record are only the ACMG/AMP guideline (PMID:25741868) and the Sherloc refinement (PMID:28492532); neither reports this variant's inheritance.
• Paper-extract review (paper_extracts.json, PMID:25741868): the only fetched full text is the ACMG/AMP 2015 standards document; variant_mentioned = false, no POLD1 or family-level data.

Still missing

• No proband/parental genotypes: whether c.1681C>T arose de novo, and whether maternity and paternity were confirmed, is unknown.
• No family history or disease-status information for the proband or first-degree relatives.

Sources

Reasoning

PS3 (Richards et al. 2015, PMID:25741868) requires a well-established functional study demonstrating a deleterious effect on the gene or gene product. No such study exists for POLD1 NM_002691.4:c.1681C>T (NP_002682.2:p.(Arg561Trp)): the case evidence contains no enzyme-activity, DNA-replication fidelity/proofreading, yeast or human cell complementation, or other functional assay result for this allele. OncoKB, queried specifically for POLD1 R561W, returned 'Unknown Oncogenic Effect' and explicitly states that no variant-specific reviewed functional evidence was identified, offering only gene-level background. Neither of the two ClinVar submissions for variation 469212 (SCV000806473, PreventionGenetics; SCV000646500, Labcorp/Invitae) reports PS3 or BS3 among applied criteria - the only criterion signal extracted from the submission text is PM2 - and both laboratories classify the variant as Uncertain significance. The two papers in the literature packet (PMID:25741868, ACMG/AMP 2015 standards, and PMID:28492532, Sherloc rule refinement) are methodology documents that neither name nor functionally test this variant; gene-context literature retrieved for POLD1 addresses other alleles (e.g. exonuclease-domain variants and mismatch-repair digenic cases), not p.Arg561Trp. Because no functional assay was performed or reported for this variant, PS3 is not satisfied, but its absence also cannot be treated as refutation of a damaging effect; the criterion is therefore recorded as not_assessed rather than not_met, and no PS3 strength calibration can be applied when there is no assay to grade. No POLD1-specific VCEP/gene-specific specification governs this case (framework_complete=false), so the generic ACMG/AMP 2015 definition applies.

Evidence considered

• PS3 requires a well-established functional study showing a damaging effect on protein function (Richards et al. 2015, ACMG/AMP, PMID:25741868). No functional study of POLD1 c.1681C>T (p.Arg561Trp) is present anywhere in the case evidence.
• OncoKB was queried directly for POLD1 R561W and returned classification 'Unknown Oncogenic Effect' with the statement that no variant-specific reviewed functional evidence was identified for this variant; its content is gene-level curated background (polymerase and 3'-to-5' exonuclease function, exonuclease-domain germline disease) and is not variant-specific.
• Neither ClinVar submission for variation 469212 (SCV000806473 PreventionGenetics, 2017; SCV000646500 Labcorp/Invitae, 2025) reports PS3 as an applied criterion; the only criterion signal extracted from the submission text is PM2, and both submissions classify the variant as Uncertain significance.
• The literature packet for this case contains only two PMIDs (25741868 ACMG/AMP 2015 standards; 28492532 Sherloc refinement). Full text was available for PMID:25741868 only; it defines PS3 categorically but names neither POLD1 nor c.1681C>T (searched for 'c.1681C>T', 'p.R561W', 'R561W', 'POLD1' - variant_mentioned=false).
• PMID:28492532 (Sherloc; Nykamp et al. 2017) reports rule weights and criteria groupings without any variant-level functional, segregation, population, or in silico data, and does not mention c.1681C>T.
• Gene-context literature retrieved for the POLD1 PVS1 gate (e.g. PMID:36291559, a digenic POLD1 p.Asp316Asn / PMS2 case with tumor mutational-signature analysis) provides functional/tumor-level information for other POLD1 alleles only; p.Arg561Trp is not mentioned, so no functional inference is transferred to it.
• No POLD1-specific ClinGen VCEP or local gene framework was found (vcep_materials: no CSPEC url, no files, framework_complete=false), so the generic ACMG/AMP 2015 PS3 definition governs and no gene-specific PS3 strength calibration exists to apply.

Still missing

• No variant-specific functional study for NM_002691.4:c.1681C>T (p.Arg561Trp) was identified - no POLD1 polymerase-activity, 3'-5' exonuclease/proofreading-fidelity, or cell-based complementation assay result.
• No ClinGen VCEP or POLD1-specific specification is available to define acceptable assay systems, controls, or PS3 strength calibration for this gene.
• PMID:28492532 (Sherloc) was cited by a ClinVar submitter but was not retrieved in full text; it is a rule-refinement methodology paper and is not expected to carry variant-level functional data.

Sources

Reasoning

PM3 (Richards et al. 2015, PMID:25741868) requires the variant to be detected in trans with a pathogenic variant in a gene/disorder with recessive inheritance. POLD1 does have a recessive-capable disease context - gene-context literature retrieved for this case notes that POLD1 and POLD2 variants have been associated with a syndromic combined immunodeficiency (T cell lymphopenia with or without intellectual disability and sensorineural hearing loss) - alongside its principal autosomal dominant germline disorder, in which heterozygous exonuclease-domain proofreading variants cause polymerase proofreading-associated polyposis and colorectal cancer susceptibility. However, this case supplies no phase information of any kind for NM_002691.4:c.1681C>T (NP_002682.2:p.Arg561Trp). There is no proband genotype, no pedigree, no relative testing and no second POLD1 allele anywhere in the case evidence: a search of review_manifest, case_summary, source_registry, prefetch, evidence, literature_pass, fulltext_map and paper_extracts (plus vcep_materials, clinvar_submission_audit and evidence_brief read directly) returns no occurrence of phase, cis, trans, zygosity, compound heterozygous or family/proband/parental terms for this variant. ClinVar variation 469212 (VCV000469212) is two single-submitter Uncertain significance records - SCV000806473 (PreventionGenetics, evaluated 2017-06-20, germline, condition 'not provided') and SCV000646500 (Labcorp Genetics/Invitae, evaluated 2025-04-29, germline, no condition listed; aggregate condition 'Colorectal cancer, susceptibility to, 10') - and neither submission reports zygosity, phase or a co-occurring pathogenic allele; the only criterion signal extracted from their submission text is PM2, and no expert-panel submission exists. The one full-text publication available for this case (PMID:25741868) is the ACMG/AMP 2015 framework itself and contains no variant-level data. Gene-level literature retrieved during the case does describe a digenic family in which two teenage siblings inherited a maternal PMS2 exon 12 deletion (NM_000535.7:c.2007-786_2174+493del1447) and a paternal POLD1 variant p.Asp316Asn (PMID:36291559), but that is a different POLD1 allele and a different partner gene, so it can neither satisfy nor refute PM3 for p.Arg561Trp. With no documented second allele, no phase and no stated recessive phenotype in this record, PM3 is recorded as not_assessed rather than not_met: the criterion is evaluable in principle but the case lacks the allelic data required. No ClinGen CSPEC and no POLD1-specific VCEP/specification exist (case_summary compact_evidence.cspec.found = false; vcep_materials.json 'no cspec url available', gene_vcep_dir = null), so the generic ACMG/AMP 2015 definition applies.

Rule

generic_acmg_2015: requires pathogenic variant in trans in a recessive gene/disorder; no phase data available -> not_assessed

Evidence considered

• PM3 definition applied from the generic ACMG/AMP 2015 guideline (Richards et al., PMID:25741868), because no ClinGen CSPEC and no POLD1-specific framework were available for this case (case_summary compact_evidence.cspec.found = false; vcep_materials.json: 'no cspec url available'; final_classification_framework.found = false, source = generic_acmg_fallback).
• ClinVar variation 469212 (VCV000469212) retrieved as 'Uncertain significance', criteria provided, multiple submitters no conflicts (2 single-submitter clinical-testing labs, 0 expert panels): SCV000806473 PreventionGenetics (germline, condition 'not provided', evaluated 2017-06-20) and SCV000646500 Labcorp Genetics/Invitae (germline, evaluated 2025-04-29). Neither submission text reports phase, zygosity, a second allele, or the PM3 criterion; the only criterion signal in the submission text is PM2.
• Case-wide search for allelic/phase evidence: review_manifest.json, case_summary.json, source_registry.json, prefetch.json, evidence.json, literature_pass.json, fulltext_map.json, paper_extracts.json, plus vcep_materials.json, clinvar_submission_audit.json and evidence_brief.json read directly - no occurrence of 'phase', 'cis', 'trans', 'zygosity', 'compound heterozygous', 'proband', 'parental', 'pedigree' or 'segregation' relating to this variant. This case is a bare variant interpretation with no attached patient.
• POLD1 disease context: heterozygous germline loss-of-function/proofreading variants in the exonuclease domain cause autosomal dominant polyposis with colorectal, endometrial and possibly brain cancer predisposition (gene_context.json gene summary and OncoKB gene background). A recessive context also exists in the gene-context literature, where POLD1/POLD2 variants have been associated with a syndromic combined immunodeficiency (T cell lymphopenia with or without intellectual disability and sensorineural hearing loss). No recessive phenotype is recorded for this case.
• Gene-level literature retrieved for the case includes a digenic family with two teenage siblings carrying a maternally inherited PMS2 exon 12 deletion together with a paternally inherited POLD1 p.Asp316Asn variant (PMID:36291559). That report concerns a different POLD1 allele and a different partner gene, and therefore provides no trans/cis information for p.Arg561Trp; it was reviewed and not transferable.
• The only full-text publication retrieved for this case, PMID:25741868, is the ACMG/AMP 2015 standards and guidelines document; its pre-computed extraction classifies it as a framework/methodology paper that does not mention c.1681C>T or p.Arg561Trp (variant_mentioned = false), so it contributes no allelic data.
• gnomAD observations (2/244578 alleles, 0 homozygotes in v2.1; 1/1601662 alleles, 0 homozygotes in v4.1; absent from gnomAD-Canada) were noted but provide no trans/cis information and were not used to infer PM3.

Still missing

• No phase information: whether c.1681C>T is in trans or in cis with any other POLD1 variant is unknown, and no parental or relative genotypes are present in the case record.
• No second POLD1 variant (pathogenic or otherwise) is reported for the same individual anywhere in the case evidence or in the two ClinVar submissions for variation 469212.
• No recessive phenotype context is documented for this case: the ClinVar conditions are 'Colorectal cancer, susceptibility to, 10' and 'not provided', and no immunodeficiency/combined-immunodeficiency phenotype is recorded.
• No ClinGen CSPEC or POLD1-specific VCEP exists to specify acceptable phase evidence, trans-allele definitions or PM3 strength calibration for this gene.

Sources

Reasoning

PM6 (Richards et al. 2015, PMID:25741868) requires an assumed de novo occurrence without confirmation of paternity and maternity — i.e. a proband in whom the variant is present, with parental testing absent or unconfirmed that nevertheless indicates a new event. This case contains no proband and no trio or parental data of any kind: searches across the review manifest, case summary, source registry, evidence, literature pass, full-text map and paper extracts, and of prefetch.json and vcep_materials.json directly, found no proband, parental, pedigree or inheritance content. The ClinVar record (VCV000469212) is two single-submitter Uncertain significance entries that say nothing about de novo status, and the only full-text paper (PMID:25741868) is the ACMG/AMP framework with no variant or gene-specific data. Because no assumed de novo event is described anywhere in this record, PM6 cannot be applied; the assumed-de-novo premise itself is absent rather than merely unconfirmed. Generic ACMG/AMP 2015 governs, as no ClinGen CSPEC or POLD1-specific specification exists.

Rule

ACMG/AMP 2015 PM6 (PMID:25741868): assumed de novo, but without confirmation of paternity and maternity

Evidence considered

• PM6 definition applied from the generic ACMG/AMP 2015 guideline (PMID:25741868); no ClinGen CSPEC or POLD1 gene-specific specification exists (case_summary compact_evidence.cspec.found = false; vcep_materials.json: 'no cspec url available', framework source = generic_acmg_fallback).
• Case-wide search for proband/parental evidence found nothing: no occurrence of 'de novo', 'assumed de novo', 'proband', 'parental', 'pedigree', 'meiosis' or 'segregation' in any case evidence file, including prefetch.json and vcep_materials.json read directly.
• ClinVar (source_registry key 'clinvar'), VCV000469212: 2 submitters, both Uncertain significance, criteria provided by single submitters; no de novo assertion, no parental testing statement, no expert-panel review (expert_panels = {}).
• Paper-extract review (paper_extracts.json, PMID:25741868): the sole fetched full text is the ACMG/AMP 2015 standards document, variant_mentioned = false; it defines PM6 as assumed de novo without confirmed parentage but reports no case data for POLD1 c.1681C>T.
• Variant context: NM_002691.4:c.1681C>T is a rare missense (gnomAD v2.1 AF 8.18e-06, gnomAD v4.1 AF 6.24e-07; ClinVar VUS), so no inherited-vs-de-novo determination can be inferred from frequency or classification alone.

Still missing

• No proband record: the variant is not tied to an affected individual for whom inheritance could be inferred.
• No parental testing information, even unconfirmed, that would allow the assumed-de-novo path to be considered.

Sources

Reasoning

PP1 (Richards et al. 2015, PMID:25741868) requires co-segregation of the variant with disease in multiple affected family members, which presupposes at least one informative pedigree with genotype data on affected relatives. This case provides no pedigree, no family members, and no meioses: an exhaustive search of the review manifest, case summary, source registry, evidence, literature pass, full-text map and paper extracts, and direct reads of prefetch.json and vcep_materials.json, found no segreg, pedigree, family-history or affected-relative content. The ClinVar record (VCV000469212) contains only two single-submitter Uncertain significance submissions, and the sole full-text paper (PMID:25741868) is the ACMG/AMP framework document with no family data. With zero informative meioses available, no co-segregation can be counted and PP1 cannot be scored. Generic ACMG/AMP 2015 applies because no ClinGen CSPEC or POLD1-specific specification exists.

Rule

ACMG/AMP 2015 PP1 (PMID:25741868): co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease

Evidence considered

• PP1 definition applied from the generic ACMG/AMP 2015 guideline (PMID:25741868); no ClinGen CSPEC or POLD1-specific framework is available for this case (case_summary compact_evidence.cspec.found = false; vcep_materials.json: framework source = generic_acmg_fallback, 'no cspec url available').
• Search of all case evidence files for segregation content ('segreg', 'co-segregation', 'pedigree', 'proband', 'meioses', 'family history', 'affected relative') returned no matches, including prefetch.json and vcep_materials.json read directly.
• ClinVar (source_registry key 'clinvar'), VCV000469212: two germline Uncertain significance submissions with 'criteria provided, single submitter' status; no family or segregation data reported by either submitter.
• Paper-extract review (paper_extracts.json, PMID:25741868): the only fetched full text (ACMG/AMP 2015 standards) is a general framework, variant_mentioned = false, and reports no co-segregation observations for this variant.

Still missing

• No pedigree or family structure for the proband.
• No genotype data on affected or unaffected relatives; zero informative meioses available for co-segregation counting.

Sources

Reasoning

PP4 requires that the patient's phenotype or family history be highly specific for a disease with a single genetic etiology. The case provides no proband-level clinical information - no HPO terms, no clinical synopsis, no age/onset, and no family history - and no affected-individual description for c.1681C>T appears in any retrieved source. The only phenotype-adjacent data are the ClinVar condition labels on VCV000469212 ('Colorectal cancer, susceptibility to, 10' and 'not provided'), which are gene-level disease associations attached to laboratory submissions rather than findings about this patient. Colorectal cancer predisposition is also not a phenotype restricted to a single genetic etiology, so a phenotype-specificity argument would additionally need case-level specificity data that are absent. With no proband phenotype to compare against the gene's disease spectrum, PP4 cannot be adjudicated and is recorded as not assessed.

Rule

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

Evidence considered

• No proband phenotype, HPO term, clinical synopsis, or family history is present anywhere in the case evidence for this variant.
• ClinVar VCV000469212 lists conditions 'Colorectal cancer, susceptibility to, 10' and 'not provided', which are gene-level disease associations attached to the laboratory submissions rather than documented features of an individual patient.
• POLD1 germline disease (proofreading-associated polyposis with colorectal, endometrial and possibly brain cancer predisposition) is not a phenotype with a single genetic etiology in the generic ACMG sense, so phenotype-based PP4 support would require case-specific specificity data that are absent.

Still missing

• No proband phenotype, HPO terms, age at onset, or family history in the case evidence; phenotype specificity for the POLD1 disease spectrum cannot be evaluated.
• ClinVar conditions on VCV000469212 ('Colorectal cancer, susceptibility to, 10'; 'not provided') are submitter-level disease labels, not patient findings.

Sources

Reasoning

BS3 (Richards et al. 2015, PMID:25741868) requires well-established functional studies showing no damaging effect on protein function. No functional assay of POLD1 NM_002691.4:c.1681C>T (NP_002682.2:p.(Arg561Trp)) was identified in the case evidence - there are no polymerase-activity, proofreading-fidelity, or cell-based complementation data for this allele - so the criterion has no evidence base in either direction. OncoKB reports no variant-specific reviewed functional evidence for POLD1 R561W, and the ClinVar record for variation 469212 contains no submitter assertion of normal or retained function; the only criterion signal extracted from the two submissions is PM2, and both submitters classify the variant as Uncertain significance. Non-functional observations cannot substitute: the gnomAD v4.1 population frequency (AF 6.24e-07, 1/1,601,662 alleles) and the somatic COSMIC observation (COSV70957138, n=2) do not measure protein function and cannot be repurposed to satisfy BS3, and neither can the in-silico scores. Because no assay was performed, the absence of a functional study is an evidence gap rather than evidence of retained function, so BS3 is recorded as not_assessed rather than not_met. Generic ACMG/AMP 2015 applies; no POLD1-specific VCEP calibration is available for this case (framework_complete=false).

Evidence considered

• BS3 requires well-established functional studies showing no damaging effect on protein function (Richards et al. 2015, ACMG/AMP, PMID:25741868). No such study was identified for POLD1 c.1681C>T (p.Arg561Trp).
• OncoKB returned 'Unknown Oncogenic Effect' for POLD1 R561W and states no variant-specific reviewed functional evidence was identified; there is therefore no curated functional result of any direction to cite.
• The ClinVar record for variation 469212 (two submissions, SCV000806473 and SCV000646500) contains no assertion of normal function and no BS3 application; both submitters report Uncertain significance and the only extracted criterion signal is PM2.
• Non-functional data present in the case cannot support BS3: gnomAD v4.1 AF 6.24e-07 (1/1,601,662 alleles, 0 homozygotes) and gnomAD v2.1 AF 8.18e-06 are population observations, the COSMIC entry (COSV70957138, n=2 somatic) is a somatic occurrence count, and REVEL (0.386) / BayesDel (0.0477) are in-silico predictions - none of these is a functional assay of protein activity.
• PMID:25741868 full text was reviewed for BS3 guidance; it defines the criterion but contains no POLD1 or c.1681C>T data (variant_mentioned=false after searching 'c.1681C>T', 'p.R561W', 'R561W', 'POLD1').
• PMID:28492532 (Sherloc) addresses criteria weighting only, reports no variant-level functional evidence, and does not mention c.1681C>T.
• No POLD1-specific VCEP/gene framework exists for this case (framework_complete=false), so no gene-specific BS3 calibration or pre-specified functional assay list is available.

Still missing

• No functional assay demonstrating normal or near-normal POLD1 function for c.1681C>T (p.Arg561Trp) was identified; the absence of a study is not evidence of retained function.
• No POLD1-specific VCEP or gene-specific specification is available to define assay validity, controls, or BS3 strength for this gene.
• No functional/tumor-level data exist for p.Arg561Trp itself; mutational-signature and proofreading-deficiency findings in the POLD1 literature concern other alleles, so no cross-variant inference was made.

Sources

Reasoning

BS4 (Richards et al. 2015, PMID:25741868) requires an observed lack of segregation in affected members of a family, i.e. affected relatives who carry the variant without co-segregating, or affected relatives shown not to carry it. No family data exists in this case: searches of the review manifest, case summary, source registry, evidence, literature pass, full-text map and paper extracts, plus direct reads of prefetch.json and vcep_materials.json, found no pedigree, proband, parental or segregation content. Absence of family data is not itself BS4 evidence — non-segregation must actually be demonstrated, and nothing here demonstrates it. The ClinVar record (VCV000469212) holds only two single-submitter Uncertain significance submissions without family data, and the only full-text paper (PMID:25741868) is the ACMG/AMP framework. BS4 therefore cannot be evaluated; the generic ACMG/AMP 2015 definition applies because no ClinGen CSPEC or POLD1-specific specification exists.

Rule

ACMG/AMP 2015 BS4 (PMID:25741868): lack of segregation in affected members of a family

Evidence considered

• BS4 definition applied from the generic ACMG/AMP 2015 guideline (PMID:25741868); no ClinGen CSPEC or POLD1-specific specification governs this case (case_summary compact_evidence.cspec.found = false; vcep_materials.json: 'no cspec url available', framework source = generic_acmg_fallback).
• Search of all case evidence files for non-segregation evidence ('segreg', 'not segregate', 'pedigree', 'affected relative', 'proband', 'family history') returned no matches; prefetch.json and vcep_materials.json read directly also contain none.
• ClinVar (source_registry key 'clinvar'), VCV000469212: 2 single-submitter germline Uncertain significance records (PreventionGenetics; Labcorp/Invitae) with no segregation or family data; conditions listed are 'Colorectal cancer, susceptibility to, 10' and 'not provided'.
• Paper-extract review (paper_extracts.json, PMID:25741868): only the ACMG/AMP 2015 standards document was fetched as full text; it is a general framework, variant_mentioned = false, and provides no segregation observations.

Still missing

• No pedigree or relative genotypes, so non-segregation can neither be shown nor excluded.
• No affected family members reported in ClinVar submissions or available literature for this variant.

Sources

Reasoning

BP2 (Richards et al. 2015, PMID:25741868) has two limbs: (1) the variant is observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or (2) it is observed in cis with a pathogenic variant in any inheritance pattern. Both limbs require a documented second variant on a known allele, and neither can be evaluated here. NM_002691.4:c.1681C>T (NP_002682.2:p.Arg561Trp) is presented as a bare variant with no proband, no pedigree, no relative genotypes and no reported co-occurring POLD1 variant; targeted searches of every case evidence file for phase, cis, trans, zygosity and compound-heterozygous terms return nothing for this variant. The ClinVar record (variation 469212) consists of two single-submitter Uncertain significance submissions - SCV000806473 (PreventionGenetics) and SCV000646500 (Labcorp/Invitae) - neither of which reports allele phase, a second variant in the same individual, or BP2 among applied criteria. POLD1's principal germline disorder is autosomal dominant (heterozygous exonuclease-domain proofreading variants causing polyposis and colorectal/endometrial cancer predisposition), which is the setting in which BP2's first limb would be meaningful, but the criterion is only satisfied by a positive observation of a pathogenic variant in trans; its absence from the record is not such an observation, and the lack of any cis/trans data means no benign inference can be drawn. The one full text available (PMID:25741868) is the ACMG/AMP framework and reports no variant-level data. BP2 is therefore not_assessed (insufficient evidence) rather than not_met, since the criterion is evaluable in principle but the record contains no allelic data. No ClinGen CSPEC or POLD1-specific VCEP/specification exists (case_summary compact_evidence.cspec.found = false; vcep_materials.json 'no cspec url available'), so the generic ACMG/AMP 2015 definition applies.

Rule

generic_acmg_2015: requires observation in cis with a pathogenic variant, or in trans for a fully penetrant dominant gene/disorder; no phase data available -> not_assessed

Evidence considered

• BP2 definition applied from the generic ACMG/AMP 2015 guideline (Richards et al., PMID:25741868), because no ClinGen CSPEC and no POLD1-specific framework were available for this case (case_summary compact_evidence.cspec.found = false; vcep_materials.json: 'no cspec url available'; final_classification_framework.found = false, source = generic_acmg_fallback).
• ClinVar variation 469212 (VCV000469212) reviewed for allelic information: two single-submitter Uncertain significance submissions (SCV000806473 PreventionGenetics; SCV000646500 Labcorp Genetics/Invitae), germline origin, conditions 'Colorectal cancer, susceptibility to, 10' and 'not provided', 0 expert-panel submissions. No phase, zygosity or second-variant information is reported, and BP2 is not among the criteria signalled in the submission text.
• Case-wide search for cis/trans evidence: review_manifest.json, case_summary.json, source_registry.json, prefetch.json, evidence.json, literature_pass.json, fulltext_map.json and paper_extracts.json, plus vcep_materials.json, clinvar_submission_audit.json and evidence_brief.json read directly - no occurrence of 'cis', 'trans', 'phase', 'zygosity' or 'compound heterozygous' for this variant; the case carries no proband or family record.
• Inheritance context: POLD1's principal germline disorder is autosomal dominant - heterozygous proofreading/exonuclease-domain variants cause polymerase proofreading-associated polyposis with colorectal, endometrial and possibly brain cancer predisposition (gene_context.json gene summary; OncoKB gene background) - which is the setting in which BP2's trans limb would apply, but no trans observation exists for this variant.
• The only full-text publication retrieved for this case, PMID:25741868, is the ACMG/AMP 2015 standards and guidelines document; per its pre-computed extraction it is a framework paper that does not mention c.1681C>T or p.Arg561Trp (variant_mentioned = false), so it provides no cis/trans data.
• Gene-level literature retrieved during the case (e.g. a digenic PMS2/POLD1 sibship, PMID:36291559, involving POLD1 p.Asp316Asn) concerns different alleles and cannot establish cis or trans status for p.Arg561Trp.

Still missing

• No cis/trans phase data for c.1681C>T: no allele-level genotyping, parental genotypes or long-read/phasing information is present in the case record.
• No co-occurring POLD1 variant is documented in the same individual, so neither BP2 limb (pathogenic variant in trans for a dominant disorder, or in cis for any inheritance pattern) can be tested.
• No proband or family data at all in this case, and the ClinVar submissions for variation 469212 provide no phase or zygosity information.
• No ClinGen CSPEC or POLD1-specific VCEP exists to define acceptable phasing evidence or BP2 usage for this gene.

Sources

Reasoning

BP5 requires that the variant be observed in a case with an alternate molecular basis for the disease. No affected individual carrying POLD1 c.1681C>T (p.Arg561Trp) is described in any retrieved source, so there is no case in which a competing cause could be documented, and the case provides no proband genetic testing data, no co-occurring pathogenic variant, and no second locus information. One retrieved paper reports teenage-onset colorectal cancer in siblings carrying a POLD1 proofreading-domain variant together with a PMS2 exon 12 deletion (PMID:36291559), but that POLD1 change is p.Asp316Asn, not c.1681C>T, so it does not establish BP5 for this variant. With no case-level data for this variant, BP5 cannot be adjudicated and is recorded as not assessed rather than met or not met.

Rule

Generic ACMG/AMP 2015 BP5 (Richards et al., PMID:25741868): the variant is found in a case with an alternate molecular basis for disease. No numeric likelihood-ratio threshold governs BP5 here (no VCEP/CSPEC specification exists for POLD1), so no <= or >= comparison applies.

Evidence considered

• No affected individual carrying POLD1 c.1681C>T is described in any retrieved source; no case exists in which an alternate molecular basis for the disease could be documented.
• PMID:36291559 reports teenage-onset colorectal cancer in siblings carrying a POLD1 proofreading-domain variant in trans with a PMS2 exon 12 deletion, but the POLD1 variant is p.Asp316Asn, not p.Arg561Trp, so that case does not support BP5 for this variant.
• The case evidence contains no additional variant, co-occurring pathogenic finding, or genetic testing data for the proband.

Still missing

• No proband-level data for this variant (no genotype, no co-occurring variants, no second-locus testing), so an alternate molecular basis for disease cannot be identified or excluded.