POLD1 c.1562G>A, p.(Arg521Gln)

Reasoning

PM2 is met at supporting strength under the generic fallback. The variant is rare rather than absent: gnomAD v4.1 AF is 0.000161129 (0.016113%) with grpmax FAF 0.00017902 (0.017902%), well below the applicable 0.1% rare-variant threshold. The low frequency is replicated in gnomAD v2.1 and gnomAD-Canada.

Rule

Generic ACMG/AMP fallback: PM2 supporting for a variant absent from controls or present at a rare frequency below 0.1%.

Evidence considered

• No POLD1-specific ClinGen VCEP specification or local gene framework was available; generic ACMG/AMP fallback applies.
• gnomAD v4.1: 260/1,613,618 alleles, AF 0.016113%, zero homozygotes, and grpmax FAF 0.017902%. gnomAD v2.1: 33/282,008 alleles, AF 0.011702%, zero homozygotes, grpmax FAF 0.016803%. gnomAD-Canada: 2/18,344 alleles, AF 0.010903%, zero homozygotes.
• Generic fallback operational population threshold: PM2 supporting is applicable when population AF is below 0.1%.

Sources

Reasoning

PS1 requires a different nucleotide change producing the identical amino acid substitution (p.Arg521Gln) that has already been established as pathogenic. No such alternate-codon comparator was identified: the exact protein change under review (NP_002682.2:p.Arg521Gln) is itself the variant being classified and is reported in ClinVar as Uncertain significance/Conflicting (7 US, 1 US, 1 LB across submitters; no P/LP submissions), and the dedicated POLD1 exonuclease-domain (ED) variant survey (PMID:32792570) classified this same substitution as likely benign, not pathogenic. No independent pathogenic reference point exists for p.Arg521Gln, so PS1 cannot be met.

Rule

PS1: same amino-acid change as an established pathogenic variant (different nucleotide change)

Evidence considered

• case_summary.json: ClinVar VariationID 239244 classification is Uncertain significance with conflicting interpretations (7 clinical labs Uncertain significance, 1 Likely benign, 1 Uncertain Significance); no pathogenic/likely pathogenic submission exists for p.Arg521Gln itself.
• PMID:32792570 (Mur et al., Genet Med 2020), Table 1: classifies POLD1 c.1562G>A;p.Arg521Gln as 'Uncertain significance (PM1 and PM2)' by automated InterVar and 'Likely benign (PM1, BS2_supporting and BP4)' by manual ACMG/AMP curation - i.e., the exact variant under review is not itself an established pathogenic reference, so no same-amino-acid-change pathogenic comparator is available.

Still missing

• No systematic search was performed across ClinVar/literature for a different NM_002691.4 codon substitution at position 1562-1563 that would also encode p.Arg521Gln and carry an independent pathogenic classification.

Sources

Reasoning

p.Arg521Gln lies within the broader POLD1 exonuclease/proofreading domain (ED, one of five ED missense variants surveyed by Mur et al.) but is explicitly annotated as located 'outside Exo' (i.e., outside the core catalytic Exo I-III active-site motifs) in PMID:32792570's structural/variant table. That same study demonstrates substantial benign/uncertain missense variation within this same outside-Exo-motif portion of the ED: of five such outside-Exo ED variants examined (p.Ile307Met, p.Ile325Val, p.Arg352Cys, p.Arg521Gln, p.Arg525Trp), none were confirmed pathogenic - p.Arg352Cys and p.Arg521Gln were classified likely benign and the remainder VUS. CancerHotspots.org also returned no significant somatic hotspot at codon R521 (evidence.json/hotspots result: 'This variant does not lie in a statistically significant hotspot'). Because documented benign/uncertain variation exists in this same non-active-site subregion of the domain, and no somatic mutational hotspot supports R521, the 'without benign variation' precondition for PM1 is not satisfied.

Rule

PM1: located in a mutational hot spot and/or critical, well-established functional domain without benign variation

Evidence considered

• PMID:32792570 Table 1 and Fig. 3: p.Arg521Gln is annotated '(outside Exo/Yes)' - located outside the core Exo catalytic motifs though within the broader exonuclease domain (ED); structural Fig. 3 places R521Q outside the DNA-binding pocket residues highlighted for POLE/POLD1 pathogenic hotspot variants.
• PMID:32792570 text: 'The five ED variants identified in POLD1, p.Ile307Met, p.Ile325Val, p.Arg352Cys, p.Arg521Gln, and p.Arg525Trp, were predicted to be neutral... only two, p.Arg352Cys and p.Arg521Gln, affected conserved amino acids in yeast. Since all gathered evidence for those two was enough to classify them as likely benign...we did not perform the yeast-based functional assay' - indicating this ED subregion tolerates missense substitutions without pathogenic consequence.
• evidence.json hotspots result (CANCERHOTSPOTS query 'POLD1 R521'): search_status 'no_result', evidence_sentence 'This variant does not lie in a statistically significant hotspot.' (no source_registry key exists for this tool result so it is not cited as an evidence_ref).

Still missing

• No ClinGen VCEP-defined POLD1 domain/hotspot map exists to confirm the precise boundaries of functionally critical exonuclease active-site residues versus flanking domain residues for R521.
• A formal cancerhotspots.org record was queried but returned no data (search_status: no_result), so absence of a somatic hotspot could not be independently corroborated beyond the single query.

Sources

Reasoning

PP2 requires the gene to have a low rate of benign missense variation and missense to be a recognized common disease mechanism. While POLD1-associated PPAP (polymerase-proofreading-associated polyposis) is indeed caused predominantly by missense variants in the exonuclease domain (e.g., recurrent p.Ser478Asn in 3 families, p.Leu474Pro per PMID:26133394), the same domain also tolerates a substantial burden of benign/uncertain missense variation: PMID:32792570's systematic ED-variant survey found that of five distinct outside-active-site ED missense variants identified across >2,800 hereditary-cancer probands (including the variant under review), none reached a confirmed pathogenic classification - two (including p.Arg521Gln) were classified likely benign and three remained VUS. This indicates benign missense variation in POLD1's functionally important domain is not rare, weakening the 'low rate of benign missense variation' precondition for PP2. No ClinGen VCEP-derived or formal gnomAD missense constraint (Z-score/o:e) metric was available in this case bundle to quantify gene-level missense intolerance (cspec.found=false, gene_vcep_dir=null).

Rule

PP2: missense variant in a gene with low rate of benign missense variation and where missense variants are a common mechanism of disease

Evidence considered

• PMID:26133394 abstract/intro: 'Two recurrent pathogenic variants, POLE p.L424V and POLD1 p.S478N, have been identified in 21 and 3 families, respectively. A novel mutation... POLD1, p.L474P, was found in a hereditary nonpolyposis CRC family' - establishing missense as the principal known POLD1 PPAP disease mechanism.
• PMID:32792570: among five surveyed outside-active-site POLD1 exonuclease-domain missense variants (p.Ile307Met, p.Ile325Val, p.Arg352Cys, p.Arg521Gln, p.Arg525Trp) identified across a >2,800-proband hereditary cancer cohort, ACMG/AMP manual curation classified two as likely benign and three as VUS, with zero reaching pathogenic - demonstrating appreciable benign/uncertain missense variation within this functionally important domain.

Still missing

• No formal ClinGen missense constraint metric (e.g., gnomAD missense Z-score or o/e ratio) was available in the case bundle to quantitatively assess POLD1's overall missense tolerance at the gene level.
• No POLD1-specific VCEP PP2 gene-applicability determination exists to consult; this assessment relies on qualitative literature evidence from a single domain-focused variant survey.

Sources

Reasoning

No POLD1-specific ClinGen VCEP CSPEC was retrieved for this case (cspec.found=false; final_classification_framework.found=false, source=generic_acmg_fallback), so the case's own generic ACMG/AMP PP3/BP4 in-silico calibration rule governs. This variant, NM_002691.4:c.1562G>A / NP_002682.2:p.(Arg521Gln), is a missense substitution, so per the governing rule exactly one mechanism applies: REVEL only, never combined with the SpliceAI splice-path score for the same variant. Local REVEL lookup gives REVEL=0.278 (chr19:50407050 G>A), which falls in the defined gray zone [0.250, 0.750] (neither PP3 nor BP4 threshold reached). PP3 is therefore not met on the missense/REVEL path. A SpliceAI score is also present for this variant (max delta score 0.75, DS_AG=0.75) but the governing rule explicitly forbids applying the splice-path threshold to a missense-classified variant, so it is not used to trigger PP3 here. BayesDel score (-0.106183) is present in the source registry but has no verified published calibration threshold available to this pipeline and per instruction must be treated as not_available for PP3/BP4 regardless of any recalled cutoff.

Rule

generic_acmg_PP3_BP4_missense_REVEL

Evidence considered

• Local REVEL predictor lookup: REVEL score = 0.278 for chr19:50407050 G>A (NM_002691.4:c.1562G>A), sourced from local REVEL v1.3 data file per source_registry 'revel' key.
• Generic ACMG/AMP PP3/BP4 in-silico calibration rule (case's own generic_acmg_classification_rules.md, cites PMID 25741868 as source basis for the combination framework): for missense variants use REVEL only; REVEL > 0.750 triggers PP3, REVEL < 0.250 triggers BP4, REVEL in [0.250, 0.750] triggers neither. 0.278 falls in this gray zone.
• Rule explicitly states a missense variant's own SpliceAI score does not substitute for its REVEL-assessed protein-level effect; SpliceAI max delta 0.75 was not applied to this missense variant for that reason.
• BayesDel score -0.106183 available in source_registry ('bayesdel' key) but treated as not_available for PP3/BP4 because no verified published calibration threshold with a named PMID is available to this pipeline.

Still missing

• No POLD1/PolE-PolD1 ClinGen VCEP CSPEC PP3/BP4 lookup table was available to override the generic REVEL gray-zone result.
• BayesDel score exists but lacks a citable, verified published threshold, so it could not be used.

Sources

Reasoning

ClinVar has no exact-variant expert-panel Pathogenic or Likely pathogenic assertion for POLD1 c.1562G>A (p.Arg521Gln); therefore PP5 cannot be applied.

Rule

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

Evidence considered

• The exact-variant ClinVar submission audit reports zero expert-panel submissions. The listed assertions are ordinary laboratory or other non-expert submissions and include uncertain significance and a single likely benign assertion.

Sources

Reasoning

BA1 is not met. The highest reliable population estimate is gnomAD v4.1 grpmax FAF 0.00017902 (0.017902%); the highest raw subgroup AF is 0.000395979 (0.039598%) in the Remaining_XX stratum. Both are far below the generic fallback BA1 threshold of 1%.

Rule

Generic ACMG/AMP fallback: BA1 requires an allele frequency greater than 1% in an appropriate population dataset.

Evidence considered

• No POLD1-specific ClinGen VCEP specification or local gene framework was available; generic ACMG/AMP fallback applies.
• gnomAD v4.1 reports 260/1,613,618 alleles (AF 0.016113%), zero homozygotes, and grpmax FAF 0.017902%; the highest raw subgroup AF is 0.039598% (13/32,830) in Remaining_XX. gnomAD v2.1 and gnomAD-Canada likewise show low frequencies (0.011702% and 0.010903%, respectively).
• Generic fallback operational population threshold: BA1 requires AF >1%.

Sources

Reasoning

BS1 is not met. gnomAD v4.1 grpmax FAF is 0.00017902 (0.017902%), and even the highest raw subgroup AF, 0.039598%, remains below the generic fallback BS1 threshold of 0.3%. No frequency excess incompatible with POLD1-associated dominant cancer predisposition is demonstrated.

Rule

Generic ACMG/AMP fallback: BS1 requires an allele frequency greater than expected for the disorder; the applicable operational threshold is >0.3%.

Evidence considered

• No POLD1-specific ClinGen VCEP specification or local gene framework was available; generic ACMG/AMP fallback applies.
• gnomAD v4.1 reports 260/1,613,618 alleles, zero homozygotes, and a grpmax FAF of 0.017902%; gnomAD v2.1 reports AF 0.011702% and gnomAD-Canada reports AF 0.010903%.
• Generic fallback operational population threshold: BS1 requires AF >0.3%.

Sources

Reasoning

BP1 applies only to genes for which disease is primarily caused by truncating variants; applying it to a missense variant in such a gene supports benign classification. POLD1's established germline cancer-predisposition mechanism (PPAP) is caused predominantly by exonuclease-domain missense variants (e.g., recurrent p.Ser478Asn, p.Leu474Pro per PMID:26133394; p.Asp316Gly/His per PMID:32792570), not by truncating variants. PMID:32792570 additionally identified only a small number (n=6) of germline loss-of-function (frameshift/nonsense) POLD1 variants in its cohort, indicating truncating variation is not the predominant disease mechanism for this gene. Because the gene-level precondition ('primarily truncating variants known to cause disease') is not met for POLD1, BP1 is not applicable/not met for this missense variant.

Rule

BP1: missense variant in a gene for which primarily truncating variants are known to cause disease

Evidence considered

• PMID:26133394: recurrent POLD1 pathogenic missense variants (p.Ser478Asn in 3 families, p.Leu474Pro) in the exonuclease/proofreading domain are the established disease-causing mechanism for POLD1-associated CRC/polyposis, not truncating variants.
• PMID:32792570, 'Germline loss-of-function POLE and POLD1 variants' section: only six heterozygous LoF POLD1/POLE variants (frameshift/nonsense) were identified across the full cohort, versus a larger and better-characterized set of exonuclease-domain missense variants driving the PPAP phenotype - indicating missense, not truncating, variation is the dominant recognized POLD1 disease mechanism.

Sources

Reasoning

Same governing generic ACMG/AMP PP3/BP4 in-silico calibration rule as PP3 (no POLD1 VCEP CSPEC available; framework_mode=generic_acmg). For this missense variant (p.Arg521Gln), REVEL=0.278 falls within the gray zone [0.250, 0.750], which is below the BP4 REVEL<0.250 threshold, so BP4 is not met. SpliceAI (max delta 0.75) is not applicable to this missense variant under the rule's mechanism-exclusivity requirement, and would in any case indicate a possible splice gain-of-function signal rather than a benign one. BayesDel (-0.106183) is not usable for BP4 for the same reason given under PP3: no verified, citable published calibration threshold is available to this pipeline.

Rule

generic_acmg_PP3_BP4_missense_REVEL

Evidence considered

• Local REVEL predictor lookup: REVEL score = 0.278 for chr19:50407050 G>A (NM_002691.4:c.1562G>A); 0.278 is above the BP4 REVEL<0.250 threshold defined in the generic rule, so it does not clear BP4.
• Generic ACMG/AMP PP3/BP4 in-silico calibration rule (case's own generic_acmg_classification_rules.md): for missense variants use REVEL only, mechanism-exclusive with SpliceAI.
• BayesDel score -0.106183 present in source_registry ('bayesdel' key) but not usable for BP4 absent a named, verified published calibration threshold.

Still missing

• No POLD1 VCEP-specific PP3/BP4 lookup table available to override the generic REVEL gray-zone result.
• BayesDel score exists but lacks a citable, verified published threshold for a benign-direction cutoff.

Sources

Reasoning

ClinVar has no exact-variant expert-panel Benign or Likely benign assertion for POLD1 c.1562G>A (p.Arg521Gln); therefore BP6 cannot be applied.

Rule

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

Evidence considered

• The exact-variant ClinVar submission audit reports zero expert-panel submissions. A Likely benign assertion from an ordinary laboratory is not eligible for BP6.

Sources

Reasoning

No confirmed de novo occurrence is documented for NM_002691.4:c.1562G>A (p.Arg521Gln). The bundle contains no parental genotypes, parentage confirmation, or phenotype information for both parents; PS2 cannot be applied.

Rule

Generic ACMG/AMP (PMID:25741868): PS2 requires a confirmed de novo occurrence in an affected individual with no family history; no POLD1-specific specification was retrieved.

Evidence considered

• No proband-parent trio, parental test result, parentage confirmation, or parental phenotype is present in the consolidated case-evidence bundle.
• Generic ACMG/AMP PS2 requires a de novo variant with maternity and paternity confirmed.

Still missing

• Variant testing in both biological parents with parentage confirmation.
• Phenotypic assessment of the proband and both parents sufficient to evaluate phenotype consistency and parental status.

Sources

Reasoning

No validated functional assay directly interrogating POLD1 p.Arg521Gln protein/enzyme function was identified. Mur et al. (PMID:32792570) performed a yeast-based exonuclease/mutator-phenotype assay for several POLD1 exonuclease-domain (ED) variants (their operational definition of PS3/BS3: PS3 = mutator phenotype observed in S. pombe assay), but explicitly did not perform this yeast assay for p.Arg521Gln, stating that other gathered evidence (REVEL benign prediction, yeast-conservation) was already sufficient to classify it likely benign without running the functional assay. No official ClinGen VCEP/CSPEC exists for POLD1 in this case (framework_mode = generic_acmg fallback), so no gene-specific PS3 rule is available either. No other publication in this case's evidence set reports a functional/enzymatic assay of this exact variant.

Evidence considered

• PMID:32792570 (Mur et al. 2020, Table 1, footnote g): defines PS3 operationally as 'functional studies are supportive of a damaging effect on the gene (supporting: when a mutator phenotype in S. pombe was observed with p=0.01-0.001; moderate: when p<0.001)'.
• PMID:32792570, main text: 'All POLD1 ED variants identified in the study were predicted benign, and only two, p.Arg352Cys and p.Arg521Gln, affected conserved amino acids in yeast. Since all gathered evidence for those two was enough to classify them as likely benign (Table 1), we did not perform the yeast-based functional assay.' -- confirms the yeast mutator assay (the paper's PS3-defining assay) was NOT run for this variant.
• GeneDx ClinVar submission (SCV000293778) signal text: 'in the absence of RNA/functional studies, the actual effect of this sequence change is unknown' -- corroborates absence of functional data from an independent clinical laboratory.

Still missing

• No exonuclease activity or polymerase-fidelity functional assay has been performed specifically for POLD1 p.Arg521Gln
• No RASA/RNA splicing functional study available
• No ClinGen POLD1/POLE VCEP specification exists to define validated assay(s) and PS3 strength calibration for this gene

Sources

Reasoning

No exact-variant case-control enrichment analysis or statistically interpretable case excess was identified for POLD1 c.1562G>A (p.Arg521Gln).

Rule

Generic ACMG/AMP PS4: prevalence in affected individuals must be significantly increased compared with controls; no POLD1-specific specification was retrieved.

Evidence considered

• Mur et al. report p.Arg521Gln in one family/proband context and tumor characterization, not an exact-variant case-control comparison.

Still missing

• A validated exact-variant case-control dataset with case and control allele counts and an association statistic is required.

Sources

Reasoning

PM5 requires a different missense change at the identical residue (e.g., p.Arg521Trp, p.Arg521Leu, p.Arg521Pro) that has been established as pathogenic/likely pathogenic. The pipeline's own PM5 comparator-harvesting step explicitly did not run (pm5_mode 'unknown', eligible_for_classic_pm5_search false, zero candidates collected) because classic same-residue PM5 semantics could not be confirmed safely for this case. Full-text review of the POLD1-focused papers available in this case (PMID:32792570, PMID:26133394, PMID:23263490, PMID:33809179) did not surface any report of an alternate amino-acid substitution at codon 521 with an independent pathogenic classification.

Rule

PM5: novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before

Evidence considered

• pm5_candidates.json: found=false, pm5_mode='unknown', eligible_for_classic_pm5_search=false, candidate_summary shows 0 total_candidates_seen and 0 same_residue_candidates; recommendation level 'not_applicable' with explicit instruction not to apply PM5 from this artifact alone.
• PMID:32792570, the most comprehensive POLD1 exonuclease-domain variant survey available in this case, reports only p.Arg521Gln at codon 521 among its cohort and cites no alternate substitution at this residue with a differing, pathogenic outcome.

Still missing

• pm5_candidates.json artifact reports pm5_mode='unknown' and explicitly instructs: 'Review whether any collected same-residue comparator variants satisfy the governing PM5 rule; do not apply PM5 from this artifact alone' - a dedicated ClinVar/literature search for other p.Arg521X substitutions at this codon was not performed within this case bundle.
• No comparator alternate-residue variant with pathogenic/likely pathogenic classification was located in the reviewed sources.

Sources

Reasoning

No de novo occurrence with unconfirmed parentage is documented for NM_002691.4:c.1562G>A (p.Arg521Gln). The bundle provides no parental test results; PM6 cannot be applied.

Rule

Generic ACMG/AMP (PMID:25741868): PM6 requires an assumed de novo occurrence without confirmation of paternity and maternity; no POLD1-specific specification was retrieved.

Evidence considered

• No proband-parent trio, parental test result, or parentage information is present in the consolidated case-evidence bundle.
• Generic ACMG/AMP PM6 is reserved for an assumed de novo occurrence when parental origin is not confirmed.

Still missing

• Parental testing showing absence of the variant; if parentage remains unconfirmed, document that limitation.
• Proband phenotype and family-history information to assess phenotype consistency.

Sources

Reasoning

No informative cosegregation or meiosis count is documented for this variant. A directly reviewed POLD1 study reports the exact variant in Family O but records no cosegregation analysis; PP1 cannot be applied.

Rule

Generic ACMG/AMP (PMID:25741868): PP1 requires cosegregation in multiple affected family members in a gene definitively known to cause the disease; no POLD1-specific specification was retrieved.

Evidence considered

• PMID:32792570 Table 1 reports POLD1 c.1562G>A (p.Arg521Gln) in Family O with the phenotype entry "CRC (×2) 34" and lists cosegregation as "n.p." (not performed).
• No pedigree, relative genotypes, or informative meioses for the exact variant are present in the consolidated case-evidence bundle.
• Generic ACMG/AMP PP1 requires cosegregation of the variant with disease in multiple affected family members.

Still missing

• Pedigree with affected and unaffected relatives, their variant genotypes, ages, and relevant cancer/polyposis phenotypes.
• Number of informative meioses and assessment of phenocopies, reduced penetrance, and age-dependent onset.

Sources

Reasoning

Early-onset colorectal cancer reported for this variant is not a highly specific phenotype for a single genetic etiology, and the reported carrier tumor lacked POLE/POLD1 exonuclease-domain-associated mutational signatures.

Rule

Generic ACMG/AMP PP4: the patient's phenotype or family history must be highly specific for a disease with a single genetic etiology; no POLD1-specific specification was retrieved.

Evidence considered

• Mur et al. report the exact variant in a family with two colorectal cancers diagnosed at age 34, but state that a CRC from a p.Arg521Gln carrier was hypermutated without POLE/POLD1-associated signatures.

Still missing

• A phenotype or tumor molecular signature shown to be highly specific for pathogenic POLD1 variation and present in an exact-variant carrier is required.

Sources

Reasoning

BS2 is not assessed. Population datasets show zero homozygotes, but no curated observations of this variant in unaffected, appropriately aged individuals with phenotype information sufficient for a penetrant POLD1-associated disorder were provided. Population allele counts alone do not establish BS2.

Rule

Generic ACMG/AMP fallback: BS2 requires observation in healthy adult individual(s) for a disorder expected to be fully penetrant at an early age; allele counts or absence of homozygotes alone are insufficient.

Evidence considered

• gnomAD v4.1, gnomAD v2.1, and gnomAD-Canada each report zero homozygotes. The gnomAD-Canada age histogram has only two heterozygote observations and does not provide disease-status adjudication.
• No POLD1-specific ClinGen VCEP specification or local gene framework was available; generic ACMG/AMP fallback applies.

Still missing

• Curated genotype-phenotype evidence documenting unaffected, appropriately aged heterozygous carriers is needed to assess BS2.

Sources

Reasoning

No completed functional assay result (damaging or benign) is available for POLD1 p.Arg521Gln under a validated assay system. Mur et al. (PMID:32792570) explicitly skipped the yeast-based exonuclease/mutator-phenotype assay for this variant (their assay defining both PS3 and BS3 in this gene context), relying instead on in silico prediction (REVEL 0.278, benign) and tumor sequencing observations. Tumor genomic sequencing of a POLD1 p.Arg521Gln carrier's CRC showed hypermutation without POLE/D1-associated mutational signatures, but the authors' own criterion mapping assigns this pattern to BP5 (alternate molecular basis) rather than to BS3/functional-assay evidence, since it is not the validated protein-function assay (yeast mutator assay) used for BS3 in their framework. No POLD1 VCEP/CSPEC is available to define a validated functional assay and BS3 calibration for this gene.

Evidence considered

• PMID:32792570 (Mur et al. 2020, Table 1, footnote g): defines BS3 operationally as 'functional studies showed no damaging effect on protein function (supporting: when a mutator phenotype in S. pombe was not observed)' -- this assay was not run for p.Arg521Gln.
• PMID:32792570, main text: 'a CRC from a POLD1 p.Arg521Gln carrier showed hypermutation but none of the POLE/D1-associated mutational signatures' -- tumor sequencing observation; per the same paper's own criterion definitions (footnote g), this pattern maps to BP5 (alternate molecular basis for hypermutation), not to BS3, since BS3 in this framework is specifically defined by the S. pombe mutator assay result.

Still missing

• No completed benign-supporting functional assay (e.g., yeast exonuclease/mutator assay) is available for this specific variant
• Tumor mutational-signature data exists but is not a validated protein-function assay applicable to BS3 per the only relevant publication's own criterion definitions
• No ClinGen POLD1/POLE VCEP specification exists to define validated assay(s) and BS3 strength calibration for this gene

Sources

Reasoning

No informative non-segregation is documented for NM_002691.4:c.1562G>A (p.Arg521Gln). The directly reviewed exact-variant report records cosegregation as not performed, not as lack of segregation; BS4 cannot be applied.

Rule

Generic ACMG/AMP (PMID:25741868): BS4 requires lack of segregation in affected members of a family; no POLD1-specific specification was retrieved.

Evidence considered

• PMID:32792570 Table 1 reports POLD1 c.1562G>A (p.Arg521Gln) in Family O and gives "n.p." for cosegregation; this is missing information rather than evidence of non-segregation.
• No relative genotype-phenotype data establishing non-segregation are present in the consolidated case-evidence bundle.
• Generic ACMG/AMP BS4 requires lack of segregation in affected members of a family.

Still missing

• Genotypes and phenotypes of multiple informative affected and unaffected relatives.
• Documentation that affected relatives lacking the variant have a comparable phenotype and that age-related penetrance and phenocopies have been considered.

Sources

Reasoning

There is no case-level observation of p.Arg521Gln in cis with a pathogenic/likely pathogenic POLD1 variant, or in trans with a pathogenic/likely pathogenic POLD1 variant in a dominant disease context. BP2 cannot be applied from the available evidence.

Rule

Generic ACMG/AMP (PMID:25741868): BP2 requires an observed qualifying cis/trans co-occurrence with a pathogenic variant; no such observation is available here.

Evidence considered

• PMID:25741868 states that, in dominant disorders, detection of a variant in trans with a pathogenic variant can support a benign impact under BP2; it also describes a pathogenic second variant in cis as supporting, but not definitive, benign evidence in the recessive setting.
• The consolidated case-evidence bundle contains no allelic co-occurrence or phase observation for NM_002691.4:c.1562G>A (p.Arg521Gln).

Still missing

• No co-occurring POLD1 pathogenic/likely pathogenic variant was reported in the same individual.
• No parental, long-read, or other phase evidence establishes cis or trans configuration.
• No affected/unaffected co-occurrence phenotype data were provided.

Sources

Reasoning

The reported carrier CRC lacked POLE/POLD1-associated signatures, but no independently established alternate molecular diagnosis explaining the patient's phenotype was provided; this does not meet BP5.

Rule

Generic ACMG/AMP BP5: the variant is found in a case with an alternate molecular basis for disease; no POLD1-specific specification was retrieved.

Evidence considered

• Mur et al. report that the CRC from a p.Arg521Gln carrier was hypermutated but had none of the POLE/POLD1-associated mutational signatures; this finding alone does not establish another molecular diagnosis.

Still missing

• An independently established alternate molecular cause of the affected individual's phenotype is required.

Sources