POLD1 c.2251-23A>G, p.?

Reasoning

No applicable POLD1-specific VCEP specification was identified, so the supplied generic BS1 threshold of allele frequency >=0.01 was applied. The variant exceeds this threshold in multiple population views: gnomAD v2.1 has overall genome AF 0.0126578 and highest subgroup AF 0.0471935 in AFR_XX, while gnomAD v4.1 has overall genome AF 0.0121687 and highest subgroup AF 0.0452842 in AFR_XX. The v2.1 and v4.1 all-comers totals are 0.00458295 and 0.00231565, respectively, and the ancestry-specific frequencies provide clear evidence of an excess population frequency for a rare disease allele. BS1 is met at strong strength under the generic framework.

Rule

{'source': 'generic ACMG/AMP population-frequency default supplied for this review', 'threshold': 'allele frequency >=0.01', 'strength': 'strong'}

Evidence considered

• No governing POLD1-specific VCEP framework was available; the supplied generic BS1 threshold is allele frequency >=0.01.
• gnomAD v2.1 reports genome AF 0.012657824257110062 and AFR_XX AF 0.047193500738552434; its overall AF is 0.00458295142071494.
• gnomAD v4.1 reports genome AF 0.012168679448598499 and AFR_XX AF 0.04528421257543313; its overall AF is 0.0023156516309417573.

Sources

Reasoning

No applicable POLD1-specific VCEP specification was identified. The variant has 27 homozygotes in gnomAD v2.1 and 86 homozygotes in gnomAD v4.1, including 86 homozygotes in the v4.1 African/African American subgroup. This is substantial homozygote observation in population data and supports BS2 under the generic ACMG/AMP framework. However, the available records are all-comers gnomAD summaries rather than a specifically phenotyped healthy-adult cohort, and POLD1-associated cancer risk may involve age-dependent penetrance; therefore the evidence is strong but warrants human review of the health and ascertainment context.

Rule

{'source': 'generic ACMG/AMP 2015', 'criterion': 'observation of the variant in healthy adults, including homozygotes when relevant', 'strength': 'strong'}

Evidence considered

• gnomAD v2.1 reports 27 homozygotes overall, including 15 exome homozygotes and 12 genome homozygotes.
• gnomAD v4.1 reports 86 homozygotes overall, including 86 in the African/African American subgroup; the v4.1 record reports 43 exome and 43 genome homozygotes.
• The generic BS2 interpretation requires observation in healthy individuals; the available gnomAD records establish homozygotes in population data but do not provide individual-level phenotype or age ascertainment.

Still missing

• No explicitly phenotyped healthy-adult or non-cancer gnomAD subset was available in the case evidence for independent confirmation of BS2 ascertainment.
• Age-dependent penetrance and disease ascertainment for POLD1-associated cancer predisposition are not quantified in the available population record.

Sources

Reasoning

The variant is intronic (NM_002691.4:c.2251-23A>G), so BP4 is evaluated through the SpliceAI path only. SpliceAI reports a maximum delta of 0.002 from component scores DS_AG 0.002, DS_AL 0.000, DS_DG 0.000, and DS_DL 0.001. This is at or below the generic SpliceAI BP4 supporting threshold of <=0.1 from the Jaganathan et al. 2019 calibration (PMID:30661751), supporting BP4 at supporting strength.

Rule

Generic SpliceAI calibration: BP4 supporting <=0.1.

Evidence considered

• The variant is intronic, so SpliceAI is the applicable BP4 path and REVEL is not applicable.
• SpliceAI component scores are DS_AG 0.002, DS_AL 0.000, DS_DG 0.000, and DS_DL 0.001; the maximum delta is 0.002.
• The generic SpliceAI BP4 supporting threshold is <=0.1, as supplied from Jaganathan et al. 2019 (PMID:30661751).

Sources

Reasoning

No applicable POLD1-specific VCEP specification was identified, so the supplied generic PM2 supporting threshold of allele frequency <=0.0001 was applied. The variant is substantially more common than this threshold in both default all-comers datasets: gnomAD v2.1 overall AF is 0.00458295 and its lowest relevant aggregate is still far above the PM2 cutoff, while gnomAD v4.1 overall AF is 0.00231565. The presence of 27 and 86 homozygotes in gnomAD v2.1 and v4.1, respectively, is also incompatible with treating this allele as absent or sufficiently rare for PM2. PM2 is not met.

Rule

{'source': 'generic ACMG/ClinGen SVI population-frequency default supplied for this review', 'threshold': 'allele frequency <=0.0001', 'strength': 'supporting'}

Evidence considered

• No governing POLD1-specific VCEP framework was available; the supplied generic PM2 threshold is allele frequency <=0.0001.
• gnomAD v2.1 reports overall AF 0.00458295142071494 from 1180/257476 alleles and 27 homozygotes.
• gnomAD v4.1 reports overall AF 0.0023156516309417573 from 3664/1582276 alleles and 86 homozygotes.

Sources

Reasoning

The variant is intronic (NM_002691.4:c.2251-23A>G), so PP3 is evaluated through the SpliceAI path only. SpliceAI reports DS_AG 0.002, DS_AL 0.000, DS_DG 0.000, and DS_DL 0.001, with a maximum delta of 0.002. This is below the generic SpliceAI PP3 supporting threshold of >=0.2 and the moderate threshold of >=0.5 from the Jaganathan et al. 2019 calibration (PMID:30661751); therefore PP3 is not met.

Rule

Generic SpliceAI calibration: PP3 supporting >=0.2; PP3 moderate >=0.5.

Evidence considered

• The variant is intronic, so SpliceAI is the applicable PP3/BP4 path and REVEL is not applicable.
• SpliceAI component scores are DS_AG 0.002, DS_AL 0.000, DS_DG 0.000, and DS_DL 0.001; the maximum delta is 0.002.
• The generic SpliceAI PP3 supporting threshold is >=0.2 and the moderate threshold is >=0.5, as supplied from Jaganathan et al. 2019 (PMID:30661751).

Sources

Reasoning

PP5 is restricted here to an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. ClinVar variation 676636 has two exact-variant laboratory submissions, classified Likely benign and Benign, with review status criteria provided/single submitter; the record reports zero expert-panel submissions. Therefore PP5 is not met. The ordinary laboratory submissions and aggregate ClinVar label cannot trigger PP5.

Rule

PP5: apply only when an exact-variant ClinVar expert panel classifies the variant as Pathogenic or Likely pathogenic; ordinary submissions, aggregate labels, and non-expert assertions do not qualify.

Evidence considered

• The exact variant NM_002691.4:c.2251-23A>G is ClinVar variation 676636.
• ClinVar reports two exact-variant submissions, Likely benign and Benign, both criteria provided/single submitter, and expert_panel_submissions = 0.
• The reviewed publication PMID:25741868 does not mention POLD1 or c.2251-23A>G and does not provide an expert-panel classification for this variant.

Sources

Reasoning

No applicable POLD1-specific VCEP specification was identified, so the generic ACMG/AMP stand-alone BA1 threshold of allele frequency >=0.05 was applied (ACMG/AMP 2015, PMID:25741868). The highest observed ancestry-specific frequency is 0.0471935 in gnomAD v2.1 AFR_XX, while gnomAD v4.1 reports 0.0452842 in AFR_XX; both are below 0.05. The combined gnomAD v2.1 and v4.1 totals are also below the threshold (0.00458295 and 0.00231565, respectively), so BA1 is not met.

Rule

{'source': 'generic ACMG/AMP 2015', 'threshold': 'allele frequency >=0.05', 'application': 'highest observed ancestry-specific or overall frequency must meet the threshold'}

Evidence considered

• No governing POLD1-specific VCEP framework was available; the generic ACMG/AMP BA1 threshold is allele frequency >=0.05 (PMID:25741868).
• gnomAD v2.1 reports the highest subgroup frequency as 0.047193500738552434 in AFR_XX; the overall frequency is 0.00458295142071494 across 1180/257476 alleles.
• gnomAD v4.1 reports the highest subgroup frequency as 0.04528421257543313 in AFR_XX; the overall frequency is 0.0023156516309417573 across 3664/1582276 alleles.

Sources

Reasoning

BP6 is restricted here to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. ClinVar variation 676636 has two exact-variant laboratory submissions, classified Likely benign and Benign, with review status criteria provided/single submitter; the record reports zero expert-panel submissions. Therefore BP6 is not met. The laboratory submissions and aggregate benign label cannot trigger BP6.

Rule

BP6: apply only when an exact-variant ClinVar expert panel classifies the variant as Benign or Likely benign; ordinary submissions, aggregate labels, and non-expert assertions do not qualify.

Evidence considered

• The exact variant NM_002691.4:c.2251-23A>G is ClinVar variation 676636.
• ClinVar reports exact-variant laboratory classifications of Likely benign and Benign, both criteria provided/single submitter, and expert_panel_submissions = 0.
• The reviewed publication PMID:25741868 does not mention POLD1 or c.2251-23A>G and does not provide an expert-panel classification for this variant.

Sources

Reasoning

No proband-level de novo observation is documented for NM_002691.4:c.2251-23A>G. The available case evidence does not report the proband's genotype in relation to either parent, parental testing results, confirmed maternity/paternity, or a de novo event; therefore PS2 cannot be assessed under the generic ACMG/AMP framework.

Evidence considered

• The case evidence contains no documented proband-parent genotype comparison or parental testing result for NM_002691.4:c.2251-23A>G.
• No publication in the available literature extract reports this specific variant in a de novo proband-parent context.

Still missing

• Proband genotype and clinical presentation linked to this variant
• Targeted testing results for both biological parents
• Confirmation of maternity and paternity
• Evidence excluding parental mosaicism if a de novo event is suspected

Reasoning

No validated functional assay was identified for NM_002691.4:c.2251-23A>G. The available evidence is limited to SpliceAI prediction (maximum delta score 0.002), which is computational rather than experimental and cannot establish a disease-relevant functional abnormality. The generic ACMG/AMP guidance states that predicted splice effects require confirmation by RNA, protein, or in vitro minigene analysis; no such assay result, controls, or biologically relevant functional readout is documented for this variant.

Evidence considered

• SpliceAI reports a maximum delta score of 0.002 for NM_002691.4:c.2251-23A>G; this is an in-silico prediction and does not satisfy PS3 functional evidence.
• The generic ACMG/AMP guidance states that predicted splice effects require confirmation by RNA or protein analysis or an in vitro minigene splicing assay; the cited paper does not report a functional assay for this variant.

Still missing

• A validated, disease-relevant functional assay for the variant is unavailable.
• Assay controls and quantitative comparison with appropriate wild-type and pathogenic or benign controls are unavailable.
• No experimental result establishes abnormal POLD1 splicing, transcript, protein, or polymerase function.

Sources

Reasoning

PS4 requires a statistically significant increase of the exact variant or an appropriate variant class in affected individuals compared with controls. The available case evidence contains no affected-case series, control comparison, odds ratio, confidence interval, or other case-control enrichment analysis for NM_002691.4:c.2251-23A>G, so PS4 cannot be assessed.

Rule

Generic ACMG/AMP PS4: prevalence is significantly increased in affected individuals compared with controls.

Evidence considered

• No case-control prevalence or enrichment data for NM_002691.4:c.2251-23A>G were identified in the supplied evidence.

Still missing

• Affected-case and control counts or a validated enrichment statistic for this variant or an appropriately defined variant class are unavailable.

Reasoning

No applicable POLD1-specific specification was identified, so the generic ACMG/AMP PM3 rule applies. PM3 requires the variant to be observed in trans with a pathogenic or likely pathogenic variant in an affected individual for a recessive disorder. The available case evidence contains no affected-proband observation, second pathogenic allele, phase result, or inheritance data establishing such a configuration. The variant's presence in population databases and its ClinVar submissions do not provide allelic-phase or affected-proband evidence. Therefore PM3 cannot be assessed from the available evidence.

Rule

Generic ACMG/AMP PM3: apply only when the variant is observed in trans with a pathogenic or likely pathogenic variant in an affected individual with a recessive disorder.

Evidence considered

• The case evidence identifies NM_002691.4:c.2251-23A>G as an intronic POLD1 variant but provides no affected-proband observation, second pathogenic allele, phase result, or inheritance data.
• Generic ACMG/AMP PM3 requires observation of the variant in trans with a pathogenic or likely pathogenic allele in an affected individual with a recessive disorder; that required allelic configuration is not documented here.

Still missing

• Affected-proband clinical observation
• A second pathogenic or likely pathogenic allele
• Validated trans-phase or inheritance data
• A confirmed recessive POLD1 disease context, if applicable

Reasoning

No likely de novo occurrence is documented for NM_002691.4:c.2251-23A>G. The case evidence provides neither a clinical proband report with absent parental testing nor sufficient family and phenotype information to support a presumed de novo interpretation; therefore PM6 cannot be assessed under the generic ACMG/AMP framework.

Evidence considered

• The available case evidence does not document a proband with this variant, unavailable or untested parents, or a presumed de novo clinical observation.
• No available paper extract reports this specific variant as a likely de novo finding.

Still missing

• A documented affected proband carrying the variant
• Clinical and phenotype details supporting a disease-relevant occurrence
• Evidence that both parents were untested or unavailable
• Any family or reproductive context supporting presumed de novo status

Reasoning

No informative family segregation data are documented for NM_002691.4:c.2251-23A>G. The available evidence identifies no affected or unaffected relatives, tested meioses, genotype-phenotype concordance, or independent families in which the variant segregates; therefore PP1 cannot be assessed under the generic ACMG/AMP framework.

Evidence considered

• The case evidence contains no family pedigree, relative testing, meiosis count, or segregation result for this variant.
• No available paper extract provides variant-specific segregation data for NM_002691.4:c.2251-23A>G.

Still missing

• Pedigree and phenotypes of informative relatives
• Genotypes of affected and unaffected family members
• Number of informative meioses and independent families
• Assessment of phenotype consistency within segregating families

Reasoning

PP4 requires a phenotype or highly specific phenotype consistent with a disease for which the gene is known to cause disease. The supplied case materials provide no patient phenotype, diagnostic features, or disease-specific clinical description that can be compared with a POLD1-associated disorder, so PP4 cannot be assessed.

Rule

Generic ACMG/AMP PP4: the patient's phenotype or family history is highly specific for a disease with a single-gene etiology.

Evidence considered

• No patient phenotype or phenotype-specific diagnostic information was identified in the supplied evidence.

Still missing

• A documented patient phenotype and the relevant POLD1-associated disease phenotype are unavailable for specificity assessment.

Reasoning

No validated functional assay was identified showing that NM_002691.4:c.2251-23A>G has a normal or benign effect. The available SpliceAI maximum delta score of 0.002 is computational and cannot establish normal biological function. The generic ACMG/AMP guidance indicates that splice effects require experimental confirmation by RNA, protein, or in vitro minigene analysis; no such assay result, assay controls, or biologically relevant normal-function readout is documented for this variant.

Evidence considered

• SpliceAI reports a maximum delta score of 0.002 for NM_002691.4:c.2251-23A>G; this computational result cannot establish normal biological function for BS3.
• The generic ACMG/AMP guidance identifies RNA, protein, or in vitro minigene testing as the relevant experimental approaches for confirming splice effects; no benign-function assay for this variant is reported.

Still missing

• A validated, disease-relevant functional assay demonstrating normal variant function is unavailable.
• Assay controls and quantitative comparison with appropriate wild-type and pathogenic controls are unavailable.
• No experimental result demonstrates normal POLD1 splicing, transcript, protein, or polymerase function.

Sources

Reasoning

No non-segregation observation is documented for NM_002691.4:c.2251-23A>G. The available case evidence does not identify an affected family in which the variant is absent, an unaffected carrier in an informative pedigree, or another phenotype- and inheritance-inconsistent result; therefore BS4 cannot be assessed under the generic ACMG/AMP framework.

Evidence considered

• The case evidence contains no affected non-carrier, unaffected carrier, or other variant-specific non-segregation observation.
• No available paper extract reports this specific variant in a family demonstrating non-segregation.

Still missing

• An informative pedigree with phenotype assignments
• Testing results for affected relatives and unaffected carriers
• Evidence that relatives were assessed at an appropriate age for the phenotype
• Evaluation of phenocopies, reduced penetrance, and variable expressivity

Reasoning

No applicable POLD1-specific specification was identified, so the generic ACMG/AMP BP2 rule applies. BP2 requires a documented allelic relationship with a pathogenic or likely pathogenic variant: in trans for a fully penetrant dominant disorder or in cis for a recessive disorder. The available case evidence contains no phase-resolved second pathogenic allele, affected-proband observation, or inheritance result establishing either configuration. Population observations and ClinVar submissions do not establish phase. Therefore BP2 cannot be assessed from the available evidence.

Rule

Generic ACMG/AMP BP2: apply only with a documented pathogenic or likely pathogenic allele in trans for a fully penetrant dominant disorder or in cis for a recessive disorder.

Evidence considered

• The case evidence provides no phase-resolved observation, second pathogenic or likely pathogenic allele, affected-proband result, or inheritance information for NM_002691.4:c.2251-23A>G.
• Generic ACMG/AMP BP2 requires a pathogenic allele in trans for a fully penetrant dominant disorder or in cis for a recessive disorder; neither configuration is documented here.

Still missing

• A phase-resolved second pathogenic or likely pathogenic allele
• Inheritance data showing trans or cis configuration
• Disease-mode confirmation needed to select the applicable BP2 configuration

Reasoning

BP5 requires a benign explanation for the patient's phenotype by a pathogenic variant in another gene, or equivalent disease-specific evidence under an applicable specification. No patient phenotype, alternative pathogenic variant, or documented alternative molecular diagnosis is supplied for this case, so BP5 cannot be assessed.

Rule

Generic ACMG/AMP BP5: a pathogenic variant in an alternative gene explains the phenotype.

Evidence considered

• No alternative pathogenic variant or alternative diagnosis explaining a documented phenotype was identified in the supplied evidence.

Still missing

• An alternative molecular diagnosis and clinical evidence showing that it explains the patient's phenotype are unavailable.