POLE c.1924-5C>T, p.?

Reasoning

No POLE-specific population rule was identified, so generic PM2 applies at supporting strength. The variant is absent from gnomAD v2.1 non-cancer and is rare in gnomAD v3.1 non-cancer at AF 6.757761288840233e-06; it is also rare in the default all-comers datasets, with AF 7.959692119108834e-06 in gnomAD v2.1 and 4.337470040474792e-06 in gnomAD v4.1. These frequencies are below the generic PM2 supporting threshold of <=0.0001, and all datasets report zero homozygotes.

Rule

Generic PM2 at Supporting strength: rare or absent from appropriate population databases at allele frequency <=0.0001; no POLE-specific population rule was provided.

Evidence considered

• gnomAD v2.1 non-cancer reports the variant as absent.
• gnomAD v3.1 non-cancer reports 1/147978 genome alleles, AF 6.757761288840233e-06, and zero homozygotes.
• Default all-comers gnomAD v2.1 reports AF 7.959692119108834e-06 and gnomAD v4.1 reports AF 4.337470040474792e-06; both report zero homozygotes.
• The generic PM2 supporting threshold is allele frequency <=0.0001, based on the ClinGen SVI recommendation cited with ACMG/AMP guidance (PMID:25741868).

Sources

Reasoning

The variant is an intronic splice-region variant (NM_006231.4:c.1924-5C>T; NP_006222.2:p.?), so the applicable computational path is SpliceAI. SpliceAI maximum delta is 0.084 (DS_AG 0.007, DS_AL 0.084, DS_DG 0.017, DS_DL 0.000), meeting the generic BP4 supporting threshold of <=0.1 from Jaganathan et al. 2019 (PMID:30661751). No exact-variant pre-assigned BP4 code was found in PATH-250-323-s003.xlsx or PATH-250-323-s004.xlsx; the POLE custom BP4 rule is missense/table-based and does not govern this splice-region variant.

Rule

Generic SpliceAI BP4 supporting: maximum delta <=0.1; POLE custom BP4 exact-table rule is limited to missense variants.

Evidence considered

• The variant is intronic/splice-region, so SpliceAI is the sole applicable PP3/BP4 predictor path; REVEL is not applicable.
• SpliceAI reported DS_AG 0.007, DS_AL 0.084, DS_DG 0.017, and DS_DL 0.000; maximum delta 0.084.
• The generic SpliceAI BP4 supporting cutoff is <=0.1, from Jaganathan et al. 2019 (PMID:30661751); 0.084 meets it.
• The POLE custom BP4 rule requires an exact missense entry in PATH-250-323-s003.xlsx or PATH-250-323-s004.xlsx with REVEL likely benign and at least four benign in-silico results; this splice-region variant has no such entry.

Still missing

• No exact entry for c.1924-5C>T or p.? was found in PATH-250-323-s003.xlsx.
• No exact entry for c.1924-5C>T or p.? was found in PATH-250-323-s004.xlsx.
• No exact variant-specific computational assignment was found in PATH-250-323.pdf.

Sources

Reasoning

PVS1 is not met. NM_006231.4:c.1924-5C>T is an intronic splice-region SNV located five bases upstream of the exon 17 boundary, not a nonsense, frameshift, or canonical ±1,2 splice-consensus variant. VariantValidator predicts NP_006222.2:p.? and provides no demonstrated exon-skipping, aberrant transcript, NMD, or protein-loss result. The generic ClinGen SVI PVS1 framework therefore does not place this variant in a default PVS1 null-variant bucket; no PVS1 downgrade tier is applicable because PVS1 is not triggered. The POLE local framework is not an applicable specification for PVS1 and does not alter this generic consequence assessment.

Rule

Generic ClinGen SVI PVS1: do not apply PVS1 to a splice-region variant outside the canonical ±1,2 splice consensus without evidence establishing a null transcript consequence.

Evidence considered

• VariantValidator places NM_006231.4:c.1924-5C>T in intronic sequence immediately upstream of exon 17 and predicts NP_006222.2:p.?.
• The generic ClinGen SVI PVS1 framework is applicable to established null-variant buckets such as nonsense, frameshift, and canonical splice-consensus variants; this -5 splice-region SNV is outside those default buckets.
• The ACMG/AMP guideline states that splice variants may cause exon skipping, exon shortening, intronic-material inclusion, or alternative splice-site use, so a specific abnormal-splicing conclusion requires functional confirmation; this paper reports no result for the exact POLE variant.

Still missing

• No RNA or other functional assay demonstrates aberrant splicing, exon loss, or NMD for this variant.

Sources

Reasoning

The governing POLE framework permits PS4_Supporting only for exact missense variants recurrent in both COSMIC and TCGA endometrial-carcinoma cohorts, with combined EC count >=10, and in the established pathogenic hotspot set. NM_006231.4:c.1924-5C>T is a splice-region intronic variant with protein consequence p.? and was not found in PATH-250-323.pdf or PATH-250-323-s002.xlsx under c.1924-5C>T, p.?, or p.?; therefore the exact-variant predicate fails before the >=10 count test. PATH-250-323-s001.tif is unavailable for text review and cannot establish recurrence.

Rule

PS4_Supporting requires exact variant recurrence in both COSMIC and TCGA endometrial-carcinoma cohorts, combined EC count >=10, and membership in the specified pathogenic hotspot set; capped at Supporting.

Evidence considered

• The POLE framework states: Apply PS4_Supporting only when the exact POLE missense variant is recurrent in both the COSMIC and TCGA endometrial carcinoma cohorts, has a combined EC count >=10, and belongs to the established/pathogenic set; it specifically names p.P286R, p.V411L, p.A456P, and p.S297F.
• The exact strings c.1924-5C>T, NP_006222.2:p.?, and p.? were not found in the converted full text of PATH-250-323.pdf or PATH-250-323-s002.xlsx.
• The case annotation identifies the variant as splice_region_variant, not missense, and COSMIC reported no prior somatic cancer occurrence.

Still missing

• No exact-variant entry was found in PATH-250-323.pdf.
• No exact-variant entry was found in PATH-250-323-s002.xlsx.
• PATH-250-323-s001.tif was unavailable for conversion and manual visual review was not performed.

Sources

Reasoning

The variant is an intronic splice-region variant (NM_006231.4:c.1924-5C>T; NP_006222.2:p.?), so the applicable computational path is SpliceAI rather than REVEL. SpliceAI maximum delta is 0.084, below the generic PP3 supporting threshold of >=0.2 from Jaganathan et al. 2019 (PMID:30661751), and no exact-variant pre-assigned PP3 code was found in PATH-250-323-s003.xlsx or PATH-250-323-s004.xlsx. The governing POLE custom PP3 rule is missense/table-based and is therefore not applicable to this splice-region variant.

Rule

Generic SpliceAI PP3 supporting: maximum delta >=0.2; POLE custom PP3 exact-table rule is limited to missense variants.

Evidence considered

• The variant is intronic/splice-region, so SpliceAI is the sole applicable PP3/BP4 predictor path; REVEL is not applicable.
• SpliceAI reported DS_AG 0.007, DS_AL 0.084, DS_DG 0.017, and DS_DL 0.000; maximum delta 0.084.
• The generic SpliceAI PP3 supporting cutoff is >=0.2, from Jaganathan et al. 2019 (PMID:30661751); 0.084 does not meet it.
• The POLE custom PP3 rule requires an exact missense entry in PATH-250-323-s003.xlsx or PATH-250-323-s004.xlsx with REVEL likely disease causing and no more than one benign in-silico result; this splice-region variant has no such entry.

Still missing

• No exact entry for c.1924-5C>T or p.? was found in PATH-250-323-s003.xlsx.
• No exact entry for c.1924-5C>T or p.? was found in PATH-250-323-s004.xlsx.
• No exact variant-specific computational assignment was found in PATH-250-323.pdf.

Sources

Reasoning

ClinVar variation 240412 contains Likely benign submissions from clinical laboratories and no expert-panel submission; there is no exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification. The ordinary laboratory submissions and aggregate ClinVar label cannot trigger PP5.

Rule

Apply PP5 only for an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification; ordinary laboratory, aggregate, and non-expert assertions do not qualify.

Evidence considered

• ClinVar variation 240412 is NM_006231.4(POLE):c.1924-5C>T and reports expert_panels as empty; submissions are single-submitter laboratory assertions.
• The ClinVar record summarizes Likely benign classifications from five clinical laboratories and Uncertain significance from one clinical laboratory, with criteria-provided/conflicting review status.
• PP5 was restricted to an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification; none is present.

Sources

Reasoning

No applicable POLE-specific population-frequency rule was identified, so the generic ACMG/AMP BA1 threshold applies. The variant's highest observed all-comers frequency is 7.959692119108834e-06 in gnomAD v2.1 and 4.337470040474792e-06 in gnomAD v4.1; the highest reported subpopulation frequency is 0.0002016942315449778 in gnomAD v3.1 non-cancer. All are far below the generic BA1 stand-alone threshold of 0.05 from ACMG/AMP guidance (PMID:25741868).

Rule

Generic ACMG/AMP BA1: stand-alone benign when allele frequency is >=0.05; no POLE-specific population rule was provided.

Evidence considered

• gnomAD v2.1 reports 2/251266 alleles, AF 7.959692119108834e-06, with zero homozygotes and East Asian AF 0.00010873110796999022.
• gnomAD v4.1 reports 7/1613844 alleles, AF 4.337470040474792e-06, with zero homozygotes and highest listed subpopulation AF 4.801536491677337e-05.
• gnomAD v3.1 non-cancer reports 1/147978 genome alleles, AF 6.757761288840233e-06, with highest listed East Asian AF 0.0002016942315449778 and zero homozygotes.
• The generic ACMG/AMP BA1 stand-alone threshold is allele frequency >=0.05 (PMID:25741868).

Sources

Reasoning

No applicable POLE-specific population-frequency rule was identified, so the generic BS1 threshold applies. The highest observed allele frequency is 0.0002016942315449778 in the gnomAD v3.1 non-cancer East Asian subgroup; all-comers frequencies are 7.959692119108834e-06 in gnomAD v2.1 and 4.337470040474792e-06 in gnomAD v4.1. These values are below the generic BS1 threshold of 0.01, so the population data do not support BS1.

Rule

Generic BS1: strong benign when allele frequency is >=0.01; no POLE-specific population rule was provided.

Evidence considered

• The highest reported frequency is 0.0002016942315449778 in the gnomAD v3.1 non-cancer East Asian subgroup.
• All-comers gnomAD frequencies are 7.959692119108834e-06 in v2.1 and 4.337470040474792e-06 in v4.1; each dataset reports zero homozygotes.
• The generic BS1 threshold is allele frequency >=0.01; no POLE-specific derivation was identified in the governing framework.

Sources

Reasoning

ClinVar variation 240412 has no expert-panel submission. Although several ordinary laboratory submissions classify the variant as Likely benign, the requested BP6 rule requires an exact-variant ClinVar expert-panel Benign or Likely benign classification, so these submissions cannot trigger BP6.

Rule

Apply BP6 only for an exact-variant ClinVar expert-panel Benign or Likely benign classification; ordinary laboratory and aggregate ClinVar labels do not qualify.

Evidence considered

• ClinVar variation 240412 reports no expert-panel submissions for NM_006231.4(POLE):c.1924-5C>T.
• The record contains Likely benign assertions from five clinical laboratories, all with criteria-provided single-submitter status, plus one Uncertain significance submission.
• BP6 was restricted to an exact-variant ClinVar expert-panel Benign or Likely benign classification; that qualifying classification is absent.

Sources

Reasoning

No case-level evidence documents the variant in a proband, testing of both biological parents, confirmed maternity and paternity, or absence of the variant in parental samples. Therefore, a confirmed or assumed de novo event cannot be established for PS2.

Rule

Generic ACMG/AMP PS2 requires a confirmed de novo variant in the affected proband with confirmed maternity and paternity; without parent testing it cannot be applied.

Evidence considered

• The case materials contain no documented proband-parent trio testing or parentage confirmation for NM_006231.4:c.1924-5C>T.
• The available literature extraction contains no variant-specific clinical case or de novo observation for this POLE variant.

Still missing

• Proband genotype report is not provided.
• Maternal and paternal variant testing results are not provided.
• Confirmed maternity and paternity are not documented.
• Parental mosaicism assessment is not documented.

Reasoning

No validated functional assay for NM_006231.4:c.1924-5C>T was identified. The variant is intronic/splice-region with predicted protein consequence p.?, and the available evidence contains no RNA transcript study, minigene assay, protein assay, or other experiment with appropriate positive and negative controls demonstrating abnormal function. SpliceAI max delta 0.084 is computational prediction, not functional evidence, and therefore cannot satisfy PS3. The local POLE framework does not specify PS3; generic ACMG/AMP PS3 requires well-established functional studies, which are absent here.

Rule

Generic ACMG/AMP PS3: functional studies showing a damaging effect can support pathogenicity when well established and appropriately controlled; no applicable POLE-specific PS3 rule was found.

Evidence considered

• The case evidence identifies NM_006231.4:c.1924-5C>T as an intronic splice-region variant with predicted protein consequence NP_006222.2:p.?.
• SpliceAI reports a maximum delta score of 0.084; this is computational prediction and does not establish PS3 functional evidence.
• PMID:25741868 states that splice effects should be confirmed by functional analysis of RNA or protein, but it does not study this POLE variant.
• PMID:26467025 describes a general variant-scoring system and provides no variant-specific functional assay or POLE-specific result for c.1924-5C>T.
• No official POLE VCEP/CSPEC specification for PS3 was available; the local POLE framework customizes PM1, PS4, PP3, and BP4, not PS3.

Still missing

• A variant-specific validated functional study is needed, preferably RNA analysis or a splice assay with appropriate normal and abnormal controls and clearly interpretable results.

Sources

Reasoning

The applicable POLE framework does not specify PM3, so generic ACMG/AMP reasoning applies. PM3 requires the variant to be observed in trans with a pathogenic or likely pathogenic variant in an affected individual with a recessive disorder, with strength determined by the number and quality of such observations. The available case evidence contains no affected-proband observation, second pathogenic allele, phase result, parental testing, or established recessive inheritance model for POLE-related disease. Therefore PM3 cannot be assessed and is not applied.

Rule

Generic ACMG/AMP PM3: consider only when the variant is observed in trans with a pathogenic or likely pathogenic variant in an affected individual with a recessive disorder; apply strength according to the number and quality of independent observations and phase evidence.

Evidence considered

• The governing León-Castillo et al. 2020 custom POLE framework customizes PM1, PS4, PP3, and BP4 only; it provides no POLE-specific PM3 rule.
• The reviewed case materials do not document an affected individual carrying this variant with a second pathogenic allele, nor do they document phase, parental testing, or recessive inheritance for the observation.

Still missing

• Affected-proband data are unavailable.
• No pathogenic or likely pathogenic second allele is documented.
• No cis/trans phase result or parental segregation data are documented.
• A recessive inheritance context applicable to this variant is not established.

Reasoning

No case-level report states that this variant occurred de novo with unconfirmed parentage, and no proband phenotype or parental testing information is available to support an assumed de novo event. PM6 is therefore not assessable.

Rule

Generic ACMG/AMP PM6 requires a variant observed de novo in an affected individual when maternity and paternity are not confirmed; no such observation is documented here.

Evidence considered

• No variant-specific de novo observation, assumed de novo designation, or parental testing limitation is reported in the available case evidence.
• The retrieved papers are general methodology or guideline papers and do not report this POLE variant in a clinical family.

Still missing

• A case report or clinical laboratory record describing the variant in a proband is not available.
• Parentage status is not documented.
• Parental testing results are not documented, even as incomplete or technically limited.

Reasoning

No affected relatives, unaffected relatives, family pedigree, variant-positive relatives, variant-negative relatives, or informative meioses are documented. Consequently, cosegregation evidence and any PP1 strength cannot be determined.

Rule

Generic ACMG/AMP PP1 requires cosegregation of the variant with disease in affected family members; strength depends on the amount and informativeness of segregation evidence.

Evidence considered

• The case materials provide no family-level segregation observations for NM_006231.4:c.1924-5C>T.
• No affected-relative phenotype concordance or informative meiosis count is available for evaluation.

Still missing

• Family pedigree and phenotype information are not provided.
• Genotypes of affected relatives are not provided.
• Genotypes of unaffected relatives are not provided.
• Number of informative meioses and segregation likelihood are unavailable.

Reasoning

No patient-level phenotype or disease-specific clinical presentation was provided that could demonstrate a highly specific phenotype for a POLE-related disorder. The reviewed POLE literature package concerns somatic endometrial-carcinoma mutation patterns and does not provide a phenotype assessment for this individual variant or patient.

Rule

PP4 requires a phenotype or family history highly specific for a disease with a single-gene etiology; no applicable phenotype-specific POLE specification was available.

Evidence considered

• The available case evidence identifies the molecular variant but does not provide a patient phenotype, clinical diagnosis, or phenotype-specific POLE syndrome findings.
• The governing POLE framework explicitly does not customize phenotype criteria, so generic PP4 requires a highly specific phenotype that is not documented here.

Still missing

• Patient-level phenotype and clinical diagnosis are not available in the reviewed evidence.

Reasoning

The population datasets report zero homozygotes in gnomAD v2.1, gnomAD v4.1, and gnomAD v3.1 non-cancer, but BS2 requires documented occurrence in healthy individuals or unaffected adults appropriate for the disease model. No such individual-level phenotype evidence is provided, and absence of homozygotes alone does not establish BS2.

Rule

BS2 requires the variant to be observed in healthy individuals or unaffected adults in a disease model where this is informative; population absence of homozygotes alone is insufficient.

Evidence considered

• gnomAD v2.1 reports homozygotes = 0, gnomAD v4.1 reports homozygotes = 0, and gnomAD v3.1 non-cancer reports homozygotes = 0.
• No case-level phenotype or unaffected-adult observation was identified; zero homozygotes in a reference population is not itself the BS2 evidence requirement.

Still missing

• Documented observation of this variant in an unaffected adult or other appropriately phenotyped healthy individual is unavailable.

Sources

Reasoning

No validated functional assay demonstrating normal function for NM_006231.4:c.1924-5C>T was identified. The available SpliceAI result (maximum delta 0.084) is computational and cannot establish normal splicing or normal protein function. There is no variant-specific RNA, minigene, protein, or other biologically relevant assay with suitable controls and a validated benign reference range to support BS3, and the local POLE framework does not specify BS3.

Rule

Generic ACMG/AMP BS3: well-established functional studies showing no damaging effect can support benign classification when appropriately controlled; no applicable POLE-specific BS3 rule or qualifying assay was found.

Evidence considered

• The case evidence identifies NM_006231.4:c.1924-5C>T as an intronic splice-region variant with predicted protein consequence NP_006222.2:p.?.
• SpliceAI reports a maximum delta score of 0.084; this computational result does not demonstrate normal splicing and cannot satisfy BS3.
• PMID:25741868 provides general ACMG guidance that splice consequences require confirmation by RNA or protein functional analysis, but it does not report a normal-function result for this variant.
• PMID:26467025 provides general rules-based scoring methodology and no variant-specific functional evidence for c.1924-5C>T.
• No official POLE VCEP/CSPEC specification for BS3 was available, and the local POLE framework does not customize BS3.

Still missing

• A variant-specific validated benign functional study is needed, with appropriate controls and a defined normal-function threshold relevant to POLE splicing or activity.

Sources

Reasoning

No affected individual known to lack the variant is identified, and no family-level non-segregation analysis is available. The absence of segregation data cannot be interpreted as evidence against causality, so BS4 is not assessable.

Rule

Generic ACMG/AMP BS4 requires the variant to be absent in affected individuals with a phenotype sufficiently specific for the disease; no such observation is documented here.

Evidence considered

• No affected variant-negative relatives or other valid non-segregation observations are reported in the available case evidence.
• Population frequency and ClinVar classifications do not establish BS4 and were not used for this criterion.

Still missing

• Affected relatives tested negative for the variant are not documented.
• Phenotype ascertainment for relatives is not documented.
• A family-based non-segregation analysis is not available.

Reasoning

The applicable POLE framework does not specify BP2, so generic ACMG/AMP reasoning applies. BP2 requires a documented co-occurrence and phase relationship with a pathogenic or likely pathogenic variant: in trans for a dominant disorder, or in cis for a recessive disorder. The available case evidence contains no second pathogenic variant, co-occurrence observation, phase result, parental testing, or sufficiently established inheritance context for this variant. Therefore BP2 cannot be assessed and is not applied.

Rule

Generic ACMG/AMP BP2: consider when the variant is observed with a pathogenic or likely pathogenic variant in trans for a dominant disorder or in cis for a recessive disorder, with the phase and inheritance model documented.

Evidence considered

• The governing León-Castillo et al. 2020 custom POLE framework customizes PM1, PS4, PP3, and BP4 only; it provides no POLE-specific BP2 rule.
• The reviewed case materials do not document a second pathogenic allele, co-occurrence in an affected individual, phase, or parental testing relevant to BP2.

Still missing

• No co-occurring pathogenic or likely pathogenic variant is documented.
• No cis/trans phase result or parental segregation data are documented.
• The inheritance model needed to interpret BP2 is not established for this observation.

Reasoning

No applicable BP5 evidence was identified. The governing POLE framework does not specify BP5, and the available evidence does not provide a validated BP5 likelihood-ratio or other BP5-specific rule for this variant. The ClinVar laboratory assertions are not BP5 evidence.

Evidence considered

• The POLE custom framework covers PM1, PS4, PP3, and BP4, not BP5.
• ClinVar contains ordinary single-submitter laboratory classifications, which are not sufficient to establish BP5 under the requested evidence policy.

Still missing

• No POLE-specific BP5 rule or applicable published BP5 threshold was identified.

Sources