POLE c.2706+5G>A, p.?

Reasoning

PM2 is met at Supporting strength. The variant is extremely rare in population databases: gnomAD v4.1 total AF 1.73509e-05 with group maximum FAF 1.454e-05, and gnomAD v2.1 total AF 7.95665e-06. There are no homozygotes. The frequency is below the 0.1% rarity threshold used for this review, supporting rarity compatible with a disease-associated variant.

Rule

Generic ACMG/AMP PM2 rule with Supporting strength for a rare variant in population databases; the reviewed operating threshold is AF <0.1%, and no POLE-specific PM2 rule was retrieved.

Evidence considered

• gnomAD v4.1 contains 28 alternate alleles among 1,613,748 alleles (AF 1.73509e-05), group maximum FAF 1.454e-05, and zero homozygotes; the highest listed population AF is 3.33267e-05 in Admixed American individuals.
• gnomAD v2.1 contains 2 alternate alleles among 251,362 alleles (AF 7.95665e-06), zero homozygotes, and highest listed population AF 2.89218e-05 in Admixed American individuals.
• The variant is below the 0.1% PM2 rarity threshold used for this review.

Still missing

• gnomAD-Canada v1.0 was unavailable because the query returned HTTP 500; ancestry-specific absence in that resource cannot be assessed.

Sources

Reasoning

PP3_Supporting is met under the generic non-missense splice-prediction fallback: this intronic +5 variant has SpliceAI maximum delta 0.381 (donor gain 0.381), exceeding the >0.2 threshold. The POLE local missense-table PP3 rule is not applicable to this intronic variant.

Rule

Generic non-missense PP3 fallback: SpliceAI maximum delta >0.2; use only the splice-impact mechanism for a non-missense variant.

Evidence considered

• SpliceAI Lookup for NM_006231.4:c.2706+5G>A reports DS_DG 0.381 and maximum delta 0.381.
• The generic non-missense workflow applies PP3_Supporting when SpliceAI maximum delta is >0.2. This threshold is attributed to Walker et al. 2023 (PMID:36413997), which provides the published calibration basis named for this threshold.

Still missing

• No RNA study is available to confirm the predicted splice consequence; this computational call does not establish the actual transcript outcome.

Sources

Reasoning

The applicable local POLE PS4 rule is limited to exact recurrent POLE missense hotspot variants meeting specified COSMIC and TCGA endometrial-cancer recurrence requirements. NM_006231.4:c.2706+5G>A is an intronic non-missense variant and was not found in COSMIC; it cannot meet that rule.

Rule

Local POLE framework: PS4_Supporting only for an exact established/pathogenic POLE missense variant recurrent in both COSMIC and TCGA endometrial-cancer cohorts with combined EC count >=10; somatic recurrence is capped at Supporting.

Evidence considered

• The local POLE framework applies PS4_Supporting only to specified exact established POLE missense variants with recurrence in both COSMIC and TCGA endometrial-cancer cohorts and a combined count of at least 10.
• The queried variant is NM_006231.4:c.2706+5G>A with predicted protein consequence p.?; it is not a missense substitution.
• The case evidence reports no COSMIC result for POLE c.2706+5G>A.

Sources

Reasoning

No exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion is present. The exact record contains only single-submitter Likely benign and uncertain-significance submissions, which are ineligible for PP5.

Rule

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

Evidence considered

• The exact ClinVar record has five submissions: four uncertain-significance and one likely-benign; its audit records zero expert-panel submissions.
• Ordinary laboratory submissions and aggregate ClinVar classifications are not used for PP5.

Sources

Reasoning

BA1 is not met. The maximum observed gnomAD v4.1 allele frequency is 3.33267e-05 in Admixed American individuals, with total allele frequency 1.73509e-05; both are far below the 1% BA1 threshold used for this review.

Rule

Generic ACMG/AMP BA1 population-frequency rule: apply BA1 when the allele frequency is greater than 1%; no POLE-specific BA1 rule was retrieved.

Evidence considered

• gnomAD v4.1 reports 28/1,613,748 alleles (AF 1.73509e-05), with the highest listed population frequency in Admixed American individuals at 2/60,012 alleles (AF 3.33267e-05).
• gnomAD v2.1 reports 2/251,362 alleles (AF 7.95665e-06), with the highest listed population frequency in Admixed American individuals at 1/34,576 alleles (AF 2.89218e-05).
• The observed frequencies are well below the 1% BA1 threshold used for this population review.

Sources

Reasoning

BS1 is not met. The highest observed allele frequency is 3.33267e-05 in gnomAD v4.1, below the 0.3% BS1 threshold used for this review; the gnomAD v4.1 group maximum FAF is 1.454e-05.

Rule

Generic ACMG/AMP BS1 population-frequency rule: apply BS1 when the allele frequency is greater than 0.3%; no POLE-specific BS1 rule was retrieved.

Evidence considered

• gnomAD v4.1 reports total AF 1.73509e-05, highest listed population AF 3.33267e-05 in Admixed American individuals, and group maximum FAF 1.454e-05.
• gnomAD v2.1 reports total AF 7.95665e-06 and highest listed population AF 2.89218e-05 in Admixed American individuals.
• All reported frequencies are below the 0.3% BS1 threshold used for this population review.

Sources

Reasoning

BS2 is not met. gnomAD v4.1 reports zero homozygotes among 1,613,748 alleles, and gnomAD v2.1 reports zero homozygotes among 251,362 alleles. No healthy homozygote observation supporting BS2 is present in the case evidence.

Rule

Generic ACMG/AMP BS2 rule: apply BS2 for observation in healthy individuals in a genotype inconsistent with a highly penetrant disorder; no such homozygote observation is available here and no POLE-specific BS2 rule was retrieved.

Evidence considered

• gnomAD v4.1 reports homozygotes = 0 overall and zero homozygotes in the listed populations, including the highest-frequency Admixed American population.
• gnomAD v2.1 reports homozygotes = 0 overall and zero homozygotes in the listed populations.
• The case bundle contains no independent observation of this variant in healthy homozygous individuals.

Sources

Reasoning

BP4 is not met. For this intronic non-missense variant, the generic splice-prediction path applies; its SpliceAI maximum delta of 0.381 is not below the BP4 threshold of 0.1 and instead supports the PP3 splice-impact path. The POLE local missense-table BP4 rule is not applicable.

Rule

Generic non-missense BP4 fallback: SpliceAI maximum delta <0.1; do not apply BP4 when the same splice predictor supports PP3.

Evidence considered

• SpliceAI Lookup for NM_006231.4:c.2706+5G>A reports maximum delta 0.381.
• The generic non-missense workflow permits BP4_Supporting only when SpliceAI maximum delta is <0.1; this threshold is attributed to Walker et al. 2023 (PMID:36413997), the named published calibration basis.

Still missing

• No RNA study is available to determine the actual splice outcome.

Sources

Reasoning

No exact-variant ClinVar expert-panel Benign or Likely benign assertion is present. Although one ordinary laboratory submission is Likely benign, non-expert submissions are ineligible for BP6.

Rule

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

Evidence considered

• The exact ClinVar record includes one Likely benign single-submitter laboratory assertion and four uncertain-significance assertions; the audit records zero expert-panel submissions.
• Ordinary laboratory submissions and aggregate ClinVar classifications are not used for BP6.

Sources

Reasoning

NM_006231.4:c.2706+5G>A is an intronic donor-region substitution, not a canonical +/-1 or +/-2 splice-site variant; its protein consequence is p.? and no RNA result establishes exon skipping, cryptic splicing, frameshift, NMD, or an in-frame outcome. Therefore a null consequence and the applicable PVS1 strength cannot be assigned.

Rule

Generic ClinGen SVI PVS1 fallback (PMC6185798): do not assign PVS1 to this non-canonical donor-region variant without evidence establishing a qualifying loss-of-function transcript and an applicable POLE loss-of-function disease mechanism.

Evidence considered

• VariantValidator normalized the variant on the MANE/refseq-select transcript as NM_006231.4:c.2706+5G>A, intron 23, with predicted protein consequence NP_006222.2:p.?; no exon or protein-level consequence is supplied.
• The ClinGen SVI PVS1 framework is the applicable generic fallback because no official POLE CSPEC/VCEP specification or local PVS1-specific rule is present. The bundled PVS1 assessment identifies this +5 variant as outside the default nonsense, frameshift, and canonical +/-1,2 splice buckets.
• SpliceAI reports donor-gain delta 0.381 (maximum delta 0.381), which is a prediction of possible splice impact but does not establish the actual transcript or a null effect.
• PMID:25741868 states that splice-site variants can have several transcript outcomes and that impact requires confirmation by RNA- or protein-based functional analysis.

Still missing

• RNA splicing evidence in an informative tissue or a validated minigene assay is needed to define the aberrant transcript(s).
• If aberrant splicing is demonstrated, determine the reading-frame consequence, predicted NMD susceptibility, and the biological importance of any affected exon(s).
• Confirm that loss of function is an established mechanism for the specific POLE-associated germline disease under assessment before applying PVS1.

Sources

Reasoning

No proband-level de novo evidence is documented in the case-evidence bundle. There is no recorded parental testing demonstrating that NM_006231.4:c.2706+5G>A is absent from both biological parents, and no information on maternity, paternity, parental mosaicism, or phenotype consistency is provided. PS2 therefore cannot be applied.

Rule

Generic ACMG/AMP PS2: confirmed de novo occurrence in a patient with the disease and no family history, with maternity and paternity confirmed; strength may be modified only when the applicable de novo framework supports such calibration.

Evidence considered

• The consolidated case-evidence bundle contains no proband/parent trio results, parental genotypes, maternity/paternity confirmation, or de novo observation for this variant.
• No applicable POLE CSPEC/VCEP specification was found; the available local POLE framework does not specify PS2, so generic ACMG/AMP PS2 logic would require confirmed de novo status that is not documented.

Still missing

• Documented proband genotype and phenotype
• Molecular confirmation of biological maternity and paternity
• Targeted parental testing showing absence of the variant in both parents
• Assessment for low-level parental mosaicism and assay sensitivity

Reasoning

No validated functional assay data for NM_006231.4:c.2706+5G>A were identified. The variant is intronic at c.2706+5, and the available evidence contains only a SpliceAI prediction, which is not a functional assay and cannot establish PS3. The applicable POLE framework does not specify PS3; therefore generic ACMG/AMP functional-evidence principles apply.

Rule

Generic ACMG/AMP PS3: apply only when well-established functional studies appropriate to the gene and disease mechanism show a damaging effect; calibrate strength to assay validity, controls, reproducibility, quantitative effect, and biological relevance. No such variant-specific study is available here.

Evidence considered

• The governing local POLE framework customizes PM1, PS4, PP3, and BP4, but does not provide a POLE-specific PS3 specification; generic ACMG/AMP principles therefore apply.
• The available case evidence reports SpliceAI max delta score 0.381, which is computational prediction rather than validated functional evidence and is insufficient for PS3.
• Richards et al. state that splice-site effects should be confirmed using RNA- or protein-based functional analysis; this paper does not report a functional result for this variant.

Still missing

• No variant-specific RNA study demonstrating abnormal splicing was provided.
• No validated protein-level or cellular assay demonstrating altered POLE function was provided.
• No assay controls, replication, quantitative effect size, or assay-to-disease-mechanism validation were provided.

Sources

Reasoning

No affected-proband, family, parental-genotype, phase, or second-variant observations are present in the case-evidence bundle. Therefore, the requirement for the variant to be observed in trans with a pathogenic or likely pathogenic variant in an appropriate recessive disease context cannot be evaluated.

Rule

Generic ACMG/AMP PM3: apply when the variant is observed in trans with a pathogenic or likely pathogenic variant in an affected individual with a recessive disorder; strength depends on the number and quality of independent informative observations and confirmed phase.

Evidence considered

• The local POLE framework explicitly customizes only PM1, PS4, PP3, and BP4 and leaves segregation, inheritance, and other generic ACMG criteria to generic ACMG/AMP assessment.
• The consolidated case-evidence bundle contains no family-level or proband-level allelic observations establishing a pathogenic variant in trans.

Still missing

• No documented affected probands carrying this variant with a pathogenic or likely pathogenic variant in trans.
• No parental testing, haplotype, or other phase evidence.
• No applicable POLE-specific PM3 specification; inheritance mode for the relevant disease context is not established in the bundle.

Reasoning

The variant is intronic and has no established protein consequence (p.?). There is no evidence of an in-frame protein length change; PM4 cannot be evaluated from the available data.

Rule

Generic ACMG/AMP PM4 applies to protein length changes from in-frame deletions/insertions or stop-loss variants; no such consequence is established here.

Evidence considered

• The normalized consequence is NP_006222.2:p.? and the bundled variant-level assessment reports unknown consequence class; no deletion, insertion, stop-loss, or in-frame alteration is documented.

Still missing

• A confirmed transcript/protein consequence is required to determine whether an in-frame protein length change occurs.

Sources

Reasoning

No case report or clinical record in the bundle describes this variant as apparently de novo without parental testing. The variant is recorded as NM_006231.4:c.2706+5G>A with an unknown protein consequence, but no proband phenotype, family history, or parental observations are supplied. PM6 cannot be applied.

Rule

Generic ACMG/AMP PM6: assumed de novo occurrence in a patient with the disease, when parental testing is unavailable or not performed; the observation and phenotype must still be documented and consistent.

Evidence considered

• The bundle provides no exact-variant case report, proband description, family history, or apparent de novo observation for NM_006231.4:c.2706+5G>A.
• The literature extraction contains no relevant paper finding for this variant or for POLE segregation/de novo evidence.

Still missing

• A published or clinical case describing the exact variant in an affected proband
• Proband phenotype consistency with the relevant POLE-related disorder
• Evidence that the variant was apparently de novo
• Clarification of whether parental testing was unavailable or simply not reported

Reasoning

No affected relatives carrying the variant, informative meioses, pedigree, or phenotype-segregation data are present in the case-evidence bundle. There is therefore no basis to evaluate cosegregation or assign PP1 at any strength.

Rule

Generic ACMG/AMP PP1: cosegregation with disease in multiple affected family members; strength should be calibrated to the number of informative meioses and the applicable disease-specific framework.

Evidence considered

• The bundle contains no pedigree, family-member genotypes, affected-relative observations, meiosis count, or segregation analysis for this variant.
• No POLE-specific PP1 rule or segregation calibration is available in the governing local framework; generic PP1 application therefore requires unavailable family data.

Still missing

• Pedigree identifying affected and unaffected relatives
• Genotypes for informative affected relatives
• Number of informative meioses
• Phenotype assessment and diagnostic criteria for each relative
• Evaluation of possible phenocopies and reduced penetrance

Reasoning

No individual-level phenotype, tumor phenotype, or phenotype-to-gene specificity assessment is supplied. The condition labels attached to the ClinVar record are not a patient phenotype and cannot establish that this individual's presentation is highly specific for a POLE-associated disorder.

Rule

Generic ACMG/AMP PP4: the patient's phenotype or family history must be highly specific for a disease with a single genetic etiology.

Evidence considered

• ClinVar lists multiple condition labels for this exact variant, including colorectal cancer susceptibility, polymerase proofreading-related adenomatous polyposis, and unrelated developmental-condition labels; these are record-level labels rather than individual-level phenotype evidence.

Still missing

• Obtain the proband's clinical phenotype, indication for testing, relevant tumor history and pathology, and assessment of whether the phenotype is highly specific for a POLE-associated condition.

Sources

Reasoning

No validated functional assay demonstrating a normal or benign effect of NM_006231.4:c.2706+5G>A was identified. The available SpliceAI result is a computational prediction and cannot establish BS3. The applicable POLE framework does not specify BS3; therefore generic ACMG/AMP functional-evidence principles apply.

Rule

Generic ACMG/AMP BS3: apply only when well-established functional studies appropriate to the gene and disease mechanism show no damaging effect, with adequate controls and assay validation. No such variant-specific study is available here.

Evidence considered

• The governing local POLE framework customizes PM1, PS4, PP3, and BP4, but does not provide a POLE-specific BS3 specification; generic ACMG/AMP principles therefore apply.
• The available case evidence reports SpliceAI max delta score 0.381, which is computational prediction rather than validated functional evidence and is insufficient for BS3.
• Richards et al. discuss the need for RNA- or protein-based functional analysis for splice-site variants, but the paper does not report a normal functional result for this variant.

Still missing

• No variant-specific RNA study showing normal splicing was provided.
• No validated protein-level or cellular assay showing preserved POLE function was provided.
• No assay controls, replication, quantitative effect size, or validated benign reference comparison were provided.

Sources

Reasoning

No unaffected relatives with reliable phenotype assessment and confirmed absence of NM_006231.4:c.2706+5G>A are documented. The absence of family data prevents evaluation of non-segregation and does not support BS4.

Rule

Generic ACMG/AMP BS4: lack of segregation in affected members of a family, or presence of the variant in unaffected relatives when the phenotype is expected to be highly penetrant and age-dependent status is adequately assessed; apply only with informative family data.

Evidence considered

• The bundle contains no unaffected-relative genotypes, clinical evaluations, pedigree, or non-segregation observations for this variant.
• No POLE-specific BS4 rule or calibration is available in the governing local framework; generic BS4 application requires documented non-segregation in informative relatives.

Still missing

• Phenotypically reliable unaffected relatives
• Genotypes for those relatives
• Adequate age and clinical follow-up to judge unaffected status
• Assessment of penetrance, variable expressivity, and phenocopies

Reasoning

No phase-resolved observation is available showing this variant in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant in a recessive disorder. The criterion therefore cannot be evaluated from the supplied evidence.

Rule

Generic ACMG/AMP BP2: apply when the variant is observed in trans with a pathogenic variant for a fully penetrant dominant disorder or in cis with a pathogenic variant for a recessive disorder, with phase and disease-model applicability established.

Evidence considered

• The local POLE framework does not specify BP2 and provides no allelic or inheritance rule for this variant.
• The consolidated case-evidence bundle contains no phase-resolved family or proband data supporting either BP2 configuration.

Still missing

• No documented pathogenic-variant co-occurrence in the same individual.
• No parental testing, haplotype, read-based phasing, or other cis/trans evidence.
• No established disease inheritance model applicable to this variant in the bundle.

Reasoning

No case-level evidence identifies an alternate molecular diagnosis that fully explains the reported phenotype. ClinVar condition labels and classifications cannot substitute for documented co-occurrence in the proband.

Rule

Generic ACMG/AMP BP5: a variant is found in a case with an alternate molecular basis for disease.

Evidence considered

• The ClinVar record provides submission-level classifications and condition labels but no proband-specific co-occurring molecular diagnosis or phenotype attribution.

Still missing

• Obtain case-level testing results and phenotype correlation to determine whether a separate molecular diagnosis fully explains the individual's phenotype.

Sources