POLE c.62+2T>G, p.?

Reasoning

PVS1 is met at full strength under the generic ClinGen SVI PVS1 framework (PMC6185798). NM_006231.4:c.62+2T>G is a canonical splice-donor +2 variant in the MANE Select POLE transcript, and the gene-level evidence package supports POLE loss of function as a germline disease mechanism. The affected position is in the first intronic boundary of the transcript, with the predicted consequence being loss of normal splicing and a premature disruptive transcript subject to nonsense-mediated decay rather than a tolerated protein-length change. No applicable POLE VCEP/CSPEC rule was identified for PVS1, and there is no evidence here that the affected transcript is irrelevant, that the splice consequence escapes NMD, or that the affected region is a non-critical distal exon. Therefore no named SVI downgrade rule applies and the strength is very strong. The SpliceAI result (maximum delta 0.989) is concordant with splice disruption but is not separately stacked as another criterion for the same splice-effect evidence.

Rule

Generic ClinGen SVI PVS1 framework (PMC6185798): canonical splice-site loss-of-function variant with relevant transcript and no applicable downgrade; full strength is very strong.

Evidence considered

• The generic ClinGen SVI PVS1 framework (PMC6185798) applies to canonical splice consensus variants and requires consideration of transcript relevance, NMD escape, exon importance, and distal-region exceptions.
• NM_006231.4 is the MANE Select POLE transcript, and the variant is annotated as a canonical splice variant at c.62+2 with predicted protein consequence p.? and no alternative protein consequence supplied.
• The gene-level assessment supports POLE loss of function as a germline disease mechanism and marks the generic PVS1 gate eligible.
• SpliceAI reports DS_DL 0.989 and a maximum delta score of 0.989, supporting a major splice effect; this is not double-counted as a separate splice-prediction criterion here.
• The available evidence does not identify NMD escape, an irrelevant transcript, a non-critical distal exon, or a repeat-region exception requiring downgrade.

Sources

Reasoning

No applicable POLE-specific PM2 rule was retrieved; the local POLE framework does not customize PM2. Under the supplied generic ACMG/ClinGen-SVI default, PM2 Supporting is met when the variant allele frequency is at most 0.0001 in an appropriate population dataset. The variant is absent from gnomAD v2.1 exomes, has AF 0 with 0/1,487,734 alleles and zero homozygotes in gnomAD v4.1, and has AF 0 with 0/142,964 alleles and zero homozygotes in the gnomAD v3.1 non-cancer genome subset. The evidence supports PM2 at Supporting strength.

Rule

Generic PM2 Supporting: apply when allele frequency is <=0.0001 in an appropriate population dataset; use the non-cancer/exome-only subset when required by a governing specification, otherwise use the default all-comers dataset.

Evidence considered

• The retrieved POLE-specific framework does not define PM2; generic ACMG/ClinGen-SVI fallback therefore applies.
• The supplied PM2 Supporting threshold is allele frequency <=0.0001 (ClinGen SVI recommendation, PMID:25741868).
• gnomAD v2.1 exomes report the variant as absent.
• gnomAD v4.1 reports total AF 0, total AC 0/1,487,734, and total homozygotes 0; the reported highest-observed ancestry group also has AF 0.
• The gnomAD v3.1 non-cancer genome-only subset reports AF 0, AC 0/142,964, grpmax FAF 0, and homozygotes 0.

Sources

Reasoning

NM_006231.4:c.62+2T>G is a canonical intronic splice-region variant with protein consequence NP_006222.2:p.?. The governing POLE materials were checked for c.62+2T>G and p.?; PATH-250-323.pdf, PATH-250-323-s003.xlsx, and PATH-250-323-s004.xlsx contain no exact entry for this variant, and their missense-specific PP3/BP4 rules therefore do not apply. Under the generic SpliceAI calibration, the applicable maximum delta score is 0.989 (DS_DL), which exceeds the moderate PP3 threshold of >=0.5 from Jaganathan et al. 2019 (PMID:30661751). The BayesDel result was not used because no generic ACMG strength calibration is established, and REVEL is not applicable to this non-missense variant.

Rule

Generic SpliceAI PP3_Moderate: apply when the maximum SpliceAI delta score is >=0.5; Jaganathan et al. 2019 (PMID:30661751).

Evidence considered

• The case normalization identifies NM_006231.4:c.62+2T>G as a canonical splice variant with predicted protein consequence NP_006222.2:p.?.
• SpliceAI reports DS_AG 0.0, DS_AL 0.0, DS_DG 0.426, and DS_DL 0.989; the maximum delta is therefore 0.989.
• The generic SpliceAI moderate PP3 threshold is >=0.5, from Jaganathan et al. 2019, Cell (PMID:30661751); 0.989 meets this threshold.
• PATH-250-323.pdf, PATH-250-323-s003.xlsx, and PATH-250-323-s004.xlsx were reviewed for c.62+2T>G and p.?; none contains an exact entry, and the POLE supplementary PP3 rule is limited to exact missense entries.
• REVEL was not used because the variant is non-missense and no REVEL score is available; BayesDel was not used because no generic ACMG strength calibration is established.

Still missing

• No exact entry for c.62+2T>G or p.? was found in PATH-250-323.pdf.
• No exact entry for c.62+2T>G or p.? was found in PATH-250-323-s003.xlsx.
• No exact entry for c.62+2T>G or p.? was found in PATH-250-323-s004.xlsx.

Sources

Reasoning

The governing POLE package was searched for c.62+2T>G and p.? and contains no entry for this variant. The POLE-specific PS4 rule in the case framework requires an exact recurrent POLE missense variant in both COSMIC and TCGA endometrial carcinoma cohorts, a combined count >=10, and membership in the established/pathogenic set; it names only p.P286R, p.V411L, p.A456P, and p.S297F. This canonical splice variant is absent from PATH-250-323.pdf and PATH-250-323-s002.xlsx, is not one of those missense variants, and no germline case-control enrichment evidence is available.

Rule

POLE-specific PS4_Supporting requires exact recurrence in both COSMIC and TCGA, combined EC count >=10, and membership in the established/pathogenic missense set; no applicable rule for this variant.

Evidence considered

• The POLE case framework limits PS4_Supporting to exact recurrent missense variants present in both COSMIC and TCGA, with combined EC count >=10, and names p.P286R, p.V411L, p.A456P, and p.S297F.
• PATH-250-323.pdf describes somatic POLE endometrial-carcinoma recurrence and lists the five hotspot missense variants, but does not provide an entry for c.62+2T>G or p.?.
• PATH-250-323-s002.xlsx lists recurrent POLE variants and counts; c.62+2T>G and p.? are absent.

Still missing

• No entry for c.62+2T>G or p.? was found in PATH-250-323.pdf.
• No entry for c.62+2T>G or p.? was found in PATH-250-323-s002.xlsx.
• PATH-250-323-s001.tif was unavailable for text conversion; it was not assumed to be empty.
• No germline case-control enrichment dataset specific to this variant was available.

Sources

Reasoning

No applicable POLE-specific population-frequency rule was retrieved; the local POLE framework customizes PM1, PS4, PP3, and BP4 only. Under the supplied generic ACMG/AMP default, BA1 requires an allele frequency of at least 0.05. The variant is absent from gnomAD v2.1 exomes and has total AF 0 (0/1,487,734 alleles) in gnomAD v4.1, with no observed alleles in any reported v4.1 ancestry group. Therefore BA1 is not met.

Rule

Generic BA1: apply stand-alone benign evidence when population allele frequency is >=0.05; no POLE-specific BA1 rule was retrieved.

Evidence considered

• The retrieved POLE-specific framework does not define BA1; generic ACMG/AMP fallback therefore applies.
• The supplied generic BA1 threshold is allele frequency >=0.05 (ACMG/AMP 2015, PMID:25741868).
• gnomAD v2.1 exomes report the variant as absent.
• gnomAD v4.1 reports exome AC 0/1,340,672, genome AC 0/147,062, total AC 0/1,487,734, total AF 0, and total homozygotes 0; every reported ancestry group also has AC 0.

Sources

Reasoning

No applicable POLE-specific population-frequency rule was retrieved; the local POLE framework customizes PM1, PS4, PP3, and BP4 only. Under the supplied generic ACMG/AMP default, BS1 requires an allele frequency of at least 0.01. The variant is absent from gnomAD v2.1 exomes and has total AF 0 (0/1,487,734 alleles) in gnomAD v4.1, including AF 0 in the highest-observed ancestry group. Therefore BS1 is not met.

Rule

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

Evidence considered

• The retrieved POLE-specific framework does not define BS1; generic ACMG/AMP fallback therefore applies.
• The supplied generic BS1 threshold is allele frequency >=0.01, with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168).
• gnomAD v2.1 exomes report the variant as absent.
• gnomAD v4.1 reports total AF 0, total AC 0/1,487,734, and AF 0 in African/African American individuals, the reported highest-observed ancestry group.

Sources

Reasoning

No applicable POLE-specific BS2 rule was retrieved. The available population datasets show no homozygous observations: gnomAD v4.1 reports total homozygotes 0, and the non-cancer gnomAD v2.1 exome and gnomAD v3.1 genome subsets also report no observed alleles and homozygotes 0. Thus the available data do not support BS2, which requires observation in healthy individuals at a frequency inconsistent with disease, including relevant homozygote observations where applicable.

Rule

Generic BS2: apply only when the variant is observed in healthy individuals at a frequency incompatible with the relevant disease model; no such observation is present.

Evidence considered

• The retrieved POLE-specific framework does not define BS2; generic ACMG/AMP interpretation applies.
• gnomAD v4.1 reports total AC 0 and total homozygotes 0 across 1,487,734 alleles, with homozygotes 0 in each reported ancestry group.
• The gnomAD v2.1 non-cancer exome subset is absent for this variant, and the gnomAD v3.1 non-cancer genome subset reports AC 0/142,964 and homozygotes 0.

Sources

Reasoning

NM_006231.4:c.62+2T>G is a canonical intronic splice-region variant, so BP4 is evaluated through SpliceAI rather than REVEL. The governing POLE materials were checked for c.62+2T>G and p.?; PATH-250-323.pdf, PATH-250-323-s003.xlsx, and PATH-250-323-s004.xlsx contain no exact entry for this variant, so their missense-specific BP4 rule does not apply. The applicable SpliceAI maximum delta is 0.989, which is above rather than at or below the generic BP4 supporting threshold of <=0.1 from Jaganathan et al. 2019 (PMID:30661751); therefore BP4 is not met.

Rule

Generic SpliceAI BP4_Supporting: apply only when the maximum SpliceAI delta score is <=0.1; Jaganathan et al. 2019 (PMID:30661751).

Evidence considered

• The case normalization identifies NM_006231.4:c.62+2T>G as a canonical splice variant with predicted protein consequence NP_006222.2:p.?.
• SpliceAI reports a maximum delta score of 0.989 (DS_DL).
• The generic SpliceAI BP4 supporting threshold is <=0.1, from Jaganathan et al. 2019, Cell (PMID:30661751); 0.989 does not meet this threshold.
• PATH-250-323.pdf, PATH-250-323-s003.xlsx, and PATH-250-323-s004.xlsx were reviewed for c.62+2T>G and p.?; none contains an exact entry, and the POLE supplementary BP4 rule is limited to exact missense entries.
• REVEL was not used because the variant is non-missense and no REVEL score is available.

Still missing

• No exact entry for c.62+2T>G or p.? was found in PATH-250-323.pdf.
• No exact entry for c.62+2T>G or p.? was found in PATH-250-323-s003.xlsx.
• No exact entry for c.62+2T>G or p.? was found in PATH-250-323-s004.xlsx.

Sources

Reasoning

No proband-level de novo evidence is documented for NM_006231.4:c.62+2T>G: parental genotypes, parentage confirmation, and testing methodology are not provided. Therefore PS2 cannot be assessed, and no de novo strength is assigned.

Evidence considered

• The available case evidence contains no documented parental genotypes, parentage confirmation, or proband-specific de novo testing result for NM_006231.4:c.62+2T>G.

Still missing

• Parental testing results for both biological parents are needed.
• Parentage confirmation and the assay or read-level evidence supporting absence in both parents are needed.

Reasoning

No variant-specific functional assay evidence was identified for NM_006231.4:c.62+2T>G. The available SpliceAI max delta score of 0.989 is a computational splice prediction, not a validated functional assay, and cannot establish PS3 without experimental results, appropriate controls, and biologically relevant readouts.

Rule

Generic ACMG/AMP PS3 requires well-established functional studies showing a damaging effect; computational splice prediction alone does not satisfy PS3.

Evidence considered

• SpliceAI reports a maximum delta score of 0.989 for NM_006231.4:c.62+2T>G, indicating predicted splice impact; this is in-silico evidence and is not sufficient for PS3.
• No variant-specific RNA, protein, cellular, biochemical, or other validated functional assay result with assay controls was available.

Still missing

• A validated variant-specific functional assay is needed, ideally measuring RNA splicing or an established POLE-relevant biological function with appropriate positive, negative, and wild-type controls.

Sources

Reasoning

No affected-proband observation, recessive disease context, or phased pathogenic/likely pathogenic variant in trans with NM_006231.4:c.62+2T>G is documented. PM3 therefore cannot be assessed from the available case evidence; parental testing, a pedigree, and phase-resolved observations would be required.

Evidence considered

• The case evidence identifies NM_006231.4:c.62+2T>G as a canonical splice variant but contains no affected-proband, compound-heterozygous, biallelic, parental, or phase-resolved observation relevant to PM3.
• The available POLE framework specifies PM1, PS4, PP3, and BP4 only; it provides no gene-specific PM3 rule, so generic ACMG/AMP PM3 logic was considered and cannot be met without evidence of a pathogenic variant in trans.

Still missing

• No affected proband or recessive disease observation is documented.
• No second pathogenic or likely pathogenic variant is documented in trans.
• No parental testing, pedigree, or phase information is available.

Reasoning

No clinical report documents NM_006231.4:c.62+2T>G as presumed de novo without parental testing, and no affected proband phenotype or case report is available to support PM6. Therefore PM6 cannot be assessed.

Evidence considered

• The literature pass contains zero candidate publications and the case evidence contains no affected-proband report or presumed-de-novo observation for NM_006231.4:c.62+2T>G.

Still missing

• A documented affected proband with phenotype information and a report of presumed de novo status is needed.
• The basis for considering the variant presumed de novo, including whether parental testing was unavailable, is needed.

Reasoning

No family segregation data are documented for NM_006231.4:c.62+2T>G: there are no informative meioses, affected relatives, unaffected relatives, or genotype-phenotype co-segregation observations. Therefore PP1 cannot be assessed.

Evidence considered

• The available case evidence contains no family structure, relative genotypes, phenotype co-segregation data, or informative meiosis count for this variant.

Still missing

• Genotypes and clinical phenotypes for informative affected and unaffected relatives are needed.
• The number of informative meioses and the segregation pattern are needed.

Reasoning

PP4 requires a highly specific phenotype or disease presentation that matches the condition associated with the variant. The available case evidence identifies the molecular variant and predicts splice impact, but supplies no patient phenotype, diagnosis, or phenotype-specific disease match for adjudication. The consulted POLE paper concerns somatic endometrial-carcinoma molecular classification and does not establish a phenotype-specific germline PP4 rule for this variant.

Rule

Generic PP4 requires a highly specific phenotype consistent with the gene-associated disease; no phenotype is available.

Evidence considered

• The case materials report NM_006231.4:c.62+2T>G with predicted splice impact (SpliceAI max delta 0.989), but no patient phenotype or disease diagnosis is provided.
• PATH-250-323.pdf addresses somatic POLE-mutated endometrial carcinoma and does not define a phenotype-specific germline PP4 rule.

Still missing

• No patient phenotype, clinical diagnosis, or phenotype-match assessment was available.
• PATH-250-323.pdf is a somatic endometrial-carcinoma paper and provides no germline PP4 specification for c.62+2T>G.

Sources

Reasoning

PP5 is restricted here to an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. The case ClinVar search found no exact ClinVar record, no variation ID, no classification, and no review status; therefore no expert-panel assertion exists to assess and PP5 cannot be applied.

Rule

Apply PP5 only for an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification; none is present.

Evidence considered

• The exact ClinVar searches returned no variation ID, no classification, no review status, and an absent-result summary for NM_006231.4:c.62+2T>G.
• Ordinary laboratory submissions, aggregate labels, and non-expert assertions are not available and would not qualify for PP5 under the requested rule.

Still missing

• No exact-variant ClinVar record was found.
• No ClinVar expert-panel classification or review status was available.

Sources

Reasoning

No variant-specific functional assay evidence was identified showing preserved normal function for NM_006231.4:c.62+2T>G. The available SpliceAI max delta score of 0.989 predicts splice disruption rather than demonstrating a normal functional result, and it cannot establish BS3 without a validated assay, appropriate controls, and biologically relevant readouts.

Rule

Generic ACMG/AMP BS3 requires well-established functional studies showing no damaging effect; computational splice prediction alone does not satisfy BS3.

Evidence considered

• SpliceAI reports a maximum delta score of 0.989 for NM_006231.4:c.62+2T>G, indicating predicted splice impact; it does not provide evidence of preserved normal function for BS3.
• No variant-specific assay demonstrating normal RNA splicing, protein function, cellular function, or another validated biologically relevant endpoint was available.

Still missing

• A validated variant-specific functional assay demonstrating preserved normal function is needed, with appropriate positive, negative, and wild-type controls and a biologically relevant readout.

Sources

Reasoning

No non-segregation evidence is documented for NM_006231.4:c.62+2T>G: no unaffected relatives carrying the variant, family phenotypes, or informative segregation observations are available. Therefore BS4 cannot be assessed.

Evidence considered

• The available case evidence contains no documented unaffected relatives carrying NM_006231.4:c.62+2T>G and no family non-segregation analysis.

Still missing

• Genotypes and phenotypes of informative unaffected relatives are needed.
• The family history and number of informative non-segregating meioses are needed.

Reasoning

No observation establishes NM_006231.4:c.62+2T>G in cis with a pathogenic variant or in trans with a pathogenic variant in a dominant disease context. BP2 therefore cannot be assessed from the available case evidence; segregation and phase-resolved genotype data would be required.

Evidence considered

• The case evidence contains no affected-family, segregation, parental, haplotype, cis, or trans observation involving NM_006231.4:c.62+2T>G and another pathogenic variant.
• The available POLE framework specifies PM1, PS4, PP3, and BP4 only; it provides no gene-specific BP2 rule, so generic ACMG/AMP BP2 logic was considered and cannot be met without documented phase and an appropriate pathogenic comparator variant.

Still missing

• No second variant relevant to BP2 is documented.
• No cis/trans phase information is available.
• No inheritance model or segregation data are available.

Reasoning

BP5 requires evidence that the patient's phenotype is better explained by an alternative molecular diagnosis or cause. The case contains no patient phenotype, competing molecular diagnosis, or alternative-cause assessment. The somatic POLE recurrence paper cannot establish BP5 for this germline splice variant.

Rule

Generic BP5 requires a documented alternative molecular explanation for the phenotype; no such evidence is available.

Evidence considered

• The available case evidence provides variant-level molecular findings but no clinical phenotype or alternative diagnosis.
• PATH-250-323.pdf is focused on somatic endometrial-carcinoma POLE classification and does not specify BP5 for this variant.

Still missing

• No patient phenotype or clinical diagnosis was available.
• No alternative molecular diagnosis or competing cause was documented.
• No POLE-specific BP5 rule was found in the governing files.

Sources

Reasoning

BP6 is restricted here to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. The case ClinVar search found no exact ClinVar record, no variation ID, no classification, and no review status; therefore no qualifying expert-panel benign assertion exists to assess and BP6 cannot be applied.

Rule

Apply BP6 only for an exact-variant ClinVar expert-panel Benign or Likely benign classification; none is present.

Evidence considered

• The exact ClinVar searches returned no variation ID, no classification, no review status, and an absent-result summary for NM_006231.4:c.62+2T>G.
• Ordinary laboratory submissions, aggregate labels, and non-expert assertions are not available and would not qualify for BP6 under the requested rule.

Still missing

• No exact-variant ClinVar record was found.
• No ClinVar expert-panel classification or review status was available.

Sources