TP53 c.840A>C, p.(Arg280Ser)

Reasoning

For TP53 NM_000546.6:c.840A>C (p.Arg280Ser), the TP53 VCEP Functional Worksheet exact-match row records non-functional Kato data and LOF in the majority of other eligible assays. This satisfies the TP53 VCEP PS3 Strong rule.

Rule

Non-functional on Kato et al. data AND loss of function (LOF) by the majority of other eligible assays

Evidence considered

• CHECKLIST ps3_1: Is the variant non-functional on Kato et al. data? — TRUE — The exact R280S row in the TP53 Functional Worksheet records Kato as Non-functional.
• CHECKLIST ps3_2: Is there loss of function (LOF) by the majority of other eligible assays? — TRUE — The exact R280S row records LOF in the majority of the other eligible assay columns.
• CHECKLIST ps3_3: Is the variant partially functional on Kato et al. data? — FALSE — The exact R280S Kato result is Non-functional, not partially functional.
• CHECKLIST ps3_4: Is there loss of function (LOF) by the majority of other available assays? — TRUE — The exact R280S row records LOF across the majority of available non-Kato assays.
• CHECKLIST ps3_5: Is the variant abnormal on Kawaguchi et al. data? — NA — The Functional Worksheet records the Kawaguchi result for R280S as NA.
• CHECKLIST ps3_6: Is the variant a small deletion? — FALSE — NM_000546.6:c.840A>C produces the missense change p.Arg280Ser.
• CHECKLIST ps3_7: For a small deletion, does it demonstrate LOF on the majority of available assays? — NA — The variant is missense rather than a small deletion, so this branch does not apply.
• The TP53 VCEP PS3 Strong rule requires non-functional Kato data AND LOF by the majority of other eligible assays; the exact R280S worksheet row meets both conditions.
• Kato et al. describes the primary systematic yeast transactivation assay used by the TP53 VCEP; its extracted publication text is not allele-specific for R280S, so the exact assay classification is taken from the VCEP Functional Worksheet.
• Giacomelli et al. and Kotler et al. describe systematic or high-throughput TP53 functional assays; their extracted text does not provide an allele-specific R280S result, so no result is inferred from their general findings.

Still missing

• The exact R280S row records Kawaguchi et al. as NA, so that assay-specific value is unavailable.
• The small-deletion branch is not applicable because this is a missense substitution.

Sources

Reasoning

PM2 Supporting is met. The exact variant NM_000546.6:c.840A>C (p.Arg280Ser) is absent from gnomAD v4.1, gnomAD v2.1, and gnomAD-Canada v1.0, satisfying the TP53 VCEP threshold of allele frequency below 0.00003 in a large sequenced population; no observed alleles are available to trigger the multiple-allele ancestry-group condition.

Rule

TP53 VCEP v2.4 PM2: apply at Supporting level when allele frequency is <0.00003 (0.003%) in gnomAD or another large sequenced population; if multiple alleles occur in an ancestry group, that group's frequency must be <0.00004 (0.004%), with specified founder-effect groups ignored.

Evidence considered

• CHECKLIST item pm2_1: Is the variant absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium? — NA — Those three legacy resources are not separately reported in the case bundle, although the exact variant is absent from the supplied gnomAD control datasets.
• CHECKLIST item pm2_2: For an indel, was the poor calling of population data by next generation sequencing considered? — FALSE — This variant is a single-nucleotide substitution, not an indel.
• CHECKLIST item pm2_3: Is PM2 applied at supporting level? — TRUE — The TP53 VCEP explicitly requires PM2 to be applied at Supporting level.
• CHECKLIST item pm2_4: Is allele frequency less than 0.00003 (0.003%) in gnomAD or another large sequenced population? — TRUE — The exact variant is absent from gnomAD v4.1, gnomAD v2.1, and gnomAD-Canada, corresponding to observed allele frequency 0 in each supplied dataset.
• CHECKLIST item pm2_5: If multiple alleles are present in any genetic ancestry group, is its allele frequency <0.00004 (0.004%)? — NA — No multiple-allele ancestry group is present because the variant is absent from the evaluated datasets.
• CHECKLIST item pm2_6: Were Ashkenazi Jewish, Finnish, Amish, Middle Eastern, and “Remaining” ignored? — TRUE — No variant-bearing ancestry group was observed, and the VCEP directs that these founder-effect groups be ignored.
• CHECKLIST item pm2_7: Does the variant meet no PM2, BA1, or BS1 population code, yet have total AF >0.00003 and no group with multiple alleles at AF >0.00004? — FALSE — The variant meets PM2 Supporting and has no observed population alleles, so the stated CHIP-recalculation prerequisite is not met.
• CHECKLIST item pm2_8: If so, was total AF recalculated from alleles with VAF >0.35 by visualizing heterozygote allele balance and totaling carriers in bars 0.35–0.65? — NA — The pm2_7 prerequisite is false and no allele-balance histogram is supplied.
• CHECKLIST item pm2_9: After excluding low-VAF likely CHIP alleles, does the revised AF support PM2_Supporting? — NA — No CHIP recalculation was indicated or necessary because the variant is absent from the evaluated datasets.
• CHECKLIST item pm2_10: Was the most recent gnomAD used, or is the alternative source necessary for optimal classification? — TRUE — gnomAD v4.1 was evaluated as the most recent supplied gnomAD dataset; the older and Canada datasets provide concordant absence but are supplementary.
• The TP53 VCEP Version 2.4 requires PM2 at Supporting level when allele frequency is <0.00003 (0.003%) in gnomAD or another large sequenced population; if multiple alleles occur in an ancestry group, that group's frequency must be <0.00004 (0.004%), with specified founder-effect groups ignored.
• gnomAD v4.1 reports chr17:7673780 T>G as absent, gnomAD v2.1 reports 17:7577098 T>G as absent, and gnomAD-Canada v1.0 reports 17:7673780 T>G as absent.

Still missing

• ESP, 1000 Genomes, and Exome Aggregation Consortium-specific records are not separately supplied; the conclusion relies on the supplied large-population gnomAD datasets.
• No allele-balance histogram or VAF carrier counts are supplied; CHIP recalculation is not triggered because the exact variant is absent from the evaluated population datasets.

Sources

Reasoning

The TP53 VCEP pre-computed PP3/BP4 worksheet directly assigns PP3_moderate to NM_000546.6:c.840A>C (p.Arg280Ser). The worksheet records Align-GVGD Class C65, BayesDel 0.471465, and maximum SpliceAI delta 0.00 for this variant. This VCEP per-variant assignment governs PP3. The low SpliceAI score does not duplicate or negate the missense computational assignment.

Rule

TP53 VCEP PP3_Moderate for missense variants: aGVGD Class C65 and BayesDel score >=0.16; a VCEP per-variant pre-assignment in Supplementary Table S2 is applied directly.

Evidence considered

• TP53 VCEP Supplementary Table S2 directly assigns PP3_moderate to c.840A>C / p.Arg280Ser, with Align-GVGD Class C65 and BayesDel 0.471465.
• SpliceAI maximum delta score is 0.004 (reported as 0.00), below the VCEP threshold for a predicted splice effect; this supports interpretation through the missense computational pathway only.

Sources

Reasoning

TP53 c.840A>C (p.Arg280Ser) is a missense substitution in exon 8 of the biologically relevant transcript NM_000546.6. It does not create a premature termination codon, frameshift, initiation-codon alteration, exon deletion, or canonical splice-site variant; therefore it is outside the TP53 VCEP PVS1 null-variant decision tree. SpliceAI predicts no splice impact (maximum delta 0.004), providing no basis to recast this apparent missense variant as a splice-mediated loss-of-function allele.

Rule

TP53 VCEP PVS1: apply the TP53-specific null-variant decision tree; this missense, non-splice-impacting variant is not an eligible PVS1 variant class.

Evidence considered

• Variant normalization identifies NM_000546.6:c.840A>C as NP_000537.3:p.(Arg280Ser), an exon 8 missense substitution.
• The TP53 VCEP PVS1 flowchart applies to null-variant categories (initiation codon, nonsense, frameshift, canonical splice, and exon deletion) and specifies NMD/truncation-position weights only for those categories.
• SpliceAI maximum delta score is 0.004 (no significant predicted splice impact).

Sources

Reasoning

BA1 is not met. The exact variant NM_000546.6:c.840A>C (p.Arg280Ser) is absent from gnomAD v4.1, gnomAD v2.1, and gnomAD-Canada v1.0, so no qualifying continental-subpopulation FAF at or above 0.001 was observed.

Rule

TP53 VCEP v2.4 BA1: filtering allele frequency ≥0.001 (0.1%) in a qualifying gnomAD continental subpopulation; the ancestry group must have ≥2,000 alleles tested and at least 2 alleles present, with founder-effect groups excluded.

Evidence considered

• CHECKLIST item ba1_1: Is the allele frequency above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium? — FALSE — The exact variant is absent from the supplied population records, with no evidence of an allele frequency above 5%.
• CHECKLIST item ba1_2: Is the FAF ≥ 0.001 or 0.1% in a gnomAD continental subpopulation of a single genetic ancestry group? — FALSE — gnomAD v4.1 and v2.1 report the exact variant as absent, so no qualifying FAF reaches 0.001.
• CHECKLIST item ba1_3: Is that group not Ashkenazi Jewish, Finnish, Amish, Middle Eastern, or “Remaining”? — NA — No variant-bearing ancestry group is present in the supplied records to evaluate this exclusion.
• CHECKLIST item ba1_4: Does that group have ≥2,000 alleles tested and a minimum of 2 alleles present? — NA — No variant-bearing ancestry group or allele-count record is available because the variant is reported absent.
• CHECKLIST item ba1_5: Is caution required because the majority of alleles have allele balance below 0.35? — NA — Allele-balance data are not supplied and no observed alleles are reported.
• CHECKLIST item ba1_6: Was the most recent gnomAD used, or is the alternative source necessary for optimal classification? — TRUE — gnomAD v4.1 was evaluated as the most recent supplied gnomAD dataset; gnomAD v2.1 and gnomAD-Canada provide concordant absence but are not needed to establish BA1 non-application.
• The TP53 VCEP Version 2.4 specifies BA1 at stand-alone strength for FAF ≥0.001 in a qualifying continental subpopulation, requiring at least 2,000 alleles tested and at least 2 variant alleles present.
• gnomAD v4.1 reports chr17:7673780 T>G as absent, gnomAD v2.1 reports 17:7577098 T>G as absent, and gnomAD-Canada v1.0 reports 17:7673780 T>G as absent.

Still missing

• No qualifying ancestry group exists in the supplied population records, so ancestry-group allele counts and allele-balance details are unavailable for direct checklist confirmation.

Sources

Reasoning

BS1 is not met. The exact variant is absent from gnomAD v4.1, gnomAD v2.1, and gnomAD-Canada v1.0, so no qualifying continental-subpopulation FAF in the TP53 VCEP range of 0.0003 to less than 0.001 was observed.

Rule

TP53 VCEP v2.4 BS1: filtering allele frequency ≥0.0003 and <0.001 in a qualifying gnomAD continental subpopulation; the ancestry group must have ≥2,000 alleles tested and at least 2 alleles present, with founder-effect groups excluded.

Evidence considered

• CHECKLIST item bs1_1: Is allele frequency greater than expected for disorder? — FALSE — The exact variant is absent from the supplied population records, providing no observed frequency greater than the expected frequency for Li-Fraumeni syndrome.
• CHECKLIST item bs1_2: Is the FAF ≥ 0.0003 but < 0.001 in a gnomAD continental subpopulation of a single genetic ancestry group? — FALSE — gnomAD v4.1 and v2.1 report the exact variant as absent, so no qualifying FAF falls in the BS1 interval.
• CHECKLIST item bs1_3: Is that group not Ashkenazi Jewish, Finnish, Amish, Middle Eastern, or “Remaining”? — NA — No variant-bearing ancestry group is present in the supplied records to evaluate this exclusion.
• CHECKLIST item bs1_4: Does that group have ≥2,000 alleles tested and a minimum of 2 alleles present? — NA — No variant-bearing ancestry group or allele-count record is available because the variant is reported absent.
• CHECKLIST item bs1_5: Is caution required because the majority of alleles have allele balance below 0.35? — NA — Allele-balance data are not supplied and no observed alleles are reported.
• CHECKLIST item bs1_6: Was the most recent gnomAD used, or is the alternative source necessary for optimal classification? — TRUE — gnomAD v4.1 was evaluated as the most recent supplied gnomAD dataset; gnomAD v2.1 and gnomAD-Canada provide concordant absence but are not needed to establish BS1 non-application.
• The TP53 VCEP Version 2.4 specifies BS1 at strong strength for FAF ≥0.0003 and <0.001 in a qualifying continental subpopulation, requiring at least 2,000 alleles tested and at least 2 variant alleles present.
• gnomAD v4.1 reports chr17:7673780 T>G as absent, gnomAD v2.1 reports 17:7577098 T>G as absent, and gnomAD-Canada v1.0 reports 17:7673780 T>G as absent.

Still missing

• No qualifying ancestry group exists in the supplied population records, so ancestry-group allele counts and allele-balance details are unavailable for direct checklist confirmation.

Sources

Reasoning

BS3 is not met. The exact TP53 VCEP worksheet row records R280S as non-functional on Kato data and LOF in the majority of other available assays, failing both TP53 VCEP benign functional pathways.

Rule

Functional on Kato et al. data AND no loss of function (LOF) by the majority of available eligible assays; OR partially functional on Kato et al. data AND no evidence of LOF by all available assays

Evidence considered

• CHECKLIST bs3_1: Is the variant a missense variant? — TRUE — NM_000546.6:c.840A>C is annotated as p.Arg280Ser, a missense substitution.
• CHECKLIST bs3_2: For a missense variant, is PP3 applied based on SpliceAI? — FALSE — SpliceAI max delta score is 0.004, below the TP53 VCEP splice-impact threshold, and no SpliceAI-based PP3 is applied here.
• CHECKLIST bs3_3: For a missense variant with PP3 applied based on SpliceAI, are the available assays done at the protein level (such as Kato et al. or Giacomelli et al.)? — NA — PP3 based on SpliceAI is not applied, so this conditional item does not apply.
• CHECKLIST bs3_4: Is there any laboratory evidence that the variant impacts splicing? — FALSE — The consolidated bundle identifies no laboratory splicing evidence for R280S, and SpliceAI predicts no significant splice impact.
• CHECKLIST bs3_5: Is the variant functional on Kato et al. data? — FALSE — The exact R280S row in the TP53 Functional Worksheet records Kato as Non-functional.
• CHECKLIST bs3_6: Is there no loss of function (LOF) by the majority of available eligible assays? — FALSE — The exact R280S row records LOF in the majority of available eligible assays.
• CHECKLIST bs3_7: Is the variant partially functional on Kato et al. data? — FALSE — The exact Kato result is Non-functional, not partially functional.
• CHECKLIST bs3_8: Is there no evidence of loss of function (LOF) by all available assays? — FALSE — LOF is recorded in multiple available assays for the exact R280S row.
• CHECKLIST bs3_9: Is the variant a small deletion? — FALSE — The variant is a missense substitution, not a deletion.
• CHECKLIST bs3_10: For a small deletion, are Kotler et al. data available? — NA — The variant is not a small deletion, so the small-deletion Kotler branch does not apply.
• CHECKLIST bs3_11: For a small deletion with available Kotler et al. data, is it loss of function (LOF) by the majority of available assays? — NA — The variant is not a small deletion, so this conditional item does not apply.
• The TP53 VCEP BS3 Strong rule requires functional Kato data AND no LOF by the majority of eligible assays; R280S fails both requirements.
• The TP53 VCEP BS3 Supporting rule requires partially functional Kato data AND no evidence of LOF by all available assays; R280S fails both requirements.
• The extracted Kato, Giacomelli, and Kotler publication findings provide assay context but no allele-specific benign R280S result; the exact functional results are taken from the VCEP Functional Worksheet.

Still missing

• The exact R280S row records Kawaguchi et al. as NA; this missing value does not alter the failure of the Kato and majority-assay BS3 requirements.
• The small-deletion Kotler branch is not applicable because R280S is a missense variant.

Sources

Reasoning

BP4 is not met because the TP53 VCEP pre-computed worksheet assigns the pathogenic-direction PP3_moderate, rather than BP4, to c.840A>C (p.Arg280Ser). Although SpliceAI predicts no splice impact, BP4 for a missense variant additionally requires the VCEP benign-direction BayesDel/Align-GVGD result, which is not present.

Rule

TP53 VCEP BP4 for missense variants requires a benign-direction BayesDel result with no predicted splice difference; do not co-apply BP4 with the assigned PP3_moderate computational evidence.

Evidence considered

• TP53 VCEP Supplementary Table S2 records c.840A>C / p.Arg280Ser as Align-GVGD Class C65, BayesDel 0.471465, preliminary code PP3_moderate, and maximum SpliceAI delta 0.00.
• SpliceAI maximum delta 0.004 establishes no predicted splice difference but cannot independently establish BP4 for this missense variant under the TP53 VCEP rule.

Sources

Reasoning

The TP53 VCEP specifies PS1 only when a variant is asserted as Pathogenic or Likely Pathogenic following the TP53 VCEP specifications. The bundle contains no qualifying prior same-amino-acid TP53 variant record that can independently establish PS1 for p.Arg280Ser; the available ClinVar submissions are non-expert-panel submissions and their classifications cannot be used as an independent criterion inference.

Rule

Can be applied to variants asserted as Pathogenic or Likely Pathogenic following the TP53 VCEP specifications; Strong for Pathogenic and Moderate for Likely Pathogenic.

Evidence considered

• TP53 VCEP v2.4 defines PS1 as Strong for variants asserted Pathogenic and Moderate for variants asserted Likely Pathogenic following the TP53 VCEP specifications.
• ClinVar variation 634747 has Likely pathogenic and Pathogenic single-submitter records but no expert-panel submission; these classifications were not treated as independent evidence for PS1.
• The same-residue comparator search reported zero useful same-residue candidates and did not establish a qualifying prior p.Arg280Ser assertion.

Still missing

• A qualifying prior TP53 VCEP-reviewed pathogenic or likely pathogenic variant with the same amino-acid change, p.Arg280Ser, was not identified.

Sources

Reasoning

The TP53 VCEP de novo point framework is applicable, but the case bundle contains no proband-level parental testing, cancer phenotype, constitutional mosaicism, or family-history data needed to establish de novo status or tally PS2 points.

Rule

TP53 VCEP PS2: ≥8 points = Very Strong; 4-7 points = Strong; 2-3 points = Moderate; 1 point = Supporting.

Evidence considered

• The governing framework is the ClinGen TP53 Expert Panel Specifications, version 2.4; its PS2 rule assigns Supporting for 1 point, Moderate for 2-3 points, Strong for 4-7 points, and Very Strong for at least 8 points.
• The TP53 PS2/PP1 cancer table is the specified source for proband cancer categories and point tallying.
• No case-level maternity confirmation is present in the consolidated evidence bundle.
• No case-level paternity confirmation is present in the consolidated evidence bundle.
• No case-level proband cancer type is present in the consolidated evidence bundle.
• No case-level constitutional mosaicism evidence is present in the consolidated evidence bundle.
• No case-level family history in the proband is present in the consolidated evidence bundle.
• CHECKLIST ps2_1: Have both maternity and paternity been confirmed? — NA — Neither maternity nor paternity confirmation is reported.
• CHECKLIST ps2_2: If only paternity has been confirmed, has this been recorded as insufficient, since confirmation of paternity only is insufficient? — NA — Paternity-only confirmation cannot be evaluated because paternity confirmation itself is not reported.
• CHECKLIST ps2_3: Have potential non-maternity circumstances been checked, including egg donation, surrogate motherhood, and errors in embryo transfer? — NA — The bundle contains no documentation that these circumstances were assessed.
• CHECKLIST ps2_4: What type of cancer(s) is seen in the proband, for tallying proband points using the specified table? — NA — The proband cancer phenotype is not reported.
• CHECKLIST ps2_5: If the proband is a confirmed constitutional mosaic, is there low TP53 VAF on blood or buccal testing and detection of the mutation in non-lymphocyte tissue and/or segregation in children? — NA — Constitutional mosaicism, VAF, tissue testing, and transmission to children are not reported.
• CHECKLIST ps2_6: For a confirmed constitutional mosaic with the disease and no family history, can the proband be counted as de novo with both maternity and paternity confirmed? — NA — Mosaic status, disease status for this purpose, family history, and both parental confirmations are not reported.
• CHECKLIST ps2_7: What total de novo proband points are tallied using the table for confirmed maternity and paternity and the type of cancer(s) seen in the proband? — NA — Required parental-confirmation and proband-cancer inputs are unavailable.

Still missing

• Maternity confirmation is not reported.
• Paternity confirmation is not reported.
• Proband cancer type is not reported.
• Constitutional mosaicism evidence is not reported.
• Family history in the proband is not reported.
• PS2 de novo proband points cannot be tallied without parental confirmation and proband cancer phenotype.

Sources

Reasoning

The applicable ClinGen TP53 VCEP Version 2.4 framework requires verified proband-level Li-Fraumeni clinical criteria and point scoring for PS4. The case bundle contains no exact-variant case-control prevalence, RR/OR, confidence interval, VCEP point total, or individual Revised Chompret assessment, so PS4 cannot be assessed.

Rule

ClinGen TP53 VCEP PS4: apply verified Li-Fraumeni clinical/cancer point scoring per proband; supporting = 1-1.5 points, moderate = 2-3.5 points, strong = 4-7.5 points, very strong = ≥8 points.

Evidence considered

• The TP53 VCEP PS4 framework assigns 0.5 point for Revised Chompret criteria, 1 point for Classical LFS criteria, and 0.5 point for HER2-positive breast cancer before age 40 when not double-counted; supporting is 1-1.5 points, moderate is 2-3.5 points, strong is 4-7.5 points, and very strong is at least 8 points.
• CHECKLIST ps4_1: Is the prevalence of the variant in affected individuals significantly increased compared to the prevalence in controls? — NA — No exact-variant case-control affected-individual or control prevalence values are provided.
• CHECKLIST ps4_2: From case-control studies, is the relative risk (RR) or odds ratio (OR) >5.0? — NA — No exact-variant case-control RR or OR is provided.
• CHECKLIST ps4_3: Does the confidence interval around the estimate of RR or OR not include 1.0? — NA — No confidence interval around an exact-variant case-control RR or OR is provided.
• CHECKLIST ps4_4: What TP53 VCEP point range is calculated: ≥ 8 points, ≥ 4-7.5 points, 2-3.5 points, or 1-1.5 points? — NA — No verified TP53 VCEP point total is available.
• CHECKLIST ps4_5: Does the individual meet the Revised Chompret criteria? — NA — No individual-level phenotype or family-history assessment is provided.

Still missing

• Affected-individual and control variant prevalence from an exact-variant case-control study.
• Case-control RR or OR and its confidence interval.
• Verified TP53 VCEP point calculation and proband-level cancer phenotype/family-history data, including Revised Chompret assessment.

Sources

Reasoning

The governing TP53 VCEP PM1 rule permits Moderate evidence for missense variants at codons 175, 245, 248, 249, 273, or 282, or for the same amino-acid change occurring at least 10 times in cancerhotspots.org; Supporting evidence is specified for 2-9 cancerhotspots.org occurrences of the same amino-acid change. p.Arg280Ser is at codon 280 and therefore is outside the listed codons. The bundle reports that R280 is a statistically significant hotspot, but provides no cancerhotspots.org occurrence count for the exact Arg280Ser change. The authoritative domain_tables object is empty, so no additional approved domain entry is available to assess.

Rule

Moderate: missense variants at codons 175, 245, 248, 249, 273, or 282, or the same amino-acid change seen in cancerhotspots.org at least 10 times. Supporting: the same amino-acid change seen 2-9 times in cancerhotspots.org.

Evidence considered

• The TP53 VCEP PM1 codon list is 175, 245, 248, 249, 273, and 282; codon 280 is not included.
• The bundle reports a statistically significant TP53 R280 hotspot, but the hotspot extraction is partial and does not provide the exact cancerhotspots.org count for p.Arg280Ser required by the VCEP rule.
• PMID:27328919 reports codon 280 as a TP53 somatic hotspot in bladder cancer but does not explicitly report p.Arg280Ser and states that the exact functional role of the hotspot was unknown.
• PMID:10761706 reports Arg-Ser substitutions at codon 280 in two esophageal-cancer tumors, but the reported nucleotide change is AGA-AGT and is not the target c.840A>C record; it was used only as contextual residue-level literature.
• PMID:29979965 and PMID:12826609 describe TP53 DNA-binding/core-domain functional landscapes, while PMID:30224644 reports differential responses involving codon 280; none provides the exact VCEP PM1 hotspot count or an approved TP53 domain-table entry for residue 280.

Still missing

• Exact cancerhotspots.org somatic occurrence count for TP53 p.Arg280Ser is not present in the bundle.
• No TP53 domain-table entries were supplied in vcep_materials.json.

Sources

Reasoning

The TP53 VCEP PM5 rule requires one or more different missense variants at the same amino-acid residue that were previously determined to be pathogenic or likely pathogenic according to the TP53 VCEP specifications, with clinical data required for a likely pathogenic comparator used at Supporting strength. The collected PM5 artifact contains zero same-residue candidates and records a soft retrieval failure due to HTTP 429, so the absence of candidates is not sufficient to conclude that no qualifying comparator exists.

Rule

Strong for at least 2 different pathogenic missense variants at the same residue; Moderate for 1 different pathogenic variant; Supporting for 1 different likely pathogenic variant with clinical pathogenicity data.

Evidence considered

• TP53 VCEP PM5 requires at least two different pathogenic same-residue missense variants for Strong, one pathogenic same-residue variant for Moderate, or one clinically supported likely pathogenic same-residue variant for Supporting.
• The PM5 candidate artifact found zero same-residue candidates and reports HTTP 429 during harvesting; it explicitly instructs that PM5 must not be applied from that artifact alone.

Still missing

• A complete review of TP53 p.Arg280 alternate missense variants and their qualifying VCEP-supported classifications is unavailable because comparator retrieval failed.

Sources

Reasoning

The TP53 combined PS2/PM6 framework describes PM6 for an assumed de novo variant without confirmation of paternity and maternity, but the bundle does not document that this variant is assumed de novo or provide the proband phenotype needed to tally the combined-rule points.

Rule

TP53 VCEP combined PS2/PM6 rule: assumed de novo without confirmation of paternity and maternity; proband points are tallied using the specified TP53 cancer table.

Evidence considered

• The governing source is the ClinGen TP53 Expert Panel Specifications, version 2.4, including the combined PS2/PM6 de novo instructions.
• No case-level assertion that the variant is assumed de novo is present in the consolidated evidence bundle.
• No case-level maternity or paternity confirmation status is present in the consolidated evidence bundle.
• No case-level proband cancer type is present in the consolidated evidence bundle.
• CHECKLIST pm6_1: Is the variant assumed de novo? — NA — The case bundle does not state that the variant is assumed de novo.
• CHECKLIST pm6_2: Is confirmation of both paternity and maternity absent? — NA — The bundle does not establish either the presence or absence of confirmation for both parents.
• CHECKLIST pm6_3: Under the referenced PS2_PM6 combined rule, what proband points are tallied based on the available parental-confirmation status and the type of cancer(s) seen in the proband? — NA — Parental-confirmation status and proband cancer type are unavailable, so points cannot be tallied.

Still missing

• Whether the variant is assumed de novo is not reported.
• Maternity and paternity confirmation status is not reported.
• Proband cancer type is not reported.
• PM6/combined-rule proband points cannot be tallied.

Sources

Reasoning

No family-level carrier testing, affected-relative phenotype data, qualifying meioses, or family counts are provided, so TP53 cosegregation evidence cannot be assessed.

Rule

TP53 VCEP PP1: ≥7 meioses across >1 family = Strong; 5-6 meioses in/across ≥1 family = Moderate; 3-4 meioses in/across ≥1 family = Supporting.

Evidence considered

• The governing TP53 VCEP PP1 thresholds are Strong for at least 7 meioses across more than 1 family, Moderate for 5-6 meioses in/across at least 1 family, and Supporting for 3-4 meioses in/across at least 1 family.
• The TP53 LFS cancer table is the specified source for deciding whether affected carriers have relevant cancers.
• No variant carrier status for relevant relatives is present in the consolidated evidence bundle.
• No relevant cancer status for potential carriers is present in the consolidated evidence bundle.
• No qualifying segregation meiosis count is present in the consolidated evidence bundle.
• No contributing family count is present in the consolidated evidence bundle.
• No information is provided on whether breast cancer is the only cancer pattern across families.
• CHECKLIST pp1_1: For each counted meiosis, does the individual both carry the variant and have a relevant cancer according to the LFS cancers table? — NA — No counted individuals, carrier results, or cancer phenotypes are reported.
• CHECKLIST pp1_2: Have qualifying meioses through unaffected obligate carriers been counted where applicable? — NA — Family structures and obligate-carrier information are not reported.
• CHECKLIST pp1_3: Is breast cancer the only cancer present across many families, requiring caution because it is a common cancer type? — NA — The cancer pattern across families is not reported.
• CHECKLIST pp1_4: What is the total number of qualifying cosegregation meioses, and are they observed across more than one family, in one or more families, or otherwise? — NA — Neither the meiosis count nor family distribution is reported.
• CHECKLIST pp1_5: Does the cosegregation evidence meet one of the stated ranges: ≥ 7 meioses across > 1 family; 5-6 meioses in/across 1 or more families; or 3-4 meioses in/across 1 or more families? — NA — Qualifying meioses and family distribution cannot be determined.

Still missing

• Variant carrier status for relevant individuals is not reported.
• Relevant cancer status for carriers is not reported.
• Qualifying segregation meioses are not reported.
• Number of families contributing cosegregation evidence is not reported.
• Whether breast cancer is the only cancer pattern across families is not reported.

Sources

Reasoning

The TP53 VCEP PP4 specification requires qualifying low-VAF observations and, for moderate evidence, at least two independent observations in the specified VAF range. No blood VAF, independent observation count, non-lymphocyte tissue result, tumor VAF, or family segregation result is present in the case bundle.

Rule

ClinGen TP53 VCEP PP4: supporting requires an observation with VAF 5-35%; moderate requires at least two independent observations with VAF 5-25%.

Evidence considered

• The TP53 VCEP PP4 specification requires an observation at VAF 5-35% for supporting evidence and at least two independent observations at VAF 5-25% for moderate evidence; constitutional mosaicism may be confirmed in non-lymphocyte tissue, tumor at higher VAF, and/or other family members.
• CHECKLIST pp4_1: Is the TP53 variant observed at a low variant allele fraction (VAF) in blood? — NA — No blood VAF is documented.
• CHECKLIST pp4_2: Could the low VAF be due to true constitutional mosaicism, confirmed by observing the variant in other non-lymphocyte tissues? — NA — No non-lymphocyte tissue observation is documented.
• CHECKLIST pp4_3: Could the low VAF be due to true constitutional mosaicism, confirmed by observing the variant in the tumor at higher VAF? — NA — No tumor VAF is documented.
• CHECKLIST pp4_4: Could the low VAF be due to true constitutional mosaicism, confirmed by the variant segregating in other family members? — NA — No segregation result is documented.
• CHECKLIST pp4_5: Are there at least 2 independent observations of the variant with VAF 5-25%? — NA — No independent observation count or qualifying VAF observations are documented.
• CHECKLIST pp4_6: Is there observation of the variant with VAF 5-35% (i.e., once or multiple times with VAF >25-35% and/or once with VAF 5-25%)? — NA — No qualifying VAF observation is documented.

Still missing

• Variant allele fraction in blood with multigene-panel testing context.
• Independent observation count and qualifying VAF values.
• Non-lymphocyte tissue observation, tumor VAF, and/or segregation in other family members to evaluate constitutional mosaicism.

Sources

Reasoning

BS2 cannot be assessed from the case bundle. The TP53 VCEP requires qualifying unrelated female carriers from a single source who reached at least age 60 without cancer, with VAF and late-sarcoma exclusions; no carrier-level cohort data for this variant are supplied.

Rule

TP53 VCEP v2.4 BS2: unrelated female carriers from a single source who reached at least age 60 without cancer; Supporting=2-3, Moderate=4-7, Strong≥8, with specified source, VAF, and late-sarcoma exclusions.

Evidence considered

• CHECKLIST item bs2_1: Was evidence derived from TP53 multigene panel testing results from two diagnostic labs comparing cancer-free individuals by age 60 in carriers versus controls? — NA — The case bundle contains no carrier-versus-control panel results for this exact variant.
• CHECKLIST item bs2_2: Are ≥ 8 unrelated females at least 60 years old without cancer in one source? — NA — No single-source qualifying carrier count is provided.
• CHECKLIST item bs2_3: Are 4-7 unrelated females at least 60 years old without cancer in one source? — NA — No single-source qualifying carrier count is provided.
• CHECKLIST item bs2_4: Are 2-3 unrelated females at least 60 years old without cancer in one source? — NA — No single-source qualifying carrier count is provided.
• CHECKLIST item bs2_5: Are cases not counted across sources? — NA — Source-level individual records are not provided, so duplicate counting cannot be evaluated.
• CHECKLIST item bs2_6: Were individuals with sarcoma diagnosed ≥ age 61 excluded from the BS2 total? — NA — Individual-level sarcoma history and exclusion status are not provided.
• The TP53 VCEP Version 2.4 defines BS2 as 2-3 qualifying unrelated females for Supporting, 4-7 for Moderate, or at least 8 for Strong, all from one source, excluding individuals with sarcoma diagnosed at age 61 or older and low-VAF observations suggestive of somatic origin.
• The case bundle contains no qualifying carrier-level BS2 observations for NM_000546.6:c.840A>C (p.Arg280Ser).

Still missing

• BS2 checklist item bs2_1 lacks the required two-laboratory TP53 multigene-panel comparison data.
• BS2 checklist item bs2_2 lacks a single-source count of at least 8 unrelated female carriers aged at least 60 without cancer.
• BS2 checklist item bs2_3 lacks a single-source count of 4-7 unrelated female carriers aged at least 60 without cancer.
• BS2 checklist item bs2_4 lacks a single-source count of 2-3 unrelated female carriers aged at least 60 without cancer.
• BS2 checklist item bs2_5 lacks source-level records sufficient to determine whether individuals were counted across sources.
• BS2 checklist item bs2_6 lacks individual-level sarcoma histories to determine whether diagnoses at age 61 or older were excluded.

Sources

Reasoning

The TP53 VCEP BS4 rule requires lack of segregation in affected family members with LFS-associated cancers, but the bundle contains no affected-relative variant results or phenotype data and does not permit assessment of phenocopies or multiple pathogenic variants.

Rule

TP53 VCEP BS4: Strong evidence for lack of segregation in affected family members diagnosed with LFS-associated cancers, with phenocopy and multiple-pathogenic-variant caveats.

Evidence considered

• The governing TP53 VCEP BS4 rule is strong evidence for lack of segregation in affected family members diagnosed with LFS-associated cancers.
• The TP53 LFS cancer table is the specified source for determining whether affected family members have LFS-associated cancers.
• No affected-family-member variant statuses are present in the consolidated evidence bundle.
• No affected-family-member cancer types are present in the consolidated evidence bundle.
• No phenocopy assessment is present in the consolidated evidence bundle.
• No assessment of more than one pathogenic variant contributing to an autosomal dominant disorder is present in the consolidated evidence bundle.
• CHECKLIST bs4_1: Is there lack of segregation in affected members of a family? — NA — Affected relatives and their variant results are not reported.
• CHECKLIST bs4_2: For affected family members considered, are they diagnosed with LFS-associated cancers as described in the Table of LFS Cancers and Points for PS2 and PP1 Code Application? — NA — No affected family members or cancer diagnoses are reported.
• CHECKLIST bs4_3: Could phenocopies for common phenotypes, including cancer or epilepsy, mimic lack of segregation among affected individuals? — NA — The affected-family phenotype data needed for phenocopy assessment are absent.
• CHECKLIST bs4_4: Could more than one pathogenic variant be contributing to an autosomal dominant disorder in the family? — NA — No family testing or assessment of additional pathogenic variants is reported.

Still missing

• Variant status of affected family members is not reported.
• Cancer types of affected family members are not reported.
• Phenocopy assessment is not reported.
• Assessment for additional pathogenic variants is not reported.

Sources