TP53 c.820G>T, p.(Val274Phe)

Reasoning

The ClinGen TP53 VCEP version 2.4 functional worksheet assigns PS3 to p.Val274Phe (NM_000546.6:c.820G>T). The exact V274F row records a non-functional result in the Kato assay column and loss-of-function results in the Funk, Giacomelli, and Kotler assay columns; the worksheet's preliminary functional code is PS3. This satisfies the TP53-specific rule requiring a non-functional Kato result and loss of function by the majority of other eligible assays.

Rule

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

Evidence considered

• The TP53 VCEP functional flowchart identifies Kato, Funk, Giacomelli, Kotler, and Kawaguchi as eligible functional studies for PS3/BS3 assessment.
• The exact VCEP Functional-worksheet.xlsx row for V274F records: Kato = Non-functional; Funk = LOF; Giacomelli = LOF; Kotler = LOF; Kawaguchi = NA; preliminary functional code = PS3.
• The TP53 VCEP version 2.4 rule is: PS3 Strong requires non-functional on Kato et al. data AND loss of function by the majority of other eligible assays.

Still missing

• The supplied paper-extract bundle does not provide a variant-specific direct quote for V274F from the individual assay publications; the PS3 assignment is supported by the VCEP Functional-worksheet.xlsx row and its assay-result columns.

Sources

Reasoning

The TP53 VCEP v2.4 PM2 rule is applied at Supporting strength when total allele frequency is <0.00003 in gnomAD or another large sequenced population; if multiple alleles occur in an ancestry group, that group's frequency must be <0.00004, with founder-effect groups and Remaining ignored. gnomAD v4.1 reports total AC=1/1,614,096 (AF=6.19542e-07), highest qualifying ancestry AF=8.47449e-07 in European non-Finnish ancestry with AC=1, and zero homozygotes. These values meet PM2_Supporting without a CHIP allele-balance recalculation.

Rule

Apply PM2 at Supporting when total AF <0.00003; if multiple alleles are present within an ancestry group, that group AF must be <0.00004; ignore founder-effect groups and Remaining.

Evidence considered

• The VCEP PM2 rule specifies Supporting strength for allele frequency <0.00003; when multiple alleles are present in an ancestry group, that group's frequency must be <0.00004. Founder-effect groups and Remaining are ignored.
• gnomAD v4.1 reports total AC=1, AN=1,614,096, AF=6.19542e-07, and homozygotes=0. The highest observed qualifying ancestry frequency is European non-Finnish AC=1, AN=1,180,012, AF=8.47449e-07, and homozygotes=0.
• The variant is absent from gnomAD v2.1 and gnomAD-Canada v1.0, providing concordant additional rarity evidence.
• Because the observed total AF is already below the VCEP PM2 threshold, the framework's optional low-VAF/CHIP recalculation is not needed for PM2 application.

Sources

Reasoning

PP3_Supporting is met under the TP53 VCEP specification. The VCEP's per-variant Supplementary Table S2 directly assigns PP3 to NM_000546.6:c.820G>T (p.Val274Phe); this assignment is based on Align-GVGD class C45 and BayesDel 0.315825. SpliceAI maximum delta score is 0.00, so there is no predicted splice effect to separately support PP3 or to conflict with the missense assignment.

Rule

TP53 VCEP PP3_Supporting: missense variant with Align-GVGD class C25-C55 and BayesDel >=0.16; apply the VCEP's direct per-variant PP3/BP4 assignment after considering predicted splicing impact.

Evidence considered

• The governing TP53 VCEP rule permits PP3_Supporting for missense variants with Align-GVGD class C25-C55 and BayesDel score >=0.16; it directs use of the PP3-BP4 per-variant spreadsheet.
• TP53 VCEP Supplementary Table S2 lists c.820G>T, p.Val274Phe as Align-GVGD Class C45, BayesDel 0.315825, with preliminary bioinformatic code PP3 and raw maximum SpliceAI delta score 0.03.
• The case SpliceAI lookup reports maximum delta score 0.00 and no significant predicted splice impact.

Sources

Reasoning

NM_000546.6:c.820G>T is a single-nucleotide missense variant, p.(Val274Phe), in exon 8. It does not create a premature termination codon, frameshift, initiation-codon change, canonical splice-site change, or exon deletion. SpliceAI maximum delta score is 0.00, providing no predicted splice alteration that could convert this apparent missense change into a loss-of-function transcript. Therefore it does not meet any TP53 VCEP PVS1 decision-tree null-variant category; NMD and C-terminal truncation rules are not reached.

Rule

TP53 VCEP PVS1 decision tree: applicable only to qualifying null or splice-altering variants; this variant is missense with no predicted splice impact.

Evidence considered

• Transcript normalization identifies NM_000546.6:c.820G>T as NP_000537.3:p.(Val274Phe), a missense substitution in exon 8.
• The TP53 VCEP PVS1 specification applies its decision tree to null variants, including nonsense, frameshift, initiation-codon, canonical splice-site, and exon-deletion events; its NMD and terminal-truncation strength rules therefore do not apply to this amino-acid substitution.
• SpliceAI reports a maximum delta score of 0.00 and no significant predicted splice impact.

Sources

Reasoning

No exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion is present. The available ClinVar record is not exact-match validated and contains only individual laboratory submissions; therefore it cannot trigger PP5.

Rule

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

Evidence considered

• ClinVar data for the candidate record list no expert panels and report criteria-provided, conflicting classifications with individual laboratory submissions. The prefetch audit also states that no exact ClinVar XML match was validated.

Sources

Reasoning

The TP53 VCEP v2.4 BA1 threshold is filtering allele frequency >=0.001 in a qualifying continental genetic-ancestry group with >=2,000 alleles tested and at least 2 variant alleles. gnomAD v4.1 reports total AC=1/1,614,096 (AF=6.19542e-07) and the highest qualifying observed frequency in European non-Finnish ancestry as AC=1/1,180,012 (AF=8.47449e-07), far below 0.001. No qualifying ancestry group reaches the BA1 threshold.

Rule

Filtering allele frequency (FAF) >= 0.001 in a single qualifying gnomAD continental ancestry group; group must have >=2,000 alleles tested and at least 2 variant alleles; exclude founder-effect groups and Remaining.

Evidence considered

• The applicable framework is the ClinGen TP53 Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines, version 2.4.
• The VCEP BA1 rule requires filtering allele frequency >=0.001 (0.1%) in one qualifying continental ancestry group, with >=2,000 alleles tested and at least 2 variant alleles; founder-effect groups and Remaining are excluded.
• gnomAD v4.1 reports NM_000546.6:c.820G>T (p.Val274Phe) at total AC=1, AN=1,614,096, AF=6.19542e-07, with 0 homozygotes. The highest observed qualifying ancestry frequency is European non-Finnish: AC=1, AN=1,180,012, AF=8.47449e-07, with 0 homozygotes.

Sources

Reasoning

The TP53 VCEP v2.4 BS1 threshold is filtering allele frequency >=0.0003 and <0.001 in a qualifying continental genetic-ancestry group with >=2,000 alleles tested and at least 2 variant alleles. The maximum observed qualifying ancestry frequency is 8.47449e-07 in European non-Finnish ancestry, based on one allele, and is below 0.0003. Therefore BS1 is not met.

Rule

Filtering allele frequency >=0.0003 but <0.001 in a single qualifying gnomAD continental ancestry group; group must have >=2,000 alleles tested and at least 2 variant alleles; exclude founder-effect groups and Remaining.

Evidence considered

• The VCEP BS1 rule uses filtering allele frequency >=0.0003 but <0.001 in one qualifying continental ancestry group; the group must have >=2,000 alleles tested and at least 2 variant alleles, with founder-effect groups and Remaining excluded.
• gnomAD v4.1 reports the highest observed qualifying ancestry frequency in European non-Finnish ancestry as AC=1/1,180,012, AF=8.47449e-07, with 0 homozygotes. This is below the VCEP BS1 lower threshold and does not meet the minimum two-allele observation requirement.

Sources

Reasoning

BS3 is not met because the exact V274F row in the ClinGen TP53 VCEP functional worksheet records loss-of-function results in the Funk, Giacomelli, and Kotler assay columns, rather than the functional or partially functional pattern required for BS3. The VCEP preliminary code for this row is PS3.

Rule

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

Evidence considered

• The exact VCEP Functional-worksheet.xlsx row for V274F records: Kato = Non-functional; Funk = LOF; Giacomelli = LOF; Kotler = LOF; Kawaguchi = NA; preliminary functional code = PS3.
• The TP53 VCEP version 2.4 BS3 rules require either functional on Kato plus no LOF by the majority of eligible assays for BS3 Strong, or partially functional on Kato plus no LOF by all available assays for BS3 Supporting; neither pattern is present.

Sources

Reasoning

BP4 is not met. The governing TP53 VCEP per-variant table assigns PP3, not BP4, to c.820G>T (p.Val274Phe). Although SpliceAI predicts no splice impact, BP4 cannot be applied because the VCEP missense computational assignment is pathogenic-direction PP3; the same computational prediction is not double-counted in opposing criteria.

Rule

TP53 VCEP BP4 missense rule: benign-direction BayesDel/aGVGD category plus SpliceAI <0.2; do not apply a competing BP4 code when the VCEP's direct per-variant assignment is PP3.

Evidence considered

• The governing TP53 VCEP rule applies BP4 only to specified benign-direction BayesDel/aGVGD combinations and requires no predicted splicing difference for missense variants.
• TP53 VCEP Supplementary Table S2 directly assigns PP3, rather than BP4, to c.820G>T (p.Val274Phe), with Align-GVGD Class C45 and BayesDel 0.315825.
• The case SpliceAI lookup reports maximum delta score 0.00; this satisfies the no-predicted-splice-impact condition but does not override the VCEP's direct PP3 assignment.

Sources

Reasoning

No exact-variant ClinVar expert-panel Benign or Likely benign assertion is present. The available candidate record has no expert-panel submission and cannot trigger BP6.

Rule

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

Evidence considered

• ClinVar data for the candidate record list no expert panels and show individual laboratory submissions only; no Benign or Likely benign expert-panel assertion is supplied.

Sources

Reasoning

The variant is TP53 NM_000546.6:c.820G>T (p.Val274Phe), but the case bundle identifies no previously established pathogenic or likely pathogenic missense variant producing the same amino-acid change at residue 274. The TP53 VCEP requires a qualifying same-amino-acid comparator and confirmation of no splicing difference; the latter is satisfied by SpliceAI max delta 0.00, but the required comparator is absent.

Rule

TP53 VCEP v2.4 PS1: same amino-acid change as a previously established pathogenic/likely pathogenic variant; rule out splicing with RNA data or SpliceAI <0.2. Strong for a pathogenic comparator and Moderate for a likely pathogenic comparator.

Evidence considered

• The TP53 VCEP PS1 rule permits comparison only with variants asserted as pathogenic or likely pathogenic under the TP53 VCEP specifications; PS1 strength is Strong for a pathogenic comparator and Moderate for a likely pathogenic comparator.
• The PM5 candidate summary reports 0 same-residue candidates and no expert-panel or non-expert majority pathogenic/likely pathogenic candidates.
• SpliceAI reports no significant splice impact for NM_000546.6:c.820G>T, with maximum delta score 0.00, satisfying the VCEP requirement to exclude a splice-driven explanation if a comparator is found.

Still missing

• A previously established pathogenic or likely pathogenic TP53 missense variant causing p.Val274X or the same p.Val274Phe amino-acid change must be identified and independently classified under the TP53 VCEP framework.

Sources

Reasoning

The TP53 VCEP specifies PS2 as the exclusive de novo code and requires tallying confirmed de novo observations using confirmed maternity and paternity plus the proband's LFS-associated cancer type. The case bundle contains no proband phenotype/cancer details, parental testing results, maternity or paternity confirmation, constitutional-mosaicism evidence, or de novo family observations. The only variant-specific paper finding is a tumor-only observation and does not establish a germline de novo event.

Rule

TP53 VCEP PS2: de novo points require the applicable proband cancer category and confirmed maternity and paternity; 1 point = supporting, 2-3 = moderate, 4-7 = strong, and >=8 = very strong.

Evidence considered

• The TP53 VCEP Version 2.4 rule states that de novo points are tallied using confirmed maternity and paternity and the proband's cancer type; total points determine PS2 strength, with 1 point supporting, 2-3 moderate, 4-7 strong, and at least 8 very strong.
• The VCEP explicitly drops PM6 and uses PS2 exclusively for TP53 de novo evidence.
• PMID:31775759 reports c.820G>T (p.Val274Phe) in one lower-alveolus oral squamous-cell-carcinoma tumor from a targeted FFPE tumor-sequencing cohort, but reports no germline testing, parental testing, de novo assessment, or segregation analysis.

Still missing

• Proband diagnosis and LFS-associated cancer category needed for VCEP point assignment
• Results of parental variant testing and confirmation of maternity and paternity
• Evidence that the variant is germline or constitutional mosaic rather than tumor-only/somatic
• Any additional independent de novo probands and their applicable VCEP points

Sources

Reasoning

The TP53 VCEP PS4 framework requires qualifying germline proband points. No eligible germline proband observations, cancer phenotypes/ages, Chompret or Classic LFS status, HER2-positive early-onset breast-cancer status, or case-control enrichment data are supplied for this variant. The one extracted exact-variant literature observation is from tumor tissue and cannot be counted as germline PS4 evidence.

Rule

TP53 VCEP v2.4 PS4: score qualifying germline probands; Supporting 1–1.5 points, Moderate 2–3.5 points, Strong 4–7.5 points, Very Strong at least 8 points.

Evidence considered

• TP53 VCEP v2.4 assigns PS4 by qualifying germline proband points: 1–1.5 points supporting, 2–3.5 moderate, 4–7.5 strong, and at least 8 very strong. The supplied bundle contains no countable germline probands or point assignments for c.820G>T (p.Val274Phe).
• PMID:31775759 reports c.820G>T (p.Val274Phe) in an FFPE oral squamous-cell-carcinoma tumor cohort. This somatic tumor observation does not establish a germline affected proband and is not eligible for TP53 VCEP PS4 scoring.
• PMID:29979965 was reviewed because it was in the PS4 literature queue; it is a functional mutation-library study and provides no variant-specific germline case count or case-control enrichment estimate for c.820G>T (p.Val274Phe).

Still missing

• Eligible unrelated germline proband-level data, including cancer type, age at diagnosis, and Chompret or Classic LFS status, are needed to calculate TP53 VCEP PS4 points.
• Any qualifying early-onset HER2-positive breast-cancer observations and a determination of whether they overlap with Chompret or Classic LFS points are needed.
• A validated case-control analysis for this exact germline variant, if available, is needed to assess enrichment independently of point-based observations.

Sources

Reasoning

Residue 274 is outside the TP53 VCEP's specified codon list for PM1 (175, 245, 248, 249, 273, and 282). The bundle reports that V274 is in a statistically significant cancer hotspot and that the exact variant has 73 COSMIC somatic observations, but the VCEP rule requires verification in cancerhotspots.org v2 of at least 10 somatic occurrences for the same amino-acid change when using the hotspot route. The collected hotspot record is explicitly partial and does not provide the required count or an auditable same-amino-acid cancerhotspots result. Therefore PM1 cannot be assigned from the current bundle.

Rule

TP53 VCEP v2.4 PM1: Moderate for missense variants in codons 175, 245, 248, 249, 273, or 282; alternatively Moderate for a germline missense variant in cancerhotspots.org v2 with >=10 somatic occurrences for the same amino-acid change, or Supporting with 2-9 occurrences.

Evidence considered

• The TP53 VCEP PM1 rule applies at Moderate strength to missense variants in codons 175, 245, 248, 249, 273, or 282, or to germline missense variants seen in cancerhotspots.org v2 with at least 10 somatic occurrences for the same amino-acid change; Supporting applies to 2-9 such occurrences.
• The target variant is p.Val274Phe, so it is not within the VCEP's enumerated codon list.
• The collected hotspot result states that the variant lies in a statistically significant hotspot, but warns that regex extraction was partial and requires screenshot verification; it contains no somatic count or same-amino-acid count.
• COSMIC reports COSV52707923 with 73 somatic observations for the exact c.820G>T variant, but this source does not establish the VCEP's cancerhotspots.org v2 criterion.

Still missing

• Verify from cancerhotspots.org v2 that TP53 p.Val274Phe has at least 10 somatic occurrences, or provide another VCEP-accepted basis for PM1.
• The COSMIC count of 73 is not substituted for the VCEP-specific cancerhotspots.org v2 threshold.

Sources

Reasoning

The TP53 VCEP requires one or more different missense variants at the same amino-acid residue that are independently classified as pathogenic or likely pathogenic under the VCEP, with the comparator reaching that classification without PM5; Grantham comparison and absence of a splice effect are also required. The case bundle reports zero same-residue comparator candidates, so no PM5 strength can be assigned.

Rule

TP53 VCEP v2.4 PM5: Strong for >=2 different pathogenic missense variants at the residue, Moderate for 1 different pathogenic missense variant, or Supporting for 1 different likely pathogenic missense variant with clinical pathogenicity data; require non-PM5 comparator classification, Grantham comparison, and SpliceAI <0.2 or RNA evidence.

Evidence considered

• The TP53 VCEP PM5 rule requires a different missense change at the same residue that is pathogenic or likely pathogenic under the VCEP and reaches that classification without PM5; the evaluated variant must be chemically equally or more different from wild type by Grantham comparison.
• The PM5 candidate summary reports 0 same-residue candidates, 0 expert-panel pathogenic/likely pathogenic candidates, and 0 non-expert majority pathogenic/likely pathogenic candidates.
• SpliceAI maximum delta score is 0.00, so lack of a predicted splice effect would not be the limiting gap if a qualifying same-residue comparator were identified.

Still missing

• Identify and independently adjudicate at least one different missense variant at TP53 residue Val274 under the TP53 VCEP, including its non-PM5 pathogenicity basis and Grantham comparison to Val274Phe.

Sources

Reasoning

The TP53 VCEP requires counting meioses in individuals who both carry the variant and have a relevant LFS-associated cancer, with 3-4 meioses for supporting, 5-6 for moderate, and at least 7 across more than one family for strong. The case bundle provides no affected relatives, variant-positive relatives, unaffected obligate carriers, family structure, or countable meioses. The tumor-only observation in PMID:31775759 does not provide familial germline segregation data.

Rule

TP53 VCEP PP1: 3-4 cosegregating meioses = supporting, 5-6 = moderate, and >=7 across >1 family = strong; countable individuals must carry the variant and have a relevant LFS-associated cancer, subject to the VCEP caveats.

Evidence considered

• The TP53 VCEP PP1 rule counts meioses for individuals who carry the variant and have a relevant cancer, permits meioses through unaffected obligate carriers, and assigns supporting strength at 3-4 meioses, moderate at 5-6, and strong at at least 7 meioses across more than one family.
• The VCEP cautions that tumor-only observations can count only under specified multi-member family circumstances when germline evidence exists in at least one family member; no such family data are present here.
• PMID:31775759 is a tumor-tissue sequencing report of one patient and reports no segregation analysis or germline family data.

Still missing

• Pedigree and family-history data for LFS-associated cancers
• Germline variant results for affected relatives or qualifying tumor results in multiple family members
• Phase/inheritance information and the number of countable meioses
• Confirmation that relatives counted have relevant LFS-associated cancers and that the family context is not confounded by phenocopies

Sources

Reasoning

For TP53, PP4 is not a generic phenotype-specificity criterion. The applicable VCEP rule concerns likely somatic TP53 variants detected on multigene panel testing: supporting requires an observation with VAF 5–35%, and moderate requires at least two independent observations with VAF 5–25%. No multigene-panel VAF observations, tissue context, blood-cancer exclusion, or independent-observation count is provided.

Rule

TP53 VCEP v2.4 PP4: Supporting for an observation with VAF 5–35%; Moderate for at least two independent observations with VAF 5–25%; do not use in blood cancer or with BA1/BS1.

Evidence considered

• TP53 VCEP v2.4 defines PP4 as evidence from likely somatic low-VAF TP53 variants found on multigene panel testing, not as the generic phenotype-specificity rule. The bundle supplies no patient-level VAF or multigene-panel observations.

Still missing

• For each potential observation, multigene-panel testing context and measured VAF are needed.
• At least two independent observations with VAF 5–25% are needed for PP4_Moderate; otherwise qualifying VAF 5–35% evidence is needed for PP4_Supporting.
• Confirmation that the individual does not have blood cancer and that the variant does not meet BA1 or BS1 is required before applying the VCEP PP4 rule.

Sources

Reasoning

No qualifying individual-level carrier observations are available in the case bundle to evaluate the TP53 VCEP BS2 rule. gnomAD reports zero homozygotes, but this does not establish the required count of unrelated females aged at least 60 years without cancer from a single source. BS2 cannot be assigned from the available population summary data.

Rule

Count qualifying unrelated female carriers aged >=60 years without cancer from one source: 2-3 Supporting, 4-7 Moderate, or >=8 Strong; exclude qualifying cases with evidence suggestive of somatic origin and do not combine sources.

Evidence considered

• The VCEP BS2 rule requires 2-3 qualifying unrelated females aged at least 60 years without cancer for Supporting, 4-7 for Moderate, or at least 8 for Strong; all must come from a single source, and low-VAF likely somatic variants must be excluded.
• gnomAD v4.1 reports zero homozygotes for the variant, but the bundle provides no individual-level carrier phenotype, age, sex, relatedness, or allele-balance data that could satisfy or refute the VCEP BS2 carrier-count rule.

Still missing

• Individual-level carrier data from a single source, including sex, age, cancer status, relatedness, and any variant allele fraction, are unavailable; therefore the required count of qualifying unrelated female carriers cannot be assessed.

Sources

Reasoning

The TP53 VCEP permits BS4 at strong strength for lack of segregation in affected family members diagnosed with LFS-associated cancers. The case bundle contains no affected-family-member genotypes, no non-segregation observations, and no pedigree or phenotype data. A single tumor-only report cannot establish lack of segregation, and the VCEP cautions that phenocopies and multiple pathogenic variants can confound this assessment.

Rule

TP53 VCEP BS4: strong evidence requires lack of segregation in affected family members diagnosed with LFS-associated cancers, with consideration of phenocopies and additional pathogenic variants.

Evidence considered

• The TP53 VCEP BS4 rule requires lack of segregation in affected family members with LFS-associated cancers and allows strong strength when that evidence is established.
• The VCEP warns that phenocopies and more than one pathogenic variant in a family can mimic lack of segregation.
• PMID:31775759 reports one tumor-tissue observation without affected-relative testing or family segregation data.

Still missing

• Affected-family-member genotypes and corresponding cancer diagnoses
• Documented non-segregation of the variant in affected relatives
• Pedigree review to assess phenocopies and possible alternative pathogenic variants

Sources