TP53 c.1066G>C, p.(Gly356Arg)

Reasoning

The TP53 VCEP requires PM2 only at supporting strength when total allele frequency is <0.00003 in gnomAD or another large sequenced population, and if multiple alleles are present in an ancestry group that group's AF must be <0.00004; founder-effect groups are ignored. gnomAD v4.1 reports total AF 4.33716e-06 (7/1,613,958 alleles), European non-Finnish AF 5.93229e-06 (7/1,179,982 alleles), grpmax FAF 2.47e-06, and zero homozygotes. Both the total and maximum eligible ancestry-group frequencies are below the VCEP cutoffs, so PM2_Supporting is met.

Rule

Apply PM2 at supporting strength when total allele frequency is <0.00003; if multiple alleles are present in any genetic ancestry group, that group's AF must be <0.00004; founder-effect groups are ignored.

Evidence considered

• TP53 VCEP v2.4 specifies PM2 at supporting strength for total AF <0.00003; where multiple alleles occur in an ancestry group, that group's AF must be <0.00004, with founder-effect groups ignored.
• gnomAD v4.1 reports 7/1,613,958 total alleles, AF 4.33716e-06, and no homozygotes.
• The highest reported eligible continental-subpopulation AF is 5.93229e-06 in European non-Finnish (7/1,179,982 alleles), below 0.00004; grpmax FAF is 2.47e-06.
• The absence of homozygotes is consistent with rarity but is not used as a separate criterion.

Sources

Reasoning

The TP53 VCEP Functional-worksheet.xlsx (Supplementary Table S3) directly assigns 'BS3' to p.(Gly356Arg)/G356R, based on Kato et al. transactivation classification 'Functional' (i.e. full/super-transactivation activity, not merely partial) and 'noLOF' status. This matches the TP53 VCEP cspec BS3 Strong rule: 'Functional on Kato et al. data AND no loss of function (LOF) by the majority of available eligible assays.' No conflicting LOF signal is present in any of the other eligible assay columns (Funk, Giacomelli, Kotler, Kawaguchi are all marked NA/not tested for this variant, so there is no assay in disagreement with the Kato result).

Rule

TP53 VCEP cspec v2.4 BS3 Strong: 'Functional on Kato et al. data AND no loss of function (LOF) by the majority of available eligible assays' -- met for G356R per VCEP Functional-worksheet.xlsx pre-assigned code BS3.

Evidence considered

• TP53 VCEP Functional-worksheet.xlsx (Supplementary Table S3) row for G356R: Kato et al. classification = Functional, LOF status = noLOF, VCEP pre-assigned code = BS3.
• TP53 VCEP cspec v2.4 BS3 Strong rule: 'Functional on Kato et al. data AND no loss of function (LOF) by the majority of available eligible assays' -- satisfied because Kato classifies G356R as Functional/noLOF and no other eligible assay (Funk, Giacomelli, Kotler, Kawaguchi) shows LOF for this variant (all marked NA/not tested).
• TP53 VCEP cspec BS3 instructionsToUse caveat: do not apply BS3 for protein-level missense assays (Kato/Giacomelli) if PP3 is applied via SpliceAI for the same variant, or if there is RNA-level splicing aberration evidence -- flagged here for cross-check against the in-silico/splicing evidence group, not resolved within this functional group.
• VCEP functional rule flowchart (Flowchart-for-application-of-functional-rule-codes.pdf) confirms Kato (PMID 12826609), Funk (PMID 39774325), Giacomelli (PMID 30224644), Kotler (PMID 29979965), and Kawaguchi (PMID 16007150) are the eligible assay set for PS3/BS3 determination in TP53.

Still missing

• Independent/non-Kato assay data (Giacomelli, Kotler, Kawaguchi, Funk) is not available for this specific variant in the VCEP worksheet, so BS3_Strong here rests on the Kato transactivation result plus the VCEP's own pre-computed worksheet call rather than cross-assay concordance.
• Per the cspec BS3 instructionsToUse caveat, BS3 should not be applied at any weight for missense variants assayed at the protein level if PP3 is separately applied to this variant based on SpliceAI; that in silico/splicing determination is out of scope for this functional-evidence group and should be checked by the PP3/BP4 group before final combination.

Sources

Reasoning

For missense variants, the TP53 VCEP cspec v2.4 BP4 rule assigns BP4_Moderate when BayesDel <= -0.008 irrespective of aGVGD score, except when aGVGD is C65 (in which case BP4_Moderate is not applied), AND no predicted differences in splicing (SpliceAI < 0.2). The local BayesDel score for this variant is -0.347259, well below the -0.008 cutoff, and SpliceAI predicts no splicing impact (max delta 0.004 < 0.2). aGVGD class for this specific variant was not retrieved from the VCEP spreadsheet in this case bundle, so the C65 exclusion cannot be directly confirmed or ruled out from available data; however a BayesDel score this strongly benign-shifted is highly discordant with an aGVGD C65 (most damaging) call. BP4_Moderate is applied based on the BayesDel and SpliceAI data available, flagged for human review to confirm aGVGD class is not C65.

Rule

TP53 VCEP cspec v2.4 BP4: missense BayesDel <= -0.008 (irrespective of aGVGD except C65) AND SpliceAI < 0.2 = BP4_Moderate.

Evidence considered

• TP53 VCEP cspec v2.4 BP4 rule: missense variants get BP4_Moderate when BayesDel <= -0.008 irrespective of aGVGD score (except C65, in which case do not apply BP4_Moderate) AND SpliceAI < 0.2 (no predicted splicing differences).
• Local BayesDel predictor lookup score for this variant is -0.347259 (source: bayesdel), well below the -0.008 BP4_Moderate cutoff.
• SpliceAI Lookup (source: spliceai) max delta score is 0.004, well under the 0.2 threshold, confirming no predicted splicing impact required for the BP4 missense rule.
• aGVGD class for this specific variant was not available in the extracted VCEP spreadsheet sample; the strongly benign-shifted BayesDel score is discordant with an aGVGD C65 call, but this was not independently confirmed.

Still missing

• aGVGD class was not retrieved for p.Gly356Arg from the VCEP PP3-BP4-codes.xlsx spreadsheet in this case bundle; per the VCEP rule, BP4_Moderate should not be applied if aGVGD class is C65. Confirming aGVGD class would remove residual uncertainty.

Sources

Reasoning

Expert panel ClinGen TP53 Variant Curation Expert Panel, ClinGen classified as Likely benign.

Rule

Exact-variant ClinVar expert-panel Benign or Likely Benign assertion supports BP6 at Supporting strength only.

Evidence considered

• ClinVar variation 234059 exactly matches NM_000546.6(TP53):c.1066G>C (p.Gly356Arg). Its expert-panel submission is SCV001949906 from the ClinGen TP53 Variant Curation Expert Panel, classified Likely Benign and reviewed by expert panel. Ordinary laboratory submissions and aggregate labels were not used.
• ClinVar expert panel classification

Sources

Reasoning

The TP53 VCEP Functional-worksheet.xlsx (Supplementary Table S3) pre-assigns the code for p.(Gly356Arg)/G356R as 'BS3' with Kato et al. transactivation classification 'Functional' and 'noLOF' status, i.e. the opposite of the loss-of-function pattern PS3 requires. Per the VCEP functional rule flowchart and cspec BS3/PS3 rules, PS3 requires the variant be non-functional or partially functional on Kato et al. data AND show loss of function on the majority of other eligible assays (Funk, Giacomelli, Kotler, Kawaguchi); none of that pattern is present here (all non-Kato assay columns for this variant are marked NA/not tested, and the single available assay class, Kato, shows full function).

Rule

TP53 VCEP cspec v2.4 PS3: not met -- Kato et al. data classifies G356R as Functional (no LOF), which does not satisfy any PS3 strength tier (all of which require non-functional or partially-functional Kato data plus LOF on other assays).

Evidence considered

• TP53 VCEP Functional-worksheet.xlsx (Supplementary Table S3) row for G356R: Kato et al. classification = Functional, LOF status = noLOF, VCEP pre-assigned code = BS3.
• TP53 VCEP cspec v2.4 PS3 rule: 'Non-functional on Kato et al. data AND loss of function (LOF) by the majority of other eligible assays' (Strong); 'Partially functional on Kato et al. data AND loss of function (LOF) by the majority of other available assays' (Moderate); 'Non-functional on Kato et al. data AND abnormal on Kawaguchi et al. data regardless of other assays' (Supporting) -- none of these patterns is met for G356R, which is Functional/noLOF on Kato data.
• VCEP functional rule flowchart (Flowchart-for-application-of-functional-rule-codes.pdf) confirms Kato (PMID 12826609), Funk (PMID 39774325), Giacomelli (PMID 30224644), Kotler (PMID 29979965), and Kawaguchi (PMID 16007150) are the eligible assay set for PS3/BS3 determination in TP53.

Still missing

• Only Kato et al. transactivation data is populated for this variant in the VCEP functional worksheet; Giacomelli, Kotler, Kawaguchi, and Funk assay columns are marked NA (no data available), so a fully independent multi-assay confirmation of no-LOF is not possible beyond the VCEP's own pre-assigned BS3 call.

Sources

Reasoning

TP53 VCEP v2.4 PM1 applies at Moderate strength only to missense variants at codons 175, 245, 248, 249, 273, or 282 (DNA-binding domain hotspots), or to variants seen in cancerhotspots.org with >=10 somatic occurrences for the same amino acid change (Supporting for 2-9 occurrences). The variant under review, p.(Gly356Arg), falls at codon 356, which is not among the listed VCEP hotspot codons (codon 356 lies in the tetramerization domain, not the DNA-binding domain). The cancerhotspots.org lookup for TP53 G356 returned no result, explicitly noting the variant does not lie in a statistically significant hotspot.

Rule

TP53 VCEP v2.4 PM1: Moderate for missense at codons 175/245/248/249/273/282 or cancerhotspots.org >=10 occurrences of same aa change; Supporting for 2-9 occurrences. Codon 356 not in this list and no cancerhotspots.org hit was found.

Evidence considered

• Codon 356 is not one of the TP53 VCEP PM1 hotspot codons (175, 245, 248, 249, 273, 282).
• cancerhotspots.org query for 'TP53 G356' returned search_status 'no_result', found=false, with evidence_sentence 'This variant does not lie in a statistically significant hotspot.'

Sources

Reasoning

Variant is NM_000546.6:c.1066G>C, NP_000537.3:p.(Gly356Arg), a missense substitution. Per the TP53 VCEP specification (cspec v2.4), PP3 for missense variants requires BayesDel score >= 0.16 (with aGVGD class C25-C55 for Supporting, or C65 for Moderate). The local BayesDel lookup for this variant returns a score of -0.347259, far below the 0.16 threshold, so the BayesDel component of PP3 is not met. SpliceAI also predicts no splicing impact (max delta score 0.004, well under the 0.2 threshold used for the splice-effect PP3 pathway), so the splice-based PP3 pathway is also not triggered. No aGVGD class was retrieved for this variant, but since BayesDel alone already fails the >=0.16 requirement for both PP3 strength tiers, aGVGD status is immaterial to the PP3 determination.

Rule

TP53 VCEP cspec v2.4 PP3: missense requires BayesDel >= 0.16 (+ aGVGD class); exonic/intronic splice-predicted variants require SpliceAI >= 0.2.

Evidence considered

• TP53 VCEP cspec v2.4 PP3 rule: missense variants require aGVGD Class C65 and BayesDel score >= 0.16 (Moderate), or aGVGD class C25-C55 and BayesDel score >= 0.16 (Supporting); splice-effect pathway requires SpliceAI >= 0.2.
• Local BayesDel predictor lookup score for this variant is -0.347259 (source: bayesdel), well below the 0.16 pathogenic threshold, per the ClinGen TP53 VCEP-approved BayesDel cutoffs stated in the governing cspec.
• SpliceAI Lookup (source: spliceai) reports max delta score 0.004 (DS_AG 0.0, DS_AL 0.002, DS_DG 0.004, DS_DL 0.0), indicating no predicted splicing effect, well under the 0.2 PP3 splice-pathway threshold.
• REVEL score (0.138) was not used because the TP53 VCEP specification designates BayesDel and aGVGD (per Fortuno et al., 2018 tool-comparison study referenced in the cspec instructionsToUse) as the selected in silico tools for TP53, not REVEL.

Still missing

• aGVGD class for p.Gly356Arg was not directly retrieved from the VCEP PP3-BP4-codes.xlsx spreadsheet (only a small sample of rows was available in the extracted bundle and did not include this variant); not required to reach a PP3 not_met call since BayesDel already fails the threshold.

Sources

Reasoning

The exact ClinVar record has an expert-panel assertion of Likely Benign, not Pathogenic or Likely Pathogenic. Therefore it cannot support PP5 under the exact-variant expert-panel-only rule.

Rule

Exact-variant ClinVar expert-panel Pathogenic or Likely Pathogenic assertion supports PP5 only.

Evidence considered

• ClinVar variation 234059 is an exact transcript-HGVS match for NM_000546.6:c.1066G>C and includes a ClinGen TP53 Variant Curation Expert Panel assertion of Likely Benign, reviewed by expert panel. Ordinary laboratory submissions were not used.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely Pathogenic assertion would be required to support PP5.

Sources

Reasoning

The TP53 VCEP BA1 threshold is gnomAD continental-subpopulation FAF >= 0.001 (0.1%) in an eligible genetic-ancestry group with at least 2,000 alleles tested and at least 2 variant alleles. gnomAD v4.1 reports total AF 4.33716e-06 (7/1,613,958 alleles), maximum observed continental-subpopulation AF 5.93229e-06 in European non-Finnish (7/1,179,982 alleles), and grpmax FAF 2.47e-06. These values are far below 0.001, so BA1 is not met.

Rule

Filtering allele frequency (FAF) >= 0.001 or 0.1% in a qualifying gnomAD continental subpopulation of a single genetic ancestry group; ancestry groups influenced by founder effects are excluded.

Evidence considered

• TP53 VCEP v2.4 specifies BA1 at stand-alone strength when eligible gnomAD continental-subpopulation FAF is >=0.001; founder-effect groups are excluded.
• gnomAD v4.1 reports NM_000546.6:c.1066G>C at total AF 4.33716e-06 with 7/1,613,958 alleles and no homozygotes.
• The highest reported continental-subpopulation AF is 5.93229e-06 in European non-Finnish, and grpmax FAF is 2.47e-06; both are below the BA1 threshold.

Sources

Reasoning

The TP53 VCEP BS1 threshold is gnomAD continental-subpopulation FAF >= 0.0003 but <0.001 in an eligible genetic-ancestry group with at least 2,000 alleles tested and at least 2 variant alleles. gnomAD v4.1 reports total AF 4.33716e-06, maximum observed continental-subpopulation AF 5.93229e-06 in European non-Finnish, and grpmax FAF 2.47e-06. The observed values are below 0.0003, so BS1 is not met.

Rule

Filtering allele frequency (FAF) >= 0.0003 but <0.001 in a qualifying gnomAD continental subpopulation of a single genetic ancestry group; ancestry groups influenced by founder effects are excluded.

Evidence considered

• TP53 VCEP v2.4 specifies BS1 at strong strength when eligible gnomAD continental-subpopulation FAF is >=0.0003 and <0.001; founder-effect groups are excluded.
• gnomAD v4.1 reports 7 variant alleles among 1,613,958 total alleles, total AF 4.33716e-06, and no homozygotes.
• The highest reported continental-subpopulation AF is 5.93229e-06 in European non-Finnish and grpmax FAF is 2.47e-06, both below the BS1 lower threshold of 0.0003.

Sources

Reasoning

PS1 (per TP53 VCEP v2.4) requires identifying a distinct nucleotide change that produces the identical amino acid change (NP_000537.3:p.(Gly356Arg)) and that has already been classified Pathogenic/Likely Pathogenic under the TP53 VCEP specifications. No alternate codon-356 variant resulting in the same p.(G356R) protein change was identified anywhere in the case bundle (source_registry, evidence.json, pm5_candidates.json, or the fetched literature).

Evidence considered

• cspec: TP53 VCEP v2.4 PS1 rule text states PS1 (Strong) applies to variants asserted Pathogenic and PS1 (Moderate) to variants asserted Likely Pathogenic under the TP53 VCEP specifications, but no same-amino-acid-change comparator variant data was located in the case bundle to apply this rule.

Still missing

• No comparator search results for alternate nucleotide substitutions yielding the identical p.(Gly356Arg) change were found in this case's evidence bundle; a dedicated ClinVar/literature search for this specific comparator has not been completed.

Reasoning

No proband-level de novo observation or documented parental testing is present in the case-evidence bundle. The TP53 VCEP assigns PS2 strength by LFS cancer points, but no eligible proband cancer phenotype or confirmed de novo status is available for this case.

Rule

Very Strong: ≥ 8 points; Strong: 4-7 points; Moderate: 2-3 points; Supporting: 1 point

Evidence considered

• The TP53 VCEP specifies PS2 as supporting for 1 point, moderate for 2-3 points, strong for 4-7 points, and very strong for at least 8 points.
• The consolidated case bundle contains no documented proband cancer phenotype, parental testing result, or confirmed de novo event for NM_000546.6:c.1066G>C (p.Gly356Arg).

Still missing

• Obtain the proband's eligible LFS-associated cancer phenotype and corresponding VCEP point value.
• Obtain parental genotypes and documentation that the variant is absent in both biological parents, with consideration of parental relationships and mosaicism.

Sources

Reasoning

The TP53 VCEP PS4 point-based framework applies, but the available evidence does not document any qualifying germline proband with this exact variant, phenotype, age, Chompret/Classic LFS status, or cancer-point total. No PS4 points can be assigned.

Rule

TP53 VCEP PS4: assign points only for qualifying independent germline probands; Supporting 1-1.5, Moderate 2-3.5, Strong 4-7.5, Very Strong at least 8 points.

Evidence considered

• TP53 VCEP v2.4 requires PS4 point assignment from qualifying probands; thresholds are Supporting 1-1.5 points, Moderate 2-3.5 points, Strong 4-7.5 points, and Very Strong at least 8 points.
• The selectively extracted full texts reviewed for PS4 either do not mention c.1066G>C/p.Gly356Arg or provide only gene-level disease context; none supplies a qualifying case observation for this variant.

Still missing

• Variant-specific germline case observations with cancer diagnoses, ages, relatedness, and Classical or Revised Chompret status.
• A qualifying case-control enrichment analysis or sufficient unrelated proband data to calculate TP53 VCEP PS4 points.

Sources

Reasoning

TP53 VCEP v2.4 PM5 requires identifying other missense variants at the same residue (codon 356) previously classified Pathogenic (Strong, if >=2 such variants) or Likely Pathogenic with supporting clinical data (Moderate for 1 Pathogenic variant, Supporting for 1 Likely Pathogenic variant with PS2/PS4/PP1-level clinical data) under TP53 VCEP specifications. The dedicated PM5 comparator search (pm5_candidates.json) failed to return usable results due to an HTTP 429 rate-limit error, leaving 'found: false' and zero same-residue candidates collected. The VCEP functional worksheet lists two other codon-356 substitutions (G356A: Functional -> BS3 (benign); G356E: Partially functional -> 'No evidence' assigned), which are functional/benign-leaning classifications, not pathogenic/likely-pathogenic classifications, so they do not support PM5 even if usable.

Evidence considered

• pm5_candidates.json: automated ClinVar comparator search for TP53 codon 356 returned found=false, same_residue_candidates=0, with error 'HTTP Error 429: Too Many Requests'; explicitly flagged as insufficient to adjudicate PM5 automatically.
• TP53 VCEP functional worksheet (Supplementary Table S3) lists G356A as Functional/noLOF -> BS3, and G356E as Partially functional/noLOF -> 'No evidence'; neither is a Pathogenic/Likely Pathogenic classification usable to support PM5.

Still missing

• PM5 same-residue comparator search (pm5_candidates.json) failed with HTTP 429 (Too Many Requests) and returned zero candidates; a successful ClinVar/literature search for other codon-356 missense variants classified Pathogenic/Likely Pathogenic under TP53 VCEP specifications has not been completed.

Reasoning

No affected relatives with confirmed testing, phenotype-consistent disease, or countable informative meioses are documented in the case-evidence bundle. Therefore cosegregation cannot be established at supporting, moderate, or strong strength.

Rule

Strong: cosegregation in ≥ 7 meioses across > 1 family; Moderate: 5-6 meioses; Supporting: 3-4 meioses

Evidence considered

• The TP53 VCEP requires 3-4 cosegregating meioses for PP1 supporting, 5-6 for moderate, and at least 7 across more than one family for strong.
• The bundle provides no family pedigree, affected-relative genotype data, phenotype-linked segregation observations, or meiosis count for this variant.

Still missing

• Obtain a pedigree identifying affected relatives and their LFS-associated cancer diagnoses.
• Obtain genotype results for affected relatives and document each informative meiosis; exclude tumor-only observations that do not establish inherited transmission.

Sources

Reasoning

For TP53, PP4 is a low-VAF multigene-panel-testing rule rather than the generic phenotype-specificity rule. No qualifying observation of c.1066G>C/p.Gly356Arg with assay type, blood VAF, and clinical context is available.

Rule

TP53 VCEP PP4: Supporting for observation(s) at VAF 5-35%; Moderate for at least 2 independent observations at VAF 5-25%, subject to the VCEP caveats.

Evidence considered

• TP53 VCEP v2.4 permits PP4_Supporting for one or more qualifying observations at VAF 5-35% and PP4_Moderate for at least two independent observations at VAF 5-25%; neither VAF evidence nor required clinical context is present in the case bundle.

Still missing

• At least one qualifying multigene-panel observation with VAF 5-35%, excluding blood-cancer cases and variants meeting BA1 or BS1.
• For Moderate strength, two independent qualifying observations with VAF 5-25%.

Sources

Reasoning

No source in the case bundle provides the TP53 VCEP-required count of unrelated females from one source who reached at least 60 years of age without cancer. Population allele counts and absence of homozygotes do not establish BS2, so this criterion is not assessed.

Rule

TP53 VCEP BS2: supporting for 2-3 unrelated females, moderate for 4-7, or strong for >=8 unrelated females, all from one source, each aged >=60 years without cancer; females with sarcoma diagnosed at >=61 years are excluded.

Evidence considered

• The case bundle contains no qualifying unaffected-older-adult carrier observations for this exact variant.
• gnomAD v4.1 reports zero homozygotes, but this is not the TP53 VCEP BS2 evidence requirement and cannot substitute for age-, sex-, cancer-status-, and source-specific observations.

Still missing

• A single-source cohort or database containing at least 2 unrelated unaffected females aged >=60 years carrying this variant is not available in the case bundle; higher BS2 strengths would require 4-7 or >=8 such individuals under the VCEP rule.
• No qualifying data are available to determine whether any individuals had sarcoma at age >=61, which must be excluded.

Reasoning

No affected family members with documented absence of the variant are identified. Lack of available family data is insufficient to establish lack of segregation.

Rule

Strong: lack of segregation in affected family members diagnosed with LFS-associated cancers

Evidence considered

• The TP53 VCEP defines BS4 as strong evidence for lack of segregation in affected family members with LFS-associated cancers.
• The case-evidence bundle contains no documented affected relative, familial cancer history, or negative genotype result in an affected family member.

Still missing

• Obtain testing results for affected family members diagnosed with TP53/LFS-associated cancers.
• Confirm that the affected relatives were adequately tested for the variant and that the phenotype is eligible under the TP53 VCEP framework.

Sources