TP53 c.760A>G, p.(Ile254Val)

Reasoning

The variant is a missense change at p.Ile254, which is outside the VCEP's explicitly listed codon hotspots 175, 245, 248, 249, 273, and 282. The authoritative vcep_materials.json domain_tables entry for TP53 is empty, so no additional approved domain-table entry applies. However, the Cancer Hotspots evidence recorded two somatic occurrences for the same amino-acid change, satisfying the TP53 VCEP's PM1 Supporting rule for 2-9 occurrences.

VCEP rule

Missense variants seen in cancerhotspots.org with 2-9 somatic occurrences for the same amino acid change.

Evidence considered

• ClinGen TP53 VCEP PM1 rules: residue 254 is not among the explicitly listed codons 175, 245, 248, 249, 273, and 282; the alternate hotspot path permits supporting strength for 2-9 somatic occurrences of the same amino-acid change.
• ClinVar expert-panel criterion signal states: "this variant has 2 somatic occurrences for the same amino acid change in cancerhotspots".
• vcep_materials.json domain_tables is empty for TP53; therefore the authoritative complete domain-table list contains no additional entry to match against residue 254.

Sources

Reasoning

PM2 is met at the VCEP-specified supporting strength. The governing rule requires an allele frequency below 0.00003 in gnomAD or another large sequenced population; if multiple alleles are present within an ancestry group, that group's frequency must be below 0.00004, with founder-effect ancestry groups ignored. The default current all-comers gnomAD v4.1 data show total AF 6.82086e-06 (11/1,612,700 alleles), highest observed European non-Finnish AF 9.32959e-06 (11/1,179,044 alleles), maximum group FAF 5.01e-06, and zero homozygotes, satisfying the stated frequency limits. The VCEP does not require a non-cancer or exome-only source for PM2, so the default gnomAD v4.1 all-comers data were used.

VCEP rule

This rule should be applied at supporting level. Variant should have an allele frequency of less than 0.00003 (0.003%) in gnomAD or another large sequenced population. If multiple alleles are present within any genetic ancestry group, allele frequency in that group must be <0.00004 (0.004%). Genetic ancestry groups influenced by founder effects (such as Ashkenazi Jewish, Finnish, Amish, Middle Eastern, and “Remaining”) should be ignored.

Evidence considered

• gnomAD v4.1 all-comers: total AF 6.82086e-06 (11/1,612,700 alleles), highest observed European non-Finnish AF 9.32959e-06 (11/1,179,044 alleles), maximum group FAF 5.01e-06, homozygotes 0.
• The governing VCEP rule does not specify a non-cancer or exome-only population source for PM2; gnomAD v4.1 all-comers is therefore the default source under the requested source-selection rule.

Sources

Reasoning

NM_000546.6:c.760A>G (p.Ile254Val/p.I254V) is a missense variant. The governing TP53 Supplementary Table S2 pre-assigns PP3 for the exact row c.760A>G, p.Ile254Val, Class C25, BayesDel 0.17079. This is the governing pre-assigned code and is applied directly. The VCEP missense PP3 supporting rule requires aGVGD class C25-C55 and BayesDel score >= 0.16; the table's Class C25 and score 0.17079 satisfy that path. SpliceAI maximum delta is 0.003, below the VCEP splice-effect threshold of 0.2, so no splice-effect PP3 path is additionally applied and the prediction is not double-counted across criteria.

VCEP rule

**Missense variants** _(See flowchart for application of PP3 and BP4 rules for missense variants)_ aGVGD class C25-C55 and BayesDel score ≥ 0.16

Evidence considered

• ClinGen TP53 VCEP Supplementary Table S2, PP3-BP4-codes.xlsx, matching row: c.760A>G | p.Ile254Val | Class C25 | BayesDel 0.17079 | PP3.
• The VCEP PP3 supporting rule for missense variants requires aGVGD class C25-C55 and BayesDel score >= 0.16; the governing table assigns Class C25 and BayesDel 0.17079.
• SpliceAI result for NM_000546.6:c.760A>G: DS_AG 0.003, DS_AL 0.000, DS_DG 0.000, DS_DL 0.000; maximum delta score 0.003, below the VCEP splice-effect threshold of 0.2.

Still missing

• No exact c.760A>G, p.Ile254Val, or p.I254V entry was found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf.txt; the flowchart provides governing scope rather than a variant lookup table.
• No exact c.760A>G, p.Ile254Val, or p.I254V entry was found in TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx.txt; that file governs single-amino-acid in-frame deletions, not this missense variant.

Sources

Reasoning

The TP53 VCEP Functional-worksheet.xlsx Supplementary Table S3 contains the exact protein-change row I254V corresponding to NM_000546.6:c.760A>G / p.Ile254Val. It records Kato as Functional, Funk as noLOF, Giacomelli as noLOF, Kotler as noLOF, Kawaguchi as NA, and pre-assigns BS3. This satisfies the VCEP strong BS3 rule: functional on Kato data and no loss of function by the majority of available eligible assays. The VCEP flowchart identifies these assay classes as eligible functional evidence. The exact variant is not explicitly named in the individual paper extracts, so the worksheet pre-assignment is the governing evidence rather than an independently reconstructed assay result.

VCEP rule

Functional on Kato et al. data **AND** no loss of function (LOF) by the majority of available eligible assays

Evidence considered

• Functional-worksheet.xlsx, Supplementary Table S3, row I254V: Kato class Functional; Funk class noLOF; Giacomelli class noLOF; Kotler class noLOF; Kawaguchi class NA; preliminary functional code BS3.
• Flowchart-for-application-of-functional-rule-codes.pdf: eligible functional studies include Kato, Funk, Giacomelli, Kotler, and Kawaguchi; no variant-specific I254V row is present.
• PMID:12826609 paper extract: the exact p.I254V variant is not explicitly mentioned; the paper describes the systematic yeast transactivation assay underlying the Kato classification.
• PMID:29979965 paper extract: the exact p.I254V variant is not explicitly mentioned; the paper describes the Kotler relative-fitness assay used in the VCEP worksheet.
• PMID:30224644 paper extract: the exact p.I254V variant is not explicitly mentioned; the paper describes the Giacomelli TP53 loss-of-function screen used in the VCEP worksheet.

Still missing

• No c.760A>G, p.Ile254Val, or p.I254V entry was found in Flowchart-for-application-of-functional-rule-codes.pdf; the variant-specific governing entry is in Functional-worksheet.xlsx.

Sources

Reasoning

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

VCEP rule

This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.

Evidence considered

• TP53 VCEP BP6 applicability entry: Not Applicable for this VCEP.
• ClinVar expert panel classification

Sources

Reasoning

The TP53 VCEP Functional-worksheet.xlsx Supplementary Table S3 contains the exact protein-change row I254V corresponding to NM_000546.6:c.760A>G / p.Ile254Val. It records Kato as Functional, Funk as noLOF, Giacomelli as noLOF, Kotler as noLOF, Kawaguchi as NA, and assigns the preliminary functional code BS3. This governing pre-assignment does not support PS3. The result is also consistent with the VCEP PS3 rules, which require non-functional or partially functional Kato data together with loss of function by the specified other eligible assays. The functional flowchart contains no variant-specific I254V entry; it provides only the general eligible-assay framework.

VCEP rule

Non-functional on Kato et al. data **AND** loss of function (LOF) by the majority of other eligible assays ... Partially functional on Kato et al. data **AND** loss of function (LOF) by the majority of other available assays ... Non-functional on Kato et al. data **AND** abnormal on Kawaguchi et al. data regardless of other assays

Evidence considered

• Functional-worksheet.xlsx, Supplementary Table S3, row I254V: Kato class Functional; Funk class noLOF; Giacomelli class noLOF; Kotler class noLOF; Kawaguchi class NA; preliminary functional code BS3.
• Flowchart-for-application-of-functional-rule-codes.pdf: eligible functional studies include Kato, Funk, Giacomelli, Kotler, and Kawaguchi; no variant-specific I254V row is present.
• PMID:12826609 paper extract: the exact p.I254V variant is not explicitly mentioned; the paper describes the systematic yeast transactivation assay underlying the Kato classification.
• PMID:29979965 paper extract: the exact p.I254V variant is not explicitly mentioned; the paper describes the Kotler relative-fitness assay used in the VCEP worksheet.
• PMID:30224644 paper extract: the exact p.I254V variant is not explicitly mentioned; the paper describes the Giacomelli TP53 loss-of-function screen used in the VCEP worksheet.

Still missing

• No c.760A>G, p.Ile254Val, or p.I254V entry was found in Flowchart-for-application-of-functional-rule-codes.pdf; the variant-specific governing entry is in Functional-worksheet.xlsx.

Sources

Reasoning

BA1 is not met. The governing TP53 VCEP rule requires a filtering allele frequency (FAF) of at least 0.001 in a qualifying continental subpopulation of one genetic ancestry group, with at least 2,000 alleles tested and at least 2 variant alleles. The default current all-comers gnomAD v4.1 data show a maximum group FAF of 5.01e-06 and the highest observed qualifying-subpopulation AF is 9.32959e-06 (11/1,179,044 in European non-Finnish), both far below 0.001; homozygotes are 0. The VCEP does not require a non-cancer or exome-only source for BA1, so the default gnomAD v4.1 all-comers data were used.

VCEP rule

Filtering allele frequency (FAF) of ≥ 0.001 or 0.1% in gnomAD continental subpopulations of a single genetic ancestry group (excluding genetic ancestry groups influenced by founder effects, such as Ashkenazi Jewish, Finnish, Amish, Middle Eastern, and “Remaining”). Genetic ancestry group must have ≥2,000 alleles tested and a minimum of 2 alleles present.

Evidence considered

• gnomAD v4.1 all-comers: total AF 6.82086e-06 (11/1,612,700 alleles), maximum group FAF 5.01e-06, highest observed European non-Finnish AF 9.32959e-06 (11/1,179,044 alleles), homozygotes 0.
• The governing VCEP rule does not specify a non-cancer or exome-only population source for BA1; gnomAD v4.1 all-comers is therefore the default source under the requested source-selection rule.

Sources

Reasoning

BS1 is not met. The governing TP53 VCEP rule requires a filtering allele frequency (FAF) of at least 0.0003 but less than 0.001 in a qualifying continental subpopulation of one genetic ancestry group, with at least 2,000 alleles tested and at least 2 variant alleles. The default current all-comers gnomAD v4.1 data show a maximum group FAF of 5.01e-06 and a highest observed qualifying-subpopulation AF of 9.32959e-06 (11/1,179,044 in European non-Finnish), below 0.0003; homozygotes are 0. The VCEP does not require a non-cancer or exome-only source for BS1, so the default gnomAD v4.1 all-comers data were used.

VCEP rule

Filtering allele frequency (FAF) of ≥ 0.0003 but < 0.001 in gnomAD continental subpopulations of a single genetic ancestry group (excluding genetic ancestry groups influenced by founder effects, such as Ashkenazi Jewish, Finnish, Amish, Middle Eastern, and “Remaining”). Genetic ancestry group must have ≥2,000 alleles tested and a minimum of 2 alleles present.

Evidence considered

• gnomAD v4.1 all-comers: total AF 6.82086e-06 (11/1,612,700 alleles), maximum group FAF 5.01e-06, highest observed European non-Finnish AF 9.32959e-06 (11/1,179,044 alleles), homozygotes 0.
• The governing VCEP rule does not specify a non-cancer or exome-only population source for BS1; gnomAD v4.1 all-comers is therefore the default source under the requested source-selection rule.

Sources

Reasoning

NM_000546.6:c.760A>G (p.Ile254Val/p.I254V) is a missense variant. The VCEP BP4 supporting rule requires BayesDel < 0.16 and > -0.008, together with no predicted splicing impact (SpliceAI < 0.2). The exact VCEP Table S2 row reports BayesDel 0.17079 and pre-assigns PP3, not BP4. Although SpliceAI maximum delta 0.003 is below 0.2, the BayesDel value is not < 0.16, so BP4 is not met. The BP4 moderate path also cannot apply because its BayesDel <= -0.008 requirement is not satisfied.

VCEP rule

**Missense variants** _(See flowchart for application of PP3 and BP4 rules for missense variants):_ BayesDel < 0.16 and > -0.008 irrespective of aGVGD score (except C65, this case do not apply BP4) AND no predicted differences in splicing (SpliceAI < 0.2)

Evidence considered

• ClinGen TP53 VCEP Supplementary Table S2, PP3-BP4-codes.xlsx, matching row: c.760A>G | p.Ile254Val | Class C25 | BayesDel 0.17079 | PP3; no BP4 assignment.
• The VCEP BP4 supporting rule requires BayesDel < 0.16 and > -0.008 plus no predicted splicing impact; the observed BayesDel score is 0.17079.
• SpliceAI result for NM_000546.6:c.760A>G: maximum delta score 0.003, below the VCEP no-predicted-splicing cutoff of 0.2; this does not overcome the failed BayesDel requirement.

Still missing

• No exact c.760A>G, p.Ile254Val, or p.I254V entry was found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf.txt; the flowchart provides governing scope rather than a variant lookup table.
• No exact c.760A>G, p.Ile254Val, or p.I254V entry was found in TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx.txt; that file governs single-amino-acid in-frame deletions, not this missense variant.

Sources

Reasoning

The variant is TP53 p.Ile254Val. The VCEP permits PS1 only when the same amino-acid change is compared with a previously established pathogenic or likely pathogenic variant under the TP53 VCEP specifications, and requires splicing to be excluded. The available PM5 comparator collection found zero same-residue candidates, but its ClinVar search ended with HTTP 429, so absence of an eligible pathogenic/likely pathogenic p.Ile254 comparator cannot be concluded from the available evidence. SpliceAI maximum delta is 0.003, below the VCEP's 0.2 exclusion threshold, but the qualifying comparator evidence is incomplete.

VCEP rule

This rule code can only be used to compare variants asserted as pathogenic or likely pathogenic following the ClinGen _TP53_ VCEP’s specifications. Must confirm there is no difference using RNA data or SpliceAI (SpliceAI < 0.2).

Evidence considered

• Variant normalization: NM_000546.6:c.760A>G, NP_000537.3:p.(Ile254Val).
• The PM5 comparator collection reported same_residue_candidates = 0 and expert_panel_plp_candidates = 0; the collection recorded HTTP Error 429: Too Many Requests.
• SpliceAI maximum delta score was 0.003, so the VCEP splicing exclusion condition is satisfied.

Still missing

• Confirm whether any different TP53 missense variant at residue Ile254 is classified as pathogenic or likely pathogenic under the TP53 VCEP specifications.

Sources

Reasoning

The TP53 VCEP assigns PS2 by de novo evidence scored with the LFS cancer points table: 1 point is Supporting, 2-3 points Moderate, 4-7 points Strong, and >=8 points Very Strong. The available case evidence contains no documented de novo proband observation, confirmed parental testing, proband cancer diagnosis, or scored PS2 observation for NM_000546.6:c.760A>G (p.Ile254Val/p.I254V), so PS2 cannot be evaluated.

VCEP rule

≥ 8 points...4-7 points...2-3 points...1 point

Evidence considered

• The case evidence bundle identifies the variant as NM_000546.6:c.760A>G, NP_000537.3:p.(Ile254Val), and NP_000537.3:p.(I254V), but provides no de novo or parental-testing record.
• Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf was searched for c.760A>G, p.Ile254Val, and p.I254V; no variant-specific entry was found.
• The TP53 VCEP CSPEC entry provides PS2 strength thresholds of Supporting = 1 point, Moderate = 2-3 points, Strong = 4-7 points, and Very Strong = >=8 points.

Still missing

• No de novo proband observation or confirmed maternity/paternity and parental testing is documented in the case evidence bundle.
• No proband cancer phenotype or LFS cancer point total is documented for this variant.
• No entry for c.760A>G, p.Ile254Val, or p.I254V was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf; this table provides the cancer categories and scoring rules, not variant observations.

Sources

Reasoning

The TP53 VCEP PS4 rule assigns strength from proband phenotype points: Supporting at 1-1.5 points, Moderate at 2-3.5 points, Strong at 4-7.5 points, and Very Strong at ≥8 points. The available case evidence contains no proband cancer phenotype, Revised Chompret or Classic LFS designation, HER2-positive breast cancer diagnosis under age 40, or point total for this variant. The governing PS4-Points-Table.pdf was searched for c.760A>G, p.Ile254Val, and p.I254V; no variant-specific entry was found.

Evidence considered

• TP53 VCEP PS4 points table: Revised Chompret = 0.5 point, Classic LFS criteria = 1 point, HER2+ breast cancer diagnosed below age 40 = 0.5 point; PS4 strength thresholds are Supporting 1-1.5, Moderate 2-3.5, Strong 4-7.5, and Very Strong ≥8 points.
• Search of PS4-Points-Table.pdf.txt for c.760A>G, p.Ile254Val, and p.I254V found no variant-specific row.

Still missing

• No patient-level cancer phenotype or PS4 point assignment is available for this variant.
• No entry for c.760A>G, p.Ile254Val, or p.I254V was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/TP53/fulltext/PS4-Points-Table.pdf.txt.

Sources

Reasoning

The variant is a missense change at amino-acid residue Ile254. The VCEP requires a different missense variant at the same residue that has independently reached pathogenic or likely pathogenic classification under the TP53 VCEP specifications, without relying on PM5 for that classification; the VCEP also requires the Grantham comparison and splicing exclusion. The available comparator collection found zero same-residue candidates and recorded an HTTP 429 retrieval failure, so the required comparator evidence is incomplete. SpliceAI maximum delta is 0.003, below 0.2, but this does not establish PM5 without a qualifying comparator.

VCEP rule

This code can be applied for a missense change at an amino acid residue where one or more pathogenic/likely pathogenic variants have been identified. The other variant must be interpreted as pathogenic or likely pathogenic following the ClinGen _TP53_ VCEP’s specifications. The previously established pathogenic/likely pathogenic variant must reach a classification of pathogenicity without PM5. ... Splicing should be ruled out with either RNA data or SpliceAI (SpliceAI < 0.2).

Evidence considered

• Variant normalization: NP_000537.3:p.(Ile254Val), a missense change at residue Ile254.
• The PM5 comparator collection reported total_candidates_seen = 7 but same_residue_candidates = 0, with no expert-panel or non-expert majority pathogenic/likely pathogenic candidates; it recorded HTTP Error 429: Too Many Requests.
• SpliceAI maximum delta score was 0.003, below the VCEP's 0.2 splicing threshold.

Still missing

• Complete a reliable search for different missense variants at TP53 residue Ile254 and verify each candidate's VCEP classification, PM5-independent pathogenicity, and Grantham comparison.

Sources

Reasoning

The TP53 VCEP requires cosegregation in 3-4 meioses for PP1 Supporting, 5-6 meioses for PP1 Moderate, or at least 7 meioses across more than 1 family for PP1 Strong. The available evidence contains no affected-family genotypes, phenotype-linked relatives, or countable meioses for NM_000546.6:c.760A>G (p.Ile254Val/p.I254V), so PP1 cannot be assessed.

VCEP rule

Cosegregation must be observed in ≥ 7 meioses across > 1 family...Cosegregation must be observed in 5-6 meioses in/across 1 or more families...Cosegregation must be observed in 3-4 meioses in/across 1 or more families

Evidence considered

• The case evidence bundle contains no affected-family segregation observations, relative genotypes, or meiosis count for NM_000546.6:c.760A>G.
• Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf was searched for c.760A>G, p.Ile254Val, and p.I254V; no variant-specific entry was found.
• The TP53 VCEP CSPEC requires cosegregation in 3-4 meioses for Supporting, 5-6 meioses for Moderate, and >=7 meioses across >1 family for Strong.

Still missing

• No family-level segregation data, affected-relative genotypes, or countable meioses are documented in the case evidence bundle.
• No family or proband cancer phenotypes are documented for applying the LFS cancer categories in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.
• No entry for c.760A>G, p.Ile254Val, or p.I254V was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.

Sources

Reasoning

The TP53 VCEP PP4 rule uses low variant allele fraction as phenotype-related evidence: Moderate requires at least 2 independent observations with VAF 5-25%, and Supporting requires observation with VAF 5-35%. The available evidence reports no qualifying VAF observations for c.760A>G (p.Ile254Val), and there is no evidence that the variant was detected on multigene panel testing in a qualifying non-blood-cancer context.

VCEP rule

At least 2 independent observations of the variant with VAF 5-25%. ... 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%)

Evidence considered

• TP53 VCEP PP4 rule: Moderate requires at least 2 independent observations with VAF 5-25%; Supporting requires observation with VAF 5-35%.
• The case evidence contains no qualifying VAF observation for NM_000546.6:c.760A>G (p.Ile254Val).

Still missing

• No patient-level VAF measurements or independent qualifying observations are available.
• No evidence confirms qualifying multigene-panel detection or excludes a blood-cancer context for PP4.

Sources

Reasoning

BS2 cannot be assessed from the available evidence. The TP53 VCEP requires counts of unrelated females aged at least 60 years without cancer, all from one source, with sarcoma diagnosed at age 61 years or older excluded; no qualifying individual-level count from a single source is available.

VCEP rule

≥ 8 unrelated females who have reached at least 60 years of age without cancer; 4-7 for moderate or 2-3 for supporting, all from a single source, excluding individuals with sarcoma diagnosed at ≥61 years.

Evidence considered

• No qualifying BS2 individual-level dataset or source-specific carrier count is present in the available case evidence.

Still missing

• A single-source, non-overlapping count of unrelated female TP53 carriers who reached age 60 or older without cancer is required.

Sources

Reasoning

The TP53 VCEP defines BS4 as lack of segregation in affected family members diagnosed with LFS-associated cancers described in the VCEP LFS cancer table. No affected family members, relevant cancer diagnoses, or family genotyping results are documented for NM_000546.6:c.760A>G (p.Ile254Val/p.I254V), so lack of segregation cannot be established.

VCEP rule

Lack of segregation in affected family members (i.e. family members diagnosed with LFS-associated cancers as described in Table of LFS Cancers and Points for PS2 and PP1 Code Application).

Evidence considered

• The case evidence bundle contains no affected-relative phenotype, genotype, or non-segregation observation for NM_000546.6:c.760A>G.
• Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf was searched for c.760A>G, p.Ile254Val, and p.I254V; no variant-specific entry was found.
• The TP53 VCEP BS4 rule is limited to lack of segregation in affected family members diagnosed with LFS-associated cancers described in the VCEP table.

Still missing

• No affected family members with LFS-associated cancers are documented.
• No family genotyping results are documented to demonstrate that the variant fails to segregate with disease.
• No entry for c.760A>G, p.Ile254Val, or p.I254V was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.

Sources