TP53 c.742C>T, p.Arg248Trp

Reasoning

The TP53 VCEP Functional-worksheet.xlsx Supplementary Table S3 row R248W assigns PS3. The row reports Kato class Non-functional, Funk LOF, Giacomelli LOF, Kotler LOF, and Kawaguchi NA. This is the VCEP pre-assigned code and is consistent with the functional flowchart: non-functional on Kato data plus loss of function by the majority of other eligible assays yields PS3. Among the four eligible non-NA assay results in the row, all four are LOF.

VCEP rule

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

Evidence considered

• Functional-worksheet.xlsx, Supplementary Table S3, row R248W: Kato = Non-functional; Funk = LOF; Giacomelli = LOF; Kotler = LOF; Kawaguchi = NA; preliminary functional code = PS3.
• Flowchart-for-application-of-functional-rule-codes.pdf, page 1: for missense variants with Kato data, Non-functional (Kato) plus LOF by the majority of available assays yields PS3.
• The exact variant was searched in the two governing functional files under c.742C>T, p.Arg248Trp, and p.R248W. The worksheet matched p.R248W; the flowchart is a rule table and has no variant-specific rows.
• The worksheet identifies the Kato assay as PMID:12826609, Funk as PMID:39774325, Giacomelli as PMID:30224644, Kotler as PMID:29979965, and Kawaguchi as PMID:16007150; the R248W row records LOF for Funk, Giacomelli, and Kotler, with Kawaguchi NA.

Sources

Reasoning

The variant is a missense change at TP53 codon 248. The TP53 VCEP specifically lists codon 248 among the codons eligible for PM1 at Moderate strength. The VCEP also names a cancerhotspots.org route, but the codon-specific route is sufficient; no unstated domain or generic hotspot rule is needed.

VCEP rule

Missense variants within the following codons using transcript NM\_00546.4: 175, 245, 248, 249, 273, 282. This code weight can also be used for germline missense variants seen in cancerhotspots.org with ≥ 10 somatic occurrences for the same amino acid change.

Evidence considered

• The normalized protein consequence is NP_000537.3:p.(Arg248Trp), so the altered residue is codon 248.
• The TP53 VCEP PM1 rule explicitly includes codons 175, 245, 248, 249, 273, and 282 for Moderate evidence.

Sources

Reasoning

The TP53 VCEP PM2 rule applies at supporting strength when the allele frequency is <0.00003 in gnomAD or another large sequenced population; when multiple alleles are present within an ancestry group, that group's allele frequency must be <0.00004. Using the default all-comers gnomAD v4.1 dataset because the VCEP rule does not require a non-cancer subset, the total AF is 5.577528262575157e-06 (9/1,613,618 alleles; homozygotes 0). The only ancestry group with multiple observed alleles is European (non-Finnish), with AF 5.9331386133746505e-06 (7/1,179,814 alleles), also below 0.00004. PM2 is met at supporting strength.

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

• ClinGen TP53 VCEP specification v2.4, PM2 rule: supporting strength, total allele frequency <0.00003; if multiple alleles occur within an ancestry group, that group's frequency must be <0.00004; founder-effect groups are ignored.
• gnomAD v4.1 all-comers: total AF 5.577528262575157e-06 (9/1,613,618 alleles; homozygotes 0). European (non-Finnish), the ancestry group with multiple observed alleles, has AF 5.9331386133746505e-06 (7/1,179,814 alleles; homozygotes 0).

Sources

Reasoning

The variant is a missense change, NM_000546.6:c.742C>T (p.Arg248Trp/p.R248W). The governing TP53 Supplementary Table S2 pre-assigns PP3_moderate for the exact row c.742C>T | p.Arg248Trp | Class C65 | BayesDel 0.533597 | PP3_moderate | 0.01. This is directly consistent with the VCEP PP3 moderate rule requiring aGVGD Class C65 and BayesDel score ≥ 0.16. The exact variant was not found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf.txt or TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx.txt; those files therefore do not add a separate exact-variant assignment. The PP3 determination relies on the governing pre-assigned table row and does not reuse the SpliceAI prediction for another criterion.

VCEP rule

**Missense variants** _(See flowchart for application of PP3 and BP4 rules for missense variants)_ aGVGD Class C65 and BayesDel score ≥ 0.16

Evidence considered

• PP3-BP4-codes.xlsx, Supplementary Table S2, matching row: c.742C>T | p.Arg248Trp | Class C65 | BayesDel score 0.533597 | PP3_moderate | 0.01.
• The TP53 VCEP PP3 moderate rule requires missense variants with aGVGD Class C65 and BayesDel score ≥ 0.16; the table's C65 classification and 0.533597 score satisfy that governing rule.
• The PP3/BP4/BP7 flowchart states that the core splice motif includes the last three nucleotides and first nucleotide of the exon; this missense variant is adjudicated by the exact pre-assigned missense table row.

Still missing

• No exact c.742C>T, p.Arg248Trp, or p.R248W entry was found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf.txt.
• No exact c.742C>T, p.Arg248Trp, or p.R248W entry was found in TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx.txt.

Sources

Reasoning

Expert panel ClinGen TP53 Variant Curation Expert Panel, ClinGen classified as Pathogenic.

VCEP rule

Not Applicable

Evidence considered

• The TP53 VCEP CSPEC criteria entry for PP5 states: Not Applicable.
• ClinVar expert panel classification

Sources

Reasoning

The variant is TP53 NM_000546.6:c.742C>T, p.Arg248Trp. The TP53 VCEP permits PS1 only when the same amino-acid change has been asserted as pathogenic or likely pathogenic under its specifications, with splicing excluded. The available evidence establishes this exact R248W variant as pathogenic, but does not identify a different nucleotide change producing the same Arg248Trp amino-acid change; the exact variant itself cannot serve as its own PS1 comparator. SpliceAI maximum delta is 0.004, below the VCEP's 0.2 exclusion threshold, but the required distinct same-amino-acid pathogenic comparator is absent.

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

• The case variant is NM_000546.6:c.742C>T, NP_000537.3:p.(Arg248Trp).
• ClinVar reports the exact c.742C>T (p.Arg248Trp) variant as Pathogenic with expert-panel review; this is the assessed variant, not a distinct same-amino-acid comparator.
• SpliceAI maximum delta score is 0.004, below the VCEP PS1 splicing threshold of 0.2.

Still missing

• A distinct, independently established pathogenic or likely pathogenic nucleotide change producing p.Arg248Trp is not documented in the available evidence.

Sources

Reasoning

The TP53 VCEP BA1 rule requires a filtering allele frequency (FAF) of ≥0.001 in a qualifying continental subpopulation, with at least 2,000 alleles tested and at least 2 variant alleles. In gnomAD v4.1, the highest observed subpopulation allele frequency is 1.6680010675206832e-05 in Admixed American (1/59,952 alleles), while the maximum group FAF is 2.47e-06; the observed allele count is below the VCEP minimum and both frequencies are far below 0.001. The criterion is therefore not met.

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

• ClinGen TP53 VCEP specification v2.4, BA1 rule: qualifying subpopulation FAF ≥0.001, with ≥2,000 alleles tested and at least 2 alleles present.
• gnomAD v4.1: total AF 5.577528262575157e-06 (9/1,613,618 alleles; homozygotes 0); highest observed subpopulation is Admixed American, AF 1.6680010675206832e-05 (1/59,952 alleles; homozygotes 0); grpmax FAF 2.47e-06.

Sources

Reasoning

The TP53 VCEP BS1 rule requires a filtering allele frequency (FAF) of ≥0.0003 but <0.001 in a qualifying continental subpopulation, with at least 2,000 alleles tested and at least 2 variant alleles. In gnomAD v4.1, the maximum group FAF is 2.47e-06 and the highest observed subpopulation allele count is 1, so the variant does not reach the frequency or minimum-observation requirements. The criterion is therefore not met.

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

• ClinGen TP53 VCEP specification v2.4, BS1 rule: qualifying subpopulation FAF ≥0.0003 but <0.001, with ≥2,000 alleles tested and at least 2 alleles present.
• gnomAD v4.1: maximum group FAF 2.47e-06; highest observed subpopulation is Admixed American with 1 variant allele among 59,952 tested alleles and homozygotes 0.

Sources

Reasoning

The TP53 VCEP Functional-worksheet.xlsx Supplementary Table S3 row R248W assigns PS3, not BS3, and reports Non-functional Kato activity with LOF in the Funk, Giacomelli, and Kotler assays. The VCEP BS3 rules require functional Kato data plus no LOF by the majority of eligible assays for BS3_Strong, or partially functional Kato data plus no LOF by all available assays for BS3_Supporting. R248W satisfies neither benign-functional path and instead meets the VCEP PS3 path.

VCEP rule

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

Evidence considered

• Functional-worksheet.xlsx, Supplementary Table S3, row R248W: Kato = Non-functional; Funk = LOF; Giacomelli = LOF; Kotler = LOF; Kawaguchi = NA; preliminary functional code = PS3, with no BS3 assignment.
• The R248W row has three non-Kato LOF results among the three non-NA assays, so it does not show no LOF by the majority of available eligible assays or no LOF by all available assays.
• Flowchart-for-application-of-functional-rule-codes.pdf, page 1: functional Kato plus noLOF by the majority yields BS3, while partially functional Kato plus noLOF by all available assays yields BS3_Supporting.

Sources

Reasoning

The available family report does not demonstrate lack of segregation in affected family members. PMID:1978757 instead reports the variant in three affected individuals in Li-Fraumeni syndrome family 1, although two unaffected carriers were also reported. Therefore the evidence does not satisfy the VCEP BS4 rule.

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

• PMID:1978757 reports c.742C>T/p.R248W in three affected individuals and two unaffected carriers in Li-Fraumeni syndrome family 1.
• The affected-carrier observations are inconsistent with a finding of lack of segregation in affected family members; the unaffected carriers indicate incomplete segregation but do not establish the BS4 rule as written.

Sources

Reasoning

The available evidence does not establish a confirmed de novo occurrence with documented maternity and paternity, nor does it provide the VCEP cancer-point data needed for PS2 scoring. A ClinVar submission labels the origin de novo, but that submission-level label has no accompanying parental-testing or proband phenotype documentation in the reviewed evidence. The variant was not found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf, which is the governing VCEP table searched for this variant.

VCEP rule

PS2 requires point-based de novo evidence under the TP53 VCEP specification.

Evidence considered

• The case evidence contains one ClinVar submission with origin recorded as de novo, but no accompanying parental-testing results, maternity/paternity confirmation, or proband cancer phenotype.
• The governing PS2/PP1 VCEP table assigns points by proband cancer type and parental confirmation; no matching c.742C>T, p.Arg248Trp, or p.R248W row was found in that table.

Still missing

• Confirmed maternity and paternity and primary parental-test results are not available.
• Proband cancer diagnoses and ages needed to assign VCEP PS2 points are not available.
• No entry for c.742C>T, p.Arg248Trp, or p.R248W was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.

Sources

Reasoning

The TP53 VCEP PS4 table assigns 0.5 points for a Revised Chompret clinical criterion, 1 point for Classic LFS criteria, and 0.5 points for HER2-positive breast cancer diagnosed before age 40, with Supporting requiring 1-1.5 points, Moderate 2-3.5 points, Strong 4-7.5 points, and Very Strong >=8 points. The searched PS4-Points-Table.pdf contains no entry for c.742C>T, p.Arg248Trp, or p.R248W. PMID:1978757 reports the exact variant in affected and unaffected members of LFS families, and PMID:17606709 reports it in 10 IARC families, 9 classified as LFS/LFL; these establish affected-family observations but do not provide enough individual proband clinical criteria, ages, or tumor-pathology details to assign the VCEP points or calculate PS4 strength. No case-control counts or matched-ancestry control comparison were reported in the reviewed variant-specific extracts, so no odds ratio or 95% CI could be computed.

VCEP rule

PS4_Supporting = 1-1.5 points ... PS4_Moderate = 2-3.5 points ... PS4 = 4-7.5 points ... PS4_Very Strong = >= 8 points

Evidence considered

• PS4-Points-Table.pdf: Clinical criteria Revised Chompret = 0.5 points; Classic LFS criteria = 1 point; HER2+ breast cancer <40 years = 0.5 points per proband; PS4_Supporting = 1-1.5 points, PS4_Moderate = 2-3.5 points, PS4 = 4-7.5 points, PS4_Very Strong = >=8 points.
• PMID:1978757, Results/Table 2: exact TP53 c.742C>T/p.R248W was identified in five members of LFS family 1, including three affected individuals and two unaffected carriers; the same codon-248 mutation was identified in a family 4 individual with soft-tissue sarcoma and osteosarcoma.
• PMID:17606709, Discussion: exact Arg248Trp was present in 10 IARC germline TP53 families, of which 9 were classified as LFS/LFL.
• The exact variant was searched in PS4-Points-Table.pdf under c.742C>T, p.Arg248Trp, and p.R248W; no pre-assigned variant row was found.

Still missing

• PS4-Points-Table.pdf was searched for c.742C>T, p.Arg248Trp, and p.R248W; no variant-specific entry was found.
• Individual proband diagnoses, ages, and tumor pathology needed to assign VCEP PS4 points are not available in the reviewed evidence.
• No affected-versus-unaffected or population-control counts were available, so an odds ratio and 95% CI could not be computed.

Sources

Reasoning

The TP53 VCEP requires a different missense variant at the same amino-acid residue that was classified as pathogenic or likely pathogenic under the TP53 VCEP specifications, with the prior classification established without PM5. The available same-residue comparator collection reports zero usable codon-248 candidates and no expert-panel or non-expert pathogenic/likely pathogenic comparator. Therefore PM5 cannot be evaluated from the available evidence without substituting an unverified external classification.

VCEP rule

Missense variant at an amino acid residue where 1 different missense variant previously determined to be pathogenic according to the TP53 VCEP’s specifications has been seen before.

Evidence considered

• The PM5 comparator collection reports total_candidates_seen = 9 but same_residue_candidates = 0, expert_panel_plp_candidates = 0, and non_expert_majority_plp_candidates = 0.
• The available evidence does not provide a qualifying alternate missense variant at TP53 residue 248 with a TP53 VCEP-based pathogenic or likely pathogenic classification.

Still missing

• A different TP53 missense variant at residue 248 with a qualifying TP53 VCEP pathogenic or likely pathogenic classification, established without PM5, is needed.

Sources

Reasoning

PMID:1978757 reports the variant in three affected individuals and two unaffected carriers in one Li-Fraumeni family and describes co-segregation with a chromosome 17p VNTR allele across three generations. However, the extract does not provide a count of phenotype-informative meioses, and the unaffected carriers prevent treating the observation as unambiguous cosegregation under the VCEP threshold. PMID:17606709 reports Arg248Trp in 10 families, with 9 classified as LFS/LFL, but explicitly provides no variant-specific segregation result. The variant was not found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.

VCEP rule

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

Evidence considered

• PMID:1978757: the variant was identified in five members of Li-Fraumeni syndrome family 1, including three affected individuals and two unaffected carriers; the mutation co-segregated with chromosome 17p VNTR allele 3 across three generations.
• PMID:17606709: the paper states, "Similar results are seen with Arg248Trp (of 10 families, 9 are LFS/LFL)." It does not provide a variant-specific segregation result.
• The TP53 VCEP requires a defined number of cosegregating meioses for PP1 strength assignment; the reviewed reports do not provide that count.

Still missing

• The number of informative meioses across the reported families is not provided.
• Family-level genotypes and phenotypes for the 10 families summarized in PMID:17606709 are not provided.
• No entry for c.742C>T, p.Arg248Trp, or p.R248W was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.

Sources

Reasoning

The TP53 VCEP PP4 rule requires observation of the variant with a specified variant allele fraction (VAF). The available case evidence identifies this as a germline missense variant and provides no qualifying VAF measurement, so the VCEP rule cannot be evaluated.

VCEP rule

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

• The VCEP PP4 rule requires at least 2 independent observations with VAF 5-25% for Moderate or observation with VAF 5-35% for Supporting; no qualifying VAF is reported.

Still missing

• No VAF measurement meeting the PP4 rule was available.

Sources

Reasoning

The TP53 VCEP BS2 rule requires counts of unrelated females who reached at least 60 years of age without cancer, all from one source, with specific exclusion of qualifying older individuals diagnosed with sarcoma. The available evidence contains population allele frequencies and homozygote counts but no source-level individual data establishing sex, age, cancer-free status, relatedness, and sarcoma history. BS2 cannot be evaluated from the available evidence.

VCEP rule

≥ 8 unrelated females who have reached at least 60 years of age without cancer. These individuals all must have come from a single source (single lab, database, etc). Cases cannot be counted across sources. Individuals with a diagnosis of sarcoma ≥ 61 years of age should not be counted towards the BS2 total.

Evidence considered

• ClinGen TP53 VCEP specification v2.4, BS2 rule requires 2-3, 4-7, or ≥8 unrelated females aged at least 60 years without cancer from a single source; individuals with sarcoma diagnosed at ≥61 years are excluded.
• gnomAD v4.1 reports homozygote count 0, but does not provide the individual-level sex, age, cancer-free status, relatedness, or sarcoma-history data needed for BS2.

Still missing

• A single source with qualifying unrelated female carriers, ages, cancer status, relatedness, and sarcoma history is required.

Sources