TP53 c.1031T>G, p.(Leu344Arg)

Reasoning

The TP53 VCEP Functional-worksheet.xlsx contains an exact p.L344R row for this missense variant and assigns the preliminary functional code PS3, with the assay table recording Non-functional and LOF results and monomeric oligomerization. This governing pre-assigned code takes precedence over independently recalculated thresholds. Variant-specific functional studies support the assignment: in p53-null H1299 cells, L344R produced no reporter signal and showed 98.2% ± 1.7% monomer fraction, while yeast-based oligomerization testing classified L344R as a monomer mutant. A separate tetramerization-domain study found L344R eluted between tetramer and monomer fractions and was classified as a dimer under its assay conditions, with thermal instability near body temperature; this does not establish preserved wild-type function and is consistent with abnormal protein function. The TP53 VCEP framework defines strong PS3 for non-functional Kato data together with loss of function by the majority of other eligible assays; the governing worksheet nevertheless directly assigns PS3 for this exact variant.

Rule

TP53 VCEP v2.4 PS3; exact worksheet pre-assignment PS3 for L344R. VCEP strong rule: non-functional on Kato et al. data AND loss of function by the majority of other eligible assays.

Evidence considered

• The exact L344R row in Functional-worksheet.xlsx records Non-functional and LOF assay results and assigns preliminary functional code PS3.
• The TP53 VCEP functional flowchart identifies Kato, Funk, Giacomelli, Kotler, and Kawaguchi as eligible functional studies and directs use of the functional worksheet for pre-assigned codes.
• In p53-null H1299 cells, L344R produced no DsRed reporter signal; the extracted study reports a 98.2% ± 1.7% monomer fraction for L344R versus 45.8% ± 3.3% for wild type.
• In a TP53 missense library, L344R was explicitly classified as a monomer mutant in the oligomerization assay.
• In a tetramerization-domain peptide assay, L344R eluted between tetramer and monomer fractions and was classified as a dimer under those conditions; the study also reported thermal instability near body temperature.
• The TP53 VCEP PS3 strong rule is non-functional on Kato data AND loss of function by the majority of other eligible assays; the exact worksheet pre-assignment is controlling for this variant.

Sources

Reasoning

The governing TP53 VCEP specifies PM1 Moderate for missense variants at codons 175, 245, 248, 249, 273, or 282, or for a same-amino-acid change seen at least 10 times in Cancer Hotspots; PM1 Supporting applies to 2-9 same-amino-acid-change hotspot occurrences. This variant is p.Leu344Arg at codon 344, outside the specified codons, but the available hotspot/COSMIC evidence reports 7 occurrences for the same amino-acid change, satisfying PM1 Supporting. The VCEP materials domain_tables key has no TP53 entry, so there is no authoritative domain-table addition to the approved PM1 list.

Rule

Moderate: missense variants at codons 175, 245, 248, 249, 273, or 282, or same-amino-acid changes with at least 10 Cancer Hotspots occurrences; Supporting: same-amino-acid changes with 2-9 Cancer Hotspots occurrences.

Evidence considered

• The TP53 VCEP PM1 Moderate rule limits codon-based application to codons 175, 245, 248, 249, 273, and 282; it also requires at least 10 same-amino-acid-change Cancer Hotspots occurrences for the alternative Moderate route.
• The TP53 VCEP PM1 Supporting rule requires 2-9 same-amino-acid-change Cancer Hotspots occurrences, and the available record reports 7 occurrences for p.Leu344Arg; the Moderate threshold is not met.
• PMID:20978130 places L344R in the TP53 tetramerization domain, but the TP53 VCEP PM1 rule does not authorize this domain alone as an additional PM1 route.
• The vcep_materials.json domain_tables object is empty for TP53.

Sources

Reasoning

The governing ClinGen TP53 VCEP version 2.4 requires PM2 to be applied only at supporting strength when the allele frequency is less than 0.00003 (0.003%) in gnomAD or another large sequenced population; if multiple alleles occur in an ancestry group, that group must have frequency below 0.00004 (0.004%), with founder-effect groups ignored. The variant is absent from gnomAD v2.1 and gnomAD v4.1, and the available gnomAD v2.1 non-cancer exome and v3.1 non-cancer genome subset queries are also absent. This is consistent with an allele frequency of zero in the queried population datasets and satisfies the TP53 PM2 supporting threshold.

Rule

Apply PM2 at supporting level when allele frequency is <0.00003 (0.003%) in gnomAD or another large sequenced population; if multiple alleles are present within an ancestry group, that group must have frequency <0.00004 (0.004%), excluding founder-effect groups.

Evidence considered

• TP53 VCEP v2.4 PM2 rule: apply at supporting strength for allele frequency <0.00003 (0.003%) in gnomAD or another large sequenced population; if multiple alleles occur in an ancestry group, that group's frequency must be <0.00004 (0.004%), excluding founder-effect groups.
• gnomAD v2.1 all-comers: variant absent.
• gnomAD v4.1 all-comers: variant absent.
• The queried gnomAD v2.1 non-cancer exome subset and gnomAD v3.1 non-cancer genome subset also report the variant as absent.

Sources

Reasoning

This is a missense variant, NM_000546.5:c.1031T>G (p.Leu344Arg/p.L344R). The governing TP53 VCEP PP3-BP4 lookup table directly assigns PP3_moderate to c.1031T>G, with BayesDel class C65 and score 0.288212. The same table reports a maximum SpliceAI delta of 0.01, below the VCEP splice-impact threshold of 0.2, so the pre-assigned PP3_moderate missense code remains applicable without a splice-based code change. The governing pre-assignment outranks generic predictor thresholds.

Rule

TP53 VCEP PP3_moderate pre-assignment for missense c.1031T>G / p.Leu344Arg in PP3-BP4-codes.xlsx; BayesDel class C65 and score >=0.16, with SpliceAI <0.2.

Evidence considered

• The TP53 VCEP PP3-BP4-codes.xlsx supplementary table entry for c.1031T>G / p.Leu344Arg assigns PP3_moderate, reports BayesDel class C65, BayesDel score 0.288212, and maximum SpliceAI delta 0.01.
• The TP53 VCEP specification states that missense variants with aGVGD class C65 and BayesDel score >=0.16 meet PP3 at moderate strength; its direct lookup-table pre-assignment is controlling here.
• The case SpliceAI result reports max delta 0.001, and the VCEP lookup table reports 0.01; both are below the VCEP predicted-splicing threshold of 0.2.

Sources

Reasoning

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

Rule

Exact-variant ClinVar expert-panel Pathogenic or Likely Pathogenic classification supports PP5 at Supporting strength.

Evidence considered

• ClinVar variation 2758378 exactly matches NM_000546.5:c.1031T>G (p.Leu344Arg) and reports a Likely Pathogenic classification by the ClinGen TP53 Variant Curation Expert Panel.
• The expert-panel classification is the qualifying evidence for PP5; clinical-laboratory submissions, the aggregate label, and non-expert assertions were not used to trigger this criterion.
• ClinVar expert panel classification

Sources

Reasoning

The governing ClinGen TP53 VCEP version 2.4 specifies BA1 at stand-alone strength when the filtering allele frequency (FAF) is at least 0.001 (0.1%) in a gnomAD continental subpopulation of a single genetic-ancestry group, excluding founder-effect groups, with at least 2,000 alleles tested and at least 2 variant alleles. The variant is absent from gnomAD v2.1 and gnomAD v4.1, and the available non-cancer subset queries are also absent; therefore no population frequency or qualifying ancestry-specific FAF reaches the TP53 BA1 threshold.

Rule

Filtering allele frequency (FAF) of >= 0.001 or 0.1% in gnomAD continental subpopulations of a single genetic ancestry group, excluding founder-effect groups; the group must have >=2,000 alleles tested and at least 2 alleles present.

Evidence considered

• TP53 VCEP v2.4 BA1 rule: FAF >=0.001 (0.1%) in a qualifying continental ancestry group, with >=2,000 alleles tested and at least 2 variant alleles; founder-effect groups are excluded.
• gnomAD v2.1 all-comers: variant absent.
• gnomAD v4.1 all-comers: variant absent.
• The queried gnomAD v2.1 non-cancer exome subset and gnomAD v3.1 non-cancer genome subset also report the variant as absent.

Sources

Reasoning

The governing ClinGen TP53 VCEP version 2.4 specifies BS1 at strong strength when the filtering allele frequency (FAF) is at least 0.0003 but less than 0.001 in a qualifying gnomAD continental subpopulation of a single genetic-ancestry group, with at least 2,000 alleles tested and at least 2 variant alleles. The variant is absent from gnomAD v2.1 and gnomAD v4.1, and the available non-cancer subset queries are also absent; therefore the evidence does not meet the TP53 BS1 frequency threshold.

Rule

Filtering allele frequency (FAF) of >= 0.0003 but < 0.001 in gnomAD continental subpopulations of a single genetic ancestry group, excluding founder-effect groups; the group must have >=2,000 alleles tested and at least 2 alleles present.

Evidence considered

• TP53 VCEP v2.4 BS1 rule: FAF >=0.0003 but <0.001 in a qualifying continental ancestry group, with >=2,000 alleles tested and at least 2 variant alleles; founder-effect groups are excluded.
• gnomAD v2.1 all-comers: variant absent.
• gnomAD v4.1 all-comers: variant absent.
• The queried gnomAD v2.1 non-cancer exome subset and gnomAD v3.1 non-cancer genome subset also report the variant as absent.

Sources

Reasoning

BS3 is not met because the governing TP53 Functional-worksheet.xlsx exact-match row for p.L344R assigns PS3 rather than BS3 and records abnormal functional results, including Non-functional and LOF outcomes with monomeric oligomerization. Variant-specific cellular and yeast assays likewise show loss or severe disruption of transcriptional and oligomerization function rather than normal protein function. The TP53 VCEP BS3 rules require functional or partially functional Kato results with no loss of function across the relevant eligible assays; those benign-function requirements are not satisfied.

Rule

TP53 VCEP v2.4 BS3; strong rule requires functional Kato data AND no LOF by the majority of eligible assays, while supporting requires partially functional Kato data AND no LOF by all available assays.

Evidence considered

• The exact L344R row in Functional-worksheet.xlsx assigns PS3, not BS3, and records Non-functional and LOF results.
• The TP53 VCEP BS3 strong rule requires functional Kato data and no loss of function by the majority of available eligible assays; the BS3 supporting rule requires partially functional Kato data and no LOF by all available assays.
• L344R showed no reporter signal and an abnormal 98.2% ± 1.7% monomer fraction in the extracted cellular functional study.
• L344R was classified as a monomer mutant in the extracted TP53 missense-library oligomerization study.
• The extracted tetramerization-domain study found abnormal intermediate oligomerization and thermal instability for L344R, not preserved benign function.

Sources

Reasoning

This is a missense variant, so BP4 is within scope. The governing TP53 VCEP PP3-BP4 lookup table assigns PP3_moderate rather than BP4 for c.1031T>G / p.Leu344Arg and reports BayesDel 0.288212. The VCEP BP4 supporting rule requires BayesDel <0.16 with no predicted splicing impact, and the moderate BP4 rule requires BayesDel <=-0.008; the observed BayesDel score satisfies neither benign threshold. The SpliceAI result is below 0.2, but absence of predicted splicing impact alone does not establish BP4 when the missense BayesDel requirement fails.

Rule

TP53 VCEP BP4 supporting requires missense BayesDel <0.16 and SpliceAI <0.2; BP4 moderate requires BayesDel <=-0.008 and SpliceAI <0.2.

Evidence considered

• The TP53 VCEP PP3-BP4-codes.xlsx supplementary table entry for c.1031T>G / p.Leu344Arg assigns PP3_moderate, not BP4, and reports BayesDel score 0.288212 with maximum SpliceAI delta 0.01.
• The TP53 VCEP BP4 supporting rule requires BayesDel <0.16 and no predicted splicing impact; its BP4 moderate rule requires BayesDel <=-0.008. BayesDel 0.288212 meets neither threshold.
• The case SpliceAI result reports max delta 0.001, below the VCEP no-predicted-splicing boundary of 0.2, but this does not overcome the failed BayesDel benign requirement.

Sources

Reasoning

The exact ClinVar record for NM_000546.5:c.1031T>G (p.Leu344Arg/p.L344R) is classified Likely Pathogenic by the ClinGen TP53 Variant Curation Expert Panel, not Benign or Likely Benign. Therefore the requested exact-variant expert-panel requirement for BP6 is not satisfied.

Rule

Exact-variant ClinVar expert-panel Benign or Likely Benign classification is required for BP6.

Evidence considered

• ClinVar variation 2758378 exactly matches NM_000546.5:c.1031T>G (p.Leu344Arg) and reports a Likely Pathogenic classification by the ClinGen TP53 Variant Curation Expert Panel.
• Because the exact expert-panel classification is not Benign or Likely Benign, the requested BP6 trigger is absent.

Sources

Reasoning

The TP53 VCEP permits PS1 at Strong for a pathogenic assertion or Moderate for a likely pathogenic assertion when the assertion follows its specifications. However, applying PS1 requires independent same-amino-acid-change evidence, and the available record provides no independently adjudicated pathogenic comparator for the L344R change. The ClinVar expert-panel classification of this same variant cannot itself be used as PS1 evidence because a criterion must not be inferred from another source's final classification.

Rule

Can be applied to variants asserted as Pathogenic following the TP53 VCEP specifications at Strong, or asserted as Likely Pathogenic following the TP53 VCEP specifications at Moderate.

Evidence considered

• The TP53 VCEP PS1 rule allows Strong for variants asserted Pathogenic and Moderate for variants asserted Likely Pathogenic following the VCEP specifications.
• No independent same-amino-acid-change pathogenic comparator for p.Leu344Arg was identified in the reviewed evidence; the ClinVar expert-panel classification is not independently reusable as PS1 evidence.

Still missing

• An independent variant record with the same amino-acid change, adjudicated pathogenic or likely pathogenic under the TP53 VCEP, is needed for PS1.

Sources

Reasoning

The TP53 VCEP requires de novo points to be tallied from documented maternity and paternity confirmation and the proband's LFS-associated cancer type, with 1 point supporting, 2-3 points moderate, 4-7 points strong, and at least 8 points very strong. The available case evidence documents no parental testing, maternity or paternity confirmation, de novo observation, proband cancer type, or applicable point total, so PS2 cannot be assigned.

Rule

De novo points should be tallied using the table for tallying proband points based on whether maternity and paternity have been confirmed and the type of cancer(s) seen in the proband. Points for all probands should be tallied to determine the strength of PS2 code application.

Evidence considered

• The TP53 VCEP Version 2.4 specifies PS2 as the exclusive de novo code, states that PM6 is dropped, and assigns strength by accumulated de novo points: supporting at 1, moderate at 2-3, strong at 4-7, and very strong at ≥8 points.
• The governing Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf was searched under c.1031T>G, p.Leu344Arg, and p.L344R; no exact-variant entry was found.
• The case evidence contains no documented parental testing, confirmed maternity or paternity, de novo status, proband cancer type, or de novo point total.

Still missing

• No documented parental testing or maternity and paternity confirmation is available.
• No documented de novo observation, proband cancer phenotype, or PS2 point calculation is available.
• No exact-variant entry was found when searching c.1031T>G, p.Leu344Arg, and p.L344R in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt; this file is a general cancer-point table rather than a variant observation table.

Sources

Reasoning

The TP53 VCEP PS4 rule assigns 0.5 points for a revised Chompret clinical criterion, 1 point for classic Li-Fraumeni syndrome criteria, and 0.5 points for HER2-positive breast cancer under age 40, with Supporting at 1-1.5 points, Moderate at 2-3.5, Strong at 4-7.5, and Very Strong at >=8 points. The searched governing file PS4-Points-Table.pdf contains no entry for c.1031T>G, p.Leu344Arg, or p.L344R, and the case evidence provides no proband phenotype, cancer history, tumor pathology, or per-proband point total from which PS4 strength can be assigned.

Rule

PS4 Supporting = 1-1.5 points; Moderate = 2-3.5 points; Strong = 4-7.5 points; Very Strong >= 8 points.

Evidence considered

• The TP53 VCEP PS4 specification states: revised Chompret clinical criteria are 0.5 points per observation, classic Li-Fraumeni syndrome criteria are 1 point per observation, and HER2-positive breast cancer under age 40 is 0.5 points unless already counted through the clinical criteria.
• The same specification defines PS4 strength thresholds as Supporting 1-1.5 points, Moderate 2-3.5 points, Strong 4-7.5 points, and Very Strong >=8 points.
• PS4-Points-Table.pdf was searched for c.1031T>G, p.Leu344Arg, and p.L344R; no variant-specific entry was found.

Still missing

• No proband phenotype, cancer history, tumor pathology, or PS4 point total is available in the reviewed case evidence.
• No variant-specific entry was found when PS4-Points-Table.pdf was searched for c.1031T>G, p.Leu344Arg, and p.L344R.

Sources

Reasoning

The TP53 VCEP requires a different missense variant at the same amino-acid residue that was previously determined Pathogenic under the VCEP specifications for PM5 Moderate or Strong, or a previously determined Likely Pathogenic variant with clinical pathogenicity data for PM5 Supporting. Functional studies identify alternate L344 substitutions, including L344Q, L344P, and L344M, and show residue-level dysfunction, but they do not provide the required VCEP pathogenic or likely pathogenic adjudication with qualifying clinical data. The collected PM5 comparator artifact contains no same-residue candidates, so the evidence is insufficient to assign a PM5 strength.

Rule

Strong: at least 2 different pathogenic missense variants at the residue; Moderate: 1 different pathogenic missense variant; Supporting: 1 different likely pathogenic missense variant with clinical pathogenicity data demonstrating PS2, PS4, or PP1.

Evidence considered

• The TP53 VCEP PM5 rules require at least one different pathogenic same-residue missense variant for Moderate, at least two for Strong, or one different likely pathogenic variant with PS2, PS4, or PP1 clinical data for Supporting.
• PMID:12826609 reports that Leu-344 was inactivated by at least three distinct substitutions in a 2,314-mutant TP53 functional survey, but it does not adjudicate those alternate variants as VCEP Pathogenic or Likely Pathogenic.
• PMID:16007150 reports L344Q, L344P, and L344R as monomer mutants and L344M as a dimer mutant, but it does not provide the VCEP-required pathogenic adjudication or qualifying clinical data for an alternate L344 variant.
• The PM5 comparator collection reports zero same-residue candidates with zero expert-panel or non-expert majority pathogenic/likely pathogenic candidates.

Still missing

• A different missense variant at residue 344 with TP53 VCEP Pathogenic or Likely Pathogenic adjudication is needed; Likely Pathogenic evidence must include PS2, PS4, or PP1 clinical data.

Sources

Reasoning

The TP53 VCEP requires observed cosegregation for PP1: 3-4 meioses for supporting, 5-6 meioses for moderate, and at least 7 meioses across more than one family for strong. The available case evidence reports no affected relatives, variant-positive relatives, variant-negative relatives, family-level segregation, or countable meioses, so PP1 cannot be assigned.

Rule

Cosegregation must be observed in 3-4 meioses for supporting, 5-6 meioses for moderate, or ≥7 meioses across >1 family for strong.

Evidence considered

• The TP53 VCEP Version 2.4 assigns PP1 supporting at 3-4 cosegregating meioses, moderate at 5-6 meioses, and strong at ≥7 meioses across more than one family.
• The governing Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf was searched under c.1031T>G, p.Leu344Arg, and p.L344R; no exact-variant entry was found.
• The case evidence contains no documented pedigree, affected-family-member genotypes, non-affected-family-member genotypes, segregation observations, or meiosis count.

Still missing

• No family pedigree or affected-relative phenotype information is available.
• No relative genotyping, segregation observations, or countable meioses are available.
• No exact-variant entry was found when searching c.1031T>G, p.Leu344Arg, and p.L344R in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt; this file is a general cancer-point table rather than a variant observation table.

Sources

Reasoning

The TP53 VCEP PP4 rule requires at least one or more independent observations of the variant with a variant allele fraction (VAF) of 5-35% for Supporting, or at least two independent observations with VAF 5-25% for Moderate. The case evidence contains no PP4-eligible observation or VAF data, so phenotype-specific PP4 cannot be assessed.

Rule

PP4 Moderate = at least 2 independent observations with VAF 5-25%; PP4 Supporting = observation with VAF 5-35%.

Evidence considered

• The TP53 VCEP PP4 specification defines Moderate as at least two independent observations with VAF 5-25%.
• The TP53 VCEP PP4 specification defines Supporting as observation of the variant with VAF 5-35%, including one or more observations with VAF above 25% and up to 35% and/or one observation with VAF 5-25%.
• No qualifying PP4 observation or VAF measurement is present in the case evidence.

Still missing

• No independent PP4-eligible observation with VAF data is available in the reviewed case evidence.

Sources

Reasoning

The TP53 VCEP version 2.4 BS2 rule requires unaffected unrelated females aged at least 60 years from one source: 2-3 individuals for supporting, 4-7 for moderate, or at least 8 for strong, with older individuals diagnosed with sarcoma excluded. The available case evidence contains no qualifying unaffected carrier observations, ages, sex information, single-source cohort, or homozygote data, so BS2 cannot be assessed.

Rule

Unrelated females who have reached at least 60 years of age without cancer, all from a single source: 2-3 for supporting, 4-7 for moderate, and >=8 for strong; individuals with sarcoma diagnosed at >=61 years should not be counted.

Evidence considered

• TP53 VCEP v2.4 BS2 rule: 2-3 qualifying unrelated females aged >=60 from one source support BS2; 4-7 support moderate BS2; >=8 support strong BS2; individuals with sarcoma diagnosed at >=61 years are excluded.
• No qualifying unaffected carrier observations or homozygote observations are present in the available case evidence.

Still missing

• A single-source cohort of unrelated female TP53 variant carriers aged at least 60 years without cancer is not available.
• Carrier-level sex, age, cancer status, source, and sarcoma-exclusion data are not available.

Sources

Reasoning

The TP53 VCEP defines BS4 as strong evidence for lack of segregation in affected family members diagnosed with LFS-associated cancers. The available case evidence contains no affected-family-member genotypes or documented non-segregation observations, so BS4 cannot be assigned.

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 TP53 VCEP Version 2.4 defines BS4 as strong evidence for lack of segregation in affected family members diagnosed with LFS-associated cancers described in the governing cancer table.
• The case evidence contains no affected-relative genotype data and no documented lack-of-segregation observation.

Still missing

• No affected family members with LFS-associated cancers are documented for this assessment.
• No variant-negative testing results or explicit non-segregation observations in affected relatives are available.

Sources