TP53 c.476C>T, p.(Ala159Val)

Reasoning

The ClinGen TP53 VCEP Functional-worksheet.xlsx Supplementary Table S3 contains the exact amino-acid entry A159V. Its row records Kato class Non-functional, Funk class LOF, Giacomelli class LOF, Kotler class LOF, Kawaguchi class NA, and the pre-assigned functional code PS3. The VCEP rule for strong PS3 is non-functional on Kato data and loss of function by the majority of other eligible assays. This variant meets that rule through the Kato non-functional result and three additional eligible LOF assay classifications. The VCEP functional flowchart identifies these assay classes as eligible functional evidence. SpliceAI shows a maximum delta score of 0.003, so the VCEP splicing caveat does not indicate a predicted splicing effect that would disqualify the protein-functional code.

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 A159V: Kato = Non-functional; Funk = LOF; Giacomelli = LOF; Kotler = LOF; Kawaguchi = NA; preliminary functional code = PS3.
• ClinGen TP53 VCEP PS3 strong rule: non-functional on Kato et al. data AND loss of function (LOF) by the majority of other eligible assays.
• Flowchart-for-application-of-functional-rule-codes.pdf: eligible functional studies include Kato, Funk, Giacomelli, Kotler, and Kawaguchi.
• SpliceAI case evidence for NM_000546.5:c.476C>T: maximum delta score 0.003, with DS_AG 0.000, DS_AL 0.000, DS_DG 0.001, and DS_DL 0.003; this is below the VCEP's 0.2 splicing-effect caveat threshold.
• The exact variant was searched as c.476C>T, p.Ala159Val, and p.A159V; the governing worksheet match was p.A159V.

Still missing

• The exact variant was not found in Flowchart-for-application-of-functional-rule-codes.pdf; that file provides the assay eligibility framework rather than a variant-specific row.
• The exact variant was not found using c.476C>T or p.Ala159Val in Functional-worksheet.xlsx; it was found under p.A159V in the A159V row.

Sources

Reasoning

The TP53 VCEP specifies PM2 at supporting strength when the variant allele frequency is below 0.00003 in gnomAD or another large sequenced population. The variant is absent from gnomAD v2.1 and gnomAD v4.1, supporting an observed allele frequency of 0 and therefore below 0.00003. The VCEP does not require a non-cancer subset for this rule, so the all-comers gnomAD results are the applicable default.

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.

Evidence considered

• TP53 VCEP PM2 rule: apply at supporting level when allele frequency is <0.00003 (0.003%) in gnomAD or another large sequenced population.
• gnomAD v2.1 all-comers result for NC_000017.10:g.7578454G>A / NM_000546.5:c.476C>T: absent, observed allele frequency 0.
• gnomAD v4.1 all-comers result for NC_000017.11:g.7675136G>A / NM_000546.5:c.476C>T: absent, observed allele frequency 0.

Sources

Reasoning

The variant is a missense substitution, so the TP53 VCEP missense PP3 pathways apply. The governing Supplementary Table S2 contains the exact row c.476C>T / p.Ala159Val / Class C0 / BayesDel 0.272813 and pre-assigns ‘No evidence’; this governing pre-assignment is the answer for PP3 and is not overridden by independently noting that the BayesDel value is at least 0.16. The same variant was not found in the single-amino-acid deletion BayesDel table, which is outside this variant’s missense form. SpliceAI max delta is 0.003, but that splice result is not a substitute for the missense PP3 protein-impact pathway and is not double-counted.

VCEP rule

**Missense variants** _(See flowchart for application of PP3 and BP4 rules for missense variants)_ aGVGD Class C65 and BayesDel score ≥ 0.16 ... **Missense variants** _(See flowchart for application of PP3 and BP4 rules for missense variants)_ aGVGD class C25-C55 and BayesDel score ≥ 0.16 ... **Exonic (including silent variants and apparent “missense” variants or “single amino acid inframe deletions” for which there is a predicted splice effect) or Intronic Splice Variants (excluding ± 1,2 positions):** SpliceAI ≥ 0.2

Evidence considered

• PP3-BP4-codes.xlsx, Supplementary Table S2 exact row: c.476C>T; p.Ala159Val; Class C0; BayesDel 0.272813; assigned code ‘No evidence’.
• Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf: the in-silico framework includes apparent missense variants with predicted splice effects and excludes ±1,2 positions; this variant is an exonic missense substitution in exon 5, not a canonical splice-site variant.
• Case SpliceAI result: DS_AG 0.000, DS_AL 0.000, DS_DG 0.001, DS_DL 0.003; max delta score 0.003.
• The exact notation search found c.476C>T only in PP3-BP4-codes.xlsx.txt; p.Ala159Val and p.A159V had no exact matches in the three governing full-text files.

Still missing

• No exact entry for c.476C>T, p.Ala159Val, or p.A159V was found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf.txt.
• No exact entry for c.476C>T, p.Ala159Val, or p.A159V was found in TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx.txt; that file covers single amino acid in-frame deletions, not this missense variant.

Sources

Reasoning

The TP53 VCEP requires a filtering allele frequency (FAF) of at least 0.001 in a qualifying gnomAD continental subpopulation, 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, so no qualifying FAF reaches the BA1 threshold.

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

• TP53 VCEP BA1 rule: qualifying continental-subpopulation FAF must be ≥0.001, with ≥2,000 alleles tested and at least 2 variant alleles.
• gnomAD v2.1 all-comers result for NC_000017.10:g.7578454G>A / NM_000546.5:c.476C>T: absent.
• gnomAD v4.1 all-comers result for NC_000017.11:g.7675136G>A / NM_000546.5:c.476C>T: absent.

Sources

Reasoning

The TP53 VCEP requires a filtering allele frequency (FAF) of at least 0.0003 but below 0.001 in a qualifying gnomAD continental subpopulation, 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, so no qualifying FAF reaches the BS1 interval.

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

• TP53 VCEP BS1 rule: qualifying continental-subpopulation FAF must be ≥0.0003 and <0.001, with ≥2,000 alleles tested and at least 2 variant alleles.
• gnomAD v2.1 all-comers result for NC_000017.10:g.7578454G>A / NM_000546.5:c.476C>T: absent.
• gnomAD v4.1 all-comers result for NC_000017.11:g.7675136G>A / NM_000546.5:c.476C>T: absent.

Sources

Reasoning

The VCEP Functional-worksheet.xlsx Supplementary Table S3 exact A159V row assigns PS3, not BS3, and reports Kato Non-functional, Funk LOF, Giacomelli LOF, and Kotler LOF. These results are contrary to the VCEP BS3 requirements for functional or partially functional Kato data with no LOF across the eligible assays. Therefore the available governing functional evidence does not support BS3 and instead supports the opposing PS3 classification.

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 A159V: Kato = Non-functional; Funk = LOF; Giacomelli = LOF; Kotler = LOF; Kawaguchi = NA; preliminary functional code = PS3, with no BS3 assignment.
• ClinGen TP53 VCEP BS3 strong rule: functional on Kato et al. data AND no loss of function (LOF) by the majority of available eligible assays.
• ClinGen TP53 VCEP BS3 supporting rule: partially functional on Kato et al. data AND no evidence of loss of function (LOF) by all available assays.
• Flowchart-for-application-of-functional-rule-codes.pdf identifies the assay set used by the governing functional worksheet as eligible functional evidence.
• SpliceAI case evidence for NM_000546.5:c.476C>T reports maximum delta score 0.003; no splice-impact signal reverses the variant-specific protein-function results.

Still missing

• Functional-worksheet.xlsx was searched for c.476C>T, p.Ala159Val, and p.A159V; the p.A159V row has no BS3 assignment and instead assigns PS3.
• The exact variant was not found in Flowchart-for-application-of-functional-rule-codes.pdf; that file provides the assay eligibility framework rather than a variant-specific row.

Sources

Reasoning

The variant is a missense substitution, so the TP53 VCEP missense BP4 pathways apply and require both the specified BayesDel range and no predicted splice difference. Supplementary Table S2 directly pre-assigns the exact c.476C>T / p.Ala159Val row as ‘No evidence’; its BayesDel score is 0.272813, which is neither ≤ -0.008 for BP4_Moderate nor < 0.16 and > -0.008 for BP4_Supporting. SpliceAI max delta 0.003 is below the VCEP’s <0.2 no-splicing threshold, but it cannot satisfy BP4 when the required BayesDel condition fails.

VCEP rule

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

• PP3-BP4-codes.xlsx, Supplementary Table S2 exact row: c.476C>T; p.Ala159Val; Class C0; BayesDel 0.272813; assigned code ‘No evidence’.
• The BayesDel value 0.272813 fails both governing missense BP4 ranges: ≤ -0.008 for Moderate and <0.16 and > -0.008 for Supporting.
• Case SpliceAI result: DS_AG 0.000, DS_AL 0.000, DS_DG 0.001, DS_DL 0.003; max delta score 0.003, satisfying the no-predicted-splicing component (<0.2) but not rescuing the failed BayesDel component.
• The exact notation search found c.476C>T only in PP3-BP4-codes.xlsx.txt; p.Ala159Val and p.A159V had no exact matches in the three governing full-text files.

Still missing

• No exact entry for c.476C>T, p.Ala159Val, or p.A159V was found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf.txt.
• No exact entry for c.476C>T, p.Ala159Val, or p.A159V was found in TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx.txt; that file covers single amino acid in-frame deletions, not this missense variant.

Sources

Reasoning

The variant is a missense substitution, but the available evidence does not establish a different nucleotide change producing the same p.Ala159Val amino-acid change that was previously determined pathogenic or likely pathogenic under the TP53 VCEP specifications. ClinVar records assert this same c.476C>T variant as pathogenic or likely pathogenic, but those assertions do not provide the required alternate-nucleotide comparator. SpliceAI maximum delta is 0.003, below the VCEP's 0.2 splice-impact threshold, but the comparator requirement remains unclosed.

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 consequence: missense, NP_000537.3:p.(Ala159Val), from NM_000546.5:c.476C>T.
• ClinVar contains pathogenic and likely pathogenic assertions for the same c.476C>T variant, but no qualifying alternate-nucleotide same-amino-acid comparator is documented.
• SpliceAI maximum delta score is 0.003.

Still missing

• A qualifying previously established pathogenic or likely pathogenic TP53 variant with the same p.Ala159Val amino-acid change but a different nucleotide change is needed.

Sources

Reasoning

The TP53 VCEP requires de novo evidence to be converted to proband points according to confirmed maternity and paternity and the proband's LFS-associated cancer. The available case evidence contains no variant-specific proband de novo observation, parental testing result, maternity or paternity confirmation, constitutional mosaicism documentation, or cancer-point tally, so PS2 cannot be evaluated.

VCEP 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.

Evidence considered

• ClinGen TP53 VCEP PS2 rule: de novo points must be tallied using confirmed maternity and paternity and the type of cancer seen in each proband; PS2 strength is Supporting at 1 point, Moderate at 2-3 points, Strong at 4-7 points, and Very Strong at 8 or more points.
• No case-specific parental, de novo, mosaicism, or proband cancer-point evidence was identified for NM_000546.5:c.476C>T (p.Ala159Val/p.A159V).

Still missing

• No variant-specific proband de novo record, parental testing result, maternity confirmation, paternity confirmation, or constitutional mosaicism evidence was available.
• No c.476C>T, p.Ala159Val, or p.A159V entry was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.
• The searched VCEP full-text files contained no variant-specific PS2 de novo entry for c.476C>T, p.Ala159Val, or p.A159V.

Sources

Reasoning

The TP53 VCEP assigns PS4 from phenotype-based points: Revised Chompret contributes 0.5 point, Classic LFS contributes 1 point, and HER2+ breast cancer diagnosed below age 40 contributes 0.5 point, with 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 does not provide a proband cancer history, LFS/Chompret assessment, HER2 status and age, or a case-control enrichment estimate, so no PS4 points can be assigned. The required governing file PS4-Points-Table.pdf was searched for c.476C>T, p.Ala159Val, and p.A159V; no variant-specific entry was found because this table is phenotype-point based.

VCEP rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... ≥ 8 points ... ≥ 4-7.5 points ... 2-3.5 points ... 1-1.5 points

Evidence considered

• PS4-Points-Table.pdf: the table gives Revised Chompret = 0.5 point, Classic LFS Criteria = 1 point, HER2+ breast cancer below age 40 = 0.5 point, and PS4 strength thresholds of Supporting 1-1.5, Moderate 2-3.5, Strong 4-7.5, and Very Strong ≥8 points.
• No c.476C>T, p.Ala159Val, or p.A159V row was found in PS4-Points-Table.pdf; the file is a phenotype-point table rather than a variant-indexed table.
• The available evidence records identify the variant and population/database results but do not supply the proband phenotype or a case-control odds ratio and confidence interval needed for PS4.

Still missing

• No proband cancer history or phenotype sufficient to determine Revised Chompret or Classic LFS points.
• No proband age and HER2 status for the VCEP's HER2+ breast-cancer point.
• No case-control enrichment estimate with confidence interval.
• No variant-specific entry was found after searching PS4-Points-Table.pdf for c.476C>T, p.Ala159Val, and p.A159V.

Sources

Reasoning

The variant is p.Ala159Val, not one of the VCEP's explicitly listed hotspot codons 175, 245, 248, 249, 273, or 282. The VCEP also permits PM1_Moderate for a germline missense variant seen in cancerhotspots.org v2 with at least 10 somatic occurrences for the same amino-acid change. The available hotspot record reports A159 as a statistically significant hotspot but is partially parsed and does not provide the required cancerhotspots same-amino-acid count. The authoritative domain_tables object is empty, so no approved domain-table match can be established.

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 VCEP PM1_Moderate rule lists codons 175, 245, 248, 249, 273, and 282, and separately permits germline missense variants in cancerhotspots.org v2 with at least 10 somatic occurrences for the same amino-acid change.
• The variant is p.Ala159Val; residue 159 is outside the six explicitly listed codons.
• The available hotspot evidence reports A159 as a statistically significant hotspot but supplies no qualifying cancerhotspots.org same-amino-acid count; the separate COSMIC record reports 106 somatic occurrences, which is not the VCEP-specified cancerhotspots.org evidence.
• The vcep_materials domain_tables object is empty for TP53, so there is no authoritative approved domain-table entry to match residue 159.

Still missing

• Verify the cancerhotspots.org v2 count for TP53 p.Ala159Val and document that it is at least 10 somatic occurrences, or provide an applicable VCEP domain-table entry.

Sources

Reasoning

The PM5 candidate collection contains zero same-residue comparator variants for residue 159. Therefore, the available evidence does not establish a different missense variant at A159 previously determined pathogenic or likely pathogenic under the TP53 VCEP specifications. The VCEP's Grantham-comparison and splice-exclusion requirements cannot be applied without a qualifying comparator.

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 VCEP PM5 rule requires a different missense variant at the same amino-acid residue previously determined pathogenic or likely pathogenic under the VCEP specifications; supporting-level PM5 additionally requires clinical data demonstrating pathogenicity through PS2, PS4, or PP1.
• The PM5 candidate artifact reports total_candidates_seen = 6 but same_residue_candidates = 0, with zero expert-panel or non-expert-majority pathogenic/likely pathogenic candidates.

Still missing

• A qualifying different missense variant at residue 159, classified under the TP53 VCEP specifications without PM5, is needed; supporting-level PM5 additionally requires PS2, PS4, or PP1 clinical data.

Sources

Reasoning

The TP53 VCEP requires meioses to be counted only for individuals who carry the variant and have a relevant LFS-associated cancer, with unaffected obligate carriers permitted. No variant-specific affected relatives, unaffected obligate carriers, germline or qualifying tumor results, family relationships, or countable meioses are documented, so PP1 cannot be evaluated.

VCEP rule

Meioses should be counted for individuals who both carry the variant and have a relevant cancer (see LFS cancers table). Meioses can be counted through unaffected obligate carriers. ... Cosegregation must be observed in 3-4 meioses in/across 1 or more families

Evidence considered

• ClinGen TP53 VCEP PP1 rule: meioses are counted for individuals who carry the variant and have a relevant cancer, and can be counted through unaffected obligate carriers; supporting requires 3-4 cosegregating meioses, moderate requires 5-6, and strong requires at least 7 across more than one family.
• No variant-specific family segregation data or countable meioses were identified for NM_000546.5:c.476C>T (p.Ala159Val/p.A159V).

Still missing

• No variant-specific family pedigree, affected-relative phenotype, unaffected obligate-carrier information, phase information, or germline testing results were available for counting meioses.
• No c.476C>T, p.Ala159Val, or p.A159V entry was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.
• The searched VCEP full-text files contained no variant-specific PP1 segregation entry for c.476C>T, p.Ala159Val, or p.A159V.

Sources

Reasoning

The TP53 VCEP PP4 rule is specifically for presumed somatic TP53 variants detected on multigene panel testing and requires a variant observation with VAF 5-35% for Supporting or at least two independent observations with VAF 5-25% for Moderate; it also excludes blood-cancer cases and confirmed constitutional mosaicism and cannot be combined with phenotype-based rules in the same individual. The available evidence contains no qualifying VAF observation, MGPT context, or individual phenotype, so PP4 is not assessed.

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

• The TP53 VCEP PP4 Supporting rule requires observation of the variant with VAF 5-35%; the Moderate rule requires at least 2 independent observations with VAF 5-25%.
• The available case evidence reports no individual VAF observation or multigene-panel context for this variant.

Still missing

• No qualifying MGPT detection context is documented.
• No VAF observation between 5% and 35% is documented.
• No two independent VAF observations between 5% and 25% are documented.
• No phenotype and blood-cancer status are documented to evaluate the PP4 caveats.

Sources

Reasoning

The TP53 VCEP requires counts of unrelated female carriers aged at least 60 years without cancer from one source, with separate thresholds for supporting, moderate, and strong evidence. The available case evidence contains no qualifying carrier-level age, sex, cancer-status, source, or sarcoma-exclusion data for this variant.

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

• TP53 VCEP BS2 requires 2–3, 4–7, or ≥8 unrelated females aged at least 60 years without cancer for supporting, moderate, or strong strength, respectively, all from one source.
• No qualifying BS2 carrier-level observations are provided in the available case evidence.

Still missing

• A single-source cohort with qualifying unrelated female carriers, age ≥60, cancer status, and sarcoma-exclusion information is needed.

Sources

Reasoning

The TP53 VCEP defines BS4 as lack of segregation in affected family members diagnosed with LFS-associated cancers. No affected family members, variant-negative affected relatives, family pedigree, or other variant-specific non-segregation evidence is documented, so BS4 cannot be evaluated.

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

• ClinGen TP53 VCEP BS4 rule: strong BS4 requires lack of segregation in affected family members diagnosed with LFS-associated cancers listed in the VCEP LFS cancers table.
• No variant-specific affected-family non-segregation evidence was identified for NM_000546.5:c.476C>T (p.Ala159Val/p.A159V).

Still missing

• No variant-specific pedigree, affected family members, family cancer diagnoses, or comparative variant testing results were available to demonstrate non-segregation.
• No c.476C>T, p.Ala159Val, or p.A159V entry was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.
• The searched VCEP full-text files contained no variant-specific BS4 non-segregation entry for c.476C>T, p.Ala159Val, or p.A159V.

Sources