TP53 c.659A>G, p.Tyr220Cys

Reasoning

The TP53 VCEP Functional-worksheet.xlsx, Supplementary Table S3, row Y220C, pre-assigns PS3 for this missense variant. The row records Kato class Non-functional, Funk class LOF, Giacomelli class LOF, Kotler class LOF, and Kawaguchi class NA. Thus the variant is non-functional in the Kato assay and LOF in all three available other eligible assays (Funk, Giacomelli, and Kotler), satisfying the VCEP strong PS3 path. The functional flowchart independently maps non-functional Kato results with LOF by the majority of available assays to PS3.

VCEP rule

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

Evidence considered

• ClinGen TP53 VCEP Functional-worksheet.xlsx, Supplementary Table S3, row Y220C: Kato class = Non-functional; Funk class = LOF; Giacomelli class = LOF; Kotler class = LOF; Kawaguchi class = NA; preliminary functional code = PS3.
• ClinGen TP53 VCEP Flowchart-for-application-of-functional-rule-codes.pdf, missense row: Non-functional (Kato) -> LOF by the majority of available assays* -> PS3.
• The available non-Kato assays listed in the Y220C worksheet row are Funk, Giacomelli, and Kotler, and all three are classified LOF.

Still missing

• No variant-specific c.659A>G, p.Tyr220Cys, or p.Y220C entry appears in Flowchart-for-application-of-functional-rule-codes.pdf.txt; that file provides the governing general functional-code flowchart and was applied with the Y220C worksheet row.

Sources

Reasoning

TP53 p.Tyr220Cys is a missense variant. Residue 220 is not among the VCEP's codon-defined hotspots 175, 245, 248, 249, 273, and 282, but the VCEP also authorizes PM1 for germline missense variants seen in cancerhotspots.org (v2) with at least 10 somatic occurrences for the same amino-acid change. The Cancer Hotspots evidence identifies this variant as lying in a statistically significant hotspot, and the linked COSMIC record reports 741 somatic occurrences for TP53 Y220C, exceeding the VCEP's threshold; therefore PM1 is met at Moderate strength.

VCEP rule

Missense variants within the following codons using transcript NM\_000546.4: 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

• TP53 VCEP PM1 rule: codon-defined hotspots are 175, 245, 248, 249, 273, and 282; an alternative Moderate route applies to germline missense variants seen in cancerhotspots.org (v2) with ≥10 somatic occurrences for the same amino-acid change.
• Cancer Hotspots result: the variant lies in a statistically significant hotspot.
• COSMIC COSV52661282: TP53 Y220C has been reported in somatic cancers 741 times.

Sources

Reasoning

The TP53 VCEP specifies PM2 at supporting strength when the allele frequency is below 0.00003 and, when multiple alleles are present in an ancestry group, that group's frequency is below 0.00004; founder-effect groups are ignored. gnomAD v4.1 reports total AF 4.95676e-06 (8/1,613,958 alleles) and the highest non-founder ancestry frequency is 5.93246e-06 in European non-Finnish individuals (7/1,179,948 alleles), with 0 homozygotes. Both requirements are met, so PM2_Supporting applies.

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: 8/1,613,958 alleles, total AF 4.95676e-06, total homozygotes 0.
• gnomAD v4.1 European non-Finnish ancestry: 7/1,179,948 alleles, AF 5.93246e-06, homozygotes 0; this is the highest reported non-founder ancestry frequency and contains multiple variant alleles.
• The total AF is below 0.00003 and the multi-allele ancestry AF is below 0.00004, satisfying PM2_Supporting.

Sources

Reasoning

The variant is a missense change, NM_000546.6:c.659A>G (p.Tyr220Cys/p.Y220C). The TP53 VCEP Supplementary Table S2 directly assigns this exact coding change to PP3_moderate: c.659A>G, p.Tyr220Cys, aGVGD Class C65, BayesDel 0.562537, PP3_moderate, SpliceAI 0.13. This governing lookup assignment outranks independent recalculation. The VCEP rule for missense PP3_moderate requires aGVGD Class C65 and BayesDel score >= 0.16; the table assignment is therefore applicable. SpliceAI max delta is 0.135, below the VCEP's <0.2 no-predicted-splicing condition for the missense protein-prediction path, so the missense PP3 assignment is not displaced by a predicted splice effect.

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.659A>G | p.Tyr220Cys | Class C65 | BayesDel 0.562537 | PP3_moderate | SpliceAI 0.13.
• The variant is NM_000546.6:c.659A>G, a missense variant encoding p.Tyr220Cys (p.Y220C).
• SpliceAI scores are DS_AG 0.0, DS_AL 0.026, DS_DG 0.135, and DS_DL 0.035; max delta score is 0.135, reported as no significant splice impact.

Still missing

• No exact c.659A>G, p.Tyr220Cys, or p.Y220C entry was found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf.txt; this file provides the governing flowchart rather than per-variant assignments.
• No exact c.659A>G, p.Tyr220Cys, or p.Y220C entry was found in TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx.txt; that table covers single amino acid in-frame deletions, not this missense variant.

Sources

Reasoning

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

VCEP rule

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

Evidence considered

• The VCEP CSPEC entry marks PP5 Not Applicable for this VCEP and states that the criterion is not for use.
• ClinVar expert panel classification

Sources

Reasoning

The variant is TP53 p.Tyr220Cys (NM_000546.6:c.659A>G). The TP53 VCEP permits PS1 only when comparing the variant with a previously established pathogenic or likely pathogenic variant producing the same amino-acid change, with no splicing difference. The available same-residue search found zero alternate missense comparator variants, so the required same-amino-acid comparison is not met; the current variant's own expert-panel Pathogenic classification cannot serve as its PS1 comparator.

VCEP rule

This rule code can only be used to compare variants asserted as Pathogenic following the _TP53_ VCEP’s specifications. ... Can be applied to variants asserted as Likely Pathogenic following the _TP53_ VCEP’s specifications.

Evidence considered

• TP53 VCEP PS1 rule: same-amino-acid comparison is restricted to variants asserted as Pathogenic or Likely Pathogenic under the TP53 VCEP specifications, with splicing differences excluded by RNA data or SpliceAI < 0.2.
• PM5 comparator candidate collection: 8 total candidates seen, 0 same-residue candidates, 0 expert-panel pathogenic/likely pathogenic candidates, and 0 non-expert-majority pathogenic/likely pathogenic candidates.
• ClinVar record NM_000546.6(TP53):c.659A>G (p.Tyr220Cys) is Pathogenic with expert-panel review; this is the assessed variant itself, not an alternate same-amino-acid comparator.

Sources

Reasoning

The TP53 VCEP PM5 rule requires a different missense variant at the same amino-acid residue that was previously established as pathogenic or likely pathogenic under the VCEP specifications, with the additional Grantham and splicing conditions applied. The available comparator collection found zero same-residue missense candidates, so no qualifying PM5 comparator exists.

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

• TP53 VCEP PM5 rule requires one or more different missense variants at the same amino-acid residue, established as Pathogenic or Likely Pathogenic under the TP53 VCEP specifications.
• PM5 comparator candidate collection: 0 same-residue candidates, 0 expert-panel pathogenic/likely pathogenic candidates, and 0 non-expert-majority pathogenic/likely pathogenic candidates.

Sources

Reasoning

The TP53 VCEP requires a filtering allele frequency (FAF) of at least 0.001 in a qualifying continental subpopulation, with at least 2,000 alleles tested and at least 2 alleles present. In gnomAD v4.1, the highest reported qualifying non-founder ancestry FAF is 2.47e-06, and the highest observed ancestry allele frequency is 1.56162e-05 in Finnish individuals, which is below 0.001. The variant therefore does not meet BA1.

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: 8/1,613,958 alleles, total AF 4.95676e-06, total homozygotes 0.
• gnomAD v4.1 highest observed ancestry frequency: Finnish, 1/64,036 alleles, AF 1.56162e-05, homozygotes 0; joint grpmax FAF 2.47e-06.
• The qualifying ancestry frequency and FAF are both below the VCEP BA1 threshold, so BA1 is not met.

Sources

Reasoning

The TP53 VCEP requires a filtering allele frequency (FAF) of at least 0.0003 but less than 0.001 in one qualifying continental ancestry group, with at least 2,000 alleles tested and at least 2 alleles present. The highest gnomAD v4.1 joint grpmax FAF is 2.47e-06, and the highest observed ancestry frequency is 1.56162e-05, both below 0.0003. BS1 is 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

• gnomAD v4.1 all-comers: total AF 4.95676e-06 from 8/1,613,958 alleles, with 0 homozygotes.
• gnomAD v4.1 joint grpmax FAF: 2.47e-06; highest observed ancestry frequency was Finnish AF 1.56162e-05 from 1/64,036 alleles.
• The available qualifying ancestry FAF is below 0.0003, so BS1 is not met.

Sources

Reasoning

The TP53 VCEP Functional-worksheet.xlsx, Supplementary Table S3, row Y220C, records Kato class Non-functional and LOF classifications in the Funk, Giacomelli, and Kotler assays, with a pre-assigned code of PS3 rather than BS3. Because the Kato result is not Functional and the majority of available other eligible assays show LOF, the VCEP BS3 strong rule is not satisfied.

VCEP rule

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

Evidence considered

• ClinGen TP53 VCEP Functional-worksheet.xlsx, Supplementary Table S3, row Y220C: Kato class = Non-functional; Funk class = LOF; Giacomelli class = LOF; Kotler class = LOF; Kawaguchi class = NA; preliminary functional code = PS3, not BS3.
• ClinGen TP53 VCEP Flowchart-for-application-of-functional-rule-codes.pdf, missense row: Functional (Kato) -> noLOF by the majority of available assays* -> BS3; Y220C does not have the required Functional Kato result and has majority LOF results instead.

Still missing

• No variant-specific c.659A>G, p.Tyr220Cys, or p.Y220C entry appears in Flowchart-for-application-of-functional-rule-codes.pdf.txt; that file provides the governing general functional-code flowchart and was applied with the Y220C worksheet row.

Sources

Reasoning

The variant is a missense change and has no predicted splice effect by the VCEP's threshold (SpliceAI max delta 0.135 < 0.2). However, the exact TP53 VCEP Supplementary Table S2 row assigns aGVGD Class C65 and BayesDel 0.562537 to PP3_moderate. The VCEP BP4 missense rule explicitly excludes Class C65 variants from BP4, and the BayesDel value is also not below 0.16. Therefore BP4 is not met despite the low splice-impact prediction; that splice result cannot negate the missense protein-impact evidence.

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

• PP3-BP4-codes.xlsx, Supplementary Table S2, matching row: c.659A>G | p.Tyr220Cys | Class C65 | BayesDel 0.562537 | PP3_moderate | SpliceAI 0.13.
• The TP53 VCEP BP4 missense rule says BP4 is not applied for aGVGD Class C65; the matching row is Class C65.
• SpliceAI max delta score is 0.135, below the VCEP's <0.2 no-predicted-splicing condition, but this does not overcome the BP4 Class C65 exclusion.

Still missing

• No exact c.659A>G, p.Tyr220Cys, or p.Y220C entry was found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf.txt; this file provides the governing flowchart rather than per-variant assignments.
• No exact c.659A>G, p.Tyr220Cys, or p.Y220C entry was found in TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx.txt; that table covers single amino acid in-frame deletions, not this missense variant.

Sources

Reasoning

The TP53 VCEP requires de novo points to be tallied from confirmed maternity and paternity and the proband's LFS-associated cancer type, with all proband points combined for strength. The available evidence does not document a de novo proband, confirmed maternity and paternity, absence of family history, qualifying cancer, or a point total. The governing LFS table was searched for c.659A>G, p.Tyr220Cys, and p.Y220C; it contains no variant-specific entry, only the cancer categories and point thresholds.

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. ... Points for all probands should be tallied to determine the strength of PS2 code application, consistent with SVI guidance. ... ≥ 8 points ... 4-7 points ... 2-3 points ... 1 point

Evidence considered

• The VCEP case instructions state that de novo points require tallying by confirmed maternity and paternity and the proband's cancer type; the case evidence does not provide those facts.
• The available case evidence includes a narrative segregation signal but no documented de novo proband, parental testing, qualifying cancer, or point calculation.
• Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf was searched for c.659A>G, p.Tyr220Cys, and p.Y220C; no variant-specific row was found.

Still missing

• No case-specific de novo observation with confirmed maternity and paternity was found.
• No qualifying proband cancer type or confirmed absence of family history was provided for point assignment.
• No entry for c.659A>G, p.Tyr220Cys, or p.Y220C was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt.

Sources

Reasoning

The TP53 VCEP PS4 rule assigns strength from proband points: Very Strong >= 8 points, Strong >= 4-7.5 points, Moderate 2-3.5 points, and Supporting 1-1.5 points. The available case evidence provides no proband cancer phenotype, Chompret or Classic LFS assessment, HER2-positive breast cancer age, case-control counts, or odds ratio for NM_000546.6:c.659A>G (p.Tyr220Cys); therefore no PS4 points can be assigned. The literature packet's PS4 candidate PMID:17508274 was checked in paper_extracts and does not mention TP53 c.659A>G or p.(Y220C), so it supplies no variant-specific case-control or phenotype evidence.

VCEP rule

≥ 8 points ... ≥ 4-7.5 points ... 2-3.5 points ... 1-1.5 points

Evidence considered

• PS4-Points-Table.pdf: the table assigns Revised Chompret 0.5 point, Classic LFS criteria 1 point, and HER2+ breast cancer below age 40 0.5 point; thresholds are PS4_Supporting 1-1.5, PS4_Moderate 2-3.5, PS4 4-7.5, and PS4_Very Strong >=8 points.
• No case phenotype or case-control data for NM_000546.6:c.659A>G (p.Tyr220Cys) are present in the reviewed case evidence.
• PMID:17508274 paper_extracts finding: the paper focuses on BRCA1/BRCA2 counseling and does not mention TP53, c.659A>G, or p.(Y220C); it therefore provides no variant-specific PS4 evidence.

Still missing

• No proband cancer phenotype, Chompret/Classic LFS status, HER2-positive breast cancer age, or other PS4 point-bearing clinical information is available.
• No case-control counts, matched-ancestry control frequency comparison, odds ratio, or 95% confidence interval is available.
• No entry for c.659A>G, p.Tyr220Cys, or p.Y220C was found when PS4-Points-Table.pdf was searched; the file contains general point definitions rather than variant rows.

Sources

Reasoning

The VCEP permits counting individuals who carry the variant and have an LFS-associated cancer, including meioses through unaffected obligate carriers, and assigns Supporting for 3-4 meioses, Moderate for 5-6, and Strong for at least 7 across more than one family. The available evidence contains a narrative statement that the variant co-segregated in at least three LFS families over two generations, but it does not identify the families, qualifying cancers, carrier status for each relative, or an exact count of meioses. Therefore no VCEP PP1 strength can be assigned. The governing LFS table was searched for c.659A>G, p.Tyr220Cys, and p.Y220C; it contains no variant-specific entry.

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 ≥ 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 available case evidence states that the variant co-segregated with disease in affected members of at least three LFS families genotyped over two generations, but does not provide an auditable meiosis count.
• The VCEP instruction requires counting individuals who both carry the variant and have a relevant cancer, with meioses countable through unaffected obligate carriers.
• Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf was searched for c.659A>G, p.Tyr220Cys, and p.Y220C; no variant-specific row was found.

Still missing

• Exact meioses and the carrier status of each relative are not documented.
• Affected relatives' cancers are not enumerated against the VCEP LFS cancer table.
• Family identifiers and source-level segregation records are not provided.
• No entry for c.659A>G, p.Tyr220Cys, or p.Y220C was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt.

Sources

Reasoning

The TP53 VCEP PP4 rule is based on low variant allele fraction observations from multigene panel testing: Moderate requires at least two independent observations with VAF 5-25%, while Supporting requires an observation with VAF 5-35%. No qualifying VAF observation for NM_000546.6:c.659A>G (p.Tyr220Cys) is available, so PP4 cannot be 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 available case evidence identifies the variant as a missense change but supplies no PP4 VAF observation, observation count, or multigene-panel testing context.

Still missing

• No multigene-panel observation with a reported VAF for this variant is available.
• The number of independent observations and whether any observation falls within VAF 5-25% are unknown.

Sources

Reasoning

The TP53 VCEP requires counts of unrelated females aged at least 60 years without cancer, all from one source, with exclusions for sarcoma diagnosed at or after age 61. The available case evidence contains gnomAD allele counts and homozygote counts but no qualifying age-, sex-, cancer-status-, relatedness-, or single-source carrier count. BS2 cannot be assessed.

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. ... 4-7 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. ... 2-3 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

• gnomAD v4.1 reports 0 homozygotes, but does not provide the VCEP-required qualifying count of unrelated cancer-free females aged at least 60 years from one source.

Still missing

• A qualifying single-source cohort count meeting the VCEP age, sex, cancer-status, relatedness, and sarcoma exclusions is required.

Sources

Reasoning

The TP53 VCEP defines BS4 as lack of segregation in affected family members diagnosed with LFS-associated cancers. No affected relative who tested negative for c.659A>G is documented, and the available segregation statement is positive rather than non-segregating. The criterion therefore cannot be evaluated as met or not met.

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 available case evidence reports positive co-segregation in affected members of LFS families and provides no negative segregation observations.
• No affected family member with a negative test for c.659A>G, p.Tyr220Cys, or p.Y220C is documented.

Still missing

• No affected family member with documented absence of the variant was identified.
• No family-level non-segregation analysis or qualifying cancer documentation was provided.

Sources