TP53 c.818G>A, p.Arg273His

Reasoning

The ClinGen TP53 VCEP Functional-worksheet.xlsx, Supplementary Table S3, contains the exact amino-acid row R273H corresponding to NM_000546.6:c.818G>A / p.Arg273His. The row assigns Kato class Non-functional, Funk LOF, Giacomelli LOF, Kotler LOF, Kawaguchi NA, and the preliminary functional code PS3. This is the governing pre-assigned code. It also directly satisfies the VCEP PS3 strong rule: non-functional on Kato and LOF by the majority of other eligible assays (three of three available other assays: Funk, Giacomelli, and Kotler). No splice-prediction substitution or non-VCEP calibration was used.

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 R273H: Kato class = Non-functional; Funk class = LOF; Giacomelli class = LOF; Kotler class = LOF; Kawaguchi class = NA; preliminary functional code = PS3.
• Supplementary Table S3 identifies the assay sources as Kato et al. (PMID:12826609), Funk et al. (PMID:39774325), Giacomelli et al. (PMID:30224644), Kotler et al. (PMID:29979965), and Kawaguchi et al. (PMID:16007150).

Still missing

• The VCEP Flowchart-for-application-of-functional-rule-codes.pdf text was searched for c.818G>A, p.Arg273His, and p.R273H; it contains no variant-specific entry. The governing variant-specific assignment is present in Functional-worksheet.xlsx.

Sources

Reasoning

The variant is a missense change at TP53 codon 273 (p.Arg273His). The TP53 VCEP specifically authorizes PM1 at Moderate strength for missense variants at codon 273, and separately names cancerhotspots.org as an eligible hotspot route. The case evidence also records TP53 R273 as a statistically significant Cancer Hotspots hotspot. The codon-specific VCEP route is sufficient; no unlisted domain or generic hotspot rule was used.

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

• TP53 VCEP specification v2.4, PM1 Moderate rule: missense variants at codons 175, 245, 248, 249, 273, or 282 qualify; the assessed variant is p.Arg273His at codon 273.
• Cancer Hotspots result for TP53 R273: the variant lies in a statistically significant hotspot; this is an independently named VCEP route, although the codon-specific route already establishes PM1.

Sources

Reasoning

The TP53 VCEP applies PM2 at Supporting strength when total allele frequency is <0.00003 and, when multiple alleles occur within an ancestry group, that group's frequency is <0.00004, ignoring founder-effect groups. gnomAD v4.1 reports total AF 1.11528e-05 (18/1,613,942), zero homozygotes, and eligible non-founder ancestry frequencies below 0.00004; for example, European non-Finnish AF is 1.18649e-05 from 14/1,179,946 alleles and African/African American AF is 1.33312e-05 from 1/75,012 alleles. Ashkenazi Jewish is ignored under the rule. PM2 Supporting is met.

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: total AF = 1.11528e-05 from 18/1,613,942 alleles; homozygotes = 0.
• gnomAD v4.1 eligible ancestry examples: European non-Finnish AF = 1.18649e-05 from 14/1,179,946 alleles; African/African American AF = 1.33312e-05 from 1/75,012 alleles; both are below 0.00004.
• gnomAD v4.1 Ashkenazi Jewish AF = 6.7563e-05 from 2/29,602 alleles; this founder-effect ancestry group is ignored under the VCEP PM2 rule.
• ClinGen TP53 VCEP v2.4 CSPEC PM2 criterion entry specifies Supporting strength, total AF <0.00003, and <0.00004 within an ancestry group when multiple alleles are present, with founder-effect groups ignored.

Sources

Reasoning

The variant is a missense substitution, NM_000546.6:c.818G>A (p.Arg273His/p.R273H). The TP53 VCEP Supplementary Table S2 directly pre-assigns PP3 for the exact row c.818G>A, p.Arg273His, BayesDel class C25, BayesDel score 0.525881, PP3, with no strength suffix; this governing lookup assignment is applied directly. The VCEP flowchart also places missense variants with BayesDel >= 0.16 and aGVGD C25-C55 at PP3, although the pre-assigned table code is the controlling result. SpliceAI returned no score, so no splice prediction is used or double-counted for PP3.

VCEP rule

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

Evidence considered

• PP3-BP4-codes.xlsx, Supplementary Table S2, matching row: c.818G>A | p.Arg273His | Class C25 | BayesDel 0.525881 | PP3 | 0.02.
• Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf, missense branch: BayesDel >= 0.16 with aGVGD C25-C55 gives PP3.
• Case SpliceAI result for NM_000546.6:c.818G>A: no SpliceAI score available; max delta is null and the result is missing data, not a zero score.

Still missing

• No c.818G>A, p.Arg273His, or p.R273H variant-specific row was found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf; that file provides the generic flowchart only.
• No c.818G>A, p.Arg273His, or p.R273H entry was found in TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx; the variant is missense rather than a single amino-acid in-frame deletion.
• SpliceAI returned no score for this variant; this missing splice result is not used as evidence.

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 TP53 VCEP CSPEC entry for PP5 states applicability: Not Applicable for this VCEP and says the criterion is not for use.
• ClinVar expert panel classification

Sources

Reasoning

The TP53 VCEP BA1 threshold is FAF >=0.001 in an eligible continental subpopulation with >=2,000 alleles tested and at least 2 variant alleles. gnomAD v4.1 reports total AF 1.11528e-05 (18/1,613,942), zero homozygotes, and grpmax FAF 6.88e-06; the highest listed subpopulation is Ashkenazi Jewish at 6.7563e-05 (2/29,602), but Ashkenazi Jewish is explicitly excluded by the VCEP rule. The eligible population frequencies therefore do 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. Caution should be exerted if the majority of alleles have a variant allele fraction ("allele balance" in gnomAD) below 0.35.

Evidence considered

• gnomAD v4.1: total AF 1.11528e-05 from 18/1,613,942 alleles; homozygotes = 0; grpmax FAF = 6.88e-06.
• gnomAD v4.1 highest listed subpopulation: Ashkenazi Jewish, AF 6.7563e-05 from 2/29,602 alleles; this ancestry group is excluded by the VCEP BA1 rule.
• ClinGen TP53 VCEP v2.4 CSPEC BA1 criterion entry: FAF >=0.001 in an eligible single-ancestry continental subpopulation, with >=2,000 alleles tested and at least 2 alleles present.

Sources

Reasoning

The TP53 VCEP BS1 threshold is FAF >=0.0003 but <0.001 in an eligible continental subpopulation with >=2,000 alleles tested and at least 2 variant alleles. gnomAD v4.1 reports grpmax FAF 6.88e-06, well below 0.0003; the highest listed Ashkenazi Jewish frequency is excluded by the rule, and no eligible ancestry group reaches the BS1 threshold. 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. Caution should be exerted if the majority of alleles have a variant allele fraction ( “allele balance” in gnomAD) below 0.35. To set the strong benign FAF cutoff, we used a Whiffin-Ware calculation using prevalence of 1 in 5,000 (Lalloo, et al., 2006 PMID: 16644204). Genetic and allelic heterogeneity were set at 100% and penetrance at 30%. In general, the most recent version of gnomAD should be used when available; however, other population databases or earlier versions of gnomAD may be utilized if they are able to provide information the curator deems necessary for optimal variant classification (e.g, they would provide superior information for a particular variant type; have a larger sample size; or better representation for certain subpopulations, etc.)

Evidence considered

• gnomAD v4.1: grpmax FAF = 6.88e-06; total AF = 1.11528e-05 from 18/1,613,942 alleles; homozygotes = 0.
• gnomAD v4.1 highest listed subpopulation: Ashkenazi Jewish, AF 6.7563e-05 from 2/29,602 alleles; this ancestry group is excluded by the VCEP BS1 rule.
• ClinGen TP53 VCEP v2.4 CSPEC BS1 criterion entry: FAF >=0.0003 but <0.001 in an eligible single-ancestry continental subpopulation, with >=2,000 alleles tested and at least 2 alleles present.

Sources

Reasoning

The ClinGen TP53 VCEP Functional-worksheet.xlsx, Supplementary Table S3, contains the exact row R273H corresponding to NM_000546.6:c.818G>A / p.Arg273His and assigns PS3, not BS3. The row records Kato Non-functional and LOF in the Funk, Giacomelli, and Kotler assays, so the variant is neither functional nor partially functional on Kato data and has clear LOF in the majority of available eligible assays. Therefore it fails both VCEP BS3 pathways.

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 R273H: Kato class = Non-functional; Funk class = LOF; Giacomelli class = LOF; Kotler class = LOF; Kawaguchi class = NA; preliminary functional code = PS3 rather than BS3.
• Supplementary Table S3 identifies the assay sources as Kato et al. (PMID:12826609), Funk et al. (PMID:39774325), Giacomelli et al. (PMID:30224644), Kotler et al. (PMID:29979965), and Kawaguchi et al. (PMID:16007150).

Still missing

• The VCEP Flowchart-for-application-of-functional-rule-codes.pdf text was searched for c.818G>A, p.Arg273His, and p.R273H; it contains no variant-specific entry. The governing variant-specific assignment is present in Functional-worksheet.xlsx.

Sources

Reasoning

BP4 is applicable to missense variants only when the VCEP's benign computational conditions are met together. The exact VCEP Supplementary Table S2 row is pre-assigned PP3, not BP4, and reports BayesDel 0.525881. This is not below 0.16 and therefore fails the missense BP4 condition; additionally, the required SpliceAI < 0.2 result is unavailable because SpliceAI returned no score. The non-calibrated Pangolin values are not used. The criterion is therefore not met, and no prediction is double-counted with PP3.

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.818G>A | p.Arg273His | Class C25 | BayesDel 0.525881 | PP3 | 0.02; no BP4 code is assigned.
• TP53 VCEP BP4 missense rule requires BayesDel < 0.16 and > -0.008, except C65, together with no predicted splicing (SpliceAI < 0.2); the observed BayesDel value fails the first condition.
• Case SpliceAI result for NM_000546.6:c.818G>A: no score available; max delta is null, and Pangolin is not calibrated for PP3/BP4 here.

Still missing

• No c.818G>A, p.Arg273His, or p.R273H variant-specific row was found in Flowchart-for-application-of-PP3%2C-BP4%2C-and-BP7.pdf; that file provides the generic flowchart only.
• The matching PP3-BP4-codes.xlsx row is present and assigns PP3, but does not pre-assign BP4.
• No c.818G>A, p.Arg273His, or p.R273H entry was found in TP53-single-amino-acid-deletions-BayesDel-scores-(1).xlsx; the variant is missense rather than a single amino-acid in-frame deletion.
• SpliceAI returned no score; therefore the required no-splicing-impact condition cannot be satisfied from a splice score.

Sources

Reasoning

The variant is TP53 p.Arg273His, but the available evidence does not identify a previously established pathogenic TP53 variant producing the same amino-acid change through a different nucleotide change. The same-variant expert-panel classification cannot itself establish the required alternate-nucleotide comparator, and the dedicated same-residue comparator collection returned no eligible candidates.

VCEP rule

Can be applied to variants asserted as Pathogenic following the TP53 VCEP’s specifications.

Evidence considered

• The assessed variant is NM_000546.6:c.818G>A, NP_000537.3:p.(Arg273His).
• The dedicated same-residue comparator collection reported same_residue_candidates = 0 and no useful candidates; the exact variant's own classification was not used as a PS1 comparator.

Still missing

• An eligible alternate nucleotide change producing TP53 p.Arg273His, asserted Pathogenic or Likely Pathogenic under the TP53 VCEP specifications, is not documented.

Sources

Reasoning

The TP53 VCEP assigns PS2 strength by de novo evidence points: 1 point is Supporting, 2-3 points Moderate, 4-7 points Strong, and >=8 points Very Strong. The exact variant is reported in a child with multiple LFS-associated malignancies and no documented family history, and the authors state that this strongly suggests a de novo mutation. However, parental testing was not reported; the family declined counseling, and absence of a family history does not establish confirmed de novo inheritance or permit assigning a VCEP PS2 point value. The governing table was searched for c.818G>A, p.Arg273His, and p.R273H; no variant-specific entry was found.

VCEP rule

1 point

Evidence considered

• Khayat and Johnston, PMID:15390294: germline TP53 codon 273 CGT>CAT was identified in a child with rhabdomyosarcoma, later mandibular osteosarcoma and adrenocortical carcinoma; the pedigree documented no family history of malignancy.
• Khayat and Johnston, PMID:15390294, Discussion: the authors state that absence of a compatible positive family history strongly suggests a de novo mutation, but they do not report parental testing and state that family counseling was declined.
• Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt was searched for c.818G>A, p.Arg273His, and p.R273H; no variant-specific entry was found.

Still missing

• Confirmed maternity and paternity and parental testing for TP53 c.818G>A are not reported.
• No entry for c.818G>A, p.Arg273His, or p.R273H was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt.

Sources

Reasoning

The TP53 VCEP PS4 table assigns 0.5 point for Revised Chompret criteria and 1 point for Classic LFS criteria, with Supporting requiring 1-1.5 points. PMID:15390294 verifies the exact germline codon 273 Arg>His variant (c.818G>A/p.R273H) in a child with rhabdomyosarcoma followed six years later by synchronous mandibular osteosarcoma and adrenocortical carcinoma, and PMID:16401470 reports the exact variant in a classic Li-Fraumeni syndrome registry family. However, the available extracts do not document the complete VCEP-defined clinical criteria for an assignable proband point, nor do they provide exact-variant case-control counts or a matched-ancestry control frequency from which an odds ratio and 95% confidence interval could be calculated. PS4 therefore cannot be evaluated reliably from the available evidence.

VCEP rule

PS4_Supporting = 1-1.5 points

Evidence considered

• PS4-Points-Table.pdf, rows Clinical criteria | Revised Chompret | 0.5, Clinical criteria | Classic LFS criteria | 1, and PS4 code strength based on total points: PS4_Supporting = 1-1.5 points.
• PMID:15390294: the exact codon 273 CGT>CAT (Arg>His), corresponding to c.818G>A/p.R273H, was found in a child with rhabdomyosarcoma followed six years later by synchronous mandibular osteosarcoma and adrenocortical carcinoma; the report does not state a complete Revised Chompret or Classic LFS point assignment.
• PMID:16401470, Table 3, Family 5: the exact codon 273 G>A, CGT (Arg)>CAT (His), missense variant was reported in a classic Li-Fraumeni syndrome registry cohort, but the available extraction does not provide a separately assignable PS4 proband point.
• The reviewed evidence does not report exact-variant case-control counts or matched-ancestry control frequency; an odds ratio and 95% confidence interval could not be computed.

Still missing

• The reviewed reports do not provide all clinical facts needed to confirm Revised Chompret or Classic LFS criteria for a specific proband and assign the VCEP PS4 point.
• No exact-variant case-control counts or matched-ancestry control frequency, odds ratio, or 95% confidence interval were available.

Sources

Reasoning

The variant is a missense change at residue 273, but the available case evidence does not document a different TP53 missense variant at residue 273 that was previously determined to be Pathogenic or Likely Pathogenic according to the TP53 VCEP specifications. The dedicated comparator collection returned no same-residue candidates, so the required one- or two-comparator threshold cannot be evaluated.

VCEP rule

Missense variant at an amino acid residue where ≥2 different missense variants previously determined to be pathogenic according to the TP53 VCEP’s specifications have been seen before.

Evidence considered

• The assessed variant is TP53 p.Arg273His at residue 273.
• The dedicated PM5 comparator collection reported total_candidates_seen = 9 but same_residue_candidates = 0; it explicitly provided no usable comparator for PM5 adjudication.

Still missing

• A different missense variant at residue 273 with a documented TP53 VCEP-based Pathogenic or Likely Pathogenic determination is not available in the assessed evidence.

Sources

Reasoning

The TP53 VCEP requires cosegregation in 3-4 meioses for Supporting, 5-6 meioses for Moderate, or at least 7 meioses across more than one family for Strong. PMID:16401470 reports the exact codon 273 Arg>His variant in the father of an affected family member in Family 5, but it does not provide enough linked affected/unaffected genotype data to establish any number of cosegregating meioses. The governing table was searched for c.818G>A, p.Arg273His, and p.R273H; no variant-specific entry was found.

VCEP rule

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

Evidence considered

• Wong et al., PMID:16401470, Table 3, Family 5: the exact TP53 codon 273 G>A / CGT (Arg)>CAT (His) variant was detected in the father of the tested affected family member.
• PMID:16401470 does not report sufficient family-member genotype/phenotype data or a cosegregating meioses count for applying a PP1 strength.
• Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt was searched for c.818G>A, p.Arg273His, and p.R273H; no variant-specific entry was found.

Still missing

• The available report does not establish the phenotype and genotype of each relevant family member or a count of cosegregating meioses.
• No entry for c.818G>A, p.Arg273His, or p.R273H was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt.

Sources

Reasoning

The TP53 VCEP PP4 rule requires observation of the variant with VAF 5-35%, or at least two independent observations with VAF 5-25% for Moderate. The available evidence documents germline clinical observations but provides no variant allele fraction, 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 available clinical reports identify germline TP53 c.818G>A/p.R273H but do not report VAF.

Still missing

• Variant allele fraction is not available for the reported observations.

Sources

Reasoning

The TP53 VCEP BS2 rule requires counts of unrelated females aged at least 60 years without cancer, all from one source, with sarcoma exclusions applied. The available gnomAD evidence reports allele counts, ancestry, and homozygote counts but does not identify individual sex, age, cancer status, relatedness, source-level carrier counts, or sarcoma history. Therefore the required BS2 evidence cannot be evaluated.

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

• Available gnomAD v4.1 data report 18 variant alleles, zero homozygotes, and ancestry-level frequencies, but not the individual age, sex, cancer status, relatedness, or sarcoma information required by BS2.
• ClinGen TP53 VCEP v2.4 CSPEC BS2 criterion entry requires unrelated females aged at least 60 years without cancer, all from a single source; individuals with sarcoma diagnosed at >=61 years are excluded.

Still missing

• A single-source count of unrelated female carriers aged >=60 years without cancer, including the VCEP sarcoma exclusion, is unavailable.

Sources

Reasoning

The TP53 VCEP defines BS4 as lack of segregation in affected family members diagnosed with LFS-associated cancers listed in its PS2/PP1 cancer table. The available reports do not document a tested affected relative who lacks the variant: PMID:15390294 describes no family history and no reported parental testing, while PMID:16401470 reports a father carrying the variant but does not provide the genotype of affected relatives sufficient to demonstrate non-segregation. The governing table was searched for c.818G>A, p.Arg273His, and p.R273H; no variant-specific entry was found.

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 Version 2.4 BS4 rule: lack of segregation must be evaluated in affected family members diagnosed with LFS-associated cancers described in the PS2/PP1 cancer table.
• Khayat and Johnston, PMID:15390294: an extensive pedigree found no documented family history of other malignancies, but this is not a tested affected-relative non-segregation observation.
• Wong et al., PMID:16401470, Table 3, Family 5: the variant was found in the father of the tested affected family member; the report does not establish a qualifying affected relative lacking the variant.
• Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt was searched for c.818G>A, p.Arg273His, and p.R273H; no variant-specific entry was found.

Still missing

• Genotypes and LFS-associated cancer phenotypes for relevant affected family members are not sufficiently reported to demonstrate lack of segregation.
• No entry for c.818G>A, p.Arg273His, or p.R273H was found in Table-of-LFS-Cancers-and-Points-for-PS2-and-PP1-Code-Application.pdf.txt.

Sources