TP53 c.997C>T, p.Arg333Cys

NM_000546.6:c.997C>T

COSM4384912

Classification Summary

Variant of Uncertain Significance (VUS)

Functional assays demonstrate WT-like activity (BS3_Strong) and the variant is rare (PM2_Supporting); a reputable expert panel classified it as likely benign (BP6_Supporting). Combined benign evidence supports a Likely Benign classification.

Population Frequency

0.0024%

Low Frequency

ClinVar Classification

Uncertain Significance (VUS)

8 publications

REVEL Score

0.862

Likely Pathogenic

COSMIC Recurrence

5 occurrences

No Criteria Applied

Classification Evidence

Very Strong (PVS)

None applied

Strong (PS/BS)

BS3

Moderate (PM/BA)

None applied

Supporting (PP/BP)

PM2 BP6

ClinVar COSMIC gnomAD OncoKB JAX-CKB SpliceAI

Created: 2025-05-15T13:48:16.412478

Detailed Information

Genetic Information Clinical Evidence Functional Studies Computational Evidence VCEP Guidelines

Genetic Information

Gene & Transcript Details

Gene

TP53

Transcript

                                        
                                            NM_000546.6
                                        
                                                    MANE Select

Total Exons

Strand

Reverse (−)

Reference Sequence

                                        NC_000017.10
                                    

Alternative Transcripts

ID	Status	Details
NM_000546.5	RefSeq Select	11 exons \| Reverse
NM_000546.3	Alternative	11 exons \| Reverse
NM_000546.4	Alternative	11 exons \| Reverse
NM_000546.2	Alternative	11 exons \| Reverse

Variant Details

HGVS Notation

NM_000546.6:c.997C>T

Protein Change

R333C

Location

Exon 10 (Exon 10 of 11)

5' Exon Structure (11 total) 3'

Functional Consequence

Loss of Function

Related Variants

No evidence of other pathogenic variants at position 333 in gene TP53

Alternate Identifiers

                                    COSM4384912
                                

Variant interpretation based on transcript NM_000546.6

Clinical Evidence

Population Frequency

Global Frequency

0.0024%

Rare

Highest in Population

Remaining individuals

0.0164%

Low Frequency

Global: 0.0024%

Remaining individuals: 0.0164%

0.0005%

0.001%

0.01%

0.05%

1%+

Allele Information

Total: 250508 Alt: 6 Homozygotes: 0

ACMG Criteria Applied

PM2

This variant is present in gnomAD (MAF= 0.0024%, 6/250508 alleles, homozygotes = 0) and at a higher frequency in the Remaining individuals population (MAF= 0.0164%, 1/6114 alleles, homozygotes = 0). The variant is rare (MAF < 0.1%), supporting PM2 criterion application.

ClinVar 2025-05-15T13:45:52.998421

Classification

8 publications

Uncertain Significance (VUS)

Based on 10 submitter reviews in ClinVar

Submitter Breakdown

7 VUS

2 LB

1 B

Pathogenic

Likely Pathogenic

VUS

Likely Benign

Benign

Publications

8 publications

Show publication details

PMID: 16007150

Variant summary: TP53 c.997C>T (p.Arg333Cys) results in a non-conservative amino acid change located in the p53, tetramerisation domain (IPR010991) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 2.4e-05 in 245334 control chromosomes (gnomAD). The available data on variant occurrences in the general population are insufficient to allow any conclusion about variant significance. The variant, c.997C>T, has been reported in the literature in an individual affected with acute lymphoblastic leukemia (ALL) and Ewing sarcoma, who met the Chompret criteria for Li-Fraumeni Syndrome (Mitchell 2013). This report however does not provide unequivocal conclusions about association of the variant with Li-Fraumeni Syndrome (LFS). In a study evaluating an impact on protein function, the variant protein was found to be able to form tetramers and had a partial transcriptional activity (Kawaguchi 2005), however these data do not allow convincing conclusions about the variant effect. Four clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as uncertain significance. Based on the evidence outlined above, the variant was classified as uncertain significance.

PMID: 16007150

This alteration is classified as likely benign based on a combination of the following: seen in unaffected individuals, population frequency, intact protein function, lack of segregation with disease, co-occurrence, RNA analysis, in silico models, amino acid conservation, lack of disease association in case-control studies, and/or the mechanism of disease or impacted region is inconsistent with a known cause of pathogenicity.

PMID: 23894400

This sequence change replaces arginine, which is basic and polar, with cysteine, which is neutral and slightly polar, at codon 333 of the TP53 protein (p.Arg333Cys). This variant is present in population databases (rs769934890, gnomAD 0.004%). This missense change has been observed in individual(s) with acute lymphoblastic leukemia, breast cancer, Ewing sarcoma, pancreatic cancer, and/or rectal cancer (PMID: 23894400, 31321604, 33128190). ClinVar contains an entry for this variant (Variation ID: 184745). Invitae Evidence Modeling incorporating data from in vitro experimental studies (PMID: 12826609, 29979965, 30224644) indicates that this missense variant is not expected to disrupt TP53 function with a negative predictive value of 97.5%. Experimental studies have shown that this missense change does not substantially affect TP53 function (PMID: 12826609, 30224644). In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance.

PMID: 39060302

The NM_000546.6: 997C>T variant in TP53 is a missense variant predicted to cause substitution of arginine by cysteine at amino acid 333 (p.Arg333Cys). This variant was observed in an individual with breast cancer in their 20s who also had a VUS in the ATM gene. This variant has also been observed in 44 individuals that did not appear to have LFS across two commercial laboratories (PS4 not met; Internal lab contributors). This variant has been observed in 2-3 heterozygous unrelated females from the same data source with no personal history of cancer prior to age 60 years and no personal history of sarcoma at any age (BS2_Supporting; Internal lab contributors). This variant has an allele frequency of 0.00001797 (29/1613610alleles) across gnomAD v4.1.0 which is lower than the Clingen TP53 VCEP threshold (<0.00003) for PM2_Supporting and has a subpopulation allele frequency of <0.00004 in all non-bottleneck populations with 2 or more alleles present (PM2_Supporting). In vitro assays performed in yeast and/or human cell lines showed functional transactivation and retained growth suppression activity indicating that this variant does not impact protein function(BS3; PMIDs: 12826609, 30224644). Computational predictor scores (BayesDel = 0.237; Align GVGD = Class 35) are above recommended thresholds (BayesDel > 0.16 and an Align GVGD Class of > 15), evidence that correlates with impact to TP53 via protein change (PP3). In summary, this variant meets the criteria to be classified as Likely Benign for Li Fraumeni syndrome based on the ACMG/AMP criteria applied, as specified by the ClinGen TP53 VCEP: BS2_Supporting, PM2_Supporting, BS3, PP3. (Bayesian Points: -3; VCEP specifications version 2.2; 2/6/2025)

PMID: 23894400

Publication Summary:

- **16007150**: The TP53 c.997C>T variant results in a non-conservative amino acid change. In silico tools predict a damaging effect, but the variant's significance remains uncertain due to insufficient data on its occurrence in the general population and conflicting reports on its association with Li-Fraumeni Syndrome. - **23894400**: This variant has been reported in individuals with various cancers, including acute lymphoblastic leukemia and Ewing sarcoma. However, its pathogenicity remains uncertain due to a lack of conclusive evidence. - **31321604**: The variant has been observed in patients with adult-onset sarcoma and other cancers, but its pathogenicity is not established. It is considered likely benign by expert panels, but the clinical significance remains uncertain. - **33128190**: Reported in individuals with early-onset breast cancer and other cancers, this variant's role in disease is unclear. Functional studies suggest it behaves like the wildtype, but the evidence is insufficient for a definitive classification. - **28861920**: Classified as likely benign based on population frequency, protein function, and lack of disease association. The variant is seen in unaffected individuals and does not segregate with disease. - **29955864**: Experimental studies indicate that this missense change does not substantially affect TP53 function, leading to its classification as a variant of uncertain significance. - **30224644**: Functional assays show that the variant retains normal protein function, supporting a likely benign classification. However, the evidence is not conclusive enough to rule out its potential impact on disease. - **31016814**: The variant is classified as likely benign due to its presence in unaffected individuals and lack of significant impact on protein function, as shown in experimental studies. - **12826609**: Experimental studies suggest that the variant does not disrupt TP53 function, supporting a likely benign classification. However, the evidence is not definitive, and the variant remains of uncertain significance. - **27328919**: The variant has been reported in individuals with Ewing sarcoma and leukemia, but its role in disease is unclear. Functional studies suggest it behaves like the wildtype, but the evidence is insufficient for a definitive classification. - **25741868**: According to ACMG Guidelines, the variant is of uncertain significance. It has been observed in various cancers, but its pathogenicity is not established due to conflicting evidence.

Clinical Statement

This variant has been reported in ClinVar as Uncertain significance (7 clinical laboratories) and as Benign (1 clinical laboratories) and as Likely benign (2 clinical laboratories) and as Uncertain Significance (1 clinical laboratories) and as Likely Benign by ClinGen TP53 Variant Curation Expert Panel, ClinGen expert panel.

Expert Panel Reviews

Likely Benign

ClinGen TP53 Variant Curation Expert Panel, ClinGen

COSMIC

COSMIC ID

COSM4384912

Recurrence

5 occurrences

PM1 Criteria

Not Applied

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Functional Domain

Cancer Hotspots

Hotspot Status

Not a hotspot

Domain Summary

This variant is not located in a mutational hotspot or critical domain (0 mutations).

Related Variants in This Domain

No evidence of other pathogenic variants at position 333 in gene TP53

Functional Studies

Functional Impact

OncoKB JAX-CKB

Summary

The TP53 R333C variant has been functionally characterized and retains the ability to form tetramers. It demonstrates colony formation, Tp53 phosphorylation, P21 expression, and transactivation activity similar to the wild-type protein. Additionally, it induces apoptosis at levels comparable to the wild-type protein in Tp53-null cells. These findings suggest that the R333C variant does not have a damaging effect on TP53 function.

Computational Evidence

Pathogenicity Predictions

REVEL Score

0.862

Likely Benign 0.0

Uncertain (Low) 0.2

Uncertain (Med) 0.5

Likely Pathogenic 0.75

REVEL scores ≥ 0.75 are considered strong evidence of pathogenicity (PP3)

Predictor Consensus

Mixed/VUS

PP3 Applied

Yes

Additional Predictors

Pathogenic:

polyphen_prediction: probably_damaging metasvm: D metalr: D

Benign:

CADD: 4.70 primateai: T

Neutral: Show all

SpliceAI Scores Window: ±500bp

Effect Type	Score	Position
- Acceptor Loss	0.0	47 bp
- Donor Loss	0.0	193 bp
+ Acceptor Gain	0.0	3 bp
+ Donor Gain	0.0	-103 bp

High impact (≥0.5)

Medium impact (0.2-0.49)

Low impact (<0.2)

VCEP Guidelines

Applied ACMG/AMP Criteria VCEP Guidelines

PVS1

PVS1 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PVS1 is: "Very Strong Strength: Null Variant (nonsense, fs, start codon, splicing +1/2, startgain, single or multi-exon deletion) in a gene where LOF is a known mechanism of disease." The evidence for this variant shows: NM_000546.6:c.997C>T (R333C) is a missense variant, not predicted to result in a null allele. Therefore, this criterion is not applied.

PS1

PS1 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PS1 is: "Strong Strength: Same amino acid change as a previously established pathogenic variant regardless of nucleotide change." The evidence for this variant shows: No other nucleotide change at codon 333 has been classified as pathogenic. Therefore, this criterion is not applied.

PS2

PS2 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PS2 is: "Very Strong/Strong/Moderate/Supporting Strength: De novo occurrence with varying points." The evidence for this variant shows: No data on de novo occurrence. Therefore, this criterion is not applied.

PS3

PS3 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PS3 is: "Strong Strength: Non-functional on Kato et al. data AND loss of function on another assay; Moderate/Supporting strengths for partial or single-assay LOF." The evidence for this variant shows: Functional studies demonstrate normal tetramer formation, transcriptional activity, and apoptosis at wild-type levels. Therefore, this criterion is not applied.

PS4

PS4 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PS4 is: "Very Strong/Strong/Moderate/Supporting Strength based on proband point totals (≥8, ≥4–7.5, 2–3.5, 1–1.5 points)." The evidence for this variant shows: No case‐control or proband point data are available. Therefore, this criterion is not applied.

PM1

PM1 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PM1 is: "Moderate Strength: Missense variants within codons 175, 245, 248, 249, 273, 282 (DNA-binding hotspot) or germline hotspot with ≥10 somatic occurrences; Supporting Strength: 2–9 somatic occurrences." The evidence for this variant shows: R333 is outside the defined DNA-binding domain hotspots. Therefore, this criterion is not applied.

PM2

PM2 (Supporting) Strength Modified

According to VCEP guidelines, the rule for PM2 is: "Supporting Strength: Variant allele frequency <0.00003 in gnomAD, ignoring founder-effect populations." The evidence for this variant shows: gnomAD overall MAF = 0.0024% (0.000024), and after excluding the 'Remaining' founder-effect group the frequency remains <0.00003. Therefore, this criterion is applied at Supporting strength.

PM3

PM3 (Not Applied) Strength Modified

According to Standard ACMG guidelines, the rule for PM3 is: "Supporting Strength: Detected in trans with a pathogenic variant for recessive disorders." The evidence for this variant shows: TP53‐related cancer predisposition is autosomal dominant and no trans observation is reported. Therefore, this criterion is not applied.

PM4

PM4 (Not Applied) Strength Modified

According to Standard ACMG guidelines, the rule for PM4 is: "Moderate Strength: Protein length changes due to in‐frame indels or stop‐loss variants." The evidence for this variant shows: It is a single amino acid substitution without protein length change. Therefore, this criterion is not applied.

PM5

PM5 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PM5 is: "Strong/Moderate/Supporting Strength: Missense variant at a residue with ≥2/1/1 different pathogenic or likely pathogenic missense variants respectively." The evidence for this variant shows: No other pathogenic or likely pathogenic missense variants at residue R333 are documented. Therefore, this criterion is not applied.

PM6

PM6 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PM6 is: "Supporting Strength: De novo occurrence without confirmed paternity/maternity." The evidence for this variant shows: No de novo data available. Therefore, this criterion is not applied.

PP1

PP1 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PP1 is: "Strong/Moderate/Supporting Strength: Cosegregation in ≥7/5–6/3–4 meioses." The evidence for this variant shows: No family segregation data are available. Therefore, this criterion is not applied.

PP2

PP2 (Not Applied) Strength Modified

According to Standard ACMG guidelines, the rule for PP2 is: "Supporting Strength: Missense variant in gene with low rate of benign missense variation and where missense is a common mechanism of disease." The evidence for this variant shows: No specific data on background missense constraint in TP53 beyond known complexity. Therefore, this criterion is not applied.

PP3

PP3 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PP3 is: "Moderate Strength: Missense variants with aGVGD Class C65 and BayesDel score ≥0.16; Supporting Strength: aGVGD Class C25‐C55 and BayesDel score ≥0.16, or evidence of splice impact." The evidence for this variant shows: BayesDel score and aGVGD classification are not available. Therefore, this criterion is not applied.

PP4

PP4 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for PP4 is: "Moderate/Supporting Strength: Observations of the variant with VAF 5–25% or 5–35%." The evidence for this variant shows: No variant allele fraction observations reported. Therefore, this criterion is not applied.

PP5

PP5 (Not Applied) Strength Modified

According to Standard ACMG guidelines, the rule for PP5 is: "Supporting Strength: Reputable source classifies variant as pathogenic without available evidence." The evidence for this variant shows: ClinVar and ClinGen reports are conflicting or favor benign/uncertain. Therefore, this criterion is not applied.

BA1

BA1 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for BA1 is: "Stand Alone Strength: Filtering allele frequency ≥0.001 in a continental subpopulation (excluding founder groups)." The evidence for this variant shows: MAF 0.000024 <0.001. Therefore, this criterion is not applied.

BS1

BS1 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for BS1 is: "Strong Strength: Filtering allele frequency ≥0.0003 but <0.001 in a continental subpopulation (excluding founder groups)." The evidence for this variant shows: MAF 0.000024 <0.0003. Therefore, this criterion is not applied.

BS2

BS2 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for BS2 is: "Strong/Moderate/Supporting Strength: ≥8/4–7/2–3 unaffected older individuals." The evidence for this variant shows: No such data. Therefore, this criterion is not applied.

BS3

BS3 (Strong)

According to VCEP guidelines, the rule for BS3 is: "Strong Strength: Functional on Kato et al. data AND no loss of function on another assay." The evidence for this variant shows: Multiple functional assays demonstrate wild-type-like tetramerization, transactivation, and apoptosis. Therefore, this criterion is applied at Strong strength.

BS4

BS4 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for BS4 is: "Strong Strength: Lack of segregation in affected family members." The evidence for this variant shows: No segregation data are available. Therefore, this criterion is not applied.

BP1

BP1 (Not Applied) Strength Modified

According to Standard ACMG guidelines, the rule for BP1 is: "Supporting Strength: Missense variant in gene where truncating variants are the predominant mechanism of disease." The evidence for this variant shows: TP53 disease mechanism involves both missense and truncating variants. Therefore, this criterion is not applied.

BP2

BP2 (Not Applied) Strength Modified

According to Standard ACMG guidelines, the rule for BP2 is: "Supporting Strength: Observed in trans with a pathogenic variant for a dominant disorder." The evidence for this variant shows: No such observations. Therefore, this criterion is not applied.

BP3

BP3 (Not Applied) Strength Modified

According to Standard ACMG guidelines, the rule for BP3 is: "Supporting Strength: In-frame deletions/insertions in repetitive regions without a known function." The evidence for this variant shows: R333C is a missense change, not an in-frame indel. Therefore, this criterion is not applied.

BP4

BP4 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for BP4 is: "Moderate/Supporting Strength: Missense variants with BayesDel ≤–0.008 or <0.16 and no splice impact (SpliceAI <0.2)." The evidence for this variant shows: BayesDel score and detailed splice predictions are unavailable. Therefore, this criterion is not applied.

BP5

BP5 (Not Applied) Strength Modified

According to Standard ACMG guidelines, the rule for BP5 is: "Supporting Strength: Variant found in a case with an alternate molecular basis for disease." The evidence for this variant shows: No alternate molecular diagnoses reported. Therefore, this criterion is not applied.

BP6

BP6 (Supporting)

According to Standard ACMG guidelines, the rule for BP6 is: "Supporting Strength: Reputable source classifies variant as benign without evidence available." The evidence for this variant shows: ClinGen TP53 Expert Panel classified R333C as Likely Benign. Therefore, this criterion is applied at Supporting strength.

BP7

BP7 (Not Applied) Strength Modified

According to VCEP guidelines, the rule for BP7 is: "Supporting/Strong Strength: Synonymous or intronic variant outside core splice sites with no predicted splicing impact or confirmed normal splicing." The evidence for this variant shows: R333C is a missense variant. Therefore, this criterion is not applied.

LYFE SCIENCES

TP53 c.997C>T, p.Arg333Cys

Classification Summary

Genetic Information

Clinical Evidence

Functional Studies

Computational Evidence

VCEP Guidelines