Variant* | MAF† | Bioinformatic predictions‡ | Functional status of the variants assessed in yeast and human cancer cell lines | ClinVar classification§ | Blood p53 functional assay¶ in p53 score, mRNA% | p53 functional assay in EBV cell lines score; mRNA% | Interpretation according to the assay | |||||
Align GVGD | PolyPhen -2 | SIFT | Bayes Del | Kato assay** | Kotler assay†† | Giacomelli assay‡‡ | ||||||
Pathogenic/likely pathogenic variants | ||||||||||||
c.524G>A p.(Arg175His) | 0.000004 | C25 | P | D | 0.5462 | Non-functional | Non-functional |
LOF
DNE | Pathogenic | Ind. 52: 3.2; 96% |
2.9; 93% 4.1; 86% | Functionally abnormal |
c.542G>A p.(Arg181His) | 0.000014 | C25 | D | D | 0.2584 | Partially functional | Functional | Unclassified | Pathogenic/likely pathogenic | Ind. 53: 3.8; 81% | Functionally abnormal | |
c.844C>T p.(Arg282Trp) | 0.000004 | C65 | D | D | 0.5418 | Non-functional | Non-functional |
LOF
DNE | Pathogenic/likely pathogenic | Ind. 54: 4.8; 102% Ind. 55: 5.0; 98% |
2.0; 104% 2.7; 116% 4.6; 76% | Functionally abnormal |
c.1010G>A p.(Arg337His) | 0.000012 | C25 | D | D | 0.1777 | Partially functional | NA | Not LOF Not DNE | Pathogenic | Ind. 56: 5.4; 101% |
5.8; 81% 6.0; 85% 6.5; 81% 6.9; 101% | Functionally abnormal |
c.375G>A p.? Splicing Alteration | NR | / | / | / | / | / | Functional | NA | Pathogenic | Ind. 57: 3.1; 61% | 5.5; 61%§§ | Functionally abnormal |
c.723del p.(Cys242Alafs*5) | NR | / | / | / | / | NA | NA | NA | NR | Ind. 58: S1: 7.1; 56% S2: 6.0; 52% | 4.2; 52%§§ | Functionally abnormal |
c.770del p.(Leu257Argfs*88) | NR | / | / | / | / | NA | Non-functional | NA | NR | Ind. 59: 4.7; 62% | Functionally abnormal | |
Benign variant | ||||||||||||
c.704A>G p.(Asn235Ser) | 0.01945 | C0 | B | T | −0.0342 | Functional | Functional | Not LOF Not DNE | Benign | Ind. 60: 12.9; 118% | Functionally normal | |
Unclassified rare variants | ||||||||||||
c.215C>A p.(Pro72His) | NR | C0 | P | D | −0.1017 | Functional | NA | Not LOF Not DNE | Uncertain significance | Ind. 61: 6.1; 86% | 7.5; 107%§§ | Functionally abnormal |
c.314G>A p.(Gly105Asp) | NR | C65 | D | D | 0.5683 | Partially functional | Functional |
LOF
DNE | Conflict. int¶¶: Likely pathogenic/ uncertain significance | Ind. 62: 2.9; 82% |
2.1; 85% 3.6; 70% | Functionally abnormal |
c.329G>A p.(Arg110His) | 0.000046 | C0 | B | T | 0.0728 | Partially functional | Functional | Not LOF Not DNE | Uncertain significance | Ind. 63: 4.1; 83% |
3.7; 92% 4.7; 91%§§ 5.2; 94% | Functionally abnormal |
c.402T>G p.(Phe134Leu) | NR | C0 | D | D | 0.3166 | Non-functional | Non-functional |
LOF
DNE | NR | Ind. 64: 3.5; 78% | 3.5; 94%§§ | Functionally abnormal |
c.472C>T p.(Arg158Cys) | 0.000008 | C65 | D | D | 0.5145 | Partially functional | Functional | Unclassified | Conflict. int: Likely pathogenic/uncertain significance | Ind. 65: 3.2; 86% | Functionally abnormal | |
c.572C>G p.(Pro191Arg) | 0.000012 | C35 | P | D | 0.0942 | Functional | Functional | Not LOF Not DNE | Uncertain significance | Ind. 66: 17.1; 90% | Functionally normal | |
c.833C>G p.(Pro278Arg) | NR | C65 | D | D | 0.6078 | Non-functional | Non-functional |
LOF
DNE | Uncertain significance | Ind. 67: 5.1; 78% | 3.9; 108%§§ | Functionally abnormal |
c.847C>T p.(Arg283Cys) | 0.000074 | C55 | B | D | 0.3309 | Functional | Functional | Not LOF Not DNE | Uncertain significance | Ind. 68: 5.3; 79% Ind. 69: 6.4; 88% |
3.1; 96% 2.5; 89%§§ 5.6; 86% 5.4; 88% | Functionally abnormal |
c.1043T>C p.(Leu348Ser) | NR | C65 | D | D | 0.5439 | Non-functional | NA | Not LOF Not DNE | NR | Ind. 70: S1: 5.8; 89% S2 : 6.1; 90% | Functionally abnormal | |
c.1054G>T p.(Asp352Tyr) | NR | C35 | D | D | 0.0844 | Functional | NA | Not LOF Not DNE | Uncertain significance | Ind. 71***: S1: 4.0; 101% S2: 5.0; 106% | 2.3; 90% | Functionally abnormal |
c.323_325del p.(Gly108_Phe109delinsVal) | NR | / | / | / | / | / | / | / | NR | Ind. 72: 3.8; 94% | Functionally abnormal | |
c.393_395del p.(Asn131del) | NR | / | / | / | / | NA | Non-functional | NA | Uncertain significance | Ind. 73: S1: 2.6; 80% S2: 4.8; 99% | Functionally abnormal | |
c.792_794del p.(Leu265del) | NR | / | / | / | / | / | NA | NA | NR | Ind. 74: 3.4; 95% | Functionally abnormal | |
c.-117G>T p.(=) (5'UTR) | NR | / | / | / | / | NA | NA | NA | NR | Ind. 75: 20.5; 100% | Functionally normal | |
Polymorphism | ||||||||||||
c.*1175A>C p.(=) (Homozygous) | 0.01189 | / | / | / | / | NA | NA | NA | Conflict. int: likely benign/uncertain significance | Ind. 76: S1: 5.5; 69% S2: 5.7; 73% | Functionally abnormal when homozygous |
*Described according to the reference transcript NM_000546.5.
†Allele frequency in the general population, as indicated in gnomAD (https://gnomad.broadinstitute.org/).
‡Prediction of the variant impact on protein, according to the Align GVGD (C0: tolerated, other classes: damaging), PolyPhen-2 (D: probably damaging, P: possibly damaging, B: benign), SIFT (D: Damaging, T: tolerated) and BayesDel (score≥0.16: damaging/score <0.16: tolerated, as indicated in Fortuno et al, 201820) algorithms.
§The ClinVar classification of the variant (https://www.ncbi.nlm.nih.gov/clinvar/) is indicated.
¶For each variant are indicated the individual who was tested and the mean values of the functionality score and of the mRNA expression, as determined using the RT- QMPSF and RT-MLPA assays (see table 1). For some individuals, two independent samples (S1 and S2) were tested.
**As classified in the International Agency for Research on Cancer (IARC) database (http://p53.iarc.fr/), according to the results obtained in the yeast transactivation assay (Kato et al 23)
††As determined in the assay developed by Kotler et al 24 and based on the quantification of the antiproliferative activity of the variant in the p53-null H1299 cancer cell line.
‡‡As determined in the assays developed by Giacomelli et al 25 and based on the ability of the variant to restore the survival of the p53-null A459 cell line after exposure to high doses of etoposide DNA breaking agents and to induce in p53-wild-type A459 cell line resistance to Nutlin-3.
§§p53 functional assay was also carried out on an EBV cell line derived from the same patient and led to the same interpretation as the blood p53 functional assay (normal score for EBV>10). Items in bold indicate results suggestive of a deleterious effect of the variant.
¶¶Conflicting interpretations of pathogenicity.
***For this individual, a second TP53 variation was identified in cis: c.1021T>A, p.(Phe314Ile), which is classified as benign.
DNE, dominant negative effect; LOF, loss of function; NA, not analysed; NR, not reported; RT-MLPA, reverse transcription–multiplex ligation probe amplification; RT-QMPSF, reverse transcription–quantitative multiplex PCR of short fluorescent fragment.