Table 1

Interpretation of germline TP53 variants integrating the blood p53 functional assay

Variant*MAF†Bioinformatic predictions‡Functional status of the variants assessed in yeast and human cancer cell linesClinVar classification§Blood p53
functional assay¶ in p53 score, mRNA%
p53
functional assay in EBV cell lines score; mRNA%
Interpretation according to the assay
Align GVGDPolyPhen
-2
SIFTBayes
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 FunctionalUnclassified 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 NANot 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/////FunctionalNA Pathogenic Ind. 57: 3.1; 61% 5.5; 61%§§ Functionally abnormal
c.723del
p.(Cys242Alafs*5)
NR////NANANANRInd. 58:
S1: 7.1; 56%
S2: 6.0; 52%
4.2; 52%§§ Functionally abnormal
c.770del
p.(Leu257Argfs*88)
NR////NA Non-functional NANRInd. 59: 4.7; 62% Functionally abnormal
Benign variant
c.704A>G
p.(Asn235Ser)
0.01945C0BT−0.0342FunctionalFunctionalNot LOF
Not DNE
BenignInd. 60: 12.9; 118%Functionally normal
Unclassified rare variants
c.215C>A
p.(Pro72His)
NRC0P D −0.1017FunctionalNANot LOF
Not DNE
Uncertain significanceInd. 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 significanceInd. 62: 2.9; 82% 2.1; 85%
3.6; 70%
Functionally abnormal
c.329G>A
p.(Arg110His)
0.000046C0BT0.0728 Partially functional FunctionalNot LOF
Not DNE
Uncertain significanceInd. 63: 4.1; 83% 3.7; 92%
4.7; 91%§§
5.2; 94%
Functionally abnormal
c.402T>G
p.(Phe134Leu)
NRC0 D D 0.3166 Non-functional Non-functional LOF
DNE
NRInd. 64: 3.5; 78% 3.5; 94%§§ Functionally abnormal
c.472C>T
p.(Arg158Cys)
0.000008 C65 D D 0.5145 Partially functional FunctionalUnclassifiedConflict. int:
Likely pathogenic/uncertain significance
Ind. 65: 3.2; 86% Functionally abnormal
c.572C>G
p.(Pro191Arg)
0.000012 C35 P D 0.0942FunctionalFunctionalNot LOF
Not DNE
Uncertain significanceInd. 66: 17.1; 90%Functionally normal
c.833C>G
p.(Pro278Arg)
NR C65 D D 0.6078 Non-functional Non-functional LOF
DNE
Uncertain significanceInd. 67: 5.1; 78% 3.9; 108%§§ Functionally abnormal
c.847C>T
p.(Arg283Cys)
0.000074 C55 B D 0.3309 FunctionalFunctionalNot LOF
Not DNE
Uncertain significanceInd. 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 NANot LOF
Not DNE
NRInd. 70:
S1: 5.8; 89%
S2 : 6.1; 90%
Functionally abnormal
c.1054G>T
p.(Asp352Tyr)
NR C35 D D 0.0844FunctionalNANot LOF
Not DNE
Uncertain significanceInd. 71***:
S1: 4.0; 101%
S2: 5.0; 106%
2.3; 90% Functionally abnormal
c.323_325del p.(Gly108_Phe109delinsVal)NR///////NRInd. 72: 3.8; 94% Functionally abnormal
c.393_395del
p.(Asn131del)
NR////NA Non-functional NAUncertain significanceInd. 73:
S1: 2.6; 80%
S2: 4.8; 99%
Functionally abnormal
c.792_794del
p.(Leu265del)
NR/////NANANRInd. 74: 3.4; 95% Functionally abnormal
c.-117G>T
p.(=) (5'UTR)
NR////NANANANRInd. 75: 20.5; 100%Functionally normal
Polymorphism
c.*1175A>C
p.(=)
(Homozygous)
0.01189////NANANAConflict. 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.