CrossMark
p53 signaling pathway polymorphisms, cancer risk and tumor phenotype in TP53 R337H mutation carriers
Gabriel S. Macedo1,2 . Igor Araujo Vieira1,2 . Fernanda Salles Luiz Vianna1,2 . Barbara Alemar1,2 . Juliana Giacomazzi3 · Ana Paula Carneiro Brandalize1,2 · Maira Caleffi4 · Sahlua Miguel Volc6 ·
Henrique de Campos Reis Galvão6 · Edenir Inez Palmero6 · Maria Isabel Achatz7 · Patricia Ashton-Prolla1,2,5,8
@ Springer Science+Business Media B.V. 2017
Abstract Li-Fraumeni and Li-Fraumeni-like syndrome (LFS/LFL) are clinically heterogeneous cancer predisposi- tion syndromes characterized by diagnosis of early-onset and often multiple cancers with variable tumor patterns and incomplete penetrance. To date, the genetic modi- fiers described in LFS/LFL have been shown to map to either TP53 or its main negative regulator, MDM2. Addi- tionally, all studies were focused on families with differ- ent TP53 germline mutations. Hence, in this study we explored the effect of the most studied polymorphisms of p53 pathway genes on clinical manifestations of individu- als carrying the founder TP53 mutation R337H (n=136)
☒ Gabriel S. Macedo gmacedoufrgs@gmail.com
1 Post-Graduate Program in Genetics and Molecular Biology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil
2 Genomic Medicine Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, Rio Grande do Sul, Brazil
3 Hospital Tacchini, Bento Gonçalves, Rio Grande do Sul, Brazil
4 Hospital Moinhos de Vento, Porto Alegre, Rio Grande do Sul, Brazil
5 Department of Genetics, UFRGS, Porto Alegre, Rio Grande do Sul, Brazil
6 Molecular Oncology Research Center, Hospital do Câncer de Barretos, Barretos, São Paulo, Brazil
7 Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, 9609 Medical Center dr., Rockville, MD 20850, USA
8 Medical Genetics Service, HCPA, Porto Alegre, Rio Grande do Sul, Brazil
and controls (n=186). Cancer-affected carriers had been diagnosed either with adrenocortical carcinoma (ACC, n=29) or breast cancer (BC, n=43). Allelic discrimation using TaqMan assay was used for genotyping MDM2 SNP 309 (rs2279744) as well as MDM4 (rs1563828) and USP7 (rs1529916) polymorphisms. We found significantly higher MDM2 SNP 309 GG genotype and G allele frequencies in the LFS cohort than in controls. Furthermore, median age at first diagnosis was earlier in MDM2 SNP309 GG carriers when compared to other genotypes for both cancers (ACC: age 1 vs. 2 years; BC: age 35 vs. 43 years, respectively), although not statistically different. The allelic and geno- typic frequencies for all SNPs did not differ between cancer affected and unaffected carriers, neither between patients with ACC or BC. In conclusion, our results suggest that MDM2 SNP 309 may contribute to the LFL phenotype and also to an earlier age at diagnosis of ACC and BC cancer in carriers of the R337H founder mutation.
Keywords P53 · R337H . Polymorphism · Li-Fraumeni syndrome
Introduction
Li-Fraumeni Syndrome (LFS) and its variant, Li-Fraumeni- like Syndrome (LFL), are clinically heterogeneous cancer predisposition syndromes characterized by an autosomal dominant inheritance pattern, diagnosis of early-onset can- cers and multiple primary tumors. The core tumors of LFS/ LFL are bone and soft-tissue sarcomas, central nervous system tumors, breast cancer (BC) and adrenocortical car- cinoma (ACC) [1-3]. Currently, germline mutations in the TP53 gene are the only known genetic alterations underly- ing LFS/LFL [4].
In Southern and Southeastern regions of Brazil, a spe- cific mutation in the TP53 gene, R337H (c.1010G>A, p.ArgR337His), has been reported to occur at a high fre- quency both at population level and among individuals with clinical criteria for LFS/LFL [5-7]. Carriers display a wide range of cancers, within and beyond the spectrum of core tumors of the syndrome, but tumor penetrance appears to be lower than that observed in carriers of DNA-binding domain (DBD) mutations [5, 7-9]. The Arginine residue at codon 337 is a critical part of an alpha-helix motif involved in the protein oligomerization. Functional data have shown that the replacement of arginine by histidine disrupts the tetramer form in a pH-dependent manner, making the domain unable to oligomerize in conditions of slightly ele- vated pH [10]. Some authors have suggested that this pecu- liarity could explain, at least in part, the reduced penetrance observed in R337H carriers [11].
The p53 protein acts as a transcriptional factor that, in response to stress, regulates the expression of an array of different genes involved in growth arrest, DNA repair, apoptosis, metabolism and senescence [12]. Variations in p53 (multiple protein isoforms and/or mutant proteins) and in their partners are thought to underlie the wide range of clinical manifestations observed in the syndrome both within and between families. For instance, single nucleo- tide polymorphisms (SNPs) in the TP53 and MDM2 genes, a negative regulator of p53, have been associated to earlier age at cancer diagnosis in carriers of TP53 germline muta- tions [13, 14].
Although significant progresses have been made in our understanding of the molecular biology of p53 and clini- cal/epidemiologic features of LFS/LFL, knowledge on risk modifiers and its effect on phenotype are still incomplete. Thus, in the present study we aimed to investigate whether selected SNPs in MDM2, MDM4 and USP7 were associ- ated with cancer risk, age at first diagnosis and specific tumor types in carriers of the germline TP53 mutation R337H.
Materials and methods
Subjects
For this study a total of 136 subjects were recruited from families attending Cancer Risk Evaluation clinics in the Hospital de Clínicas de Porto Alegre (Porto Alegre, Bra- zil), Hospital do Câncer A.C. Camargo (São Paulo, Brazil) and Hospital do Câncer de Barretos (Barretos, São Paulo). The LFL group included cancer-unaffected R337H muta- tion carriers (n=60); R337H mutation carriers with a previous diagnosis of ACC (n=29), BC (n=43) or other tumors (n=4). In addition, a control group consisted of
cancer-unaffected individuals with no family history of cancer in first or second degree and without the R337H mutation (n= 186). Controls were recruited from a commu- nity based BC prevention program in Southern Brazil [15]. The institutional ethics committees of participating institu- tions approved the study and all participants provided writ- ten informed consent before recruitment.
Polymorphism analyses
Genomic DNA was extracted from white blood cells or non-tumoral tissue using commercial kits (Illustra Blood genomicPrep Mini Spin Kit, GE Healthcare and DNA FFPE Kit, FFPE Qiagen). Mutation testing was previously performed using Sanger sequencing of the entire coding region (exons 2-11) of TP53 according to standard proto- cols (http://p53.iarc.fr/download/tp53_directsequencing iarc.pdf).
TaqMan allelic discrimination analyses were per- formed according to Applied Biosystems standard proto- cols (Applied Biosystems, Carlsbad, USA). The analyzed SNPs were as follows: MDM4 rs1563828 (C_9493064_10), USP7 rs1529916 (C_9688119_1), and MDM2 rs2279744 for which a custom-made TaqMan assay was made, using forward primer 5’-CGGGAGTTCAGGGTAAAGGT-3’, reverse primer 5’-ACAGGCACCTGCGATCATC-3’, VIC probe 5’-CTCCCGCGCCGAAG-3’ and FAM probe 5’-TCCCGCGCCGCAG-3’ (Applied Biosystems). PCR cycling reactions were performed on an ABI StepOne Sys- tem (Applied Biosystems) and consisted of initial dena- turation at 95 ℃ for 15 min, 40 cycles with denaturation 95 ℃ for 15 s, and then annealing and extension at 60 ℃ for 1 min.
Statistical analyses
Descriptive statistics was used to determine allelic and genotypic frequencies. Differences in the genotype distri- bution and Hardy-Weinberg equilibrium were assessed by Chi square analysis. Comparison of the age at first cancer diagnosis according to polymorphism status was assessed by the non-parametric Kruskal-Wallis and Mann-Whitney tests. A p value of <0.05 was considered statistically sig- nificant. SPSS V.18.0 (SPSS Inc., Chicago, IL) was used for data handling and for all analyses.
Results
Clinical characteristics of TP53 R337H carriers and non- carriers enrolled in the study are shown in Table 1. Of the 136 carriers, 60 (44%) were cancer-unaffected, 29 (21%) had a previous diagnosis of ACC, 43 (32%) of BC and 4
| R337H mutation carriers* | Controls | |||
|---|---|---|---|---|
| ACC | BC | Cancer-unaffected | ||
| Number of patients | 29 | 43 | 60 | 186 |
| Age at siagnosis/recruit- ment, median (IQR) | 2.5 (1-6.7) | 42 (36-50) | 33 (IQR 24-45) | 52 (IQR 47-57) |
ACC adrenocortical carcinoma, BC breast cancer
*Four patients developed other tumors
(3%) developed other tumors. As expected, the median age of ACC diagnosis was earlier [2.5 years; InterQuar- tile Range (IQR) 1.0-6.7] than that found in BC cases (42 years; IQR 36-50). In cancer-unaffected and controls, the median age at recruitment was 33 (IQR 24-45) and 52 (IQR 47-57) years, respectively.
The genotypic and allele frequencies of MDM2, MDM4 and USP7 SNPs among R337H carriers (with and with- out cancer) and controls are presented in Table 2. First, we compared the distribution of genotypes and alleles between all R337H mutation carriers, regardless of personal history of cancer, and non-carriers (control group). The genotypic and allelic distribution between groups did not differ sig- nificantly for MDM4 and USP7 SNPs. In contrast, MDM2 SNP309 (T>G) was significantly associated to presence of the mutation for both, allelic (p=0.014) and genotypic (p=0.042) frequencies.
Second, we analyzed the overall impact of each SNP on cancer risk in the group of R337H mutation carriers, irre- spective of the cancer type. No statistically significant dif- ferences in the allelic and genotypic frequencies of each SNP were found between R337H carriers with and without cancer, indicating similar frequencies among these groups (Table 2). Since the mean age at recruitment of cancer- unaffected R337H mutation carriers was earlier than that found in the BC group, meaning that some of these sub- jects may still develop BC, when we investigated the role of the SNPs on cancer risk we considered two different sce- narios: (1) cancer unaffected versus all cancer-affected; and (2) cancer unaffected versus ACC group. Similar allelic and genotypic frequencies were observed in these comparisons.
Moreover, median age at first diagnosis was earlier in GG carriers (MDM2 SNP309) when compared to others genotypes for both cancers (age of 23 vs. 37 years), and
| Polymorphisms | TP53 R337H carriers (cases) | Controls | p cancer versus no cancer | p cases versus controls | |
|---|---|---|---|---|---|
| Cancer-unaf- fected, N (%) | Cancer-affected, N (%) | ||||
| MDM2 | |||||
| TT | 18 (30.0) | 25 (34.7) | 79 (42.5) | 0.600 | 0.042* |
| TG | 26 (43.3) | 33 (45.8) | 83 (44.6) | ||
| GG | 16 (26.7) | 14 (19.4) | 24 (12.9) | ||
| TG+GG | 42 (70.0) | 47 (65.3) | 107 (57.5) | 0.697 | 0.074 |
| G | 0.483 | 0.427 | 0.352 | 0.014* | |
| MDM4 | |||||
| TT | 12 (20.0) | 16 (21.3) | 25 (13.5) | 0.799 | 0.115 |
| TC | 28 (46.7) | 38 (50.7) | 87 (47.0) | ||
| CC | 20 (33.3) | 21 (28.0) | 73 (35.5) | ||
| TC+TT | 40 (66.7) | 54 (72.0) | 112 (60.5) | 0.630 | 0.119 |
| T | 0.433 | 0.466 | 0.370 | 0.217 | |
| HAUSP | |||||
| AA | 5 (8.3) | 5 (6.6) | 13 (7) | 0.792 | 0.983 |
| AG | 26 (43.3) | 30 (39.5) | 75 (40.5) | ||
| GG | 29 (48.4) | 41 (53.9) | 97 (52.4) | ||
| GA+AA | 31 (51.7) | 35 (46.1) | 88 (47.6) | 0.633 | 0.954 |
| A | 0.300 | 0.263 | 0.273 | 0.648 | |
*Comparison of the MDM2 rs2279744 genotypic and allelic frequencies between cases and controls (Chi square test)
also separately (ACC: age of 1 vs. 2 years; BC: age of 35 vs. 43 years, respectively), although with no significant dif- ference. For MDM4 and USP7 gene SNPs, we did not find any pattern regarding to age at first diagnosis (Table 3).
Finally, we investigated the distribution of genotypes/ alleles of the three SNPs according to tumor type among cancer-affected R337H mutation carriers. Although there were no differences on genotypic and allelic frequencies between ACC- and BC-affected carriers (Table 4) for any of the polymorphisms investigated, the USP7 rs1529916 AG genotype was found in about 52% of ACC cases in comparison to 30% of the BC cases. Hardy-Weinberg equi- librium was achieved for all polymorphisms in both cases and control group.
Discussion
Mdm2, Mdm4 and Usp7 are three critical p53 regulators. Mdm2 and Mdm4 degrade p53 through the binding and polyubiquitination of the protein, blocking its activity as transcriptional factor [16]. On the other hand, Usp7 play a role as a deubiquitinase, regulating the stability of p53 and the p53-binding protein Mdm2 [17-19]. Given the central role of these proteins on p53 signaling, in this study we investigated the impact of the most studied SNPs in the MDM2, MDM4 and USP7 genes on clinical manifestation (cancer risk, age at first diagnosis and tumor type) of TP53 R337H mutation carriers.
| Polymorphisms | Adrenocortical carcinoma | Breast cancer | p |
|---|---|---|---|
| MDM2 | |||
| TT | 7 (27.6) | 15 (38.5) | 0.408 |
| TG | 13 (44.8) | 18 (46.2) | |
| GG | 8 (27.6) | 6 (15.4) | |
| TG+GG | 21 (72.4) | 24 (61.5) | 0.498 |
| MDM4 | |||
| TT | 5 (17.2) | 11 (26.2) | 0.672 |
| TC | 15 (51.7) | 19 (45.2) | |
| CC | 9 (31) | 12 (28.6) | |
| TC+TT | 20 (69.0) | 30 (71.4) | 1.000 |
| HAUSP | |||
| AA | 2 (6.9) | 3 (7.0) | 0.172 |
| AG | 15 (51.7) | 13 (30.2) | |
| GG | 12 (41.4) | 27 (62.8) | |
| GA+AA | 17 (58.6) | 16 (37.2) | 0.122 |
To date, only a few genetic alterations have been shown to modify the LFS/LFL phenotype. Among these, a 16 bp duplication in the TP53 gene, PIN3 (polymorphism intron 3), has been described as the germline variant with strong- est modifier effect. In a study published by Marcel et al. the authors have found that cancer diagnosis occurred 19 years later in TP53 germline mutation carriers with the 16 bp
| Polymorphisms | Adrenocortical carcinoma | p | Breast cancer | p | All cancer cases | p | |||
|---|---|---|---|---|---|---|---|---|---|
| n (%) | Median (IQR) | n (%) | Median (IQR) | n (%) | Median (IQR) | ||||
| MDM2 | |||||||||
| TT | 8 (28.5) | 2 (0.7-5.25) | 0.415* | 15 (38.5) | 43 (37-48) | 0.638* | 23 (34.3) | 37 (3-46) | 0.481* |
| TG | 13 (46.5) | 3 (1.5-10.5) | 18 (46.2) | 42 (35-52) | 31 (46.3) | 32 (6-44) | |||
| GG | 7 (25.0) | 1 (1.0-5.0) | 6 (15.4) | 35 (25-60) | 13 (19.4) | 23 (1-41) | |||
| TG+GG | 20 (72.4) | 2.5 (1.5-9.2) | 0.412 ** | 24 (61.5) | 41 (32-56) | 0.452 ** | 44 (65.3) | 28 (3.2-42) | 0.534* |
| MDM4 | |||||||||
| TT | 4 (14.3) | 2.5 (1.2-38.2) | 0.620* | 11 (26.2) | 44 (19-57) | 0.891* | 15 (21.4) | 40 (22-50) | 0.456* |
| TC | 15 (53.6) | 2 (1-6) | 19 (45.2) | 42 (37-50) | 34 (48.5) | 36 (2-43) | |||
| CC | 9 (32.1) | 5 (0.8-11) | 12 (28.6) | 40 (36-54) | 21 (30.0) | 32 (5.5-42) | |||
| TC+TT | 19 (67.9) | 2 (1-6) | 0.487 ** | 30 (71.4) | 42 (36-50) | 0.770 ** | 49 (70.0) | 36 (3-45.5) | 0.667 ** |
| HAUSP | |||||||||
| AA | 2 (7.1) | 3-22 | 0.446* | 3 (7.0) | 36-39 | 0.821* | 5 (7.0) | 39 (19.5-42.5) | 0.442* |
| AG | 14 (50.0) | 1.5 (1-10.2) | 13 (30.2) | 40 (36-54) | 27 (38.0) | 29 (1-42) | |||
| GG | 12 (42.9) | 3 (1-5.7) | 27 (62.8) | 43 (32-47) | 39 (55.0) | 36 (6-46) | |||
| GA+AA | 16 (57.1) | 2 (2.40.2) | 0.888 ** | 16 (37.2) | 39.5 (36.2-37.7) | 0.900 ** | 32 (45.1) | 36 (2-42) | 0.293 ** |
IQR interquartile range
*Kruskal-Wallis test
** Mann-Withney test
duplicated allele [13]. With regard to SNPs in TP53-related genes, although conflicting results, several publications have demonstrated the impact of MDM2 SNP309 (T>G), a SNP located in the promoter region of MDM2, on earlier age of tumor onset in LFS patients carrying predominantly TP53 DBD mutations [20-23].
Here, although not statistically significant, MDM2 G/G genotype was found in higher frequency in ACC cases, usu- ally a tumor of early-onset, than BC cases (27.6 vs. 15.4%, respectively). In the same way, age at diagnosis was lower in MDM2 SNP309 GG carriers when compared to other genotypes. The fact that none statistically significant differ- ences were observed between these comparisons might be explained by the lack of statistic power. In this context, it would be required to genotype at least 35 subjects in each genotype in order to identify a difference of 10 years in the age at diagnosis, taking into account the dispersal of ages observed in this study (with 80% power and 5% alpha). We can also hypothesize that MDM2 SNP309 has a limited contribution when isolated assessed. In fact, Renaux-Petel et al. have only observed an effect of MDM2 SNP309 GG genotype on age of tumor onset when haplotype analyses were performed. The authors showed that MDM2 SNP309 along with MDM2 SNP285 (MDM2 285-309 G-G) develop tumors 5 years earlier than patients harbouring other haplotypes [20]. In contrast, Wu et al. using a robust and reliable statistical method to evaluate cancer risk attrib- utable to a measured hereditary susceptibility gene in fam- ily studies did not find a statistically significant interaction between MDM2 SNP309 G allele and TP53 mutation on cancer incidence [24].
Interestingly, we observed an enrichment of the MDM2 SNP309 G allele in the R337H mutation carriers group (regardless of personal history of cancer) when compared to controls (p=0.014), suggesting that this SNP may con- tribute to the LFL phenotype in families carrying the R337H germline mutation. Similar to our findings, Ruijs et al. showed higher percentage of SNP309 homozygotes (G/G) in TP53-negative LFS and LFS-related patients when compared to the general populations. The authors suggested that SNP309 G polymorphism act as an addi- tional disease-causing factor [23].
We also did not observe a significant association between MDM4 rs1563828 and USP7 rs1529916 poly- morphisms with cancer risk, age at first diagnosis and tumor type in R337H mutation carriers. Although we have found a higher frequency of USP7 rs1529916 AG genotype in ACC cases, its potential role on cancer type in R337H mutations should be investigated in a larger series of patients and with other TP53 germline mutation in order to confirm its potential role as a genetic modifier.
If confirmed, these findings may have important clinical implications.
Our study has several limitations that must be con- sidered in the interpretation of the results. Although the relatively small sample size, due to the rarity of the syndrome, this is one of the largest cohorts used in the context of genetic modifiers of LFS/LFL. In addition, we investigated just one SNP of each gene and the haplotype analyses have not been performed at this time. Finally, differently of MDM2 SNP309, the mechanistic basis of a potential negative effect of MDM4 and USP7 on p53 signaling remains unknown. On the other hand, a posi- tive aspect of our study is the homogeneity of the cases. Here, all patients were carriers of a particular germline mutation, the TP53 R337H. Due the particularities found in the clinical presentation of DBD mutation and R337H carriers, we believe that different SNPs in genes of the p53 pathway may affect the p53 function in different ways. To our knowledge, this is the first study to investi- gate genetic modifiers in LFL patients carrying the same germline mutation.
The information about polymorphisms with modifier effect of the LFS/LFL phenotype may have important implications in the cancer risk assessment. Although several reports showing the impact of MDM2 SP309 on clinical manifestations of LFS families carrying mostly TP53 DBD mutations, our data indicate that this func- tional SNP may also play a role on age at first diagnosis in patients carrying the R337H germline mutation. The enrichment of G allele and GG genotype in LFL patients, regardless of personal cancer history, also suggest that MDM2 SP309 may have an additive impact on LFL phe- notype. Haplotype analyses as well as a larger number of patients are needed in order to confirm our findings.
Acknowledgements We are grateful to Patricia Silva, Diego Pasku- lin and Cristina Brinckmann Netto for their valuable contributions and support. The authors also wish to thank Flávia Giusti and Rafael Bringhenti from Unidade de Patologia Experimental, Centro de Pes- quisa Experimental. This work was supported by fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and funded in part by Fundação de Amparo à Pes- quisa do Estado do Rio Grande do Sul (FAPERGS-PPSUS) (Grant # 09/0103-0), FAPERGS-PRONEX (Grant 0051-9), Fundo de Incentivo à Pesquisa do Hospital de Clínicas de Porto Alegre (FIPE- HCPA) and CNPQ (Grant 2012-4).
Compliance with ethical standards
Conflict of interest The authors have no conflict of interest to de- clare.
References
1. Li FP, Fraumeni JF, Mulvihill JJ et al (1988) A cancer family syn- drome in twenty-four kindreds. Cancer Res 48(18):5358-5362
2. Li FP, Fraumeni JF (1969) Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Intern Med 71(4):747-752
3. Li FP, Fraumeni JF (1969) Rhabdomyosarcoma in children: epidemiologic study and identification of a familial cancer syn- drome. J Natl Cancer Inst 43(6):1365-1373
4. Malkin D, Li FP, Strong LC et al (1990) Germ line p53 muta- tions in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250(4985):1233-1238
5. Palmero EI, Schüler-Faccini L, Caleffi M et al (2008) Detection of R337H, a germline TP53 mutation predisposing to multiple cancers, in asymptomatic women participating in a breast cancer screening program in Southern Brazil. Cancer Lett 261(1):21- 25. doi:10.1016/j.canlet200710.044
6. Custódio G, Parise GA, Kiesel Filho N et al (2013) Impact of neonatal screening and surveillance for the TP53 R337H muta- tion on early detection of childhood adrenocortical tumors. J Clin Oncol 31(20):2619-2626. doi:10.1200/JCO.2012.46.3711
7. Achatz MI, Olivier M, Le Calvez F et al (2007) The TP53 muta- tion, R337H, is associated with Li-Fraumeni and Li-Fraumeni- like syndromes in Brazilian families. Cancer Lett 245(1-2):96- 102. doi:10.1016/j.canlet200512.039
8. Ribeiro RC, Sandrini F, Figueiredo B et al (2001) An inherited p53 mutation that contributes in a tissue-specific manner to pediatric adrenal cortical carcinoma. Proc Natl Acad Sci USA 98(16):9330-9335. doi:10.1073/pnas.161479898
9. Giacomazzi J, Selistre SG, Rossi C et al (2013) Li-Fraumeni and Li-Fraumeni-like syndrome among children diagnosed with pediatric cancer in Southern Brazil. Cancer 119(24):4341-4349. doi: 10.1002/cncr.28346
10. DiGiammarino EL, Lee AS, Cadwell C et al (2002) A novel mechanism of tumorigenesis involving pH-dependent destabi- lization of a mutant p53 tetramer. Nat Struct Biol 9(1):12-16. doi: 10.1038/nsb730
11. Malkin D (2011) Li-fraumeni syndrome. Genes Cancer 2(4):475-484. doi:10.1177/1947601911413466
12. Vousden KH, Prives C (2009) Blinded by the light: the grow- ing complexity of p53. Cell 137(3):413-431. doi:10.1016/j. cell200904.037
13. Marcel V, Palmero EI, Falagan-Lotsch P et al (2009) TP53 PIN3 and MDM2 SNP309 polymorphisms as genetic modifiers in the Li-Fraumeni syndrome: impact on age at first diagnosis. J Med Genet 46(11):766-772 doi:10.1136/jmg.2009.066704
14. Bougeard G, Baert-Desurmont S, Tournier I et al (2006) Impact of the MDM2 SNP309 and p53 Arg72Pro polymorphism on age of tumour onset in Li-Fraumeni syndrome. J Med Genet 43(6):531-533. doi:10.1136/jmg.2005.037952
15. Caleffi M, Ribeiro RA, Duarte Filho DL et al (2009) A model to optimize public health care and downstage breast cancer in limited-resource populations in southern Brazil. (Porto Alegre Breast Health Intervention Cohort). BMC Public Health 9:83. doi: 10.1186/1471-2458-9-83
16. Lee JT, Gu W (2010) The multiple levels of regulation by p53 ubiquitination. Cell Death Differ 17(1):86-92. doi:10.1038/ cdd.2009.77
17. Brooks CL, Li M, Hu M, Shi Y, Gu W (2007) The p53-Mdm2- HAUSP complex is involved in p53 stabilization by HAUSP. Oncogene 26(51):7262-7266. doi:10.1038/sj.onc.1210531
18. Li M, Brooks CL, Kon N, Gu W (2004) A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol Cell 13(6):879-886
19. Perry ME (2010) The regulation of the p53-mediated stress response by MDM2 and MDM4. Cold Spring Harb Perspect Biol 2(1):a000968. doi:10.1101/cshperspect.a000968
20. Renaux-Petel M, Sesboüé R, Baert-Desurmont S et al (2014) The MDM2 285G-309G haplotype is associated with an earlier age of tumour onset in patients with Li-Fraumeni syndrome. Fam Cancer 13(1):127-130. doi:10.1007/s10689-013-9667-2
21. Pinto C, Veiga I, Pinheiro M et al (2009) TP53 germline muta- tions in Portugal and genetic modifiers of age at cancer onset. Fam Cancer 8(4):383-390. doi:10.1007/s10689-009-9251-y
22. Tabori U, Nanda S, Druker H, Lees J, Malkin D (2007) Younger age of cancer initiation is associated with shorter telomere length in Li-Fraumeni syndrome. Cancer Res 67(4):1415-1418. doi: 10.1158/0008-5472.CAN-06-3682
23. Ruijs MW, Schmidt MK, Nevanlinna H et al (2007) The single- nucleotide polymorphism 309 in the MDM2 gene contributes to the Li-Fraumeni syndrome and related phenotypes. Eur J Hum Genet 15(1):110-114. doi:10.1038/sj.ejhg.5201715
24. Wu CC, Krahe R, Lozano G et al (2011) Joint effects of germ- line TP53 mutation, MDM2 SNP309, and gender on cancer risk in family studies of Li-Fraumeni syndrome. Hum Genet 129(6):663-673. doi:10.1007/s00439-011-0957-1