An identical, complex TP53 mutation arising independently in two unrelated families with diverse cancer profiles: the complexity of interpreting cancer risk in carriers

Abstract

Most inherited TP53 mutations have been identified in individuals with a family cancer predisposition syndrome, in which the activity of p53 mutants is severely reduced. However, germline p53 mutants in children with 'sporadic' adrenocortical or choroid plexus tumors exhibit a wide range of functional activity. Here, we demonstrate the occurrence of a complex germline TP53 mutation in two unrelated families with different cancer phenotypes, neither fulfilling the classic criteria for Li-Fraumeni syndrome. The TP53 mutation consists of a duplication of 7 bp in exon 4, resulting in a frame shift and premature stop signal. Haplotype analysis indicated that the mutation arose independently in the two families. Analysis of the DNA secondary structure predicts the TP53 mutation occurred within a hairpin loop. Additional germline complex mutations occurring within the same region of exon 4 have been identified in the IARC database. Our findings suggest that certain TP53 regions are prone to intrinsic genetic alterations, possibly through defects in DNA replication or repair. Further, carriers of the same TP53 mutation can have diverse cancer profiles, illustrating the complexity of genetic counseling and risk prediction.

Citation: Oncogenesis (2012) 1, e1; doi:10.1038/oncsis.2012.1

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www.nature.com/oncsis

ORIGINAL ARTICLE

An identical, complex TP53 mutation arising independently in two unrelated families with diverse cancer proles:the complexity of interpreting cancer risk in carriers

EM Pinto1,2, RC Ribeiro1,3, J Li4, L Taja-Chayeb5, LF Carrasco6, M de Lourdes Pea-Torres5, S Vidal-Milln5, H Maldonado-Mtz5, A Dueas-Gonzlez5,7, L McGregor3 and GP Zambetti2

Most inherited TP53 mutations have been identied in individuals with a family cancer predisposition syndrome, in whichthe activity of p53 mutants is severely reduced. However, germline p53 mutants in children with sporadic adrenocortical or choroid plexus tumors exhibit a wide range of functional activity. Here, we demonstrate the occurrence of a complex germline TP53 mutation in two unrelated families with different cancer phenotypes, neither fullling the classic criteria for Li-Fraumeni syndrome. The TP53 mutation consists of a duplication of 7 bp in exon 4, resulting in a frame shift and premature stop signal. Haplotype analysis indicated that the mutation arose independently in the two families. Analysis of the DNA secondary structure predicts the TP53 mutation occurred within a hairpin loop. Additional germline complex mutations occurring within the same region of exon 4 have been identied in the IARC database. Our ndings suggest that certain TP53 regions are prone to intrinsic genetic alterations, possibly through defects in DNA replication or repair. Further, carriers of the same TP53 mutation can have diverse cancer proles, illustrating the complexity of genetic counseling and risk prediction.

Oncogenesis (2012) 1, e1; doi:http://dx.doi.org/10.1038/oncsis.2012.1

Web End =10.1038/oncsis.2012.1 ; published online 20 February 2012

Subject Category: molecular oncology

Keywords: TP53 mutation; adrenocortical tumor; haplotype; copy number variation

INTRODUCTIONInherited mutations in the TP53 tumor suppressor gene are associated with susceptibility to cancer.1 Most of these mutations have been identied in individuals from families with Li-Fraumeni syndrome (LFS), Li-Fraumeni-like syndrome or TP53-associated malignancies such as pediatric adrenocortical tumors (ACTs)2,3 and

choroid plexus carcinoma.4,5 In familial cancer syndromes, functional studies have shown severe reduction in the activity of p53 mutants.6 Conversely, the p53 mutants show varying degrees of functional activity in children with ACT or choroid plexus carcinoma, and some have wild-type (WT) activity.2 In carriers of the latter p53 mutations, the spectrum and the onset of malignancies differ from those in carriers belonging to LFS families.7,8 Varley et al.9 and Birch et al.10 suggested the existence of inherited low-penetrance TP53 mutations that are associated with ACT but not with a specic familial cancer susceptibility syndrome. The tumorigenic effect of such low-penetrance mutants may depend on cooperating age-and tissue-specic genetic changes. Therefore, detailed and active life-long geno-type/phenotype studies in the families of individuals carrying lowpenetrance mutations may reveal genetic abnormalities and pathways associated with tumorigenesis. Here, we report a child with ACT who carried a complex, inherited TP53 mutation identical to a mutation previously reported in a young woman with breast

cancer from a family that did not t the strict LFS criteria.11

Detailed haplotype analysis of these families conrmed that the mutation arose independently in each.

RESULTS Case report

We studied two apparently unrelated families that shared no common surnames or known relationships. The rst family was that of a patient entered into the International Pediatric Adrenocortical Tumor Registry (IPACTR) (http://www.stjude.org/ipactr

Web End =http://www.stjude.org/ http://www.stjude.org/ipactr

Web End =ipactr ). A previously healthy 2-year-old girl in Honduras had a 4-week history of hirsutism, acne, pubarche and strong adult body odor. The patient weighed 12.5 kg (50th percentile) and was 82 cm tall (25th percentile). Her blood pressure was 90/60 mm Hg. She had a verrucous clitoris and Tanner stage III pubarche. A mass was palpable in her left hemi-abdomen. The family history of cancer did not t the criteria for LFS. Her father had surgical resection of a lung mass (unknown histology) at the age of 2 years, her paternal grandmother had uterus (cervix) cancer, a great grandmother had lung cancer at age 67, and the paternal great grandfather had skin cancer (melanoma). Laboratory tests revealed adrenocortico-tropic hormone o5 rg/ml (normal range o46 rg/ml); morning cortisol 12.9 mg/dl (normal range 5--25 mg/dl); dehydroepian-

1International Outreach Program, St Jude Childrens Research Hospital, Memphis, TN, USA; 2Department of Biochemistry, St Jude Childrens Research Hospital, Memphis, TN, USA;

3Department of Oncology, St Jude Childrens Research Hospital, Memphis, TN, USA; 4Hartwell Center for Bioinformatics and Biotechnology, St Jude Childrens Research Hospital, Memphis, TN, USA; 5Instituto Nacional de Cancerologa, Mexico City, Mxico; 6Hospital Escuela, Servicio de Hematooncologia Pediatrica, Tegucigalpa, Honduras and 7Unidad de Investigacin Biomdica en Cncer, Instituto de Investigaciones Biomdicas, Universidad Nacional Autnoma de Mxico, Mexico City, Mxico. Correspondence: Dr EM Pinto, International Outreach Program, Department of Biochemistry, Mail Stop 276, St Jude Childrens Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-2794, USA. E-mail: mailto:[email protected]

Web End [email protected]

Received 31 August 2011; revised 27 December 2011; accepted 29 December 2011

Complex TP53 mutation arising two unrelated families

EM Pinto et al

2 drosterone sulfate 652 mg/dl (normal range 35--430 mg/dl) and

17a-hydroxyprogesterone 1370 ng/dl (normal range 3--51 ng/dl).

Abdominal ultrasonography revealed a left adrenal mass that was surgically removed. Grossly, it measured 4.4 cm and weighed 15 g. Histopathological ndings revealed nuclear grade 3, clear cell, abnormal mitosis o25%, diffuse architecture and no capsular or venous invasion or necrosis. This family is referred to as HON001. Clinical and genetic studies of the second family, which resided in Mexico (MEX001), were recently reported.11

The index case was a 23-year-old woman with breast carcinoma (lobular carcinoma plus inltrating ductal carcinoma). Her father, carrier of this mutation, had leiomyosarcoma at age 67 years, and her half-sister died of bronchioloalveolar carcinoma at age 25 years. The patient had a healthy brother in his 20s who carried the mutation. The pedigrees of both families are shown in Figure 1.

Sequence analysis of TP53TP53 sequence analysis of genomic DNA from family HON001 revealed a duplication of 7 bp (GTTTCCG) (c.329_330 ins7) affecting codons 108--110 in exon 4, which generated a frame shift and a premature stop signal at position 150. The mutation was heterozygous in the germline of the ACT patient and her father (Figure 2a). The mother was WT for TP53 sequence. The ACT underwent loss of heterozygosity (LOH) tissue with deletion of the WT allele. The same 7 bp duplication in exon 4 was observed in the germline of the Mexican family, including the father with leiomyosarcoma (67 years), the daughter with early-onset breast cancer (25 years) and additional family members who were healthy (8). As shown in Figure 2b, the leiomyosarcoma remained heterozygous for the mutation (Figure 2b, panel II), whereas the breast tumor underwent LOH and selected the mutant allele (Figure 2b, panel III).

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Figure 1. Genealogy of the two families cancer history. (a) The Honduran family. The pediatric patient with ACT is a carrier of the duplication of 7 bp (GTTTCCG) (c.329_330 dupl GGTTTCC) affecting codons 108--110 in exon 4 of the TP53 gene. Her father is also a carrier of this mutation. (b) The Mexican family. The patient with breast cancer, her father with sarcoma and a healthy brother are carriers of the same mutation observed in the Honduran family. The circles represent females, whereas the squares represent males. Open symbols indicate no presence of tumor and lled symbols represent those with tumor; crossed symbols indicate deceased individuals. Arrows indicate proband. TP53 status is identied above those cases that biological material was accessed.

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Figure 2. Sequence analyses of TP53. (a) Chromatogram showing a 7-bp duplication, in heterozygous state, in the TP53 gene. (b) Lossof heterozygosity (LOH) analysis. Upper panel (I): the WT allele in a homozygous state; center panel (II): the WT and mutant alleles in a heterozygous state; lower panel (III): an allelic imbalance indicating LOH. (c) Genetic proling of the index case in each family, using 15 single tandem repeat markers. This analysis shows only alleles reported to be most common in the overall populations of their respective countries. The patients share only four alleles, red ones (the most common in both populations).

Haplotype analysisWe analyzed the structure of the TP53 locus to precisely identify the haplotype carrying the complex TP53 mutation. The HON001 ACT patient was heterozygous for the VNTRp53 marker (127/132) and homozygous for the p53(CA)n (122/122), allele G (rs1642785)

in intron 2, arginine in codon 72 (rs1042522) and C and T for single-nucleotide polymorphisms rs12947788 and rs12951053 in intron 7. Therefore, the patient harbored the carrier haplotype represented by allele 122 for p53(CA)n and 127 for VNTRp53; this haplotype was also observed in the ACT DNA and in the germline of her fathers DNA (Figure 3a).

The MEX001 breast tumor patient was homozygous for all markers, with allele 122 for p53(CA)n, allele 132 for VNTRp53, allele

C for single-nucleotide polymorphism rs1642785 in intron 2 and allele C (proline) at codon 72 (rs1042522). Single-nucleotide polymorphisms rs12947788 and rs12951053 at intron 7 were T and G, respectively. The carrier haplotype was also observed in this patients father germline DNA. The paternal half-sister (39 years) and two brothers (22 and 20 years) of the patient were also analyzed. Only the younger brother, who is healthy and cancer free, inherited the carrier haplotype (Figure 3b).

These data demonstrate a different haplotype for the inherited 7 bp duplication TP53 mutation occurring in the Honduras and Mexican families. Corroborating these ndings, the two index cases (HON001 ACT and MEX001 breast cancer) were proled by using a set of 15 single tandem repeat markers and only four alleles, all of which were commonly found in the populations of the patients respective countries, were matched suggesting no close ancestry (Figure 2c).

Copy-number variation (CNV) analysisNine genomic DNA samples were tested for CNV by multiplex ligation-dependent probe amplication (MLPA). All probes for the TP53 gene revealed extensive CNV in the ACT DNA and no CNV in the leiomyosarcoma and breast tumor tissue (Figure 4).

Analysis of DNA secondary structure of WT TP53 exon 4The DNA secondary structure of WT TP53 exon 4 was predicted using the Mfold program.12 Because of the limitations of the mathematical model and uncertainties in the thermodynamic parameters used in these methods, Mfold predicted multiple suboptimal structures with similar free energy values. The program predicted a secondary structure between codons 107 and 110, suggesting that the duplication event occurred within a hairpin structure (Figure 5).

p53 ImmunostainingThe pediatric ACT showed strong to weak cytoplasmic staining for the p53 protein in almost all tumor cells. By contrast, p53 immunostaining was negative in the leiomyosarcoma and breast tumor tissue (Figure 6).

DISCUSSIONHere we report an identical, inherited, complex germline mutation in the TP53 gene arising independently in two unrelated individuals from families with different cancer proles. Because of the uniqueness of the mutation and the shared Hispanic

& 2012 Macmillan Publishers Limited

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Figure 3. Detailed haplotype analysis for the TP53 locus. (a) The haplotype in the Honduran family. (b) The haplotype in the Mexican family.

heritage, we considered the possibility of a founder effect. In population genetics, founder events occur when a new population is established from a few individuals descended from a large ancestral population.13 The founder effect has been reported only once for a TP53 mutation (Arg337His).14 Our detailed TP53 analyses in both families denitively showed different haplotypes in each family,

ruling out the possibility of a common founder. Corroborating evidence was provided by genetic proling of the two index cases with 15 single tandem repeat markers, clearly showing that they shared only alleles commonly identied in the populations of their respective countries.15,16 We observed no shared rare alleles that would increase the likelihood of common ancestry.

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CNV for TP53 locus

PMP22 EFNB3_1 EFNB3_2 TP53 EXON1 TP53 EXON1_2 TP53 EXON2 TP53 EXON3 TP53 EXON4 TP53 EXON5 TP53 EXON6 TP53 EXON7 TP53 EXON8 TP53 EXON9 TP53 EXON11 TP53 EXON12 ATP1B2_1 ATP1B2_2 SHBG MPDU1_1 MPDU1_2 POLR2A_1 POLR2A_2

In the late 1960s, Li and Fraumeni observed a high concentration of cancers in four families, in which siblings or cousins had childhood sarcoma; a high frequency of soft-tissue sarcoma and breast carcinoma was noted in the ancestral line of one parent in each family. Acute leukemia, brain tumor and carcinoma of the lung, pancreas and skin frequently occurred in rst- and second-degree relatives, and adrenocortical carcinomas were often diagnosed in children.17 This nonrandom aggregation of cancer is termed Li-Fraumeni syndrome. Classic LFS criteria include (1) diagnosis of sarcoma before age 45 years; (2) at least one rst-degree relative with a cancer of any kind before age 45; and (3) A

Figure 4. Heat map derived from MLPA analysis of the TP53 locus and nearby genes in nine samples. No signicant CNV was observed between germline (1) and breast tumor tissue (1A) or between germline (2) and sarcoma tissue (2A). However, extensive CNV was observed between germline (4) and tumor tissue (4A) from the patient with ACT. Healthy individuals from both families were included in this analysis (3,5,6). Two (3 and 5) were carriers of the mutation, and their germline samples showed no signicant CNV. Sample 6, from the mother of the patient with ACT (4), had the WT TP53 sequence. Red indicates duplications (41.3 ratio to normal controls). Blue indicates deletions (o0.7 ratio to normal controls).

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Figure 6. Immunohistochemical staining pattern for p53 protein. (a) Strong to weak cytoplasmic staining is seen in almost all ACT cells. Staining was negative in the breast tumor (b) and sarcoma (c) specimens. Appropriate positive and negative controls were implied.

third family member (a rst- or second-degree relative) with cancer under age 45 or with sarcoma at any age.18 Although the family histories of our cases did not t these criteria, the presence of ACT in one family and early-onset breast cancer in the other are an acceptable indication for TP53 mutation testing.19 This less stringent recommendation should identify TP53 mutations that may be associated with an increased cancer predisposition even in the absence of a dened familial pattern.

LOH at the TP53 locus was observed in both the ACT and breast tumor, but not in the leiomyosarcoma (Figure 2). CNV supports LOH ndings in ACT and leiomyosarcoma (Figure 4). However, the loss of the p53 allele in the breast tumor was not accounted for by the MLPA suggesting a neutral copy number, possibly due to gene conversion. Consistent with the loss of the nuclear localization signals in the C-terminus of p53, the truncated mutant p53 protein was expressed at detectable levels within the cytoplasm of the ACT (Figure 5). Although the other tumors (breast and leiomyosarcoma) harbored the same mutation, the samples were negative for p53 staining, indicating that immunohistochemistry alone is not always informative for assessing TP53 status.20

The TP53 mutation observed in our Honduran pediatric ACT and Mexican breast cancer patients comprises a seven nucleotide duplication, affecting codons 108--110, resulting in a frame shift and premature stop codon at position 150. The observation that this complex mutation exists as different TP53 haplotypes in these two families, demonstrates that this mutation arose independently and suggests that this region of exon 4 is susceptible to genetic alterations. Consistent with this possibility, the Mfold program predicted a potential hairpin loop encompassing codons 107--110, which corresponds to the 7 bp duplication in exon 4. The identication of multiple complex mutations in breast carcinomas, ovarian cancer, rhabdomyosarcomas and other adrenocortical carcinomas further suggests that this region is prone to genetic alterations (Figure 7).10,21--24

The heterogeneity of cancer phenotype in our two study families can be partially attributed to constitutional and acquired genetic changes. Our ndings suggest that this complex TP53

mutation could be a driver of oncogenesis. The ability to differentiate between drivers and passengers will be crucial to better understanding the process of tumorigenesis and dening age- and tissue-specic factors. Registries for specic tumors associated with TP53 mutations can provide invaluable clinical and biological correlates, allowing studies of the effects of environment, lifestyle and modier genes on the penetrance and spectrum of tumor types associated with seemingly identical genetic lesions.

MATERIALS AND METHODSThe patients parents provided informed consent for her participation in the IPACTR (http://www.stjude.org/ipactr

Web End =http://www.stjude.org/ipactr), whose research protocol was approved by the St Jude Childrens Research Hospital Institutional Review Board. Mexican family members provided signed informed consent with approval from the local ethics and scientic committees (Comite de Bioetica, Instituto Nacional de Cancerologa, with: Reg. no. COFEPRIS 103300538X0301, Ofce for Human Research Protections IORG0006100). We also obtained St Jude Institutional Review Board approval to use genetic material from this Mexican family.

Genotyping of TP53DNA was isolated from peripheral blood lymphocytes and tumor tissue by standard procedures. TP53 exons 2 through 11, including each exons anking intron sequence, were amplied by PCR and sequenced on a high-throughput 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Two highly informative polymorphic markers, VNTRp53 and p53(CA)n, were also amplied by PCR. VNTRp53 is a pentanucleotide repeat (AAAAT)n within intron 1 of the human TP53 gene,25 whereas p53(CA)n is a dinucleotide repeat polymorphism located 30 kb upstream of the 50 start site of the gene.26 The forward oligonucleotide primers for these two markers were labeled with uorescent dye (FAM). Genomic DNA was amplied in a 15-ml reaction mixture consisting of 9 ml of True Allele PCR

Premix (Applied Biosystems), 1 ml of primer mix (5 mM each), 4 ml of water and 1 ml of genomic DNA (50 ng). The PCR reaction mixture was denatured

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Figure 7. Schematic representation of TP53 exon 4. In this representation, all germline TP53 mutations included were registered at IARC database.27 Upper: complex mutations, including insertions and deletions. Down: TP53 point mutations including missense or nonsense mutations. Number 1 indicates HON001 family and number 2 indicates MEX001 family.

for 12 min at 95 1C, followed by a 10-cycle program (94 1C for 15 s, 55 1C for 30 s and 72 1C for 30 s), a 20-cycle program (89 1C for 15 s, 55 1C for 15 s and 72 1C for 30 s) and a nal 10-min extension at 72 1C. Genescan 400HD ROX size standard (Applied Biosystems) and 2 ml of PCR products were added to 24 ml of formamide (Invitrogen, Carlsbad, CA, USA). The mixture was separated by capillary electrophoresis in a 3730xl DNA Analyzer. Data were analyzed by using GeneMapper v4.0 software (Applied Biosystems). The same procedure was used to amplify a short fragment of exon 4 containing the mutation. Forward primer was also labeled with FAM. LOH was assessed by comparing the peak height for each allele amplied (188 bp corresponded to the WT allele and 195 bp to the mutated allele) in tumor versus blood DNA. Homozygosity was dened as the identication of only one allele in both blood and tumor tissue. Heterozygosity was dened as an allelic imbalance ratio of 0.5:2; a ratio o0.5 or 42.0 was dened as LOH.

Genetic prolingGenetic proling analysis used the 15 single tandem repeat loci included in the PowerPlex 16 kit (Promega, Madison, WI, USA). A multiplex PCR reaction was performed using uorescent dye-linked primers and two-color detection according to the manufacturers manual. The amplied PCR products were separated by capillary electrophoresis on a 3730xI DNA Analyzer and analyzed by using GeneMapper software.

Multiplex ligation-dependent probe amplicationMLPA of genomic blood and tumor DNA (100 ng) was performed by using the SALSA P056 TP53 MLPA Kit (MRC Holland, Amsterdam, the Netherlands), which contains probes for each of the 11 exons of the TP53 gene on 17p13.1 and several probes for nearby exons telomeric and centromeric of the TP53 gene. MLPA reactions were performed as recommended by the manufacturer. All fragments were separated by capillary electrophoresis on the 3730xl DNA Analyzer and analyzed by using GeneMapper software. CNV (that is, copy number that deviated from the population median) was measured by normalizing the target probe to all reference probes used in the run. The mean and standard deviation values for the probe of interest were calculated by using a panel of 12 unaffected blood donors (HRC-1 panel, Sigma, St Louis, MO, USA) to establish the z score for the tumor samples. We found that the peak height provided the most consistent results. Normal peaks showed a 0.8:1.2 ratio with normal controls, deletions showed a ratio o0.7, and duplications showed a ratio 41.3. A heat map was created by using the Spotre DecisionSite for Functional Genomics software (Tibcoo Spotre, Somerville, MA, USA).

Analysis of secondary structureDNA folding was analyzed by using the DNA Mfold server (http://mfold.rna.albany.edu

Web End =http://

http://mfold.rna.albany.edu

Web End =mfold.rna.albany.edu 12) with DNA free energy parameters. Folding was predicted at 37 1C.

Immunohistochemical detection of p53 protein Immunohistochemical detection of p53 expression was performed on deparafnized tissue sections from ACT, sarcoma and breast tumors by using the avidin--biotin complex method. Slides were incubated with monoclonal antibodies to p53 protein (DO-7; Dako, Carpinteria, CA, USA; dilution 1:50). Staining was classied as negative (no staining), weak (up to 25% of cells stained), moderate (26--70% of cells stained) or strong (more than 70% of cells stained); cellular localization was classied as cytoplasmic or nuclear.

CONFLICT OF INTEREST

The authors declare no conicts of interest.

ACKNOWLEDGEMENTS

We thank Sharon Naron and David Galloway for reviewing and editing the manuscript content and Dr Jesse Jenkins for immunohistochemical analysis. This work was supported in part by grant CA-21765 from the National Institutes of Health (US Department of Health and Human Services), by a Center of Excellence grant from the State of Tennessee, and by the American Lebanese Syrian Associated Charities (ALSAC).

REFERENCES

1 Malkin D, Li FP, Strong LC, Fraumeni Jr JF, Nelson CE, Kim DH et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 1990; 250: 1233 -- 1238.

2 Ribeiro RC, Sandrini F, Figueiredo B, Zambetti GP, Michalkiewicz E, Lafferty AR et al. An inherited p53 mutation that contributes in a tissue-specic manner to pediatric adrenal cortical carcinoma. Proc Natl Acad Sci USA 2001; 98: 9330 -- 9335.

3 Varley JM. Germline TP53 mutations and Li-Fraumeni syndrome. Hum Mutat 2003; 21: 313 -- 320.

4 Tabori U, Shlien A, Baskin B, Levitt S, Ray P, Alon N et al. TP53 alterations determine clinical subgroups and survival of patients with choroid plexus tumors. J Clin Oncol 2010; 28: 1995 -- 2001.

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8

5 Seidinger AL, Mastellaro MJ, Fortes FP, Assumpcao JG, Cardinalli IA, Ganazza MA et al. Association of the highly prevalent TP53 R337H mutation with pediatric choroid plexus carcinoma and osteosarcoma in Southeast Brazil. Cancer 2011; 117: 2228--2235.

6 Brown CJ, Lain S, Verma CS, Fersht AR, Lane DP. Awakening guardian angels: drugging the p53 pathway. Nat Rev Cancer 2009; 9: 862 -- 873.

7 Figueiredo BC, Sandrini R, Zambetti GP, Pereira RM, Cheng C, Liu W et al. Penetrance of adrenocortical tumours associated with the germline TP53 R337H mutation. J Med Genet 2006; 43: 91 -- 96.

8 Ognjanovic S, Oliver M, Bergemann TL, Hainaut P. Sarcomas in TP53 germline mutation carriers: a review of the IARC TP53 database. Cancer (e-pub ahead of print 11 August 2011; doi:10.1002/cncr.26390).

9 Varley JM, McGown G, Thorncroft M, James LA, Margison GP, Forster G et al. Are there low-penetrance TP53 Alleles? Evidence from childhood adrenocortical tumors. Am J Hum Genet 1999; 65: 995 -- 1006.

10 Birch JM, Hartley AL, Tricker KJ, Prosser J, Condie A, Kelsey AM et al. Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Res 1994; 54: 1298 -- 1304.

11 Taja-Chayeb L, Vidal-Millan S, Gutierrez-Hernandez O, Trejo-Becerril C, Perez-Cardenas E, Chavez-Blanco A et al. Identication of a novel germ-line mutation in the TP53 gene in a Mexican family with Li-Fraumeni syndrome. World J Surg Oncol 2009; 7: 97.

12 Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003; 31: 3406 -- 3415.

13 de la Chapelle A, Wright FA. Linkage disequilibrium mapping in isolated populations: the example of Finland revisited. Proc Natl Acad Sci USA 1998; 95: 12416 -- 12423.

14 Pinto EM, Billerbeck AE, Villares MC, Domenice S, Mendonca BB, Latronico AC. Founder effect for the highly prevalent R337H mutation of tumor suppressor p53 in Brazilian patients with adrenocortical tumors. Arq Bras Endocrinol Metabol 2004; 48: 647 -- 650.

15 Juarez-Cedillo T, Zuniga J, Acuna-Alonzo V, Perez-Hernandez N, Rodriguez-Perez JM, Barquera R et al. Genetic admixture and diversity estimations in the Mexican Mestizo population from Mexico City using 15 STR polymorphic markers. Forensic Sci Int Genet 2008; 2: e37 -- e39.

16 Matamoros M, Pinto Y, Inda FJ, Garcia O. Population genetic data for 15 STR loci (Identiler kit) in Honduras. Leg Med (Tokyo) 2008; 10: 281 -- 283.

17 Li FP, Fraumeni Jr JF. Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Intern Med 1969; 71: 747 -- 752.

18 Li FP, Fraumeni Jr JF, Mulvihill JJ, Blattner WA, Dreyfus MG, Tucker MA et al. A cancer family syndrome in twenty-four kindreds. Cancer Res 1988; 48: 5358--5362.

19 Frebourg T, Abel A, Bonaiti-Pellie C, Brugieres L, Berthet P, Bressac-de Paillerets B et al. [Li-Fraumeni syndrome: update, new data and guidelines for clinical management]. Bull Cancer 2001; 88: 581 -- 587.

20 Steels E, Paesmans M, Berghmans T, Branle F, Lemaitre F, Mascaux C et al. Role of p53 as a prognostic factor for survival in lung cancer: a systematic review of the literature with a meta-analysis. Eur Respir J 2001; 18: 705 -- 719.

21 Chappuis PO, Estreicher A, Dieterich B, Bonnefoi H, Otter M, Sappino AP et al. Prognostic signicance of p53 mutation in breast cancer: frequent detection of non-missense mutations by yeast functional assay. Int J Cancer 1999; 84: 587 -- 593.

22 Nagai MA, Schaer Barbosa H, Zago MA, Araujo Silva Jr W, Nishimoto IN, Salaorni S et al. TP53 mutations in primary breast carcinomas from white and African-Brazilian patients. Int J Oncol 2003; 23: 189 -- 196.

23 Dansonka-Mieszkowska A, Ludwig AH, Kraszewska E, Kupryjanczyk J. Geographical variations in TP53 mutational spectrum in ovarian carcinomas. Ann Hum Genet 2006; 70: 594 -- 604.

24 Vegran F, Boidot R, Oudin C, Defrain C, Rebucci M, Lizard-Nacol S. Association of p53 gene alterations with the expression of antiapoptotic survivin splice variants in breast cancer. Oncogene 2007; 26: 290 -- 297.

25 Hahn M, Fislage R, Pingoud A. Polymorphism of the pentanucleotide repeat d(AAAAT) within intron 1 of the human tumor suppressor gene p53 (17p13.1). Hum Genet 1995; 95: 471 -- 472.

26 Jones MH, Nakamura Y. Detection of loss of heterozygosity at the human TP53 locus using a dinucleotide repeat polymorphism. Genes Chromosomes Cancer 1992; 5: 89 -- 90.

27 Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P et al. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat 2007; 28: 622 -- 629.

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