Familial Cancer DOI 10.1007/s10689-014-9754-z

ORIGINAL ARTICLE

Further evidence for pathogenicity of the TP53 tetramerization domain mutation p.Arg342Pro in Li–Fraumeni syndrome Anna Etzold • Julia C. Schro¨der • Oliver Bartsch Ulrich Zechner • Danuta Galetzka

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Ó Springer Science+Business Media Dordrecht 2014

Abstract Li–Fraumeni syndrome (LFS) is a rare genetic disease with a highly significant predisposition to multiple early-onset neoplasms. These neoplasms include adrenocortical carcinoma, sarcoma, leukemia and CNS tumors in children and sarcoma, breast cancer and lung cancer in adults. LFS is inherited in an autosomal dominant manner. In most patients germline mutations in the tumor suppressor gene TP53 are found. As the majority of known mutations affect the DNA-binding domain of the p53 protein, there are only a few case reports showing the clinical presentation of mutations outside of this mutational hotspot. Here we present a family with a typical LFS pedigree with patients suffering from early-onset lung cancer, bilateral breast cancer and osteosarcoma. TP53 sequence analysis of the index patient revealed the germline mutation c.1025G [ C in a heterozygous state, resulting in an amino acid exchange from arginine to proline (p.Arg342Pro) in the tetramerization domain of p53. Using DNA from an old bedside blood typing test, the same mutation was found in the mother of the index patient, who had died of breast cancer 29 years ago. In conclusion, we provide evidence for the co-segregation of a TP53 tetramerization domain mutation and cancer phenotypes, but also report pre-symptomatic mutation carriers Anna Etzold and Julia C. Schro¨der have contributed equally to this work. A. Etzold (&)
J. C. Schro¨der
O. Bartsch
U. Zechner (&)
D. Galetzka Institute of Human Genetics, University Medical Centre of the Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany e-mail: [email protected] U. Zechner e-mail: [email protected]

within the family. We review published recommendations for clinical management and surveillance of high-risk members in Li–Fraumeni kindreds. Keywords Li–Fraumeni syndrome
Germline TP53 mutation
p53 Tetramerization domain
Cancer predisposition syndrome
LFS management
Presymptomatic testing

Introduction Li–Fraumeni syndrome (LFS) is a rare hereditary cancer predisposition disorder displaying a wide clinical heterogeneity. It is characterized by early-onset tumors [1], often occurring syn- or metachronously [2–4] in one individual and several affected individuals within the family. The most common cancers typically diagnosed in pediatric LFS patients are adrenocortical carcinoma, soft tissue sarcoma, leukemia and tumors of the central nervous system. In adulthood affected individuals most frequently suffer from osteosarcoma, breast cancer and lung carcinoma [5, 6]. Within the past decade ‘‘Chompret criteria’’ [7, 8] were developed and revised to facilitate clinical diagnosis of LFS and to define LFS-like syndrome (LFL), which resembles the cancer predisposition phenotype of LFS but differs in family history characteristics. Both LFS and LFL are transmitted in an autosomal dominant manner with high penetrance [9]. In 1990 germline mutations in the gene TP53 could be established to be the genetic cause of LFS. Although such mutations are only found in 77 % of classical LFS and in 40 % of LFL cases [9], the contribution of other genes, e.g. CHEK2, could not be confirmed to date. The gene TP53 encodes a nuclear phosphoprotein which in many ways acts as a tumor suppressor. It is known to often be

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Fig. 1 Pedigree of the studied Li–Fraumeni family. Cancer affected individuals are marked with filled symbols and diagnoses are given with age of onset. Current ages or death ages (d) are given as known. Results of mutational analysis are given as ± for familial mutation (p.Arg342Pro) present in heterozygous state and -/- for familial mutation not detected. LuCa lung cancer, BrCa breast cancer, OS osteosarcoma

mutated in cancer development. p53 activity is usually reduced in tumor material of LFS patients by alterations of both alleles: a germline mutation is coupled with one developed at the somatic level. Germline as well as somatic mutations in TP53 are mainly found in exons 5–8 [10], with a mutation hotspot in exon 8 [11]. These mutations hit the DNAbinding domain (DBD). Meanwhile, however, several in vitro studies [12–15] and some clinical reports [16–18] have also shown that mutations in the tetramerization domain (TD) of p53 may have a destructive effect on protein functionality and are of pathological relevance. Herein, we describe a family matching the revised Li– Fraumeni Chompret criteria. The index case and his mother are carriers of the germline mutation, p.Arg342Pro, in the tetramerization domain of p53. We provide first evidence for co-segregation of this mutation and disease, and discuss the related clinical phenotypes. Moreover, we review published recommendations for clinical management and surveillance of high-risk family members in Li–Fraumeni kindreds.

Materials and methods Case presentation A young man (index patient, Fig. 1III: 5) was seen for genetic counselling after he had been diagnosed with an

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osteosarcoma of the right os ilium at the age of 28 years. The histological analysis of the tumor biopsy showed a highgrade osteosarcoma with mainly chondroblastic, partly osteoblastic and focal fibroblastic differentiation. Neoadjuvant chemotherapy in accordance with the European and American Osteosarcoma Study Group (EURAMOS-1) was initiated and he subsequently received hemipelvectomy and reconstructive plastic surgery. After surgery, adjuvant chemotherapy was reinitiated and in the course of time he recovered well. Staging examinations yielded no indication for metastases of the lung or other organs. Family anamnesis revealed that the index patient’s mother (Fig. 1II: 2) had been affected by bilateral invasiveductal breast cancer with osseous and hepatic metastases. She had been treated by radiotherapy of the vertebrae, bilateral mastectomy, chemotherapy and anti-oestrogen therapy, but died a few months after diagnosis in 1985 at the age of 30 years. The deceased has an older brother who has two sons, 39 and 36 years of age—all of them were not affected by cancer. The father (Fig. 1II: 1) of the index patient was not affected by cancer. The maternal mother (Fig. 1I: 2) died from lung cancer in 1958 at 40 years of age without having been a smoker at any time during her life. The index patient has three brothers 37, 36 and 32 years of age (Fig. 1III: 2/3/4), all having no signs of cancer. The oldest brother has two healthy daughters at an age of 3 years and 2 months (Fig. 1IV: 1/2). All three brothers of the index patient decided to undergo predictive genetic testing. As the family met clinical criteria, we diagnosed Li– Fraumeni syndrome in the index patient and a mutation analysis of the TP53 gene was initiated. DNA isolation In the medical files of the index patient´s deceased mother an old bedside test (SerafolÒ, BIOTEST-SERUM-INSTITUT GmbH) was found, that had been used to confirm her blood type before a blood transfusion in 1984. Such paper card tests are used by mixing patient´s whole blood with antibodies against A and respectively B blood type surface antigens onto the test areas of the paper cards. By observing agglutination the blood type of the patient can be concluded. DNA from the bedside test was isolated using the QIAmpÒ DNA Mini Kit (Qiagen) using the enclosed protocol ‘‘DNA Purification from Dried Blood Spots’’ adapted to the special requirements of the material. Briefly, the cardboard holding the dried blood was cut into small pieces and lysed with 600 ll of buffer ATL per test. During the following procedure volumes of reagents were increased accordingly and incubation times were extended until complete dissolution of blood. Frequent vortexing was performed to avoid adhesion of board snippets. The cell lysates of both test fields were combined on one column and

TP53 tetramerization domain mutation p.Arg342Pro

finally DNA was eluted with 30 ll prewarmed buffer AE. DNA from blood samples was extracted using the GentraÒ PuregeneÒ Blood Kit (Qiagen) according to the manufacturer’s protocol. DNA extraction from saliva was performed using the OrageneÒ DNA Purifier (DNA Genotek). Sequence analysis The TP53 gene was analyzed for aberrations referring to the NCBI reference sequence NM_001126114.2. In detail, intronic primers tagged with M13 were used to amplify the coding sequence plus flanking regions. Cycling conditions were 95 °C for 10 min, 34 cycles of 95 °C for 30 s, 59 °C for 30 s and 72 °C for 40 s and finally 72 °C for 5 min. Primer sequences used for exon 10 were: TP53-Ex10_F: tgtaaaacgacggccagtTCAAACAATTGTAACTTGAACCAT C and TP53-Ex10_R: caggaaacagctatgaccGAAGGCAGGA TGAGAATGGA. A direct bi-directional cycle sequencing reaction was performed and resulting products were analyzed on an automated capillary sequencer (Beckman Coulter CEQTM 8000 Genetic Analysis System). Sequences were examined using Mutation Surveyor, version 3.2 (SoftGenetics, State College, Pa., USA). The pathological potential of the found genetic alteration was assessed by means of literature and database search.

Results The molecular analysis of blood of the index patient revealed the heterozygous base exchange c.1025G [ C in exon 10 of the TP53 gene. The resulting alteration CGA [ CCA at codon 342 causes a substitution of arginine by proline (Fig. 2) and affects the tetramerization domain of the protein. Family history suggested that this missense mutation was passed on to the index patient by his mother (Fig. 1II: 2) who died 29 years ago on bilateral breast cancer. Indeed, the mutation could also be detected in the mother’s DNA extracted from a 30-year-old bed side test found in her medical record folder. No sample of the proband´s grandmother (Fig. 1I: 2), who had died from lung cancer in the 1950s, was available for analysis. Three brothers (Fig. 1III: 2/3/4) and two nieces (Fig. 1IV: 1/2) of the proband, all having no history of cancer to date, underwent presymptomatic testing. The familial mutation could be detected in all cases except for one of the index patient´s brothers (Fig. 1III: 3).

Discussion Germline mutations in TP53 are the only well-established genetic cause of Li–Fraumeni syndrome. Referring to the

Fig. 2 Sanger sequencing result from exon 10 of TP53 of index patient III:5. Analysis shows heterozygous base exchange G [ C at position c.1025. This affects codon 342 resulting in a change of amino acid arginine (R) to proline (P) p.Arg342Pro. aa amino acid

‘‘International Agency for Research on Cancer TP53 Mutation database’’, 636 germline mutations in the TP53 gene are recognized up to date (R17, status of November 2013) [19]. The majority of reported mutations affect the DBD [19]. This domain consists of 199 residues and covers the major part of the protein, which in sum consists of 392 amino acids. It is evident that DBD mutations alter the ability of the p53 protein to specifically recognize and bind its target sequences and to execute transcriptional regulation onto downstream genes. The TD of p53 consists of only 30 amino acids. Although the most common TP53 mutation ever described, p.Arg337His, is located in this domain, other TD mutations are infrequent and their functional effects were widely under-researched and neglected in the past [20]. In the meantime, it has become well-established that protein tetramerization is essential for p53-mediated tumor suppressor activity in a multilayered manner. One reason is that each subunit of the p53 tetramer recognizes five nucleotides of the 20 nucleotide long DNA target sequence, and therefore steric proximity of all four subunits is essential for specific binding [21]. Furthermore, only p53 oligomers allow acetylation of C-terminal lysines, which plays a critical role in protein stability and transcriptional activity of p53 [22]. The same is also true for other types of post-translational modifications such as phosphorylation and ubiquitination [23–25]. The germline mutation c.1025G [ C (p.Arg342Pro) described in this report affects a residue that is significantly conserved throughout mammals [20] and part of an alphahelix motif of p53 in the TD. An amino acid exchange from arginine to proline is very likely to destabilize the protein structure of the p53 protein, as proline is a known helix breaker. This is supported by in vitro studies that showed

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that introduction of a proline at this position completely eliminates tetramer formation leading to affected peptides only existing as unfolded monomers [14]. Accordingly, a comprehensive yeast-based functional study conducted by Kato et al. [12, 13] as well as functional in vitro assays with mutated protein by Rollenhagen et al. demonstrated that changing p.Arg342 residue results in a functional inactivation of p53 and therefore implicate its pathogenicity. The described Li–Fraumeni syndrome family has a recorded cancer history that extends three generations and consists of early onset lung cancer, bilateral breast cancer, and osteosarcoma. Revised Chompret criteria are fully fulfilled as all occurred malignancies belong to the LFS spectrum and disease onset was in the late second to mid third decade of life [8]. The missense change p.Arg342Pro in the TP53 gene identified in our index patient has been reported only once before in a Polish family with LFS. The pedigree of the Polish family showed early onset LFStypical cancer types including female reproductive organ cancer, leukemia, and osteosarcoma. In this family pediatric neoplasms occurred, namely a synovial sarcoma at the age of 8 and an osteosarcoma at 12 years of age in one individual. In the study of Fiszer-Maliszewska et al. [16] it was assumed that the p.Arg342Pro mutation was causally related to the Li–Fraumeni phenotype, but co-segregation of mutation and disease could not be shown because of lack of family members available for testing. In our study, we succeeded to extract and analyze old genomic DNA from a blood typing bedside test of the index patient’s mother who had died 29 years ago of bilateral breast cancer. The molecular study showed the presence of the p.Arg342Pro mutation and therefore confirmed the co-occurrence of mutation and disease in the index patient and a second individual in that family. In summary, the reported together with the previous clinical case and experimental data dispel any doubts that the germline TP53 mutation p.Arg342Pro is pathogenic and causative for cancer susceptibility associated with LFS in the presented family. For the affected cancer patient, treatment implications that arise from knowledge of the mutation mainly consist of avoidance of DNA-damaging agents. Necessity of radiation therapy should be carefully evaluated as the risk of developing a second malignancy after radiotherapy in LFS patients is about 57 % [4]. A major challenge that results from genetic confirmation of clinical LFS diagnosis is the management of unaffected relatives who also are mutation carriers. The exceptional high life time cancer risk for TP53 mutation carriers [26] and the need for close monitoring gave reason for predictive genetic testing of all three healthy brothers of the index patient. Two of them were tested positive for the pathogenic mutation and were advised to enroll in a closely

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monitored surveillance program according to consensusbased screening guidelines published by the National Comprehensive Cancer Network (NCCN) and other scientific recommendations [27]. These include comprehensive annual physical examination including careful skin and neurologic examinations, abdominal and pelvic ultrasound examination and differential blood count half yearly. Furthermore, for pre-symptomatic detection of tumors a rapid whole-body MRI and brain MRI should be performed once a year. For female mutation carriers annual clinical examination of the breast as well as mammography and MRI of the breasts were recommended starting at the age of 20 years. The lack of clearly defined guidelines made it necessary to discuss the question of pre-symptomatic testing of the index patient’s 3-year old and newborn nieces, in particular as the familial mutation was also detected in a minor child of the Polish family that had already developed neoplasms. Even though it is known that the risk for a LFS individual to develop a malignancy before adulthood is as high as 20 % [11], this question is still discussed controversially in the literature [28, 29]. Critics argue that testing does not lead to any evidently proven benefit in terms of reduction of morbidity or mortality that would give reason to curtail the minor’s autonomy and its right for self-determination [28]. However, there is data that impressively, even though not representatively, show that a surveillance program in minor and adult LFS individuals may impact positively the medical history and may increase survival chances [27, 30, 31]. In addition, first experiences in predictive LFS testing during childhood have not shown any deleterious psychosocial consequences neither in the child nor in the parents [29, 32]. In conclusion, we have presented a case report that adds evidence to the pathogenicity of the rare TP53 tetramerization domain mutation p.Arg342Pro. Moreover, this case impressively shows that genetic analysis of ancient medical material alienated from its original purpose can be a possibility to overcome the lack of living affected relatives, in cases where co-segregation has to be proved for diagnostic purposes. Acknowledgments The authors wish to thank the patient’s family for consent to share their case with the scientific community. This study was supported by the FAZIT-STIFTUNG Gemeinnu¨tzige Verlagsgesellschaft mbH. We thank Prof. Dr. Susann Schweiger and Dr. Dennis Strand for careful and critical reading of the manuscript. Conflict of interest of interest.

The authors declare that they have no conflict

Ethical standards All patients gave their informed consent prior to their inclusion in that report. Molecular genetic testing was performed as a clinical service according to the ethical guidelines of the institution (University Medical Center Mainz).

TP53 tetramerization domain mutation p.Arg342Pro

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