SHORT COMMUNICATION
Richard Hamelin . Fernande Barichard . Isabelle Henry Claudine Junien · Gilles Thomas
Single base pair germ-line deletion in the p53 gene in a cancer predisposed family
Received: 15 November 1993
Abstract A family with an aggregation of adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, and early onset breast cancer was referred to our laboratory. Be- cause this aggregation was reminiscent of Li-Fraumeni syndrome, germ-line mutation of the p53 tumor suppres- sor gene was sought in the DNA of two affected members. The highly conserved regions spanning exons 5 to 8 of the p53 gene were screened by a previously validated de- naturing gradient gel electrophoresis method. A single base pair deletion at codon 215 was detected in constitu- tional DNA of the two patients, and in the DNA extracted from an adrenocortical carcinoma tumor specimen of the propositus. This deletion is predicted to lead to the forma- tion of a truncated p53 protein, a relatively rare event in Li-Fraumeni families. The spectrum of tumors observed in this family does not differ markedly from the spectrum observed in families with missense p53 mutations.
Introduction
Li-Fraumeni syndrome (LFS) is a dominantly inherited disease in which affected individuals are predisposed to develop malignancies at unusually early ages (Li and Fraumeni 1969). The spectrum of cancers associated with this syndrome was initially described as including mainly soft tissue sarcoma, oesteosarcoma, brain tumors, leuke- mia, adrenocortical tumors, and pre-menopausal breast cancer (Li et al. 1988). In a subset of LFS families, germ- line mutations of the p53 gene have also been reported to be associated with the disease (Malkin et al. 1990; Santi-
banez-Koref et al. 1991; Srivastava et al. 1990). Recent- ly, germline p53 mutations have been found in familial aggregations of cancer that diverge from the initial de- scription of LFS (Eeles et al. 1993; Prosser et al. 1992; Metzger et al. 1991). It is therefore important to obtain additional data in order to achieve a precise description of the phenotypic consequences of the presence of the p53 mutation in affected families. We report one such family.
Patients and methods
Patients
The following family was brought to our attention (Fig. 1). The propositus presented at age 12 with adrenocortical carcinoma. In his family, his brother developed a rhabdomyosarcoma at 6 years of age and an osteosarcoma at 11, his mother developed breast cancer at age 34 (and died at 36), his maternal grand-mother died of breast cancer at age 49 (age of initial diagnosis unknown), and one of his maternal uncle had hepatic cancer at age 36. Blood sam- ples were obtained from the propositus and his brother, and tumor tissue from the propositus.
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R. Hamelin . Gilles Thomas ☒ Laboratoire de Génétique des Tumeurs, Institut Curie,
26 Rue d’Ulm, F-75231 Paris Cedex, France
F. Barichard . I. Henry . C. Junien INSERM U383, Hôpital Necker-Enfants malades, 149 Rue de Sèvres, F-75015 Paris, France
Denaturing gradient gel electrophoresis
DNA was isolated by proteinase K digestion and phenol-chloro- form extraction according to standard protocols. DNA was ampli- fied in a DNA thermocycler (Perkin-Elmer Cetus) in a 25-ul reac- tion mixture containing 200 ng genomic DNA. DNA fragments amplified by the polymerase chain reaction (PCR) were analyzed by denaturing gradient gel electrophoresis (DGGE) (Fischer and Lerman 1983). Samples were loaded onto 6.5% polyacrylamide gel (acrylamide/bisacrylamide = 29/1) containing the appropriate denaturent gradient parallel to the direction of electrophoresis (100% denaturant corresponds to 7 M urea and 40% deionized formamide). Sequences of amplimers for exons 5 to 8, PCR con- ditions, gradient concentrations, and running conditions for each amplified DNA fragments were as described (Hamelin et al. 1993).
Single strand DNA presenting altered profiles in DGGE were synthesized by asymmetric PCR, purified from the remaining primers by centrifugation through Centricon-100 columns (Ami- con), and used as the template in a dideoxy-termination sequenc- ing procedure using a Sequenase version 2.0 kit (US Biochemi- cals).
Results
The familial aggregation of rhabdomyosarcoma, osteosar- coma, and early onset breast cancer observed in the fam- ily shown in Fig. 1 was reminiscent of the syndrome de- scribed by Li and Fraumeni (1969), and prompted us to search for the presence of a germ line p53 mutation. We took advantage of a previously validated screening meth- od for the detection of p53 mutations using DGGE (Hamelin et al. 1993). A total of five different PCR reac- tions performed on the DNA of the propositus and of his brother were used to screen the coding sequence con- tained within exons 5 to 8 and their corresponding splice junctions. One of the oligonucleotide sets showed an al- tered DGGE profile for both the propositus and his brother (Fig. 2). This set allowed the detection of muta- tions in two non-adjacent regions: from 50 bp upstream of the exon 5 acceptor site to the first 40 bp of exon 5, and from 20 bp upstream of exon 6 to 35 bp downstream of the exon 6 donor splice site. The abnormal DGGE profile was clearly different from that generated by a known polymorphism at position 213 (Carbone et al. 1991) (Fig. 2). When amplified with a set of primers specific for exon 5, these DNAs did not demonstrate abnormal behavior, thus indicating that the mutation was located in exon 6 or in the flanking intronic sequences.
Direct sequencing after asymmetric PCR of p53 exon 6 from DNAs of peripheral lympocytes from the propositus and from his brother demonstrated a single base deletion (Fig. 3). Indeed, both sequence ladders were normal until the guanosine at the second position of codon 215. After this position, a superposition of two sequences was evi- dent and could be derived from one another by a single base pair shift. This shift was attributable to a thymidine deletion at the third position of codon 215, a deletion leading to a termination codon at position 246 in exon 7. When an identical protocol was performed on the DNA extracted from the adrenocortical carcinoma of the pro- positus, the normal sequence was barely visible, suggest-
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ing that the normal p53 allele was deleted in the cancer cells. No blood or tumor samples were available from other members of the family.
Discussion
Germ-line nonsense or frame shift mutations in the coding region of the p53 gene have previously been reported on five occasions. Tumors developing in the corresponding affected patients were reported to be osteosarcoma, lung cancer, breast cancer, and fibrous histiocytoma (Togu- chida et al. 1992), and adrenocortical carcinoma (Same- shima et al. 1992). A large variety of tumours was also observed in the relatives of these cases. We report, for the
first time, the development of a rhabdomyosarcoma in a patient with a germ-line frameshift mutation. All these tu- mor types have been observed in families with missense p53 mutations, suggesting that patients in which the germ line p53 mutation leads to a truncated p53 protein do not demonstrate a markedly different spectrum of tumor pre- disposition.
Acknowledgements This work was supported by grants from the Ligue Nationale contre le Cancer and from the Institut Curie. We thank Dr. L. Lerman for providing the Melt87 and SQHTX com- puter programs, and Drs. L. Brugiere (Institut Gustave Roussy, Villejuif) and J-L. Chaussain (Hôpital Saint-Vincent de Paul, Paris) for providing blood samples and the tissue specimen.
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