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Japanese Journal of Clinical Oncology Pages 513-516

Germline p53 Mutation in a Case of Li-Fraumeni Syndrome Presenting Gastric Cancer
Case Report And Genetic Analysis
Methods For Mutation Detection
Acknowledgment
References
Germline p53 Mutation in a Case of Li-Fraumeni Syndrome Presenting Gastric Cancer

Germline p53 Mutation in a Case of Li-Fraumeni Syndrome Presenting Gastric Cancer

Kokichi Sugano1,2, Takahiro Taniguchi2, Morihiro Saeki3, Yukiko Tsunematsu4, Utano Tomaru5 and Tadakazu Shimoda5

1Division of Gene Testing and DNA Analysis, Tochigi Cancer Center, Tochigi, 2Clinical Laboratory Division, National Cancer Center Hospital, Tokyo, 3Department of Surgery, National Children's Hospital, Tokyo, 4Department of Hematology, National Children's Hospital, Tokyo and 5Department of Pathology, National Cancer Center Hospital, Tokyo, Japan

Disorder: Li-Fraumeni syndrome
Ethnicity of patients: Japanese
Gene: TP53
GenBank accession number: NM 000546
Chromosomal assignment: 17p13.1
Type of DNA variant: A germline missense mutation
Mutation: CGT (Arg, wild type) to CAT (His) substitution at codon 273 of the p53 gene
Allelic frequency: Not tested
Method of mutation detection: PCR/direct sequencing

Key words: p53 - missense mutation - Li-Fraumeni syndrome - gastric cancer - hepatosarcoma

CASE REPORT AND GENETIC ANALYSIS

Li-Fraumeni syndrome is a relatively rare disease entity characterized by sarcomas of the soft tissues, bone and miscellaneous tumors of juvenile onset and frequent occurrence of metachronous tumors (1). Germline mutation of the p53 tumor suppressor gene has been reported in approximately 70% of the Li-Fraumeni kindreds (2). Here we report a case of Li-Fraumeni syndrome presenting a gastric cancer in the proband and a hepatosarcoma in his son.

The proband was a 38-year-old Japanese male referred to a clinic with a diagnosis of gastric carcinoma with multiple liver metastases. There was no previous history of malignant disorders, but his 11-year-old son suffered from a liver tumor diagnosed as a hepatosarcoma and underwent surgical resection followed by systemic chemotherapy (Fig. 1). The proband received systemic chemotherapy comprising seven courses of 5FU and MTX. However, the disease was progressive during the course. The proband died 3 months after admission and was autopsied.


Figure 1. Gene testing revealed the proband and two sons were heterozygous [E+ (mut/wt)] for the p53 germline mutation and his mother and sister were homozygous for the wild-type p53 gene (E-). `E' stands for Evaluation and is used to represent clinical and/or test information on the pedigree. An arrow indicates the proband. HCC, hepatocellular carcinoma. An asterisk indicates the kindreds examined for the p53 gene mutation at the National Cancer Center Hospital. The pedigree was constructed according to the recommendations of the Pedigree Standardization Task Force of the National Society of Genetic Counselors (3).

The family history of pediatric cancer and the relatively young onset of the gastric carcinoma prompted genetic analysis of the p53 gene with suspicion of Li-Fraumeni syndrome. PCR/direct sequencing analysis revealed a germline missense mutation in exon 8 at codon 273 of the p53 gene, causing a substitution of CAT (His) for CGT (Arg) (Fig. 2). Analyses of the primary tumor and liver metastasis showed overexpression of the p53 protein in immunohistochemistry (Fig. 3) and loss of heterozygosity (LOH) in the opposing allele carrying the wild-type p53 gene (Fig. 4). In segregation analysis of the kindred, the same mutation was detected in his two children. However, his mother and sister were negative for the germline p53 mutation (Fig. 1). His father died at 63 years of age due to a hepatocellular carcinoma (HCC) which was caused by viral hepatitis. Hence it is likely that the mutation was a new germline mutation arising at the proband. At autopsy, the tumor was limited to the stomach and the liver and there was no other tumor originating from the other organs.

So far, four cases of Li-Fraumeni syndrome have been reported showing the mutation at codon 273 of the p53 gene, i.e. two TGT (Cys), one GGT (Gly) and one CAT (His) mutation (5-8). To our knowledge, this is the second report of the missense mutation to CAT (His) at codon 273 in exon 8 of the p53 gene.


Figure 2. Direct sequencing of the PCR product using the forward primer revealed a missense mutation at codon 273 of the p53 gene, substituting CAT (His) for CGT (Arg).

   A
   B

Figure 3. H-E stain and immunostaining of the primary gastric cancer (a, b) and liver metastasis (c, d). (a) Histological diagnosis was a moderately differentiated adenocarcinoma of the stomach; (b) immunohistochemistry of the gastric cancer using an anti-p53 antibody (RSP53) showed intense nuclear staining of tumor cells; (c) histological diagnosis was a moderately differentiated adenocarcinoma that was compatible with the metastatic gastric carcinoma (a); (d) immunohistochemistry using the anti-p53 antibody (RSP53) showed overexpression of the p53 protein at cell nuclei.


Figure 4. Germline mutation of the p53 gene and allelic status of the p53 locus. (a) PCR/SSCP analysis of the p53 gene exon 8. Arrowheads indicate mutated alleles. Signals from wild-type p53 allele were faint in tissues of gastric cancer and liver metastasis (lanes 2 and 3), indicating LOH in the allele carrying wild-type p53 gene. (b) Allelic status of the p53 locus was analyzed by blunt-end single-strand DNA conformation polymorphism analysis, using three polymorphic markers (Ex4, Int7 and Int1) in the p53 gene (4). In analysis of peripheral blood lymphocyte (PBL), the proband was heterozygous for all three markers (panel 1), while the analyses of gastric cancer tissue and liver metastasis indicate the allelic loss of the p53 locus (panels 2 and 3). An asterisk indicates the decreased signal derived from the lost allele.

METHODS FOR MUTATION DETECTION

PCR/direct sequencing was performed with the following conditions and parameters:

PCR primer, forward: 5[prime]CCT ATC CTG AGT AGT GGT AA3[prime]

PCR primer, reverse: 5[prime]CCA AGA CTT AGT ACC TGA AG3[prime]

Size of PCR product: 331 bp

Thermal cycle profile:

    Initial denaturation: 94°C, 5 min

    30 cycles of 94°C, 30 s/58°C, 30 s/72°C, 60 s

    Final extension: 72°C, 7 min

Sequencing primer: the same as the PCR primers.

Acknowledgment

This work was supported in part by Grants-in-Aid for Cancer Research and for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan.

References

1. Varley JM, Evans DG, Birch JM. Li-Fraumeni syndrome - a molecular and clinical review. Br J Cancer 1997;76:1-14. MEDLINE Abstract

2. Online Mendelian Inheritance in Man, OMIM (TM). Baltimore, MD: Johns Hopkins University 1999. MIM Number: 191170:1999.9.18. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim.

3. Bennett RL, Steinhaus KA, Uhrich SB, O'Sullivan CK, Resta RG, Lochner-Doyle D, et al. Recommendations for standardized human pedigree nomenclature. Am J Hum Genet 1995;56:745-52. MEDLINE Abstract

4. Sugano K, Tsutsumi M, Nakashima Y, Yamaguchi K, Ohkura H, Kakizoe T, et al. Diagnosis of bladder cancer by analysis of the allelic loss of the p53 gene in urine samples using blunt-end single-strand conformation polymorphism. Int J Cancer 1997;74:403-6. MEDLINE Abstract

5. Brugieres L, Gardes M, Moutou C, Chompret A, Meresse V, Martin A, et al. Screening for germ line p53 mutations in children with malignant tumors and a family history of cancer. Cancer Res 1993;53:452-5. MEDLINE Abstract

6. Kovar H, Auinger A, Jug G, Muller T, Pillwein K p53 mosaicism with an exon 8 germline mutation in the founder of a cancer-prone pedigree. Oncogene 1992;7:2169-73. MEDLINE Abstract

7. Eeles RA, Warren W, Knee G, Bartek J, Averill D, Stratton MR, et al. Constitutional mutation in exon-8 of the p53 gene in a patient with multiple primary tumours - molecular and immunohistochemical findings. Oncogene 1993;8:1269-76. MEDLINE Abstract

8. Frebourg T, Barbier N, Yan YX, Garber JE, Dreyfus M, Fraumeni J, et al. Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome. Am J Hum Genet 1995;56:608-15. MEDLINE Abstract

Cancer Genetics Report is a new section for JJCO and presents a brief, 1-2 page report focusing on genetic analysis of a case which merits publication. Specifically,

  1. Typically, the section reports a previously undescribed germline mutation or polymorphism of a gene which is associated with a cancer. A case report with known mutation or polymorphism may also be considered, if the report can be expected to contribute substantially to the advancement and/or accumulation of our knowledge in the field of clinical cancer genetics.
  2. The nucleotide sequence of the mutation or polymorphism must be defined on the genomic DNA.
  3. The method of the mutation/polymorphism detection should be described explicitly, such as PCR conditions and primer sequences.
  4. Whenever appropriate, a pedigree (family tree) must be presented. The pedigree should be drawn according to the `Recommendations for Standardized Human Pedigree Nomenclature', Am J Hum Genet 1995;56:745-52.
  5. The privacy of a patient and any other relevant family member(s) should be strictly protected in the manuscript and it is the responsibility of author(s) to obtain appropriate informed consent for publication.

A manuscript for Cancer Genetics Report should be prepared according to the Instructions to Authors for regular papers, except: (1) Running Head, Abstract and Mini-abstract are not required, (2) a genetic Summary should be provided describing Disorder, Ethnicity, Gene and its GenBank, EMBL or DDBJ accession number and Chromosomal assignment, Type of DNA variant, Mutation, Allelic frequency and Method of mutation detection, etc., (3) the text may have a minimum number of sections, but a Methods section is mandatory and (4) two copies of the manuscript with original figures should be sent to the Editorial Office with a covering letter signed by all co-authors.

Received August 10, 1999; accepted August 11, 1999
For reprints and all correspondence: Kokichi Sugano, Division of Gene Testing and DNA Analysis, Tochigi Cancer Center, 4-9-13 Yohnan, Utsunomiya, Tochigi 320-0834, Japan. E-mail: ksugano{at}tcc.pref.tochigi.jp

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification: 1 Dec 1999
Copyright© 1999 Foundation for Promotion of Cancer Research.
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