The First Documentation of Li-Fraumeni Syndrome in Korea
Yung-Jue Bang, M.D.,1),2) Shin-Hyeok Kang, M.D.,2) Tae-You Kim, M.D.,1) Chul-Won Jung, M.D.,1) Se-Min Oh, M.D.,3) Kuk Jin Choe, M.D.,3) Noe Kyeong Kim, M.D.,1),2)
1)Department of Internal Medicine,
2)Laboratory of Anticancer Drug Evaluation, Cancer Research Center, and
3) Department of Surgery, Seoul National University College of Medicine, Seoul, Korea
Li-Fraumeni syndrome(LFS) is an autosomal dominant disorder that predis- poses individuals to multiple forms of cancer including breast cancer, soft tissue sarcoma, brain tumor, osteosarcoma, leukemia, and adrenocortical carcinoma. Recently, germ-line mutation of the p53 tumor suppressor gene has been implicated in this familial disorder.
We report a case of a 25-year old woman who presented with bilateral breast cancer and uterine leiomyoma. Her mother had died of early-onset bilateral breast cancer. And her younger sister had breast carcinoma as well, which was identified at the age of 22, indicating her strong familial history. To test for the presence of the p53 germ-line mutation, we analyzed the genomic DNA from the peripheral blood of the proband and her sister by PCR-SSCP analysis of exon 5 through exon 8 of the p53 gene. As a result, a p53 mutation in exon 7 was detected in an allele, and it was shared with her sister as the same pattern. Sequencing analysis determined the altered nucleotide at codon 248(CGG>TGG) which is one of the most frequent mutation sites related to LFS. Therefore, this patient has the most consistent characteristic features of LFS phenotype and it is believed that this case is the first report of a family with Li-Fraumeni syndrome carrying the p53 germ-line mutation in Korea.
The abbreviations used are : LFS, Li-Fraumeni Syndrome ; PCR, polymerase chain reaction ; SSCP, single strand conformation polymorphism.
Key Words : Li-Fraumeni syndrome, Breast cancer, p53, Germ-line mutation, PCR-SSCP
INTRODUCTION
In 1969, Li and Fraumeni observed several families with an aggregation of specific cancer types such as childhood sarcoma and breast cancer, and sug-
gested a new familial cancer syndrome, Li-Fraumeni syndrome(LFS) (Li and Fraumeni, 1969). LFS is an autosomal dominantly inherited familial cancer syn- drome characterized by a high susceptibility to di- verse malignant tumors. The spectrum of cancers in the syndrome has been recognized to include breast cancer, soft tissue sarcoma, brain tumor, osteosarco- ma, leukemia, and adrenocortical carcinoma. These diverse tumor types in family members characteristi- cally develop at unusually early ages and multiple primary tumors are frequent(Malkin, 1993). To define
☒
Br 29,43
C-1
C-2
BBr, Lm 25
Br, 22
the genetic background of LFS, several efforts have been made to search for the candidate gene of LFS. In 1990, inherited mutation within a defined region of the p53 tumor suppressor gene was suggested to underlie the defect in families with LFS(Malkin et al., 1990 ; Srivastava et al., 1990).
We recently identified a breast cancer prone family, in which cancers occurred at early ages. Subsequent- ly, we also determined the presence of a germ-line mutation of the p53 tumor suppressor gene in the proband and her sister by PCR-SSCP and sequenc- ing. It is thought that this case displaying characteris- tic features of LFS was the first documentation of Li-Fraumeni syndrome with germ-line mutation of the p53 gene in Korea.
CASE
A 25-year-old woman was admitted to Seoul National University Hospital due to palpable bilateral breast masses. One year before, she first noticed a left breast mass. The size of the mass increased slowly and 11 months later, she felt another mass in the right breast. There were left axillary masses as well. Her mother had suffered from breast cancer at age 29 and died of the disease at age 43(Fig. 1). Her younger sister had also had unilateral breast cancer at the age of 22 and had received a modified radical mastectomy followed by adjuvant chemotherapy at another hospital. The patient was single and denied other illnesses. On examination, both breast masses were nontender, hard, and movable. The size of the right and left masses were about 2cmX2cm and 9cmX7cm, respectively. Two left axillary masses were hard and fixed, and 1cm×1cm in size. Abdo- minal palpation and pelvis ultrasonography suggested a large pelvic mass. Bone scintigraphy revealed no bony abnormality. She underwent bilateral modified radical mastectomy and total abdominal hysterectomy with bilateral salphingoophorectomy. Pathologic ex- amination showed infiltrating ductal carcinoma of bi- lateral breasts with metastasis to the left axillary lymph nodes and uterine leiomyoma.
MATERIALS AND METHODS
1. Tissue Samples and DNA Extraction
Blood samples were obtained from the proband, her sister, and two other young sarcoma patients with family history. Tested individuals in this study are listed in Table 1. The proband’s primary tissues and lymph nodes were also obtained at the time of surgery as follows : both breast carcinomas, both axillary lymph nodes, both ovaries, and uterus. Fresh tissues were immediately frozen in liquid nitrogen and stored
| No. | Key | Sex/Age | Cancer Types | Comments |
|---|---|---|---|---|
| 1 | Wt | M/25 | normal | |
| 2 | a | F/17 | chondrosarcoma | |
| 3 | b | F/15 | osteosarcoma | |
| 4 | c-1 | F/25 | breast carcinoma | proband |
| 5 | c-2 | F/22 | breast carcinoma | proband's sister |
| 6 | KG-1 | leukemic cell line | p53 point mutation in exon 6 | |
| 7 | HL-60 | leukemic cell line | partial deletion in the p53 |
| exon | PCR products(bp) | primer sequence |
|---|---|---|
| exon 5 | 325 | 5'-TTCCTCTTCCTGCAGTACTC-3 5'-GCAAATTTCCTTCCACTCGG-3' |
| exon 6 | 236 | 5'-ACCATGAGCGCTGCTCAGAT-3' 5'-AGTTGCAAACCAGACCTCAG-3' |
| exon 7 | 139 | 5'-GTGTTGTCTCCTAGGTTGGC-3' 5'-CAAGTGGCTCCTGACCTGGA-3' |
| exon 8 | 330 | 5'-CCTATCCTGAGTAGTGGTAA-3 5'-CCAAGACTTAGTACCTGAAG-3 |
at -70℃. High molecular weight DNA was isolated from blood and frozen tissues according to the standard protocol(Sambrook et al., 1989). As positive controls for p53 gene mutation, the DNA samples from KG-1 and HL-60, leukemic cell lines, were also used.
2. PCR-SSCP Analysis of p53
Portions of the p53 gene including exons 5 to 8 were amplified by polymerase chain reaction. The oligonucleotide primers used for amplification are shown in Table 2. Each amplification reaction was carried out in a 25 ul reaction volume containing 200 ng of template DNA, 10mM Tris-HCl(pH8.3), 1.5mM MgCl2, 50mM KCI, 10 pmol of each primer, deoxynucleoside triphosphates dATP, dGTP, dTTP, and dCTP at 200 uM each, 1 uCi of [x-p32] dCTP(3,000 Ci/mmol), and 1 unit of Taq
polymerase(Boehringer Mannheim, Germany). Reac- tion mixtures were subjected to 35 cycles of which each cycle consisted of denaturation at 95 ℃ for 40 sec, annealing at 55 ℃ or 60 ℃(for exon 7) for 1 min, followed by polymerization at 72 ℃ for 1 min. The PCR products were diluted 1:24 in loading solution(95% formamide, 10mM EDTA, 0.05% bro- mophenol blue, 0.05% xylene cyanol), denatured at 95 ℃ for 5 min and loaded onto 6% non-denaturing polyacrylamide gel with 10% glycerol. Samples were electrophoresed at room temperature for 3-4 hr at 30W(Orita et al., 1989). Alternative gel condition used for confirmative detection of gene mutation was 50% MDE gel(AT Biochem, Inc., Malvem, PA, US) with 0.6X TBE. The samples were run as described above except for the voltage condition of 20 V/cm. Gels were transferred to 3MM Whatman paper, dried and autoradiographed with X-ray film at -70℃ for 4-18 hr.
HL 60
Wt
c-1
c-2
c-1
c-2
KG 1
c-1
c-2
KG 1
HL 60
b
Wt
a
b
Wt
a
b
Wt
c-1
c-2
KG 1
HL 60
a
b
EXON 5
EXON 6
EXON 7
EXON 8
3. Sequencing Analysis
To confirm the SSCP results and determine the altered nucleotide sequence, the samples that were identified as mutants were reamplified by PCR and purified. The products were then subcloned into ddT-tailed vectors prepared as previously de- scribed(Holton and Graham, 1990). Sequencing analysis was performed using Sequenase kit(United States Biochemical, Cleveland, Ohio, US).
RESULTS
1. PCR-SSCP Analysis
We initially analyzed DNA samples from peripheral blood. On electrophoresis, mobility shifted bands were detected in the exon 7 of p53 in the samples from the proband and her sister. There was no mobility shift in the other exons. The DNAs from the other patients showed no mobility shift in the exons tests(Fig. 2). The samples with altered mobility band showed additional bands with normal mobility, suggesting the single allele germ-line mutation. This finding was confirmed with alternative gel condition(Fig. 3). We next ex- amined DNA samples from various surgical speci- mens from the proband. Mobility shifted bands for exon 7 were present in all the tissues tested, which showed the same pattern as peripheral blood(Fig. 4).
2. Sequencing Analysis
Sequencing analysis for two DNA samples with
Wt
c-1
c-2
KG 1
HL 60
a
b
Rt. Breast
Lt. Ax. LN
Uterine Mass
Wt-1
Wt-2
Lt. Breast
Rt. Ovary
Lt. Ovary
PB
TCGA TCGA
5>>9059999
T
C-2 C-1
altered mobility showed the base change of C to T at the first position in codon 248 of exon 7(Fig. 5). The resulting amino acid transition was arginine to tryp- tophan, which is one of the most common p53 mutations in LFS families.
DISCUSSION
Li-Fraumeni syndrome is a clinical diagnosis based on the aggregation of diverse forms of cancer includ- ing breast cancer in a proband, and two or more close relatives. Approximately 50% of cancers in reported Li-Fraumeni families occur before 30 years of age. In young adults, premenopausal breast can- cer is, by far, the most common neoplasm whereas soft tissue sarcoma and osteosarcoma are prevalent in children and in adolescence, respectively(Li et al., 1992). In our case, three members of the family had early-onset breast cancer(Fig.1) and presented a history that was consistent with LFS.
Human breast cancer occurs usually as a result of somatic mutation. However, hereditary breast cancer occurs and accounts for approximately 10% of all of breast cancer(Anderson, 1992). Four inherited types of breast cancer have been identified. Among them, LFS involves early-onset of breast cancer, bilaterality, and associated tumor in the family such as soft tissue sarcoma(Borresen, 1992).
Recently, germ-line mutations of the p53 gene have been found in affected members and obligate carriers in families with LFS(Malkin et al., 1990; Srivastava et al., 1990) indicating that the p53 germ- line mutation is the genetic basis for the LFS. The p53 tumor suppressor gene is located on the short arm of chromosome 17, band p13.1 and alterations of the gene are the most frequently encountered genetic events so far both in sporadic and hereditary human malignancies(Sager, 1989). The germ-line mutation of p53 seems to be distinct from the somatic mutation in that it occurs in a more defined region(between codon 245 and 258), which is within conserved region IV of the p53 gene. The mutations of this region are believed to functionally inactivate p53 protein and thereby provide an increased risk for cancer(Malkin et al., 1990; Frebourg et al., 1992; Malkin, 1993). It has become clearer that not all families with classic LFS have detectable germ-line mutations of the p53 gene and germ-line p53 muta- tion is not the sine qua non of LFS. However, little is known about the frequency of germ-line p53 muta- tions in hereditary breast cancer patients outside families with LFS. Although one study found 1 of 126 early-onset breast cancer patients to have carried mutant p53(Sidransky et al., 1992), most studies failed to find the germ-line mutation without LFS phenotype- (Malkin, 1993). In addition, the mutation of the carrier
without LFS turned out to occur outside the conserved region and thereby it might be functionally silent(Fre- bourg et al., 1992). These results suggested that germ-line p53 mutations occur rarely in early-onset breast cancer outside the LFS(Borresen et al., 1992 ; Sidransky et al., 1992).
In this study, the PCR-SSCP result for lymphocyte DNA of the proband and her sister indicated that the family carry the germ-line mutation of the p53 gene- (Fig. 2 and 3). Mobility shifted bands were also detected in all the samples including peripheral blood, tumor tissues, and benign tissues as the same pat- tern, confirming that it is constitutional mutation(Fig. 4). Sequencing analysis showed that the p53 mutation is C to T change at the CpG dinucleotide moiety of codon 248 in exon 7, which leads to substitute amino acid arginine for tryptophan. This mutation is the most common p53 gene alteration in not only sporadic tumors but also hereditary tumors. And this site was previously suggested to be a mutational ‘hot spot’ in LFS(Santibanez et al., 1991 ; Eng and Ponder, 1993 ; Birch et al., 1994). This mutation is believed to have functional significance.
The family in our case had the most consistent characteristic features of LFS phenotype. It is believed that this is the first report of a family with Li-Fraumeni syndrome carrying the p53 germ-line mutation in Korea. In addition, our observations further confirm that this inherited p53 mutation may predispose mem- bers of the LFS family to increased susceptibility to cancer. However, it is not the time of occurrence of mutations, but rather accumulations, that is the most important in tumor development. In Li-Fraumeni syn- drome, it is thought that p53 gene mutations occur first and other mutations follow(Vogelstein, 1990). Therefore, further studies regarding the subsequent genetic defects and its biological significance should be assessed.
ACKNOWLEDGMENTS
This study was supported by a grant from the Korea Science Engineering Foundation(KOSEF- SRC-56-CRC-93-23) and Seoul National University College of Medicine.
REFERENCES
Anderson DE. Familial versus sporadic breast cancer. Can- cer(suppl.) 1992 ; 70 : 1740-6.
Birch JM, Hartley AL, Tricker KJ, Prosser J, Condie, A, Kelsey AM, Harris M, Jones PHM, Binchy A, Crowther D, Craft AW, Eden OB, Evans DGR, Thompson E, Mann JR, Mratin J, Mitchel LD, Santibanez-Koref MF. Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Res 1994 ; 54 : 1298-304.
Borresen A-L. Role of genetic factors in breast cancer susceptibility. Acta oncologica 1992 ; 31 : 51-5.
Borresen A-L, Anderson TI, Garber J, Barbier-Piraux N, Thorlacius S, Eyfjord J, Ottestas L, Smith-Sorensen B, Hovig E, Malkin D, Friend SH. Screening for germ-line TP53 mutations in breast cancer patients. Cancer Res 1992 ; 53 : 3234-6.
Eng C, Ponder BAJ. The role of gene mutations in the genesis of familial cancers. FASEB 1993 ; 7 : 910-9.
Frebourg T, Kassel J, Lam KT, Gryka MA, Barbier N, Anderson TI, Borresen A-L, Friend SH. Germ-line muta- tions of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein. Proc Natl Acad Sci USA 1992; 89 : 6413-7.
Holton TA, Graham MW. A simple and efficient method for direct cloning of PCR products using ddT-tailed vec- tors. Nucl. Acids Res 1990 ; 9 : 1156.
Li FP, Fraumeni JF Jr. Soft tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Intern Med 1969 ; 71 : 747-52.
Li FP, Garber JE, Friend SH, Strong LC, Patenaude AF, Juengst ET, Reilly PR, Correa P, Fraumeni JF. Recom- mendations on predictive testing for germ-line p53 mutations among cancer-prone individuals. J Natl Can-
cer Inst 1992 ; 84 : 1156-60.
Malkin D. The Li-Fraumeni syndrome. Principles & Practice of Oncology 1993 ; 7 : 1-14.
Malkin D, Li FP, Strong LC, Fraumeni JF Jr, Nelson CE, Kim DH, Kassel J, Gryka MA, Bischoff FZ, Tainsky MA, Friend SH. Germ-line p53 mutations in a familial syn- drome of breast cancer, sarcomas, and other neo- plasms. Science 1990 ; 250 : 1233-8.
Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989; 5 :874-9.
Sager R. Tumor suppressor genes : The puzzle and the promise. Science 1989 ; 246 : 1406-12.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning : A laboratory manual. 2nd ed. New York, Cold Spring Harbor Laboratory Press, 1989 ; 9.17-19.
Santibanez-Koref MF, Birch JM, Hartely AL, Jones PHM, Craft AW, Eden T, Crowther D, Kelsey AM, Harris M. p53 germline mutations in Li-Fraumeni syndrome. Lan- cet 1991 ; 338 : 1490-1.
Sidransky D, Tokino T, Helzlsouer K, Zehabauer B, Rausch G, Shelton B, Prestiniacomo L, Vogelstein B, Davidson N. Inherited p53 gene mutations in breast cancer. Cancer Res 1992 ; 52 : 2984-6.
Srivastava S, Zou Z, Pirollo K, Blattner W, Chang EH. Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome. Nature 1990 ; 348 : 747-9.
Vogelstein B. A deadly inheritance. Nature 1990; 348 : 681-2.