Loss of heterozygosity on chromosome 17 and mutation of the p53 gene in retinoblastoma

Cancer Letters 106 (1996) 75-82

Loss of heterozygosity on chromosome 17 and mutation of the ~53 gene in retinoblastoma Mitsuo V. Katoa,b,*,Takashi Shimizub, Kanji Ishizakic, Akihiro Kanekod, David W. Yandelle, Junya Toguchidaf, Masao S. Sasakib Tsukuba Life Science Center; Institute of Physical and Chemical Research (RIKEN). Koyadui, Tsukuba, Ibaraki 305, Japan bRadiation Biology Centet; Kyoto University, Yoshida-konoecho, Sakyo-ku, Kyoto 606, Japan ‘Laboratory of Experimental Radiology, Aichi Cancer Centel; Research Institute, Chikusa-ku, Nagoya 464, Jupan dDepartment of Ophthalmology, National Cancer Center Hospital, Tsukiji, Chuo-ku, Tokyo 105, Japan eDepartment of Pathology, University qf Vermont, Burlington, vTO5405, USA fResearch Centerfor Biomedical Engineering, Kyoto University, Sakyo-ku, Kyoto 606, Japan

“lnhoratory

of Molecular

Oncology,

Received2 April 1996;accepted2 May 1996

Abstract Loss of heterozygosity (LOH) on chromosome 17 and mutations of the ~53 gene were examined in 25 retinoblastomas (RB), consisting of three. familial tumors, nine hereditary tumors without family history, 11 non-hereditary tumors, one recurrent tumor and one lung-metastatic tumor. LOH on chromosome 17 was detected in only one of the 23 primary RB. No mutations of the ~5’3 gene wete detected in the primary tumors. A recurrent tumor showed LOH on the short arm region of chromosome 17. LDH on chromosome 17 and a point mutation of the p.53 gene were also detected in a metastatic tumor. These results suggest that LOH on chromosome 17 and mutation of the ~53 gene may not be associated with the development of primary Rl3, but may play a role in the progression of RB. KeJlwords:

Retinoblastoma; Loss of heterozygosity; Chromosome 17; p53

1. Introduction Functional inactivation of tumor suppressor gene is critical in the development of human cancers. The ~53 gene is the tumor suppressor gene that is mutated in human cancers most frequently [l]. Functional

* Corresponding author. Tel.: + 81 298 365265; fax: +81 298 369020:e-mail: [email protected]

inactivation of both alleles of the tumor suppressor gene known as the two-mutation paradigm, is required for tumorigenesis [2]. Functional inactivation of the ~53 gene is also involved in the two-mutation paradigm, while dominant negative mutations of this gene had also been reported. Loss of heterozygosity (LOH) on chromosome 17 detected as a result of loss of the entire chromosome, partial deletion of the chromosome, and mitotic recombinations, has also been detected in various cancers. These chromosomal

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phenomena are considered to make mutations homologous in tumor cells. Germ-line mutations of the ~5.3 gene were detected in cancer-prone families, such as those with Li-Fraumeni syndrome [3,4]. Sarcomas and thymomas developed in mice lacking the ~53 gene [5]. Mutations of the ~53 gene appear to be quite important in the development of cancers. On the other hand, mutations of the ~53 gene were also reported in metastatic tumors, suggesting that alterations of the ~53 gene may be considered as a an indicator for determining the prognosis of patients

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also analyzed. Samples of these RB and skin biopsies for use as non-tumorous somatic cells were obtained from the National Cancer Center Hospital and Kyoto University Hospital [9,10]. All tumor cells were enucleated at stage Va or Vb [I l] and immediately frozen, Primary tumor cells were directly subjected to DNA isolation. Fibroblast cells expanded from skin biopsies of patients were cultured in modified Eagle’s minimum essential medium (Irvine) supplemented with 10% fetal bovine serum (HyClone).

Ill. Retinoblastoma (RB) is a pediatric malignant eye tumor. Genetic alterations of the RB gene are critical in the development of RB [6]. Aside from mutations of the RB gene or deletions of chromosome 13, other abnormalities that have been detected in tumors include trisomies of the long arm of chromosome 1, isochromosomes of the short arm of chromosome 6, amplifications of the N-myc gene, amplifications of the int-I gene and the absence of transforming growth factor-#? receptors [6]. Although mutations of the ~53 gene had been detected in the majority of cancers, LOH on chromosome 17, and mutations of the ~53 gene have been reported in only one patient with bilateral RB [7]. Moreover, loss of function of ~53 has been reported as necessary for the development of murine RB [8]. To determine whether the mutation of the ~53 gene is essential in the development and/or progression of human RB, we analyzed LOH on chromosome 17 and the mutation of the ~53 gene in 23 primary, one recurrent and one lungmetastatic RB and discussed the role of the ~53 gene in the development of RB.

The polymorphic probes used for LOH analysis on chromosome 17 were pR4-2 homologous to TP53 [13], pYNZ22 to D17S30, pMCT35.1 to D17S31 and THH59 to D17S4 [ 141. pR4-2 contains a cDNA clone of the human ~53 gene [13]. Chromosomal locations [ 151 and specific restriction endonucleases of these probes are shown in Table 2.

2. Materials

2.4. PCR-SSCP analysis

and methods

2. I. Samples of tumors We investigated 23 cases of primary RB, consisting of three familial cases, nine hereditary cases defined by either bilateral occurrence or constitutional deletion of chromosome 13, and 11 sporadic unilateral cases supposed to be non-hereditary (Table 1) [9]. In one sporadic bilateral case (RB188), recurrent tumor (RB188TR) was also examined. Primary cell culture established from lung-metastatic tumor (RB 134TM) of one hereditary case (RB 134) was

2.2. Southern-blot analysis LOH on chromosome 17 was analyzed by Southern-blot hybridization. High molecular weight DNA was isolated from cultured skin fibroblasts and tumor cells by a previously described method [12]. DNA samples were digested by the appropriate restriction endonucleases listed in Table 2, fractionated in 0.7% agarose gel, and transferred to a nylon membrane filter (HybondTM N; Amersham). After hybridization with 32P-labeled probes, signals were detected by autoradiography. 2.3. DNA probes

Genomic regions from exon 4 to 9 of the ~53 gene were amplified by polymerase chain reaction (PCR), and mutations were screened by the single strand conformation polymorphism (SSCP) method. Each exon of the ~53 gene was amplified by the same method using the same PCR primers previously reported [ 161. PCR was performed in the presence of a [a-32P]-dCTP (Amersham) and NTP mix (200 mM dATP, 200 mM dTTP, 200 mM dGTP and 20 mM dCTP) using a Taq polymerase (Takara). Labeled PCR products were diluted with 4 ~01s. of dilution

M. V. Kutn et ul. I Cuncer Letters Table

106 (I 996) 75-82

77

I

Summary ---

of characteristics

‘Tumor

Primafy

of tumors analyzed

Laterality

Age (days) at operation

Tumor stage

LOH #13”

LOH #I7

RB mutation

-

Ex. 24b

-

p53 mutation

tumwc

Hereditary RB~l34T RB21h’I RB273T

cases with family FB F% FB

i-Iereditary RR 182T RBl8ST RR2081 RB214T RB229-f RB2347 KB247T RB260T RR2661

cases without SU SB SB SR SB SB SB SB SB

son-hereditary RR I X3T RB196T RI321 1-r RB226T RB22XT RB231T RB232T RBKuRiT RB27OT 138276-f RB298T Recurrent

in this study

cases su su su su SU su su SU SU su su

history

family

340 48 107

Va Va Va

513 285 230 284 316 139 516 421 543

Va Va Va Vb Va Va Vb Va Va

NI t -

181 562 972 692 519 1036 1505 1171 218 1462 894

Va Va Vb Vb Va Vb Vb ND Va Va Va

t Nl

-

+

-

history

+ -

-

-

Ex. 13b

t -

Ex. 17’

+

Ex. 11’

+ +

t t + + +

+ ._

+

t

t

t

Ex. 13’ Ex. 16’ Ex. 12’ Ex. 15’

tumor

RB188TR Lung-metastutic RB134TM

tumor Ex. 24b

Ex. 5

FB, familial bilateral; SB, sporadic bilateral; SU, sporadic unilateral; ND, not determined; NI, not informative for LOH; Ex., exon. Va and Vb stages both indicate massive tumors invoking over half of the retina, while tumors at stage Vb have seeded in the vitreous body [ 1 I]. a Ref. [Y] bRef. [lo]. ‘Ref. [24]

buffer (0.1% SDS, 10 mM EDTA) and with 5 ~01s. of loadmg buffer (95% formamide, 20 mM EDTA, 0.05% xykme cyan01 and bromophenol blue). Diluted samples were denatured at 94°C for 5 min, and then applied to 6% polyacrylamide gel. Electrophoresis was performed at 30 W for approximately 6 h at 22°C. The gel was dried on filter paper and exposed to X-ray film at room temperature.

2.5. Direct sequencing analysis

PCR products were purified by Suprec 02 (Takara), denatured, and then annealed with a primer labeled with [y-32P]ATP (NEN) using polynucleotide kinase (Takara). Nucleotide sequences of PCR fragments were determined using a A Tth DNA polymerase (Toyobo).

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3. Results

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other primary tumors that had retained heterozygosity.

3. I. LOH on chromosome17 in primary RB Fig. 1 shows examples of LOH on chromosome 17. LOH on chromosome 17 was detected in only one (RB232T) of the 23 primary RB (approximately 4%). The distal region, D17S30 (17~13.3) of the short arm of chromosome 17 was also lost in the tumor cells (Table 2) as was the region of the ~53 gene, TP53 (Fig. 1, right panel). LOH on chromosome 17 in primary RB appears to be uncommon. RB232T is the primary tumor with the latest onset (age at the time of operation is 1505 days after birth of the patient), although no statistically significant difference existed in ages of the patients at operation and in the stages (Va or Vb) between a primary tumor (RB232T) that had lost heterozygosity on chromosome 17 and

3.2. LOH on chromosomeI7 in a recurrent tumor and a lung-metastatic tumor While a primary tumor (RB188T) of a patient RB 188 retained heterozygosity on chromosome 17, a recurrent tumor (RB 188TR) of the same patient lost one allele of the short arm region, D17S31 (17p13.1-p11.2), of chromosome 17 (Fig. 1, left panel) but retained both alleles of the long arm region, D17S4 (17q23-q25.3) (Fig. 1, middle panel). LOH on D17S30 was also detected in RB 188TR (Table 2), suggesting that one allele of the short arm of chromosome 17 may be lost in RB 188TR. A lung-metastatic tumor (RB 134TM) also lost one chromosome 17, although a primary tumor (RB134T) in the same patient did not exhibit LOH on chromosome 17 (Table 2). 3.3. PCR-SSCPand direct sequencinganalysis

pMCT35.1 Msp I

pTHH59 Taq I

pR4-2 Bgl II

Fig. 1. Examples of LOH analysis on chromosome 17. LOH on the short arm region (p13.1-~11.2; pMCT35.1 homologous to D17S31) of chromosome 17 was detected in a recurrent tumor (RB188TR; left panel), while LOH was not detected in a primary tumor (RB188T) in the same patient and heterozygosity was retained on the long arm region (q23q25.3; pTHH59 homologous to D17S4) of chromosome 17 in the same recurrent tumor and the primary tumor (middle panel). LOH in the ~53 gene locus (pR4-2 homologous to TP53) was detected in a primary RB (RB232T; right panel).

As LOH on chromosome 17 was detected in a primary (RB232T), a recurrent (RBl88TR) and a lung-metastatic (RB134TM) RB, mutations of the ~53 gene were screened by the PCR-SSCP method. While the genomic regions from exon 4 to 9 of the ~53 gene were analyzed in all 25 of the RB tumors studied in the present paper, altered bands were detected only in the lung-metastatic RB (RB134TM) at exon 5 (Fig. 2A). Direct sequencing analysis revealed a single point mutation: TGC (Cys) to TTC (Phe) substitution at codon 135 (Fig. 2B). No band with normal mobility was detected by SSCP analysis and no normal sequence (TGC at codon 135) was detected by direct sequenceanalysis in RB134TM. This result was consistent with the result of LOH analysis as described above (Tables 1 and 2). 4. Discussion LOH on chromosome 17 was detected in only one (RB232T) of the 23 primary RB (approximately 4%). No altered band was detected by the SSCP analysis of the genomic regions of the ~53 gene in any of the primary RB. Since LOH on

I’:thle 2 LOH -

on chromosome

17

‘T’urnor

I’rimcrp

Dl7S30 Taq 1

TPS3 (13.1) Bgl I1

Dl7S31 Msp I

-

-

(p13.1-~11.2)

D I7S4 (q23-q25.3) Taq 1

tlrn1or.c

Hereditary RH 133T Rn2lh’r KT(27731

cases with family

tferedhary KB 182T RHIX8T RH208T RBZl4T lu3’2YT Iik234T RR247T RR260T RB266T

cases without

history

family

-

history

-

Non-hereditary RB I 83T RB 196T RB21 IT RB226-f RB228T KB231T RB232T RBKuRiT KB270T RB216T RB298T Recurrent RB I88TR

(p13.3)

cases

i

+ -

tumrtr

Lung-metustatic RB 134TM

i-

+

+

+

tumor +

Cfuomosomal assignments were made according to the Third International Workshop on Chromosome 17 Mapping [IS]. Restriction endonucleases~ were indicated under the name of the probes used in this study. + indicates that LOH at the locus was detected. - indicates that heterozygoslty was retained at the locus. No information was obtained at open columns.

chromosome 17 and mutations of the ~53 gene have not been reported in primary RB, these may not be important factors in the development of primary RB. Aithough the loss of function of p-53 has been reported as necessary for the development of murine RB [8], no mutation of the p-53 gene was found in the human primary RB examined in the present study. RB did not develop in RB-deficient mice, in which the RB gene had been disrupted by gene-targeting [I 71. Therefore, the mechanism of tumorigenesis of the human RB may be different from that of the

murine RB. An alternative mechanism equivalent to the inactivation of the ~53 gene, such as an amplification of the MDM2 gene [ 181, might be involved in the development of human RB. The age at operation of the patient RB232, whose primary tumor showed LOH on chromosome 17, was the oldest of all patients with primary RB in this study. Onsets of RB are affected by LOH on chromosome 13 [9] and parental origin of the lost allele on chromosome 13 [19]. Two possibilities concerning the relationship between

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EXON

B

GATC

RB134

5

GATC

RB134TM

Fig. 2. (A) Examples of SSCP analysis of the ~53 gene (exon 5). Altered bands were detected only in a lung-metastatic tumor (RB 134TM). (B) Direct sequence analysis of the PCR product of exon 5 of the ~53 gene. Left panel shows the sequence analysis of normal fibroblast cells of the patient (RB134) and right panel shows the analysis of a lung-metastatic tumor (RB134TM) in the same patient. The ~53 gene in the tumor cells were homozygous for a point mutation: TGC (Cys) to TTC (Phe) substitution.

late onset and LOH on chromosome 17 should be considered. The first is that genetic alterations (LOH on chromosome 17, mutations of the ~53 gene, etc.) other than that of the RB gene may easily occur in tumors that have a long latent period. LOH on chromosome 13 in a primary tumor (RB 188T) was also detected in a recurrent tumor (RB188TR; Table 1) from the same patient [9], but LOH on chromosome 17 was detected only in the recurrent tumor (RB188TR). LOH on chromosome 17 of this tumor might have occurred after the initial operation. Tumors that have lost one allele on chromosome 17 might have a growth advantage, and only tumor cells that have lost one chromosome might grow dominantly. Another possibility is that other genetic alterations might

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be necessary for late-initiated tumors to develop. Therefore, other genetic alterations such as LOH on chromosome 17 and mutations of the ~53 gene may be necessary for tumorigenesis with late onset. Indeed, RB do not develop in the adult human. Such a difference may also help to explain the difference in the mechanism of the development of RB between the human and the mouse, as described above. LOH on chromosome 17 and a point mutation, TGC (Cys) to TTC (Phe) substitution at codon 135 of the ~53 gene, were detected in the lungmetastatic tumor (RB 134TM). Abnormalities of the ~53 gene have been reported in the metastasis of breast cancers, prostate cancers, gastric cancers, ovarian cancer, non-small cell lung cancers and head-and-neck squamous cell carcinoma [l]. Mutations of the p.53 gene might also be associated with the metastasisof RB. p53 is a transcription factor that binds to specific DNA sequencesand regulates the expression of genes that contain p53-responsive elements in their genomic region [20]. The mutation detected in the lung-metastatic tumor (RB134TM) is localized in the sequence-specific DNA-binding domain of p53 protein [20]. Loss of normal DNAbinding ability in the mutant protein may result in the acquisition of the metastatic capacity in the tumor. In fact, p53 can regulate the expression of the genes that are directly associated with angiogenesis. Wild-type p53 up-regulates the expression of the thrombospondin-1 (TSP-1) gene [21], which exhibits an anti-angiogenic activity. In addition, mutant p53 protein can induce and potentiate the expression of vascular endothelial growth factor (VEGF) [22]. Although the expression of these genes was not checked in the lungmetastatic tumor (RB134TM), down-regulation of the TSP-I gene and up-regulation of the VEGF gene by the mutant ~53 gene may be associated with the acquisition of metastatic potential in the tumor. The patient RB134 had a family history, and the germ-line mutation of the RB gene has already been identified [lo]. The primary tumor (RB134T) retained heterozygosity both on chromosomes 13 and 17 (Tables 1 and 2). In familial cases with germ-line mutations, multiple RB develop in one patient [23]. Therefore, RB134TM

M.V. Kate et (11. I Cancer

developed independently from the primary tumor (RB 134T), while both tumors harbored the same germ-line mutation: deletion of exon 24 of the RB gene [ 101. RB 134TM may be derived from one tumor cell that lost normal ~53 activity, since we could not detect any mutation in the ~53 gene in primary RB. Because of small number of such advanced cases, further investigations are required in order to clarify whether the analysis of mutations of the ~53 gene can be used to predict the prognosis of RB patients. However, it is an intriguing matter to analyze the mutation of the ~5.3 gene in a number of advanced cases, and it might allow us to assess the risk of recurrence or metastasis of RB. Acknowledgements The authors are very grateful to Dr. Y. Nakamura for his generous donation of the DNA probes, Ms. K. Sudo for her technical assistance and Ms. S. Kobayashi for preparation of the manuscript. This work was supported by a Cancer Research Grant from the Ministry of Health and Welfare of Japan, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan References I! j Greenblatt, MS., Bennett. W.P., Hollstein, M. and Harris, CC. (!994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res.. 51. ~1855-4878 f2] Knudson, Jr.. A.G. (1985) Hereditary cancer, oncogenes and anttoncogenes. Cancer Res., 45, 1437-1443. [.%I Malkin, I), Lt. F.P.. Strong, L.C., Fraumeni, J.J., Nelson C.E , Km. D.H.. Kassel. .I., Gryka. M.A., Bischoff, F.Z.. ‘Tamsky. M.A. and Friend, S.H. (1990) Germ line pS3 mutatmns tn a familial syndrome of breast cancer. sarcomas and other neoplasms. Science, 250, 1233-1238. !3] Srivastnva. S., Zou, Z.Q.. Pirollo, K., Blattner, W. and Chang, E.H. (1990) Germ-line transmission of a mutated p.53 gene m a cancer-prone family with Li-Fraumeni syndrome Nature, 348, 747~-749. ]S] Donchower, L.A., Harvey, M., Slagle, B.L., McArthur, M.J., Montogomery, Jr.. C.A., Butel, J.S. and Bradley, A. (1992) Mice deticient for pS3 are developmentally normal but susceptible to spontaneous tumors. Nature, 356, 215-221. 161 Horsthemke. 8. (1992) Genetics and cytogenetics of retinoblastoma Cancer Genet. Cytogenet.. 63. 1-7.

Letters [7]

[8]

[9]

[IO]

[I I] [ 121

[ 131

[ 141

[IS]

[I61

] 171 [ 181

[19]

1201 [2l]

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Hovig, E., Andreassen,A., Fangan, B.M. and Borresen, A.L. (1992) A TP53 mutation detected in cells established from an osteosarcoma, but not in the retinoblastoma of a patient with bilateral retinoblastoma and multiple primary osteosar comas. Cancer Genet. Cytogenet., 64, 178-182. Howes, K.A., Ransom, N., Papermaster, D.S., Lasudry, J.G.H., Albert, D.M. and Windle, J.J. (1994) Apoptosis or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the presence or absence of ~53. Genes Dev.. 8, 1300-1310. Kato. M.V., Ishizaki, K., Ejima, Y., Kaneko, A., Tanooka, H. and Sasaki, M.S. (1993) Loss of heterozygosity on chromosome 13 and its association with delayed growth of retinoblastoma. Int. J. Cancer, S4, 922-926. Kato, M.V., Ishizaki. K., Toguchida, J., Kaneko. A.. Takayama. J., Tanooka, H., Kato, T., Shim& T. and Sasaki. M.S. (1994) Mutations in the retinoblastoma gene and their expression in somatic and tumor cells of patients with hereditary retinoblastoma. Hum. Mutat.. 3, 4451. Reese, A.B. (1976) Tumors in the Eye, 3rd edn., pp. 90-124. Harper and Row, New York. Sambrook, J., Fritch, J. and Maniatis. T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Harlow, E., Williamson, N.M., Ralston, R., Helfman, D.M. and Adams, T.E. (1985) Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen ~53. Mol. Cell. Biol., 5, 1601-1610. Nakamura, Y., Lathrop, M.. O’Connell. P., Leppert, M.. Barker, D., Wright, E., Skolnick, M., Kondoleon, S.. Litt, M., Lalouel, J. and White, R. (1988) A mapped set of DNA markers for human chromosome 17. Genomics, 2. 302309. Fain, P.R. (1992) Third International Workshop on Human Chromosome 17 Mapping. Cytogenet. Cell Genet.. 60, 17X186. Toguchida, J., Yamaguchi. T.. Ritchie, B., Beauchamp, R.L., Dayton, S.H., Herrera, G.E.. Yamumuro, T., Kotoura, Y., Sasaki, M.S.. Little, J.B., Weichsellbaum, R.R., Ishizaki, K. and Yandell, D.W. (1992) Mutation spectrum of the pS3 gene in bone and soft tissue sarcomas. Cancer Res., S2, 61944199 Harlow. E. (1992) Retinoblastoma: for our eyes only. Nature, 3S9. 270. Oliner, J.D., Kinzler, K.W., Meltzer, P.S., George, D.L. and Vogelstein, B. (1992) Amplification of a gene encoding a pS3-associated protein in human sarcomas. Nature, 358. 8083. Kato, M.V.. lshizaki, K., Shimizu, T., Toguchida, J., Kaneko, A. and Sasaki, MS. (1995) Delayed development of retinoblastoma associated with loss of a maternal allele on chromosome 13. Int. I. Cancer, 64, 3-X. Friend, S. (1994) ~53: a glimpse at the puppet behind the shadow play. Science, 265, 334-335. Dameron, K.M., Volpert, O.V., Tainsky, M.A. and Bouck, N. (1994) Control of angiogenesis in fibroblasts by p53 regulation of ThrombospondinI. Science, 265. 1582-1584.

82 [22]

[23]

M. V. Kato et al. / Cancer Kieser, A., Weich, H.A., Brandner, G., Marme, D. and Koich, W. (1994) Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression. Oncogene, 9.963-969. Vogel, F. (1979) Genetics of retinoblastoma. Hum. Genet., 52, 1-54.

Letters [24]

IO6 (I 996) 75-82 Shim& T., Toguchida, J., Kato, zaki, K. and Sasaki, MS. (1994) of the RBl gene in retinoblastoma exon PCR-SSCP analysis. Am. J. 800.

M.V., Kaneko, A., IshiDetection of mutations patients using exon-byHum. Genet., 54, 793-