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Polymorphisms in the p53 gene in thyroid tumours and blood samples of children from areas in Belarus
Mutation Research 381 Ž1997. 201–207
Polymorphisms in the p53 gene in thyroid tumours and blood samples of children from areas in Belarus S. Hillebrandt a , C. Streffer a
a,)
, E.P. Demidchik b, J. Biko c , Chr. Reiners
c
Institut fur Essen, Hufelandstr. 55, 45122 Essen, Germany ¨ Medizinische Strahlenbiologie, UniÕersitatsklinikum ¨ b Centre for Thyroid Tumours, Minsk, Belarus c Klinik fur Wurzburg, Josef Schneider Str. 2, Wurzburg, Germany ¨ Nuklearmedizin des UniÕersitatsklinikum ¨ ¨ ¨ Received 22 April 1997; revised 22 April 1997; accepted 23 July 1997
Abstract We present changes in the p53 gene in a group of 70 thyroid tumours and 40 blood samples obtained from children from Belarus. Three thyroid tumours show a polymorphism in exon 6 Žcodon 213. and 5 tumours show a polymorphism in intron 6, 37 bp upstream to the 5X-end of exon 7. Only one patient has a mutation in exon 7 Žcodon 258. resulting in an amino acid substitution in the protein p53. The distribution of polymorphisms in the 40 blood samples was as follows: three patients had a polymorphism in exon 6 and two persons had a polymorphism in intron 6. One polymorphism in intron 6 was also found in the group of 30 healthy children from Belarus. The fact that the differences in the sequence in p53 found in the tumours was also seen in the blood of these patients demonstrates that they are polymorphisms not induced by radiation exposure. It is difficult to conclude, if the polymorphisms found by us could be associated with the predisposition to radiation-induced cancer. q 1997 Elsevier Science B.V.
1. Introduction The structure and function of the p53 tumour suppressor gene have been extensively studied and it was found to be the most commonly mutated gene in human cancers. The majority of p53 mutations found are missense alterations within exons 5–8 of the gene w1–3x. In thyroid tumours, mutations of the p53 gene occur rarely w4–6x suggesting, that alterations in this gene may not be important for the development of thyroid tumours. On the other hand, there are some studies describing a relatively high frequency Ž13–25%. of mutations in exons 5–8 of the p53 gene )
Corresponding author. Fax: q49 Ž201. 723-5966.
in thyroid tumours w7,8x. The reasons for the different results are not known. In addition to mutations occurring in the p53 exons, there are several examples of genetic changes in intronic sequences of the p53 gene that result in splicing mutations w9–12x. These intronic mutations include insertions, deletions or base substitutions in regions adjacent to donor or acceptor splice sites of the affected intron. Polymorphisms are additional DNA sequence alterations observed in exons and introns of the p53 gene of normal tissues as well as that of tumours. They do not affect the structure of the wild-type protein. Some polymorphisms were detected in the various exons and introns with no obvious effect on p53 structure w13–15x. However, a
0027-5107r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 1 6 9 - 3
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S. Hillebrandt et al.r Mutation Research 381 (1997) 201–207
possible association of polymorphisms with cancer predisposition and susceptibility was discussed by Peller et al. w16x. It has been reported that alterations in the p53 gene may be a potential marker for radiation-induced cancer w17x. The authors described p53 mutation hotspots in radon-associated lung cancers from uranium miners. Mutations occurring specifically in such tumours may be potential markers for radiation-induced cancer and could help to distinguish those tumours from tumours that develop spontaneously. For this reason, more investigations on tumours derived from patients exposed to radioactivity are needed. Spontaneous thyroid carcinoma in children is a rare occurrence and its frequency in Belarus before the Chernobyl accident was between one and two per year w18,19x. The reports of increasing numbers of childhood thyroid cancer cases in the years between 1986 and 1991 first appeared in 1991. The number of cases has roughly doubled each year from 1988, and reached more than 50 per year in 1991 w18,19x. By far the greatest increase was seen in the Gomel region Ž38 cases in 1991., and a less obvious increase was seen in the Brest and Grodno regions. The Gomel region is situated immediately to the north of Chernobyl and is known to have received a high level of radioactivity as fallout after the breakdown of the reactor. In this study, p53 gene alterations in cells of papillary thyroid carcinomas derived from 70 children who lived in different areas of Belarus during the reactor accident of Chernobyl in 1986 were analysed. Base-substitutions in exon 6 Žcodon 213. and in intron 6 Ž37 bp upstream to the 5X-end of exon 7. of the p53 gene have been observed. In our previous study, mutations in the p53 gene in thyroid tumours of children from areas highly contaminated by the Chernobyl accident were described w20x. Some of these mutations have been identified as polymorphisms. These polymorphisms appear to contribute to the high frequency of mutations reported by us relative to those described in other studies of thyroid tumours from patients without analogous exposures to ionizing radiation. Thus, our previous conclusion that the number of p53 mutations observed in thyroid carcinomas of children from areas contaminated by the Chernobyl accident
is induced by ionizing radiation appears to be incorrect. However, the polymorphisms already present before the radiation exposure may support the development of thyroid carcinomas.
2. Materials and methods 2.1. Tumour samples and blood The donors who were included in this study are male and female children from Belarus ranging from 6 to 18 years. The children lived in different areas of the republic during the reactor accident of Chernobyl. The areas are located around 150 km ŽGomel., 450 km ŽBrest., 350 km ŽMinsk., 500 km ŽGrodno. from Chernobyl. The children received thyroid surgery at the Centre for Thyroid Tumours in Minsk. Clinical data and tumour pathology were available from this institution. Blood samples of the children from Belarus were obtained prior to radiotherapy from the Clinic of Nuclear Medicine in Wurzburg, where the children ¨ received radioiodine for the therapy of disseminated thyroid tumours. As controls, blood samples from healthy Belarussian children were used. 2.2. DNA preparation Tumour tissues were pressed through a Nylon net and the cells were fixed in 96% ethanol. Those suspensions containing cells derived from one part of each tumour were obtained from the Centre for Thyroid Tumours in Minsk. Since sections taken from different parts of the same tumour were not available, the level of heterogeneity within individual tumours could not be tested. No cytogenetic analysis has been performed. The cells were incubated in 200 ml 0.5% SDS solution at 378C for 1 h. Then the same volume of a digestion buffer Ž100 mM Tris-HCl, pH 8.5, 1 mM EDTA, 0.5% Tween 20, 100 mgrml proteinase K. was added and the solution was stirred at 378C for 12 h. The DNA was precipitated with 100% ethanol and diluted with 100 ml nuclease-free water. No phenol–chloroform extraction was performed. DNA from blood was prepared with the Split Second Kit from Boehringer ŽMannheim..
S. Hillebrandt et al.r Mutation Research 381 (1997) 201–207
2.3. PCR conditions The PCR conditions for the detection of polymorphisms in exons 4–9 and the adjacent introns of the p53 gene with the TGGE Žtemperature gradient gel electrophoresis. were described by Hillebrandt et al. w20x. For sequencing of exons 6 and 7, and the adjacent introns the primers used in PCR reaction were: exon 6 q introns, the 5X-primer: 5X-TGGTTGCCCAGGGTCCCCAG-3X , the 3X -primer: 5X -CCAGT TGCAAACCAGACC-3X ; exon 7 q introns, the 5Xprimer: 5X-CTTGCCACAGGTCTCCC CA-3X , the 3X-primer: 5X-TGGCAAGTGGCTCCTGAC-3X . PCR reactions were carried out in 100 ml solution containing genomic DNA, 50 pmol of each primer, 2 mmol each of the four deoxynucleotide triphosphates, 2.5 U of Taq DNA polymerase ŽBoehinger, Mannheim., 10 mM Tris-HCl, pH 8.3, 25 mM KCl, 1.5 mM MgCl 2 . An Omni-Gene thermal cycler was used for the amplification. The PCR program was as follows: one cycle of 10 min at 948C, 35 cycles of 30 s at 948C, min at 608C, 30 s at 728C, ending with one cycle of 7 min at 728C. 2.4. Identification of polymorphisms with the help of temperature gradient gel electrophoresis (TGGE) This method, described in detail by Hillebrandt et al. w20x, is based on the principle that heteroduplexes of DNA molecules have a different melting temperature than DNA–homoduplexes: a heterozygote mutation could be identified as 4 bands and a homorhemizygote mutation as one band showing a different electrophoretic mobility comparing to a fragment without mismatch. A fragment with a mismatch is retarded at a slightly different temperature and produces a distinct band on the gel. The TGGE was performed as described by the manufacturer ŽTGGE Handbook, Diagen.. Several PCR products were run at the same time. The molecules exhibited different electrophoretic mobilities according to their melting temperatures. The optimal running time and the temperature gradient were calculated from the parameters of the perpendicular TGGE with the help of a formula given in the manufacturer’s handbook. In each case, tumour DNA with a known p53 mutation was used as a
203
positive control. For analysis of point mutations in ds DNA, an extremely high detection rate of ) 95% is routinely achieved with TGGE. Thus, we can detect the majority of polymorphisms in the analysed area. Exons and the adjacent introns showing polymorphisms identified by the TGGE were analysed by the direct sequencing as described below. 2.5. Direct sequence analysis Sequence analysis was carried out by solid-phase sequencing of single-strand PCR products. The antisense primer of the primer pair was biotinylated, allowing immobilization of the PCR product on streptavidin-coated beads ŽDynabeads M-280 Streptavidin, Dynal.. Denaturation of the immobilized double-stranded DNA was achieved by incubation with NaOH. For sequencing, a PRISM Sequenase Terminator Sequencing Kit ŽApplied Biosystems. was used. Its performance relies on four dye-labelled dideoxynucleotides ŽT, C, A, and G dye terminators. which allow all four termination reactions to be performed in one single tube. Gel electrophoresis, data collection and analysis were performed with an Applied Biosystems Model 373 DNA Sequencing System.
3. Results 3.1. Distribution of polymorphisms in thyroid tumour samples Exons and the adjacent introns amplified by PCR were analysed for polymorphisms with the TGGE. The distribution of polymorphisms in 70 papillary thyroid carcinomas Žshown in Table 1. reveals that three tumours Ž4.2%. show an A:T ™ G:C substitution in codon 213 of exon 6. This substitution changing CGA ™ CGG does not result in an amino acid alteration in the p53 protein. Fig. 1A shows the wild-type sequence of exon 6 and indicates the polymorphic site. The sequence analysis of exon 6 was performed for the tumour samples A4, A6, A8 and for human fibroblasts used as wild-type. The results of the sequence analysis demonstrate, that there is a peak for ‘A’ and a peak for ‘G’ at the position corresponding to the ‘A’-peak in the wild-type se-
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Table 1 Polymorphisms and mutations in the p53 gene determined in 70 children with papillary thyroid carcinomas as well as in the blood cells Case Sex Area
Level of differentiation Intron or exonrcodon polymorphismrmutation Coding effect Polymorphism in the blood cells
O7 A4 A6 A7 A8 B3 D1 4N 7N 16N
high high moderate high low high high high moderate high
a b
F M F M M F M F M F
Gomel Minsk Gomel Gomel Grodno Brest Brest Gomel Grodno Brest
intron 6 exon 6r213 exon 6r213 exon 7r258 exon 6r213 intron 6 intron 6 exon 1 intron 6 intron 6
G:C ™ C:G A:T ™ G:C A:T ™ G:C G:C ™ A:T A:T ™ G:C G:C ™ C:G G:C ™ C:G –a G:C ™ C:G G:C ™ C:G
none none none Glu ™ Lys none none none –a none none
0b 0 0 0 exon 6rA:T™ G:C intron 6rG:C™ C:G intron 6rG:C™ C:G 0 intron 6rG:C™ C:G 0
No sequencing was performed. 0, blood samples were not available.
quence indicating a heterozygote base substitution in the tumour sample Ždata not shown.. Furthermore, five out of 70 patients Ž7.1%. demonstrate a G:C ™ C:G substitution in intron 6, located 37 bp upstream to exon 7. The wild-type sequence of the part of intron 6 indicating the polymorphic site is shown by Fig. 1B. The sequence analysis was performed for the tumour samples 07, B3, D1, 16N, 7N. The results demonstrate a homozygote Žor hemizygote. base substitution in tumour sample 7N and a heterozygosity for both alleles in the other tumours. In only one case, the GAA ™ AAA substitution in codon 258 of exon 7 occurs and is leading to the amino acid change Glu ™ Lys. The wild-type sequence of exon 7 indicating the mutated position is shown by Fig. 1C. The G ™ A homozygote substitution observed in the tumour sample A7 is a result of the loss of the wild-type sequence. Using the TGGE method, we have found a polymorphism in exon 1 of one tumour. However, a
sequence analysis could not be performed in this case. Blood was available from 4 patients showing polymorphisms in the thyroid tumours ŽB3, D1, 7N, A8.. The polymorphism found in intron 6 in tumours B3, D1, 7N, and the polymorphism in exon 6 found in patient A8 were also observed in the blood samples of those patients. This indicates that they are present in constitutional, normal cells. Fig. 2 represents the results of the TGGE demonstrating a polymorphism in intron 6 and in exon 6. 3.2. Analysis of polymorphisms in peripheral blood cells from children with thyroid tumours and from healthy children Screening of blood cells from 40 children with thyroid cancer indicates that 12.5% Ž5r40. display a polymorphism in the p53 gene. Thyroid tumour samples were not available from these children. The distribution of the polymorphic patterns in the blood Table 2 Polymorphisms in the p53 gene determined in the blood samples from 40 children with thyroid carcinomas as well as from 30 healthy children from Belarus
Fig. 1. Wild-type sequences of exon 6 ŽA. of the relevant part of X intron 6 and the 5 -end of exon 7 ŽB. and of exon 7 ŽC.. The arrowheads show positions for which polymorphisms Žmutation. were observed.
Case
Sex Tumour Exon or intronrcodon Polymorphism
BI GO BA NA KO KOW
F F F F F F
q q q q q y
exon 6r213 exon 6r213 exon 6r213 intron 6 intron 6 exon 6
A:T ™G:C A:T ™G:C A:T ™G:C G:C ™C:G G:C ™C:G A:T ™G:C
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Fig. 2. PCR–TGGE analysis of peripheral blood samples demonstrating a polymorphism in intron 6 Žleft. and a polymorphism in exon 6 Žright..
is as follows: three patients show the A:T ™ G:C polymorphism in codon 213 of exon 6 and two patients demonstrate the G:C ™ C:G polymorphism in intron 6. The polymorphic patterns are as shown in Fig. 2. Blood cells from 30 healthy children from Belarus were used as controls. In this group, the G:C ™ C:G polymorphism in intron 6 was observed in one child. The frequency of polymorphisms in the p53 gene in the group of 40 blood samples obtained from children with thyroid tumours is not significantly higher compared with the group of 30 healthy children Ž p s 0.23, Fisher’s exact test.. Table 2 summarizes the distribution of polymorphisms in the blood samples from 40 children with a thyroid tumour and 30 healthy children from Belarus.
4. Discussion In this study, we analysed DNA of thyroid tumours and of peripheral blood cells from children
with and without thyroid tumours from Belarus. Three out of 70 tumour samples and three out of 40 blood samples show an A ™ G substitution in codon 213 of exon 6. Previous studies have indicated that some polymorphisms in the p53 gene were found to be distributed in typical patterns of various geographical locations and in various human races: it was observed that 10% from 50 unrelated individuals from South America and 3% from 30 unrelated individuals from North America have a polymorphism in codon 213 of the p53 gene w21,22x. However, there are no data on its occurrence in the population of Belarus. Correlations between a specific distribution of polymorphic patterns and cancer development have been studied: an analysis of mutations in the p53 gene in Burkitt’s lymphomas shows that 2% of the tumours have the silent A ™ G substitution in codon 213 w23x. The p53 codon 213 polymorphism was also studied in a healthy Turkish population and in Turks with different types of tumours. The data revealed no association between this polymorphism and the development of tumours w24x.
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A second polymorphism observed in the analysed group of thyroid tumours and blood samples is a G ™ C substitution found in intron 6, 37 bp upstream of exon 7. Five out of 70 thyroid tumours, two out of 40 blood samples from children with those tumours and one blood sample in the group of 30 healthy children showed this intronic polymorphism, which is a new polymorphism which has not been previously described. Nikiforov et al. w25x analysed 33 papillary thyroid carcinomas, one follicular carcinoma and 22 benign lesions removed from radiation-exposed children from the regions around Chernobyl and screened them for point mutations in p53 Žexons 5–8. using single-strand conformation polymorphism ŽSSCP. analysis. The authors found no intron 6 or exon 6rcodon 213 polymorphisms, but one missense mutation in exon 5 and one silent mutation in codon 182 in the group of 33 papillary thyroid carcinomas. They conclude that p53 mutations do not appear to be important in the development of post-Chernobyl thyroid papillary carcinomas, but may, in some cases, have a role in progression to a more aggressive phenotype. Intronic point mutations of the gene could lead to abnormal pre-mRNA splicing and defective protein. Mutations at consensus sequences at either the 5X- or 3X-splice site of intron 6 and 7 were found in human hepatocellular carcinomas w26x. Splicing mutations leading to the production of abnormal p53 protein were also described for lung cancer w27x. The polymorphism found by us in intron 6 is not located at the consensus sequence of the splice sites, but in the region of the branch site also involved in splicing. However, we do not know, if this polymorphism could be involved in the splicing process. The functions of the intronic sequences are not fully known. Control sequences affecting transcription and the level of the p53 protein could be located in the intronic regions. Studies on a polymorphism in the Ki-ras oncogene suggest, that polymorphisms in intronic sequences may affect the gene regulatory role, leading to susceptibility to cancer w27x. Thus, sequence alterations in introns could influence binding of transcription factors to the DNA and could change the expression of tumour suppressor genes. Furthermore, a helical distortion as a consequence of intronic sequence alterations could prevent binding
of proteins to the DNA. The possibility that the regions of intronic polymorphisms could control the stability or half-life of proteins was discussed by Peller et al. w16x. However, these assumptions should be investigated further. The differences in the p53 sequence found in the thyroid tumours of children from the regions around Chernobyl are also observed in the blood cells Žlymphocytes. of these patients. Thus, they have not been induced by radiation exposure and may be polymorphisms specific for the population in Belarus. Correlations between a specific distribution of a polymorphic pattern and cancer development have not been established so far. It was predicted, that some intronic polymorphisms may predispose towards coding-region mutations that increase the likelihood of a deleterious phenotype w28x. In summary, it is difficult to conclude if the development of thyroid cancer in children after the Chernobyl accident is influenced by the germ-line polymorphisms described in this study.
Acknowledgements This work was supported by the GAST project ‘Scientists Help Chernobyl Children’ sponsored the Vereinigung Deutscher Elektrizitatswerke, Germany. ¨
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S. Hillebrandt et al.r Mutation Research 381 (1997) 201–207 w7x M.S. Greenblatt, W.P. Bennett, M. Hollstein, C.C. Harris, Mutations in the p53 tumour suppressor gene: clues to cancer etiology and molecular pathogenesis, Cancer Res. 54 Ž1994. 4855–4878. w8x M. Zou, Y. Shi, N.R. Farad, p53 mutations in all stages of thyroid carcinomas, J. Clin. Endocrinol. Metab. 77 Ž1993. 1054–1058. w9x I. Chiba, T. Takahashi, N.M. Nau, D. D’Amico, D.T. Curiel, T. Mitsudomi, D.L. Buchhagen, D. Carbone, J.D. Minna, Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer, Oncogene 5 Ž1990. 1603–1610. w10x A. Foti, M. Bar-Eli, H.G. Ahuja, M.J. Cline, A splicing mutation accounts for the lack of p53 expression in a CML blast crisis cell line: a novel mechanism of p53 gene inactivation, Br. J. Haematol. 76 Ž1990. 143–145. w11x T. Takahashi, D. D’Amico, I. Chiloa, D.L. Buchagen, J. Minna, Identification of intronic point mutations as an alternative mechanism for p53 inactivation in lung cancer, J. Clin. Invest. 86 Ž1990. 363–369. w12x C.H. Hensel, R.H. Xiang, A.J. Sakaguchi, S.L. Naylor, Use of single conformation polymorphism technique and PCR to detect p53 gene mutations in small cell lung cancer, Oncogene 6 Ž1991. 1067–1071. w13x U. Masharani, D. Wolf, Bgl-II restriction fragment length polymorphism at the human p53 gene locus, Hum. Genet. 86 Ž1990. 244. w14x D. Carbone, I. Chiba, T. Mitsudomi, Polymorphism at codon 213 within the p53 gene, Oncogene 6 Ž1991. 1691–1692. w15x T.D. McDaniel, T. Carbone, Takahashi, P. Chumatov, E.H. Chang, K.F. Pirollo, J. Yin, Y. Huang, S.J. Meltzer, The MspI polymorphism in intron 6 of p53 detected by digestion of PCR products, Nucleic Acids Res. 19 Ž1991. 4796. w16x S. Peller, Y. Kopilova, S. Slutzki, A. Halevy, K. Kvitko, V. Rotter, A novel polymorphism in intron 6 of the human p53 gene: a possible association with cancer predisposition and susceptibility, DNA Cell Biol. 14 Ž1995. 983–990. w17x J.A. Taylor, M.A. Watson, T.R. Devereux, R.Y. Michels, G. Saccomanno, M. Anderson, p53 mutation hotspot in radonassociated lung cancer, Lancet 343 Ž1994. 86–87.
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w18x K. Baverstock, P. Egloft, A. Pinchera, C. Ruchti, D. Williams, Thyroid cancer after Chernobyl, Nature 359 Ž1992. 21–22. w19x D. Williams, A. Pinchera, A. Karaouglu, K.H. Chadwick, Thyroid cancer in children living near Chernobyl, Commission of the European Communities, Radiation Protection Research and Training Programme, 1993, pp. 1–108. w20x S. Hillebrandt, C. Streffer, Chr. Reiners, E. Demidchik, Mutations in the p53 tumour suppressor gene in thyroid tumours of children from areas contaminated by the Chernobyl accident, Int. J. Radiat. Biol. 69 Ž1996. 39–45. w21x A. Serra, G.L. Gaidano, D. Revello, A. Guerrasio, P. Ballerini, R. Dalla Favera, G. Saglio, A new TaqI polymorphism in the p53 gene, Nucleic Acids Res. 20 Ž1992. 4796. w22x K. Bhatia, M.I. Gutierrez, K. Huppi, I.T. Magrath, PCR detection of a neutral CGArCGG dimorphism in exon 6 of the human p53 gene, Nucleic Acids Res. 20 Ž1992. 4796. w23x G. Gaidano, Proc. Natl. Acad. Sci. USA 88 Ž1991. 5413– 5417. w24x I. Ilhan, S. Erekul, S. Atesalp, O. Ihan, N. Ankar, p53 codon 213 ŽA–G. polymorphism in a Turkish population, Pediatr. Hematol. Oncol. 12 Ž1995. 499–501. w25x Y.E. Nikiforov, M.N. Nikiforova, D.R. Gnepp, J.A. Fagin, Prevalence of mutations of ras and p53 in benign and malignant thyroid tumors from children exposed to radiation after the Chernobyl nuclear accident, 1996. w26x M.-Y. Lai, H.-C. Chang, H.-P. Li, C.-K. Ku, P.-J. Chen, J.-C. Sheu, G.-Z. Huang, P.H. Lee, D.S. Chen, Splicing mutations of the p53 gene in human hepatocellular carcinoma, Cancer Res. 53 Ž1993. 1653–1656. w27x T. Takahashi, D. D’Amico, I. Chiba, D.L. Buchhagen, J.D. Minna, Identification of intronic point mutations as an alternative mechanism for p53 inactivation in lung cancer, J. Clin. Invest. 89 Ž1990. 363–369. w28x A.M. Malkinson, Y. Ming, The intronic structure of cancerrelated genes regulates susceptibility to cancer, Mol. Carcinogen. 10 Ž1994. 61–65.
Polymorphisms in the p53 gene in thyroid tumours and blood samples of children from areas in Belarus S. Hillebrandt a , C. Streffer a
a,)
, E.P. Demidchik b, J. Biko c , Chr. Reiners
c
Institut fur Essen, Hufelandstr. 55, 45122 Essen, Germany ¨ Medizinische Strahlenbiologie, UniÕersitatsklinikum ¨ b Centre for Thyroid Tumours, Minsk, Belarus c Klinik fur Wurzburg, Josef Schneider Str. 2, Wurzburg, Germany ¨ Nuklearmedizin des UniÕersitatsklinikum ¨ ¨ ¨ Received 22 April 1997; revised 22 April 1997; accepted 23 July 1997
Abstract We present changes in the p53 gene in a group of 70 thyroid tumours and 40 blood samples obtained from children from Belarus. Three thyroid tumours show a polymorphism in exon 6 Žcodon 213. and 5 tumours show a polymorphism in intron 6, 37 bp upstream to the 5X-end of exon 7. Only one patient has a mutation in exon 7 Žcodon 258. resulting in an amino acid substitution in the protein p53. The distribution of polymorphisms in the 40 blood samples was as follows: three patients had a polymorphism in exon 6 and two persons had a polymorphism in intron 6. One polymorphism in intron 6 was also found in the group of 30 healthy children from Belarus. The fact that the differences in the sequence in p53 found in the tumours was also seen in the blood of these patients demonstrates that they are polymorphisms not induced by radiation exposure. It is difficult to conclude, if the polymorphisms found by us could be associated with the predisposition to radiation-induced cancer. q 1997 Elsevier Science B.V.
1. Introduction The structure and function of the p53 tumour suppressor gene have been extensively studied and it was found to be the most commonly mutated gene in human cancers. The majority of p53 mutations found are missense alterations within exons 5–8 of the gene w1–3x. In thyroid tumours, mutations of the p53 gene occur rarely w4–6x suggesting, that alterations in this gene may not be important for the development of thyroid tumours. On the other hand, there are some studies describing a relatively high frequency Ž13–25%. of mutations in exons 5–8 of the p53 gene )
Corresponding author. Fax: q49 Ž201. 723-5966.
in thyroid tumours w7,8x. The reasons for the different results are not known. In addition to mutations occurring in the p53 exons, there are several examples of genetic changes in intronic sequences of the p53 gene that result in splicing mutations w9–12x. These intronic mutations include insertions, deletions or base substitutions in regions adjacent to donor or acceptor splice sites of the affected intron. Polymorphisms are additional DNA sequence alterations observed in exons and introns of the p53 gene of normal tissues as well as that of tumours. They do not affect the structure of the wild-type protein. Some polymorphisms were detected in the various exons and introns with no obvious effect on p53 structure w13–15x. However, a
0027-5107r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 1 6 9 - 3
202
S. Hillebrandt et al.r Mutation Research 381 (1997) 201–207
possible association of polymorphisms with cancer predisposition and susceptibility was discussed by Peller et al. w16x. It has been reported that alterations in the p53 gene may be a potential marker for radiation-induced cancer w17x. The authors described p53 mutation hotspots in radon-associated lung cancers from uranium miners. Mutations occurring specifically in such tumours may be potential markers for radiation-induced cancer and could help to distinguish those tumours from tumours that develop spontaneously. For this reason, more investigations on tumours derived from patients exposed to radioactivity are needed. Spontaneous thyroid carcinoma in children is a rare occurrence and its frequency in Belarus before the Chernobyl accident was between one and two per year w18,19x. The reports of increasing numbers of childhood thyroid cancer cases in the years between 1986 and 1991 first appeared in 1991. The number of cases has roughly doubled each year from 1988, and reached more than 50 per year in 1991 w18,19x. By far the greatest increase was seen in the Gomel region Ž38 cases in 1991., and a less obvious increase was seen in the Brest and Grodno regions. The Gomel region is situated immediately to the north of Chernobyl and is known to have received a high level of radioactivity as fallout after the breakdown of the reactor. In this study, p53 gene alterations in cells of papillary thyroid carcinomas derived from 70 children who lived in different areas of Belarus during the reactor accident of Chernobyl in 1986 were analysed. Base-substitutions in exon 6 Žcodon 213. and in intron 6 Ž37 bp upstream to the 5X-end of exon 7. of the p53 gene have been observed. In our previous study, mutations in the p53 gene in thyroid tumours of children from areas highly contaminated by the Chernobyl accident were described w20x. Some of these mutations have been identified as polymorphisms. These polymorphisms appear to contribute to the high frequency of mutations reported by us relative to those described in other studies of thyroid tumours from patients without analogous exposures to ionizing radiation. Thus, our previous conclusion that the number of p53 mutations observed in thyroid carcinomas of children from areas contaminated by the Chernobyl accident
is induced by ionizing radiation appears to be incorrect. However, the polymorphisms already present before the radiation exposure may support the development of thyroid carcinomas.
2. Materials and methods 2.1. Tumour samples and blood The donors who were included in this study are male and female children from Belarus ranging from 6 to 18 years. The children lived in different areas of the republic during the reactor accident of Chernobyl. The areas are located around 150 km ŽGomel., 450 km ŽBrest., 350 km ŽMinsk., 500 km ŽGrodno. from Chernobyl. The children received thyroid surgery at the Centre for Thyroid Tumours in Minsk. Clinical data and tumour pathology were available from this institution. Blood samples of the children from Belarus were obtained prior to radiotherapy from the Clinic of Nuclear Medicine in Wurzburg, where the children ¨ received radioiodine for the therapy of disseminated thyroid tumours. As controls, blood samples from healthy Belarussian children were used. 2.2. DNA preparation Tumour tissues were pressed through a Nylon net and the cells were fixed in 96% ethanol. Those suspensions containing cells derived from one part of each tumour were obtained from the Centre for Thyroid Tumours in Minsk. Since sections taken from different parts of the same tumour were not available, the level of heterogeneity within individual tumours could not be tested. No cytogenetic analysis has been performed. The cells were incubated in 200 ml 0.5% SDS solution at 378C for 1 h. Then the same volume of a digestion buffer Ž100 mM Tris-HCl, pH 8.5, 1 mM EDTA, 0.5% Tween 20, 100 mgrml proteinase K. was added and the solution was stirred at 378C for 12 h. The DNA was precipitated with 100% ethanol and diluted with 100 ml nuclease-free water. No phenol–chloroform extraction was performed. DNA from blood was prepared with the Split Second Kit from Boehringer ŽMannheim..
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2.3. PCR conditions The PCR conditions for the detection of polymorphisms in exons 4–9 and the adjacent introns of the p53 gene with the TGGE Žtemperature gradient gel electrophoresis. were described by Hillebrandt et al. w20x. For sequencing of exons 6 and 7, and the adjacent introns the primers used in PCR reaction were: exon 6 q introns, the 5X-primer: 5X-TGGTTGCCCAGGGTCCCCAG-3X , the 3X -primer: 5X -CCAGT TGCAAACCAGACC-3X ; exon 7 q introns, the 5Xprimer: 5X-CTTGCCACAGGTCTCCC CA-3X , the 3X-primer: 5X-TGGCAAGTGGCTCCTGAC-3X . PCR reactions were carried out in 100 ml solution containing genomic DNA, 50 pmol of each primer, 2 mmol each of the four deoxynucleotide triphosphates, 2.5 U of Taq DNA polymerase ŽBoehinger, Mannheim., 10 mM Tris-HCl, pH 8.3, 25 mM KCl, 1.5 mM MgCl 2 . An Omni-Gene thermal cycler was used for the amplification. The PCR program was as follows: one cycle of 10 min at 948C, 35 cycles of 30 s at 948C, min at 608C, 30 s at 728C, ending with one cycle of 7 min at 728C. 2.4. Identification of polymorphisms with the help of temperature gradient gel electrophoresis (TGGE) This method, described in detail by Hillebrandt et al. w20x, is based on the principle that heteroduplexes of DNA molecules have a different melting temperature than DNA–homoduplexes: a heterozygote mutation could be identified as 4 bands and a homorhemizygote mutation as one band showing a different electrophoretic mobility comparing to a fragment without mismatch. A fragment with a mismatch is retarded at a slightly different temperature and produces a distinct band on the gel. The TGGE was performed as described by the manufacturer ŽTGGE Handbook, Diagen.. Several PCR products were run at the same time. The molecules exhibited different electrophoretic mobilities according to their melting temperatures. The optimal running time and the temperature gradient were calculated from the parameters of the perpendicular TGGE with the help of a formula given in the manufacturer’s handbook. In each case, tumour DNA with a known p53 mutation was used as a
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positive control. For analysis of point mutations in ds DNA, an extremely high detection rate of ) 95% is routinely achieved with TGGE. Thus, we can detect the majority of polymorphisms in the analysed area. Exons and the adjacent introns showing polymorphisms identified by the TGGE were analysed by the direct sequencing as described below. 2.5. Direct sequence analysis Sequence analysis was carried out by solid-phase sequencing of single-strand PCR products. The antisense primer of the primer pair was biotinylated, allowing immobilization of the PCR product on streptavidin-coated beads ŽDynabeads M-280 Streptavidin, Dynal.. Denaturation of the immobilized double-stranded DNA was achieved by incubation with NaOH. For sequencing, a PRISM Sequenase Terminator Sequencing Kit ŽApplied Biosystems. was used. Its performance relies on four dye-labelled dideoxynucleotides ŽT, C, A, and G dye terminators. which allow all four termination reactions to be performed in one single tube. Gel electrophoresis, data collection and analysis were performed with an Applied Biosystems Model 373 DNA Sequencing System.
3. Results 3.1. Distribution of polymorphisms in thyroid tumour samples Exons and the adjacent introns amplified by PCR were analysed for polymorphisms with the TGGE. The distribution of polymorphisms in 70 papillary thyroid carcinomas Žshown in Table 1. reveals that three tumours Ž4.2%. show an A:T ™ G:C substitution in codon 213 of exon 6. This substitution changing CGA ™ CGG does not result in an amino acid alteration in the p53 protein. Fig. 1A shows the wild-type sequence of exon 6 and indicates the polymorphic site. The sequence analysis of exon 6 was performed for the tumour samples A4, A6, A8 and for human fibroblasts used as wild-type. The results of the sequence analysis demonstrate, that there is a peak for ‘A’ and a peak for ‘G’ at the position corresponding to the ‘A’-peak in the wild-type se-
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Table 1 Polymorphisms and mutations in the p53 gene determined in 70 children with papillary thyroid carcinomas as well as in the blood cells Case Sex Area
Level of differentiation Intron or exonrcodon polymorphismrmutation Coding effect Polymorphism in the blood cells
O7 A4 A6 A7 A8 B3 D1 4N 7N 16N
high high moderate high low high high high moderate high
a b
F M F M M F M F M F
Gomel Minsk Gomel Gomel Grodno Brest Brest Gomel Grodno Brest
intron 6 exon 6r213 exon 6r213 exon 7r258 exon 6r213 intron 6 intron 6 exon 1 intron 6 intron 6
G:C ™ C:G A:T ™ G:C A:T ™ G:C G:C ™ A:T A:T ™ G:C G:C ™ C:G G:C ™ C:G –a G:C ™ C:G G:C ™ C:G
none none none Glu ™ Lys none none none –a none none
0b 0 0 0 exon 6rA:T™ G:C intron 6rG:C™ C:G intron 6rG:C™ C:G 0 intron 6rG:C™ C:G 0
No sequencing was performed. 0, blood samples were not available.
quence indicating a heterozygote base substitution in the tumour sample Ždata not shown.. Furthermore, five out of 70 patients Ž7.1%. demonstrate a G:C ™ C:G substitution in intron 6, located 37 bp upstream to exon 7. The wild-type sequence of the part of intron 6 indicating the polymorphic site is shown by Fig. 1B. The sequence analysis was performed for the tumour samples 07, B3, D1, 16N, 7N. The results demonstrate a homozygote Žor hemizygote. base substitution in tumour sample 7N and a heterozygosity for both alleles in the other tumours. In only one case, the GAA ™ AAA substitution in codon 258 of exon 7 occurs and is leading to the amino acid change Glu ™ Lys. The wild-type sequence of exon 7 indicating the mutated position is shown by Fig. 1C. The G ™ A homozygote substitution observed in the tumour sample A7 is a result of the loss of the wild-type sequence. Using the TGGE method, we have found a polymorphism in exon 1 of one tumour. However, a
sequence analysis could not be performed in this case. Blood was available from 4 patients showing polymorphisms in the thyroid tumours ŽB3, D1, 7N, A8.. The polymorphism found in intron 6 in tumours B3, D1, 7N, and the polymorphism in exon 6 found in patient A8 were also observed in the blood samples of those patients. This indicates that they are present in constitutional, normal cells. Fig. 2 represents the results of the TGGE demonstrating a polymorphism in intron 6 and in exon 6. 3.2. Analysis of polymorphisms in peripheral blood cells from children with thyroid tumours and from healthy children Screening of blood cells from 40 children with thyroid cancer indicates that 12.5% Ž5r40. display a polymorphism in the p53 gene. Thyroid tumour samples were not available from these children. The distribution of the polymorphic patterns in the blood Table 2 Polymorphisms in the p53 gene determined in the blood samples from 40 children with thyroid carcinomas as well as from 30 healthy children from Belarus
Fig. 1. Wild-type sequences of exon 6 ŽA. of the relevant part of X intron 6 and the 5 -end of exon 7 ŽB. and of exon 7 ŽC.. The arrowheads show positions for which polymorphisms Žmutation. were observed.
Case
Sex Tumour Exon or intronrcodon Polymorphism
BI GO BA NA KO KOW
F F F F F F
q q q q q y
exon 6r213 exon 6r213 exon 6r213 intron 6 intron 6 exon 6
A:T ™G:C A:T ™G:C A:T ™G:C G:C ™C:G G:C ™C:G A:T ™G:C
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Fig. 2. PCR–TGGE analysis of peripheral blood samples demonstrating a polymorphism in intron 6 Žleft. and a polymorphism in exon 6 Žright..
is as follows: three patients show the A:T ™ G:C polymorphism in codon 213 of exon 6 and two patients demonstrate the G:C ™ C:G polymorphism in intron 6. The polymorphic patterns are as shown in Fig. 2. Blood cells from 30 healthy children from Belarus were used as controls. In this group, the G:C ™ C:G polymorphism in intron 6 was observed in one child. The frequency of polymorphisms in the p53 gene in the group of 40 blood samples obtained from children with thyroid tumours is not significantly higher compared with the group of 30 healthy children Ž p s 0.23, Fisher’s exact test.. Table 2 summarizes the distribution of polymorphisms in the blood samples from 40 children with a thyroid tumour and 30 healthy children from Belarus.
4. Discussion In this study, we analysed DNA of thyroid tumours and of peripheral blood cells from children
with and without thyroid tumours from Belarus. Three out of 70 tumour samples and three out of 40 blood samples show an A ™ G substitution in codon 213 of exon 6. Previous studies have indicated that some polymorphisms in the p53 gene were found to be distributed in typical patterns of various geographical locations and in various human races: it was observed that 10% from 50 unrelated individuals from South America and 3% from 30 unrelated individuals from North America have a polymorphism in codon 213 of the p53 gene w21,22x. However, there are no data on its occurrence in the population of Belarus. Correlations between a specific distribution of polymorphic patterns and cancer development have been studied: an analysis of mutations in the p53 gene in Burkitt’s lymphomas shows that 2% of the tumours have the silent A ™ G substitution in codon 213 w23x. The p53 codon 213 polymorphism was also studied in a healthy Turkish population and in Turks with different types of tumours. The data revealed no association between this polymorphism and the development of tumours w24x.
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A second polymorphism observed in the analysed group of thyroid tumours and blood samples is a G ™ C substitution found in intron 6, 37 bp upstream of exon 7. Five out of 70 thyroid tumours, two out of 40 blood samples from children with those tumours and one blood sample in the group of 30 healthy children showed this intronic polymorphism, which is a new polymorphism which has not been previously described. Nikiforov et al. w25x analysed 33 papillary thyroid carcinomas, one follicular carcinoma and 22 benign lesions removed from radiation-exposed children from the regions around Chernobyl and screened them for point mutations in p53 Žexons 5–8. using single-strand conformation polymorphism ŽSSCP. analysis. The authors found no intron 6 or exon 6rcodon 213 polymorphisms, but one missense mutation in exon 5 and one silent mutation in codon 182 in the group of 33 papillary thyroid carcinomas. They conclude that p53 mutations do not appear to be important in the development of post-Chernobyl thyroid papillary carcinomas, but may, in some cases, have a role in progression to a more aggressive phenotype. Intronic point mutations of the gene could lead to abnormal pre-mRNA splicing and defective protein. Mutations at consensus sequences at either the 5X- or 3X-splice site of intron 6 and 7 were found in human hepatocellular carcinomas w26x. Splicing mutations leading to the production of abnormal p53 protein were also described for lung cancer w27x. The polymorphism found by us in intron 6 is not located at the consensus sequence of the splice sites, but in the region of the branch site also involved in splicing. However, we do not know, if this polymorphism could be involved in the splicing process. The functions of the intronic sequences are not fully known. Control sequences affecting transcription and the level of the p53 protein could be located in the intronic regions. Studies on a polymorphism in the Ki-ras oncogene suggest, that polymorphisms in intronic sequences may affect the gene regulatory role, leading to susceptibility to cancer w27x. Thus, sequence alterations in introns could influence binding of transcription factors to the DNA and could change the expression of tumour suppressor genes. Furthermore, a helical distortion as a consequence of intronic sequence alterations could prevent binding
of proteins to the DNA. The possibility that the regions of intronic polymorphisms could control the stability or half-life of proteins was discussed by Peller et al. w16x. However, these assumptions should be investigated further. The differences in the p53 sequence found in the thyroid tumours of children from the regions around Chernobyl are also observed in the blood cells Žlymphocytes. of these patients. Thus, they have not been induced by radiation exposure and may be polymorphisms specific for the population in Belarus. Correlations between a specific distribution of a polymorphic pattern and cancer development have not been established so far. It was predicted, that some intronic polymorphisms may predispose towards coding-region mutations that increase the likelihood of a deleterious phenotype w28x. In summary, it is difficult to conclude if the development of thyroid cancer in children after the Chernobyl accident is influenced by the germ-line polymorphisms described in this study.
Acknowledgements This work was supported by the GAST project ‘Scientists Help Chernobyl Children’ sponsored the Vereinigung Deutscher Elektrizitatswerke, Germany. ¨
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