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An Ava I polymorphism in the TP53 gene
Molecular and Cellular Probes (1999) 13, 393–395 Article No. mcpr.1999.0256, available online at http://www.idealibrary.com on
Polymorphism Report
An Ava I polymorphism in the TP53 gene D. Graziani,1 S. Romagnoli,1∗ B. Cassani,1 R. M. Alfano,1 M. Roncalli2 and G. Coggi1 1
II Department of Pathology, University of Milan School of Medicine, Via A di Rudinı` 8, 20142 Milano, Italy and 2Humanitas Clinical Institute, Rozzano, Milano, Italy (Received 11 January 1999, Accepted 22 June 1999)
TP53 gene plays a major role in the process of malignant transformation and tumour progression so that abnormalities such as point mutation or allelic loss of this gene are a common finding in different tumour types. Most of the mutations identified cover a conserved region of the gene, spanning from exon 4 to exon 9. The present report describes a novel polymorphism, 12 nucleotides downstream the splicing junction of exon/intron 9 identified in a cohort of 103 Italian healthy blood donors. The polymorphism results in the creation of a new restriction site for Ava I. 1999 Academic Press
KEYWORDS: p53 gene polymorphism, p53 intron 9, SSCP, RFLP, Ava I polymorphism.
INTRODUCTION In a large number of human tumours, TP53 gene (MIM 191170) abnormalities have been detected:1–3 both allelic losses and point mutations are involved in malignant transformation and tumour progression4,5 and in some cases related to both prognosis of different tumour types and their response to chemotherapy.6–8 Awareness of TP53 genetic polymorphisms is critical in the evaluation of possible clinical significance. Further, polymorphisms that are easily recognizable by restriction endonucleases can further simplify the screening. So far a number of TP53 gene polymorphisms have been reported in the literature, some of them in the coding region (two in exon 4, one in exon 5 and one in exon 6) and some in the introns of the gene.9–13
We report the finding of a new polymorphism of TP53, near the splicing site between exon 9 and intron 9, identified on 103 Italian healthy blood donors. MATERIALS AND METHODS Peripheral blood from 103 healthy blood donors was examined: genomic DNA was extracted from leucocytes using conventional phenol/chloroform techniques. Polymerase chain reaction (PCR) was performed using intronic primers flanking p53 exon 9:14 Upstream:
5′ GGT GGA GGA GAC CAA GGG TGC AGT T 3′
Downstream:
5′ CTG GAA ACT TTC CAC TTG AT 3′
∗ Author to whom correspondence should be addressed at: II Department of Pathology, University of Milan School of Medicine, Via A di Rudinı` 8, 20142 Milano, Italy. Tel: +39 2813 5366; Fax: +39 28912 2032; E-mail: [email protected]
0890–8508/99/050393+03 $30.00/0
1999 Academic Press
394
D. Graziani et al.
Fluorescent sequencing of the abnormal band, excised from an SSCP gel, detected a common T to C transition of TP53 gene 12 bp downstream of the splicing site between exon 9 and intron 9. As a consequence, a new restriction site, recognized by Ava I was created [ct(t→c) ggg], as documented by RFLP analysis performed on samples showing the same SSCP pattern (Fig. 1). Assuming Hardy–Weinberg equilibrium, the allele frequencies were estimated to be: 0·9806 for the common T allele and 0·01942 for the C allele, Ava I variant. The demonstration of this restriction polymorphism is important when analysing molecular alterations of TP53 encountered in neoplastic transformation.
REFERENCES Fig. 1. Restriction fragment length polymorphism analysis: samples heterozygous for the polymorphic allele (4, 5, 6) are cut in two bands of 155 and 31 bp, respectively, while homozygotes for the wild-type allele (1, 2, 3) are not cleaved by the enzyme.
Polymerase chain reaction conditions were as follows: 0·5 l of each primer, 1·5 m MgCl2 and 55°C annealing temperature; after 35 cycles the final 186 bp amplification product was visualized on an ethidium bromide stained 2% agarose gel. Screening for p53 gene mutations was performed using non-radioactive single strand conformation polymorphism (SSCP), as previously described.15 DNA bands showing abnormal mobility as compared to controls, were excised, eluted from the gel and amplified again, in order to enrich the amount of the mutated allele, using the same primers, but with a 5′ tail sequence of M13. Samples were finally sequenced with M13 dye primer chemistry using an ABI PRISM 310 automatic sequencer (Perkin Elmer). For restriction fragment length polymorphism (RFLP) analysis 10 ll (about 2 lg) of PCR products were digested overnight with 5 U of Ava I at 37°C. After digestion, products were visualized on agarose gel. Samples with the polymorphism appeared as two fragments of 155 and 31 bp, respectively.
RESULTS The analysis was performed on 103 non-related Italian healthy blood donors: in four cases, three males and a female, a common SSCP pattern of altered mobility was detected.
1. Levine, A. J., Momand, J. & Finlay, C. A. (1991). The p53 tumor suppressor gene. Nature 351, 453–456. 2. Hollstein, M., Sidransky, D., Vogelstein, B. & Harris, C. C. (1991). p53 mutation in human cancers. Science 253, 49–53. 3. Hainaut, P., Hernandez, T., Robinson, A. et al. (1998). IARC database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualization tools. Nucleic Acid Research 26, 205–213. 4. Fearon, E. R. & Vogelstein, B. (1990). A genetic model for colorectal tumorigenesis. Cell 61, 759–767. 5. Greenblatt, M. S., Bennet, W. P., Hollstein, M. & Harris, C. C. (1994). Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Research 54, 4855–4878. 6. Levesque, M. A., Katsaros, D., Yu, H., Zola, P., Sismondi, P., Giardina, G. & Diamandis, E. P. (1995). Mutant p53 protein overexpression is associated with poor outcome in patients with well or moderately differentiated ovarian carcinoma. Cancer 75, 1327– 1338. 7. Nabeya, Y., Loganzo, F., Maslak P. et al. (1995). The mutational status of p53 protein in gastric and esophageal adenocarcinoma cell lines predicts sensitivity to chemotherapeutic agents. International Journal of Cancer 64, 37–46. 8. Righetti, S. C., Della Torre, G., Pilotti, S. et al. (1996). A comparative study of p53 gene mutation, protein accumulation, response to cisplatin-based chemotherapy in advanced ovarian carcinoma. Cancer Research 56, 689–693. 9. Carbone, D., Chiba, I. & Mitsudomi, T. (1991). Polymorphism at codon 213 within the p53 gene. Oncogene 6, 1691–1692. 10. Mazars, G. R., Jeanteur, P., Lynch, H. T., Lenoir, G. & Theillet, C. (1992). Nucleotide sequence polymorphism in a hotspot mutation region of the p53 gene. Oncogene 7, 781–782. 11. Felix, C. A., Brown, D. L., Mitsudomi, T. et al. (1994). Polymorphism at codon 36 of the p53 gene. Oncogene 9, 327–328. 12. Oliva, M. R., Saez, G. T., Latres, E. & Cordon-Cardo,
An Avaa I polymorphism in the TP53 gene C. (1995). A new polymorphic site in intron 2 of TP53 characterizes LOH in human tumors by PCR-SSCP. Diagnostic Molecular Pathology 4, 54–58. 13. Ito, T., Seyama, T., Hayashi, T. et al. (1994). HaeIII polymorphism in intron 1 of the human p53 gene. Human Genetics 93, 222. 14. Marchetti, A., Buttitta, F., Merlo, G. et al. (1993). p53 alteration in non-small cell lung cancers correlate with
395
metastatic involvement of hilar and mediastinal lymph nodes. Cancer Research 53, 2846–2851. 15. Bosari, S., Marchetti, A., Buttitta, F. et al. (1995). Detection of p53 mutations by Single-Strand Conformation Polymorphisms (SSCP) gel electrophoresis. A comparative study of radioactive and nonradioactive Silver-stained SSCP analysis. Diagnostic Molecular Pathology 4, 249–255.
Polymorphism Report
An Ava I polymorphism in the TP53 gene D. Graziani,1 S. Romagnoli,1∗ B. Cassani,1 R. M. Alfano,1 M. Roncalli2 and G. Coggi1 1
II Department of Pathology, University of Milan School of Medicine, Via A di Rudinı` 8, 20142 Milano, Italy and 2Humanitas Clinical Institute, Rozzano, Milano, Italy (Received 11 January 1999, Accepted 22 June 1999)
TP53 gene plays a major role in the process of malignant transformation and tumour progression so that abnormalities such as point mutation or allelic loss of this gene are a common finding in different tumour types. Most of the mutations identified cover a conserved region of the gene, spanning from exon 4 to exon 9. The present report describes a novel polymorphism, 12 nucleotides downstream the splicing junction of exon/intron 9 identified in a cohort of 103 Italian healthy blood donors. The polymorphism results in the creation of a new restriction site for Ava I. 1999 Academic Press
KEYWORDS: p53 gene polymorphism, p53 intron 9, SSCP, RFLP, Ava I polymorphism.
INTRODUCTION In a large number of human tumours, TP53 gene (MIM 191170) abnormalities have been detected:1–3 both allelic losses and point mutations are involved in malignant transformation and tumour progression4,5 and in some cases related to both prognosis of different tumour types and their response to chemotherapy.6–8 Awareness of TP53 genetic polymorphisms is critical in the evaluation of possible clinical significance. Further, polymorphisms that are easily recognizable by restriction endonucleases can further simplify the screening. So far a number of TP53 gene polymorphisms have been reported in the literature, some of them in the coding region (two in exon 4, one in exon 5 and one in exon 6) and some in the introns of the gene.9–13
We report the finding of a new polymorphism of TP53, near the splicing site between exon 9 and intron 9, identified on 103 Italian healthy blood donors. MATERIALS AND METHODS Peripheral blood from 103 healthy blood donors was examined: genomic DNA was extracted from leucocytes using conventional phenol/chloroform techniques. Polymerase chain reaction (PCR) was performed using intronic primers flanking p53 exon 9:14 Upstream:
5′ GGT GGA GGA GAC CAA GGG TGC AGT T 3′
Downstream:
5′ CTG GAA ACT TTC CAC TTG AT 3′
∗ Author to whom correspondence should be addressed at: II Department of Pathology, University of Milan School of Medicine, Via A di Rudinı` 8, 20142 Milano, Italy. Tel: +39 2813 5366; Fax: +39 28912 2032; E-mail: [email protected]
0890–8508/99/050393+03 $30.00/0
1999 Academic Press
394
D. Graziani et al.
Fluorescent sequencing of the abnormal band, excised from an SSCP gel, detected a common T to C transition of TP53 gene 12 bp downstream of the splicing site between exon 9 and intron 9. As a consequence, a new restriction site, recognized by Ava I was created [ct(t→c) ggg], as documented by RFLP analysis performed on samples showing the same SSCP pattern (Fig. 1). Assuming Hardy–Weinberg equilibrium, the allele frequencies were estimated to be: 0·9806 for the common T allele and 0·01942 for the C allele, Ava I variant. The demonstration of this restriction polymorphism is important when analysing molecular alterations of TP53 encountered in neoplastic transformation.
REFERENCES Fig. 1. Restriction fragment length polymorphism analysis: samples heterozygous for the polymorphic allele (4, 5, 6) are cut in two bands of 155 and 31 bp, respectively, while homozygotes for the wild-type allele (1, 2, 3) are not cleaved by the enzyme.
Polymerase chain reaction conditions were as follows: 0·5 l of each primer, 1·5 m MgCl2 and 55°C annealing temperature; after 35 cycles the final 186 bp amplification product was visualized on an ethidium bromide stained 2% agarose gel. Screening for p53 gene mutations was performed using non-radioactive single strand conformation polymorphism (SSCP), as previously described.15 DNA bands showing abnormal mobility as compared to controls, were excised, eluted from the gel and amplified again, in order to enrich the amount of the mutated allele, using the same primers, but with a 5′ tail sequence of M13. Samples were finally sequenced with M13 dye primer chemistry using an ABI PRISM 310 automatic sequencer (Perkin Elmer). For restriction fragment length polymorphism (RFLP) analysis 10 ll (about 2 lg) of PCR products were digested overnight with 5 U of Ava I at 37°C. After digestion, products were visualized on agarose gel. Samples with the polymorphism appeared as two fragments of 155 and 31 bp, respectively.
RESULTS The analysis was performed on 103 non-related Italian healthy blood donors: in four cases, three males and a female, a common SSCP pattern of altered mobility was detected.
1. Levine, A. J., Momand, J. & Finlay, C. A. (1991). The p53 tumor suppressor gene. Nature 351, 453–456. 2. Hollstein, M., Sidransky, D., Vogelstein, B. & Harris, C. C. (1991). p53 mutation in human cancers. Science 253, 49–53. 3. Hainaut, P., Hernandez, T., Robinson, A. et al. (1998). IARC database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualization tools. Nucleic Acid Research 26, 205–213. 4. Fearon, E. R. & Vogelstein, B. (1990). A genetic model for colorectal tumorigenesis. Cell 61, 759–767. 5. Greenblatt, M. S., Bennet, W. P., Hollstein, M. & Harris, C. C. (1994). Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Research 54, 4855–4878. 6. Levesque, M. A., Katsaros, D., Yu, H., Zola, P., Sismondi, P., Giardina, G. & Diamandis, E. P. (1995). Mutant p53 protein overexpression is associated with poor outcome in patients with well or moderately differentiated ovarian carcinoma. Cancer 75, 1327– 1338. 7. Nabeya, Y., Loganzo, F., Maslak P. et al. (1995). The mutational status of p53 protein in gastric and esophageal adenocarcinoma cell lines predicts sensitivity to chemotherapeutic agents. International Journal of Cancer 64, 37–46. 8. Righetti, S. C., Della Torre, G., Pilotti, S. et al. (1996). A comparative study of p53 gene mutation, protein accumulation, response to cisplatin-based chemotherapy in advanced ovarian carcinoma. Cancer Research 56, 689–693. 9. Carbone, D., Chiba, I. & Mitsudomi, T. (1991). Polymorphism at codon 213 within the p53 gene. Oncogene 6, 1691–1692. 10. Mazars, G. R., Jeanteur, P., Lynch, H. T., Lenoir, G. & Theillet, C. (1992). Nucleotide sequence polymorphism in a hotspot mutation region of the p53 gene. Oncogene 7, 781–782. 11. Felix, C. A., Brown, D. L., Mitsudomi, T. et al. (1994). Polymorphism at codon 36 of the p53 gene. Oncogene 9, 327–328. 12. Oliva, M. R., Saez, G. T., Latres, E. & Cordon-Cardo,
An Avaa I polymorphism in the TP53 gene C. (1995). A new polymorphic site in intron 2 of TP53 characterizes LOH in human tumors by PCR-SSCP. Diagnostic Molecular Pathology 4, 54–58. 13. Ito, T., Seyama, T., Hayashi, T. et al. (1994). HaeIII polymorphism in intron 1 of the human p53 gene. Human Genetics 93, 222. 14. Marchetti, A., Buttitta, F., Merlo, G. et al. (1993). p53 alteration in non-small cell lung cancers correlate with
395
metastatic involvement of hilar and mediastinal lymph nodes. Cancer Research 53, 2846–2851. 15. Bosari, S., Marchetti, A., Buttitta, F. et al. (1995). Detection of p53 mutations by Single-Strand Conformation Polymorphisms (SSCP) gel electrophoresis. A comparative study of radioactive and nonradioactive Silver-stained SSCP analysis. Diagnostic Molecular Pathology 4, 249–255.