Glioblastoma is one of the most malignant tumors in humans. This tumor is thought to develop as a result of the accumulation of genetic abnormalities, mainly focused on the loss of heterozygosity on chromosome 10. O^sup 6^-methylguanine-DNA methyltransferase (MGMT), which is one of the most important DNA repair proteins, has also been reported that enzymatic activity, as well as the methylation status of the promoter region of the MGMT gene, contributes to the therapeutic response of alkylating agents. We previously found three allelic variants in the MGMT gene and assayed the characteristics of these polymorphic proteins. We designed a case-control study to investigate the role of MGMT genotypic risk factors for primary brain tumors. We compared the distributions of MCMT genotypes in primary brain tumors and normal controls. The frequencies of MGMT genotypes in examined primary brain tumors were not different from normal subjects. However, the combined heterozygote of V1 and a wild allele (V1/W) was frequently detected in de novo glioblastoma group with significant difference. Interestingly, among glial tumors, the V1/W genotype was dominantly detected in the patients with de novo glioblastoma. This study suggests that the V1/W genotype of the MGMT gene may contribute to the de novo occurrence of glioblastoma. [Neurol Res 2003; 25: 875-879]
Keywords: O^sup 6^-methylguanine-DNA methyltransferase; genotype; polymorphism; brain tumor; glioblastoma
Glioblastoma multiforme (GBM) is the most malignant glial brain tumor in humans. Genetic alterations such as point mutations, loss of heterozygosity, excess activation of a particular gene and so on, are reported in many sites of chromosomes in GBMs, but intrinsic risk factors are currently unknown. Exposure to exogenous alkylating agents, particularly N-nitroso compounds, has been associated with increased incidence of primary human brain tumors1, so the individual ability to repair the DNA damage induced by alkylating agents must be considered with regard to the tumorigenesis of GBM. A previous report2 demonstrated that the histological normal brain tissue adjacent to primary brain tumors lacked detectable O^sup 6^-methylguanine-DNA methyltransferase (MGMT) activity [methyl excision repair-defective (Mer-) status]. MGMT is known to be one of the most important DNA repair enzymes, and catalyzes the transfer of the methyl group from O^sup 6^-methylguanine, as well as O^sup 4^-methylthymine adducts of double-stranded DNA induced by the alkylating agents to the cysteine residue in its own molecule and thus prevent the G:C to A:T transition3,4. This enzyme is also known to locate in the chromosome 10q26(5), where heterozygous deletion is often observed in GBM patients. So it is important to study the relationship between the genetic and enzymatic status of the MGMT and GBMs. Previously, as a result of a Japanese population survey, we reported three allelic variants for the MGMT gene named V1, V2, and V36. V1 has a C-T transition at nt. 262, thus causing a single amino acid change (Leu-84Phe) combined with an additional silent C-T transition at nt. 171 in exon 3. V2 has a G-C transversion at nt. 207 in exon 3, which is thus considered to cause Trp-65-Cys. V3 has a silent G-A transition at nt. 579 in exon 5. The allelic frequencies of V1 and V2 were estimated to be 0.162 and 0.002, respectively. Furthermore, we investigated the enzymatic characteristics of the polymorphic MGMT protein7.
In this report, we survey the frequencies of the MGMT genotype in histologically verified primary brain tumor patients. As a result, the combined heterozygote of V1 and wild type MGMT was detected at a higher frequency in the patients with GBM with statistical significance than in the control subjects.
SUBJECTS AND METHODS
The surgical specimens were obtained from 74 patients treated in the Department of Neurosurgery, Oita Medical University, Oita, Japan, between 1998 to 2001 (mean age ± SD; 45.5 ± 19.23). Two hundred and fifty-five healthy Japanese volunteers (mean age ± SD; 46.1 ± 10.49) were included in this study. Informed consent was obtained from all participants. As shown in Table 1, patients with various brain tumors were included in this study, although the majority of the tumors were of glial origin. Twenty-two patients with de novo (primary) glioblastoma had a clinical history of less than three months, without a prior biopsy or excision for low-grade or anaplastic astrocytomas.
In principle, genomic DNA was isolated from the subjects' peripheral blood by standard phenol/chloroform extraction and ethanol precipitation protocols following overnight protease K digestion8,9, or by using ISOTISSUE (Nippon Gene Co., Ltd, Toyama, Japan) according to the manufacturer's protocols. When we could not obtain peripheral blood, we extracted the DNA from frozen-stocked or freshly prepared brain tumor tissues
The MGMT coding region extends over four exons, i.e. Exon 2, 3, 4 and 5. PCR amplification for exon 3 was carried out separately using 50 ng of genomic DNA, 2.5 pmol of the sense and antisense primers described in a previous report6, dNTP at 0.2 mM each, 2 mM MgCl2 and 0.45 unit of Taq DNA poiymerase (Wako, Osaka, Japan) in the presence of 10 mM Tris HCl, pH8.3, 50 mM KCI, 0.1 % Triton X-10O in a final volume of 10 I for 35 cycles. The PCR annealing temperatures were 58°C. Following amplification, 3I of the reaction mixture was mixed with 3 I of 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, 1OmM NaOH, and 20 mM EDTA. The mixture was then heated at 94°C for 2 min, immediately cooled on ice and applied to MDE gel (AT Biochem Inc., Malvern, PA, USA) in a 1TBE buffer (Tris/borate/EDTA), pH 8.0 at 110-130 volts at room temperature for 12h. After completing electrophoresis, the MDE gel was processed by silver staining using Silver Stain DAIICHI (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan) according to the manufacturer's protocol. We judged the genotype of MGMT according to the previously reported patterns of mobility shifts6. When an unknown shift pattern was detected, we performed PCR-based direct sequencing of the samples, using an automatic DNA sequencer type 377 (Perkin Elmer, Tokyo, Japan).
Differences in the frequency of each genotype, allele or phenotype between the patients and controls were analyzed using the chi-square test. Relative risk (RR) was calculated as the strength of association between the polymorphism and brain tumor by the odds ratio, which was calculated from 22 contingency tables.
Radiolabeled PCR-SSCP in exon 3 clearly demonstrated a mobility shift in V1 and V2 from the wild type (W)6. The silver staining method was confirmed to be comparable to the radiolabeled SSCP analysis (data not shown).
The allele frequencies in patients with primary brain tumors were 0.87 for the wild type (W), 0.12 for V1 and 0.01 for V2. Those in normal subjects were 0.84 for W, 0.16 for V1 and 0.00 for V2. The results of normal subjects were cited from a previous report6 by Abe, who is one of the co-authors of this article.
The distribution of MGMT genotypes in patients with primary brain tumors and the control subjects is presented in Table 2. Deviations from the Hardy-Weinberg's distribution were not significant in either group (X^sup 2:^ 1.7643 and 2.4891, df=3, p > 0.10). No significant difference was observed in the distribution of genotype between the two groups.
We further investigated the difference in the distribution of genotype between 58 cases of patients with glioma and control subjects. The data is shown in Table 3. The frequencies of genotype in patients were 0.74 for W/W, 0.24 for W/V1, 0.02 for W/V2, and there was no significant difference between the two groups.
However, when we investigated the distribution of 22 patients with de novo glioblastoma, the W/V1 genotype was detected with a significantly higher incidence than control group (Table 4). The frequencies in de novo glioblastoma were 0.50 for W/W and 0.50 for W/V1. The odds ratio for W/V1 was 2.91 (95% CI; 1.19 to 7.08). Furthermore, when compared with a population of 36 cases containing low grade (WHO grade 1 and 2), as well as anaplastic glioma (WHO grade 3), the V1/W genotype was detected dominantly in de novo glioblastoma with a significant difference (odds ratio 10.67, 95% CI 2.51 to 45.42) (Table 5).
We previously surveyed the frequencies of MGMT polymorphisms in colorectal cancer6, Parkinson's disease (data not shown) and Alzheimer's disease (data not shown). However, we could not find significant differences between those disease and normal subjects. In this study, a combined heterozygote of V1 and wild type MGMT was detected frequently in de novo glioblastoma (GBM) with a significant difference. Although some investigations mentioned the relationships between the enzymatic activity of MGMT and the clinical response to alkylating agents10-12, we could not find any report mentioning an ecogenetic study of glioblastoma risk.
It has been reported that loss of chromosome 10q occurred in the vast majority of GBM13-15. Recently, the PTEN/MMAC1 (Phosphatase and Tensin homolog deleted on chromosome TEN/Mutated in Multiple Advanced Cancers 1) gene, a tumor suppressor, was discovered on chromosome 10q 23.3(16-18) and mutations of this gene have been detected in glioblastoma cell lines and tumors17-19. DMBT1 (deleted in malignant brain tumors), a new member of the scavenger receptor cysteine-rich (SRCR) superfamily, was also detected as a putative tumor-suppressor gene implicated in the carcinogenesis of glioblastoma multiforme20. Other putative tumor suppressor genes were suggested on chromosome 10q25-qter covering DMBT1 and FGFR2 loci5. From these reports, the long arm of chromosome 10 is thought to be the most important chromosomal location in the tumorigenesis of GBM. O^sup 6^-methylguanine-DNA methyltransferase (MGMT) is also located in the chromosome 10q, exactly 10q 26 downstream from the above mentioned tumor suppressor genes, so the MGMT is suggested to be an important gene in the carcinogenesis of GBM.
It was also demonstrated that a V1/VV genotype was detected frequently with a significant difference in the de novo GBM among glioma patients including low grade (grade 1 or 2), anaplastic (grade 3) and glioblastoma. It is believed that glioblastoma occurs as either a de novo or secondary form as the final stage of tumor progression from low-grade astrocytoma22,23 and that the genetic background is different between the de novo and secondary form. The dominant detection of the VI/W genotype in de novo glioblastoma may be important evidence of the different genetic background between the two types.
So far, some polymorphisms have been reported to be risk factors for some diseases, such as the APOE gene in Alzheimer disease, the CYP17 gene in prostate cancer24 and so on. No risk factor for glioblastoma, however, has been reported. This is the first study to report a risk factor for glioblastoma. The mechanism of how an MGMT polymorphism induces glioblastoma is not understood and remains to be investigated. However, previous reports25,26 suggested the possibility that some polymorphisms induce the loss of heterozygosity (LOH) in related chromosomal locations. In respect to these reports, the relationship between MGMT polymorphisms and LOH in chromosome 10 must be further investigated to clarify the significance of W/V1 in the carcinogenesis of GBM.
The V1/W genotype of the MGMT gene is supposed to contribute to the de novo occurrence of glioblastoma.
This work was performed according to protocols approved by the ethical committee of Oita Medical University (approval number 48) and supported by a grant-in-aid for a Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (No. 13770766). The authors are grateful to Ms Fujita, Ms Anami and Ms Tokunaga for technical assistance.
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Ryo Inoue*, Mitsuo Isono*, Masako Abe[dagger], Tatsuya Abe* and Hidenori Kobayashi*
*Department of Neurosurgery, Oita Medical University, Oita, Japan
[dagger]Department of Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan
Correspondence and reprint requests to: Ryo Inoue, Department of Neurosurgery, Oita Medical University, 1-1 Idaigaoka, Hasama, Oita 879-5593, Japan, [firstname.lastname@example.org] Accepted for publication May 2003.
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