Find information on thousands of medical conditions and prescription drugs.


A glioma is a type of primary central nervous system (CNS) tumor that arises from glial cells. The most common site of involvement of a glioma is the brain, but they can also affect the spinal cord, or any other part of the CNS, such as the optic nerves.

Gardner's syndrome
Gastric Dumping Syndrome
Gastroesophageal reflux
Gaucher Disease
Gaucher's disease
Gelineau disease
Genu varum
Geographic tongue
Gerstmann syndrome
Gestational trophoblastic...
Giant axonal neuropathy
Giant cell arteritis
Gilbert's syndrome
Gilles de la Tourette's...
Gitelman syndrome
Glanzmann thrombasthenia
Glioblastoma multiforme
Glucose 6 phosphate...
Glycogen storage disease
Glycogen storage disease...
Glycogen storage disease...
Glycogenosis type IV
Goldenhar syndrome
Goodpasture's syndrome
Graft versus host disease
Graves' disease
Great vessels transposition
Growth hormone deficiency
Guillain-Barré syndrome


[List your site here Free!]

A genotype of the polymorphic DNA repair gene MGMT is associated with de novo glioblastoma
From Neurological Research, 12/1/03 by Inoue, Ryo

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.


Study participants

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.

DNA extraction

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

PCR-SSCP analysis

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).

Statistical analysis

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.


1 Boeing H, Schlehofer B, Blettner M, Wahrendorf J. Dietary carcinogens and the risk for glioma and meningioma in Germany. Int J Cancer 1993; 53: 561-565

2 Silber JR, Blank A, Bobola MS, Mueller BA, Kolstoe DD, Ojemann GA, Berger MS. Lack of the DNA repair protein O^sup 6^-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary human brain tumors. Proc Natl Acad Sci USA 1996; 93: 6941-6946

3 Pegg AE, Byers TL. Repair of DNA containing O^sup 6^-alkylguanine. FA5EB J 1992; 6: 2302-2310

4 Sekiguchi M, Nakabeppu Y. Adaptive response: Induced synthesis of DNA-repair enzymes by alkylating agents. Trends Genet 1987; 3: 51-54

5 Natarajan AT, Vermeulen S, Darroudi F, Valentine MB, Brent TP, Mitra S, Tano K. Chromosomal localization of human O^sup 6^-methylguanine-DNA methyltransferase (MGMT) gene by in situ hybridization. Mutagenesis 1992; 7: 83-85

6 Otsuka M, Abe M, Nakabeppu Y, Sekiguchi M, Suzuki T. Polymorphisms in the human O^sup 6^-methylguanine-DNA methyltransferase gene detected by PCR-SSCP analysis. Pharmacogenetics 1996; 6: 361-363

7 Inoue R, Abe M, Nakabeppu Y, Sekiguchi M, Mori T, Suzuki T. Characterization of human polymorphic DNA repair methyltransferase. Pharmacogenetics 2000; 10: 59-66

8 Blin N, Stafford DW. A general method for isolation of high molecular DNA from eukaryotes. Nucleic Acids Res 1976; 3: 2303-2308

9 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. A laboratory manual, 2nd edn. MA: Cold Spring Harbor Laboratory Press, 1989

10 Hotta T, Saito Y, Fujita H, Mikami T, Kurisu K, Kiya K, Uozumi T, Isowa G, Ishizaki K, Ikenaga M. O^sup 6^-Alkylguanine-DNA alkyltransferase activity of human malignant glioma and its clinical implications. J Neuro Oncol 1994; 21: 135-140

11 Manel E, Jesus GF, Esther A, Steven NG, Oscar FH, Vicente V, Stephen B, James GH. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000; 343: 1350-1354

12 Mineura K, Izumi I, Kuwahara N, Kowada M. O^sup 6^-methylguanineDNA methyltransferase activity in cerebral gliomas. A guidance for nitrosourea treatment? Acta Oncologica 1994 ; 33: 29-32

13 Albarosa R, Colombo BM, Roz L, Magnani I, Polio B, Cirenei N, Giani C, Conti AMF, DiDonato S, Finocchiaro D. Deletion mapping of gliomas suggests the presence of two small regions for candidate tumor-suppressor genes in a 17-cM interval on chromosome 10q. Am J Hum Genet 1996; 58: 1260-1267

14 Karlbom AE, James CD, Boethius J, Cavenee WK, Collins VP, Norden SM, Larsson C. Loss of heterozygosity in malignant gliomas involves at least three distinct regions on chromosome 10. Hum Cenef 1993; 92: 169-174

15 Rasheed BKA, Fuller GN, Friedman AH, Darrel D, Bigner DD, Bigner SH. Eoss of heterozygosity for 10q loci in human gliomas. Genes Chromosomes Cancer 1992; 5: 75-82

16 Li D, Sun H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor [beta]. Cancer Res 1997; 57: 2124-2129

17 Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Pue J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, lttmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997; 275: 1943-1947

18 Steck PA, Pershouse MA, Jasser SA, Yung WKA, Lin H, Ligon AH, Langford LA, Baumgard ME, Hattier T, Davis T, Frye C, Hu R, Swedlund B, Teng DHF, Tavtigian SV. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nature Genef 1997; 15: 356-362

19 Wang SI, Puc J, Li J, Bruce JN, Cairns P, Sidransky D, Parsons R. Somatic mutations of PTEN in glioblastoma multiforme. Cancer Res 1997; 57: 4183-4186

20 Mollenhauer J, Wiemann S, Scheurlen W, Korn B, Hayashi Y, Wilgenbus KK, Andreas von Deimling, Poustka A. DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain tumors. Nature Genet 1997; 17: 32-39

21 Fujisawa H, Kurrer M, Reis RM, Yonekawa Y, Kleihues P, Ohgaki H. Acquisition of the Glioblastoma Phenotype during astrocytoma progression is associated with loss of heterozygosify on 10q25-qter. Am J Pathol 1999; 55: 387-394

22 Kleihues P, Ohgaki H. Primary and secondary glioblastoma: From concept to clinical diagnosis. Neuro-Oncology 1999; 1: 44-51

23 Lang FF, Miller DC, Koslow M, Newcomb EW. Pathways leading to glioblastoma multiforme: A molecular analysis of genetic alterations in 65 astrocytic tumors. J Neurosurg 1994; 81: 427-436

24 Andrea G, Gabriele B, Sonja H, Gerald H, Georg S, Stephan M, Michael M, Christian V, Michael M. A polymorphism in the CYP17 gene is associated with prostate cancer risk. Int J Cancer 2000; 87: 434-437

25 Noll WW, Belloni DR, Rutter JL, Storm CA, Schned AR, TitusErnstoff L, Ernstoff MS, Brinckerhoff CE. Loss of heterozygosity on chromosome 11q22-23 in melanoma is associated with retention of the insertion polymorphism in the matrix metalloproteinase-1 promoter. Am J Pathol 2001; 158: 691-769

26 Schnakenberg E, Ehlers C, Feyerabend W, Werdin R, Hubotter R, Dreikorn K, Schloot W. Genotyping of the polymorphic N-acetyltransferase (NAT2) and loss of heterozygosity in bladder cancer. Clin Genet 1998; 53: 396-440

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, [] Accepted for publication May 2003.

Copyright Forefront Publishing Group Dec 2003
Provided by ProQuest Information and Learning Company. All rights Reserved

Return to Glioblastoma
Home Contact Resources Exchange Links ebay