Department of Preventive Medicine/Biostatistics and Medical Decision Making, Nagoya University Graduate School of Medicine, Nagoya,
1 Nagoya Kita Health Center, Nagoya and
2 Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Japan
(Received 28 January 2003; first review notified 17 March 2003; in revised form 3 April 2003; accepted 11 April 2003)
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ABSTRACT |
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INTRODUCTION |
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Alcoholism was reported to have associations with ADH2 47Arg and ALDH2 487Glu in Chinese people (Muramatsu et al., 1995; Chen et al., 1999
) and in Japanese people (Higuchi et al., 1995
). The protective alleles, ADH2 47His and ALDH2 487Lys are more prevalent in Oriental people than in other ethnic groups (Goedde et al., 1992
; Hamajima et al., 2002a
). Because alcohol tolerance depends on these genotypes, ADH2 and ALDH2 genotyping may be useful in realizing the doseresponse of alcoholism.
Both polymorphisms have been simultaneously examined in several studies (Higuchi et al., 1995; Muramatsu et al., 1995
; Wan et al., 1998
; Chen et al., 1999
; Chao et al., 2000
; Wall et al., 2003
). The common genotyping method for polymorphisms is polymerase chain reactionrestriction fragment length polymorphism (PCRRFLP). For ADH2 Arg47His, the restriction enzyme is MaeIII (Goedde et al., 1992
). ALDH2 Gly487Lys has been genotyped by a PCRRFLP method using MboII (Yokoyama et al., 1998
) or a mismatch primer making a restriction site of Ksp632I (Takeshita et al., 1994
). We genotyped ALDH2 Gly487Lys with PCR with confronting two-pair primers (PCRCTPP) (Hamajima et al., 2000
; Matsuo et al., 2001
). This paper reports a newly developed PCRCTPP for allele-specific DNA amplification of ADH2 Arg47His and ALDH2 Glu487Lys simultaneously in one tube. The duplex PCRCTPP was applied in hundreds of subjects to demonstrate that its use was practical and applicable.
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MATERIALS AND METHODS |
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Buffy coat was applied 72 h after blood draw on the residual blood (stored at 4°C after routine laboratory tests for health, and preserved at -80°C). DNA was extracted from the buffy coat using QIAamp DNA Blood Mini Kit (QIAGEN Inc., Valencia, CA, USA).
PCRCTPP method
The basis of PCRCTPP for single nucleotide polymorphisms is described in previous papers (Hamajima et al., 2000, 2002b
; Hamajima, 2001
). Four allele-specific primers were designed, as follows: F1 for the sense primer of allele X; R1 for the anti-sense primer of allele X with the anti-sense base of polymorphism site at the 3' end; F2 for the sense primer of allele Y with the sense base of polymorphism site at the 3' end; and R2 for the anti-sense primer of allele Y (Fig. 1
). The difference in the size of amplified DNA allows to genotype the single nucleotide polymorphism. Duplex PCRCTPP is a PCRCTPP method amplifying allele-specific DNA for two independent, single-nucleotide polymorphisms, at the same time in one tube.
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RESULTS |
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DISCUSSION |
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PCRCTPP is based on competitive DNA amplification, which usually gives a wider range of optimal annealing temperatures than allele-specific PCR for each allele (Hamajima et al., 2002b). The finding that the duplex PCRCTPP made clearer the contrast of the amplified DNA for some polymorphisms than did single PCRCTPP adds potential to the use of multiplex PCRCTPP.
Because PCRCTPP does not need incubation time with a restriction enzyme, the time and costs of the digestion of PCR products can be saved. This method is applicable for any laboratory where PCR can be performed. PCRCTPP conditions were established for tens of polymorphisms (Hamajima et al., 2002c). However, there are several limitations for PCRCTPP designing; namely (i) the sequence around the targeted polymorphism should allow four primers with a similar melting temperature (Hamajima et al., 2002b
); (ii) monotonous or repeated sequences make it difficult to amplify allele-specific DNA; and (iii) the existence of a pseudogene makes it difficult to find primers specific to the polymorphism under study, though this is a common problem for any PCR.
To date, we have reported duplex PCRCTPP for two sets of polymorphisms (Hamajima, 2001). One was for interleukin 1B C-31T and interleukin 1RN 86-bp variable number of tandem repeat polymorphisms. The other was complicated PCRCTPP for Se, sej, and se5 alleles of fucosyltransferase 3. They are not duplex for two independent PCRCTPP, but the combination of PCRCTPP and ordinary PCR. These successful examples provide further possible inexpensive and time-saving methods with which to genotype a set of polymorphisms. With ADH2 Arg47His and ALDH2 Glu487Lys, separated genotyping with PCRRFLP is common. As the elimination of the incubation time shortens the overall genotyping time to less than half, the present duplex PCRCTPP reduces the overall time to less than one-quarter.
Genotyping of both polymorphisms could be applied to alcoholism, as well as to the risk estimation of alcohol-related diseases (Yokoyama et al., 2002). Persons aware of their own genotypes could adjust alcohol intake to avoid risk. Behaviour changes resulting from the recognition of ones genotype would be an interesting theme for examination by those involved in health promotion.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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Chen, C. C., Lu, R. B., Chen, Y. C., Wang, M. F., Chang, Y. C., Li, T. K. and Yin, S. J. (1999) Interaction between the functional polymorphisms of the alcohol-metabolism genes in protection against alcoholism. American Journal of Human Genetics 65, 795807.[CrossRef][ISI][Medline]
Goedde, H. W., Agarwal, D. P., Fritze, G., Meier-Tackmann, D., Singh, S., Beckmann, G., Bhatia, K., Chen, L. Z., Fang, B. and Lisker, R. (1992) Distribution of ADH2 and ALDH2 genotype in different populations. Human Genetics 88, 344346.[ISI][Medline]
Hamajima, N. (2001) PCRCTPP: a new genotyping technique in the era of genetic epidemiology. Experimental Reviews in Molecular Diagnosis 1, 119123.
Hamajima, N., Saito, T., Matsuo, K., Kozaki, K., Takahashi, T. and Tajima, K. (2000) Polymerase chain reaction with confronting two-pair primers for polymorphism genotyping. Japanese Journal of Cancer Research 91, 865868.[ISI][Medline]
Hamajima, N., Takezaki, T. and Tajima, K. (2002a) Allele frequencies of 25 polymorphisms pertaining to cancer risk for Japanese, Koreans and Chinese. Asian Pacific Journal of Cancer Prevention 3, 197206.
Hamajima, N., Saito, T., Matsuo, K. and Tajima, K. (2002b) Competitive amplification and unspecific amplification in polymerase chain reaction with confronting two-pair primers (PCRCTPP). Journal of Molecular Diagnosis 4, 103107.
Hamajima, N., Saito, T., Matsuo, K., Suzuki, T., Nakamura, T., Matsuura, A., Okuma, K. and Tajima, K. (2002c) Genotype frequencies of 50 polymorphisms for 241 Japanese non-cancer patients. Journal of Epidemiology 12, 229236.
Higuchi, S., Matsushita, S., Murayama, M, Takagi, S. and Hayashida, M. (1995) Alcohol and aldehyde dehydrogenase polymorphisms and the risk for alcoholism. American Journal of Psychiatry 152, 12191221.[Abstract]
Matsuo, K., Hamajima, N., Shinoda, M., Hatooka, S., Inoue, M., Takezaki, T. and Tajima, K. (2001) Geneenvironment interaction between an aldehyde dehydrogenase-2 (ALDH2) polymorphism and alcohol consumption for the risk of esophageal cancer. Carcinogenesis 22, 913916.
Matsuo, Y., Yokoyama, R. and Yokoyama, S. (1989) The genes for human alcohol dehydrogenases ß1 and ß2 differ by only one nucleotide. European Journal of Biochemistry 183, 317320.[Abstract]
Muramatsu, T., Wang, Z. C., Fang, Y. R., Hu, K. B., Yan, H., Yamada, K., Higuchi, S., Harada, S. and Kono, H. (1995) Alcohol and aldehyde dehydrogenase genotypes and drinking behavior of Chinese living in Shanghai. Human Genetics 96, 151154.[ISI][Medline]
Takeshita, T., Morimoto, K., Mao, X., Hashimoto, T. and Furuyama, J. (1994) Characterization of the three genotypes of low Km aldehyde dehydrogenase in a Japanese population. Human Genetics 94, 217223.[ISI][Medline]
Takeshita, T., Mao, X. Q. and Morimoto, K. (1996) The contribution of polymorphism in the alcohol dehydrogenase ß-subunit to alcohol sensitivity in a Japanese population. Human Genetics 97, 409413.[CrossRef][ISI][Medline]
Wall, T. L., Carr, L. G. and Ehlers, C. L. (2003) Protective association of genetic variation in alcohol dehydrogenase with alcohol dependence in Native American Mission Indians. American Journal of Psychiatry 160, 12.
Wan, Y. J., Poland, R. E. and Lin, K. M. (1998) Genetic polymorphism of CYP2E1, ADH2, and ALDH2 in Mexican-Americans. Genetics Testing 2, 7983.
Yin, S. J., Bosron, W. F., Magnes, L. J. and Li, T. K. (1984) Human liver alcohol dehydrogenase: purification and kinetic characterization of the ß2ß2, ß2ß1, ß2, and ß2
1 Oriental isoenzymes. Biochemistry 23, 58475853.[ISI][Medline]
Yokoyama, A., Muramatsu, T., Ohmori, T., Yokoyama, T., Okuyama, K., Takahashi, H., Hasegawa, Y., Higuchi, S., Murayama, K., Shirakura, K. and Ishii, H. (1998) Alcohol-related cancers and aldehyde dehydrogenase-2 in Japanese alcoholics. Carcinogenesis 19, 13831387.[Abstract]
Yokoyama, A., Kato, H., Yokoyama, T., Tsujinaka, T., Muto, M., Omori, T., Haneda, T., Kumagai, Y., Igaki, H., Yokoyama, M., Watanabe, H., Fukuda, H. and Yoshimizu, H. (2002) Genetic polymorphisms of alcohol and aldehyde dehydrogenases and glutathione S-transferase M1 and drinking, smoking, and diet in Japanese men with esophageal squamous cell carcinoma. Carcinogenesis 23, 18511859.