Geneenvironment interaction between an aldehyde dehydrogenase-2 (ALDH2) polymorphism and alcohol consumption for the risk of esophageal cancer
Keitaro Matsuo1,4,5,
Nobuyuki Hamajima1,
Masayuki Shinoda2,
Shunzo Hatooka3,
Manami Inoue1,
Toshiro Takezaki1 and
Kazuo Tajima1
1 Division of Epidemiology and Prevention,
2 Department of Rehabilitation Service, and
3 Department of Thoracic Surgery, Aichi Cancer Center, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 464-8681 and
4 Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan
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Abstract
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Aldehyde dehydrogenase-2 (ALDH2) degrades acetaldehyde metabolized from ethanol. Its encoding gene ALDH2 has a functional polymorphism: ALDH2 Glu487Lys. An association between this polymorphism and esophageal cancer among alcoholics has been reported. To further evaluate the geneenvironment interaction, a hospital-based casecontrol study was conducted. Cases were 102 patients with histologically confirmed esophageal cancer and controls were 241 non-cancer outpatients of Aichi Cancer Center. ALDH2 genotypes were examined by a PCRCTPP method developed in our laboratory, which does not require a digestion stage. Logistic regression analysis was employed for estimation of relative risk and geneenvironment interaction. The allele frequency for ALDH2 Lys487 was 0.28, consistent with previous reports. The age, sex, smoking and drinking status adjusted odds ratio for the ALDH2 Glu/Lys and Lys/Lys genotypes as compared with the Glu/Glu genotype was 3.43 (95% CI 1.746.75). The odds ratio for heavy drinking was 49.6 (14.5169.4) among Lys487 carriers and 7.84 (2.7722.2) for the Glu/Glu genotype. The geneenvironment interaction between alcohol drinking and the ALDH2 Lys487 allele was 6.84 (2.3919.6), whereas no significant interaction was obtained with smoking status. Although limited because of its prevalent casecontrol design, our study revealed a strong geneenvironment interaction between ALDH2 polymorphism and heavy alcohol consumption. Taking the observed high risk of esophageal cancer in association with the ALDH2 Lys487 allele into consideration, reducing alcohol intake may be most protective among Lys487 allele carriers of this polymorphism.
Abbreviations: ALDH2, aldehyde dehydrogenase-2; CI, confidence interval; OR, odds ratio.
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Introduction
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Many epidemiological studies have consistently shown heavy alcohol drinking to be a risk factor for esophageal cancer (1). The oxidative metabolite of ethanol, acetaldehyde, is recognized to be carcinogenic for animals (2) and is suspected of having similar effects in man. Aldehydes have the potential to bind to cellular proteins and DNA, thus leading to carcinogenesis (3).
Aldehyde dehydrogenase-2 (ALDH2) generates acetic acid from acetaldehyde metabolism and its activity correlates with the in vivo concentration of acetaldehyde (4). A polymorphism of ALDH2 (Glu487Lys) is prevalent in Asians (5) and the Lys487 allele is associated with enzyme activity (4). Most studies, conducted mainly in Japan, have revealed associations between the ALDH2 polymorphism and esophageal cancer risk (68). A recent study showed an association with multiple esophageal dysplasia (9). Yokoyama et al. reported that the ALDH2*2 (Lys487) allele leads to increased susceptibility to esophageal cancer among male alcoholics (6,8). To our best knowledge, however, there has been no report directly confirming a geneenvironmental interaction between the ALDH2 polymorphism and alcohol consumption.
We therefore conducted the present hospital-based casecontrol study to evaluate this question.
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Materials and methods
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Subjects
A total of 102 Japanese esophageal cancer patients (86 male and 16 female, median age 61, age range 4076) and 241 non-cancer controls (118 male and 123 female, median age 58, age range 3969) who visited Aichi Cancer Center in 19992000 were recruited. Cases were first diagnosed as having esophageal cancers between 1984 and 2000. Controls were outpatients without a history of cancer who underwent gastroscopy and were confirmed to have no esophageal or stomach malignancies. All subjects gave written informed consent to participate in this study, completed a self-administered questionnaire and provided blood. The questionnaire included items on smoking status and alcohol consumption. Smoking status was classified as smoker, ex-smoker or never smoked and the level of exposure was expressed in pack years. Alcohol consumption was classified into two categories, drinkers taking more than 3 gou (equivalent to 75 ml ethanol)/day
5 times/week and others.
Genotyping procedure
DNA was extracted from the buffy coat fraction using a QIAamp blood mini kit (Qiagen, Valencia, CA) and the ALDH2 Glu487Lys (1543 G
A substitution, accession no. NM_000690) polymorphism was characterized by a PCRCTPP method developed in our laboratory (10). Each 25 µl reaction tube contained 30100 ng DNA, 0.12 mM dNTPs, 12.5 pmol each primer, 0.5 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA) and 2.5 µl of 10x PCR buffer including 15 mM MgCl2. Briefly, the genotyping by PCRCTPP is as follows. Primers used were: F1, 5'-TCA TGC CAT GGC AAC TCC AGC-3'; R1, 5'-CCC ACA CTC ACA GTT TTC TCT TC-3'; F2, 5'-TAC GGG CTG CAG GCA TAC ACT A-3'; R2, 5'-TGA TCC CCA GCA GGT CCT GAA-3'. Primer pairs F1 and R1 for the G allele (Glu487), F2 and R2 for the A allele (487Lys) (R1 included a guanine antibase at the 3'-end and F2 included adenine at the 3'-end) produced allele-specific bands of 296 and 203 bp for the G and A alleles, as well as a common 455 bp band between F1 and R2. The setting for the thermal cycler was 30 cycles of 1 min at 95°C, 1 min at 60°C and 1 min at 72°C, followed by 5 min extension at 72°C.
Statistics
All statistical analyses were performed using STATA v.6 software (STATA, College Station, TX). Accordance with the HardyWeinberg equilibrium, which indicates an absence of discrepancies between genotype and allele frequencies, was checked for controls with the
2 test. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using an unconditional logistic regression model. The factors employed for multivariate analyses were as follows: age as a continuous variable; sex; smoking status, never smoked, ex-smoker, smoker (<50 and
50 pack years); alcohol consumption, heavy drinker [consumption of 3 gou (equivalent to 75 ml alcohol)/day
5 days/week]. Geneenvironment interactions with the ALDH2 polymorphism were estimated by the logistic model (11), which included an interaction term as well as variables for exposure (alcohol drinking or smoking), genotype (ALDH2) and potential confounders (sex and age). The interaction in this study was defined as the ratio of the OR among the Lys allele carriers to the OR among the individuals with the Glu/Glu genotype.
This study was approved by the Institutional Review Board of Aichi Cancer Center in 1999.
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Results
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Table I
summarizes subject characteristics. The age distribution was slightly older among cases. The median period from diagnosis to entry was 1 year. Heavy smokers (
50 pack years) constituted 33.7% of cases and 6.2% of the controls. Alcohol was also more frequently and heavily consumed by cases (those consuming >3 gou/day for
5 days/week, 66.7% for cases and 10.8% for controls). Among the 26 heavy drinking controls aged 3968 years eight subjects (30.1%) were younger than 50 years of age. The proportion was similar to that for the other controls; 47 (21.9%) of 215.
Figure 1
shows the representative result of genotyping by the PCRCTPP method. The ALDH2 Lys487 allele frequencies for controls and cases were 0.28 and 0.33 and the genotype distribution among controls was in accordance with the HardyWeinberg equilibrium law: Glu/Glu, 52.3%; Glu/Lys, 39.8%; Lys/Lys, 7.9%. The distribution among cases was: Glu/Glu, 34.3%; Glu/Lys, 64.7%; Lys/Lys, 1.0%.

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Fig. 1. Representative results for the ALDH2 Glu487Lys polymorphism by the PCRCTPP method. DNA fragments stained with ethidium bromide are shown. Lane M, markers; lane 1, Glu/Glu; lane 2, Glu/Lys; lane 3, Lys/Lys.
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As shown in Table II
, the crude OR for the mutant genotype (Glu/Lys and Lys/Lys) relative to the homozygous wild-type (Glu/Glu) was 2.10 (95% CI 1.303.39, P = 0.003). The age, gender, smoking status and alcohol consumption adjusted OR was 3.43 (955 CI 1.746.75, P < 0.001). When focusing on the Lys/Lys genotype the crude and adjusted ORs were not statistically significant but showed a decreased risk tendency. Crude ORs for years from diagnosis did not differ (<1 year versus
1 year; data not shown), therefore, we analyzed all cases together. When subjects were analyzed according to alcohol consumption the ORs for Glu/Lys and Lys/Lys compared with Glu/Glu were much higher for heavy drinkers (OR for Glu/Lys and Lys/Lys 11.5, 95% CI 3.5337.4, P < 0.001) than others (OR 1.51, 95% CI 0.723.18, P = 0.27) (Table III
). Table IV
gives the ORs for lifestyle factors according to ALDH2 genotype. The impact of smoking in subjects with the Glu/Glu genotype appeared higher than with the Glu/Lys and Lys/Lys genotypes. Adjusted ORs for alcohol drinking were 49.6 (95% CI 14.5169.4, P < 0.001) for Glu/Lys and Lys/Lys and 7.84 (95% CI 2.7722.2, P < 0.001) for Glu/Glu.
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Table III. Adjusted odds ratios and 95% CI for ALDH2 Lys allele carriers relative to the ALDH2 Glu/Glu genotype according to alcohol consumption
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The OR for the geneenvironment interaction between the ALDH2 Lys487 allele and alcohol drinking was 6.84 (95% CI 2.3919.6, P < 0.001), while no statistically significant interaction existed with regard to current smoking (OR 2.04, 95% CI 0.676.20, P = 0.211).
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Discussion
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In this study we found the ALDH2 Lys487 allele to have a statistically significant geneenvironment interaction with heavy drinking with respect to risk of esophageal cancer. In addition, we confirmed an increased risk with the ALDH2 Lys487 allele and heavy drinking.
Previous studies showed ORs for esophageal cancer in Lys487 allele carriers (68) of from 4 to 12. ORs >10 were only observed in alcoholics (6,8). The OR for Lys487 allele carriers compared with Glu/Glu in this study was thus consistent with the literature. Although detailed biological mechanisms require clarification, we can draw the conclusion that diminished ALDH2 enzyme activity and resultant higher concentrations of acetaldehyde are definite risk factors for esophageal cancer.
Of major interest in this context was the difference in the effect of the Lys487 allele between heavy drinkers and others. The risk was thus dramatically changed with high ethanol exposure and low ALDH2 activity, in line with expectation.
Estimation of the geneenvironment interaction with the ALDH2 polymorphism demonstrated statistical significance only for heavy drinking, and not for smoking. The observed significant interaction means that the OR for the heavy drinkers with Lys487 relative to non-heavy drinkers with the Glu/Glu genotype was [OR for heavy drinking]x[OR for Lys487 allele]x6.84, implying that the effects of the Lys487 allele and heavy drinking are enhanced if both factors coexist. Individuals with the ALDH2 Lys487 allele in particular should be targeted with regard to prevention efforts aimed at reducing alcohol exposure.
We should mention the risk trend for Lys/Lys genotype subjects, although it was not statistically significant. Contrary to the basal hypothesis, these subjects showed a decreased risk for esophageal cancer. Previous studies on the association between the ALDH2 polymorphism and drinking behavior revealed that individuals with the Lys/Lys genotype are unlikely to be alcohol dependent (12,13). Compatible with this observation, there were no Lys/Lys genotype heavy drinkers among either cases or controls. The decreased risk for the Lys/Lys genotype seemed due to the confounding factor that persons with the genotype were likely to be non-drinkers, a low risk group for esophageal cancer.
We have applied a PCRCTPP method we have developed for genotyping of single nucleotide polymorphisms (10) and proved that it is applicable to genotyping of the ALDH2 Glu487Lys polymorphism. This approach does not require restriction enzyme digestion so that the time and cost are reduced. Although skill is necessary in designing primers, the logic itself is simple and it is of practical use without high technology instrumentation.
The present results should be interpreted with care, because this study was of prevalent casecontrol type. We analyzed all cases, including prevalent cases, together because absence of a survival effect has been reported for the ALDH2 genotype (14). To confirm the absence of a difference in attribution of the polymorphism between prevalent and incident cases we examined the ORs according to years from diagnosis to enrolment (<1 year versus
1 year); there did not appear to be any difference, suggesting that the OR reflected mainly risk, not prognosis. Although further prospective studies are desirable, results of our and former studies provide strong evidence for our interpretation.
In summary, the present study revealed a strong geneenvironment interaction between the ALDH2 polymorphism and alcohol drinking. Taking the observed higher risk of esophageal cancer among ALDH2 Lys487 allele carriers into consideration, protective effects of reducing alcohol consumption may be higher in this population.
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Notes
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5 To whom correspondence should be addressed 
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Acknowledgments
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The authors are grateful to Ms Naomi Takeuchi, Ms Keiko Asai and Ms Hiroko Fujikura for their technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (C) in 20002003 from the Ministry of Education, Science, Sports and Culture, Japan.
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Received January 3, 2001;
revised February 28, 2001;
accepted March 2, 2001.