Association between Smoking Habits and Dopamine Receptor D2 TaqI A A2 Allele in Japanese Males: a Confirmatory Study

Our previous study showed that A2 allele of dopamine D2 (DRD2) TaqI A polymorphism related to smoking habits, which was opposed to the results of studies for Caucasians. In order to confirm our finding, a similar study was conducted for the first-visit outpatients of Aichi Cancer Center Hospital, who participated in HERPACC-II (Hospital-based Epidemiologic Research Program at Aichi Cancer Center - II). Among consecutive 1,577 first-visit patients between November 2000 and February 2001, 800 patients provided a 7ml of peripheral blood. Smoking habit data were available for 798 participants. Excluding five participants aged < 20 years or ≥ 80 years, the remaining 793 participants (346 males and 447 females) were analyzed. The DRD2 genotype was determined by a new method, polymerase chain reaction with confronting two-pair primers (PCR-CTPP). In males, current smokers were 35.3% of individuals with A1A1 genotype, 43.1% of individuals with A1A2 genotype, and 57.0% of individuals with A2A2 genotype, while in females, they were 19.6%, 14.6%, and 10.9%, respectively. Age-adjusted odds ratio (OR) of current smoking relative to A1A1 was 1.61 (95% confidence interval, 0.71-3.46) for A1A2 and 2.32 (1.02-5.29) for A2A2 in males, and 0.72 (0.32-1.61) and 0.51 (0.22-1.18) in females, respectively. The present study indicated that Japanese males with A2A2 genotype have a higher risk of being current smokers. No association for Japanese females suggested that female smoking behavior is differently affected in biological and/or psychological manner.

The associations with genetic polymorphisms propose a new insight that smoking behavior may be constitutionally influenced through biological mechanisms.1 To date, polymorphisms pertaining to neurotransmitters,2-9 nicotine metabolism, 10  , while the other one reported an insignificant association with A2 allele. 12 We previously reported a significant association with A2 allele of DRD2 TaqI A for Japanese. 15 The finding suggests that there are different links to other DRD2 polymorphisms or other genes responsible to smoking behavior. Another possible explanation is interactions with other genes or lifestyle/psychological factors, which convert the effect of the alleles. Although associations with the different allele could be observed among different ethnic groups, confirming the association for different Japanese subjects is the first step to discuss the above listed possibilities. This study aimed to confirm our previous finding that the A2 allele relates to smoking behavior for Japanese. The previ-ous study was conducted for re-visit non-cancer outpatients of Aichi Cancer Center Hospital, and this time for first-visit outpatients of the same hospital. In this study, never smokers were defined as those who smoked less than 100 cigarettes in the past, current smokers as those who smoked in the past one year, and former smokers as those who quit smoking more than one year before the questionnaire study.
DRD2 has been reported to have 20 polymorphisms; TaqI A, TaqI B, TaqI D, EcoRI, BclI, MboI, and HincII restriction fragment length polymorphisms (RFLP), GA and GT tandem repeat polymorphisms, a C-to-G polymorphism not genotyped by PCR-RFLP listed in Genbank (Accession No. AF050737), as well as a functional polymorphism -141C Ins/Del in the promoter region,16 three missense polymorphisms (Val96A1a, Pro3l0Ser, and Ser3l lCys),17 and six silent variants (44Leu,17 141Leu,18 255Val,19 319Pro,20 313His,18 and 367Lys19). The TaqI A and TaqI B are linked closely ,4,15 and we found that -141C Ins/Del polymorphism was not associated with smoking habits. 15 In this paper, MboI polymorphism located in intron 2 was examined for a subset of the subjects on an exploratory purpose, as well as TaqI A in the 3' untranslated sequence of exon 8.

Study subjects
Subjects were first-visit patients of Aichi Cancer Center Hospital who were consecutively invited to lifestyle questionnaire and peripheral blood donation in the framework of HER-PACC-II. 21 The participants in HERPACC-II during November 2000 and February 2001 were sampled. Among 1,577 first-visit outpatients, 800 provided a 7m1 of peripheral blood. Data on smoking habits were available for all but two. Four participants aged less than 20 and one aged 80 years or over were excluded from the analysis. The remaining were 793 participants (346 males with mean age 55.8 years and standard deviation 12.1 years, and 447 females with mean age 50.2 years and standard deviation 13.1 years). The first 395 outpatients (174 males and 221 females) were used for exploratory analysis of MboI polymorphism. In this hospital, cancer patients are about 20% of the first-visit outpatients. Since this study examined the association with smoking habits defined at the time one year before their visit, cancer patients were included in the study subjects.
This study had been approved by the Ethical Committee at Aichi Cancer Center before the study started (Ethical Committee Approval Numbers 41-2).

Genotyping
DNA was extracted from 200, 1 buffy coat preserved at -80 C by QIAamp DNA Blood Mini Kit (QIAGEN Inc., Valencia, CA). The genotyping was conducted by a novel PCR technique, PCR-CTPP (polymerase chain reaction with confronting two-pair primers) .22,23 The primers were Fl: 5' TGA GCC ACC ACG GCT GG, R 1: 5' CAT CCT CAA AGT  GCT GGT CG, F2: 5' AGC TGG GCG CCT GCC TT, and  R2: 5' CTC TTG GAG CTG TGA ACT GG for TaqI A polymorphism, and Fl: 5' GAG AAA TGA TGC TTT CGG  Genomic DNA (30ng to 100ng) was used in a volume of 25 1 with 0.1mM dNTPs, 12.5 pmol of each primer, 0.5 units of "AmpliTaq Gold" (Perkin-Elmer Corp., Foster City, CA), and 2.5 1 10 PCR Buffer including 15mM MgC12. A 2.5, 1 of glycerol was added in genotyping for TaqI A polymorphism, not for MboI polymorphism. PCR for TaqI A was conducted as follows; a 10 min of initial denature at 95 C, 30 cycles of 1 min at 95 C, 1 min at 56 C, and 1 min 72 C, and a 5 min of final extension at 72 C. The condition for MboI was same but annealing temperature at 60 C.
All PCR products were visualized on a 2% agarose gel with ethidium bromide staining. Genotyping of TaqI A is 292 by for Al (T) allele and 207 by for A2 (C) allele with a 493-bp common band, and that of MboI 196 by for A allele and 154 by for T allele with a 310-bp common band, as demonstrated in Figure 1.

Statistical analysis
Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by an unconditional logistic model with computer program STATA Version 7 (STATA Corporation, College Station, TX). Age-adjustment for the ORs was conducted as a continuous variable. The fitness for Hardy-Weinberg equilibrium was examined by "genhwi" command of the STATA.

RESULTS
Current smokers were 47.7% in males and 13 .7% in females. The percentages according to age group are shown in Table 1. The genotype frequency for DRD2 TaqI A was in Hardy-Weinberg equilibrium for both sexes (X 2=0 .861, p=0.353 for males and X 2=0.868, p=0.352 for females). When both sexes were combined, A1A1 genotype was 12 .9%, A1A2 46.2%, and A2A2 41.0%.
As shown in Table 2, the percentage for current smokers was lowest among males with AlAl genotype (35 .3%), and highest among males with A2A2 genotype (57 .0%). However, the percentage was larger among females with A1A1 than among females with A2A2 genotype (19 .6% and 10.9%, respectively). When the two groups were combined , the difference was cancelled .
The ORs relative to AlA1 genotype were calculated in four different settings of case-control design; current smoker cases compared with non-current (former + never) smoker controls ,  ever (current + former) smoker cases with never smoker controls, current smoker cases with never smoker controls, and current smoker cases with former smoker controls ( Table 3). The age-adjusted OR of A2A2 genotype was significantly elevated for current smokers compared with non-current or never smokers in males; OR=2.33 (1.19-4.53) with non-current smokers and OR=2.32 (1.02-5.29) with never smokers. The OR of current smoker cases with former smoker controls was also significant for A2A2 genotype in males. The corresponding ORs for A1A2 were almost in the mid of that for A2A2. In females, there were no significant ORs observed, though the ORs for A2A2 genotype were less than unity and smaller than for A1A2 genotype.
Genotyping of MboI polymorphism was conducted for 395 participants including one individual whose DNA was not amplified by PCR-CTPP. As shown in Table 2, the great majority had the AA genotype. The distribution was in Hardy-Weinberg equation (x 2=0.407, p=0.524 for males, and x2 =1.090, p=0.297 for females). There was no association with smoking habits. Accordingly, no further analysis was conducted. Fifty-six individuals with ALAI genotype of TaqI A were all with AA genotype of MboI, while 128 (81.5%) out of 157 with A2A2 had the AA genotype and 166 (91.7%) out of 181 with A1A2 had the AA genotype.

DISCUSSION
This is a confirmatory study for the hypothesis that smoking habits are associated with the A2A2 genotype in Japanese. We estimated the sample size for the comparison between A1A1 and A2A2 as follows; two-sided alpha error = 0.05, statistical power 90%, from the previous study, smokers 12% (5/41) for AlA1 and 29% (39/136) for A2A2, and genotype frequency, 0.123 for AlA1, 0.467 for A1A2, and 0.410 for A2A2, resulting in 692 participants (85, 323 and 284, respectively). Since the male/female ratio and smoking percentage were unknown, the sample size was set to be 800 at the start of the present study. The genotype frequency in this study (12.9% for A1Al, 46.2% for A1A2 and 41.0% for A2A2) was similar to that for the previous study as mentioned above (n=332). The percentage of current smokers was slightly higher in males (47.7%) and same in females (13.7%) as the previous study (34.2%, n=155, and 13.6%, n=177, respectively). These figures indicated that the sample size estimation was appropriate.
The hypothesis that the polymorphism is associated with smoking habits was not confirmed when both sexes were combined. However, the findings for males were quite consistent. The association for females was not significant in the previous study. The OR was a little smaller than the previous study (OR=3.72 for current vs. non-current smokers), but this study with a larger sample size demonstrated a significant association with the A2 allele. The p-value for OR=2.33 for current vs. non-current smokers was 0.013, so the adjustment of multiple comparisons for three tests (analysis for males, females, and combined) allows a significant result (0.013 3=0.039 < 0.05). Accordingly, it could be concluded that the association exists for males. In the present dataset, male current smokers also showed a significant OR compared with former smokers.
Since this finding was not hypothesized in this study, further studies are required to evaluate the effect of the genotype on quitting smoking. There are several different reasons to quit smoking, the reasons should be taken into account in such studies.