An Association Between the Serotonin Transporter Gene Promoter Polymorphism and Smoking Cessation Among Japanese Males

Abstract

The objective of this study was to replicate the associations between the serotonin transporter genotypes and smoking cessation and between these genotypes and long-term smoking cessation success (for 2 years or more). Two case–control designs were used in this study. The first case–control design consisted of 47 ex-smokers (cases) and 94 smokers (controls). The second case–control design consisted of the 12 ex-smokers who had succeeded in long-term smoking cessation (cases) and the 24 smokers (controls). In the first design, there was no significant difference in allele frequency and genotype distribution between cases and controls. In the second design, the S/S genotype frequency was higher in cases than in controls, but the difference did not reach slightly statistical significance. The present study suggests that individuals with the S/S genotype are more inclined to achieve long-term smoking cessation than others.

Keywords

serotonin transporter gene polymorphism genotype long-term smoking cessation success smoking cessation program

Introduction

Smoking is one of the most important risk factors for serious diseases, including cancer, chronic obstructive pulmonary diseases, and cardiovascular diseases. In addition, it has been shown that tobacco smoking moderately increases the risk (by approximately 1.5 times) of total cancers in the Japanese population compared with the nonsmoking Japanese population.¹ However, smoking behavior persists in the Japanese population.^2-4 In Japan, the incidence of smoking is 39.5% among males, and the incidence of smoking among females (20-30 years old) has increased recently.⁴ It is strongly recommended that smokers should cease smoking for good health. However, there are reasons as to why smokers are unable to quit smoking. It is now recognized that cigarette smoking is primarily a manifestation of nicotine addiction and that smokers have individual tendencies in their level of nicotine intake. Smokers control the way they puff and inhale to achieve their desired nicotine dose. Therefore, to conduct an effective smoking cessation program we need to adapt the cessation program to each individual.

Studies on twins indicated that genetic factors were more significant than environmental factors in influencing smoking habits.⁵ Carmelli et al⁶ reported that the concordance rate was significantly higher among monozygotic than dizygotic twins for smoking, not smoking, and quitting smoking. Therefore, to conduct a smoking cessation program effectively it may be beneficial to personalize the program based on the individual’s genetic background. It will, therefore, be useful to clarify the genetic polymorphisms associated with smoking cessation and smoking behavior. Smoking has been associated with the serotonin transporter–linked polymorphic region (5-HTTLPR), which influences serotonin transporter (5-HTT) function.

An association between 5-HTTLPR genotype and smoking cessation has been reported.^7-9 The 5-HTT gene is located on human chromosome 17q11.1-17q12.¹⁰ In the 5-HTTLPR, there are 2 common alleles, a 44-bp insertion (L allele) or deletion (S allele). Compared with the L allele, the S allele is associated with reduced transcription.^11,12 5-HTT activity was shown to be decreased in vivo in the S/S genotype in comparison with those with the S/L and L/L genotypes.¹² After smoking cessation, diminished serotoninergic transmission may mediate mood disturbances associated with nicotine withdrawal.¹³ Furthermore, preliminary clinical data suggested that serotonin reuptake inhibitors, such as fluoxetine hydrochloride, may facilitate smoking cessation.¹⁴ Therefore, we predicted that individuals with the S/S genotype or the S allele could easily stop smoking than others.

Japanese individuals with the S/S genotype were less inclined to smoking and/or could quit more easily smoking than others.⁸ Conversely, there is a report suggesting that American individuals with either the S/S or the S/L genotype and a high level of neuroticism experienced difficulty in quitting.⁹ However, Lerman et al⁷ were unable to report any association between 5-HTTLPR genotype and a 12-month posttreatment quit rate following a minimal-contact smoking cessation program among Caucasian or African American smokers. A recent study found no association between the 5-HTTLPR genotype and smoking behavior in Caucasians.¹⁵ Moreover, no work has focused on the association of the 5-HTTLPR genotypes with long-term smoking cessation success.

The objective of this study was to replicate the association between the 5-HTTLPR genotypes and smoking cessation and that between these genotypes and long-term smoking cessation success; that is, cessation for 2 years or more. Considering the gender difference in the prevalence of smoking, only male subjects were examined.

Methods

Subjects

The subjects included 363 male smokers recruited during 1999. We followed them until 2003. They were a group of employees aged between 25 and 50 years, working in Yamanashi. Their smoking habits were assessed annually by questionnaire, for example, “Do you currently smoke?” and “Did you smoke in the past?” At the last assessment in 2003, there were 47 ex-smokers (ie, those who had stopped smoking) and 316 others, who had continued smoking. Among the 47 ex-smokers, 12 ex-smokers had succeeded in long-term smoking cessation for 2 years or more.

In this study, we used 2 case–control designs. In these designs, each case was matched with a control for age and number of cigarettes smoked per day. The first case–control design consisted of the 47 ex-smokers (cases) and the 94 smokers (controls). The ratio of controls to cases was 1:2. The second case–control design consisted of the 12 ex-smokers who had succeeded in long-term smoking cessation (cases) and the 24 smokers (controls). In addition, the ratio of controls to cases was 1:2.

We obtained informed consent from all the subjects. The study was approved by the Ethics Committee of the University of Yamanashi, Faculty of Medicine.

Genotyping

Genomic DNA was extracted from whole-blood samples by standard procedures using the FlexiGene DNA kit (Qiagen, Hilden, Germany). Genotyping was carried out according to a method reported previously, with a minor modification.¹² In brief, the fragment including the polymorphism was amplified by polymerase chain reaction (PCR) using oligonucleotide primers 5′-GGC GTT GCC GCT CTG AAT GC-3′ and 5′-GAG GGA CTG AGC TGG ACA ACC A-3′ in a final volume of 15 µL containing approximately 50 ng genomic DNA, 3 pmol of each primer, 3 µL 5× AccuPrime (GC-Rich Buffer, Invitrogen, Carlsbad, CA), which contains 300 mM Tris–HCl (pH 9.2), 10 mM MgSO₄, 150 mM NaCl, 1 mM dGTP, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, thermostable AccuPrime proteins and enhancers, and 0.6 units GC-Rich DNA Polymerase (AccuPrime) with a thermocycler (GeneAmp PCR System 9700; PE Applied Biosystems, Carlsbad, CA). PCR was performed with an initial step at 95°C for 3 minutes, followed by 30 cycles at 95°C for 30 seconds, annealing at 61°C for 30 seconds, and an extension at 72°C for 1 minute with a final extension at 72°C for 10 minutes. To distinguish the alleles, all the amplicons were separated using 2% agarose gel electrophoresis and stained with ethidium bromide.

Statistical Procedures

Deviations of genotype distribution from Hardy–Weinberg equilibrium were assessed using χ² test. The relationships between the 5-HTTLPR genotypes and smoking status were analyzed using the conditional logistic regression model. The relationships between 5-HTTLPR allele frequency and smoking status were analyzed using unconditional logistic regression model. Genotypes were divided into S/S and S/L + L/L genotypes because whole-blood serotonin levels were significantly lower in individuals with the S/S genotype than in those with other genotypes¹⁶ and the L/L genotype being uncommon among the Japanese. A P value of .05 was considered to be significant.

Results

In the first case–control design, there were 47 cases and 94 controls (Table 1). In the 47 cases, the average age was 35.3 years and the average number of cigarettes smoked per day was 17.7. In the 94 controls, the average age was 35.4 years and the average number of cigarettes smoked per day was 18.1. A χ² test by the calculated genotypes on observed alleles and the observed genotypes showed that the observed genotypes both in cases (P = .95) and controls (P = .45) were in Hardy–Weinberg equilibrium. There was no significant difference in allele frequency and genotype distribution between the 47 cases and the 94 controls (Table 2).

Table 1.

Characteristics of Ex-Smokers and Controls: First Case–Control Design

	Ex-Smokers^a; Mean (SD)	Controls; Mean (SD)
Age in years	35.3 (6.1)	35.4 (5.9)
Cigatettes/day	17.7 (7.9)	18.1 (6.2)

Ex-smokers include those who had succeeded in long-term smoking cessation for 2 years or more.

Table 2.

Comparison of Genotype Distribution and Allele Frequencies Between Ex-Smokers and Controls: First Case–Control Design

	Ex-Smokers^a; n (%)	Controls; n (%)
Genotype	N = 47	N = 94
S/S	31 (66.0)	56 (59.4)
S/L	14 (30.0)	34 (36.2)
L/L	2 (4.3)	2 (2.1)
XL/S	0 (0.0)	2 (2.1)
Allele	N = 94	N = 188
S	76 (80.9)	148 (78.7)
L^b	18 (19.1)	40 (21.3)
Hazard ratio (95% CI)^b,c S/S vs S/L + L/L	1.1 (0.9-2.6)
Odds ratio (95% CI)^b,c S vs L	1.1 (0.6-2.1)

Abbreviations: L, the insertion allele of the 5HTTLPR; S, the deletion allele; LL, homozygotes for the L allele; LS, heterozygotes for the L and S alleles; CI, confidence interval.

Ex-smokers include those who had succeeded in long-term smoking cessation for 2 years or more.

L allele includes XL allele.

Ex-smokers versus controls.

In the second case–control design, there were 12 cases and 24 controls (Table 3). In the 12 cases, the average age was 36.6 years and the average number of cigarettes smoked per day was 17.9. In the 24 controls, the average age was 36.8 years and the average number of cigarettes smoked per day was 18.3. The observed genotypes both in cases (P = .99) and controls (P = .45) were in Hardy–Weinberg equilibrium. As shown in Table 4, the frequency of the S/S genotype was higher in the 12 cases than in the 24 controls, but the difference did not reach statistical significance (hazard ratio = 8.3, 95% confidence interval = 0.98-71.00). However, no significant association was found between the S allele and long-term smoking cessation success for 2 years or more (odds ratio = 6.8, 95% confidence interval = 0.80-56.50).

Table 3.

Characteristics of Ex-Smokers and Controls: Second Case–Control Design

	Ex-Smokers^a; Mean (SD)	Controls; Mean (SD)
Age in years	36.6 (4.0)	36.8 (4.0)
Cigatettes/day	17.9 (3.3)	18.3 (4.3)

Ex-smokers are those who had succeeded in long-term smoking cessation for 2 years or more.

Table 4.

Comparison of Genotype Distribution and Allele Frequencies Between Ex-Smokers and Controls: Second Case–Control Design

	Ex-Smokers^a; n (%)	Controls; n (%)
Genotype	N = 12	N = 24
S/S	11 (91.7)	13 (54.2)
S/L	1 (8.3)	11 (45.8)
L/L	0 (0.0)	0 (0.0)
XL/S	0 (0.0)	0 (0.0)
Allele	N = 24	N = 48
S	23 (95.8)	37 (77.1)
L^b	1 (4.2)	11 (22.9)
Hazard ratio (95% CI)^b,c S/S vs S/L + L/L	8.3 (0.98-71.00)
Odds ratio (95% CI)^b,c S vs L	6.8 (0.80-56.50)

Abbreviations: L, the insertion allele of the 5HTTLPR; S, the deletion allele; LL, homozygotes for the L allele; LS, heterozygotes for the L and S alleles; CI, confidence interval.

Ex-smokers include those who had succeeded in long-term smoking cessation for 2 years or more.

L allele includes XL allele.

Ex-smokers versus controls.

The 5-HTTLPR genotype frequencies in the subjects included in this study who experienced smoking (ie, ex-smokers and smokers) were similar to those in previously reported subjects who experienced smoking.⁷ The frequencies of the S/S, S/L, and L/L genotypes in this study were 61.7%, 35.5%, and 2.8%, respectively, and 67.9%, 28.2%, and 3.9% in the previous study subjects.

The uncommon allele, XL, was found in 2 of the 188 alleles in the smokers, whereas no XL allele was found in the 47 ex-smokers.

Discussion

This study suggests that the 5-HTTLPR genotype is associated with long-term smoking cessation success; that is, cessation of smoking for 2 years or more. The S/S genotype frequency was higher in ex-smokers who had succeeded in long-term smoking cessation than in controls but the difference did not reach slightly statistical significance (hazard ratio = 8.3, 95% confidence interval = 0.98-71.00). This finding indicates that having the S/S genotype may be an important factor for success in long-term smoking cessation. However, no association between the 5-HTTLPR genotype and smoking cessation was observed.

We considered a neurochemical mechanism for the association. Nicotine increases serotonin secretion in the brain, whereas nicotine withdrawal has the opposite effect, suggesting that the appetite and mood disturbances associated with nicotine withdrawal may be mediated by diminished serotoninergic transmission. Treatment with fluoxetine, a 5-HT uptake inhibitor, effectively prevents the increased food intake and weight gain in smokers who reduce their nicotine intake.¹⁷ The S allele is associated with lower transcriptional activity, resulting in decreased 5-HTT expression and serotonin uptake.¹² In addition, Narita et al¹⁸ have suggested that reuptake activity of 5-HTT in the brains of individuals with the L allele may be higher and that the 5-HT concentration in the extracellular space may be maintained at a lower level in individuals with the L allele compared with those with the S allele. Therefore, individuals with the S allele or S/S genotype may easily stop smoking than others. However, the results of the present study do not support this hypothesis.

We found no association between the 5-HTTLPR genotype and smoking cessation. On the other hand, the S/S genotype may be associated with long-term smoking cessation success for 2 years or more. A possible reason for this difference is the greater contribution of the 5-HTTLPR genotype to long-term smoking cessation success than to temporary smoking cessation. Dependence on tobacco smoking is a complex behavior, and both genetic and environmental factors contribute to the variance. Table 2 shows that the frequencies of the S/L + L/L genotypes were almost the same in the ex-smokers and in controls. This result may indicate that other factors, such as peer influence, rather than the 5-HTTLPR genotype contribute more strongly to the difficulty of quitting. On the other hand, as shown Table 4, the frequency of the S/S genotypes differed widely between the ex-smokers who had succeeded in long-term smoking cessation and their controls. This result may indicate that the L allele is associated with the difficulty of successful long-term smoking cessation. This result is consistent with a previous Japanese study.⁸

We cannot compare the present study with previous research because of the differences in recruitment methods and genotype grouping. For example, Lerman et al⁷ reported no association between 5-HTT genotypes and a 12-month posttreatment quit rate among Caucasian or African American smokers. However, these smokers, more than half of whom were females, were recruited through media advertisement for a smoking cessation program; these smokers may be eager to quit smoking but were unable to quit on their own. On the other hand, the subjects in the present study were male employees whose smoking behavior was assessed annually. Furthermore, the L allele frequency is known to be lower in Japanese (0.19) than in European Americans (0.60) and African Americans.¹⁹

It should be noted that a previous study in which subjects were randomly selected Japanese men has suggested that 4 single nucleotide polymorphisms in the 5-HTT gene may influence the difficulty of quitting among Japanese men.²⁰ Furthermore, a 17-bp variable number of tandem repeats at the second intron in the 5-HTT gene also seems to modulate the transcription of this gene.²¹ Therefore, further studies are needed with regard to these polymorphisms to confirm whether 5-HTT gene polymorphisms influence smoking cessation.s

Our study has several limitations. The subjects were followed for 5 years to assess self-reports of smoking behavior; therefore, their precise smoking behavior was not known after the assessment. It is possible that some of them may get back into the habit of smoking. It should also be noted that we used self-reports of smoking behavior for the 5 years; therefore, an accurate assessment of their smoking cessation was not possible. However, to minimize this inaccuracy we conducted a yearly survey through questionnaires. The other limitation was that we were unable to obtain objective measurements indicating the amount of nicotine intake and the nicotine dependence level among the subjects. However, to minimize this problem we evaluated the mean number of cigarettes smoked per day, which indicated the subjects’ nicotine dependence level.

Our study indicated an association between the S/S genotype and long-term smoking cessation success for 2 years or more. In other words, we showed that individuals with the S/L or L/L genotype may experience more difficulty in quitting than individuals with the S/S genotype. Therefore, smokers with the S/L or L/L genotype require a more rigorous smoking cessation program. Further studies are required to clarify the extent to which the ease of smoking cessation is attributable to the effect of the 5-HTTLPR genotype, to establish an appropriate smoking cessation program.

In conclusion, the present study suggests that individuals with the S/S genotype are more likely to continue smoking cessation than others.

Footnotes

The first two authors contributed equally to this publication.

The author(s) declared no potential conflicts of interest with respect to the authorship and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a grant-in-aid (Zentaro Yamagata, #17390182) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References

Inoue

Tsuji

Wakai

. Evaluation based on systematic review of epidemiological evidence among Japanese populations: tobacco smoking and total cancer risk. Jpn J Clin Oncol. 2005;35: 404-411.

Shibuya

. Change in mortality and years of life lost attributable to tobacco in Japan, 1985 and 1995. Asia Pac J Public Health. 1999;11:65-70.

Ariyoshi

. A case report of anti-smoking measures at a Japanese small-scale worksite. Asia Pac J Public Health. 2008;20(suppl):18-22.

Health and Welfare Statistics Association. Kokumin eisei no doukou 2009 [in Japanese]. Kousei no Shihyou. 2009;56(9):93.

Maes

Sullivan

Bulik

. A twin study of genetic and environmental influences on tobacco initiation, regular tobacco use and nicotine dependence. Psychol Med. 2004;34:1251-1261.

Carmelli

Swan

Robinette

Fabsitz

. Genetic influence on smoking: a study of male twins. N Engl J Med. 1992;327:829-833.

Lerman

Shields

Audrain

. The role of the serotonin transporter gene in cigarette smoking. Cancer Epidemiol Biomarkers Prev. 1998;7:253-255.

Ishikawa

Ohtsuki

Ishiguro

. Association between serotonin transporter gene polymorphism and smoking among Japanese males. Cancer Epidemiol Biomarkers Prev. 1999;8:831-833.

Brody

Fisher

. Interaction between the serotonin transporter gene and neuroticism in cigarette smoking behavior. Mol Psychiatry. 2000;5:181-188.

10.

Ramamoorthy

Bauman

Moore

. Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization. Proc Natl Acad Sci U S A. 1993;90:2542-2546.

11.

Heils

Teufel

Petri

. Allelic variation of human serotonin transporter gene expression. J Neurochem. 1996;66:2621-2624.

12.

Lesch

Bengel

Heils

. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science. 1996;274:1527-1531.

13.

Ribeiro

Bettiker

Bogdanov

Wurtman

. Effects of systemic nicotine on serotonin release in rat brain. Brain Res. 1993;621:311-318.

14.

Hitsman

Pingitore

Spring

. Anti-depressant pharmacotherapy helps some smokers more than others. Paper presented at: 18th Annual Meeting of the Society of Behavioral Medicine; April 1997; San Francisco, CA.

15.

Trummer

Köppel

Wascher

. The serotonin transporter gene polymorphism is not associated with smoking behavior. Pharmacogenomics J. 2006;6:397-400.

16.

Hanna

Himle

Curtis

. Serotonin transporter and seasonal variation in blood serotonin in families with obsessive-compulsive disorder. Neuropsychopharmacology. 1998;18:102-111.

17.

Pomerleau

Morrell

Lowenbergh

. Effects of fluoxetine on weight gain and food intake in smokers who reduce nicotine intake. Psychoneuroendocrinology. 1991;16:433-440.

18.

Narita

Takashima

Nakayama

Nagai

Okado

. Serotonin transporter gene variation is s risk factor for sudden infant death syndrome in the Japanese population. Pediatrics. 2001;107:690-692.

19.

Gelernter

Kranzler

Cubells

. Serotonin transporter protein (SLC6A4) allele and haplotype frequencies and linkage disequilibria in African- and European-American and Japanese populations and in alcohol-dependent subjects. Hum Genet. 1997;101:243-246.

20.

Liu

Yoshimura

Hanaoka

. Association of habitual smoking and drinking with single nucleotide polymorphism (SNP) in 40 candidate genes: data from random population-based Japanese samples. J Hum Genet. 2005;50:62-68.

21.

Hranilovic

Stefulj

Schwab

. Serotonin transporter promoter and intron 2 polymorphisms: relationship between allelic variants and gene expression. Biol Psychiatry. 2004;55:1090-1094.