The SLC6A3 gene possibly affects susceptibility to late-onset alcohol dependence but not specific personality traits in a Han Chinese population
The SLC6A3 gene possibly affects susceptibility to late-onset alcohol dependence but not specific personality traits in a Han Chinese population
Chang-Chih Huang 1 2 3
Shin-Chang Kuo 0 2 3
Yi-Wei Yeh 0 2 3
Chun-Yen Chen 0 2 3
Che-Hung Yen 2 3
Chih-Sung Liang 3 5
Pei-Shen Ho 3 5
Ru-Band Lu 3 4
San-Yuan Huang 0 2 3
0 Department of Psychiatry, Tri-Service General Hospital , Taipei, R.O.C , 4 Department Division of Neurology, Department of Internal Medicine, Chiayi Yang-Ming Hospital , Chiayi , Taiwan, R.O.C
1 Department of Psychiatry, Taipei Buddhist Tzu Chi General Hospital , New Taipei City, R.O.C
2 Graduate Institute of Medical Sciences, National Defense Medical Center , Taipei , Taiwan, R.O.C
3 Editor: Zhicheng Carl Lin, Harvard Medical School , UNITED STATES
4 Department of Psychiatry, Institute of Behavior Medicine, College of Medicine, National Cheng Kung University , Tainan , R.O.C
5 Department of Psychiatry, Beitou Branch, Tri-Service General Hospital , Taipei , Taiwan, R.O.C
Dopaminergic dysfunction has an important role in the pathoetiology of alcohol dependence (AD). The purpose of this study was to determine whether the solute carrier family 6 member 3 (SLC6A3) gene (also known as the dopamine transporter DAT gene) was associated with AD, and whether variants in the SLC6A3 locus were associated with specific personality traits in patients with AD. Sixteen polymorphisms in SLC6A3 were analyzed using 637 patients with AD and 523 healthy controls. To reduce clinical heterogeneity, patients were classified into two subgroups: early-onset AD (EOAD) and late-onset AD (LOAD). The Tridimensional Personality Questionnaire was used to assess the personality traits novelty seeking (NS) and harm avoidance (HA) in the patients with AD. Using allele frequency and genotype distribution comparisons and logistic regression analysis, we found evidence of association between rs6350 and AD (P < 0.05). Following subgroup analysis, we confirmed evidence of an association in patients with LOAD (P = 0.003), but not in patients with EOAD. Heterozygous carriers of the A allele have a nearly 3 times greater risk to develop LOAD compared to individuals who do not have an A allele. Although we found that patients with AD had higher NS and HA scores compared to controls (P < 0.001), we did not find evidence of association between SLC6A3 polymorphisms and either NS or HA in patients with AD using linear regression analysis. The findings from our study indicate that the SLC6A3 gene may have a role in susceptibility to late-onset AD in the Han Chinese population.
Data availability statement: Relevant data are
contained in the manuscript and supporting
information files. Data that could be used to identify
study participants is available upon request from
the corresponding author.
100) to SYH, and the Medical Affairs Bureau,
Ministry of National Defense, Taiwan
(MAB-104073, MAB-106-120) to SYH. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
Alcohol dependence (AD) is a complex disorder presenting a broad variability of clinical
manifestations that involve multifactorial etiologies including genetic, behavioral, environmental,
and personality factors [
]. The heritability of AD is estimated to be moderate to high ranging
from 51% to 64% [
]. In attempts to identify the genetic influence on AD susceptibility,
genes involved in the dopaminergic system have drawn much attention during the recent
decades. Evidence suggests that alcohol administration alters dopamine release [4±7]. In
medicating dopaminergic activity, the dopamine transporter plays a central role in the human
brain. Alcohol may directly affect the function of the dopamine transporter, although with
inconsistent results. For example, in vitro studies found that alcohol increased dopamine
release via inhibition of the dopamine transporter [
], whereas a subsequent animal study
reported an increase in efficiency of the dopamine transporter caused by alcohol
]. These differences may be accounted for by site-specific dopamine transporter
responses towards alcohol in different brain regions [
]. Therefore, dysfunction or altered
function of the dopamine transporter may contribute towards the pathophysiology of AD.
The solute carrier family 6 member 3 (SLC6A3) gene (also known as the dopamine
transporter DAT gene) consists of 15 exons and is found on chromosome 5p15.3. Polymorphisms
at the SLC6A3 locus have been implicated in gene expression, transcriptional activity, and
protein availability [
]. Because of conflicting findings regarding an association between
SLC6A3 and substance dependence [
], the role of the SLC6A3 gene in the pathogenesis of
AD remains unclear. Evidence of an association between SLC6A3 and AD was limited using
few genetic markers. Meta-analyses that explored whether one of the most frequently tested
SLC6A3 polymorphisms, a 40-bp variable number tandem repeat (VNTR) in the 30
untranslated region, was associated with AD have shown inconsistent results [
]. In contrast,
other studies found that several single nucleotide polymorphisms (SNPs) located in exons of
the SLC6A3 gene may functionally contribute to AD. For example, the A allele of rs27072
located in exon 15 was identified as a risk allele for AD in a Japanese population .
Furthermore, Lind et al. [
] found that the A allele of rs6350 located in exon 2 had a protective effect
against harmful drinking behavior in a male Finnish population. However, because of the
inconsistent findings supporting a role of the SLC6A3 gene in AD, further study using a larger
sample size and a different ethnic population is warranted.
Different subtypes of AD may have different underlying neurobiological etiologies [
furthermore, genetic contributions affect a narrowly defined phenotype of affected individuals (a
subgroup) instead of the entire range of phenotypes that form the clinical characteristics of
complex brain disorders [
]. A major factor for addiction may be the age of onset. Previous
research has demonstrated that age of onset in patients with AD is associated with different
genetic variants and activity of the serotonin transporter [
], response to antidepressants
], and clinical characteristics [
]. Schmid and colleagues found that variants in SLC6A3
were associated with increased alcohol consumption by individuals that started drinking early
in life and proposed that gene-environment interactions may contribute to the development of
]. Compared to patients with AD characterized by an early onset of AD (EOAD),
patients with late onset AD (LOAD) have a prolonged interval from regular drinking to the
development of AD, and they may be influenced largely by environmental factors [
Furthermore, patients with LOAD show reduced alcohol-drinking behavior with sertraline
], a finding that indicates a relationship between alcohol drinking behavior and
depression in this subgroup of patients. Based on these findings, we posit that further
investigation to determine whether SLC6A3 variants are associated with AD in subgroups defined by
the age of onset is warranted.
2 / 17
Personality factors may be major predictors for either the course or the outcome of AD [
]. Using the model of Cloninger's biosocial theory of personality, patients with AD have
higher novelty seeking (NS) and harm avoidance (HA) scores than that found in controls .
Studies have found an association between the SLC6A3 gene and personality traits defined by
the Cloninger's model in healthy individuals [
] and patients with depression .
However, evidence demonstrating a relationship between the SLC6A3 gene and personality traits of
Cloninger's model in patients with AD remains lacking. Therefore, the present study evaluated
personality traits determined by the Tridimensional Personality Questionnaire (TPQ) in
patients with AD to determine whether there was evidence of genetic contribution to
In the present study, we performed a comprehensive association study using 16 genetic
markers from the SLC6A3 gene in a large sample of a Han Chinese population to investigate
whether there was evidence of an association between SLC6A3 variants with age of onset and
specific personality traits in patients with AD.
Materials and methods
The study protocol was approved by the Ethics Committee of Tri-Service General Hospital
(TSGH) for the Protection of Human Subjects (TSGHIRB 096-05-073-A). All of the patients
were recruited from the outpatient or inpatient departments of TSGH in Taipei, Taiwan.
Following a detailed description of the study procedure, written informed consent was obtained
from all participants. All of the study participants were recruited from July 1, 1997 to
December 31, 2015 and they were unrelated to each other and ethnically Han Chinese.
The patient group consisted of 637 AD patients, of whom 229 are classified with EOAD
(age < 24 y/o) and 408 with LOAD (age 25 y/o). Each patient was diagnosed AD based on
the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV-TR) by a
general psychiatrist initially in clinical inpatient or outpatient settings and subsequently
interviewed by either a well-trained psychologist or a psychiatrist using a Chinese Version of the
Modified Schedule of Affective Disorder and Schizophrenia-Life Time (SADS-L) [
with a history of other substance dependence (except nicotine), organic brain disease, severe
medical illness, or any concomitant major psychiatric disorders were excluded from this study.
The control group comprised 436 healthy volunteers from the community. We used
SADS-L to exclude past or present psychiatric illnesses including affective disorders,
schizophrenia, anxiety disorders, personality disorders, and substance use disorders in this group.
Moreover, subjects with positive family history of psychiatric disorders presented in
firstdegree relatives were excluded.
Selection of genetic variants and genotyping methods of the SLC6A3 gene
Genomic DNA from the participants was extracted from peripheral blood leukocytes by a
commercial kit (DNAzol; Invitrogen, Carlsbad, CA, USA). The study variants were selected
according to the human SLC6A3 polymorphisms in the HapMap database (www.hapmap.org),
NCBI SNP database (www.ncbi.nlm.nih.gov/projects/SNP/), and a relevant review of literature
]. A total of 16 SNP polymorphisms were investigated to cover a region of 56 kb in the
SLC6A3 gene: three SNPs [rs2550948, rs2652511, and rs2975226] in the promoter region, one
[rs6350] in exon 2, one [rs2981359] in intron 2, two [rs403636, rs460000] in intron 3, two
[rs460700, rs464049] in intron 4, one [rs37020] in intron 6, one [rs37022] in intron 7, one
3 / 17
[rs27048] in intron 8, one [rs6347] in exon 9, one [rs11133767] in intron 13, one [rs40184] in
intron 14, and one [rs27072] in exon 15. All SNPs of the SLC6A3 gene were genotyped by
TaqMan assays (Applied Biosystems, Foster City, CA) with Applied Biosystems StepOne™ software
and StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA).
Assessment of personality traits
The Chinese version of the Tridimensional Personality Questionnaire (TPQ) was used to
evaluate personality traits in patient and control groups [
]. We analyzed only NS (32 items,
Cronbach's α = 0.70) and HA (34 items, Cronbach's α = 0.87) dimensions but not
rewarddependence dimension (RD, 34 items, Cronbach's α = 0.54) because of low inter-rater
reliability of RD among Han Chinese in Taiwan [
]. 198 AD patients and 252 controls who
completed the TPQ were eligible for subsequent analysis. Subjects in AD group were assessed after
a withdrawal period in order to avoid confounding effects as a result of alcohol withdrawal
We employed an independent sample t test and two-tailed Pearson test to analyze the
differences in mean age, gender, allele and genotype frequencies for each SNPs between the AD
patients and controls. Fisher's exact test was substituted for chi-square test when the sample
size was smaller than expected. Multivariate logistic regression analysis was used to correct the
effect of possible covariates such as age, gender, and gene-to-gene interactions in the risk for
AD. For personality trait assessment, a linear regression analysis was conducted using each
SNP as an independent variable, with patient/control group, gender, and age as covariates, and
NS or HA score as dependent variables. All statistical analyses were performed using SPSS
statistical software (version 19, SPSS Inc., Chicago, IL, USA).
For haplotype association analysis, we analyzed the linkage disequilibrium (LD) coefficients
(D'), haplotype frequency, haplotype block, haplotype association, and Hardy-Weinberg
equilibrium for each variant using Haploview V4.1 software (Broad Institute, Cambridge, MA) [
in which we defined a haplotype block as a set of contiguous SNPs with an average D' greater
than 0.9. All tests were two-tailed, and alpha was set at 0.05. To correct the halplotype analysis
for multiple testing, 10,000 permutations were performed for haplotype-specific P-values.
Of the 1,185 participants enrolled in this study, 25 were excluded because of incomplete
genotype data. The final 1,160 subjects that were used in our analysis included 637 patients with
AD and 523 healthy controls. The mean age of onset for AD group was 30.62 ± 9.52 years.
There were no differences in age between the two groups (mean age of 39.36 ± 11.23 years for
patients vs. mean age of 39.84 ± 12.32 years for controls, P = 0.490); however, our AD cohort
had a higher proportion of males than females compared to the control group (88.2% vs.
69.2% respectively, P < 0.001). Furthermore, 256 patients (40.19%) with AD were recruited
from inpatient department and there was no difference in gender and age compared to those
recruited from outpatient department (P > 0.05).
SLC6A3 variants in patients with AD
Genotype distributions and allele frequencies in AD and control cohorts are summarized in
Table 1. All variants were in Hardy-Weinberg equilibrium in the control group. We found an
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9 9 9 (00 18 14 78 79 45 95 101 (6
1 5 7 23 26
1 6 4 8 5 0 1 5 7 3 1 6 1 3 6 7
b 9 2 3 0 4 6 1 5 4 3 1 1 1 3 4 6
.00 .01 .02 .00 .07 .09 .02 .01 .09 .06 .01 .09 .03 .04 .08 .01 ;nAD ighh roD
iecseno a)(% lAD .5178 .6156 .6156 .283 .4403 .3320 .8451 .9484 .6350 .6334 .7433 .5186 .1115 .1013 .2512 .2465 i,It;rnno cyenuqe ittshnAw ttt.scea
freuq FAM trono .0031 .3441 .6341 .241 .3134 .9823 .0864 .0474 .4263 .3753 .3085 .6581 .4231 .1191 .5532 .4742 xoen iftrh iteap 'rxse
, w e
E s i h
lle lleea 2 G T T G C C T C G C A C A C C C ;re lllee ise isF
a lA 1 A C A A G A G T A A T T G T T T to a c y
51 16 38 c30 50 06 13 52 09 39 10 89 18 08 21 07
increased frequency of the rs6350 A allele and of the A/G genotype in patients with AD
compared to that found in controls (uncorrected P = 0.008 and P = 0.008, respectively). We found
in our subgroup analysis of genotype distribution comparisons that this association was with
the LOAD group (uncorrected P = 0.003), but not with the EOAD group (P = 0.346, as shown
in S1 Table). Following conservative adjustments for multiple comparisons, our finding in the
LOAD subgroup remained significant (adjusted P = 3.13 × 10−3). To account for potentially
confounding effects because of differences in gender distribution between patient and control
cohorts, we performed multivariate logistic regression analysis in our efforts to identify
putative risk alleles from 16 SNPs in the SLC6A3 gene associated with AD and its subgroups
(Table 2). We confirmed that rs6350 in the SLC6A3 gene was associated with the pathogenesis
of AD (P = 0.012), and the association became more significant in the LOAD subgroup
(P = 0.005); heterozygous carriers of the A allele have a nearly 3 times greater risk to develop
LOAD compared to individuals who do not have an A allele.
Haplotype analysis in the SLC6A3 gene
SNP locations at the SLC6A3 gene, linkage disequilibrium (LD) structure, and D0 values for all
variants used in this study are shown in Fig 1. A haplotype block was defined as a set of
contiguous SNPs with an average D0 greater than 0.9. We identified two major haplotype blocks using
these 16 polymorphisms at the SLCC6A3 locus in our cohorts (Table 3). Block 1 is 20 kb in size
and includes five SNPs (rs37020, rs464049, rs460700, rs460000, and rs403636) spanning the
region between intron 3 and intron 6, while block 2 is 4.8 kb in size and includes three SNPs
(rs2975226, rs2652511, and rs2550948) spanning the promoter region. Among all subjects, no
significant difference was observed for these two blocks between patients with AD and controls.
Relationship between SLC6A3 variants and personality scores
One hundred and ninety-eight patients with AD and two hundred and fifty-two controls
completed the TPQ and were available for gene-personality analysis. The scores for both HA and
NS were higher in the patient cohort than that found in the control cohort (17.24 ± 4.91 vs.
12.61 ± 4.63 respectively, P < 0.001 for NS; 16.63 ± 6.22 vs. 10.58 ± 5.28 respectively, P < 0.001
for HA). No difference were observed in the HA scores between patients from the EOAD
(N = 68) and LOAD (N = 130) subgroups who completed the TPQ (16.65 ± 5.32 for EOAD vs.
16.62 ± 6.67 for LOAD, P = 0.978). However, patients with EOAD had higher NS scores than
the patients with LOAD (18.60 ± 4.76 vs. 16.53 ± 4.85 respectively, P = 0.004). Using one-way
ANOVA to analyze all three groups together (EOAD, LOAD, and controls), we found a
decreasing NS score in the order EOAD > LOAD > controls (P < 0.001).
To exclude potential effects from age of onset and gender bias, we performed linear
regression analysis to investigate further whether SLC6A3 variants were associated with specific
personality traits in patients with AD (Table 4). Of the 16 SLC6A3 polymorphisms tested,
rs464049 and rs37020 were associated with NS (P = 0.024 and P = 0.008, respectively), and
several SNPs were associated with HA (P = 0.013 for rs2550948, P = 0.016 for rs2652511,
P = 0.016 for rs2975226, and P = 0.036 for rs403636). However, none of these associations
remained following Bonferroni corrections for multiple comparisons. Furthermore, gender
was not associated with either NS or HA scores; in contrast, we found that age of onset was
negatively correlated with NS in patients with AD (P < 0.001).
Although the role of the dopamine transporter in dopaminergic neurotransmission makes the
SLC6A3 gene an appealing candidate gene for AD, evidence of an association and
6 / 17
AD: Alcohol dependence; LOAD: late-onset alcohol dependence; EOAD: early-onset alcohol dependence.
Age and gender are used as covariates.
a Genotype within parenthesis indicates the reference group of genotype.
b The minor genotype frequencies are combined if the frequency is less than 10%.
c Odds ratio is given with 95% CI after using a logistic regression analysis compared with controls.
7 / 17
Fig 1. The linkage disequilibrium (LD) structure between 16 SNP polymorphisms in SLC6A3 gene. The upper panel shows the
location of 16 polymorphisms in SLC6A3 gene and lower panel shows the output of Haploview version 4.1. D' (left) and r2 value (right) were
shown within each square represents a pairwise linkage disequilibrium relationship between two polymorphisms. Red squares indicate
statistically significant LD between the pair of polymorphisms. Darker colors of red indicate higher values of D' or r2, up to a maximum of 1
and white squares indicate pairwise D' values with no statistically significant difference of LD. The two haplotype blocks generated under
confidence interval algorithm of haploview are marked. The definitions of the abbreviations: Ex, exon; In, intron; P, promoter.
identification of susceptibility alleles to AD has been controversial and non-reproducible [
]. To our knowledge, this is the first case-control study using single marker and haplotype
analyses with multiple SNPs to determine whether there is evidence of a role of the SLC6A3
gene in patients with AD in a Han Chinese population. Using allele frequency and genotype
distribution comparisons, and logistic regression analysis, we found evidence of an association
with the A allele of the exonic SNP rs6350 in patients with AD compared to that found in
controls. However, not all findings remained statistically significant following corrections for
Harm Avoidance a
AD, alcohol dependence; TPQ, Tridimensional Personality Questionnaire.
a The association between all SLC6A3 variants and TPQ score are corrected for age, gender, and age of onset as covariates using linear regression
b The p value for the relationship between TPQ score and each SLC6A3 variant.
multiple comparisons. In fact, our major finding was in patients from the LOAD subgroup,
but not the EOAD subgroup. In the present study, the minor A allele of rs6350 was considered
a risk allele for LOAD and that heterozygous carriers have a nearly 3 times increased risk to
develop LOAD compared to individuals who do not have the A allele. Our findings are in
contrast to the findings of Lind et al. [
] in which the rs6350 A allele was a protective allele
associated with decreased severity of problem drinking and incidence of AD. Several factors may
account for these observed differences between our studies. First, there can be ethnic- and
population-specific differences in allele frequencies and genotype distributions of genetic
polymorphisms. The minor allele frequency of rs6350 was approximately 2.2% in our Han Chinese
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controls, a frequency that is considerably lower than the 10.5% found in controls from a
Finnish population [
]. To decrease the risk of bias because of sample selection and to increase the
statistical power of our study, we used a large sample size recruiting 1,160 individuals. In
addition, we recruited both males and females with AD instead of specifically testing only affected
males in our investigation of the role of the SLC6A3 gene in AD. We also used restricted
inclusion criteria for our control subjects, excluding individuals if they met the diagnosis of AD and
showed alcohol abuse as determined using the SADS-L. However, individuals recruited in the
control cohort of the study by Lind et al. were assessed solely by the Alcohol Use Disorders
Identification Test (AUDIT) and furthermore, they were categorized as either pure controls or
problem drinkers. This heterogeneity in exclusion and inclusion criteria between the control
cohorts of our two studies may also contribute to the differences in their respective findings.
Finally, the contradictory AD allele of rs6350 in the two populations might be derived from
high recombination between this SNP and a disease locus. However, both Lind et al. and we
conclude that rs6350 at the SLC6A3 gene has an association with AD. Whether this
synonymous exonic SNP is in linkage disequilibrium with other SLC6A3 variants that could alter
transporter availability or function in the human brain warrants further investigation.
Genetic polymorphisms at the 50 UTR promoter region of SLC6A3 have been proposed to
be functional and known to alter transcriptional activity [
], and that these variants found in
the promoter region may have a role in several psychiatric disorders, including attention
deficit-hyperactivity disorder [
], bipolar disorder [
], and schizophrenia [
]. In the present
study, we used three common SNPs to explore the significance of SLC6A3 promoter for AD.
However, neither single marker nor haplotype analyses revealed an association between
patients with AD and controls. Therefore, despite the fact that SLC6A3 promoter variants are
excellent candidates for linkage and association studies, we did not find evidence supporting
their role in AD in our cohort of Han Chinese population. For the most investigated 3' UTR
VNTRs in relation to AD, two meta-analyses showed inconsistent results [
contradictory findings might be attributable to differences in study design such as data sampling and
statistical methods. In the latter study, for example, Ma et al.  concluded that the 9-repeat
allele increased the risk for AD in different populations using a dominant genetic model with a
larger sample size. However, the authors did not include two studies that considered that the
9-repeat allele was protective for AD in Japanese and Han Chinese populations [
Another frequently tested polymorphism located in the 30 region of SLC6A3 that is tested for
association with psychiatric disorders is rs27072, which is in exon 15. Ueno et al. [
investigated the role of rs27072 in AD in a Japanese population and found that the minor A allele was
a risk allele in their AD cohort. In comparison, we found that the minor allele frequency
(MAF) of rs27072 was comparable between our affected cohort (24.7%) and that of Ueno et al.
(26.6%). However, they had a markedly lower MAF in their control group (17.3%) compared
to that found in ours (27.4%). Therefore, it is likely that the MAF differences between the
control groups accounts for the differences in findings between the two studies. Based on our
findings, we conclude that polymorphisms from either the 50 or 30 region of the SLC6A3 gene are
not associated with AD in our Han Chinese population.
Another factor that may account for the observed heterogeneity between findings in genetic
association studies is that interactions between different genetic and environmental factors
modulate susceptibility to AD. Therefore, subgroup analysis using age of onset of AD has been
used as a means to resolve conflicting findings and increase homogeneity in genetic
association studies [
]. In the present study, and following adjustments for multiple
comparisons, the rs6350 polymorphism was identified to confer risk to develop AD, specifically in
patients from the LOAD subgroup, but not in patients from the EOAD subgroup. In
Cloninger's model of alcoholism, type I alcoholism is characterized by anxiety proneness, loss of
10 / 17
control and excessive guilt over alcohol intake, over 25 years of age, and progressive severity of
alcohol abuse. Individuals with type I alcoholism initially inhibit drinking behavior because of
their proneness to anxiety and therefore, experience a later onset of alcoholism. However, they
may be characterized as having a binge drinking pattern with a rapid progression, and
accompanied by the development of alcoholic liver disease later in life . It is proposed that
individuals with type I alcoholism have a greater environmental predisposition than individuals
with type II alcoholism. For example, adoptive studies found that both males and females were
equally likely to develop type I alcoholism, and that they may have more severe and frequent
alcohol abuse if their adoptive fathers have a low occupational status [
]. In addition, there
may be neurobiological differences underlying type I and II alcoholism, owing to which
individuals with type I alcoholism are more vulnerable to dopaminergic deficits, and individuals
with type II alcoholism have deficits in serotonergic neurotransmission [
]. We hypothesize
that patients in our LOAD subgroup have deficits in the dopaminergic system because of
dysfunction or altered function of the dopamine transporter. Depression is another potential
factor that may confound the role of SLC6A3 in patients with LOAD. Previous studies found that
depressive symptoms are risk factors for LOAD [
], and that patients with LOAD have
significantly reduced alcohol drinking and favorable outcomes following treatment with
]. Although Kranzler et al. [
] posited a role for the serotonergic system towards
susceptibility of AD among patients with LOAD, we propose that dopaminergic deficits
contribute to both depression and addictive disorders, and are responsible for the comorbidity
found between depression and AD. We hypothesize that individuals with the rs6350 A allele
show dysfunction or altered dopaminergic function, and that this genetic predisposition
increases vulnerability to environmental stressors or exacerbates a predisposition to
depression in patients with LOAD. Consequently, we speculate that these patients adopted a
selfmedication pattern of alcohol drinking behavior for their sub-clinical depression, which
occurs later in life and contributes to the late-onset of AD found in these patients. In the
present study, we could not examine environmental factors such as life stressors or childhood
negative experiences, or evaluate the presence or severity of depressive symptoms in our cohorts.
Nevertheless, it is warranted that future studies are performed to investigate these factors.
Because pathologies that underlie comorbid personality traits may contribute or have an
effect on the course or prognosis of AD, specific personality traits may reflect a genetic
predisposition for substance abuse [
]. Our patients with AD had higher NS and HA scores
compared to the healthy controls, findings that are in line with those from an earlier study
. In our subgroup analysis, we found that patients with EOAD had the highest NS scores,
followed by patients with LOAD, and controls. In contrast, no association was found between
our subgroups and controls using HA scores. It was proposed that NS is a highly inherited
personality trait [
], and similar to addiction, is modulated by the dopaminergic system [
Individuals with a high NS score are more likely to experiment with drugs at an early age, to be
attracted to novel stimuli, and to respond rapidly to cues for reward despite potential
punishment. Therefore, it was considered that a high NS score was a predictor for an increased risk of
relapse, cravings, and the compulsive use of alcohol in patients with AD [
evidence demonstrating that NS and addictive behaviors are affected by dysfunction of altered
function of the dopaminergic system because of variants in SLC6A3 is limited. For healthy
controls, both association and non-association between NS and SLC6A3 polymorphisms have
been reported [
30, 50, 51
]. Therefore, researchers argued that the impact of genes on specific
personality traits might be due to gene-gene interactions rather than a single genetic
polymorphism. To minimize or avoid potential confounding effects caused by age of onset or gender,
we used linear regression analysis instead of one-way ANOVA in our evaluation. Although
evidence of associations between two SLC6A3 intronic variants (rs464049 and rs37020) and
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NS was found, the findings were not significant following corrections for multiple testing.
However, our finding that there is no association is comparable to previous studies that did
not find evidence of association in patients with substance use disorders such as amphetamine
] and heroin dependence [
]. Anghelescu and colleagues proposed that the 30
VNTR polymorphism of the SLC6A3 gene may affect NS in Caucasian patients with AD. They
found that a small subgroup of patients homozygous for the 10 allele had lower NS tendencies
compared to that found in other patients with AD and controls [
]. Although we did not
directly test the 30 VNTR in our study, we used multiple genetic markers to comprehensively
cover the SLC6A3 gene. In addition, we performed strict statistical analyses incorporating
adjustments for multiple comparisons, and used a large sample size to confirm that there was
no evidence of an association between the SLC6A3 gene and NS scores in our cohort of
patients with AD. In line with the current understanding that HA is determined by the
serotonergic system of the brain, we found no evidence of an association between our tested SNPs in
SLC6A3 and HA scores in our cohort of patients with AD. Therefore, although we found
higher scores for NS and HA personality traits in patients with AD compared to that found in
controls, we found no evidence of a role for the SLC6A3 gene in influencing these specific
personality traits in an AD cohort from the Han Chinese population.
We identified several limitations in the present study. First, despite enrolment of a relatively
large number of participants, there may be insufficient power to robustly detect associations
within patient subgroups and in particular, the EOAD subgroup. Our total sample size
(N = 752) for EOAD subgroup analysis only had the power of 0.69 to detect a small effect.
Second, we observed a disequilibrium in the sex ratio between our controls and that found in our
AD cohort. To account for this observed gender bias, we analyzed our data only in male
subjects (as shown in S2 Table). Although the results were similar to that obtained using total
subjects, we only detected nominal association for rs6350 after multiple corrections between case
and control groups, including that for LOAD subgroup. This finding might be attributable to
the insufficient sample size for male subjects and warrants further investigation. Third, we
used a dichotomized method with an age of onset at 25 years, rather than a detailed age
distribution, for the subgroup analysis, which may not be sufficient to apply in real situation.
Fourth, although personality scores were not determined while patients were intoxicated or in
a state of withdrawal, the possible confounding effects of medications such as soporifics or
sedatives on personality traits cannot be ruled out. In addition, we used Cloninger's biosocial
theory that considers NS and HA, but not other personality traits in the Temperament and
Character Inventory such as persistence, self-directedness, cooperativeness, and
self-transcendence. Therefore, the lack of evidence supporting genetic contribution to personality in our
study does not represent the entire personality dimension, but is specific to that of NS and HA
in our patient cohort. Moreover, ethnic or population differences in allele or genotype
frequencies of genetic variants or environmental factors may have important roles in the
pathogenesis of AD, particularly, of LOAD. However, the influence of environmental factors on the
disease was not examined in the present study. Finally, the MAF of rs6350 was very low (less
than 3%) and we did not find any individuals homozygous for the A allele in our study.
Further investigation to determine whether this exonic SLC6A3 gene variant has a role in the
development of AD in individuals later in life is warranted.
Using single marker and haplotype analyses, we found evidence of an association between the
SLC6A3 gene and susceptibility to AD. Following adjustments for multiple comparisons,
evidence of this association remained in patients from the LOAD subgroup, but not the EOAD
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subgroup in our study of a Han Chinese population. In addition, we found no evidence for a
role of the SLC6A3 gene in personality traits in our cohort of patients with AD. Further
geneenvironment association studies are warranted to confirm our findings.
S1 Fig. The linkage disequilibrium (LD) structure between 16 SNP polymorphisms in
SLC6A3 gene for patient with alcohol dependence (AD). The upper panel shows the location
of 16 polymorphisms in SLC6A3 gene and lower panel shows the output of Haploview version
4.1. D' (left) and r2 value (right) were shown within each square represents a pairwise linkage
disequilibrium relationship between two polymorphisms. The definitions of the abbreviations:
Ex, exon; In, intron; P, promoter.
S2 Fig. The linkage disequilibrium (LD) structure between 16 SNP polymorphisms in
SLC6A3 gene for controls. The upper panel shows the location of 16 polymorphisms in
SLC6A3 gene and lower panel shows the output of Haploview version 4.1. D' (left) and r2 value
(right) were shown within each square represents a pairwise linkage disequilibrium
relationship between two polymorphisms. The definitions of the abbreviations: Ex, exon; In, intron; P,
S1 Table. Genotype distributions of the polymorphisms in the SLC6A3 (DAT) gene
between patients with early-onset alcohol dependence (EOAD) and controls in a Han
S2 Table. Genotype distributions and allelic frequencies of the polymorphisms in the
SLC6A3 (DAT) gene between male patients with alcohol dependence and controls in a Han
This work was supported by the Ministry of Science and Technology, Taiwan
(MOST1042314-B-016-012-MY3, MOST103-2325 -B-016-001) to SYH, the National Science Council
(NSC101-2325-B-016-003) to SYH, the Tri-Service General Hospital (TSGH-C105-124,
TSGH-C-106-100) to SYH, and the Medical Affairs Bureau, Ministry of National Defense,
Taiwan (MAB-104-073, MAB-106-120) to SYH. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Conceptualization: SYH CCH.
Data curation: SYH CCH.
Formal analysis: SYH CCH.
Funding acquisition: SYH.
Investigation: SYH CCH.
Methodology: SYH CCH PSH SCK CYC YWY CHY CSL.
13 / 17
Project administration: SYH CCH.
Supervision: SYH RBL.
Validation: SYH CCH PSH SCK CYC YWY CHY CSL.
Visualization: SYH CCH.
Writing ± original draft: SYH CCH.
Writing ± review & editing: SYH CCH.
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