Original article 1
Further evidence for genetic variation at the serotonin
transporter gene SLC6A4 contributing toward anxiety
Andreas J. Forstnera,b,e,f,g,*, Stefanie Rambauc,*, Nina Friedricha,b,
Kerstin U. Ludwiga,b, Anne C. Bhmera,b, Elisabeth Mangolda,b, Anna Maasera,b,
Timo Hessa,b, Alexandra Kleimanc, Antje Bittnerd, Markus M. Nthena,b,
Jessica Beckera,b, Franziska Geiserc,*, Johannes Schumachera,b,*
and Rupert Conradc,*
Objectives Social anxiety disorder (SAD) is a common and
heritable psychiatric disorder. However, genetic studies in
SAD are rare and only a few candidate genes have been
implicated so far. In the present study, we investigated
whether single-nucleotide polymorphisms (SNPs)
associated with other psychiatric disorders also contribute
toward the development of SAD and followed up variants
associated with SAD on the phenotypic level.
Patients and methods We genotyped a total of 24 SNPs in
a German sample of 321 SAD patients and 804 controls. We
carried out single-marker analyses as well as quantitative
association analyses of SAD severity and harm avoidance.
Results None of the variants investigated showed an
association with SAD in our casecontrol sample after
Bonferroni correction. Two SNPs reached nominal
significance (rs818702, P = 0.032; rs140701, P = 0.048). Of
these, only rs140701 showed an association in the same
allelic direction as reported previously. This SNP is located
within the serotonin transporter gene SLC6A4, which is the
primary target of selective-serotonin reuptake inhibitors
used for the treatment of depressive and anxiety disorders.
The quantitative association analysis of all cases with
available data on symptom severity showed four SNPs with
a nominal significant association. Among these SNPs,
rs10994359 showed the strongest association (P = 0.001)
and was located near the ANK3 gene. In addition,
Introduction
Social anxiety disorder (SAD) is a common psychiatric
disorder with a lifetime prevalence of about 1213%
(Stein and Stein, 2008; Kessler et al., 2012). It is char-
acterized by marked and persistent fear and related
avoidance of interpersonal situations in which the affec-
ted individuals fear to act in a way that will be humi-
liating or embarrassing. Individuals with SAD show a
significant reduction in quality of life (Eng et al., 2005)
Supplemental digital content is available for this article. Direct URL citations
appear in the printed text and are provided in the HTML and PDF versions of this
article on the journal's website (www.psychgenetics.com).
0955-8829 Copyright 2017 Wolters Kluwer Health, Inc. All rights reserved.
Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
rs10994359 was nominally associated with harm avoidance
scores (P = 0.001).
Conclusion Our results provide further evidence for an
involvement of the serotonin transporter gene SLC6A4 in the
etiology of anxiety-related traits. Furthermore, our study
implicates that genetic variation at the genome-wide
associated bipolar disorder locus ANK3 might influence
anxiety-related personality traits. Psychiatr Genet
00:000000 Copyright 2017 Wolters Kluwer Health, Inc. All
rights reserved.
Psychiatric Genetics 2017, 00:000000
Keywords: harm avoidance, personality, serotonin,
single-nucleotide polymorphism, SLC6A4, social anxiety disorder
a
Institute of Human Genetics, bDepartment of Genomics, Life and Brain Center,
c
d
Clinic for Psychosomatic Medicine and Psychotherapy, University of Bonn, Bonn,
Department of Psychotherapy and Psychosomatic Medicine, University Hospital
Carl Gustav Carus, Technische Universitaet Dresden, Dresden, Germany,
e
Department of Psychiatry (UPK), fHuman Genomics Research Group,
Department of Biomedicine, University of Basel and gInstitute of Medical Genetics
and Pathology, University Hospital Basel, Basel, Switzerland
Correspondence to Johannes Schumacher, MD, Institute of Human Genetics,
University of Bonn, Sigmund-Freud-Street 25, 53127 Bonn, Germany
Tel: + 49 228 287 51028; fax: + 49 228 287 51011;
e-mail: johannes.schumacher@uni-bonn.de
*Andreas J. Forstner, Stefanie Rambau, Franziska Geiser, Johannes Schumacher,
and Rupert Conrad contributed equally to the writing of this article.
Received 27 July 2016 Revised 23 January 2017 Accepted 20 February 2017
and find themselves exposed to adverse social and
economic consequences (Patel et al., 2002).
SAD is a multifactorial disorder for which both genetic
and environmental factors contribute toward disease
susceptibility (Bandelow et al., 2004). Family studies
showed an over threefold increase of SAD among
first-degree relatives of SAD patients (Fyer et al., 1993;
Stein et al., 1998; Coelho et al., 2007; Smoller et al., 2008)
and, on the basis of twin studies, the estimated herit-
ability of SAD ranges between 20 and 50% (Kendler
et al., 1999, 2001; Skre et al., 2000). However, genetic
studies in SAD are rare (Smoller et al., 2008; Hamilton,
2009) and only a few candidate genes have been
DOI: 10.1097/YPG.0000000000000171
 2 Psychiatric Genetics 2017, Vol 00 No 00
implicated so far (Donner et al., 2008; Sipila et al., 2010;
Strug et al., 2010; Jensen et al., 2014). These include the
serotonin transporter gene SLC6A4, which encodes an
integral membrane protein that is the primary target of
selective-serotonin reuptake inhibitors used for the
treatment of SAD (Kent et al., 1998; Strug et al., 2010).
In addition, SAD shows high comorbidity with other
anxiety disorders (Fehm et al., 2005). Hettema et al.
(2005) reported that the genetic risk for SAD partly
overlaps with the risk for generalized anxiety disorder,
panic disorder (PD), and agoraphobia. In addition, a
genetic overlap between anxiety disorders has been
suggested by Jensen et al. (2014). SAD is also frequently
comorbid with other psychiatric disorders. These include
major depressive disorder (Rush et al., 2005; Stein and
Stein, 2008) and bipolar disorder (Simon et al., 2004; Stein
and Stein, 2008).
It is noteworthy that a shared genetic risk profile has
already been observed for other psychiatric disorders.
For example, in a large mega-analysis of genome-wide
association study (GWAS) data from more than 32 000
patients and 46 000 controls by the Psychiatric Genomics
Consortium, shared genetic effects have been identified
between the five major psychiatric disorders, that is,
schizophrenia, bipolar disorder, autism, major depressive
disorder, and attention deficit-hyperactive disorder
(Lee et al., 2013). Another large cross-disorder study of
the Psychiatric Genomics Consortium identified that
single-nucleotide polymorphisms (SNPs) at four chro-
mosomal loci showed genome-wide association across the
five above-mentioned major psychiatric disorders
(Cross-Disorder Group of the Psychiatric Genomics
Consortium, 2013). As most of these SNPs showed
pleiotropic effects on two or more of the five psychiatric
disorders, we investigated their possible contribution
toward SAD in the present study.
Recent studies suggest that a high number of patients
with SAD also show dysfunctional personality traits,
particularly a high comorbidity with avoidant personality
disorder (Friborg et al., 2013) as well as with personality
traits associated with cluster C personality disorders
(Cloninger et al., 1993; Conrad et al., 2007). Of particular
interest in this context is harm avoidance (HA) as defined
by the psychobiological personality model of Cloninger
et al. (1993). According to this model, HA is part of the
temperamental or emotional core of personality and
refers to a specific sensitivity to fear and worry. In this
line, high HA has consistently been found in patients
with SAD (Kampman et al., 2014). Moreover, in suc-
cessfully treated SAD patients, the reduction of social
anxiety is correlated with decreases in HA (Mortberg
et al., 2007). HA is a complex trait with an estimated
heritability of 36% (Verweij et al., 2012). One of the most
extensively studied candidate genes in HA is SLC6A4
(Verweij et al., 2010). Some studies provided evidence for
Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
an association between a polymorphism (5-HTTLPR) in
the promotor region of SLC6A4 and HA (Lesch et al.,
1996), whereas other studies were unsuccessful in repli-
cating this association (Herbst et al., 2000; Munafo et al.,
2009). Notably, a large meta-analysis of association stu-
dies of Cloningers temperament scales that comprised
more than 11 000 individuals did not show any genome-
wide significant SNP associated with HA, suggesting that
it will be challenging to identify such associations at a
genome-wide level (Service et al., 2012).
In the present study, we (i) investigated whether SNPs
associated with psychiatric diseases being comorbid with
SAD also contribute toward the development of SAD and
(ii) followed up variants associated with SAD on the
phenotypic level. For this purpose, we genotyped a total
of 24 SNP markers in a German sample of 321 patients
with SAD and 804 controls. The 321 SAD patients were
newly recruited and have not been published in any
previous study.
Patients and methods
Sample description
Our sample consisted of 321 SAD cases and 804 controls.
SAD patients (44.9% men, mean age at recruitment =
44.6 years) were recruited at the Clinic for Psychosomatic
Medicine and Psychotherapy at the University of Bonn
(Bonn, Germany) through own clinical services (out-
patients and inpatients). Inclusion in the SAD group
required a diagnosis of lifetime SAD (generalized or
nongeneralized form) confirmed by the Structured
Clinical Interview for Diagnostic and Statistical Manual of
Mental Disorders, 4th ed. (DSM-IV) Axis I disorders
(SCID-I; German version: Wittchen et al., 1997) assessed
by trained interviewers. In addition, all participants had
to be 18 years of age or older. Exclusion criteria were
insufficient German language skills or somatic and/or
mental difficulties in completing inserted questionnaires.
Controls were chosen randomly from a sample recruited
among voluntary blood donors, in cooperation with the
Institute of Experimental Haematology and Transfusion
Medicine, University of Bonn (Birnbaum et al., 2009).
Sex distribution was 366/438 = male/female and the mean
age at recruitment was 33.4 years. Controls had not been
screened with respect to SAD.
Both cases and controls were of Central European des-
cent (i.e. four grandparents born in Central Europe). All
participants signed informed consent and the study was
approved by the ethics committee of the University of
Bonn (Bonn, Germany).
Phenotypic assessment
Demographic data (including sex, age, partnership,
education level) were documented from patients. The
severity of social anxiety symptoms was assessed using
the Social Phobia Inventory (SPIN; German version:
Sosic et al., 2008). The SPIN is a brief self-report

questionnaire measuring the behavioral, physiological,
and cognitive symptoms associated with social anxiety.
The SPIN has good psychometric properties and is
highly economic (Sosic et al., 2008). To assess the
severity of depression, the Beck Depression Inventory
(German version: Hautzinger et al., 1994) was used. The
Beck Depression Inventory is a short, widely used, and
well-validated self-report questionnaire (Hautzinger
et al., 1994) reflecting participants personal agreement on
depression symptoms over the past week. HA was
assessed using the Temperament and Character
Inventory (German version: Richter et al., 1999), which
distinguishes between four temperament (novelty seek-
ing, HA, reward dependence, and persistence) and three
character (self-directedness, cooperativeness, and self-
transcendence) dimensions using a dichotomous item
response format. HA is distinguished into four subscales
split onto 35 items: anticipatory worry, fear of uncer-
tainty, shyness/shyness with strangers, fatigability/fatig-
ability, and asthenia. HA has satisfactory psychometric
properties (Richter et al., 2000). Diagnoses of SAD as well
as relevant comorbid Axis I diagnoses were made with
the SCID-I (Wittchen et al., 1997). The SCID is a valid
and reliable semi-structured interview for making DSM-
IV Axis I diagnoses (Lobbestael et al., 2011). Overall, 14
SAD cases with a comorbid diagnosis of schizophrenia or
bipolar disorder were excluded from the present study.
Selection of candidate variants and genotyping
Lymphocyte DNA was isolated from venous blood sam-
ples using standard methods. A total of 24 variants were
selected for the genotyping analysis (Supplementary
Table 1, Supplemental digital content 1, http://links.lww.
com/PG/A184). The selection criteria were as follows:
Table 1 Social anxiety disorder sample description (n = 303)
Mean (SD)
Demographics
Male
Female
Age (years) 44.25 (13.58)
Partnership (yes) (n = 255)
Level of education (n = 267)
No formal education
Secondary school
A levels/college
Axis I comorbidities (lifetime)
Panic disorder (n = 278)
Generalized anxiety disorder (n = 264)
Specific anxiety disorder (n = 261)
Major depression (n = 292)
Alcohol abuse or dependency (n = 287)
Drug abuse or dependency (n = 263)
Medicine abuse or dependency (n = 257)
Severity measures
Severity of social anxiety symptoms (SPIN), sum 40.77 (11.50)
score (n = 284)
Depression severity (BDI), sum score (n = 286) 19.52 (10.80)
Personality
Harm avoidance (TCI), raw score (n = 281) 26.64 (5.58)
BDI, Beck Depression Inventory; TCI, Temperament and Character Inventory.
Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Genetic variation at SLC6A4 and anxiety Forstner et al. 3
(i) genome-wide significant associations in GWAS of
bipolar disorder (n = 11 SNPs, Sullivan et al., 2012; Green
et al., 2013; Muhleisen et al., 2014) or in GWAS across five
psychiatric disorders (n = 4 SNPs, Cross-Disorder Group of
the Psychiatric Genomics Consortium, 2013) or (ii) pre-
viously reported association in studies of SAD (n = 4
SNPs), PD (n = 4 SNPs), and phobias (n = 1 SNP) (Donner
et al., 2008, 2012; Sipila et al., 2010; Strug et al., 2010;
Erhardt et al., 2012; Jensen et al., 2014; Nivard et al., 2014).
All 24 variants were genotyped in the 307 patients
and 804 controls using the iPLEX Gold Sequenom
MassARRAY system (Sequenom, San Diego, California,
USA). The iPLEX primer sequences and assay conditions
are available upon request. For quality control (QC), we
genotyped intraplate and interplate duplicates. In addition,
we added negative controls (H2O) on each 384-well plate
to exclude contamination. Cluster plots of each SNP were
visually checked and manually corrected if necessary.
Genotyping data were subjected to different QC steps
(HardyWeinberg equilibrium P > 0.001, minor allele fre-
quency > 5%, SNP call rate > 95%). After applying these
criteria, all 24 SNPs remained for association testing.
Following the exclusion of four patients and seven controls
with a call rate below 90%, the sample consisted of
303 SAD cases (43.6% men, Table 1) and 797 controls
(45.0% men).
Association analysis
Single-marker association analyses were carried out in the
entire casecontrol sample. In addition, we performed
association testing for (i) SAD cases without PD (n = 188)
against all controls (n = 797). Notably, information on
comorbid PD was not available for 25 SAD patients. In
addition, (ii) a quantitative association analysis of all cases
with available data on symptom severity (n = 284, mea-
sured with SPIN) was carried out and (iii) a quantitative
association analysis of all cases with available data on
HA (n = 281) as measured by the Temperament and
n (%)
Character Inventory was carried out. QC, single marker,
132 (43.6)
and quantitative association analyses were carried out
171 (56.4) using PLINK software (Purcell et al., 2007). Although too
conservative (as the different association tests are not
139 (54.5)
independent), the P values calculated were Bonferroni-
1 (0.4) corrected for multiple testing according to the number
106 (39.7) of investigated SNPs and performed association tests
160 (59.9)
(n = 24 4 = 96).
90 (32.4)
45 (17.0)
85 (32.6)
Results
221 (75.7) After QC, all 24 SNPs were tested for association with
66 (23.0) SAD. None of the tested variants showed an association
21 (8.0)
21 (8.2) with SAD in our casecontrol sample after Bonferroni
correction. Two SNPs reached nominal significance
(rs818702, P = 0.032, Pcorr > 0.999; rs140701, P = 0.048,
Pcorr > 0.999; Table 2). Of these, only one variant
(rs140701) showed an association in the same allelic
direction as reported previously (risk allele: A, Strug et al.,
2010). Interestingly, this variant is located within the
 4 Psychiatric Genetics 2017, Vol 00 No 00

Table 2 Results of the association analysis

SNP Chr Positiond A1 A2 P SAD vs. controlsb OR Nearby gene/s Phenotypec AD
rs818702 9 116 144 973 G A 0.032 1.269 ALAD SAD
rs140701 17 28 538 532 A G 0.048 1.215 SLC6A4 SAD, PD +
rs12576775 11 79 077 193 G A 0.140 0.822 ODZ4 BD
rs17826816 5 7 519 298 G A 0.176 1.159 ADCY2 BD +
rs2306073 12 27 555 837 A G 0.195 0.873 ARNTL2 SAD +
rs7131056 11 113 329 774 A C 0.261 1.112 DRD2 SAD +
rs2239547 3 52 855 229 G A 0.298 0.895 ITIH3 BD, SCZ +
rs10994359 10 62 222 107 C T 0.339 1.177 ANK3 BD, SCZ +
rs7309727 12 129 955 359 T C 0.355 1.106 TMEM132D PD +
rs1064395 19 19 361 735 A G 0.377 0.880 NCAN BD
rs11191454 10 104 660 004 G A 0.395 1.153 AS3MT Cross disordera
rs7240351 18 25 554 456 A G 0.407 0.921 CDH2 SAD +
rs4765913 12 2 419 896 A T 0.440 1.092 CACNA1C BD +
rs9371601 6 152 790 573 T G 0.446 0.926 SYNE1 BD, MDD
rs769407 2 171 693 708 C G 0.450 0.922 GAD1 Phobias
rs2799573 10 18 601 928 G A 0.452 1.084 CACNB2 Cross disorder
rs2535629 3 52 833 219 T C 0.509 0.935 ITIH3 Cross disorder +
rs1133503 6 96 054 588 C T 0.520 0.941 MANEA SAD, PD
rs1344706 2 185 778 428 G T 0.687 1.041 ZNF804A BD, SCZ
rs1024582 12 2 402 246 T C 0.687 0.960 CACNA1C BD, SCZ
rs6550435 3 36 864 489 G T 0.706 1.037 TRANK1 BD +
rs1012176 5 11 320 538 A C 0.738 0.965 CTNND2 SAD, PD, GAD NA
rs12202969 6 98 576 223 A G 0.816 0.978 MIR2113, POU3F2 BD
rs4765905 12 2 349 584 C G 0.848 0.980 CACNA1C BD, SCZ

Results of the association analysis of the 24 investigated single-nucleotide polymorphisms (SNPs) in the entire social anxiety disorder (SAD) casecontrol sample.
+ / , same/opposite allelic direction as reported previously; A1, minor allele (on the basis of the entire sample); A2, other allele; AD, allelic direction; BD, bipolar disorder;
Chr, chromosome; GAD, generalized anxiety disorder; MDD, recurrent major depressive disorder; NA, not available; OR, odds ratio predicted to A1; PD, panic disorder;
SCZ, schizophrenia.
a
Association across the five psychiatric disorders: autism spectrum disorder, attention deficit-hyperactivity disorder, bipolar disorder, major depressive disorder, and
schizophrenia.
b
P value of the association analysis.
c
Association previously reported in the given phenotypes.
d
Chromosomal position according to hg19.

serotonin transporter gene SLC6A4 on chromosome 17.
The other SNP (rs818702) showed an association in the
opposite direction as reported previously (Donner et al.,
2008).
In addition, we carried out an association analysis of cases
without PD as well as quantitative analyses on SAD
severity and HA (Supplementary Table 2, Supplemental
digital content 1, http://links.lww.com/PG/A184). No mar-
ker showed association with any of the investigated
subphenotypes after correction for multiple testing
(Supplementary Table 3, Supplemental digital content 1,
http://links.lww.com/PG/A184).
However, in the analysis of SAD cases without PD, two
SNPs (rs140701, rs12576775) showed a nominal associa-
tion with SAD (P = 0.021, Pcorr > 0.999 and P = 0.028,
Pcorr > 0.999, respectively). Interestingly, the association
for rs140701 at SLC6A4 became even more pronounced
in respective SAD cases compared with all SAD cases,
although the sample size of this phenotypic subcohort
was considerably smaller (188 vs. 303 patients).
The quantitative association analysis of all cases with
available data on symptom severity showed four SNPs
(rs10994359, rs818702, rs1133503, rs2306073) with a
nominally significant association (Supplementary Table 2,
Supplemental digital content 1, http://links.lww.com/
PG/A184). Thereby, rs10994359 showed the strongest
association (P = 0.001, Pcorr = 0.136). The SNP rs10994359
is located near the ankyrin 3 (ANK3) gene on chromosome
10. In addition, this variant was nominally associated with
HA scores (P = 0.001, Pcorr = 0.083).
Discussion
In the present study, we investigated the contribution of
psychiatric disorder-associated SNPs toward the devel-
opment of SAD in a casecontrol sample of SAD patients
that have not been published in any previous study.
The analysis showed a nominally significant association
of rs140701 with SAD. SNP rs140701 is located in intron
9 of the serotonin transporter gene SLC6A4, which
encodes an integral membrane protein that transports the
neurotransmitter serotonin from synaptic spaces into
presynaptic neurons. This protein is the primary target of
selective-serotonin reuptake inhibitors used for the
treatment of depressive and anxiety disorders (Strug
et al., 2010). Serotonergic dysfunction has been impli-
cated in many psychiatric disorders and traits, especially
in affective disorders and anxiety-related phenotypes
(Gelernter, 2014). Interestingly, a recent imaging study
found increased serotonin synthesis and transporter
availability in patients with SAD compared with healthy
controls, suggesting that alterations in the presynaptic
serotonin system might contribute toward the develop-
ment of SAD (Frick et al., 2015).

Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

Since the era of candidate gene studies, SLC6A4 is one of
the few genes that have consistently been reported as
being associated with anxiety-related traits (Lesch et al.,
1996; Schumacher and Deckert, 2010). In most studies,
the association was reported for a polymorphism found in
the promoter region of SLC6A4 (5-HTTLPR, Lesch
et al., 1996). However, also SNPs at SLC6A4 that are not
in linkage disequilibrium with 5-HTTLPR (r2 < 0.4)
have found to be associated with anxiety-related dis-
orders, including rs140701 (Strug et al., 2010; Vinkhuyzen
et al., 2011). Interestingly, in the present study, the
association for rs140701 became even more pronounced
in the analysis of SAD cases without PD, although the
sample size was considerably smaller compared with the
entire sample. This provides evidence that SLC6A4 is
associated with SAD independent of comorbid PD
(Lesch et al., 1996). On the basis of our results, future
studies are required to investigate whether SAD without
anxiety disorder comorbidities may represent a more
homogenous phenotype compared with all SAD patients.
The other nominally associated SNP in the casecontrol
analysis, rs818702, showed an association in the opposite
direction as reported in a previous genetic association
study of SAD patients (Donner et al., 2008). Therefore,
the investigation of this SNP in larger samples of SAD
patients and controls is required before definitive con-
clusions on its possible involvement in the development
of SAD can be drawn.
The quantitative analysis of SAD symptom severity
showed four SNPs (rs10994359, rs818702, rs1133503,
rs2306073) with a nominal association. This includes the
bipolar disorder-associated ANK3 gene at rs10994359,
which has been implicated previously in the regulation of
anxiety-related behavior and stress reactivity (Leussis
et al., 2013). This SNP was also nominal significantly
associated with HA scores. As high HA scores have
consistently been found in patients with SAD (Kampman
et al., 2014), our results may suggest that rs10994359 at
ANK3 contributes toward the development of SAD on
the level of anxiety-related personality subdimensions.
Interestingly, patients with bipolar disorder also score
higher on HA (Engstrom et al., 2004; Loftus et al., 2008;
Jylha et al., 2011). Given the high prevalence of
SAD in offspring of parents with bipolar disorder
(Hirshfeld-Becker et al., 2006; Duffy et al., 2013), further
research on the possible overlapping contribution of
genes implicated in anxiety-related personality traits to
SAD and affective disorders is required.
Limitations
The present study has some limitations. The control
individuals were chosen randomly from a sample recruited
among voluntary blood donors at the University of Bonn
(Bonn, Germany). These controls were not screened for
the presence of SAD. The use of unscreened controls
may have led to a reduction of statistical power to detect
Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Genetic variation at SLC6A4 and anxiety Forstner et al. 5
associations in the present study. However, as the lifetime
prevalence of SAD is about 1213% in the general popu-
lation (Stein and Stein, 2008; Kessler et al., 2012), the
power of the present association study should not have
been considerably reduced by the use of unscreened
controls according to Moskvina et al. (2005). Furthermore,
we have to note that a considerable proportion of our
SAD cases had a comorbid major depressive disorder.
Thus, we cannot estimate to what extent this comorbidity
has influenced the observed associations. To address this
point, the investigation of larger samples of SAD cases
without psychiatric comorbidities is required for the future.
None of the variants tested showed an association with
SAD or any investigated subphenotype after correction
for multiple testing. This may reflect the fact that the size
of the investigated sample is limited to detection of an
association of common variants with small effect as it has
already been observed in other complex psychiatric dis-
orders (Visscher et al., 2012). Therefore, the investigation
of larger samples of SAD patients and controls is required
for the future.
In addition, we focused on single marker associations and
investigated 24 SNPs that were located at 21 indepen-
dent chromosomal loci (chromosomal distance between
the loci >1 Mb). Therefore, we cannot exclude that the
investigated loci might be associated with SAD on a
multimarker haplotype level.
Conclusion
In the present study we systematically investigated the
contribution of SNPs toward SAD by performing geno-
typing of 24 SNPs that have been reported previously in
SAD or other psychiatric disorders. This showed an
association between rs140701 at SLC6A4 and SAD,
especially in SAD patients without PD. Although this
association does not withstand a Bonferroni correction,
our finding adds further evidence for an involvement of
the serotonin transporter gene SLC6A4 in the etiology of
anxiety-related traits. Furthermore, our study implicates
that genetic variation at the genome-wide associated
bipolar disorder locus ANK3 influences anxiety-related
personality traits.
Acknowledgements
The authors are grateful to all of the patients and control
participants who contributed toward this study. In addition,
they would like to thank Christine Wissussek for her
assistance with patient recruitment. They also thank Prof.
Bernd Ptzsch (Institute of Experimental Hematology and
Transfusion Medicine, University of Bonn) for help with
collection of DNA samples from anonymous blood donors.
The study was supported by the German Federal Ministry
of Education and Research (BMBF) through the Integrated
Network IntegraMent (Integrated Understanding of Causes
and Mechanisms in Mental Disorders), under the auspices
 6 Psychiatric Genetics 2017, Vol 00 No 00
of the e:Med Programme (grant 01ZX1314A to M.M.N. and
J.S.). M.M.N. is a member of the DFG-funded Excellence-
Cluster ImmunoSensation. M.M.N. also received support
from the Alfried Krupp von Bohlen und Halbach-Stiftung.
S.R. is supported by a scholarship for doctoral students from
Evangelisches Studienwerk e.V. Villigst, Germany.
Conflicts of interest
There are no conflicts of interest.
References
Bandelow B, Charimo Torrente A, Wedekind D, Broocks A, Hajak G, Ruther E
(2004). Early traumatic life events, parental rearing styles, family history of
mental disorders, and birth risk factors in patients with social anxiety disorder.
Eur Arch Psychiatry Clin Neurosci 254:397405.
Birnbaum S, Ludwig KU, Reutter H, Herms S, Steffens M, Rubini M, et al. (2009).
Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate
on chromosome 8q24. Nat Genet 41:473477.
Cloninger CR, Svrakic DM, Przybeck TR (1993). A psychobiological model of
temperament and character. Arch Gen Psychiatry 50:975990.
Coelho HF, Cooper PJ, Murray L (2007). A family study of co-morbidity between
generalized social phobia and generalized anxiety disorder in a non-
clinic sample. J Affect Disord 100:103113.
Conrad R, Schilling G, Bausch C, Nadstawek J, Wartenberg HC, Wegener I, et al.
(2007). Temperament and character personality profiles and personality dis-
orders in chronic pain patients. Pain 133:197209.
Cross-Disorder Group of the Psychiatric Genomics Consortium (2013).
Identification of risk loci with shared effects on five major psychiatric dis-
orders: a genome-wide analysis. Lancet 381:13711379.
Donner J, Pirkola S, Silander K, Kananen L, Terwilliger JD, Lonnqvist J, et al.
(2008). An association analysis of murine anxiety genes in humans implicates
novel candidate genes for anxiety disorders. Biol Psychiatry 64:672680.
Donner J, Sipila T, Ripatti S, Kananen L, Chen X, Kendler KS, et al. (2012).
Support for involvement of glutamate decarboxylase 1 and neuropeptide Y in
anxiety susceptibility. Am J Med Genet B Neuropsychiatr Genet 159B:
316327.
Duffy A, Horrocks J, Doucette S, Keown-Stoneman C, McCloskey S, Grof P
(2013). Childhood anxiety: an early predictor of mood disorders in offspring of
bipolar parents. J Affect Disord 150:363369.
Eng W, Coles ME, Heimberg RG, Safren SA (2005). Domains of life satisfaction
in social anxiety disorder: relation to symptoms and response to cognitive-
behavioral therapy. J Anxiety Disord 19:143156.
Engstrom C, Brandstrom S, Sigvardsson S, Cloninger R, Nylander PO (2004).
Bipolar disorder: I. Temperament and character. J Affect Disord 82:131134.
Erhardt A, Akula N, Schumacher J, Czamara D, Karbalai N, Muller-Myhsok B, et al.
(2012). Replication and meta-analysis of TMEM132D gene variants in panic
disorder. Transl Psychiatry 2:e156.
Fehm L, Pelissolo A, Furmark T, Wittchen HU (2005). Size and burden of social
phobia in Europe. Eur Neuropsychopharmacol 15:453462.
Friborg O, Martinussen M, Kaiser S, Overgard KT, Rosenvinge JH (2013).
Comorbidity of personality disorders in anxiety disorders: a meta-analysis of
30 years of research. J Affect Disord 145:143155.
Frick A, Ahs F, Engman J, Jonasson M, Alaie I, Bjorkstrand J, et al. (2015).
Serotonin synthesis and reuptake in social anxiety disorder: a positron emis-
sion tomography study. JAMA Psychiatry 72:794802.
Fyer AJ, Mannuzza S, Chapman TF, Liebowitz MR, Klein DF (1993). A direct
interview family study of social phobia. Arch Gen Psychiatry 50:286293.
Gelernter J (2014). SLC6A4 polymorphism, population genetics, and
psychiatric traits. Hum Genet 133:459461.
Green EK, Grozeva D, Forty L, Gordon-Smith K, Russell E, Farmer A, et al. (2013).
Association at SYNE1 in both bipolar disorder and recurrent major depres-
sion. Mol Psychiatry 18:614617.
Hamilton SP (2009). Linkage and association studies of anxiety disorders.
Depress Anxiety 26:976983.
Hautzinger M, Bailer M, Worall H, Keller F (1994). Beck-Depressions-Inventar
(BDI) Testhandbuch. Bern: Hans Huber.
Herbst JH, Zonderman AB, McCrae RR, Costa PT Jr (2000). Do the dimensions
of the temperament and character inventory map a simple genetic archi-
tecture? Evidence from molecular genetics and factor analysis. Am J
Psychiatry 157:12851290.
Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Hettema JM, Prescott CA, Myers JM, Neale MC, Kendler KS (2005). The structure
of genetic and environmental risk factors for anxiety disorders in men
and women. Arch Gen Psychiatry 62:182189.
Hirshfeld-Becker DR, Biederman J, Henin A, Faraone SV, Dowd ST, De
Petrillo LA, et al. (2006). Psychopathology in the young offspring of parents
with bipolar disorder: a controlled pilot study. Psychiatry Res 145:155167.
Jensen KP, Stein MB, Kranzler HR, Yang BZ, Farrer LA, Gelernter J (2014). The
alpha-endomannosidase gene (MANEA) is associated with panic disorder
and social anxiety disorder. Transl Psychiatry 4:e353.
Jylha P, Mantere O, Melartin T, Suominen K, Vuorilehto M, Arvilommi P, et al.
(2011). Differences in temperament and character dimensions in patients with
bipolar I or II or major depressive disorder and general population subjects.
Psychol Med 41:15791591.
Kampman O, Viikki M, Jarventausta K, Leinonen E (2014). Meta-analysis of anxiety
disorders and temperament. Neuropsychobiology 69:175186.
Kendler KS, Karkowski LM, Prescott CA (1999). Fears and phobias: reliability and
heritability. Psychol Med 29:539553.
Kendler KS, Myers J, Prescott CA, Neale MC (2001). The genetic epidemiology of
irrational fears and phobias in men. Arch Gen Psychiatry 58:257265.
Kent JM, Coplan JD, Gorman JM (1998). Clinical utility of the selective serotonin
reuptake inhibitors in the spectrum of anxiety. Biol Psychiatry 44:812824.
Kessler RC, Petukhova M, Sampson NA, Zaslavsky AM, Wittchen HU (2012).
Twelve-month and lifetime prevalence and lifetime morbid risk of anxiety and
mood disorders in the United States. Int J Methods Psychiatr Res 21:
169184.
Lee SH, Ripke S, Neale BM, Faraone SV, Purcell SM, Perlis RH, et al. (2013).
Genetic relationship between five psychiatric disorders estimated from gen-
ome-wide SNPs. Nat Genet 45:984994.
Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, et al. (1996).
Association of anxiety-related traits with a polymorphism in the serotonin
transporter gene regulatory region. Science 274:15271531.
Leussis MP, Berry-Scott EM, Saito M, Jhuang H, de Haan G, Alkan O, et al.
(2013). The ANK3 bipolar disorder gene regulates psychiatric-related beha-
viors that are modulated by lithium and stress. Biol Psychiatry 73:683690.
Lobbestael J, Leurgans M, Arntz A (2011). Inter-rater reliability of the Structured
Clinical Interview for DSM-IV Axis I Disorders (SCID I) and Axis II Disorders
(SCID II). Clin Psychol Psychother 18:7579.
Loftus ST, Garno JL, Jaeger J, Malhotra AK (2008). Temperament and character
dimensions in bipolar I disorder: a comparison to healthy controls. J Psychiatr
Res 42:11311136.
Mortberg E, Bejerot S, Aberg Wistedt A (2007). Temperament and character
dimensions in patients with social phobia: patterns of change following
treatments? Psychiatry Res 152:8190.
Moskvina V, Holmans P, Schmidt KM, Craddock N (2005). Design of case-
controls studies with unscreened controls. Ann Hum Genet 69:566576.
Muhleisen TW, Leber M, Schulze TG, Strohmaier J, Degenhardt F, Treutlein J,
et al. (2014). Genome-wide association study reveals two new risk loci for
bipolar disorder. Nat Commun 5:3339.
Munafo MR, Freimer NB, Ng W, Ophoff R, Veijola J, Miettunen J, et al. (2009).
5-HTTLPR genotype and anxiety-related personality traits: a meta-analysis and
new data. Am J Med Genet B Neuropsychiatr Genet 150B:271281.
Nivard MG, Mbarek H, Hottenga JJ, Smit JH, Jansen R, Penninx BW, et al. (2014).
Further confirmation of the association between anxiety and CTNND2:
replication in humans. Genes Brain Behav 13:195201.
Patel A, Knapp M, Henderson J, Baldwin D (2002). The economic consequences
of social phobia. J Affect Disord 68:221233.
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. (2007).
PLINK: a tool set for whole-genome association and population-based linkage
analyses. Am J Hum Genet 81:559575.
Richter J, Eisemann M, Richter G, Cloninger CR (1999). The Temperament and
Character Inventory (TCI). Frankfurt am Main: Swets Test Services.
Richter J, Eisemann M, Richter G (2000). The German version of the Temperament
and Character Inventory (TCI). Zeitschrift fr Klinische Psychologie und
Psychotherapie 29:117126.
Rush AJ, Zimmerman M, Wisniewski SR, Fava M, Hollon SD, Warden D, et al.
(2005). Comorbid psychiatric disorders in depressed outpatients: demo-
graphic and clinical features. J Affect Disord 87:4355.
Schumacher J, Deckert J (2010). Serotonin transporter polymorphisms and panic
disorder. Genome Med 2:40.
Service SK, Verweij KJ, Lahti J, Congdon E, Ekelund J, Hintsanen M, et al. (2012).
A genome-wide meta-analysis of association studies of Cloningers
Temperament Scales. Transl Psychiatry 2:e116.
Simon NM, Otto MW, Wisniewski SR, Fossey M, Sagduyu K, Frank E, et al.
(2004). Anxiety disorder comorbidity in bipolar disorder patients: data from
the first 500 participants in the Systematic Treatment Enhancement Program
for Bipolar Disorder (STEP-BD). Am J Psychiatry 161:22222229.

Sipila T, Kananen L, Greco D, Donner J, Silander K, Terwilliger JD, et al. (2010). An
association analysis of circadian genes in anxiety disorders. Biol Psychiatry
67:11631170.
Skre I, Onstad S, Torgersen S, Philos DR, Lygren S, Kringlen E (2000). The
heritability of common phobic fear: a twin study of a clinical sample. J Anxiety
Disord 14:549562.
Smoller JW, Gardner-Schuster E, Covino J (2008). The genetic basis of panic and
phobic anxiety disorders. Am J Med Genet C Semin Med Genet 148C:118126.
Sosic Z, Gieler U, Stangier U (2008). Screening for social phobia in medical in- and
outpatients with the German version of the Social Phobia Inventory (SPIN).
J Anxiety Disord 22:849859.
Stein MB, Stein DJ (2008). Social anxiety disorder. Lancet 371:11151125.
Stein MB, Chartier MJ, Hazen AL, Kozak MV, Tancer ME, Lander S, et al. (1998).
A direct-interview family study of generalized social phobia. Am J Psychiatry
155:9097.
Strug LJ, Suresh R, Fyer AJ, Talati A, Adams PB, Li W, et al. (2010). Panic
disorder is associated with the serotonin transporter gene (SLC6A4) but not
the promoter region (5-HTTLPR). Mol Psychiatry 15:166176.
Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Genetic variation at SLC6A4 and anxiety Forstner et al. 7
Sullivan PF, Daly MJ, ODonovan M (2012). Genetic architectures of psychiatric
disorders: the emerging picture and its implications. Nat Rev Genet
13:537551.
Verweij KJ, Zietsch BP, Medland SE, Gordon SD, Benyamin B, Nyholt DR, et al.
(2010). A genome-wide association study of Cloningers temperament scales:
implications for the evolutionary genetics of personality. Biol Psychol
85:306317.
Verweij KJ, Yang J, Lahti J, Veijola J, Hintsanen M, Pulkki-Raback L, et al. (2012).
Maintenance of genetic variation in human personality: testing evolutionary
models by estimating heritability due to common causal variants and investi-
gating the effect of distant inbreeding. Evolution 66:32383251.
Vinkhuyzen AA, Dumenil T, Ryan L, Gordon SD, Henders AK, Madden PA, et al.
(2011). Identification of tag haplotypes for 5HTTLPR for different genome-
wide SNP platforms. Mol Psychiatry 16:10731075.
Visscher PM, Brown MA, McCarthy MI, Yang J (2012). Five years of GWAS
discovery. Am J Hum Genet 90:724.
Wittchen HU, Wunderlich U, Gruschwitz S, Zaudig M (1997). SCID-I Structured
Clinical Interview for DSM-IV Axis I Disorders (SCID-I). Gttingen: Hogrefe.