A Large Case–Control Study of Common Functional SLC6A4
and BDNF Variants in Obsessive–Compulsive Disorder
Jens R Wendland*,1, Matthew R Kruse1, Kiara C Cromer1,2and Dennis L Murphy1
1Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA;2Department of
Psychology, Florida State University, Tallahassee, FL, USA
Both serotonin transporter (SLC6A4) and brain-derived neurotrophic factor (BDNF) genes have shown positive associations with
obsessive–compulsive disorder (OCD) and some other psychiatric disorders, but these results have not been consistently replicated. To
explore the hypothesis that this variability might result from the effects of differing combinations of overlooked variants within SLC6A4
together with small OCD and control sample sizes, we studied three common functional polymorphisms (5-HTTLPR, STin2, and the
newly discovered SNP, rs25531) in the largest sample size of OCD patients (N¼347) and controls (N¼749) ever investigated. During
methods development, we found evidence for potential SLC6A4 genotyping problems with earlier methodology, a third possible
contributor to variability in earlier studies. A fourth possible explanation might be SLC6A4?BDNF interactions, which prompted us to
investigate combined genotypes of BDNF V66M with the three SLC6A4 loci. Except for a nominal association with rs25531 alone, which
did not survive correction for multiple comparisons, we found no evidence for any of these other variants being associated alone or
together with OCD, and we therefore also examined clinical OCD subtypes within the sample to evaluate clinical heterogeneity.
Subgroups based on the age of OCD onset, gender, familiality, factor analysis-derived symptom dimensions, or comorbidity with other
psychiatric disorders failed to identify SLC6A4- or BDNF-associated phenotypes, with one exception of overall number of comorbid
anxiety disorders being significantly associated with 5-HTTLPR/rs25531. We conclude that despite their attractiveness as candidate genes
in OCD, our data provide no support for association in this large OCD patient sample and point toward the need to examine other
genes as candidates for risk determinants in OCD.
Neuropsychopharmacology (2007) 32, 2543–2551; doi:10.1038/sj.npp.1301394; published online 21 March 2007
Keywords: 5-HTT; psychiatric genetics; serotonin transporter; SERT
Obsessive–compulsive disorder (OCD) is a debilitating
chronic psychiatric illness with a lifetime prevalence of 2–
3% of the worldwide population (Hasler et al, 2006; Kessler
et al, 2005; Weissman et al, 1994). This disorder is
characterized by recurrent and intrusive thoughts (obses-
sions) and repetitive behaviors (compulsions) meant to
reduce distress associated with obsessions. There is a
substantial body of evidence from twin and family studies
indicating that OCD has a genetic component (Hettema
et al, 2001; Rasmussen and Tsuang, 1984). Complex
segregation analyses, though not entirely conclusive, point
toward a complex pattern of inheritance dominated by a few
genes of major effect (Hemmings and Stein, 2006). This is in
keeping with results from two genome-wide scans of OCD
probands which provided suggestive evidence for suscept-
ibility loci on 9p, 3q, 7p, 1q, 15q, and 6q (Hanna et al, 2002;
Shugart et al, 2006).
In addition to the above-mentioned studies, a number of
candidate genes for OCD have been investigated through
association analyses (Hemmings and Stein, 2006). Particular
emphasis has been placed on functional variants in the
serotonin transporter gene (SERT, 5-HTT, SLC6A4), given
an etiopathological hypothesis based on the effectiveness of
serotonin reuptake inhibitors in the treatment of OCD.
Studies have investigated common non-coding variants
affecting transcriptional efficiency, such as the 43bp
promoter indel referred to as 5-HTTLPR and STin2, a
variable number of tandem repeats polymorphism in intron
2 (Heils et al, 1995; Hranilovic et al, 2004; Hu et al, 2006;
Lesch et al, 1995; Lovejoy et al, 2003; MacKenzie and Quinn,
1999). Rare coding mutations such as the gain-of-function
I425V have also been reported as segregating in families
with OCD probands (Ozaki et al, 2003). Whereas this
SLC6A4 I425V polymorphism was subsequently found in
additional probands with a predominantly OCD-like
phenotype (Delorme et al, 2005; Wendland et al, in press),
data linking OCD with common SLC6A4 variants have been
generally inconclusive. Although a few reports found a
Received 30 August 2006; revised and accepted 14 February 2007
*Correspondence: Dr JR Wendland, Laboratory of Clinical Science,
National Institute of Mental Health, NIH, 10 Center Dr., MSC 1264,
Building 10, Room 3D41, Bethesda, MD 20892, USA, Tel: +1 301 594
0219, Fax: +1 301 402 0188, E-mail: email@example.com
Neuropsychopharmacology (2007) 32, 2543–2551
& 2007 Nature Publishing Group All rights reserved 0893-133X/07 $30.00
positive association for the gain-of-function 5-HTTLPR L
allele and OCD (Bengel et al, 1999; McDougle et al, 1998),
the majority of studies have been negative (Camarena et al,
2001; Cavallini et al, 2002; Chabane et al, 2004; Frisch et al,
2000; Kinnear et al, 2000; Meira-Lima et al, 2004; Walitza
et al, 2004). An association between OCD and the STin2
higher-expressing 12-repeat allele was reported in one study
(Ohara et al, 1999).
Another potential explanation for the inconsistent find-
ings seen in association studies might be a lack of
appreciation of known variants. In particular, this may be
relevant to 5-HTTLPR, as additional variants have been
identified for both the L and S allele (Nakamura et al, 2000).
In their recent report, Hu et al (2006) demonstrated that a
substantial fraction of L alleles, designated LG by the
authors (which refers to the haplotype of L at 5-HTTLPR
and G at dbSNP rs25531, corresponding to the originally
reported allele 16-D, GenBank accession number AB031254)
acts similar to the low-expressing S allele, and that only the
LA(16-A, AB031251) allele is the actual higher expressing
variant. On the basis of genotyping using these functional
data, a significant association between OCD and the higher
expressing LAallele and the LALAgenotype in both a case–
control and a family-based investigation of trios was found
(Hu et al, 2006).
One additional factor that might explain the lack of
consistent findings in association analyses is the substantial
clinical heterogeneity of OCD (Pato et al, 2002). It is thought
that the diverse phenomenological and treatment presenta-
tion of this disorder may reflect, in turn, a heterogeneity of
susceptibility genes. Attempts at better defining more
homogeneous OCD subgroups have been made at the level
of symptom presentation (Hasler et al, 2006), age of OCD
onset (Shugart et al, 2006), family history (Denys et al,
2006), and comorbidity patterns (Leckman et al, 2003).
Despite the theoretical plausibility of these subgroups,
studies assessing candidate genes in light of more unified
OCD sub-phenotypes have also been marked by incon-
clusive findings and a lack of replication.
The aim of this investigation was to analyze the SLC6A4
STin2 and refined 5-HTTLPR polymorphisms in the largest
OCD sample to date and in an ethnically matched control
sample. In addition, we assessed the brain-derived neuro-
trophic factor (BDNF) V66M polymorphism in our sample,
given the recent positive finding of its involvement in OCD
(Hall et al, 2003). We first validated our multiplexed
SLC6A4 genotyping method (Wendland et al, 2006b) in
light of a recent report showing magnesium chloride-
dependent preferential amplification of the S allele (Yonan
et al, 2006), and then performed a number of single locus,
expression grouping, haplotype, and combined genotype
analyses. Moreover, we conducted separate exploratory
analyses of hypothesized OCD sub-phenotypes.
MATERIALS AND METHODS
We genotyped a total of 347 OCD probands and 749 healthy
control individuals. Among these, we selected 295 self-
reported Caucasian adult OCD probands and 657 ethnically
matched Caucasian controls for statistical comparisons.
Probands were recruited through an on-going, IRB-
approved adult outpatient OCD program at the NIMH
Intramural Research Program in Bethesda, MD, with
nationwide and international recruitment. Inclusion criteria
for participation included being at least 18 years old and
having a primary OCD diagnosis based on the Structured
Clinical Interview for DSM-IV (SCID). Exclusion criteria
included active schizophrenia or psychosis, severe mental
retardation that does not permit an evaluation to char-
acterize OCD, or OCD symptoms that occur exclusively in
the context of depression (LaSalle et al, 2004). Peripheral
blood was collected by venipuncture after written informed
consent. DNA was extracted by standard procedures.
Control genomic DNA originated from three independent
sources: (1) human variation panel purchased from Coriell
cell repository (N¼200 self-declared healthy US Cauca-
sians); (2) human random control, panels 1 and 2 (N¼192
apparently healthy, randomly selected UK Caucasian blood
donors) purchased from the European Collection of Cell
Cultures (Sigma-Aldrich, St Louis, MO, USA); and (3)
undergraduate students (N¼357 total, of which N¼265
were self-declared healthy Caucasians and were used in this
study) from a large Southeastern university which attracts a
diverse student body from the entire US as well as B10%
international students; these students participated in a
separate study of genes and personality in return for partial
course credit. Since none of these three control groups was
clinically evaluated, we cannot rule out the possibility of
individuals present in the control sample having OCD or OC
symptoms, although this fraction is unlikely to be higher
than the general population prevalence of 2–3% (Kessler
et al, 2005; Weissman et al, 1994). Allelic and genotypic
frequencies did not significantly differ between these three
control groups (data not shown).
The current report assessed seven distinct OCD sub-
phenotypes within the clinical sample. The magnitude of
obsessions and compulsions was measured with the Yale-
Brown Obsessive Compulsive Scale (Y-BOCS), a frequently
used assessment tool for the severity of obsessive–
compulsive symptoms (Goodman et al, 1989). The average
symptom severity for the sample as a whole, as measured by
the Y-BOCS, was 21.63 (SD¼9.34). We next classified the
sample into either childhood or adult OCD onset groups.
Consistent with the literature, those individuals with an
onset younger than 18 years of age were classified as
childhood onset (Samuels et al, 2006). The third sub-
phenotype we assessed was familial history of OCD. The
sample was dichotomized into either a familial group, which
included those with a first-degree relative with OCD, or a
non-familial group. Consistent with the literature (Cromer
et al, in press; Hasler et al, 2005), we furthermore derived
factor scores for the symptom dimensions of OCD using the
Y-BOCS symptom checklist. Specifically, principal compo-
nent analysis was applied to the 13 a priori Y-BOCS
categories and initial factor solutions were then rotated
using the Varimax procedure (Hasler et al, 2005; Leckman
et al, 1997). The factor analysis generated four factor
scores for each subject representing the correlation of the
symptom profile of the subject with each factor. The four
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Supplementary Information accompanies the paper on the Neuropsychopharmacology website (http://www.nature.com/npp)
Common SLC6A4 and BDNF variants in OCD
JR Wendland et al