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Association of major depression with rare functional variants in norepinephrine transporter and serotonin1A receptor genes.

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Neuropsychiatric Genetics
Association of Major Depression With Rare Functional
Variants in Norepinephrine Transporter and
Serotonin1A Receptor Genes
Britta Haenisch,1 Karoline Linsel,1 Michael Br€uss,1 Ralf Gilsbach,1,2 Peter Propping,3
Markus M. N€othen,3 Marcella Rietschel,4,5 Rolf Fimmers,6 Wolfgang Maier,4 Astrid Zobel,4
Susanne H€ofels,4 Vera Guttenthaler,4 Manfred G€othert,1 and Heinz B€onisch1*
Institute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany
Institute of Experimental and Clinical Pharmacology, University of Freiburg, Freiburg, Germany
Institute of Human Genetics, University of Bonn, Bonn, Germany
Department of Psychiatry, University of Bonn, Bonn, Germany
Central Institute of Mental Health, Mannheim, Germany
Institute of Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany
Received 28 April 2008; Accepted 12 November 2008
Dysregulations of central noradrenergic and serotonergic neurotransmission have been suggested to contribute to the pathogenesis of neuropsychiatric disorders such as depression. The
norepinephrine transporter (NET; SLC6A2) and the serotonin
(5-HT)1A receptor (5-HT1A receptor; HTR1A) play an important
role in central nervous monoaminergic homeostasis. As shown
previously, variations in the human NET and 5-HT1A receptor
genes can alter noradrenergic and serotonergic signaling in the
brain: a single nucleotide polymorphism (SNP) in the coding
region of the NET gene resulting in a F528C substitution increased plasma membrane expression of this NET variant, and a
SNP in the human 5-HT1A receptor gene leading to the R219L
receptor variant almost abolished cellular signal transduction
subsequent to receptor activation. The present study aimed at
investigating whether these NET and 5-HT1A receptor variants
are associated with major depression (MD). The sample comprised 426 patients suffering from unipolar MD as well as 643
healthy control subjects for the variants of the 5-HT1A receptor
and the NET. Both SNPs were shown to be associated with MD. In
conclusion, our results favor the hypothesis that monoaminergic
neurotransmission in general and the F528C NET and R219L 5HT1A receptor variants in particular are involved in the pathogenesis of depression. 2008 Wiley-Liss, Inc.
Key words: norepinephrine transporter; 5-HT1A receptor; unipolar depression; association analysis; single nucleotide polymorphism
Major depression (MD) is a common and debilitating psychiatric
illness with a lifetime prevalence of up to 20%. Despite the limited
knowledge about the etiology and pathogenesis of MD, effective
antidepressant drugs are available. The ability of most of the
clinically used antidepressants (AD) to enhance noradrenergic
2008 Wiley-Liss, Inc.
How to Cite this Article:
Haenisch B, Linsel K, Br€
uss M, Gilsbach R,
Propping P, N€
othen MM, Rietschel M,
Fimmers R, Maier W, Zobel A, H€
ofels S,
Guttenthaler V, G€
othert M, B€
onisch H. 2009.
Association of Major Depression With Rare
Functional Variants in Norepinephrine
Transporter and Serotonin1A Receptor Genes.
Am J Med Genet Part B 150B:1013–1016.
and/or serotonergic neurotransmission by inhibiting the respective
neurotransmitter inactivation (mainly by blockade of the corresponding reuptake transporter) was the basis for the classical
monoamine hypothesis of depression [Schildkraut, 1965; Coppen,
1967]. According to this more than 40 years old concept, depression
was suggested to be due to a deficiency of central noradrenergic and/
or serotonergic neurotransmission and the ADs mentioned above
would compensate for this deficiency by enhancing neurotransmission. An alternative therapeutic measure would be a direct
Britta Haenisch and Karoline Linsel contributed equally to this work.
Grant sponsor: Deutsche Forschungsgemeinschaft; Grant sponsor:
*Correspondence to:
Heinz B€
onisch, Institute of Pharmacology and Toxicology, University of
Bonn, Reuterstrasse 2b, 53113 Bonn, Germany.
Published online 22 December 2008 in Wiley InterScience
DOI 10.1002/ajmg.b.30912
stimulation of the neurotransmitter receptor which is the target of
the respective synaptic transmission. Although the monoamine
hypothesis of depression fails to explain the delayed onset of the
antidepressant effect, this hypothesis is still an important basis for
research in the field of MD. Thus, based on this hypothesis
functionally significant single nucleotide polymorphisms (SNPs)
in the genes of relevant neurotransmitter transporters and/or
receptors may contribute to the pathogenesis of MD. The present
article is focused on the norepinephrine (NE) transporter (NET;
SLC6A2) and the serotonin (5-HT)1A receptor (5-HT1A receptor;
HTR1A) which play an important role in CNS noradrenergic and
serotonergic signaling, respectively.
The NET mediates rapid re-uptake of released NE into noradrenergic neurons [B€
onisch and Br€
uss, 2006]. It is one of the main
targets of important ADs (e.g., reboxetine) causing an elevation of
synaptic NE. In the NET gene a naturally occurring, non-synonymous SNP has been identified [Halushka et al., 1999] resulting in
the F528C NET variant. This variant displays an increased plasma
membrane expression associated with increased NE uptake compared to the wild-type NET [Hahn et al., 2005]. Accordingly, this
variant may be expected to lead to decreased levels of NE in the
synaptic cleft. Thus, this functional variant may be involved in the
pathogenesis of depression, while our previous study on other NET
missense variants did not show any association with major psychiatric disorders [St€
ober et al., 1996].
5-HT1A receptors coupled to Gi/Go proteins occur both in the
somadendritic area of the serotonergic neurons in the raphe nuclei
where they act as autoreceptors, and on non-serotonergic neurons
of the forebrain where they are expressed as somadendritic postsynaptic receptors. Inhibitory 5-HT1A receptors on the cell bodies
and/or synaptic boutons of inhibitory GABAergic interneurons
impinge on noradrenergic nerves. Thus, activation of these receptors is indirectly involved in facilitation of NE release in the
hippocampus and cerebral cortex [Fink and G€
othert, 2007]. Several
lines of evidence suggest that not only the NET but also the 5-HT1A
receptor is involved in the pathogenesis of anxiety and depression
[Hamon, 1997; Strobel et al., 2003]. For example, in patients
suffering from depression ligand binding to 5-HT1A receptors
measured by positron emission tomography was reduced in cortical
regions and in the midbrain raphe [Drevets et al., 1999; Sargent
et al., 2000]. As in the case of the NET gene, several SNPs have been
found in the coding region of the 5-HT1A receptor gene [see,
e.g., G€
othert et al., 2001]. Among them was a SNP resulting in the
R219L 5-HT1A receptor variant. Expression of this variant which
was first identified in a Tourette0 s syndrome patient [Lam et al.,
1996] almost abolished cellular signal transduction subsequent to
receptor activation [Br€
uss et al., 2005]. On the basis of the data
summarized here, it was conceivable that the R219L 5-HT1A
receptor variant may also play a role in the pathogenesis of
Taken together, the aim of the present study was to investigate
whether genetic variation in two main targets of central monoamine homeostasis, the NET and the 5-HT1A receptor, may contribute to the development of MD. To obtain a hint for this
hypothesis, we determined the allele frequency of the F528C NET
and R219L 5-HT1A receptor variants in patients with MD and in
healthy controls.
The present study included 426 patients with unipolar affective
disorder (68% women and 32% men, mean age of 49.26 13.40)
comprising 14 individuals with dysthymic disorder, and 643
healthy control subjects (54% women and 46% men, mean age of
43.67 13.92). Within the MD sample, 339 patients suffered from
unipolar recurrent disorder, and 87 from unipolar single episode
disorder. The average age of onset was 36.37 13.36 for women and
37.99 13.36 for men (for further details, see Table I). All subjects
were of German descent. Patients were systematically recruited
from consecutive admissions to the Department of Psychiatry at the
University of Bonn. Lifetime consensus diagnosis according to
DSM-IV criteria was based on multiple sources of information
including a personal structured interview [SCID I; First et al.,
1997], medical records, and the family history method. Best
estimate diagnoses were obtained by at least two experienced
psychiatrists/psychologists. Comorbidities were recorded as
DSM-IV Axis I diagnosis and are presented in Table II. Control
individuals were questioned for a history of psychiatric disorder.
Four control individuals had a history of MD and were excluded
from the analysis. The study was approved by the ethical review
board of the Medical Faculty at the University of Bonn.
Genotyping was carried out by employing 50 nuclease assays with
fluorescent allele-specific TaqMan probes. We used Custom TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA)
for each SNP. The probes used in the bi-allelic system were
synthesized with the fluorescent reporter dye FAM (6-carboxyfluorescein) or VIC attached to the 50 end and a NFQ (non-fluorescent quencher) at the 30 end. Allelic discrimination was assayed
on a Mx3000P real-time cycler (Stratagene, Amsterdam, the
Netherlands). Each assay included negative controls (no-template
controls) and positive controls (allele 1 or 2 controls). Genotype
frequencies for cases and controls were in Hardy–Weinberg equilibrium. To determine the significance of the genotype distribution,
we used the two-tailed Fisher’s exact test.
The results of our study are documented in Table III. Both
variants are relatively rare, with allele frequencies of 0.3% and
0.2% in the general population and 1.2% and 1.1% in patients for
the F528C NET and R219L 5-HT1A receptor variant, respectively.
Both variants show significant association with MD (P ¼ 0.017 and
0.024; OR ¼ 4.6 and 3.8; 95% confidence intervals 1.2–17.1 and
1.2–12.3). Logistic regression analysis was used to check for the
influence of sex and age. Though we found clear sex and age effects
in the combined case and control sample, there was no evidence for
an interaction between age and genetic factors as well as between sex
TABLE I. Age of Onset in the MD Sample (n ¼ 416)
Age of onset
(n ¼ 281)
5 (1.8%)
61 (21.7%)
79 (28.1%)
63 (22.4%)
47 (16.7%)
21 (7.5%)
5 (1.8%)
(n ¼ 135)
0 (0%)
26 (19.3%)
40 (29.6%)
30 (22.2%)
18 (13.3%)
20 (14.8%)
1 (0.7%)
TABLE II. Comorbidities in the MD Sample: 149 of 426 Patients Showed Comorbidities
Anxiety disorder
Anxiety disorder (not otherwise specified)
Panic disorder without agoraphobia
Panic disorder with agoraphobia
Agoraphobia without history of panic disorder
Social phobia
Specific phobia
Obsessive-compulsive disorder
Substance abuse/dependence
Alcohol abuse/dependence
Sedative-, hypnotic- or anxiolytic abuse/dependence
Cannabis abuse/dependence
Eating disorder
Anorexia nervosa
Bulimia nervosa
Somatization disorder
Somatoform disorder (not otherwise specified)
Body dysmorphic disorder
Pain disorder associated with psychological factors
Pain disorder associated with both psychological factors and a general medical condition
and genetic factors with respect to the development of MD. The
impact of the genetic factors in logistic modeling with age and sex as
covariates (no interaction assumed) was comparable to the results
in Table III. These findings are in line with the hypothesis that both
variants are involved in the pathogenesis of depression—a contention which is supported by additional findings reported in the
(1) As already mentioned, the F528C NET variant is expressed at
increased density in the plasma membrane leading to increased NE
uptake [Hahn et al., 2005]. This, in turn, may be assumed to result in
a decreased NE concentration in the synaptic cleft and at the
adrenoceptors in the respective synapses—a finding which conforms to the monoamine hypothesis of depression. (2) This conclusion is also in line with the observation that the serotonin
transporter, which is closely related to the NET, is in a hyperfunctional state during depression in seasonal affective disorder [Willeit
et al., 2008]. (3) A further mechanism potentially underlying the
involvement of the F528C NET variant in the pathogenesis of MD is
the insensitivity of this variant to regulation by protein kinase C
(PKC)-linked pathways [Hahn et al., 2005]. PKC-mediated downregulation (internalization) of the wild-type NET can be induced
through stimulation of Gq-coupled receptor such as muscarinic
receptors co-located with the NET in the neuronal plasma
membrane [Apparsundaram et al., 1998]. The F528C NET variant
may be insensitive to physiological down-regulation, and this may
contribute to sustained overactivity of this NET variant and thus the
severity of depression. Furthermore, the F528C NET variant has
been shown to be eightfold less sensitive to inhibition by the NETselective tricyclic antidepressant desipramine [Hahn et al., 2005].
This, in turn, may contribute to partial resistance to treatment with
NET-selective antidepressants of patients expressing this NET gene
Likewise, the suggestion that the 5-HT1A receptor is involved in
the pathogenesis of MD, derived from the association between the
R219L 5-HT1A receptor variant and MD, is in line with data
reported in the literature. (1) The position of the SNP and the
TABLE III. Allele and Genotype Distribution of Two Non-Synonymous SNPs Within the Coding Regions of the SLC6A2
and HTR1A Genes Among MD Patients and Control Subjects
Genotype distribution
Gene (protein)
Amino acid
Sample size
MD (n ¼ 426)
Controls (n ¼ 643)
MD (n ¼ 426)
Controls (n ¼ 643)
0 (0.000)
0 (0.000)
0 (0.000)
0 (0.000)
Second allele is the rare allele.
Fisher’s exact test (allele based); both test results remain significant even after applying Bonferroni correction.
Allelic odds ratio with confidence intervals.
9 (0.021)
3 (0.005)
10 (0.023)
4 (0.006)
417 (0.979)
640 (0.995)
416 (0.977)
639 (0.994)
OR** [CI]
4.6 [1.2–17.1]
3.8 [1.2–12.3]
replaced amino acid in the R219L variant is critical for the function
of the receptor since the R219L 5-HT1A receptor variant exhibited
an almost complete abolition of cellular signal transduction subsequent to receptor stimulation [Br€
uss et al., 2005]. (2) Downregulation and reduced responsiveness of 5-HT1A receptors have
been found in patients suffering from MD [Sargent et al., 2000;
Lesch et al., 2003]. (3) 5-HT1A receptor agonists such as ipsapirone,
gepirone, and other azapirones have been developed and investigated as potential antidepressant drugs but, in spite of the widespread recognition of their clinical efficiency, their introduction
into the clinic has been hampered by their undesirable pharmacokinetic properties [Blier and Ward, 2003]. In support of an antidepressant effect, long-term treatment of rats with such compounds
resulted in desensitization of the somadendritic 5-HT1A autoreceptors; this, in turn, leads to an increase in 5-HT release [Hamon
1997]. Blockade of the inhibitory somadendritic 5-HT1A autoreceptors resulting in acceleration of effects of antidepressant drugs
by enhancement of 5-HT release is (one of) the mechanism(s)
underlying the use of the b-adrenoceptor/5-HT1A/1B receptor
antagonist pindolol for adjunct treatment of MD [Artigas et al.,
2001; Celada et al., 2004]. (4) Stimulation of 5-HT1A receptors on
GABAergic interneurons innervating noradrenergic neurons increases NE release in the hippocampus and cerebral cortex by
inhibiting the release of the inhibitory neurotransmitter GABA
[Fink and G€
othert, 2007]. The increase in 5-HT and NE release as
potential therapeutic mechanisms conforms to the monoamine
hypothesis of depression.
In spite of the limited sample size, it could be shown that these rare
functional variants have larger effects than is normally observed for
susceptibility variants with higher frequencies.
In conclusion, the present study shows for the first time that
variants in the coding regions of the NET and the 5-HT1A receptor
genes are associated with MD and, thus, may be involved in the
pathogenesis of this disorder. Given our limited sample size,
however, our finding requires independent replication before it
can be regarded as robust.
This work was supported by grants of the Deutsche Forschungsgemeinschaft and BONFOR; Britta Haenisch is a recipient of a
scholarship from the Studienstiftung des Deutschen Volkes.
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