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Detailed analysis of the serotonin transporter gene (SLC6A4) shows no association with bipolar disorder in the Northern Swedish population.

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BRIEF RESEARCH COMMUNICATION
Neuropsychiatric Genetics
Detailed Analysis of the Serotonin Transporter Gene
(SLC6A4) Shows no Association With Bipolar Disorder
in the Northern Swedish Population
Maaike Alaerts,1,2 Shana Ceulemans,1,2 Diego Forero,1,2 Lotte N. Moens,1,2 Sonia De Zutter,1,2
Lien Heyrman,1,2 An-Sofie Lenaerts,1,2 Karl-Fredrik Norrback,3,4 Dirk Goossens,1,2 Peter De Rijk,1,2
Lars-G€oran Nilsson,5 Rolf Adolfsson,3,4 and Jurgen Del-Favero1,2*
1
Applied Molecular Genomics Group, Department of Molecular Genetics, VIB, Antwerp, Belgium
2
University of Antwerp (UA), Antwerp, Belgium
Department of Clinical Sciences, Psychiatry, Umea University, Umea, Sweden
3
4
Department of Psychiatry, Hospital of Sunderby, Lulea, Sweden
5
Department of Psychology, Stockholm University, Stockholm, Sweden
Received 25 April 2008; Accepted 5 August 2008
Through active reuptake of serotonin into presynaptic neurons,
the serotonin transporter (5-HTT) plays an important role in
regulating serotonin concentrations in the brain, and it is the site
of binding for tricyclic antidepressants and selective serotonin
reuptake inhibitors (SSRIs). Therefore it has been hypothesized
that this transporter is involved in the etiology of bipolar (BP)
disorder. Inconsistent association study results for the SLC6A4
gene encoding 5-HTT reported in literature emphasize the need
for more systematic and detailed analyses of this candidate gene.
We performed an extensive analysis of SLC6A4 on DNA of 254
BPI patients and 364 control individuals from a Northern
Swedish isolated population. This analysis consisted of a
HapMap LD-based association study including three widely
investigated polymorphisms (5-HTTVNTR, 5-HTTLPR, and
rs3813034), a copy-number variation (CNV) analysis and a
mutation analysis of the complete coding sequence and the
30 -UTR of SLC6A4. No single marker showed statistically significant association with BPI, nor did any of the haplotypes. In the
mutation analysis 13 novel variants were detected, including 2
amino acid substitutions M389V and I587L, but these are probably not implicated in risk for BP. No deletions or duplications
were detected in the CNV analysis. We conclude that variation in
the SLC6A4 gene or its regulatory regions does not contribute to
the susceptibility for BP disorder in the Northern Swedish
population. Ó 2008 Wiley-Liss, Inc.
Key words: serotonin transporter; SLC6A4; bipolar disorder;
linkage disequilibrium; mutation analysis; copy-number
variation
INTRODUCTION
The neurotransmitter serotonin (5-HT) has been implicated in a
variety of behavioral and psychological processes relevant to the
Ó 2008 Wiley-Liss, Inc.
How to Cite this Article:
Alaerts M, Ceulemans S, Forero D, Moens LN,
De Zutter S, Heyrman L, Lenaerts A-S,
Norrback K-F, Goossens D, De Rijk P, Nilsson
L-G, Adolfsson R, Del-Favero J. 2009.
Detailed Analysis of the Serotonin
Transporter Gene (SLC6A4) Shows no
Association With Bipolar Disorder in the
Northern Swedish Population.
Am J Med Genet Part B 150B:585–592.
symptomatology of bipolar (BP) disorder. Therefore it has been
hypothesized that a dysfunction in brain serotonergic systems
contributes to its etiology. The serotonin transporter (5-HTT)
regulates the synaptic serotonin concentration through active
Additional supporting information may be found in the online version of
this article.
Grant sponsor: Swedish Medical Research Council; Grant number: K200121X-10412-09A; Grant sponsor: County Council of V€asterbotten and
Norrbotten, Sweden; Grant sponsor: Fund for Scientific Research
Flanders (FWO-F); Grant sponsor: Industrial Research Fund (IWT);
Grant sponsor: Special Research Fund of the University of Antwerp,
Belgium.
*Correspondence to:
Prof. Dr. Ir. Jurgen Del-Favero, Ph.D., Applied Molecular Genomics
Group, Department of Molecular Genetics, VIB, University of Antwerp
(UA), Campus Drie Eiken, Universiteitsplein 1, B-2610 Antwerp, Belgium.
E-mail: jurgen.delfavero@ua.ac.be
Published online 15 September 2008 in Wiley InterScience
(www.interscience.wiley.com)
DOI 10.1002/ajmg.b.30853
585
586
AMERICAN JOURNAL OF MEDICAL GENETICS PART B
reuptake of the neurotransmitter. In addition, it is the site of
binding for tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs), making this transporter a strong candidate
for involvement in the pathogenesis of BP.
The 5-HTT protein is encoded by SLC6A4, located on chromosome 17q11.2. The gene is organized in 15 exons spanning 37.8 kb
(Fig. 1). Two functional SLC6A4 polymorphisms, 5-HTTLPR and
5-HTTVNTR, have already been extensively investigated in affective disorders. The long (L) variant of 5-HTTLPR is more efficiently
transcribed than the short (S) variant and is responsible for a higher
rate of 5-HT uptake in vitro [Heils et al., 1996; Lesch et al., 1996;
Greenberg et al., 1999]. 5-HTTVNTR acts as a transcriptional
regulatory element of 5-HTT, with the 12 repeat variant being the
strongest enhancer [Lesch et al., 1994; Fiskerstrand et al., 1999;
MacKenzie and Quinn, 1999]. Both polymorphisms have been used
in BP association studies with positive [Collier et al., 1996; Furlong
et al., 1998; Mynett-Johnson et al., 2000; Rotondo et al., 2002], as
well as negative results [Rees et al., 1997; Gutierrez et al., 1998;
Hoehe et al., 1998; Vincent et al., 1999; Mundo et al., 2000; Lotrich
and Pollock, 2004; Mendlewicz et al., 2004; Ikeda et al., 2005]. Three
recent meta-analyses revealed significant association of the
5-HTTLPR (OR ¼ 1.13) [Lasky-Su et al., 2005] or both 5-HTTLPR
(OR ¼ 1.14; OR ¼ 1.12) and 5-HTTVNTR (OR ¼ 1.18; OR ¼ 1.12)
[Anguelova et al., 2003; Cho et al., 2005] with BP disorder.
However, odds ratios are very low, indicating only a small effect
of these variants in BP.
Another polymorphism with potential functional significance is
a SNP, rs3813034, within a polyadenylation signal in the 30 -UTR of
SLC6A4, but no association has yet been found with BP [Battersby
et al., 1999; Mynett-Johnson et al., 2000; Ikeda et al., 2005]. We
genotyped the three described functional polymorphisms and
analyzed single and multiple marker association in a sample consisting of 254 BPI patients and 364 control individuals from an
isolated Northern Swedish population.
To cover the variation content in the whole gene, we conducted a
detailed LD-analysis of SLC6A4 and surrounding regions based on
the information provided by the International HapMap-project
(www.hapmap.org). A set of SNPs fully characterizing the LDstructure of the gene region was selected and consists of 20 haplotype tagging SNPs (htSNPs) within 3 LD-blocks and 6 tagging SNPs
(tSNPs) in the recombination hotspot (Fig. 1). With this strategy
common variants that confer risk to disease phenotype should
be captured, but we also considered the hypothesis that multiple
rare risk alleles exist that contribute to the disease. Therefore we
performed a mutation analysis on the complete coding sequence of
SLC6A4 and of its complete 30 -UTR to investigate gain or loss in
miRNA binding sites.
Finally, we searched for SLC6A4 copy-number variations
(CNVs) because this type of structural genetic variation may also
contribute to disease susceptibility.
To our knowledge this is the first complete HapMap LD-based
association study of the serotonin transporter. In addition it is the
first time that miRNA binding sites and CNVs in SLC6A4 are
investigated.
SUBJECTS AND METHODS
Association Sample
FIG. 1. LD-structure of the SLC6A4 genomic region. At the left
haplotype blocks in the region around SLC6A4 are shown, derived
from HapMap Data Release #20 (PhaseII) of January 2006 and
calculated with Haploview. All the genotyped polymorphisms are
shown in correct genomic order in the middle of the figure. Black
boxes represent the LD-block boundaries. Location of SLC6A4
relative to the blocks (arrow) and its organization in exons and
introns (figure at the right) is given. Filled boxes represent the
coding region, open boxes represent UTR-regions. For
polymorphisms located in SLC6A4, the position in the gene is
indicated. In light gray lines at the left of the exons, the 12 target
amplicons of the MAQ analysis are drawn.
The association sample is comprised of 254 unrelated BPI patients
and 364 control individuals recruited from the V€asterbotten region
in Northern Sweden. Diagnoses were made according to the
DSM-IV criteria [American Psychiatry Association, 1994]. All
participants signed an informed consent and the study was approved by the Medical Ethical Committees of the universities of
Umea and Antwerp. The association sample was controlled for
population stratification by analyzing 37 STR-markers with
STRUCTURE (http://pritch.bsd.uchicago.edu/structure.html) and
no population substructure was observed.
Genotyping
Genomic DNA was extracted from peripheral blood using standard
methods. The polymerase chain reaction (PCR) was used to amplify
ALAERTS ET AL.
all genomic regions of interest. Primer sequences are given in
Supplementary Table SI and detailed information on protocols is
available on request.
5-HTTLPR and 5-HTTVNTR fluorescent-labeled PCR-products were sized on an ABI 3730xl Sequencer (Applied Biosystems,
Foster City, CA). Genotypes were assigned and scored using
LGV, an in-house developed software program (http://www.
vibgeneticservicefacility.be—section technology). Genotyping of
rs12601963 was performed on a PSQ HS96 pyrosequencer
(http://www.biotage.com). Genotyping of the other SNPs was
carried out using the MassARRAY system, following the protocol
provided by Sequenom (www.sequenom.com).
Data Analysis
SNPs were selected from the HapMap Data Release #20 (PhaseII) of
January 2006. LD-measures were calculated using Haploview
(http://www.broad.mit.edu/mpg/haploview) with the CEU population as reference. SNPs with minor allele frequency (MAF) below
5% were excluded and blocks were defined based on confidence
intervals as proposed by Gabriel et al. [2002]. Haploview LDanalysis of the SLC6A4 region resulted in 3 haplotype blocks ranging
from 83.4 kb 50 -upstream to 473.8 kb 30 -downstream of the gene
(chromosome 17: 25075265-25670183; Fig. 1). All haplotypes with
an estimated overall frequency 1.5% were considered in the
analyses and htSNPs were chosen to represent these haplotypes.
In the recombination hotspot tSNPs were selected using Tagger
(http://www.broad.mit.edu/mpg/tagger). Haploview was also used
to calculate LD-blocks specific for the Northern Swedish
population.
GENEPOP v3.3 (http://wbiomed.curtin.edu.au/genepop) was
used to calculate Hardy–Weinberg equilibrium and investigate
allelic and genotypic single marker association. Haplotype
frequencies and associations were calculated using Haplo Stats
v1.2.1 (http://mayoresearch.mayo.edu/mayo/research/biostat/
schaid.cfm). To avoid false positive findings due to multiple testing,
empirical simulated P-values were calculated using 5,000 random
permutations of patient and control labels.
Mutation Analysis
The 13 coding exons of SLC6A4 (exon 3–15), corresponding
exon–intron boundaries and the 30 -UTR were examined for mutations by direct PCR-sequencing in 180 BPI patients, allowing
us to detect variants with a frequency of 0.0028. SNPBox
(www.snpbox.org) was used to design PCR-primers. Sequencing
reactions were run on an ABI 3730xl Sequencer and resulting trace
files were analyzed with novoSNP [Weckx et al., 2005]. All the
detected SNPs were subsequently typed with the Sequenom MassARRAY system in the complete association sample.
Multiplex Amplicon Quantification
To explore SLC6A4 for CNVs, an in-house developed technique for
multiplex amplicon quantification (MAQ) was used [Suls et al.,
2006]. Twelve target amplicons comprising exons of SLC6A4 and
11 reference amplicons were used (Supplementary Table SII; Fig. 1).
587
The comparison of normalized peak areas between patient and
control individuals results in a dosage quotient (DQ) of the target
amplicon, calculated using the in-house developed software package MAQs (http://www.vibgeneticservicefacility.be/maq.htm). A
DQ between 1.3 and 1.7 was considered indicative of a heterozygous
duplication, a DQ between 0.3 and 0.7 was considered indicative of
a heterozygous deletion.
RESULTS
Selection of the SLC6A4 htSNPs
The HapMap Generic Genome Browser was used to view SLC6A4
and surrounding genomic region and Haploview analysis resulted
in three LD-blocks (Fig. 1). We selected a panel of 20 htSNPs,
representing all haplotypes with a frequency above 1.5% in the
blocks, and a set of 6 tSNPs in the recombination hotspot between
blocks 2 and 3 (Table I), in order to represent as much variation as
possible.
Single Marker and Haplotype Association Analysis
We performed single marker allelic and genotypic association
analysis for all the markers in a Northern Swedish association
sample consisting of 254 BPI patients and 364 control individuals.
One of the tSNPs, rs16965623, was not polymorphic in our
population and was removed from further analysis. All polymorphisms were in Hardy–Weinberg equilibrium in the control population (P 0.01). No single marker association with BP disorder
was observed (Table I).
For the 3 putative functional polymorphisms, 5-HTTVNTR,
5-HTTLPR and rs3813034, we performed 2- and 3-marker haplotype association analysis, but no statistically significant association
was found (Table II; Supplementary Table SIII).
Subsequently we performed a haplotype association analysis
based on the HapMap defined LD-blocks (Fig. 1). No significant
difference in haplotype distribution between BPI patients and
control individuals was observed (Table I; Supplementary Table
SIV). LD-block-structure specific for the Northern Swedish population was very similar to that for the CEU population (Table I).
Haplotype association analysis based on these new blocks also did
not yield significant results (Table I).
In a subgroup of 109 BPI patients with an early age at onset
(AAO 21 years) following the cut-off of Lin et al. [2006], no single
marker or haplotype showed statistically significant association (all
P > 0.05; data not shown).
Mutation Analysis of SLC6A4
In total 1893 bp of coding sequence (exon 3–15), 123 bp 50 -UTR and
4438 bp of flanking intronic sequence of the SLC6A4 gene were
sequenced in 180 BPI patients. 645 bp 30 -UTR, encompassing a
second putative polyadenylation signal where SNP rs3813034 is
located, was sequenced in all the patients. Mutation analysis led to
the discovery of three novel exonic SNPs, eight novel intronic SNPs
and two novel SNPs and one small deletion in the 30 -UTR. We also
detected eight known SNPs. Gain or loss of miRNA target sites or
splice-sites was checked. All these variants were subsequently typed
588
AMERICAN JOURNAL OF MEDICAL GENETICS PART B
TABLE I. Genomic Localization of the Genotyped Polymorphisms and Organization into LD-Blocks
Polymorphism
rs2628179
rs3794730
rs12601963
rs4567782
rs4436830
rs11650871
rs3813034
rs3794808
rs140701
rs4583306
rs140700
rs2020942
5-HTTVNTR
rs2020936
rs12150214
rs2066713
rs4251417
rs16965623
5-HTTLPR
rs8073965
rs2020933
rs4392119
rs2020930
rs1050565
rs2129785
rs3816828
rs7215330
rs11080123
Bp position
on chr 17a
25095922
25174485
25270484
25292926
25407835
25481175
25548930
25555919
25562658
25562841
25567515
25571040
25572734
25574940
25575014
25575791
25575984
25577112
25582977
25583308
25585881
25589489
25590167
25600202
25614656
25622843
25627811
25641782
SLC6A4
location
30 -UTR
intron 13
Intron 9
Intron 9
Intron 6
Intron 3
Intron 3
Intron 1
Intron 1
Intron 1
Intron 1
Intron 1
Intron 1
Intron 1
Intron 1
Alleles
C/G
A/G
A/G
A/C
A/G
A/G
A/C
A/G
A/G
A/G
A/G
A/G
9/10/12b
C/T
C/G
C/T
A/G
A/G
S/Lc
G/T
A/T
C/T
A/G
A/G
A/G
G/T
C/T
C/T
Allelic association
P-value
0.67
0.80
0.63
0.51
0.63
0.65
0.33
0.23
0.18
0.09
0.83
0.31
0.70
0.16
0.14
0.39
0.58
—
0.41
0.10
0.70
1.00
0.86
0.71
0.58
0.26
0.52
0.51
Genotypic association
P-value
0.69
0.80
0.65
0.51
0.62
0.64
0.31
0.23
0.18
0.10
0.91
0.34
0.72
0.19
0.18
0.40
0.58
—
0.40
0.11
0.71
1.00
0.87
0.72
0.53
0.28
0.55
0.52
P-value for CEU
LD-blocksd
P-value for N-Sw
LD-blockse
} }}
0.51
0.53
}
0.14
0.25
}
0.57
}
0.57
Results of single marker allelic and genotypic and haplotype association analysis in the Northern Swedish BPI patient-control sample.
a
NT010799, build 36.2.
b
9, 10, and 12 repeat allele of the 5-HTTVNTR.
c
Short (S) and long (L) variant of the 5-HTTLPR.
d
Simulated haplotype association P-value for LD-blocks in the HapMap CEU population.
e
Simulated haplotype association P-value for LD-blocks in the Northern Swedish population.
in the complete association sample (Table III). Four of the 22
polymorphisms were only found in 1 or 2 BPI patients and not in the
Northern Swedish control individuals or an ethnically diverse
reference sample of 450 individuals [Glatt et al., 2001]. Two of
these are exonic SNPs that cause an amino acid substitution,
M389V and I587L, the other two are an intronic SNP and a 3bp
deletion in the 30 -UTR. For all the other variants frequency of the
TABLE II. Haplotype Association Analysis of Combinations of
the Three Functional Polymorphisms
minor allele in patients and control individuals was comparable
(Table III).
MAQ Analysis
We explored the SLC6A4 gene for CNVs in the 254 BPI patients and
364 control individuals using the MAQ technique. The target
amplicons covered a total of 2676 bp including 12 exons of SLC6A4
(Fig. 1). No complete gene or exonic deletion or duplication was
observed.
DISCUSSION
Marker combination
rs3813034–5-HTTVNTR
rs3813034–5-HTTLPR
5-HTTVNTR–5-HTTLPR
rs3813034–5-HTTVNTR–5HTTLPR
Global simulated P-value
0.66
0.22
0.22
0.48
The serotonin transporter is a long standing important candidate
gene for BP disorder and a lot of research effort has been put in it.
However, consistent associations did not emerge, following the
unfortunate story of many candidate genes for complex disorders.
These inconsistencies could be due to genetic heterogeneity and the
problem of power to detect risk variants with small effects but even
ALAERTS ET AL.
589
TABLE III. Results of the Mutation Analysis of SLC6A4
Location in
SLC6A4
Intron 2 pos 587
Chr 17 pos
25573376
Variant
G>C
Intron 2 pos 627
Exon 3 pos 196
25573336
25573030
T>C
G>A
Exon3 pos 290
25572936
G>C
rs6355
Intron 5 pos 728
Intron 6 pos 795
Exon 7 pos 87
25568534
25567515
25567274
C>T
G>A
T>C
rs140700
Intron 7 pos 253
Intron 8 pos 83
Intron 8 pos 151
Exon 9 pos 89
25566973
25566672
25566604
25563923
C>T
C>T
C>G
A>G
Intron 9 pos 1116
Intron 9 pos 1226
Intron 11 pos 148
Intron 13 pos 4360
25562768
25562658
25561511
25554516
C>T
G>A
A>G
G>A
Exon 14 pos 109
Exon 14 pos 165
30 -UTR pos 415
25554375
25554319
25549185
A>C
A>C
G>C
rs6352
30 -UTR pos 463
25549137
T>G
rs1042173
30 -UTR pos 564
30 -UTR pos 593
30 -UTR pos 670
25549036
25549007
25548930
T>C
delGTT
T>G
DbSNP
Prot pos
and amino
acid
G25R
G56A
G308G
Comment
Creates binding site
for miR-34a/449
Intracellular N-terminal
amino acidb
Intracellular N-terminal
amino acidb
Amino acid between
TM5 and EL3b
rs28914829
M389V
Amino acid between
TM7 and EL4ab
rs140701
I587L
K605N
rs3813034
Creates binding site
for miR-302b*
Amino acid in TM12b
Amino acid in TM12b
In binding site for
hsa-let7i
In binding site for
miR-376a*
Frequency
(%) in
Glatt’s
samplea
(no. chroms)
Frequency
(%) in 254
patients
(no. chroms)
0.2 (1)
Frequency
(%) in 364
controls
(no. chroms)
0.5 (4)
0.4 (2)
0.2 (1)
0.0 (0)c
0.1 (1)
1.4 (7)
1.5 (11)
0.4 (4)
0.8 (4)
8.5 (43)
0.2 (1)
0.8 (6)
8.1 (59)
0.0 (0)
0.1 (1)
0.2 (1)
5.9 (30)
0.2 (1)
0.2 (1)
0.1 (1)
3.7 (27)
0.1 (1)
0.0 (0)c
2.6 (13)
42.5 (216)
0.6 (3)
1.6 (8)
2.1 (15)
46.6 (339)
0.4 (3)
1.8 (13)
0.2 (1)
1.8 (9)
0.4 (2)
0.0 (0)c
2.3 (17)
0.5 (4)
48.6 (247)
43.1 (314)
1.2 (6)
0.2 (1)
48.2 (245)
0.7 (5)
0.0 (0)c
51.1 (372)
0.1 (1)
Gain or loss of miRNA target sites or splice-sites was checked. Location, nature, frequency and consequence of the variants in patients and control individuals are given.
TM ¼ transmembrane segment, EL ¼ extracellular loop.
a
Reference sample of 450 ethnically diverse individuals (Glatt et al., 2001).
b
Location of the amino acid in the secondary structure of 5-HTT as determined by Yamashita et al. [2005].
c
The 4 variants that are absent in control chromosomes are indicated in bold.
so to low genetic variation coverage in the majority of studies, with
low LD between the ‘‘real’’ risk variant(s) and the studied polymorphisms. This emphasizes the need for well-structured, systematic and more extensive studies of candidate genes, especially now
information on large numbers of polymorphisms is publicly available. In this study we performed a detailed LD-based association
analysis with tagging SNPs covering a maximum amount of genetic
variation, a mutation analysis and a CNV analysis of the serotonin
transporter gene to investigate its implication in BPI disorder in a
Northern Swedish population.
We tackled the heterogeneity problem in two ways, aiming to
increase the power to detect susceptibility variants. First we used
individuals from a geographically isolated population with a more
homogeneous genetic and environmental background and second
we included only patients diagnosed with the narrow and welldefined phenotype BPI disorder, increasing etiological homogeneity [Faraone et al., 2006]. This sample of 254 BPI patients and 364
control individuals has 90% power to detect a susceptibility factor
with frequency 0.15 and a relative risk of 1.7.
5-HTTLPR and 5-HTTVNTR influence the expression of
SLC6A4 and rs3813034 is located within a potential polyadenylation signal [Battersby et al., 1999]. We genotyped these three
functional polymorphisms, but statistical analysis showed no association with BP. Triallelic genotyping of 5-HTTLPR has been
proposed [Hu et al., 2006], but functionality is doubted [Sakai et al.,
2002; Martin et al., 2007; Philibert et al., 2007] and we covered all
common variation with our tSNPs, so we used the S/L genotyping.
Since it is possible that only specific combinations of alleles of the
functional polymorphisms cause a functional effect that influences
the risk for BP disorder, we also performed 2 and 3 marker
haplotype association analysis. None of the combinations showed
statistical significant association (Table II). To investigate
Hranilovic’s finding of a combined effect of 5-HTTLPR and
5-HTTVNTR on the expression of SLC6A4 [Hranilovic et al.,
590
2004], we divided our samples into the described genotype groups,
but no association of the allelic combination of the polymorphisms
with BP was observed (P ¼ 0.329; Supplementary Table SV).
To date only three groups examined association of SLC6A4 with
BP disorder based on LD, selecting polymorphisms randomly and
calculating LD afterwards to define haplotype blocks [Sun et al.,
2004; Ikeda et al., 2005; Mansour et al., 2005]. None of these studies
found a haplotype conferring risk for bipolar disorder. We used the
LD-information provided by the HapMap project to select 26
(haplotype) tagging SNPs that represent a maximum of other
known and unknown variations. None of the single markers was
found to be significantly associated and haplotype analysis based on
block-boundaries specific for the CEU or the Northern Swedish
population also did not yield significant results (Table I; Supplementary Table SIV). In a subgroup of patients with an early age at
onset 21 years [Lin et al., 2006], none of the single polymorphisms
or haplotypes showed significant association.
From our LD-based association approach we can conclude that
common variants in SLC6A4 with a relative risk 1.7 are unlikely to
contribute to the susceptibility for BPI disorder in the Northern
Swedish population, but we cannot exclude that common variants
with a smaller effect or multiple rare variants on different haplotypic backgrounds confer risk to the disease. Therefore we
performed a mutation analysis on the coding sequence of SLC6A4
as well as the 30 -UTR of the gene to search for variants in miRNA
target sites, since these could have an important influence on the
final amount of gene product [Bartel, 2004]. Mutation analysis of
the coding region of SLC6A4 has been reported before in four
samples of bipolar patients, without identification of risk variants
[Lesch et al., 1995; Di Bella et al., 1996; Ikeda et al., 2005; Mansour
et al., 2005]. We discovered 13 novel SNPs and one small deletion
and detected 8 known SNPs (Table III). Four of the 22 polymorphisms were only found in patients: a SNP in intron 2 and a
3 bp deletion in the 30 -UTR with no obvious functional consequence, and 2 nonsynonymous SNPs, M389V and I587L. These
amino acids are located outside the substrate or ion binding/
transport part of the protein, which consists of transmembrane
segments 1, 3, 6, and 8 [Yamashita et al., 2005]. In addition they are
not particularly conserved in the family of Naþ/Cl dependent
neurotransmitter transporters, a Valine is found at position 389 in
the Glycine transporter and a Leucine at position 587 in the GABA
transporter. Still the amino acid changes could have an effect on
protein structure and function of the serotonin transporter and the
other two polymorphisms on transcriptional regulation, but this
remains to be determined experimentally. For all the other variants
frequencies of the minor allele in patients and control individuals
are comparable, so they can be excluded as disease risk variants for
BPI disorder.
Finally, we searched SLC6A4 for CNVs, since evidence is accumulating that these variants constitute a major type of genetic
variation and they are likely to make an important contribution to
disease susceptibility [Feuk et al., 2006; Nadeau and Lee, 2006]. No
exonic nor genomic deletions or duplications were detected, so in
our studied population CNVs in the serotonin transporter gene do
not play a role in the development of BP disorder.
Since the serotonin transporter is the site of binding for tricyclic
antidepressants and SSRIs our finding is in fact consistent with
AMERICAN JOURNAL OF MEDICAL GENETICS PART B
recent pharmacological data doubting the efficacy of antidepressants [Kirsch et al., 2008] and with current treatment guidelines that
advise minimal use of antidepressants in BP disorder [Kahn et al.,
2000; Ghaemi et al., 2003].
Taken together our findings from the association study, mutation analysis and CNV analysis, we can conclude that the serotonin
transporter is unlikely to be a risk factor for BPI disorder in the
Northern Swedish population. Since in different populations,
different genes could be contributing to the vulnerability for BP
disorder, our conclusion might not be extendable to other populations and we recommend more HapMap LD-based haplotype
association studies of the serotonin transporter to be performed in
different populations.
ACKNOWLEDGMENTS
We acknowledge the contribution of the personnel of the VIB
Genetic Service Facility (http://www.vibgeneticservicefacility.be/)
for the genetic analyses. The research was funded by the Swedish
Medical Research Council K2001-21X-10412-09A and the County
Council of V€asterbotten and Norrbotten, Sweden, as well as by
grants from the Fund for Scientific Research Flanders (FWO-F), the
Industrial Research Fund (IWT) and the Special Research Fund of
the University of Antwerp, Belgium. Eva Lundberg and Annelie
Nordin are thankfully acknowledged for their help and expertise in
ascertaining the BP1 patient sample. M.A. holds a PhD-fellowship
of the FWO-F.
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