Detailed analysis of the serotonin transporter gene (SLC6A4) shows no association with bipolar disorder in the Northern Swedish population.код для вставкиСкачать
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: email@example.com 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. . 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. , 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. . 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. REFERENCES American Psychiatry Association. 1994. Diagnostic and Statistical Manual of Mental Disorders, 4th edition. Washington, DC: American Psychiatric Press. Anguelova M, Benkelfat C, Turecki G. 2003. 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