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  • Human Molecular Genetics A novel, putative gain-of-function haplotype at SLC6A4 associates with obsessive-compulsive disorder Jens R. Wendland, Pablo R. Moya, Matthew R. Kruse, Renee F. Ren-Patterson, Catherine L. Jensen, Kiara R. Timpano and Dennis L. Murphy Hum. Mol. Genet. 17:717-723, 2008. First published 30 Nov 2007; doi:10.1093/hmg/ddm343 The full text of this article, along with updated information and services is available online at References This article cites 28 references, 8 of which can be accessed free at Reprints Reprints of this article can be ordered at Email and RSS alerting Sign up for email alerts, and subscribe to this journal’s RSS feeds at PowerPoint® Images from this journal can be downloaded with one click as a PowerPoint slide. image downloads Journal information Additional information about Human Molecular Genetics, including how to subscribe can be found at Published on behalf of Oxford University Press Downloaded from at Columbia University Libraries on 22 September 2008
  • Human Molecular Genetics, 2008, Vol. 17, No. 5 717–723 doi:10.1093/hmg/ddm343 Advance Access published on November 30, 2007 A novel, putative gain-of-function haplotype at SLC6A4 associates with obsessive-compulsive disorder Jens R. Wendland1,{,à , Pablo R. Moya1,{, Matthew R. Kruse1, Renee F. Ren-Patterson2, Catherine L. Jensen1, Kiara R. Timpano3 and Dennis L. Murphy1 1 Laboratory of Clinical Science and 2Clinical Brain Disorders Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA and 3Department of Psychology, Florida State University, Tallahassee, FL 32306, USA Received August 2, 2007; Revised and Accepted November 22, 2007 Obsessive-compulsive disorder (OCD) is a disabling neuropsychiatric illness with strong segregation data indicative of major genetic contributions. Association analyses of common functional variants of the serotonin transporter gene (SLC6A4), a long-standing OCD candidate, have so far been inconsistent. Here, we set out to investigate the role of additional functional SLC6A4 loci in OCD. We describe a common, func- tional C > T single nucleotide polymorphism, rs25532, located less than 150 nucleotides centromeric of the serotonin transporter-linked polymorphic region indel known as 5-HTTLPR. The minor allele of rs25532 sig- nificantly decreased luciferase reporter gene expression levels by 15 – 80%, depending on 5-HTTLPR allele background and cell type. Haplotype-based testing of rs25532 and all other known non-coding functional SLC6A4 variants revealed a highly significant omnibus association with OCD in a large case – control sample. Remarkably, the haplotype significantly overrepresented in probands contained the higher-expres- sing allele at each locus, supporting the notion of increased serotonin transporter functioning being patho- genetically involved in OCD. Conditional haplotype analyses with the software WHAP revealed that this association is primarily driven by 5-HTTLPR, rs25532 and rs16965628. Our results contribute to a better understanding of SLC6A4 expression genetics and provide a functional haplotype framework for future serotonin-related studies. INTRODUCTION 5-HTTLPR, have been extensively investigated in OCD. While two initial studies (8,9) observed an association with Altered functioning of the serotonin transporter (SERT, 5-HTT, the greater-expressing L allele and LL genotype, a number SLC6A4) has long been postulated to be involved in the patho- of reports from other groups were inconclusive or negative genesis of obsessive-compulsive disorder (OCD). Support for (10 – 15). Recently, Hu et al. (16) demonstrated that a possible this hypothesis stems from the therapeutic efficacy of serotonin explanation for the lack of consistent association was the re-uptake inhibitors in OCD (1,2) and from the recent identi- until-then unappreciated presence of a single nucleotide poly- fication of a rare gain-of-function missense mutation of morphism (SNP), rs25531, within the repetitive region that SLC6A4, I425V (3) (termed ‘OCD1’ in Online Mendelian comprises the 5-HTTLPR. The minor allele (G) of rs25531 Inheritance in Man), which results in a constitutive transporter is almost always in phase with the long (L) allele of the activation (4,5) and a primarily OCD-like phenotype (6,7). 5-HTTLPR and attenuates the initially reported (17) Non-coding functional polymorphisms of SLC6A4, in par- gain-of-function relative to the short (S) allele. Thus, modu- ticular the serotonin transporter-linked polymorphic region, lation of 5-HTTLPR by rs25531 results in three common à To whom correspondence should be addressed at: Laboratory of Clinical Science, National Institute of Mental Health, NIH, Building 10, Room 3D41, 10 Center Dr, MSC 1264, Bethesda, MD 20892, USA. Tel: þ1 3015940219; Fax: þ1 3014020188; Email: † The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Published by Oxford University Press 2007
  • 718 Human Molecular Genetics, 2008, Vol. 17, No. 5 alleles: LA (highest-expressing), LG and S (both low- We then genotyped 295 Caucasian OCD probands and 657 expressing). With this refined understanding of 5-HTTLPR ethnically matched control individuals for rs25532 but functionality, Hu et al. (16) found an association in two observed no significant allelic association of this locus when independent samples, both case– control and family-based, tested individually (Table 2, bottom line). Next, we used the of the higher-expressing LA allele and LALA genotype with software WHAP: haplotype-based association analysis (22) OCD. In an immediate replication attempt using a large to analyze rs25532 as part of a haplotype with the triallelic case – control design, however, we were unable to corroborate 5-HTTLPR, and detected a strong omnibus association of this finding with statistical significance surviving correction rs25532 with OCD (x2 ¼ 19.3, df ¼ 4, P , 0.0007; Table 1). for multiple testing, although we also observed an increased This association was heavily driven by the rare LAT haplotype, frequency of the LA allele and LALA genotype in OCD pro- which was twice as common in controls compared with OCD bands (18). This prompted us to determine whether additional probands. To rule out possibly spurious results related to rare functional variation of SLC6A4 could have contributed to this haplotypes, we re-analyzed our data without LAT (by exclud- lack of replication. Specifically, we pursued screening of the ing haplotypes with ,5% frequency), and found that the entire 5-HTTLPR for additional common and possibly func- omnibus association remained significant, although less tional variants and also sought to analyze two recently- strongly so (x2 ¼ 10.1, df ¼ 3, P , 0.018; Table 1). As can described SNPs associated with allelic expression imbalance further be seen in Table 1 and in line with the hypothesis of of SLC6A4 in lymphoblastoid cell lines, rs2020933 and increased SLC6A4 functioning in OCD, the LAC haplotype rs16965628 (19). We describe a heretofore-uninvestigated (which corresponds to the luciferase construct with the SNP, rs25532, which we found to modulate functionality of highest expression) was more frequent in OCD probands than the 5-HTTLPR and which is part of a presumed in controls (52 versus 47.5%), although this did not reach stati- gain-of-function haplotype associated with OCD. stical significance in the haplotype-specific test (Table 1). In light of the small effect size of rs25532 and the 5-HTTLPR in general, we then investigated two additional SNPs, rs2020933 and rs16965628, that were recently shown to predict allelic RESULTS gene expression imbalance of SLC6A4 in lymphoblastoid cell As an initial step, we re-sequenced 40 SS and 40 LL lines (19). As with rs25532, these SNPs individually were not 5-HTTLPR individuals from our OCD and control samples (rs2020933) or only moderately (rs16965628, P , 0.04) signi- for the entire repetitive region comprising the 5-HTTLPR. ficantly associated with OCD (Table 2). We observed a common C . T polymorphism in 20 out of We then studied triallelic 5-HTTLPR, rs25532, rs2020933, 80 S alleles and in 5 out of 80 L alleles, that is known to rs16965628 and the SLC6A4 intron 2 variable number of dbSNP as rs25532. This SNP is located 176 bp (L allele) or tandem repeat polymorphism as 5-locus haplotypes. Using 133 bp (S allele) centromeric of rs25531 in the third-last default parameters of WHAP, we observed a strong omnibus repetitive element of the 5-HTTLPR which Nakamura et al. association of these five loci with OCD (x2 ¼ 25.4, df ¼ 9, (20) termed the m element (Fig. 1). Consistent with their sys- P , 0.003; Table 2). Remarkably, the haplotype that was sig- tematic nomenclature, we termed these alleles 14-E (S allele nificantly associated with an increased odds ratio of 1.60 for with A at rs25531 and T at 25532) and 16-G (L allele with OCD when tested individually against all other haplotypes A at rs25531 and T at 25532). We submitted the sequences (H6 in Table 2) is the haplotype containing the higher- of these two novel alleles to the National Center for Biotech- expressing allele at each of the five polymorphisms. nology Information’s GenBank database as accession numbers Lastly, in order to dissect this haplotypic association, we EU035982 (14-E) and EU035981 (16-G). We did not observe took advantage of the ability to specify different alternate the rs25532 T allele on the SG or LG background. In the fol- and null models in WHAP for conditional analyses (22) and, lowing, to facilitate readability and in line with the nomencla- again, excluded rare haplotypes of ,5% frequency. Systema- ture introduced by Hu et al. (16), we will refer to the alleles or tic testing of all five polymorphisms through individual exclu- haplotypes at 5-HTTLPR studied herein as LAC (16-A with sion from the null model as well as controlling for each locus Nakamura et al.’s nomenclature), LAT (16-G), LG (16-D), by excluding all other polymorphisms from the null model SAC (14-A) and SAT (14-E), where the capital letter refers to revealed that three loci (triallelic 5-HTTLPR, rs25532 and the 43 bp indel, the first subscript letter to rs25531 and the rs16965628) with five common haplotypes suffice for an second to rs25532. Further, we will refer to the molecular hap- overall strong association with OCD (x2 ¼ 13.8, df ¼ 4, lotype of the 43 bp indel and rs25531 only as ‘triallelic P , 0.008; Table 3). As earlier, the haplotype conferring an 5-HTTLPR’ with the common alleles S, LA and LG as our gen- increased odds ratio of 1.63 is the one containing the higher- otyping method for these two loci is phase-certain (21). expressing allele at each of the three loci. We generated four reporter gene constructs to determine whether rs25532 modulates 5-HTTLPR-conferred expression levels in vitro. As can be seen in Figure 2, expression of both DISCUSSION the S and L constructs was significantly attenuated by the T allele of rs25532 in RN46A, PC12 and JAR cell lines. The The present study investigated the involvement of multiple effect size of the T allele at rs25532 differed between S and L common, non-coding functional variants of SLC6A4 in alleles and between cell lines: the mean normalized firefly/ OCD. We demonstrated functionality in vitro for a previously Renilla ratio was reduced by 15–30% in S constructs (SAT rela- reported SNP, rs25532, which interacts with the 5-HTTLPR. tive to SAC) and by 25–80% in L constructs (LAT relative to LAC). Consistent with the pathogenetic hypothesis of increased
  • Human Molecular Genetics, 2008, Vol. 17, No. 5 719 Figure 1. Schematic overview of the repetitive region containing the 5-HTTLPR indel and two functional SNPs, rs25531 and rs25532. Individual elements of the repetitive region are depicted by Greek letters according to the nomenclature introduced by Nakamura et al. (20). The two previously described functional poly- morphisms depicted here are a 43 bp indel known as the 5-HTTLPR (grey shading) and the rs25531 A/G SNP. Note that, as we have reported previously, the definition of the exact beginning and end of the indel varies between authors (21). A previously uncharacterized C . T SNP known as rs25532 is located ,150 bp from the indel in the third-last repetitive element. The T allele of this SNP occurred much more frequently on S than on L allele background, and both alleles of rs25532 were always in phase with A at rs25531 in this study. Three chromatograms from amplified genomic DNA of all three genotypes at rs25532 (arrowheads) are shown in the bottom part. Figure 2. Luciferase reporter gene assay analyses of rs25532 in RN46A, PC12 and JAR cell lines in the context of the 5-HTTLPR. Columns and error bars represent mean normalized luciferase / Renilla luciferase ratios+SEM for four different constructs, SAC, SAT, LAC and LAT, that were used to assess the func- tional impact of the rs25532 in transfected rat medullary raphe (RN46A), adrenal pheochromocytoma (PC12) and human choriocarcinoma (JAR) cell lines. The T allele significantly decreased expression by 15–30% in S constructs (SAT relative to SAC) and by 25–80% (LAT relative to LAC) in L alleles. N ¼ 5 –6 transfec- tions per construct for RN46A and JAR cells, N ¼ 13–15 transfections per construct for PC12 cells. One-way analysis of variance for all four constructs: RN46A cells, F(3,18) ¼ 169.0, P , 0.0001; PC12 cells, F(3,51) ¼ 58.2, P , 0.0001; JAR cells, F(3,20) ¼ 56.3, P , 0.0001; Newman-Keuls post hoc test: RN46A cells, P , 0.001 for every pairwise comparison; PC12 cells, P , 0.001 for every pairwise comparison with the exception of SAC versus SAT, where P , 0.05; JAR cells, P , 0.001 for every pairwise comparison with the exception of SAC versus SAT and LAT versus SAT where P , 0.01. serotonin transporter activity in OCD, we showed that a hap- The serotonin transporter gene encodes the single molecule lotype containing the higher-expressing allele at each of that terminates serotonergic neurotransmission and has there- several SLC6A4 loci is significantly associated with increased fore been an intensely studied candidate. Allelic variation in genetic susceptibility to OCD. Importantly, the association this transporter’s gene expression, regulated by a 43 bp indel with OCD was observed only when multiple variants were in the upstream region termed 5-HTTLPR, was initially analyzed as haplotypes, but not as single loci. Our work con- associated with anxiety-related traits (23) and has since been tributes to a more detailed understanding of SLC6A4 investigated in many fields of serotonin-related human gen- expression genetics in OCD and provides a molecular frame- etics (24). Association results have generally been marked work to explain, at least in part, inconsistent previous associa- by at least some degree of inconsistency, for which a tions of 5-HTTLPR and other functional SLC6A4 number of explanations such as underpowered samples, polymorphisms in serotonin-related neuropsychiatric genetics. small effect size, epistasis, polygenic contributions and
  • 720 Human Molecular Genetics, 2008, Vol. 17, No. 5 Table 1. Haplotype analysis of 5-HTTLPR and rs25532 in OCD replicated in OCD and in neighboring fields such as OCD spectrum disorders, autism or serotonin re-uptake inhibitor Triallelic rs25532 Haplotype Haplotype-specific response, especially given the recent report of an association 5-HTTLPR frequency tests (df ¼ 1) of coding gain-of-function SLC6A4 variants with rigid- OCD Controls P-value Odds ratio compulsive behaviors in autism (25). H1 LA C 0.510 0.466 ns 1.21 Three limitations in our study pose caveats to the interpret- H2 S C 0.302 0.328 ns 0.89 ation and warrant further analyses. First, our case– control H3 S T 0.128 0.106 ns 1.24 association study design is generally susceptible to population H4 LG C 0.046 0.072 0.024 0.61 stratification, and although we addressed this issue by match- H5 LA T 0.013 0.028 0.007 0.22 ing ethnicities between cases and controls, spurious associa- tion can only be ruled out definitively through family-based Omnibus X2 ¼ 19.3 (df ¼ 4); P , 0.0007. Omnibus test excluding H5: X2 ¼10.1 (df ¼ 3); P , 0.018. studies or through analysis of a large number of N ¼ 295 OCD probands and N ¼ 657 control individuals. ancestry-informative markers, which was not feasible in the present work. Second, although the haplotype associated with OCD in this study contains the allele associated with population stratification have been suggested. From a molecu- higher-expression at each of its loci, it remains to be function- lar perspective, unknown additional functional variation ally demonstrated that all of these loci interact additively in a within SLC6A4 could further contribute to inconsistent way that renders this haplotype as highest-expressing and results. Hu et al. (16) have recently shown that a significant others as lower-expressing. These analyses can currently not fraction of 10 – 25% (depending on ethnicity) of L alleles in readily be undertaken given the very large distance between the 5-HTTLPR are in fact low-expressing due to contributions these loci, but they will be crucial to determine the relative from an interacting SNP, rs25531. We were able to further contribution of each of these polymorphisms in their broader refine the functionality of this locus by confirming and analyz- genomic context. Lastly, the two recently-identified SNPs ing another SNP, rs25532, located ,150 bp from the indel. associated with allelic expression imbalance, rs16965628 Our luciferase expression results are in line with previous and rs2020933, could of course merely be markers in studies of this region that have shown an up to 2-fold differ- linkage disequilibrium not only with each other [as reflected ence between S and L alleles (16,17). Our data and the work in Table 2 and as noted previously by Martin et al. (19)] but by Hu et al. (16) demonstrate that the classic attribution of also with the actual functional locus or loci since they have S and L alleles as low- and high-expressing, respectively, not yet been independently analyzed in reporter gene or can no longer be viewed as valid without appreciation of the other in vitro assays. In summary, our work shows that allow- modulating effects of rs25531 and rs25532. More importantly, ing for multiple putatively or refined functional variants and however, it will be crucial to determine (1) how this functional their analysis as haplotypes can be crucial for the detection locus interacts with the allelic expression imbalance markers of association, and we hope it contributes to a better under- downstream (and which the actual locus conferring allelic standing of SLC6A4 expression functionality. expression imbalance is, see in what follows) (2), what the relative contribution to a net effect on serotonin transporter gene expression is in vivo and (3) how (if at all) this translates MATERIALS AND METHODS into protein abundance and actual genetically determined differential functioning of the serotonergic synapse. To this Subjects end, it will be essential to investigate the role of these novel We studied a total of 295 Caucasian OCD probands and 657 SLC6A4 variants in mRNA levels and protein abundance in Caucasian control individuals. Both samples have recently dorsal raphe nuclei as well as in peripheral cells known to been described in detail (18,26). Briefly, probands were express the serotonin transporter, such as lymphocytes, mega- recruited through an adult outpatient OCD program at the karyocytes and lymphoblastoid cell lines. National Institute of Mental Health (NIMH) in Bethesda, The conditional haplotype analyses conducted herein MD, USA. All probands were at least 18 years old at unveiled that the LA alleles at 5-HTTLPR can be further subdi- inclusion, have a primary OCD diagnosis on the basis of the vided at least 2-fold (1): by interaction with rs25532 into LAC Structured Clinical Interview for DSM-IV (SCID) and gave and LAT; and (2) on the basis of different rs16965628 alleles. written informed consent. Control samples originated from While the LAT haplotype was too rare to be included in our stat- three independent sources: (i) Coriell cell repository istical tests, appreciation of rs16965628 revealed that it is the (Camden, NJ, USA; N ¼ 200 self-declared healthy US Cauca- presumably higher-expressing LAC haplotype with C at sians); (ii) European Collection of Cell Cultures rs16965628 (H4 in Table 3) that is significantly more frequent (Sigma-Aldrich, St Louis, MO, USA; N ¼ 192 apparently in OCD than in controls. In contrast, the more common LAC healthy UK Caucasian blood donors); and (iii) undergraduate haplotype with G at rs16965628 (H1 in Table 3) was not signifi- students (N ¼ 265 self-declared healthy Caucasians) from a cantly different. This subdivision of LA alleles on the basis of large Southeastern university who participated in a separate rs16965628 is a plausible explanation for our previous non- study of genes and personality in return for partial course replication of association of LA with OCD (18), and it illustrates credit. All studies were conducted under a protocol approved the usefulness of haplotypic approaches with multiple func- by the Institutional Review Board of the NIMH Division of tional markers to complex genetic disorders. It will be of high Intramural Research Programs in Bethesda, MD, and by the interest to see whether our haplotypic association can be Human Subjects Committee at Florida State University. As
  • Human Molecular Genetics, 2008, Vol. 17, No. 5 721 Table 2. Single locus and haplotype association analyses of multiple functional polymorphisms of SLC6A4 in OCD Triallelic rs25532 rs2020933 rs16965628 STin2 Haplotype frequency Haplotype-specific 5-HTTLPR tests (df ¼ 1) OCD Controls P-value Odds ratio H1 S C A G 12 0.251 0.274 ns 0.89 H2 LA C A G 10 0.270 0.240 ns 1.19 H3 LA C A G 12 0.166 0.184 ns 0.85 H4 S T A G 12 0.074 0.068 ns 1.12 H5 S T A G 10 0.063 0.046 ns 1.48 H6 LA C T C 12 0.074 0.047 0.026 1.60 H7 LG C A G 12 0.036 0.049 ns 1.45 H8 S C A G 10 0.048 0.047 ns 1.02 H9 LG C A G 10 0.012 0.025 0.019 028 H10 LA T A G 10 0.007 0.020 0.005 0.13 Functionalitya LA . LG . S C.T T.A C.G 12 . 10 Omnibus X2 ¼25.3 (df ¼ 9), MAF OCD LG, 0.046 T, 0.142 T, 0.086 C, 0.099 10, 0.405 P , 0.003 MAF controls 0.072 0.134 0.064 0.070 0.378 Single locus testb ns ns ns P , 0.040 ns N ¼ 295 OCD probands and N ¼ 657 control individuals. A haplotype significantly associated with increased OCD risk, H6 (bold-face values), contains the higher-expressing allele at each locus. MAF, minor allelic frequency. a Functionality classification is based on the works by Heils et al. (17) and Hu et al. (16) for triallelic 5-HTTLPR, MacKenzie and Quinn (29) for STin2, Martin et al. (19) for rs2020933 and rs16965628 and the present work for rs25532. b Single locus association analyses for triallelic 5-HTTLPR and STin2 have been published previously (18). Table 3. Three-locus haplotype association of SLC6A4 with OCD after Genotyping procedures and genotype results for 5-HTTLPR/ conditional effect tests rs25531 and STin2 have been published previously by us (18) and were used here only as part of the haplotype analyses. Triallelic rs25532 rs Haplotype Haplotype-specific 5-HTTLPR 16965628 frequency tests (df ¼ 1) The three SNPs rs25532, rs2020933 and rs16965628 were OCD Controls P-value Odds ratio genotyped using 5’-exonuclease assays (TaqMan SNP geno- typing assays-by-design; Applied Biosystems, Foster City, H1 LA C G 0.433 0.422 ns 1.04 CA) under standard conditions described previously (27) and H2 S C G 0.287 0.326 ns 0.84 H3 S T G 0.140 0.118 ns 1.21 with the following primer and probe sequences: rs25532, H4 LA C C 0.093 0.059 0.010 1.63 forward primer CTG CAC CCC CCA GCA T, reverse H5 LG C G 0.047 0.073 0.027 0.62 primer GGT AGG GTG CAA GGA GAA TGC, VIC probe CCG GcA TCC CCC CT, FAM probe CCC GGt ATC CCC Omnibus X2 ¼ 13.8 (df ¼ 4); P , 0.0008. CCT; rs2020933, forward primer TGT ATG TAT TTT TAC N ¼ 295 OCD probands and N ¼ 657 control individuals. As in Table 2, the CAT CAG TTT TGT CCA GAA, reverse primer GAG AGT haplotype with the higher-expressing allele at each locus is significantly TAG CTA GCA GGC TCA TAA AT, VIC probe CAT associated with increased susceptibility to OCD. Note also that rs16965628 distinguishes between a common (H1) and a rare (H4, bold-face values) LAC TGA CCa GGT TCA C, FAM probe CAT TGA CCt GGT haplotype, and that only the rare H4 haplotype with the higher-expressing C TCA C; and rs16965628, forward primer GGC CTC AGT allele at rs16965628 significantly differs between OCD and controls. TTC CCT GCT A, reverse primer GTT GAT GTC ACT ATC ACC ACC ATA CA, VIC probe AAC CCA TTg TGC only the third control group was evaluated by self-report and CTT T, FAM probe AAC CCA TTc TGC CTT T. The standard scales, we cannot rule out the occurrence of OCD overall genotype completion rate exceeded 94% for each within the other groups, although frequency above the assay, samples analyzed in duplicate and no-template-controls general population prevalence of 2 – 3% would seem unlikely consistently yielded expected results. and thus should not have significantly impacted our results. Allelic and genotypic frequencies did not significantly differ between these three control groups; moreover, none of the Sequencing of the 5-HTTLPR polymorphisms significantly deviated from Hardy – Weinberg equilibrium as determined by contingency table statistics A total of 30 –50 ng of genomic DNA were amplified in a total (data not shown). reaction volume of 20 ml using final concentrations of 1Â multiplex master mix (Qiagen, Valencia, CA) and 500 nM each of oligonucleotide primers GCC AGC ACC TAA CCC CTA AT and GAG GGA CTG AGC TGG ACA AC Genotyping (Operon, Huntsville, AL). Thermocycling consisted of Deoxyribonucleic acid was extracted from whole blood 15 min initial hot start activation at 958C followed by 38 obtained through peripheral venipuncture or from saliva cycles of denaturation at 968C (10 s), annealing at 628C samples (Oragene discs, DNA Genotek, Ottawa, ON). (25 s) and extension at 728C (50 s); final extension was
  • 722 Human Molecular Genetics, 2008, Vol. 17, No. 5 performed at 728C for 5 min. Specific amplification of a as 10-repeat alleles. After detection of a significant main 468 bp (SS) or 511 bp (LL) product was confirmed by effect, we performed conditional analyses in WHAP as per agarose gel electrophoresis and ethidium bromide staining the author’s website instructions (http://pngu.mgh.harvard. (Sigma). Amplicons were purified using MultiScreen HTS edu/~purcell/whap/condtut.shtml). Each of the five loci was filter plates (Millipore, Bedford, MA) attached to a vacuum both dropped from the null model (to detect whether it has manifold (Millipore), and the concentration was determined an independent effect after controlling for everything else) spectrophotometrically (NanoDrop, Wilmington, DE). A and specified as the sole null model (to control for it and total of 60 ng of purified amplicon DNA was subsequently test whether it can explain the total association). For these con- sequenced bidirectionally with amplification primers using ditional analyses, the haplotype frequency threshold was BigDye Terminator chemistry on an ABI 3100 automated raised to 5% with the -at 5 flag. Haplotype-specific tests sequence analyzer (Applied Biosystems) at the National Insti- with df ¼ 1 and single locus tests with df ¼ 2 for triallelic tute of Neurological Disorders and Stroke DNA sequencing 5-HTTLPR/rs25531 and df ¼ 1 for all other loci were core facility. Raw chromatograms were analyzed separately carried out with the -hs and -alt options, respectively. Odds for SS and LL samples with the phred/phrap/consed suite ratios were calculated by raising Euler’s number to the and PolyPhred version 6.02 beta (28). power of the regression coefficients as returned by WHAP. A P-value of 0.05 was considered significant. Reporter gene assays We created 5-HTTLPR reporter gene constructs for LAC, LAT, ACKNOWLEDGEMENTS SAC and SAT. Genomic DNA was amplified with primers and We gratefully thank all the participants in this study. We are under conditions described above (Sequencing of the indebted to Diane Kazuba, Brenda Justement and Michael 5-HTTLPR). Amplicons were cloned into pCRII-TOPO Wheaton for conducting patient interviews; to Teresa Tolliver vector (Invitrogen, Carlsbad, CA) and then subcloned into for technical assistance in whole blood DNA extraction; and pGL4.10 (Promega, Madison, WI). All pGL constructs were to Prof Dr K.-P. Lesch, from the University of Wurzburg, for ¨ bidirectionally sequenced with primers AGT GCA GGT helpful discussion and comments on the manuscript. We also GCC AGA ACA TT and TCT TCC ATG GTG GCT TTA thank James Nagle and Debbie Kauffman, from the NINDS CC to check for proper orientation and to confirm sequence DNA sequencing core facility, for excellent technical assist- specificity and absence of artificial mutations. Undifferen- ance with sequencing the serotonin transporter gene promoter tiated rat raphe medullary raphe cells (RN46A, a kind gift as part of a ‘large sequencing project’ granted to J.R.W.; and from Dr. Scott R. Whittemore, University of Louisville), as Dr Scott R. Whittemore, University of Louisville, for providing well as rat adrenal pheochromocytoma (PC12) and human us with the RN46A cell line. choriocarcinoma (JAR) cells grown under standard conditions were cotransfected with the pGL4.10 constructs and pRL Conflict of Interest statement. None declared. (Renilla luciferase, Promega) using lipofectamine 2000 reagent (Invitrogen). Twenty-four hours after transfection, cells were harvested and lysed, and luciferase activity was measured using Dual Luciferase Assay (Promega) following FUNDING the manufacturer’s protocol in a 20/20n luminometer This research was supported by the Intramural Research (Turner Biosystems, Sunnyvale, CA). Renilla luminescence Program of the National Institute of Mental Health, National was used to correct for variable transfection efficiency. Each Institutes of Health. of the four constructs was transfected 3 – 5 times in each of four experiments for a total of 13 – 15 independent transfec- tions for PC12 cells; for RN46A and JAR cells, each of the REFERENCES constructs was transfected 5 – 6 times; the empty pGL4.10 1. Pigott, T.A., Pato, M.T., Bernstein, S.E., Grover, G.N., Hill, J.L., Tolliver, T.J. vector was transfected thrice for control purposes (data not and Murphy, D.L. (1990) Controlled comparisons of clomipramine and shown). Firefly/Renilla ratios were normalized to the geo- fluoxetine in the treatment of obsessive-compulsive disorder. Behavioral metric mean of SAC ratios. Normalized ratios for the four con- and biological results. Arch. Gen. Psychiatry, 47, 926 – 932. structs were then analyzed by one-way analysis of variance 2. Greist, J.H., Jefferson, J.W., Kobak, K.A., Katzelnick, D.J. and Serlin, R.C. (1995) Efficacy and tolerability of serotonin transport inhibitors in followed by Newman – Keuls test to compare all pairwise obsessive-compulsive disorder. A meta-analysis. Arch. Gen. Psychiatry, group means with Prism 4 for Windows (GraphPad Software, 52, 53–60. San Diego, CA). 3. Ozaki, N., Goldman, D., Kaye, W.H., Plotnicov, K., Greenberg, B.D., Lappalainen, J., Rudnick, G. and Murphy, D.L. (2003) Serotonin transporter missense mutation associated with a complex neuropsychiatric Statistical genetics phenotype. Mol. Psychiatry, 8, 933– 936. 4. Kilic, F., Murphy, D.L. and Rudnick, G. (2003) A human serotonin All analyses were carried out using the software WHAP: transporter mutation causes constitutive activation of transport activity. haplotype-based association analysis version 2.09 (22). We Mol. Pharmacol., 64, 440–446. analyzed 5-HTTLPR/rs25531 as one triallelic locus with LA, 5. Prasad, H.C., Zhu, C.B., McCauley, J.L., Samuvel, D.J., Ramamoorthy, S., Shelton, R.C., Hewlett, W.A., Sutcliffe, J.S. and Blakely, R.D. (2005) LG and SA alleles (two SG alleles (21) were treated as SA) Human serotonin transporter variants display altered sensitivity to protein using the -usat command. Nine-repeat alleles (38 out of kinase G and p38 mitogen-activated protein kinase. Proc. Natl Acad. Sci. 1904 chromosomes) at the STin2 polymorphism were treated USA, 102, 11545–11550.
  • Human Molecular Genetics, 2008, Vol. 17, No. 5 723 6. Delorme, R., Betancur, C., Wagner, M., Krebs, M.O., Gorwood, P., Pearl, P., 16. Hu, X.Z., Lipsky, R.H., Zhu, G., Akhtar, L.A., Taubman, J., Greenberg, B.D., Nygren, G., Durand, C.M., Buhtz, F., Pickering, P. et al. (2005) Xu, K., Arnold, P.D., Richter, M.A., Kennedy, J.L. et al. (2006) Support for the association between the rare functional variant I425V of Serotonin transporter promoter gain-of-function genotypes are linked to the serotonin transporter gene and susceptibility to obsessive compulsive obsessive-compulsive disorder. Am. J. Hum. Genet., 78, 815–826. disorder. Mol. Psychiatry, 10, 1059–1061. 17. Heils, A., Teufel, A., Petri, S., Stober, G., Riederer, P., Bengel, D. and 7. Wendland, J.R., DeGuzman, T.B., McMahon, F., Rudnick, G., Lesch, K.P. (1996) Allelic variation of human serotonin transporter gene Detera-Wadleigh, S. and Murphy, D.L. SERT Ileu425Val (OCD1) and expression. J. Neurochem., 66, 2621–2624. other uncommon gene variants and chromosomal anomalies associated 18. Wendland, J.R., Kruse, M.R., Cromer, K.C. and Murphy, D.L. (2007) with an obsessive-compulsive disorder phenotype. Psychiatr. Genetics, A large case-control study of common functional SLC6A4 and BDNF in press. variants in obsessive-compulsive disorder. Neuropsychopharmacology, 8. McDougle, C.J., Epperson, C.N., Price, L.H. and Gelernter, J. (1998) 32, 2543–2551. Evidence for linkage disequilibrium between serotonin transporter protein 19. Martin, J., Cleak, J., Willis-Owen, S.A., Flint, J. and Shifman, S. (2007) gene (SLC6A4) and obsessive compulsive disorder. Mol. Psychiatry, 3, Mapping regulatory variants for the serotonin transporter gene based on 270–273. allelic expression imbalance. Mol. Psychiatry, 12, 421–422. 9. Bengel, D., Greenberg, B.D., Cora-Locatelli, G., Altemus, M., Heils, A., 20. Nakamura, M., Ueno, S., Sano, A. and Tanabe, H. (2000) The human Li, Q. and Murphy, D.L. (1999) Association of the serotonin transporter serotonin transporter gene linked polymorphism (5-HTTLPR) shows ten promoter regulatory region polymorphism and obsessive-compulsive novel allelic variants. Mol. Psychiatry, 5, 32–38. disorder. Mol. Psychiatry, 4, 463– 466. 21. Wendland, J.R., Martin, B.J., Kruse, M.R., Lesch, K.P. and Murphy, D.L. 10. Frisch, A., Michaelovsky, E., Rockah, R., Amir, I., Hermesh, H., Laor, N., (2006) Simultaneous genotyping of four functional loci of human Fuchs, C., Zohar, J., Lerer, B., Buniak, S.F. et al. (2000) Association SLC6A4, with a reappraisal of 5-HTTLPR and rs25531. Mol. Psychiatry, between obsessive-compulsive disorder and polymorphisms of genes 11, 224– 226. encoding components of the serotonergic and dopaminergic pathways. 22. Purcell, S., Daly, M.J. and Sham, P.C. (2007) WHAP: haplotype-based association analysis. Bioinformatics, 23, 255– 256. Eur. Neuropsychopharmacol., 10, 205–209. 23. Lesch, K.P., Bengel, D., Heils, A., Sabol, S.Z., Greenberg, B.D., Petri, S., 11. Kinnear, C.J., Niehaus, D.J., Moolman-Smook, J.C., du Toit, P.L., van Benjamin, J., Muller, C.R., Hamer, D.H. and Murphy, D.L. (1996) Kradenberg, J., Weyers, J.B., Potgieter, A., Marais, V., Emsley, R.A., Association of anxiety-related traits with a polymorphism in the serotonin Knowles, J.A. et al. (2000) Obsessive-compulsive disorder and the transporter gene regulatory region. Science, 274, 1527– 1531. promoter region polymorphism (5-HTTLPR) in the serotonin transporter 24. Murphy, D.L., Lerner, A., Rudnick, G. and Lesch, K.P. (2004) Serotonin gene (SLC6A4): a negative association study in the Afrikaner population. transporter: gene, genetic disorders, and pharmacogenetics. Mol. Interv., Int. J. Neuropsychopharmacol., 3, 327– 331. 4, 109–123. 12. Camarena, B., Rinetti, G., Cruz, C., Hernandez, S., de la Fuente, J.R. and 25. Sutcliffe, J.S., Delahanty, R.J., Prasad, H.C., McCauley, J.L., Han, Q., Nicolini, H. (2001) Association study of the serotonin transporter gene Jiang, L., Li, C., Folstein, S.E. and Blakely, R.D. (2005) Allelic polymorphism in obsessive-compulsive disorder. Int. J. Neuropsychophar heterogeneity at the serotonin transporter locus (SLC6A4) confers macol., 4, 269–272. susceptibility to autism and rigid-compulsive behaviors. Am. J. Hum. 13. Chabane, N., Millet, B., Delorme, R., Lichtermann, D., Mathieu, F., Genet., 77, 265–279. Laplanche, J.L., Roy, I., Mouren, M.C., Hankard, R., Maier, W. et al. 26. LaSalle, V.H., Cromer, K.R., Nelson, K.N., Kazuba, D., Justement, L. and (2004) Lack of evidence for association between serotonin transporter Murphy, D.L. (2004) Diagnostic interview assessed neuropsychiatric gene (5-HTTLPR) and obsessive-compulsive disorder by case control and disorder comorbidity in 334 individuals with obsessive-compulsive family association study in humans. Neurosci. Lett., 363, 154– 156. disorder. Depress. Anxiety, 19, 163– 173. 14. Meira-Lima, I., Shavitt, R.G., Miguita, K., Ikenaga, E., Miguel, E.C. and 27. Wendland, J.R., Kruse, M.R. and Murphy, D.L. (2006) Functional Vallada, H. (2004) Association analysis of the catechol-o-methyltransfer- SLITRK1 var321, varCDfs and SLC6A4 G56A variants and susceptibility ase (COMT), serotonin transporter (5-HTT) and serotonin 2A receptor to obsessive-compulsive disorder. Mol. Psychiatry, 11, 802–804. (5HT2A) gene polymorphisms with obsessive-compulsive disorder. 28. Bhangale, T.R., Stephens, M. and Nickerson, D.A. (2006) Automating Genes Brain Behav., 3, 75– 79. resequencing-based detection of insertion-deletion polymorphisms. 15. Walitza, S., Wewetzer, C., Gerlach, M., Klampfl, K., Geller, F., Barth, N., Nat. Genet., 38, 1457–1462. Hahn, F., Herpertz-Dahlmann, B., Gossler, M., Fleischhaker, C. et al. 29. MacKenzie, A. and Quinn, J. (1999) A serotonin transporter gene intron 2 (2004) Transmission disequilibrium studies in children and adolescents polymorphic region, correlated with affective disorders, has with obsessive-compulsive disorders pertaining to polymorphisms of allele-dependent differential enhancer-like properties in the mouse genes of the serotonergic pathway. J. Neural. Transm., 111, 817– 825. embryo. Proc. Natl Acad. Sci. USA, 96, 15251–11525.