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Human Molecular Genetics Advance Access originally published online on November 30, 2007
Human Molecular Genetics 2008 17(5):717-723; doi:10.1093/hmg/ddm343
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Published by Oxford University Press 2007

A novel, putative gain-of-function haplotype at SLC6A4 associates with obsessive-compulsive disorder

Jens R. Wendland1,{dagger},*, Pablo R. Moya1,{dagger}, Matthew R. Kruse1, Renee F. Ren-Patterson2, Catherine L. Jensen1, Kiara R. Timpano3 and Dennis L. Murphy1

1 Laboratory of Clinical Science and 2 Clinical Brain Disorders Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA and 3 Department of Psychology, Florida State University, Tallahassee, FL 32306, USA

* 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: +1 3015940219; Fax: +1 3014020188; Email: wendlandj{at}mail.nih.gov

Received August 2, 2007; Accepted November 22, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
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, functional 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 significantly 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-expressing allele at each locus, supporting the notion of increased serotonin transporter functioning being pathogenetically 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
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Altered functioning of the serotonin transporter (SERT, 5-HTT, SLC6A4) has long been postulated to be involved in the pathogenesis of obsessive-compulsive disorder (OCD). Support for this hypothesis stems from the therapeutic efficacy of serotonin re-uptake inhibitors in OCD (1,2) and from the recent identification of a rare gain-of-function missense mutation of SLC6A4, I425V (3) (termed ‘OCD1’ in Online Mendelian Inheritance in Man), which results in a constitutive transporter activation (4,5) and a primarily OCD-like phenotype (6,7).

Non-coding functional polymorphisms of SLC6A4, in particular the serotonin transporter-linked polymorphic region, 5-HTTLPR, have been extensively investigated in OCD. While two initial studies (8,9) observed an association with the greater-expressing L allele and LL genotype, a number of reports from other groups were inconclusive or negative (10–15). Recently, Hu et al. (16) demonstrated that a possible explanation for the lack of consistent association was the until-then unappreciated presence of a single nucleotide polymorphism (SNP), rs25531, within the repetitive region that comprises the 5-HTTLPR. The minor allele (G) of rs25531 is almost always in phase with the long (L) allele of the 5-HTTLPR and attenuates the initially reported (17) gain-of-function relative to the short (S) allele. Thus, modulation of 5-HTTLPR by rs25531 results in three common alleles: LA (highest-expressing), LG and S (both low-expressing). With this refined understanding of 5-HTTLPR functionality, Hu et al. (16) found an association in two independent samples, both case–control and family-based, of the higher-expressing LA allele and LALA genotype with OCD. In an immediate replication attempt using a large case–control design, however, we were unable to corroborate this finding with statistical significance surviving correction for multiple testing, although we also observed an increased frequency of the LA allele and LALA genotype in OCD probands (18). This prompted us to determine whether additional functional variation of SLC6A4 could have contributed to this lack of replication. Specifically, we pursued screening of the entire 5-HTTLPR for additional common and possibly functional variants and also sought to analyze two recently-described SNPs associated with allelic expression imbalance of SLC6A4 in lymphoblastoid cell lines, rs2020933 and rs16965628 (19). We describe a heretofore-uninvestigated SNP, rs25532, which we found to modulate functionality of the 5-HTTLPR and which is part of a presumed gain-of-function haplotype associated with OCD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
As an initial step, we re-sequenced 40 SS and 40 LL 5-HTTLPR individuals from our OCD and control samples for the entire repetitive region comprising the 5-HTTLPR. We observed a common C > T polymorphism in 20 out of 80 S alleles and in 5 out of 80 L alleles, that is known to dbSNP as rs25532. This SNP is located 176 bp (L allele) or 133 bp (S allele) centromeric of rs25531 in the third-last repetitive element of the 5-HTTLPR which Nakamura et al. (20) termed the µ element (Fig. 1). Consistent with their systematic nomenclature, we termed these alleles 14-E (S allele with A at rs25531 and T at 25532) and 16-G (L allele with A at rs25531 and T at 25532). We submitted the sequences of these two novel alleles to the National Center for Biotechnology Information's GenBank database as accession numbers EU035982 (14-E) and EU035981 (16-G). We did not observe the rs25532 T allele on the SG or LG background. In the following, to facilitate readability and in line with the nomenclature introduced by Hu et al. (16), we will refer to the alleles or haplotypes at 5-HTTLPR studied herein as LAC (16-A with Nakamura et al.'s nomenclature), LAT (16-G), LG (16-D), SAC (14-A) and SAT (14-E), where the capital letter refers to the 43 bp indel, the first subscript letter to rs25531 and the second to rs25532. Further, we will refer to the molecular haplotype of the 43 bp indel and rs25531 only as ‘triallelic 5-HTTLPR’ with the common alleles S, LA and LG as our genotyping method for these two loci is phase-certain (21).


Figure 1
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  		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 polymorphisms 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.

 
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 the S and L constructs was significantly attenuated by the T allele of rs25532 in RN46A, PC12 and JAR cell lines. The effect size of the T allele at rs25532 differed between S and L alleles and between cell lines: the mean normalized firefly/Renilla ratio was reduced by 15–30% in S constructs (SAT relative to SAC) and by 25–80% in L constructs (LAT relative to LAC).


Figure 2
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  		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 functional 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 = 5–6 transfections per construct for RN46A and JAR cells, N = 13–15 transfections per construct for PC12 cells. One-way analysis of variance for all four constructs: RN46A cells, F(3,18) = 169.0, P < 0.0001; PC12 cells, F(3,51) = 58.2, P < 0.0001; JAR cells, F(3,20) = 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.

 
We then genotyped 295 Caucasian OCD probands and 657 ethnically matched control individuals for rs25532 but observed no significant allelic association of this locus when tested individually (Table 2, bottom line). Next, we used the software WHAP: haplotype-based association analysis (22) to analyze rs25532 as part of a haplotype with the triallelic 5-HTTLPR, and detected a strong omnibus association of rs25532 with OCD ({chi}2 = 19.3, df = 4, P < 0.0007; Table 1). This association was heavily driven by the rare LAT haplotype, which was twice as common in controls compared with OCD probands. To rule out possibly spurious results related to rare haplotypes, we re-analyzed our data without LAT (by excluding haplotypes with <5% frequency), and found that the omnibus association remained significant, although less strongly so ({chi}2 = 10.1, df = 3, P < 0.018; Table 1). As can further be seen in Table 1 and in line with the hypothesis of increased SLC6A4 functioning in OCD, the LAC haplotype (which corresponds to the luciferase construct with the highest expression) was more frequent in OCD probands than in controls (52 versus 47.5%), although this did not reach statistical significance in the haplotype-specific test (Table 1).


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  		Table 1. Haplotype analysis of 5-HTTLPR and rs25532 in OCD

 


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  		Table 2. Single locus and haplotype association analyses of multiple functional polymorphisms of SLC6A4 in OCD

 
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 gene expression imbalance of SLC6A4 in lymphoblastoid cell lines (19). As with rs25532, these SNPs individually were not (rs2020933) or only moderately (rs16965628, P < 0.04) significantly associated with OCD (Table 2).

We then studied triallelic 5-HTTLPR, rs25532, rs2020933, rs16965628 and the SLC6A4 intron 2 variable number of tandem repeat polymorphism as 5-locus haplotypes. Using default parameters of WHAP, we observed a strong omnibus association of these five loci with OCD ({chi}2 = 25.4, df = 9, P < 0.003; Table 2). Remarkably, the haplotype that was significantly associated with an increased odds ratio of 1.60 for OCD when tested individually against all other haplotypes (H6 in Table 2) is the haplotype containing the higher-expressing allele at each of the five polymorphisms.

Lastly, in order to dissect this haplotypic association, we took advantage of the ability to specify different alternate and null models in WHAP for conditional analyses (22) and, again, excluded rare haplotypes of <5% frequency. Systematic testing of all five polymorphisms through individual exclusion from the null model as well as controlling for each locus by excluding all other polymorphisms from the null model revealed that three loci (triallelic 5-HTTLPR, rs25532 and rs16965628) with five common haplotypes suffice for an overall strong association with OCD ({chi}2 = 13.8, df = 4, P < 0.008; Table 3). As earlier, the haplotype conferring an increased odds ratio of 1.63 is the one containing the higher-expressing allele at each of the three loci.


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  		Table 3. Three-locus haplotype association of SLC6A4 with OCD after conditional effect tests

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
The present study investigated the involvement of multiple common, non-coding functional variants of SLC6A4 in OCD. We demonstrated functionality in vitro for a previously reported SNP, rs25532, which interacts with the 5-HTTLPR. Consistent with the pathogenetic hypothesis of increased serotonin transporter activity in OCD, we showed that a haplotype containing the higher-expressing allele at each of several SLC6A4 loci is significantly associated with increased genetic susceptibility to OCD. Importantly, the association with OCD was observed only when multiple variants were analyzed as haplotypes, but not as single loci. Our work contributes to a more detailed understanding of SLC6A4 expression genetics in OCD and provides a molecular framework to explain, at least in part, inconsistent previous associations of 5-HTTLPR and other functional SLC6A4 polymorphisms in serotonin-related neuropsychiatric genetics.

The serotonin transporter gene encodes the single molecule that terminates serotonergic neurotransmission and has therefore been an intensely studied candidate. Allelic variation in this transporter's gene expression, regulated by a 43 bp indel in the upstream region termed 5-HTTLPR, was initially associated with anxiety-related traits (23) and has since been investigated in many fields of serotonin-related human genetics (24). Association results have generally been marked by at least some degree of inconsistency, for which a number of explanations such as underpowered samples, small effect size, epistasis, polygenic contributions and population stratification have been suggested. From a molecular perspective, unknown additional functional variation within SLC6A4 could further contribute to inconsistent results. Hu et al. (16) have recently shown that a significant fraction of 10–25% (depending on ethnicity) of L alleles in the 5-HTTLPR are in fact low-expressing due to contributions from an interacting SNP, rs25531. We were able to further refine the functionality of this locus by confirming and analyzing another SNP, rs25532, located <150 bp from the indel. Our luciferase expression results are in line with previous studies of this region that have shown an up to 2-fold difference between S and L alleles (16,17). Our data and the work by Hu et al. (16) demonstrate that the classic attribution of S and L alleles as low- and high-expressing, respectively, can no longer be viewed as valid without appreciation of the modulating effects of rs25531 and rs25532. More importantly, however, it will be crucial to determine (1) how this functional locus interacts with the allelic expression imbalance markers downstream (and which the actual locus conferring allelic 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 into protein abundance and actual genetically determined differential functioning of the serotonergic synapse. To this end, it will be essential to investigate the role of these novel SLC6A4 variants in mRNA levels and protein abundance in dorsal raphe nuclei as well as in peripheral cells known to express the serotonin transporter, such as lymphocytes, megakaryocytes and lymphoblastoid cell lines.

The conditional haplotype analyses conducted herein unveiled that the LA alleles at 5-HTTLPR can be further subdivided at least 2-fold (1): by interaction with rs25532 into LAC and LAT; and (2) on the basis of different rs16965628 alleles. While the LAT haplotype was too rare to be included in our statistical tests, appreciation of rs16965628 revealed that it is the presumably higher-expressing LAC haplotype with C at rs16965628 (H4 in Table 3) that is significantly more frequent in OCD than in controls. In contrast, the more common LAC haplotype with G at rs16965628 (H1 in Table 3) was not significantly different. This subdivision of LA alleles on the basis of rs16965628 is a plausible explanation for our previous non-replication of association of LA with OCD (18), and it illustrates the usefulness of haplotypic approaches with multiple functional markers to complex genetic disorders. It will be of high interest to see whether our haplotypic association can be replicated in OCD and in neighboring fields such as OCD spectrum disorders, autism or serotonin re-uptake inhibitor response, especially given the recent report of an association of coding gain-of-function SLC6A4 variants with rigid-compulsive behaviors in autism (25).

Three limitations in our study pose caveats to the interpretation and warrant further analyses. First, our case–control association study design is generally susceptible to population stratification, and although we addressed this issue by matching ethnicities between cases and controls, spurious association can only be ruled out definitively through family-based studies or through analysis of a large number of 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 higher-expression at each of its loci, it remains to be functionally demonstrated that all of these loci interact additively in a way that renders this haplotype as highest-expressing and others as lower-expressing. These analyses can currently not readily be undertaken given the very large distance between these loci, but they will be crucial to determine the relative contribution of each of these polymorphisms in their broader genomic context. Lastly, the two recently-identified SNPs associated with allelic expression imbalance, rs16965628 and rs2020933, could of course merely be markers in linkage disequilibrium not only with each other [as reflected in Table 2 and as noted previously by Martin et al. (19)] but also with the actual functional locus or loci since they have not yet been independently analyzed in reporter gene or other in vitro assays. In summary, our work shows that allowing for multiple putatively or refined functional variants and their analysis as haplotypes can be crucial for the detection of association, and we hope it contributes to a better understanding of SLC6A4 expression functionality.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Subjects
We studied a total of 295 Caucasian OCD probands and 657 Caucasian control individuals. Both samples have recently been described in detail (18,26). Briefly, probands were recruited through an adult outpatient OCD program at the National Institute of Mental Health (NIMH) in Bethesda, MD, USA. All probands were at least 18 years old at inclusion, have a primary OCD diagnosis on the basis of the Structured Clinical Interview for DSM-IV (SCID) and gave written informed consent. Control samples originated from three independent sources: (i) Coriell cell repository (Camden, NJ, USA; N = 200 self-declared healthy US Caucasians); (ii) European Collection of Cell Cultures (Sigma-Aldrich, St Louis, MO, USA; N = 192 apparently healthy UK Caucasian blood donors); and (iii) undergraduate students (N = 265 self-declared healthy Caucasians) from a large Southeastern university who participated in a separate study of genes and personality in return for partial course credit. All studies were conducted under a protocol approved by the Institutional Review Board of the NIMH Division of Intramural Research Programs in Bethesda, MD, and by the Human Subjects Committee at Florida State University. As only the third control group was evaluated by self-report and standard scales, we cannot rule out the occurrence of OCD within the other groups, although frequency above the general population prevalence of 2–3% would seem unlikely 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 polymorphisms significantly deviated from Hardy–Weinberg equilibrium as determined by contingency table statistics (data not shown).

Genotyping
Deoxyribonucleic acid was extracted from whole blood obtained through peripheral venipuncture or from saliva samples (Oragene discs, DNA Genotek, Ottawa, ON). Genotyping procedures and genotype results for 5-HTTLPR/rs25531 and STin2 have been published previously by us (18) and were used here only as part of the haplotype analyses. The three SNPs rs25532, rs2020933 and rs16965628 were genotyped using 5’-exonuclease assays (TaqMan SNP genotyping assays-by-design; Applied Biosystems, Foster City, CA) under standard conditions described previously (27) and with the following primer and probe sequences: rs25532, forward primer CTG CAC CCC CCA GCA T, reverse primer GGT AGG GTG CAA GGA GAA TGC, VIC probe CCG GcA TCC CCC CT, FAM probe CCC GGt ATC CCC CCT; rs2020933, forward primer TGT ATG TAT TTT TAC CAT CAG TTT TGT CCA GAA, reverse primer GAG AGT TAG CTA GCA GGC TCA TAA AT, VIC probe CAT TGA CCa GGT TCA C, FAM probe CAT TGA CCt GGT TCA C; and rs16965628, forward primer GGC CTC AGT TTC CCT GCT A, reverse primer GTT GAT GTC ACT ATC ACC ACC ATA CA, VIC probe AAC CCA TTg TGC CTT T, FAM probe AAC CCA TTc TGC CTT T. The overall genotype completion rate exceeded 94% for each assay, samples analyzed in duplicate and no-template-controls consistently yielded expected results.

Sequencing of the 5-HTTLPR
A total of 30–50 ng of genomic DNA were amplified in a total reaction volume of 20 µl using final concentrations of 1x 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 (Operon, Huntsville, AL). Thermocycling consisted of 15 min initial hot start activation at 95°C followed by 38 cycles of denaturation at 96°C (10 s), annealing at 62°C (25 s) and extension at 72°C (50 s); final extension was performed at 72°C for 5 min. Specific amplification of a 468 bp (SS) or 511 bp (LL) product was confirmed by agarose gel electrophoresis and ethidium bromide staining (Sigma). Amplicons were purified using MultiScreen HTS filter plates (Millipore, Bedford, MA) attached to a vacuum manifold (Millipore), and the concentration was determined spectrophotometrically (NanoDrop, Wilmington, DE). A total of 60 ng of purified amplicon DNA was subsequently sequenced bidirectionally with amplification primers using BigDye Terminator chemistry on an ABI 3100 automated sequence analyzer (Applied Biosystems) at the National Institute of Neurological Disorders and Stroke DNA sequencing core facility. Raw chromatograms were analyzed separately for SS and LL samples with the phred/phrap/consed suite and PolyPhred version 6.02 beta (28).

Reporter gene assays
We created 5-HTTLPR reporter gene constructs for LAC, LAT, SAC and SAT. Genomic DNA was amplified with primers and under conditions described above (Sequencing of the 5-HTTLPR). Amplicons were cloned into pCRII-TOPO vector (Invitrogen, Carlsbad, CA) and then subcloned into pGL4.10 (Promega, Madison, WI). All pGL constructs were bidirectionally sequenced with primers AGT GCA GGT GCC AGA ACA TT and TCT TCC ATG GTG GCT TTA CC to check for proper orientation and to confirm sequence specificity and absence of artificial mutations. Undifferentiated rat raphe medullary raphe cells (RN46A, a kind gift from Dr. Scott R. Whittemore, University of Louisville), as well as rat adrenal pheochromocytoma (PC12) and human choriocarcinoma (JAR) cells grown under standard conditions were cotransfected with the pGL4.10 constructs and pRL (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 the manufacturer's protocol in a 20/20n luminometer (Turner Biosystems, Sunnyvale, CA). Renilla luminescence was used to correct for variable transfection efficiency. Each of the four constructs was transfected 3–5 times in each of four experiments for a total of 13–15 independent transfections for PC12 cells; for RN46A and JAR cells, each of the constructs was transfected 5–6 times; the empty pGL4.10 vector was transfected thrice for control purposes (data not shown). Firefly/Renilla ratios were normalized to the geometric mean of SAC ratios. Normalized ratios for the four constructs were then analyzed by one-way analysis of variance followed by Newman–Keuls test to compare all pairwise group means with Prism 4 for Windows (GraphPad Software, San Diego, CA).

Statistical genetics
All analyses were carried out using the software WHAP: haplotype-based association analysis version 2.09 (22). We analyzed 5-HTTLPR/rs25531 as one triallelic locus with LA, LG and SA alleles (two SG alleles (21) were treated as SA) using the -usat command. Nine-repeat alleles (38 out of 1904 chromosomes) at the STin2 polymorphism were treated as 10-repeat alleles. After detection of a significant main effect, we performed conditional analyses in WHAP as per the author's website instructions (http://pngu.mgh.harvard.edu/~purcell/whap/condtut.shtml). Each of the five loci was both dropped from the null model (to detect whether it has an independent effect after controlling for everything else) and specified as the sole null model (to control for it and test whether it can explain the total association). For these conditional analyses, the haplotype frequency threshold was raised to 5% with the -at 5 flag. Haplotype-specific tests with df = 1 and single locus tests with df = 2 for triallelic 5-HTTLPR/rs25531 and df = 1 for all other loci were carried out with the -hs and -alt options, respectively. Odds ratios were calculated by raising Euler's number to the power of the regression coefficients as returned by WHAP. A P-value of 0.05 was considered significant.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This research was supported by the Intramural Research Program of the National Institute of Mental Health, National Institutes of Health.


   ACKNOWLEDGEMENTS
 
We gratefully thank all the participants in this study. We are indebted to Diane Kazuba, Brenda Justement and Michael Wheaton for conducting patient interviews; to Teresa Tolliver for technical assistance in whole blood DNA extraction; and to Prof Dr K.-P. Lesch, from the University of Würzburg, for helpful discussion and comments on the manuscript. We also thank James Nagle and Debbie Kauffman, from the NINDS DNA sequencing core facility, for excellent technical assistance with sequencing the serotonin transporter gene promoter as part of a ‘large sequencing project’ granted to J.R.W.; and Dr Scott R. Whittemore, University of Louisville, for providing us with the RN46A cell line.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

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