Original Article

Molecular Psychiatry (2011) 16, 108–120; doi:10.1038/mp.2009.100; published online 6 October 2009

Association of SLC6A4 variants with obsessive-compulsive disorder in a large multicenter US family study

E Voyiaziakis1, O Evgrafov1, D Li2, H-J Yoon3, P Tabares4, J Samuels5, Y Wang5, M A Riddle5, M A Grados5, O J Bienvenu5, Y Y Shugart6, K-Y Liang7, B D Greenberg8, S A Rasmussen8, D L Murphy9, J R Wendland9, J T McCracken10, J Piacentini10, S L Rauch11, D L Pauls12, G Nestadt5, A J Fyer4 and J A Knowles1,3

  1. 1Department of Psychiatry and the Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
  2. 2Division of Biostatistics, Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
  3. 3Zilkha Neurogenetics Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
  4. 4Department of Psychiatry, College of Physicians and Surgeons at Columbia University and the New York State Psychiatric Institute, New York, NY, USA
  5. 5Department of Psychiatry and Behavioral Sciences, Johns Hopkins University, School of Medicine, Baltimore, MD, USA
  6. 6Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
  7. 7Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
  8. 8Department of Psychiatry and Human Behavior, Brown Medical School, Butler Hospital, Providence, RI, USA
  9. 9Laboratory of Clinical Science, NIMH, NIH, Bethesda, MD, USA
  10. 10Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, School of Medicine, Los Angeles, CA, USA
  11. 11Department of Psychiatry, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
  12. 12Psychiatric and Neurodevelopmental Genetics Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

Correspondence: Dr E Voyiaziakis, Department of Psychiatry and the Behavioral Sciences, Keck School of Medicine, University of Southern California, MC-2821, 1501 San Pablo, ZNI 401, Los Angeles, CA 90089, USA. E-mail: emanuelv@keck.usc.edu

Received 26 November 2008; Revised 1 June 2009; Accepted 18 August 2009; Published online 6 October 2009.

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Abstract

Genetic association studies of SLC6A4 (SERT) and obsessive-compulsive disorder (OCD) have been equivocal. We genotyped 1241 individuals in 278 pedigrees from the OCD Collaborative Genetics Study for 13 single-nucleotide polymorphisms, for the linked polymorphic region (LPR) indel with molecular haplotypes at rs25531, for VNTR polymorphisms in introns 2 and 7 and for a 381-bp deletion 3′ to the LPR. We analyzed using the Family-Based Association Test (FBAT) under additive, dominant, recessive and genotypic models, using both OCD and sex-stratified OCD as phenotypes. Two-point FBAT analysis detected association between Int2 (P=0.0089) and Int7 (P=0.0187) (genotypic model). Sex-stratified two-point analysis showed strong association in females with Int2 (P<0.0002), significant after correction for linkage disequilibrium, and multiple marker and model testing (PAdj=0.0069). The SLC6A4 gene is composed of two haplotype blocks (our data and the HapMap); FBAT whole-marker analysis conducted using this structure was not significant. Several noteworthy nonsignificant results have emerged. Unlike Hu et al., we found no evidence for overtransmission of the LPR LA allele (genotype relative risk=1.11, 95% confidence interval: 0.77–1.60); however, rare individual haplotypes containing LA with P<0.05 were observed. Similarly, three individuals (two with OCD/OCPD) carried the rare I425V SLC6A4 variant, but none of them passed it on to their six OCD-affected offspring, suggesting that it is unlikely to be solely responsible for the ‘OCD plus syndrome’, as reported by Ozaki et al. In conclusion, we found evidence of genetic association at the SLC6A4 locus with OCD. A noteworthy lack of association at the LPR, LPR-rs25531 and rare 425V variants suggests that hypotheses about OCD risk need revision to accommodate these new findings, including a possible gender effect.

Keywords:

OCD genetics; family study; affected sib-pair study; molecular haplotype analysis; serotonin transporter; heterogeneity analysis

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Introduction

Twin and family studies suggest that obsessive-compulsive disorder (OCD) has a genetic basis, but a major causative gene(s) for the disorder has not yet been identified.1, 2 Segregation analyses suggest that OCD is transmitted as either an autosomal dominant or codominant trait, particularly in those with disease onset before adulthood.3, 4, 5 The first linkage study of OCD found evidence for suggestive linkage (LOD (logarithm (base 10) of odds)=2.25) to chromosome 9p24 in 56 individuals from 7 pedigrees at marker D9S288.6 Additional evidence of linkage to this locus (HLOD (heterogeneity LOD)=2.26) was observed in a sample of 50 pedigrees with markers D9S1813 and D9S1792 that are located within 0.5cM (<350kb) of D9S288,7 and two of the markers tested provided modest evidence of association (P=0.046 and P=0.02, respectively). The gene encoding the neuronal glutamate transporter SLC1A1 is located in the 9p24 linkage region and is found to have genetic association with OCD by several groups.8, 9, 10, 11 It is likely that there are additional genomic loci for OCD as a substantially larger genome-wide linkage scan observed suggestive linkage to chromosomes 3q, 7p, 15q, 6q and 1q, although it did not strongly support linkage to 9p24.12

The gene for another transporter of neurochemical importance, SLC6A4 (serotonin transporter, 5-HTT or SERT), located on chromosome 17q11.1–q12, has also been studied extensively for genetic association with OCD. SLC6A4 is the molecular target of the selective serotonin reuptake inhibitors (SSRIs), namely fluoxetine, fluvoxamine, sertraline, paroxetine and citalopram. SSRIs collectively represent the most clinically effective and widely studied pharmacological treatment for OCD; they are effective in reducing both the thought (obsessions) and behavioral (compulsions) components of OCD.13 Imaging studies have suggested that individuals with OCD have decreased serotonin transporter availability in the midbrain/brainstem.14, 15, 16

A large amount of genetic variation has been observed in SLC6A4, including both rare and common single-nucleotide polymorphisms (SNPs),17 repeat polymorphisms in introns 2 and 7, as well as an extensively studied functional 44-base pair indel polymorphism in the promoter region (5-HTTLPR). Both the 5-HTTLPR and the intron 2 polymorphism have been shown to affect the transcription of SLC6A4.18, 19 Although the initial reports suggested an association between the linked polymorphic region (LPR) and OCD,20, 21 most subsequent studies have been negative.22, 23, 24, 25, 26, 27, 28, 29 More recently, Dickel et al.10 failed to detect association with OCD when examining select polymorphisms in SLC6A4, including the LPR. They carried out a meta-analysis constituted of five previous reports consisting of both positively and negatively associated findings that did not support a combined-sex association for either the long (L) or the short (S) allele, after correction. However, they found nominally significant excess transmission of the L allele in females but not in males. Lin30 expanded the scope of investigation by a meta-analysis of association findings of the LPR and OCD by pooling results from 13 independent case–control association studies with 3445 subjects (1242 OCD patients and 2203 controls). All 13 studies used an OR (odds ratio) estimator of risk, and random-effects modeling showed OCD associated with the SS genotype and inversely associated with the LS genotype (OR=1.21 and P=0.04 vs OR=0.79 and P=0.03, respectively). No association was detected for the LL genotype or solely for the allelic L variant.

Most recently, further meta-analysis19 analyzed a larger set of case–control and, additionally, family-based association findings. No evidence of association with variation at the LPR locus and OCD was detected in the overall meta-analysis. However, stratified meta-analysis showed a significant association between the L allele and OCD in family-based association testing and in studies involving children (child-onset OCD) and Caucasians. However, the authors note that no adjustments were made to the significance threshold for multiple comparisons.

The simple model of the 5-HTTLPR having a long allele (L) with higher expression and a lower-expressing short (S) allele has been refined with the discovery of additional nearby variation that influences transcription. Hu et al.1 showed that a G allele at rs25531 located within the LPR on an L background (designated LG) creates a functional AP2 transcription-factor binding site and has the serotonin reuptake activity of an S allele. This group then found that the gain-of-function LA/LA genotype was twice as common in OCD cases (n=169) as in ethnically matched controls (n=263) and that the LA allele showed a twofold overtransmission in OCD-affected trios (n=175), leading to a 1.8-fold increased risk of OCD.

Rare genetic variation in SLC6A4 also has been implicated in the pathogenesis of OCD. A SNP in exon 8 alters amino acid 425 from isoleucine to valine (I425V), resulting in a protein with an increase in Vmax and decrease in KM.31 This variation was observed to segregate with OCD in two OCD-dense families, and all six of the individuals with 425V who could be psychiatrically assessed had either OCD or OCPD (obsessive–compulsive personality disorder).2 Additional studies have found a prevalence of 425V of 1.5% in OCD cases (n=530) as compared with that of 0.2% in controls (n=1300), with P=0.004 and an OR=6.54.32

Given the large (n=1241), multicenter US family sample that we collected, we investigated comprehensively whether the genetic association of OCD with multiple SLC6A4 polymorphisms and haplotypes are observed using family-based statistical approaches.

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Materials and methods

Human subjects

The OCD Collaborative Genetics Study (OCGS) is an ongoing, NIMH (National Institute of Mental Health)-funded collaboration among investigators at seven sites in the United States (namely Brown University, Columbia University, University of Southern California, Johns Hopkins University, Massachusetts General Hospital, University of California at Los Angeles and the NIMH). The methods of the study are described in detail elsewhere33 and are summarized below.

The OCGS targeted families that comprised at least one sibling pair in which both siblings were affected with OCD and extended these pedigrees when possible through affected first- and second-degree relatives. Family history interviews were conducted to determine whether there were at least two OCD-affected siblings in the family who were willing to participate and to identify additional affected relatives. All first- and second-degree relatives were considered for inclusion, and families were extended through the first-degree relatives of all the affected cases.

To be considered affected, a participant had to meet the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4th edition) OCD diagnostic criteria at any time in his or her life. Probands were included if, in addition to meeting the DSM-IV criteria, their first onset of obsessions and/or compulsions occurred before 18 years of age. Probands with schizophrenia, severe mental retardation, Tourette's disorder, or secondary OCD (OCD occurring exclusively in the context of depression) were excluded. Participants had to be at least 7 years of age to participate in the study.

SNP genotyping

A total of 13 SNPs were genotyped using SNPlex chemistry according to the manufacturer's instructions (protocol P/N 4360858 Revision B), and genotype calls were made using Applied Biosystems (ABI, Foster City, CA, USA) GeneMapper v4.0 Software. A SNP set was assembled using data from phase I of the HapMap Project (http://hapmap.org), and a ‘tag’ set of SNPs was generated using the Tagger server (http://broad.mit.edu/mpg/tagger/index.php), using parameters of pairwise tagging, r2 of 0.99 and a minor allele frequency threshold of 1%. Additional non-synonymous SNPs were selected with preference given to those with reported allele frequencies. The overall genotyping completion rate for the 13 SNPlex-assayed SNPs was 98.5% (96.7–99.0%). The discordance rate for 48 randomly plated blind duplicates was 3 genotypes in a total of 624 genotypes (0.5%).

381 Base pair deletion genomic DNA amplification

Amplification was performed in a final volume of 5μl containing 20ng genomic DNA template, 200μM dNTPs, 1.5M betaine (Sigma-Aldrich, St Louis, MO, USA), 50mM KCl, 20mM Tris-HCl (pH 8.4), 2.5mM MgCl2, 200nM primers (F-CTCCAGCATTCTCCTTGCAC and R-TGAGCCCAGGAATTCAAGAC (Invitrogen, Carlsbad, CA, USA)) and 0.25Units Platinum Taq DNA polymerase (Invitrogen), then cycled at 95°C for 1min, followed by 10 cycles at 95°C for 20s, 61–56°C for 20s (decreased by 0.5-°C intervals per cycle) and 72°C for 1min, followed by 35 cycles at 95°C for 20s, 56°C for 20s and 72°C for 1min, with a final 30min at 72°C on a DNA Engine Dyad PTC-220 thermal cycler (MJ Research, Waltham, MA, USA). Amplified samples were run on 2% agarose in 1 × TBE (Tris/borate/EDTA) with EtBr (Cambrex Bio Science, Rockland, ME, USA) and examined under ultraviolet fluorescence.

Microsatellite and LPR genotyping

A PCR ‘triplex’ for SLC6A4 LPR, intron 2 and intron 7 was performed in 2.5μl containing 25ng genomic DNA template, 400μM dNTPs, 1.5mM 10 × PCR Buffer IV (Abgene, Surrey, UK), 3.0mM MgCl2, 200nM primers for the LPR (F-FAM-ATGCCAGCACCTAACCCCTAATGT; R-GGACCGCAAGGTGGGCGGGA), 200nM primers for intron 2 (F-FAM-GTCAGTATCACAGGCTGCGAG and R-TGTTCCTAGTCTTACGCCAGTG), 100nM primers for intron 7 (F-HEX-ACCGCACCCCGTCTCTCTCTTT and R-ACACCTGTAAGCACAGCCACTTG) (Invitrogen) and 1.0Units Platinum Taq DNA polymerase (Invitrogen), then cycled according to the following protocol: at 96°C for 5min, followed by 35 cycles of 96°C for 45s, 68°C for 30s and 72°C for 1min, followed by a final extension at 72°C for 10min on a DNA Engine Dyad PTC-220 thermal cycler. Samples were then subjected to capillary electrophoresis on an ABI 3100 fragment analyzer (ABI) and the following size ranges were observed: 5-HTTLPR (375 or 419bp); Int2 VNTR (variable number tandem repeat) (260–310bp); and Int7 GAAA (263–288bp). Microsatellite genotyping calls were made using Gene Marker v1.5 software (SoftGenetics, LLC, State College, PA, USA). A total of 48 randomly distributed blind duplicates showed no discordances. This assay achieved the following genotype completion rates: LPR (99.6%); Int2 (98.2%); and Int7 (99.6%).

LPR-rs25531 (A/G) SNP variant

Phase-certain haplotyping of the LPR variants LA, LG, SA and the rare SG allele was performed by a two-step protocol as follows. Step I: determination of the LPR L or S allele, as mentioned above; and step II: digestion of this amplicon with MspI restriction endonuclease. The assay was designed to include an invariant MspI digest site located 94bp from the end of the LPR amplicon to provide an internal control for digestion/partial digestion (Supplementary Figure 1). After separation of the digestion products by capillary electrophoresis and analysis using Gene Marker v1.5, the following restriction fragment allele sizes were obtained: SA (281bp), LA (325bp) and SG and LG (151bp). When these data are combined with the results from the undigested fragment data, unambiguous haplotypes can be deduced (see legend to Supplementary Figure 1). All of the rare SG alleles were confirmed by capillary DNA sequencing. The 48 randomly distributed blind duplicates were 100% concordant. Completion rate for the LPR-rs25531 genotyping (step II) was 96.5%.

Statistics

Family-based association analysis
 

We used the FBAT and PDT (Pedigree disequilibrium test) (http://www.chg.duke.edu/software/pdt.html)34, 35, 36 to run two-point SNP analyses under additive, dominant, recessive, and genotypic models and for haplotype-based association analysis.

Corrections for multiple testing
 

Two-point P-values from the FBAT were corrected for multiple-marker testing in the presence of linkage disequilibrium (LD), and also multiple-model assessments, but not gender. The PACT (P-values adjusted for correlated tests) approach adjusts for multiple comparisons by calculating the null distribution of the test statistics through numerical integration of their asymptotic joint distribution. This approach has been shown to be reliable and highly efficient for the adjustment of multiple comparisons.37

Haplotype analysis and haplotype phasing
 

We used Haploview 4.1 (www.biostat.harvard.edu) to assess LD structure at the SLC6A4 locus using the OCGS genotype data (17 markers). We adopted two LD blocks defined by the solid spine approach (extension with D′> 0.95) that encompassed our set of polymorphic markers for all haplotype analysis in the FBAT (Supplementary Figure 2). The HapMap phase III data set supported this LD block structure (Supplementary Figure 3). Haplotype analysis was conducted on only the Caucasian portion of the sample by removing 47 individuals who reported non-Caucasian ethnicity, which included 5 entire pedigrees (see Table 4, pedigree ethnicity summary).


In our haplotype analysis, we assigned the most likely estimated haplotype to each individual. Given that the haplotypes were estimated within haplotype blocks with tight LD structure for SNPs within a block, haplotype diversity was low with estimated haplotype posterior probabilities within each block >92%. Thus, the method of assigning the most likely haplotype yields similar results to an approach using the expectation or haplotype dosage.38, 39, 40 The permutation method implemented in the FBAT was used to compute haplotype whole-marker (minimal) P-values (http://biostat.harvard.edu/~fbat/default.html).

Genotype relative risk
 

Conditional logistic analysis was carried out using the STATA package (Cambridge Institute for Medical Research, Addenbrooke's Hospital, Cambridge, UK) (http://www-gene.cimr.cam.ac.uk/clayton/software/stata).

Power calculations
 

In separate calculations assessed in PBAT (http://www.goldenhelix.com/SNP_Variation/PBAT/), the power to detect association assuming an additive model of inheritance with a disease allele frequency of 0.05 and a population prevalence of 0.03 using the 278 families (459 nuclear families) already collected is roughly 0.60 (if the D′ between the disease locus and candidate SNP is 1.0).

Sex-stratified analyses
 

We stratified the data set by the gender status of male or female OCD-affected individuals in each family. We coded the affection status of either all female OCD-affected individuals as ‘unknown,’ or all male affected individuals as ‘unknown,’ and analyzed using the FBAT. P-values for sex-stratified analyses were not corrected for the three hypotheses tested (all, male-only and female-only).

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Results

We genotyped 278 pedigrees containing 459 nuclear families and 1241 individuals (>96% Caucasian pedigrees; see Table 4) with 14 SNPs, the LPR indel and 2 VNTR polymorphisms in introns 2 and 7 within SLC6A4 to determine whether they are in LD with OCD (Table 1, Supplementary Figure 4). Three SNPs, rs6353 (minor allele frequency=0), I425V and rs140699 (minor allele frequency=0.003) with low minor allele frequencies did not have a sufficient number of families to calculate the two-point results using the FBAT and were also excluded from the haplotype analysis. In addition, we also failed to detect a 381-bp deletion variant located immediately 3′ of the LPR41 in 354 randomly selected samples from 102 families. All other SNP data were confirmed to be in the Hardy–Weinberg equilibrium and were used to determine the haplotype structure in the region, which was in agreement with the haplotype structure of SLC6A4 observed with HapMap Project phase III data (Supplementary Figures 2 and 3).


We conducted two-point analyses using the FBAT under additive, dominant, recessive and genotypic models and the results for the additive model are presented in Table 2, parts a and c. All allele-model combinations for the whole sample that yielded nominal P-values <0.05 are presented in Table 3. Specifically, under an additive model, we observed nominal evidence of positive association with allele 5 of the microsatellite repeat in intron 7 (P=0.0330; 14 informative families). Under a genotypic model, the intron 2 genotype containing alleles 9/10 showed a positive association (P=0.0089; 13 families), and the intron 7 genotype containing alleles 3/7 was inversely associated with OCD (that is, with reduced risk) (P=0.0187; 105 families). Given that a number of correlated tests were conducted and that significant LD existed within the set of markers (see Supplementary Figure 2), we used the method described in the study by Conneely and Boehnke37 to correct jointly for multiple marker comparisons, in the setting of LD. On the full sample, none of the resulting two-point analysis adjusted P-values (PAdj) were <0.05 (Table 3). The results did not change appreciably when analyzed using the PDT.



The most notable negative results were at the LPR locus. We found no evidence of association with either the LPR polymorphism or with the molecular haplotype of LPR-rs25531 with any of the genetic models (additive model data shown in Table 2, Supplementary Table 1). We observed 10 individuals with the SG variant, all of which were confirmed by DNA sequencing. Two of these individuals had a LPR variant similar to that as reported previously (see variant 14B in Nakamura et al.42) (data not shown). We investigated whether these results were influenced by either genotypic (for example, LPR allele frequencies) or phenotypic heterogeneity (for example, %females, ethnicity, mean age of onset of symptoms) across the different clinical sites. No evidence of either genotypic or phenotypic heterogeneity was observed (Table 4, parts a and b). Furthermore, we did not observe association of the LPR-rs25531 haplotype when each site was analyzed individually.

Three individuals with the rare 425V variant were detected, and we examined their pedigrees in greater detail. In all three, the individual with 425V was the biological parent of an OCD-affected sib-pair. Two of the carriers were fathers: one with OCD (1808–3), one with OCPD (2113–69); the third individual was an unaffected mother (2947–68). However, none of these individuals passed the 425V variant to their OCD-affected sib-pair offspring (confirmed by DNA sequencing). This nonsegregation of 425V to the six OCD-affected offspring was unexpected; such segregation by chance alone should only occur 1/64th of the time. Two of the 425V carriers were diagnosed with depressive disorders (1808–3 with probable MDD (major depressive disorder) and 2947–68 with recurrent MDD/dysthymia), two had skin picking (1808–3 (probable) and 2947–68 (definite)); and two had probable alcohol dependence (1808–3 and 2113–69) (see Supplementary Table 2).

We also analyzed the data stratified by sex using FBAT (similar PDT results), under the hypothesis that genetic loci for OCD may be sex specific (Tables 2 and 3). One SNP, rs2020930, located 5′ to the LPR had a nominal P-value of <0.05 in males, but this did not withstand correction for multiple testing (PAdj=0.3379). The most statistically significant two-point findings were obtained using a genotypic model and showed association in females with intron 2 alleles 9/10 (P<0.0002; 10 informative families). With PAdj=0.0054, this finding withstood the joint correction for multiple marker comparison and LD. We then further adjusted the statistical significance for the testing of multiple genetic models, and the result remained significant (PMAdj=0.0069) (Table 3). Even after using a conservative Bonferroni correction for the testing of three sex-based models (all, male-only and female-only), this result is still significant after correction for all the conducted tests (P<0.017).

Finally, we carried out a haplotype analysis with FBAT for the two observed haplotype blocks using the entire and sex-stratified Caucasian sample. The 5′-block (rs2020930:rs4392119:LPR-rs25531:rs25533:rs2020933) contained the LPR and the first exon, whereas the 3′-block (rs6355:Int2:rs140700:Int7:rs4583306:rs7224199) included the rest of the gene (Table 2, Supplementary Figures 2 and 3). Neither block achieved whole-marker significance using a permutation test. In the 5′-block, four individual haplotypes had P<0.05 of the 18 observed haplotypes. Interestingly, both individual haplotypes containing a high-expressing LA allele conferred risk (ACLATA and GTLATT, although in different sexes), whereas the two individual haplotypes containing low-expressing alleles (GTSATT and GCLGTT, principally in males) were protective. We note that the individual haplotypes GTLATT and GTSATT are identical except at LPR-rs25531, yet have opposite effects on OCD risk. In the 3′-block of 6 markers (including 2 microsatellites) and 25 observed individual haplotypes, 3 haplotypes with P<0.05 were observed. Two of these contain the 267-bp allele at intron 2, which is part of the 250/267 genotype that was significant after correction. The set of 10 female families (250/267 genotype, Table 3) overlapped with the set of 8 female families (267-bp allele-containing haplotype, Table 5) in only a single female. We note that individual haplotype P-values are uncorrected for multiple testing.


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Discussion

The serotonin transporter, SLC6A4, is the molecular target of the SSRIs, which are the current medications of choice for the treatment of OCD. There have been multiple genetic association studies of OCD and SLC6A4 and although some of these have been positive, overall, the results have been equivocal. In an effort to determine whether variation in SLC6A4 alters the probability of an individual developing OCD, we genotyped 1241 DNA samples from the OCGS OCD-affected sib-pair study (278 pedigrees, 459 nuclear families, 1598 total persons) with 17 marker loci.

After correction for multiple testing with several models and marker loci, none of the two-point results using the entire sample remained statistically significant. Given the suggestion of gender differences that have been observed in genetic association studies of OCD and HTR2A, COMT and MAOA,43, 44, 45 as well as in segregation studies,3 we also analyzed our data stratified by sex, under the hypothesis that the genetic loci for OCD may be sex specific. In these analyses, genotype 9/10 of the Int2 VNTR was associated with OCD in females both before (P<0.0002) and after correction for LD structure and multiple marker comparisons (PAdj=0.0054). This result remained significant after we further corrected for the testing of additive, dominant, recessive and genotypic genetic models (PMAdj=0.0069). There have been suggestions of sex-specific OCD findings for SLC6A4, for the LPR, in a Dutch sample46 and in a recent meta-analysis;10 the latter did not withstand multiple-marker nor multiple-analysis corrections, whereas the former may have. Although we approached sex differences in the conferring of OCD risk as separate hypotheses, we note that even with additional correction, findings remain significant with P<0.0167.

In the OCGS sample, there seems to be little difference in the OCD symptomatology between males and females. There are no sex differences in Y-BOCS (Yale–Brown Obsessive Compulsive Scale) severity scores, numbers of different categories of obsessions or compulsions or in the proportions treated for their OCD symptoms. There are sex differences in the prevalence of several Axis I disorders, females showing more recurrent major depression, hypomania, dysthymia, hypochondriasis, skin picking, panic disorder, agoraphobia, social phobia, generalized anxiety disorder, anorexia and bulimia and less Tourette's disorder, alcohol dependence and Asperger's syndrome (AS).33 Serotonin has been hypothesized to have a role in many of these disorders. Given that the majority of OCD-affected individuals in the OCGS sample are females (66%), it is possible that the observation of the association with alleles 9/10 of the Int2 VNTR in the OCGS females may be because of the presence of comorbid Axis I disorders, rather than OCD.

There have been previous observations of association of alleles at the Int2 marker with a number of neuropsychiatric disorders. Allele 9 has been associated with both unipolar depression47, 48 and depression in the context of bipolar disorder.48 The Int2 allele 12 has been associated with bipolar disorder,49 schizophrenia (meta-analysis),50 autism51 and anxiety disorders with and without OCD, in Japanese subjects, but not in Caucasians.52 In addition, less favorable SSRI treatment response was observed in Asians with major depression and the Int2 10/12 genotype in a meta-analysis.53 More recently, the study of OCD in a Spanish Caucasian sample found an excess of 12/12 and 12/10 genotypes in cases.54

The functional mechanism driving the associations of these Int2 alleles in vivo may be complex and may involve combined effects with the LPR polymorphism.55 VNTR copy number variation has been shown to exert effects on transcription,56 as have VNTR sequence variants on differential reporter gene expression.57 Recent investigation of the Int2 VNTR as a target for mediating a transcriptional response to LiCl by the transcription factors CTCF and YB-1, found that in vivo transcriptional variation was correlated with differential binding of both these transcription factors to the three distinct VNTR variants after exposure to LiCl, suggesting differential allelic gene expression.18

Although there is evidence that the Int2 VNTR can influence the expression of SLC6A4, it is the LPR indel that has been most extensively studied. Early studies of the LPR by Lesch et al.58 observed that the transcriptional activity of the L allele was two- to threefold higher than that of the S allele in lymphoblastoid cell cultures. The L allele was associated with higher platelet serotonin uptake.59, 60 Consistent with many previous reports, we failed to detect an association of the LPR with OCD in the OCGS sample, either in the combined-sex data or when analyzing by sex.

It seems that additional variation within the LPR affects the strength of the SLC6A4 promoter. Hu et al.1 showed that the A-to-G base change at rs25531 within the LPR forms a haplotype (LG) that has a comparable low activity with the S allele. They also found that the LA allele was associated with OCD in both case–control and trio samples and conferred a 1.8-fold increased risk (OR) of developing the disorder. Another published report of the haplotype found equivocal replication with LA/LA, or LA alone, in a case–control study of 347 OCD-affected individuals vs 749 population-matched controls, and results did not withstand the correction for multiple testing.61 We generated phase-certain haplotypes of the LPR-rs25531, but failed to observe any evidence that the LA/LA genotype conferred risk for OCD. In addition, we did not observe an association with either LA or LG under additive, dominant, recessive or genotypic models, either in the total data set or when stratified by sex. In our sample, the genotype relative risk for the LA/LA genotype vs all others was 1.11 (95% confidence interval: 0.77–1.60, P=0.58). Although positive, this risk estimate is markedly less than that reported by Hu et al. (see also Supplementary Table 1).

Phenotypic and/or genotypic heterogeneity may have contributed to the lack of replication in this multisite association study. Factors such as ethnicity, method of ascertainment, site of patient recruitment, mean age at the time of patient recruitment and mean age at the onset of OCD symptoms all may have a contributory role. We did not observe significant differences when analyzing these parameters by site of patient recruitment. We then assessed for genetic heterogeneity in our sample by examining LPR L and S alleles, and LPR-rs25531 haplotype frequencies in the OCGS sample by site. We did not detect any significant differences.

Alternatively, it is possible that the results of our study differ from those of Hu et al.1 because of additional unmeasured genetic variation in the SLC6A4 gene that has a functional effect on the promoter of the gene. Two SNPs in the first intron of SLC6A4, namely rs16965628 and rs2020933, were shown to be correlated with allelic expression imbalances62 and rs25532 has been suggested to affect expression.61 The latter report described a functional C to T SNP (rs25532) located near the LPR; its minor allele significantly decreased luciferase reporter gene expression levels. Haplotype-based testing of rs25532 and all other known noncoding functional SLC6A4 variants showed significant overrepresentation in probands who had the higher-expressing allele at each locus, supporting the notion of increased serotonin transporter functioning being pathogenetically involved in OCD. Conditional haplotype analyses showed that this association was driven by the LPR, rs25532 and rs16965628 in concert. Finally, our results may differ from those of Hu et al. because of the characteristics of the respective samples. The OCGS sample is composed entirely of familial OCD cases,34 whereas the case–control and trio samples contain a higher proportion of sporadic cases that may have a different genetic basis.

We also investigated several rare variants within SLC6A4 to determine whether they were associated with OCD in our sample. We found no individuals with either rs6353 (T439T) or the 381-bp deletion variant located immediately 3′ of the LPR that has been previously shown to contain several canonical transcription-factor binding sites. rs6353 is believed to be functional57, 17 and has been associated with both autism and depression.63, 64 We also investigated the rare SNP that caused a non-synonymous change from isoleucine to valine at position 425 of SLC6A4, which has been suggested to contribute to an uncommon familial form of OCD in two unrelated pedigrees with OCD and other serotonin-related comorbidities.2 We observed three individuals from three pedigrees with 425V, all of whom were parents of OCD-affected sib-pairs. Collectively, these three individuals had six OCD-affected offspring, none of whom received the 425V allele from their parents, a result that should occur in only 1/64th of such segregations. These genetic data are not supportive of the hypothesis that 425V is solely responsible for the ‘OCD plus syndrome.’2, 65 We also tested the ‘double-hit’ hypothesis proposed by Ozaki et al.,2 in which homozygosity of the L allele plus the 425V variant increases both transcription and functional activity, and hence increases the risk for OCD. Consistent with this hypothesis, the single 425V carrier who was affected with OCD is homozygous for the LPR L allele. Haplotype analysis of LPR-rs25531 showed that this individual is LG/LA, and hence a functional heterozygote. Haplotype analysis of the three pedigrees segregating the 425V variant found that this rare variant was found on an LPR LA background (the other two 425V carriers were S/LA), suggesting one mutation event, possibly as a shared ancestral event in these pedigrees. The 425V allele has been observed in controls66, 65 and in another study in families with pervasive developmental disorders, eating disorders and OCD, but other large studies on various types of patients and controls have failed to detect it.67, 68 Non-transmittal of the 425V allele to six definite OCD cases proves that those OCD cases in high-density OCD families have some other cause of their OCD.

Ozaki et al. have suggested the 425V variant as a possible mediator of SSRI treatment response or as a mediator/exacerbator of neuropsychiatric comorbidity.2 They hypothesized an ‘atypical OCD plus,’ a syndrome incorporating OCD plus AS/autism/social phobia and eating disorders. We failed to find support for any of these hypotheses, as we observed no instances of 425V in the 32 self-reported SSRI treatment nonresponders, and although each of the three 425V carriers do have a complex neuropsychiatric phenotype, it differs from ‘atypical OCD plus.’ We suggest that 425V might predispose to a spectrum of complex serotonergic phenotypes that is wider than that observed by Ozaki et al., and that OCD may not be a necessary component of this spectrum.

Family-based haplotype reconstruction has shown higher reliability when compared with haplotype reconstruction from unrelated individuals.69 When analyzing haplotypes, we excluded the small number of non-Caucasian pedigrees that may have contributed to heterogeneity. Several less common individual haplotypes do show the LA allele associated with OCD risk, and the SG and SA alleles comparably associated with inverse risk. In contrast, the two-point analysis at the LPR showed no association with OCD. Investigation of the COMT gene recently showed that although single variants might fail to show association (for example, COMT V158M), functional haplotypes containing these alleles can show strong association (for example, TMJ (temporomandibular joint) disorder and pain sensitivity).70 The selection of haplotype blocking algorithm in the context of degree of LD at the given locus is known to affect association findings and type 1 error rates.71 We noted that several of the blocking algorithms (for example, Gabriel et al. and Patil et al.) gave marginally significant findings after correction for the number of LD blocks tested in the 3′-haplotype block, notably when a significant portion of the marker set was excluded (for example, Gabriel algorithm) or when haplotype ‘windows’ approached small numbers of markers (for example, Patil algorithm). Hence, the full significance of these individual haplotypes is yet to be determined; some may confer appreciable risk in a genotypic subset of this sample.

In conclusion, we found evidence of genetic association at the SLC6A4 locus with OCD. The lack of association at the LPR, the LPR-rs25531 haplotype and the rare 425V variant is noteworthy. Current hypotheses about the risk of specific variants need revision to accommodate these new findings, including a possible gender effect.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank the many families who have participated in the OCGS; the OCF; Ann Pulver, PhD, Kathleen Merikangas, PhD, David Houseman, MD and Alec Wilson, PhD, for consultation; and clinicians and coordinators at each OCGS site: Providence (Maria Mancebo, PhD, Richard Marsland, RN and Shirley Yen, PhD); New York (Renee Goodwin, PhD, Joshua Lipsitz, PhD and Jessica Page, PsyD); Baltimore (Laura Eisen, BS, Karan Lamb, PsyD, Tracey Lichner, PhD, Yung-mei Leong, PhD and Krista Vermillion, BA); Boston (Dan Geller, MD, Anne Chosak, PhD, Michelle Wedig, BS, Evelyn Stewart, MD, Michael Jenike, MD, Beth Gershuny, PhD and Sabine Wilhelm, PhD); Bethesda (Lucy Jestement, Diane Kazuba, V Holland LaSalle-Ricci and Theresa B DeGuzman); and Los Angeles (R Lindsey Bergman, PhD, Susanna Chang, PhD, Audra Langley, PhD and Amanda Pearlman, BA). In addition, we wish to thank Jason Briggs at ABI for his technical assistance during set-up of the SNPlex assay in a high-throughput, 384-well robotized format, Ming-Chen Chien, MS, at the Columbia University Genome Center, Dave Conti, USC, Keck School, Biostatistics and Dr Jonathan Liu and colleagues, Soft Genetics, Inc. for early design versions of Gene Marker & Mutation Surveyor softwares.

Funding Source: This study is supported by NIMH R01 MH50214 and NIH/NCRR/OPD-GCRC RR00052 (GN). EV is a recipient of a 2006 grant award from the Obsessive Compulsive Foundation (OCF) and a departmental fellowship award (USC, Keck School, Psychiatry). OE received a Young Investigator Award from NARSAD. YYS initiated her contribution to this report while working at JHU, and is currently working at the Genomic Research Branch, NIMH. Part of this work was completed as her outside activity, and as such the views expressed in this article do not necessarily represent the views of the NIMH, NIH, HHS, or the US Government. SAR and DLP are funded by the NIMH. SLR has received honoraria and/or consultation fees from Neurogen, Sepracor, Novartis and Medtronic, and has conducted research funded by Medtronic, Cyberonics, Cephalon and Northstar. JTM has received research support from Eli Lilly, Bristol Myers, Squibb, Seaside Pharmaceuticals, and Aspect. Other relevant NIMH awards: K23-MH066284 (MG) & K23-MH64543 (OJB). JAK is funded by the NIMH, CMREF and NARSAD, and this project was funded by NIMH grants MH079494 & MH050214. He is a member of the scientific advisory boards of SoftGenetics, Inc. & Life Technologies, Inc. and has received compensation from the latter.

Supplementary Information accompanies the paper on the Molecular Psychiatry website