in the serotonin transporter gene

Obsessive–compulsive disorder and the promoter region polymorphism (5-HTTLPR) in the serotonin transporter gene (SLC6A4) : a negative association study in the Afrikaner population

B R I E F R E P O RT

International Journal of Neuropsychopharmacology (2000), 3, 327–331. Copyright # 2000 CINP

Craig J. Kinnear1, Dana J. H. Niehaus2, Johanna C. Moolman-Smook1, Pieter L. du Toit2, Jeanine van Kradenberg2, Jakobus B. Weyers1, Annemarie Potgieter2, Vanessa Marais2, Robin A. Emsley2, James A. Knowles3, Valerie A. Corfield1, Paul A. Brink1 and Dan J. Stein2 " MRC\US Centre for Molecular and Cellular Biology, University of Stellenbosch, PO Box 19063, Tygerberg 7505 # MRC Unit on Anxiety and Stress Disorders, University of Stellenbosch, PO Box 19063, Tygerberg 7505 $ Columbia Genome Centre, Columbia University and the New York State Psychiatric Institute, 1051 Riverside Drive, New York, NY 10032

Abstract A polymorphism (5-HTTLPR) in the promoter region of the serotonin transporter gene (SLC6A4) has been reported to have functional significance and to be associated with obsessive–compulsive disorder (OCD). However, other studies have generated confounding results. A study was undertaken to re-evaluate this association in subjects drawn from the relatively genetically homogeneous Afrikaner population of South Africa. Fifty-four OCD patients of Afrikaner descent and 82 ethnically matched control individuals were phenotyped and genotyped. No significant association was found between the distribution of the 5-HTTLPR genotypes at the SLC6A4 locus and OCD. A similar result ( p l 0.108) was generated when a meta-analysis of the 5-HTTLPR polymorphism, combining the current study with a previously reported Caucasian group, was performed ; the meta-study comprised 129 OCD patients and 479 control individuals. However, both studies lacked power. Therefore, evidence that variation in SLC6A4 plays a significant role in the development of OCD in the population groups studied is inconclusive. Future association studies in Caucasian populations may extend the power of such meta-analyses and assist in delineating the role of SLC6A4 in OCD. Received 25 January 2000 ; Reviewed 3 April 2000 ; Revised 21 June 2000 ; Accepted 29 June 2000 Key words : Serotonin, serotonin transporter, polymorphism, Afrikaner.

Introduction Obsessive–compulsive disorder (OCD) is a prevalent (Karno et al., 1988) and disabling (Murray et al., 1996) psychiatric disorder characterized by recurrent intrusive obsessions and neutralizing compulsive behaviour. There is growing evidence that OCD has a specific neurochemical and neuroanatomical basis (Stein, 1996). Furthermore, genetic mechanisms may contribute to the pathogenesis of OCD ; in family studies, there is a higher concordance rate for monozygotic twins compared with dizygotic twins (Rasmussen and Tsuang, 1986), and up to

Address for correspondence : Professor D. J. Stein, MRC Unit on Anxiety and Stress Disorders, Department of Psychiatry, University of Stellenbosch, Medical School, PO Box 19063, Tygerberg 7505, South Africa. Tel. : j27219389161 Fax : j27219335790 E-mail : djs2!gerga.sun.ac.za

35 % of first-degree relatives of OCD probands also suffer from this disorder (Carey and Gottesman, 1981). Although the majority of candidate gene approaches have found no strong association between any particular polymorphism and OCD (Altemus et al., 1996 ; Brett et al., 1995 ; Nicolini et al., 1996 ; Novelli et al., 1994), Karayiorgou et al. (1997) reported an association between a functional polymorphism, resulting in a 3- to 4-fold reduction in enzyme activity, in the catechol-O-methyltransferase (COMT) gene and OCD. Homozygosity for the low-activity COMT genotype conferred a higher risk of OCD in North American Caucasian males (Karayiorgou et al., 1997). However, in a study conducted in the Afrikaner population of South Africa, it was reported that the heterozygous COMT genotype was associated with OCD, independently of gender (Niehaus et al., In Press). The serotonin transporter gene (SLC6A4) is an attractive candidate gene for involvement in OCD. First, as considerable evidence implicates the serotonin (5-HT)

328

C. J. Kinnear et al.

system in the development of OCD, including the finding that OCD responds selectively to 5-HT reuptake inhibitors (Murray et al., 1996). Secondly, a 44 bp insertion\ deletion (l\s) polymorphism (5-HTTLPR) in the promoter region of SLC6A4 has been shown to have a significant effect on blood 5-HT content (Hanna et al., 1998 ; Lesch et al., 1996). However, investigations of a possible role for SLC6A4 polymorphisms in OCD pathogenesis have generated confounding results. Whereas the 5-HTTLPR functional variant was implicated in playing a role in the development of OCD in two independent European– American population studies (Bengel et al., 1999 ; McDougle et al., 1998), re-evaluation of these findings in a study of a Mexican population revealed no significant association (Nicolini et al., 1999). Furthermore, Billet et al. (1997) had previously concluded that 5-HTTLPR variants did not appear to influence response to pharmacotherapy in OCD, thus raising some doubt about the functional importance of the association studies. In addition, early reports of an association between SLC6A4 polymorphisms and anxious and depressive traits (Lesch et al., 1996) have not been replicated (Gelernter et al., 1998). A strategy used to reduce aetiological complexity, which may underlie the divergent results encountered in association studies such as those discussed above, is to investigate candidate genes in genetically homogeneous population groups. The Afrikaners are a group of Afrikaans-speaking South Africans of northern European descent, whose history over the past 150 years has contributed to their genetic isolation and relative genetic homogeneity (Moolman-Smook et al., 1999 ; Niehaus et al., 1999 ; Simonic et al., 1998). This population has been used successfully in a comparative genome scan of patient and control groups (Simonic et al., 1998), to identify genetic markers associated with Tourette’s syndrome, a disorder that is believed to share a genetic basis with OCD (Pauls et al., 1995). Other studies have also employed this population to delineate genetic risk factors in a variety of disorders (Moolman-Smook et al., 1999 ; Starfield et al., 1997). Consequently, we studied the distribution of genotypes of 5-HTTLPR in Afrikaners with OCD. We then included previously reported data in a meta-analysis. Neither analysis detected association between 5-HTTLPR and OCD, but both were lacking in power.

Methods Subjects Fifty-four unrelated patients with OCD (26 male and 28 female) were recruited from the MRC Research Unit on

Anxiety and Stress Disorders. Eighty-two unrelated control individuals were recruited from the community. All subjects were of Afrikaner descent based on home language and reported descent. The protocol was approved by the University of Stellenbosch Institutional Review Board, and all subjects provided written, informed consent. All subjects underwent a structured interview by a trained research clinician. The interview included the SCID-I (First et al., 1994), the Yale–Brown Obsessive Compulsive Scale (YBOCS) and the Yale Global Tic Severity Scale (YGTSS) (Goodman et al., 1989 ; Leckman et al., 1989). All patients met the DSM-IV criteria for OCD, and control individuals had no history of an anxiety, mood or psychotic disorder. Four OCD patients, but no control individuals, had current or past tics.

Subjects included in meta-analysis The meta-analysis of 5-HTTLPR was performed using a combination of the Afrikaner subjects and data generated by Bengel et al. (1999), the latter consisting of 75 unrelated Caucasian OCD subjects, all of whom underwent the Structured Clinical Interview for DSM-III-R, as well as the YBOCS, and 397 unrelated controls (Table 1). Two other association studies were excluded from the meta-analysis, namely, the dataset generated with Japanese subjects (Ohara et al., 1998), in which allele distributions at 5-HTTLPR in the control group differed markedly from the European–American and Afrikaner control groups (Tables 1 and 2, respectively), and the data of McDougle et al. (1999), in which a TDT strategy was used.

Genotyping The region containing the 5-HTTLPR polymorphism was amplified by polymerase chain reaction (PCR) using published primer sequences (Gelernter et al., 1997). The amplification was performed in a 50 µl reaction containing 0.1 µg genomic DNA, 75 µ each of dATP, dGTP, dCTP and dGTP (Promega Corp., Madison, WI, USA), 5 µl of a 10iTaq DNA polymerase buffer (Bioline UK Ltd, London, UK, 1.5 m MgCl , 0.5 U Taq DNA polymerase # (Bioline UK Ltd) and 5 % glycerol (Sigma Chemical Co., St Louis, MO, USA). Amplification was performed in a thermocycler (Gene E, Techne Ltd, Cambridge, UK) with cycling parameters set as follows : 94 mC for 5 min (1 cycle) followed by 30 cycles of 94 mC for 30 s, 66 mC for 30 s, 72 mC for 30 s. This was followed by a final extension cycle of 72 mC for 10 min. The l and s 5-HTTLPR PCRamplified alleles of SLC6A4 were size-separated by

5-HTTLPR polymorphism and OCD

329

Table 1. Distribution of 5-HTTLPR genotypes in previously reported studies Study 1*

OCD subjects Control subjects

Study 2†

l\l

l\s

s\s

Total

1 (7.7 %) 8 (7.2 %)

2 (15.4 %) 40 (36.4 %)

10 (76.9 %) 62 (56.4 %)

13 110

l\l

l\s

s\s

35 (46.6 %) 98 (24.7 %)

29 (38.7 %) 214 (53.9 %)

11 (14.7 %) 85 (21.4 %)

Total 75 397

* Ohara et al. (1998) (excluded from meta-analysis). † Bengel et al. (1999) (included in meta-analysis). Table 2. Distribution of 5-HTTLPR genotypes and alleles in OCD subjects and control individuals of Afrikaner descent Genotype distribution l\l OCD subjects Control subjects

l\s

11 27 (20.3 %) (50 %) 27 39 (32.9 %) (47.6 %) χ# l 3.29, 2 d.f., p l 0.19

Allele frequencies s\s

Total

l

s

16 (29.7 %) 16 (19.5 %)

54

0.55

0.45

82

0.43

0.57

χ# l 2.88, 2 d.f., p l 0.09

electrophoresis in a 12 % non-denaturing polyacrylamide gel and visualized by silver staining. Statistical analysis Experimental and control groups were tested for Hardy– Weinberg equilibrium and genotypes of affected vs. control groups from the Afrikaner and pooled datasets were compared using χ# analysis. Results

between the observed genotype distribution ( p l 0.19) and allele frequencies ( p l 0.09) of the 5-HTTLPR polymorphism in the two groups (Table 2). With 54 OCD cases, it was not possible to collect a control group of sufficient size to generate 80 % power to detect a difference of 20 % in distribution of 5-HTTLPR genotypes between cases and controls.

Meta-analysis

Analysis of Afrikaner data Both OCD-affected and control groups were in Hardy– Weinberg equilibrium. There was no statistical difference

The results of the meta-analysis showed no significant difference in the distribution of 5-HTTLPR genotypes between patient and control groups ( p l 0.10) (Table 3).

Table 3. Meta-analysis of distribution of 5-HTTLPR genotypes and alleles in OCD subjects and control individuals Genotype distribution l\l OCD subjects Control subjects

l\s

46 56 (35.7 %) (43.4 %) 155 253 (32.4 %) (52.8 %) χ# l 4.57, 2 d.f., p l 0.10

* Data from present study and Bengel et al. (1999).

Allele frequencies s\s

Total*

l

s

27 (20.9 %) 71 (14.8 %)

129

0.57

0.43

479

0.58

0.42

χ# l 0.02, 2 d.f., p l 0.88

330

C. J. Kinnear et al.

The calculated sample size required to detect a difference of 20 % between 5-HTTLPR genotype distributions with 80 % power at 5 % significance for the 129 OCD subjects in the pooled dataset was 1444 control individuals. Discussion The data generated in this case-control study did not support the hypothesis of an association between the distribution of genotypes of the functional 5-HTTLPR polymorphism in the SLC6A4 gene and OCD in the Afrikaner population (Table 2). However, as the power of the study failed to provide support at the 80 % level, a meta-analysis was performed. The latter analysis also did not detect any significant association between 5-HTTLPR genotypes and OCD (Table 3) but again lacked sufficient power to reject the alternative hypothesis that the 5HTTLPR polymorphism is involved in the development of OCD. Association studies are susceptible to errors that can generate false-positive or -negative results, thus making it important to re-evaluate findings in other circumstances. Previous association studies, conducted in other population groups to assess the role of the 5-HTTLPR polymorphism in the SLC6A4 gene in the pathogenesis of OCD, have produced discrepant results. In the present study, when the data of Bengel et al. (1999), which had previously supported involvement of the l allele of 5HTTLPR in the development of OCD, was included in the meta-analysis, the reported association was negated. Difficulties in confirming positive or negative associations detected between a specific candidate gene and a disease in different studies are often ascribed to underlying genetic differences between populations (Gelernter et al., 1999). Thus, it could be speculated that the influence of 5-HTTLPR in triggering OCD in susceptible individuals in diverse populations is affected by a complex interplay of polymorphic variants in a diverse spectrum of genes, acting in concert with environmental factors. Alternatively, it may be that the role played by variation SLC6A4 in disease pathogenesis of OCD is negligible or non-existent in the populations studied. Under these circumstances, random factors, which cannot be controlled in the design of the study, or other non-investigated and generally unknown influences, or both, may generate spurious results, which may be responsible for the discrepant datasets generated in reevaluation of the influence of SLC6A4 in OCD (Bengel et al., 1999). In summary, in the present study, neither the data generated in the homogeneous Afrikaner sub-population nor the meta-analysis, provided the power to exclude the possibility that SLC6A4 variants may play a role in the

pathogenesis. Future association studies in Caucasian populations may extend the power of such meta-analyses and assist in delineating the role of SLC6A4 in OCD. Furthermore, the interaction of identified genetic influences with other mediating factors, such as postinfective auto-immunity (Niehaus et al., 1999 ; Swedo et al., 1994), in precipitating OCD should be investigated. A larger cohort of clinically well-characterized OCDaffected individuals and a carefully matched control group, both drawn from the relatively homogeneous and genetically distinct Afrikaner population, will provide a valuable resource in continuing studies to identify susceptibility factors underlying this prevalent and incapacitating disease. Acknowledgements This work is supported by grants from the Obsessive– compulsive Foundation, the National Research Foundation and the MRC Unit on Anxiety and Stress Disorders (South Africa). References Altemus M, Murphy DL, Greenberg B, Lesch KP (1996). Intact coding region of the serotonin transporter gene in obsessive–compulsive disorder. American Journal of Medical Genetics 26, 409–411. Bengel D, Greenberg BD, Cora' -Locatelli G, Altemus M, Heils A, Li Q, Murphy DL (1999). Association of the serotonin transporter promoter regulatory region polymorphism and obsessive–compulsive disorder. Molecular Psychiatry 4, 463–466. Billet EA, Richter MA, King N, Heils A, Lesch KP, Kennedy JL (1997). Obsessive–compulsive disorder, response to serotonin reuptake inhibitors and the serotonin transporter gene. Molecular Psychiatry 2, 403–406. Brett PM, Curtis D, Robertson MM, Gurling HM (1995). Exclusion of the 5-HT1A serotonin neuroreceptor and tryptophan oxygenase genes in a large British kindred multiply affected with Tourette’s syndrome, chronic motor tics, and obsessive–compulsive behavior. American Journal of Psychiatry 152, 437–440. Carey G, Gottesman II (1981). Twin and family studies of anxiety, phobic and obsessive disorders. In Klein DF, Rabkin J (Eds.), Anxiety : New Research and Changing Concepts. New York : Raven Press. First MB, Spitzer RL, Gibbon M, Williams JB (1994). Structured Clinical Interview for Axis I DSM-IV Disorders. Biometrics Research Department, New York State Psychiatric Institute. Gelernter J, Cubells JF, Kidd JR, Pakstis AJ, Kidd KK (1999). Population studies of the polymorphism of the serotonin protein transporter gene. American Journal Medical Genetics 88, 61–66.

5-HTTLPR polymorphism and OCD Gelernter J, Kranzler H, Coccaro EF, Siever LJ, New AS (1998). Serotonin transporter protein gene polymorphism and personality measures in African American and European American subjects. American Journal of Psychiatry 155, 1332–1338. Gelernter J, Kranzler H, Cubes JE (1997). Serotonin transporter protein (SLC6A4) allele and haplotype frequencies and linkage disequilibria in African- and European-American and Japanese populations and in alcohol-dependent subjects. Human Genetics 101, 243–246. Goodman WK, Price LH, Rasmussen SA, Mazure C, Fleischmann RL, Hill CL, Heninger GR, Charney DS (1989). The Yale–Brown Obsessive Compulsive Scale I. Development, use, and reliability. Archives of General Psychiatry 46, 1006–1011. Hanna GL, Himle JA, Curtis GC, Koram DQ, Weele JV, Leventhal BL, Cook Jr. ER (1998). Serotonin transporter and seasonal variation in blood serotonin in families with obsessive–compulsive disorder. Neuropsychopharmacology 18, 102–111. Karayiorgou M, Altemus M, Galke BL, Goldman D, Murphy DL, Ott J, Gogos JA (1997). Genotype determining low catechol-O-methyltransferase activity as a risk factor for obsessive–compulsive disorder. Proceedings of the National Academy of Sciences USA 94, 4572–4575. Karno M, Goldin JM, Sorenson SB, Burnam MA (1988). The epidemiology of obsessive–compulsive disorder in five US communities. Archives of General Psychiatry 45, 1094–1099. Leckman JF, Riddle MA, Hardin MT, Ort SI, Swartz KL, Stevenson J, Cohen DJ (1989). The Yale Global Tic Severity Scale (1989). Initial testing of a clinician-rated scale of tic severity. Journal of the American Academy of Child Adolescence Psychiatry 28, 566-573. Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri s, Benjamin J, Muller CR, Hamer DH, Murphy DL (1996). Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274, 1527–1531. McDougle CJ, Epperson CN, Price LH, Gelernter J (1998). Evidence for linkage disequilibrium between serotonin transporter protein gene (SLC6A4) and obsessive–compulsive disorder. Molecular Psychiatry 3, 270–273. Moolman-Smook JC, DeLange WJ, Bruwer E, Brink PA, Corfield VA (1999). The origin of hypertrophic cardiomyopathy-causing mutation in two South African sub-populations : a unique profile of both independent and founder events. American Journal of Human Genetics 65, 1308–1320. Murray CJL, Lopez AD (Eds.) (1996). Global Burden of Disease : A Comprehensive Assessment of Mortality and Morbidity from

331

Diseases, Injuries and Risk Factors in 1990 and Projected to 2020, vol. I. Harvard : World Health Organization. Nicolini H, Camarena B, Dı' az A, Rinetti G (1999). Analysis of the insertion\deletion promoter SCL6A4 gene polymorphism in Mexican subjects with obsessive–compulsive disorder or major depression (MD). Molecular Psychiatry 4 (Suppl. 1), S111 [Abstract]. Nicolini H, Cruz C, Camarena B (1996). DRD2, DRD3, and 5-HT2A receptor genes polymorphisms in obsessive–compulsive disorder. Molecular Psychiatry 152, 461–465. Niehaus DJH, Kinnear CJ, Corfield VA, du Toit PL, Van Kradenberg J, Moolman-Smook JC, Weyers JB, Seedat S, Emsley RA, Knowles JA, Brink PA, Stein DJ (In Press). Association between a catechol-O-methyl transferase polymorphism and obsessive–compulsive disorder in the Afrikaner population. Journal of Affective Disorders. Niehaus DJH, Knowles JA, van Kradenberg J, du Toit W, Kaminer D, Seedat S, Daniels W, Cotton M, Brink P, Beyers AD, Bouic P, Chapman F, Zabriskie JB, Stein DJ (1999). D8\17 in obsessive–compulsive disorder and trichotillomania. South African Medical Journal 89, 755–756. Novelli EM, Nibile M, Diaferia G (1994). A molecular investigation suggests no relationship between obsessive–compulsive disorder and the dopamine D2 receptor. Neuropsychobiology 29, 61–63. Ohara K, Nagai M, Suzuki Y, Ochiai M (1998). No association between anxiety disorders and catechol-Omethyltransferase polymorphism. Psychiatry Research 80, 145–148. Pauls DL, Alsobrook II JP, Goodman W, Rasmussen S, Leckman JF (1995). A family study of obsessive–compulsive disorder. American Journal of Psychiatry 152, 76–84. Rasmussen SA, Tsuang MT (1986). Clinical characteristics and family history in DSM-III obsessive–compulsive disorder. American Journal of Psychiatry 143, 317–322. Simonic I, Gericke GS, Ott J, Weber JL (1998). Identification of genetic markers associated with Gilles de la Tourette’s Syndrome in an Afrikaner population. American Journal of Human Genetics 63, 839–846. Starfield M, Hennies HC, Jung M, Jenkins T, Wienker T, Hull P, Spurdle A, Kuster W, Ramsay M, Reis A (1997). Localization of the gene causing keratolytic winter erythema to chromosome 8p22-p23, and evidence for a founder effect in South African Afrikaans-speakers. American Journal Human Genetics 61, 370–378. Stein DJ (1996). The neurobiology of obsessive–compulsive disorder. The Neuroscientist 2, 300–305. Swedo SE, Leonard HL, Kiessling LS (1994). Speculations on antineuronal antibody-mediated neuropsychiatric disorders of childhood. Pediatrics 93, 323–326.