Association of serotonin transporter gene polymorphisms with obsessive-compulsive disorder (OCD) in a south Indian population.
Good therapeutic efficacy of serotonin reuptake inhibitors (SRIs) in OCD (7,8) has prompted search for association between OCD and variations in the SLC6A4 gene. A functional polymorphism in the 5' regulatory promoter region 5-HTTLPR (5-HTT gene-linked polymorphic region), involving two common alleles that correspond to a 43-base pair insertion (L allele) or deletion (S allele), has been reported (9). The S allele of 5-HTTLPR polymorphism reduces transcription efficiency for the SLC6A4 gene, resulting in decreased gene expression, and thus, decreased serotonin uptake in lymphoblast cell lines (10). Recent research shows varied results with respect to association between 5-HTTLPR alleles and OCD. In a meta-analysis by Lin (11), only two studies (12,13) showed a significant association of LL genotype with OCD. The overall result of the meta-analysis demonstrated an association of OCD with SS genotype and a reverse association with L/S genotype. Denys et al (14), found an association of S allele of 5-HTTLPR with female OCD patients. However, Dickel et al (15), supported a nominally significant over-transmission of the L allele in female patients. A recent meta-analysis suggests the possibility that the L allele may be associated with OCD in specific subgroups such as childhood-onset OCD, and in Caucasians (16).
A possible association between the Stin2.12 allele of the other polymorphism 5-HTTVNTR in the second intron of SLC6A4 and OCD has been suggested (17,18). Role of combined effect of 5-HTTLPR and VNTR polymorphism on the expression of the serotonin transporter has also been reported (19).
Hu et al (13) demonstrated that possible lack of consistent association with 5-HTTLPR was the overlooked contribution of a single nucleotide polymorphism (SNP), rs25531, within the repetitive region that comprises the 5-HTTLPR. The modulation of 5-HTTLPR by rs25531 results in three common alleles: [L.sub.A] (highest-expressing), [L.sub.G] and S (both low expressing). The [L.sub.G] allele creates a functional AP2 transcription-factor binding site which is one of the nuclear factors that function as transcriptional activators or repressors (20) and reduces transcription of the transporter protein. They found an association in two independent samples, both case-control and family-based, of the higher-expressing [L.sub.A] allele and [L.sub.A][L.sub.A] genotype with OCD.
Replication in a large case-control design was unable to corroborate this, although an increased frequency of the LA allele and [L.sub.A][L.sub.A] genotype in OCD probands was observed (21). Wendland et al (22) reported a highly significant haplotype-based omnibus association of all the known non-coding functional SLC6A4 variants with OCD in a large case-control sample. The haplotype significantly overrepresented in probands contained the higher-expressing L allele at each locus, supporting the notion of increased serotonin transporter functioning being pathogenetically involved in OCD.
Existing evidences of involvement of SLC6A4 in OCD, prompted us to study the polymorphisms at this region in a south Indian population of OCD subjects and controls. To reduce the phenotypic heterogeneity, we included only adults with OCD who did not have co-morbid tic disorder.
Material & Methods
The institutional Ethics Committee approved the study protocol. Participants gave written informed consent for participation in the study.
Subjects: Consecutive subjects fulfilling DSM-IV criteria for OCD (23) were recruited from the OCD Clinic and the clinical services of the National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore during April 2006 to January 2008. Those with a history of substance abuse and co-morbid tic disorder were excluded. All patients and controls were adults, unrelated and of south Indian origin. The Mini-International Neuropsychiatric Interview (MINI) version 4.4, a brief structured interview (24), was used for the DSM-IV diagnosis of 93 OCD patients, and for the exclusion of any Axis-I diagnosis in 92 healthy controls. All patients were further assessed using the Yale-Brown Obsessive Compulsive Scale (YBOCS) symptom checklist and Y-BOCS severity rating scale for symptom profile and severity of OCD (25,26). Clinical Global Impression CGI-S and CGI-I (27) were applied to assess the global severity and improvement. The patients and controls were matched for age and gender.
The 5-HTTVNTR, and 5-HTTLPR (A/G) polymorphism was genotyped in OCD patients (n=93) and healthy controls (n= 92). The demographic and clinical details of the case control sample are shown in Table I.
DNA methods: Venous blood (10 ml) was drawn from patients and controls under aseptic precautions. DNA was isolated from peripheral leucocytes using salting out method (28). Oligonucleotide primers flanking the 5-HTTVNTR were used to generate fragments corresponding to the 12 and 10 alleles (29). The 5-HTTLPR/(A/G) polymorphisms were genotyped as described in Wendland et al (30). Five unlinked Alu polymorphisms were genotyped in OCD patients (n=93), and control (n=50) subjects to rule out the impact of stratification in the case-control association study. There was no differential distribution of alleles between the cases and controls at any of the Alu loci ruling out population stratification in the cohort (data not shown).
Statistical analysis: The allelic distribution and genotype frequency in cases and controls were compared using chi square test after checking the Hardy-Weinberg Equilibrium. Logistic regression was applied to test for effects of confounding factors. In order to test for the effects of genotype on heterogeneity of the illness, linear regression was performed (using SPSS v13.0) on the 5-HTTLPR and rs25531 genotype data with severity index and co-morbid depression as dependent variable and the genotypes as independent variables. The effect of genotype 5-HTTLPR(A/G) on YBOCS score was assessed by testing the regression coefficient ([beta]) under three different genetic models separately with two sided test. Three models dominant, co-dominant and recessive were assumed with the genotypes at both loci. Haplotype based association analysis was carried out using the 2LD calculator (31).
There was no deviation from Hardy-Weinberg Equilibrium at either of the polymorphisms studied. There was no association of the 5-HTTVNTR polymorphisms of the SLC6A4 gene both with the genotypic and allelic frequencies between OCD patients and healthy control samples (Table II).
Since prior evidence from gene expression studies indicated that the "low expressing" [SL.sub.G] and SS genotypes are not distinguishable biochemically, the distribution of the [L.sub.A][L.sub.A], S/[L.sub.A], [L.sub.A][L.sub.G] and the combined [SL.sub.G] and SS genotypes was compared between case and controls, but there was no significant difference (Table II). The allele frequencies were found to differ between the patients and controls with a modest higher frequency of the [L.sub.A] allele in the cases compared with the controls (28% vs. 25%); further, the frequency of [L.sub.G] allele differed between the cases and controls (8% vs 13%). However, none of these differences were statistically significant. Logistic regression was applied to test for effects of age and sex. No significant contribution was seen on assuming any of the three inheritance models (data not shown). No significant difference was found in frequencies of haplotypes in cases and controls (Table III).
On clinical evaluation, 58 per cent of the cases had a co-morbid psychiatric illness, of which 41 per cent had major depression. There was a family history of psychiatric illness in 44 per cent of cases and family history of OCD was found in 13 per cent. There was no significant difference in the allele or genotype frequencies in any of the above subgroups.
On linear regression analysis, there was no significant association of the 5-HTTLPR locus with both phenotypes (severity index and co-morbid depression). However, a statistically significant association (P=0.036) of the dominant model of 5-HTTLPR (A/G) (non-risk allele: [L.sub.A]; risk alleles: [S.sub.A], [S.sub.G], [L.sub.G]) was seen with the severity index or YBOCS score (Table IV). Similar analysis for association of the SNP with comorbid depression did not yield any significant result (data not shown).
In the present study involvement of the 5-HTTVNTR and 5-HTTLPR (A/G) gene polymorphism with OCD was examined. There was no association with the 5-HTTVNTR polymorphism. At the 5-HTTLPR (A/G) locus no significant association was observed. Also no significant association was observed on comparing the combined frequencies of lower expressing genotypes (SS, SL, [L.sub.G][L.sub.G]) and intermediate expressing genotypes ([SL.sub.A], [L.sub.A][L.sub.G]) with higher expressing ([L.sub.A][L.sub.A]) genotypes. However, there was a modest association of the dominant model of 5-HTTLPR (A/G) with the severity index of OCD. This may indicate that the promoter SNP rs25531 which has a role in the expression efficiency of the gene (SLC6A4), may partly mediate the pathophysiology of OCD at least in terms of severity of symptoms.
The S allele frequency in our control population (0.60) was more than that reported in White European origin populations (0.35-0.40) (13). However, it varies across other populations, varying from 0.454 in a Brazilian sample to 0.64-0.66 in American Indians (32). It is noteworthy that in the populations which have shown an association of L allele with OCD, the L allele frequency was higher (0.592 and 0.60 to 0.65 respectively) (12,13) than in our sample (L= 0.39).
Association analyses of common functional variants of the SLC6A4 gene, a long-standing OCD candidate, have so far been inconsistent. Our findings lend support for the putative association of serotonin transporter variations to severity of OCD. The variation in allele frequencies across populations with possible different contributions to disease phenotypes across ethnicities underlines the need for comparative studies, at both phenotypic and genotypic levels, to further explore the biology of this syndrome. Genotyping additional SNPs/novel SNPs around the complex 5-HTTLPR and 5-HTTVNTR region and inclusion of these in LD (Linkage disequilibrium) based association will facilitate identification of risk alleles/haplotypes for OCD in the population.
One possible explanation for the equivocal results across populations could be the presence of sex-related differences and co-morbid diagnosis. Effect of gender has been reported in OCD previously (33,34). In our sample cohort, the representation of males in cases and controls was high. However, the small sample size could be a confounding reason responsible for lack of significance on logistic regression. The small size and the consequent lack of statistical power is the major limitation of our study. Thus, further examination of these loci on a larger sample set with adoption of a family based association design may also help rule out other confounding factors like ethnicity.
The results from this study and earlier association studies indicate that OCD resembles other complex psychiatric disorders in being aetiologically heterogeneous. It is possible that the modest significance derived on linear regression with severity is a reflection of such a phenomenon. Genetic contributions may involve highly penetrant alleles and/or more common rare alleles, as well as a polygenetic inheritance involving multiple polymorphisms in serotonergic candidate genes. The understanding of these contributions requires further studies under the triallelic model and replication in a larger sample size.
The authors acknowledge the Indian Council of Medical Research, New Delhi, for financial support.
Received February 10, 2010
(1.) Kessler RC, Berglund P, Demler O, Jin R, Merikangas KR, Walters EE. Lifetime prevalence and age-of-onset distributions of DSM-IV disorders in the National Comorbidity Survey Replication. Arch Gen Psychiatry 2005; 62 : 593-602.
(2.) Lochner C, Stein DJ. Heterogeneity of obsessive-compulsive disorder : a literature review. Harv Rev Psychiatry 2003; 11 : 113-32.
(3.) Grados MA, Riddle MA, Samuels JF, Liang KY, Hoehn-Saric R, Bienvenu OJ, et al. The familial phenotype of obsessive-compulsive disorder in relation to tic disorders : the Hopkins OCD family study. Biol Psychiatry 2001; 50 : 559-65.
(4.) Miguel EC, Leckman JF, Rauch S, do Rosario-Campos MC, Hounie AG, Mercadante MT, et al. Obsessive-compulsive disorder phenotypes : implications for genetic studies. Mol Psychiatry 2005; 10 : 258-75.
(5.) Jaisoorya TS, Janardhan Reddy YC, Srinath S. Is juvenile obsessive-compulsive disorder a developmental subtype of the disorder?--Findings from an Indian study. Eur Child Adolesc Psychiatry 2003; 12 : 290-7.
(6.) Jaisoorya TS, Reddy YC, Srinath S, Thennarasu K. Obsessive-compulsive disorder with and without tic disorder : a comparative study from India. CNS Spectr 2008; 13 : 705-11.
(7.) McDougle CJ, Goodman WK, Leckman JF, Price LH. The psychopharmacology of obsessive compulsive disorder. Implications for treatment and pathogenesis. Psychiatr Clin North Am 1993; 16 : 749-66.
(8.) Soomro G, Altman D, Rajagopal S, Oakley-Browne M. Selective serotonin re-uptake inhibitors (SSRIs) versus placebo for obsessive compulsive disorder (OCD). Cochrane Database Syst Rev 2008 : CD001765.
(9.) Heils A, Teufel A, Petri S, Stober G, Riederer P, Bengel D, et al. Allelic variation of human serotonin transporter gene expression. J Neurochem 1996; 66 : 2621-4.
(10.) Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, et al. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 1996; 274 : 1527-31.
(11.) Lin PY. Meta-analysis of the association of serotonin transporter gene polymorphism with obsessive-compulsive disorder. Prog Neuropsychopharmacol Biol Psychiatry 2007; 31 : 683-9.
(12.) Bengel D, Greenberg BD, Cora-Locatelli G, Altemus M, Heils A, Li Q, et al. Association of the serotonin transporter promoter regulatory region polymorphism and obsessive-compulsive disorder. Mol Psychiatry 1999; 4 : 463-6.
(13.) Hu XZ, Lipsky RH, Zhu G, Akhtar LA, Taubman J, Greenberg BD, et al. Serotonin transporter promoter gain-of-function genotypes are linked to obsessive-compulsive disorder. Am J Hum Genet 2006; 78 : 815-26.
(14.) Denys D, Van Nieuwerburgh F, Deforce D, Westenberg HG. Association between serotonergic candidate genes and specific phenotypes of obsessive compulsive disorder. J Affect Disord 2006; 91 : 39-44.
(15.) Dickel DE, Veenstra-VanderWeele J, Bivens NC, Wu X, Fischer DJ, Van Etten-Lee M, et al. Association studies of serotonin system candidate genes in early-onset obsessive-compulsive disorder. Biol Psychiatry 2007; 61 : 322-9.
(16.) Bloch MH, Landeros-Weisenberger A, Sen S, Dombrowski P, Kelmendi B, Coric V, et al. Association of the serotonin transporter polymorphism and obsessive-compulsive disorder : systematic review. Am J Med Genet B Neuropsychiatr Genet 2008; 147B : 850-8.
(17.) Baca-Garcia E, Vaquero-Lorenzo C, Diaz-Hernandez M, Rodriguez-Salgado B, Dolengevich-Segal H, Arrojo-Romero M, et al. Association between obsessive-compulsive disorder and a variable number of tandem repeats polymorphism in intron 2 of the serotonin transporter gene. Prog Neuropsychopharmacol Biol Psychiatry 2007; 31 : 416-20.
(18.) Saiz PA, Garcia-Portilla MP, Arango C, Morales B, Bascaran MT, Martinez-Barrondo S, et al. Association study between obsessive-compulsive disorder and serotonergic candidate genes. Prog Neuropsychopharmacol Biol Psychiatry 2008; 32 : 765-70.
(19.) Hranilovic D, Stefulj J, Schwab S, Borrmann-Hassenbach M, Albus M, Jernej B, et al. Serotonin transporter promoter and intron 2 polymorphisms : relationship between allelic variants and gene expression. Biol Psychiatry 2004; 55 : 1090-4.
(20.) Jiang JG, DeFrances MC, Machen J, Johnson C, Zarnegar R. The repressive function of AP2 transcription factor on the hepatocyte growth factor gene promoter. Biochem Biophys Res Commun 2000; 272 : 882-6.
(21.) Wendland JR, Kruse MR, Cromer KR, Murphy DL. A large case-control study of common functional SLC6A4 and BDNF variants in obsessive-compulsive disorder. Neuropsychopharmacology 2007; 32 : 2543-51.
(22.) Wendland JR, Moya PR, Kruse MR, Ren-Patterson RF, Jensen CL, Timpano KR, et al. A novel, putative gain-of-function haplotype at SLC6A4 associates with obsessive-compulsive disorder. Hum Mol Genet 2008; 17 : 717-23.
(23.) American Psychiatric Association (APA). Diagnostic and statistical manual of mental disorders, 4th ed., text revision. Washington, DC: APA; 2000.
(24.) Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E, et al. The Mini-International Neuropsychiatric Interview (M.I.N.I.) : the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD10. J Clin Psychiatry 1998; 59 (Suppl 20) : 22-33; quiz 34-57.
(25.) Goodman WK, Price LH, Rasmussen SA, Mazure C, Fleischmann RL, Hill CL, et al. The Yale-Brown Obsessive Compulsive Scale. I. Development, use, and reliability. Arch Gen Psychiatry 1989; 46 : 1006-11.
(26.) Goodman WK, Price LH. Assessment of severity and change in obsessive compulsive disorder. Psychiatr Clin North Am 1992; 15 : 861-9.
(27.) Guy W. ECDEU Assessment manual for psychopharmacology. Washington DC: US Department of Health, Education and Welfare; 1976. p. 218-22.
(28.) Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16 : 1215.
(29.) Ogilvie AD, Battersby S, Bubb VJ, Fink G, Harmar AJ, Goodwim GM, et al. Polymorphism in serotonin transporter gene associated with susceptibility to major depression. Lancet 1996; 347 : 731-3.
(30.) Wendland JR, Martin BJ, Kruse MR, Lesch KP, Murphy DL. Simultaneous genotyping of four functional loci of human SLC6A4, with a reappraisal of 5-HTTLPR and rs25531. Mol Psychiatry 2006; 11 : 224-6.
(31.) Zhao JH. 2LD, GENECOUNTING and HAP : Computer programs for linkage disequilibrium analysis. Bioinformatics 2004; 20 : 1325-6.
(32.) Marques FZ, Hutz MH, Bau CH. Influence of the serotonin transporter gene on comorbid disorders among alcohol-dependent individuals. Psychiatr Genet 2006; 16 : 125-31.
(33.) Labad J, Menchon JM, Alonso P, Segalas C, Jimenez S, Jaurrieta N, et al. Gender differences in obsessive-compulsive symptom dimensions. Depress Anxiety 2008; 25 : 832-8.
(34.) Torresan RC, Ramos-Cerqueira AT, de Mathis MA, Diniz JB, Ferrao YA, Miguel EC, et al. Sex differences in the phenotypic expression of obsessive-compulsive disorder: an exploratory study from Brazil. Compr Psychiatry 2009; 50 : 63-9.
Reprint requests: Dr Sanjeev Jain, Molecular Genetics Laboratory, Department of Psychiatry, National Institute of Mental Health & Neurosciences, Hosur Road, Bangalore 560 029, India e-mail: firstname.lastname@example.org
Prashant Tibrewal (+), Kiran Kumar H.B. (+),**, Shubha G.N. (+), Subhashree D. (+), Meera Purushottam (+), Thennarasu K. *, Reddy Y.C.J. (+) & Sanjeev Jain (+)
Departments of * (+) Psychiatry & * Biostatistics, National Institute of Mental Health & Neurosciences, Bangalore, India & ** Department of Biology, The University of Western Ontario, London, Canada
Table I. Demographic information of the case-control sample set No. (%) Cases (N=92): Mean age (yr) 28.8 [+ or -] 9.04 Number of males 62 Age of onset (yr) 21.14 [+ or -] 7.98 Onset before 18 yr of age 42 [+ or -] 4 Duration of illness (month) 98.59 [+ or -] 9.5 Duration of untreated illness (month) 53.13 [+ or -] 7.0 Family history of any psychiatric illness 42 (44.2) (i) OCD 13 (13.7) (ii) Substance abuse 3 (3.2) (iii) Psychosis 11 (11.6) (iv) Affective illness 12 (12.6) (v) Anxiety disorder 3 (3.2) (vi) Suicide 10 (10.5) (vii) Others 5 (5.3) Controls (N=92): Age (yr) 29.47 [+ or -] 9.5 Number of males 62 Table II. Genotypic and allelic distribution at serotonin transporter loci Genotype frequency Controls OCD cases (n=92) (n=93) Genotypic and allelic distribution at 5-HTTVNTR: 10 10 12 (0.103) 11 (0.118) 10 12 31 (0.336) 30 (0.322) 12 12 49 (0.532) 52 (0.559) Chi square=0.14 df=2 P=0.93 Genotypic and allelic distribution of 5-HTTLPR (L/S) polymorphism and rs25531(A/G): [L.sub.A][L.sub.A] 4 (0.04) 8 (0.08) [L.sub.A][L.sub.G] 6 (0.06) 7 (0.07) [L.sub.G][L.sub.G] 1 (0.01) 1 (0.01) S [L.sub.A] 33 (0.35) 30 (0.32) S [L.sub.G] 17 (0.18) 7 (0.075) SS 31 (0.33) 40 (0.43) Chi square=6.855 df=5 P=0.23 Expression based grouping of 5-HTTLPR (L/S) polymorphism and rs25531(A/G) genotypes: SS, [SL.sub.G], [L.sub.G] [L.sub.G] (Low) 49 (0.53) 48 (0.51) [L.sub.A] [L.sub.G], [SL.sub.A] (Medium) 39 (0.42) 37 (0.39) [L.sub.A][L.sub.A] (High) 4 (0.04) 8 (0.08) Chi square=1.39 df=2 P=0.49 Allele frequency Controls OCD cases (n=184) (n=186) 10 55 (0.298) 52 (0.279) 12 129 (0.701) 134 (0.720) Chi square=.09 df=1 P=0.76 [L.sub.A] 47 (0.25) 53 (0.28) [L.sub.G] 25 (0.13) 16 (0.08) S 112 (0.60) 117 (0.63) Chi square=2.43 df=2 P=0.296 S or [L.sub.G] (Low) 137 (0.74) 133 (0.71) [L.sub.A] (High) 47 (0.25) 53 (0.28) Chi square=0.27 df=1 P=0.6 Table III. Estimated haplotype frequencies of the 5-HTTVNTR and 5-HTTLPR polymorphisms 5HTTLPR 5HTTVNTR ([L.sub.A]) ([L.sub.G]) Controls 10 0.1430 0.0311 12 0.1450 0.0504 Cases 10 0.1246 0.0784 12 0.1281 0.0614 Combined 10 0.1325 0.0568 12 0.1378 0.0540 5HTTLPR 5HTTVNTR ([S.sub.A]) ([S.sub.G]) Controls 10 0.5380 0.0107 12 0.0761 0.0056 Cases 10 0.4959 0.0000 12 0.1008 0.0108 Combined 10 0.5180 0.0035 12 0.0874 0.0100 Table IV. Linear regression analysis with severity index as dependent and 5-HTTLPR (A/G) genotype as independent variable Genetic model Beta Std error t P value Dominant 0.23 1.85 2.14 0.036 * Co-dominant 0.17 0.84 1.53 0.13 Recessive 0.09 1.14 0.78 0.44 * Significant
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|Author:||Prashant, Tibrewal; Kiran, Kumar H.B.; Shubha, G.N.; Subhashree, D.; Meera, Purushottam; Thennarasu,|
|Publication:||Indian Journal of Medical Research|
|Date:||Dec 1, 2010|
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