- Email: [email protected]
Association of GRIN2B gene polymorphism and Obsessive Compulsive disorder and symptom dimensions: A pilot study
Psychiatry Research 243 (2016) 152–155
Contents lists available at ScienceDirect
Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Association of GRIN2B gene polymorphism and Obsessive Compulsive disorder and symptom dimensions: A pilot study Fabiana Barzotti Kohlrausch a,n, Isabele Gomes Giori a, Fernanda Brito Melo-Felippe a, Tamiris Vieira-Fonseca a, Luis Guillermo Coca Velarde b, Juliana Braga de Salles Andrade c,d, Leonardo Franklin Fontenelle c,d,e a
Departamento de Biologia Geral, Instituto de Biologia, Universidade Federal Fluminense (UFF), Niterói, Brazil Departamento de Estatística, Instituto de Matemática, Universidade Federal Fluminense (UFF), Niterói, Brazil c Programa de Transtornos Obsessivo-Compulsivos e de Ansiedade, Instituto de Psiquiatria, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil d Instituto D′Or de Pesquisa e Ensino (IDOR), Rio de Janeiro, Brazil e School of Psychological Sciences, MONASH University, Melbourne, Australia b
art ic l e i nf o
a b s t r a c t
Article history: Received 6 January 2016 Received in revised form 11 May 2016 Accepted 19 June 2016 Available online 25 June 2016
The etiology of OCD is largely unknown, but neuroimaging and pharmacological studies suggest that glutamatergic system plays a significant role on OCD development. We genotyped one polymorphism at GRIN2B (rs1019385) by real time Polymerase Chain Reaction in a sample of Brazilian Obsessive-Compulsive patients and healthy controls, and evaluated its influence on OCD. We found the T-allele and TT genotype to be significantly associated with OCD and ordering dimension. The T-allele was also significantly associated with checking. These preliminary results demonstrated that the GRIN2B gene may confer to some extent the susceptibility to OCD and its symptoms. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Genetics Symptom dimensions Brazil
1. Introduction Obsessive-compulsive disorder (OCD) is a frequent and disabling neuropsychiatric condition characterized by recurrent, intrusive thoughts (obsessions) and repetitive behaviors (compulsions). There is no consensus on the etiology of OCD and the identification of genetic factors in the pathophysiology of the disorder remains unclear. Glutamatergic neurotransmission, as well as cortico-striatal-thalamo-cortical (CSTC) abnormalities probably play a role in OCD (Wu et al., 2012). However, the precise relationship between the expression of OCD symptoms and dysregulation of CSTC and glutamate is still not well characterized. Saxena and Rauch (2000) suggested that OCD is caused by an imbalance between excitatory and inhibitory pathways in the CSTC, resulting in hyperactivation of the orbitofrontal-subcortical pathway and consequent appearance of obsessive-compulsive symptoms. As result, exaggerated danger or hygiene concerns may cause obsessions mediated by orbitofrontal cortex (OFC). This circuit is involved in motor, cognitive and affective system, and therefore related to OCD symptoms (Brem et al., 2014). n
Corresponding author. E-mail address: [email protected] (F.B. Kohlrausch).
http://dx.doi.org/10.1016/j.psychres.2016.06.027 0165-1781/& 2016 Elsevier Ireland Ltd. All rights reserved.
Candidate gene studies in OCD have focused mainly on genes involved in the serotonergic, dopaminergic, and glutamatergic pathways, immune pathways, or in neurotrophic and neurodevelopmental genes (Murphy et al., 2013; Pauls, et al., 2014). Glutamate is the major excitatory neurotransmitter in adult brains (Shepherd, 2004). Since animal models and candidate gene studies suggest that post-synaptic dysfunction of glutamate system plays a central role in circuits consistently implicated in OCD (Ting and Feng, 2008), this system is a promising candidate to association studies. Glutamate transporter the N-methyl-D-aspartate (NMDA) receptors are probably involved in neurodevelopmental processes of the nervous system (Grünblatt et al., 2014) and dysfunction of NMDA receptors neurotransmission activity were suggested to be associated to OCD (Richter et al., 2012). Additionally, the NMDA antagonist memantine has been shown to be effective in OCD treatment (Murphy et al., 2013). Two different subunits, NR1 and NR2 or NR3, compose these receptors (Villmann and Becker, 2007). The NR2 subunit is the predominant excitatory neurotransmitter receptor in the mammalian brain and is also the agonist binding site for glutamate (Grünblatt et al., 2014). GRIN2B (glutamate receptor, ionotropic, N-methy1-D-aspartate 2B) gene is located in 12p12 and is highly expressed in adult brain
F.B. Kohlrausch et al. / Psychiatry Research 243 (2016) 152–155
regions implicated in the pathogenesis of OCD (Su et al., 2002). Arnold et al. described an association between a Single Nucleotide Polymorphism (SNP) in GRIN2B (-200T/G; rs1019385) and decreased anterior cingulate cortex (ACC) glutamatergic concentration in psychotropic-naïve pediatric OCD patients (Arnold et al., 2009a). Patients with GG genotype showed decreased ACC glutamate concentration when compared with individuals carrying the T allele. Arnold et al. (2009b) also found an association with rs1805476, suggesting that GRIN2B may be associated with regional volumetric alterations in OFC, ACC, and thalamus in children with OCD. Wu et al., (2013) found several SNPs in GRIN2B associated with total thalamus volume in pediatric OCD. Analyzing OCD subphenotypes, Alonso et al. (2012) observed that SNPs in GRIN2B were significantly associated with the presence of contamination obsessions and cleaning compulsions. Besides Arnold et al. (2009a), no other data about the influence of rs1019385 on OCD risk was reported in the literature. Therefore, we evaluated the possible association of this SNP at GRIN2B gene with risk of OCD or OCD clinical features, in a well-characterized population of Brazilian OCD patients.
153
n ¼274, 69%), pardo (brown, n ¼106, 26%) and preto (black, n ¼19, 5%). No statistically significant difference was observed between patients (69% white, 26% brown and 5% black) and controls (68% white, 27% brown and 5% black) (P¼0.95). Since the color distribution in both groups was uniform, statistical analyses were performed considering only the total sample. Genotyping of rs1019385 polymorphism was performed using s a custom Taqman SNP genotyping assay (Life Technologies), with primers and probes as follows: forward primer 5′-GGAGTCAGGGTGTGCGA-3′, reverse primer 5′-CGCGCACACACACACA-3′; probe allele G 5‘-CACACACACCCCCTACC-3′, and probe allele T 5′ACACACACACCCTACC-3′. The genotyping protocol followed the manufacturer’s instruction, running the samples in the CFX96 real time PCR system (Bio-Rad Laboratories). Deviations from HardyWeinberg equilibrium, and differences in genotype and allele frequencies between OCD and healthy controls were assessed by χ2 test or Fisher exact test. Multivariate logistic regression was performed in order to assess the odds ratio (OR) and 95% confidence interval (95%CI), as well as to control for confounding factors (gender and age of onset). Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 20.0, and a significant P value was set as o0.05.
2. Methods A hundred and ninety nine unrelated Brazilian OCD patients (mean age7 S.D. 32.38 713.45 years; mean age of onset 7 S.D. 14.91 77.45 years; 47.7% male) were recruited from the Anxiety and Depression Research Program at Institute of Psychiatry of the Federal University of Rio de Janeiro, and the Association of family, friends and people with Obsessive Compulsive Disorder in Rio de Janeiro. All patients received a comprehensive assessment by Board-Certified Psychiatrists and met clinical criteria for OCD according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR). Two hundred unrelated healthy controls (mean age7S.D. 38.10 713.26 years; 37.0% male) were recruited from the general Brazilian population and were evaluated with a modified Mini International Neuropsychiatric Interview (MINI) to exclude presence of OCD or other psychiatric illness. Detailed diagnostic procedures and samples characteristics were described previously (Melo-Felippe et al., 2016). Symptom dimensions were obtained from the Obsessive-Compulsive Inventory Reviewed (OCI-R), consisting of checking, washing, ordering, hoarding, obsession, and neutralization. Age of first symptoms was obtained from the Florida Obsessive-Compulsive Inventory (FOCI). This study was approved by the Brazilian National Commission of Research Ethics (CONEP) and all subjects provided written informed consent. The subjects were categorized according to the Brazilian Census, which relies on self-perception of ‘race/color’ as branco (white,
3. Results The genotype distribution of rs1019385 was in Hardy–Weinberg equilibrium in the total sample (P ¼0.26), as well in cases and controls (P ¼0.45 and 0.28, respectively), and the minor allele frequency (MAF) of rs1019385 (T allele) was 0.39. We observed statistically significant differences in genotype (P ¼0.04) and allele distribution (P ¼0.01; OR¼1.43 95%CI 1.07– 1.90) between patients and controls (Table 1). A recessive effect of the T-allele was observed in OCD patients (P¼ 0.04, TT genotype OR¼1.82 95%CI 1.02–3.25). Associations between rs1019385 and age of onset or presence of specific symptoms dimensions were also evaluated (Table 1). Significant associations between ordering dimension and genotype (P ¼0.03), and the T-allele (P ¼0.01, OR¼1.79 95%CI 1.12–2.85) were observed. A recessive effect of the T-allele on ordering dimension was also observed (P ¼0.03, TT genotype OR¼ 3.20 95%CI 1.04–13.09). Logistic regression analysis confirmed that the TT genotype was predictor of ordering in our sample after controlling for risk factors (P¼ 0.04, OR¼3.11 95%CI 1.03–9.44), and no effect was observed for gender and age of onset (P¼ 0.83 and P¼ 0.14, respectively). Additionally, we observed a statistically significant association between the T-allele and checking (P¼ 0.03, OR ¼1.71 95%CI 1.04–2.80), but no significant differences were observed in the genotype distribution (P ¼0.10) (Table 1) or in the recessive
Table 1 Genotype and allele distribution between the studied groups. Groups Case-Control Patients Controls OR (95% CI) Symptom dimensions Ordering Present Absent OR (95% CI) Checking Present Absent OR (95% CI)
GG
GT
TT
P
G
T
P
61 (0.31) 81 (0.41) –
103 (0.52) 98 (0.49) 1.39 (0.90–2.15)
35 (0.17) 21 (0.10) 2.21 (1.17–4.17)
0.04
225 (0.57) 260 (0.65) –
173 (0.43) 140 (0.35) 1.43 (1.07–1.90)
0.01
39 (0.27) 22 (0.41) –
76 (0.52) 27 (0.51) 1.59 (0.80–3.14)
30 (0.21) 4 (0.07) 4.23 (1.32–13.59)
0.03
154 (0.53) 71 (0.67) –
136 (0.47) 35 (0.33) 1.79 (1.12–2.85)
0.01
42 (0.27) 19 (0.42) –
81 (0.53) 22 (0.49) 1.66 (0.81–3.41)
30 (0.20) 4 (0.09) 3.39 (0.98–14.94)
0.10
165 (0.54) 60 (0.67) –
141 (0.46) 30 (0.33) 1.71 (1.04–2.80)
0.03
154
F.B. Kohlrausch et al. / Psychiatry Research 243 (2016) 152–155
model of the T-allele (P ¼0.12). Furthermore, non-significant results were observed with washing (P ¼0.34), hoarding (P ¼0.17), obsession (P ¼1.00), neutralization (P ¼0.35), and age of onset (P ¼0.89) (data not shown).
4. Discussion The rs1019385 polymorphism is located in the promoter region of GRIN2B in a Sp1 binding site and there is only one study that showed 30-folds higher activity in the T-allele through a luciferase activity experiment with nerve growth factor (Miyatake et al., 2002). The results of Arnold et al. (2009a) are partially consistent with this finding, since patients with the GG genotype showed reduced glutamate concentration in ACC when compared to individuals carrying the T allele. Therefore, our results suggest that the presence of the T-allele implies in an excessive increase of NR2 subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor, enabling an increase in the receptor copy numbers. As the literature suggests that OCD may be caused by hyperactivity of the glutamatergic system and associated over-activity of the direct pathway (Carlsson, 2001; Chakrabarty et al., 2005; Wu et al., 2012), patients carrying two copies of the T-allele would have a higher gene transcription, resulting in a higher number of receptors, which could influence glutamatergic dysregulation. Additionally, as we stated in the introduction, some studies have previously observed significant associations between other GRIN2B polymorphisms and OCD or glutamate concentration in different brain areas, reinforcing the importance of this gene in the CSTC and OCD (Alonso et al., 2012; Arnold et al., 2004, 2009a, 2009b). Concerning other nervous system diseases and rs1019385, Hokyo et al. (2010) observed a significant decrease on habituation after acoustic stimulus in schizophrenia patients with the TT genotype. Miyatake et al. (2002) reported that the transcriptionally less competent G allele was significantly more prevalent in schizophrenics than controls. A potential involvement of several GRIN2B polymorphisms (including rs1019385) in the risk for Alzheimer's disease (AD) was evaluated by Jiang and Jia (2009), and a significant association between rs3764028 and AD was observed in a sample of North Han Chinese populations. Che et al. (2015) examined two GRIN2B functional polymorphisms (rs1805476 and rs1805502) in Tourette syndrome, and found an over-transmission of the A allele in rs1805476 and the T allele in rs1805502 from parents to their affected children. Since OCD and TS probably share common etiologic factors (Grados, 2010), this is an additional evidence about the importance of GRIN2B on OCD etiology. However, since there are no accessible data about GRIN2B linkage covering the rs1019385, more association and linkage studies are needed to confirm the involvement of GRIN2B and this SNP on OCD. Discrepancies among studies possibly reflect methodological issues such as sample size or study design. Our sample size is not large; thus, these data require additional confirmation in a larger sample. Other features, as ethnicity and comorbidities, could also contribute to conflicting results. We can not exclude that our findings might be biased by hidden genetic heterogeneity present in the Brazilian population, since the MAF varies across populations. However, we observed a uniform distribution of black, brown and white people in our sample, and the MAF observed (0.39) was very similar to those described in 1000 Genomes and HapMap for European populations (0.43–0.46), and much higher than those for African populations (0.02–0.06). Associated comorbidities may also contribute to the heterogeneity of OCD etiology. If in future comorbid patients were assigned to different
subgroups, the search for factors influencing these groups would be facilitated. Besides these limitations, our preliminary results demonstrated that the GRIN2B gene might confer to some extent the susceptibility to OCD and its symptoms. Nevertheless, functional studies and independent replication in different cohorts from the same population are essential to confirm our results.
Conflict of interest The authors declared no conflict of interest.
Acknowledgments Financial support was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil, grant number 4776582009_1), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil, grant number E-26/110.080/2010) and Pró-Reitoria de Pesquisa, Pós Graduação e Inovação from Universidade Federal Fluminense (Proppi/PDI/UFF, grant INFRALABPESQ-2011). The funding source(s) had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
References Arnold, P.D., Rosenberg, D.R., Mundo, E., Tharmalingam, S., Kennedy, J.L., Richter, M. A., 2004. Association of a glutamate (NMDA) (subunit receptor gene (GRIN2B) with obsessive-compulsive disorder: a preliminary study. Psychopharmacology 174, 530–538. Arnold, P.D., Macmaster, F.P., Richter, M.A., Hanna, G.L., Sicard, T., Burroughs, E., Mirza, Y., Easter, P.C., Rose, M., Kennedy, J.L., Rosenberg, D.R., 2009a. Glutamate receptor gene (GRIN2B) associated with reduced anterior cingulate glutamatergic concentration in pediatric obsessive-compulsive disorder. Psychiatry Res. 172, 136–139. Arnold, P.D., Macmaster, F.P., Hanna, G.L., Richter, M.A., Sicard, T., Burroughs, E., Mirza, Y., Easter, P.C., Rose, M., Kennedy, J.L., Rosenberg, D.R., 2009b. Glutamate system genes associated with ventral prefrontal and thalamic volume in pediatric obsessive-compulsive disorder. Brain Imaging Behav. 3, 64–76. Alonso, P., Gratacós, M., Segalàs, C., Escaramís, G., Real, E., Bayés, M., Labad, J., LópezSolà, C., Estivill, X., Menchón, J.M., 2012. Association between the NMDA glutamate receptor GRIN2B gene and obsessive-compulsive disorder. J. Psychiatry Neurosci. 37, 273–281. Brem, S., Grünblatt, E., Drechsler, R., Riederer, P., Walitza, S., 2014. The neurobiological link between OCD and ADHD. Atten. Defict Hyperact. Disord. 6, 175–202. Carlsson, M.L., 2001. On the role of prefrontal cortex glutamate for the antithetical phenomenology of obsessive compulsive disorder and attention deficit hyperactivity disorder. Prog. Neuropsychopharmacol. Biol. Psychiatry 25, 5–26. Chakrabarty, K., Bhattacharyya, S., Christopher, R., Khanna, S., 2005. Glutamatergic dysfunction in OCD. Neuropsychopharmacology 30, 1735–1740. Che, F., Zhang, Y., Wang, G., Heng, X., Liu, S., Du, Y., 2015. The role of GRIN2B in Tourette syndrome: results from a transmission disequilibrium study. J. Affect. Disord. 187, 62–65. Grados, M.A., 2010. The genetics of obsessive-compulsive disorder and Tourette syndrome: an epidemiological and pathway-based approach for gene discovery. J. Am. Acad. Child. Adolesc. Psychiatry 49, 810–819. Grünblatt, E., Hauser, T.U., Walitza, S., 2014. Imaging genetics in obsessive-compulsive disorder: linking genetic variations to alterations in neuroimaging. Prog. Neurobiol. 121, 114–124. Hokyo, A., Kanazawa, T., Uenishi, H., Tsutsumi, A., Kawashige, S., Kikuyama, H., Glatt, S.J., Koh, J., Nishimoto, Y., Matsumura, H., Motomura, N., Yoneda, H., 2010. Habituation in prepulse inhibition is affected by a polymorphism on the NMDA receptor 2B subunit gene (GRIN2B). Psychiatr. Genet. 20, 191–198. Jiang, H., Jia, J., 2009. Association between NR2B subunit gene (GRIN2B) promoter polymorphisms and sporadic Alzheimer’s disease in the North Chinese population. Neurosci. Lett. 450, 356–360. Melo-Felippe, F.B., de Salles Andrade, J.B., Giori, I.G., Vieira-Fonseca, T., Fontenelle, L. F., Kohlrausch, F.B., 2016. Catechol-O-methyltransferase gene polymorphisms in specific obsessive-compulsive disorder patients’ subgroups. J. Mol. Neurosci. 58, 129–136. Miyatake, R., Furukawa, A., Suwaki, H., 2002. Identification of a novel variant of the human NR2B gene promoter region and its possible association with
F.B. Kohlrausch et al. / Psychiatry Research 243 (2016) 152–155
schizophrenia. Mol. Psychiatry 7 (10), 1101–1106. Murphy, D.L., Moya, P.R., Fox, M.A., Rubenstein, L.M., Wendland, J.R., Timpano, K.R., 2013. Anxiety and affective disorder comorbidity related to serotonin and other neurotransmitter systems: obsessive-compulsive disorder as an example of overlapping clinical and genetic heterogeneity. Philos. Trans. R. Soc. Lond. B Biol. Sci. 368, 435. Pauls, D.L., Abramovitch, A., Rauch, S.L., Geller, D.A., 2014. Obsessive-compulsive disorder: an integrative genetic and neurobiological perspective. Nat. Rev. Neurosci. 15, 410–424. Richter, M.A., de Jesus, D.R., Hoppenbrouwers, S., Daigle, M., Deluce, J., Ravindran, L. N., Fitzgerald, P.B., Daskalakis, Z.J., 2012. Evidence for cortical inhibitory and excitatory dysfunction in obsessive compulsive disorder. Neuropsychopharmacology 37, 1144–1151. Saxena, S., Rauch, S.L., 2000. Functional neuroimaging and the neuroanatomy of obsessive-compulsive disorder. Psychiatr. Clin. N. Am. 23, 563–586. Shepherd, G.M., 2004. The Synaptic Organization of the Brain, fifth. Oxford
155
University Press, NewYork. Su, A.I., Cooke, M.P., Ching, K.A., Hakak, Y., Walker, J.R., Wiltshire, T., Orth, A.P., Vega, R.G., Sapinoso, L.M., Moqrich, A., Patapoutian, A., Hampton, G.M., Schultz, P.G., Hogenesch, J.B., 2002. Large-scale analysis of the human and mouse transcriptomes. Proc. Natl. Acad. Sci. USA 99 (7), 4465–4470. Ting, J.T., Feng, G., 2008. Glutamatergic synaptic dysfunction and obsessive–compulsive disorder. Curr. Chem. Genom. 2, 62–75. Villmann, C., Becker, C.M., 2007. On the hypes and falls in neuroprotection: targeting the NMDA receptor. Neuroscientist 13, 594–615. Wu, K., Hanna, G.L., Rosenberg, D.R., Arnold, P.D., 2012. The role of glutamate signaling in the pathogenesis and treatment of obsessive-compulsive disorder. Pharm. Biochem Behav. 100 (4), 726–735. Wu, K., Hanna, G.L., Easter, P., Kennedy, J.L., Rosenberg, D.R., Arnold, P.D., 2013. Glutamate system genes and brain volume alterations in pediatric obsessivecompulsive disorder: a preliminary study. Psychiatry Res. 211, 214–220.
Contents lists available at ScienceDirect
Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Association of GRIN2B gene polymorphism and Obsessive Compulsive disorder and symptom dimensions: A pilot study Fabiana Barzotti Kohlrausch a,n, Isabele Gomes Giori a, Fernanda Brito Melo-Felippe a, Tamiris Vieira-Fonseca a, Luis Guillermo Coca Velarde b, Juliana Braga de Salles Andrade c,d, Leonardo Franklin Fontenelle c,d,e a
Departamento de Biologia Geral, Instituto de Biologia, Universidade Federal Fluminense (UFF), Niterói, Brazil Departamento de Estatística, Instituto de Matemática, Universidade Federal Fluminense (UFF), Niterói, Brazil c Programa de Transtornos Obsessivo-Compulsivos e de Ansiedade, Instituto de Psiquiatria, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil d Instituto D′Or de Pesquisa e Ensino (IDOR), Rio de Janeiro, Brazil e School of Psychological Sciences, MONASH University, Melbourne, Australia b
art ic l e i nf o
a b s t r a c t
Article history: Received 6 January 2016 Received in revised form 11 May 2016 Accepted 19 June 2016 Available online 25 June 2016
The etiology of OCD is largely unknown, but neuroimaging and pharmacological studies suggest that glutamatergic system plays a significant role on OCD development. We genotyped one polymorphism at GRIN2B (rs1019385) by real time Polymerase Chain Reaction in a sample of Brazilian Obsessive-Compulsive patients and healthy controls, and evaluated its influence on OCD. We found the T-allele and TT genotype to be significantly associated with OCD and ordering dimension. The T-allele was also significantly associated with checking. These preliminary results demonstrated that the GRIN2B gene may confer to some extent the susceptibility to OCD and its symptoms. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Genetics Symptom dimensions Brazil
1. Introduction Obsessive-compulsive disorder (OCD) is a frequent and disabling neuropsychiatric condition characterized by recurrent, intrusive thoughts (obsessions) and repetitive behaviors (compulsions). There is no consensus on the etiology of OCD and the identification of genetic factors in the pathophysiology of the disorder remains unclear. Glutamatergic neurotransmission, as well as cortico-striatal-thalamo-cortical (CSTC) abnormalities probably play a role in OCD (Wu et al., 2012). However, the precise relationship between the expression of OCD symptoms and dysregulation of CSTC and glutamate is still not well characterized. Saxena and Rauch (2000) suggested that OCD is caused by an imbalance between excitatory and inhibitory pathways in the CSTC, resulting in hyperactivation of the orbitofrontal-subcortical pathway and consequent appearance of obsessive-compulsive symptoms. As result, exaggerated danger or hygiene concerns may cause obsessions mediated by orbitofrontal cortex (OFC). This circuit is involved in motor, cognitive and affective system, and therefore related to OCD symptoms (Brem et al., 2014). n
Corresponding author. E-mail address: [email protected] (F.B. Kohlrausch).
http://dx.doi.org/10.1016/j.psychres.2016.06.027 0165-1781/& 2016 Elsevier Ireland Ltd. All rights reserved.
Candidate gene studies in OCD have focused mainly on genes involved in the serotonergic, dopaminergic, and glutamatergic pathways, immune pathways, or in neurotrophic and neurodevelopmental genes (Murphy et al., 2013; Pauls, et al., 2014). Glutamate is the major excitatory neurotransmitter in adult brains (Shepherd, 2004). Since animal models and candidate gene studies suggest that post-synaptic dysfunction of glutamate system plays a central role in circuits consistently implicated in OCD (Ting and Feng, 2008), this system is a promising candidate to association studies. Glutamate transporter the N-methyl-D-aspartate (NMDA) receptors are probably involved in neurodevelopmental processes of the nervous system (Grünblatt et al., 2014) and dysfunction of NMDA receptors neurotransmission activity were suggested to be associated to OCD (Richter et al., 2012). Additionally, the NMDA antagonist memantine has been shown to be effective in OCD treatment (Murphy et al., 2013). Two different subunits, NR1 and NR2 or NR3, compose these receptors (Villmann and Becker, 2007). The NR2 subunit is the predominant excitatory neurotransmitter receptor in the mammalian brain and is also the agonist binding site for glutamate (Grünblatt et al., 2014). GRIN2B (glutamate receptor, ionotropic, N-methy1-D-aspartate 2B) gene is located in 12p12 and is highly expressed in adult brain
F.B. Kohlrausch et al. / Psychiatry Research 243 (2016) 152–155
regions implicated in the pathogenesis of OCD (Su et al., 2002). Arnold et al. described an association between a Single Nucleotide Polymorphism (SNP) in GRIN2B (-200T/G; rs1019385) and decreased anterior cingulate cortex (ACC) glutamatergic concentration in psychotropic-naïve pediatric OCD patients (Arnold et al., 2009a). Patients with GG genotype showed decreased ACC glutamate concentration when compared with individuals carrying the T allele. Arnold et al. (2009b) also found an association with rs1805476, suggesting that GRIN2B may be associated with regional volumetric alterations in OFC, ACC, and thalamus in children with OCD. Wu et al., (2013) found several SNPs in GRIN2B associated with total thalamus volume in pediatric OCD. Analyzing OCD subphenotypes, Alonso et al. (2012) observed that SNPs in GRIN2B were significantly associated with the presence of contamination obsessions and cleaning compulsions. Besides Arnold et al. (2009a), no other data about the influence of rs1019385 on OCD risk was reported in the literature. Therefore, we evaluated the possible association of this SNP at GRIN2B gene with risk of OCD or OCD clinical features, in a well-characterized population of Brazilian OCD patients.
153
n ¼274, 69%), pardo (brown, n ¼106, 26%) and preto (black, n ¼19, 5%). No statistically significant difference was observed between patients (69% white, 26% brown and 5% black) and controls (68% white, 27% brown and 5% black) (P¼0.95). Since the color distribution in both groups was uniform, statistical analyses were performed considering only the total sample. Genotyping of rs1019385 polymorphism was performed using s a custom Taqman SNP genotyping assay (Life Technologies), with primers and probes as follows: forward primer 5′-GGAGTCAGGGTGTGCGA-3′, reverse primer 5′-CGCGCACACACACACA-3′; probe allele G 5‘-CACACACACCCCCTACC-3′, and probe allele T 5′ACACACACACCCTACC-3′. The genotyping protocol followed the manufacturer’s instruction, running the samples in the CFX96 real time PCR system (Bio-Rad Laboratories). Deviations from HardyWeinberg equilibrium, and differences in genotype and allele frequencies between OCD and healthy controls were assessed by χ2 test or Fisher exact test. Multivariate logistic regression was performed in order to assess the odds ratio (OR) and 95% confidence interval (95%CI), as well as to control for confounding factors (gender and age of onset). Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 20.0, and a significant P value was set as o0.05.
2. Methods A hundred and ninety nine unrelated Brazilian OCD patients (mean age7 S.D. 32.38 713.45 years; mean age of onset 7 S.D. 14.91 77.45 years; 47.7% male) were recruited from the Anxiety and Depression Research Program at Institute of Psychiatry of the Federal University of Rio de Janeiro, and the Association of family, friends and people with Obsessive Compulsive Disorder in Rio de Janeiro. All patients received a comprehensive assessment by Board-Certified Psychiatrists and met clinical criteria for OCD according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR). Two hundred unrelated healthy controls (mean age7S.D. 38.10 713.26 years; 37.0% male) were recruited from the general Brazilian population and were evaluated with a modified Mini International Neuropsychiatric Interview (MINI) to exclude presence of OCD or other psychiatric illness. Detailed diagnostic procedures and samples characteristics were described previously (Melo-Felippe et al., 2016). Symptom dimensions were obtained from the Obsessive-Compulsive Inventory Reviewed (OCI-R), consisting of checking, washing, ordering, hoarding, obsession, and neutralization. Age of first symptoms was obtained from the Florida Obsessive-Compulsive Inventory (FOCI). This study was approved by the Brazilian National Commission of Research Ethics (CONEP) and all subjects provided written informed consent. The subjects were categorized according to the Brazilian Census, which relies on self-perception of ‘race/color’ as branco (white,
3. Results The genotype distribution of rs1019385 was in Hardy–Weinberg equilibrium in the total sample (P ¼0.26), as well in cases and controls (P ¼0.45 and 0.28, respectively), and the minor allele frequency (MAF) of rs1019385 (T allele) was 0.39. We observed statistically significant differences in genotype (P ¼0.04) and allele distribution (P ¼0.01; OR¼1.43 95%CI 1.07– 1.90) between patients and controls (Table 1). A recessive effect of the T-allele was observed in OCD patients (P¼ 0.04, TT genotype OR¼1.82 95%CI 1.02–3.25). Associations between rs1019385 and age of onset or presence of specific symptoms dimensions were also evaluated (Table 1). Significant associations between ordering dimension and genotype (P ¼0.03), and the T-allele (P ¼0.01, OR¼1.79 95%CI 1.12–2.85) were observed. A recessive effect of the T-allele on ordering dimension was also observed (P ¼0.03, TT genotype OR¼ 3.20 95%CI 1.04–13.09). Logistic regression analysis confirmed that the TT genotype was predictor of ordering in our sample after controlling for risk factors (P¼ 0.04, OR¼3.11 95%CI 1.03–9.44), and no effect was observed for gender and age of onset (P¼ 0.83 and P¼ 0.14, respectively). Additionally, we observed a statistically significant association between the T-allele and checking (P¼ 0.03, OR ¼1.71 95%CI 1.04–2.80), but no significant differences were observed in the genotype distribution (P ¼0.10) (Table 1) or in the recessive
Table 1 Genotype and allele distribution between the studied groups. Groups Case-Control Patients Controls OR (95% CI) Symptom dimensions Ordering Present Absent OR (95% CI) Checking Present Absent OR (95% CI)
GG
GT
TT
P
G
T
P
61 (0.31) 81 (0.41) –
103 (0.52) 98 (0.49) 1.39 (0.90–2.15)
35 (0.17) 21 (0.10) 2.21 (1.17–4.17)
0.04
225 (0.57) 260 (0.65) –
173 (0.43) 140 (0.35) 1.43 (1.07–1.90)
0.01
39 (0.27) 22 (0.41) –
76 (0.52) 27 (0.51) 1.59 (0.80–3.14)
30 (0.21) 4 (0.07) 4.23 (1.32–13.59)
0.03
154 (0.53) 71 (0.67) –
136 (0.47) 35 (0.33) 1.79 (1.12–2.85)
0.01
42 (0.27) 19 (0.42) –
81 (0.53) 22 (0.49) 1.66 (0.81–3.41)
30 (0.20) 4 (0.09) 3.39 (0.98–14.94)
0.10
165 (0.54) 60 (0.67) –
141 (0.46) 30 (0.33) 1.71 (1.04–2.80)
0.03
154
F.B. Kohlrausch et al. / Psychiatry Research 243 (2016) 152–155
model of the T-allele (P ¼0.12). Furthermore, non-significant results were observed with washing (P ¼0.34), hoarding (P ¼0.17), obsession (P ¼1.00), neutralization (P ¼0.35), and age of onset (P ¼0.89) (data not shown).
4. Discussion The rs1019385 polymorphism is located in the promoter region of GRIN2B in a Sp1 binding site and there is only one study that showed 30-folds higher activity in the T-allele through a luciferase activity experiment with nerve growth factor (Miyatake et al., 2002). The results of Arnold et al. (2009a) are partially consistent with this finding, since patients with the GG genotype showed reduced glutamate concentration in ACC when compared to individuals carrying the T allele. Therefore, our results suggest that the presence of the T-allele implies in an excessive increase of NR2 subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor, enabling an increase in the receptor copy numbers. As the literature suggests that OCD may be caused by hyperactivity of the glutamatergic system and associated over-activity of the direct pathway (Carlsson, 2001; Chakrabarty et al., 2005; Wu et al., 2012), patients carrying two copies of the T-allele would have a higher gene transcription, resulting in a higher number of receptors, which could influence glutamatergic dysregulation. Additionally, as we stated in the introduction, some studies have previously observed significant associations between other GRIN2B polymorphisms and OCD or glutamate concentration in different brain areas, reinforcing the importance of this gene in the CSTC and OCD (Alonso et al., 2012; Arnold et al., 2004, 2009a, 2009b). Concerning other nervous system diseases and rs1019385, Hokyo et al. (2010) observed a significant decrease on habituation after acoustic stimulus in schizophrenia patients with the TT genotype. Miyatake et al. (2002) reported that the transcriptionally less competent G allele was significantly more prevalent in schizophrenics than controls. A potential involvement of several GRIN2B polymorphisms (including rs1019385) in the risk for Alzheimer's disease (AD) was evaluated by Jiang and Jia (2009), and a significant association between rs3764028 and AD was observed in a sample of North Han Chinese populations. Che et al. (2015) examined two GRIN2B functional polymorphisms (rs1805476 and rs1805502) in Tourette syndrome, and found an over-transmission of the A allele in rs1805476 and the T allele in rs1805502 from parents to their affected children. Since OCD and TS probably share common etiologic factors (Grados, 2010), this is an additional evidence about the importance of GRIN2B on OCD etiology. However, since there are no accessible data about GRIN2B linkage covering the rs1019385, more association and linkage studies are needed to confirm the involvement of GRIN2B and this SNP on OCD. Discrepancies among studies possibly reflect methodological issues such as sample size or study design. Our sample size is not large; thus, these data require additional confirmation in a larger sample. Other features, as ethnicity and comorbidities, could also contribute to conflicting results. We can not exclude that our findings might be biased by hidden genetic heterogeneity present in the Brazilian population, since the MAF varies across populations. However, we observed a uniform distribution of black, brown and white people in our sample, and the MAF observed (0.39) was very similar to those described in 1000 Genomes and HapMap for European populations (0.43–0.46), and much higher than those for African populations (0.02–0.06). Associated comorbidities may also contribute to the heterogeneity of OCD etiology. If in future comorbid patients were assigned to different
subgroups, the search for factors influencing these groups would be facilitated. Besides these limitations, our preliminary results demonstrated that the GRIN2B gene might confer to some extent the susceptibility to OCD and its symptoms. Nevertheless, functional studies and independent replication in different cohorts from the same population are essential to confirm our results.
Conflict of interest The authors declared no conflict of interest.
Acknowledgments Financial support was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil, grant number 4776582009_1), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil, grant number E-26/110.080/2010) and Pró-Reitoria de Pesquisa, Pós Graduação e Inovação from Universidade Federal Fluminense (Proppi/PDI/UFF, grant INFRALABPESQ-2011). The funding source(s) had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
References Arnold, P.D., Rosenberg, D.R., Mundo, E., Tharmalingam, S., Kennedy, J.L., Richter, M. A., 2004. Association of a glutamate (NMDA) (subunit receptor gene (GRIN2B) with obsessive-compulsive disorder: a preliminary study. Psychopharmacology 174, 530–538. Arnold, P.D., Macmaster, F.P., Richter, M.A., Hanna, G.L., Sicard, T., Burroughs, E., Mirza, Y., Easter, P.C., Rose, M., Kennedy, J.L., Rosenberg, D.R., 2009a. Glutamate receptor gene (GRIN2B) associated with reduced anterior cingulate glutamatergic concentration in pediatric obsessive-compulsive disorder. Psychiatry Res. 172, 136–139. Arnold, P.D., Macmaster, F.P., Hanna, G.L., Richter, M.A., Sicard, T., Burroughs, E., Mirza, Y., Easter, P.C., Rose, M., Kennedy, J.L., Rosenberg, D.R., 2009b. Glutamate system genes associated with ventral prefrontal and thalamic volume in pediatric obsessive-compulsive disorder. Brain Imaging Behav. 3, 64–76. Alonso, P., Gratacós, M., Segalàs, C., Escaramís, G., Real, E., Bayés, M., Labad, J., LópezSolà, C., Estivill, X., Menchón, J.M., 2012. Association between the NMDA glutamate receptor GRIN2B gene and obsessive-compulsive disorder. J. Psychiatry Neurosci. 37, 273–281. Brem, S., Grünblatt, E., Drechsler, R., Riederer, P., Walitza, S., 2014. The neurobiological link between OCD and ADHD. Atten. Defict Hyperact. Disord. 6, 175–202. Carlsson, M.L., 2001. On the role of prefrontal cortex glutamate for the antithetical phenomenology of obsessive compulsive disorder and attention deficit hyperactivity disorder. Prog. Neuropsychopharmacol. Biol. Psychiatry 25, 5–26. Chakrabarty, K., Bhattacharyya, S., Christopher, R., Khanna, S., 2005. Glutamatergic dysfunction in OCD. Neuropsychopharmacology 30, 1735–1740. Che, F., Zhang, Y., Wang, G., Heng, X., Liu, S., Du, Y., 2015. The role of GRIN2B in Tourette syndrome: results from a transmission disequilibrium study. J. Affect. Disord. 187, 62–65. Grados, M.A., 2010. The genetics of obsessive-compulsive disorder and Tourette syndrome: an epidemiological and pathway-based approach for gene discovery. J. Am. Acad. Child. Adolesc. Psychiatry 49, 810–819. Grünblatt, E., Hauser, T.U., Walitza, S., 2014. Imaging genetics in obsessive-compulsive disorder: linking genetic variations to alterations in neuroimaging. Prog. Neurobiol. 121, 114–124. Hokyo, A., Kanazawa, T., Uenishi, H., Tsutsumi, A., Kawashige, S., Kikuyama, H., Glatt, S.J., Koh, J., Nishimoto, Y., Matsumura, H., Motomura, N., Yoneda, H., 2010. Habituation in prepulse inhibition is affected by a polymorphism on the NMDA receptor 2B subunit gene (GRIN2B). Psychiatr. Genet. 20, 191–198. Jiang, H., Jia, J., 2009. Association between NR2B subunit gene (GRIN2B) promoter polymorphisms and sporadic Alzheimer’s disease in the North Chinese population. Neurosci. Lett. 450, 356–360. Melo-Felippe, F.B., de Salles Andrade, J.B., Giori, I.G., Vieira-Fonseca, T., Fontenelle, L. F., Kohlrausch, F.B., 2016. Catechol-O-methyltransferase gene polymorphisms in specific obsessive-compulsive disorder patients’ subgroups. J. Mol. Neurosci. 58, 129–136. Miyatake, R., Furukawa, A., Suwaki, H., 2002. Identification of a novel variant of the human NR2B gene promoter region and its possible association with
F.B. Kohlrausch et al. / Psychiatry Research 243 (2016) 152–155
schizophrenia. Mol. Psychiatry 7 (10), 1101–1106. Murphy, D.L., Moya, P.R., Fox, M.A., Rubenstein, L.M., Wendland, J.R., Timpano, K.R., 2013. Anxiety and affective disorder comorbidity related to serotonin and other neurotransmitter systems: obsessive-compulsive disorder as an example of overlapping clinical and genetic heterogeneity. Philos. Trans. R. Soc. Lond. B Biol. Sci. 368, 435. Pauls, D.L., Abramovitch, A., Rauch, S.L., Geller, D.A., 2014. Obsessive-compulsive disorder: an integrative genetic and neurobiological perspective. Nat. Rev. Neurosci. 15, 410–424. Richter, M.A., de Jesus, D.R., Hoppenbrouwers, S., Daigle, M., Deluce, J., Ravindran, L. N., Fitzgerald, P.B., Daskalakis, Z.J., 2012. Evidence for cortical inhibitory and excitatory dysfunction in obsessive compulsive disorder. Neuropsychopharmacology 37, 1144–1151. Saxena, S., Rauch, S.L., 2000. Functional neuroimaging and the neuroanatomy of obsessive-compulsive disorder. Psychiatr. Clin. N. Am. 23, 563–586. Shepherd, G.M., 2004. The Synaptic Organization of the Brain, fifth. Oxford
155
University Press, NewYork. Su, A.I., Cooke, M.P., Ching, K.A., Hakak, Y., Walker, J.R., Wiltshire, T., Orth, A.P., Vega, R.G., Sapinoso, L.M., Moqrich, A., Patapoutian, A., Hampton, G.M., Schultz, P.G., Hogenesch, J.B., 2002. Large-scale analysis of the human and mouse transcriptomes. Proc. Natl. Acad. Sci. USA 99 (7), 4465–4470. Ting, J.T., Feng, G., 2008. Glutamatergic synaptic dysfunction and obsessive–compulsive disorder. Curr. Chem. Genom. 2, 62–75. Villmann, C., Becker, C.M., 2007. On the hypes and falls in neuroprotection: targeting the NMDA receptor. Neuroscientist 13, 594–615. Wu, K., Hanna, G.L., Rosenberg, D.R., Arnold, P.D., 2012. The role of glutamate signaling in the pathogenesis and treatment of obsessive-compulsive disorder. Pharm. Biochem Behav. 100 (4), 726–735. Wu, K., Hanna, G.L., Easter, P., Kennedy, J.L., Rosenberg, D.R., Arnold, P.D., 2013. Glutamate system genes and brain volume alterations in pediatric obsessivecompulsive disorder: a preliminary study. Psychiatry Res. 211, 214–220.