Genetic Contribution of BDNF Gene in Obsessive Compulsive Disorder in Pakistani Punjabi Population

Javeria1, Masroor Ellahi Babar2,*, Tanveer Hussain2, Rashid Saif2, Sadaf Rashid3, Hanan Sarfraz4, Abbas Ali Shah3, Muhammad Wasim1 and Muhammad Abdullah1 1Institute of Biochemistry and Biotechnology,, University of Veterinary and Animal Sciences, Lahore, Pakistan 2Faculty of Science and Technology, Virtual University of Pakistan, Lahore, Pakistan 3Department of Psychiatry, Sir Ganga Ram Hospital, Lahore, Pakistan 4Punjab Institute of Mental Health, Lahore, Pakistan Article Information Received 12 August 2016 Revised 07 October 2016 Accepted 01 December 2016 Available online 26 June 2017

B rain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is a glutamate neurotrophic factor that plays an important role in promotion and maintenance of cell differentiation (maturation), nerve cell survival (neurans) and cell death (Huang and Reichardt, 2001a). In the brain, the BDNF protein paly significant role in connections and communications between nervenerve cells (synopses) and helps in regulation of synaptic plasticity, which is important for memory and learning. BDNF is produced as pro-form that can be cleaved intracellular later in order to release mature secreted ligands. These ligands then bind to Trk family members of receptor tyrosine kinases that prop up Trk mediated differentiation and survival (Friedman and Greene, 1999). Pro-neurotrophins are active precursors, since they can be secreted and cleaved extracellularly and serve as high affinity ligands for p75 NTR, which encourages apoptosis in neurons and oligo-dendrocytes (Lee et al., 2001). The BDNF gene is located on chromosome 11p13, and it encodes pre-protein of 247 amino acids that is later cleaved to produce mature protein of 120 amino acids. The genetic polymorphisms identified in this gene have been associated with increased risk of developing psychiatric disorders such as obsessive compulsive disorder (OCD), Anxiety, eating disorder, schizophrenia, and bipolar disorder. A single nucleotide polymorphism (SNP) in the BDNF gene at position c.196 (G/A) has been reported which replace valine with methionine (Valine/Met, rs6265) in the region which encodes the prodomain. This change has been characterized with impairs the BDNF protein's function. Some studies supported this polymorphism with various psychiatric disorders (Hall et al., 2003;Hemmings et al., 2008), however, some previous and recent studies did not support these finding of association (Wang et al., 2015;Alonso et al., 2007;Wendland et al., 2007). This is controversial and unclear whether this polymorphism is related to these disorders, however, the other genetic and environmental factors are remain unknown. OCD is a fourth most common deliberate psychiatric disorder. Its worldwide prevalence is 1-3% of children and adults and equally affects both males and females (Sarvet, 2013). OCD is a mental disorder, according to the Diagnostic Manual of Mental Disorders (DSM-IV) criteria, this disease is characterized by intermittent and interfering obsessive thoughts and cyclic compulsive behaviors which reduce anxiety that is associated with obsessions. Previous studies on twin and family's genetic showed that OCD is a chronic disorder and established irrespective of sex, race, marital status, intelligence, socioeconomic status, nationality or religions (Hettema et al., 2001;Sampaio et al., 2013). Patients with OCD experience intrusive and repetitive thoughts known as obsession and the uncontrollable urge to repeatedly acts or rituals compulsions to reduce the anxiety produced by obsessional thoughts (American Psychiatric Association, 1994). Several studies revealed that OCD is associated mainly with dysregulation and less concentration of 5-HT neurotransmission, which may play a possible role in the pathogenesis of OCD (Zohar et al., 2000;Westenberg et al., 2007).

Materials and methods
Genomic DNA was extracted from the blood using a standard procedure (Sambrook and Russel, 2001). Primers were designed through Primer3 software to amplify the complete BDNF coding region (Table I). The complete coding region of BDNF gene was directly sequenced using ABI 3130xl automated sequencer (Applied Biosystem Inc, Foster City, CA). The sequenced data was aligned and analyzed using Blast Local search Alignment tool. Chi square (X 2 ) test was used for categorical variables and student's t-test was used for continuous variables. In-silico prediction analysis was performed using mutation taster, Polyphin2 and Exon splicing enhancer software.

Results and discussion
In this study, we investigated the genetic variation of OCD individuals in the Punjab ethnic group in Pakistan. we aimed at exploring the existence of possible genetic interaction between BDNF gene and clinical variables in samples of OCD patients (Fig 1). The demographic distribution of the both groups is illustrated in Table II. In both groups the distribution of male and female was similar. We observed one variation G>A, resulting in an amino acid change from valine to methionine at 66 amino acid position. Two patients were found homozygous for Val>Met and heterozygous for Val>Met at c.196 position in BDNF gene. In-silico analysis of this genetic variation was performed using mutation taster (http://www.mutationtaster.org/) and Exon Splicing Enhancer software (http://www.umd.be/ HSF3/4DACTION/AW_QuickMutant). Resulting data

O n l i n e F i r s t A r t i c l e
predicted it as disease causing with a site broken effect. We collected blood samples of other healthy family members of the patients observed with Val>Met polymorphism, but found as negative for this variation. Several studies supported the hypothesis that BDNF gene Val66Met genotype has been found to be associated with the clinical features of OCD in various ethnic groups in the world (Hall et al., 2003;Hemmings et al., 2008Hemmings et al., , 2013. Our results support most of previous and recent studies. BDNF Val66Met polymorphism has been extensively studied, the first study that associates Val66Met was conducted in 2003 (Hall et al., 2003), that study reported that the Met66 allele revealed a protective effect on OCD (Hall et al., 2003). Other previous studies reported Val66Met was a risk allele (Hemmings et al., 2008(Hemmings et al., , 2013. BDNF polymorphism Val66Met has been studied in several other mental disorders such as schizophrenia, bipolar disorder, depression, drug abuse and anorexia nervosa, most of them did not associate between the Val66Met and the disease (Kanazawa et al., 2007;Verhagen et al., 2008;Brandys et al., 2013;Gyekis et al., 2013;González-Castro et al., 2015). Some found the association between OCD and Val66Met, showed inconsistent results (Wang et al., 2015;Alonso et al., 2007;Wendland et al., 2007;Zai et al., 2005).
It has been found that there are significant ethnic differences in BDNF Val66Met polymorphism (Pivac et al., 2009). Caucasian carriers with Val/Val genotype have higher anxiety scores than carriers of heterozygous Val/ Met genotype or homozygous Met/Met genotype (Lang et al., 2005). Comparison of genotype frequency Val/Val, Val/ Met and Met/Met reported as significant ethnic differences in Val66Met variation in the BDNF gene with a similar distribution of Val/Met alleles among Korean individuals and the most frequent genotype in Caucasian population was Val/Val (Pivac et al., 2009). Homozygous Val/Val (G/G) genotype is also considered as an increased risk for sporadic Alzheimer's disease (AD) and homozygous A (Met) allele with the increased risk of poorer episodic memory (Ventriglia et al., 2002;Egan et al., 2003). Also evidence that the Val66 allele might increase the risk of depression as it was specially transmitted to bipolar volunteers in families (Neves-Pereira et al., 2002;Sklar et al., 2002). However, a study conducted on Japanese population, found no association of bipolar disorder and BDNF polymorphism (Nakata et al., 2003).
As BDNF gene plays a significant role in regulating neuronal proliferation and function and participating in the synaptic plasticity (Huang and Reichardt, 2001b), many previous and recent studies found and support that its peripheral levels were down regulated in various mental disorders such as OCD (Wang et al., 2015), anorexia nervosa (Saito et al., 2009), depression (Sen et al., 2008) and schizophrenia (Chen et al., 2014). It might be suggested that the lower peripheral blood level is a common feature and might be a marker reflecting impairment of synaptic plasticity and neuronal function in various mental disorders.

Conclusion
Our finding suggests that polymorphism Val66Met (rs6265) have some possible role of OCD development in Punjabi ethnic group. Genetic association of this polymorphism can be validated by screening at large scale.