Genetic Polymorphisms of Nervous System Development and the Risk of Posttraumatic Stress Disorder

Background: Posttraumatic stress disorder (PTSD) is a complex severe polygenic psychiatric disease, influenced by environmental and genetic factors. PTSD development and progression is characterized by cognitive impairment, which may result in altered processes of nervous system development and synaptic plasticity, where a number of growth factors and their receptors were shown to play important role. Since neurotrophins play an essential role in the development of central nervous system, it is widely implicated in psychiatric disorders. The aim of this study is to investigate the potential association functional polymorphisms of genes encoding netrin G1 (NTNG1), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and its receptor (NGFR) with PTSD. Methods: Study groups consisted of 200 combat veterans with PTSD and an equal number of controls with no family or past history of any psychiatric disorders. The DNA samples were genotyped for NTNG1 rs62811; BDNF rs6265; NGF rs6330, rs4839435; NGFR rs11466155, rs734194 SNPs using polymerase chain reaction with sequence specific primers. Results: According to the results, NGF rs6330 was overrepresented in patients with PTSD compared to controls. Furthermore, negative association for BDNF rs6265, NGF rs4839435 and NGFR rs734194 was observed in PTSD patients. Conclusions: In summary, BDNF rs6265, NGF rs6330, rs4839435 and NGFR rs734194 are implicated in PTSD in Armenian population. However, further research is required to provide the definitive evidence of selected polymorphism association with gene expression.


Introduction
Posttraumatic stress disorder (PTSD) is a complex severe multifactorial polygenic psychiatric disease (ICD-10 code: 43.1; DSM-V code: 309.81), influenced by environmental and genetic factors [1] [2].Although most people experience a traumatic event during their lives and many of them experience multiple traumatic events, only a minority will develop PTSD [3] [4], suggesting that individual vulnerability and resilience factors are important in PTSD pathophysiology.PTSD is heritable [5] [6] [7] [8], suggesting that these individual differences might, in part, be explained by genetic factors.However, our understanding of biological mechanisms underlying PTSD is still incomplete.The results of many studies show that genes related to the physiological stress response (e.g., glucocorticoid receptor activity, neuroendocrine release) [9] [10] [11], learning and memory (e.g., plasticity) [12] [13], mood, and pain perception are tied to neural endophenotype associated with PTSD.These genes are associated with and can predict the structure and the function of neurons in brain areas responsible for such functions, as attention, decision-making, memory, cognition, response to the pain and other threats.Evidence suggests these risk polymorphisms and neural intermediate phenotypes are vulnerabilities toward developing PTSD in the aftermath of trauma, or vulnerabilities toward particular symptoms once PTSD has developed [14] [15].PTSD development and progression is characterized by cognitive impairment, which may result in altered processes of nervous system development and synaptic plasticity [16], where a number of growth factors and their receptors were shown to play important role.
Netrins including netrin G1 (NTNG1) are known to be axon guidance factors in the developing brain.They could be very important contributors to the genetic risk for psychosis [17].Neurotrophin family is another important class of signaling molecules in the brain recognized for their nerve growth promoting function and is recently identified as crucial factors in regulating neuronal activity in the central and peripheral nervous systems.The family members including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are the essential mediators of synaptic and morphological plasticity, neuronal growth, survival, and differentiation, especially in the developing brain.It is thought they may play an important role in pathogenesis of PTSD.We examined the hypothesis that allelic variations in NTNG1, NGF, NGFR, as well as BDNF contribute to the risk of development of PTSD.

Study Subjects
Study groups consisted of 200 combat veterans with PTSD (mean age: M ± SD = 54.52 ± 11.0 years) and an equal number of controls (mean age: M ± SD = 43.6 ± 9.1 years) with no family or past history of any mental disorders, as well as disorders characterized by alterations in apoptosis and synaptic plasticity.Clinical diagnosis was made according to DSM-IV-TR (American Psychiatric Association) criteria [18].The study was verified and approved by the Ethics Committee of the Institute of Molecular Biology (IRB #00004079).The informed consents from all study subjects were collected for these studies.

Blood Sampling and Genomic DNA Extraction
The experiments were performed using genomic DNA samples of study subjects.
Genomic DNA was prepared from peripheral venous blood using standard phenol-chloroform extraction [19] and stored at −30˚C until use.
The presence/absence of allele-specific amplicons in the PCR products was visualized in 2% agarose gel stained with ethidium bromide fluorescent dye using DNA molecular weight markers as a reference.To check the reproducibility of results, randomly selected DNA samples (10% of total) were genotyped twice.

Data Analysis
The distributions of genotypes for selected SNPs were checked for correspondence to Hardy-Weinberg equilibrium.The significance of differences in genotype and allele frequencies and minor allele carriage between patients and healthy subjects was determined using Pearson's Chi-square test.P-values < 0.05 were considered statistically significant.P-values adjusted by Bonferroni multiple

Results
Cases and controls were well matched in terms of gender and ethnicity.Genotype frequencies did not deviate from HWE.The results of genotyping are presented in Table 3. Bonferroni correction, difference in allele frequency between the patient and control groups for these minor alleles remained significant.

Discussion
The etiology and pathology of PTSD is not fully understood.Neurobiological abnormalities in PTSD patients are numerous and probably occur as a result of dysregulation of several stress-mediating systems when exposed to psychological trauma.In certain extreme conditions, people with genetic predispositions have higher probability of developing these pathologic changes.
Many observations suggest that PTSD is due to a series of genetic anomalies in neural development and differentiation [10] [21] [22].Neurotrophins NGF and BDNF, which are known for their classical role in neurogenesis and synaptic plasticity, are most probably involved in trauma memory and cognitive dysfunction in PTSD.
The present study investigates the BDNF val66met (rs6265), the NTNG1 rs628117, the NGF rs6330 and rs4839435, and the NGFR rs11466155 and rs734194 variants and the risk of development of PTSD in combat veterans in Armenian population.
BDNF is part of the neurotrophin family of growth factors, such as the NGF, neurotrophins 3 and 4 (NT-3, NT-4).They are responsible for enhancing progenitor-cell proliferation and differentiation, cell growth, regeneration processes, neuronal survival, synaptic regulation and remodeling, the regulation of plasticity, and repair and connectivity in the brain [23].Investigations of biological factors commonly associated with learning and memory formation have indicated that BDNF may be a promising candidate.BDNF is highly expressed in the mamma-American Journal of Molecular Biology lian brain, especially in the hippocampus, which is functionally associated with learning and memory processes [24] [25].Its binding to TrkB (tyrosine receptor kinase) causes intracellular cascades affecting neuronal development, plasticity, long-term potentiation, and apoptosis [26] [27].The polymorphism rs6265, also known as Val66Met, which results in a change from valine to methionine in the precursor protein, of BDNF has been hypothesised to be important in fear learning and has shown some promising associations in animal models [28].
There are several studies with controversial results of BDNF rs6265 in association with PTSD susceptibility [25] [29].Our study showed the involvement of BDNF rs6265 in PTSD.It shows a potential protective factor of the minor allele carriers for PTSD.However further research is required to provide the definitive evidence of BDNF rs6265 polymorphism association with BDNF level.
Netrins including NTNG1 are known to be axon guidance factors in the developing brain.They could be very important contributors to the genetic risk for psychosis.Moreover, studies suggested that NTNG1 genetic polymorphisms probably are implicated in pathogenesis of schizophrenia [30] [31] [32], ischemic stroke as well as other neurodevelopment disorders [33].In our study, the NTNG1 rs628117 genotypes were equally distributed in the groups of PTSD patients and controls, so it is not associated with PTSD in Armenian population.
Nerve growth factor is important for the development and maintenance of the sympathetic and sensory nervous systems.Extracellular ligand for the NTRK1 and NGFR receptors activates cellular signalling cascades through corresponding receptor tyrosine kinase cascade to regulate neuronal proliferation, differentiation and survival [34].In the literature, there are a limited number of studies evaluating the relationship between NGF gene and PTSD.However, there are several studies show association between NGF and anxiety and psychiatric disorders [35] [36] [37] [38].A non-synonymous single-nucleotide polymorphism in the NGF gene, rs6330, produces an alanine to valine replacement at amino acid position [36], and is thought to involve intracellular processing and secretion of NGF.Allelic variations at the rs6330 locus have previously shown associations with anxiety-related traits and affective disorders [37] [38] [39].
These neurotrophins exert their actions through binding to the NGFR that belongs to the tumor necrosis factor receptor super family and has similar affinity for all neurotrophins as well as to members of neurotrophic tyrosine receptor kinase family, each selectively binding a different neurotrophin [40] [41] [42].
The results of the present study demonstrated a positive association between PTSD and the rs6330 SNP of the NGF gene.Also, a negative association between this disorder and rs4839435 SNP of the NGF gene as well as the rs734194 SNP of the NGFR gene was found.

Conclusion
In summary, our results demonstrate the association of BDNF rs6265, NGF

Table 2 .
The PCR conditions.

Table 3 .
Distribution of genotypes, alleles and minor allele carriage frequencies of the selected SNPs in patients with PTSD and controls.According to the results obtained, for NTNG1 rs628117 no significant differences in the allele and genotype frequency were found between PTSD patients and controls.In the case of the BDNF gene the rs6265 * A allele was lower in pa- a. p corrected values for comparison of mutant allele frequency between PTSD patients and controls.b. p corrected values for comparison of mutant allele carriage between PTSD patients and controls.American Journal of Molecular Biology However, further research is required to provide the definitive evidence of selected polymorphism association with gene expression.