Özet:

INTRODUCTION The worldwide prevalence of anxiety disorders range from 10.6% to 16.6% (1). Although the etiopathogenesis still cannot be clearly explained, there are on-going investigations on potential factors including psychosocial theories, neuroanatomical models, endocrine processes, and autonomic nervous system dysregulation. Recently, the researches on genetic etiology have gained momentum. Available genetic studies reported the prevalence of generalized anxiety disorders (GAD) to be nearly 3-fold higher among the first-degree relatives of GAD probands (2,3). The heritability rate of anxiety disorders has been reported to be moderate, ranging from 32% to 48% (4,5). Neuropeptide Y (NPY) is a 36-amino acid peptide which is a member of the NPY family of biologically active peptides. NPY has an essential role in many physiologic functions such as food intake, energy homeostasis, circadian rhythm, and cognition. Additionally, NPY has anxiolytic properties, and it is recommended as a critical component in the stress response (6), reported as a marker of stress-resilience (7-9). NPY has an endogenous anxiolytic activity. In addition to the NPY Y1 and Y2 receptors, the Y4 and Y5 subtypes could have essential roles in anxiety-related behaviors (10). The biological actions of NPY are moderated by the Y1, Y2, Y4, and Y5 receptors in mammals. The Y1 receptor activation is anxiolytic whereas Y2 receptor stimulation is anxiogenic (7). Previous studies aimed to demonstrate (demonstrated) that NPY has an important effect on stress-related psychiatric disorders and may be effective in the etiopathogenesis of both anxiety and depression as well as that of post-traumatic stress disorder (PTSD) (11-13). Even low serum NPY levels have been shown to improve after treatment in patients with anxiety symptoms and depressive symptoms (14). Additionally, NPY was thought to be in close association with substance abuse, alcohol addiction, and smoking (15-18). Since NPY is considered to be closely related to stress, researches have begun to focus on NPY gene studies. The NPY gene is one of the nine genes (NPY, Brain derived neurotrophic factor (BDNF), B-cell Lypmhoma-2 (BCL2), DED2, Regulator of G protein signaling 2 (RGS2), 5-hydroxytryptamine receptor 1A (HTR1A), Mono amine oxidase A (MAO-A), Estrogen receptor 2 (ERS2) and Serotonin transporter (5-HTT)), which have been found to be closely associated with generalized anxiety disorder in genetic studies (19). It is thought that there may be a close relationship between the susceptibility to anxiety disorders and heredity pattern of NPY heredity (20). A study conducted in humans suggests that NPY expression modulates stress reactions and emotional responses (21). The NPY rs16147 modifies risk of post-disaster GAD under conditions of high stressor exposure (22). NPY has robust anxiolytic properties and is reduced in patients with anxiety disorders. However, the mechanisms, by which NPY modulates circuit function to reduce anxiety behavior, are not known. Anxiolytic effects of NPY are mediated in the CA1 region of hippocampus, and the NPY injection into hippocampus alleviates anxiety symptoms in the Predator Scent Stress model of stress-induced anxiety. The mechanisms that regulate NPY release and its effects on CA1 synaptic function are not fully understood (23). Increased anxiety levels were reported in rats that were given NPY Y1 receptor antagonist (24), whereas the anxiety levels decreased when NPY or NPY Y1 receptor agonist was injected in the cerebrospinal fluid (25). While the NPY Y5 receptor agonist led to anxiolytic effect in rats, the administration of NPY Y5 receptor antagonist resulted in anxiety-like behavior (26-28). Current data indicate the potential of the NPY system as a target for new pharmacological treatments of stress-related disorders, including anxiety and depression. The development of Y2 antagonists currently seems to offer the most promising strategy for developing these clinical treatments (29). In a recent study, the use of intranasal NPY has been shown to be effective in the prevention and treatment of PTSD (30). Genetic studies have examined the role of the single gene variant -399 C / T (rs16147) NPY gene (7p15.1) in panic disorder; however, they have not yielded significant results (31). Nevertheless, such studies demonstrated rs16147:T>C to be associated with anxiety and depressive symptoms (32). In a study performed among patients with anxious depression, the less active NPY rs16147 −399C allele conferred slow response to treatment and failure to achieve remission (33). In a recent study, it is shown that NPY polymorphisms modifies the effect of childhood adversities on anxiety susceptibility (Rs 16142, Rs 2023890, Rs 17374047) (15). It is known that the genes encoding the NPY Y1, Y2 and Y5 receptors in humans are located in the 4q31.3-q32 chromosome region. A genetic linkage study performed in 2006 demonstrated a strong association between the chromosome region 4q31-q34 and anxiety disorders including panic disorder, and specified this chromosome region as the risk locus for anxiety disorders (34). Ultimately, the genes encoding the NPY receptors were revealed to be in the region, which is considered as the risk locus for anxiety disorders. In another study designed in the light of this information, researchers examined the polymorphisms of the NPY Y1, Y2 and Y5 receptor genes within the scope of panic disorders and reported significant results regarding the region of the NPY Y5 gene receptor (33). In the present study, we aimed to determine the relationship between the NPY gene polymorphisms and clinical variables in consideration of the results obtained from a few studies in the literature addressing the association between the NPY gene polymorphisms and anxiety disorders. In line with this objective, we made a comparison between the patients with generalized anxiety disorder and those with panic disorder as well as and healthy controls. METHODS Study Participants The study population consisted of patients and healthy controls, who presented to the Gülhane School of Medicine’s Psychiatry Outpatient Clinic between July, 2015 and April, 2016. Ninety-three patients (48 men, 45 women), who were diagnosed with panic disorder or generalized anxiety disorder according to the Diagnostic and Statistical Manual of Mental Disorders (DSM–5), and 76 healthy controls were enrolled into the study. The patients, who had new-onset disorders; who were diagnosed with first-time disorder and scheduled for treatment; who were previously given treatment but not on medication anymore, and those, who were still suffering from the symptoms despite their on-going medication (except for benzodiazepines),were included in the study. The inclusion criteria for the control group were not having a psychiatric diagnosis according to DSM-5, not having a psychiatric complaint, willingness for participation in the study, and having sociodemographic characteristics consistent with the patient group. All participants provided written consent for participation in the study. The study was approved by the GATA Clinical Research Ethics Committee (KAEK-2015/24, 25.02.2015). Psychometric Measurements Sociodemographic Data Form: This form aimed to obtain information on patients’ age, gender, educational status, occupation, economic status, height and weight, smoking status, medical illness, and the history of psychiatric illness in the family. Beck Anxiety Inventory: The Beck Anxiety Inventory (BAI), which was developed by Beck et al. in 1988 to measure the severity of anxiety in the psychiatric population, is a Likert type self-report scale (35). The validity and reliability study of the Turkish version of BAI was performed by Ulusoy et al. in 1998. (36). The Cronbach’s Alpha reliability coefficient was 0.93 in the present study. Panic Agoraphobia Scale: This scale, which was developed by Bandelow and published in 1995, measures the severity of panic disorder (37). The Panic Agoraphobia Scale, which contains 5 sub-scales, namely panic attacks, agoraphobic avoidance, anticipatory anxiety, disability, and health concerns, consists of a total of 13 yes-no questions evaluating the severity of panic disorder and agoraphobia. It is employed in clinical researches. The validity and reliability study of the scale was conducted by Tural et al. (38). Internal consistency (Cronbach’s alpha) was 0.86. Test-retest reliability was 0.70 (p<0.001). Panic Disorder Severity Scale: The Panic Disorder Severity Scale was developed by Shear et al. in 1997 (39). The scale was adapted to Turkish by Monkul et al. in 2004. In the validity and reliability study of the Turkish version, the correlation between the scores of the observer‐rated Panic Disorder Severity Scale (P&Ao) (r=0.87 and r=0.65) and the self‐rating Panic and Agoraphobia Scale (P&Asr) (r=0.87 and 0.58) was analyzed for the whole group (n=174) and the panic disorder group (n=104). There was significant correlation between the results (r=0.89, p<0.0001) (40). The internal consistency (Cronbach's alpha) of the PDSS was 0.92-94. The inter-rater correlation coefficient was 0.79. The test-retest correlation coefficient after 4 weeks was 0.63. The cut-off point was six/seven, associated with high sensitivity (99%) and specificity (98%). This study confirmed the objectivity, reliability and validity of the Turkish version of the PDSS. Somatosensory Amplification Scale: The Somatosensory Amplification Scale (SSAS) was developed by Barsky et al. in 1990 (41). It is an assessment tool that measures how patients experience somatic symptoms and their susceptibility to somatization. This scale is applicable to patients with psychiatric or medical conditions as well as healthy people. It is a single page, self-report questionnaire consisting of 10 items rated on a 1 (not at all) to 5 (all the time) point Likert scale. The cut-off score of the scale was not calculated and the scale can be used in comparative studies. Barsky et al. determined the test-retest reliability coefficient as r=0.79 and the internal consistency value as 0.82. The reliability and validity of the Turkish form of the somatosensory amplification scale (SSAS) was performed by Güleç and Sayar in 2007. In the validity analysis, there was a significant difference between the averages of the patient group and control group (42). The SSAS scores were normally distributed, and had acceptable test-retest reliability (r=0.73) and internal consistency (alpha, 0.62-0.76). Item to scale correlations varied from 0.10 to 0.72, and most were highly significant. Test-retest, internal reliability, and item-total score correlation, discriminating power for specific groups and criterion-related validity of the SSAS showed that the scale had acceptable reliability and validity for the Turkish population. Anxiety Sensitivity Index, ASI-3: It was first developed in 1986 by Reiss et al. (43) to measure anxiety sensitivity. The validity and reliability study of the translated form was performed (44). Later, a revised version of the scale and the Anxiety Sensitivity Profile were also developed. The recent version of the scale, namely the Anxiety Sensitivity Index - ASI-3, was developed by Taylor at al. in 2007. The validity and reliability of the scale was conducted (45). In this study, it was found that ASI-3 had a high internal consistency (Cronbach α=0.93) and the scale had a fairly good test-retest reliability (r=0.64, p<0.001). While the Beck Anxiety Inventory (BAI) was administered to all participants, the Panic and Agoraphobia Scale (PAS) and the Panic Disorder Severity Scale (PDSS) were administered only to the panic disorder patients. In this way, we intended to analyze the severity of the disorder. Additionally, all participants were asked to complete the Somatosensory Amplification Scale (SAS) and the Anxiety Sensitivity Index-3 (ASI-3). SNP Selection and Genotyping The single-nucleotide polymorphisms (SNPs), covering the gene region of NPY Y1, Y2, and NPY Y5 at the risk locus on chromosome 4q31-q34, which were shown to be involved in the pathogenesis of panic disorder (34), were included in the present study based on previous association findings in the pathogenesis of panic disorder at non-mental diseases as well as known functional relevance: rs12507653, rs12510104, rs7687423 (46), rs4691075 at NPY Y1; rs11099992, rs12507396, rs1047214, rs11728843 at NPY Y2 (47-48); rs11724320, rs11946004 at NPY Y5 (33,47,48). Blood samples (2 mL) were taken from each patient into EDTA-containing tubes, and genomic DNA was isolated from whole blood using High Pure PCR Template Preparation Kit (Roche, Germany). A real-time PCR device Light Cycler 2.0 (Roche) was used for the detection of polymorphisms in the study. The rs 12507653, rs 12510104, rs 7687423, rs 4691075 SNP regions for NPYR1, rs12507396, rs1047214, rs11728843, rs 4234955, rs 11099992, rs 12507396 SNP regions for NPYR2; rs11946004, rs 11724320 SNP regions for NPYR5 were genotyped using the end-point genotyping method with TaqMan®Genotyping Assays (Applied Biosystems, Foster City, CA) Statistical Analysis The IBM SPSS 22.00 package program was used for data analysis. We used numbers, and the percentage, mean and standard deviation values for the descriptive statistics of the study data. Normal distribution of continuous variables was assessed with the Kolmogorov-Smirnov test. The Chi-square test was employed for inter-group comparisons of the intermittent variables while the Kruskal-Wallis test was used for multiple comparisons of continuous variables. For post-hoc tests, we performed the Mann-Whitney U test with Bonferroni correction. In the comparison of two groups, we preferred the T test for normally distributed variables, but used the Mann-Whitney U test when variables were not normally distributed. The Pearson Correlation test was utilized to evaluate linear relationships between variables. Considering the comparison of the patient and control groups, we performed the Multivariate Logistic Regression Analysis to determine the effect of genotype without the influence of other variables. Univariate and multivariate logistic regression analysis performed by SPSS for Windows. The variables that have lower p-value than 0.1 in univariate analysis were added in multivariate analysis. The Forward-Stepwise method was used in multivariate analysis. p<0.05 was considered statistically significant. Additionally, the Hardy–Weinberg equilibrium was checked to assess the consistency of the genetic data distribution of the study population with the expectations. RESULTS Our patient group consisted of 45 male (48.4%) and 48 (51.6%) female patients while there were 41 men (53.9%) and 35 women (46.1%) in the control group. The average age of the patients was 37.1±11.1 years, whereas the average age of the controls was 36.2±8.6 years. There was no significant difference between the groups regarding the sociodemographic variables including age, gender, educational background and smoking. Within the scope of the study, we analyzed 11 SNPs (Single Nucleotide Polymorphisms) of the NPY Y1, Y2 and Y5 receptors. However, only the allele and genotype were considered in the evaluation of genetic variants. There was a significant difference (p≤0.05) between the patient group and the control group in 6 of 11 SNPs analyzed. The distribution of alleles and genotypes in the patient and control groups is shown for each SNP in Table 1. We also examined whether the polymorphisms varying between the patient group and the control group differentiated between the panic disorder and generalized anxiety disorder groups. As a result, we found a significant difference between the two groups for the NPY-Y2-SNP4 (rs11728843) (Table 2). This study analyzed the association between the clinical variables of the patient group (the scores of the Beck Anxiety Inventory, the Panic Disorder Severity Scale, the Panic and Agoraphobia Scale, the Anxiety Sensitivity Index-3 and the Somatosensory Amplification Scale) and polymorphisms. The correlation of the scores of ASI-3 and SSAS to polymorphisms was also assessed for the control group. This assessment was conducted by analyzing whether the mean scores of the scales varied according to each variant. Table 3 shows the SNPs, in which the mean scale scores were significantly different for each variant. The patients with variant genotype CC in the region of NPY-Y1-SNP4 (rs4691075) had higher scores in the BAI and ASI-3 as compared to the patients that had other variants (p≤0.05). Moreover, the BAI scores of the patients with CC variant in the region of NPY-Y2-SNP3 (rs1047214) were higher than those of the patients with other variants (p≤0.05). The patients that had AA and GG variants in the region of NPY-Y2-SNP4 (rs11728843), nevertheless, got higher BAI scores than the patients with other variants (p≤0.05). The regression analysis indicated that the presence of GG genotype in the region of NPY-Y1-SNP3 (rs7687423) increased the risk of anxiety disorder development by 65.8 fold as compared to the genotype AA (p=0.034) (%95 CI: 1.37-3158.61) (Table 4). We examined whether the genetic data obtained on the relevant polymorphism regions complied with the Hardy–Weinberg equilibrium, and found that the data regarding the NPY-Y1-SNP3 (rs7687423) region pertained to the Hardy–Weinberg equilibrium while the data on other regions does not. DISCUSSION In this study, a total of 11 SNPs encoding the NPY receptors were examined both in an anxiety disorder group consisting of patients with panic disorder and generalized anxiety disorder and in a control group of healthy individuals. Subsequently, we evaluated the potential association between genetic findings and anxiety severity, anxiety sensitivity, and somatosensory. In short, this study indicated that the genetic polymorphisms of NPY-Y1-SNP3 (rs7687423), NPY-Y1-SNP4 (rs4691075), NPY-Y2-SNP2 (rs12507396), NPY-Y2-SNP3 (rs1047214), NPY-Y2-SNP4 (rs11728843) and NPY-Y5-SNP3 (rs11946004) differentiated between the anxiety disorder group and the control group. It was observed when a comparison was made among the patients in the anxiety disorder group that the NPY-Y2-SNP4 (rs11728843) polymorphism varied between the PD and GAD patients. Additionally, the genotype CC in the NPY-Y1-SNP4 (rs4691075) region was associated not only with the BAI scores, but also with the ASI-3 scores. Furthermore, the genotype CC in the region of NPY-Y2-SNP3 (rs1047214) was related with the BAI scores while the genotype GG in the NPY-Y2-SNP4 (rs11728843) region was found to be related with the BAI scores. A study conducted by Domschke et al. in 2008 researched the link between panic disorder and NPY polymorphisms (41). In that study, a total of 16 SNPs covering the gene region of NPY, NPY Y1, Y2, and NPY Y5 (namely, NPY- rs16157, rs16147, rs16139, rs9785023, rs16474/ NPY Y1- rs12507653, rs12510104, rs7687423, rs4691075/ NPY Y2- rs11099992, rs12507396, rs1047214, rs11728843/ NPY Y5- rs4234955, rs11724320, rs11946004) were analyzed. Domschke et al. could not find an association between the gene variants encoding the NPY, NPY Y1 and NPY Y2 receptors and panic disorder; however, both the allele and genotype of the rs11946004 polymorphism encoding the NPY Y5 receptor were significantly associated with panic disorder. The authors suggested that their results supported the theory that the NPY Y5 receptor variants have an influence on the etiology of panic disorder in a potentially gender-specific manner and can be considered a risk locus for anxiety disorders. In the present study, however, there was a difference between panic disorder and generalized anxiety disorder groups regarding the NPY-Y5-SNP3 (rs11946004). Although these findings support the study by Domschke et al., our study suggests that the NPY-Y5-SNP3 (rs11946004) gene region is not only associated with panic disorder, but also with generalized anxiety disorder. In addition to the results of Domschke et al., we showed a relationship with other SNPs encoding the NPY Y1 and NPY Y2 receptors (namely, rs7687423, rs4691075, rs12507396, rs1047214, rs11728843). The fact that the rs4691075 (NPY Y1-SNP4), rs1047214 (NPY Y2-SNP3) and rs11728843 (NPY Y2-SNP4) variants differentiated between the patient group and the control group and also in terms of the mean scores of the clinical scales proved the significance of these variants. These findings are consistent with the results of the study by Kaabi et al. referring the chromosomal region 4q31-q34 as a risk locus for anxiety disorders (41). In our study, the variant of rs11728843 (NPY Y2-SNP4) showed a difference between the generalized anxiety disorder and panic disorder groups. This is a new finding, which should be supported with further studies. When a regression analysis was carried out to clarify the association between gene polymorphism and anxiety disorder, the presence of GG genotype in the region of NPY-Y1-SNP3 (rs7687423) was observed to raise the risk of anxiety disorder by 65.8 fold (as compared to AA genotype). This is a recent finding, and since the only SNP region complying with the Hardy-Weinberg principle was NPY-Y1-SNP3 (rs7687423), this new finding may be significant. However, the regression analysis performed separately for the panic disorder and generalized anxiety disorder groups did not show such increased risk. This study has certain limitations to be addressed. The most important limitation is the relatively small size of the sample group, which limits the statistical strength and the validity of the results. Since the frequencies of the variants to be analyzed were not available for Turkish society in the planning phase of the study, we were not able to calculate the sample size. When a power analysis was performed with the values obtained from the study, the power of our study (power value) was found to range from 0.54 to 0.99 which shows the size of our study sample to be partially limited. Moreover, we assessed the compliance of the genetic data distribution to the Hardy-Weinberg equation and found that none of the variants, other than the rs7687423 (NPY-Y1-SNP3), complied with that equilibrium, suggesting that the data that do not conform to the principle cannot be generalized to the population. This may have resulted from the fact that we conducted our study with a limited population. Another limitation is that although the participants were given the Beck-A Index for subjective assessment of the severity of anxiety, their assessments were not supported by a clinician through objective anxiety assessment methods. As far as we know, this is the first study investigating the polymorphisms of the NPY gene receptor through a comparison between the panic disorder and generalized anxiety groups and analyzing their association with clinical variables. CONCLUSIONS In sum, we examined the NPY gene polymorphisms and compared the variants between the anxiety disorder group and the control group. We explored whether the somatosensory amplification and anxiety sensitivity, which are clinical indicators of disease severity, significantly differed depending on the variants. In this way, we intended to reveal the genetic aspects of these factors and gained an insight about their relationship with the NPY system. NPY is one of the new areas of interest in biological psychiatry and particularly addressed due to its potential role in anxiety. Clarification of the association of NPY with anxiety disorders will ensure better understanding of the biological aspects of the disorders and be guiding in the development of new treatment methods. Acknowledgments: We thank the Health Sciences University Gülhane School of Medicine for providing laboratory materials. Ethics Committee Approval: The study was approved by the GATA Clinical Research Ethics Committee (KAEK-2015/24, 25.02.2015). Conflict of Interest: No potential conflict of interest was reported by the authors. Financial Disclosure: No financial support was reported by the authors. REFERENCES 1.Somers JM, Goldner EM, Waraich P, Hsu L. Prevalence and incidence studies of anxiety disorders: A systematic review of the literature. Can J Psychiatry 2006;51(2);100-13. 2.Goldstein RB, Weissman MM, Adams PB, Horwath E, Lish JD, Charney D et al. Psychiatric disorders in relatives of probands with panic disorder and/or major depression. 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