Association between serotonin 2A receptor genetic

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Gene 658 (2018) 191–197

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Research paper

Association between serotonin 2A receptor genetic variations, stressful life events and suicide Asghar Ghasemia,1, Morteza Seifib,

⁎,1

, Fatemeh Baybordic, Nasim Danaeid, Bahram Samadi Radc,

T



a

Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Medicine, University of Alberta, Edmonton, AB, Canada c Legal Medicine Research Center, Legal Medicine Organization, Tabriz, Iran d School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Polymorphisms Serotonin 2A receptor Stressful life Suicide

Aim: Life events are series of events that disrupt a person's psychological equilibrium and may enhance the development of a disorder such as suicide. Several studies have assessed a relationship between 5-hydroxytryptamine (serotonin) 2A receptor (5-HTR2A) gene polymorphisms with an increased risk of suicide. However, there has been no study about the association between three 5-HTR2A gene polymorphisms, A1438G (rs6311), T102C (rs6313) and C1354T (rs6314), suicide, stressful life, and loss events in a same time. Methods: Relatives of 191 suicide victims were interviewed using a semi-structured questionnaire designed according to Iranian culture. Venous blood was taken from all subjects for DNA isolation. 5-HTR2A polymorphisms in a total of 191 suicide victims and 218 healthy controls were genotyped using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Chi-squared and Fisher's exact tests were used to compare genotype and allele frequencies between suicide and control groups. Correction for multiple comparisons was calculated using Bonferroni correction. Results: There was a significant association between the 102 C/C genotype of 5-HTR2A gene and suicide (к2 = 8.700, P = 0.012). Furthermore, we found that suicide victims with a 102 C/C genotype had a significantly higher number of stressful life and loss events (P < 0.05). Genotype and allele distributions of A1438G (rs6311) and C1354T (rs6314) polymorphisms of 5-HTR2A gene showed no differences between suicide victims and control participants and there was no association between genotype distribution and higher number of stressful life and loss events (P > 0.05). Conclusion: Our results suggest that C102T (rs6313) polymorphism of 5-HTR2A gene may be involved in the susceptibility to suicide, higher number of stressful life and loss events, but A1438G (rs6311) and C1354T (rs6314) polymorphisms of 5-HTR2A gene were not associated with suicide, higher number of stressful life and loss events.

1. Introduction Suicide is a serious problem worldwide. The extent of this problem is well reflected in the World Health Organisation (WHO) statistics, which reveal suicide as being responsible for 2% of deaths worldwide (Gray et al., 2014). Several recent studies have been carried out that support a possible relationship between genetic factors and suicide (Zalsman et al., 2008; Wasilewska et al., 2017; Ducci and Goldman, 2012; Currier and Mann, 2008). During the last three decades, evidence indicating that altered serotonergic neurotransmission influences

suicide has been accumulated. One of the more consistent findings is the observation of low cerebrospinal fluid (CSF) levels of 5-hydroxyindole-acetic-acid (5HIAA), a serotonin metabolite, among suicide victims (Träskman et al., 1981; Engström et al., 1999). Consequently, research regarding specific genes that may influence suicide has mostly, focused on the serotonergic system. Fourteen known serotonin receptor subtypes are involved in serotonin action (Yoon and Kim, 2009). One candidate gene in the study of suicide is the gene encoding the serotonin 2A receptor (5-HTR2A). There is evidence that the density of 5-HTR2A is upregulated in parietal

Abbreviations: 5-HTR2A, 5-hydroxytryptamine receptor 2A; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; (SNPs), single nucleotide polymorphisms; GXE, gene and environment ⁎ Corresponding authors. E-mail addresses: mseifi@ualberta.ca (M. Seifi), [email protected] (B. Samadi Rad). 1 Contributed equally to this article. https://doi.org/10.1016/j.gene.2018.03.023 Received 21 September 2017; Received in revised form 14 February 2018; Accepted 7 March 2018 Available online 09 March 2018 0378-1119/ © 2018 Elsevier B.V. All rights reserved.

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worthlessness, and poor self-control), interview risk profile (e.g., suicide ideation, suicide plan, and past suicide attempt), and risk buffers (e.g., reason for living, internal strengths for managing risk, and external strengths for managing risk). Suicide methods were as follows: Drugs, the most common method of committed suicide, (58.0%); selfburning (18%); poisoning (15%); hanging (6%); and of suicide victims with firearms (3.0%). The control subjects consisted of randomly selected healthy individuals who visited the Tabriz University Madani hospital for regular health screenings. Subjects all were unrelated Iranian population confirmed as physically healthy on first medical evaluation. The controls with manifestation of somatic diseases, psychiatric problems or physical problems or history of suicidal attempts as assessed in brief interviews by two psychiatrists were excluded from study. Written consents from control subjects and victims' relatives were obtained. Decomposition of samples caused non-reliable and inaccurate genotyping were excluded from criteria.

cortical regions of depressed suicide victims and this increase has been suggested to be at least partly genetically regulated (Turecki et al., 1999). The 5-HTR2A gene is located at 13q14-q21, and several single nucleotide polymorphisms (SNPs) have been identified within this region, a few of which determine amino acid substitutions and may thus be relevant from a functional point of view (Jakubczyk et al., 2013). Two very frequent polymorphisms have been described in this gene include A1438G (rs6311) and T102C (rs6313). Several studies, but not all, suggest a complete linkage disequilibrium between the two polymorphisms and findings suggest that A1438G (rs6311) polymorphism could have functional effects on the expression of this receptor conservative C/T transition at position 1354 of the third exon of the 5HTR2A gene (C1354T, rs6314) (Saiz et al., 2008a). This substitution has variedly been associated with several psychiatric disorders (Beretta et al., 2008). To date, however, genetic association studies of the 5HTR2A gene gave inconclusive results, and the specificity of its potential involvement in suicide is still unclear. in the brain (Peñas-Lledó et al., 2007; Saiz et al., 2008a; Walitza et al., 2012; Yildiz et al., 2013). Among other substitutions, one of the best characterized is the non-. The term stressful life events are commonly shown to play pivotal role in the mental and physical health of a person. Stressful life events can be independent of the personal responsibility of the individual (e.g., loss of loved one) or to be dependent directly and/or indirectly to the person (e.g., job loss) (Cleland et al., 2016). There is a hypothesis that genetic and environment interaction plays an important role in suicide (Roy et al., 2009). This hypothesis, stress-diathesis model, suggests that some level of vulnerability is present for any given psychiatric disorder in each individual. Therefore, suicide may differ depending on the degree of pre-dispositional risk factors and experienced stressful life and loss events (Roy et al., 2009). To the best of our knowledge, there are no studies to investigate the association of 5-HTR2A genotypes with stressful life and loss events. Therefore, in this study, we aimed to determine the possible association between 3 serotonergic polymorphisms of 5-HTR2A gene (A1438G, T102C and C1354T) and suicide in suicide victims. As well, since the importance of environmental stress on various components of the serotonergic system has been supported by studies performed in the past years (Roy et al., 2009), we aimed to evaluate the association of 5-HTR2A polymorphisms and stressful life and loss events in a year before suicide completion in suicide victims.

2.2. Assessment of life events The 12 categories in the list of Threatening Experiences, which was originally developed by Brugha et al., have been used for assessment of stressful life events (Brugha et al., 1985). The items were modified according to our understanding of Iranian culture. Since there are previous reports of the significance of the contribution of loss events to the risk of suicide (Portzky et al., 2005; Cheng et al., 2000), the association of polymorphisms and loss events were analyzed separately, and other stressful events were pooled together. The loss events were identified in the interviews using the Life Events and Difficulties Schedule (LEDS) scale. This scale classifies loss events into four major types including loss of health, person, a cherished idea and employment/material possessions. To consider life events, we used the initial binary scoring system; in which a score of 1 indicated if the life event had happened over the past 12 months, and 0 if it had not. The numbers of all life events, were pooled together and recorded separately for each suicide victims during a period of 12 months prior to suicide. It was scored on the basis that the more life events in a subject, the greater the likelihood of psychiatry disorders including suicide. Table 1 represents stressful and loss event in the year before suicide and in the month before suicide for suicide victims.

2. Material and method 2.1. Study subjects

2.3. Genotyping

Venous blood was taken from all subjects and stored at −70 °C before DNA isolation. 191 suicide victims (141 men and 50 women) with a mean age of 46.6 ± 16.2 years and 218 control persons (132 males, 86 females) with a mean age of 44.8 ± 18.2 years from Iranian origin were enrolled in the study which was approved by the Madani Hospital research ethics review board, the study protocol by University of Tabriz ethics review board, and the Ethics Committee at the Iranian Legal Medicine Organization. Samples were collected in the course of autopsy at the Legal Medicine Organization, Iran. The study was performed in accordance with the ethical standards of Declaration of Helsinki. Psychiatric diagnoses were examined by the specialized clinicians using the Mini International Neuropsychiatric Interview (MINI). Interview time from victims' relatives was around 20 min. One part of this instrument is devoted to the evaluation of suicidal risk, stating questions about past and current suicidality. As well, Victims' relatives completed a socio-demographic interview at intake, the Beck Hopelessness Scale (BHS) (Beck et al., 1974), the Suicidal History SelfRating Screening Scale (SHSS) (Innamorati et al., 2011), and the Global Assessment of Functioning Scale (GAF) (Jones et al., 1995). The questions from the questionnaire for assessment of suicide include individual risk profile (e.g., demographic, family history, and medical illness), symptom risk profile (e.g., depression, intense emotion,

Genomic DNA was extracted from venous blood samples using the Promega commercial kit. For the identification of the A1438G Table 1 Information on stressful and loss event in the year before suicide and in the month before suicide for suicide victims. Stressful and loss event

12 Months before suicide

1 Month before suicide

1. Interpersonal conflicts 2. Separation/divorce

75 suicide: No. 1, 4, 6, 9 45 suicide: No. 1, 2, 5, 6

3. Physical illness 4. Unemployment 5. Problems at work 6. Financial problems 7. Serious injury or attack 8. Mourning the person loss 9. Domestic violence 10. Problems with the law

35 suicide: No. 5, 7, 10 20 suicide: No. 4, 6, 10 10 suicide: No. 1, 3, 8 6 suicide: No. 2, 8, 9

85 suicide: No. 1, 4, 5, 9 34 suicide: No. 2, 3, 6, 10 25 suicide: No. 2, 6, 8 20 suicide: No. 6, 7 17 suicide: No. 4, 6 7 suicide: No. 1, 5 3 suicide: 3, 10

Initial binary scoring system was used to determine the number of life events; in which a score of 1 indicated if the life event had happened over the past 1, and 12 months, and 0 if it had not. The numbers of all life events, were pooled together and recorded separately for each suicide victims during a period of 1, and 12 months prior to suicide. N: number.

192

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polymorphism (rs6311) of 5-HTR2A gene PCR was performed using the following primers; sense primer 5′-AAGGTAGCAACAGCCAGGAG-3′ and antisense primer 5′-TCATTACTGGTGGGGGAAAA-3′. 100 ng of DNA was amplified with 0.4 μmol/L of each primer, 200 μmol/L dNTPs and 1 unit of Taq DNA polymerase for 30 cycles, with denaturation at 94.0 °C for 1:00 min, annealing at 57.0 °C for 0.55 min and extension at 72.0 °C for 2:00 min. For the determination of the T102C (rs6313) polymorphism in the 5-HTR2A gene genomic DNA was amplified with sense primer: 5′-TCTGCTACAAGTTCTGGCTT-3′and anti-sense primer: 5′-CTGCAGCTTTTTCTCTAGGG-3′. The mixture was denatured initially at 96.0 °C for 10:00 min, followed by 35 cycles with 0:50 min at 94.0 °C, 0:50 min of annealing at 55.0 °C, 0:45 min at 72.0 °C. Then, the PCR products (497 bp and 342 bp for the A1438G and T102C polymorphisms respectively) was digested with MspI restriction enzyme (New England Biolabs, Mannheim Germany). After electrophoresis in a 3% ethidium bromide-stained agarose gel, the PCR products were visualized under UV light. The C1354T (rs6314) polymorphism of 5-HTR2A gene was genotyped using the following primers; sense primer 5′-CAAAGCAAGATG CCAAGACA-3 and antisense primer 5′-GGCATACAGATATGATCGT TGG-3′. The PCR reaction was performed using 100 ng of genomic DNA, 0.5 μM of both forward and reverse primer, 200 μM dNTPs, and 1 unit of Taq DNA polymerase. The PCR program consisted of 30 cycles of 95.0 °C for 0:45 min, 57.0 °C for 0:40 min, and 72.0 °C for 0:30 min with a final extension period of 72.0 °C for 5:00 min. The PCR product was then digested with BbvI restriction endonuclease (New England Biolabs, Mannheim Germany).

Table 2 Demographic characteristics of suicide victims. Characteristic

Marital status (%) Single† Married Divorced

Suicide victims No. of cases

P

69 (36%) 63 (33%) 59 (31%) 0.72

Residence (%) Urban Rural

137 (72%) 54 (28%) *0.018

Education (%) Illiterate High school (< 12 years) University (≥12 years)

60 (31%) 64 (34%) 67 (35%) 0.38

Economic status (%) Low Moderate High

101 (53%) 39 (20%) 51 (27%) *0.034

Occupation (%) Housewife Unemployed Student

67 (35%) 64 (34%) 60 (31%) 0.44

† Never married; * one way- ANOVA or t-student test where applicable; 5-HTR2A: 5Hydroxytryptamine receptor 2A.

2.4. Statistical analysis

significantly different (P = 0.012) and the C/C genotype was associated with a suicide. The A1438G (rs6311) and C1354T (rs6314) genotypes did not show a significant association (P = 0.940 and P = 0.913, respectively), but the low powers of the latter tests prevent the drawing of any conclusions. Table 4 lists stressful life events, loss events, age, and gender of suicide victims according to the genotypes of 5-HTR2A gene polymorphisms. The prevalence of stressful life and loss events was higher in suicide victims subjects carrying the C/C genotype (P = 0.014 and 0.035, respectively), compared with those with C/G or G/G genotypes. In suicide victims with C/G or G/G genotypes there were no significant differences in the stressful life and loss events with regard to the studied genotypes of A1438G (rs6311) and C1354T (rs6314) polymorphisms (P = 0.33 and P = 0.58 for A1438G and P = 0.26 and P = 0.60 for C1354T, respectively). The power of the latter analysis, however, was very low. To check for lack of sampling bias for age and gender, we tested the present cases and controls for genotype frequencies across different ages and genders and did not find any significant differences. Since we found that suicide can be affected by low income and urban area as covariates, ANCOVA test was performed to study the independent effect of CC genotype on suicide. As Table 5 shows F-test value for CC genotype was still significant, and subjects with higher number of CC genotype tend to have more risk to suicide.

Chi-squared and Fisher's exact tests were used to compare genotype and allele frequencies between suicide and control groups. We used gene counting method and chi-squire test to determine allele frequencies and Hardy–Weinberg equilibrium, respectively. The mean number of life events was calculated for each genotype of 5-HTR2A and compared using one-way ANOVA. Correction for multiple comparisons was calculated using Bonferroni correction, and subsequently, the Pvalue was set at P = 0.005. ANCOVA (Analysis of Covariate) was used to determine the effect of CC genotype of 5-HTR2A gene on suicide considering other covariates involved in suicide. P < 0.05 was considered significant and all calculations were performed using SPSS Statistics 22.00 (SPSS, Chicago, IL, USA). For comparisons involving genotype frequencies in suicide victims, PGA version 2.0 (Biostatistics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA; http://dceg.cancer.gov/bb/tools/pga) was used for calculation of power assuming a dominant mode of inheritance for the SNP and a perfect linkage disequilibrium (D' = 1) between the marker and disease. For comparisons involving genotype frequencies in different genders, ages, and life events, power was calculated using G*Power 3.1.0 (University of Kiel, Kiel, Germany). 3. Results

4. Discussion

Table 2 shows the demographic properties of suicide victims. As Table 2 represents, there were statistically differences in suicide victims regarding residence and economic status. Data on suicide victims' residence showed that suicide were more common in an urban area (72%) compared to rural area (28%). On average 53% of suicides took place in those with low income. The rates of suicide in those with moderate and with high income were 20% and 27%, respectively. However, we found no statistically differences in suicide victims considering marital status, education and occupation (P > 0.05). Table 3 shows the genotype and allele frequencies of the polymorphisms of 5-HTR2A gene. Allele frequencies were determined by the gene counting method, and Hardy–Weinberg's equilibrium was checked by a chi-square test. The distribution of T102C (rs6313) genotypes among the two groups was

Our study confirms the implication of T102C (rs6313) polymorphism of 5-HTR2A gene in suicide and shows that the C/C genotype is associated with suicide, stressful life and loss events. Due to the low power of the present study for the A-1438G (rs6311) and C1354T (rs6314) polymorphism, no association was found between the A1438G (rs6311) and C1354T (rs6314) polymorphisms of 5 HTR2A gene and suicide, stressful life and loss events. However, the possibility of involvement of these polymorphisms in suicide cannot be ruled out. Several lines of research have focused on the serotonin system, suggesting that this system may be involved in the pathogenesis of suicide, aggression, and impulsivity (Wang et al., 2016; Lee et al., 2015; 193

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Table 3 Genotype and allele frequencies of 5-HTR2A polymorphisms. Genotype and alleles

Suicide victims n (%)

Control subjects n (%)

T102C

a

CC CT TT C T A1438G

110 (57.59%) 66 (34.55%) 15 (7.85%) 286 96

AA AG GG A G HC1354T

32 (16.75%) 85 (44.50%) 74 (38.74%) 149 233

HCC HCT TTT HC TT

160 (83.76%) 21 (10.99%) 10 (5.23%) 341 41

P

Pa corrected

ҡ2

Powerb (%)

OR (95%CI)

OR (95%CI)

0.012

0.031

8.700

81.32

If C is the risk allele 1.443944553 (1.053855598–1.978426528)

If T is the risk allele 0.692547368 (0.505452179–0.948896606)

0.940

1.000

0.123

4.1

If A is the risk allele 1.011077601 (0.755844473–1.352497706)

If G is the risk allele 0.989043768 (0.739372788–1.3230235)

0.913

1.000

0.180

4.3

If C is the risk allele 1.133050548 (0.726241772–1.767735751)

If T is the risk allele 0.882573158 (0.56569541–1.376951917)

90 (41.28%) 98 (44.95%) 30 (13.76%) 278 158

41 (18.80%) 95 (43.57%) 82 (37.61%) 177 259

173 (79.35%) 23 (10.55%) 22 (10.09%) 369 67

P-values after Bonferroni's correction for multiple tests

b

Calculated using PGA (version 2.0); CI, confidence interval; 5-HTR2A, serotonin 2A receptor; OR, odds ratio; n:number.

(rs63134) polymorphism of 5-HTR2A gene and suicide. The C1354T (rs6314) is a functional polymorphism that regulates the 5-HTR2A receptor activity throughout the regulation of Ca2+ fluctuation in cells (Ozaki et al., 1997) and activation of phospholipases C and D (Hazelwood and Sanders-Bush, 2004). This polymorphism has been associated with several psychiatric disorders and rule-breaking components of antisocial behavior in adolescence, but not physical aggression. It has been indicated that the different results from the studies may be due to the genetic heterogeneity in relation to the 5-HTR2A gene polymorphisms (de Quervain et al., 2003). Furthermore, there are differences in the study design spatially patients' groups. For example, Zhang et al. have studied patients with psychiatric disorders and suicide (Zhang et al., 2008); Wrzosek et al. studied patients with attempted suicides and alcohol dependence (Wrzosek et al., 2011); other studies have analyzed suicide in patients with schizophrenia (Ran et al., 2004; Kao and Liu, 2011; De Luca et al., 2006; Correa et al., 2002), and finally, there are studies such as ours that have analyzed only suicide victims. Although it has been shown that genetic susceptibility may contribute to suicide (Bondy et al., 2006) but, besides well-known genetic background, recent life events have been associated with increased risk of suicide (Liu and Miller, 2014). In the first study Paykel et al. revealed that suicide victims had a more life events in 6 months before suicide when compared to the controls (Paykel et al., 1975). Similarly, several case-control designed studies confirmed such a relationship between suicide and life events (Fjeldsted et al., 2017). However, besides the independent influences of genetic background and environmental factors, life events, the gene and environment (GXE) interaction play a critical role in psychiatric problems including suicide. Caspi et al. first demonstrated a GXE interaction between serotonin transporter genotypes, life events and suicide (Caspi et al., 2003). The authors found a significant interaction between allele distribution, life events in producing depressive symptoms from the age of 21–26 years, and suicidal ideation or suicide attempts. They hypothesized that serotonin transporter genotypes moderates the influence of current and earlier life events. In present study, we found a high number of life events in suicide victims with C/C genotype. This result may be reasonable due to the low expression level of 5-HTR2A

Glick, 2015; Bani-Fatemi et al., 2016); because binding studies showed alterations in this receptor type in patients suffering from psychiatric disorders. To date, however, genetic association studies of the 5-HTR2A receptor and suicide gene gave inconclusive results, and the specificity of its potential involvement in psychiatric disorders including suicide is still unclear. Several studies on the association of T102C (rs6313) polymorphism of 5-HTR2A gene with suicide have shown that the C/C genotype is associated with increased risk of suicide (Khait et al., 2005; Illi et al., 2009; Bjork et al., 2002). However, the association between this polymorphism and suicide isn't still conclusive. Some studies have revealed a preponderance of the T/T genotype in the cases group, while others have not found a significant association (Saiz et al., 2008a; VaqueroLorenzo et al., 2008a; Sato et al., 2002; Li et al., 2006; Choi et al., 2005). In a latest meta-analysis which included 23 studies of different population there was no association between T102C (rs6313) polymorphism of 5-HTR2A gene and suicide (González-Castro et al., 2013). Overall, the meta-analysis included 13 studies in Caucasian populations, 6 studies comprising Asian populations and 4 from other ethnic origins. The T102C (rs6313) polymorphism is located in the first exon of the HTR2A gene. The substitution of base thymine (T) with cytosine (C) encodes in both cases for the amino acid serine and do not change the amino-acid sequence of the protein. However, T102C (rs6313) polymorphism positioned close to the promoter region and could be involved in gene regulation (Serretti et al., 2007) by changing the secondary structure of transcript or by prevention of gene expression by methylation of cytosine in position 102 (Vaquero-Lorenzo et al., 2008b). These results suggest why in our study C/C genotype presents with higher frequency in suicide victims. In the present study, no association was found between the A1438G (rs6311) and C1354T (rs6314) polymorphisms of 5-HTR2A gene and suicide, stressful life and loss events. On the other hand, the association between A1438G (rs63131) polymorphism of 5-HTR2A gene and suicide is compared impulsive suicidal attempter with the non-impulsive group (Saiz et al., 2008b). Other studies, however, have failed to replicate these results and found no association between these polymorphism and suicide (Videtic et al., 2006). Our results provide no evidence of association between C1354T 194

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Table 4 Characteristic and life events in suicide victims vs 5-HTR2A polymorphisms. Characteristics

Life events Gender, n (%)

Genotypes

T102C CC CT TT d

P P correctede ҡ2 Power (%)f A1438G AA AG GG Pd P correctede ҡ2 Power (%)f HC1354T HCC HCT TTT d

P P correctede ҡ2 Power (%)f

Male (n = 141)

101 (71.63%) 27 (54.0%) 28 (19.85%) (36.0%) 12 (8.51%) (10%) 0.904d 1.000 0.269c 11 22 (15.60%) (20.0%) 64 (45.39%) 21 (42.0%) 55 (39.0%) (38.0%) 0.766838303 1.000 0.530958636 11 119 (84.39%) (82.0%) 15 (10.63%) (12.0%) 7 (4.96%) 3 (6.0%) 0.835c 1.000 0.378c 11

Age, n (%)

No. occurrences (mean ± SD)

30 years (n = 63)

> 30 years (n = 128)

a

38 (60.3%)

72 (56.2%)

4.03 ± 1.39

3.31 ± 1.30

18

20 (31.7%)

46 (35.9%)

1.02 ± 1.21

1.23 ± 1.10

5

5 (7.9%)

10 (7.8%)

1.45 ± 1.25

1.56 ± 1.43

0.014

0.035

99

99

Female (n = 50)

0.845030562 1.000 0.336764971 11 10

19

Stressful life events

b

Loss events

9 (14.2%)

23 (17.9%)

1.02 ± 1.52

1.56 ± 1.36

29 (46.0%)

56 (43.7%)

1.8 ± 1.56

1.72 ± 145

25 (39.6%)

49 (38.2%)

1.43 ± 1.32

1.65 ± 1.58

0.33

0.58

5

5

0.813579641 1.000 0.412622915 11 41

52 (82.5%)

108 (84.3%)

1.65 ± 1.29

1.69 ± 1.25

6

8 (12.6%)

13 (10.1%)

1.54 ± 1.41

1.78 ± 1.53

3 (4.7%)

7 (5.4%)

1.48 ± 1.15

1.55 ± 1.42

0.26

0.60

5

5

c

0.900 1.000 0.395c 11

a

Stressful life events were evaluated according to the list of threatening experiences that originally developed by the Bruga et al study. These threatening experiences are a subset of 12 categories of life events with considerable long-term contextual threat. b The Life Events and Difficulties Schedule (LEDS) scale was used in the interviews to determine the loss events. According to this scale loss events are classified into four major types including loss of health, person, a cherished idea and employment/material possessions. c Fisher's exact test, n: number; 5HTR2A: 5-Hydroxytryptamine receptor 2A. d Fisher's exact test. e P-values after Bonferroni's correction for multiple tests. f Calculations done using G*Power (version 3.1.0). n: number; 5HTR2A: 5-Hydroxytryptamine receptor 2A.

polymorphisms in 5-HTR2A gene. It will be interesting to identify if these polymorphisms have a role in the manifestation and development of other psychological diseases including bipolar abnormality, schizophrenia disorder, and other depressive disorders. Another limitation of our study which may be addressed is that as we found no association between two other polymorphisms of 5-HTR2A, suicide, and life events, the hypothesis of GXE interaction is confined to a specific polymorphism in a single gene. Therefore, further studies designed to evaluate the effect of different genes on life event are needed to extract an accurate hypothesis about GXE interaction existed in suicide victims.

gene related to C/C genotype. As this genotype also associated with suicide victims, it may be suggested that high frequency of C/C genotype in suicide victims affects life events which in turn modulates suicide. However, it must be noted that this is only a hypothesis derived by a population based and a small number case-control study. Further studies are warranted to clear and confirm the function of this gene in modulating life events and the association of this GXE interaction with suicide. In summary, our data suggest an association of the C102T (rs6313) polymorphism of 5-HTR2A gene and suicide, stressful life and loss events. No significant association between the A1438G (rs6311) and C1354T (rs6313) polymorphisms of HTR2A gene and suicide, stressful life and loss events was found in the present study, the low power of the present study does not allow the drawing of a definite conclusion. However, further studies are necessary to confirm such findings.

Conflict of interests The authors declare that they have no conflict of interests.

5. Study limitation Funding This work was a small study, and these results should be validated in a large scale prospective study to confirm the associative effect of these polymorphisms with suicide victims. As well, we analyzed only three

This work was supported by the Research Center of Forensic Medicine [grant number: 206702031]. 195

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Table 5 The effect of the CC genotype of 5-HTR2A gene on suicide considering covariates. Covariates Marital status Single† Married Divorced Residence Urban Rural Education Illiterate High school (< 12 years) University (≥12 years) Economic status Low Moderate High Occupation Housewife Unemployed Student CC genotype of 5-HTR2A

F

P

12.2

0.72

1.89

*0.018

8.8

0.38

2.6

*0.034

health and well-being survey. BMC. Res. Notes 9, 470. http://dx.doi.org/10.1186/ s13104-016-2278-x. Correa, H., Duval, F., Mokrani, M.C., Bailey, P., Tremeau, F., Staner, L., et al., 2002. Serotonergic function and suicidal behavior in schizophrenia. Schizophr. Res. 56 (1–2), 75–85. Currier, D., Mann, J.J., 2008. Stress, genes and the biology of suicidal behavior. Psychiatr. Clin. North Am. 31 (2), 247–269(Jun, [cited 2017 Jan 8]. Available from: http:// www.ncbi.nlm.nih.gov/pubmed/18439448). De Luca, V., Tharmalingam, S., Müller, D.J., Wong, G., de Bartolomeis, A., Kennedy, J.L., 2006. Gene-gene interaction between MAOA and COMT in suicidal behavior: analysis in schizophrenia. Brain Res. 1097 (1), 26–30. de Quervain, D.J.-F., Henke, K., Aerni, A., Coluccia, D., Wollmer, M.A., Hock, C., et al., 2003. A functional genetic variation of the 5-HT2a receptor affects human memory. Nat. Neurosci. 6 (11), 1141–1142(Available from: http://www.ncbi.nlm.nih.gov/ pubmed/14566344). 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Suicidal behavior and insight into illness among patients with schizophrenia spectrum disorders. Psychiatry Q. 82 (3), 207–220. Khait, V.D., Huang, Y., Zalsman, G., Oquendo, M.A., Brent, D.A., Harkavy-Friedman, J.M., et al., 2005. Association of serotonin 5-HT2A receptor binding and the T102C polymorphism in depressed and healthy Caucasian subjects. Neuropsychopharmacology 30 (1), 166–172. Lee, H.-Y., Hong, J.-P., Hwang, J.-A., Lee, H.-J., Yoon, H.-K., Lee, B.-H., et al., 2015. Possible association between serotonin transporter gene polymorphism and suicide behavior in major depressive disorder. Psychiatry Investig. 12 (1), 136(Jan [cited 2017 Jan 8]. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25670956). Li, D., Duan, Y., He, L., 2006. Association study of serotonin 2A receptor (5-HT2A) gene with schizophrenia and suicidal behavior using systematic meta-analysis. Biochem. Biophys. Res. Commun. 340 (3), 1006–1015. Liu, R.T., Miller, 2014. Life events and suicidal ideation and behavior: a systematic review. Clin. Psychol. Rev. 34 (3), 181–192. http://dx.doi.org/10.1016/j.cpr.2014.01. 006. (Epub 2014 Feb 4). Ozaki, N., Manji, H., Lubierman, V., Lu, S.J., Lappalainen, J., Rosenthal, N.E., et al., 1997. A naturally occurring amino acid substitution of the human serotonin 5-HT2A receptor influences amplitude and timing of intracellular calcium mobilization. J. Neurochem. 68 (5), 2186–2193(May, [cited 2017 Jan 8]. Available from: http:// www.ncbi.nlm.nih.gov/pubmed/9109547). Paykel, G., Prusoff, B., Myers, J., 1975. Suicide attempts and recent life events: a controlled comparison. Arch. Gen. Psychiatry 32, 327–333. Peñas-Lledó, E.M., Dorado, P., Cáceres, M.C., De La Rubia, A., Llerena, A., 2007. Association between T102C and A-1438G polymorphisms in the serotonin receptor 2A (5-HT2A) gene and schizophrenia: relevance for treatment with antipsychotic drugs. Clin. Chem. Lab. Med. 45 (7), 835–838. Portzky, G., Audenaert, K., van Heeringen, K., 2005. Suicide among adolescents: a psychological autopsy study of psychiatric, psychosocial and personality-related risk factors. Soc. Psychiatry Psychiatr. Epidemiol. 40 (11), 922–930. Ran, M.-S., Wu, Q.-H., Conwell, Y., Chen, E.Y.-H., Chan, C.L.-W., 2004. Suicidal behavior among inpatients with schizophrenia and mood disorders in Chengdu, China. Suicide

0.44 9.3

1.6

0.029

† Never married; * any differences were calculated by ANCOVA (Analysis of Covariance); 5-HTR2A: 5-Hydroxytryptamine receptor 2A.

Authors' contributions Study concept and design: Bahram Samadi Rad. Supervising the project: Bahram Samadi Rad. Acquisition, Analysis and interpretation of data: Asghar Ghasemi and Morteza Seifi. Drafting of the manuscript: Asghar Ghasemi and Morteza Seifi. Carrying out the experiments: Asghar Ghasemi and Morteza Seifi. Statistical analysis: Asghar Ghasemi, Morteza Seifi, Fatemeh Baybordi, Nasim Danaei. Technical, and material support: Nasim Danaei, Sample preparation: Nasim Danaei, Fatemeh Baybordi. All authors discussed the results and contributed to the final manuscript. References Bani-Fatemi, A., Howe, A.S., Matmari, M., Koga, A., Zai, C., Strauss, J., et al., 2016. Interaction between methylation and CpG single-nucleotide polymorphisms in the HTR2A gene: association analysis with suicide attempt in schizophrenia. Neuropsychobiology 73 (1), 10–15(Jan 27 [cited 2017 Jan 8]. Available from: http:// www.ncbi.nlm.nih.gov/pubmed/26812280). Beck, A.T., Weissman, A., Lester, D., Trexler, L., 1974. The measurement of pessimism: the hopelessness scale. J. Consult. Clin. Psychol. 42, 861–865(http://www.ncbi.nlm. nih.gov/pubmed/4436473). Beretta, L., Cossu, M., Marchini, M., Cappiello, F., Artoni, A., Motta, G., et al., 2008. A polymorphism in the human serotonin 5-HT2A receptor gene may protect against systemic sclerosis by reducing platelet aggregation. Arthritis Res. Ther. 10 (5), R103([cited 2017 Jan 8]. Available from: http://arthritis-research.biomedcentral. com/articles/10.1186/ar2495). Bjork, J.M., Gerard Moeller, F., Dougherty, D.M., Swann, A.C., Machado, M.A., Hanis, C.L., 2002. Serotonin 2a receptor T102C polymorphism and impaired impulse control. Am. J. Med. Genet. Neuropsychiatr. Genet. 114 (3), 336–339. Bondy, B., Buettner, A., Zill, P., 2006. Genetics of suicide. Mol. Psychiatry 11 (4), 336–351. Brugha, T., Bebbington, P., Tennant, C., Hurry, J., 1985. The list of threatening experiences: a subset of 12 life event categories with considerable long-term contextual threat. Psychol. Med. 15 (1), 189–194. Caspi, A., Sugden, K., Moffitt, T.E., Taylor, A., Craig, I.W., Harrington, H., et al., 2003. Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene. Sci. N. Ser. 301 (5631), 386–389. Cheng, A.T.A., Chen, T.H.H., Chen, C.C., Jenkins, R., 2000. Psychosocial and psychiatric risk factors for suicide: case-control psychological autopsy study. Br. J. Psychiatry 177 (OCT), 360–365. Choi, M.J., Kang, R.H., Ham, B.J., Jeong, H.Y., Lee, M.S., 2005. Serotonin receptor 2A gene polymorphism (−1438A/G) and short-term treatment response to citalopram. Neuropsychobiology 52 (3), 155–162. Cleland, C., Kearns, A., Tannahill, C., Ellaway, 2016. The impact of life events on adult physical and mental health and well-being: longitudinal analysis using the GoWell

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