Association of 5HTR1A Gene Variants with Suicidal Behavior: Case-Control Study and Updated Meta-Analysisq

Journal of Psychiatric Research 47 (2013) 1665e1672

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Association of 5HTR1A gene variants with suicidal behavior: Case-control study and updated meta-analysisq

Thelma Beatriz González-Castro a, Carlos Alfonso Tovilla-Zárate b,*, Isela Juárez-Rojop a, Sherezada Pool García c, Alma Genis d, Humberto Nicolini d, Lilia López Narváez e a Universidad Juárez Autónoma de Tabasco, División Académica de Ciencias de la Salud, Villahermosa, Tabasco, Mexico b Universidad Juárez Autónoma de Tabasco, División Académica Multidisciplinaria de Comalcalco, Comalcalco, Tabasco, Mexico c Hospital General de Comalcalco, Tabasco, Secretaría de Salud, Comalcalco, Tabasco, Mexico d Grupo de Estudios Médicos y Familiares Carracci, México, D.F., Mexico e CIGEN, Centro de Investigación Genómica, Comalcalco, Tabasco, Mexico article info abstract

Article history: Introduction: The gene encoding the serotonin 1A receptor (5HTR1A) has been a candidate gene asso- Received 15 February 2013 ciated with suicidal behavior in case-control and meta-analysis studies. We carried out a meta-analysis Received in revised form and a case-control study on the 5HTR1A gene to examine the association of this gene with suicidal 6 April 2013 behavior. Accepted 11 April 2013 Methods: We performed a systematic search in electronic databases to study meta-analytically the association of 5HTR1A gene with suicidal behavior; we found 9 published genetic association studies Keywords: concerning the rs6295 polymorphism. To get a comprehensive knowledge of this association we con- Suicide Serotonin receptor ducted a case-control study on the following polymorphisms: rs1423691, rs6295, and rs878567 in a Genetic association study Mexican population; the sample was composed of 152 suicide attempters and 264 healthy subjects. Meta-analysis Results: The meta-analysis revealed that the rs6295 polymorphism is not associated with suicidal behavior. Similarly, no signiﬁcant association for polymorphisms rs6295 and rs878567 was found in the case-control study. The polymorphism rs1423691 was excluded of the association analysis because cases and control groups were in HardyeWeinberg disequilibrium. Conclusion: The meta-analysis of functional rs6295 polymorphisms produced no association. Likewise, the analysis in our case-control study in a Mexican population resulted in lack of association of polymorphisms rs6295 and rs878567 with suicidal behavior. However, further studies assessing different populations, as well as larger samples are necessary to obtain conclusive outcomes. Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

1. Background and prevention of suicidal behavior. The implication of the serotonergic system in suicidal behavior has been studied for several Suicide is a significant public health issue and one of the most years (Arango et al., 2001; Tovilla-Zárate et al., 2011). common causes of death throughout the world (Anguelova et al., At present, several studies have investigated the association be- 2003; Kamali et al., 2001). Genetic studies are trying to identify tween various 5HTR1A polymorphisms and suicide and suggest that possible genetic markers that will be helpful for the early detection this gene is playing a role in suicidal behavior (Ohtani et al., 2004; Samadi Rad et al., 2012; Serretti et al., 2007, 2009; Videtic et al., 2009; Wasserman et al., 2006). Even though in our most recent Abbreviations: PRISMA, preferred reporting items for systematic reviews and meta-analysis we could not find any association with suicidal meta-analyses; DSM-IV, diagnostic and statistical manual of mental disorders-IV. q behavior, the analysis only included 4 studies comprising 957 pa- This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and tients and 957 controls. Therefore, we decided to undertake another reproduction in any medium, provided the original author and source are credited. study in which a larger sample size and more specific selection * Corresponding author. Tel.: þ52 9933581500x6900. criteria were included (Angles et al., 2012). On the other hand, E-mail addresses: [email protected] (T.B. González-Castro), alfonso_ concomitant to suicidal behavior we observed several clinical fea- [email protected] (C.A. Tovilla-Zárate), [email protected] (I. Juárez- tures such as the presence of impulsivity, substance abuse (alcohol Rojop), [email protected] (S. Pool García), [email protected] (A. Genis), [email protected] (H. Nicolini), [email protected] dependence), and even major depression. As a result, various genetic (L. López Narváez). studies are taking into consideration the analysis of suicidal

0022-3956/$ e see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jpsychires.2013.04.011 1666 T.B. González-Castro et al. / Journal of Psychiatric Research 47 (2013) 1665e1672 behavior in association with other diseases (Benko et al., 2010; Two investigators (González-Castro TB, Tovilla-Zárate CA) Czesak et al., 2012; Koller et al., 2006; Lemonde et al., 2003; Pitchot working independently screened each of the titles, abstracts, and et al., 2005; Videtic et al., 2009; Wang et al., 2009; Wrzosek et al., full texts to determine inclusion. The results were compared and 2011; Yoon and Kim, 2009; Zhang et al., 2008; Zupanc et al., 2010). disagreements were resolved by consensus. 5HTR1A receptors are located both at post-synaptic and pre- synaptic levels. In the first case, they mediate the action of sero- 2.3. Data extraction tonin in cortical and limbic neurons; in the second case, they act as auto-receptors in serotonergic neurons in the raphe nuclei and The following data were obtained for each of the studies: au- prevent the release of serotonin by a negative feedback (Czesak thors, year of publication, region, number of cases and controls, et al., 2012; Yoon and Kim, 2009). gender, age, psychiatric diagnosis, ethnical origin, sample size and The5HTR1Areceptorgeneislocatedonthelongarmofchromo- genotype and allele frequencies. These data were not always somal region 5q11.2-13; it is intronless, codes for a 422 amino acid, available for all studies, but when needed, we contacted the authors and has a number of single nucleotide polymorphisms (SNPs) (Angles to ask for genotype distributions not included in the papers. The et al., 2012; Samadi Rad et al., 2012; Videtic et al., 2009). However, only study by Benko et al. (2010) did not report a control group but we a small number of the latter have been investigated so far as candidate made an adjustment accordingly based on the other studies in the variants in suicidal behavior. One of the most common SNPs is the literature that included a control group. Briefly, we calculated the functional C-1019G variant (rs6295) (Chen et al., 2004; Kim et al., weighted frequency of a particular genotype from studies that 2011; Koller et al., 2006). This polymorphism is located within the included controls and applied it to the study not including a control 26-bp palindromic region, which bounds a single repressor, the nu- group. We considered the number of the “virtual” control group as clear DEAF-1 related (NUDR) protein. The G allele abolishes repression equal to the number of patients in a specific study. Then, the hy- by NUDR and produces higher expression of 5HTR1A, enhances the pothetical number of subjects with the particular genotype fre- negative feedback inhibition of serotonergic raphe neurons exerted by quency was assigned in proportion to the percentage of the same 5HTR1A autoreceptors, and leads to lower serotonergic neurotrans- genotype that was obtained from the weighted analysis (Illi et al., mission (Benko et al., 2010; Lemonde et al., 2003). For these reasons 2009; Kishi et al., 2011). The outcomes of the meta-analysis were the allele G of C-1019G polymorphism may be considered an allele risk built by taking into consideration the following categories: (a) manifest in suicide attempters. The polymorphism rs1423691 is exposed sick, (b) exposed not-sick, (c) not-exposed sick and (d) not- located downstream the band 5q12.3, in which the change of C to T exposed not-sick. The “sick” term refers to subjects exhibiting does not produce a different amino acid. With regard to the rs878567 suicidal behavior and the “exposed” term to the allele of risk. The polymorphism, it is located in the same band of rs1423691. Likewise, information of the selected publications was extracted indepen- the change of C to T does not produce a change in amino acid. Other dently by the same two authors (González-Castro TB, Tovilla-Zárate less studied polymorphisms of the 5HTR1A gene associated with CA) in accordance with the inclusion criteria. suicidal behavior are rs1423691 and rs878567. Currently, there are only few reports showing an association between suicidal behavior 2.4. Data analysis and these polymorphisms (Serretti et al., 2007, 2009). In this study, we conducted an updated meta-analysis of all For the meta-analysis procedures, we used the EPIDAT 3.1 pro- published data on the rs6295 polymorphism. Also, we investigated gram (http://dxsp.sergas.es). This software is freely available for the possible association between a panel of markers of the 5HTR1A epidemiologic analysis of tabulated data. Data was analyzed with the gene (rs6295, rs1423691 and rs878567) in a Mexican sample of random-effects model following the reports in the literature (Angles suicide attempters and controls. et al., 2012; Tovilla-Zárate et al., 2011). Sample heterogeneity was analyzed with the Dersimonian and Laird’s Q test. The result of the Q 2. Methods test was complemented with graphs to help visualize those studies favoring heterogeneity. The results of the meta-analysis are 2.1. Meta-analysis expressed as odds ratios (ORs). To address the problem of publication bias, the Egger’s test and funnel plots were calculated with the This meta-analysis followed the Preferred Reporting Items for EPIDAT 3.1 software. This plotting standardizes the effect of each of Systematic Reviews and Meta-Analyses (PRISMA) criteria (Moher the published studies on the vertical axis and its correspondent et al., 2009; Swartz, 2011). precision on the horizontal axis. Finally, a chi-squared (c2) analysis was used to calculate the HardyeWeinberg equilibrium to evaluate 2.2. Identification and selection of publications genotype distribution. Studies deemed for inclusion in the systematic review were scored for methodological quality using the The literature included in the current study was selected from Newcastle-Ottawa Assessment Scale (NOS). A score of six was taken Pubmed and EBSCO databases with the keywords: “HTR1A and as the cut-off point to distinguish higher from lower quality studies. suicidal behavior”, “HTR1A and suicide”, “rs6295 and suicide”, “5- Quality assessment was done by the same two authors (González- hydroxytryptamine-receptor 1A” and “serotonin receptor 1A”. Castro TB, Tovilla-Zárate CA) based on the NOS instrument. The literature search was performed in September 2012. The dates of publication of the searched papers comprised from March 2003 2.5. Case-control study to August 2012. To be selected, the publications had to fulfill the following criteria: (1) to be published in peer-reviewed journals, (2) The group of patients consisted of 152 unrelated suicide to be written in English, (3) to contain independent data, (4) to be attempters. They were consecutively recruited in the outpatient case-control association studies in which the frequencies of three service from the General Hospital of Comalcalco in the state of genotypes were clearly stated or could be calculated, and (5) the Tabasco, Mexico. In addition, 264 healthy volunteers attending the use of healthy individuals as controls. Besides, we included one Blood Donor Center were included as controls. All subjects came article consisting only of cases, because the sample in this study exclusively from Comalcalco. The participants in this study was large and raised the detection power in the meta-analysis descended from Mexican parents and grandparents to reduce study (Benko et al., 2010). ethnic variation and stratification effects. T.B. González-Castro et al. / Journal of Psychiatric Research 47 (2013) 1665e1672 1667

2.6. Clinical evaluation 186 potentially relevant articles (EBSCO 88 Pubmed= 98) Patients with attempted suicide were evaluated by a trained psychiatrist or clinical psychologists with at least a master’s degree level. Initially, a clinical interview helped us to determine the most Excluded based on title relevant symptoms and the diagnostic was stabilized for DSM-IV in n= 104 which disorders pertaining to Axis I, II and III were considered. DSM-IV lifetime diagnoses of mental disorders among the patients were classiﬁed as: schizophrenia spectrum disorder (30.2%), anxiety disorder (50.0%), and undiagnosed (19.8%). We deﬁned suicide Screening of 49 references based on attempt as a self-harm behavior with at least some intent to end their abstracts one’s life (Mann, 2003). Subjects were excluded when self-injury behaviors were determined to have no suicidal intention or ideation based on the assessment of suicidal intention, using the Scale for Suicide Ideation in the Spanish version (Beck et al., 1979). Excluded n= 6 review articles On the other hand, each control was interviewed using sys- n= 16 no outcome of interest tematic forms to obtain a detailed medical and psychiatric history in order to exclude subjects who had relatives with a lifetime history of a mental disorder or suicidal behavior. 12 relevant studies retrieved for detailed 2.7. Ethics statement evaluation

Written informed consent was obtained from all subjects after they were given a detailed and extensive description of the study; Excluded n= 4 they did not receive any economical remuneration. The study was approved by the local Ethics and Research Committee DAMC-UJAT (UJAT-DAMC-2012-02). The study was carried out in accordance with the ethical standards laid down in the 1964 Declaration of 9 studies in the meta-analysis Helsinki. including our study

2.8. Genotype assays Fig. 1. Flow-chart showing the different stages of the meta-analysis.

Peripheral blood samples were drawn from all subjects; DNA of Our meta-analysis consisted of 2366 cases and 2943 controls leukocytes was extracted using a modified version of the protocol (Table 1). We enlarged a previous meta-analysis on rs6295 (C- by Lahiri and Nurnberger (1991). The polymerase chain reaction 1019G) (Videtic et al., 2009) with 1217 cases and 1986 controls that (PCR) end-point method was used in all patients to analyze the included samples of 7 Caucasian populations, 1 Asian population, following 5HTR1A genotypes: rs6295(C-1019G), rs1423691, and and our case-control association study. rs878567. The complete protocol appeared recently in a previous We measured the HardyeWeinberg equilibrium in all geno- meta-analysis (Tovilla-Zárate et al., 2011). All genotyping was per- typed populations, both groups, cases and controls, were in equi- formed blind to patient outcome. As a quality control in our gen- librium (p > 0.05) with the exception of the studies by Benko et al. otyping analyses we used random blind duplicates. (2010) with p < 0.001 in the control group and Samadi Rad et al. (2012) with a p < 0.0001 in control and patient groups. Also, 2.9. Statistical analysis Lemonde et al. (2003) reported disequilibrium (p < 0.0001) in the patient group only. Similarly, when we calculated all populations in The presence of HardyeWeinberg equilibrium was tested using a combined way we encountered p ¼ 0.60 in the controls and Pearson’s goodness-of-fit chi-squared test. Genotype and allele p ¼ 0.0002 in the cases. As a result, we measured the Hardye frequencies between groups were compared using Chi-squared and Weinberg equilibrium after heterogeneity was discarded and ob- Fisher’s Exact tests. Tests for association using multi-marker hap- tained p ¼ 0.33 in controls and p ¼ 0.68 in suicide attempters. lotypes were performed utilizing Thesias version 3.1 software. The The pooled OR derived from all studies showed a non-significant power to detect associations was analyzed with the Quanto 1.2 association of the G allele in the C-1019G polymorphism with sui- software. By way of an example: for rs6295 with minor allele cidal behavior (Random effects: OR: 1.33; 95% CI: 0.87e2.03; frequency ¼ 0.3, dominant model, effect size ¼ 1.5, we obtained a p(Z) ¼ 0.72) and presence of heterogeneity in the studies power of 0.43. Significance was set at p < 0.05. The Haploview 4.2 (Q ¼ 203.50, df ¼ 9; p ¼ <0.0001). The Egger’s test provided no program (Barrett et al., 2005) was employed to calculate the linkage evidence for publication bias (t ¼1.62, df ¼ 8; p ¼ 0.14) (Fig. 2A). disequilibrium (LD) of the markers. Given that some studies were not in HardyeWeinberg equilibrium we conducted an analysis in which the study giving rise to het- 3. Results erogeneity was discarded. Even then we encountered no association between rs6295 and suicidal behavior (OR: 0.93; 95% CI: 0.83e 3.1. Meta-analysis study 1.04; p(Z) ¼ 0.1.3) (Fig. 2B). We carried out a sub-analysis in the Caucasian populations and As for the literature search, a total of 186 studies were identified, found no association [Egger’s test; Random effects model; OR: 1.47; but only 9 were used including our case-control study. Fig. 1 shows 95% CI: 0.91e2.39 with heterogeneity (Fig. 3A) and OR: 0.94; 95% the selection process of the meta-analysis (Benko et al., 2010; CI: 0.83e1.08 without heterogeneity (Fig. 3B)]. We could not Lemonde et al., 2003; Samadi Rad et al., 2012; Serretti et al., 2007, perform an analysis in the Asian population because we only had a 2009; Videtic et al., 2009; Wrzosek et al., 2011; Yoon et al., 2009). single study on the association of rs6295 with suicidal behavior. 1668 T.B. González-Castro et al. / Journal of Psychiatric Research 47 (2013) 1665e1672

Table 1 Descriptive characteristics of 9 studies on the role of the C-1019G polymorphism of the 5HTR1A gene in suicidal behavior.

Study (Year) Sample size, n Location Diagnosis Genotypes Alleles Gender (Males) Mean age (Cases-Controls) GG GC CC G C Cases Controls Cases Controls

Videtic et al. (2009) 323/190 Slovenian Suicide victims 91 160 72 342 182 48 49 Caucasians Benko et al. (2010) 725/1103 Hungarian Suicide attempters 193 368 163 754 1590 129 30 0 Caucasians in general population Serretti et al. (2009) 111/289 German Suicide attempters 26 55 30 107 291 43 123 39 45 Caucasians Samadi Rad et al. (2012) 191/218 Iranian Suicide victims 56 53 82 217 342 47 45 Caucasians Lemonde et al. (2003) 102/116 Quebec Suicide victims 17 30 55 64 196 53 55 32 34 Caucasians with major depression Serretti et al. (2007) 259/312 German Suicide attempters 61 132 66 254 317 39 48 Caucasians Serretti et al. (2007) 92/163 Italian Suicide attempters 17 50 25 84 152 39 48 Caucasians and victims Wrzosek et al. (2011) 38/112 Poland Suicide attempters 10 16 12 36 113 >18 >18 Caucasian in alcohol dependence Yoon and Kim (2009) 181/176 Korean Suicide attempters 93 79 9 120 89 82 80 40 40 Asians with major depression González-Castro 152/264 Mexican Suicide attempters 26 58 68 265 303 56 97 25 31 (see Section 3.2) Latin Americans

3.2. Case-control study rs878567 (Fig. 4). Also, we measured the LD with the MEX panel analysis, but there were no references of any of the following The mean age of the 152 patients (56 males, 96 females) was polymorphisms: rs6295, rs1423691 and rs878567. 25.5 (9.56) years old (range: 14e56 years). The possibility of We calculated the genotype counts and frequency distributions childhood abuse was not evaluated. The mean age of the 264 for polymorphisms rs6295 (C-1019G) and rs878567 of the 5HTR1A controls (97 males, 167 females) was 33.1 (13.0) years of age (range: gene in the 152 suicide attempters and control group. The results 14e61 years). Socio-demographic features of suicide attempters are are shown in Table 3. presented in Table 2. The analysis for the rs6295 (C-1019G) polymorphism yielded no The HardyeWeinberg equilibrium was measured in all geno- signiﬁcant differences in genotype (c2 ¼ 0.89, p ¼ 0.64, df ¼ 2) or typed populations. The polymorphisms rs6295 and rs878567 were allele (c2 ¼ 0.78, p ¼ 0.37, df ¼ 1) frequencies between controls and in equilibrium in both groups (p > 0.05). We encountered two cases. The same results were obtained for the rs878567 poly- exceptions: the rs1423691 polymorphism was not in equilibrium in morphism: no association was found in genotype (c2 ¼ 2.83, the control group (p ¼ 0.0003) or in the group of patients p ¼ 0.24, df ¼ 2) or allele (c2 ¼ 2.73, p ¼ 0.09, df ¼ 1) frequencies (p ¼ 0.006). In consequence, only the rs6295 (C-1019G) and between controls and cases. rs878567 polymorphisms could be analyzed. The LD (Linkage- We performed a second analysis in the sample of suicide Disequilibrium) in our Mexican sample taken as a whole was attempters and control subjects with respect to gender. The geno- complete (r2 ¼ 1.0), as well as when taken separately in controls type and allele distribution is shown in Table 4. The analysis of the and suicide attempters for variants rs6295, rs1423691 and rs878567 polymorphism in the female gender yielded a signiﬁcant

Fig. 2. Odds ratios and forest plots for all the models in overall studies. (A) G allele vs C allele with heterogeneity and (B) G allele vs C allele without heterogeneity. T.B. González-Castro et al. / Journal of Psychiatric Research 47 (2013) 1665e1672 1669

Funnel plot Funnel plot

0 estimation estimation Effect Effect

0 0.5 0.1 0.15 0.2 0.25 0 0.5 0.1 0.15 0.2 0.25 Standard error Standard error A B

Fig. 3. Egger’s funnel plot indicating publication bias for studies on suicidal behavior. (A) G allele vs C allele with heterogeneity and (B) G allele vs C allele without heterogeneity. association in the allele frequency (c2 ¼ 5.54, p ¼ 0.01, df ¼ 1). With analysis without heterogeneity. We also carried out a sub-analysis regard to the rs6295 (C-1019G) polymorphism we observed the in Caucasian populations. Our findings of the G allele in the C- same results as on the first analysis, no association was found in 1019G (rs6295) polymorphism support previous results, showing genotype and allele frequencies both in males and females. In this no association between suicidal behavior and the rs6295 (C-1019G) Mexican sample, haplotype analysis in relation to attempted sui- polymorphism when genotypes or allele frequencies were consid- cide did not reveal any significant association either (Table 5). ered. Even when we undertook an explorative analysis including only Caucasian samples, no significant association was found either. 4. Discussion These results are in agreement with our previous meta-analysis of the 5-HTR1A gene stating the lack of association (Angles et al., In this study we analyzed the possible association between the 2012), though this time our sample consisted of 1217 cases and 5-HTR1A gene and suicidal behavior based on a case-control study 1986 controls more and it also included our case-control study in a and an updated meta-analysis. We have already published a meta- Mexican population. In addition, the literature search for the pre- analysis of the 5-HTR1A gene (Angles et al., 2012), but we consid- sent analysis was performed three years later than the previous ered necessary to perform another meta-analysis with updated published results that consisted of a larger sample size and more specific selection criteria, in which various of the 5HTR1A polymorphisms were studied in relation to suicidal behavior. The hypothesis that the presence of a single G allele in the C- 1019G polymorphism could result in an elevated risk for suicide attempts was investigated by means of a meta-analysis. Thus, we performed an analysis with all reports published to date. The studies presented heterogeneity; however, we conducted a further

Table 2 Socio-demographic features of Mexican controls and suicide attempters.

Socio-demographic features Controls Cases c2 df p

Gender [n (%)] Males 97 (36.7) 56 (36.8) 0.004 1 0.98 Females 167 (63.3) 96 (63.2) Marital status [n (%)] Single 117 (44.3) 77 (50.6) 4.63 3 0.20 Married 124 (46.9) 63 (41.4) Separated/divorced 10 (3.8) 9 (5.9) Widowed 13 (5.0) 3 (2.1) Socio-economic level [n (%)] High 3 (1.3) 2 (2.4) 7.31 2 0.02a Middle 171 (64.7) 78 (50.3) Low 90 (34.0) 72 (47.3) Fig. 4. Linkage disequilibrium in 5HTR1A markers (rs6295, rs1423691 and rs878567) a p Value signiﬁcant. in suicide attempters and control group. 1670 T.B. González-Castro et al. / Journal of Psychiatric Research 47 (2013) 1665e1672

Table 3 Genotypes, allele counts and frequency distributions for rs6295 (C-1019G), rs1423691 and rs878567 polymorphisms of the 5HTR1A gene in suicide attempters and control group in a Mexican population.

Genotypes Cases Controls c2 df p Alleles Cases Controls c2 df p

rs6295 rs6295 GG, n (%) 26 (0.17) 55 (0.21) 0.89 2 0.64 G, n (%) G: 120 (0.39) G: 225 (0.43) 0.78 1 0.37 CG, n (%) 68 (0.45) 115 (0.43) C, n (%) C: 184 (0.61) C: 303 (0.57) CC, n (%) 58 (0.38) 94 (0.36) rs878567 rs878567 TT, n (%) 38 (0.25) 79 (0.30) 2.83 2 0.24 T, n (%) T: 146 (0.48) T: 285 (0.54) 2.73 1 0.09 CT, n (%) 70 (0.46) 127 (0.48) C, n (%) C: 158 (0.52) C: 243 (0.46) CC, n (%) 44 (0.29) 58 (0.22)

one. The above evidence suggests that the rs6295 (C-1019G) poly- victims, but not with suicidality among depressed patients. Simi- morphism is not associated with suicidal behavior. Future studies larly, the study by Serretti et al. (2007) showed an association of the comprising larger samples of suicidal behavior are important to G allele with suicide attempt only in female groups. Data by determine this result conclusively. Sawiniec et al. (2007) dealing with suicide attempts and, recently, In addition, we performed a case-control study in a Mexican Samadi Rad et al. (2012) in suicide victims also support the hy- population. To our knowledge this is the ﬁrst study addressing the pothesis of the association between the G allele and suicidal genetic association between various polymorphisms (rs6295 and behavior. Alternatively, the studies of Videtic et al. (2009), Benko rs878567) of the 5-HTR1A gene with suicidal behavior in a Mexican et al. (2010), Wrzosek et al. (2011), and Yoon et al. (2009) showed population. Our case control study consisted in looking for the asso- a lack of association of either the C allele or the G allele in this ciation of polymorphisms rs6295 and rs878567 with attempted polymorphism (rs6295) of 5HTR1A with suicidal behavior. In 2009, suicide, but when we analyzed genotype and allele frequencies of a new and larger study of Serretti et al. (2009) showed no associ- polymorphisms rs878567 and rs6295 we did not encounter any as- ation of genotype, allele, or haplotype frequency between the sociation. The possible role of 5-HTR1A receptors in the pathophys- rs6295 (C-1019G) polymorphism of 5HTR1A and suicidal behavior. iology of suicidal behavior has been assessed in case-control studies Our analysis of rs6295 (C-1019G) is in agreement with their results. based on familial and postmortem data, but with highly controversial However, no conclusive outcomes have been yet attained. Finally, results (Arango et al., 2001; Cheetham et al., 1989; Lowther et al., with respect to the rs878567 polymorphism, we could not ﬁnd an 1997; Stockmeier, 1997). With regard to the post-mortem studies, association with suicide attempters in our case-control study in the some of them found a reduction in 5-HTR1A receptor distribution Mexican sample, nor in the Italian and German population samples volume and a decrease in the total number of 5-HTR1A receptors of Serretti et al. (2007, 2009). (given as an index) in the dorsal raphe nucleus of suicide victims The discrepancies in the results of association studies may be compared with the control group. These studies also reported that 5- explained as follows. First, there are differences in diagnosis in the HTR1A receptor binding capacity in the medium raphe nucleus was population of patients. Lemonde et al. (2003) and Yoon et al. (2009) lower in suicide victims compared with control subjects; they also recruited patients with major depression; Wrzosek et al. (2011) suggested that this reduction in 5-HTR1A auto receptors could be an worked with alcohol dependent patients, and Benko et al. (2010) adaptive mechanism, whose function is to increase serotonin activity studied attempted suicides in the general population. Second, (Huang et al., 2004; Lowther et al., 1997; Stockmeier, 1997). there are differences in genetic heterogeneity in the populations. The most studied polymorphism of the 5HTR1A gene in relation Several studies have shown that allele frequencies of the various to suicidal behavior is C-1019G (rs6295). Lemonde et al. (2003) polymorphisms of the 5-HTR1A gene may be ethnic-dependent. reported an association of the G allele of the C-1019G poly- One of the strengths of our study is that it only included subjects morphism with a sample consisting of French-Canadian suicide from Comalcalco in the state of Tabasco, Mexico, with parents and

Table 4 Genotypes, allele counts and frequency distributions for rs6295 (C-1019G) and rs878567 polymorphisms of the 5HTR1A gene in suicide attempters and control group in a Mexican population by gender.

5HTR1A genotypes Males Genotypes Females

Cases Controls c2 df p Cases Controls c2 df p

rs6295 rs6295 GG, n (%) 11 (19.6) 24 (24.7) 0.96 2 0.61 GG, n (%) 15 (15.6) 31 (18.5) 0.44 2 0.80 CG, n (%) 20 (35.7) 37 (38.1) CG, n (%) 48 (50.0) 78 (46.7) CC, n (%) 25 (44.7) 36 (37.2) CC, n (%) 33 (34.4) 58 (34.8) rs878567 rs878567 TT, n (%) 21 (37.5) 31 (32.1) 0.74 2 0.69 TT, n (%) 17 (17.7) 48 (28.7) 5.62 2 0.06 CT, n (%) 21 (37.5) 43 (44.3) CT, n (%) 49 (51.0) 84 (50.2) CC, n (%) 14 (25.0) 23 (23.6) CC, n (%) 30 (31.3) 35 (21.1)

Alleles Cases Controls c2 df p Alleles Cases Controls c2 df p

rs6295 rs6295 G, n (%) 42 (37.5) 85 (43.8) 1.16 1 0.28 G, n (%) 78 (40.6) 140 (41.9) 0.083 1 0.77 C, n (%) 70 (62.5) 109 (56.2) C, n (%) 114 (59.4) 194 (58.1) rs878567 rs878567 T, n (%) 63 (56.2) 105 (54.1) 0.12 1 0.71 T, n (%) 83 (43.2) 180 (53.9) 5.54 1 0.01a C, n (%) 49 (43.8) 89 (45.9) C, n (%) 109 (56.8) 154 (46.1)

a p Value signiﬁcant. T.B. González-Castro et al. / Journal of Psychiatric Research 47 (2013) 1665e1672 1671

Table 5 Anguelova M, Benkelfat C, Turecki G. A systematic review of association studies Haplotypes for 5HTR1A markers -rs6295 (G/C), rs1423691 (C/T) and rs878567 (C/T)- investigating genes coding for serotonin receptors and the serotonin trans- in controls and suicide attempters. porter: II. Suicidal behavior. Molecular Psychiatry 2003;8:646e53. Arango V, Underwood MD, Boldrini M, Tamir H, Kassir SA, Hsiung S, et al. Serotonin 5HTR1A haplotypes Suicide attempters Controls Or (CI95%) p 1A receptors, serotonin transporter binding and serotonin transporter mRNA (frequency) (Frequency) expression in the brainstem of depressed suicide victims. Neuro- psychopharmacology 2001;25:892e903. Reference TC 0.55 0.52 Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and e CG 0.38 0.41 0.89(0.67 1.19) 0.46 haplotype maps. Bioinformatics 2005;21:263e5. CC 0.04 0.04 0.83(0.45e1.54) 0.56 Beck AT, Kovacs M, Weissman A. Assessment of suicidal intention: the scale for TG 0.01 0.01 0.66(0.17e2.53) 0.55 suicide ideation. Journal of Consulting and Clinical Psychology 1979;47: 343e52. Benko A, Lazary J, Molnar E, Gonda X, Tothfalusi L, Pap D, et al. Significant associ- grandparents of Mexican descent. Our sample is relatively ation between the C(-1019)G functional polymorphism of the HTR1A gene and impulsivity. American Journal of Medical Genetics Part B: Neuropsychiatric homogenous. Genetics 2010;5:592e9. To evaluate our case-control study let us describe its limitations. Cheetham SC, Crompton MR, Czudek C, Horton RW, Katona CL, Reynolds GP. Se- The size of our case-control sample is small compared with studies rotonin concentrations and turnover in brains of depressed suicides. Brain e involving other diseases and may not have sufficient power to Research 1989;502:332 40. Chen TJ, Yu YW, Hong CJ, Chen MC, Tsai SJ. Association analysis for the C-1019G detect an association with suicidal behavior. We did not report promoter variant of the 5-HT1A receptor gene with auditory evoked potentials psychopathologies related to suicide attempters. Also, we did not in major depression. Neuropsychobiology 2004;50:292e5. evaluate possible endophenotypes which could be increasing the Czesak M, Le Francois B, Millar AM, Deria M, Daigle M, Visvader JE, et al. Increased serotonin-1A (5-HT1A) autoreceptor expression and reduced raphe serotonin risk for suicide attempts. Finally, we could not exclude the genetic levels in deformed epidermal autoregulatory factor-1 (Deaf-1) gene knock-out heterogeneity in the sample as a whole. On a broader perspective, mice. The Journal of Biological Chemistry 2012;287:6615e27. the number of studies in the meta-analysis is small compared to Huang YY, Battistuzzi C, Oquendo MA, Harkavy-Friedman J, Greenhill L, Zalsman G, et al. Human 5-HT1A receptor C(-1019)G polymorphism and psychopathology. analyses dealing with other types of diseases. Also, we did not The International Journal of Neuropsychopharmacology 2004;7:441e51. perform a sub-analysis of suicide attempters and suicide com- Illi A, Setala-Soikkeli E, Viikki M, Poutanen O, Huhtala H, Mononen N, et al. 5- pleters, or an analysis based on gender because not all the studies HTR1A, 5-HTR2A, 5-HTR6, TPH1 and TPH2 polymorphisms and major depres- e fi sion. Neuroreport 2009;20:1125 8. speci ed these variables explicitly. Kamali M, Oquendo MA, Mann JJ. Understanding the neurobiology of suicidal In conclusion, our case-control study in a Mexican population behavior. Depression and Anxiety 2001;14:164e76. showed no association of the functional rs6295 or rs878567 poly- Kim HK, Kim SJ, Lee YJ, Lee HJ, Kang SG, Choi JE, et al. Influence of the interaction between the serotonin 1A receptor C-1019G polymorphism and negative life morphismwith suicidal behavior. The meta-analysis of the functional stressors on the development of depression. Neuropsychobiology 2011;64:1e8. rs6295 (C-1019G) polymorphism supports the outcomes of our pre- Kishi T, Okochi T, Tsunoka T, Okumura T, Kitajima T, Kawashima K, et al. Serotonin vious meta-analysis and of the present case-control study, that is, a 1A receptor gene, schizophrenia and bipolar disorder: an association study and e lack of association. To gain a comprehensive knowledge of the asso- meta-analysis. Psychiatry Research 2011;185:20 6. Koller G, Bondy B, Preuss UW, Zill P, Soyka M. The C(-1019)G 5-HT1A promoter ciation between 5-HTR1A polymorphisms and suicidal behavior it is polymorphism and personality traits: no evidence for significant association in necessary to include larger samples and integral studies. alcoholic patients. Behavioral and Brain Functions 2006;2:7. Lahiri DK, Nurnberger Jr JI. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Research 1991;19. Role of funding source Lemonde S, Turecki G, Bakish D, Du L, Hrdina PD, Bown CD, et al. Impaired repression at a 5-hydroxytryptamine 1A receptor gene polymorphism associated with major depression and suicide. The Journal of Neuroscience 2003;23:8788e99. The collection of data and the genotyping of subjects were Lowther S, De Paermentier F, Cheetham SC, Crompton MR, Katona CL, Horton RW. carried out thanks to the support of grants from the UJAT-DAMC- 5-HT1A receptor binding sites in post-mortem brain samples from depressed 2012-02 and CONACYT CB-2012-177459. suicides and controls. Journal of Affective Disorders 1997;42:199e207. Mann JJ. Neurobiology of suicidal behaviour. Nature Reviews Neuroscience 2003;4: 819e28. Authors’ contributions Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Open Medicine 2009;3:21. TZC and GCTB conceived the study, participated in its design and Ohtani M, Shindo S, Yoshioka N. Polymorphisms of the tryptophan hydroxylase helped to draft the manuscript. TZC, JRI, LNL and NH helped to gene and serotonin 1A receptor gene in suicide victims among Japanese. The perform the statistical analysis and to draft the manuscript. PGS Tohoku Journal of Experimental Medicine 2004;202:123e33. Pitchot W, Hansenne M, Pinto E, Reggers J, Fuchs S, Ansseau M. 5- recruited participants and helped with data integration and analysis. Hydroxytryptamine 1A receptors, major depression, and suicidal behavior. GA, TZC and HN coordinated and supervised the integration of data. Biological Psychiatry 2005;58:854e8. Samadi Rad B, Ghasemi A, Seifi M, Samadikuchaksaraei A, Baybordi F, Danaei N. Serotonin 1A receptor genetic variations, suicide, and life events Competing interests in the Iranian population. Psychiatry and Clinical Neurosciences 2012;66: 337e43. The authors declare not to have any competing interests. Sawiniec J, Borkowski K, Ginalska G, Lewandowska-Stanek H. Association between 5-hydroxytryptamine 1A receptor gene polymorphism and suicidal behavior. Przegla˛d Lekarski 2007;64:208e11. Acknowledgments Serretti A, Calati R, Giegling I, Hartmann AM, Moller HJ, Rujescu D. Serotonin receptor HTR1A and HTR2C variants and personality traits in suicide attempters and controls. Journal of Psychiatric Research 2009;43:519e25. The authors gratefully acknowledge our research volunteers Serretti A, Mandelli L, Giegling I, Schneider B, Hartmann AM, Schnabel A, et al. who helped to recruit the participants in this study. The collection HTR2C and HTR1A gene variants in German and Italian suicide attempters and of data and the genotyping of subjects were carried out thanks to completers. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 2007;5:291e9. the support of grants from the University of Tabasco (UJAT-DAMC- Stockmeier CA. Neurobiology of serotonin in depression and suicide. Annals of the 2012-02) and CONACYT (CB-2012-177459). New York Academy of Sciences 1997;836:220e32. Swartz MK. The PRISMA statement: a guideline for systematic reviews and meta- analyses. Journal of Pediatric Health Care 2011;25(1):1e2.. http://dx.doi.org/ References 10.1016/j.pedhc.2010.09.006. Tovilla-Zárate C, Juárez-Rojop I, Ramón-Frías T, Villar-Soto M, Pool-García S, Angles MR, Ocana DB, Medellín BC, Tovilla-Zárate C. No association between the Medellín BC, et al. No association between COMT val158met polymorphism HTR1A gene and suicidal behavior: a meta-analysis. Revista Brasileira de Psi- and suicidal behavior: meta-analysis and new data. BMC Psychiatry quiatria 2012;34:38e42. 2011;11:151. 1672 T.B. 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