BRIEF RESEARCH COMMUNICATION Neuropsychiatric Genetics Angiotensin I-Converting Enzyme I/D Polymorphism and Suicidal Behaviors D. Larry Sparks,1 John C. Hunsaker III,2 Philippe Amouyel,3,4* Alain Malafosse,5,6 Franck Bellivier,7,8,9 Marion Leboyer,7,8,9 Philippe Courtet,6,10,11 and Nicole Helbecque3 1 Sun Health Research Institute, Sun City, Arizona 2 Division of Medical Examiner’s Services, Kentucky Justice Cabinet, Frankfort, Kentucky INSERM, U744, Lille, France; Institut Pasteur de Lille, Lille, France; University of Lille 2, Lille, France 3 4 Centre Hospitalier et Universitaire de Lille, Lille, France 5 Department of Medical Genetics and Development, School of Medicine, Geneva, Switzerland INSERM, U888; University of Montpellier I, Montpellier, France 6 7 INSERM, U841; IMRB, Department of Genetics, Psychiatry Genetics, Creteil, France 8 Universite Paris 12, Faculty of Medicine, IFR10, Creteil, France AP-HP, Groupe Henri Mondor-Albert Chenevier, Departement Hospitalo-Universitaire de Psychiatrie, Creteil, France 9 10 University of Montpellier I, Montpellier, France 11 Department of Psychological Medicine and Psychiatry, Lapeyronie Hospital, University Hospital of Montpellier, Montpellier, France Received 27 February 2008; Accepted 28 April 2008 Suicide is one of the ten most common causes of death in Western countries. It involves genetic vulnerability factors and is often associated with major depression. A Japanese team reported an association between the insertion allele of the angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism with completed suicide. The ACE I/D polymorphism was investigated in two independent case-control studies, one involving 64 suicide completers and 90 controls who all underwent forensic investigations, the second one consisting of 588 suicide attempters and 639 controls. In the two population samples studied a statistically significant risk of suicidal behavior was observed for subjects bearing the DD genotype. These results suggest a possible role of the renin-angiotensin system in suicidal behavior. 2008 Wiley-Liss, Inc. Key words: angiotensin-converting enzyme; association study; genetics; suicide In Western countries, suicide is the first cause of death among people aged 25–34 years and the second cause of death among 15- to 24-year-old and college students. Each year there are 500,000 suicide attempts in the US and 220,000 in France. Worldwide, 1.8% of all deaths are due to suicide and psychiatric illness is involved in more than 90% of cases [Mann, 2003]. Mood disorders, more particularly in combination with drug abuse or alcohol, are by far the most common psychiatric disorders leading to suicide [Jamison, 2000]. Patients with bipolar disorders, particularly during depressive episodes, have a high risk of committing suicide [Angst et al., 1999]. For instance among patients with manic depressive illness the rate of suicide attempts varies between 2008 Wiley-Liss, Inc. How to Cite this Article: Sparks DL, Hunsaker JC, Amouyel P, Malafosse A, Bellivier F, Leboyer M, Courtet P, Helbecque N. 2009. Angiotensin IConverting Enzyme I/D Polymorphism and Suicidal Behaviors. Am J Med Genet Part B 150B:290–294. 24% and 50% [Jamison, 2000]. The treatment of depression usually involves the classical tricyclic drugs, monoamine oxidase inhibitors, and selective serotonin-reuptake inhibitors. Angiotensinconverting enzyme (ACE) inhibitors have also been reported effective in the treatment of depression [Bosio et al., 1990; Michalsen et al., 2001; Braszko et al., 2003]. This observation is sustained by animal models: mice lacking angiotensinogen showed a reduction of depressive-like behavior [Okuyama et al., 1999]. Affective disorders, particularly bipolar disorders, are linked with cardiac mortality, suggesting an association between hypertension Grant sponsor: Suiss National Foundation; Grant number: #32-112084. *Correspondence to: Prof. Philippe Amouyel, Service d’Epidemiologie et de Sante Publique, INSERM, U744, Institut Pasteur de Lille, 1 rue Calmette, BP 245, 59019 Lille Cedex, France. E-mail: email@example.com Published online 2 June 2008 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/ajmg.b.30793 290 SPARKS ET AL. 291 and a variety of symptoms, such as anxiety or depression [Rudisch and Nemeroff, 2003]. Hypertensive subjects receiving ACE inhibitors reported general well-being and increased work performances after 6 months of treatment, suggesting a possible impact on mood [Testa et al., 1993]. Conversely other hypotensive drugs did not exhibit such effects, prompting us to look at possible relationships between the renin-angiotensin system (RAS) and mood disorders. ACE belongs to the RAS whose components are detected in the central nervous system [Phillips et al., 1979]. Previously angiotensin-receptor blockers were identified as a possible risk factor for suicide (OR ¼ 3.52) [Callreus et al., 2007]. ACE generates angiotensin-II, a potent vasoconstrictor, from its precursor angiotensinogen and degrades bradykinin a vasodilator. The circulating and cellular levels of ACE are partly genetically determined through an insertion (I)/deletion (D) genetic polymorphism located in intron 16 of the gene: subjects bearing the D allele have higher levels of enzyme than the I allele bearers [Rigat et al., 1990]. This polymorphism has been associated with completed suicide in Japanese subjects [Hishimoto et al., 2006]. In this work we tested a possible association of this polymorphism in two independent population samples, one including 64 suicide completers, the other dealing with 588 suicide attempters. Study 1 included 154 subjects selected from a population of unrelated Americans who all underwent forensic evidences (Table I). Sixty-four of them committed suicide. Controls (n ¼ 90) were individuals without cardiovascular problems detected at autopsy. Due to the limited number of subjects who underwent autopsy, we included subjects aged less than 35 years in our control population sample, although suicide is rather frequent in this age range. Suicide attempts were classified as violent according to the criteria proposed by Asberg et al. . Hanging attempts, the use of firearms, and drowning were classified as violent attempts; drug overdoses or poisoning were considered to be non-violent suicide attempts. Study 2 included 588 patients consecutively admitted to psychiatric departments after a suicide attempt and 639 healthy controls without cardiovascular problems recruited in a general population (Table I). In order to minimize their morbid risk by suicide, only controls older than 35 years were included. All subjects gave informed consent. A suicide attempt was defined as intentional self-harm that was not self-mutilatory in nature and required medical evaluation and treatment in an emergency or intensive care unit [Mann, 1998]. Suicide attempts were classified as violent according to the criteria proposed by Asberg et al. . For all TABLE I. Characteristics of the Two Populations Studied Males (%) Age (years)a Caucasians (%) Study 1 Controls (n ¼ 90) Suicide (n ¼ 64) Study 2 Controls (n ¼ 639) Suicide (n ¼ 588) a Mean SD. 66.7 84.1 57 19 41 17 88.9 95.3 52.3 31.4 45 5 39 12 100 100 patients, we investigated whether there was a past history of violent suicide attempts. Concerning the control group, no information concerning psychiatric illness or previous suicide attempts had been collected. Patients with a history of violent suicide attempts were classified as violent suicide attempters, regardless of the nature of the current suicide attempt. All patients were interviewed by trained psychiatrists or psychologists, using the French version of the Mini International Neuropsychiatric Interview (MINI). Lifetime diagnosis (DSM-IV-TR) were made by the interviewer and then blindly rated by an independent psychiatrist according to medical case notes and MINI. Eighty two percent of the suicide attempters suffered from depression, 10% from bipolar disorders, 2% from schizophrenia, 2.8% from various psychiatric pathologies, and 3.2% from none. To minimize population heterogeneity, we ensured that both the patients and controls were entirely of West European Caucasian origin for at least two generations. The study was approved by the institutional ethics committees. Genomic DNA was extracted from postmortem brain samples (study 1) using phenol-chloroform or from blood samples collected into ethylenediaminetetraaceticacid (EDTA)-treated tubes (study 2) using a salt-out procedure [Parra et al., 1992]. All tissues analyzed in study 1 were collected as part of the routine coroner authorized autopsy procedure. ACE polymorphism was detected as previously described, except for addition of dimethylsulfoxide (DMSO) to enhance amplification of the I allele [Fogarty et al., 1994]. The laboratory personnel were blind to clinical information concerning the subjects tested. All gel results were analyzed by two different investigators. Statistical analyses were conducted with the SAS Software release 8.0 (SAS Institute, Inc., Cary, NC). Differences between groups of suicide victims (or attempters) and control subjects were assessed using Pearson’ c2 The relationship between suicidal behavior or completed suicide and ACE genotype was examined using multiple linear regression models and adjusted systematically for gender and age (and ethnic origin for study 1). Statistical interactions between ACE genotype and covariates were systematically explored. The relative risks were estimated from this multivariate analysis by the odds ratios (OR) with their 95% confidence intervals. Significance levels were set at P < 0.05. Table I presents the main characteristics of the two populations studied and Table II shows the genotype distribution and allele frequencies of the ACE I/D polymorphism in suicide victims or suicide attempters and control subjects in the two independent studies. They were in Hardy–Weinberg equilibrium in all samples. For study 1 (completed suicide) a weak statistically significant difference was observed in genotype (P ¼ 0.05) and allele (P ¼ 0.04) frequencies between cases and controls (Table II). The relative risk for completed suicide (as expressed by the odds ratio OR) was 2.4 (95% CI ¼ 1.2–4.8) (DD vs. II þ ID bearers) (P ¼ 0.02). After adjustment for confounding variables (age, gender, and ethnicity when necessary), the risk of suicide was significant for DD genotype bearers (Table III). No statistically significant interaction with covariate was found in this population sample. For study 2 (suicide attempts) a statistically significant difference was observed in allele (P ¼ 0.02) frequencies between cases and controls, whereas there was only a trend for genotype frequencies (P ¼ 0.06) (Table II). The relative risk for attempting suicide for DD 292 AMERICAN JOURNAL OF MEDICAL GENETICS PART B TABLE II. Distribution of the ACE I/D Polymorphism in Suicide Victims or Suicide Attempters and Control Subjects Genotypes (n, %) Study 1 Control subjects Suicide victims Violent suicide Non violent suicide Caucasian control subjects Caucasian suicide victims Study 2 Control subjects Suicide attempters Violent suicide Non violent suicide Alleles (n, %) n DD ID II P 90 64 54 10 80 61 20 (22.2) 26 (40.6) 20 (37.0) 6 (60.0) 19 (23.8) 24 (39.3) 50 (55.6) 27 (42.2) 25 (46.3) 2 (20.0) 43 (53.7) 27 (44.3) 20 (22.2) 11 (17.2) 9 (16.7) 2 (20.0) 18 (22.5) 10 (16.4) 639 588 155 384 189 (29.6) 209 (35.5) 55 (35.5) 136 (35.4) 315 (49.3) 276 (46.9) 70 (45.2) 184 (47.9) 135 (21.1) 103 (17.5) 30 (19.3) 64 (16.7) D I 0.13 90 (50.0) 79 (61.7) 65 (60.2) 14 (70.0) 81 (50.6) 75 (61.5) 90 (50.0) 49 (38.3) 43 (39.8) 6 (30.0) 79 (49.4) 47 (38.5) 0.07 0.06 0.36 0.08 693 (54.2) 694 (59.0) 180 (58.1) 456 (59.4) 585 (45.8) 482 (41.0) 130 (41.9) 312 (40.6) 0.02 0.22 0.02 0.05 0.15 0.03a P 0.04 0.09 0.10a a 2 c exact test. genotype bearers was 1.3 (95% CI ¼ 1.0–1.7, P ¼ 0.03). After adjustment for confounding variables (age and gender), the risk of suicide was significant for DD genotype bearers (Table III) and the OR was similar when excluding subjects suffering from schizophrenia, whose genotype distribution differed widely from the others. No statistically significant interaction with covariate was found. From the results presented here dealing with two independent population samples, Caucasians bearing the ACE DD genotype are at risk for suicide. This is in accordance with a previous observation of an increase of ACE activity in the substantia nigra of suicide completers [Arregui et al., 1980]. The ACE I/D genotype distribution observed in controls from study 1 is rather unusual, when compared to values previously published in various population samples. However it did not differ when excluding black subjects from the sample (Table II) and remained balanced when taking the same cut-off for controls (35 years) as in study 2 (36 years and over: 18 DD, 40 ID, 17 II). The statistical power in study 1 was 83.3% to detect an OR of 2.8 (the one observed after adjustment in the global population in study 1). When considering only Caucasians in the calculations, the statistical power was 71.2% to detect an OR of 2.5 (the one observed after adjustment in the Caucasian subsample in study 1). A previous work reported an impact of the I allele on suicide completion in Japanese male subjects [Hishimoto et al., 2006], a result opposite to our findings. The observed discrepancy could be explained (i) by differences in genotype distribution of the ACE I/D polymorphism between Japanese and Caucasian populations [Ishigami et al., 1995] or (ii) by the fact that ACE should be in linkage disequilibrium with another gene which is truly associated to suicide. Subjects attempting suicide are usually depressed. Although in study 2 the ACE genotype distribution in the group of suicide attempters was similar whatever the psychiatric disease they suffered from (except for schizophrenia), it is interesting to note that the majority of them (82%) suffered from major depression. Several hypotheses could explain the observed association between the ACE I/D polymorphism and the risk of suicide. ACE colocalized with substance P-containing neurons in brain regions TABLE III. Odds Ratios for the ACE I/D Polymorphism in the Two Populations Study 1 All Violent suicide Caucasian subjects Study 2 All Violent suicide Non violent suicide ID vs. II OR (95% CI) P D vs. Ia OR (95% CI) P 2.8 (1.3–6.4)b 2.4 (1.0–5.7)b 2.5 (1.1–5.8)c 0.01 0.04 0.03 1.6 (1.0–2.6) 1.5 (0.9–2.5) 1.6 (0.9–2.6) 0.04 0.09 0.07 1.3 (1.0–1.7)c 1.2 (0.8–1.9)c 1.3 (0.9–1.8)c 0.03 0.28 0.08 1.2 (1.0–1.4) 1.2 (0.9–1.5) 1.2 (1.0–1.5) 0.02 0.22 0.02 P DD vs. II þ ID OR (95% CI) 2.8 (1.0–8.2)b 2.6 (0.8–8.0)b 2.8 (0.9–8.5)c 0.05 0.10 0.06 1.5 (1.1–2.1)c 1.1 (0.7–1.9)c 1.6 (1.1–2.4)c 0.02 0.65 0.02 P DD vs. II OR (95% CI) 1.0 (0.4–2.6)b 1.1 (0.4–3.0)b 1.2 (0.4–3.2)c 0.99 0.86 0.76 1.2 (0.9–1.7)c 0.9 (0.5–1.4)c 1.4 (0.9–2.0)c 0.31 0.59 0.12 Due to the low number of subjects, ORs were not calculated in the case of non violent suicide for study 1. a ORs without any adjustment. b ORs are adjusted for age, sex, and ethnicity. c ORs are adjusted for age and sex. SPARKS ET AL. that coordinate stress responses. In guinea pigs and humans substance P antagonists have been shown to exert antidepressant effects [Kramer et al., 1998]. An increase in ACE levels associated with the DD genotype [Arinami et al., 1996] will lead to an increase in substance P and thus of stress and/or anxiety, possibly increasing the risk of suicide [Ebner and Singewald, 2006]. Another mechanism involves serotonin, a neurotransmitter which has been implicated in psychiatric disorders, particularly mood disorders. Serotonin may also induce vasoconstriction in patients either directly [Hillis and Lange, 1991] or through amplification of the release and activity of other vasoconstrictors, such as angiotensin II. Previous reports of an association between suicide and a functional polymorphism in the regulatory region of the serotonin transporter gene [Anguelova et al., 2003; Helbecque et al., 2006], already known to be associated with bipolar disorders, are in favor of this mechanism, suggesting an association between depression and/or suicide and vasoconstriction. Previous investigations have suggested some links between depression and cardiovascular mortality. This hypothesis is sustained by animal models. ACE-deficient mice have low blood pressure [Esther et al., 1996] and mice lacking angiotensinogen show a reduction of depressive-like behavior [Okuyama et al., 1999], suggesting a possible link between angiotensinogen or its metabolites and depression. Angiotensinogen and ACE are part of the RAS. In vivo studies in rats have shown that ACE interferes with the secretion of pituitary hormones such as corticotropin (ACTH) and potentiates the stimulatory effects of corticotropin releasing hormone (CRH) [Jezova et al., 1998], suggesting that the brain RAS is involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis [Aguilera et al., 1995]. Dysregulation of the activity of the HPA system is one of the major neuroendocrine abnormalities observed in depression. An increase in ACE levels associated with the DD genotype will lead to an hyperactivation of the HPA axis response [Baghai et al., 2002]. This in turn could be associated with a higher risk of suicide as previously suggested by Mann . In humans, the observed association between the ACE I/D polymorphism and the risk of suicide could involve angiotensin itself or the angiotensin type 1 receptor (AGTR1) [Callreus et al., 2007]. The use of ACE inhibitors may be of benefit as an antidepressant treatment strategy. The antidepressant activity may be related to reduced angiotensin II activity since ACE inhibitors which elevate mood in depressed patients reduce angiotensin II production. A previous study suggested that the ACE D allele, associated with higher ACE levels, might be associated with a positive outcome of various antidepressant therapies, at least in major depressed women [Baghai et al., 2004]. On the other hand antidepressant drugs induce a reduction of responses to angiotensin II, their effect being at the AGTR1 [Gard et al., 1999]. These drugs may act either directly or by interference with post-receptor events. A genetic A1166C polymorphism located in the 30 -nontranslated region of the AGTR1 gene, was associated with hypertension [Baudin, 2005], and subjects bearing the CC genotype respond better to an antidepressant treatment than the others [Bondy et al., 2005]. However no significant differences in genotype or allele frequencies of this polymorphism have been observed between cases and controls in our population samples (data not shown), neither in the Japanese study [Hishimoto et al., 2006]. 293 These findings still reinforce the existence of common genetic factors for depression and cardiovascular disease [Bondy, 2007]. Although the underlying mechanism is still unknown, recent studies suggest that gene–gene or gene–environment interactions must be investigated in order to delineate more precisely the impact of each component. Our study has several limitations, in particular the small number of subjects in study 1, due to the difficulty to recruit suicide victims. However results from this preliminary study were confirmed by study 2, suggesting a potential role of the ACE DD genotype on the risk of suicide. 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