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Association of 2A-adrenergic receptor gene polymorphism with susceptibility to suicide in Japanese females
Masaaki Fukutake a,, Akitoyo Hishimoto a,b , Naoki Nishiguchi a , Hideyuki Nushida c , Yasuhiro Ueno c , Osamu Shirakawa d , Kiyoshi Maeda a
a

Department of Psychiatry, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-Ku, Kobe, 650-0017, Japan b Molecular Neurobiology Branch, NIDA-IRP, NIH, Baltimore, USA c Department of Legal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan d Department of Neuropsychiatry, Kinki University School of Medicine, Osaka, Japan Received 30 November 2007; received in revised form 31 January 2008; accepted 8 February 2008 Available online 16 February 2008

Abstract Objectives: It has been suggested that noradrenergic system abnormalities are involved in suicide. Postmortem brain studies have shown that molecular and functional alterations in 2A-adrenergic receptor-induced signal transduction are associated with suicide and depression. Recently, a single nucleotide polymorphism (SNP) within a coding region of the 2A-adrenergic receptor gene (ADRA2A), which results in an Asn-to-Lys change at amino acid 251 (N251K), has been implicated in susceptibility to suicide in Caucasians. The aim of our study is to determine whether genetic variants of the ADRA2A gene are also associated with suicide in a Japanese population. Methods: Three SNPs, C-1291G, N251K and rs3750625C/A, and one insertion/deletion polymorphism in the ADRA2A gene were genotyped in 184 completed suicides and 221 control subjects with the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. Results: Neither variation of the N251K SNP nor the insertion/deletion polymorphism was found in our Japanese samples. The C-1291G SNP in the promoter region was found to be significantly associated with suicide in females (P = 0.043 and 0.013 for genotypic and allelic comparisons, respectively). One of the common haplotypes, CC of C-1291G and rs3750625C/A, was also associated with suicide in females (P = 0.015). These associations were also significant in the female violent suicide victims (P = 0.009 and 0.009 for allelic and CC haplotypic comparisons, respectively). Although the significance was nominal, it was maintained even after correction for multiple comparisons. By contrast, neither of these two SNPs showed any association with violent and/or non-violent suicide in males. Conclusion: Our results raise the possibility that promoter genetic variation in the ADRA2A gene is associated with either suicide or violent suicide in females. 2008 Elsevier Inc. All rights reserved.
Keywords: 2A-adrenergic receptor; Haplotype; Japanese; Single nucleotide polymorphism; Suicide

1. Introduction
Abbreviations: 5HTT, serotonin transporter; ACE, angiotensin converting enzyme; ADRA2A, 2A-adrenergic receptor gene; bp, base pair; COMT, catechol-O-methyltransferase; DNA, deoxyribonucleic acid; HPA axis, hypothalamicpituitaryadrenal axis; LD, linkage disequilibrium; Met, methionine; mRNA, messenger ribonucleic acid; NaI, sodium iodide; NCBI, National Center for Biotechnology Information; PCR, polymerase chain reaction; PCRRFLP, polymerase chain reaction and restriction fragment length polymorphism; PS, power and sample size calculation; SNP, single nucleotide polymorphism; SPSS, Statistical Package for the Social Sciences; TPH, tryptophan hydroxylase; Val, valine. Corresponding author. Tel.: +81 78 382 6065; fax: +81 78 382 6079. E-mail address: masaaaki@med.kobe-u.ac.jp (M. Fukutake). 0278-5846/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.02.003

Suicide is a major public health problem. Genetic factors for suicide have been observed in family, twin, and adoption studies (Roy et al., 1997) and completed suicides are thought to be more homogeneous than suicide attempters in terms of inheritance of suicidal behavior (Brent and Mann, 2005). Even though 90% of suicide victims or suicide attempters have a diagnosable psychiatric illness (Beautrais et al., 1996; Robins et al., 1959; Shaffer et al., 1996), most patients never attempt suicide, indicating a predisposition to suicidal behavior that is

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independent of the main psychiatric disorder (Mann, 1998, 2003). Several lines of evidence have suggested that some genetic variations in the serotonergic system are associated with suicide (Bondy et al., 2006), but the results are inconsistent. There is also a possibility that various signaling abnormalities are involved in the pathogenesis of suicide (Hishimoto et al., 2006, in press; Yanagi et al., 2005). It has been suggested that noradrenergic system abnormalities, one of several types of neurochemical dysfunctions, are involved in the pathogenesis of suicide. Postmortem radioligand receptor binding studies of the brains of depressed suicide victims have found up-regulation in the density of 2-adrenergic receptor agonist and antagonist binding sites (De Paermentier et al., 1997; Gonzlez et al., 1994; Meana et al., 1992; Meana and Garca-Sevilla, 1987; Ordway et al., 1994). Three different subtypes of human 2-adrenergic receptors have been identified (2A, 2B, and 2C) by molecular cloning, and the 2Aadrenergic receptor subtype is predominant in the frontal cortex of human brain (Grijalba et al., 1996). Of special interest is that selective enhancement of 2A-adrenergic receptor binding, immunolabeled levels and mRNA levels has been observed in the brain of suicide victims (Callado et al., 1998; Escrib et al., 2004; Garca-Sevilla et al., 1999; Gonzlez-Maeso et al., 2002). These alterations further support the hypothesis that 2Aadrenergic receptor supersensitivity partly explains the biological pathogenesis of suicide (Garca-Sevilla et al., 1986). These functional alterations may originate from alterations in the 2A-adrenergic receptor gene (ADRA2A). The ADRA2A gene consists of an intronless, single 3650-base pair exon and is located on chromosomes 10q24q26, the region of which has been linked to suicidal attempts independent of disease phenotypes (Cheng et al., 2006). Several genetic variants of the ADRA2A gene, including promoter genetic variants, have been identified (Feng et al., 1998). One of these, the N251K SNP, generates functional change (Small et al., 2000; Small and Liggett, 2001) and has been reported to be implicated in susceptibility to suicide completion (Sequeira et al., 2004). However, Martn-Guerrero et al. (2006) could find no association between the N251K SNP and completed suicides. The C-1291G SNP in the promoter region of the ADRA2A gene has been associated with a range of personality traits related to irritability, hostility and impulsivity (Comings et al., 2000). In the study presented here, we examined the possible association between suicide and genetic variants of the ADRA2A gene using a casecontrol association study of 184 completed suicides and 221 control subjects in a Japanese population. This constitutes the first such study in a Japanese population. 2. Methods 2.1. Subjects The study population consisted of 184 suicide victims (124 males: mean age SD, 49.2 16.9 years; 60 females: 45.7 19.0 years), whose autopsies were conducted at the Department of Legal Medicine, Kobe University Graduate School of Medicine. The methods of suicide were hanging (87), jumping

from a height (57), drowning (9), drug overdose (8), several deep cuts (7), jumping in front of a vehicle (4), burning (3), gas poisoning (2), and other methods (7). Suicide methods were classified as violent or non-violent according to Heil et al. (1997). Of suicide victims, 91.1% of males and 80.0% of females died by violent methods (i.e., hanging, jumping from heights, cutting, jumping in front of a vehicle, and burning). Accurate information about the clinical backgrounds of the suicide victims could not be obtained under our ethical code for genetic studies. The controls consisted of 221 unrelated volunteers (134 males: 41.9 16.1 years; 87 females: 45.1 16.4 years). They were recruited from the main island of Japan with their consent after the purpose and procedures of the study had been explained. All were healthy and of Japanese descent and none of them manifested psychiatric problems in unstructured interviews conducted by psychiatrists. This study was approved by the Kobe University Graduate School of Medicine Ethics Committee for Genetic Studies. 2.2. Genotyping Blood samples were taken from suicide victims and controls, and the leukocyte DNA was extracted according to standard procedures (Wang et al., 1994) with the sodium iodide (NaI) method using a DNA Extractor WB Kit (Wako Pure Chemical Industries, Ltd. Tokyo, Japan). We genotyped three SNPs: C1291G (rs1800544), N251K (rs1800035), rs3750625C/A, and one 18 base pair (bp) insertion/deletion polymorphism in a region of the gene corresponding to amino acids 255260 of the 2A-adrenergic receptor (Fig. 1). The three SNPs were genotyped with PCR-RFLP techniques. The C-1291G and N251K SNPs were genotyped with the method of Ohara et al. (2000) and Martn-Guerrero et al. (2006), respectively. For the rs3750625C/A SNP, the target sequences, including this SNP, were amplified by polymerase chain reaction (PCR) with the forward primer: 5-GCTCTTCAGAGCAAGCACTG-3 and the reverse primer: 5-GGGCTGATGTGAGGCAGAAA-3. The PCR products were digested with Bts I at 37 C for 16 h and the digestion products were visualized by ethidium bromide staining after electrophoresis in a 2% agarose gel. The PCR product size was 523 bp and the A allele was cleaved into 391 bp and 132 bp fragments, while the C allele was not digested. The insertion/deletion polymorphism was genotyped with the method of Hamann et al. (2001). 2.3. Statistical analysis The genotype distribution or allele frequency for the suicide victims and the controls was compared by using two-tailed Fisher's exact test. HardyWeinberg equilibrium was assessed with the chi-square test. These statistical analyses were performed with the SNPAlyze program v 4.1 (http://www. dynacom.co.jp/e/products/package/snpalyze/index.html). Linkage disequilibrium (LD) and haplotype frequencies, as well as any association between haplotype and suicide, were determined with the Haploview software program v3.32 (Barrett et al., 2005) (http://www.broad.mit.edu/mpg/haploview/).

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Table 1 Distribution of polymorphisms in the ADRA2A gene Marker Controls Minor allele frequency Suicide victims Minor allele frequency Statistics genotype P value Statistics allele 2 P value

Male and female C-1291G CC (n = 185) 0.086 rs3750625 CC (n = 183) 0.536 Male C-1291G (n = 112) rs3750625 (n = 120) Female C-1291G (n = 73) rs3750625 (n = 63)

CG 0.422 CA 0.404

GG 0.492 AA 0.060

C 0.297 A 0.262

(n = 171) (n = 181)

CC 0.117 CC 0.607

CG 0.474 CA 0.343

GG 0.409 AA 0.050

C 0.354 A 0.221

0.264 0.397

2.589 1.693

0.108 (0.226) 0.196 (0.376)

CC 0.089 CC 0.550

CG 0.446 CA 0.392

GG 0.465 AA 0.058

C 0.312 A 0.254

(n = 118) (n = 122)

CC 0.093 CC 0.631

CG 0.458 CA 0.328

GG 0.449 AA 0.041

C 0.322 A 0.205

0.980 0.426

0.048 1.66

0.826 (0.979) 0.198 (0.386)

CC 0.082 CC 0.508

CG 0.384 CA 0.429

GG 0.534 AA 0.063

C 0.274 A 0.278

(n = 53) (n = 59)

CC 0.170 CC 0.559

CG 0.509 CA 0.373

GG 0.321 AA 0.068

C 0.425 A 0.254

0.043 0.844

6.228 0.173

0.013 (0.027) 0.678 (0.977)

This column shows nominal P values and corrected P values for multiple testing (in parentheses). Boldface represents significant differences between the suicide victims and the controls.

Permutation tests based on 10,000 replications were also performed to calculate corrected P values of allele or haplotype analyses for multiple testing with the Haploview software program. The SPSS program (version 10.0; SPSS, Chicago, IL) was used for the odds ratio analysis and the PS v2.1.3.1 program (Dupont and Plummer, 1998) for the power analysis. The level of significance for all statistical results was set at P b 0.05. 3. Results Allele frequencies and genotype distributions for the C1291G SNP and rs3750625C/A SNPs are shown in Table 1. The genotype distributions of the two SNPs did not differ from the HardyWeinberg equilibrium for either the male/female control or the male/female suicide victim groups (data not shown). The two SNPs were in tight linkage disequilibrium [D =0.961 (95% confidence intervals 0.811.0), LOD = 12.44, r2 = 0.14 (P b 10 6)]. Neither variation of the N251K SNP nor the insertion/deletion polymorphism was found in our Japanese samples (data not shown). When the results for both sexes were combined, no association with suicide was detected in the allele frequencies for either the C-1291G SNP [P = 0.108 (corrected P = 0.226)] or the rs3750625C/A SNP [P = 0.196 (corrected P = 0.376)]. Based on the observed allele frequencies of the C-1291G SNP and rs3750625C/A SNP, the current samples yielded powers of 0.209 and 0.149, respectively, for the detection of nominally significant results. Haplotype frequencies of these two SNPs for suicide victims and controls are shown in Table 2. Three common haplotypes, GC, CC, and GA, were observed in our Japanese population. There were no significant differences in the frequencies of these haplotypes between the suicide victims and the controls (Table 2).

For females, the genotype distribution of the C-1291G SNP showed significant differences between the suicide victims and the controls (2 = 6.244, df = 2, P = 0.043) (Table 1). The C allele frequency of the C-1291G SNP was significantly greater for female suicide victims than for female controls [2 = 6.228, df = 1, P = 0.013 (corrected P = 0.027)] (Table 1). The odds ratio for phenotype was 1.96 (95% confidence intervals 1.153.32) for females. Based on observed allele frequencies of the C1291G SNP in females, the current samples yielded a power of 0.95 for the detection of nominally significant results. The frequency of the CC haplotype for females was also significantly greater for suicide victims than for controls [2 = 5.892, df = 2, P = 0.015 (corrected P = 0.032)] (Table 2). These signifTable 2 Haplotype distribution of the ADRA2A gene Haplotype Controls Suicide victims 0.430 0.347 0.222 2 0.197 2.807 1.494 P value * 0.657 (0.914) 0.094 (0.201) 0.222 (0.426)

Male and female GC 0.446 CC 0.292 GA 0.258 Male GC CC GA Female GC CC GA

0.431 0.311 0.258

0.431 0.323 0.207

0.772 0.094 1.878

0.380 (0.649) 0.759 (0.956) 0.171 (0.332)

0.465 0.260 0.260

0.349 0.394 0.248

3.925 5.892 0.054

0.048 (0.103) 0.015 (0.032) 0.816 (0.999)

Boldface represents significant differences between the suicide victims and the controls. This column shows nominal P values and corrected P values for multiple testing (in parentheses).

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Fig. 1. Relative position of the four genotyped polymorphisms. Exon 1 is shown as a solid gray block and the coding region is shown in dark gray in exon 1.

icances were maintained even after correction for multiple comparisons. By contrast, there was no association between the frequency of genotype, allele or haplotype of the two SNPs and male suicide victims (Tables 1, 2). Furthermore, we compared the frequency of genotype, allele or haplotype for the C-1291G SNP and rs3750625C/A SNP between 161 violent suicide victims and the controls in male or female subjects. For females, significant differences were found between the violent suicide victims and the controls in either the genotype distribution (2 = 6.395, df = 2, P = 0.041) or the allele frequencies [2 = 6.835, df = 1, P = 0.009 (corrected P = 0.024), Odds ratio 2.10, 95% confident interval 1.203.67] (Table 3). The frequency of the CC haplotype for female violent suicide victims (0.413) was also significantly greater than that for female controls (0.260) [2 = 6.809, df = 2, P = 0.009 (corrected P = 0.028)]. By contrast, there was no association between the frequency of genotype, allele or haplotype of the two SNPs and female non-violent suicide victims (Table 3). Finally, there was also no association between the frequency of genotype, allele or haplotype of the two SNPs and male violent/non-violent suicide victims (data not shown).

4. Discussion In this study, we found that the C-1291G SNP was associated with suicide in a Japanese female population. The frequencies of the C-1291 allele and the CC haplotype of C-1291G and rs3750625C/A SNPs were significantly higher for female suicide victims (0.425 and 0.394, respectively) than for female controls (0.274 and 0.260, respectively). Furthermore, the frequencies of the C-1291 allele and the CC haplotype of these two SNPs were also higher for female violent suicide victims (0.442 and 0.413, respectively). For males, on the other hand, the C-1291G SNP showed no association with either total suicide or violent/nonviolent suicide groups. These results were consistent with those reported by Sequeira et al. (2004), who found no association in male Caucasians of FrenchCanadian origin between three SNPs in the promoter region of the gene, including the C-1291G SNP, and suicide victims. There are ethnically differences of allelic distributions of the C-1291G SNP with the C allele frequency of 0.700.73 in Caucasians and 0.290.43 in African Americans (Belfer et al., 2005; Kurnik et al., 2006; Small et al., 2006), and 0.280.36 in Japanese that is similar to the frequency of our

Table 3 Distribution of polymorphisms in the ADRA2A gene in the female violent/non-violent suicide victims Female controls C-1291G Genotype CC CG GG Allele C G rs3750625 Genotype CC CA AA Allele C A Female violent suicide victims 2 P value * Female non-violent suicide victims 2 P value *

(vs. female controls)

(vs. female controls)

6 (0.082) 28 (0.384) 39 (0.534) 40 (0.274) 106 (0.726)

9 (0.209) 20 (0.465) 14 (0.326) 38 (0.442) 48 (0.558)

6.395

0.041

0 (0.000) 7 (0.700) 3 (0.300) 7 (0.350) 13 (0.650)

3.864

0.145

6.835

0.009 (0.024)

0.501

0.479 (0.766)

32 (0.508) 27 (0.429) 4 (0.063) 91 (0.722) 35 (0.278)

31 (0.660) 12 (0.255) 4 (0.085) 74 (0.787) 20 (0.213)

3.533

0.171

2 (0.200) 8 (0.800) 0 (0.000) 12 (0.600) 8 (0.400)

4.875

0.087

1.214

0.271 (0.551)

1.241

0.265 (0.487)

Boldface represents significant differences between the female violent suicide victims and the female controls. This column shows nominal P values and corrected P values for multiple testing (in parentheses).

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control subject (Matsunaga et al., 2007; Ohara et al., 2000, 1998). In our study, we could not detect any variants of the N251K SNP, a minor allele of which, 251K, was found only in the male Canadian suicide victims (Sequeira et al., 2004). The frequency of the minor 251K allele has been reported as 0.004 in Caucasians and 0.05 in African Americans (Belfer et al., 2005), while MartnGuerrero et al. (2006) failed to detect this allele in Caucasians. Because all our Japanese subjects had the N251 allele, this SNP is considered to be a rare polymorphism in Japanese. The insertion/ deletion polymorphism was not also found in our Japanese samples, while it was found in German with an allele frequency of 0.0120.017 (Hamann et al., 2001). The findings of our LD and haplotype analyses of the C1291G and rs3750625C/A SNPs confirmed the existence of a strong LD (D = 0.961) between these two SNPs and resulted in the detection of three common haplotypes, GC, CC, and GA. Belfer et al. (2005) also found one restricted haplotype block with a strong LD encompassing the whole ADRA2A gene locus and detected common three haplotypes as detected in our study by genotyping nine SNPs in Caucasian sample. Data from the International HapMap project (http://www.hapmap.org/) also show a strong LD block at the ADRA2A gene locus, and common three haplotypes were produced from five SNPs in a Japanese population. It may therefore be sufficient to detect these three haplotypes for the ADRA2A gene locus by genotyping two SNPs only. We found the frequency of the CC haplotype was higher for female suicide victims (0.394) than for female controls (0.260). However, in view of the differences in the frequency of the vulnerable C-1291 allele between the case (0.425) and control (0.274) groups, it can be assumed that the differences in frequency of the CC haplotype between the two groups could be largely determined with the C-1291G SNP. The functional change in the C-1291G SNP remains unclear. To the best of our knowledge, no in vitro study of the function of C-1291G SNP has been conducted. The C-1291G SNP is assumed to be located at the promoter region of the ADRA2A gene (Small and Liggett, 2001). The NCBI data base (http:// www.ncbi.nlm.nih.gov/projects/SNP/) showed that the C1291G SNP was located within a CpG island of the 5-flanking region of the gene and the C-1291 nucleotide constructed a CpG site with a neighbor nucleotide G. The C-to-G nucleotide substitution of this SNP may therefore change the CpG methylation status and thus affect transcript regulation of the gene (Cedar, 1988). In addition, the C-1291G SNP might be implicated in the hypothalamicpituitaryadrenal (HPA) axis response, dysregulation of which may be involved in the pathophysiology of suicide (Mann 1998, 2003; Mann and Currier, 2007), because the C-1291G SNP is suggested to affect cortisol response to the dexamethasone suppression test (Rosmond et al., 2002). However, the HPA axis is affected by many other systems, including the serotonergic system, and the relationship between the C-1291G SNP in the ADRA2A gene and the HPA axis should be clarified for the future. Because the rate of suicide is generally higher for males than females (Mann, 2003), there may be gender differences in the biological susceptibility of suicidal behavior. In our previous studies, the COMT 158Val/Met or the ACE insertion/deletion

polymorphisms, which is also thought to influence the HPA axis, was found to be associated with suicide, especially in males (Hishimoto et al., 2006; Ono et al., 2004). Our finding that an association between suicide and the ADRA2A gene polymorphism was observed particularly in females implies that some genes susceptible to suicide may not be shared by the sexes. The activity of the noradrenergic system exhibits gender differences at physiological and biochemical levels across species (Schmidt et al., 1997). The female-specific association of the ADRA2A gene polymorphism with suicide observed in our study may thus be related to these gender differences in the noradrenergic system. In addition, gender differences in suicidal behavior are suggested to be associated with higher levels of aggression and impulsivity. Several genetic variations in the serotonergic transmission related genes such as TPH1 or 5HTT genes have been reported to be associated with violent suicidal behavior so far (Bondy et al., 2006). The findings that significant association between the female suicide victims and the ADRA2A gene polymorphism was also found in the subjects with violent methods imply that dysregulation of the noradrenergic transmission via 2A-adrenergic receptor may impact on aggressive and impulsive behavior especially in females. Our study has certain limitations. First, psychiatric diagnoses of the suicide victims were not available in this study under our ethical code for genetic studies. We can therefore not exclude the possibility that our results may be related to specific mental disorders such as mood disorder, drug dependence, and personality disorder rather than suicide. Second, the sample size of our study population may be insufficient to reach any robust conclusions. Retrospective power analyses for allele frequencies of the C-1291G and rs3750625C/A SNPs, especially in males, indicated that our study has powers of 0.053 and 0.148, respectively, to yield a statistically significant result for these frequencies. We can therefore not exclude the possibility that our failure to find an association between the ADRA2A gene and suicide in males may be due to a type II error. 5. Conclusion Our results raise the possibility that presence of the C-1291G SNP of the ADRA2A gene increases the susceptibility to either suicide or violent suicide in females. Presence of the vulnerable C-1291 allele may increase the risk of suicide by affecting transcript regulation for the ADRA2A gene, although it should be kept in mind that the pathophysiology of most psychiatric disorders are polygenic and the etiology of most psychiatric conditions is multifactorial. Further replication studies specifically focusing on SNPs in the promoter region of the gene in independent populations and using larger samples will be required to determine the validity of this association between the ADRA2A gene and suicide. Acknowledgements We thank Mrs. Y. Nagashima for her technical assistance. This research was supported by a Research Grant for Nervous and Mental Disorders and Health Sciences from the Ministry of

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Health, Labor and Welfare, Japan, and by grants from the Ministry of Education, Science, Culture and Sports, Japan. References
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