The tryptophan hydroxylase 1 (TPH1) gene schizophrenia susceptibility and suicidal behavior A multi-centre caseЦcontrol study and meta-analysis.код для вставкиСкачать
RESEARCH ARTICLE Neuropsychiatric Genetics The Tryptophan Hydroxylase 1 (TPH1) Gene, Schizophrenia Susceptibility, and Suicidal Behavior: A Multi-Centre Case–Control Study and Meta-Analysis† Peter Saetre,1* Per Lundmark,2 August Wang,3 Thomas Hansen,4,5 Henrik B. Rasmussen,4 Srdjan Djurovic,6,7,8 Ingrid Melle,6,7,8 Ole A. Andreassen,6,7,8 Thomas Werge,4 Ingrid Agartz,1,6,9 Hakan Hall,1 Lars Terenius,1 and Erik G. J€onsson1 1 Department of Clinical Neuroscience, HUBIN Project, Karolinska Institutet and Hospital, Stockholm, Sweden 2 Department of Medical Sciences, Molecular Medicine, Uppsala University, Uppsala, Sweden Mental Health Center Amager, Copenhagen University Hospital, Copenhagen, Denmark 3 4 Research Institute of Biological Psychiatry, Copenhagen University Hospital, Mental Health Centre Sct. Hans, Roskilde, Denmark 5 Centre for Pharmacogenomics, University of Copenhagen, Copenhagen N, Denmark Institute of Psychiatry, University of Oslo, Oslo, Norway 6 7 Department of Medical Genetics, Ulleval University Hospital, Oslo, Norway 8 Department of Psychiatry, Ulleval University Hospital, Oslo, Norway 9 Department of Psychiatric Research, Diakonhjemmet Hospital, Oslo, Norway Received 23 January 2009; Accepted 7 May 2009 How to Cite this Article: Serotonin (5-hydroxytryptamin; 5-HT) alternations has since long been suspected in the pathophysiology of schizophrenia. Tryptophan hydroxylase (tryptophan 5-monooxygenase; TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT, and sequence variation in intron 6 of the TPH1 gene has been associated with schizophrenia. The minor allele (A) of this polymorphism (A218C) is also more frequent in patients who have attempted suicide and individuals who died by suicide, than in healthy control individuals. In an attempt to replicate previous findings, five single nucleotide polymorphisms (SNPs) were genotyped in 837 Scandinavian schizophrenia patients and 1,473 controls. Three SNPs spanning intron 6 and 7, including the A218C and A779C polymorphisms, were associated with schizophrenia susceptibility (P ¼ 0.019). However there were no differences in allele frequencies of these loci between affected individuals having attempted suicide at least once and patients with no history of suicide attempts (P ¼ 0.84). A systematic literature review and meta-analysis support the A218C polymorphism as a susceptibility locus for schizophrenia (odds ratio 1.17, 95% confidence interval 1.07–1.29). Association studies on suicide attempts are however conflicting (heterogeneity index I2 ¼ 0.54) and do not support the A218C/A779C polymorphisms being a susceptibility locus for suicidal behavior among individuals diagnosed with a psychiatric disorder (OR ¼ 0.96 [0.80–1.16]). We conclude that the TPH1 A218/A779 locus increases the susceptibility of schizophrenia in Caucasian and Asian populations. In addition, the data at hand suggest that the locus contributes to the liability of psychiatric disorders characterized by elevated suicidal rates, rather than affecting Ó 2009 Wiley-Liss, Inc. Saetre P, Lundmark P, Wang A, Hansen T, Rasmussen HB, Djurovic S, Melle I, Andreassen OA, Werge T, Agartz I, Hall H, Terenius L, J€ onsson EG. 2010. The Tryptophan Hydroxylase 1 (TPH1) Gene, Schizophrenia Susceptibility, and Suicidal Behavior: A Multi-Centre Case–Control Study and Meta-Analysis. Am J Med Genet Part B 153B:387–396. † This article was published online on 12 June 2009. An error was subsequently identified. August Wang was not included in the list of authors. This notice is included in the online and print versions to indicate that both have been corrected 11 February 2010. Additional Supporting Information may be found in the online version of this article. Grant sponsor: Copenhagen Hospital Corporation Research Fund; Grant sponsor: Danish National Psychiatric Research Foundation; Grant sponsor: Danish Agency for Science; Grant sponsor: Research Council of Norway; Grant numbers: 147787, 167153; Grant sponsor: Eastern Norway Health Authority (Helse Øst RHF); Grant number: 123/2004; Grant sponsor: Swedish Research Council; Grant number: K2007-62X15077-04-1; Grant sponsor: The Knut and Alice Wallenberg Foundation; Grant number: KAW 2005.0256. *Correspondence to: Dr. Peter Saetre, R5:00, Stockholm SE-171 76, Sweden. E-mail: email@example.com Published online 12 June 2009 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/ajmg.b.30991 387 388 suicidal behavior of individuals suffering from a psychiatric disorder. Ó 2009 Wiley-Liss, Inc. Key words: tryptophan hydroxylase 1 gene (TPH1); schizophrenia; suicidal behavior; case–control association study; metaanalysis INTRODUCTION A role for serotonin (5-hydroxytryptamin; 5-HT) alterations in the pathophysiology of schizophrenia has long been suspected. 5-HT agonists have psychogenic effects [Lieberman et al., 1998; Abi-Dargham, 2007], and positive symptoms, like delusions and hallucinations, are thought to occur after activation of the 5-HT2 receptors [Aghajanian, 1994; Schotte et al., 1996; Sodhi and Sanders-Bush, 2004]. Atypical anti-psychotic drugs are potent 5-HT2A receptor antagonists, but relatively weak dopamine D2 receptor antagonists [Schotte et al., 1996]. In addition, postmortem samples from schizophrenia patients show a reduced density of 5 -HT2A receptors in prefrontal cortex, suggesting that 5-HT dysfunction is a part of the pathophysiology of the disease [Burnet et al., 1996; Dean et al., 1999; Matsumoto et al., 2005]. Suicidal behavior is a well-established clinical feature of schizophrenia [Tandon et al., 2008]. The lifetime suicide prevalence in schizophrenia has been estimated to about 5.6%, and young patients, early in the course of schizophrenia, are more likely to commit suicide than older patients [Palmer et al., 2005]. However, the suicide risk remain high also later in life [Carlborg et al., 2008]. Poor adherence to treatment, previous depressive disorders and/or suicide attempts, fear of mental disintegration and affective symptoms increases the risk for future suicide attempts [Hawton et al., 2005]. The curable effects of anti-depressive and anti-anxiety drugs that modulate 5-HT, suggests that 5-HT dysfunction may underlie suicidal behavior associated with schizophrenia and other psychiatric disorders. Moreover, low cerebrospinal fluid (CSF) concentrations of the major 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA) in suicide attempters [Asberg, 1997; Mann and Malone, 1997], as well as the pronounced differences with respect to the density of 5-HT transporter sites and several 5-HT receptor subtypes among suicidal individuals [Arango et al., 1995; Mann et al., 2000; Anisman et al., 2008], provide additional support for a general link between 5-HT dysfunction and suicidal behavior. Tryptophan hydroxylase (tryptophan 5-monooxygenase; TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT. Consequently, dysfunction in this enzyme has been hypothesized to be involved in pathological disturbances of the 5-HT system. There are two genes encoding TPH: TPH1 (11p15.3–p14) and TPH2 (12q21.1). In rodents TPH2 is the predominant isoform of the enzyme in the adult central nervous system (CNS), whereas TPH1 is primarily active in the periphery [Sakowski et al., 2006]. However, TPH1 is abundantly expressed in the brain during the late prenatal period, and appears to modulate the effect of serotonin on neuron number and growth [Rind et al., 2000; Nakamura et al., 2006]. In humans both TPH1 and TPH2 are expressed in cortex, hypothalamus, thalamus, hippocampus, amygdala, cerebellum, and raphe nuclei [Zill et al., 2007]. AMERICAN JOURNAL OF MEDICAL GENETICS PART B TPH1 is the best studied of the two genes so far, and numerous studies have demonstrated association between single nucleotide polymorphisms (SNPs) in the TPH1 gene and the liability of psychiatric disorders and suicidal behavior. A218C (rs1800532) and A779C (rs1799913) are two polymorphisms located in intron 6 of the gene, which are in complete, or almost complete, linkage disequilibrium (LD) in Caucasian populations. The A218-allele has consistently been associated with an increased liability of schizophrenia across multiple ethnic groups [Watanabe et al., 2007], and the association show strong epidemiological credibility [Allen et al., 2008]. The association between A218C/A779C and suicidal behavior has been extensively studied in both European and Asian populations [Rujescu et al., 2003; Bellivier et al., 2004]. Also the latest meta-analysis, contrasting allele frequencies between suicide attempters suffering from different psychiatric disorders or individuals who died by suicide (often of unknown mental health status) and unaffected controls in 34 independent studies, demonstrates an overall association between the A218C/A779C polymorphisms and suicidal behavior [Li and He, 2006]. This supports the involvement of TPH1 also in the pathogenesis of suicidal behavior. In the present study five TPH1 single nucleotide polymorphisms (SNPs) were genotyped in three Scandinavian case–control samples comprising 837 patients suffering from schizophrenia and related disorders and 1,473 controls. For patients with retrospective information on suicidal behavior (n ¼ 738), TPH1 polymorphisms were also analyzed for an association with suicide attempts. In addition we reviewed the literature for TPH1 association studies with schizophrenia and suicidal attempts among patients diagnosed with a psychiatric disorder, and meta-analyzed previously published reports. Our study is in agreement with the previously published literature, suggesting that the A218C locus is associated with schizophrenia but not with suicidal behavior among individuals affected by schizophrenia, affective, alcoholism, or personality related disorders. MATERIALS AND METHODS Clinical Samples The clinical samples originate from the Scandinavian Collaboration on Psychiatric Etiology (SCOPE) and were collected in Denmark (DK), Norway (NO), and Sweden (SE). Affected individuals diagnosed with schizophrenia (n ¼ 734), schizoaffective disorder (n ¼ 87), or schizophreniform disorder (n ¼ 16), according to ICD-10 (DK) or DSM-III-R/DSM-IV (NO and SE), were compared to unrelated matched controls (Table I). All individuals in the study were of Caucasian origin and detailed descriptions of the samples have previously been reported [Hansen et al., 2007; J€ onsson et al., 2008a; K€ahler et al., 2008]. One hundred fifty one affected and 130 control individuals from the Swedish sample were included in a previous association analysis between TPH1 and schizophrenia [Zaboli et al., 2006b]. Patients (n ¼ 738) were assessed for presence or absence of suicide attempts based on record reviews (DK, SE) and interview data (NO, SE). Any previous self-harm, in combination with suicidal ideation, documented or reported was regarded as a suicide SAETRE ET AL. 389 TABLE I. Characteristics of Danish (DK), Norwegian (NE), and Swedish (SE) Samples Analyzed for Association Between Tryptophan Hydroxylase 1 (TPH1) Gene Variants and Schizophrenia and Suicide Attempt Among Affected (Case) Country DK NO SE Characteristic N Gender (%women) Agea Age onseta Suicide attempters (%) N Gender (%women) Age Age onset Suicide attempters (%) N Gender (%women) Age Age onset Suicide attempters (%) Case 420 42.6 43.9 12.3 27.2 8.9 43.5 162 46.3 37.2 10.7 27.6 8.7 27.6 255 37.2 54.1 15.2 26.3 7.2 44.8 Control 1004 41.5 43.2 11.8 177 55.4 38.7 10.3 292 37.7 50.3 10.1 a Mean standard deviation given for quantitative variables. attempt. For the present analysis subjects were divided into those who had made at least one suicide attempt and those who had not performed any suicide attempt. The Danish Scientific Committees, the Danish Data Protection Agency, the Norwegian Scientific-Ethical Committees, the Norwegian Data Protection Agency, the Ethical Committee of the Karolinska Hospital, the Stockholm Regional Ethical Committee and the Swedish Data Inspection Board all approved the study. All participants had given informed consent prior to inclusion in the study. SNP Selection and Genotyping Based on previous published literature we selected five SNPs for genotyping. These markers are spanning the TPH1 from the upstream region (6.5 kbp; rs4537731), through intron 2 (rs211105) and intron 6 (rs1800532, rs1799913) to intron 7 (rs7933505). Genomic DNA was extracted from whole blood samples, and the selected SNPs were genotyped at the SNP Technology Platform in Uppsala, Sweden (www.genotyping.se), using the Illumina BeadStation 500GX and the 1536-plex Illumina Golden Gate assay (Illumina, Inc., San Diego, CA). The sample success rate was on average 99.7% for the genotyped SNPs and the reproducibility of the genotyping was 100% according to duplicate analysis of 2.6% of the genotypes. Literature Search We performed a meta-analysis of published case–control studies examining the association between the TPH1 A218C polymorphism (rs1800532) and schizophrenia. In addition we metaanalyzed the association between A218C/A779C polymorphisms and suicide attempt among individuals diagnosed with a psychiatric disorder. Data were obtained by searching the National Library of Medicine’s database PubMed up to August 2008, using the search terms ‘‘tryptophan hydroxylase,’’ ‘‘TPH,’’ and ‘‘schizophrenia’’ or ‘‘suicidal behavior’’ and ‘‘suicide’’, respectively. References cited in identified studies and reviews were scrutinized to identify additional work not identified by our initial PubMed search. We included case–control studies, which were published in a peerreviewed journal, contained independent data that was sufficient to calculate the odds ratio (OR) and its confidence interval, and where psychiatric disorder (including schizophrenia) and/or suicidal behavior had been diagnosed according to the International Classification of Diseases (ICD), Diagnostic and Statistical manual of Mental disorders (DSM) or Chinese Classification of Mental Disorders (CCMD) systems. Since our aim was to examine the association between TPH1 and suicidal behavior independently of mental health status, we only included studies where genotype frequencies were available for both suicide (SA) and non-suicide attempters (NSA) diagnosed with the same psychiatric disorder. The A218C and A779C polymorphisms are in complete LD (R2 1) in populations of European descent and thus association studies of the A218C and A779C (rs1799913) polymorphisms conducted in Caucasian populations were treated synonymously. In total we identified 11 studies comparing the frequency of A218C/A779C in schizophrenia and healthy controls. Of these, five were excluded: the study of Chotai et al.  and the study of Zaboli et al. [2006b], which overlapped with the study by Serretti et al.  and the present Swedish sample, respectively, and the studies of Shinkai et al. , Chao and Richardson , and Semwal et al. , who genotyped A779C in Japanese, African-American, and Indian populations, where theA218C and A779C loci are not known to be in complete LD. We identified 16 studies that compared the frequency of A218C/A779C in suicide and nonsuicide attempters diagnosed with the same psychiatric disorder. If insufficient data were given in the reports, we tried to contact the corresponding authors by e-mail for additional information. Despite these efforts, four studies were excluded from the analysis due to insufficient data. That is, for the studies conducted by Furlong et al.  and Ho et al. , no data on the numbers of individuals per TPH1 genotype and suicide/nonsuicide attempters were listed in the original reports. In the study by Kunugi et al. , the numbers of individuals per TPH1 genotype of non-suicide attempters diagnosed with bipolar disorder or unipolar depression were not listed. Souery et al.  matched controls and patients with respect to ethno-geographic origin, but the numbers of individuals per A218C genotype for matched suicide and non-suicide patients was not provided. Statistical Analysis Hardy–Weinberg (HW) equilibrium was tested in the control samples using Fisher’s exact test as implemented in PEDSTATS [Wigginton and Abecasis, 2005]. Linkage disequlibrium (D0 and R2) between SNP pairs, and haplotype block structure [Gabriel et al., 2002] were determined with Haploview 4.0 [Barrett et al., 2005] (Supplementary Fig. 1). The fixation index (FST) was 390 AMERICAN JOURNAL OF MEDICAL GENETICS PART B calculated for each SNP separately, and all loci combined, in the control sample with FSTAT, grouping controls by country of origin. No evidence of population stratification was evident from the data: the fixation index for all combined loci was 0.001 0.001 (95% bootstrap confidence interval). Association analysis comparing allele and genotype frequencies between affected individuals and controls, and between suicide attempters and non-attempters within the patient group, were performed with UNPHASED (version 3.0.9) [Dudbridge, 2003, 2008]. To account for possible population stratification, we included country of origin as a confounding factor in the association analyses. Test for heterogeneity between countrieswas performed in a separate analysis, specifying country as a modifier. Haplotype analysis was carried out with UNPHASED using all five SNPs, which belonged to one haplotype block. We chose 0.05 as the level of nominal significance. Correction for multiple testing was accomplished by permuting the affection status and presence/absence of suicide attempts (among affected only), respectively, and recalculating P values for the permuted data (1,000 permutations). Empirical P values were calculated for each of the two hypotheses separately, (compensating for five tests), as the fraction of permutations yielding P values less or equal to the observed P values. For the meta-analysis we first calculated crude study-specific odds ratios (ORs) and 95% confidence intervals (95% CI) for each study, comparing the minor versus major alleles. We obtained summary ORs and 95% CI with the random-effects model [DerSimonian and Laird, 1986], which utilizes weights that incorporate both the within-study and between-study variance. Between -study heterogeneity was assessed with Cochran’s Chi-squarebased Q statistic, and by the I2 heterogeneity metric. Publication bias was assessed by examining funnel plot asymmetry [Egger et al., 1997]. That is, the standard normal deviate was regressed against the OR estimate precision, and a t-test was used to detect if the intercept deviated significantly from zero. RESULTS Association With Schizophrenia Three out of five tested markers attained nominal significant association with the disease (P 0.05), namely rs1800532 (A218C), rs1799913 (A779C), and rs7933505 (Table II). These markers, which are located in a 1.8 kb segment spanning introns 6 and 7, were in almost complete LD (D0 ¼ 1, R2 0.99) (Fig. S1), and it was the minor alleles (A) that were over-represented among affected individuals. The odds ratio (OR) for the most significant marker rs1799913 was 1.18 for the combined sample with a 95 confidence interval (95 CI) between 1.04 and 1.34. The OR tended to be larger for the Swedish sample (OR ¼ 1.42) than for the Norwegian and Danish samples (1.18 and 1.09, respectively). However, the difference in association strength between countries of origin was not statistically significant (P ¼ 0.20), and may well have occurred by chance alone. As expected, the results for the two markers rs1800532 and rs7933505, which were in very strong LD with rs1799913, were similar (Table II). The association signal from the three nominally significant SNPs was globally significant, and the empirical P-value for attaining three markers with an equally strong (or stronger) disease association was 0.019 (determined from 1,000 permutations). Five haplotypes accounted for 99.6% of the segregating haplotypes in the population. As expected the haplotype retaining the A alleles of rs1800532, rs1799913 and rs7933505 (ATAAA) was associated with an increased risk of the disease (freqSCZ ¼ 0.41, freqCTRL ¼ 0.37; P ¼ 0.019), and a haplotype lacking these alleles (GTCCG) tended to be protective (freqSCZ ¼ 0.17, freqCTRL ¼ 0.20; P ¼ 0.049). Association With Suicidal Behavior in Affected Individuals To examine whether the TPH1 polymorphisms were associated with suicidal behavior in affected individuals, we tested allele and TABLE II. Allele Frequencies, Genotype Counts, and Association Statistics for Single Nucleotide Polymorphisms (SNPs) in the Tryptophan Hydroxylase 1 (TPH1) Gene and Schizophrenia in Swedish (SE), Norwegian (NO) and Danish (DK) Case–Control Samples Polymorphism SNP ID rs4537731 rs211105 rs1800532 rs1799913 rs7933505 1/2b A/G T/G C/A C/A G/A MAFc 0.43 0.25 0.37 0.37 0.38 HWd 0.92 0.09 0.82 0.54 1.00 Case/control 1/1 304/470 468/839 290/570 289/570 289/565 1/2 373/725 309/520 391/691 391/694 391/690 *Indicate nominal significance (P < 0.05). a OR corresponds to allele association. Major/Minor allele. Minor allele frequency. d P-value from test of Hardy–Weinberg equilibrium among control subjects. e P-value for test of heterogeneity between samples. b c Disease association (P-value) 2/2 157/275 53/102 149/203 145/196 154/211 Allele 0.169 0.991 0.016* 0.010* 0.015* Genotype 0.075 0.836 0.043* 0.024* 0.042* Odds ratioa All 0.92 1.00 1.17 1.18 1.17 SE 0.82 1.04 1.39 1.42 1.35 NO 1.03 1.17 1.18 1.18 1.19 DK 0.93 0.93 1.07 1.09 1.08 Heterogeneitye 0.56 0.41 0.19 0.20 0.30 SAETRE ET AL. 391 TABLE III. Genotype Counts and Association Statistics for Single Nucleotide Polymorphisms (SNPs) in the Tryptophan Hydroxylase 1 (TPH1) Gene and Suicidal Behavior in Swedish (SE), Norwegian (NO), and Danish (DK) Schizophrenia Patients SA/NSAa SNP ID rs4537731 rs211105 rs1800532 rs1799913 rs7933505 Suicide attempt association (P-value) Allelec A/G T/G C/A C/A G/A 1/1 105/154 163/247 99/154 99/154 99/153 1/2 136/202 110/163 150/202 150/202 150/202 2/2 58/80 24/25 50/77 49/77 50/82 Allele 0.654 0.263 0.842 0.896 0.905 Genotype 0.761 0.342 0.848 0.830 0.749 Odds ratiob All 1.05 1.15 1.02 1.01 0.99 SE 0.72 0.92 1.55 1.55 1.49 NO 1.93 1.82 0.76 0.76 0.76 DK 1.08 1.10 0.86 0.85 0.82 Heterogeneityd 0.04* 0.24 0.06 0.06 0.06 *Indicate nominal significance (P < 0.05). a Suicide attempt/No suicide attempt. OR corresponds to allele association. Major/minor allele (1/2). d P-value for test of heterogeneity between samples. b c genotype association between the five genotyped SNPs and presence/absence of suicide attempts in affected individuals (n ¼ 738). The association between TPH1 and suicidal behavior was contradictory in the three samples. That is, four of the five investigated SNPs showed divergent effects between countries of origin (Pheterogeneity 0.06, Table III), and no evidence was found for a general association between any of the SNPs and presence of suicide attempts among affected individuals in the Scandinavian population (P-value for allele and genotype association >0.25, Table III). Nor did haplotype analysis reveal any segregating segment that was consistently associated with suicide attempts among affected individuals. Meta-Analysis of A218C and Schizophrenia Our meta-analysis of seven independent studies [Paik et al., 2000; Hong et al., 2001; Serretti et al., 2001; Sekizawa et al., 2004; Liu et al., 2006; Watanabe et al., 2007; present study], including a total of FIG. 1. Retrospective meta-analysis of the association between the tryptophan hydroxylase 1 (TPH1) A218C allele and schizophrenia. Odds ratios (on a logarithmic scale) are presented for seven sequential analyses of accumulating data. The first point represents the odds ratio from the study of Paik et al.  only, the second point the odds ratio from a meta-analysis of the combined data from Paik et al.  and Hong et al. . The last point (This study) represents the pooled estimate from all included studies. The size of the circle is inversely proportional to the variance of the estimate, and whiskers represent the 95% confidence intervals. 392 AMERICAN JOURNAL OF MEDICAL GENETICS PART B FIG. 2. Forest plot of ln (odds ratio) from the association between the tryptophan hydroxylase 1 (TPH1) A218C/A779C alleles and presence of suicidal attempts among individuals diagnosed with a psychiatric disorder (1, alcohol abuse/dependence; 2, major depression; 3, bipolar affective disorder; *, schizophrenia). The size of the circle is inversely proportional to the variance of the estimate, and whiskers represent the 95% confidence intervals. The summary estimate of all studies is indicated by the diamond to the right. 2,513 cases and 3,093 controls, indicated that the A218 allele is associated with schizophrenia (P ¼ 0.0008) (Table SI). A retrospective analysis of cumulative data show that the odds ratio of the combined data is starting to converge at an estimate of 1.17 (95 CI: 1.07–1.29; Fig. 1), which is similar to the findings of the present study on its own. In all seven individual studies the A allele was more frequent in affected individuals than in controls, and the between study heterogeneity was limited (I2 ¼ 21.0, PQ ¼ 0.27). No evidence of publication bias was evident from the test of funnel plot asymmetry (P ¼ 0.57). Meta-Analysis of A218C/A779C and Suicidal Behavior Among Individuals Affected With a Psychiatric Disorder The meta analysis of 12 studies [Nielsen et al., 1994, 1998; Mann et al., 1997; Bellivier et al., 1998; Tsai et al., 1999; Paik et al., 2000; Hong et al., 2001; Koller et al., 2005; Liu et al., 2006; Viana et al., 2006; Wilson et al., 2009; present study], including 1,272 suicide attempters and 1,727 non suicide attempters diagnosed with a psychiatric disorder, showed no evidence for a consistent association between the A218C/A779C locus and suicidal behavior (P ¼ 0.70, Fig. 2). The patients included in the meta-analysis were diagnosed with schizophrenia (three studies), bipolar disorder (two), major depression (two), alcohol abuse/dependence (three), or borderline personality disorder (one). Two studies included samples of patients from several diagnostic categories (Table SII). The summary odds ratio from all studies was close to one (0.96, 95 CI: 0.80–1.16), and results from individual studies were contradictory with a large heterogeneity in response (I2 ¼ 53.6, PQstudy ¼ 0.01). Although there were differences in the fraction of patients that had attempted suicide between diagnostic groups, there were no significant differences in allele association with suicidal behavior between diagnostic groups (PQdiagnosis ¼ 0.12). In addition, we found no evidence of publication bias in the test of funnel plot asymmetry (P ¼ 0.41). Thus the observed heterogeneity in odds ratio of the association between the A alleles of the A218C/A779C locus and suicidal behavior is likely to reflect random within and between study variation (Pheterogeneity with random model ¼ 0.19). DISCUSSION There was a significant association between TPH1 and schizophrenia in the examined Scandinavian population, and it was the minor alleles of the three tightly linked markers spanning intron six and seven (rs1800532, rs1799913, and rs7933505) that were overrepresented in affected individuals, in both single-marker and haplotype analysis. These results are in agreement with our metaanalysis of previously reported studies (in total 2,513 cases and 3,093 controls), where the A218 (rs1800532 A) allele shows a consistent association with increased susceptibility of schizophrenia. The estimated odds ratio of 1.17 (95 CI 1.03–1.32) from our Scandinavia population is similar to the summary estimate from all published studies (including this). Moreover, as of the two last well-powered studies, the estimated effect of the locus appears to be converging towards an odds ratio close to 1.17. As expected these results are similar to another recent meta-analyses of the A218C polymorphism [Watanabe et al., 2007], and the accumulated evidence strongly suggest that there is an association between TPH1 gene variation and susceptibility of schizophrenia. However no function of the A218C locus, or any other polymorphisms in LD with this SNP, is known. In addition, mouse models give limited clues to a possible link between TPH1 and mental health in humans, as near depletion of 5-HT in the central and/or peripheral nervous system of knockout TPH1 and TPH2 mice, appears to cause only SAETRE ET AL. subtle changes in behavior [Cote et al., 2003; Savelieva et al., 2008]. Thus whereas the epidemiological evidence for TPH1 as a susceptibility gene for schizophrenia appears to be solid, we are still awaiting the identification of a causative mutation and the biological mechanisms by which it affects schizophrenia susceptibility. There was no evidence for a consistent association between TPH1 and suicidal behavior in individuals affected with schizophrenia in the Scandinavian population. Although these results are in agreement with previous studies on suicidal behavior in schizophrenia, which reported no differences in A218C allele frequencies between suicide attempters and non-attempters [Paik et al., 2000; Hong et al., 2001; Liu et al., 2006], the results are at odds with the suggestion that TPH1 polymorphisms affects suicidal behavior independently of mental health status [Courtet et al., 2005]. Over 90% of suicide victims or suicide attempters have been diagnosed with a psychiatric illness, most commonly a mood disorder [Beautrais et al., 1996; Shaffer et al., 1996]. Consequently, association studies contrasting healthy controls with suicide attempters or individuals who died by suicide will almost inevitable confound mental health status and suicidal behavior. To investigate whether the established association between A218C/A779C and suicidal behavior, based on comparisons between patients who performed a suicide attempt or died by suicide and healthy controls [Li and He, 2006], has primarily been driven by a statistical confounding, or whether there are evidence of an independent effect of the locus on suicidal behavior, we searched the literature for studies which had compared allele frequencies in suicidal and non-suicidal patients diagnosed with a psychiatric illness. We identified 12 studies, which had compared the allele frequencies of A218C/A779C in the suicidal attempters and nonattempters diagnosed with a psychiatric disorder. A meta-analysis of in total 3,000 patients provided no evidence for an association between A218C/A779C and suicidal behavior that was independent of mental health status. In fact, the pooled odds ratio was close to 1 and the heterogeneity between studies appeared to reflect random between study variation, rather than a diagnostic dependent association. In the light of these results, the simplest interpretation of the previously reported association between TPH1 and suicidal behavior is that it has been driven by a statistical confounding with poor mental health status. If the allele frequencies are consistently elevated in suicidal individuals suffering from any psychiatric disorder, then this suggests to us that TPH1 is not only a susceptibility gene for schizophrenia, but also for several other psychiatric disorders characterized by elevated suicidal behavior. The concept of shared biological susceptibility to two or more co-morbid disorders is not new [Palomo et al., 2007], and medication that affects 5-HT neurotransmission are used to treat a suit of psychiatric disorders, including major depression, anxiety disorders, and schizophrenia. Reduced cerebrospinal fluid 5-HIAA levels have been reported to be associated not only with suicide attempts but also with susceptibility for psychiatric disorders more generally [J€ onsson et al., 2008b]. Moreover, studies that address the coinheritance of psychiatric disorders have shown shared genetics behind, for example, schizophrenia and bipolar disorder [Yip et al., 2008; Lichtenstein et al., 2009] and between major depression and alcohol dependence [Fu et al., 2002a]. It has also been reported that 393 the heritability of suicidal behavior can be partly attributed to genetic components underlying psychiatric disorders [Fu et al., 2002b]. If TPH1 gene variants are associated with mental disorders characterized by a high suicide rate, then association should occur between the same TPH1 variants and other psychiatric disorders than schizophrenia. The relationship between TPH1 A218C/A779C variation and bipolar disorder, major depression, alcohol dependence and borderline personality disorder has been investigated with various results. However, hitherto only a restricted number of studies have been performed for each of these diagnostic categories, for example, only two studies with in total about 200 patients have assessed borderline personality disorder [Zaboli et al., 2006a; Wilson et al., 2009], making meta-analyses premature given the low odds ratios anticipated. In the future, the correctness of our hypothesis will be possible to test in meta-analyses with larger number of studies and samples. In conclusion, TPH1 is a susceptibility gene for schizophrenia in the Scandinavian population, but it is not associated with suicidal behavior within the patient group. These findings are supported by meta-analyses of previously published reports. The data suggests that the established association between TPH1 and suicidal behavior may primarily be driven by statistical confounding of suicidal behavior and mental health status. Thus, another interpretation of the reported association is that the TPH1 A218C/A779C polymorphisms are associated with increased susceptibility for psychiatric disorders in general, which in turn are characterized by an increased incidence of suicide. ACKNOWLEDGMENTS We thank patients and controls for their participation and express our gratitude towards health professionals who facilitated our work. This study was financed in Denmark by grants to TW from the Copenhagen Hospital Corporation Research Fund, the Danish National Psychiatric Research Foundation, the Danish Agency for Science, and Technology and Innovation (Centre for Pharmacogenetics), in Norway from the Research Council of Norway (147787, 167153), the Eastern Norway Health Authority (Helse Øst RHF 123/2004), Ulleval University Hospital, and University of Oslo, and in Sweden from the Swedish Research Council (K2007-62X -15077-04-1, K2007-62X-15078-04-3, K2008-62P-20597-01-3), the regional agreement on medical training and clinical research between Stockholm County Council and the Karolinska Institutet, Wallenberg Foundation, and the HUBIN project. We thank Alexandra Tylec, Agneta Gunnar, Monica Hellberg, and Kjerstin Lind for technical assistance. We also thank Kristina Larsson, Tomas Axelsson and Ann-Christine Syv€anen at the SNP Technology Platform in Uppsala for performing the genotyping. 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