Original article 151 Case–control and linkage disequilibrium studies of the tryptophan hydroxylase gene polymorphisms and major depressive disorder Ene-Choo Tana, Angelina O. M. Chanb, Chay-Hoon Tanc, Rathi Mahendranb, Adrian Wangb and Hong-Choon Chuab Objectives Alterations in the level of the serotonin, serotonin uptake and the number of binding sites have been linked to affective illness. We investigated the association of tryptophan hydroxylase gene polymorphisms and unipolar depression in a case–control study design. search for the causative variant directly involved in the susceptibility to unipolar depression in Chinese as this polymorphism within the intron might not be the true susceptibility variant. Psychiatr Genet 13:151–154 c 2003 Lippincott Williams & Wilkins. Methods Patients and ethnically matched controls were genotyped for three polymorphisms of the tryptophan hydroxylase gene. Psychiatric Genetics 2003, 13:151–154 Results Significant difference in genotype frequency between patient and control groups was observed for the IVS7 + 218A > C polymorphism but not for the two promoter polymorphisms – 1067G > A and – 347T > G. Strong linkage disequilibrium among the three polymorphisms was also observed. Conclusions As the sample size was small, the positive association would need to be replicated by family-based association studies or in a larger set of samples. As our results did not indicate association with either of the two promoter polymorphisms, there is a need to continue the Introduction Tryptophan hydroxylase (EC 1.14.16.4) is the ratelimiting enzyme in the biosynthesis of serotonin, which has been implicated in the pathogenesis of depression and a number of psychiatric disorders. The gene is therefore a strong candidate for the genetic contribution to depression. Results from investigations on two intron 7 polymorphisms, IVS7 + 218A > C and IVS7 + 779C > A, have suggested that tryptophan hydroxylase might be involved in suicidality (Nielsen et al., 1994, 1998), alcoholism (Nielsen et al., 1998), bipolar disorder (Bellivier et al., 1998), violence, impulsivity and aggression (New et al., 1998; Manuck et al., 1999), smoking initiation (Sullivan et al., 2001), and the control of anxiety (Du et al., 2001). Although there were also negative findings, they could be due to insufficient power, sample size limitation, population stratification and the complexity of the phenotypes involved that could have multiple aetiological factors. It must be noted that, unlike chance findings, the 0955-8829 c 2003 Lippincott Williams & Wilkins Keywords: serotonin transporter, polymorphism, linkage disequilibrium, unipolar depression, Chinese a Defence Medical Research Institute, Defence Science and Technology Agency, Republic of Singapore, bInstitute of Mental Health and Woodbridge Hospital, Republic of Singapore and cDepartment of Pharmacology, National University of Singapore, Republic of Singapore. Sponsorship: This study was funded by grant number 0286/1998 to A.O.M.C. from the National Medical Research Council. Correspondence to Ene-choo Tan, Defence Medical Research Institute, Defence Science and Technology Agency, Clinical Research Centre #04-07, 10 Medical Drive, Singapore 117597, Singapore. Tel: + 65 67794726; fax: + 65 67791677; e-mail: nmiv27@nus.edu.sg Received 23 May 2002 Accepted 25 September 2002 association was replicated in a significant number of studies, such as for completed suicide (Roy et al., 2001; Zalsman et al., 2001), violent suicide (Abbar et al., 2001), suicidal behaviour in major depression (Mann et al., 1997; Souery et al., 2001), schizophrenia (Paik et al., 2000), and bipolar disorder without co-morbid panic disorder (Rotondo et al., 2002). There was also a number of studies reporting association approaching statistical significance, such as with depressive symptoms (Serretti et al., 2001), violent suicide (Turecki et al., 2001) and suicidal attempts (Geijer et al., 2000). In previous studies with Han Chinese, positive association was found between IVS7 + 218A > C and major depression, suicidal behaviour in depressed patients (Tsai et al., 1999) and schizophrenia (Hong et al., 2001). In the present study, we tested the hypothesis that this intron 7 polymorphism and two other polymorphisms of the tryptophan hydroxylase gene might be associated with major depressive disorder in Chinese patients. The allele and genotype frequencies and the pattern of DOI: 10.1097/01.ypg.0000066960.66429.db Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 152 Psychiatric Genetics 2003, Vol 13 No 3 linkage disequilibrium were investigated in a collection of major depressive patients and healthy controls of Chinese ancestry. Materials and methods Table 1 Demographic data on subjects Males Females Mean age ± standard deviation (years) Psychiatric diagnosis Controls Patients 56 83 44.99 ± 11.52 Unipolar depression 33 58 43.55 ± 16.02 None Subjects Study subjects were 91 patients (33 males and 58 females) with unipolar depression and 139 controls (56 males and 83 females) without any history of mental disorder. Unrelated individuals with unipolar depression were recruited from the outpatient clinics of Woodbridge Hospital. All subjects were classified as having major depressive disorder according to DSM-IV diagnostic criteria, and patients with other co-existing psychiatric diagnoses were excluded. All subjects were residents of Singapore, and most were second-generation or thirdgeneration Chinese Singaporeans descended from the Han. The majority originated from the Southern provinces of Fujian and Guangdong of China. Ethnically matched controls that did not manifest psychiatric symptoms and had no history of mental illness were recruited from staff of the hospital. All gave informed consent for the study. DNA extraction and genotyping Peripheral blood was collected in ethylenediamine tetraacetic acid tubes. DNA was then extracted using the QIAamp Blood Kit according to the manufacturer’s instructions (Qiagen GmbH, Hilden, Germany). Genotyping for the IVS7 + 218A > C polymorphism was carried out by polymerase chain reaction according to Nielsen et al. (1997). Genotyping of the two promoter polymorphisms ( – 1067G > A and – 347T > G) was carried out according to Paoloni-Giacobino et al. (2000). Amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Statistical analysis Mean ages for the two groups were compared using Levene’s Test for Equality of Variances. Gender distribution, genotype and allele frequencies were compared between patients and controls using the chi-square test. Genotypes of all groups were also checked for conformation to Hardy–Weinberg equilibrium. All tests were performed using SPSS for Windows (Statistical Package for the Social Sciences, version 10.0 SPSS Inc. Chicago, USA). Haplotype frequencies and linkage disequilibrium were estimated using the EH program (http:// linkage.rockefeller.edu/ott/eh.htm). For measurement of linkage disequilibrium, D0 between markers was calculated according to Reich et al. (2001). from the same ethnic group and did not differ significantly in mean age at 43.5497 years [standard deviation (SD) = 16.0257] for the control group and 44.9885 years (SD = 11.5199) for the patients. There was also no significant difference in gender distribution between the two groups (w2 = 0.354, degrees of freedom = 1, P = 0.588). Genotype and allele frequencies of the three polymorphisms for both patients and controls are summarized in Table 2. There was no deviation from Hardy–Weinberg equilibrium for the distribution of all three polymorphisms for both patient and control groups. For the – 1067G > A promoter polymorphism, distribution of the two alleles and three genotypes were very similar between the patient and control groups. The major allele was the A–1067 allele for both control and patient groups, with the frequencies at 0.764 and 0.725, respectively. For the other promoter polymorphism at – 347, allele frequencies for the more common (G–347) allele were 0.781 for the control group and 0.738 for the patient group. No statistically significant difference in either allelic or genotypic distributions was found between the two groups for either of these two promoter polymorphisms. For the IVS7 + 218A > C polymorphism, there was significant difference in genotype distribution between control and patient groups (w2 = 6.915, degrees of freedom = 2, P = 0.032). However, difference in allele frequencies did not reach statistical significance. Estimated haplotype frequencies are presented in Table 3. Overall frequency comparisons did not reveal any statistically significant difference between cases and controls at the haplotype level. However, linkage disequilibrium between the two promoter polymorphisms was almost complete with the D0 value at 0.865. There was also strong linkage disequilibrium between the IVS7 + 218A > C polymorphism and the two promoter polymorphisms (IVS7 + 218A > C and –1067G > A, D0 = 0.754; IVS7 218A > C and –347T > G, D0 = 0.702). Discussion Results The demographic and clinical data for the subjects are summarized in Table 1. All patients and controls were In the present study, we found a difference in the distribution of the three genotypes for the IVS7 + 218A > C polymorphism for patients with unipolar Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Case–control and linkage disequilibrium studies Tan et al. 153 Comparisons of genotype and allele frequencies of the three polymorphisms in the TPH gene in controls and patients with unipolar depression Table 2 Genotype frequency G–1067A Controls (n = 123) Patients (n = 91) T–347G Controls (n = 135) Patients (n = 82) 218A > C Controls (n = 138) Patients (n = 85) Table 3 G/A 44 (35.8%) 34 (37.4%) T/G 49 (36.3%) 27 (32.9%) A/C 62 (44.9%) 53 (62.4%) Estimated haplotype frequencies for cases and controls Haplotypes – 1067G – 1067G – 1067G – 1067G – 1067A – 1067A – 1067A – 1067A G/G 7 (5.7%) 8 (8.8%) T/T 5 (3.7%) 8 (9.8%) A/A 37 (26.8%) 13 (15.3%) – 347 T 218A – 347 T 218C – 347G 218A – 347G 218C – 347 T 218A – 347 T 218C – 347G 218A – 347G 218C Cases Controls 0.193304 0.012100 0.020878 0.007051 0.018504 0.036092 0.293981 0.418090 0.193419 0.018567 0.018643 0.005482 0.038012 0.000002 0.259185 0.466690 major depression compared with healthy controls. The frequency of the A-containing genotypes (AA and AC) is much lower for patients. This is consistent with a reported finding that the A-containing variants may confer protection against depressive symptoms for males with mood disorders (Serretti et al., 2001). A previous study in another Chinese population also found an association between this polymorphism and unipolar depression (Tsai et al., 1999). However, the association was with the A allele for both unipolar depression and suicidal behaviour. As studies in several different populations have found positive association but with different alleles of this polymorphism, it is probable that it is not the true susceptibility locus, but merely in linkage disequilibrium with the actual causative allele. Linkage disequilibrium among three promoter polymorphisms for the gene (–1067G > A, –347T > G, and an additional marker further upstream at –1606T > C not included in this study) was reported to be complete in Western Europeans (Paoloni-Giacobino et al., 2000). We found the linkage disequilibrium to be present, but incomplete with D0 at 0.865. Allele frequencies were also less equitable, with A–1067 and G–347 being the more common variants at frequencies of 0.764 and 0.781, respectively, in the control population. A study on four polymorphisms further upstream found all to be in complete linkage disequilibrium in the Finns and almost complete in Italians and American Caucasians. Significant linkage disequilibrium was also observed between one of the polymorphisms ( – 6526A > G) and the + 779C > A polymorphism within intron 7 in Finns and Italians but not Southwestern American Indians (Rotondo et al., 1999). The availability of allele frequencies and linkage Allele frequency A/A 72 (58.5%) 49 (53.8%) G/G 81 (60.0%) 47 (57.3%) C/C 39 (28.3%) 19 (22.4%) G 0.236 0.275 T 0.219 0.262 A 0.493 0.465 A 0.764 0.725 G 0.781 0.738 C 0.507 0.535 disequilibrium measures for variants within this gene will facilitate identification of the causative variant and role of the gene in psychiatric disorders. The results of the present study show an association between major depression (with or without suicidal behaviour) and the IVS7 + 218A > C polymorphism of the tryptophan hydroxylase gene. This polymorphism is in complete linkage disequilibrium with the 779A > C polymorphism within the same intron, but neither of them results in a change in amino acid sequence or has been shown to affect transcription or splicing (Nielsen et al., 1997). In addition, although there was significant linkage disequilibrium between all three polymorphisms investigated in this study, the two promoter polymorphisms were not associated with the diagnosis, indicating that neither of them was the causative variant. Although results from this study and that of Tsai et al. (1999) found the IVS7 + 218A > C polymorphism to be associated with unipolar depression, the at-risk allele identified was different. It is thus probable that another polymorphism in the TPH gene is directly involved. Alternatively, the causative polymorphism could even be that from another gene that is in linkage disequilibrium with the 218A > C polymorphism. Acknowledgements The authors thank Catherine Hong, Joshua Goh and Geraldine Toh for technical assistance, and Fang Dong for statistical analysis. References Abbar M, Courtet P, Bellivier F, Leboyer M, Boulenger JP, Castelhau D, et al. (2001). Suicide attempts and the tryptophan hydroxylase gene. Mol Psychiatry 6:268–273. 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