摘要
　　血清素是一種中樞神經系統重要的神經傳遞物質。在生理上，它參與如情緒，睡眠，食慾，性慾等功能的調節。因此，當中樞神經的血清素系統異常時，可能會導致精神疾病的發生。在血清素的生物合成中，色胺酸氫氧化脢是此合成路徑的速率限制脢。因此，在許多精神疾病的致病基因定位的研究上，人類色胺酸氫氧化脢的基因自然成為重要的研究主題。目前已知的人類色胺酸氫氧化脢基因有兩個，一個是主要表現在周邊組織的人類第一型色胺酸氫氧化脢基因；另一個是最近才被發現 主要表現在中樞神經的人類第二型色胺酸氫氧化脢基因。先前已經有研究發現人類第二型色胺酸氫氧化脢基因與憂鬱症、自殺與抗憂鬱症藥物的反應之間有關聯性。本實驗的目的就是以新發現的人類第二型色胺酸氫氧化脢基因為候選基因，在台灣地區的不同族群中做遺傳分析，來了解此基因在血清素相關的精神疾病的病源學上所扮演的角色。研究可分為兩部分，第一部分是尋找此基因的所有表現序列、5端啟動子、3端不轉錄區域上的變異。再利用其中五個單一核甘酸多型性標竿，來建立台灣漢族與三個原住民族群中基因型與對偶基因頻率的分佈情形。此一資料可作為以後不同族群的精神疾病的病源學探討基礎。我們已經發現不論是對偶基因或基因型的頻率，在不同台灣族群間的分佈有差異，且具有統計上的顯著。代表台灣族群之間演化上來源的不同。本研究的第二部分利用實驗組-對照組研究模式來分析在台灣漢族群中，人類第二型色胺酸氫氧化脢基因與躁鬱症之關聯性。我們已經找到八個單一核甘酸多型性，其中單一核甘酸多型性 C2755A，對偶基因的頻率，在實驗組與對照組中的分佈情形有差異，具有統計上的顯著。代表人類第二型色胺酸氫氧化脢基因與躁鬱症之間存在有關聯性。進一步地，包含此單一核甘酸多型性單套體的頻率也在實驗組與對照組中的分佈情形有差異，具有統計上的顯著，提供更多人類第二型色胺酸氫氧化脢基因與躁鬱症之間存在有關聯性的証據。除此外，我們針對啟動子單一核甘酸多型性T-473A，利用報導基因系統進行基因表現的研究，我們的結果證實位於啟動子區域的單一核甘酸多型性具有調節人類第二型色胺酸氫氧化脢基因表現的功能。

ABSTRACT
　Serotonin is one of the monoamine neurotransmitters and the expression of serotonin in central nervous system (CNS) plays a role in the regulation of emotion, sleep, appetite and sexual activity. The TPH gene encodes the rate-limiting enzyme of serotonin biosynthesis and is the candidate gene for the etiology of bipolar disease (BPD). Previous association studies of TPH1 and BPD revealed inconclusive results. The second TPH isoform (TPH2, also known as neuronal TPH) was identified recently and showed predominantly expressed in the CNS. This discovery provides another window to examine the role of TPH gene in serotonin-related psychiatric disorders. The TPH2 gene contains 11 exons that span about 93.5 kb on chromosome 12q15. In addition to some studies have documented the expression pattern of this gene, limited information regarding the TPH2 gene and its effects on psychiatric disorders has been reported. This study aims to search the sequence variants of TPH2 locus and to study the possible association of this gene with BPD in Taiwan. Furthermore, we want to study allelic distribution of TPH2 polymorphisms in three different aboriginal populations. We have completed polymorphism screening of sequences in regulatory and exon regions. Eight single nucleotide polymorphisms (SNPs) were identified that include two in the promoter region, five in exons and one in intron regions. Five selected SNPs have been genotyped using high throughput genotyping system in normal controls, BPD patients and three aboriginal populations. The genotype data of these SNPs from different populations are collected and have been statistically analyzed. The allele frequency and genotype frequency of four SNPs in TPH2 gene are significantly different between four Taiwanese populations that showed the evolutional origin is different among the four populations. In addition, the allele frequency and haplotype frequency of SNP C2755A in TPH2 gene are significantly different between BPD patient and normal control populations indicated a strong association with BPD. Luciferase reporter assays were performed to check the gene expression regulation by different allele of the promoter SNPs. Results from this study has provided the evidences that the polymorphism on promoter region can affect the expression of TPH2 gene. Besides, the document of TPH2 gene polymorphisms in Taiwan populations will help the future association study for another trait related to serotonin system in CNS.

Reference
Afonina, I., M. Zivarts, I. Kutyavin, E. Lukhtanov, H. Gamper, and R.B. Meyer. 1997. Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res 25: 2657-60.
Azmitia, E.C. 1986. Re-engineering the brain serotonin system:localized application of neurotoxins and fetal neurons. Adv. Neurol. 43: 493-507.
Bellivier F, L.M. 1998. Association between the tryptophan hydroxylase gene and manic-depressive illness. Arch Gen Psychiatry 55: 33-37.
Bevan, D. 2004. Genes, race and drugs. Clin Invest Med 27: 5-6.
Bloche, M.G. 2004. Race-based therapeutics. N Engl J Med 351: 2035-7.
Boadle-Biber, M.C. 1980. Activation of tryptophan hydroxylase from slices of rat brain stem incubates with N6, 02'-dibutyryl adenosine-3':5'-cyclic monophosphate. Biochem Pharmacol 29: 669-72.
Breidenthal, S.E., D.J. White, and C.E. Glatt. 2004. Identification of genetic variants in the neuronal form of tryptophan hydroxylase (TPH2). Psychiatr Genet 14: 69-72.
Carlson, N.R. 2001. Physiology of behavior (7th ed.). Allyn and Bacon, London.
Cheng, T.A. 1995. Mentle illness and suicide. Archives of general psychiatry 52: 594-603.
Cheng, T.A. and M. Hsu. 1993. Sex differences in minor psychiatric morbidity among three aboriginal groups in Taiwan: the effects of lineage. Psychol Med 23: 949-56.
Coon, H., D. Dunn, J. Lainhart, J. Miller, C. Hamil, A. Battaglia, R. Tancredi, M.F. Leppert, R. Weiss, and W. McMahon. 2005. Possible association between autism and variants in the brain-expressed tryptophan hydroxylase gene (TPH2). Am J Med Genet B Neuropsychiatr Genet.
Coppen, A. 1967. The biochemistry of affective disorders. Br J Psychiatry 113: 1237-1264.
Curzon, G. 1974. Availability of tryptophan to the brain and some hormonal and drug influences on it. Adv Biochem Psychopharmacol 10: 263-71.
De Luca, V., D.J. Mueller, S. Tharmalingam, N. King, and J.L. Kennedy. 2004. Analysis of the novel TPH2 gene in bipolar disorder and suicidality. Mol Psychiatry 9: 896-7.
Deakin, J.F. 1994. Strategies for studying brain disorders.
Devlin, B. and N. Risch. 1995. A comparison of linkage disequilibrium measures for fine-scale mapping. Genomics 29: 311-22.
Fallin, D., A. Cohen, L. Essioux, I. Chumakov, M. Blumenfeld, D. Cohen, and N.J. Schork. 2001. Genetic analysis of case/control data using estimated haplotype frequencies: application to APOE locus variation and Alzheimer's disease. Genome Res 11: 143-51.
Fallin, D. and N.J. Schork. 2000. Accuracy of haplotype frequency estimation for biallelic loci, via the expectation-maximization algorithm for unphased diploid genotype data. Am J Hum Genet 67: 947-59.
Feighner, J.P. 1999. Overview of antidepressants currently used to treat anxiety disorders. J Clin Psychiatry 60 Suppl 22: 18-22.
Fils-Aime ML, E.M., George DT. 1996. Early-onset alcoholics have lower cerebrospinal fluid 5-hydroxyindoleacetic acid levels than late-onset alcoholics. Arch Gen Psychiatry 53: 211-216.
Graeff, F.G. 1993a. The elevated T-maze, a new experimental model of anxiety and memory: Effect of diazepam. Braz J Med Biol Res 26: 67-70.
-. 1993b. Role of 5-HT in defensive behavior and anxiety. Reviews in the neurosciences 4: 181-211.
-. 1996. Role of 5-HT in stress, anxiety, and depression. Pharmacology, biochemistry, and behavior. 54: 129-141.
Harvey, M., E. Shink, M. Tremblay, B. Gagne, C. Raymond, M. Labbe, D.J. Walther, M. Bader, and N. Barden. 2004. Support for the involvement of TPH2 gene in affective disorders. Mol Psychiatry 9: 980-1.
Holland, P.M., R.D. Abramson, R. Watson, and D.H. Gelfand. 1991. Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88: 7276-80.
Hu, X., G. Oroszi, J. Chun, T.L. Smith, D. Goldman, and M.A. Schuckit. 2005. An expanded evaluation of the relationship of four alleles to the level of response to alcohol and the alcoholism risk. Alcohol Clin Exp Res 29: 8-16.
Jiang, G.C., G.J.t. Yohrling, J.D. Schmitt, and K.E. Vrana. 2000. Identification of substrate orienting and phosphorylation sites within tryptophan hydroxylase using homology-based molecular modeling. J Mol Biol 302: 1005-17.
Johansen, P.A., I. Jennings, R.G. Cotton, and D.M. Kuhn. 1996. Phosphorylation and activation of tryptophan hydroxylase by exogenous protein kinase A. J Neurochem 66: 817-23.
Kandel, E.R. 2000. Principles of neural science (4th ed.). New York, NY; London: McGraw-Hill.
Ko, T.M., L.H. Tseng, T.A. Cheng, H.L. Hwa, Y.K. Chang, S.M. Chuang, and T.Y. Lee. 1994. Prevalence and molecular studies of thalassemia in five aboriginal groups in Taiwan. J Formos Med Assoc 93: 379-82.
Kuhn, D.M., R.J. Arthur, and S. JC. 1997. Phosphorylation and activation of brain tryptophan hydroxylase: identification of serine-58 as a substrate site for protein kinase A. A. J Neurochem 69: 1738-45.
Kumer, S.C., S.M. Mockus, P.J. Rucker, and K.E. Vrana. 1997. Amino-terminal analysis of tryptophan hydroxylase: protein kinase phosphorylation occurs at serine-58. J Neurochem 69: 1738-45.
Lai, T.J., C.Y. Wu, H.W. Tsai, Y.M. Lin, and H.S. Sun. 2005. Polymorphism screening and haplotype analysis of the tryptophan hydroxylase gene (TPH1) and association with bipolar affective disorder in Taiwan. BMC Med Genet 6: 14.
Lee, K.C. 2003. The study of alcoholism and serotnin transporter gene in
Hsin-Yi township. In Graduate institute of environmental medicine, public health college, pp. 51. China medical university, Taichung.
Lesch, K.P. 1996. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 247: 1527-1531.
Lewontin, R.C. 1964. The Interaction of Selection and Linkage. Ii. Optimum Models. Genetics 50: 757-82.
Lin, L.I., Wei, C. C., Chiang, F. T. 1996. Population Genetics Analysis of the VNTR Loci in 8 Taiwan Populations. In the Symposium on Cultural as well as Biological Affinities among the Indigenous Peoples of Taiwan and Southeast Asia. Academia Sinica, Taipei.
Lin, M., C.C. Chu, H.L. Lee, S.L. Chang, J. Ohashi, K. Tokunaga, T. Akaza, and T. Juji. 2000. Heterogeneity of Taiwan's indigenous population: possible relation to prehistoric Mongoloid dispersals. Tissue Antigens 55: 1-9.
Lin, Y.M., H.C. Yang, T.J. Lai, C.S. Fann, and H.S. Sun. 2003. Receptor mediated effect of serotonergic transmission in patients with bipolar affective disorder. J Med Genet 40: 781-6.
Livak, K.J. 1999. Allelic discrimination using fluorogenic probes and the 5' nuclease assay. Genet Anal 14: 143-9.
Livak, K.J., S.J. Flood, J. Marmaro, W. Giusti, and K. Deetz. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4: 357-62.
Mamedov, Z.G., G.G. Gasanov, and T.T. Gulieva. 1992. [Serotonin-induced changes in the responses of electrically stimulated neuronal membrane of the mollusk]. Neirofiziologiia 24: 286-90.
Martinez, A., P.M. Knappskog, and J. Haavik. 2001. A structural approach into human tryptophan hydroxylase and its implications for the regulation of serotonin biosynthesis. Curr Med Chem 8: 1077-91.
Matsumoto, H. 1989. Characteristics of Mongoloid populations and immunogenetics of various diseases based on the genetic markers of human immunoglobulins. Exp Clin Immunogenet 6: 68-87.
MC, B.-B. 1980. Activation of tryptophan hydroxylase from slices of rat brain stem incubates with N6, 02'-dibutyryl adenosine-3':5'-cyclic monophosphate. Biochem Pharmacol 29: 669-72.
McKinney, J., P.M. Knappskog, and J. Haavik. 2005. Different properties of the central and peripheral forms of human tryptophan hydroxylase. J Neurochem 92: 311-20.
McKinney, J., K. Teigen, N.A. Froystein, C. Salaun, P.M. Knappskog, J. Haavik, and A. Martinez. 2001. Conformation of the substrate and pterin cofactor bound to human tryptophan hydroxylase. Important role of Phe313 in substrate specificity. Biochemistry 40: 15591-601.
Melton, T., S. Clifford, J. Martinson, M. Batzer, and M. Stoneking. 1998. Genetic evidence for the proto-Austronesian homeland in Asia: mtDNA and nuclear DNA variation in Taiwanese aboriginal tribes. Am J Hum Genet 63: 1807-23.
MuGufin, P. 1988. Major genes for major affective disorder. British J Psychiatry 153: 591-596.
Nielsen, D.A., M. Dean, and D. Goldman. 1992. Genetic mapping of the human tryptophan hydroxylase gene on chromosome, using an intronic conformational polymorphism. Am J Hum Genet 51: 1366-1371.
Nielsen, D.A., Jenkins GL, and S. KM. 1997. Sequence, splice site and population frequency distribution analyses of the polymorphic human tryptophan hydroxylase intron 7. Am J Hum Genet 45: 145-148.
Nutt, D. 1990. The neurochemistry of anxiety: an update. Progress Neuro-Psychopharma&Bio Psychiatry 14: 737-752.
Ohara, K., Nagai, M., Tani, K. 1999. Schizophrenia and the serotonin-2A receptor promoter polymorphism. Psychiatry Res 85: 221-224.
Roy, A. 1988. Acting out hostility in normal volunteers: negative correlation with levels of 5HIAA in cerebrospinal fluid. Psychiatry Res 24: 187-194.
-. 1989. Cerebrospinal fluid monoamine metabolites and suicidal behavior in depressed patients. A 5-year follow-up study. Arch Gen Psychiatry 46: 609-612.
Schanfield, M.S., K. Ohkura, M. Lin, R. Shyu, and H. Gershowitz. 2002. Immunoglobulin allotypes among Taiwan aborigines: evidence of malarial selection could affect studies of population affinity. Hum Biol 74: 363-79.
Sekar, V. 1987. A rapid screening procedure for the identification of recombinant bacterial clones. Biotechniques 5: 11-13.
Shaw, C.K., L.L. Chen, A. Lee, and T.D. Lee. 1999. Distribution of HLA gene and haplotype frequencies in Taiwan: a comparative study among Min-nan, Hakka, Aborigines and Mainland Chinese. Tissue Antigens 53: 51-64.
Sun, H.S., C.S. Fann, H.Y. Lane, Y.T. Chang, C.J. Chang, Y.L. Liu, and A.T. Cheng. 2005. A functional polymorphism in the promoter region of the tryptophan hydroxylase gene is associated with alcohol dependence in one aboriginal group in Taiwan. Alcohol Clin Exp Res 29: 1-7.
Sun, H.S., H.W. Tsai, H.C. Ko, F.M. Chang, and T.L. Yeh. 2004a. Association of tryptophan hydroxylase gene polymorphism with depression, anxiety and comorbid depression and anxiety in a population-based sample of postpartum Taiwanese women. Genes Brain Behav 3: 328-36.
Sun, H.S., H.C. Wang, T.J. Lai, T.J. Wang, and C.M. Li. 2004b. Sequence variants and haplotype analysis of serotonin transporter gene and association with bipolar affective disorder in Taiwan. Pharmacogenetics 14: 173-9.
Tajima, A., C.S. Sun, I.H. Pan, T. Ishida, N. Saitou, and S. Horai. 2003. Mitochondrial DNA polymorphisms in nine aboriginal groups of Taiwan: implications for the population history of aboriginal Taiwanese. Hum Genet 113: 24-33.
Tang, T.K., W.Y. Huang, C.J. Tang, M. Hsu, T.A. Cheng, and K.H. Chen. 1995. Molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in three Taiwan aboriginal tribes. Hum Genet 95: 630-2.
Walther, D.J. and M. Bader. 2003. A unique central tryptophan hydroxylase isoform. Biochem Pharmacol 66: 1673-80.
Walther, D.J., J.U. Peter, S. Bashammakh, H. Hortnagl, M. Voits, H. Fink, and M. Bader. 2003. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299: 76.
Yamauchi T, Nakata H, and F. H. 1981. A new activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+/calmodulin-dependent protein kinase. Purification and characterization. J Biol Chem 256: 5404-9.
Yohrling, G.J.t., G.C. Jiang, S.M. Mockus, and K.E. Vrana. 2000. Intersubunit binding domains within tyrosine hydroxylase and tryptophan hydroxylase. J Neurosci Res 61: 313-20.
Zhang, X., J.M. Beaulieu, T.D. Sotnikova, R.R. Gainetdinov, and M.G. Caron. 2004. Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305: 217.
Zhang, X., R.R. Gainetdinov, J.M. Beaulieu, T.D. Sotnikova, L.H. Burch, R.B. Williams, D.A. Schwartz, K.R. Krishnan, and M.G. Caron. 2005. Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45: 11-6.
Zill, P., T.C. Baghai, P. Zwanzger, C. Schule, D. Eser, R. Rupprecht, H.J. Moller, B. Bondy, and M. Ackenheil. 2004a. SNP and haplotype analysis of a novel tryptophan hydroxylase isoform (TPH2) gene provide evidence for association with major depression. Mol Psychiatry 9: 1030-6.
Zill, P., A. Buttner, W. Eisenmenger, H.J. Moller, B. Bondy, and M. Ackenheil. 2004b. Single nucleotide polymorphism and haplotype analysis of a novel tryptophan hydroxylase isoform (TPH2) gene in suicide victims. Biol Psychiatry 56: 581-6.