臺灣博碩士論文加值系統

精神分裂症 (schizophrenia)是一種嚴重、慢性的精神疾病。 在家族、雙胞胎及領養的研究指出精神分裂症是一種複雜性疾病並有很高的遺傳因素。 許多分子遺傳研究指出染色體22號長臂區域對精神分裂症來說是的熱門的區域，許多位置在染色體二十二號長臂 (chromosome 22q)的基因已被研究是否跟精神分裂症有相關，例如COMT，ARVCF，和 SYN3等等，但是到底是哪些基因會造成精神分裂症或者跟這疾病有相關性，至今並沒有結論性的答案。 MAPK1基因在22號染色體長臂11.2位置上，其轉譯出來的蛋白質參與真核生物細胞訊息傳遞並可啟動或調節基因的表現，另外神經元中的MEK1/ERK1/2 訊息傳遞路徑在學習與記憶的過程中也扮演重要的角色。 SHANK3基因位在22號染色體長臂13.3上，SHANK3蛋白在突觸後神經元細胞中是主要的鷹架蛋白，可以和突觸後神經元膜上的NMDA (N-Methyl-D-Aspartic acid) receptor、 glutamate receptor交互作用或者跟細胞骨架連結。 SHNAK3基因的缺失與22q13缺失症候群等神經性病變有極大的關聯。 本實驗將MAPK1和SHANK3基因當做精神分裂症的位置候選基因(positional candidate gene)，實驗的目的是想要找出精神分裂症患者在這兩個基因上是否有變異的產生，並利用病例對照分析來研究MAPK1和SHANK3基因是否是造成精神分裂症的相關基因之一。 本實驗初期系統性的篩檢100個精神分裂症患者其MAPK1和SHANK3基因的變異，分別在MAPK1上找到4個變異，在SHANK3找到14個變異。 並挑選位在SHANK3基因上的3個變異點，分別是c.961 T>C、 c.1250 G>A、 c.4721 G>A， 這三個分子變異皆會造成胺基酸的改變，是一種missense mutation; 其分別造成SHANK3蛋白質第321位置上的Serine被Proline取代 (S321P)、 第417位置上的Arginine被Glutamine取代 (R417Q)、 第1574位置上的Glycine被Aspartate取代 (G1574D)。 更進一步增加實驗組 (450名)與對照組 (434名)的樣本數目來進行genotyping。 病例對照相關研究的結果顯示，在SHANK3的S321P、R417Q、G1574D這三個變異點上，精神分裂症患者與對照組之間其基因型頻率或對偶基因頻率並沒有任何差異。 但這三個變異點 (S321P, R417Q, G1574D)是種missense mutation，是否會造成SHANK3蛋白質表現量或功能上的異常，則可利用functional assay進一步的證實。 目前本實驗的結果顯示SHANK3基因與精神分裂症沒有關聯。 儘管如此，本實驗所發現的分子變異點或許將有助於SHANK3基因結構功能或基因調控的研究，並增加對神經精神疾病的更深層認知。

Schizophrenia is a severe, chronic mental illness. Family, twin and adoption studies have demonstrated that schizophrenia is a complex disease with a high genetic component in its etiology. Many molecular genetic studies have demonstrated that the chromosome 22q is a hot region for schizophrenia. Several genes in 22q have been studied, such as COMT, ARVCF and SYN3 etc, but no conclusive gene for schizophrenia was identified. MAPK1 locates on the chromosome 22q11.2 and encodes a protein involved in the RAF/MAK/ERK pathway and regulates gene expressions. This RAF/MAK/ERK pathway within neuron could play an important role in the processes of learning and memory. SHANK3 locates on the chromosome 22q 13.3 and translates the scaffold protein in the postsynaptic neuron. SHANK3 protein is a multi-domain protein that can interact with receptors of the postsynaptic membrane including NMDA and glutamate receptors, and the actin-based cytoskeleton. SHANK3 gene could be a causative factor for 22q13 deletion syndrome with neurological defects. The purpose of this study is to detect whether genetic mutations of MAPK1 and SHANK3 have association with schizophrenia. We first systemically identify for mutations in the MAPK1 and SHANK3 gene in 100 schizophrenia patients from Taiwan. Four molecular variants were detected in MAPK1; 14 molecular variants were detected in SHANK3. We focus on the three variants in SHANK3, including a T to C substitution in exon 6 (c.961 T>C) and two G to A substitutions in exon 9 (c.1250 G>A) and exon 22 (c.4721 G>A), because the three variants can cause the amino acid change (S321P, R417Q and G1574D, respectively). Further increase sample size and genotyping. Case-control association study in 450 patients and 434 controls shows that there is no association between these variants (S321P, R417Q and G1574D) with schizophrenia. So far, our result suggests that MAPK1 and SHANK3 genes could not be the major susceptibility genes for schizophrenia. Nevertheless, the identification of these molecular variants will contribute to the study of gene regulation and function of MAPK1 and SHANK3, and a deeper understanding of the neuropsychiatric disorder.

目 錄
誌 謝………………………………………………………1
中文摘要………………………………………………………2
Abstract………………………………………………………4
緒 言……………………………………………………6
實驗目的………………………………………………13
材料與方法…………………………………………………14
結 果…………………………………………28
討 論……………………………………………………42
附 錄……………………………………………………45
參考文獻……………………………………………………49

1. Freedman R. 2003. Schizophrenia. N. Engl. J. Med. 349: 1738-1749.
2. Craddock N, O'Donovan MC, Owen MJ. 2005. The genetics of schizophrenia and bipolar disorder: dissecting psychosis. J. Med. Genet. 42: 193-204.
3. 孔繁鐘編譯。1997。DSM-IV精神疾病的診斷與統計。合計圖書出版社。
4. Park HJ, Levitt J, Shenton ME, Salisbury DF, Kubicki M, Kikinis R, Jolesz FA, McCarley RW. 2004. An MRI study of spatial probability brain map differences between first-episode schizophrenia and normal controls. Neuroimage 22:1231-1246.
5.Rapoport JL, Addington AM, Frangou S, Psych MR. 2005. The neurodevelopmental model of schizophrenia: update 2005. Mol. Psychiatry 10: 434-449.
6.Kirov G, O'Donovan MC, Owen MJ. 2005. Finding schizophrenia genes. J. Clin. Invest. 115: 1440-1448.
7.Bassett AS, Chow EW, Waterworth DM, Brzustowicz L. 2001. Genetic insights into schizophrenia. Can. J. Psychiatry 46: 131-7.
8.Badner JA, Gershon ES. 2002. Meta-analysis of whole-genome linkage scans of bipolar disorder and schizophrenia. Mol. Psychiatry 7: 405-411.
9.Kohn Y, Lerer B. 2002. Genetics of schizophrenia: a review of linkage findings. Isr. J. Psychiatry Relat. Sci. 39: 340-351.
10. McGuffin P, Tandon K, Corsico A. 2003. Linkage and association studies of schizophrenia. Curr. Psychiatry Rep. 5: 121-7.
11. Williams NM, Norton N, Williams H, Ekholm B, Hamshere ML, Lindblom Y, Chowdari KV, Cardno AG, Zammit S, Jones LA, Murphy KC, Sanders RD, McCarthy G, Gray MY, Jones G, Holmans P, Nimgaonkar V, Adolfson R, Osby U, Terenius L, Sedvall G, O'Donovan MC, Owen MJ. 2003. A systematic genomewide linkage study in 353 sib pairs with schizophrenia. Am. J. Hum. Genet. 73: 1355-67.
12. Levinson DF. 2005. Meta-analysis in Psychiatric Genetics. Curr. Psychiatry Rep. 7: 143-151.
13. Takahashi S, Cui YH, Kojima T, Han YH, Zhou RL, Kamioka M, Yu SY, Matsuura M, Matsushima E, Wilcox M, Arinami T, Shen YC, Faraone SV, Tsuang MT. 2003. Family-based association study of markers on chromosome 22 in schizophrenia using African-American, European-American, and Chinese families. Am. J. Med. Genet. B Neuropsychiatr. Genet. 120: 11-17.
14. van Amelsvoort T, Henry J, Morris R, Owen M, Linszen D, Murphy K, Murphy D. 2004. Cognitive deficits associated with schizophrenia in velo-cardio-facial syndrome. Schizophr. Res. 70: 223-32.
15. Maynard TM, Haskell GT, Peters AZ, Sikich L, Lieberman JA, LaMantia AS. 2003. A comprehensive analysis of 22q11 gene expression in the developing and adult brain. Proc. Natl. Acad. Sci. U. S. A. 100: 14433-14438.
16. Hoogendoorn B, Coleman SL, Guy CA, Smith SK, O'Donovan MC, Buckland PR. 2004. Functional analysis of polymorphisms in the promoter regions of genes on 22q11. Hum. Mutat. 24: 35-42.
17. Williams NM, Owen MJ. 2004. Genetic abnormalities of chromosome 22 and the development of psychosis. Curr. Psychiatry Rep. 6: 176-82.
18. Horowitz A, Shifman S, Rivlin N, Pisante A, Darvasi A. 2005. A survey of 22q11microdeletion in a large cohort of schizophrenia patients. Schizophr. Res. 73: 263-267.
19. Wilson HL, Wong AC, Shaw SR, Tse WY, Stapleton GA, Phelan MC, Hu S, Marshall J, McDermid HE. 2003. Molecular characterisation of the 22q13 deletion syndrome supports the role of haploinsufficiency of SHANK3/PROSAP2 in the major neurological symptoms. J. Med. Genet. 40: 575-584.
20. Chen CH, Lee YR, Chung MY, Wei FC, Koong FJ, Shaw CK, Yeh JI, Hsiao KJ. 1999. Systematic mutation analysis of the catechol O-methyltransferase gene as a candidate gene for schizophrenia. Am. J. Psychiatry 156: 1273-1275.
21. Chen HY, Yeh JI, Hong CJ, Chen CH. 2005. Mutation analysis of ARVCF gene on chromosome 22q11 as a candidate for a schizophrenia gene. Schizophr. Res. 72: 275-277.
22. Sinibaldi L, De Luca A, Bellacchio E, Conti E, Pasini A, Paloscia C, Spalletta G, Caltagirone C, Pizzuti A, Dallapiccola B. 2004. Mutations of the Nogo-66 receptor (RTN4R) gene in schizophrenia. Hum. Mutat. 24: 534-535.
23. Saito S, Ikeda M, Iwata N, Suzuki T, Kitajima T, Yamanouchi Y, Kinoshita Y, Takahashi N, Inada T, Ozaki N. 2005. No association was found between a functional SNP in ZDHHC8 and schizophrenia in a Japanese case-control population. Neurosci. Lett. 374: 21-24.
24. Verma R, Kubendran S, Das SK, Jain S, Brahmachari SK. 2005. SYNGR1 is associated with schizophrenia and bipolar disorder in southern India. J. Hum. Genet. 50: 635-640.
25. Hung CC, Chen YH, Tsai MT, Chen CH. 2001. Systematic search for mutations in the human tissue inhibitor of metalloproteinases-3 (TIMP-3) gene on chromosome 22 and association study with schizophrenia. Am. J. Med. Genet. 105: 275-278.
26. Wonodi I, Hong LE, Avila MT, Buchanan RW, Carpenter WT Jr, Stine OC, Mitchell BD, Thaker GK. 2005. Association between polymorphism of the SNAP29 gene promoter region and schizophrenia. Schizophr. Res. 78: 339-341.
27. Li T, Ma X, Sham PC, Sun X, Hu X, Wang Q, Meng H, Deng W, Liu X, Murray RM, Collier DA. 2004. Evidence for association between novel polymorphisms in the PRODH gene and schizophrenia in a Chinese population. Am. J. Med. Genet. B Neuropsychiatr. Genet. 129: 13-15.
28. McGhee KA, Morris DW, Schwaiger S, Nangle JM, Donohoe G, Clarke S, Meagher D, Quinn J, Scully P, Waddington JL, Gill M, Corvin A. 2005. Investigation of the apolipoprotein-L (APOL) gene family and schizophrenia using a novel DNA pooling strategy for public database SNPs. Schizophr. Res. 76: 231-238.
29. Tsai MT, Hung CC, Tsai CY, Liu MY, Su YC, Chen YH, Hsiao KJ, Chen CH. 2002. Mutation analysis of synapsin III gene in schizophrenia. Am. J. Med. Genet. 114: 79-83.
30. OMIM 176948. Mitogen-activated protein kinase 1; MAPK1.
31. Selcher JC, Atkins CM, Trzaskos JM, Paylor R, Sweatt JD. 1999. A necessity for MAP kinase activation in mammalian spatial learning. Learn Mem. 6: 478-490.
32. Shalin SC, Zirrgiebel U, Honsa KJ, Julien JP, Miller FD, Kaplan DR, Sweatt JD. 2004. Neuronal MEK is important for normal fear conditioning in mice. J. Neurosci. Res. 75: 760-770.
33. Thomas GM, Huganir RL. 2004. MAPK cascade signalling and synaptic plasticity. Nat. Rev. Neurosci. 5: 173-183.
34. Kyosseva SV. 2004. Differential expression of mitogen-activated protein kinases and immediate early genes fos and jun in thalamus in schizophrenia. Prog. Neuropsychopharmacol. Biol. Psychiatry 28: 997-1006.
35. Fumagalli F, Frasca A, Sparta M, Drago F, Racagni G, Riva MA. 2006. Long-term exposure to the atypical antipsychotic olanzapine differently up-regulates extracellular signal-regulated kinases 1 and 2 phosphorylation in subcellular compartments of rat prefrontal cortex. Mol. Pharmacol. 69: 1366-1372.
36. 黃佳興。 2004。應用微陣列晶片技術尋找精神分裂症的生物標記。慈濟大學人類遺傳學研究所碩士論文。
37. Kim HJ, Bar-Sagi D. 2004. Modulation of signalling by Sprouty: a developing story. Nat. Rev. Mol. Cell Biol. 5: 441-450.
38. Boeckers TM, Bockmann J, Kreutz MR, Gundelfinger ED. 2002. ProSAP/Shank proteins - a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease. J. Neurochem. 81: 903-910.
39. Kreienkamp HJ, Soltau M, Richter D, Bockers T. 2002. Interaction of G-protein-coupled receptors with synaptic scaffolding proteins. Biochem. Soc. Trans. 30: 464-468.
40. Uchino S, Wada H, Honda S, Nakamura Y, Ondo Y, Uchiyama T, Tsutsumi M, Suzuki E, Hirasawa T, Kohsaka S. 2006. Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor. J. Neurochem. 97: 1203-1214.
41. Bonaglia MC, Giorda R, Borgatti R, Felisari G, Gagliardi C, Selicorni A, Zuffardi O. 2001. Disruption of the ProSAP2 gene in a t (12; 22) (q24.1; q13.3) is associated with the 22q13.3 deletion syndrome. Am. J. Hum. Genet. 69: 261-268.
42. Bonaglia MC, Giorda R, Mani E, Aceti G, Anderlid BM, Baroncini A, Pramparo T, Zuffardi O. 2005. Identification of a recurrent breakpoint within the SHANK3 gene in the 22q13.3 deletion syndrome. J. Med. Genet.
43. Wilson HL, Wong AC, Shaw SR, Tse WY, Stapleton GA, Phelan MC, Hu S, Marshall J, McDermid HE. 2003. Molecular characterisation of the 22q13 deletion syndrome supports the role of haploinsufficiency of SHANK3/PROSAP2 in the major neurological symptoms. J. Med. Genet. 40: 575-584.
44. Wolford JK, Blunt D, Ballecer C, Prochazka M. 2000. High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC). Hum. Genet. 107: 483-487.
45. den Dunnen JT, Antonarakis SE. 2001. Nomenclature for the description of human sequence variations. Hum. Genet. 109: 121-124.
46. Wang J, Chuang K, Ahluwalia M, Patel S, Umblas N, Mirel D, Higuchi R, Germer S. 2005. High-throughput SNP genotyping by single-tube PCR with Tm-shift primers. Biotechniques 39: 885-893.
47. Wang WY, Barratt BJ, Clayton DG, Todd JA. 2005. Genome-wide association studies: theoretical and practical concerns. Nat. Rev. Genet. 6: 109-118.