臺灣博碩士論文加值系統

第八型脊髓小腦運動失調症(簡稱SCA8)是一種會導致小腦運動能力失調的體染色體顯性遺傳之神經退化性疾病，但是其外顯率並不完全。SCA8相關基因是在染色體13q21的位置，包含了ATXN8OS CUG重複擴增RNA及ATXN8多麩醯胺(polyQ)擴增的蛋白。先前本實驗室研究SCA8重複序列在台灣人族群中的分佈情形，結果發現在巴金森氏症族群中的等位基因的比例較正常人族群高(8/593=1.3%相較於3/659=0.2%)，雖然未達顯著性(Wu et al., 2009)。另外，我們在ATXN8的啟動子上發現一新穎的-62 G/A多型性，神經腫瘤細胞及胚胎腎細胞的螢光酵素報告檢測顯示，包含-62G等位基因的啟動子轉錄活性顯著高於-62A等位基因(Wu et al., 2009)。本論文探討-62 G/A多型性對帕金森氏症感受性與分子機轉。首先針對此多型性點進行帕金森氏症患者及性別、年齡相當的正常人族群的病例-對照組研究，結果發現AA基因型者有顯著低的疾病罹患風險。其次探究-62 G/A多型性對ATXN8基因的啟動子調控，cDNA過度表現及luciferase reporter檢測顯示在SK-N-SH細胞中，轉錄因子C/EBPA會增強ATXN8啟動子的活性，但CEBPA與ATXN8 -62G、-62A啟動子的結合檢測，並未看出顯著的差異。

Spinocerebellar ataxia type 8 (SCA8) is a hereditary neurodegenerative disorder, manifesting itself as a slowly progressive cerebellar ataxia. The penetrance of SCA8 is incomplete. SCA8 involves the expression of a CTG/CAG expansion mutation from opposite strands producing CUG expansion transcripts (ATXN8OS) and a polyglutamine expansion protein (ATXN8) on chromosome 13q21 known to be pathogenic on other disorders. Previously we assessed the SCA8 repeat size ranges in Taiwanese population and found an increase (although not significant) in the proportion of the individuals carrying SCA8 larger alleles in Parkinson’s disease (PD) (8/593=1.3%) as compared to that in the control subjects (3/659=0.2%) (Wu et al., 2009). In addition, a novel -62 G/A promoter SNP was identified with significantly higher transcriptional activity in the luciferase reporter construct containing the -62G allele than that containing the -62A allele in both neuroblastoma and embryonic kidney cells (Wu et al., 2009). The purpose of this study is to investigate the ATXN8 -62 G/A polymorphism and the risk of Parkinson's disease using a case-control study. The results demonstrate that individuals carrying AA genotype exhibited a decrease risk of Taiwanese PD. cDNA over-expression and luciferase reporter assay revealed that CEBPA binds to the ATXN8 proximal promoter to up-regulate ATXN8 expression in neuroblastoma SK-N-SH cells. Nevertheless, no significant difference was observed upon examining the binding of CEBPA to ATXN8 -62G、-62A.

目錄 I
摘要 III
Abstract IV
圖表目錄 V
壹、緒論 1
一、脊髓小腦運動失調症(SCA) 1
(一) 臨床病徵 1
(二) 病因學 2
二、第八型脊髓小腦運動失調症(SCA8) 3
三、SCA8的致病機制 5
(一) 調節KLHL1基因表現 5
(二) RNA gain-of function 6
(三) PolyQ擴增蛋白 7
(四) 外遺傳改變 8
貳、研究目的 9
參、研究材料與方法 10
一、ATXN8 -62 G/A多型性的遺傳分析 10
(一) 研究樣品 10
(二) ATXN8 -62 G/A多型性檢測 10
(三) 統計分析 11
二、ATXN8 -62 G/A多型性功能分析 12
(一) 電腦模擬 12
(二) 凝膠電泳遷移檢測(EMSA) 12
(三) cDNA過度表現及螢光酵素報告基因檢測 15
(四) 內生性C/EBPA表現及與ATXN8啟動子的結合 17
肆、結果 19
一、ATXN8 -62 G/A基因多型性分析 19
二、ATXN8 -62 G/A基因啟動子功能性分析 20
(一) 電腦模擬 20
(二) 凝膠電泳遷移檢測 20
(三) cDNA過度表現及螢光酵素報告基因檢測 21
(四) 內生性C/EBPA表現及與ATXN8啟動子的結合 22
伍、討論 23
一、ATXN8 -62 G/A基因多型性分析 23
二、ATXN8 -62 G/A基因啟動子功能性分析 23
陸、參考文獻 27
柒、附錄圖表 37

圖表目錄

圖一、ATXN8、ATXN8 OS與KLHL1的基因構造圖。 37
圖二、ATXN8 -62 G/A多型性檢測。 38
圖三、ATXN8啟動子序列。 39
圖四、DNA探針製備。 40
圖五、核蛋白萃取檢測。 41
圖六、凝膠電泳遷移檢測。 42
圖七、cDNA過度表現及螢光酵素報告基因檢測。 43
圖八、內生性C/EBPA表現及與ATXN8啟動子的結合。 44
表一、帕金森氏症患者(PD)與正常人族群(Control) ATXN8 -62 G/A基因的多型性及罹病風險分析 45

高士寰, 第八型脊髓小腦運動失調症：遺傳及啟動子功能性分析. 國立臺灣師範大學生命科學系碩士論文. 2008年11月.
Baba, Y., Uitti, R. J., Farrer, M. J., and Wszolek, Z. K. (2005). Sporadic SCA8 mutation resembling corticobasal degeneration. Parkinsonism Relat Disord 11, 147-150.
Benzow, K. A., and Koob, M. D. (2002). The KLHL1-antisense transcript (KLHL1AS) is evolutionarily conserved. Mamm Genome 13, 134-141.
Bowman, A. B., Yoo, S. Y., Dantuma, N. P., and Zoghbi, H. Y. (2005). Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation. Hum Mol Genet 14, 679-691.
Brook, J. D., McCurrach, M. E., Harley, H. G., Buckler, A. J., Church, D., Aburatani, H., Hunter, K., Stanton, V. P., Thirion, J. P., Hudson, T. (1992). Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 68, 799-808.
Charlet-B, N., Savkur, R. S., Singh, G., Philips, A. V., Grice, E. A., and Cooper, T. A. (2002). Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol Cell 10, 45-53.
Chen, W. L., Lin, J. W., Huang, H. J., Wang, S. M., Su, M. T., Lee-Chen, G. J., Chen, C. M., and Hsieh-Li, H. M. (2008). SCA8 mRNA expression suggests an antisense regulation of KLHL1 and correlates to SCA8 pathology. Brain Res 1233, 176-184.
Chen, I. C., Lin, H. Y., Lee, G. C., Kao, S. H., Chen, C. M., Wu, Y. R., Hsieh-Li, H. M., Su, M. T., and Lee-Chen, G. J. (2009). Spinocerebellar ataxia type 8 larger triplet expansion alters histone modification and induces RNA foci. BMC Mol Biol 10, 9.
David, G., Abbas, N., Stevanin, G., Duerr, A., Yvert, G., Cancel, G., Weber, C., Imbert, G., Saudou, F., Antoniou, E., Drabkin, H., Gemmill, R., Giunti, P., Benomar, A., and Brice, A. (1997). Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion. Nat Genet 17, 65-70.
Duenas, A. M., Goold, R., and Giunti, P. (2006). Molecular pathogenesis of spinocerebellar ataxias. Brain 129,1357-1370.
Erickson, R. L., Hemati, N., Ross, S. E., and MacDougald, O. A. (2001). p300 coactivates the adipogenic transcription factor CCAAT/enhancer- binding protein alpha. J Biol Chem 276, 16348-16355.
Fardaei, M., Rogers, M. T., Thorpe, H. M., Larkin, K., Hamshere, M. G., Harper, P. S., and Brook, J. D. (2002). Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum Mol Genet 11, 805-814.
Fu, Y. H., Pizzuti, A., Fenwick, R. G., Jr., King, J., Rajnarayan, S., Dunne, P. W., Dubel, J., Nasser, G. A., Ashizawa, T., and de Jong, P. (1992). An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 255, 1256-1258.
He, Y., Zu, T., Benzow, K. A., Orr, H. T., Clark, H. B., and Koob, M. D. (2006). Targeted deletion of a single Sca8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits. J Neurosci 26, 9975-9982.
Holmes, S. E., O'Hearn, E. E., McInnis, M. G., Gorelick-Feldman, D. A., Kleiderlein, J. J., Callahan, C., Kwak, N. G., Ingersoll-Ashworth, R. G., Sherr, M., Sumner, A. J., Sharp, A. H., Ananth, U., Seltzer, W. K., Boss, M. A., Vieria-Saecker, A. M., Epplen, J. T., Riess, O., Ross, C. A., and Margolis, R. L. (1999). Expansion of a novel CAG trinucleotide repeat in the 5' region of PPP2R2B is associated with SCA12. Nat Genet 23, 391-392.
Hong S., Lee M. Y., and Cheong J. (2001). Functional interaction of transcriptional coactivator ASC-2 and C/EBPalpha in granulocyte differentiation of HL-60 promyelocytic cell. Biochem Biophys Res Commun 282, 1257-1262.
Hung C. C., Liu X, Kwon M. Y., Kang Y. H., Chung S. W., and Perrella M. A. (2010). Regulation of heme oxygenase-1 gene by peptidoglycan involves the interaction of Elk-1 and C/EBPalpha to increase expression. Am J Physiol Lung Cell Mol Physiol 298, L870-L879.
Ikeda, Y., Shizuka, M., Watanabe, M., Okamoto, K., and Shoji, M. (2000). Molecular and clinical analyses of spinocerebellar ataxia type 8 in Japan. Neurology 54, 950-955.
Ikeda, Y., Dalton, J. C., Moseley, M. L., Gardner, K. L., Bird, T. D., Ashizawa, T., Seltzer, W. K., Pandolfo, M., Milunsky, A., Potter, N. T., Shoji, M., Vincent, J. B., Day, J. W., and Ranum, L. P. (2004). Spinocerebellar ataxia type 8: molecular genetic comparisons and haplotype analysis of 37 families with ataxia. Am J Hum Genet 75, 3-16.
Ikeda, Y., Daughters, R. S., and Ranum, L. P. (2008). Bidirectional expression of the SCA8 expansion mutation: one mutation, two genes. Cerebellum 7, 150-158.
Ito, H., Kawakami, H., Wate, R., Matsumoto, S., Imai, T., Hirano, A., and Kusaka, H. (2006). Clinicopathologic investigation of a family with expanded SCA8 CTA/CTG repeats. Neurology 67, 1479-1481.
Juvonen, V., Kairisto, V., Hietala, M., and Savontaus, M. L. (2002). Calculating predictive values for the large repeat alleles at the SCA8 locus in patients with ataxia. J Med Genet 39, 935-936.
Kawaguchi, Y., Okamoto, T., Taniwaki, M., Aizawa, M., Inoue, M., Katayama, S., Kawakami, H., Nakamura, S., and Kakizuka, A. (1994). CAG expansions in a novel gene for machado-joseph disease at chromosome 14q32.1. Nat Genet 8, 221-228.
Koeppen, A. H. (2005). The pathogenesis of spinocerebellar ataxia. Cerebellum 4, 62-73.
Koide, R., Ikeuchi, T., Onodera, O., Tanaka, H., Igarashi, S., Endo, K., Takahashi, H., Kondo, R., Ishikawa, A., Hayashi, T., Saito, M., Tomoda, A., Miike, T., Naito, H., Ikuta, F., and Tsuji, S. (1994). Unstable expansion of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA). Nat Genet 6, 9-13.
Koide, R., Kobayashi, S., Shimohata, T., Ikeuchi, T., Maruyama, M., Saito, M., Yamada, M., Takahashi, H., and Tsuji, S. (1999). A neurological disease caused by an expanded CAG trinucleotide repeat in the TATA-binding protein gene: A new polyglutamine disease? Hum Mol Genet 8, 2047-2053.
Koob, M. D., Moseley, M. L., Schut, L. J., Benzow, K. A., Bird, T. D., Day, J. W., and Ranum, L. P. (1999). An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). Nat Genet 21, 379-384.
Kumar, N., and Miller, G. M. (2008). White matter hyperintense lesions in genetically proven spinocerebellar ataxia 8. Clin Neurol Neurosurg 110, 65-68.
Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., Barcelo, J., and O'Hoy, K. (1992). Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science 255, 1253-1255.
Maltecca, F., Filla, A., Castaldo, I., Coppola, G., Fragassi, N. A., Carella, M., Bruni, A., Cocozza, S., Casari, G., Servadio, A., and De Michele, G. (2003). Intergenerational instability and marked anticipation in SCA-17. Neurology 61, 1441-1443.
Manto, M. U. (2005). The wide spectrum of spinocerebellar ataxias (SCAs). Cerebellum 4, 2-6.
Matsuura, T., Yamagata, T., Burgess, D. L., Rasmussen, A., Grewal, R. P., Watase, K., Khajavi, M., McCall, A. E., Davis, C. F., Zu, L., Achari, M., Pulst, S. M., Alonso, E., Noebels, J. L., Nelson, D. L., Zoghbi, H. Y., and Ashizawa, T. (2000). Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10. Nat Genet 26, 191-194.
McCampbell, A., Taylor, J. P., Taye, A. A., Robitschek, J., Li, M., Walcott, J., Merry, D., Chai, Y., Paulson, H., Sobue, G., and Fischbeck, K. H. (2000). CREB-binding protein sequestration by expanded polyglutamine. Hum Mol Genet 9, 2197-2202.
Michalik, A., and Van Broeckhoven, C. (2003). Pathogenesis of polyglutamine disorders: aggregation revisited. Hum Mol Genet 12 Spec No 2, R173-186.
Miller, J. W., Urbinati, C. R., Teng-Umnuay, P., Stenberg, M. G., Byrne, B. J., Thornton, C. A., and Swanson, M. S. (2000). Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J 19, 4439-4448.
Moseley, M. L., Schut, L. J., Bird, T. D., Koob, M. D., Day, J. W., and Ranum, L. P. (2000). SCA8 CTG repeat: en masse contractions in sperm and intergenerational sequence changes may play a role in reduced penetrance. Hum Mol Genet 9, 2125-2130.
Moseley, M. L., Zu, T., Ikeda, Y., Gao, W., Mosemiller, A. K., Daughters, R. S., Chen, G., Weatherspoon, M. R., Clark, H. B., Ebner, T. J., Day, J. W., and Ranum, L. P. (2006). Bidirectional expression of CUG and CAG expansion transcripts and intranuclear polyglutamine inclusions in spinocerebellar ataxia type 8. Nat Genet 38, 758-769.
Mosemiller, A. K., Dalton, J. C., Day, J. W., and Ranum, L. P. (2003). Molecular genetics of spinocerebellar ataxia type 8 (SCA8). Cytogenet Genome Res 100, 175-183.
Mutsuddi, M., Marshall, C. M., Benzow, K. A., Koob, M. D., and Rebay, I. (2004). The spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila. Curr Biol 14, 302-308.
Mutsuddi, M., and Rebay, I. (2005). Molecular genetics of spinocerebellar ataxia type 8 (SCA8). RNA Biol 2, 49-52.
Nemes, J. P., Benzow, K. A., Moseley, M. L., Ranum, L. P., and Koob, M. D. (2000). The SCA8 transcript is an antisense RNA to a brain-specific transcript encoding a novel actin-binding protein (KLHL1). Hum Mol Genet 9, 1543-1551.
Orr, H.T., Chung, M., Banfi, S., Kwiatkowski, T. J., Jr, Servadio, A., Beaudet, A. L., McCall, A. E., Duvick, L. A., Ranum, L. P. W., and Zoghbi, H. Y. (1993). Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nat Genet 4, 221-226.
Perez, M. K., Paulson, H. L., Pendse, S. J., Saionz, S. J., Bonini, N. M., and Pittman, R. N. (1998). Recruitment and the role of nuclear localization in polyglutamine-mediated aggregation. J Cell Biol 143, 1457-1470.
Pulst, S. M., Nechiporuk, A., Nechiporuk, T., Gispert, S., Chen, X., Lopes-Cendes, I., Pearlman, S., Starkman, S., Orozoco-Diaz, G., Lunkes, A., DeJong, P., Rouleau, G. A., Auburger, G., and Sahba, S. (1996). Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2. Nat Genet 14, 269-276.
Ravikumar, B., Vacher, C., Berger, Z., Davies, J. E., Luo, S., Oroz, L. G., Scaravilli, F., Easton, D. F., Duden, R., O'Kane, C. J., and Rubinsztein, D. C. (2004). Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36, 585-595.
Robinson, D. N., and Cooley, L. (1997). Drosophila kelch is an oligomeric ring canal actin organizer. J Cell Biol 138, 799-810.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. 2nd ed. New York: Cold Spring Harbor.
Sato, N., Amino, T., Kobayashi, K., Asakawa, S., Ishiguro, T., Tsunemi, T., Takahashi, M., Matsuura, T., Flanigan, K. M., Iwasaki, S., Ishino, F., Saito, Y., Murayama, S., Yoshida, M., Hashizume, Y., Takahashi, Y., Tsuji, S., Shimizu, N., Toda, T., Ishikawa, K., and Mizusawa, H. (2009). Spinocerebellar ataxia type 31 is associated with "inserted" penta-nucleotide repeats containing (TGGAA)n. Am J Hum Genet 85, 544-557.
Saveliev, A., Everett, C., Sharpe, T., Webster, Z., and Festenstein, R. (2003). DNA triplet repeats mediate heterochromatin-protein-1- sensitive variegated gene silencing. Nature 422, 909-913.
Savkur, R. S., Philips, A. V., and Cooper, T. A. (2001). Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 29, 40-47.
Schols, L., Szymanski, S., Peters, S., Przuntek, H., Epplen, J. T., Hardt, C., and Riess, O. (2000). Genetic background of apparently idiopathic sporadic cerebellar ataxia. Hum Genet 107, 132-137.
Schols, L., Bauer, I., Zühlke, C., Schulte, T., Kölmel, C., Bürk, K., Topka, H., Bauer, P., Przuntek, H., and Riess, O. (2003). Do CTG expansions at the SCA8 locus cause ataxia? Ann Neurol 54, 110-115.
Schols, L., Bauer, P., Schmidt, T., Schulte, T., and Riess, O. (2004). Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis. Lancet Neurol 3, 291-304.
Seng, S., Avraham, H. K., Jiang, S., Venkatesh, S., and Avraham, S. (2006). KLHL1/MRP2 mediates neurite outgrowth in a glycogen synthase kinase 3beta-dependent manner. Mol Cell Biol 26, 8371-8384.
Silveira, I., Alonso, I., Guimaraes, L., Mendonca, P., Santos, C., Maciel, P., Fidalgo De Matos, J. M., Costa, M., Barbot, C., Tuna, A., Barros, J., Jardim, L., Coutinho, P., and Sequeiros, J. (2000). High germinal instability of the (CTG)n at the SCA8 locus of both expanded and normal alleles. Am J Hum Genet 66, 830-840.
Sobrido, M. J., Cholfin, J. A., Perlman, S., Pulst, S. M., and Geschwind, D. H. (2001). SCA8 repeat expansions in ataxia: a controversial association. Neurology 57, 1310-1312.
Soong, B. W., Lu, Y. C., Choo, K. B., and Lee, H. Y. (2001). Frequency analysis of autosomal dominant cerebellar ataxias in Taiwanese patients and clinical and molecular characterization of spinocerebellar ataxia type 6. Arch Neurol 58, 1105-1109.
Stevanin, G., Herman, A., Durr, A., Jodice, C., Frontali, M., Agid, Y., and Brice, A. (2000). Are (CTG)n expansions at the SCA8 locus rare polymorphisms? Nat Genet 24, 213; author reply 215.
Teive, H. A. (2009). Spinocerebellar ataxias. Arq Neuropsiquiatr 67, 1133-1142.
Worth, P. F., Houlden, H., Giunti, P., Davis, M. B., and Wood, N. W. (2000). Large, expanded repeats in SCA8 are not confined to patients with cerebellar ataxia. Nat Genet 24, 214-215.
Wu, Y. R., Lin, H. Y., Chen, C. M., Gwinn-Hardy, K., Ro, L. S., Wang, Y. C., Li, S. H., Hwang, J. C., Fang, K., Hsieh-Li, H. M., Li, M. L., Tung, L. C., Su, M. T., Lu, K. T., and Lee-Chen, G. J. (2004). Genetic testing in spinocerebellar ataxia in Taiwan: expansions of trinucleotide repeats in SCA8 and SCA17 are associated with typical Parkinson's disease. Clin Genet 65, 209-214.
Wu, Y. R., Chen, I. C., Soong, B. W., Kao, S. H., Lee, G. C., Huang, S. Y., Fung, H. C., Lee-Chen, G. J., and Chen, C. M. (2009). SCA8 repeat expansion: large CTA/CTG repeat alleles in neurological disorders and functional implications. Hum Genet 125, 437-444.
Wullner, U. (2003). Genes implicated in the pathogenesis of spinocerebellar ataxias. Drugs Today 39, 927-937.
Yamada, M., Sato, T., Shimohata, T., Hayashi, S., Igarashi, S., Tsuji, S., and Takahashi, H. (2001). Interaction between neuronal intranuclear inclusions and promyelocytic leukemia protein nuclear and coiled bodies in CAG repeat diseases. Am J Pathol 159, 1785-1795.
Zhuchenko, O., Bailey, J., Bonnen, P. Ashizawa, T., Stockton, D. W., Amos, C., Dobyns, W. B., Subramony, S. H., Zoghbi, H. Y., and Lee, C. C. (1997). Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet 15, 62-69.