2012-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/686910摘要：研究背景:巴金森氏症(巴病) 約佔65歲以上老年人口的1-2%，目前已知有11個基因的突變已被證實是引起少數遺傳性巴病的致病因，其中，high temperature requirement A2 (HTRA2,又名Omi) 基因近日被發現其突變與巴病的發生有很強的相關性，學者發現Gly399Ser與Ala141Ser會顯著的增加罹患巴病的危險性，其臨床的表徵為典型的晚發型巴病。HTRA2基因轉錄的蛋白質為一粒線體中的serine protease，目前已知其功能與細胞凋亡反應(apoptosis)有關，當細胞接收到凋亡訊號時，HTRA2會經由autoprocessing活性將靠近N端的133個胺基酸切除，曝露出與inhibitor of apoptosis protein (IAP)結合的motif並釋放到細胞間質中，藉由與IAP結合達到影響apoptosis pathway的作用；另一方面，HTRA2也藉由其protease作用來分解細胞中累積的不正常蛋白質以達到保護細胞的效果，值得注意的是，HTRA2的protease活性會受到phosphorylation 磷酸化的調控，最近發現PINK1 (另一個巴病致病基因)可以透過p38 kinase磷酸化HTRA2蛋白，被磷酸化的HTRA2蛋白結構會由inactive homotrimer 轉變為activation form，進而影響apoptosis pathway以及分解細胞中累積的不正常蛋白質的作用。這些發現使我們推測在HTRA2突變的巴病患者中，粒腺體功能失常是導致其病理機轉的重要原因。基於以上之背景說明，目前對於HTRA2基因在巴病病患中所佔角色仍所知甚少，其突變所導致的粒線體功能變化仍未明目前。研究目的: 本研究具有以下三個子目的:1. 探討台灣漢人巴金森氏症患者是否具有HTRA2基因的突變。2. 利用SH-SY5Y neuroblastoma cell line作為多巴胺神經細胞模式並探討該突變點的功能性變化，以了解HTRA2基因突變對於神經細胞粒線體功能的影響。3. 以表現突變HTRA2蛋白的SH-SY5Y細胞株作為藥物篩選平台，探討Coenzyme Q10保護神經細胞的作用。研究方法: 分為三階段來進行，第一階段利用direct sequencing方式探討在200位年輕型及遺傳性巴病患者以及200位年齡與性別match的正常對照組中HTRA2基因的突變率，分析是否有台灣漢人族群獨特的突變熱點，並將大規模的驗證在第二批500位老年型巴病患者及500位正常對照組中；第二階段我們將以SH-SY5Y cell line 作為多巴胺神經細胞模式，利用transfection 技術，分別將帶有野生型與突變型的HTRA2cDNA的載體送入SH-SY5Y細胞中，以進ㄧ步分析神經細胞細胞凋亡、粒線體功能變化以及細胞內氧化自由基的累積程度。第三階段我們將進一步以表現突變HTRA2蛋白的SH-SY5Y細胞株作為藥物篩選平台，探討Coenzyme Q10保護神經細胞的作用。初步研究結果:我們在第一階段的年輕型及遺傳性巴病病患分析中發現了兩個過去沒有被報告過的突變點，包括在exon 1上以及在exon 3與exon 4 交界的+3 IVS G>A。我們將進行大規模的病患以及對照組分析以及第二、三階段的功能性分析與藥物篩選。<br> Abstract: The primary cause of Parkinson’s disease (PD) is still unknown. Currently, eleven genes have been identified for familial PD: α-synuclein, UCH-L1, LRRK2, GIGYF2 and HTRA2 in autosomal dominant forms of PD, Parkin, DJ-1, PINK1, ATP13A2, PLA2G6 and FBXO7 in autosomal recessive forms of early-onset PD (AREP). We have previously performed a comprehensive analysis of mutations in parkin and DJ-1 in a cohort of 41 Taiwanese/Ethnic Chinese patients with early-onset dopa-responsive parkinsonism (Lockhart et al., 2004; Wu et al., 2002, 2005). The frequency of parkin mutation was 6% (5 of 82 alleles), none of DJ-1 mutation and 1.7% of ATP13A2 mutation. The genetic causes in the remaining 90% of patients were still unclear.Very recently, the identification of high temperature requirement A2 (HTRA2) mutations predisposing to sporadic PD confirmed once again mitochondrial dysfunction as an important contributor to PD pathogenesis. In healthy cells HtrA2 resides in the intermembrane space of the mitochondria. On an apoptotic stimulus, it is released into the cytosol where it binds to cytosolic inhibitor of apoptosis proteins (IAPs) and inactivates their caspase-inhibitory activity. HTRA2 proteolytic activity has also been suggested to trigger a caspase-independent cell death pathway. To date, two heterozygous HTRA2 missense mutations—c.1195G4A (p.Gly399Ser) and c.420G4T (p.Ala141Ser), located respectively in the PDZ domain and N-terminus of mature HTRA2—have been identified in PD patients. Both mutations decreased in vitro activation of the protease activity and compromised mitochondrial function and morphology.All together, HtrA2 may be a protein with multiple critical functions in the central nervous system and may be particularly relevant in parkinsonian neurodegeneration. Given the biological and genetic evidence for a role of HtrA2 in PD pathogenesis, we decided to perform a detailed mutation analysis and genetic association study in 500 PD patients and 500 age- and gender-matched control individuals to assess the contribution of HTRA2 genetic variation to risk for PD in the Taiwanese population. Our study will lead to a better understanding of the underlying genetic mechanism of PD in Taiwanese, and perhaps a better understanding in the ethnic difference between oriental and white populations of PD. This might ultimately lead to understand the ethnic difference in the pharmacogenetics of Parkinson’s disease.