- 學位論文
ETFDH突變引起氧化壓力導致粒線體功能障礙
Molecular Insights into the Electron Transfer Flavoprotein Dehydrogenase (ETFDH) Mutation Induced Mitochondrial Dysfunction
摘要
多發性醯基輔酶A去氫酶缺乏症 (Multiple Acyl-CoA Dehydrogenase Deficiency, MADD)是一種粒線體中脂肪酸代謝異常的體染色體隱性遺傳疾病，又被稱為戊二酸尿症第Ⅱ型 (glutaric aciduria type II) 。在大多數的臨床病例中，MADD是由於電子傳遞黃素蛋白 (electron transfer flavoprotein (ETF)) 或電子傳遞黃素蛋白脫氫酶 (electron transfer flavoprotein dehydrogenase (ETFDH)) 基因突變，造成的電子傳遞黃素蛋白(ETF)與ETF:泛菎氧化還原酶(electron transfer flavoprotein: ubiquinone oxidoreductase (ETF-QO)) 功能異常或缺乏，其中又以ETFDH基因的突變更為常見。ETF/ ETF-QO扮演著連結脂肪酸β-oxidation代謝與電子傳遞鏈的角色。 ETFDH及ETF/ETF-QO的變異使MADD患者常有脂肪代謝異常的症狀，於症狀較輕微M-MADD患者上可見肌肉細胞內脂肪油滴堆積造成肌肉無力的狀況。此外，ETF-QO的不足也與活性氧化物 (reactive oxygen species)的生成與細胞受到氧化傷害有關。 我們認為ETFDH基因的突變也會造成粒線體的功能不全進而促使MADD的發病。在本研究中，我們針對樣本進行組織病理染色檢查、電子顯微鏡的超微結構檢查、血獎中脂肪代謝物分析, 以及基因突變分子診斷。我們發現兩種點位異常c.92C>T 和 c.250G>A於ETFDH基因的第二外顯子。我們使用c.92C> T和 c.250G>A突變的淋巴球母細胞作為細胞模型，進行生物能 (bioenergetics)與粒線體功能 (biogenesis) 的分析。同時藉由脂肪酸的添加培養 (Hexanoic acid (C:6)、Capric acid (C:10)與Palmitic acid (C:16))，測試ETFDH基因突變細胞株對於不同長度脂肪酸其粒線體代謝能力的影響。本研究發現ETFDH基因突變細胞經過脂肪酸的添加培養會造成ETFDH表現量的下降、ATP生成的不足、粒線體膜電位的減少、細胞內油滴及脂肪過氧化物堆積增加，以及無法對應脂肪酸添加處理引發更多的粒線體功能缺損。具有連接β-oxidation與電子傳遞鏈的功能的輔酶─Coenzyme Q10以及Riboflavin的添加培養，可以減緩由ETFDH基因突變所引發的細胞內油滴及脂肪過氧化物堆積。本研究計畫測試Coenzyme Q10及Riboflavin的添加與粒線體功能改善的關係，以提供臨床MADD的病理機制更完善的了解。
並列摘要
Abstract Multiple acyl-CoA dehydrogenase deficiency (MADD), glutaric aciduria type II, is an autosomal recessive inherited disorder of fatty acid metabolism. In most cases MADD is caused by variations in one of two genes encoding electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The ETF/ETF-QO system serves as a short electron transfer pathway and linking the oxidation of fatty acids and some amino acids to the mitochondrial respiratory system. The most mutation is in ETFDH gene (electron transfer flavoprotein dehydrogenase), which encodes ETF-QO and causes lipid storage myopathy. Moreover, ETF-QO deficiency serves as a source of reactive oxygen species which has become unable to reoxidize the ubiquinone pool and produce oxidative damages. We proposed that ETFDH mutation induced mitochondrial dysfunction contributing to MADD pathogenesis. In this study, the plasma fatty acid profile, histopathological staining, transmission electron microscopy, and mutation screening were performed to make a molecular identification. Two types of ETFDH gene mutations were identifies in the affected tissues, one is c.92C>T and the other is c.250G>A mutation. We used the lymphoblastoid cells with c.92C>T and c.250G>A mutations in ETFDH gene as cell model. We challenged the cells with three types of fatty acids, hexanoic (C:6), capric (C:10) and palmitic acid (C:16). We found the reduced expression levels of ETFDH mRNA and ETF-QO, decreased ATP synthesis, dissipated mitochondrial membrane potentials, reduced mitochondrial bioenergetics, induced oil droplets and lipid peroxides, and declined response to fatty acid-induced mitochondrial stress in the ETFDH-mutated cell lines. Additionally, CoQ10 and riboflavin which links the mitochondrial fatty acid β-oxidation to the respiratory chain attenuated the lipid-induced mitochondrial stress in the ETFDH-mutated cell lines. With respect to recapitulate clinical relevant mutations, we search for better understand the pathology of MADD.