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在本論文第一部份的研究中，我們探討了一個罹患MELAS症候群的女性病人粒線體DNA突變及其母系親屬的臨床病徵、生化缺陷及細胞功能變異。病人除表現典型的MELAS症候群外，另合併有糖尿病、甲狀腺機能亢進及心臟肌病變等症狀。內分泌功能分析顯示病人具有甲狀腺素T3、T4以及TSH抗體等指數較高的現象。經由分析病人及其家族成員各種不同組織中之粒線體DNA ，我們發現這位病人及其母親和三個兒子的體細胞在tRNALeu(UUR)基因上有A3243G的點突變發生，同時他們也都有一個260 bp tandem duplication的長度突變在粒線體的D-loop區域發生，而此種突變尚未在其它MELAS病人身上發現過。另外，我們也藉由長距鏈鎖反應(long-PCR)技術來檢視是否有大幅度的斷損突變(large-scale deletion)存在，結果顯示病人的組織並沒有這種粒線體DNA突變。病人的肌肉及頭髮中分別有含有80%及52%的A3243G突變型粒線體DNA。而血球細胞中的A3243G突變型粗線體DNA含量，則隨著取樣的時間不同而有20%到42%的變化。另一方面，我們運用semi-quantitative PCR及南方墨漬法(Southern blot)分析，發現具260 bp tandem duplication突變的粒線體DNA在肌肉中有38%約含量。值得注意的是，我們也發現這個病人和她的母親的體細胞具有一個新的C3093G點突變，這個突變位在粒線體DNA的16S rRNA基因上。而且，此突變型粒線體DNA在肌肉組織中的含量為47%，其中的70%與A3243G點突變存在同一粒線體DNA分子上。相較於肌肉組織中較高的含量及具有與A3243G同時發生在同一條粒線體DNA上的情況， C3093G突變在病人的血球及毛囊細胞中分別含有10%及21%，且是單獨存在，並不與A3243G突變同時存在於同一粒線體DNA分子上。病人的3個小孩的組織中都有高於50%的A3243G突變型粒線體DNA，但卻沒有C3093G突變的存在。C3093G突變型粒線體DNA存在不同的組織及不同的家族成員中有不同的分配方式(segregation pattern)。此一發現顯示突變型粒線體DNA在這個家族中有一個組織特異性的分配(tissue-specific segregation)以及在一代之間粒線體 DNA基因型態發生了快速轉換(rapiddrift。在另一方面，我們也利用病人、病人的母親及其三個小孩的皮膚檢體培養纖維母細胞。將病人的皮膚型纖維母細胞與去掉粒線體DNA的p°。細胞進行融合後，可得到含有突變型粒線體DNA的融合細胞(cybrid)，而且不同的cybrid細胞含有不同比例的突變型粒線體DNA。利用這些纖維母細胞及cybrid細胞，我們研究這些粒線體DNA突變對粗線體呼吸酵素及細胞生長的影響。結果顯示病人的纖維母細胞之粒線體呼吸酵素Complex I活性有明顯的下降，而且Complex II及Complex III的酵素活性反而有些微的上升。
我們發現含有260 bp tandem duplication突變及C3093G點突變的粒線體DNA在初代培養的過程中會快速的消失。相對於上述的兩種突變，A3243G粗線體DNA突變則可在細胞中維持一定的比例。為了要瞭解這些突變對粒線體呼吸功能影響的機制，我們也探討了細胞中的含有突變粒線體DNA的cybrid細胞其粒線體基因的表現。我們由病人萃取RNA經曲RNase protection分析，可以偵測到C3093G突變粒線體DNA在相對應的RNA分子的確有表現。我們也發現細胞中含有大量未經修飾的polycistronic mtRNNA。這是第一個被發現同時具有兩個粒線體DNA點突變及一個260 bp tandem duplication長度突變的罹患MELAS症候群病人。這些在MELAS病人上的多重粒線體DNA突變會導致粒線體氧化磷酸化功能的嚴重失常，並導致病人病灶組織中的代謝活性低於能量需求。這些發現將有助於解釋這位罹患MELAS症候群病人在臨床上具有較典型MELAS病人表現更多而嚴重的多重臨床症狀。
在另一部份的研究，我們檢測一群不同年齡且沒有粒線體疾病的成人其肌肉粒線體中各種呼吸酵素的活性，結果顯示cytochrome c oxidase酵素活性隨著年齡的增加有顯著下降的趨勢，NADH-cytochrome c reductase酵素活性略有下降，但succinate-cytochrome c reductase酵素活性則沒有明顯的變化。同時，我們也利用PCR技術來分析肌肉粒線體DNA中的斷損突變，我們發現三種與老化相關的大幅度斷損突變，它們的斷損長度分別是4，977bp、6，603bp及7，436bp。4，977bp斷損粒線體DNA在36歲以上的個體中即開始出現，超過60歲的個體有78%的人肌肉粒線體中存在此種斷損的粒線體DNA。6，063bp斷損粗線體DNA在25歲的個體中開始出現突變，超過60歲的個體有91%的人其肌肉粒線體中存在有此種斷損的粒線體DNA。然而，7，436bp的粒線體DNA斷損則較少見於肌肉組織中，超過60歲的個體只有47.2%的人其肌肉粒線體中有此種斷損突變的粒線體DNA。利用半定量PCR方法，我們發現7，436bp斷損粒線體DNA在細胞中的含量隨著年齡而增加。我們在少部份的個體發現含有多種斷損突變粒線體DNA共同存在，而部份個體則只含有某一種斷損突變粒線體DNA。這些斷損突變粒線體DNA並 不具有組織特異性，因為它們也可以在肝臟、心肌、睪丸、皮膚、肺臟及其它組織中被偵測到。這些斷損突變的部位涵蓋了一些NADH dehydrogenaSe，Cytochrome C oxidaSe及tRNA的基因。廷些基因被刪除的結果會影響到呼吸鏈酵素Complex I及Complex IV的功能。我們認為隨著年齡增加的各種粒線體DNA的斷損突變可導致伴隨年齡增加所引起的粒線體呼吸功能異常。
We investigated a female patient and her maternal relatives in a family with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. The proband also had noninsulin-dependent diabetes mellitus, hyperthyroidism and cardiomyopathy. Endocrinological studies demonstrated a high titer of TSH receptor antibody and elevated levels of thyroid hormones T3 and T4 in the blood of the proband. Analysis of mitochondrial DNA (mtDNA) showed an A-to-G transition at nucleotide position (np)3243 in the tRNALeu(UUR) gene (A3243G mutation) in various tissues of the three-generation members of the family. A previously described 260 bp tandem duplication in the D-loop region of mtDNA was also found in the proband and her maternal relatives, but at different abundance. Such kind of tandem duplication has never been reported in the patients with MELAS syndrome. We did not find any large-scale deletions of mtDNA in various tissues of the proband by long-PCR. The proportions of the mtDNA with the A3243G point mutation in the muscle and hair follicles of the proband were 80% and 52%, respectively. The proportions of the A3243G mutant mtDNA varied from 20% to 42% in the blood cells obtained from different periods of time. The proportion of the mtDNA with the 260 bp tandem duplication was determined to be about 38% in the muscle by semi-quantitative PCR and Southern blot analysis. Most interestingly, we found a novel C to G point mutation at np 3093 (C3093G mutation) in the 16S rRNA gene of mtDNA in various tissues of the proband. The proportion of the C3093G mutant mtDNA was approximately 47%, and 70% of them coexisted with the A3243G mutation on the same mtDNA molecules in the muscle biopsies of the proband. In contrast to the mtDNA of muscle biopsies, 10% and 21% of the C3093G mutant mtDNA existed alone in the blood cells and hair follicles, respectively. The blood cells of the 3 sons of the proband harbored more than 50% A3243G mutant mtDNA but had no C3093G point mutation. The C3093G mutant mtDNA molecules segregated differently within different tissues and between the proband and her offsprings. These findings indicate a tissue-specific segregation and rapid switching of mtDNA genotype within one generation of the family.
On the other hand, primary cultures of skin fibroblasts were prepared from the proband and mother and three sons of the proband. The fibroblasts were then used for the construction of cybrids using cytoplasrnic transfer of patient-derived enucleated fibroblasts to the human osteosarcoma-derived mtDNA-less p°cells. Various cybrids containing different proportions of the mutant mtDNA were cloned. Using the fibroblasts and cybrids, we investigated the effects of these mtDNA mutations, alone or in combination, on mitochondrial respiratory enzyme functions and growth kinetics of the cell. The results showed that the cell growth is retarded and the electron transport function is impaired in the cells harboring these mtDNA mutations. Among the respiratory enzymes, the activity of Complex I was found to decrease significantly in the primary culture fibroblast of the proband. The mtDNA molecules with the 260 bp tandem duplication and the C3093G mutation disappeared very quickly in the primary culture and during the subculture process. By contrast, the A3243G mutation could be maintained at a relatively stable level during the subculture process. In order to better understand the mechanism by which these mtDNA mutations affect the respiratory function of the cell, we also studied the mitochondrial gene expression of the cybrids harboring the mutant mtDNA. RNA isolated from the cybrids was subjected to RNase protection, and the 3093 mutation on the RNA molecule corresponding to the mutant mtDNA template was detected. Furthermore, abnormalities in the processing of the polycistronic precursor mtRNA were noted in the cybrids harboring high proportion of the mutant rntDNAs. An incompletely processed RNA species, termed RNA 19, was found to accumulate at greater abundance in the mutant cybrids. This is the first case in which two heteroplasmic point mutations and a tandem duplications of mtDNA coexist in the target and peripheral tissues of the patient with the MELAS syndrome and multi-system disorders. The multiple mtDNA mutations in this MELAS patient may elicit a synergistic impairment of oxidative phosphorylation and cause the metabolic activity to fall below a energy threshold required by the target tissues of the patient. These findings may explain the clinical observations that the MELAS patient exhibited much more complicated multi-system clinical features than those of the patients with the typical MELAS syndrome.
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