小腦脊髓運動失調症的分子診斷及其第三型和第七型致病機轉之研究

人類小腦脊髓運動失調症候群是一種「體染色體顯性的遺傳性疾病」。造成此疾病的原因是由一段不穩定的CAG三核酸重覆序列倍增突變(amplification mutation)，轉譯出的多麩醯胺酸突變蛋白(polyglutamine)所形成。這些小腦脊髓運動失調症候群的遺傳疾病有一個明顯的特徵就是遺傳的期望值，它可以傳給子代且發病年齡越早漸進性神經退化越嚴重。目前我們的實驗室已鑑定7種疾病包括spinocerebeller ataxia type 1 (SCA1), SCA2,SCA3/Machado-Joseph disease (MJD), SCA6, SCA7, SCA8, SCA12 除了SCA8和SCA12外，它們大都屬於CAG三核酸重覆序列倍增突變(trinucleotides repeat expansion)。從我們實驗室收集的病人檢體中利用PCR的檢測方法，一共分析211個體染色體顯性遺傳的病人，其中有202個病人來自81個體染色體顯性遺傳的家族而另外9個為偶發性的病人，其結果顯示在台灣SCA3/ MJD這型的病人佔最多，它約佔32% (26個家族)，其次為SCA2 (11%，9個家族)，SCA1和SCA7佔最少(1 .2%，1個家族)。此外，我們是台灣第一個在產前診斷中成功的鑑定出胎兒具有MJD的突變基因，利用胎兒的羊水DNA鑑定其在MJD的基因座上有74個CAG核酸重覆序列倍增，並且從引產中取得胎兒的纖維母細胞，並鑑定出在胎兒早期即有此種突變的ataxin-3蛋白。另一方面我們想要了解MJD和細胞凋亡路徑的關係，實驗中利用不同促使細胞凋亡的誘導劑如tunicamycin， brefeldin A (ER stress)和staurosporine (non-ER stress)，結果顯示出突變的ataxin-3蛋白在扮演細胞凋亡的角色中是走non-ER stress路徑，有趣的是我們發現到在未做任何處的條件下，在西方點墨法和免疫細胞化學組織染色法中發現含突變的ataxin-3的細胞株其Bcl-2蛋白表現量下降，且細胞質內的cytochrome c蛋白表現量增加。同樣Bcl-2蛋白表現量下降也在MJD胎兒的纖維母細胞中發現。另外，這種多麩醯胺酸突變蛋白所造成疾病常和不正常的蛋白聚集堆積有關係，而molecular chaperones對於此種漸進性神經退化疾病扮演著重要的角色。所以我們取得了兩個SCA7病人的淋巴母細胞株各包括100個和41個CAG核酸重覆序列，用來研究多麩醯胺酸擴增對於熱休克蛋白是否會有影響。在西方點墨法和免疫細胞化學組織染色法中發現病人的淋巴母細胞株內的Hsp27 和Hsp70蛋白表現量明顯下降(和正常人的淋巴母細胞株作比較)，然而，我們的結果雖顯示Hsp27 和Hsp70蛋白在SCA7病人的淋巴母細胞株是有缺陷，但它們對於熱休克的刺激卻有正常的反應。這結果推測出突變的ataxin-7造成細胞內的Hsp27 和Hsp70蛋白減少，可能會引起結構摺疊異常的蛋白堆積和細胞毒性的壓力。綜合以上的研究，我們的結果顯示出含有多麩醯胺酸的突變蛋白會使抗細胞凋亡的蛋白如Bcl-2和Hsp27及molecular chaperones Hsp70的蛋白表現降低，這在研究多麩醯胺酸的突變蛋白和細胞凋亡過程的探討或許扮演著重要的角色。

The spinocerebellar ataxias (SCAs) are inherited autosomal dominant diseases. An expanded CAG repeat within the disease gene encodes a polyQ repeat in the mutant proteins. Compelling features of SCAs are the genetic anticipation with an earlier age at onset and a more sever progression of disease in successive generations. Seven different loci causing spinocerebellar ataxia (SCA) have been studied in our laboratory, including SCA1, SCA2, SCA3/MJD (Machado-Joseph disease), SCA6, SCA7, SCA8 and SCA12 loci. Most of these diseases (except SCA8 and SCA12) are caused by CAG trinucleotide expansion in the coding region of the respective gene. To determine the relative contributions of known ataxia genes in the patient population sent to our laboratory, we genetically evaluated 202 patients with dominantly inherited ataxia from 81 families and 9 patients with sporadic ataxia by the use of radioactive genomic polymerase chain reaction (PCR). Our results showed that SCA3/MJD was the most common genotype in our collection, accounting for 32% of cases (26 families). SCA2 was the second frequent genotype (11%, 9 families), SCA1 and SCA7 were much less frequent (1.2%, 1 family for each genotype). Furthermore, the first case of MJD prenatal diagnosis in Taiwan was successfully performed in our laboratory. We identified the presence of a 74 CAG expansions on MJD gene from the amniocentesis sampling by the prenatal diagnosis. MJD fibroblast cells were obtained from the aborted fetus after termination of pregnancy. The expression of mutant ataxin-3 was also identified in the fetal fibroblast cells. On the other hand, we are interested in the apoptotic pathway involved in the MJD. The results from cells treated with different apoptosis inducers, including tunicamycin, brefeldin A (ER stress) and STS (non-ER stress), indicated that mutant ataxin-3 plays role(s) in the non-ER stress induced apoptosis. Interestingly, under basal conditions, Western blot and immunocytochemical analysis showed a significant decrease of Bcl-2 protein expression and an increase of cytochrome c in cells containing expanded ataxin-3 when compared with those of the parental cells. The same reduction of Bcl-2 was further confirmed in fibroblast cells with mutant ataxin-3. In addition, because the polyglutamine disease is associated with abnormal protein aggregation, the molecular chaperones may play a role in disease progression. Two SCA7 lymphoblastoid cell lines with 100 and 41 polyglutamine repeats were utilized to examine the effects of polyglutamine expansion on heat shock proteins. A significant decrease of Hsp 27 and Hsp70 protein expression was observed through Western blot and immunocytochemical analysis. However, our results showed that even though the protein expression of Hsp27 and Hsp70 is defective, a normal heat shock response is present in lymphoblastoid cells expressing mutant ataxin-7. The results suggested that the reduction of Hsp 27 and Hsp70 proteins in the presence of mutant ataxin-7 might lead to misfolded protein accumulation and cell-toxicity stress. Taken together, our results indicated that the reduction of the anti-apoptotic proteins, Bcl-2 and Hsp27, and molecular chaperones, Hsp70, in the presence of polyglutamine proteins may contribute together to the cell apoptotic process.