Wang W, Zhang J, Guo Y, Guo Z, Ren H
Institute of Basic Medical Sciences, CAMS, Beijing.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 1997 Aug;19(4):278-83.
To characterize the deletions of mitochondrial DNA (mtDNA) in Chinese patients with Kearns-Sayre syndrome (KSS) and chronic progressive external ophthalmolegia (CPEO) and identify deletion mutations of mtDNA be the etiology of these diseases.
Patients and preparation of total DNA: Two patients with KSS and two with CPEO and ten with other mitochondrial myopathies or encephalomyopathies were determined by histological and biochemical assays. Total DNA was isolated from 100 mg to 150 mg of frozen muscle obtained by biopsy using the methods described by Zeviani et al. (1988). Preparation of mtDNA probes: MtDNA were isolated and purified according to Palva's method (1985). The mtDNA was linearized by digestion with the restriction endonuclease Pvu II and separated by electrophoresis through low melting agarose gel. The 16.5 kb DNA band was recovered from the gel and labeled with alpha-p32 dCTP by random primer labeling. Southern blot analysis: Portions of five micrograms total DNA obtained from the patients and controls were digested with Pvu II, EcoR I, Hind III And Xba I respectively. The digested DNA was separated by agarose gel electrophoresis and transferred to nitrocellulose membrane and then hybridized with P32 labeled mtDNA as previously described (Zhang, 1991).
A large-scale deletions of mtDNA were identified in two patients with KSS and two patients with CEPO. The deletions ranged in size from 2.4 kb to 5.5 kb. The proportion of mutated mtDNA in each patients ranged from 54.6% to 84.6% of total mtDNA. No detectable deletions were found in mtDNA of ten patients with other mitochondrial myopathies or encepholomyopathies and normal controls.
Deletions of mtDNA were found in all KSS and CEPO patients detected. These results supported The view point of the deletions are important causes of physical defect in KSS and CEPO.
对中国克尔恩斯-塞尔综合征（KSS）和慢性进行性眼外肌麻痹（CPEO）患者线粒体DNA（mtDNA）的缺失情况进行特征分析，并确定mtDNA的缺失突变是否为这些疾病的病因。
患者及总DNA的制备：通过组织学和生化检测确定了2例KSS患者、2例CPEO患者以及10例患有其他线粒体肌病或脑肌病的患者。使用Zeviani等人（1988年）描述的方法，从经活检获得的100毫克至150毫克冷冻肌肉中分离总DNA。mtDNA探针的制备：根据Palva的方法（1985年）分离和纯化mtDNA。用限制性内切酶Pvu II消化使mtDNA线性化，并通过低熔点琼脂糖凝胶电泳分离。从凝胶中回收16.5 kb的DNA条带，并用α-p32 dCTP通过随机引物标记法进行标记。Southern印迹分析：分别用Pvu II、EcoR I、Hind III和Xba I消化从患者和对照中获得的5微克总DNA的部分。消化后的DNA通过琼脂糖凝胶电泳分离，转移到硝酸纤维素膜上，然后如先前所述（Zhang，1991年）与P32标记的mtDNA杂交。
在2例KSS患者和2例CPEO患者中鉴定出mtDNA的大规模缺失。缺失大小范围为2.4 kb至5.5 kb。每位患者中突变mtDNA的比例占总mtDNA的54.6%至84.6%。在10例患有其他线粒体肌病或脑肌病的患者以及正常对照的mtDNA中未发现可检测到的缺失。
在所检测的所有KSS和CPEO患者中均发现了mtDNA的缺失。这些结果支持了缺失是KSS和CPEO身体缺陷重要原因的观点。
