Leber遗传性视神经病三种组织间mtDNA基因型差异性的研究

采用ＰＣＲ技术对我国两个Ｌｅｂｅｒ遗传性视神经病（ＬＨＯＮ）家系成员外周血、头发和口腔粘膜３种组织线粒体ＤＮＡ进行扩增，限制性酶切分析、以探讨组织间ｍｔＤＮＡ基因型是否存在差异。结果发现一例患者ｍｔＤＮＡ基因型杂合，并且３种组织间突变型ｍｔＤＮＡ所占比例不同，证明线粒体ＤＮＡ组织间存在差异性。     

一例MELAS的线粒体DNA突变

为了检测中国ＭＥＬＡＳ患者的致病因素，采用Ｓｏｕｔｈｅｒｎ印迹杂交、ＰＣＲ、亚克隆及ＤＮＡ测序等技术，对１例中国线粒体脑肌病、乳酸中毒、中风样发作综合征（ｍｉｔｏｃｈｏｎｄｒｉａｌｅｎｃｅｐｈａｌｏｍｙｏｐａｔｈｙｗｉｔｈｌａｃｔｉｃａｃｉｄｏｓｉｓａｎｄｓｔｒｏｋｅ－ｌｉｋｅｅｐｉｓｏｄｅｓ，ＭＥＬＡＳ）患者的线粒体ＤＮＡ（ｍｔＤＮＡ）进行了突变分析。检测到ＭＥＬＡＳ患者特有的Ａ→Ｇ３２４３碱基替换，这一替换导致了一个新的ＡｐａⅠ酶切位点的形成。此突变型ｍｔＤＮＡ在检测的骨骼肌及血液标本中均被发现。突变型ｍｔＤＮＡ在检测的骨骼肌及血液ｍｔＤＮＡ标本中所占的比例分别为８０％和６０％。推测这一影响到ｍｔＤＮＡ中ｔＲＮＡｌｅｕ（ＵＵＲ）基因二氢尿嘧啶环的核苷酸突变是此例ＭＥＬＡＳ的重要致病因素。此外，在所测ｍｔＤＮＡ５６１ｂｐ（２９５０～３５１０）范围内还发现了１个Ｇ→ＴＮＤ１３４２３中性突变。     

线粒体肌病和脑肌病患者的肌细胞线粒体DNA突变分析

从１７例原发性线粒体肌病和脑肌病患者骨骼肌活检标本中，经ＤＮＡ抽提，以线粒体ＤＮＡ全长为探针进行了ＤＮＡ杂交，并通过聚合酶链式反应，确认两例慢性进行性眼外肌瘫痪者有线粒体ＤＮＡ改变。其中一例是改变了内切酶ＰｖｕⅡ限制位点的点突变；另一例的线粒体ＤＮＡ存在有约５ｋｂ的“共有缺失”大段。     

Leber遗传性视神经病三种组织间mtDNA基因型差异…

采用ＰＣＲ技术对我国两个Ｌｅｂｅｒ遗传性视神经病家系成员外周血，头发和口腔粘膜３种组织线粒体ＤＮＡ进行扩增，限制性酶切分析，以探计组织间ｍｔＤＮＡ基因型是否存在差异，结果发现一例患者ｍｔＤＮＡ基因型杂合，并且３种组织间突变型ｍｔＤＮＡ所占比例不同，证明线粒体ＤＮＡ组织间存在差异性。     

脂质沉积性肌病10例临床病理分析

目的 检测抗线粒体抗体(anti-mitochondrial antibody,AMA)、BCL-2、Bax在脂质沉积性肌病(lipid storage myopathy,LSM)患者骨骼肌中的表达,探讨AMA在LSM中的诊断价值及细胞凋亡在LSM发病机制中的作用.方法 对18例患者肌肉组织活检标本进行AMA、BCL-2、Bax免疫组化染色,其中经临床、肌肉活检病理确诊的LSM患者10例,正常肌肉组织3例及非线粒体肌病组织5例作为对照组.结果 光镜下,LSM患者骨骼肌AMA免疫组化染色可见Ⅰ型肌纤维中有大量棕黄色细颗粒沉积;BCL-2在18例患者的肌纤维和小血管中有不同程度的表达,Bax在5例非线粒体肌病组织中的肌纤维和小血管中有表达.结论 LSM患者的骨骼肌纤维中存在有异常线粒体增多现象,AMA可作为诊断LSM的一个病理学指标.LSM和非线粒体肌病的发病机制中均存在凋亡因素的异常调节.     

丙型肝炎肝外组织内病毒抗原的存在及意义

迄今为止，对丙型肝炎病毒（ＨＣＶ）感染肝外组织情况的研究报道甚少。本研究用免疫组化法检测丙型肝炎肝外组织内３种病毒抗原，旨在探讨ＨＣＶ感染泛嗜性的存在及其意义。９例丙型肝炎患者（肝组织内ＨＣＶ抗原均阳性）尸解肾、心、胰腺和肠（仅５例）等共３２份标本为...     

中国人食管癌 p16 基因突变分析

为了解ｐ１６基因与中国人食管癌遗传易感性的关系，应用ＰＣＲ－ＳＳＣＰ银染法和ＤＮＡ序列测定法对４０例食管癌及相应手术切端正常组织ＤＮＡ标本ｐ１６基因进行研究，结果在４０例食管癌标本ＤＮＡ中共发现ＤＮＡ变异１４例，其中９例存在有ｐ１６基因的错义突变和剪接位点突变，５例分别在密码子４１和１４０存在与白种人已发现的相同的正常多态现象，而相应的正常手术切端组织标本中未发现有错义突变和剪接位点突变。提示：（１）中国人食管癌中存在较高频率的ｐ１６基因突变，并以错义突变为主；（２）高发区和普通人群食管癌标本中ｐ１６基因突变频率未见差异；（３）正常对照手术切端组织未发现相应体细胞突变。表明ｐ１６基因突变在中国人食管癌发展过程中属较晚期的改变。     

线粒体DNA突变与人类疾病

近几年来，线粒体ＤＮＡ（ｍｔ ＤＮＡ）突变与人类疾病的关系越来越受到人们的重视。目前发现线粒体ＤＮＡ突变不但可累及脑、心脏、骨骼肌、肾脏和内分泌腺、造成人类多种疾病，而且还和人类的衰老过程有关。从分子水平上深入探讨ｍｔ ＤＮＡ突变的发生情况，阐明突变ｍｔ ＤＮＡ的传递规律及线粒体ＤＮＡ与核ＤＮＡ的相互关系，都有助于揭开线粒体病的本质，为人类诊断、预防和治疗这些疾病作出贡献。     

弱精子症病人精子线粒体DNA4977—bp缺失研究

本文采用聚合酶链反应（ＰＣＲ） 技术对４１ 例标本,其中２０ 例弱精子症病人和２１ 例精子活力正常人进行了精子线粒体ＤＮＡ（ｍｔＤＮＡ）缺失的研究。结果发现２０ 例弱精子症病人中１８ 例有缺失,而２１ 例精子活力正常人中仅有两例有缺失。说明线粒体ＤＮＡ４９７７ｂｐ 缺失在弱精子症的发病中起重要作用。     

Wistar大鼠不同组织线粒体DNA相结含量的测定

为了比较Ｗｉｓｔａｒ大鼠各组织线粒体ＤＮＡ的含量，探讨其与组织功能状态的关系，本文应用定量Ｓｏｕｔｈｅｒｎ杂交法，以线粒体ＤＮＡ与核１８ＳｒＤＮＡ的密度比值作为观察指标，检测Ｗｉｓｔａｒ大鼠骨骼肌，心肌，脑，肾及肝组织线粒体ＤＮＡ的相对含量。     

Adult onset limb-girdle type mitochondrial myopathy with a mitochondrial DNA np8291 A-to-G substitution

We analyzed mitochondrial DNA (mtDNA) from 7 patients in four families with adult onset limb-girdle type mitochondrial myopathy to clarify their genetic background. The patients, 2 men and 5 women, showed common clinical features, characterized by isolated skeletal myopathy, high serum creatine kinase level, ragged-red fibers and cytochrome c oxidase-defective fibers. Analysis of muscle biopsy specimens indicated that cytochrome c oxidase activity was decreased relative to that of citrate synthase in 5 of the 7 patients. Southern blotting and direct sequence analyses showed an A-to-G homoplasmic transition at np8291 and intergenic COII/tRNA(Lys) 9bp deletion in all patients. This substitution was detected in only 2 of 600 control individuals including healthy subjects and patients with other neuromuscular disorders; these 2 individuals had diabetes mellitus and myotonic dystrophy, respectively. Consequently, the mtDNA transition at np8291 was a rare polymorphism. However, the 7 patients we studied had identical clinical, pathological, biochemical, and genetic features. Therefore, limb-girdle type mitochondrial myopathy with this rare polymorphism may form a subgroup of adult onset mitochondrial myopathy. Received: December 3, 1998 / Accepted: February 18, 1999     

Histopathological changes in skeletal muscle associated with chronic ischaemia

Muscle biopsy is an essential part in the diagnostic workup in patients with suspected neuromuscular disorders. It is therefore important to be aware of morphological alterations that can be caused by systemic factors or natural ageing. Chronic limb ischaemia is frequent in elderly individuals. This study was performed to examine histopathological and mitochondrial changes in muscle in patients with chronic critical limb ischaemia. Muscle biopsy of skeletal muscle of the lower limb of patients with chronic ischaemia leading to amputation was performed and compared with muscle biopsies of healthy, age‐matched controls. The histopathological abnormalities included fibrosis, necrosis, atrophy, glycogen depletion, internal nuclei, rimmed vacuoles, fibre type grouping, cytochrome c oxidase deficient fibres, MHC‐I upregulation, and signs of microangiopathy. The only alteration found in age‐matched controls was a few cytochrome c oxidase deficient fibres. There were also increased levels of multiple mitochondrial DNA deletions in ischaemic muscles compared with controls. Critical limb ischaemia is associated with significant histopathological changes in muscle tissue and also increased levels of mitochondrial DNA deletions. Since the alterations mimic different primary myopathic changes, chronic ischaemia is important to consider as a differential diagnosis in elderly individuals, investigated with muscle biopsy for muscle disease.     

Approaches to mitochondrial gene therapy

Since their discovery during the end of the 80's the number of diseases found to be associated with defects in the mitochondrial genome has grown significantly. Organs affected by mutations in mitochondrial DNA (mtDNA) include in decreasing order of vulnerability the brain, skeletal muscle, heart, kidney and liver. Hence neuromuscular and neurodegenerative diseases represent the two largest groups of mtDNA diseases. Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is however no satisfactory treatment available to the vast majority of patients. This is largely due to the fact that most of these patients have respiratory chain defects, i.e. defects that involve the final common pathway of oxidative metabolism, making it impossible to bypass the defect by administering alternative metabolic carriers of energy. Conventional biochemical treatment having reached an impasse, the exploration of gene therapeutic approaches for patients with mtDNA defects is warranted. For now mitochondrial gene therapy appears to be only theoretical and speculative. Any possibility for gene replacement is dependent on the development of an efficient mitochondrial transfection vector. In this review we describe the current state of the development of mitochondria-specific DNA delivery systems. We summarize our own efforts in exploring the properties of dequalinium and other similar cationic bolaamphiphiles with delocalized charge centers, for the design of a vector suited for the transport of DNA to mitochondria in living cells. Further, we outline some unique hurdles that need to be overcome if the development of such delivery systems is to progress.     

Morphological Findings of Extraocular Myopathy with Chronic Progressive External Ophthalmoplegia

Mitochondrial diseases are a large group of disorders resulting from mutations of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Patients present clinically with multiple manifestations, including myopathies and multiple system disorders. Establishing a specific diagnosis often requires extensive clinical and laboratory evaluation. In this study of 2 adult patients with presumptive mitochondrial disease, the authors have identified distinctive morphological changes in medial rectus muscle biopsies that confirm the diagnosis of chronic progressive external ophthalmoplegia (CPEO). These findings demonstrate the usefulness of electron microscopy using medial rectus muscle in the diagnosis of adult patients with a slowly progressive course of mild skeletal weakness and CPEO.     

Infantile onset encephalomyopathy,retinopathy, optic atrophy,and mitochondrial DNA depletion associated with a novel pathogenic DHX16 variant

We studied a patient with mitochondrial DNA depletion in skeletal muscle and a multiorgan phenotype, including fatal encephalomyopathy, retinopathy, optic atrophy, and sensorineural hearing loss. Instead of pathogenic variants in the mitochondrial maintenance genes, we identified previously unpublished variant in DHX16 gene, a de novo heterozygous c.1360C>T (p. Arg454Trp). Variants in DHX16 encoding for DEAH-box RNA helicase have previously been reported only in five patients with a phenotype called as neuromuscular oculoauditory syndrome including developmental delay, neuromuscular symptoms, and ocular or auditory defects with or without seizures. We performed functional studies on patient-derived fibroblasts and skeletal muscle revealing, that the DHX16 expression was decreased. Clinical features together with functional data suggest, that our patient's disease is associated with a novel pathogenic DHX16 variant, and mtDNA depletion could be a secondary manifestation of the disease.     

Experimental myasthenia gravis, myositis and myocarditis in guinea-pigs immunized with subcellular fractions of calf thymus or calf skeletal muscles in freund''s complete adjuvant

Experimental thymitis and a partial neuromuscular block was produced in guinea-pigs by immunization with the subcellular fractions of calf thymus and skeletal muscle homogenates. A significant incidence of thymitis was observed in guinea-pigs immunized with the soluble or with the microsomal fractions of thymus or of skeletal muscle. The mitochondrial fraction of thymus or skeletal muscle was less effective in producing thymitis. In guinea-pigs immunized with the subcellular fractions of skeletal muscle additional evidence of experimental myositis (60%) and myocarditis (40%) was shown. All three skeletal muscle fractions were equally effective in the production of experimental muscle lesions.

A close correlation was found between the presence of thymitis and the evidence of a partial neuromuscular block. In contrast, a partial neuromuscular block was not observed in animals with experimental myositis but without thymitis. This finding gives further support to the hypothesis that the inflamed thymus is a source of a factor which blocks neuromuscular transmission in experimental myasthenia gravis.

     

Wistar大鼠不同组织线粒体DNA相对含量的测定

为了比较Wistar大鼠各组织线粒体DNA的含量,探讨其与组织功能状态的关系,本文应用定量Southern杂交法,以线粒体DNA与核18SrDNA的密度比值作为观察指标,检测Wistar大鼠骨骼肌、心肌、脑、肾及肝组织线粒体DNA的相对含量。结果表明：（1）骨骼肌的线粒体DNA相对含量最高,心、脑及肝肾依次减少（P＜0．05）;（2）肝和肾的线粒体DNA相对含量无显著性差别。说明各组织的线粒体DNA相对含量是有区别的,与其功能状态密切相关,了解各组织线粒体DNA含量的差别有助于研究氧化磷酸化疾病和衰老。同时也提供了一个较好的研究方法。     

The histochemical localization of reduced glutathione in skeletal muscle under different pathophysiological conditions.

The localization of reduced glutathione in skeletal muscle fibres of patients with inherited or acquired neuromuscular diseases and of subjects with no apparent disease of the neuromuscular system was studied histochemically. In healthy human skeletal muscle fibres, the level of reduced glutathione is higher in aerobic type I fibres than in anaerobic type II fibres. This finding suggests that glutathione in these healthy fibres is held in the reduced state chiefly by the activity of the decarboxylating and NADPH regenerating enzyme NADP(+)-dependent isocitrate dehydrogenase. In diseased muscle fibres, there is generally a positive relationship between the activity of the NADPH producing enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the level of reduced glutathione. This positive relationship suggests that glutathione in these diseased fibres is held in the reduced state chiefly by the activity of both enzymes of the pentose phosphate pathway.     

Respiratory chain deficiency in aged spinal motor neurons

Sarcopenia, muscle wasting, and strength decline with age, is an important cause of loss of mobility in the elderly individuals. The underlying mechanisms are uncertain but likely to involve defects of motor nerve, neuromuscular junction, and muscle. Loss of motor neurons with age and subsequent denervation of skeletal muscle has been recognized as one of the contributing factors. This study investigated aspects of mitochondrial biology in spinal motor neurons from elderly subjects. We found that protein components of complex I of mitochondrial respiratory chain were reduced or absent in a proportion of aged motor neurons–a phenomenon not observed in fetal tissue. Further investigation showed that complex I-deficient cells had reduced mitochondrial DNA content and smaller soma size. We propose that mitochondrial dysfunction in these motor neurons could lead to the cell loss and ultimately denervation of muscle fibers.