线粒体脑肌病伴高乳酸血症和卒中样发作综合征的线粒体DNA突变特点

目的探讨线粒体脑肌病伴高乳酸血症和卒中样发作(MELAS综合征)的分子遗传学特点.方法用聚合酶链反应-限制片段长度多态性(PCR-RFLP)方法检测来自7个家庭的9例MELAS患者及其部分母系亲属的肌肉和(或)外周血细胞的mtDNA的A3243G和T3271C点突变,并进行突变型mtDNA的定量.结果在9例患者和1例亲属的肌肉和(或)外周血细胞中检测到A3243G点突变,未检测到T3271C突变.在这10例A3243G阳性标本中,外周血细胞(9例)的突变型mtDNA的比例为26.8%～50.3%;肌肉组织(4例)的突变型mtDNA的比例为46.8%～61.0%;对3例患者同时进行了肌肉和血细胞标本的检测,突变型mtDNA的比例肌肉组织均高于血细胞.对6个家庭的部分母系亲属的血细胞研究表明:只有1例先证者的同胞有此突变;另外3例先证者的母亲及2例先证者的同胞均未检测到此突变.另外有2例先证者的儿子临床表现符合MELAS,血中也检测到此突变.结论 mtDNA A3243G突变在本组MELAS综合征中的发生率较高,并且可在不同组织中检测到此突变,与国外文献报道一致;但国外报道多为母系遗传,而我们的病例以散发的居多,推测是由于新生突变所致.     

肌阵挛癫痫合并破碎红纤维综合征的线粒体DNA突变特点

目的　探讨肌阵挛癫痫伴破碎红纤维综合征 ( MERRF)的分子遗传学特点。方法　用聚合酶链反应-限制片段长度多态性 ( PCR- RFL P)方法检测 6例 MERRF患者及其部分母系亲属的肌肉和 (或 )外周血细胞的mt DNA的 A8344 G点突变 ,并进行突变型 mt DNA的定量分析。结果　在 2例患者的肌肉和外周血细胞中检测到A8344 G点突变。但其母亲的外周血细胞中未能检测到此突变。这 2例 A8344 G阳性标本中 ,肌肉组织的突变型mt DNA的比例分别为 79.0 %和 86 .8%,而在外周血细胞中分别为 5 9.7%和 72 .9%,突变型 m t DNA的比例在肌肉组织中高于外周血细胞中。结论　在 MERRF患者不同组织中检测到 mt DNA A8344 G点突变 ,与国外报道一致。但国外报道 MERRF多为母系遗传 ,而我们的病例未能有此发现 ,须扩大样本量进一步分析。     

线粒体DNA A3243G点突变在成年患者中的临床特点

目的 探讨mtDNA A3243G点突变在成年患者的临床表现特点.方法 对发病年龄在18岁以上的36例患者的临床资料进行分析(5个家系28例,散发8例),其中23例行mtDNAA3243G点突变检查(5个家系15例,散发8例),14例行头颅影像学检查,10例进行了骨骼肌病理检查.结果 5个家系的28例患者中出现糖尿病17例(60.7%)、耳聋16例(57.1%)、卒中样发作9例(32.1%),三者可以并存或单独出现,少见表现包括心肌病和肾脏损害.23例mtDNA A3243G点突变患者中线粒体脑肌病伴随乳酸血症和卒中样发作(MELAS)14例(61.0%),其主要表现为认知功能障碍、言语障碍和头痛;其余9例包括无症状的基因突变携带者3例(13.0%)、线粒体糖尿病和(或)神经性耳聋2例(8.7%)、自主神经功能异常2例(8.7%)、糖尿病伴不孕症1例(4.3%)和心肌病1例(4.3%).14例MELAS患者的头颅影像学检查显示以枕叶和颞叶受累为主,额叶最少;10例肌肉病理检查发现9例存在不整红边纤维.mtDNA A3243G点突变比例均值在MELAS患者为28.75%±13.69%,非MELAS患者为25.08%±11.54%,两者差异没有统计学意义.结论 mtDNAA3243G点突变在成年患者主要累及中枢神经、胰岛以及听神经.认知功能障碍、言语障碍和头痛是成年MELAS的主要临床表现.家族中多例患者出现糖尿病和耳聋,提示该突变并非MELAS突变,应当关注mtDNA A3243G点突变家系中非MELAS患者的存在.     

应用实时荧光定量聚合酶链反应结合突变阻滞形成系统进行线粒体脱氧核糖核酸A3243G突变负荷检测

目的 应用实时荧光定量PCR结合突变阻滞形成系统(RT ARMS-qPCR)技术检测线粒体DNA A3243G突变负荷,并探讨线粒体脑肌病伴高乳酸血症和脑卒中样发作(MELAS)综合征患者不同组织中A3243G突变负荷的分布情况.方法 分别构建含线粒体DNA3243位点的野生型(A)和突变型(G)序列的质粒,将这2种质粒按一定比例混合构建成13个含有不同A3243G突变负荷的标准对照,应用PCR-限制性片段多态性技术(RFLP)和RT ARMS-qPCR技术检测13个标准对照及存在A3243G突变的7例MELAS患者、1名携带者以及53名健康对照的外周血、肌肉、毛发及尿沉渣等组织的A3243G突变负荷.结果 标准对照的突变负荷经PCR-RFLP和RT ARMS-qPCR检测的结果均与预期值呈直线相关,决定系数分别为R21=0.885、R22=0.991,RT ARMS-qPCR检测的结果更接近预期值;7例MELAS患者及1名携带者不同组织的突变负荷经RT ARMS-qPCR检测的结果比经PCR-RFLP检测出的高,且RT ARMS-qPCR比PCR-RFLP更容易检测出低负荷的A3243G突变,并发现MELAS患者的肌肉、尿沉渣、头发的A3243G突变负荷均高于外周血.结论 应用RT ARMS-qPCR可以快速、灵敏、准确地检测不同组织的A3243G突变及其突变负荷.     

Leigh综合征的线粒体DNA突变分析

目的：了解中国人Leigh综合征的线粒体DNA（mtDNA）突变特点。方法：对12例LS患者用Southern杂交和PCR－限制性内切酶分析的方法检测有无mtDNA的缺失及T8993G、T8993C、T9176C、A8344G、A3243G等点突变。结果：在4例患者中发现mtDNA点突变，包括T8993G1例、T8993C1例、A8344G2例，定量分析表明突变型mtDNA的比例均较高，为87．2％－97．8％，未发现mtDNA的大片段缺失及T9176C、A3243G点突变。结论：LS在遗传方面有显著的异质性，根据不同的病因，临床表现略有差异。     

线粒体脑肌病患者的基因突变研究

目的 探讨线粒体脑肌病患者骨骼肌细胞线粒体DNA基因突变情况及发病机制。方法 观察总结5例线粒体脑肌病患者的临床表现,影像学变化特点,并应用PCR、限制性内切酶BglⅠ、ApaⅠ酶切,PAGE电泳鉴定DNA片段长度的方法,检测5例患者骨骼肌细胞中mtDNA是否发生nt3243和8344位点A→G突变。结果 5例患者（3例MELAS和2例MERRF）在临床表现和影像学改变等方面均与国外学者的研究结果相符。1例MELAS患者仅存在3243A→G点突变,1例MERRF患者存在8344A→G点突变,1例MERRF上述2个位点均存在突变。另2例呈家系起病的MELAS患者这2个位点都无突变。结论 3243及8344位点突变分别与MELAS和MERRF的发病有关,MERRF患者可以同时存在上述2个位点的突变。临床表现仍是确诊和分类的主要依据。Ⅰ     

线粒体脑肌病伴高乳酸血症和脑卒中样发作综合征一家系临床特征及线粒体基因A3243G位点点突变异质性水平

目的 调查1个疑似患有母系遗传性线粒体脑肌病伴高乳酸血症和脑卒中样发作(MELAS)综合征家系的临床表现、生物化学检测数据和影像学资料,并探索其与血细胞线粒体基因突变异质性水平的关联性.方法 收集先证者和11位其母系家系成员的一般情况、抽搐及脑卒中样发作等病史,检测家系成员的血常规和运动前后血浆乳酸水平等生化指标,并做头颅磁共振检查.用聚合酶链反应(PCR)-限制性内切酶片段长度多态和DNA测序法检测其成员是否存在线粒体基因组A3243G点突变,并用荧光实时定量PCR定量该突变的水平.结果 该家系部分成员存在抽搐、脑卒中样发作和高乳酸血症等MELAS综合征典型症状,以及身材矮小、运动不耐受和发热、偏头痛等非典型症状.发作期头颅磁共振成像符合MELAS综合征的典型特点,且普遍存在小脑萎缩.母系亲属均存在线粒体基因的A3243G位点点突变,突变异质性水平越高,症状越典型且严重.结论 该调查家系确诊母系遗传性MELAS综合征,其致病基因为线粒体A3243G点突变.外周血血细胞线粒体基因突变异质性水平与亲缘关系、抽搐早现性和血乳酸值等临床表型存在相关性.     

线粒体3243位点基因点突变异质性水平与MELAS病临床特征的家系分析

背景 线粒体脑肌病伴高乳酸血症及卒中样发作（MELAS）是线粒体疾病中最常见的一种,它主要由线粒体基因组第3243号位点的腺嘌呤突变为鸟嘌呤所导致的。本研究的目的即是调查一个疑似患有母系遗传性MELAS病家系的临床表现、生化学检测数据和影像学资料,并探索其与血细胞线粒体基因突变异质性水平的关联性。 方法 收集先征者和其母系家系12人的自然情况、抽搐及卒中样发作等病史,检测家系成员的血常规和运动前后血浆乳酸水平等生化指标,并做头部磁共振检查。之后用聚合酶链反应-限制性内切酶片段长度多态性（PCR-RFLP）法和DNA测序法检测并验证其成员存在线粒体基因组的m.3243A>G点突变,并用荧光实时定量PCR法（Real-time PCR）来定量m.3243A>G的水平。 结果 该家系部分成员存在抽搐、卒中样发作和高乳酸血症等MELAS病典型症状和身材矮小、运动不耐受和发热、偏头痛等非典型症状。发作期头磁共振符合MEALS病影像学资料的典型特点,且普遍存在小脑萎缩表现。母系亲属均存在线粒体基因的3243位点点突变,同时突变异质性水平越高,症状越典型且严重。 结论 该调查家系确诊母系遗传性MELAS病,其致病因素为线粒体A3243G点突变。突变异质性水平与亲缘关系、抽搐早现性和血乳酸值等临床表型存在相关性。     

线粒体基因G13513A突变导致的线粒体脑肌病六例临床表型分析

目的 报告6例mtDNA G13513A点突变引起的线粒体脑肌病患者的临床、影像学特点,总结mtDNA G13513A突变所致的线粒体病的临床表型.方法 对35例mtDNA常见突变(包括大片段缺失及A3243G、T3271C、A8344G、T8993G/C点突变)检查为阴性的线粒体脑肌病患者,用线粒体DNA全长测序和(或)聚合酶链反应-限制性片段长度多态法检测mtDNA G13513A点突变,分析阳性患者的临床特点,复习文献报道的mtDNA G13513A所致线粒体病的病例.结果 35例患者中有6例存在mtDNA G13513A突变.该6例患者均出现偏盲、轻偏瘫或偏身感觉障碍等卒中样发作表现,其中3例成人发病者以卒中样发作为主要症状,伴随癫痫、头痛、身材矮小、神经性耳聋等,头颅MRI显示以顶-枕-颢叶受累为主的大片病灶,符合成人型线粒体脑肌病伴高乳酸血症和卒中样发作(MELAS)的临床和影像学特点;3例青少年发病者除卒中样发作外,还有构音障碍、共济失调、眼外肌瘫痪等脑干受累的症状,MRI检查可见枕-颞叶大脑皮质非对称性病灶,以及双侧基底节和脑干的对称性病灶,符合青少年型MELAS-Leigh叠加综合征的临床和影像学特点.肌肉病理检查在5例患者发现不整红边纤维.经复习文献,发现mtDNA G13513A突变患者还存在婴幼儿型Leigh或Leigh样综合征表型.结论 mtDNA G13513A点突变是线粒体脑肌病较常见的致病性突变,主要导致Leigh综合征、MELAS-Leigh叠加综合征或MELAS综合征,其临床表型具有年龄依赖性.

Abstract：

Objective To report 6 Chinese patients with mitochondrial encephalomyopathy caused by mitochondrial DNA(mtDNA)G13513A mutation and discuss the mitochondrial phenotype associated with this mutation based on the data of our patient series as well as the reports by others.Methods Direct sequencing of polymerase chain reaction(PCR)products or PCR-RFLP analysis Was performed to screen mtDNA G13513A mutation in 35 cases with mitoehondrial encephalomyopathy.who carried no mtDNA common mutations(1arge 8eale deletion,A3243G,T3271 C,A8344G,or T8993G/C).The clinical features,MRI changes were retrospectively collected and analyzed.Published studies of all patients with mtDNA G13513A mutation were also reviewed.Results Six patients were identified carrying mtDNA G13513A mutation.All patients presented stroke-like episodes with hemianopsia.hemiparesis or hemiparesthesia.Three adult patients presented clinical and radiological features of adult-onset mitochondrial myopathy,encephalopathy,lactic acidosis,and stroke-like episodes(MELAS),including stroke-like episodes,epilepsy,headache,short stature,sensorineural deafness,multifocal lesions on parietal,occipital and temporal lobes on cranial MRI scans.Three iuvenile.onset patients presented the clinical and brain MRI features of MELAS-Leigh syndrome(LS)overlap syndrome.In addition to the stroke-like episodes,they also showed brain stem lesions with dysarthria,ataxia,and ophthalmopJegia. Brain MRI revealed asymmetrical lesions in the cortex of the oecipital and temporal lobes,as well as symmetrical lesions in the bilateral basal ganglia and brainstem.Muslce biopsy showed ragged redfibem in 5 patients.The infant-onset LS or Leigh-like syndrome with mtDNA G135 13A was described in the English literature.Conclusions mtDNA G13513A mutation is a common pathogenic mutmion for mitochondrial encephalomyopathy,which can result in Leigh syndrome,MELAS-LS overlap syndrome and adult MELAS.The onset of various phenotypes is relatively age-dependent.     

MELAS型线粒体脑肌病一例报告

线粒体脑肌病是一组由线粒体结构和功能异常所导致的多系统疾病。线粒体脑肌病伴高乳酸血症和脑卒中样发作综合征（MELAS）作为线粒体脑肌病中最为常见的一种类型，已证实其临床表现与21种mtDNA点突变相关，其中A3243G最为多见。此文报道1例MELAS型线粒体脑肌病。     

线粒体肌病与线粒体脑肌病的临床分析

目的探讨神经肌肉系统线粒体病的发病机制、临床与病理特征及诊断。方法对7例确诊为线粒体病患者的临床表现、病理检查、实验室与影像学资料进行了回顾性分析。结果该组患者诊断为线粒体肌病3例,线粒体脑肌病4例;其中2例患者血乳酸水平升高;7例患者肌电图均有异常发现,肌肉活检均有特征性的改变;4例线粒体脑肌病患者头部影像学均有异常改变。结论线粒体病主要累及肌肉及中枢神经系统,诊断要求多种手段结合,以临床和病理表现为主,近年来基因方面的研究及影像学诊断发展迅速,目前对本病主要采取对症治疗。     

In situ hybridization of muscle mitochondrial mRNA in mitochondrial myopathies

Summary To determine whether a mitochondrial mRNA deficiency exists in mitochondrial myopathies, muscle biopsies from a patient with chronic progressive external ophthalmoplegia (CPEO) and a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) were studied using in situ hybridization. Histochemistry and immunohistochemistry were performed along with hybridization. Hybridization reactions were widely distributed over the sarcoplasm of all muscle fibers in the patient with MELAS. In the patient with CPEO, 80% of the fibers showed a marked decrease in density of autoradiographic grains. This marked decrease corresponded to the histochemical and immunohistochemical findings of a very weak staining of cytochromec oxidase (CCO). The isotope-labeled cDNA probe used in in situ hybridization in this study complements a part of subunit I of CCO and a part of subunit II of complex I in the mitochondrial gene. Our results suggest a defect in the mRNA in this CPEO patient.Supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education and by a grant (62-2-05) from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

AZT-induced mitochondrial myopathy

Histochemical, electron microscopy and biochemical studies were performed on muscle biopsy specimens from 11 AIDS patients treated with zidovudine. A peculiar association of structural abnormalities and mitochondrial dysfunction was found. Focal cytochrome c oxidase (COX) deficiency was evident in muscle sections from 9 patients, 8 of whom had received long-term treatment while one had been treated for 1 month only. Electron microscopy showed changes in number, size and structure of mitochondria. Biochemical studies proved partial COX and succinate cytochrome c reductase (SCR) deficiency in 4 patients; one patient had only reduced SCR activity. Our data confirm that AZT therapy can cause toxic myopathy with mitochondrial dysfunction.Paper presented at the National Congress at Sorrento in 1991 and selected by the Editorial Board of the Journal     

线粒体脑肌病八例患者临床与神经影像学分析

目的分析线粒体脑肌病患者的临床及神经影像学特点。方法对8例病理确诊线粒体脑肌病患者进行回顾性分析研究。结果本病的主要临床表现为抽搐、运动耐受差、发育迟缓和智能障碍;主要影像学特点为幕上多发脑叶病灶,同时合并其他部位的病变,病灶广泛。结论具有上述临床特征和神经影像学改变者,提示线粒体脑肌病的可能。     

线粒体肌病和脑肌病患者骨骼肌细胞线粒体DNA缺失分析

目的为了检测线粒体肌病和脑肌病患者的骨骼肌细胞的线粒体ＤＮＡ的缺失情况。方法从６例原发性线粒体肌病和１例脑肌病患者的骨骼肌活检标本中，提取总ＤＮＡ，以线粒体ＤＮＡ全长为探针进行分子杂交。结果发现１例ＭＥＲＲＦ患者有５ｋｂ的线粒体ＤＮＡ基因缺失，另１例线粒体肌病患者有１５ｋｂ的线粒体ＤＮＡ基因缺失，剂量分析表明缺失型线粒体ＤＮＡ分别占总线粒体ＤＮＡ的１９．３％和１０．７％。结论线粒体ＤＮＡ基因缺失是线粒体疾病的重要病因之一     

Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion

Charcot–Marie–Tooth neuropathy type 2A (CMT2A) is associated with heterozygous mutations in the mitochondrial protein mitofusin 2 (Mfn2) that is intimately involved with the outer mitochondrial membrane fusion machinery. The precise consequences of these mutations on oxidative phosphorylation are still a matter of dispute. Here, we investigate the functional effects of MFN2 mutations in skeletal muscle and cultured fibroblasts of four CMT2A patients applying high-resolution respirometry. While maximal activities of respiration of saponin-permeabilized muscle fibers and digitonin-permeabilized fibroblasts were only slightly affected by the MFN2 mutations, the sensitivity of active state oxygen consumption to azide, a cytochrome c oxidase (COX) inhibitor, was increased. The observed dysfunction of the mitochondrial respiratory chain can be explained by a twofold decrease in mitochondrial DNA (mtDNA) copy numbers. The only patient without detectable alterations of respiratory chain in skeletal muscle also had a normal mtDNA copy number. We detected higher levels of mtDNA deletions in CMT2A patients, which were more pronounced in the patient without mtDNA depletion. Detailed analysis of mtDNA deletion breakpoints showed that many deleted molecules were lacking essential parts of mtDNA required for replication. This is in line with the lack of clonal expansion for the majority of observed mtDNA deletions. In contrast to the copy number reduction, deletions are unlikely to contribute to the detected respiratory impairment because of their minor overall amounts in the patients. Taken together, our findings corroborate the hypothesis that MFN2 mutations alter mitochondrial oxidative phosphorylation by affecting mtDNA replication.     

Amyloid-beta-induced mitochondrial dysfunction

As an important molecule in the pathogenesis of Alzheimer's disease (AD), amyloid-beta (Abeta) interferes with multiple aspects of mitochondrial function, including energy metabolism failure, production of reactive oxygen species (ROS) and permeability transition pore formation. Recent studies have demonstrated that Abeta progressively accumulates within mitochondrial matrix, providing a direct link to mitochondrial toxicity. Abeta-binding alcohol dehydrogenase (ABAD) is localized to the mitochondrial matrix and binds to mitochondrial Abeta. Interaction of ABAD with Abeta exaggerates Abeta-mediated mitochondrial and neuronal perturbation, leading to impaired synaptic function, and dysfunctional spatial learning/memory. Thus, blockade of ABAD/Abeta interaction may be a potential therapeutic strategy for AD.     

Age-related mitochondrial DNA point mutations in patients with mitochondrial myopathy

Mutations in the control region (D-loop) of mitochondrial DNA (mtDNA) have been described in normal old individuals and it is suggested that they originated from oxidative damage. Respiratory chain defects may lead to increased free radical generation, increased susceptibility to oxidative damage and further increased accumulation of age-related mutations. The objective of this study was to verify whether patients with a mitochondrial disease are more predisposed to accumulate the A189G and T408A mutations in the D-loop and confirm their age-associated nature. We evaluated the presence and levels of heteroplasmy of these two mutations in muscle DNA of 52 individuals with different ages (21 age-matched controls and 31 patients with single or multiple mtDNA deletions). The frequency of both mutations was significantly increased with age, but no differences were observed comparing the group of patients with their age-matched controls. We could not observe correlation of levels of heteroplasmy with age. Our results confirm the age-related nature of the A189G and T408A mutations in the D-loop in controls and patients with mitochondrial disease, but do not suggest that patients are more predisposed to the development of age-related point mutations.