线粒体脑肌病临床病理及部分基因突变研究

目的 探讨线粒体脑肌病患者骨骼肌组织病理特点及线粒体DNA(mitochondrial DNA,mtDNA)在3243、8344和8993位点的点突变,为疾病的诊断和鉴别诊断提供依据.方法 取8例患者及10例对照者骨骼肌活检标本速冻后切片,常规和酶组化学染色,光镜下观察其病理特点,并提取骨骼肌中的总DNA ,以相应的引物行PCR扩增,分别应用BglⅠ、ApaI 、SmaⅠ和MspⅠ酶酶切后,琼脂糖电泳判定,检测mtDNA中3243、8344和8993位点是否存在基因突变.结果 8例患者的肌肉病理均存在不整边红纤维;其中2例患者肌肉中mtDNA存在A3243G点突变,1例存在A8344G点突变,8例患者均未发现T8993G和T8993C突变.结论 肌肉组织病理学方法和分子生物学研究可以为线粒体脑肌病的诊断和鉴别诊断提供依据.     

线粒体脑肌病的线粒体3271T＞C、8356T＞C、9176T＞C/G和13513G＞A位点突变筛查

目的 了解中国线粒体脑肌病患儿的线粒体DNA(mtDNA)3271T＞C、8356T＞C、9176T＞C/G和13513G＞A位点的突变情况.方法 选择2005年10月至2009年10月500例线粒体脑肌病患儿,提取外周血DNA,用PCR-限制性片段长度多态性(RFLP)方法进行mtDNA 3271T＞C、8356T＞C、9176T＞C/G和13513G＞A位点的突变筛查分析;用DNA直接测序方法验证PCR-RFLP的结果.结果 在500例线粒体脑肌病患儿中未发现3271T＞C、8356T＞C、9176T＞C/G和13513G＞A位点的突变.结论 在中国线粒体脑肌病患儿中线粒体3271T＞C、8356T＞C、9176T＞C/G和13513G＞A位点不是常见突变.

Abstract：

Objective To investigate the spectrum of mitochondrial DNA (deoxyribonucleic acid)3271T＞C, 8356T ＞ C, 9176T ＞ C/G and 13513G ＞ A mutations in Chinese patients with mitochondrial encephalomyopathies. Methods Peripheral blood samples were collected from 500 mitochondrial encephalomyopathic patients clinically diagnosed as mitochondrial encephalomyopathy lactic acidosis & stroke-like episodes (MELAS), myoclonus epilepsy & ragged-red fibers (MERRF) or Leigh's syndrome from October 2005 to October 2009. The methods of PCR- polymerase chain reaction-restriction fragment length polymorphism ( RFLP ) and PCR-sequencing were performed to identify the mutations. Results No patients with the 3271T ＞ C, 8356T ＞ C, 9176T ＞ C/G or 13513G ＞ A mutations were identified.Conclusion The mutations of 3271T ＞ C, 8356T ＞ C, 9176T ＞ C/G and 13513G ＞ A are rare causes of mitochondrial encephalomyopathies in Chinese patients.     

线粒体脑肌病患者骨骼肌mtDNA点突变与发病类型的关系

目的：探讨线粒体脑肌病患者骼肌mtDNA在3243及8344位点的点突变与发病类型的关系,方法：提取5例患者骨骼肌中的总DNA,对2对寡核苷酸为引物行PCR扩增,分别应用BglI和Apal酶酶切后,琼脂糖电泳判定,结果：2例患者nt3243位点出出A→G点突变,座中样发病型（MELAS）和不整红边纤维型9MERRF）各1例,2例MERRF患者nt8344位点出现A→G点突变,其中1例同时存在着3243位点突变,结论：nt3243及nt8344位点突变分别与MELAS和MERRF的发病有关,1例MERRF中层得同时在上述2个位点的突变,这可能与线粒体脑肌病临床表现的多样化有关。     

线粒体脑肌病患者线粒体DNA突变分析

目的：分析线粒体脑肌病患者骨骼肌细胞线粒体DNA的突变情况,为疾病诊断提供依据.方法：用常规苏木精-伊红（H-E）、酶组织化学染色和电镜检查等方法对线粒体脑肌病进行诊断,用聚合酶链反应（PCR）/限制性内切酶酶切方法对线粒体脑肌病伴高乳酸血症和卒中样发作综合征（MELAS）和肌阵挛性癫痫伴肌红纤维断裂综合征（MERRF）的患者进行线粒体基因分析.结果：3例患者均被确诊为线粒体脑肌病,其中例1和例2 tRNA^leu基因发生A3243G杂合突变,例3发生A8344G杂合突变.结论：线粒体DNA中的tRNA基因突变,是线粒体脑肌病的重要病因之一.     

特异性寡核苷酸探针反向杂交在mtDNA点突变检测中的应用研究

目的：建立一种快捷、有效的mtDNA点突变检测体系。方法：线粒体病患者15例，根据临床表现分为3组，线粒体脑肌病伴高乳酸血症、卒中样发作（MELAS）组7例，单纯型线粒体肌病组4例，慢性进行性眼外肌麻痹（CPEO）组4例。用苯酚／氯仿法提取外周全血DNA，采用PCR／ASO反向杂交技术。用PCR扩增mtDNA的突变“热区”——tRNA^Leu（UUR）基因，在下游引物的5’端标记上地高辛，与点在尼龙膜上的5对寡核苷酸（AS0）探针：A3243／A3243G（野生型／突变型）、T3271／T3271C、G3460／G3460A、T3291／T3291C、C3256／C3256T进行反向杂交，根据杂交显色信号，确定有无突变。同时利用PCR／RFLP方法加以验证。结果：MELAS组中有A3243G点突变3例，但没有其他4个位点的突变。单纯型线粒体肌病组和CPEO组均未发现以上5个位点的突变。PCR/ASO反向杂交法与PCR／RFLP法结果一致。结论：PCR/ASO反向杂交技术，适用于已知mtDNA点突变的检测，具有敏感、节时、节省费用的特点。尤其适用于大批量标本的筛选。     

线粒体DNA A3243G点突变的临床异质性表现

目的探讨线粒体DNA A3243G点突变的临床表现特点.方法以25例临床怀疑为线粒体病,经血或肌肉线粒体DNA检查证实有A3243G点突变的线粒体脑肌病患者为研究对象,分析其临床表现、脑影像学特点、血乳酸水平和肌肉病理检查结果.结果基因检测证实25例患者均存在比例不同的线粒体DNA A3243G点突变,但临床表型有很大不同,其中19例为线粒体脑肌病伴乳酸血症和卒中样发作(MELAS),2例为无法分类的线粒体脑病,2例为松软儿,1例为Kearns-Sayer综合征(KSS),1例为线粒体胃肠肌病.大部分患者脑影像学检查可见病灶,肌肉活检可见蓬毛样红纤维改变,所有患者均有血乳酸水平增高.结论线粒体DNA A3243G点突变的临床表现和脑影像学均呈高度的异质性.对表现为多系统受累的患者,如果同时合并高乳酸血症,就应考虑线粒体病的可能,应进行线粒体DNA突变的检查以明确诊断.     

线粒体DNA突变糖尿病的特点

对线粒体DNA突变，尤其是转移核糖核酸亮氨酸tRNA（Leu，UUR）基因nt3243A→G点突变在糖尿病发病机制中的作用作一综述．nt3243A→G点突变常常是导致MIDD（母系遗传糖尿病伴耳聋）、MELAS（线粒体脑肌病、乳酸酸中毒、癫痫样发作综合征）及肾衰的常见病因．由于mtDNA突变影响ATP生成及可改变细胞内线粒体代谢产物的含量，从而可能导致与mtDNA突变有关的特殊类型糖尿病的发生，临床上出现一系列的特殊表现．     

2 型糖尿病患者线粒体常见基因突变PCR检测的局限性

目的:研究北京地区汉族人群中线粒体DNA(m itochondrial DNA,m tDNA)多位点突变频率与2型糖尿病之间的相关性。方法:采用直接PCR、PCR-限制性片段长度多态性(restriction fragm ent length polymorph ism,RFLP)、PCR-时相温度梯度凝胶电泳(temporal temperature grad ient gel electrophoresis,TTGE)法筛查m tDNA 4977片段缺失、线粒体tRNALeu(UUR)A3243G突变和m tDNA T14577C突变。结果:在250例2型糖尿病患者及142例健康对照外周血基因组总DNA中,未发现m tDNA 4977缺失、线粒体tRNALeu(UUR)A3243G突变及m tDNA T14577C突变。结论:m tDNA 4977缺失、线粒体tRNALeu(UUR)A3243G突变及m tDNA T14577C突变在外周血中没有检出,采用常规PCR法检测2型糖尿病患者外周血线粒体DNA突变具有较大的局限性。     

线粒体基因突变所致糖尿病发病机制及治疗进展

对线粒体DNA突变、尤其是转移核糖核酸亮氨酸tRNALeu(UUR)基因nt3243A→G点突变在糖尿病发病机制中的作用进行综述。nt3243A→G点突变常常是导致母系遗传糖尿病伴耳聋、MELAS(线粒体脑肌病、乳酸酸中毒、癫痫样发作综合征)常见病因。由于mtDNA突变影响ATP生成及可改变细胞内线粒体代谢产物的水平,从而可能导致与mtDNA突变有关的特殊类型糖尿病的发生,临床上出现一系列的特殊表现。基于其发病机制的特殊性,其诊断及治疗也有一定的特殊性。     

线粒体脑肌病中线粒体DNA的部分缺失

在2例Kearns－Sayre综合征（KSS）和2例慢性进行性眼外肌麻痹（CPEO）患者的骨骼肌线粒体DNA（mtDNA）中发现存在单一的大片段缺失突变。缺失的长度2．5～5．5kb，突变只发生在部分线粒体DNA中，突变型mtDNA占全部mtDNA的60．5％～84．6％。在10例其它的线粒体脑肌病患者骨骼肌标本和外周血标本及数例正常骨骼肌和胎肝标本的mtDNA中均未发现缺失突变。上述发现支持mtDNA的缺失突变为KSS和CPEO主要病因的观点。     

Point mutations of muscle mitochondrial DNA from patients with mitoch ondrial encephalomyopathies

Objective To study the relation between point mutations at nt3243 and nt8344 of muscle mit ochondrial DNA from patients with mitochondrial encephalomyopathies and phenotyp es. Methods DNA was extracted from muscle specimens from 5 patients with mitochondrial encep halomyopathies and amplified by PCR method, using corresponding oligonucleotide primers. DNA fragments were digested with restriction enzymes BglⅠ and ApaⅠ, then the digested DNA fragments were analyzed with an electrophoresis method. Results The point mutation at nt3243 of mtDNA was found in 2 patients, one with mitochon drial encephalomyopathy, lactic acidosis and stroke- like episodes (MELAS) and a nother with myoclonic epilepsy with ragged red fibers (MERRF). The point mutati on at nt8344 was found in 2 patients with MERRF, including the one with point mu tation at nt3243. Conclusion The point mutation of DNA at nt3243 correlated with MELAS and nt8344 correlated with MERRF. In addition, the detection of point mutations at both nt3243 and nt 8344 in a patient with MERRF shows the association of mutation with diversity i n clinical manifestations of mitochondrial encephalomyopathies.     

Detection of A3243G point mutation in mitochondrial DNA from 10 cases of MELAS

目的：对10例MELAS型线粒体脑肌病患者进行线粒体DNA A3243G点突变的检测。方法：用PCR-限制性内切酶分析法（restriction analysis）,检测10例MELAS患者及其8名母系亲属的肌肉和/或外周血细胞中有无mtDNA的A3243G点突变,并进行突变型mtDNA的定量。结果：在10例患者的肌肉和/血细胞中,均检测到A3243点突变。突变型mtDNA的比例在血细胞（7例）中为10.8％-47.8％,在肌肉（5例）中为39.4％-67.7％。有2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。在血细胞中,年轻患者的突变型比例通常较高。在1个家系中可证实为母系遗传 。但在3例先证者的母亲及2例先证者抽胞均未检测到此突变。结论：10例MELAS综合征患者均携有mtDNA A3243G点突变。在6个家庭中,只有1个家庭可证实为母系遗传,另外5个家庭中此突变可能为散发性,提示在中国人MELAS的发病机制中,mtDNAA3243G点突变为新生突变的居多。     

Clinical and laboratory survey of 65 Chinese patients with Leigh syndrome

Background Leigh syndrome is an inherited neurodegenerative disease that emerges in infancy and childhood and presents with a clinically heterogeneous variety of neuromuscular and non-neuromuscular disorders. It can result from the inheritance of mutations in either nuclear or mitochondrial DNA. In the current study, we performed a retrospective study in 65 patients in order to investigate the clinical and genetic characteristics of Leigh syndrome in Chinese patients. Methods Sixty-five unrelated cases （35 men and 30 women） who were hospitalized in the past 12 years were reviewed. Diagnosis was based on both the clinical presentation and the characteristic neuropathologic findings of bilateral symmetric necrotizing lesions in the basal ganglia and brain stem as detected using cranial computed tomography （CT） scan or magnetic resonance imaging （MRI）. The differential diagnosis of organic acidurias and fatty acid IS-oxidation defects were performed. Specific point mutations and deletions in mitochondrial DNA （T8993G, T8993C, T9176C, A8344G, A3243G） were screened by PCR-restriction analysis and Southern blot. The SURF1 gene was sequenced. Skeletal muscle biopsies were performed in 17 （26.2%） of the patients. The diagnosis was confirmed by autopsy in 6 （9.2%） patients. Results The patients had various forms of metabolic encephalomyopathy. Filly-nine （90.8%） of the patients had the typical neuroradiological features of Leigh syndrome, including symmetrical necrotizing lesions scattered within the basal ganglia, thalamus and brain stem. Twenty （30.8%） patients were confirmed by genetic, biochemical analysis and autopsy. Specific point mutations in mitochondrial DNA were found in 5 cases （7.7%）. Of these, the A8344G mutation was detected in 2 patients. The T8993G T8993C, and A3243G point mutations were identified in 3 other patients, respectively. SURF1 mutations associated with cytochrome c oxidase deficiency were identified in 8 （12.3%） families by DNA sequencing. A G604C mutation was identified in 6 （9.2%） patients. The genotypes of 52 patients remained unknown. Conclusions Leigh syndrome presents as a diverse array of clinical features and can result from specific mutations in nuclear or mitochondrial DNA. In this study, SURF1 mutations associated with cytochrome c oxidase deficiency were identified in 8 （12.3%） out of 65 patients with Leigh syndrome. It indicates that SURF1 mutations might be a common cause of Leigh syndrome in China. The etiology of Leigh syndrome in Chinese patients represents a persistent challenge to clinicians.     

线粒体DNA 3243、3316、3394位点突变与精神分裂症相关分析

目的分析线粒体tRNALeu(UUR)基因3243位点及ND1基因3316、3394位点突变对精神分裂症(SZ)发病的影响。方法采用PCR扩增、限制性内切酶消化、琼脂糖凝胶电泳分型检测、DNA测序等方法,对随机抽取的无亲缘关系的250例患者(SZ组)和292例对照组的外周血mtDNA进行3243、3316和3394位点的突变检测。结果在SZ组中发现8例3316G/A突变,对照组中3例,两组相比差异无统计学意义(P=0.138)。在SZ组中发现15例3394T/C突变,对照组中有4例,两组相比差异有统计学意义(P=0.007)。在精神分裂症患者组和对照组中均未发现3243A/G突变。结论 mtDNA3394T/C突变可能与SZ发生有一定关系,mtDNA 3243A/G、3316G/A突变可能与SZ发生无关。     

Mitochondrial gene mutations and type 2 diabetes in Chinese families

Background Numerous mitochondrial DNA mutations are significantly correlated with development of diabetes. This study investigated mitochondrial gene, point mutations in patients with type 2 diabetes and their families.
Methods Unrelated patients with type 2 diabetes （n=826） were randomly recruited; unrelated and nondiabetic subjects （n=637） served as controls. The clinical and biochemical data of the participants were collected. Total genome was extracted from peripheral leucocytes. Polymerase chain reaction, restriction fragment length poiymorphism （PCR-RFLP） and cloning techniques were used to screen mitochondrial genes including np3316, np3394 and np3426 in the ND1 region and np3243 in the tRNA^Leu（UUR）.
Results In 39 diabetics with one or more mitochondrial gene point mutations, the prevalence （4.7%, 39/826） of mtDNA mutations was higher than that （0.7%, 5/637） in the controls. The identical mutation was found in 23 of 43 tested members from three pedigrees. Affected family members presented with variable clinical features ranging from normal glucose tolerance to impaired glucose tolerance （IGT） （n=2）, impaired fasting glucose （IFG） （n=1） to type 2 diabetes （n=13） with 3 family members suffering from hearing loss.
Conclusions Type 2 diabetes in China is associated with several mitochondrial gene mutations. Aged patients with diabetic family history had a higher prevalence of mutation and various clinical pictures. Mitochondrial gene mutation might be one of the genetic factors contributing to diabetic familial clustering.     

MELAS综合征无创性基因突变分析方法研究

目的 研究携带A3243G突变的线粒体脑病-乳酸酸中毒-卒中样发作(MELAS)综合征患者及其母系亲属的外周血、尿、毛囊、唾液和部分患者的肌肉组织A3243G基因突变率,找到用于检测突变率更敏感的组织,建立更佳的、无创性的检查手段.方法 收集25例MELAS患者及其母系亲属33例的外周血、尿、毛囊、唾液和部分患者的肌肉组织提取DNA,利用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测各组织中A3243G突变,根据酶切产物电泳条带密度值问的比例计算突变型线粒体DNA的含量,比较各组织A3243G突变率的差异.结果 25例患者外周血、尿、毛囊、唾液均榆测到A3243G突变,尿液中A3243G突变率(62%±9%)明显高于外周血巾的突变率(36%±10%)(t=-11.13,P<0.01).5例患者进行了肌活检,肌肉突变率44.9%～75.1%,尿液中A3243G突变率和肌肉中A3243G突变率存在正相关,(r=0.900,P=0.037).33例母系亲属中有28例在至少一种组织中检测到A3243G突变,尿液中A3243G突变率33.0%(5.O%-70.4%)明显高于外周血中的突变率8.0%(0-33.3%)(z=-4.197,P<0.01).患者及其母系亲属唾液和毛囊组织中A3243G突变率与外周血相比筹别均不大.结论 在MELAS患者及其母系亲属巾,尿液中A3243G突变率均明显高于外周血,尿液A3243G突变率分析可能是优于外周血线粒体分析的一种无创性方法.     

Maternally inherited diabetes and deafness (MIDD) syndrome: a clinical and molecular genetic study of a Taiwanese family

We report on a case of a 48-year-old woman presenting with maternally inherited diabetes mellitus and deafness (MIDD) syndrome. Molecular genetic analysis and clinical evaluation were conducted in the patient and her 4 children to investigate the interrelation between an MIDD-associated mitochondrial DNA (mtDNA) mutation and clinical manifestations. Various symptoms and markers of MIDD, including seizures, migraines, short stature, mental retardation, and stroke-like episodes, were reviewed. Diabetes mellitus (DM) was studied by oral glucose tolerance and glucagon stimulation tests. Hearing impairment was determined by standard hearing tests and a brainstem auditory evoked potential test. The A3243G and T3271C transitional mutations of mtDNA were investigated from muscle and/or leukocytes and hair follicles. Mitochondrial-related symptoms were not found in the children, although they all harbored a heteroplasmic A3243G transition of mtDNA, as detected in screened samples. For the patient, the proportion of mutant mtDNA was highest in muscle cells followed by hair follicles and then leukocytes. Moreover, the proportion of mutant mtDNA was also higher in hair follicles than in leukocytes for asymptomatic family members. This Taiwanese MIDD family was found to have the A3243G point mutation as revealed from molecular genetic studies of leukocytes, hair follicles, and muscle tissue. However, no correlation was found between the proportion of mutant mtDNA and clinical features of any family member.