A maternal line study investigating the 4977-bp mitochondrial DNA deletion

The most frequently reported species of mitochondrial DNA (mtDNA) damage associated with ageing is the 4977-bp 'Common Deletion'. However, recent observations have raised several issues within the deletion debate namely: the significance of the 4977-bp deletion (CD) as a universal DNA marker of ageing and mitochondrial dysfunction; and the possibility for maternal transmission of deletions in humans. Previous attempts at answering these questions have been limited because many investigations have been cross-sectional studies of unrelated individuals. With the unique feature of the maternal inheritance of mtDNA, our study overcomes some of these limitations by investigating the CD in human maternal lines, which represent 21 families spanning four generations. Using a highly sensitive PCR methodology, we identified the presence of the CD in leukocytes from all 71 individuals (age range-8 months-99 years) including all infants and children (n=15) which in addition were free of any known mitochondrial diseases. This is important because the few reports of the CD in infants have been linked to mitochondrial disease. These results question the significance of the CD as a universal DNA marker of ageing and subsequent mitochondrial dysfunction and provide support for the possibility for maternal transmission of deletions.     

8-甲氧补骨脂素及长波紫外线诱导培养皮肤成纤维细胞线粒体DNA突变的研究

目的 探讨线粒体DNA4977bp缺失突变与皮肤成纤维细胞光老化之间的关系。方法 采用8－甲氧补骨脂素及长波紫外线（8－MOP／UVA）共同作用诱导培养皮肤成纤维细胞光老化，一步法提取线粒体DNA（mtDNA），三引物PCR的方法检测皮肤成纤维细胞线粒体DNA4977bp缺失突变，密度扫描半定量分析。结果 8－MOP／UVA作用下，培养皮肤成纤维细胞迅速出现细胞老化的形态学改变；28d时，8－MOP／UVA组、UVA组4977bp缺失突变的发生率及占总mtDNA中的比较分别为100％、11％和0.253、0.053；8－MOP组、正常对照组均为阴性，8－MOP／UVA组珉春他各组比较差异均有显著性（P＜0.01）。结论 线粒体DNA4977bp缺失突变累积与皮肤光老化密切相关，可能作为衡量皮肤老化程度的分子生物学标志。     

Myasthenia gravis with concomitant severe paraspinal muscle degeneration and mitochondrial DNA4977 deletion

Recent reports have discussed the many causes of dropped head syndrome and bent spine syndrome. We described a case of myasthenia gravis with concomitant severe degeneration of spinal muscle, mitochondrial DNA4977 deletion and sensorineural deafness. These associations were thought to be independent, however this is an important case to consider the etiology of bent spine syndrome.     

The relation between D-galactose injection and mitochondrial DNA 4834 bp deletion mutation

Since,D-galactose (D-gal) overload model has been used as a premature aging model, we hypothesized that it may also lead to accelerated aging in the inner ear. Furthermore, though the mitochondrial DNA (mtDNA) 4834 bp deletion mutation has been considered as the marker of aging, there is no information available in the literature concerning the mtDNA 4834 bp deletion mutation condition of the D-gal induced premature aging model. We investigate the changes in inner ear enzymatic activity, the occurring of mtDNA 4834 bp deletion in inner ear and other tissues and the relating hearing thresholds after the administration of high dosage (150 mg/kg per day) and low dosage (50 mg/kg per day) of D-gal to rats. Furthermore, the incidence of the mtDNA 4834 bp deletion in different tissues as well as in blood sample was compared. The results showed that daily subcutaneous injections of D-gal into rats for 8 weeks could lead to the biochemical defects and mtDNA 4834 bp deletion in the inner ear tissue and other tissues, which represent the typical aging animals, but the relating hearing threshold shifts (TS) were nearly identical in the three groups. This study also indicates that using of blood samples to detect mtDNA 4834 bp deletion in clinical research might lead to a 'false negative' result. A higher sensitive result could be gained using tissue biopsy to examine mtDNA 4834 bp deletion.     

线粒体DNA 4977 bp缺失与冠心病的关系

目的:探讨人外周血线粒体DNA 4 977 bp(mtDNA 4 977)缺失与冠心病(CAD)传统危险因素的关系以及与CAD发病的关系.方法:选择90例经冠状动脉造影证实的无亲属关系的CAD患者和60例年龄与CAD组相匹配的健康受试者作为对照组.所有受试者均采用巢式PCR法测定外周血mtDNA 4 977缺失相对数量,检测TC、TG、LDL-C、HDL-C、空腹血糖(FPG)、餐后2 h血糖(2hPG)水平以及收缩压(SBP)、舒张压(DBP)、体质指数(BMI)等相关指标,同时询问吸烟史、高血压病史及糖尿病史,研究它们与mtDNA 4 977缺失的关系.结果:①CAD患者外周血mtDNA 4 977缺失发生率和相对数量显著高于正常对照组(P＜0.01).②CAD组高血压、糖尿病和血脂异常者的mtDNA 4 977缺失相对数量显著升高(P＜0.05).mtDNA 4 977缺失相对数量与年龄、SBP、DBP、TC、LDL-C、FPG、2 hPG呈明显正相关,与HDL-C呈明显负相关,与吸烟、BMI、TG无相关性.③Logistic多元回归分析显示,调整其他传统危险因素后,mtDNA 4 977缺失仍与CAD显著相关.结论:CAD患者外周血mtDNA 4 977缺失发生率和相对数量显著升高,与年龄、SBP、DBP、TC、LDL-C、FPG、2hPG呈明显正相关,与HDL-C呈明显负相关.mtDNA 4 977缺失与CAD的发生机制有关.     

The frequency of 4977 base pair deletion of mitochondrial DNA in various types of liver disease and in normal liver

Using a polymerase chain reaction (PCR) method, we tested for the hepatic mitochondrial DNA (mtDNA) deletion in 40 hepatic tumors (28 hepatocellular carcinomas [HCcs], 9 other malignant tumors, and 3 benign tumors) and in the livers of 71 patients, including 16 pediatric patients with end-stage liver disease who underwent living related donor liver transplantation and 16 liver donors. A 4977 base pair (bp) deletion of mtDNA was detected in 36 of 55 specimens of non-tumor portions of adult liver (65.5%). However, none of the specimens obtained from cirrhotic livers of the 16 pediatric patients younger than 13 years of age had the 4977 bp deletion. The frequency of mtDNA deletion was significantly decreased compared with normal liver in HCCs (7 of 28) and other malignant liver tumors (2 of 9). The frequency of this deletion was unrelated to the presence of liver cirrhosis, patient's gender, hepatitis B virus surface antigen status, and hepatitis C virus antibody status.     

松花粉对衰老成纤维细胞线粒体DNA缺失突变的影响

目的 通过研究松花粉对衰老细胞线粒体(mt) DNA4977缺失突变的影响,探讨松花粉抗衰老的作用机制.方法 将细胞分为青年组、衰老细胞组、含240 mg/dl松花粉的衰老细胞处理组,三组细胞分别用不同培养基培养后,抽提线粒体DNA,进行PCR检测mtDNA4977缺失突变,以线粒体DNA中保守序列PCR扩增结果作为内参,比较各组mtDNA4977缺失突变在总线粒体DNA中的比例;同时对各组细胞进行β-半乳糖苷酶染色;并测定各组细胞超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量.结果 松花粉能够改善衰老成纤维细胞的衰老变化,并能降低衰老细胞mtDNA4977的缺失突变,提高细胞SOD活性及降低其MDA含量(P＜0.05).结论 松花粉可能通过减轻成纤维细胞的氧化损伤,从而保护细胞mtDNA延缓成纤维细胞的衰老.     

充血性心力衰竭患者线粒体DNA缺失的意义

为探讨线粒体DNA（mtDNA）5．0kb缺失与充血性心力衰竭（CHF）发病的关系，应用聚合酶链反应（PCR）技术分析72例CHF患者的mtDNA。结果显示，CHF患者mtDNA5．0kb缺失率明显高于对照组（0.254％±0．08％比0．032％±0．01％，P＜0．01）；CHF组内扩张型心肌病组及冠心病组mtDNA5．0kb缺失率高于风心病及高血压心脏病组（P＜0．05）。研究中还发现，随着心功能分级增加，mtDNA5．0kb缺失率增高（P＜0．05）。提示mtDNA缺失影响心肌细胞线粒体呼吸功能，心肌能量供应障碍与CHF的发生、发展有关。     

一种新发现的线粒体DNA大片段缺失与衰老的关系初探

目的 在人外周血细胞中筛选新的线粒体DNA（mtDNA）大片段缺失突变,探讨mtDNA尤其在片段缺失突变与衰老的关系。方法 以人外周血细胞DNA为模板,进行聚合酶链反应（PCR）扩增,产物克隆、测序。用PCR半定量方法测定1条长达13．1kb的mtDNA缺失突变,＜60岁组和≥60岁组突变型与野生型的比例,差异有显著性。结论 该突变以前未见报道,可能是一种与衰老有关的缺失突变。     

A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues.

An assay that selectively amplifies a specific deletion of the mitochondrial genome has been used to study the extent of the deletion's accumulation in a variety of human tissues. The deletion occurs at much higher levels in nervous and muscle tissues than in all other tissues studied. The variation in deletion level between the same tissues in different persons of similar age appears to be less than the variation among tissues within an individual. Tests for artifactual explanations of the level differences were each negative. Three cellular parameters that are correlated with the level of the deletion are identified. The preferential accumulation of deleterious mitochondrial mutations in a restricted subset of aging human tissues may compound deficiencies of function in those tissues that accrue with age.     

恩替卡韦致外周血单个核细胞中线粒体DNA损伤的研究

目的 探讨长期服用恩替卡韦是否会导致慢性乙型肝炎(CHB)患者外周血单个核细胞(PBMCs)中线粒体损伤.方法 CHB患者52例,分为3组:(1)2年组:恩替卡韦单药治疗2年,共17例;(2)3年组:恩替卡韦单药治疗3年,共17例;(3)对照组:未经抗病毒治疗的首次发病的CHB患者18例.实时定量PCR检测各组患者外周血单个核细胞中线粒体DNA含量;酶联免疫吸附法检测血浆丙二醛、F2-isoprostanes含量;分光光度法检测血浆总抗氧化能力.满足方差齐性的数据,采用方差分析方法进行组间差异的比较;不满足方差齐性的数据,用秩转换的方差分析思想进行组间差异的比较.计数资料采用x2检验或Fisher's确切概率法进行组间差异的比较. 结果 3组间RQ值(线粒体DNA/核DNA)差异有统计学意义(F=5.233,P=0.009).3年组RQ值为0.5±0.3,低于对照组1.4±1.2,两组比较,差异有统计学意义(P=0.022);2年组RQ值0.4±0.2与对照组相比,P=0.004,差异有统计学意义.3组间F2-isoprostanes含量差异有统计学意义(F=6.266,P=0.004).3年组F2-isoprostanes为(1.2±0.5)ng/ml,显著低于对照组的(3.6±2.9) ng/ml和2年组的(2.4±1.3) ng/ml,P值分别为0.002,0.007.3组间血浆总抗氧化能力差异有统计学意义(F=4.326,P=0.019).3年组血浆总抗氧化能力为(2.6± 1.2) U/ml,显著低于对照组的(5.0±3.0) U/ml,差异有统计学意义(P=0.005),与2年组的血浆总抗氧化能力(3.2±1.6)U/ml相比,差异无统计学意义(P=0.227).血浆丙二醛3组间差异无统计学意义(P=0.749).结论 服用恩替卡韦治疗CHB达3年时总的效应是可能会导致外周血单个核细胞中线粒体DNA数量减少,但对线粒体功能的损伤在机体代偿范围内,不会损害线粒体功能.     

Fatal dilated cardiomyopathy associated with a mitochondrial DNA deletion

A 27-year-old man was admitted to hospital because of severe cardiac failure. Investigation revealed dilated cardiomyopathy with a left ventricular ejection fraction of 15-20%. During adolescence the patient had been investigated for growth retardation and he also had progressive external ophthalmoplegia. There had been no symptoms of cardiac disease until 2 weeks before admittance. An endomyocardial biopsy showed cardiomyocytes deficient in cytochrome c oxidase (COX) in a mosaic pattern. A skeletal muscle biopsy showed mitochondrial myopathy with COX-deficient ragged-red fibers. Molecular genetic analysis revealed a heteroplasmic, 3.8-kb, mitochondrial DNA (mtDNA) deletion in heart and muscle. PCR-based quantification of the proportion of mtDNA with deletion showed 47% mutated mtDNA in the myocardial biopsy and 68% in muscle. In spite of treatment, the condition deteriorated and the patient died 5 days after admittance. This case demonstrates that mtDNA deletions may occasionally be the cause of severe dilated cardiomyopathy, and that morphological and molecular genetic diagnosis may be obtained by endomyocardial biopsy.     

Primary adrenal insufficiency in a child with a mitochondrial DNA deletion

Mitochondrial disorders can affect any organ system, but certain tissues, such as skeletal muscle, heart, and brain are more susceptible to oxidative phosphorylation defects because of their high energy requirements. Endocrinological manifestations, especially diabetes mellitus, are common but they rarely dominate the clinical picture. We describe a 5-year-old girl who died of primary adrenal insufficiency with a mitochondrial disease. Biochemical studies in muscle showed decreased respiratory chain enzyme activities. We detected a novel 7.0 kb mtDNA deletion in muscle from the proband, but not in her mother's white blood cells. Our findings further enlarge the spectrum of clinical presentation associated with mitochondrial DNA deletions.     

Atrial fibrillation is associated with accumulation of aging-related common type mitochondrial DNA deletion mutation in human atrial tissue

STUDY OBJECTIVE: Accumulation of somatic mutations of mitochondrial DNA (mtDNA) contributes to the aging process and progressive organ dysfunction. We investigated the mitochondrial DNA with 4977-base-pair mtDNA deletion mutation (mtDNA(4977)) in human atrial tissue and correlated the amount of mtDNA(4977) to clinical atrial fibrillation (AF). METHODS AND RESULTS: Atrial tissue from the right atrial appendage was obtained in 88 patients during open-heart surgery (22 children/adolescents and 66 adults). The amount of mtDNA(4977) was measured using a nested polymerase chain reaction protocol and normalized to wild-type mtDNA. We found that the mtDNA(4977) was absent in all 22 pediatric/adolescent patients. In the adult group, the relative amount of mtDNA(4977) was significantly higher in patients with AF than in patients without AF (0.55 +/- 0.26 vs 0.35 +/- 0.29, p < 0.007) [mean +/- SD]. The amount of mtDNA(4977) was also positively associated with age (r = 0.29, p < 0.01). Left and right atrial pressures, left atrial dimension, hypertension, and cardiac diagnosis did not influence the amount of mtDNA(4977) significantly. Further multivariate analysis showed that both aging and AF contributed independently to the accumulation of mtDNA(4977). CONCLUSION: AF is associated with an increase of mtDNA(4977). This change is similar to the aging process of atrial tissue and might contribute to atrial dysfunction in AF.     

Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts

Methods of rapidly extracting chromosomal DNA from human pathogenic yeasts were used in mitochondrial DNA (mtDNA) studies. This paper is concerned with rapid and reliable methods of extracting mtDNA for sequence analysis for species or strain identification, and epidemiological study of medically important fungi and fungal infections. To determine the optimal method of mtDNA extraction without isolating mitochondria, we examined three commonly used methods: 1). boiling, 2). glass bead disruption, and 3). a commercially available kit. We assessed the amount and quality of DNAs using a spectrophotometer and specific polymerase chain reaction (PCR). The DNA yield depended on the extraction method used and the yeast species. An adequate amount of mtDNA was obtained with both glass beads and a commercially available kit to amplify the mitochondrial gene using PCR without isolating the mitochondria. These techniques are convenient for extracting DNA from a variety of small-scale samples.