线粒体脑肌病相关的线粒体DNA突变

<正>线粒体DNA(mtDNA)或核DNA突变可造成线粒体结构形态或(和)功能发生异常,由此导致的以神经肌肉系统(脑和肌肉组织)受累为主的多系统疾病,称为线粒体脑肌病。mtDNA突变是导致神经肌肉系统发生病变的重要原因之一。早在上世纪70年代,有研究者在患有脑肌病儿童的肌肉组织中首次发现了线粒体形态的畸变和呼吸链功能的缺陷,提示线粒体功能异常可能与该疾病有关[1]。1988年,Holt等[2]首先报     

线粒体DNA突变与阿尔茨海默病的相关性

阿尔茨海默病(Alzheimerdisease,AD)是一种以进行性痴呆为特征的脑变性疾病,很多研究显示它的病变与脑内的能量代谢衰减有密切关系。线粒体是动物细胞中除细胞核外惟一的含有DNA的细胞器,同时也是细胞活动的能源工厂。线粒体形态及功能的缺陷必将引发一系列的细胞功能失调。近年来研究发现阿尔茨海默病患者的脑细胞中的线粒体形态以及线粒体DNA发生了很大的改变,尤其是线粒体DNA缺失率和突变率都明显高于正常人。有关阿尔茨海默病患者的细胞中线粒体功能改变以及线粒体DNA突变的研究已有较大进展,现作初步阐述。     

线粒体DNA突变与骨髓增生异常综合征

骨髓增生异常综合征是以造血干细胞的异常克隆性增生、血细胞减少、骨髓出现病态造血为特征的一类疾病。最近的研究发现：环形铁粒幼细胞作为MDS的一个重要病态造血现象，可存在于各型MDS中，并且具有明显的异质性。这种克隆内的异质性现象与线粒体DNA突变有关，线粒体DNA突变主要引起呼吸链异常，继而导致多种病理生理改变，在MDS的发病机制中可能有重要的作用。     

线粒体tRNALeu(UUR)基因A3243G突变糖尿病家系的基因诊断

目的确认一个由线粒体tRNA^Leu（UUR）基因A3243G点突变引起的母系遗传糖尿病家系，并分析其临床特征。方法采用PCR产物直接测序法对53例无血缘关系的有家族史的2型糖尿病（T2DM）患者线粒体DNA（mitochondria DNA，mtDNA）片段（nt3153-nt3551）进行点突变筛选，然后对筛选到的A3243G异质性突变样本作DHPLC分析确认。详细调查此突变糖尿病家系发病特征，并做线粒体基因组全序列分析。结果在53例样本中检测到1例A3243G突变，对其家系调查发现7例母系成员中5例患病（1男4女），患者多数伴听力损害，体重指数减低，口服降糖药效果不佳最终需用胰岛素治疗，呈母系遗传，发病年龄不等，平均32．75岁。线粒体全基因组测序表明患者存在包括A3243G突变的共33个变异位点，但除A3243G突变外其余都不是进化上高度保守的位点，因此A3243G突变是与这个糖尿病家系发病有关的惟一的mtDNA突变。结论线粒体tRNA^Leu（UUR）基因A3243G突变是这个三代母系遗传糖尿病家系发病的原因，其临床特点是发病年龄轻、不胖、母系遗传、耳聋发生率和使用胰岛素比率高等。     

苏中地区母系遗传性高血压病患者线粒体DNA 3777～4679区域基因突变相关研究

目的探讨苏中地区母系遗传性高血压病(MIEH)患者发病与线粒体DNA(mtDNA)突变的关系。方法实验分为研究组和对照组。收集300例MIEH患者作为研究组,调查发病年龄、进行超声心动图检查。选择同期体检证实的300例健康人群作为对照组。抽取受试者外周静脉血,对序列3777⁴679位置的mtDNA进行直接基因测序,通过与标准人类mtDNA序列的剑桥序列进行对比,分析正常人群与MIEH患者mtDNA突变情况;根据mtDNA有无突变,对发病年龄和超声心动图资料进行对比分析。结果 (1)与正常人群相比,突变集中在一些呼吸链NADH氧化还原酶亚单位1(ND1)、NADH氧化还原酶亚单位2(ND2),高突变位点为ND1C3970T突变。(2)MIEH中mtDNA突变者发病年龄明显低于非突变者(P<0.05),并且突变者舒张末期左心室内径(LVIDd)、收缩末期左心室内径(LVIDs)平均值明显高于无突变者(P<0.05);左心室射血分数(LVEF)平均值低于无突变者(P<0.05)。结论 mtDNA结构改变可以导致其相应功能异常,进而影响心脏靶器官结构和功能,参与MIEH的发生和发展。     

有氧运动延缓衰老线粒体DNA突变的机制

目的:线粒体DNA突变在衰老过程中起核心作用。总结有氧运动在延缓机体衰老过程中对线粒体DNA突变的影响,探讨有氧运动延缓衰老的机制。资料来源:应用计算机检索Medline数据库1996-01/2006-01期间关于线粒体DNA突变与有氧运动延缓衰老的文章,检索词为"aerobics exercise,aging,mtDNA mutation",限定文章语言种类为English。同时计算机检索中国期刊全文数据库、万方数据库1994-01/2006-12期间的有氧运动、衰老和线粒体DNA突变相关的文章,检索词"衰老,线粒体DNA突变,有氧运动",并限定文章语言种类为中文。资料选择:对资料进行初审,纳入标准:①有氧运动延缓衰老关系的理论研究。②线粒体DNA突变与衰老关系的基础与临床研究。③有氧运动对线粒体DNA突变的影响研究。资料提炼:共收集到36篇线粒体DNA突变与有氧运动延缓衰老相关的文献,均为全文,32篇符合纳入标准,排除4篇重复性研究。资料综合:①有氧运动可以通过减少线粒体DNA突变,从而达到延缓机体的衰老。有氧运动可能通过影响自由基及抗氧化系统、细胞凋亡、线粒体的结构和功能,对衰老进程产生重要影响。②线粒体DNA突变随增龄而积累,达到一定阈值后,可导致细胞能量供应的严重障碍,从而造成组织器官生理功能的减退。③长期有氧运动可通过刺激心肌、骨骼肌线粒体的生成及蛋白质的合成而延缓线粒体形态结构的改变,有利于维持线粒体功能以满足机体对其能源的需求。结论:中、低强度有氧运动可以提高机体有氧工作能力,增进线粒体氧化磷酸化的功能,对延缓衰老具有一定的积极作用。有氧运动可以减少心肌、骨骼肌等线粒体DNA突变,提示有氧运动在延缓衰老机制中,减少线粒体DNA突变是可能机制之一。     

线粒体DNA突变与阿尔茨海默病的相关性

阿尔茨海默病（Alxheimer disease,AD）是一种以进行性痴呆为特征的脑变性疾病，很多研究显示它的病变与脑内的能量代谢衰减有密切关系。线粒体是动物细胞中除细胞核外惟一的含有DNA的细胞器，同时也是细胞活动的能源工厂。线粒体形态及功能的缺陷必将引发一系列的细胞功能失调。近年来研究发现阿尔茨海默病患的脑细胞中的线粒体形态以及线粒体DNA发生了很大的改变。尤其是线粒体DNA缺失率和突变率都明显高于正常人。有关阿尔茨海默病患的细胞中线粒体功能改变以及线粒体DNA突变的研究已有较大进展，现作初步阐述。     

线粒体肌病患者的肌细胞线粒体DNA突变检测

目的:分析线粒体肌病患者骨骼肌线粒体DNA的突变情况,为疾病诊断提供依据.方法:用聚合酶链反应(polymerase chain reaction,PCR)扩增线粒体22个tRNA基因,利用变性高效液相色谱分析技术(denaturing high-performance liquid chromatography,DHPLC)对PCR产物进行突变筛选,出现异常峰型的tRNA基因进行核苷酸序列测定,明确突变位点.结果:例1 tRNA-Val基因发生A1625G纯合突变,例2tRNA-Val基因发生A1625G/A杂合突变,例3tRNA-Arg基因发生A10411C/A杂合突变.结论:线粒体DNA中的tRNA基因突变是线粒体肌病的重要病因之一.     

RT-ARMS-qPCR定量测定线粒体耳聋患者mtDNA A1555G位点突变

目的 探讨RT-ARMS-qPCR(real time amplification refractory mutation system quantitativePCR)系统定量测定线粒体DNA(mitochondrial DNA,mtDNA)A1555G位点突变在线粒体耳聋发病机制研究中的价值.方法 以PeR扩增含mtDNA 1555位点的片段,并将其克隆到pGEM-T Easy载体上,构建质粒标准品;在引物3'端插入错配碱基AC建立RT-ARMS-qPCR系统,对含突变型和野生型mtDNA 1555位点片段的12例线粒体耳聋患者进行定量检测.结果 RT-ARMS-qPCR系统在检测1个含野生型mtDNA 1555的重组质粒DNA模板时,其批内变异系数(CV)为1.34%,批间CV为1.96%,线性范围为102-108拷贝/ul;突变型或野生型引物只特异扩增相对应的序列,特异性好;突变型/野生型mtDNA拷贝数及突变型所占的百分比与耳聋的严重程度相关(r=0.771,P=0.003),突变型mtDNA所占的比例越高,耳聋程度越严重.结论 RT-ARMS-qPCR系统适合于定量检测mtDNAA1555G点突变的线粒体DNA片段,结果特异、稳定、准确.线粒体耳聋的严重程度与含突变型和野生型mtDNA 1555位点的拷贝数比例有关.     

突变敏感性分子开关检测线粒体DNA A1555G位点的条件优化

目的对高保真酶介导的突变敏感性分子开关技术检测线粒体DNA(mt DNA)A1555G位点条件进行优化。方法利用3'硫代磷酸化修饰的突变型引物和野生型引物作为下游引物,在其上游设计一条公共引物分别构成突变引物对和野生引物对,以构建好的包含mt DNA A1555G位点的突变型质粒和野生型质粒为模板,进行高保真聚合酶介导的双向引物延伸反应,对PCR体系中的退火温度、引物浓度、模板浓度等条件优化,通过凝胶成像系统对其PCR结果进行分析确定最佳反应条件。结果分子开关技术检测mt DNA A1555G位点的最佳PCR条件:退火温度为61.0℃,引物浓度为0.6μmol/L,检测模板浓度为103~106copies/μL。结论确定了分子开关技术检测mt DNA A1555G位点的最佳反应条件,为该技术在线粒体DNA的突变筛查中的应用提供依据。     

遗传性蛋白C缺陷症家系的一个基因突变

目的 研究一个遗传性蛋白C（PC）缺陷症家系的遗传表型及基因特征。方法 PC活性用凝固法测定，PC抗原用ELISA方法测定。用PCR扩增2代家系12个成员中4个PC活性及抗原减低的PCⅡ-Ⅸ号外显子片段，用单链构象多态性（SSCP）分析cDNA变性后的差异，用测序法检测突变点。用限制性酶切验证突变点，同时分析家系的基因型。结果 该家系2代4名成员PC抗原水平在34．3％－67．8％（参考值80％－120％）。PC活性在22％－49％（参考值70％－130％），较正常参考范围明显减低。限制性酶切分析该家系12名成员时发现9名成员存在基因的突变。基因突变位点在Ⅶ号外显子第6219位核苷酸G→A突变，使正常编码的CGG精氨酸突变为CAG谷氨酰胺。结论 该家系为I型PC缺陷症，基因分析证明先证为杂合子型。在PCⅦ号外显子上第6219位核苷酸G→A突变，在蛋白质合成过程中第169位精氨酸被谷氨酰胺替代（R→Q），为目前国内献中尚未报道的一个基因突变点。     

T2182C mutation in 23S rRNA is associated with clarithromycin resistance in Helicobacter pylori isolates obtained in Bangladesh

Twelve clarithromycin-resistant (MIC, > or = 1 microg/ml) Helicobacter pylori isolates were analyzed for point mutations in the 23S rRNA gene. Sequence analysis of all of the resistant isolates revealed a T-to-C transition mutation at position 2182. Transformation experiments confirmed that a single T-to-C transition mutation at position 2182 is associated with clarithromycin resistance.     

GNAS1 T393C polymorphism is associated with migraine

Migraineurs have an interictal sympathetic nervous system (SNS) hypofunctionality and hypersensitivity to adrenergic amines. The GNAS1 T393C polymorphism has been associated with a distinct SNS sensitivity in healthy subjects. We tested GNAS1 T393C variant in two independent sets of subjects. In the case-control subset, 365 migraine patients [194 with aura (MA)] vs. 347 healthy controls were studied. A significant excess of the CC genotype was found in migraneurs (31.2%) as opposed to controls (20.2%; P=0.003). Using a logistic regression model corrected for sex, the CC genotype conferred a general risk for migraine twice [odds ratio (OR) 1.79, 95% confidence interval (CI) 1.27-2.53; P=0.001] higher than CT/TT genotypes. Using parents from 117 migraine families, a marginally significant trend for association could be observed (P=0.025), but the transmission disequilibrium test for alleles maternally transmitted failed to demonstrate familial association. In this subgroup, CC genotype conferred a risk for migraine over twice (OR 2.20; 95% CI 1.14-4.40; P=0.019) higher than TT/TC genotypes. In conclusion, the GNAS1 T393C variant is associated with migraine, which suggests a genetic basis for its higher SNS sensitivity.     

一个遗传性全白甲中国家系FZD5、LOC729607基因突变分析

背景：遗传性全白甲是一种少见的常染色体显性遗传病，其致病基因目前尚未发现。目的：对一个中国汉族遗传性全白甲家系的2个候选基因进行突变筛查检测，研究遗传性全白甲的分子遗传基础。设计、时间及地点：候选基因突变筛查检测，于2007—01／06在中国人类基因组北方研究中心实验室完成。材料：搜集一遗传性全白甲家系患者静脉血，制备基因组DNA。方法：采用定位候选克隆法，在已经定位的致病基因区域内，选择FZD5，LOC729607基因进行突变筛查。应用PCR扩增、PCR产物直接测序的方法检测和对比遗传性全白甲家系内3例患者和一个正常人候选基因的外显子区及邻近内含子区域碱基顺序，并将测序结果与NCBI数据库内标准系列对比。主要观察指标：检测和对比遗传性全白甲家系内患者和正常人候选基因的外显子区及邻近内含子区域碱基顺序，并将测序结果与NCBI数据库内标准系列对比，发现患者、正常人、标准系列碱基顺序的差异，分析这些差异的意义。结果：FZD5基因exon706位碱基A→G：LoC729607基因exon5下游51661位缺T，exon6上游57257位C→T，exon7上游71187位AG杂合。所检测到的变异在家系中患者和正常人中都存在，在NCBI数据库中能查到相应的位点，是单核苷酸多态。结论：在FZD5（frizzled5），LOC729607基因编码区域及邻近非编码区域中未发现遗传性全白甲家系的致病突变。     

Gain-of-function mutation in the KCNMB1 potassium channel subunit is associated with low prevalence of diastolic hypertension

Hypertension is the most prevalent risk factor for cardiovascular diseases, present in almost 30% of adults. A key element in the control of vascular tone is the large-conductance, Ca(2+)-dependent K(+) (BK) channel. The BK channel in vascular smooth muscle is formed by an ion-conducting alpha subunit and a regulatory beta(1) subunit, which couples local increases in intracellular Ca(2+) to augmented channel activity and vascular relaxation. Our large population-based genetic epidemiological study has identified a new single-nucleotide substitution (G352A) in the beta(1) gene (KCNMB1), corresponding to an E65K mutation in the protein. This mutation results in a gain of function of the channel and is associated with low prevalence of moderate and severe diastolic hypertension. BK-beta(1E65K) channels showed increased Ca(2+) sensitivity, compared with wild-type channels, without changes in channel kinetics. In conclusion, the BK-beta(1E65K) channel might offer a more efficient negative-feedback effect on vascular smooth muscle contractility, consistent with a protective effect of the K allele against the severity of diastolic hypertension.     

IL-2 gene C/T polymorphism is associated with prostate cancer

Cytokines are reported to be associated with the formation of prostate cancer. Our aim was to investigate whether C/T polymorphisms of the interleukin-2 (IL-2) gene and IL-2 receptor beta (IL-2RB) gene are associated with prostate cancer. We compared the frequency of the polymorphisms of the IL-2 gene and the IL-2RB gene between 96 patients with prostate cancer and 105 healthy male volunteers from the same area (age >60 years). They were followed for at least 5 years. There was a significant difference in distribution of the genotype of the IL-2 gene polymorphism between the prostate cancer group and the control group (P = 0.017). The distribution of the TT homozygote of the IL-2 gene was significantly higher in the cancer group (32.3%) than in the control group (16.2%). However, no significant statistical difference was found between the polymorphism of the IL-2 gene and prostate cancer in survival analysis during a 5-year follow up period (log rank test; P = 0.19). There was no significant difference in the distribution of the genotype of the IL-2RB gene polymorphism between controls and cancer patients (P = 0.388). This study suggests that the IL-2 gene may be associated with susceptibility to prostate cancer in the Taiwan population.     

Pyrethroid resistance in Sitophilus zeamais is associated with a mutation (T929I) in the voltage-gated sodium channel

The maize weevil, Sitophilus zeamais, is the most important pest affecting stored grain in Brazil and its control relies heavily on the use of insecticides. The intensive use of compounds such as the pyrethroids has led to the emergence of resistance, and previous studies have suggested that resistance to both pyrethroids and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) may result from reduced sensitivity of the insecticide target, the voltage-gated sodium channel. To identify the molecular mechanisms underlying pyrethroid resistance in S. zeamais, the domain II region of the voltage-gated sodium channel (para-orthologue) gene was amplified by PCR and sequenced from susceptible and resistant laboratory S. zeamais strains that were selected with a discriminating dose of DDT. A single point mutation, T929I, was found in the para gene of the resistant S. zeamais populations and its presence in individual weevils was strongly associated with survival after DDT exposure. This is the first identification of a target-site resistance mutation in S. zeamais and unusually it is a super-kdr type mutation occurring in the absence of the more common kdr (L1014F) substitution. A high-throughput assay based on TaqMan single nucleotide polymorphism genotyping was developed for sensitive detection of the mutation and used to screen field-collected strains of S. zeamais. This showed that the mutation is present at low frequency in field populations and is a useful tool for informing control strategies.