Leber遗传性视神经病三个家系患者线粒体DNA突变位点的研究

目的对Leber遗传性视神经病（Leber’s hereditary optic neuropathy，LHON）家系的原发突变位点11778与继发突变位点9804、13708、13730、15257进行突变分析，探讨两者之间相关性及对LHON的影响。方法应用聚合酶链反应一单链构象多态性和DNA测序对3个LHON家系37位母系成员和47名正常人的线粒体DNA（mitochondrial DNA，mtDNA）进行检测。结果16例患者及其母系亲属均存在11778位点突变，未发现9804、13708、13730、15257位点突变，但DNA测序发现13759、13928、13942、15301、15326、15323这6个新突变位点。结论3个家系都存在mtDNA11778位点突变，在13759位点患者突变率远高于正常人，差异有统计学意义（P〈0．001），表明13759是LHON新的继发突变位点。     

中国人LHON患者mtDNA继发突变的筛查

目的分析中国人Leber遗传性视神经病变(Leber's hereditary optic neuropathy,LHON)线粒体DNA 4个继发突变位点与LHON发病的关系.方法分别用突变特异性引物聚合酶链反应,异源双链-单链构象多态性,限制性片段长度多态性和DNA测序方法,对137例LHON患者、60例不明原因球后视神经炎进行mtDNA3394C、9438A、13708A、4216C 4个继发位点的检测,并以100例正常人作对照.结果在4例LHON患者(包括3例11778突变、1例3460突变)、2例不明原因球后视神经炎患者及1例正常人中发现存在13708位点突变(G→A),引起ND5蛋白第458位中度保守丙氨酸变成苏氨酸(A458T).经χ2检验,无统计学意义.在1例正常人中检测到3394位点T→C突变,造成ND1蛋白第30位高度保守酪氨酸变成组氨酸(Y30H).137例LHON患者及60例不明原因球后视神经炎患者均未发现此位点突变.在137例LHON患者、60例不明原因球后视神经炎患者及100例正常人中未检测到9438及4216位点突变.结论我们的研究结果与日本、韩国研究结果相似,初步排除了在中国人kber遗传性视神经病变患者中13708A、3394C、9438A、4216C四种突变协同原发突变发病的可能性.     

Leber遗传性视神经病变相关的线粒体G11778A,T14484C和G3460A突变筛查

目的探讨Leber遗传性视神经病变(Leber's hereditary optic neuropathy,LHON)相关的线粒体DNA致病性的突变位点,为LHON的分子诊断和早期预防提供理论依据。方法收集2016年6月至2018年1月在杭州市第一人民医院就诊的LHON患者100例以及80例性别、年龄相仿的正常对照。使用PCR-Sanger测序法检测线粒体G11778A,T14484C和G3460A这三个原发性突变位点。结果经过测序比对,我们共发现有5例患者携带这3个致病性线粒体突变位点,其中携带线粒体G11778A突变的患者2例,T14484C突变的患者2例,携带G3460A突变的个体1例,这些突变位点在正常人群中均未发现。结论线粒体G11778A,T14484C以及G3460A突变是LHON相关的致病性突变位点,在临床上开展这些突变位点的早期筛查显得非常有必要,这对于LHON的预防和分子诊断具有较好的指导作用。     

中国人Leber 遗传性视神经病变的原发突变及临床特征

目的 分析中国人Leber遗传性视神经病变(Leber’s hereditary optic neuropathy,LHON)线粒体DNA3个原发致病基因突变遗传及其临床特征。方法 分别用突变特异性引物聚合酶链反应,异源双链-单链构象多态性,限制性片段长度多态性和DNA测序方法,对110个家系的156例LHON患者进行11778A、3460A、14484C 3个原发位点检测,并收集患者病史及其临床资料,进行统计学分析。结果 110例LHON先证者中,11778位点突变者100例,占90．9％;3460位点突变者2例,占1．8％;14484位点突变者8例,占7,3％。不同突变位点的LHON患者发病时视力分布：125人(250眼)11778位点突变患眼中发病时视力≤0．01(占17．6％),视力介于0.01至0．1之间(占52．1％),视力≥0．1(占30．3％);28人(56眼)14484位点突变患眼中无患眼视力低于0．01视力介于001至0．1之间(占12．7％),视力≥0．1(占87．3％);3人(6眼)3460位点突变患眼视力均介于0．03至0．08之间。视力恢复情况：250只11778位点突变的患眼中,6．97％的眼视力有所恢复,平均最终视力0．03(指数～0．07);56只14484位点突变的患眼中,50％的眼视力有所恢复,占50％,平均最终视力0．8(0．3～1．2)。结论 中国人LHON患者mtDNA 3个原发致病突变中,以11778A位点突变为主、14484C位点突变较少、3460A罕见。LHON的临床表现与致病突变位点有关,14484C突变患者的发病视力及视力恢复情况明显好于11778A患者。     

Leber遗传性视神经病变线粒体DNA三个原发性突变位点的研究

目的 通过对Leber遗传性视神经病变(Leber's hereditary optic neuropathy,LHON)线粒体DNA ND4 11778G＞A、ND1 3460G＞A和ND6 14484T＞C 3个原发性突变位点序列分析,阐明LHON患者发病的分子机理.方法 PCR扩增35例患者的上述3个原发性突变位点所在的区段,PCR产物直接测序分析.结果 35例患者中,6例存在ND4 11778 G＞A突变位点,1例存在ND1 3460 G＞A突变位点,未检出ND6 14484 T＞C突变位点,3个原发性突变位点的检出率为20.0％(7/35).35例患者均存在ND4 11719G＞A同义突变,除此突变位点外,23例(65.7％)患者共计筛出21个突变位点,2种突变类型.其中13例患者存在单个突变位点,8例存在2个突变位点,2例存在3个突变位点.21个突变位点中,ND4 11778G＞A突变频率最高,为28.6％(6/21); ND1 3552 T＞A、ND6 14470 T＞C、ND4 11794 T＞C、ND1 3497 C＞T和3644 T＞C位点突变频率依次为19.0％(4/21)、19.0％(4/21)、14.3％(3/21)、9.5％(2/21)和9.5％(2/21).3例存在ND4 11794 T＞C突变位点的患者,2例为异质性突变,1例为同质性突变.结论 LHON患者线粒体DNA ND4 11778G＞A、ND1 3460G＞A和ND6 14484T＞C 3个原发性致病突变中,以ND4 11778 G＞A为主;继发性突变位点ND1 3552 T＞A或ND1 3644 T＞C单独或协同原发性突变位点ND4 11778 G＞A导致LHON的发生,与单纯携带ND4 11778 G＞A的患者相比视力受损较弱.     

线粒体tRNAThr A15951G可能是与Leber遗传性视神经病变相关的基因突变

目的 进一步分析中国汉族Leber遗传性视神经病变(Leber's hereditary optic neuropathy,LHON)家系的临床和分子遗传学特征,阐明LHON的分子致病机制.方法 对2例具有典型LHON临床特征的先证者和家系其他成员进行眼科学及其临床检查.对这2个家系先证者使用24对有部分重叠的引物进行线粒体DNA(mitochondrial DNA,mtDNA)全序列扩增分析.结果 检查发现这些家系成员中视力损害的外显率分别为5.3％(1/19)、18.2％(4/22).经mtDNA测序分析,并没有发现mtDNA G11778A、G3460A和T14484C 3个常见的突变,在tRNAThr上发现了A15951G同质性突变位点.线粒体DNA全序列分析显示2个家系呈现mtDNA多态性,都属于东亚单倍型D4b1.A15951G突变位于线粒体tRNAThr高度保守区(通用位点为71位),可能导致tRNA空间结构和稳定性发生改变,线粒体蛋白合成功能受损,最终发生视力损害.结论 线粒体tRNAThr A15951G可能是与Leber遗传性视神经病变相关的致病性线粒体基因突变.     

Leber''''s遗传性视神经病线粒体DNA突变二例的检测

目的检测两例Leber's遗传性视神经病的突变位点.方法常规酚-氯仿法提取2名LHON患者基因组DNA,PCR扩增后对mtDNA11778进行检测.结果mtDNA11778位点处存在G→A突变.     

Leber＇s遗传性视神经病线粒体DNA突变二例的检测

目的检测两例Leber＇s遗传性视神经病的突变位点.方法常规酚-氯仿法提取2名LHON患者基因组DNA,PCR扩增后对mtDNA11778进行检测.结果mtDNA11778位点处存在G→A突变.     

中国Leber遗传性视神经病变G11696A突变两个家系分析

目的对两个中国Leber遗传性视神经病变(Leber’shereditary optic neuropathy,LHON)家系的临床和分子遗传学特征进行分析。方法眼科临床检查发现在这两个家系中只有先证者1人出现视力障碍,发病年龄分别为10岁和17岁。对这两个家系先证者使用24对有部分重叠的引物进行线粒体DNA(mitochondrial DNA,mtDNA)全序列扩增分析。结果没有发现mtDNAG11778A、G3460A和T14484C3个常见的突变位点,而发现了与LHON相关的ND4G11196A同质性突变位点的存在,在167名正常对照只发现1例G11696A突变。结论线粒体DNA全序列分析发现两个家系呈现独特的mtDNA多态性,都属于东亚单体型D4。不完全外显率和正常对照频率(1/167)表明G11696A突变本身不足以导致LHON的发生,说明其它因素在这两个LHON家系的表型表达中也起一定的作用。在这些家系mtDNA中缺乏影响重要功能突变位点的存在,排除了线粒体背景对LHON临床表型的影响。因此,核修饰基因、环境因素可能对两个中国G11696A突变家系的外显率和发病严重程度起促进作用。     

线粒体DNA11778突变所致Leber遗传性视神经病变外显率分析

目的 分析携带线粒体DNA（mitochondrialDNA,mtDNA)11778突变者视神经病变的外显率。方法 对经基因诊断确定为mtDNA11778突变的Leber遗传性视神经病变（Leber hereditary optic neuropathy,LHON)家系进行分析。确定mtDNA11778突变携带者及患者。结果 16个家系中mtDNA11778突变携带者130人,其中男65人,女65人,130人突变携带者中43人患病,外显率33.1%。男性患者34人,男性外显率52.3%,女性患者9人,女性外显率13.8%,男女患病比率3.8:1,患者中男性占79%。结论 携带纯合性mtDNA11778位点突变的中国人,LHON外显率近1/3。     

The effects of storage of blood and isolated DNA on the integrity of DNA

Long-term storage of DNA is required for a number of genetic studies; prior to extraction, blood samples may be subject to elevated temperatures for variable intervals. We have studied the effect of temperatures ranging from ?70°C to +65°C on human blood and on DNA extracted from it. DNA in solution stored at ambient temperatures up to 37°C for 6 months was digestible by three different restriction endonucleases, whereas storage at 45°C is deleterious after 6-7 weeks. DNA can be extracted from blood samples stored at ?70°C for at least 2 months or at 23°C for a week or more, but blood stored at these temperatures may yield less high-molecular-weight DNA. Cell pellets from which plasma has been removed also can serve as a source of DNA. Isolated DNA stored dry for years (up to 30) is difficult to dissolve and may appear degraded, but a sample stored dry for 13 years and then in solution at ?20°C for 7 years appeared to be intact.     

DNA homologies between the genomes of Choristoneura fumiferana and Autographa californica nuclear polyhedrosis viruses

The DNA sequence homology between the genomes of Choristoneura fumiferana and Autographa californica multicapsid nuclear polyhedrosis viruses (CfMNPV and AcMNPV) were compared by hybridization of nick-translated [32P]CfMNP[V DNA to restricted AcMNPV genome. In the presence of 5 x SSC and 50% formamide the CfMNPV DNA exhibited extensive homology to the AcMNPV genome. When the stringency conditions of hybridization were lowered, we observed hybridization to almost all the EcoRI fragments of AcMNPV. We then utilized the cloned EcoRI fragments from both genomes to obtain more detailed information, and to localize the hybridizing fragments on the EcoRI physical map of AcMNPV. It was clear that some CfMNPV clones hybridized to more than one fragment of the AcMNPV genome indicating that there has been some DNA sequence rearrangement in the AcMNPV genome.     

Specific probes for Trypanosoma (Trypanozoon) evansi based on kinetoplast DNA minicircles

Trypanosoma evansi is difficult to distinguish from other members of subgenus Trypanozoon, save for its inability to develop cyclically in the tsetse fly and its characteristic kinetoplast DNA (kDNA). We have used cloned kDNA minicircle fragments as specific probes to distinguish T. evansi from other trypanosomes of subgenus Trypanozoon. Two probes were required, each specific for one of the subgroups of T. evansi previously described. Probe A reacted only with the major isoenzyme group of T. evansi stocks, which have minicircle type A and occur in South America, Kenya, Sudan, Nigeria and Kuwait. The probe did not hybridise with various Trypanosoma brucei spp. stocks, Trypanosoma vivax, Trypanosoma congolense or Trypanosoma simiae, nor with trypanosomes of the minor isoenzyme group of T. evansi stocks found in Kenya with type B minicircles. Probe B was specific for the latter. The probes were sensitive down to a level of 100 trypanosomes in a dot blot. These probes thus provide a simple means of distinguishing T. evansi from T. brucei spp. using comparatively few trypanosomes and without resort to tsetse transmission experiments.     

Life span is related to the free energy of mitochondrial DNA

Broadly speaking, the mitochondrial theory of aging relates aging to the rate of damage to mitochondria. In this work, I concentrate on a DNA sequence property, the free energy, which can be interpreted as a factor in the susceptibility of mitochondrial DNA (mtDNA) to mutation. I show that life spans across a broad range of species are a function of the mtDNA free energy and are proportional to the probability of opening of bubbles of single-stranded mtDNA of approximately 20 base pairs in length, in agreement with the measured nucleation size of these bubbles. These transient separations of the mtDNA strands are a possible aging mechanism, through increased mtDNA mutations. In comparisons of species with similar life spans, avian mtDNA has more negative free energy than does mammalian mtDNA, suppressing the predicted probability of mtDNA bubble formation in birds by over 80% and thus protecting them against mutation. Based on these results I propose three hypotheses about the conflicting evolutionary forces that have acted on the free energy of mtDNA.     

孕妇血浆中的游离胎儿DNA及其意义

孕妇血浆中已证实存在着胎儿来源的游离DNA。作为一种胎儿新的检测材料,游离胎儿DNA的发现为非侵入性产前基因诊断提供了可能。有关游离胎儿DNA的临床应用以及其生物学包括来源、动力学和性质等,近年来引起人们很大关注。实时定量PCR技术是目前检测孕妇血浆中游离胎儿DNA的主要方法。实验室的一些技术因素会影响游离胎儿DNA的检测。尽管有些问题还不很清楚,但母体血浆中游离胎儿DNA的检测对于胎儿遗传病和妊娠相关性疾病的非侵入性产前诊断仍具有重要临床意义。讨论孕妇血浆中游离胎儿DNA的最新研究状况。     

DNA diagnosis of human genetic individuality

DNA studies of the human genome have shown polymorphic variation at thousands of sites, defining an absolute genetic uniqueness for each individual. There are many circumstances in which it may be desirable to diagnose this molecular individuality, as for instance, in criminal investigations or paternity testing. Several techniques can be used for this DNA diagnosis and we can choose among them the one that best suits the specific problem at hand. In this review we describe the main methodologies in current use to investigate human DNA polymorphisms, discussing the best application of each option, as well as their advantages and disadvantages.Abbreviations LSSP Low-stringency single specific primer - PCR Polymerase chain reaction - RFLP Restriction fragment length polymorphism - SSP Sequence specific primer - SSO Sequence specific oligonucleotide - VNTR Variable number of tandem repeats     

The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart

The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.     

卵巢癌血浆DNA含量及其与肿瘤基因表达的相关性分析

目的： 探讨上皮性卵巢癌血浆DNA改变特点及其与肿瘤基因表达的相关性。方法： 采用DNA定量检测试剂盒检测35例原发性上皮性卵巢癌、20例良性上皮性卵巢肿瘤和20例健康对照血浆DNA含量，并以免疫组织化学方法检测卵巢癌肿瘤组织Survivin、FHIT和nm23-H1的表达状况。结果： 健康组、良性组及恶性组血浆DNA水平分别为16.5 μg/L、87.6 μg/L和630.2 μg/L，恶性组与健康组、良性组相比，均有明显差异（P<0.01）；上皮性卵巢癌组血浆DNA水平与有无伴发腹水及淋巴结转移有明显相关性，其血浆DNA平均水平在术后明显下降。上皮性卵巢癌肿瘤组织nm23-H1和Survivin蛋白表达的阳性率分别为68.57%和60.00%，FHIT蛋白的缺失率为17.14%，血浆DNA水平明显差异仅可见于nm23-H1蛋白不同表达组别间。结论： 上皮性卵巢癌患者循环DNA含量明显高于对照组，而其水平变化则可能与肿瘤的侵袭转移能力相关。