干血斑滤纸片检测Leber''s视神经萎缩线粒体突变

目的 建立干血斑滤纸片分析Leber's遗传性视神经萎缩(LHON)线粒体DNA(mtDNA)的方法，为更加广泛开展LHON的基因诊断和为进一步研究LHON积累资料。方法 收集了81例视神经炎、视神经萎缩或临床怀疑LHON患者的干血斑滤纸片，提取DNA，用突变特异性引物PCR(MSP—PCR)和异源双键—单链构象多态性(HA—SSCP)检测其mtDNA的三个常见原发致病突变位点。结果 81例受检者中，检出51例l1778位点突变阳性；1例3460位点突变阳性；14484位点突变全部阴性。结论 应用干血滤纸片检测LHON的mtDNA，具有微量、准确、有效、邮寄方便的特点，值得推广。     

Leber遗传性视神经病一家系的遗传学研究

目的对Leber遗传性视神经病一家系进行线粒体DNA基因突变检测,并探讨其临床特点。方法完成家系调查和系谱分析,收集家系成员的临床资料,并进行眼科专科检查(视力、视野和眼底检查)。利用聚合酶链式反应及DNA测序方法对线粒体DNA三个原发突变位点G3460A、G11778A和T14484C进行突变检测。结果该家系表现为典型的母系遗传特征。突变检测结果提示:先证者及家系患者均存在线粒体DNA的G11778A原发突变位点,该位点突变后导致ND4基因第340位氨基酸由精氨酸变为组氨酸。在家系健康成员及100名健康对照中均未发现该突变位点。此外,在先证者及患者中进行3460和14484原发突变位点检测时未发现突变。结论该家系先证者及患者中均发现线粒体DNA的G11778A原发突变位点。结合临床特征及基因突变检测结果,该家系可明确诊断为Leber遗传性视神经病。     

应用HRM分析Leber遗传性视神经病变家系mtDNA突变

目的揭示一个Leber遗传性视神经病变（Leber＇s hereditary optic neuropathy,LHON）家系的遗传基础。方法对家系成员提取线粒体DNA（mtDNA）,直接进行测序分析,用分子克隆法为每个人构建单克隆群,再用高分辨熔解曲线技术和DNA测序的方法,对家系中成员的单克隆群分析统计,计算该家系成员的线粒体DNA突变比例。结果该家系患者mtDNA上11778位核苷酸发生G到A的突变。家系成员中G11778A突变比例分别为：先证者（112）91．67％;父亲（12）0％;3位母系家属正常人依次为：（11）90．83％、（111）53．16％、（113）49．16％。结论G11778A的同质体（即：突变比例达到90％以上）女性仍可不患Leber遗传性视神经病变,该女性后代的平均突变比例远小于先前的报道。     

母系遗传性糖尿病伴耳聋3个家系报道

目的探讨母系遗传性糖尿病伴耳聋（MIDD）的发病机制、临床特点以及线粒体mtDNA A3243G点突变在这类家系病例中的发生率。方法收集3个MIDD家系患者，外周血白细胞标本提取DNA，PCR扩增线粒体DNA目的片段，以ApaⅠ限制性内切酶检测mtDNA A3243G点突变。结果3个家系18例患者中，平均发病年龄32岁，其中MIDD7例（39％）；线粒体糖尿病3例（17％）；遗传性耳聋4例（22％）；线粒体脑肌病合并乳酸血症及卒中发作（MELAS）4例（22％）；糖尿病患者主要表现为消瘦和乏力；8例患者行胰岛素治疗，遗传性耳聋患者的主要症状为进行性的听力下降；11例患者进行了基因检查，其中10例发现mtDNA A3243G点突变（91％）；所有患者均为母系亲属。结论MIDD家系中mtDNA A3243G点突变有较高的发生率，mtDNA A3243G点突变筛查对这类糖尿病和／或耳聋的诊断有重要意义。     

中国人非综合征型耳聋患者线粒体DNA A1555G突变分析

目的分析线粒体DNA A1555G（mitoehondrial DNA A1555G，mtDNA A1555G）在中国人非综合征型耳聋（NSHI）患者中的突变率及突变性质。方法用PCR．限制性内切酶切分析（-RFLP）和PER产物直接测序法对325例中国人NSHI患者、50名健康体检者的血样进行mtDNA A1555G突变检测及测序分析。结果325例NSHI患者mtDNA A1555G突变率为14．5％（47／325），47例mtDNA A1555G突变中28例为均质性突变，19例为异质性突变；50名健康体检者均未发现同样突变。结论NSHI患者mtDNA A1555G突变率较高，并为均质性和异质性两种突变，这对进一步研究中国人耳聋相关基因突变与临床表型的关系奠定了基础。     

海南省71例黎、汉族葡萄糖-6-磷酸脱氢酶缺乏症患者的基因突变型分析

目的分析海南省29例汉族和42例黎族葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症患者的G6PD基因突变类型。方法采用突变特异性扩增系统法筛查海南省人群中G6PD缺乏症的G1376T、G1388A和A95G三种常见突变位点；运用DNA测序技术鉴定未知突变标本G6PD基因外显子2至外显子13的基因突变类型。结果在29例汉族G6PD缺乏症患者中，发现G1376T 11例（37．9％）、G1388A 2例（6．9％）、G1376T复合G1388A突变1例（3．4％）和G1376T复合A95G突变1例（3．4％），G1376T、G1388A、A95G三种常见突变及其复合突变共占汉族G6PD缺乏症患者的51．7％；在42例黎族G6PD缺乏症患者中，发现G1376T 25例（59．5％）、G1388A 6例（14．3％）、A95G 2例（4．8％）、G1376T复合G1388A突变4例（9．5％）和G1376T复合A95G突变1例（2．4％），G1376T、G1388A、A95G三种常见突变及其复合突变共占黎族G6PD缺乏症患者的90．5％；18例（汉族14例，黎族4例）未发现G1376T、G1388A、A95G。对这18例标本的测序发现：1例G6PD内含子5第636或637位核苷酸T缺失（IVS-5 636或637 T del）突变；2例C1311T复合IVS-11 T93C突变，其余标本未发现突变。结论G1376T和G1388A是海南省人群中最常见的基因突变型；在我国人群中存在IVS-5636或637 T del突变型；在海南省黎族人群中首次报道G1376T／A95G复合突变型。     

MS基因A2756G多态性位点与青年缺血性脑卒中相关性研究

目的 研究人体MS基因A2756G多态性与青年缺血性脑卒中的遗传相关性。方法 采用限制性内切酶片段长度多态性方法（PCR-RFLP），对98例脑卒中病人和116例健康人的MS2756多态性位点进行检测。结果病例组A等位基因纯合子（A／A）、杂合子（A／G）和G等位基因纯合子基因型（G／G）所占比例分别为77．55％，18．37％和4．08％。对照组A／A纯合子、A／G杂合子和G／G纯合子基因型比例分别为79.31％，15．52％和5．17％。两组无显著性差异（X^2=0．41，P〉0．05）。ORAA／GG=1．23；ORA／G=1．02。结论 汉族人群MSA2756G位点多态性与青年缺血性脑卒中无明显相关性。     

云南玉溪葡萄糖-6-磷酸脱氧酶缺乏症基因突变研究

目的：调查云南玉溪G6PD缺乏症的发病率．分析鉴定云南玉溪G6PD缺乏症的基因突变类型及特征，探讨检测G6PD缺乏症基因突变的有效方法。方法：NBT纸片法定性筛查玉溪当地居民450人，NBT定量酶活性，PCR-ARMS法检测中国人中最常见的三种突变，作PCR-SSCP分析，最后用DNA测序分析和证实。结果：（1）450例当地居民筛查发现G6PD缺乏症患者48例（男31例，女17例）。（2）PCR-ARMS发现G1388A 21例（43．75％）、G1376T4例（8．33％），未见A93G。（3）PCR-SSCP发现27例异常，PCR—DNA测序结果与PCR—ARMS结果吻合。2份未知突变样本测序分析证实为C1311T。（4）测序发现复合型突变：1例G1376T／IVS-11T93C，1例G1376T／C1311T和1例G1388A／IVS-11＇I＇93C。结论：（1）云南玉溪G6PD缺乏症发病率为10．67％，男性13．78％．女性7．56％；男、女性别比例1．82。（2）云南玉溪G6PD缺乏症基因突变以G1388A突变最常见，其次是G1376T突变，两种突变共占52．08％，未发现A95G突变。（3）研究云南玉溪G6PD缺乏症基因突变型对指导优生、优育，预防该病的遗传传播．提高我省各地州人口素质具有一定的社会意义。对开展G6PD缺乏症的基因诊断具有一定的应用价值和指导意义     

三个遗传性血小板无力症家系临床表型和基因表型诊断的研究

目的对3个遗传性血小板无力症（glanzmann thrombasthenia，GT）家系进行血小板膜糖蛋白Ⅱb、Ⅲa（GPⅡb／GPⅢa）基因突变的检测。方法应用PCR对先证者GPⅡb／GPⅢa基因所有外显子及其侧翼序列进行扩增；PCR产物纯化后直接测序，检测其突变基因。突变位点经直接测序证实排除基因多态性。结果3个家系的先证者PLT均正常，血小板形态分散，出血时间（BT）延长，凝血象正常，对二磷酸腺苷（ADP）、凝血酶、肾上腺素、胶原、花生四烯酸等多种诱聚剂反应低下，而对瑞斯托霉素反应基本正常；家系1和家系2先证者的血小板膜表面CD41，（GPⅡb）／CD61（GPⅢa）的含量极度降低，分别为0．16％／1．8％、0．9％／3．7％，家系3先证者的血小板膜表面GPⅡb阳性血小板为10．1％，GPⅢa阳性血小板为12．8％。免疫印迹法几乎检测不到家系1和家系3先证者的α／Ⅱb。蛋白，家系2先证者的α／Ⅱb蛋白含量明显降低。家系1先证者在GPⅡb基因存在T2255G和C2671T复合杂合突变，家系2先证者在GPⅡb基因存在A2334C纯合突变，家系3先证者在GPⅡb基因存在C1750T和69-79del复合杂合突变。结论T2255G和C2671T复合杂合突变是导致家系1先证者发生GT的原因，A2334C纯合突变是导致家系2先证者发生GT的原因，C1750T和69-79del复合杂合突变是导致家系3先证者发生GT的原因。     

氨基糖苷类抗生素导致mtDNA A1555G和mtDNA A7445G突变的相关遗传药理分析

Mitochondrial DNA A1555G（mtDNA1555）和mitochondrial DNA A7445G（mtDNA 7445）位点突变是氨基糖苷类抗生素（aminoglycoside antibiotics，AmAn）诱导的耳聋（aminoglycoside antibiotics induced deafness，AAID）的主要遗传基础。mtDNA7445位点的突变与糖尿病合并耳聋有关。线粒体基因突变糖尿病是糖尿病单基因致病类型，属于B细胞遗传缺陷疾病。     

The epidemiology and mutation types of Leber’s hereditary optic neuropathy in Thailand

PurposeLeber’s hereditary optic neuropathy (LHON), the most common mitochondrial optic neuropathy, causes visual loss, especially in young adults. Due to the absence of epidemiological data in Southeast Asia, we aimed to determine Thai LHON patients’ characteristics (demographic data, mutation types, and prognoses) as the first study in this region.MethodsThis retrospective chart review enrolled all Thai LHON patients confirmed by three mitochondrial DNA mutations (G11778A, T14484C, and G3460A) between January 1997 and December 2016. Patients with more than one year of follow-up were included in a visual progression analysis. The Mann–Whitney U-test was applied to compare groups, and prognosis-associated factors were analysed with the generalized estimating equation.ResultsIn all, 229 patients were enrolled, with only nineteen females. Most mutations were of the G11778A type (91%), with T14484C accounting for the remainder. The age at onset of G11778A (21.9 years; interquartile range [IQR] 14.9, 33.5) was younger than that of T14484C (33.0 years; IQR 19.4, 37.5). Of 45 patients, the T14484C group demonstrated good vision recovery, whereas the G11778A group did not improve (difference in logMAR −0.7 and IQR −1.5, −0.2 versus logMAR 0.0 and IQR −0.3, 0.2, respectively; P value .001). The G11778A mutation, male, and older age were related to poor prognoses.ConclusionsThe leading mutation in Thai LHON patients is the G11778A missense, followed by T14484C, while G3460A was not detected. The vast majority of patients were young adult males. The G11778A mutation, older age, and male gender are associated with poor vision outcomes.

Key message

• The G11778A missense mutation is the most common among Thai LHON patients, followed by T14484C, while G3460A was not found. The G11778A mutation, older age, and male gender are associated with poor vision outcomes.

     

Rapid quantification of the heteroplasmy of mutant mitochondrial DNAs in Leber's hereditary optic neuropathy using the Invader technology

PURPOSE: To quantify the degree of heteroplasmy of a mitochondrial DNA (mtDNA) mutation in Leber's hereditary optic neuropathy (LHON) a biplex Invader assay was applied. METHODS: To determine the optimum condition for the Invader assay, mtDNAs were assayed in various amounts of total DNA in 1-4-h incubations at 63 degrees C. To evaluate the suitability of the Invader assay to detect the three mutations, G3460A, G11778A, and T14484C, 10 ng of DNAs from 224 patients with bilateral optic atrophy was assayed. To quantify mtDNA heteroplasmy, a standard curve of known mixture ratios of mutation against calculation by the Invader assay was constructed. Seventy-two of the 224 patients had one of the three mutations, which corresponded with the mutation detected earlier by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. The percentages of mutant mtDNAs were calculated by the Invader assay in five heteroplasmic families, including 30 individuals with the G11778A mutation. The results were compared with those calculated earlier by labeled polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis. RESULTS: In 1-8 ng of DNA, the fluorescence intensity increased near linearly during a 4-h assay. With more than 16 ng of DNA, the intensities were saturated even at the 2-h assay. A linear relationship was observed between the results obtained from separate mixtures and from the Invader assay analysis. Because two fluorescent intensities are not always the same, one of the two intensities was modified to adjust to that of the other. Complete concordance was observed between PCR-RFLP analysis and Invader assay genotyping for the 224 patients. Results of percentage of heteroplasmy in five LHON families obtained by the Invader assay were consistent with those by the PCR-SSCP analysis. CONCLUSIONS: Invader assay is a simple, rapid, and reliable method of genotyping mtDNA mutations as well as quantifying heteroplasmy simultaneously under optimum conditions.     

多重连接探针扩增技术检测常见线粒体疾病

  目的  探讨多重连接探针扩增技术(multiplex ligation-dependent probe amplification, MLPA)用于常见线粒体疾病基因检测的可行性。  方法  收集2010年5月至2012年8月北京协和医院281例可疑线粒体异常病例, 包括神经科存在神经系统损伤的患者233例及眼科疑诊Leber遗传性视神经病变的患者48例, 运用MLPA法对常见线粒体疾病突变位点进行检测, 并使用线粒体基因序列测定方法进行验证。  结果  281例标本中共38例检测到线粒体基因突变, 总检出率为13.5%。眼科48例标本中, 3460G > A、11778G > A和14484T > C 3种突变共检测到19例, 检出率为39.6%;神经科233例标本中, 3243A > G、8344A > G、8993T > G和大片段缺失共检测到19例, 检出率为8.2%。除1例大片段缺失暂无PCR测序验证外, 其余MLPA结果均经序列测定验证为相符。  结论  MLPA法是一种可行的快速、准确、简便的基因诊断方法, 可作为筛查常见线粒体疾病的检测工具, 为临床诊断和治疗提供依据。     

Screening the three LHON primary mutations in the general Chinese population by using an optimized multiplex allele-specific PCR

BackgroundLeber hereditary optic neuropathy (LHON) is one of the most common mitochondrial diseases, which is mainly caused by three mitochondrial DNA (mtDNA) mutations (m.3460G> A, m.11778G> A and m.14484T> C). Incomplete penetrance suggests that there might be asymptomatic carriers in general populations. These asymptomatic carriers are clinically important as they are potential future patients and the female carriers could transfer the pathogenic mutations to their offspring. Thus, screening the three LHON primary mutations in general populations is important for genetic counseling.MethodsWe optimized a multiplex allele-specific PCR method based on previous studies, and the sensitivity was evaluated. The three LHON primary mutations were screened by using this MAS-PCR method in 1571 subjects from general Chinese populations that are without symptoms or family history of optic neuropathy.ResultsThe optimized MAS-PCR approach can detect a heteroplasmy level at 5%, 5%, and 20% for m.3460G> A, m.11778G> A and m.14484T> C, respectively. None of the three LHON primary mutations was detected in the 1571 subjects.ConclusionThe three LHON primary mutations are rare in general Chinese populations. The optimized MAS-PCR assay provides an easier, faster and more cost-effective method for detection of the three LHON primary mutations, making it practical for clinical diagnosis.     

线粒体基因异常糖尿病临床预报的探讨

目的　拟建立基因序列检测的糖尿病 (DM )临床预报方法。方法　对 10 4例糖尿病患者和 5 0例正常无DM家族史对照者 ,用聚合酶链反应扩增 3 161～ 3 610基因片段直接测序。结果　 10 4例DM患者有 2 .88%同时出现mtDNA3 497C→T、3 5 71C→T点突变 ,和 3 2 90T→C点突变 ;1.92 %出现 3 42 1G→A基因突变。DM组与正常对照组比较有显著性差异 (P <0 .0 1)。结论　 3 497C→T、3 5 71C→T位点同时突变 ,或同时合并其他基因突变 ;3 3 16G→A位点突变合并 3 2 90T→C或其他错义基因突变时与DM有关。     

Study of mitochondrial DNA mutations in patients with migraine with prolonged aura

Ten patients with migraine with prolonged aura were studied for the presence of mitochondrial DNA point mutations utilizing DNA isolated from blood and hair samples. We analyzed for nine point mutations reported in patients with MELAS (A3243G, C3256T, T3271C, T3291C, A5814G, T8356C, T9957C, G13513A, and A13514G) and three secondary LHON mutations (T4216C, A4917G, and G13708A). None of the patients tested had any of these mutations in mitochondrial DNA. However, one patient was found to have a tRNA(Gln) A4336G mitochondrial DNA variant. From this study it appears that migraine with prolonged aura is not an oligosymptomatic form of MELAS and is not related to secondary LHON mutations. The significance of the tRNA A4336G variant is unknown.     

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.     

线粒体新变异与中国北方人群中2型糖尿病的关联性的关联性

目的:探讨线粒体DNA 3537A/G、5351A/G位点突变与中国北方人群2型糖尿病发生之间的关系.方法:全部对象均来自大连市,2型糖尿病患者包括大连地区2型糖尿病家系调查收集的患者61例,大连市中心医院内分泌科收治的患者497例及国家"95"攻关"中老年糖尿病普查"大连地区散发患者56例.另选国家"95"攻关"中老年糖尿病普查"中糖耐量正常的344人作为正常对照.在614例2型糖尿病患者和334名非糖尿病对照群体中检测线粒体DNA 3537A/G、5351A/G位点的突变情况.在测序基础上,从线粒体DNA中排序选择2个有较高可能性与2型糖尿病相关的候选SNP,然后使用限制性片断长度多态性聚合酶链反应技术进行基因分型.结果:线粒体DNA 3537A/G、5351A/G在2型糖尿病患者中的突变频率分别为2.0%和2.6%,在对照人群中则分别为2.1%和4.2%,两者之间的差异无显著性意义(P＞0.05).按照体质量指数和血压进行分层之后,发现5351A/G在肥胖的2型糖尿病患者中突变频率为1.61%,在肥胖的对照中频率为15.38%,两者之间的差异具有显著性意义(P_(Fisher)=0.02,OR=2.76);但是A 3537G在各组中的差异无显著性意义(P＞0.05).结论:线粒体DNA ND2基因5351A/G可能与中国北方人群中2型糖尿病发病风险相关.