线粒体DNA G7444A突变可能影响A1555G突变的表型表达

线粒体12S rRNA和tRNASer(UCN) 基因是导致非综合征型听力损失的两个突变热点区域。作者收集了1个母系遗传感音神经性聋家系, 该家系同时携带线粒体DNA (mtDNA) A1555G和G7444A突变。临床资料分析表明, 该家系包括药物致聋的耳聋外显率(所有耳聋患者/所有母系成员)为58%, 而非药物致聋的耳聋外显率(非药物性聋患者/所有母系成员)为25%, 明显高于其他携带A1555G突变的耳聋家系。先证者的线粒体全序列分析表明, 该线粒体基因组共有28个多态位点, 属于东亚人群B4c1单体型。在这些多态位点中, 除A1555G和G7444A突变外, 未发现其他有功能意义的突变。这表明mtDNA G7444A突变可能加重由A1555G突变造成的线粒体功能缺失, 从而增加耳聋的外显率。     

温州地区2型糖尿病患者线粒体DNA 3243、3316位点的突变筛查

为了解浙江省温州地区2型糖尿病病人中线粒体DNA tRNALeu (UUR)基因A3243G及NADH 脱氢酶亚单位1 (ND1)基因G3316A位点突变的发生频率, 并探讨突变与2型糖尿病主要临床指标出现的相关性。对随机收集的无血缘关系的244例温州地区2型糖尿病患者进行研究, 同时选择156例无 DM 家族史的糖耐量正常者作为对照组, 用聚合酶链反应及限制性片段长度多态性分析技术进行点突变筛选, 筛选到的异质性突变样本经T-A克隆后再作测序和变性高效液相色谱(DHPLC)确证。结果在244例的2型糖尿病患者中检出A3243G突变1例(0.410%), 156例对照者中未检出该突变, 突变发生率在两组间差异无统计学意义(P>0.05); 2型糖尿病患者中检出G3316A突变4例(1.639%), 156例对照者中检出突变2例(1. 282%), 突变发生率在两组间差异无统计学意义(P>0.05)。结果表明线粒 体tRNALeu (UUR) 基因A3243G突变在浙江温州2型糖尿病人群中发生频率低, 不是温州人群中2型糖尿病的常见病因。线粒体ND1基因G3316A突变在糖尿病人群中的发生频率也较低, 且在正常人群中也有出现, 可能仅为人群中线粒体DNA的基因多态性。     

线粒体基因突变与糖尿病的相关性研究进展

线粒体DNA(mtDNA)突变可引起多种遗传性疾病,其中包括糖尿病.与mtDNA突变相关的糖尿病中最常见的变异是tRNALeu(UUR)] 3243 A→G.文章描述了mtDNA线粒体突变与糖尿病的相关性,介绍了线粒体糖尿病的临床特点和发病机制,概括了线粒体糖尿病相关变异基因位点,重点介绍了m.3243A→G、3310C→T、16189T→C基因突变与线粒体糖尿病病理生理的联系.文章认为mtDNA突变位点的研究为糖尿病的发生机制提供新的视角,也为糖尿病的治疗提供了新方向.     

与耳聋相关的线粒体12S rRNA突变

线粒体DNA突变是引起听力损伤的重要原因之一. 其中,线粒体12S rRNA基因突变与综合征型耳聋和非综合征型耳聋相关. 导致综合征型耳聋的线粒体DNA突变多为异质性,然 而对于非综合征型耳聋突变则多以同质性或高度异质性存在,说明这种分子致病性需要较高的阈值. 位于12S rRNA解码区的A1555G和C1494T突变是造成氨基糖甙类抗生素耳毒性和 非综合征型耳聋常见的分子机制. 这些突变可能造成12S rRNA二级结构的改变,影响线粒体蛋白质的合成,降低细胞内ATP的产生,由此引起的线粒体功能障碍导致耳聋. 但是多数 基因突变的致病机制还仅处于推测阶段. 其它修饰因子如氨基糖甙类抗生素、线粒体单体型、核修饰基因参与了线粒体12S rRNA基因A1555G和C1494T突变相关的耳聋表型表达.     

MELAS和MERRF综合征相关mtDNA突变位点检测集成芯片的建立

摘要: 文中建立了一种新型的寡核苷酸芯片, 用于线粒体脑肌病伴高乳酸血症和卒中样发作(Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes, MELAS)和肌阵挛性癫痫伴发不规整红纤维(Myoclonic epilepsy with ragged red fibers, MERRF)线粒体DNA所有已知突变位点的集成检测。将31对allele位点特异性的寡核苷酸探针包被在醛基修饰的载玻片表面, 以多重不对称PCR方法制备Cy5荧光标记靶基因。利用此芯片对5例MELAS患者、5例MERRF患者及20例健康对照进行筛查, 结果发现, MELAS患者均为MT-T1基因A3243G突变; 在MERRF患者组, MT-TK基因A8344G突变4例, T8356C突变1例; 健康对照组均未发现31种相关mtDNA突变。芯片检测与DNA测序结果完全一致。结果表明, 这种寡核苷酸芯片可以对MELAS和MERRF综合征已知突变位点进行同步快速检测, 具有较高的灵敏度和特异性。这一模式的基因芯片经过适当改装后也可用于其他人类线粒体疾病的基因诊断。     

两个携带线粒体tRNA~(Met)/tRNA~(Gln)A4401G和tRNA~(Cys)G5821A突变的中国汉族原发性高血压家系的临床及分子遗传学特征

线粒体tRNA基因可能是原发性高血压发病相关的突变热点区域。文章报道了两个具有母系遗传特征的中国汉族原发性高血压家系的临床和分子遗传学特征。家系先证者和其他成员的临床数据表明,家系中母系成员高血压发病的严重程度存在差异,发病年龄也从36~79岁不等。对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全序列扩增分析,结果发现这两个先证者均携带同质性的tRNAMet/tRNAGlnA4401G和tRNACysG5821A突变,多态性变异位点都属于东亚单体型C。A4401G突变可能通过影响tRNAMet和tRNAGln前体的加工引起线粒体tRNA代谢水平的改变,而tRNACysG5821A突变位于tRNACys氨基酸受体臂,该突变使tRNACys氨基酸受体臂上原有的G6-C67配对消失,可能影响tRNACys空间结构和功能的稳定性,致使线粒体功能障碍。因此,tRNAMet/tRNAGlnA4401G和tRNACysG5821A突变可能是这两个原发性高血压家系的分子致病基础。     

中国儿童线粒体病的临床特征及分子遗传学研究

线粒体病具有高度临床异质性和基因异质性,对于临床医生来说诊断非常困难.本研究分析了中国线粒体病患儿的临床特征和基因特点,为临床医生的诊断提供了一定的参考依据.本文总结了2012年10月~2015年1月就诊北京儿童医院的141例已进行二代测序基因检测的疑似线粒体病患儿,分析基因确诊病例的临床特征以及遗传学特点.本研究共收集了40例基因确诊病例,包括8种不同类型的线粒体病,而Leigh综合征是最常见类型,其次是线粒体脑肌病伴乳酸酸中毒和卒中样发作(MELAS)综合征.不同的线粒体病发病年龄各异,但是普遍临床早发.40例基因确诊病例中, 25例(62.5%)为mtDNA突变, 15例(37.5%)为nDNA突变. m.3243AG突变7例,在mtDNA突变中最为常见, nDNA突变包括SURF1 7例, PDHA1 2例, NDUFV1, NDUFAF6, SUCLA2, SUCLG1, RRM2B,以及C12orf65各一例.     

线粒体tRNALeu(UUR)基因A3243G突变型糖尿病患者的家系分析及随访

目的:探讨线粒体糖尿病家系中的基因突变位点及临床转归。方法:收集1例线粒体糖尿病患者家系的临床资料,采用PCR、DNA直接测序法对家系成员进行线粒体基因突变高发区域t RNA~(Leu(UUR))检测,以了解mtDNA3243位点突变情况,并随访8年进一步了解研究对象的临床转归情况及胰岛功能变化。结果:6例家系成员中有5例携带mtDNA3243A→G位点的突变,其中4例为糖尿病患者且伴发不同程度的双侧听力受损(神经性耳聋),1例父系患者后代未检测出突变位点。随访过程中,先证者死亡,余3例糖尿病患者除常规治疗外,长期口服辅酶Q10,血糖控制较为稳定、尚未出现严重并发症但双侧听力严重下降,胰岛分泌功能明显下降,1例携带者已出现糖耐量受损。结论:线粒体tRNA~(Leu(UUR))点突变与糖尿病具有显著相关性。     

7例携带线粒体tRNAAlaC5601T突变的Leber遗传性视神经病变家系的相关研究

文章收集了7例携带线粒体tRNAAl。C5601T突变的中国Leber遗传性视神经病变(Leber’s hereditary opticneuropathy,LHON)的家系,通过眼科检查和遗传学分析,发现7个家系的外显率很低,分别为9.5%、14.3%、4.5%、8.3%、10.0%、22.2%和25.0%。用24对有部分重叠的引物对7个先证者线粒体DNA(Mitochondrial DNA,mtDNA)全序列进行扩增,并进行相关的分子生物学分析,结果发现这些家系均未携带G11778A、G3460A和T14484C这3个常见的原发突变位点,而在tRNAAla上发现了C5601T同质性突变,多态性位点分析分别属于东亚线粒体单体型G2、G2a1、G2a1、G2、G2b、G2a1、G2。C5601T突变位于线粒体tRNAAla的高度保守区(通用位点为59位),可能引起tRNA空间结构和稳定性发生改变,继而影响tRNA的代谢,导致线粒体蛋白和ATP合成障碍,最终导致视力损害。因此,tRNAAlaC5601T突变可能是与LHON相关的线粒体突变位点。同时低外显率提示其他因素(包括核修饰基因、环境因素)可能影响这7个中国C5601T突变家系的表型表达。     

线粒体tRNAGlu A14693G可能是与Leber遗传性视神经病变相关的基因突变

收集了3个具有典型临床特征的中国汉族Leber遗传性视神经病变(Leber's hereditary optic neuropathy, LHON)家系。通过对先证者和家系其他成员进行眼科临床(如视力损害程度和发病年龄)检查, 发现这些家系成员中视力损害的外显率很低, 经mtDNA测序分析, 在tRNA^Glu 上发现了A14693G同质性突变位点, 多态性位点分别属于东亚单体型Y1b、Y1和Y1, 没有发现其他高度保守和有功能意义的突变位点。A14693G突变位于线粒体tRNAGlu高度保守区(通用位点为54位), 可能导致tRNA空间结构和稳定性发生改变, 继而影响tRNA的代谢, 导致线粒体蛋白合成功能受损和ATP障碍, 最终导致视力损害。所以, tRNAGlu A14693G突变可能是与视神经病变相关的致病性线粒体突变位点。     

The study of mitochondrial A3243G mutation in different samples

Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS) is the most frequent syndromic manifestation of A3243G mutation in mitochondrial DNA. Detection of A3243G mutation in blood is less helpful for the diagnosis of MELAS and the carriers, and the mutation ratio in blood correlates only in a limited extent with the severity of the disease. Here we compared the ratio of A3243G mutation in four easily available samples (blood, urine, hair follicle and saliva) in patients with MELAS carrying A3243G mutation as well as their maternal relatives from 32 families, to find out the samples appropriate for the detection of the patients and carriers and useful for the evaluation of clinical severity from their mutation ratio. In MELAS patients and the carriers with minor symptoms or normal phenotype, A3243G mutation ratio was significantly higher in urine than in blood. A close correlation between A3243G mutation ratio in blood and that in urine, hair follicles and saliva was found in the probands and their relatives. Clinical features closely correlated with the mutation ratio in urine. Measurement of A3243G mutation ratio in urine is a non-invasive, convenient and rapid method with its diagnostic meaning superior to blood testing.     

A whole mitochondrial genome screening in a MELAS patient: a novel mitochondrial tRNA(Val) mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA(Val). This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

Mitochondrial tRNA gene mutations in patients having mitochondrial disease with lactic acidosis

Lactic acidosis has been associated with a variety of clinical conditions and can be due to mutation in nuclear or mitochondrial genes. We performed mutations screening of all mitochondrial tRNA genes in 44 patients who referred as hyperlactic acidosis. Patients showed heterogeneous phenotypes including Leigh disease in four, MELAS in six, unclassified mitochondrial myopathy in 10, cardiomyopathy in five, MERRF in one, pure lactic acidosis in six, and others in 12 including facio-scaplo-femoral muscular dystrophy (FSFD), familial cerebellar ataxia, recurrent Reye syndrome, cerebral palsy with mental retardation. We measured enzymatic activities of pyruvate dehydrogenase complex, and respiratory chain enzymes. All mitochondrial tRNA genes and known mutation of ATPase 6 were studied by single strand conformation polymorphism (SSCP), automated DNA sequence and PCR-RFLP methods. We have found one patient with PDHC deficiency and six patients with Complex I+IV deficiency, though the most of the patients showed subnormal to deficient state of respiratory chain enzyme activities. We have identified one of the nucleotide changes in 29 patients. Single nucleotide changes in mitochondrial tRNA genes are found in 27 patients and one in ATPase 6 gene in two patients. One of four pathogenic point mutations (A3243G, C3303T, A8348G, and T8993G) was identified in 12 patients who showed the phenotype of Leigh syndrome, MELAS, cardimyopathy and cerebral palsy with epilepsy. Seventeen patients have one of the normal polymorphisms in the mitochondrial tRNA gene reported before. SSCP and PCR-RFLP could detect the heteroplasmic condition when the percentage of mutant up to 5, however, it cannot be observed by direct sequencing method. It is important to screen the mtDNA mutation not only by direct sequence but also by PCR-RFLP and the other sensitive methods to detect the heroplasmy when lactic acidosis has been documented in the patients who are not fulfilled the criteria of mitochondrial disorders.     

Clinical phenotype, prognosis and mitochondrial DNA mutation load in mitochondrial encephalomyopathies

We studied 42 individuals, including 8 patients with either complete or partial syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), 8 patients with either complete or partial syndrome of myoclonic epilepsy with ragged-red fibers (MERRF) and 26 maternal family members who carried either the A3243G or A8344G mutation of mitochondrial DNA (mtDNA). Clinical manifestations and prognosis were followed up in the patients harboring the A3243G or A8344G mutation. The relationship between clinical features and proportions of mutant mtDNAs in muscle biopsies, blood cells and/or hair follicles was studied. In the 8 regularly followed patients with the A3243G mutation, 4 died within 1 month to 7 years due to status epilepticus and/or recurrent stroke-like episodes. Two patients developed marked mental deterioration and 2 remained stationary. All of the patients harboring the A8344G mutation were stable or deteriorated slightly, except for 1 patient who died due to brain herniation after putaminal hemorrhage. The A3243G and A8344G mtDNA mutations were heteroplasmic in the muscle biopsies, blood cells and hair follicles of both the probands and their maternal family members. The mean proportion of A3243G mutant mtDNA in the muscle biopsies of the patients with MELAS syndrome (68.5 ± 21.3%, range 33–92%) was significantly higher than that of the asymptomatic family members (37.1 ± 12.6%, range 0–51%). The average proportions of A8344G mutant mtDNA in the muscle biopsies (90.1 ± 3.9%, range 89–95%) and hair follicles (93.9 ± 6.4%, range 84–99%) of the patients with MERRF syndrome were also significantly higher than those of the asymptomatic family members (muscle: 40.3 ± 39.5%, range 1–80%; hair follicles: 51.0 ± 44.5%, range 0.1–82%). We concluded that measurement of the proportion of mutant mtDNA in muscle biopsies may provide useful information in the identification of symptomatic patients with mitochondrial encephalomyopathies. For patients with the A3243G mutation, the prognosis was related to status epilepticus and the number of recurrent stroke-like episodes and was much worse than for patients with the A8344G mutation of mtDNA, who had stable or slowly deteriorating clinical courses.     

A whole mitochondrial genome screening in a MELAS patient: A novel mitochondrial tRNA mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA^Val. This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

Mitochondrial DNA haplogroups do not play a role in the variable phenotypic presentation of the A3243G mutation

Thirty-five mitochondrial (mt) DNAs from Spain that harbor the mutation A3243G in association with either MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) syndrome or a wide array of disease phenotypes (ranging from diabetes and deafness to a mixture of chronic progressive external ophthalmoplegic symptoms and strokelike episodes) were studied by use of high-resolution restriction fragment length polymorphism analysis and control-region sequencing. A total of 34 different haplotypes were found, indicating that all instances of the A3243G mutation are probably due to independent mutational events. Haplotypes were distributed into 13 haplogroups whose frequencies were close to those of the general Spanish population. Moreover, there was no statistically significant difference in haplogroup distribution between patients with MELAS and those with disease phenotypes other than MELAS. Overall, these data indicate that the A3243G mutation harbors all the evolutionary features expected from a severely deleterious mtDNA mutation under strong negative selection, and they reveal that European mtDNA backgrounds do not play a substantial role in modulating the mutation's phenotypic expression.     

Screening of common mitochondrial mutations in Chinese patients with mitochondrial encephalomyopathies

To investigate the spectrum of common mitochondrial mutations in Northern China during the years of 2000-2005, 552 patients of mitochondrial encephalomyopathies clinically diagnosed as MELAS, MERRF or Leigh's syndrome, 14 cases of LHON and 46 cases of aminoglycoside induced deafness along with their family members, accepted routine point mutation tests at nucleotide positions 3243, 8344, 8993, 11778 or 1555 in mitochondrial genome. PCR-RFLP analysis, site-specific PCR and PCR-sequencing methods were used to identify the mutations. Fifty-seven cases with A3243G mutation, 4 cases with A8344G, 2 cases with T8993C and 1 case with T8993G were identified from the 552 encephalomyopathy patients. In addition, one case with G11778A was found from the 14 cases of LHON, and 5 cases with A1555G from the 46 cases of aminoglycoside ototoxicity patients. Additional screening for T8356G and T3271C merely had limited significance for the diagnosis of MERRF and MELAS. Differential diagnosis among mitochondrial encephalomyopathies was often complicated due to many similar clinical manifestations. For A3243G mutation, the proportion of mutant mtDNA was not related to severity of the disease but to the age of onset.     

Epidemiology of A3243G, the mutation for mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes: prevalence of the mutation in an adult population.

Mitochondrial diseases are characterized by considerable clinical variability and are most often caused by mutations in mtDNA. Because of the phenotypic variability, epidemiological studies of the frequency of these disorders have been difficult to perform. We studied the prevalence of the mtDNA mutation at nucleotide 3243 in an adult population of 245,201 individuals. This mutation is the most common molecular etiology of MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes), one of the clinical entities among the mitochondrial disorders. Patients with diabetes mellitus, sensorineural hearing impairment, epilepsy, occipital brain infarct, ophthalmoplegia, cerebral white-matter disease, basal-ganglia calcifications, hypertrophic cardiomyopathy, or ataxia were ascertained on the basis of defined clinical criteria and family-history data. A total of 615 patients were identified, and 480 samples were examined for the mutation. The mutation was found in 11 pedigrees, and its frequency was calculated to be >=16. 3/100,000 in the adult population (95% confidence interval 11.3-21. 4/100,000). The mutation had arisen in the population at least nine times, as determined by mtDNA haplotyping. Clinical evaluation of the probands revealed a syndrome that most frequently consisted of hearing impairment, cognitive decline, and short stature. The high prevalence of the common MELAS mutation in the adult population suggests that mitochondrial disorders constitute one of the largest diagnostic categories of neurogenetic diseases.     

Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing

Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations.