中国人非综合征性耳聋患者线粒体基因组C1494T突变筛查

目的进行非综合征性聋患者的线粒体DNA C1494T突变的分子流行病学调查。方法对中国人群中20例氨基糖甙类药物致聋患者、136例散发的非综合征性聋患者以及50例非综合征性聋家系先证者，以聚合酶链反应结合限制性片段长度多态性分析法检测线粒体DNA C1494T突变的发生情况。结果全部受检者无线粒体DNA C1494T突变的发生。结论中国人群中非综合征性聋患者的线粒体DNA C1494T突变的发生率较低，且明显低于线粒体DNA A1555G的突变发生率。通过聋病分子流行病学调查，提示线粒体DNA C1494T突变不是氨基糖甙类药物致聋的主要病因。     

线粒体12S rRNA基因突变与2型糖尿病

目的 观察线粒体12S rRNA 1310、1438及1442位点在中国汉族2型糖尿病患者群体中的突变情况,同时筛查该区域与2型糖尿病发生有关的突变。方法 采用PDR－SSCP及PCR产物直接测序等技术对86例2型糖尿病患者及70名正常对照个体的血细胞线粒体DNA进行突变分析。结果 发现1例患者线粒体DNA 12S rRNA基因1438位点存在G→A的点突变,另1例存在1442位点G→A的点突变,所有对照个体均未发现该两位点的突变。未发现线粒体基因12S rRNA 1310位点C→T点突变。结论 1438位点G→A、1442位点G→A的突变可能与2型糖尿病的发生相关,该两位点突变的具体意义如何尚需进一步研究。1310位点C→T在血细胞中的突变率可能较低,进一步说明2型糖尿病的发生在线粒体遗传上具有一定的异质性。     

修饰因子对线粒体DNA突变致聋的影响

线粒体DNA突变是引起感音神经性耳聋的重要原因之一,这些突变主要位于线粒体12SrRNA和tRNA基因上.其中12S rRNA基因上的同质性A1555G和C1494T突变与氨基糖甙类抗生素造成的耳聋相关.携带这两个突变的个体对耳毒性药物高度敏感,导致临床上常见的"一针致聋"现象.但携带A1555G或C1494T突变的个体在没用药的情况下也能产生非综合征型耳聋,而且同一家系内和不同家系间的母系成员在听力损失程度、发病年龄及听力曲线上存在很大差异.这些数据表明A1555G或C1494T突变是导致非综合征型耳聋发生的首要因子,其他修饰因子包括氨基糖甙类抗生素、线粒体DNA单倍型和核修饰基因等,在线粒体12S rRNA A1555G或C1494T突变相关的耳聋表型表达上起协同作用.作者简要介绍了这些因素对线粒体DNA突变致聋的影响以及母系遗传性耳聋发生的可能致病机制.     

修饰因子对线粒体DNA突变致聋的影响

线粒体DNA突变是引起感音神经性耳聋的重要原因之一,这些突变主要位于线粒体12SrRNA和tRNA基因上.其中12S rRNA基因上的同质性A1555G和C1494T突变与氨基糖甙类抗生素造成的耳聋相关.携带这两个突变的个体对耳毒性药物高度敏感,导致临床上常见的"一针致聋"现象.但携带A1555G或C1494T突变的个体在没用药的情况下也能产生非综合征型耳聋,而且同一家系内和不同家系间的母系成员在听力损失程度、发病年龄及听力曲线上存在很大差异.这些数据表明A1555G或C1494T突变是导致非综合征型耳聋发生的首要因子,其他修饰因子包括氨基糖甙类抗生素、线粒体DNA单倍型和核修饰基因等,在线粒体12S rRNA A1555G或C1494T突变相关的耳聋表型表达上起协同作用.作者简要介绍了这些因素对线粒体DNA突变致聋的影响以及母系遗传性耳聋发生的可能致病机制.     

2型糖尿病患者线粒体基因8个突变位点的研究

目的：探讨线粒体DNA tRNA Leu (UUR)基因及其相邻的ND1基因8个位点的突变与2型糖尿病的关系。 方法： 采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）的分析方法，对174例2型糖尿病患者和207名健康对照的mtDNA的3 243、3 256、3 290、3 316、3 394、3 421、3 426、3 460和3 593共9个已报道的突变位点同时进行筛选，并采用测序、反向斑点杂交和基因芯片进行方法学比较和确证，对检出的突变位点采用DNASTAR和Antherprot软件进行分析。 结果： 糖尿病组检出3 316(G→A) 突变5例(2.9%)，其中2例为多个位点突变：1例伴随3 256(C→T)和3 688(G→C)，另1例伴随3 606(A→G)；3 394(T→C)突变4例(2.3%)，3 593(T→C)、3 290(T→C)和3 618(T→C)突变各1例(0.6%)； 其中3 606(A→G)、3 618(T→C)和3 688(G→C) 为新突变位点，GenBank登录号为DQ092356；对照组只检出3 316(G→A)突变1例(0.5%)。两组间仅3 394(T→C) 突变率差异显著(P<0.05)。以上8个突变位点的测序、反向斑点杂交和基因芯片检测结果完全一致。3 606(A→G)为Leu的无意义突变，3 618(T→C)为Phe的无意义突变，3 688(G→C)为有意义突变，Ala→Pro,导致ND1蛋白的二级结构发生改变。 结论： 线粒体DNA ND1基因3 316(G→A)突变伴随3 688(G→C)突变，3 394(T→C) 突变可能与2型糖尿病发病有关。     

线粒体DNA 3243、3316点突变与2型糖尿病

目的研究2型糖尿病患者中线粒体tRNA^Leu(UUR)基因3243A／G突变和NADH脱氢酶亚单位1基因(ND1)基因3316G／A突变的发生频率及其与2型糖尿病的相关性。方法应用聚合酶链反应及限制性片段长度多态性技术检测225例中国云南2型糖尿病患者和195名无糖尿病家族史的健康对照者有无3243A／G突变和3316G／A突变，并经DNA直接测序确证。结果2型糖尿病患者中3316G／A突变者5例(2．22％)，195例对照者中突变者2例(1．03％)，突变发生率在两组间差异无统计学意义(P=0．4576)；两组中无线粒体3243A／G突变。结论线粒体tRNA^Leu(UUR)基因3243A／G突变在中国云南2型糖尿病人群中发生频率低，可能不是云南人群中2型糖尿病的常见病因。线粒体ND1基因3316G／A突变可能仅为人群中线粒体基因组的正常多态。其他的遗传、环境及子宫内因素需要进一步研究。     

遗传性共济失调一家系中发现的线粒体DNA突变

目的探索遗传性共济失调（hereditary ataxia，HA）家系中的线粒体DNA突变。方法采用聚合酶链反应扩增两个HA家系及35名健康对照者外周血白细胞的线粒体DNA，并对PCR产物进行单链构象多态性分析，对出现异常条带者进行线粒体DNA片段测序。结果其中一家系中的2例患者及1例无临床症状亲属检测到线粒体DNA11893（A→G）点突变。结论遗传性共济失调的发生、发展可能与线粒体DNA11893（A→G）点突变有关。     

早发糖尿病线粒体基因突变的研究

目的研究天津地区早发糖尿病(发病年龄≤45岁)中线粒体基因突变的发生率及其相关性。方法随机选取无血缘关系、发病年龄≤45岁的糖尿病患者348例;对照组207名,收集相应临床资料,提取外周血基因组DNA,应用聚合酶链反应-限制性片段长度多态性及克隆技术检测线粒体基因点突变。结果糖尿病组线粒体基因点突变总的发生率为8.3%,明显高于对照组(3.3%)(P<0.05),其中17例ND412026A→G突变(4.9%);10例ND1点突变(2.9%),其中包括4例3316G→A突变(1.2%),5例3394T→C突变(1.4%),1例3426A→G突变(0.3%);2例3243A→G突变,检出率为0.6%。在对照组中发现4例12026A→G(1.9%);2例3394T→C突变(0.9%);1例G3316G→A突变(0.5%);未发现3243A→G突变和3426A→G突变。结论天津地区早发糖尿病患者存在多种线粒体基因点突变。     

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.     

修饰因子对线粒体DNA突变致聋的影响

线粒体DNA突变是引起感音神经性耳聋的重要原因之一,这些突变主要位于线粒体12SrRNA和tRNA基因上.其中12S rRNA基因上的同质性A1555G和C1494T突变与氨基糖甙类抗生素造成的耳聋相关.携带这两个突变的个体对耳毒性药物高度敏感,导致临床上常见的"一针致聋"现象.但携带A1555G或C1494T突变的个体在没用药的情况下也能产生非综合征型耳聋,而且同一家系内和不同家系间的母系成员在听力损失程度、发病年龄及听力曲线上存在很大差异.这些数据表明A1555G或C1494T突变是导致非综合征型耳聋发生的首要因子,其他修饰因子包括氨基糖甙类抗生素、线粒体DNA单倍型和核修饰基因等,在线粒体12S rRNA A1555G或C1494T突变相关的耳聋表型表达上起协同作用.作者简要介绍了这些因素对线粒体DNA突变致聋的影响以及母系遗传性耳聋发生的可能致病机制.

Abstract：

Mutations in the mitochondrial DNA have been found to be one of the most important causes of sensorineural hearing loss. In particular, these mutations often occur in the mitochondrial 12S rRNA and tRNA genes. Of these, the homoplasmic A1555G and C1494T mutations in the 12S rRNA have been associated with both aminoglycoside induced and nonsyndromic hearing impairment in many families worldwide. Children carrying the A1555G or C1494T mutation are susceptible to the exposure of ototoxic drugs, thereby inducing or worsening hearing loss. Individuals harboring A1555G or C1494T mutation can also develop hearing loss even in the absence of aminoglycoside exposure. However, matrilineal relatives of intra-families or inter-families carrying the A1555G or C1494T mutation exhibit a wide range of severity,age-at-onset, and audiometric configuration of hearing impairment. These indicate that the A1555G or C1494T mutation is a primary factor underlying the development of deafness but insufficient to produce the clinical phenotype. Thus, other modifier factors, such as aminoglycoside (s), mitochondrial DNA haplotype(s) or nuclear modifier gene(s), play a role in the phenotypic expression of the deafness-associated mitochondrial 12S rRNA A1555G or C1494T mutation. In this review, we summarize the modifier factors for the phenotypic expression of deafness-associated 12S rRNA A1555G and C1494T mutations and propose the molecular pathogenetic mechanism of maternally inherited deafness.     

Novel mitochondrial DNA mutations associated with myopathy, cardiomyopathy, renal failure, and deafness

Patients with mitochondrial disease usually manifest multisystemic dysfunction with a broad clinical spectrum. When the tests for common mitochondrial DNA (mtDNA) point mutations are negative and the mtDNA defects are still hypothesized, it is necessary to screen the entire mitochondrial genome for unknown mutations in order to confirm the diagnosis. We report an 8-year-old girl who had a long history of ragged-red fiber myopathy, short stature, and deafness, who ultimately developed renal failure and fatal cardiac dysfunction. Respiratory chain enzyme analysis on muscle biopsy revealed deficiency in complexes I, II/III, and IV. Whole mitochondrial genome sequencing analysis was performed. Three novel changes: homoplasmic 15458T > C and 15519T > C in cytochrome b, and a near homoplasmic 5783G > A in tRNA(cys), were found in the proband in various tissues. Her mother and asymptomatic sibling also carry the two homoplasmic mutations and the heteroplasmic 5783G > A mutation in blood, hair follicles, and buccal cells, at lower percentage. The 5783G > A mutation occurs at the T arm of tRNA(cys), resulting in the disruption of the stem structure, which may reduce the stability of the tRNA. 15458T > C changes an amino acid serine to proline at a conserved alpha-helix, which may force the helix to bend. These two mutations may have pathogenic significance. This case emphasizes the importance of pursuing more extensive mutational analysis of mtDNA in the absence of common mtDNA point mutations or large deletions, when there is a high suspicion of a mitochondrial disorder.     

A homoplasmic mtDNA variant can influence the phenotype of the pathogenic m.7472Cins MTTS1 mutation: are two mutations better than one?

Mutations in mitochondrial tRNA (mt-tRNA) genes are well recognized as a common cause of human disease, exhibiting a significant degree of clinical heterogeneity. While these differences are explicable, in part, by differences in the innate pathogenicity of the mutation, its distribution and abundance, other factors, including nuclear genetic background, mitochondrial DNA (mtDNA) haplotype and additional mtDNA mutations may influence the expression of mt-tRNA mutations. We describe the clinical, biochemical and molecular findings in a family with progressive myopathy, deafness and diabetes and striking respiratory chain abnormalities due to a well-characterized heteroplasmic mt-tRNA mutation in the mt-tRNA(Ser(UCN)) (MTTS1) gene. In addition to the m.7472Cins mutation, all individuals were homoplasmic for another variant, m.7472A > C, affecting the adjacent nucleotide in the mt-tRNA(Ser(UCN)) structure. In addition to available patient tissues, we have analysed transmitochondrial cybrid clones harbouring homoplasmic levels of m.7472A > C and varying levels of the m.7472Cins mutation in an attempt to clarify the precise role of the m.7472A > C transversion in the underlying respiratory chain abnormality. Evidence from both in vivo and in vitro studies demonstrate that the m.7472A > C is able to modify the expression of the m.7472Cins mutation and would suggest that it is not a neutral variant but appears to cause a biochemical defect by itself, confirming that homoplasmic mtDNA variants can modulate the phenotypic expression of pathogenic, heteroplasmic mtDNA mutations.     

Mitochondrial myopathy associated with a novel 5522G>A mutation in the mitochondrial tRNATrp gene

We report a novel pathogenic mutation of the mitochondrial transfer RNA (tRNA) gene for tryptophan in a patient with isolated myopathy and persistently elevated creatine kinase. Muscle studies revealed ragged red fibres and decreased activity of respiratory chain complex I and cytochrome c oxidase (COX). Sequencing of the 22 mitochondrial tRNA genes revealed a mutation m.5522G>A, which alters a conserved base pairing in the D-stem of the tRNA for tryptophan. The mutation was heteroplasmic with a mutational load between 88 and 99% in COX-negative fibres. This case contributes to the genetic heterogeneity of mitochondrial diseases caused by mutations in mitochondrial tRNA genes.     

两个非综合征型耳聋家系中线粒体DNA 12S rRNA基因A827G突变

目的探讨线粒体DNA（mitochondrial DNA，mtDNA）12S rRNA基因与中国人非综合征型遗传性耳聋的关系。方法对两个母系遗传性的非综合征型耳聋家系中20名成员及32例散发耳聋患者外周血DNA进行12S rRNA、tRNA^ser（UCN）以及GJB2基因PCR扩增，产物通过限制性片段多态性分析及基因测序，进行突变检测和分析。结果所有研究对象的基因区域均扩增成功。12S rRNA全序列测定发现两家系中所有受检的母系成员（包括12例耳聋患者）均存在nt827A→G转换，并表现为同质性突变。而非母系成员该位点序列正常。32例散发耳聋中有1例A827G突变阳性。未检测到GJB2基因、tRN^ser（UCN） A7445G及12S rRNA A1555G突变。结论再次验证了mtDNA 12S rRNA基因突变在母系遗传性非综合征型耳聋发病中的重要性。首次发现mt DNA 12S rRNA nt827A→G转换是导致两个中国家系耳聋遗传易感性的分子基础。     

A patient with two mitochondrial DNA mutations causing PEO and LHON

We report a 22-year-old man with PEO and optic atrophy. PEO developed before the onset of optic atrophy. The patient showed mitochondrial myopathy with cytochrome c oxidase deficient fibers.In skeletal muscle the patient was homoplasmic for the mtDNA G11778A Leber hereditary optic neuropathy (LHON) mutation and heteroplasmic for the mtDNA 5 kb “common” deletion mutation. In blood only the homoplasmic LHON mutation was identified.The occurrence of two pathogenic mtDNA mutations is exceedingly rare. The clinical findings in this patient indicate that the combination of the two mtDNA mutations resulted in the expected combined phenotype since the mtDNA deletion mutation accounted for the PEO and the mtDNA G11778A point mutation for the optic atrophy.     

A simple oligonucleotide biochip capable of rapidly detecting known mitochondrial DNA mutations in Chinese patients with Leber's hereditary optic neuropathy (LHON)

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease. Clinically, no efficient assay protocols have been available. In this study, we aimed to develop an oligonucleotide biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing LHON and to investigate frequencies of LHON relevant variants in Anhui region of China. Thirty-two pairs of oligonucleotide probes matched with the mutations potentially linked to LHON were covalently immobilized. Cy5-lablled targets were amplified from blood DNA samples by a multiplex PCR method. Two kinds of primary mutations 11778 G > A and 14484 T > C from six confirmed LHON patients were interrogated to validate this biochip format. Further, fourteen Chinese LHON pedigrees and twenty-five unrelated healthy individuals were investigated by the LHON biochip, direct sequencing and pyrosequencing, respectively. The biochip was found to be able efficiently to discriminate homoplasmic and heteroplasmic mtDNA mutations in LHON. Biochip analysis revealed that twelve of eighteen LHON symptomatic cases from the 14 Chinese pedigree harbored the mutations either 11778G > A, 14484T > C or 3460G A, respectively, accounting for 66.7%. The mutation 11778G > A in these patients was homoplasmic and prevalent (55.5%, 10 of 18 cases). The mutations 3460G > A and 3394T > C were found to co-exist in one LHON case. The mutation 13708G > A appeared in one LHON pedigree. Smaller amount of sampling and reaction volume, easier target preparation, fast and high-throughput were the main advantages of the biochip over direct DNA sequencing and pyrosequencing. Our findings suggested that primary mutations of 11778G > A, 14484T > C or 3460G > A are main variants of mtDNA gene leading to LHON in China. The biochip would easily be implemented in clinical diagnosis.     

Exhaustive scanning approach to screen all the mitochondrial tRNA genes for mutations and its application to the investigation of 35 independent patients with mitochondrial disorders

To gain a better understanding of the molecular basisof mitochondrial (mt) encephalomyopathies, a highly heterogeneous condition, we developed a denaturing gradient gel electrophoresis-based approach that allows rapid and exhaustive screening for mutations of all 22 mt tRNA- encoding genes and their flanking regions in large cohorts of patients. This method, that detects heteroplasmy (i.e. co-existence of mutant and wild-type mtDNA species in various ratios) directly, was applied to the investigation of 35 independent patients with a disease phenotype compatible with a mitochondrial encephalomyopathy. Twenty-five of the 35 patients investigated displayed a sequence variation in at least one tRNA gene. A total of 46 different sequence variations (41 point mutations, four short insertions and one short deletion), among which 20 are new, were characterized. Forty of them were present in a homoplasmic state, whereas six were heteroplasmic. Twenty-two were located in tRNA genes, among which 10 are new homoplasmic or heteroplasmic sequence variations; 24 were located in flanking regions (12 in mRNA-encoding genes, seven of them leading to missense sequence variations; two in rRNA genes; and 10 in non-coding regions). This study demonstrates (i) the high frequency of homoplasmic tRNA gene sequence variations in our patient sample, and (ii) the existence of several polymorphic sites in tRNA gene regions that may be helpful for defining haplogroups in different populations. It relies on a screening method that can now be applied easily to other population samples.