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。     

25个携带线粒体12S rRNA A1555G 突变的中国汉族非综合征型耳聋家系

线粒体12S rRNA A1555G突变是引起氨基糖甙类药物诱导的非综合征型耳聋的重要原因之一。文章对收集的25个携带A1555G突变的中国汉族非综合征型耳聋家系进行了临床和分子遗传学评估。结果表明,这25个家系的母系成员在耳聋外显率、听力损失严重程度和发病年龄上存在较大差异。当包括和不包括氨基糖甙类药物使用史时,耳聋的平均外显率分别为28.1%和21.5%,排除氨基糖甙类药物时,耳聋的平均发病年龄从1~15岁不等。线粒体全序列分析发现了16个新变异,不同的线粒体DNA多态性位点显示这25个家系分别属于东亚人群A、B、D、F、G、M、N和R单倍型,其中线粒体单倍型B的家系耳聋外显率和表现度较其他单倍型高。此外,7个继发突变位点和21个高保守性位点突变可能增加了这些家系的耳聋外显率。GJB2基因上未检测到与耳聋相关的突变,表明在本研究的耳聋家系中,GJB2基因可能没有参与A1555G突变的表型表达。以上各方面提示,线粒体单倍型和其他因素可能参与了这25个家系耳聋患者的表型修饰。     

一个母系遗传非综合征耳聋大家系mtDNA序列分析

通过分析本家系mtDNA序列,探讨淮阴一非综合征耳聋大家系患病的分子遗传学机制.采用聚合酶链反应(PCR)扩增mtDNA与非综合征耳聋相关位点nt1555、nt7445的区域和人类种群研究的D-loop区、PCR-异源双链分析、PCR-RFLP、PCR产物克隆序列测定等技术对该家系进行了系统的研究.发现该家系中全部母系亲属有mtDNAA1555G突变,而家系中非母系个体、对照组(100例正常个体)的mtDNA1555位点均为A.该家系mtDNA7445位点无突变;该家系属于II型线粒体;发现家系D-loop区存在未见报道的碱基插入.提示mtDNAA1555G位点突变可能是导致该家系患者致聋的主要因素之一.遗传背景可能对家系疾病的表型存在一定程度的影响。 Abstract:We find an extensive nonsyndromic sensorineural deafness family in Huaiyin,and investigate the possible molecular genetic mechanism of matrilineal nonsyndromic sensorineural deafness.We use PCR,combined with PCR-heteroduplex analysis,PCR-RFLP and sequencing techniques to examine part of 12S rRNA,tRNAser(UCN),and D-loop region of this pedigree.1)We found an A to G transition at position 1555(A1555G) of the mitochondrial 12S rRNA from all the patients and four matrilineal.2)An new nucleotide insertion was indentified in D-Loop region.3)According to the polymorphism of D-loop,this pedigree belong to mitochondrial type II.The study showed that the A1555G mutation may be one of major factors in progressive inherited deafness of this family and genetic background should be investigated in the future.     

一个母系遗传非综合征耳聋大家系mtDNA序列分析

通过分析本家mtDNA序列,探讨淮阴一非综合耳聋大家患病的分子遗传学机制。采用聚合酶链反应（PCR）扩增mtDNA与非综合征耳聋相关位点nt1555,nt7445的区域和人类种群研究的D－loop区,PCR－异源双链分析,PCR－RFLP、PCR产物克隆序列测定等技术对该家系进行了系统的研究。发现该家系中全部母系亲属有mtDNAA1555G突变,而家系中非母 个体,对照组（100例正常个体）的mtDNA1555位点均为A。该家系mtDNA7445位点无突变;该系属于Ⅱ型线性体;发现家系D－loop区存在未见报道的碱基插入。提示mtDNAA1555G位点突变可能是导致该家系患致聋的主要因素之一。遗传背景可能对家系疾病的表现存在一定程度的影响。     

线粒体tRNAThr G15927A突变可能影响耳聋相关的12S rRNA A1555G突变的表型表达

摘要: 对1个中国汉族耳聋家系进行了临床和分子遗传学特征分析。家系中听力下降的母系成员表现为程度不等、听力图形态不同的听力损害, 但同为双侧对称的感觉神经性耳聋。该家系耳聋外显率很高, 包括药物致聋的耳聋外显率为75%, 而非药物致聋的外显率为41.7%。对母系成员进行线粒体DNA(mtDNA)全序列扩增分析, 发现了耳聋相关12S rRNA A1555G同质性突变位点和多态性位点, 属于东亚人群B5b单体型。在这些变异位点中, mtDNA 15927位点的G-A碱基变化破坏tRNA^Thr反密码子结构上十分保守的C-G碱基对, 这可能加重由A1555G突变造成的线粒体功能缺陷。这表明tRNAThrG15927A突变可能增强携带12S rRNA A1555G的中国汉族耳聋家系的外显率和表现度。     

非综合征耳聋大家系永生细胞系的建立及几种EB病毒转化外周血淋巴细胞建系方法的探讨

永久保存珍贵的家系材料,是对该家系进行深入研究的基础,为此采用EB病毒（Epstein-Barr virus,EBV）转化淋巴细胞的方法对中国江苏淮阴地区非综合征耳聋大家系行建系工作。该家系患者呈典型的母系遗传特征,且研究发现患者中均具有线粒体DNA 12s RNA A1555G突变,是迄今世界上最大的非综合征耳聋家系之一,在该家系的建系过程中 使用了4种不同的方法。建系结果分别为：微量全血法1株,冻存全血法1株,冻存白细胞法14株及环孢霉素A（CyA）法36株,共计52株。本文就建系工作及这四种转化方法作一简单探讨。     

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.     

携带线粒体tRNA~(Thr)15910C>T突变和12S rRNA 1555A>G突变的非综合征型耳聋家系的线粒体功能研究

该研究旨在探讨一个非综合征型耳聋(nonsyndromic hearing impairment,NSHI)家系中线粒体t RNAThr 15910CT和12S r RNA 1555AG基因突变共同作用对线粒体功能的影响。该研究建立了同时携带线粒体t RNAThr 15910CT和12S r RNA 1555AG基因突变(双突变组)、仅携带12S r RNA 1555AG基因突变(单突变组)和对照组的永生化淋巴细胞,这3组细胞系的线粒体DNA单体型均属于R单体型。对该家系的临床资料进行分析,当包括使用氨基糖苷类抗生素(aminoglycoside antibiotics,Am An)的药物性耳聋家系成员时,此家系耳聋外显率为37.5%;当排除用药的耳聋成员时,耳聋外显率是25.0%;相比之下,在用药和未用药的情况下,已报道的14个m.1555AG的耳聋家系的平均外显率只有12.8%和6.1%。通过对双突变组、单突变组和对照组永生化细胞系的线粒体功能进行研究,结果发现与对照组相比,双突变和单突变组细胞系的ROS水平分别上升了19.08%(P=0.005 4)和9.05%(P=0.003 7);ΔΨm水平分别下降了47.78%(P=0.006 3)和35.39%(P=0.0245);复合体II活力分别下降了8.26%(P=0.721 1)和19.48%(P=0.004 9),复合体IV活力分别下降了32.75%(P=0.033 5)和27.44%(P=0.180 5)。m.1555AG与m.15910CT共同作用,导致ROS生成量升高,ΔΨm水平下降以及线粒体呼吸链复合体IV活力降低等线粒体功能缺陷,提示m.15910CT可能是m.1555AG导致耳聋的继发性突变。     

携带线粒体tRNA~(Thr) 15943T>C协同12S rRNA 1555A>G突变的非综合征性耳聋致病机理的初步研究

该研究通过构建携带线粒体tRNA~(Thr )15943TC协同12S rRNA 1555AG突变(双突变组)的永生化淋巴母细胞系,同时建立仅含12S rRNA 1555AG突变(单突变组)和正常对照组永生化淋巴母细胞系,探究线粒体tRNA~(Thr )15943TC协同12S rRNA 1555AG突变与耳聋发病的关系,以了解线粒体突变致聋的分子机制。对该家系的临床资料进行分析的结果表明,当包括使用氨基糖苷类抗生素(aminoglycoside antibiotic,AmAn)的药物性耳聋家系成员时,此家系耳聋外显率为26%;当排除用药的耳聋成员时,此家系耳聋外显率是10%;相比之下,已报道的14个m.1555AG的耳聋家系的平均外显率在用药和未用药的情况下分别仅为13%和6%。利用Northern blot和Western blot分别检测三组细胞中线粒体tRNA和多肽的表达量,结果表明相比于正常对照组,tRNA~(Thr)在双突变组中的表达量显著降低,而在单突变组中的表达量无显著变化,tRNA~(Trp)、tRNA~(Ala) 、tRNA~(Tyr) 、tRNA~(Cys)和tRNA~(Pro)的稳态水平在三组细胞中没有显著性差异;CO2、CO3和A6在双突变组中的表达量显著降低,而在单突变组中的表达量无显著性差异;其他蛋白多肽在三组细胞中的表达量没有显著差异,说明m.15943TC突变降低了tRNA~(Thr)的稳态水平,致使线粒体部分多肽表达水平下降,从而影响了线粒体呼吸链复合体的功能和稳定性进而导致了线粒体代谢障碍,提示线粒体tRNA~(Thr) 15943TC可能与m.1555AG突变引起的耳聋相关。     

线粒体T12338C突变可能是与Leber遗传性视神经病变相关的突变位点

Leber遗传性视神经病变变(Leber’s hereditary optic neuropathy,LHON)是一种与线粒体DNA(Mito-chondrial DNA,mtDNA)突变相关的母系遗传性眼科疾病。文章报道了两例具有典型LHON临床、分子遗传特征的中国汉族家系。首先通过对家系先证者和其他成员进行眼科相关检查,发现两个家系成员中视力都仅有先证者一人损害严重,即外显率很低。经常规的方法对母系成员进行mtDNA测序及相关软件分析,结果发现携带ND4 G11696A和ND5 T12338C同质性突变位点,多态性变异位点均属于东亚单体型F2。线粒体DNA ND4 G11696A是一个已知的与LHON相关的突变位点,而T12338C位于线粒体氧化磷酸化复合体I亚基ND5的第2个碱基,该突变使起始密码子由蛋氨酸转变成苏氨酸,并且紧连tRNALeu(CUN)的3′末端。这可能影响tRNA Leu(CUN)空间结构和稳定性发生改变,以及起始密码子改变导致线粒体ND5蛋白合成功能受损和ATP障碍,最终导致需求能量高的视神经受损和视力损害。因此,线粒体ND4 G11696A和ND5 T12338C突变可能协同作用Leber遗传性视神经病变的发生,是与LHON相关的mtDNA突变位点,但外显率很低说明突变本身不足以造成LHON的表型表达,提示其他修饰因子(核修饰基因、环境等)可能对这两个家系发病起协同作用。     

Functional characterization of the mitochondrial 12S rRNA C1494T mutation associated with aminoglycoside-induced and non-syndromic hearing loss

In this study, we report the biochemical characterization of the deafness-associated mitochondrial 12S rRNA C1494T mutation using 27 cybrid cell lines constructed by transferring mitochondria from 9 lymphoblastoid cell lines derived from a Chinese family into human mitochondrial DNA (mtDNA)-less (ρ°) cells. Six cybrids derived from two asymptomatic members, and nine cybrids derived from three symptomatic members of the Chinese family carrying the C1494T mutation exhibited ~38 and 43% decrease in the rate of mitochondrial protein labeling, respectively, compared with twelve cybrids derived from four Chinese control individuals. These defects are apparently a primary contributor to significant reductions in the rate of overall respiratory capacity or the rate of malate/glutamate promoted respiration, or succinate/G3P-promoted respiration, or TMPD/ascorbate-promoted respiration in mutant cybrid cell lines derived from either symptomatic or asymptomatic individuals. Furthermore, the very significant/nearly identical increase in the ratio of doubling times in DMDM medium in the presence/absence of high concentration of paromomycin was observed in symptomatic or asymptomatic cybrid cell lines carrying the C1494T mutation as compared with the average rate in control cell lines. These observations provide the direct biochemical evidences that the C1494T mutation is a pathogenic mtDNA mutation associated with aminoglycoside-induced and non-syndromic hearing loss. In addition, these data provide the first biochemical evidence that nuclear background plays a critical role in the phenotypic manifestation of non-syndromic hearing loss and aminoglycoside toxicity associated with the C1494T mutation.     

Maternally inherited aminoglycoside-induced and nonsyndromic deafness is associated with the novel C1494T mutation in the mitochondrial 12S rRNA gene in a large Chinese family

We report here the characterization of a large Chinese family with maternally transmitted aminoglycoside-induced and nonsyndromic deafness. In the absence of aminoglycosides, some matrilineal relatives in this family exhibited late-onset/progressive deafness, with a wide range of severity and age at onset. Notably, the average age at onset of deafness has changed from 55 years (generation II) to 10 years (generation IV). Clinical data reveal that the administration of aminoglycosides can induce or worsen deafness in matrilineal relatives. The age at the time of drug administration appears to be correlated with the severity of hearing loss experienced by affected individuals. Sequence analysis of mitochondrial DNA in this pedigree identified a homoplasmic C-to-T transition at position 1494 (C1494T) in the 12S rRNA gene. The C1494T mutation is expected to form a novel U1494-1555A base pair, which is in the same position as the C1494-1555G pair created by the deafness-associated A1555G mutation, at the highly conserved A site of 12S rRNA. Exposure to a high concentration of paromomycin or neomycin caused a variable but significant average increase in doubling time in lymphoblastoid cell lines derived from four symptomatic and two asymptomatic individuals in this family carrying the C1494T mutation when compared to four control cell lines. Furthermore, a significant decrease in the rate of total oxygen consumption was observed in the mutant cell lines. Thus, our data strongly support the idea that the A site of mitochondrial 12S rRNA is the primary target for aminoglycoside-induced deafness. These results also strongly suggest that the nuclear background plays a role in the aminoglycoside ototoxicity and in the development of the deafness phenotype associated with the C1494T mutation in the mitochondrial 12S rRNA gene.     

The A1555G mitochondrial DNA mutation in Greek patients with non-syndromic, sensorineural hearing loss

Mitochondrial DNA mutations are undoubtedly a factor that contributes to sensorineural, non-syndromic deafness. One specific mutation, the A1555G, is associated with both aminoglycoside-induced and non-syndromic hearing impairment. The mutation is considered to be the most common of all mitochondrial DNA deafness-causing mutations but its frequency varies between different populations. Here we report on the first large screening of the A1555G mitochondrial DNA mutation in the Greek population. The aim of this study was to determine the frequency of the A1555G mutation in Greek sensorineural, non-syndromic deafness patients, with childhood onset. We screened 478 unrelated Greek patients with hearing loss of any degree and found two individuals harboring the A1555G mutation (0.42%). Both cases had been subjected to aminoglycosides. They were prelingual, familial and homoplasmic for the A1555G mutation. One of the cases was also found heterozygous for the frequent GJB2 35delG mutation, while the other case was negative. The A1555G mutation seems to be less common than in other European populations.     

Mitochondrial non-syndromic sensorineural hearing loss: a clinical, audiological and pathological study from Italy, and revision of the literature

Over the last decade, a number of distinct mutations in the mtDNA (mitochondrial DNA) have been found to be associated with both syndromic and non-syndromic forms of hearing impairment. Their real incidence as a cause of deafness is poorly understood and generally underestimated. Among the known mtDNA mutations, the A1555G mutation in the 12S gene has been identified to be one of the most common genetic cause of deafness, and it has been described to be both associated to non-syndromic progressive SNHL (sensorineural hearing loss) and to aminoglycoside-induced SNHL. In the present study, we have investigated the presence of mtDNA alterations in patients affected by idiopathic non-syndromic SNHL, both familiar and sporadic, in order to evaluate the frequency of mtDNA alterations as a cause of deafness and to describe the audiological manifestations of mitochondrial non-syndromic SNHL. In agreement with previous studies, we found the A1555G mutation to be responsible for a relevant percentage (5.4%) of cases affected with isolated idiopathic sensorineural hearing impairment.     

Co-occurrence of A1555G and G11778A in a Chinese family with high penetrance of Leber's hereditary optic neuropathy

Co-occurrence of double pathogenic mtDNA mutations with different claimed pathological roles in one mtDNA is infrequent. It is tentative to believe that each of these pathogenic mutations would have its own deleterious effect. Here we reported one three-generation Chinese family with a high penetrance of LHON (78.6%). Analysis of the complete mitochondrial genome in the proband revealed the presence of the LHON primary mutation G11778A in the NADH dehydrogenase 4 (ND4) gene and a deafness-associated mutation A1555G in the 12S rRNA gene. The other mtDNA variants in this family suggested a haplogroup status G2b. Although A1555G has long been confirmed to be a primary mutation for aminoglycoside-induced and non-syndromic hearing loss, none of the maternally related members in this family showed hearing impairment. It thus seems that the occurrence of A1555G in this family had no pathological manifestation. However, whether A1555G has a synergistic effect with G11778A and contribute to the high penetrance of LHON remained an open question. To our knowledge, this is the first report that identified the co-existence of a deafness mutation A1555G and a primary LHON mutation G11778A in one family.     

Cochlear alterations in deaf and unaffected subjects carrying the deafness-associated A1555G mutation in the mitochondrial 12S rRNA gene

The A1555G mutation in the mitochondrial small ribosomal RNA gene (12S rRNA) has been associated with aminoglycoside-induced, nonsyndromic hearing loss. However, the clinical phenotype of A1555G carriers is extremely variable. In the present study, we have performed an audiological evaluation of a group of deaf patients and hearing carriers of mutation A1555G with the aim to assess the prevalence of the mutation and determine the associated cochlear alterations. Fifty-four patients affected of nonsyndromic hearing loss were screened for the presence of the A1555G mitochondrial mutation. Nine of the familial cases (21%) carried the A1555G mutation, whereas the mutation was not found in any of the sporadic cases. The positive cases and some of their family members underwent a clinical study consisting in a clinical evaluation and audiological testing. The phenotype of A1555G patients varied in age of onset and severity of hearing loss, ranging from profound deafness to completely normal hearing. The audiometric alterations showed bilateral hearing loss, being more severe at high frequencies. Otoacoustic emissions were absent in deaf A1555G carriers, and auditory brainstem response indicated a prolonged Wave I, suggesting a cochlear dysfunction without any effect of the auditory nerve. Moreover, all hearing carriers of A1555G also presented alterations in cochlear physiology. In conclusion, the A1555G mitochondrial mutation causes a cochlear form of deafness, characterized by a more severe loss of hearing at high frequencies. Although the expression of the mutation is variable, cochlear alterations are present in all carriers of mutation A1555G.     

Candidate locus for a nuclear modifier gene for maternally inherited deafness

Maternally inherited deafness associated with the A1555G mutation in the mitochondrial 12S ribosomal RNA (rRNA) gene appears to require additional environmental or genetic changes for phenotypic expression. Aminoglycosides have been identified as one such environmental factor. In one large Arab-Israeli pedigree with congenital hearing loss in some of the family members with the A1555G mutation and with no exposure to aminoglycosides, biochemical evidence has suggested the role of nuclear modifier gene(s), but a genomewide search has indicated the absence of a single major locus having such an effect. Thus it has been concluded that the penetrance of the mitochondrial mutation appears to depend on additive effects of several nuclear genes. We have now investigated 10 multiplex Spanish and Italian families with 35 members with the A1555G mutation and sensorineural deafness. Parametric analysis of a genomewide screen again failed to identify significant evidence for linkage to a single autosomal locus. However, nonparametric analysis supported the role of the chromosomal region around marker D8S277. The combined maximized allele-sharing LOD score of 3.1 in Arab-Israeli/Spanish/Italian families represents a highly suggestive linkage result. We suggest that this region should be considered a candidate for containing the first human nuclear modifier gene for a mitochondrial DNA disorder. The locus operates in Arab-Israeli, Spanish, and Italian families, resulting in the deafness phenotype on a background of the mitochondrial A1555G mutation. No obvious candidate genes are located in this region.     

Isolation and characterization of the putative nuclear modifier gene MTO1 involved in the pathogenesis of deafness-associated mitochondrial 12 S rRNA A1555G mutation

The human mitochondrial 12 S rRNA A1555G mutation has been found to be associated with aminoglycoside-induced and non-syndromic deafness. However, putative nuclear modifier gene(s) have been proposed to regulate the phenotypic expression of this mutation. In yeast, the mutant alleles of MTO1, encoding a mitochondrial protein, manifest respiratory-deficient phenotype only when coupled with the mitochondrial 15 S rRNA P(R)454 mutation corresponding to human A1555G mutation. This suggests that the MTO1-like modifier gene may influence the phenotypic expression of human A1555G mutation. Here we report the identification of full-length cDNA and elucidation of genomic organization of the human MTO1 homolog. Human Mto1 is an evolutionarily conserved protein that implicates a role in the mitochondrial tRNA modification. Functional conservation of this protein is supported by the observation that isolated human MTO1 cDNA can complement the respiratory deficient phenotype of yeast mto1 cells carrying P(R)454 mutation. MTO1 is ubiquitously expressed in various tissues, but with a markedly elevated expression in tissues of high metabolic rates including cochlea. These observations suggest that human MTO1 is a structural and functional homolog of yeast MTO1. Thus, it may play an important role in the pathogenesis of deafness-associated A1555G mutation in 12 S rRNA gene or mutations in tRNA genes.