Novel m.4268T>C mutation in the mitochondrial tRNA^(Ile) gene is associated with hearing loss in two Chinese families

BACKGROUND Herein,we report the genetic,clinical,molecular and biochemical features of two Han Chinese pedigrees with suggested maternally transmitted non-syndromic hearing loss.AIM To investigate the pathophysiology of hearing loss associated with mitochondrial tRNA mutations.METHODS Sixteen subjects from two Chinese families with hearing loss underwent clinical,genetic,molecular,and biochemical evaluations.Biochemical characterizations included the measurements of tRNA levels using lymphoblastoid cell lines derived from five affected matrilineal relatives of these families and three control subjects.RESULTS Three of the 16 matrilineal relatives in these families exhibited a variable seriousness and age-at-onset(8 years)of deafness.Analysis of mtDNA mutation identified the novel homoplasmic tRNA^(Ile) 4268T>C mutation in two families both belonging to haplogroup D4j.The 4268T>C mutation is located in a highly conserved base pairing(6U–67A)of tRNA^(Ile).The elimination of 6U–67A basepairing may change the tRNA^(Ile) metabolism.Functional mutation was supported by an approximately 64.6%reduction in the level of tRNA^(Ile) observed in the lymphoblastoid cell lines with the 4268T>C mutation,in contrast to the wild-type cell lines.The reduced level of tRNA was below the proposed threshold for normal respiration in lymphoblastoid cells.However,genotyping analysis did not detect any mutations in the prominent deafness-causing gene GJB2 in any members of the family.CONCLUSION These data show that the novel tRNA^(Ile) 4268T>C mutation was involved in maternally transmitted deafness.However,epigenetic,other genetic,or environmental factors may be attributed to the phenotypic variability.These findings will be useful for understanding families with maternally inherited deafness.

Mitochondrial rRNA and tRNA and hearing function

The human ear is a delicate sensory apparatus of hearing for normal communication, and its proper functioning is highly dependent on mitochondrial oxidative phosphorylation. The first mitochondrial point mutation for nonsyndromic and aminoglycoside-induced hearing loss was identified in 1993. Since then a number of inherited mitochondrial mutations have been implicated in hearing loss. Most of the molecular defects responsible for mitochondrial disorder-associated hearing loss are mutations in the 12S rRNA gene and tRNA genes. In this review, after a short description of normal hearing mechanisms and mitochondrial genetics, we outline the recent advances that have been made in the identification of deafness-associated mitochondrial mutations, and discuss how mitochondrial dysfunction contributes to hearing loss.

线粒体tRNA^(Ile) A4317G突变可能影响12S rRNA A1555G突变相关的耳聋表型表达

线粒体12S rRNA基因A1555G突变与非综合征型耳聋和氨基糖甙类抗生素(Aminoglycoside antibiotics,AmAn)致聋相关。文章通过对一个携带线粒体12S rRNA A1555G突变的中国汉族母系遗传耳聋大家系成员进行听力学检查和遗传学分析,发现该家系耳聋外显率很高,包括AmAn使用史的耳聋外显率为81%,不包括AmAn使用史的耳聋外显率66.7%,明显高于其他携带A1555G突变的耳聋家系。对该家系进行线粒体基因组全序列分析发现存在同质性的tRNAIleA4317G突变和38个多态位点,属于东亚线粒体B4c1b2单体型。进一步分析发现A4317G突变位于tRNAIle的tRNAIleTΨC环区的高保守性区域(第59通用位点),该突变可能影响tRNAIle二级结构和功能,从而导致线粒体功能缺陷,且在961例正常对照中未发现该突变。同时,其他线粒体DNA并未发现有功能意义的突变位点。因此,A4317G突变可能影响tRNAIle的代谢并加重A1555G突变导致的线粒体功能缺陷,最终导致耳聋的外显率增高。从而推测线粒体tRNA Ile A4317G突变可能是一个影响12S rRNAA1555G突变的耳聋表型表达的因素。

线粒体tRNA^(Thr) G15927A突变可能影响耳聋相关的12S rRNA A1555G突变的表型表达

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12SrRNAA1555G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,mtDNA15927位点的G-A碱基变化破坏tRNAThr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12SrRNAA1555G的中国汉族耳聋家系的外显率和表现度。

Mitochondrial tRNA^(Glu) A14693G variant may modulate the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in a Han Chinese family

Mutations in mitochondrial 12S rRNA gene are one of the most important causes of aminoglycoside-induced and nonsyndromic hearing loss. Here we report the characterization of one Han Chinese pedigree with aminoglycoside-induced and nonsyndromic hearing loss. This Chinese family carrying the 12S rRNA A1555G mutation exhibited high penetrance and expressivity of heating impairment. In particular, penetrances of hearing loss in this family pedigree were 43.8% and 25%, respectively, when aminoglycoside-induced heating loss was included or excluded. Mutational analysis of entire mitochondrial genomes in this family showed the homoplasmic A1555G mutation and a set of variants belonging to haplogroup Y2. Of these, the A14693G variant occurred at the extremely conserved nucleotide （conventional position 54） of the TψC-loop of tRNA^Clu and was absent in 156 Chinese controls. Nucleotides at position 54 of tRNAs are often modified, thereby contributing to the structural formation and stabilization of functional tRNAs. Thus, the structural alteration of tRNA by the A14693G variant may lead to a failure in tRNA metabolism and impair mitochondrial protein synthesis, thereby worsening mitochondrial dysfunctions altered by the A1555G mutation. Therefore, the tRNA^Glu A14693G variant may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated A1555G mutation in this Chinese pedigree.

非综合征型耳聋患者mtDNA A1555G异质性突变比例与临床表型的关系

目的定量检测非综合征型耳聋患者线粒体DNA(mitochondrial DNA,mtDNA)1555突变型/野生型的拷贝数,探讨突变型/野生型比例与临床表型之间的关系。方法建立实时定量PCR技术和扩增阻滞突变系统(Real-time quantitative PCR和Amplification refractory mutation system,RT-ARMS-qPCR系统)对含突变型和野生型mtDNA 1555位点的拷贝数进行定量检测并计算比例。共检测散发组12例、家系组7例异质性突变患者,结合耳聋患者的临床资料,分析突变型与野生型的比例与耳聋严重程度的关系。结果散发组mtDNA A1555G异质性突变的患者中,突变型mtDNA所占的比例与耳聋轻重程度相关(r=0.771,P=0.003);家系组患者中,突变型mtD-NA所占的比例亦与耳聋轻重程度相关(r=0.850,P=0.015)。结论突变型mtDNA占所有mtDNA的比例与耳聋的严重程度密切相关,是非综合征型耳聋临床表型多样性的分子基础。

线粒体tRNA^Ser（UCN）突变及其致聋机制的研究

线粒体tRNA^Ser（UCN）基因突变与感音神经耳聋密切相关。tRNA^Ser（UCN）基因上游部分的突变将影响tRNA^Ser（UCN）结构以及转录后的加工，例如位于tRNA^Ser（UCN）前体3’端的线粒体7444G〉A等突变，以及位于tRNA^Ser（UCN）二级结构茎以及环上的线粒体7472insC和7511T〉C等突变可改变tRNA^Ser（UCN）的稳定性，进而影响线粒体内多肽的合成，降低ATP的产量，最终导致耳聋。本文主要讨论与耳聋相关的线粒体tRNA^Ser（UCN）基因突变及其致聋的机理。

七个非综合征型耳聋家系患者的mtDNAA1555G突变性质与特点

目的探讨非综合征型耳聋家系患者mtDNAA1555G突变性质及其特点，探索l临床表型多样性的分子遗传学基础。方法应用聚合酶链反应一限制性片段长度多态和实时荧光-扩增阻碍突变系统一定量PCR（realtime—amplificationrefractorymutationsystem—quantitativePCR，RT—ARMS—qPCR）检测7个非综合征型耳聋家系71个成员的mtDNAA1555G突变，并收集、分析其临床资料。结果7个家系中所有受检的母系成员mtDNAA1555G突变均为阳性，突变性质含同质性和异质性两种；非母系成员及配偶该突变为阴性。7个家系mtDNAA1555G同质性突变的拷贝数与耳聋轻重程度相关（R=0．341，P=0．022）；mtDNAA1555G异质性突变的拷贝数与耳聋轻重程度相关（R=0．85，P=0．015）。结论mtDNAA1555G突变可导致非综合征型耳聋和氨基糖甙类抗生素致聋，其突变性质含同质性和异质性两种，且含mtDNAA1555G位点的突变型与野生型的比例与耳聋的严重程度密切相关。

台州地区耳聋相关的线粒体12S rRNA C1494T突变筛查

目的探讨线粒体12SrRNA基因C1494T突变和耳聋的相关性,为了耳聋的防治提供理论依据。方法在台州地区收集耳聋患者200例以及100例性别和年龄相仿的正常对照,使用聚合酶链反应(PCR)和测序法筛选线粒体C1494T突变,并进行临床和分子遗传学的评估。结果测序结果显示有2名耳聋患者携带同质性的C1494T突变,检出率为1%,其中只有1位患者具有明显的家族遗传。该家系的外显率较高,线粒体全基因组测序发现携带tRNASer(UCN)T7505C突变,该突变在进化上高度保守,可能会引起线粒体功能损伤,进而加剧了耳聋的外显率和表现度。结论线粒体C1494T突变是药物性耳聋相关的重要分子基础,继发突变T7505C可能会影响C1494T突变的表型表达,因此,本研究对耳聋的防治有重要的临床指导意义。