Mitochondrial DNA mutations associated with aminoglycoside induced ototoxicity

Aminoglycosides(Am An) are widely used for their great efficiency against gram-negative bacterial infections. However, they can also induce ototoxic hearing loss, which has affected millions of people around the world. As previously reported, individuals bearing mitochondrial DNA mutations in the 12 S rRNA gene, such as m.1555A>G and m.1494C>T, are more prone to Am An-induced ototoxicity. These mutations cause human mitochondrial ribosomes to more closely resemble bacterial ribosomes and enable a stronger aminoglycoside interaction. Consequently,exposure to Am An can induce or worsen hearing loss in these individuals. Furthermore, a wide range of severity and penetrance of hearing loss was observed among families carrying these mutations. Studies have revealed that these mitochondria mutations are the primary molecular mechanism of genetic susceptibility to Am An ototoxicity, though nuclear modifier genes and mitochondrial haplotypes are known to modulate the phenotypic manifestation.

Mitochondrial variants may influence the phenotypic manifestation of Leber's hereditary optic neuropathy-associated ND4 G11778A mutation

We report here the characterization of a five-generation Han Chinese family with Leber＇s hereditary optic neuropathy （LHON）. Strik- ingly, this Chinese family displayed high penetrance and expressivity of visual loss. The average age-of-onset of vision loss was 18 years in this family. Nineteen （11 males/8 females） of 29 matrilineal relatives in this family developed visual loss with a wide range of severity, ranging from blindness to normal vision. Sequence analysis of mitochondrial genome in this pedigree revealed the presence of the ND4 G 11778A mutation and 44 other variants belonging to Asian haplogroup M7b. The G 11778A mutation is present at homoplasmy in matri- lineal relatives of this Chinese family. Of other variants, the C01 G6480A, ND5 T12811C and Cytb A15395G located at highly conserved residues of corresponding polypeptides. In fact, these variants were implicated to be involved in other clinical abnormalities. Here, these variants may act in synergy with the primary LHON-associated Gl1778A mutation. Thus, the mitochondrial dysfunction caused by the primary ND4 G11778A mutation may be worsened by these mitochondrial variants. The results imply that the G6480A, T12811C and A15395G variants might have a potential modifier role in increasing the penetrance and expressivity of the primary LHON-associated G11778A mutation in this Chinese family.

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.

A peep into mitochondrial disorder： multifaceted from mitochondrial DNA mutations to nuclear gene modulation

Mitochondrial genome is responsible for multiple human diseases in a maternal inherited pattern, yet phenotypes of patients in a same pedigree frequently vary largely. Genes involving in epigenetic modification, RNA processing, and other biological pathways, rather than ＂threshold effect＂ and environmental factors, provide more specific explanation to the aberrant phenotype. Thus, the double hit theory, mutations both in mito- chondrial DNA and modifying genes aggravating the symptom, throws new light on mitochondrial dysfunc- tion processes. In addition, mitochondrial retrograde signaling pathway that leads to reconfiguration of cell metabolism to adapt defects in mitochondria may as well play an active role. Here we review selected examples of modifier genes and mitochondrial retrograde signaling in mitochondrial disorders, which refine our understanding and will guide the rational design of clinical therapies.

The role of mitochondria in osteogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells （MSCs） are progenitors of con- nective tissues, which have emerged as important tools for tissue engineering due to their differentiation potential along various cell types. In recent years, accumulating evidence has suggested that the regulation of mitochon- dria dynamics and function is essential for successful dif- ferentiation of MSCs. In this paper, we review and provide an integrated view on the role of mitochondria in MSC dif- ferentiation. The mitochondria are maintained at a relatively low activity level in MSCs, and upon induction, mtDNA copy number, protein levels of respiratory enzymes, the oxygen consumption rate, mRNA levels of mitochondrial biogene- sis-associated genes, and intracellular ATP content are increased. The regulated level of mitochondrial ROS is found not only to influence differentiation but also to con- tribute to the direction determination of differentiation. Understanding the roles of mitochondrial dynamics during MSC differentiation will facilitate the optimization of differ- entiation protocols by adjusting biochemical properties, such as energy production or the redox status of stem cells, and ultimately, benefit the development of new pharmacologic strategies in regenerative medicine.

哺乳动物内耳毛细胞的体外培养（英文）

由于内耳的血脑屏障作用,药物渗入到内耳比较困难。新生小鼠内耳毛细胞的体外培养体系的建立,为体外进行支持细胞转分化机制的研究和进行体外药物损伤毛细胞实验等提供实验技术的前提。为了避免毛细胞体外培养过程中污染杂菌,解剖内耳耳蜗的整个过程十分重要。处死小鼠后,将其浸泡在75%酒精中1～3分钟,防止鼠毛污染培养基。打开内耳耳蜗之前,选用添加了青霉素的磷酸盐缓冲液(1×PBS);培养过程中使用的是仅仅添加青霉素的培养基来减少对毛细胞的损伤。在基底膜培养的第一步,选用DMEM(包含5%马血清体积比和5%胎牛血清)作为组织粘附培养的培养基,保证足够的营养,同时更好地维持整个基底膜培养状态下的形态。在之后的培养中,选用DMEM(添加了10%胎牛血清、1%N2和1%B27)作为长期的培养基。使用含有表皮生长因子的N2和B27的培养基进行基底膜以及椭圆囊之后的培养,有助于维持毛细胞的体外生长时间。选用鼠尾胶包被盖玻片后培养,可以增加基底膜和椭圆囊的粘附作用,保证毛细胞静纤毛的向上生长。该文章展现了哺乳动物毛细胞的体外培养的具体方法,能够较好地维持耳蜗基底膜在体外培养的形态,并增加毛细胞体外培养的存活时间。

中国人群中非综合征耳聋相关MARVELD2(DFNB49)基因新单核苷酸多态性位点分析

目的:探究MARVELD2在中国非综合征耳聋(NSHL)人群中的突变频谱和突变频率。创新点:发现MARVELD2突变频谱具有明显种族特异性。中国NSHL人群中的突变位点及频率不同于已报道的其他人群,并首次筛选到新致聋候选突变MARVELD2 c.730G>A。本研究有助于进一步阐释MARVELD2在NSHL中的作用。方法:收集283例NSHL患者外周血,提取基因组DNA,涉及9对引物覆盖MARVELD2基因编码区,经聚合酶链反应(PCR)扩增后Sanger测序。测序结果与参考序列比对,获得的MARVELD2变异位点通过正常人群频率比较、氨基酸保守性分析、氨基酸性质分析、SIFT和PolyPhen有害性预测及蛋白结构功能预测分析等进一步筛选得到耳聋候选突变位点。结论:中国NSHL人群的MARVELD2突变位点与巴基斯坦人群,以及斯洛伐克、匈牙利和捷克罗马人群不同,具有明显的种族特异性。本研究在283个NSHL病例中共鉴定了11个变异位点。其中,c.730G>A突变可能影响MARVELD2蛋白的正常功能,与NSHL致病有较高的相关性,是一个候选致聋突变。