The Clinical And Molecular Research Of Mitochondrial Inherited Deafness And Nuclear Modified Genes

PART?:The 1555A>G mutation is the most common cause of aminoglycoside-induced and non-syndromic deafness. However, the variable clinical phenotype and incomplete penetrance of 1555A>G-induced hearing loss complicate our understanding of this mutation. Environmental factors, nuclear genes, mitochondrial haplotypes/variants and a possible threshold effect have been reported to may be involved in its manifestation. Here, we performed a clinical, molec?Lar, genetic and phylogenic analysis in a six-generation Chinese family. A clinical evaluation revealed that affected individuals without aminoglycoside exposure developed hearing loss extending gradually from 12000 Hz to 8000 Hz and then to 4000 Hz. Using pyrosequencing, we detected an identical homoplasmic 1555A>G mutation in all individuals except one. We did not find any correlation between the mutation load and the severity of hearing loss. T123N coexisted with the 1555A>G mutation in six affected subjects in our pedigree. Analysis of the complete mtDNA genome of this family revealed that this family belonged to haplotype B4C1C and exhibited high penetrance. Upon the inclusion of subjects that had been exposed to aminoglycosides, the penetrance of the hearing loss was 63.6%.; without exposure to aminoglycosides, it was 51.5%. This pedigree and another reported Chinese pedigree share the same haplotype (B4C1C) and lack functionally significant mitochondrial tRNA variants, but nevertheless they exhibit a different penetrance of hearing loss. Our res?Lts imply that the factors responsible for the higher penetrance and variable expression of the deafness associated with the 1555A>G mutation in this pedigree may not be mtDNA haplotype/variants, but rather nuclear genes and/or aminoglycosides. Furthermore,12000 Hz was the first frequency to be affected in subjects with 1555A>G-induced hearing loss especially in those without a clinical syndrome and aminoglycoside exposure. PART II:Mitochondrial maternally inherited deafness is one of the inherited deafness. Phenotypic heterogeneity is an unsolved problem of mitochondrial disorders. The 1555A>G mutation is the most common cause of aminoglycoside-induced and non-syndromic deafness, but accurate pathological mechanism of mitochondria deafness at present is not clear. Nuclear genes may be the modifier of the clinical phenotype due to A1555G mutation. The most ideal method looking for the nuclear modifier genes of the A1555G mutations is the study of the cochlear cells expression in different people who have different clinical phenotype due to same A1555G mutation, but we can't get cochlear organization. Early pedigree linkage analysis located TRMU, MTO1, TFB1M, GTPBp3 these four nuclear genes were relevant to mitochondrial RNA modified. And mtTFB1M and mitochondrial deafness syndrome have potential relationship, because in E. coli, lack of the KsgA methyltransferase leads to resisitance to the aminoglycoside kasugamycin.but when give mt-TFBIM, mt-TFB1M can functionally replace the homologous KsgA dimethyltransferase and restore the susceptibility to aminoglycoside in E. coli.Transcription of mtDNA requires a small number of nucleus-encoded proteins including a single RNA polymerase(POLRMT), auxiliary factors necessary for promoter recognition (TFB1M, TFB2M) and activation (Tfam), and a termination factor (mTERF).TFB1M and TFB2M are important factors involved in regulation of mitochondrial gene expression in mammalian cells.Both of them are nucleus encoded and imported to mitochondrial. Their expression is tightly regulated by nucler transcription factors.Studies have found that A1555G mtDNA mutations like TFB1M over express, can lead to 12S rRNA hypermethylation, result in similar faulty retrograde mitochondrial synthetic signals and predisposescell to stress-induced cell death. And methylation of 12S rRNA is necessary for stability of the mammalian mitochondrial ribosomes,12S rRNA hypermethylation may be a unrealized novel mitochondrial biogenesis signal.Since there is not a real model of mouse which can be used as A1555G mitochondrial DNA mutations, so we try to observe the relationship between nuclear genes TFB1M, TRMU, cochlear mitochondria, and hair cell apoptosis in neomycin-induced hair cell death in the mouse in vitro. Namely existence of exogenous compounds in hair cell, whether there is interaction between nucleus and mitochondria which cause hair cell apoptosis occurrence, development or death.First,we establish practical method of auditory epithelia culture model in vitro for P1?P2 mice.Second we detect the mitochondrial membrane potential of auditory epithelia.Then we detect the expression of nuclear gene TFBM, TRMU and apoptosis related gene in auditory epithelia of mice. During the first 4 h in culture with neomycin treatment, we found the morphogenesis of hair cell in middle turn were aligned, the mitochondrial membrane potential was intact in the majority of the cells. Except few hair cell was indicated as by a slight decrease their membrane potential Subsequently, after about 3 hours, the hair cell of middle turn and basical turn lose their mitochondrial membrane potential.Meanwhile we found hair cell death as detected by PI staining of the nuclei. During the 12h in culture, majority of the hair cell in middle turn and basical turn lost. The expression of nuclear and mitochondrial TFB1M was elevated. During the 24h in culture, almost all mitochondria of OHCs had lost their membrane potential, the level of Caspase9, caspase8 were increased. Auditory epithelia were thoroughly washed after 24 hour of neomycin treatment.Two days later, we found that the level of Caspase9, Caspase7, Caspase8 were increased, Caspase3 and XIAP were reduced.There was no difference in the expression of TRMU and TFB1M. Our results showed that the mitochondrial membrane potential decreased before cell death occurred and apoptosis, there may be elution period. There also may be another potential pathway for activation of apoptosis in hair cells with neomycin treatment during elution period. TFB1M During the 12h in culture, majority of the hair cell in middle turn and basical turn lost. The elevated.expression of nuclear and mitochondrial TFB1M was accompanied with the death of hair cell and change of mitochondrial membrane potential.So the over expression of TFB1M may be relevant to the death of hair cell.