The Biological Function And Deaf Mechanism Of DFNA5

DFNA5(Deafness, Autosomal Dominant Nonsyndromic Sensorineural5) is one of the few deafness genes whose biological function and deafness mechanism remains unclear so far among all deafness genes that have been cloned. In1995, DFNA5is targeted at Area15, chromosome7(7p15), as the fifth one that is positioned of autosomal dominant hereditary deafness sites. In1998, DFNA5gene was cloned with its mutation sites were also found. So far, many DFNA5gene mutation were found to cause deafness. Although these sites are not the same at the genomic level, interestingly, all DFNA5gene mutations that cause deafness lead to the skipping of exon8in mRNA levels which cause a frameshift and thus an early termination of translation.It is now generally considered that DFNA5gene mutations causing deafness is a gain of function mutation, and may be related to the41amino acids which is translated after the skipping of exon8The reasons are listed as below:1) Deafness-causing DFNA5mutations are all dominant mutations;2) When the deafness-related DFNA5mutant type was transfected in vitro mammalian cells, it is capable of causing the increase in cell death and can cause cell cycle arrest in the transformed yeast cells;3) DFNA5gene exon5mutation can also cause an early termination of the open reading frame, but does not cause hearing lossTo simulate the deafness mutation of DFNA5in mouse, Van Laer L and colleagues knocked out the exon8of DFNA5using homologous recombination, which leads to a frameshift causing an early termination after translating43amino acids.(Notably, the wrongly-translated43amino acids in mouse are not homological with the41amino acids in human). ABR results showed the mouse were not deaf. This suggests that the41amino acids translated after the skipping of exon8of DFNA5in human may be very important to the occurrence of deafness. In this study, we focus on the biological functions and deafness mechanisms of DFNA5using mammalian cells and other materials and methods.In this thesis, we studied the biological functions of DFNA5at the cellular level and discussed preliminarily the deafness and mechanisms of DFNA5mutation. In the first part of this article, the DFNA5biological functions were studied.We obtained expression vectors for cells transfection, the yeast two-hybrid, GST-pull down by molecular cloning.We transfected DFNA5WT and MU to COS7cells and found that DFNA5WT positioned in the cytoplasm and cells were in a healthy state. But after transfecting DFNA5MU to cells, there were less adherent cells and these cells were unhealthy. In the meantime, we found DFNA5MU localized unevenly in cells, aggregating into group. We tried to identify interactive proteins of DFNA5through yeast two-hybrid, GST-pull down and IP. Regretfully, we havn’t found any so far because of DFNA5cytotoxicity, protein inactivated easily and other reasons.The etoposide-resistant human melanoma cell line MeWo ETO1was transfected with a DFNA5-encoding expression vector construct by Hermann Lage et al. They found transfected clones showed a30-35%reduced etoposide susceptibility by comparing the values of these DFNA5clones with those displayed by the non-transfected, etoposide-resistant melanoma cell line MeWo ETO1and controls. Furthermore, they found a decrease in DNFA5mRNA expression level is associated with an increased etoposide resistance in melanoma cells due to an elevated cellular susceptibility to trigger a caspase3-depending signal pathway leading to programmed cell death. In the second part of this paper, we furtherly detected the regulation of DFNA5to cell death by western blot. At the same time, to learn more about the effect to aopotosis mechanism of DFNA5mutation, we used high-throughput sequencing methods to screen downstream regulated genes of DFNA5...