Molecular Mechanism Study Of A Novel Variation Of Hereditary Hearing Loss Pathogenic Gene CEACAM16

Background:DFNA4is an gene locus of autosomal dominant non-syndromic hereditary hearing loss. It was named due to the discovery of MYH14gene mutation which is located on chromosome19q13.33. In2011, Zheng J et al first found that the mutation of CEACAM16gene is also responsible for the dominant form of human deafness, DFNA4, in Family1070, and identified functions of CEACAM16protein through bioinformatic analysis and in vitro experiments. In2013, our team investigated a five generation family from Hunan province of China with autosomal dominant sensorineural hearing loss (SY-026), and identified a noval pathogenic mutation c.505G>A (p.G169R) in the CEACAM16gene by taking the traditional whole-genome scan linkage analysis with the latest exome sequencing technology. This mutation is significant because it’s the second case in the world and the first case in our country.In this study, we performed a series of in vitro studies on the novel mutation of CEACAM16which is first found by our team, in order to understand the functional consequences brought by this mutation. Objective:To investigate the possible deafness-causing mechanism of the mutation G169R in the CEACAM16gene through measurements of the expression levels and the localization of the wild-type (WT) and mutant-type(MT) proteins in vitro.Methods:We first constructed two eukaryotic expression plasmids pRK5-CEACAM16-Flag (WT) and pRK5-G169R-Flag (MT) with C-terminal Flag using the plasmid kindly provided by Professor Wolfgang Zimmermann as a template.293T cells were transfected with WT and MT plasmids to detect their intracellular and extracellular expressions after48hours by Western blot. Immunostaining and confocal microscopy were used to detect the subcellular localization of WT and MT proteins.Results:1.The two plasmids pRK5-CEACAM16-Flag (WT) and pRK5-G169R-Flag (MT) were successfully constructed and identified by double digestion and sequencing.2. Both of WT and MT CEACAM16proteins could be detected in the cell pellets and cell-culture medium, forming oligomers. The resulting bands on the SDS-PAGE gel were of the expected molecular weights. But the MT protein secreted to medium was significantly reduced.3. Immunofluorescence assay indicated that the WT and MT CEACAM16proteins were mainly distributed in the cytoplasm without regional aggregation. And there was no notable distinction between them.Conclusion:The G169R mutation of CEACAM16gene can cause a significant reduction in the amount of protein secreted into the cell-culture medium, but it dose’t affect the distribution of mutant protein in cells.