Chinese Population-Specific Deafness Related Genes SLC26A4Gene Variation Of Pathogenic Identification And Their Deaf Mechanism

Objective:Large vestibular aqueduct syndrome (LVAS) or enlarged vestibular aqueduct syndrome(EVAS) is autosomal recessive nonsyndromic hearing loss which is characterized as enlarged vestibular aqueduct and sensorineural hearing loss(SNHL). The present study shows that, for large vestibular aqueduct is associated with SLC26A4gene mutation,which is first discovered by Everett in an autosomal recessive genetic disease-Pendred syndrome pedigrees. Abroad in recent years a number of studies have shown that the SLC26A4gene mutations in Pendred syndrome and vestibular aqueduct alone to expand and inner ear malformations are closely related. It is estimated that worldwide children’s pre-lingual deaf patients4%to10%caused by the Pendred syndrome Pendred syndrome in China reported fewer, but enlarged vestibular aqueduct in deaf patients to take up a considerable proportion. SLC26A4gene encoding780amino acid protein Pendrin, Pendrin is a transmembrane protein of SO42-, HCO3-, I-, Cl-, and other multiple unit price or divalent cation transporter in the balance of the maintenance of the ionic composition of the bodyplay an important role. Up to160kinds of the gene (open reading frame2343bp), ex on up to21mutations of a broad spectrum of mutations, it was reported as of2010, Pendrin was mainly expressed in the thyroid, and also have distribution in the inner ear, the fetal kidney and fetal brain. Pendrin expression in within the lymphatic vessels, and endolymphatic sac, the utricle, balloon, etc. in the inner ear. Everett, etc. found in the embryo of13days, the mice within the lymphatic and endolymphatic sac there is strong expression of the SLC26A4gene mRNA, lymphatic and endolymphatic sac endolymph metabolism, acid-base balance of the endolymph by H (+)-ATPase and Cl (-)/HCO3(-) to maintain. Pendrin is likely as a Cl (-)/HCO3(-) symporter regulate endolymph ion balance, as both of HCO3(-) transporter in the stria vascularis space of HCO3(-) secretion into the endolymph, HCO3(-) accumulation will be limited to the generation of protective glutathione increased free radical pressure, removal of HCO3(-) to help protect blood vessels pattern from free radical damage, to maintain its normal function. The specific mechanism of SLC26A4gene mutations cause the disease phenotype is not completely clear.20008published the JMG’s a South Korean study that different mutations in the Pendrin protein conformation and ion transport dysfunction caused by different mechanisms, the study found that most of the mutations of the abnormal protein product remain in the cells, while wild-type protein in the plasma membrane thus affecting the Cl (-)/HCO3(-) ion-exchange function. In addition, the solution of the mutant protein showed significant heterogeneity, for example, the H723R mutant protein product was mainly present in the endoplasmic reticulum of cells, and abnormal modification of low-temperature incubation of this protein product and damage to the ion transport function to a large degree of recovery L236P mutant protein product is the main stay in the center of the cell body is not sensitive to the same temperature treatment. The the deaf disease molecular epidemiology survey showed that the genetic variation Chinese deaf population with a wide range of heterogeneity, some of which have not been reported mutations in other ethnic groups. So, the new variant can cause the precise disease phenotype urgent function studies confirmed.This study intends to establish a set of SLC26A4gene variation pathogenic identification system to analyze new found variability and pathogenicity, at the protein level of SLC26A4gene induced deafness mechanisms, illuminate China population specific SLC26A4genotype phenotype correlation, guide clinical SLC26A4related deafness gene diagnosis and prenatal diagnosis.Method:1.Cloned wild-type of SLC26A4, and constructed in the pEGFP-N1 plasmid, so that it can express Pendrin fusion proteinDesign primers to amplify the SLC26A4gene cDNA sequence and its connection with a pCR Ⅱ-TA plasmid. Target genes will then be constructed in the pEGFP-Nl plasmid to enable them to fusion protein expression of Pendrin.2.SLC26A4gene-directed mutagenesis, cloned variant of SLC26A4, and constructed in the pEGFP-Nl plasmid, so that it can express the variation the Pendrin fusion protein.Response to the above are most likely to impact Pendrin protein10kinds of SLC26A4gene variants were designed to contain a pair of primers of the base variable sites (positive and negative), and mature application development QuikChange Site-directed Mutagenesis kit for site-directed mutagenesis. Annealing with the template plasmid using PfuTurbo polymerase cycle extension "pros and cons of annealing to the primer extension product, paired with notch open-loop plasmid. Dpn I, digested extension product, the original template plasmid from the conventional E. coli, modified by dam methylation sensitive to Dpn I, was chopped, synthesized in vitro with the mutant sequence of plasmid absence of methylation rather than was cut, and the subsequent conversion of the successful transformation can be obtained by the cloning of mutant plasmids and mutant plasmids for sequencing screening validation.3.The wild-type SLC26A4and SLC26A4variants were transfected into HEK293cells and HeLa cells.Cultured HEK293cells and HeLa cells, the transfection kit application, the wild-type SLC26A4and SLC26A4variants were transfected into HEK293cells and HeLa cells to analyze the fluorescence signal of the green fluorescent protein (GFP) to identify the transfection efficiency.Pendrin expression in normal HeLa cells transfected with wild-type SLC26A4, SLC26A4variant, you can use the immunofluorescence by laser scanning confocal microscope wild-type Pendrin and a variety of abnormal type Pendrin positioning in HeLa cells. Observed in HEK293cells, cells of wild-type Pendrin and a variety of abnormalities Pendrin CI (-)/HCO3(-) transporter. 4.Measure the intracellular chloride ion flow by isotope labeling method.Results:1.Construction of wild-type and mutant plasmid pEGFP-of SLC26A4were sequenced and exactly the same as the sequence published on the NCBI GenBank NM000441.12.Wild type of pEGFP-of SLC26A4plasmid express the the Pendrin fusion protein by transfection of cells, Western blot detect the87kDa protein3. Through a variety of plasmid transfected cells and found that wild-type SLC26A4gene product Pendrin is expressed on the cell membrane, the mutant SLC26A4gene expression product of Pendrin some expression in the cell membrane, and some expression in the cytoplasm, and some in the cell membrane andwere expressed in cytoplasm4. By the isotope labeling method to measure the flow of formate ions in the various transfected cells, experiments have shown that mutations in the gene expression of Pendrin protein for formate ions transport efficiency decreased in varying degrees.Conclusion:1. In this study, mutations in SLC26A4expression to change the positioning of the product Pendrin protein in the cell, some expression in the cell membrane, and some expression in the cytoplasm, some were expressed in the cell membrane and cytoplasm, which is also its transport function changefoundation.2.10kinds of mutant SLC26A4expression product of this study transporter function were decreased in varying degrees, even though some mutant SLC26A4expression product Pendrin protein expression in the cell membrane, its encoding Pendrin protein unable to complete the normal anion exchange function of formate ions transport function decreased in varying degrees, resulting in the occurrence of deafness.