Molecular Analysis Of Human Hereditary Deafness

National Institute On Deafness And Other Communication Disorders

Investigators

Linked publications, trials & patents

Abstract

(1) We used a gene candidate approach and exome sequencing to study the etiology of nonsyndromic hearing loss and EVA in families of our cohort, which do not have detectable SLC26A4 mutations. We identified likely causal variants in two genes which had not been previously associated with such phenotype. Computational modeling of the wild-type and mutated proteins supported the hypothesis of the deleterious effects of these variants. We were also able to show the expression of these genes in the endolymphatic sac and its duct in mice using various methods (manuscript about to be submitted). To further study the mechanisms leading to hearing loss and EVA in the patients carrying variants in one of these genes, and the potential role of the endolymphatic sac and duct in this process, we used CRISPR/Cas9 genome editing to obtain a mouse model with the same missense variant we identified in one of the families, introduced in the orthologue gene (collaboration with Dr. Dong at NEI/NIH). We are also testing using different mouse models already available (collaboration with Dr. Friedman at NIDCD/NIH), if the presence of an EVA phenotype is variant-dependent or not, and whether or not the auditory phenotype of these mice is influenced by their genetic background. To identify the molecular mechanisms leading to hearing loss in patients, we are also in the process of testing whether the missense variant we identified alters protein-protein interactions previously reported to occur in the domain of the protein where this variant is present. (2) We used CRISPR/Cas9 genome editing to create several mouse lines segregating alleles of Slc26a4 with missense mutations identified in human patients and expected to lead to the expression of hypofunction SLC26A4 protein (pendrin) with various degrees of residual function. We are finalizing the characterization of the auditory and vestibular function of these mice on different genetic backgrounds, in case the genetic background influences the phenotype (publications in preparation). The goal of this study was to generate mouse models with hearing loss phenotypes that are less severe than those of the Slc26a4-null line and mimic more closely the phenotype seen in patients. In collaboration with the team of Dr. Chien at the NIDCD/NIH, we tested the ability of several AAVs serotypes to infect the cells of the endolymphatic sac (manuscript in press), a prerequisite to develop gene therapy strategies for genetic forms of hearing loss affecting genes expressed in the endolymphatic sac, such as SLC26A4, but also for developing functional assays to study the endolymphatic sac function. In collaboration with Dr. Chien and his team and with the help of Dr. Hoa's team at NIDCD/NIH, we have been investigating possible gene therapy approaches using dox-inducible Slc26a4-insufficient mouse model of EVA which we had previously reported. (3) We identified potential partners of SLC26A4 protein using a yeast two-hybrid approach. We focused our work on one of these proteins due to its potential role in the regulation of SLC26A4 surface expression and thus its potential for therapy for patients carrying hypomorph variants of SLC26A4. We first showed that both proteins are expressed in the same cells, at the same time, and have overlapping distribution in the endolymphatic sac. We then used both yeast and mammalian cell-based assays to confirm and characterize the interaction of SLC26A4 and this protein. We also used structural approaches to model their binding at the atomic level. We started developing functional assays to test whether the expression of SLC26A4 at the plasma membrane of the cells of the endolymphatic sac could indeed be increased by modulating this interaction. To this effect, we developed primary culture of the endolymphatic sac epithelium and identified strategies to express molecules of interest in these explants using either a BioRad Helios gene gun approach or AAV infections. To monitor changes of SLC26A4 expression at the plasma membrane in different experimental conditions, we developed a chimeric protein with an extracellular HA tag. After validating the targeting to the membrane of this chimeric protein in vitro, we used AAV in vivo injection in the inner ear to express our reporter protein and allow for its expression in the endogenous ionic environment of the inner ear. Micro-dissected AAV infected endolymphatic sacs could then be used in ex vivo preparations to test our hypothesis using pharmacology. We are currently repeating these experiments in endolymphatic sac preparations with an endogenous level of SLC26A4 as we could obtain CRISPR/Cas9 edited mice with an HA tag introduced in the gene Slc26a4 (collaboration with Dr. Dong at NEI) to further validate our observations (manuscript about to be submitted). Further experiments involving genetic manipulation of the level of the protein partner are also in process.