Genomic approach identifies novel proteins necessary for inner ear function and development across multiple species. O. Diaz-Horta1, M. Grati2, C. Abad1, A. DeSmidt3, G. Bademci1, A. Subasioglu-Uzak4, J. Foster II1, S. Tokgoz-Yilmaz4, D. Duman4, F. B. Cengiz4, S. H. Blanton1, X. Z. Liu2, A. Farooq5, Z. Lu3, K. Walz1, M. Tekin1 1) University of Miami Miller School of Medicine John P. Hussman Institute for Human Genomics, Miami, FL; 2) Department of Otolaryngology, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; 3) Department of Biology, University of Miami, Miami, FL, 33146, USA; 4) Division of Pediatric Genetics, Ankara University School of Medicine, Ankara, 06100, Turkey; 5) Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.

   The concerted action of thousands of proteins is required for inner ear development and function. Many of these proteins are currently unknown. In this study, we used a genomic approach to identify novel components of inner ear development and function. We present two such proteins that have not been previously recognized to be involved in hearing. The first protein was identified through studying a large Turkish kindred with autosomal recessive non-syndromic deafness. Whole exome sequencing (WES) in this family detected a splice site mutation (c.102-1G>A) in FAM65B (MIM611410) that co-segregates with the phenotype and is absent in 330 ethnicity-matched controls. The mutation leads to skipping of an exon and deletion of 52 amino acid residues in a membrane localization domain. We show that wild type Fam65b is expressed during embryonic and postnatal developmental stages in murine cochlea, and that the protein localizes to the plasma membranes of the stereocilia of inner and outer hair cells. The wild type protein targets the plasma membrane, whereas the mutant protein accumulates in cytoplasmic inclusion bodies and does not reach the membrane. In zebrafish, knockdown of fam65b leads to significant reduction in the number of saccular hair cells and neuromasts, and to hearing loss. We conclude that FAM65B is a plasma membrane-associated protein of hair cell stereocilia that is essential for hearing. The second protein was identified through studying another multiplex Turkish family with autosomal recessive non-syndromic deafness. Affected individuals had common cavity inner ear anomaly. Autozygosity mapping and WES identified a missense mutation in a gene encoding a tyrosine kinase-like receptor. In vitro studies showed that the mutant protein is unable to reach the plasma membrane, the natural localization of the wild type receptor. In zebrafish, knockdown of the orthologous gene with splice-blocking morpholinos led to anatomic changes in the ear and to hearing loss. Investigations of knock-out mouse demonstrate profound deafness along with cochlear anomalies. We will disclose the name of the second gene at the meeting. We conclude that genomic studies along with animal models are effective strategies to characterize the remaining proteins that are necessary for hearing.

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