Hearing loss is an economically and socially important cause of human morbidity. The genetic factors are one of the main etiologies causing hearing loss. While more than 100 genes for non-syndromic hearing loss [NSHL) have been mapped and a growing number have been cloned, little is known about the molecular mechanisms underlying hereditary inner ear defect. The long-term objective is to better understand the cellular and molecular basis of hereditary deafness so that effective genetic counseling and successful treatment strategies for hereditary deafness can be developed. During the previous grant period, we have identified several genes and mapped two novel loci for non-syndromic hearing loss, which opened new avenues to genes cloning and functional investigations. Given the facts that little is known about how mutations in these genes produce similar phenotypes and that many deafness pedigrees still fail to show linkage to any of these loci, it is therefore important for us to continue identifying human deafness genes and to investigate function of identified genes. Interestingly, as shown in our preliminary studies, we have confirmed further heterogeneity of NSHL, narrowed the critical regions of the DFNA41 and DFNA53 loci, successfully created the Ush1c knockout mouse model and double mutant mice for Cdh23 and Pcdh15, and identified positive ES cell clones for the Slc26a5 knockin mouse model. These interesting preliminary results have thus led us to continue cloning novel genes for NSHL and fully investigate the molecular mechanisms underlying NSHL (short-term objective) towards identifying potential therapeutic approaches for hereditary inner ear diseases. Aim 1. Localize and clone additional genes for NSHL. Aim 2. Clone DFNA41/DFNA53 genes. Aim 3. Determine cellular and molecular mechanism underlying inner ear defects using the Ush1c knockout mice and functional interactions in vivo of Ush1c with other Ush1 genes. Aim 4. Characterize the structural and functional consequences of the IVS2-2A>G mutation of the human prestin gene in the Slc26a5 knockin mice. Significance: The studies proposed in the current proposal together with the studies completed during the previous grant period will provide new insights into the pathological mechanisms underlying genetic hearing loss in humans, which can uncover new development of strategies for therapeutic intervention of hearing impairment in NSHL.