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Non-Syndromic Hereditary Hearing Loss (NSHHL) is a genetically heterogeneous sensory disorder with about 120 genes already associated. Through exome sequencing (ES) and data aggregation, we identified a family with six affected individuals and one unrelated NSHHL patient with predicted-to-be deleterious missense variants in USP48. We also uncovered an eighth patient presenting unilateral cochlear nerve aplasia and a de novo splice variant in the same gene. USP48 encodes a ubiquitin carboxyl-terminal hydrolase under evolutionary constraint. Pathogenicity of the variants is supported by in vitro assays that showed that the mutated proteins are unable to hydrolyze tetra-ubiquitin. Correspondingly, three-dimensional representation of the protein containing the familial missense variant is situated in a loop that might influence the binding to ubiquitin. Consistent with a contribution of USP48 to auditory function, immunohistology showed that the encoded protein is expressed in the developing human inner ear, specifically in the spiral ganglion neurons, outer sulcus, interdental cells of the spiral limbus, stria vascularis, Reissner's membrane and in the transient Kolliker's organ that is essential for auditory development. Engineered zebrafish knocked-down for usp48, the USP48 ortholog, presented with a delayed development of primary motor neurons, less developed statoacoustic neurons innervating the ears, decreased swimming velocity and circling swimming behavior indicative of vestibular dysfunction and hearing impairment. Corroboratingly, acoustic startle response assays revealed a significant decrease of auditory response of zebrafish lacking usp48 at 600 and 800 Hz wavelengths. In conclusion, we describe a novel autosomal dominant NSHHL gene through a multipronged approach combining ES, animal modeling, immunohistology and molecular assays. influence the binding to ubiquitin. Consistent with a contribution of USP48 to auditory function, immunohistology showed that the encoded protein is expressed in the developing human inner ear, specifically in the spiral ganglion neurons, outer sulcus, interdental cells of the spiral limbus, stria vascularis, Reissner’s membrane and in the transient Kolliker’s organ that is essential for auditory development. Engineered zebrafish knocked-down for usp48, the USP48 ortholog, presented with a delayed development of primary motor neurons, less developed statoacoustic neurons innervating the ears, decreased swimming velocity and circling swimming behavior indicative of vestibular dysfunction and hearing impairment. Corroboratingly, acoustic startle response assays revealed a significant decrease of auditory response of zebrafish lacking usp48 at 600 and 800 Hz wavelengths. In conclusion, we describe a novel autosomal dominant NSHHL gene through a multipronged approach combining ES, animal modeling, immunohistology and molecular assays.

Melanocytes are present in various parts of the inner ear, including the stria vascularis in the cochlea and the dark cell areas in the vestibular organs, where they contribute to endolymph homeostasis. Developmental studies describing the distribution of vestibular melanocytes are scarce, especially in humans. In this study, we investigated the distribution and maturation of the vestibular melanocytes in relation to the developing dark cell epithelium in inner ear specimens from week 5 to week 14 of development and in surgical specimens of the adult ampulla. Vestibular melanocytes were located around the utricle and the ampullae of the semicircular canals before week 7 and were first seen underneath the transitional zones and dark cell areas between week 8 and week 10. At week 10, melanocytes made intimate contact with epithelial cells, interrupting the local basement membrane with their dendritic processes. At week 11, most melanocytes were positioned under the dark cell epithelia. No melanocytes were seen around or in the saccule during all investigated developmental stages. The dark cell areas gradually matured and showed an adult immunohistochemical profile of the characteristic ion transporter protein Na+/K+‐ATPase α1 by week 14. Furthermore, we investigated the expression of the migration‐related proteins ECAD, PCAD, KIT, and KITLG in melanocytes and dark cell epithelium. This is the first study to describe the spatiotemporal distribution of vestibular melanocytes during the human development and thereby contributes to understanding normal vestibular function and pathophysiological mechanisms underlying vestibular disorders.