Usher syndrome type 1 (USH1) is an autosomal recessive sensory defect involving congenital profound sensorineural deafness, vestibular dysfunction and blindness (due to progressive retinitis pigmentosa)1. Six different USH1 loci have been reported. So far, only MYO7A (USH1B), encoding myosin VIIA, has been identified as a gene whose mutation causes the disease. Here, we report a gene underlying USH1C (MIM 276904), a USH1 subtype described in a population of Acadian descendants from Louisiana and in a Lebanese family. We identified this gene (USH1C), encoding a PDZ-domain-containing protein, harmonin, in a subtracted mouse cDNA library derived from inner ear sensory areas. In patients we found a splice-site mutation, a frameshift mutation and the expansion of an intronic variable number of tandem repeat (VNTR). We showed that, in the mouse inner ear, only the sensory hair cells express harmonin. The inner ear Ush1c transcripts predicted several harmonin isoforms, some containing an additional coiled-coil domain and a proline- and serine-rich region. As several of these transcripts were absent from the eye, we propose that USH1C also underlies the DFNB18 form of isolated deafness.

A 3,673-bp murine cDNA predicted to encode a glycosylphosphatidylinositol-anchored protein of 1,088 amino acids was isolated during a study aimed at identifying transcripts specifically expressed in the inner ear. This inner ear-specific protein, otoancorin, shares weak homology with megakaryocyte potentiating factor͞ mesothelin precursor. Otoancorin is located at the interface between the apical surface of the inner ear sensory epithelia and their overlying acellular gels. In the cochlea, otoancorin is detected at two attachment zones of the tectorial membrane, a permanent one along the top of the spiral limbus and a transient one on the surface of the developing greater epithelial ridge. In the vestibule, otoancorin is present on the apical surface of nonsensory cells, where they contact the otoconial membranes and cupulae. The identification of the mutation (IVS12؉2T>C) in the corresponding gene OTOA in one consanguineous Palestinian family affected by nonsyndromic recessive deafness DFNB22 assigns an essential function to otoancorin. We propose that otoancorin ensures the attachment of the inner ear acellular gels to the apical surface of the underlying nonsensory cells. T he mammalian inner ear consists of the cochlea, the organ of hearing, and the vestibule, which is responsible for balance. The vestibule is composed of five separate organs, the saccule, the utricle, and three cristae of the semicircular canals. The apical surface of each sensory organ is covered by an acellular gel. The tectorial membrane (TM) lies over the surface of the organ of Corti in the cochlea, an otoconial membrane covers the macula of the utricle and saccule, and a cupula sits on top of each crista in the ampullae of the semicircular canals. Relative motion generated between the apical surface of the sensory epithelium and the overlying acellular gel, in response to sound-induced basilar membrane motion in the cochlea or head motion in the vestibule, results in deflection of the hair cell's stereociliary bundle, thereby modulating the gating of mechanotransducer channels.With the exception of prestin (1), all of the inner ear-specific proteins described so far in mammals, namely ␣-and ␤-tectorin (2), otogelin (3), and otoconin-95 (4), are components of these acellular gels. In the mouse inner ear, ␣-and ␤-tectorin are components of the tectorial and otoconial membranes but are not present in the cupulae (2). Otogelin is present in all of the acellular gels (3). Otoconin-95 is the major component of the otoconia, dense biominerals that load the otoconial membrane and provide inertial mass (4, 5), and is also present in the cupulae. The mammalian TM is a complex structure composed of collagen fibrils imbedded in a collagenase-insensitive striated-sheet matrix (6). Otogelin is associated mainly with the collagen fibril bundles (3, 7), whereas ␣-and ␤-tectorin are major components of the striated-sheet matrix (8, 9). Collagens are not prominent components of either the otoconial membranes or cupulae. Thus far, little is known about ...

Although the cochlea is an amplifier and a remarkably sensitive and finely tuned detector of sounds, it also produces conspicuous mechanical and electrical waveform distortions1. These distortions reflect non-linear mechanical interactions within the cochlea. By allowing one tone to suppress another (masking effect), they contribute to speech intelligibility2. Tones can also combine to produce sounds with frequencies not present in the acoustic stimulus3. These sounds compose the otoacoustic emissions that are extensively used to screen hearing in newborns. As both cochlear amplification and distortion originate from the outer hair cells, one of the two types of sensory receptor cells, it has been speculated that they stem from a common mechanism. Here, the non-linearity underlying cochlear waveform distortions is shown to rely on the presence of stereocilin, a protein defective in a recessive form of human deafness4. Stereocilin was detected in association with horizontal top connectors5-7, lateral links that join adjacent stereocilia within the outer hair cell’s hair bundle, and these links were absent in stereocilin-null mutant mice. These mice become progressively deaf. At the onset of hearing, however, their cochlear sensitivity and frequency tuning were almost normal, although masking was much reduced and both acoustic and electrical waveform distortions were completely lacking. From this unique functional situation, we conclude that the main source of cochlear waveform distortions is a deflection-dependent hair bundle stiffness resulting from constraints imposed by the horizontal top connectors, and not from the intrinsic non-linear behaviour of the mechanoelectrical transducer channel.

Hearing impairment affects about 1 in 1,000 children at birth. Approximately 70 loci implicated in non-syndromic forms of deafness have been reported in humans and 24 causative genes have been identified (see also http://www.uia.ac.be/dnalab/hhh). We report a mouse transcript, isolated by a candidate deafness gene approach, that is expressed almost exclusively in the inner ear. Genomic analysis shows that the human ortholog STRC (so called owing to the name we have given its protein-stereocilin), which is located on chromosome 15q15, contains 29 exons encompassing approximately 19 kb. STRC is tandemly duplicated, with the coding sequence of the second copy interrupted by a stop codon in exon 20. We have identified two frameshift mutations and a large deletion in the copy containing 29 coding exons in two families affected by autosomal recessive non-syndromal sensorineural deafness linked to the DFNB16 locus. Stereocilin is made up of 1,809 amino acids, and contains a putative signal petide and several hydrophobic segments. Using immunohistolabeling, we demonstrate that, in the mouse inner ear, stereocilin is expressed only in the sensory hair cells and is associated with the stereocilia, the stiff microvilli forming the structure for mechanoreception of sound stimulation.

During the course of a study aimed at identifying inner ear-specific transcripts, a 1,906-bp murine cDNA predicted to encode a secreted 469-aa protein with two domains of homology with the secreted phospholipases A 2 was isolated. This transcript is specifically expressed in the inner ear from embryonic day 9.5. The encoded 95-kDa glycoprotein is the major protein of the utricular and saccular otoconia and thus was named otoconin-95. By immunohistof luorescence, otoconin-95 also was detected in the cupulae of the semicircular canals and in previously undescribed transient granular structures of the cochlea. Otoconin-95 was found to be synthesized by various nonsensory cell types, but not by the supporting cells of the sensory epithelia, which produce the otoconial precursor vesicles. In addition, multiple isoforms generated by differential splicing were observed in different combinations during development. Based on the present results, we propose a model for the formation of the otoconia.The mammalian inner ear is composed of two sensory organs: the cochlea, which is the auditory component, and the vestibule, responsible for the control of balance. The latter consists of the saccule and the utricle, which detect linear acceleration, and the three semicircular canals sensitive to angular acceleration. Each sensory epithelium is covered by an acellular gelatinous membrane, namely the tectorial membrane in the cochlea, the cupulae in the ampullae of the semicircular canals, and the otoconial membranes in the saccule and the utricle. The displacement of the acellular membrane relative to the sensory epithelia leads to the deflection of the stereocilia of sensory hair cells, which in turn opens the mechanotransduction channels, leading to cell depolarization.The otoconial membranes of the saccule and utricle are overlayed with dense biominerals made of a filamentous organic matrix and calcium carbonate (CaCO 3 ). These biominerals are found as large deposits (otoliths) in most fish and as numerous small crystals (otoconia) in all other vertebrates. Otoliths and otoconia have been classified in three groups according to the crystalline form of CaCO 3 , i.e., vaterite, aragonite, or calcite. In mammals, otoconia are calcitic, whereas in amphibians they are calcitic in the utricle and aragonitic in the saccule (1-4). Each type of otoconia is characterized by a distinct set of proteins collectively named otoconins (3). The major protein of aragonitic otoconia has been purified from Xenopus laevis saccule and sequenced (5). In contrast, none of the otoconins of the calcitic otoconia have been characterized so far. In mice, the rate of otoconia production is highest between embryonic days 15 and 17 (E15-E17), although the biominerals continue to grow until postnatal day 7 (P7) (6, 7). Different hypotheses for the genesis of otoconia have been proposed (reviewed in ref. 8). It is now generally accepted that otoconia are produced from protrusions of the saccular and utricular supporting cells that form vesicular str...

Stereocilin is defective in a recessive form of deafness, DFNB16. We studied the distribution of stereocilin in the developing and mature mouse inner ear and analyzed the consequences of its absence in stereocilin-null (Strc −/− ) mice that suffer hearing loss starting at post-natal day 15 (P15) and progressing until P60. Using immunofluorescence and immunogold electron microscopy, stereocilin was detected in association with two cell surface specializations specific to outer hair cells (OHCs) in the mature cochlea: the horizontal top connectors that join the apical regions of adjacent stereocilia within the hair bundle, and the attachment links that attach the tallest stereocilia to the overlying tectorial membrane. Stereocilin was also detected around the kinocilium of vestibular hair cells and immature OHCs. In Strc −/− mice, the OHC hair bundle was structurally and functionally normal until P9. Top connectors, however, did not form and the cohesiveness of the OHC hair bundle progressively deteriorated from P10. The stereocilia were still interconnected by tip links at P14, but these progressively disappeared from P15. By P60, the stereocilia, still arranged in a V-shaped bundle, were fully disconnected from each other. Stereocilia imprints on the lower surface of the tectorial membrane were also not observed in Strc −/− mice, thus indicating that the tips of the tallest stereocilia failed to be embedded into this gel. We conclude that stereocilin is essential to the formation of horizontal top connectors. We propose that these links, which maintain the cohesiveness of the mature OHC hair bundle, are required for tip-link turn over.

Otoconia are biominerals of the vestibular system that are indispensable for the perception of gravity. Despite their importance, the process of otoconia genesis is largely unknown. Reactive oxygen species (ROS) have been recognized for their toxic effects in antimicrobial host defense as well as in aging and carcinogenesis. Enzymes evolved for ROS production belong to the recently discovered NADPH oxidase (Nox) enzyme family . Here we show that the inactivation of a regulatory subunit, NADPH oxidase organizer 1 (Noxo1), resulted in the severe balance deficit seen in the spontaneous mutant "head slant" (hslt) mice whose phenotype was rescued by Noxo1 transgenes. Wild-type Noxo1 was expressed in the vestibular and cochlear epithelia and was required for ROS production by an oxidase complex. In contrast, the hslt mutation of Noxo1 was biochemically inactive and led to an arrest of otoconia genesis, characterized by a complete lack of calcium carbonate mineralization and an accumulation of otoconial protein, otoconin-90/95 (OC-90/95). These results suggest that ROS generated by a Noxo1-dependent vestibular oxidase are critical for otoconia formation and may be required for interactions among otoconial components. Noxo1 mutants implicate a constructive developmental role for ROS, in contrast to their previously described toxic effects.

We have recently shown that USH1C underlies Usher syndrome type 1c (USH1C), an USH1 subtype characterized by profound deafness, retinitis pigmentosa, and vestibular dysfunction. USH1C encodes a PDZ-domain-containing protein, harmonin. Eight different Ush1c transcripts were identified in the mouse inner ear. Moreover, transcripts containing seven alternatively spliced exons (A-F, G/G) were found to be expressed in the inner ear, but not in the eye. These findings suggested that mutations involving USH1C might also be the cause of DFNB18, a form of non-syndromic deafness, which maps to a chromosomal region that includes USH1C. We screened 32 Chinese multiplex families with non-syndromic recessive deafness for USH1C mutations. In one family, congenital profound deafness without RP was associated with a C to G transversion in the alternatively spliced exon D, predicting an arginine to proline substitution at codon 608 in the proline-, serine- and threonine-rich region of harmonin. We also screened 320 deaf probands from other ethnic background and found three who were heterozygous for changes in the alternately spliced exons including Gly431Val in exon B, Arg620Leu and Arg636Cys in exon D. None of these mutations were detected in DNA from 200 control subjects with normal hearing including 110 Chinese. We also screened 121 non-Acadian probands with type 1 Usher syndrome. None carried any mutations in these exons of USH1C. Our findings show that USH1C mutations can also cause non-syndromic deafness and that some harmonin isoforms are specifically required for inner ear function.