Bradley A. Schulte

Deafness is a highly heterogenous disorder with over 100 genes known to underlie human non-syndromic hearing impairment. However, many more remain undiscovered, particularly those involved in the most common form of deafness: adult-onset progressive hearing loss. Despite several genome-wide association studies of adult hearing status, it remains unclear whether the genetic architecture of this common sensory loss consists of multiple rare variants each with large effect size or many common susceptibility variants each with small to medium effects. As next generation sequencing is now being utilised in clinical diagnosis, our aim was to explore the viability of diagnosing the genetic cause of hearing loss using whole exome sequencing in individual subjects as in a clinical setting. We performed exome sequencing of thirty patients selected for distinct phenotypic sub-types from well-characterised cohorts of 1479 people with adult-onset hearing loss. Every individual carried predicted pathogenic variants in at least ten deafness-associated genes; similar findings were obtained from an analysis of the 1000 Genomes Project data unselected for hearing status. We have identified putative causal variants in known deafness genes and several novel candidate genes, including NEDD4 and NEFH that were mutated in multiple individuals. The high frequency of predicted-pathogenic variants detected in known deafness-associated genes was unexpected and has significant implications for current diagnostic sequencing in deafness. Our findings suggest that in a clinic setting, efforts should be made to a) confirm key sequence results by Sanger sequencing, b) assess segregations of variants and phenotypes within the family if at all possible, and c) use caution in applying current pathogenicity prediction algorithms for diagnostic purposes. We conclude that there may be a high number of pathogenic variants affecting hearing in the ageing population, including many in known deafness-associated genes. Our findings of frequent predicted-pathogenic variants in both our hearing-impaired sample and in the larger 1000 Genomes Project sample unselected for auditory function suggests that the reference population for interpreting variants for this very common disorder should be a population of people with good hearing for their age rather than an unselected population.

Voltage-gated chloride channels (ClCs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify ClC-K variants in types II, IV, and V fibrocytes of the rodent spiral ligament. We have now confirmed the expression of ClC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K⁺ recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat spiral ligament underlying and inferior to the spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,Cl-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both ClC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl⁻ currents in the cultured cells. One, an inwardly rectifying Cl⁻ current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of ClC-2. The other, a weak outwardly rectifying Cl⁻ current regulated by extracellular pH, Cl⁻, and Ca²⁺ resembled the channel characteristics of ClC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV spiral ligament fibrocytes.

The most common causes of hearing loss are exposure to noise, aging, and the combined effects of the two. Anatomically, noise-induced hearing loss (NIHL) is characterized by a loss of sensory cells and secondary neural degeneration. Physiologically, NIHL is characterized by threshold elevations of the compound action potential of the auditory nerve, auditory brainstem response, and higher-level potentials. The endocochlear potential is most commonly unaffected. A distinctive feature of NIHL is the loss of cochlear nonlinearities, including otoacoustic emissions and two-tone rate suppression measured in single fibers of the auditory nerve. Degeneration of the stria vascularis is typically not associated with NIHL. The pathological anatomy and physiology of the aging ear is different from that associated with NIHL. The most dominant pathology is degeneration of the stria vascularis/lateral wall of the cochlea with a reduction in the endolymphatic potential from 90 to 100 mV to values as low as 10 to 20 mV. Blood supply is compromised and correlated with the extent of strial degeneration. A distinctive anatomic feature of the aging ear is the presence and normality of sensory cells except in the most basal and most apical regions. A distinctive physiological feature is the presence of otoacoustic emissions and two-tone rate suppression. There is significant degeneration of spiral ganglion cells in the presence of a normal population of sensory cells, thus suggesting an age-related primary degeneration of the auditory nerve. Thus, age-related hearing loss is not a sensorineural problem, as is NIHL, but is a vascular, metabolic, neural disorder.

Ultrastructural examination revealed an epithelium of about five tectal cells (TCs) roofing the outer tunnel (OT) in the mid to upper, but not the basal, region of gerbil and chinchilla cochlea. Structures in TCs that are apparently specialized for retrieval of K⁺ released into tunnel fluid from outer hair cells (OHCs) include surface fimbriae in the gerbil and canalicular reticulum in the chinchilla. A tunnel roof of organelle‐rich TCs appeared to be better equipped for ion resorption than a roof composed of organelle‐poor Hensen cells (HCs). Fimbriae, filopodia, and the cell body of TCs descended to contact the third Deiters cell (DC3) in the gerbil, and the hypertrophied DC3 phalanx rose to contact TCs in the chinchilla, which suggests a solute exchange between TCs and DCs. Previously unrecognized structures that are speculated to provide ATP ligand for cochlear purinoreceptors occurred in the chinchilla DC and gerbil TC. The observation of a microtubule stalk in DCs indicated that they also function in cochlear mechanics. A newly delineated lateral tunnel cell (LTC) intervened between the DC3 and HC in both species. The apicomedial plasmalemma of all DCs fitted closely to the base of OHCs and enveloped afferent nerves. The morphologic specializations reported here provide further support for the proposed transcellular lateral flow route for K⁺ currents generated by sound exposure and neural activity. The previously demonstrated expansion of Boettcher cells, outer sulcus cell roots, type Il and IV fibrocytes, and apical microvilli on HCs and Claudius cells (CCs) in the base of the cochlea is postulated here to mediate a basal parallel current that could supply the increased K⁺ transport required for the basally elevated electric potential (EP). Anat Rec Part A 271A:342–359, 2003.

Objective To gain insight into molecular and cellular mechanisms regulating cochlear potassium (K⁺) recycling, including the possible effects of mutations in the GJB2 gene, which encodes the gap junction protein connexin 26. Intercellular K⁺ flux was manipulated in vivo by infusion of the gap junction uncoupler proadifen (SKF-525A) into perilymph. Functional and structural alterations induced by gap junction blockade were assessed by electrophysiological and morphologic analysis. Study Design Laboratory study using an animal model. Methods Physiological effects of acute and chronic uncoupling of gap junctions in the Mongolian gerbil inner ear were evaluated by measurement of compound action potential (CAP) thresholds and input-output (I/O) functions, distortion product otoacoustic emissions (DPOAE), and the endocochlear potential (EP). Morphologic changes were assessed by electron microscopy. Results Acute exposures to proadifen resulted in large decreases in EP values, DPOAE magnitudes, and CAP I/O maximum amplitudes and an increase in high-frequency CAP thresholds. These physiological changes were accompanied by vacuolization of type II and type V fibrocytes in the lateral wall of the cochlea. Chronic treatments revealed some recovery in EP values and CAP thresholds, which showed a relatively flat 15- to 20-dB elevation across frequencies. Conclusions Gap junctions play a significant role in normal cochlear function. In particular they appear to be essential for maintaining the EP, an activity that could be related to their participation in K⁺ recycling. Thus, hearing losses associated with mutations in the GJB2 gene that alter the expression or function of connexin 26 may result from a diminished capacity to recycle K⁺ from perilymph back to the stria vascularis and a consequent decline in the EP.

Four months after the selective ablation of inner hair cells by carboplatin, the interdental cell epithelium exhibited dilated intercellular spaces and cytosolic vacuoles not seen in controls. In addition, the wide, often electron-lucent phalanges observed in the interdental cells of the normal chinchilla collapsed into a dense stratum that projected enlarged polypoid profiles into the limbal zone of the tectorial membrane. Carboplatin treatment also resulted in the restructuring of the tectorial membrane overlying the limbus. Changes in this membrane included a variable accumulation of the basal matrix, the rearrangement of intermediate lucent spaces, and the disappearance of a superimposed filamentous mesh. These three strata are, under normal conditions, apparently involved in events underlying tectorial membrane renewal. The post-carboplatin changes in the interdental cells and tectorial membrane occurred exclusively in the proposed medial pathway for K⁺ diffusion from inner hair cells and presumably resulted from a reduced flow of ions and fluid secondary to the ablation of these cells.