Allelic Mutations of KITLG, Encoding KIT Ligand, Cause Asymmetric and Unilateral Hearing Loss and Waardenburg Syndrome Type 2.

Linkage analysis combined with whole-exome sequencing in a large family with congenital and stable non-syndromic unilateral and asymmetric hearing loss (NS-UHL/AHL) revealed a heterozygous truncating mutation, c.286_303delinsT (p.Ser96Ter), in KITLG. This mutation co-segregated with NS-UHL/AHL as a dominant trait with reduced penetrance. By screening a panel of probands with NS-UHL/AHL, we found an additional mutation, c.200_202del (p.His67_Cys68delinsArg). In vitro studies revealed that the p.His67_Cys68delinsArg transmembrane isoform of KITLG is not detectable at the cell membrane, supporting pathogenicity. KITLG encodes a ligand for the KIT receptor. Also, KITLG-KIT signaling and MITF are suggested to mutually interact in melanocyte development. Because mutations in MITF are causative of Waardenburg syndrome type 2 (WS2), we screened KITLG in suspected WS2-affected probands. A heterozygous missense mutation, c.310C>G (p.Leu104Val), that segregated with WS2 was identified in a small family. In vitro studies revealed that the p.Leu104Val transmembrane isoform of KITLG is located at the cell membrane, as is wild-type KITLG. However, in culture media of transfected cells, the p.Leu104Val soluble isoform of KITLG was reduced, and no soluble p.His67_Cys68delinsArg and p.Ser96Ter KITLG could be detected. These data suggest that mutations in KITLG associated with NS-UHL/AHL have a loss-of-function effect. We speculate that the mechanism of the mutation underlying WS2 and leading to membrane incorporation and reduced secretion of KITLG occurs via a dominant-negative or gain-of-function effect. Our study unveils different phenotypes associated with KITLG, previously associated with pigmentation abnormalities, and will thereby improve the genetic counseling given to individuals with KITLG variants.

Somatosensory projections to cochlear nucleus are upregulated after unilateral deafness.

The cochlear nucleus (CN) receives innervation from auditory and somatosensory structures, which can be identified using vesicular glutamate transporters, VGLUT1 and VGLUT2. VGLUT1 is highly expressed in the magnocellular ventral CN (VCN), which receives auditory nerve inputs. VGLUT2 is predominantly expressed in the granule cell domain (GCD), which receives nonauditory inputs from somatosensory nuclei, including spinal trigeminal nucleus (Sp5) and cuneate nucleus (Cu). Two weeks after unilateral deafening VGLUT1 is significantly decreased in ipsilateral VCN while VGLUT2 is significantly increased in the ipsilateral GCD (Zeng et al., 2009), putatively reflecting decreased inputs from auditory nerve and increased inputs from nonauditory structures in guinea pigs. Here, we wished to determine whether the upregulation of VGLUT2 represents increases in the number of somatosensory projections to the CN that are maintained for longer periods of time. Thus, we examined concurrent changes in VGLUT levels and somatosensory projections in the CN using immunohistochemistry combined with anterograde tract tracing three and six weeks following unilateral deafening. The data reveal that unilateral deafness leads to increased numbers of VGLUT2-colabeled Sp5 and Cu projections to the ventral and dorsal CN. These findings suggest that Sp5 and Cu play significant and unique roles in cross-modal compensation and that, unlike after shorter term deafness, neurons in the magnocellular regions also participate in the compensation. The enhanced glutamatergic somatosensory projections to the CN may play a role in neural spontaneous hyperactivity associated with tinnitus.

Reversible conductive hearing loss: restored activity in the central auditory system.

The effect of a reversible, unilateral hearing loss on 2-deoxyglucose (2-DG) uptake in the central auditory system was studied using young gerbils. All animals had a unilateral conductive hearing loss (CHL), induced by atresia, on postnatal day 21 (P21). One week later, on P28, animals had their atresia repaired (CHL/R), or not repaired (CHL/NR), and CHL/NR animals entered the 2-DG experiments. CHL/R animals were allowed a 1-week period of restored binaural hearing experience prior to entering 2-DG experiments on P35. Animals in each group were injected with 2-DG and exposed to ambient sounds for 45 min prior to sacrifice. Uptake of 2-DG was measured in the anteroventral cochlear nucleus (AVCN), the medial superior olive (MSO), and the inferior colliculus (IC) on both sides of the brain. In CHL/NR animals, there were significant differences in uptake between the AVCN, MSO, and IC ipsilateral versus contralateral to the manipulated ear, indicating an imbalance in ascending afferent activity. In CHL/R animals, there were no significant differences, suggesting that 1 week after CHL repair, the appearance of balanced afferent activity had been restored.

Response of the flat cochlear epithelium to forced expression of Atoh1.

Following hair cell elimination in severely traumatized cochleae, differentiated supporting cells are often replaced by a simple epithelium with cuboidal or flat appearance. Atoh1 (previously Math1) is a basic helix-loop-helix transcription factor critical to hair cell differentiation during mammalian embryogenesis. Forced expression of Atoh1 in the differentiated supporting cell population can induce transdifferentiation leading to hair cell regeneration. Here, we examined the outcome of adenovirus mediated over-expression of Atoh1 in the non-sensory cells of the flat epithelium. We determined that seven days after unilateral elimination of hair cells with neomycin, differentiated supporting cells are absent, replaced by a flat epithelium. Nerve processes were also missing from the auditory epithelium, with the exception of infrequent looping nerve processes above the habenula perforata. We then inoculated an adenovirus vector with Atoh1 insert into the scala media of the deafened cochlea. The inoculation resulted in upregulation of Atoh1 in the flat epithelium. However, two months after the inoculation, Atoh1-treated ears did not exhibit clear signs of hair cell regeneration. Combined with previous data on induction of supporting cell to hair cell transdifferentiation by forced expression of Atoh1, these results suggest that the presence of differentiated supporting cells in the organ of Corti is necessary for transdifferentiation to occur.

Consequences of unilateral hearing loss: time dependent regulation of protein synthesis in auditory brainstem nuclei.

Conductive hearing impairment results in marked changes in neuronal activity in the central auditory system, particularly in young animals [Tucci, D.L., Cant, N.B., Durham, D., 1999. Conductive hearing loss results in a decrease in central auditory system activity in the young gerbil. Laryngoscope 109, 1359-1371]. To better understand the effects of conductive hearing loss (CHL) on cellular metabolism, incorporation of (3)H-leucine was used as a measure of protein synthesis in immature postnatal day 21 gerbils subjected to either unilateral CHL by malleus removal or profound sensorineural hearing loss by cochlear ablation. (3)H-leucine uptake was measured after survival times of 6 or 48h. Protein synthesis values were standardized to measurements from the abducens nucleus and compared with measurements from sham animals at similar age/survival times. Protein synthesis in the medial superior olive (MSO) was found to be significantly down-regulated (bilaterally) after CHL in animals surviving 48h. However, 6h after CHL manipulation, protein synthesis is up-regulated in MSO (bilaterally) and in the ipsilateral medial nucleus of the trapezoid body.