ABR/DPOAE publications

Cochlear and vestibular hair cells are highly specialized sensory receptors for hearing and balance. Here, we report a serendipitous identification of a hair-cell-specific organelle in neonatal mouse inner ear, which we name "apicosome." The apicosome is ∼500 nm in diameter and shows itinerant nature and transient appearance during development in cochlear hair cells. In contrast to cochlear hair cells, the apicosome persists in vestibular hair cells even in adult. The timing of apicosome translocation and disappearance in cochlear hair cells during development is correlated with kinocilium development and maintenance. The apicosome is not seen in supporting cells despite the fact that nascent supporting cells have microvilli and a primary cilium. Interestingly, transdifferentiated hair cells from supporting cells also contain apicosome, suggesting that it is unique to hair cells. Thus, our study identifies a previously undescribed organelle in hair cells and lays the foundation for further characterization of this specialized structure.

Hearing loss is a hallmark of aging, typically initially affecting the higher frequencies. In echolocating bats, the ability to discern high frequencies is essential. However, nothing is known about age-related hearing loss in bats, and they are often assumed to be immune to it. We tested the hearing of 47 wild Egyptian fruit bats by recording their auditory brainstem response and cochlear microphonics, and we also assessed the cochlear histology in four of these bats. We used the bats’ DNA methylation profile to evaluate their age and found that bats exhibit age-related hearing loss, with more prominent deterioration at the higher frequencies. The rate of the deterioration was ∼1 dB per year, comparable to the hearing loss observed in humans. Assessing the noise in the fruit bat roost revealed that these bats are exposed to continuous immense noise—mostly of social vocalizations—supporting the assumption that bats might be partially resistant to loud noise. Thus, in contrast to previous assumptions, our results suggest that bats constitute a model animal for the study of age-related hearing loss.

Evidence shows that females have increased supra-threshold peripheral auditory processing compared to males. This is indicated by larger auditory brainstem responses (ABR) wave I amplitude, which measures afferent spiral ganglion neuron (SGN)-auditory nerve synchrony. However, the underlying molecular mechanisms of this sex difference are mostly unknown. We sought to elucidate sex differences in ABR wave I amplitude by examining molecular markers known to affect synaptic transmission kinetics. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate fast excitatory transmission in mature SGN afferent synapses. Each AMPAR channel is a tetramer composed of GluA2, 3, and 4 subunits ( Gria2, 3 , and 4 genes), and those lacking GluA2 subunits have larger currents, are calcium-permeable, and have faster gating kinetics. Moreover, alternatively spliced flip and flop isoforms of each AMPAR subunit affect channel kinetics, having faster kinetics those AMPARs containing Gria3 and Gria4 flop isoforms. We hypothesized that SGNs of females have more fast-gating AMPAR subunit mRNA than males, which could contribute to more temporally precise synaptic transmission and increased SGN synchrony. Our data show that the index of Gria3 relative to Gria2 transcripts on SGN was higher in females than males (females: 48%; males: 43%), suggesting that females have more SGNs with higher Gria3 mRNA relative to Gria2 . Analysis of the relative abundance of the flip and flop alternatively spliced isoforms showed that females have a 2-fold increase in fast-gating Gria3 flop mRNA, while males have more slow-gating (2.5-fold) of the flip . We propose that Gria3 may in part mediate greater SGN synchrony in females. Significance Statement: Females of multiple vertebrate species, including fish and mammals, have been reported to have enhanced sound-evoked synchrony of afferents in the auditory nerve. However, the underlying molecular mediators of this physiologic sex difference are unknown. Elucidating potential molecular mechanisms related to sex differences in auditory processing is important for maintaining healthy ears and developing potential treatments for hearing loss in both sexes. This study found that females have a 2-fold increase in Gria3 flop mRNA, a fast-gating AMPA-type glutamate receptor subunit. This difference may contribute to greater neural synchrony in the auditory nerve of female mice compared to males, and this sex difference may be conserved in all vertebrates.

Background Third window syndrome is a vestibular-cochlear disorder in humans in which a third mobile window of the otic capsule creates changes to the flow of sound pressure energy through the perilymph/endolymph. The nature and location of this third mobile window can occur at many different sites (or multiple sites); however, the most common third mobile window is superior semicircular canal dehiscence (SSCD). There are two essential objective diagnostic characteristics needed to validate a model of SSCD: the creation of a pseudoconductive hearing loss and cVEMP increased amplitude and decreased threshold. Methods Adult Mongolian gerbils ( n = 36) received surgical fenestration of the superior semicircular canal of the left inner ear. ABR and c+VEMP testing were carried out prior to surgery and over acute (small 1 mm SSCD, 1–10 days) or prolonged (large 2 mm SSCD, 28 days) recovery. Because recovery of function occurred quickly, condenser brightfield stereomicroscopic examination of the dehiscence site was carried out for the small SSCD animals post-hoc and compared to both ABRs and c+VEMPs. Micro-CT analysis was also completed with representative samples of control, day 3 and 10 post-SSCD animals. Results The SSCD created a significant worsening of hearing thresholds of the left ear; especially in the lower frequency domain (1–4 kHz). Left (EXP)/right (CTL) ear comparisons via ABR show significant worsening thresholds at the same frequency representations, which is a proxy for the human pseudoconductive hearing loss seen in SSCD. For the c+VEMP measurements, increased amplitude of the sound-induced response (N1 2.5 ms and P1 3.2 ms) was observed in animals that received larger fenestrations. As the bone regrew, the c+VEMP and ABR responses returned toward preoperative values. For small SSCD animals, micro-CT data show that progressive osteoneogenesis results in resurfacing of the SSCD without bony obliteration. Conclusion The large (2 mm) SSCD used in our gerbil model results in similar electrophysiologic findings observed in patients with SSCD. The changes observed also reverse and return to baseline as the SSCD heals by bone resurfacing (with the lumen intact). Hence, this model does not require a second surgical procedure to plug the SSCD.

Cochlear sound encoding depends on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), but reliance on specific pore-forming subunits is unknown. With 5-week-old male C57BL/6J Gria3 -knockout mice (i.e., subunit GluA3 KO ) we determined cochlear function, synapse ultrastructure, and AMPAR molecular anatomy at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons. GluA3 KO and wild-type (GluA3 WT ) mice reared in ambient sound pressure level (SPL) of 55–75 dB had similar auditory brainstem response (ABR) thresholds, wave-1 amplitudes, and latencies. Postsynaptic densities (PSDs), presynaptic ribbons, and synaptic vesicle sizes were all larger on the modiolar side of the IHCs from GluA3 WT , but not GluA3 KO , demonstrating GluA3 is required for modiolar–pillar synapse differentiation. Presynaptic ribbons juxtaposed with postsynaptic GluA2/4 subunits were similar in quantity, however, lone ribbons were more frequent in GluA3 KO and GluA2-lacking synapses were observed only in GluA3 KO . GluA2 and GluA4 immunofluorescence volumes were smaller on the pillar side than the modiolar side in GluA3 KO , despite increased pillar-side PSD size. Overall, the fluorescent puncta volumes of GluA2 and GluA4 were smaller in GluA3 KO than GluA3 WT . However, GluA3 KO contained less GluA2 and greater GluA4 immunofluorescence intensity relative to GluA3 WT (threefold greater mean GluA4:GluA2 ratio). Thus, GluA3 is essential in development, as germline disruption of Gria3 caused anatomical synapse pathology before cochlear output became symptomatic by ABR. We propose the hearing loss in older male GluA3 KO mice results from progressive synaptopathy evident in 5-week-old mice as decreased abundance of GluA2 subunits and an increase in GluA2-lacking, GluA4-monomeric Ca 2+ -permeable AMPARs.

Cisplatin is a widely used and efficacious chemotherapeutic agent for treating solid tumors, yet it causes systemic end-organ damage that is often irreversible and detrimental to quality of life. This includes severe sensorineural hearing loss, hepatotoxicity, and renal injury. Based on the hard-soft acid-base theory, we recently developed two acetophenone-derived, enol-based compounds that directly interfere with the side effects of cisplatin. We investigated organ-specific and generalized toxicity in order to define dose-dependent responses in rodents injected with cisplatin with or without the protective compounds. All metrics that were used as indicators of toxicity showed retention of baseline or control measurements when animals were pre-treated with acetophenones prior to cisplatin administration, while animals injected with no protective compounds showed expected elevations in toxicity measurements or depressions in measurements of organ function. These data support the further investigation of novel acetophenone compounds for the prevention of cisplatin-induced end-organ toxicity.

The cochlea’s ability to discriminate sound frequencies is facilitated by a special topography along its longitudinal axis known as tonotopy. Auditory hair cells located at the base of the cochlea respond to high-frequency sounds, whereas hair cells at the apex respond to lower frequencies. Gradual changes in morphological and physiological features along the length of the cochlea determine each region’s frequency selectivity, but it remains unclear how tonotopy is established during cochlear development. Recently, sonic hedgehog (SHH) was proposed to initiate the establishment of tonotopy by conferring regional identity to the primordial cochlea. Here, using mouse genetics, we provide in vivo evidence that regional identity in the embryonic cochlea acts as a framework upon which tonotopy-specific properties essential for frequency selectivity in the mature cochlea develop. We found that follistatin (FST) is required for the maintenance of apical cochlear identity, but dispensable for its initial induction. In a fate-mapping analysis, we found that FST promotes expansion of apical cochlear cells, contributing to the formation of the apical cochlear domain. SHH, in contrast, is required both for the induction and maintenance of apical identity. In the absence of FST or SHH, mice produce a short cochlea lacking its apical domain. This results in the loss of apex-specific anatomical and molecular properties and low-frequency-specific hearing loss.

Hearing loss is the most common sensory deficit, of which genetic etiologies are a frequent cause. Dominant and recessive mutations in TMC1 , a gene encoding the pore-forming subunit of the hair cell mechanotransduction channel, cause DFNA36 and DFNB7/11, respectively, accounting for ∼2% of genetic hearing loss. Previous work has established the efficacy of mutation-targeted RNAi in treatment of murine models of autosomal dominant non-syndromic deafness. However, application of such approaches is limited by the infeasibility of development and validation of novel constructs for each variant. We developed an allele-non-specific approach consisting of mutation-agnostic RNAi suppression of both mutant and WT alleles, co-delivered with a knockdown-resistant engineered WT allele with or without the use of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to augment transgene expression. This therapeutic construct was delivered into the mature murine model of DFNA36 with an AAV vector and achieved robust hair cell and auditory brainstem response preservation. However, WPRE-enhanced Tmc1 expression resulted in inferior outcomes, suggesting a role for gene dosage optimization in future TMC1 gene therapy development.

The medial nucleus of the trapezoid body (MNTB) is an integral component of the auditory brainstem circuitry involved in sound localization. The giant presynaptic nerve terminal with multiple active zones, the calyx of Held (CH), is a hallmark of this nucleus, which mediates fast and synchronized glutamatergic synaptic transmission. To delineate how these synaptic structures adapt to reduced auditory afferents due to aging, we acquired and reconstructed circuitry-level volumes of mouse MNTB at different ages (3 weeks, 6, 18, and 24 months) using serial block-face electron microscopy. We used C57BL/6J, the most widely inbred mouse strain used for transgenic lines, which displays a type of age-related hearing loss. We found that MNTB neurons reduce in density with age. Surprisingly we observed an average of approximately 10% of poly-innervated MNTB neurons along the mouse lifespan, with prevalence in the low frequency region. Moreover, a tonotopy-dependent heterogeneity in CH morphology was observed in young but not in older mice. In conclusion, our data support the notion that age-related hearing impairments can be in part a direct consequence of several structural alterations and circuit remodeling in the brainstem.

The study aimed to investigate the potential role of lysine-specific demethylase 5A (KDM5A) in cisplatin-induced ototoxicity. The effect of the KDM5A inhibitor CPI-455 was assessed by apoptosis assay, immunofluorescence, flow cytometry, seahorse respirometry assay, and auditory brainstem response test. RNA sequencing, qRT-PCR, and CUT&Tag assays were used to explore the mechanism underlying CPI-455-induced protection. Our results demonstrated that the expression of KDM5A was increased in cisplatin-injured cochlear hair cells compared with controls. CPI-455 treatment markedly declined KDM5A and elevated H3K4 trimethylation levels in cisplatin-injured cochlear hair cells. Moreover, CPI-455 effectively prevented the death of hair cells and spiral ganglion neurons and increased the number of ribbon synapses in a cisplatin-induced ototoxicity mouse model both in vitro and in vivo. In HEI-OC1 cells, KDM5A knockdown reduced reactive oxygen species accumulation and improved mitochondrial membrane potential and oxidative phosphorylation under cisplatin-induced stress. Mechanistically, through transcriptomics and epigenomics analyses, a set of apoptosis-related genes, including Sos1 , Sos2 , and Map3k3 , were regulated by CPI-455. Altogether, our findings indicate that inhibition of KDM5A may represent an effective epigenetic therapeutic target for preventing cisplatin-induced hearing loss.

Progressive functional deterioration in the cochlea is associated with age-related hearing loss (ARHL). However, the cellular and molecular basis underlying cochlear aging remains largely unknown. Here, we established a dynamic single-cell transcriptomic landscape of mouse cochlear aging, in which we characterized aging-related transcriptomic changes in 27 different cochlear cell types across five different time points. Overall, our analysis pinpoints loss of proteostasis and elevated apoptosis as the hallmark features of cochlear aging, highlights unexpected age-related transcriptional fluctuations in intermediate cells localized in the stria vascularis (SV) and demonstrates that upregulation of endoplasmic reticulum (ER) chaperon protein HSP90AA1 mitigates ER stress-induced damages associated with aging. Our work suggests that targeting unfolded protein response pathways may help alleviate aging-related SV atrophy and hence delay the progression of ARHL.

Inhibitory G proteins (GNAI/Gα i ) bind to the scaffold G protein signaling modulator 2 (GPSM2) to form a conserved polarity complex that regulates cytoskeleton organization. GPSM2 keeps GNAI in a guanosine diphosphate (GDP)-bound state, but how GPSM2-GNAI is generated or relates to heterotrimeric G protein signaling remains unclear. We find that RGS12, a GTPase-activating protein (GAP), is required to polarize GPSM2-GNAI at the hair cell apical membrane and to organize mechanosensory stereocilia in rows of graded heights. Accordingly, RGS12 and the guanine nucleotide exchange factor (GEF) DAPLE are asymmetrically co-enriched at the hair cell apical junction, and Rgs12 mouse mutants are deaf. GPSM2 and RGS12 share GoLoco motifs that stabilize GNAI(GDP), and GPSM2 outcompetes RGS12 to bind GNAI. Our results suggest that polarized GEF/GAP junctional activity might dissociate heterotrimeric G proteins, generating free GNAI(GDP) for GPSM2 at the adjacent apical membrane. GPSM2-GNAI(GDP), in turn, imparts asymmetry to the forming stereocilia to enable sensory function in hair cells.

Background Uncoupling protein 2 (UCP2), activated by excessive reactive oxygen species (ROS) in vivo, has the dual effect of reducing ROS to protect against oxidative stress and reducing ATP production to regulate cellular metabolism. Both the UCP2 and ROS are increased in cochleae in age-related hearing loss (ARHL). However, the role of UCP2 in sensory hair cells in ARHL remains unclear. Methods Male C57BL/6 J mice were randomly assigned to an 8-week-old group (Group 1), a 16-week-old group (Group 2), a 16-week-old + adeno-associated virus-inner ear (AAV-ie) group (Group 3), and a 16-week-old + AAV-ie-UCP2 group (Group 4). Mice aged 8 weeks were administrated with AAV-ie-GFP or AAV-ie-UCP2 via posterior semicircular canal injection. Eight weeks after this viral intervention, hearing thresholds and wave-I amplitudes were tested by auditory brainstem response (ABR). Subsequently, the cochlear basilar membrane was dissected for investigation. The number of hair cells and inner hair cell (IHC) synapses, the level of ROS, and the expression of AMP-activated protein kinase α (AMPKα), were assessed by immunofluorescence staining. In addition, mitochondrial function was determined, and the expression of AMPKα and UCP2 proteins was further evaluated using western blotting. Results Mice with early-onset ARHL exhibited enhanced oxidative stress and loss of outer hair cells and IHC synapses, while UCP2 overexpression aggravated hearing loss and cochlear pathophysiological changes in mice. UCP2 overexpression resulted in a notable decrease in the number of IHCs and IHC synapses, caused ATP depletion and excessive ROS generation, increased AMPKα protein levels, and promoted IHC apoptosis, especially in the apical and middle turns of the cochlea. Conclusion Collectively, our data suggest that UCP2 overexpression may cause mitochondrial dysfunction via energy metabolism, which activates mitochondrion-dependent cellular apoptosis and leads to IHC loss, ultimately exacerbating ARHL.

The lateral superior olive (LSO) is a key structure in the central auditory system of mammals that exerts efferent control on cochlear sensitivity and is involved in the processing of binaural level differences for sound localization. Understanding how the LSO contributes to these processes requires knowledge about the resident cells and their connections with other auditory structures. We used standard histological stains and retrograde tracer injections into the inferior colliculus (IC) and cochlea in order to characterize two basic groups of neurons: (1) Principal and periolivary (PO) neurons have projections to the IC as part of the ascending auditory pathway; and (2) lateral olivocochlear (LOC) intrinsic and shell efferents have descending projections to the cochlea. Principal and intrinsic neurons are intermixed within the LSO, exhibit fusiform somata, and have disk-shaped dendritic arborizations. The principal neurons have bilateral, symmetric, and tonotopic projections to the IC. The intrinsic efferents have strictly ipsilateral projections, known to be tonotopic from previous publications. PO and shell neurons represent much smaller populations (<10% of principal and intrinsic neurons, respectively), have multipolar somata, reside outside the LSO, and have non-topographic, bilateral projections. PO and shell neurons appear to have widespread projections to their targets that imply a more diffuse modulatory function. The somata and dendrites of principal and intrinsic neurons form a laminar matrix within the LSO and share quantifiably similar alignment to the tonotopic axis. Their restricted projections emphasize the importance of frequency in binaural processing and efferent control for auditory perception. This study addressed and expanded on previous findings of cell types, circuit laterality, and projection tonotopy in the LSO of the mouse.

Centrosomal protein dysfunction might cause ciliopathies. However, the role of centrosomal proteins in male infertility remains poorly defined. Here, we identified a pathogenic splicing mutation in CEP78 in male infertile patients with severely reduced sperm number and motility, and the typical multiple morphological abnormalities of the sperm flagella phenotype. We further created Cep78 knockout mice, which showed an extremely low sperm count, completely aberrant sperm morphology, and approximately null sperm motility. The infertility of the patients and knockout mice could not be rescued by an intracytoplasmic sperm injection treatment. Mechanistically, CEP78 might regulate USP16 expression, which further stabilizes Tektin levels via the ubiquitination pathway. Cep78 knockout mice also exhibited impairments in retina and outer hair cells of the cochlea. Collectively, our findings identified nonfunctional CEP78 as an indispensable factor contributing to male infertility and revealed a role for this gene in regulating retinal and outer hair cell function in mice.

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4 ) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

Foraging on nocturnally migrating birds is one of the most challenging foraging tasks in the animal kingdom. Only three bat species (e.g., Ia io) known to date can prey on migratory birds. However, how these bats have exploited this challenging dietary niche remains unknown. Here, we demonstrate that I. io hunts at the altitude of migrating birds during the bird migration season. The foraging I. io exhibited high flight altitudes (up to 4945 m above sea level) and high flight speeds (up to 143.7 km h-1). I. io in flight can actively prey on birds in the night sky via echolocation cues. Genes associated with DNA damage repair, hypoxia adaptation, biting and mastication, and digestion and metabolism have evolved to adapt to this species' avivorous habits. Our results suggest that the evolution of behavioral innovation and genomic novelty are associated with the exploitation of challenging dietary opportunities.

The transduction of acoustic information by hair cells depends upon mechanosensitive stereociliary bundles that project from their apical surface. Mutations or absence of the stereociliary protein EPS8 cause deafness in humans and mice, respectively. Eps8 knockout mice (Eps8 -/- ) have hair cells with immature stereocilia and fail to become sensory receptors. Here, we show that exogenous delivery of Eps8 using Anc80L65 in P1-P2 Eps8 -/- mice in vivo rescued the hair bundle structure of apical-coil hair cells. Rescued hair bundles correctly localize EPS8, WHIRLIN, MYO15, and BAIAP2L2, and generate normal mechanoelectrical transducer currents. Inner hair cells with normal-looking stereocilia re-expressed adult-like basolateral ion channels (BK and KCNQ4) and have normal exocytosis. The number of hair cells undergoing full recovery was not sufficient to rescue hearing in Eps8 -/- mice. Adeno-associated virus (AAV)-transduction of P3 apical-coil and P1-P2 basal-coil hair cells does not rescue hair cells, nor does Anc80L65-Eps8 delivery in adult Eps8 -/- mice. We propose that AAV-induced gene-base therapy is an efficient strategy to recover the complex hair-cell defects in Eps8 -/- mice. However, this therapeutic approach may need to be performed in utero since, at postnatal ages, Eps8 -/- hair cells appear to have matured or accumulated damage beyond the point of repair.

Background: Cryopyrin-associated periodic syndrome (CAPS) is an inherited autoinflammatory disease caused by a gain-of-function mutation in NLRP3. Although CAPS patients frequently suffer from sensorineural hearing loss, it remains unclear whether CAPS-associated mutation in NLRP3 is associated with the progression of hearing loss. Methods: We generated a mice with conditional expression of CAPS-associated NLRP3 mutant (D301N) in cochlea-resident CX3CR1 macrophages and examined the susceptibility of CAPS mice to inflammation-mediated hearing loss in a local and systemic inflammation context. Findings: Upon lipopolysaccharide (LPS) injection into middle ear cavity, NLRP3 mutant mice exhibited severe cochlear inflammation, inflammasome activation and hearing loss. However, this middle ear injection model induced a considerable hearing loss in control mice and inevitably caused an inflammation-independent hearing loss possibly due to ear tissue damages by injection procedure. Subsequently, we optimized a systemic LPS injection model, which induced a significant hearing loss in NLRP3 mutant mice but not in control mice. Peripheral inflammation induced by a repetitive low dose of LPS injection caused a blood-labyrinth barrier disruption, macrophage infiltration into cochlea and cochlear inflammasome activation in an NLRP3-dependent manner. Interestingly, both cochlea-infiltrating and -resident macrophages contribute to peripheral inflammation-mediated hearing loss of CAPS mice. Furthermore, NLRP3-specific inhibitor, MCC950, as well as an interleukin-1 receptor antagonist significantly alleviated systemic LPS-induced hearing loss and inflammatory phenotypes in NLRP3 mutant mice. Interpretation: Our findings reveal that CAPS-associated NLRP3 mutation is critical for peripheral inflammation-induced hearing loss in our CAPS mice model, and an NLRP3-specific inhibitor can be used to treat inflammation-mediated sensorineural hearing loss. Funding: National Research Foundation of Korea Grant funded by the Korean Government and the Team Science Award of Yonsei University College of Medicine.

Clinical exome sequencing has yielded extensive disease-related missense single-nucleotide variants (SNVs) of uncertain significance, leading to diagnostic uncertainty. KCNQ4 is one of the most commonly responsible genes for autosomal dominant nonsyndromic hearing loss. According to the gnomAD cohort, approximately one in 100 people harbors missense variants in KCNQ4 (missense variants with minor allele frequency > 0.1% were excluded), but most are of unknown consequence. To prospectively characterize the function of all 4085 possible missense SNVs of human KCNQ4 , we recorded the whole-cell currents using the patch-clamp technique and categorized 1068 missense SNVs as loss of function, as well as 728 loss-of-function SNVs located in the transmembrane domains. Further, to mimic the heterozygous condition in Deafness nonsyndromic autosomal dominant 2 (DFNA2) patients caused by KCNQ4 variants, we coexpressed loss-of-function variants with wild-type KCNQ4 and found 516 variants showed impaired or only partially rescued heterogeneous channel function. Overall, our functional classification is highly concordant with the auditory phenotypes in Kcnq4 mutant mice and the assessments of pathogenicity in clinical variant interpretations. Taken together, our results provide strong functional evidence to support the pathogenicity classification of newly discovered KCNQ4 missense variants in clinical genetic testing.