Combined AAV-mediated gene replacement therapy improves auditory function in a mouse model of human DFNB42 deafness.

Hearing loss is a common disorder affecting nearly 20% of the world's population. Recently, studies have shown that inner ear gene therapy can improve auditory function in several mouse models of hereditary hearing loss. In most of these studies, the underlying mutations affect only a small number of cell types of the inner ear (e.g., sensory hair cells). Here, we applied inner ear gene therapy to the Ildr1Gt(D178D03)Wrst (Ildr1w-/-) mouse, a model of human DFNB42, non-syndromic autosomal recessive hereditary hearing loss associated with ILDR1 variants. ILDR1 is an integral protein of the tricellular tight junction complex and is expressed by diverse inner ear cell types in the organ of Corti and the cochlear lateral wall. We simultaneously applied two synthetic adeno-associated viruses (AAVs) with different tropism to deliver Ildr1 cDNA to the Ildr1w-/- mouse inner ear: one targeting the organ of Corti (AAV2.7m8) and the other targeting the cochlear lateral wall (AAV8BP2). We showed that combined AAV2.7m8/AAV8BP2 gene therapy improves cochlear structural integrity and auditory function in Ildr1w-/- mice.

Transcription co-factor LBH is necessary for the survival of cochlear hair cells.

Hearing loss affects ∼10% of adults worldwide. Most sensorineural hearing loss is caused by the progressive loss of mechanosensitive hair cells (HCs) in the cochlea. The molecular mechanisms underlying HC maintenance and loss remain poorly understood. LBH, a transcription co-factor implicated in development, is abundantly expressed in outer hair cells (OHCs). We used Lbh-null mice to identify its role in HCs. Surprisingly, Lbh deletion did not affect differentiation and the early development of HCs, as nascent HCs in Lbh knockout mice had normal looking stereocilia. The stereocilia bundle was mechanosensitive and OHCs exhibited the characteristic electromotility. However, Lbh-null mice displayed progressive hearing loss, with stereocilia bundle degeneration and OHC loss as early as postnatal day 12. RNA-seq analysis showed significant gene enrichment of biological processes related to transcriptional regulation, cell cycle, DNA damage/repair and autophagy in Lbh-null OHCs. In addition, Wnt and Notch pathway-related genes were found to be dysregulated in Lbh-deficient OHCs. Our study implicates, for the first time, loss of LBH function in progressive hearing loss, and demonstrates a critical requirement of LBH in promoting HC survival in adult mice.

ROR1 is essential for proper innervation of auditory hair cells and hearing in humans and mice.

Hair cells of the inner ear, the mechanosensory receptors, convert sound waves into neural signals that are passed to the brain via the auditory nerve. Little is known about the molecular mechanisms that govern the development of hair cell-neuronal connections. We ascertained a family with autosomal recessive deafness associated with a common cavity inner ear malformation and auditory neuropathy. Via whole-exome sequencing, we identified a variant (c.2207G>C, p.R736T) in ROR1 (receptor tyrosine kinase-like orphan receptor 1), cosegregating with deafness in the family and absent in ethnicity-matched controls. ROR1 is a tyrosine kinase-like receptor localized at the plasma membrane. At the cellular level, the mutation prevents the protein from reaching the cellular membrane. In the presence of WNT5A, a known ROR1 ligand, the mutated ROR1 fails to activate NF-κB. Ror1 is expressed in the inner ear during development at embryonic and postnatal stages. We demonstrate that Ror1 mutant mice are severely deaf, with preserved otoacoustic emissions. Anatomically, mutant mice display malformed cochleae. Axons of spiral ganglion neurons show fasciculation defects. Type I neurons show impaired synapses with inner hair cells, and type II neurons display aberrant projections through the cochlear sensory epithelium. We conclude that Ror1 is crucial for spiral ganglion neurons to innervate auditory hair cells. Impairment of ROR1 function largely affects development of the inner ear and hearing in humans and mice.

A dominant variant in the PDE1C gene is associated with nonsyndromic hearing loss.

Identification of genes with variants causing non-syndromic hearing loss (NSHL) is challenging due to genetic heterogeneity. The difficulty is compounded by technical limitations that in the past prevented comprehensive gene identification. Recent advances in technology, using targeted capture and next-generation sequencing (NGS), is changing the face of gene identification and making it possible to rapidly and cost-effectively sequence the whole human exome. Here, we characterize a five-generation Chinese family with progressive, postlingual autosomal dominant nonsyndromic hearing loss (ADNSHL). By combining population-specific mutation arrays, targeted deafness genes panel, whole exome sequencing (WES), we identified PDE1C (Phosphodiesterase 1C) c.958G>T (p.A320S) as the disease-associated variant. Structural modeling insights into p.A320S strongly suggest that the sequence alteration will likely affect the substrate-binding pocket of PDE1C. By whole-mount immunofluorescence on postnatal day 3 mouse cochlea, we show its expression in outer (OHC) and inner (IHC) hair cells cytosol co-localizing with Lamp-1 in lysosomes. Furthermore, we provide evidence that the variant alters the PDE1C hydrolytic activity for both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Collectively, our findings indicate that the c.958G>T variant in PDE1C may disrupt the cross talk between cGMP-signaling and cAMP pathways in Ca2+ homeostasis.

Indispensable Role of Ion Channels and Transporters in the Auditory System.

Ear is a complex system where appropriate ionic composition is essential for maintaining the tissue homeostasis and hearing function. Ion transporters and channels present in the auditory system plays a crucial role in maintaining proper ionic composition in the ear. The extracellular fluid, called endolymph, found in the cochlea of the mammalian inner ear is particularly unique due to its electrochemical properties. At an endocochlear potential of about +80 mV, signaling initiated by acoustic stimuli at the level of the hair cells is dependent on the unusually high potassium (K+ ) concentration of endolymph. There are ion channels and transporters that exists in the ear to ensure that K+ is continually being cycled into the stria media endolymph. This review is focused on the discussion of the molecular and genetic basis of previously and newly recognized ion channels and transporters that support sensory hair cell excitation based on recent knock-in and knock-out studies of these channels. This article also addresses the molecular and genetic defects and the pathophysiology behind Meniere's disease as well as how the dysregulation of these ion transporters can result in severe defects in hearing or even deafness. Understanding the role of ion channels and transporters in the auditory system will facilitate in designing effective treatment modalities against ear disorders including Meniere's disease and hearing loss. J. Cell. Physiol. 232: 743-758, 2017. © 2016 Wiley Periodicals, Inc.

Signaling in the Auditory System: Implications in Hair Cell Regeneration and Hearing Function.

Ear is a sensitive organ involved in hearing and balance function. The complex signaling network in the auditory system plays a crucial role in maintaining normal physiological function of the ear. The inner ear comprises a variety of host signaling pathways working in synergy to deliver clear sensory messages. Any disruption, as minor as it can be, has the potential to affect this finely tuned system with temporary or permanent sequelae including vestibular deficits and hearing loss. Mutations linked to auditory symptoms, whether inherited or acquired, are being actively researched for ways to reverse, silence, or suppress them. In this article, we discuss recent advancements in understanding the pathways involved in auditory system signaling, from hair cell development through transmission to cortical centers. Our review discusses Notch and Wnt signaling, cell to cell communication through connexin and pannexin channels, and the detrimental effects of reactive oxygen species on the auditory system. There has been an increased interest in the auditory community to explore the signaling system in the ear for hair cell regeneration. Understanding signaling pathways in the auditory system will pave the way for the novel avenues to regenerate sensory hair cells and restore hearing function. J. Cell. Physiol. 232: 2710-2721, 2017. © 2016 Wiley Periodicals, Inc.

Neurotransmitters: The Critical Modulators Regulating Gut-Brain Axis.

Neurotransmitters, including catecholamines and serotonin, play a crucial role in maintaining homeostasis in the human body. Studies on these neurotransmitters mainly revolved around their role in the "fight or flight" response, transmitting signals across a chemical synapse and modulating blood flow throughout the body. However, recent research has demonstrated that neurotransmitters can play a significant role in the gastrointestinal (GI) physiology. Norepinephrine (NE), epinephrine (E), dopamine (DA), and serotonin have recently been a topic of interest because of their roles in the gut physiology and their potential roles in GI and central nervous system pathophysiology. These neurotransmitters are able to regulate and control not only blood flow, but also affect gut motility, nutrient absorption, GI innate immune system, and the microbiome. Furthermore, in pathological states, such as inflammatory bowel disease (IBD) and Parkinson's disease, the levels of these neurotransmitters are dysregulated, therefore causing a variety of GI symptoms. Research in this field has shown that exogenous manipulation of catecholamine serum concentrations can help in decreasing symptomology and/or disease progression. In this review article, we discuss the current state-of-the-art research and literature regarding the role of neurotransmitters in regulation of normal GI physiology, their impact on several disease processes, and novel work focused on the use of exogenous hormones and/or psychotropic medications to improve disease symptomology. J. Cell. Physiol. 232: 2359-2372, 2017. © 2016 Wiley Periodicals, Inc.

A missense mutation in DCDC2 causes human recessive deafness DFNB66, likely by interfering with sensory hair cell and supporting cell cilia length regulation.

Hearing loss is the most common sensory deficit in humans. We show that a point mutation in DCDC2 (DCDC2a), a member of doublecortin domain-containing protein superfamily, causes non-syndromic recessive deafness DFNB66 in a Tunisian family. Using immunofluorescence on rat inner ear neuroepithelia, DCDC2a was found to localize to the kinocilia of sensory hair cells and the primary cilia of nonsensory supporting cells. DCDC2a fluorescence is distributed along the length of the kinocilium with increased density toward the tip. DCDC2a-GFP overexpression in non-polarized COS7 cells induces the formation of long microtubule-based cytosolic cables suggesting a role in microtubule formation and stabilization. Deafness mutant DCDC2a expression in hair cells and supporting cells causes cilium structural defects, such as cilium branching, and up to a 3-fold increase in length ratios. In zebrafish, the ortholog dcdc2b was found to be essential for hair cell development, survival and function. Our results reveal DCDC2a to be a deafness gene and a player in hair cell kinocilia and supporting cell primary cilia length regulation likely via its role in microtubule formation and stabilization.

FAM65B is a membrane-associated protein of hair cell stereocilia required for hearing.

In a large consanguineous Turkish kindred with recessive nonsyndromic, prelingual, profound hearing loss, we identified in the gene FAM65B (MIM611410) a splice site mutation (c.102-1G>A) that perfectly cosegregates with the phenotype in the family. The mutation leads to exon skipping and deletion of 52-amino acid residues of a PX membrane localization domain. FAM65B is known to be involved in myotube formation and in regulation of cell adhesion, polarization, and migration. We show that wild-type Fam65b is expressed during embryonic and postnatal development stages in murine cochlea, and that the protein localizes to the plasma membranes of the stereocilia of inner and outer hair cells of the inner ear. The wild-type protein targets the plasma membrane, whereas the mutant protein accumulates in cytoplasmic inclusion bodies and does not reach the membrane. In zebrafish, knockdown of fam65b leads to significant reduction of numbers of saccular hair cells and neuromasts and to hearing loss. We conclude that FAM65B is a plasma membrane-associated protein of hair cell stereocilia that is essential for hearing.

MYO3A Causes Human Dominant Deafness and Interacts with Protocadherin 15-CD2 Isoform.

Hereditary hearing loss (HL) is characterized by both allelic and locus genetic heterogeneity. Both recessive and dominant forms of HL may be caused by different mutations in the same deafness gene. In a family with post-lingual progressive non-syndromic deafness, whole-exome sequencing of genomic DNA from five hearing-impaired relatives revealed a single variant, p.Gly488Glu (rs145970949:G>A) in MYO3A, co-segregating with HL as an autosomal dominant trait. This amino acid change, predicted to be pathogenic, alters a highly conserved residue in the motor domain of MYO3A. The mutation severely alters the ATPase activity and motility of the protein in vitro, and the mutant protein fails to accumulate in the filopodia tips in COS7 cells. However, the mutant MYO3A was able to reach the tips of organotypic inner ear culture hair cell stereocilia, raising the possibility of a local effect on positioning of the mechanoelectrical transduction (MET) complex at the stereocilia tips. To address this hypothesis, we investigated the interaction of MYO3A with the cytosolic tail of the integral tip-link protein protocadherin 15 (PCDH15), a core component of MET complex. Interestingly, we uncovered a novel interaction between MYO3A and PCDH15 shedding new light on the function of myosin IIIA at stereocilia tips.

Tectorins crosslink type II collagen fibrils and connect the tectorial membrane to the spiral limbus.

All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and ß tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and ß-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4±1.5nm, width), resembling another collagen type protein, or chains of globular elements (15±3.2nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and ß-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.

Molecular Structure and Regulation of P2X Receptors With a Special Emphasis on the Role of P2X2 in the Auditory System.

The P2X purinergic receptors are cation-selective channels gated by extracellular adenosine 5'-triphosphate (ATP). These purinergic receptors are found in virtually all mammalian cell types and facilitate a number of important physiological processes. Within the past few years, the characterization of crystal structures of the zebrafish P2X4 receptor in its closed and open states has provided critical insights into the mechanisms of ligand binding and channel activation. Understanding of this gating mechanism has facilitated to design and interpret new modeling and structure-function experiments to better elucidate how different agonists and antagonists can affect the receptor with differing levels of potency. This review summarizes the current knowledge on the structure, activation, allosteric modulators, function, and location of the different P2X receptors. Moreover, an emphasis on the P2X2 receptors has been placed in respect to its role in the auditory system. In particular, the discovery of three missense mutations in P2X2 receptors could become important areas of study in the field of gene therapy to treat progressive and noise-induced hearing loss. J. Cell. Physiol. 231: 1656-1670, 2016. © 2015 Wiley Periodicals, Inc.

Intricate Functions of Matrix Metalloproteinases in Physiological and Pathological Conditions.

Matrix metalloproteinases (MMPs) are a diverse group of proteolytic enzymes and play an important role in the degradation and remodeling of the extracellular matrix (ECM). In normal physiological conditions, MMPs are usually minimally expressed. Despite their low expression, MMPs have been implicated in many cellular processes ranging from embryological development to apoptosis. The activity of MMPs is controlled at three different stages: (1) transcription; (2) zymogen activation; and (3) inhibition of active forms by tissue inhibitor metalloproteinases (TIMPs). They can collectively degrade any component of ECM and basement membrane, and their excessive activity has been linked to numerous pathologies mainly including, but not limited to, tumor invasion and metastasis. The lack of information about several MMPs and the steady stream of new discoveries suggest that there is much more to be studied in this field. In particular, there is a need for controlling their expression in disease states. Various studies over the past 30 years have found that each MMP has a specific mode of activation, action, and inhibition. Drugs specifically targeting individual MMPs could revolutionize the treatment of a great number of health conditions and tremendously reduce their burden. In this review article, we have summarized the recent advances in understanding the role of MMPs in physiological and pathological conditions. J. Cell. Physiol. 231: 2599-2621, 2016. © 2016 Wiley Periodicals, Inc.

CRISPR: a versatile tool for both forward and reverse genetics research.

Human genetics research employs the two opposing approaches of forward and reverse genetics. While forward genetics identifies and links a mutation to an observed disease etiology, reverse genetics induces mutations in model organisms to study their role in disease. In most cases, causality for mutations identified by forward genetics is confirmed by reverse genetics through the development of genetically engineered animal models and an assessment of whether the model can recapitulate the disease. While many technological advances have helped improve these approaches, some gaps still remain. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated), which has emerged as a revolutionary genetic engineering tool, holds great promise for closing such gaps. By combining the benefits of forward and reverse genetics, it has dramatically expedited human genetics research. We provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples.

A mutation in SLC22A4 encoding an organic cation transporter expressed in the cochlea strial endothelium causes human recessive non-syndromic hearing loss DFNB60.

The high prevalence/incidence of hearing loss (HL) in humans makes it the most common sensory defect. The majority of the cases are of genetic origin. Non-syndromic hereditary HL is extremely heterogeneous. Genetic approaches have been instrumental in deciphering genes that are crucial for auditory function. In this study, we first used NADf chip to exclude the implication of known North-African mutations in HL in a large consanguineous Tunisian family (FT13) affected by autosomal recessive non-syndromic HL (ARNSHL). We then performed genome-wide linkage analysis and assigned the deafness gene locus to ch:5q23.2-31.1, corresponding to the DFNB60 ARNSHL locus. Moreover, we performed whole exome sequencing on FT13 patient DNA and uncovered amino acid substitution p.Cys113Tyr in SLC22A4, a transporter of organic cations, cosegregating with HL in FT13 and therefore the cause of ARNSHL DFNB60. We also screened a cohort of small Tunisian HL families and uncovered an additional deaf proband of consanguineous parents that is homozygous for p.Cys113Tyr carried by the same microsatellite marker haplotype as in FT13, indicating that this mutation is ancestral. Using immunofluorescence, we found that Slc22a4 is expressed in stria vascularis (SV) endothelial cells of rodent cochlea and targets their apical plasma membrane. We also found Slc22a4 transcripts in our RNA-seq library from purified primary culture of mouse SV endothelial cells. Interestingly, p.Cys113Tyr mutation affects the trafficking of the transporter and severely alters ergothioneine uptake. We conclude that SLC22A4 is an organic cation transporter of the SV endothelium that is essential for hearing, and its mutation causes DFNB60 form of HL.

Spectrum of DNA variants for non-syndromic deafness in a large cohort from multiple continents.

Hearing loss is the most common sensory deficit in humans with causative variants in over 140 genes. With few exceptions, however, the population-specific distribution for many of the identified variants/genes is unclear. Until recently, the extensive genetic and clinical heterogeneity of deafness precluded comprehensive genetic analysis. Here, using a custom capture panel (MiamiOtoGenes), we undertook a targeted sequencing of 180 genes in a multi-ethnic cohort of 342 GJB2 mutation-negative deaf probands from South Africa, Nigeria, Tunisia, Turkey, Iran, India, Guatemala, and the United States (South Florida). We detected causative DNA variants in 25 % of multiplex and 7 % of simplex families. The detection rate varied between 0 and 57 % based on ethnicity, with Guatemala and Iran at the lower and higher end of the spectrum, respectively. We detected causative variants within 27 genes without predominant recurring pathogenic variants. The most commonly implicated genes include MYO15A, SLC26A4, USH2A, MYO7A, MYO6, and TRIOBP. Overall, our study highlights the importance of family history and generation of databases for multiple ethnically discrete populations to improve our ability to detect and accurately interpret genetic variants for pathogenicity.

Genetic analysis of Tunisian families with Usher syndrome type 1: toward improving early molecular diagnosis.

PURPOSE: Usher syndrome accounts for about 50% of all hereditary deaf-blindness cases. The most severe form of this syndrome, Usher syndrome type I (USH1), is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. Six USH1 genes have been identified, MYO7A, CDH23, PCDH15, USH1C, SANS, and CIB2, encoding myosin VIIA, cadherin-23, protocadherin-15, harmonin, scaffold protein containing ankyrin repeats and a sterile alpha motif (SAM) domain, and calcium- and integrin-binding member 2, respectively. METHODS: In the present study, we recruited four Tunisian families with a diagnosis of USH1, together with healthy unrelated controls. Affected members underwent detailed audiologic and ocular examinations. We used the North African Deafness (NADf) chip to search for known North African mutations associated with USH. Then, we selected microsatellite markers covering USH1 known loci to genotype the DNA samples. Finally, we performed DNA sequencing of three known USH1 genes: MYO7A, PCDH15, and USH1C. RESULTS: Four biallelic mutations, all single base changes, were found in the MYO7A, USH1C, and PCDH15 genes. These mutations consist of a previously reported splicing defect c.470+1G>A in MYO7A, three novel variants, including two nonsense (p.Arg3X and p.Arg134X) in USH1C and PCDH15, respectively, and one frameshift (p.Lys615Asnfs*6) in MYO7A. CONCLUSIONS: We found a remarkable genetic heterogeneity in the studied families with USH1 with a variety of mutations, among which three were novel. These novel mutations will be included in the NADf mutation screening chip that will allow a higher diagnosis efficiency of this extremely genetically heterogeneous disease. Ultimately, efficient molecular diagnosis of USH in a patient's early childhood is of utmost importance, allowing better educational and therapeutic management.

Mutation of the ATP-gated P2X(2) receptor leads to progressive hearing loss and increased susceptibility to noise.

Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X(2) receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G > T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X(2) receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X(2) receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2-null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X(2) function is required for life-long normal hearing and for protection from exposure to noise.

Novel mutations confirm that COL11A2 is responsible for autosomal recessive non-syndromic hearing loss DFNB53.

Hearing loss (HL) is a major public health issue. It is clinically and genetically heterogeneous.The identification of the causal mutation is important for early diagnosis, clinical follow-up, and genetic counseling. HL due to mutations in COL11A2, encoding collagen type XI alpha-2, can be non-syndromic autosomal-dominant or autosomal-recessive, and also syndromic as in Otospondylomegaepiphyseal Dysplasia, Stickler syndrome type III, and Weissenbacher-Zweymuller syndrome. However, thus far only one mutation co-segregating with autosomal recessive non-syndromic hearing loss (ARNSHL) in a single family has been reported. In this study, whole exome sequencing of two consanguineous families with ARNSHL from Tunisia and Turkey revealed two novel causative COL11A2 mutations, c.109G > T (p.Ala37Ser) and c.2662C > A (p.Pro888Thr). The variants identified co-segregated with deafness in both families. All homozygous individuals in those families had early onset profound hearing loss across all frequencies without syndromic findings. The variants are predicted to be damaging the protein function. The p.Pro888Thr mutation affects a -Gly-X-Y- triplet repeat motif. The novel p.Ala37Ser is the first missense mutation located in the NC4 domain of the COL11A2 protein. Structural model suggests that this mutation will likely obliterate, or at least partially compromise, the ability of NC4 domain to interact with its cognate ligands. In conclusion, we confirm that COL11A2 mutations cause ARNSHL and broaden the mutation spectrum that may shed new light on genotype-phenotype correlation for the associated phenotypes and clinical follow-up.

Exome sequencing identifies a novel CEACAM16 mutation associated with autosomal dominant nonsyndromic hearing loss DFNA4B in a Chinese family.

Autosomal dominant nonsyndromic hearing loss (ADNSHL/DFNA) is a highly genetically heterogeneous disorder. Hitherto only about 30 ADNSHL-causing genes have been identified and many unknown genes remain to be discovered. In this research, genome-wide linkage analysis mapped the disease locus to a 4.3 Mb region on chromosome 19q13 in SY-026, a five-generation nonconsanguineous Chinese family affected by late-onset and progressive ADNSHL. This linkage region showed partial overlap with the previously reported DFNA4. Simultaneously, probands were analyzed using exome capture followed by next-generation sequencing. Encouragingly, a heterozygous missense mutation, c.505G>A (p.G169R) in exon 3 of the CEACAM16 gene (carcinoembryonic antigen-related cell adhesion molecule 16), was identified via this combined strategy. Sanger sequencing verified that the mutation co-segregated with hearing loss in the family and that it was not present in 200 unrelated control subjects with matched ancestry. This is the second report in the literature of a family with ADNSHL caused by CEACAM16 mutation. Immunofluorescence staining and western blots also prove CEACAM16 to be a secreted protein. Furthermore, our studies in transfected HEK293T cells show that the secretion efficacy of the mutant CEACAM16 is much lower than that of the wild type, suggesting a deleterious effect of the sequence variant.