Embryonic and Neonatal Mouse Cochleae Are Susceptible to Zika Virus Infection.

Congenital Zika Syndrome (CZS) is caused by vertical transmission of Zika virus (ZIKV) to the gestating human fetus. A subset of CZS microcephalic infants present with reduced otoacoustic emissions; this test screens for hearing loss originating in the cochlea. This observation leads to the question of whether mammalian cochlear tissues are susceptible to infection by ZIKV during development. To address this question using a mouse model, the sensory cochlea was explanted at proliferative, newly post-mitotic or maturing stages. ZIKV was added for the first 24 h and organs cultured for up to 6 days to allow for cell differentiation. Results showed that ZIKV can robustly infect proliferating sensory progenitors, as well as post-mitotic hair cells and supporting cells. Virus neutralization using ZIKV-117 antibody blocked cochlear infection. AXL is a cell surface molecule known to enhance the attachment of flavivirus to host cells. While Axl mRNA is widely expressed in embryonic cochlear tissues susceptible to ZIKV infection, it is selectively downregulated in the post-mitotic sensory organ by E15.5, even though these cells remain infectible. These findings may offer insights into which target cells could potentially contribute to hearing loss resulting from fetal exposure to ZIKV in humans.

A New Pathogenic Variant in POU3F4 Causing Deafness Due to an Incomplete Partition of the Cochlea Paved the Way for Innovative Surgery.

Incomplete partition type III (IP-III) is a relatively rare inner ear malformation that has been associated with a POU3F4 gene mutation. The IP-III anomaly is mainly characterized by incomplete separation of the modiolus of the cochlea from the internal auditory canal. We describe a 71-year-old woman with profound sensorineural hearing loss diagnosed with an IP-III of the cochlea that underwent cochlear implantation. Via targeted sequencing with a non-syndromic gene panel, we identified a heterozygous c.934G > C p. (Ala31Pro) pathogenic variant in the POU3F4 gene that has not been reported previously. IP-III of the cochlea is challenging for cochlear implant surgery for two main reasons: liquor cerebrospinalis gusher and electrode misplacement. Surgically, it may be better to opt for a shorter array because it is less likely for misplacement with the electrode in a false route. Secondly, the surgeon has to consider the insertion angles of cochlear access very strictly to avoid misplacement along the inner ear canal. Genetic results in well describes genotype-phenotype correlations are a strong clinical tool and as in this case guided surgical planning and robotic execution.

Long-range cis-regulatory elements controlling GDF6 expression are essential for ear development.

Molecular mechanisms governing the development of the mammalian cochlea, the hearing organ, remain largely unknown. Through genome sequencing in 3 subjects from 2 families with nonsyndromic cochlear aplasia, we identified homozygous 221-kb and 338-kb deletions in a noncoding region on chromosome 8 with an approximately 200-kb overlapping section. Genomic location of the overlapping deleted region started from approximately 350 kb downstream of GDF6, which codes for growth and differentiation factor 6. Otic lineage cells differentiated from induced pluripotent stem cells derived from an affected individual showed reduced expression of GDF6 compared with control cells. Knockout of Gdf6 in a mouse model resulted in cochlear aplasia, closely resembling the human phenotype. We conclude that GDF6 plays a necessary role in early cochlear development controlled by cis-regulatory elements located within an approximately 500-kb region of the genome in humans and that its disruption leads to deafness due to cochlear aplasia.

CDK5RAP2 primary microcephaly is associated with hypothalamic, retinal and cochlear developmental defects.

BACKGROUND: Primary hereditary microcephaly (MCPH) comprises a large group of autosomal recessive disorders mainly affecting cortical development and resulting in a congenital impairment of brain growth. Despite the identification of >25 causal genes so far, it remains a challenge to distinguish between different MCPH forms at the clinical level. METHODS: 7 patients with newly identified mutations in CDK5RAP2 (MCPH3) were investigated by performing prospective, extensive and systematic clinical, MRI, psychomotor, neurosensory and cognitive examinations under similar conditions. RESULTS: All patients displayed neurosensory defects in addition to microcephaly. Small cochlea with incomplete partition type II was found in all cases and was associated with progressive deafness in 4 of them. Furthermore, the CDK5RAP2 protein was specifically identified in the developing cochlea from human fetal tissues. Microphthalmia was also present in all patients along with retinal pigmentation changes and lipofuscin deposits. Finally, hypothalamic anomalies consisting of interhypothalamic adhesions, a congenital midline defect usually associated with holoprosencephaly, was detected in 5 cases. CONCLUSION: This is the first report indicating that CDK5RAP2 not only governs brain size but also plays a role in ocular and cochlear development and is necessary for hypothalamic nuclear separation at the midline. Our data indicate that CDK5RAP2 should be considered as a potential gene associated with deafness and forme fruste of holoprosencephaly. These children should be given neurosensory follow-up to prevent additional comorbidities and allow them reaching their full educational potential. TRIAL REGISTRATION NUMBER: NCT01565005.

Identification of MYO6 copy number variation associated with cochlear aplasia by targeted sequencing.

Copy number variation is an extensively studied cause of hereditary diseases. However, its role in hereditary sensorineural deafness has been rarely reported. Using targeted sequencing, SNP array and qPCR, we found a novel 622.2 kb duplication of 6q14.1 in a patient with congenital sensorineural hearing loss and cochlear aplasia. The duplication included MYO6 and IMPG1 genes. FISH study confirmed that this duplication was inherited from the patient's mosaic mother.

Degeneration of saccular hair cells caused by MITF gene mutation.

BACKGROUND: Waardenburg syndrome (WS) is the consequence of an inherited autosomal dominant mutation which causes the early degeneration of intermediate cells of cochlear stria vascularis (SV) and profound hearing loss. Patients with WS may also experience primary vestibular symptoms. Most of the current WS studies did not discuss the relationship between WS and abnormal vestibular function. Our study found that a spontaneous mutant pig showed profound hearing loss and depigmentation. MITF-M, a common gene mutation causes type WS which affect the development of the intermediate cell of SV, was then identified for animal modeling. RESULTS: In this study, the degeneration of vestibular hair cells was found in pigs with MITF-M. The morphology of hair cells in vestibular organs of pigs was examined using electron microscopy from embryonic day E70 to postnatal two weeks. Significant hair cell loss in the mutant saccule was found in this study through E95 to P14. Conversely, there was no hair cell loss in either utricle or semi-circular canals. CONCLUSIONS: Our study suggested that MITF-M gene mutation only affects hair cells of the saccule, but has no effect on other vestibular organs. The study also indicated that the survival of cochlear and saccular hair cells was dependent on the potassium release from the cochlear SV, but hair cells of the utricle and semi-circular canals were independent on SV.

Effects of cochlear synaptopathy on middle-ear muscle reflexes in unanesthetized mice.

Cochlear synaptopathy, i.e. the loss of auditory-nerve connections with cochlear hair cells, is seen in aging, noise damage, and other types of acquired sensorineural hearing loss. Because the subset of auditory-nerve fibers with high thresholds and low spontaneous rates (SRs) is disproportionately affected, audiometric thresholds are relatively insensitive to this primary neural degeneration. Although suprathreshold amplitudes of wave I of the auditory brainstem response (ABR) are attenuated in synaptopathic mice, there is not yet a robust diagnostic in humans. The middle-ear muscle reflex (MEMR) might be a sensitive metric (Valero et al., 2016), because low-SR fibers may be important drivers of the MEMR (Liberman and Kiang, 1984; Kobler et al., 1992). Here, to test the hypothesis that narrowband reflex elicitors can identify synaptopathic cochlear regions, we measured reflex growth functions in unanesthetized mice with varying degrees of noise-induced synaptopathy and in unexposed controls. To separate effects of the MEMR from those of the medial olivocochlear reflex, the other sound-evoked cochlear feedback loop, we used a mutant mouse strain with deletion of the acetylcholine receptor required for olivocochlear function. We demonstrate that the MEMR is normal when activated from non-synaptopathic cochlear regions, is greatly weakened in synaptopathic regions, and is a more sensitive indicator of moderate synaptopathy than the suprathreshold amplitude of ABR wave I.

Single nucleotide polymorphisms in DNA glycosylases: From function to disease.

Oxidative stress is associated with a growing number of diseases that span from cancer to neurodegeneration. Most oxidatively induced DNA base lesions are repaired by the base excision repair (BER) pathway which involves the action of various DNA glycosylases. There are numerous genome wide studies attempting to associate single-nucleotide polymorphisms (SNPs) with predispositions to various types of disease; often, these common variants do not have significant alterations in their biochemical function and do not exhibit a convincing phenotype. Nevertheless several lines of evidence indicate that SNPs in DNA repair genes may modulate DNA repair capacity and contribute to risk of disease. This overview provides a convincing picture that SNPs of DNA glycosylases that remove oxidatively generated DNA lesions are susceptibility factors for a wide disease spectrum that includes besides cancer (particularly lung, breast and gastrointestinal tract), cochlear/ocular disorders, myocardial infarction and neurodegenerative disorders which can be all grouped under the umbrella of oxidative stress-related pathologies.

Mouse models for pendrin-associated loss of cochlear and vestibular function.

The human gene SLC26A4 and the mouse ortholog Slc26a4 code for the protein pendrin, which is an anion exchanger expressed in apical membranes of selected epithelia. In the inner ear, pendrin is expressed in the cochlea, the vestibular labyrinth and the endolymphatic sac. Loss-of-function and hypo-functional mutations cause an enlargement of the vestibular aqueduct (EVA) and sensorineural hearing loss. The relatively high prevalence of SLC26A4 mutations provides a strong imperative to develop rational interventions that delay, ameliorate or prevent pendrin-associated loss of cochlear and vestibular function. This review summarizes recent studies in mouse models that have been developed to delineate the role of pendrin in the physiology of hearing and balance and that have brought forward the concept that a temporally and spatially limited therapy may be sufficient to secure a life-time of normal hearing in children bearing mutations of SLC26A4.

Variation in genes related to cochlear biology is strongly associated with adult-onset deafness in border collies.

Domestic dogs can suffer from hearing losses that can have profound impacts on working ability and quality of life. We have identified a type of adult-onset hearing loss in Border Collies that appears to have a genetic cause, with an earlier age of onset (3-5 years) than typically expected for aging dogs (8-10 years). Studying this complex trait within pure breeds of dog may greatly increase our ability to identify genomic regions associated with risk of hearing impairment in dogs and in humans. We performed a genome-wide association study (GWAS) to detect loci underlying adult-onset deafness in a sample of 20 affected and 28 control Border Collies. We identified a region on canine chromosome 6 that demonstrates extended support for association surrounding SNP Chr6.25819273 (p-value = 1.09 × 10(-13)). To further localize disease-associated variants, targeted next-generation sequencing (NGS) of one affected and two unaffected dogs was performed. Through additional validation based on targeted genotyping of additional cases (n = 23 total) and controls (n = 101 total) and an independent replication cohort of 16 cases and 265 controls, we identified variants in USP31 that were strongly associated with adult-onset deafness in Border Collies, suggesting the involvement of the NF-κB pathway. We found additional support for involvement of RBBP6, which is critical for cochlear development. These findings highlight the utility of GWAS-guided fine-mapping of genetic loci using targeted NGS to study hereditary disorders of the domestic dog that may be analogous to human disorders.

Cochlear gene therapy.

PURPOSE OF REVIEW: This review highlights recent advances in cochlear gene therapy over the past several years. Cochlear gene therapy has undergone tremendous advances over the past decade. Beginning with some groundbreaking work in 2005 documenting hair cell regeneration using virally mediated delivery of the mouse atonal 1 gene, gene therapy is now being explored as a possible treatment for a variety of causes of hearing loss. RECENT FINDINGS: Recent advances in cochlear gene therapy include improved methods of gene delivery with a better delineation of viral vectors that are suitable for this purpose, additional improvements in hair cell regeneration, and directed research toward autoimmune hearing loss, ototoxicity, spiral ganglion survival, and genetic forms of hearing loss. SUMMARY: If successful, cochlear gene therapy will dramatically alter our ability to treat a variety of forms of acquired and genetic hearing loss.

Cochlear involvement in Familial Mediterranean Fever: a new feature of an old disease.

OBJECTIVES: In this study we first aimed to assess the cochlear functions in children with Familial Mediterranean Fever. The second aim was to investigate the correlation between the hearing levels and some clinical features of Familial Mediterranean Fever including the duration of the disease, age at onset, genetic analysis and colchicine use. METHODS: Thirty-four children with Familial Mediterranean Fever and 27 age matched children were included in the study. Following otologic examination, all children underwent audiometric evaluation, including Pure Tone Average measurements and Distortion Product Otoaoustic Emission testing. Audiological results of the two groups were compared and correlation between the audiologic status and clinical parameters of the disease like the duration of disease, age at onset, mutations and colchicine treatment were studied. RESULTS: Pure tone audiometry hearing levels were within normal levels in both groups. Hearing thresholds of Familial Mediterranean Fever patients were found to be increased at frequencies 8000, 10,000, 12,500 and 16,000 (p<0.05). In otoacoustic emission evaluation, distortion products and signal-noise ratio of FMF children were lower in the tested frequencies, from 1400 Hz to 4000 Hz (p<0.05). Interaction of the disease duration and age of disease onset was found to predict hearing levels, distortion products and signal-noise ratios of children with Familial Mediterranean Fever (F value=2.034; p=0.033). CONCLUSIONS: To our knowledge this is the first study demonstrating cochlear involvement in children with Familial Mediterranean Fever which showed increased hearing thresholds at higher frequencies in audiometry together with decreased distortion products and signal-noise ratios demonstrated by distortion product otoacoustic emission testing. Similar studies must be carried out on adult patients to see if a clinical hearing impairment develops. The possible mechanisms that cause cochlear involvement and the effect of colchicine treatment on cochlear functions must be enlightened.

T-cadherin in the mammalian cochlea.

OBJECTIVES/HYPOTHESIS: Cadherins are a superfamily of transmembrane glycoproteins, which mediate calcium-dependent intercellular adhesions. T-cadherin is an atypical member of the cadherin family in regard to its structure; it acts as a signalling receptor rather than an adhesion molecule. In this study we examine the role of T-cadherin in the mammalian cochlea. STUDY DESIGN: This study investigated the expression of T-cadherin in the inner ear under physiologic and pathologic conditions. METHODS: Expression of T-cadherin in the rat cochlea was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, and immunohistochemistry. RESULTS: We detected T-cadherin mRNA expression in three different components in the cochlea of postnatal mouse, namely the organ of Corti (OC), the spiral ganglion (SG), and the stria vascularis (SV). The SG and SV showed a higher T-cadherin mRNA level than the OC. T-cadherin protein was detected by Western blotting in the OC, SG, and SV. Immunofluorescence microscopy of adult mouse cochlea revealed the presence of T-cadherin in the apical parts of the inner and outer hair cells as well as in the SV and SG. OCs treated with gentamicin for 3, 6, or 12 hours did not show any change in T-cadherin gene expression compared to control explants maintained in culture medium alone. CONCLUSIONS: T-cadherin is expressed within the cochlea. T-cadherin seems to have a wide variety of functions in the inner ear, ranging from mechanical functions to functions in response to hair cell damage and loss.

Selective cochlear degeneration in mice lacking the F-box protein, Fbx2, a glycoprotein-specific ubiquitin ligase subunit.

Little is known about the role of protein quality control in the inner ear. We now report selective cochlear degeneration in mice deficient in Fbx2, a ubiquitin ligase F-box protein with specificity for high-mannose glycoproteins (Yoshida et al., 2002). Originally described as a brain-enriched protein (Erhardt et al., 1998), Fbx2 is also highly expressed in the organ of Corti, in which it has been called organ of Corti protein 1 (Thalmann et al., 1997). Mice with targeted deletion of Fbxo2 develop age-related hearing loss beginning at 2 months. Cellular degeneration begins in the epithelial support cells of the organ of Corti and is accompanied by changes in cellular membrane integrity and early increases in connexin 26, a cochlear gap junction protein previously shown to interact with Fbx2 (Henzl et al., 2004). Progressive degeneration includes hair cells and the spiral ganglion, but the brain itself is spared despite widespread CNS expression of Fbx2. Cochlear Fbx2 binds Skp1, the common binding partner for F-box proteins, and is an unusually abundant inner ear protein. Whereas cochlear Skp1 levels fall in parallel with the loss of Fbx2, other components of the canonical SCF (Skp1, Cullin1, F-box, Rbx1) ubiquitin ligase complex remain unchanged and show little if any complex formation with Fbx2/Skp1, suggesting that cochlear Fbx2 and Skp1 form a novel, heterodimeric complex. Our findings demonstrate that components of protein quality control are essential for inner ear homeostasis and implicate Fbx2 and Skp1 as potential genetic modifiers in age-related hearing loss.

Vertical corneal striae in families with autosomal dominant hearing loss: DFNA9/COCH.

PURPOSE: Investigation of a possible association between vertical corneal striae and mutations in the COCH gene, observed in four DFNA9 families with autosomal dominant hearing loss and vestibular dysfunction. DESIGN: Prospective case series. METHODS: Ophthalmologic examinations with photography of the cornea after instillation of fluorescein were performed in 98 family members with 61 mutation carriers of four DFNA9 families at the Radboud University Nijmegen Medical Centre. Families 1 and 2 harbor the Pro51Ser mutation, and families 3 and 4 harbor the Gly88Glu and the Gly87Trp mutation, respectively. Statistical analysis was performed to find an association between the vertical corneal striae and the COCH mutation for each family and to test whether the four families were different in this respect. RESULTS: The vertical corneal striae were exclusively visible after instillation of fluorescein. They caused minor problems, as dry eye symptoms, and were not present in the general Dutch ophthalmologic population. The striae were present from an age of 47 years in 32 individuals, of whom 27 individuals had a COCH mutation. Statistical analysis on the striae and the COCH mutations showed a significant association in families 1, 2, and 3 (P = .0006), but not in family 4 (P = .63). CONCLUSIONS: Data analysis demonstrated a significant association between vertical corneal striae and the Pro51Ser and Gly88Glu mutations in the COCH gene in DFNA9 families 1, 2, and 3 with cochleovestibular dysfunction. Our findings suggest that the vertical corneal striae and cochleovestibular dysfunction may be caused by the same COCH mutations.

How to prevent a stapes gusher.

A stapes gusher is the result of a congenital inner ear anomaly showing at tone audiometry a conductive or mixed hearing loss. The conductive part of the hearing loss could lead to the thought to explore the middle ear. The congenital origin should lead to a high resolution. CT-scanning to evaluate a widening of the internal acoustic canal. Repeated audiometry could show especially a large conductive impairment in the lowest frequencies with a closure of the airbone gap at 2 khz and a high sensorineural high frequency loss at 4 and 8 khz. Contralateral stapedial reflexes may be present. Since the x-recessive mixed deafness syndrome (DFN3) frequently involves males with an early childhood hearing impairment, clinical suspicion should be high. When stapes surgery is considered a precise medical history is essential regarding on the start of the hearing impairment. A continuous suspicion will guide to the audiological, radiological and molecular genetic clues to trace the correct diagnosis before embarking on stapes surgery.

Defective carbohydrate metabolism in mice homozygous for the tubby mutation.

Tub is a member of a small gene family, the tubby-like proteins (TULPs), with predominant expression in neurons. Mice carrying a mutation in Tub develop retinal and cochlear degeneration as well as late-onset obesity with insulin resistance. During behavioral and metabolic testing, we found that homozygous C57BL/6J-Tub(tub) mice have a lower respiratory quotient than C57BL/6J controls before the onset of obesity, indicating that tubby homozygotes fail to activate carbohydrate metabolism and instead rely on fat metabolism for energy needs. In concordance with this, tubby mice show higher excretion of ketone bodies and accumulation of glycogen in the liver. Quantitation of liver mRNA levels shows that, during the transition from light to dark period, tubby mice fail to induce glucose-6-phosphate dehydrogenase (G6pdh), the rate-limiting enzyme in the pentose phosphate pathway that normally supplies NADPH for de novo fatty acid synthesis and glutathione reduction. Reduced G6PDH protein levels and enzymatic activity in tubby mice lead accordingly to lower levels of NADPH and reduced glutathione (GSH), respectively. mRNA levels for the lipolytic enzymes acetyl-CoA synthetase and carnitine palmitoyltransferase are increased during the dark cycle and decreased during the light period, and several citric acid cycle genes are dysregulated in tubby mice. Examination of hypothalamic gene expression showed high levels of preproorexin mRNA leading to accumulation of orexin peptide in the lateral hypothalamus. We hypothesize that abnormal hypothalamic orexin expression leads to changes in liver carbohydrate metabolism and may contribute to the moderate obesity observed in tubby mice.

p27(Kip1) deficiency causes organ of Corti pathology and hearing loss.

p27(Kip1) (p27) has been shown to inhibit several cyclin-dependent kinase molecules and to play a central role in regulating entry into the cell cycle. Once hair cells in the cochlea are formed, p27 is expressed in non-sensory cells of the organ of Corti and prevents their re-entry into the cell cycle. In one line of p27 deficient mice (p27(-/-)), cell division in the organ of Corti continues past its normal embryonic time, leading to continual production of cells in the organ of Corti. Here we report on the structure and function of the inner ear in another line of p27 deficient mice originating from the Memorial Sloan-Kettering Cancer Center. The deficiency in p27 expression of these mice is incomplete, as they retain expression of amino acids 52-197. We determined that mice homozygote for this mutation had severe hearing loss and their organ of Corti exhibited an increase in the number of inner and outer hair cells. There also was a marked increase in the number of supporting cells, with severe pathologies in pillar cells. These data show similarities between this p27(Kip1) mutation and another, previously reported null allele of this gene, and suggest that reducing the inhibition on the cell cycle in the organ of Corti leads to pathology and dysfunction. Manipulations to regulate the time and place of p27 inhibition will be necessary for inducing functionally useful hair cell regeneration.

Vestibular deterioration precedes hearing deterioration in the P51S COCH mutation (DFNA9): an analysis in 74 mutation carriers.

OBJECTIVES: To analyze cochleovestibular impairment features in P51S COCH mutation carriers (n = 22) in a new, large Dutch family and to compare the results to those obtained in previously identified similar mutation carriers (n = 52). To evaluate age-related features between progressive hearing and vestibular impairment of all mutation carriers (n = 74). STUDY DESIGN: Family study. METHODS: Regression analysis was performed in relation to age to outline the development of hearing thresholds, speech recognition scores, and vestibulo-ocular reflex time constant as the key vestibular response parameter. RESULTS: Pure tone thresholds, phoneme recognition scores, and vestibular responses of the mutation carriers in the new family were essentially similar to those previously established in all other mutation carriers. Hearing started to deteriorate in all mutation carriers from 43 years of age onwards, whereas deterioration of vestibular function started from age 34. CONCLUSION: Vestibular impairment starts earlier, progresses more rapidly, and, eventually, is more complete than hearing impairment in P51S COCH mutation carriers.