KCNQ4 is frequently mutated in autosomal dominant non-syndromic hearing loss (NSHL), a typically late-onset, initially high-frequency loss that progresses over time (DFNA2). Most KCNQ4 mutations linked to hearing loss are clustered around the pore region of the protein and lead to loss of KCNQ4-mediated potassium currents. To understand the contribution of KCNQ4 variants to NSHL, we surveyed public databases and found 17 loss-of-function and six missense KCNQ4 variants affecting amino acids around the pore region. The missense variants have not been reported as pathogenic and are present at a low frequency  in the population. We examined the functional impact of these variants, which, interestingly, induced a reduction in potassium channel activity without altering expression or trafficking of the channel protein, being functionally similar to DFNA2-associated KCNQ4 mutations. Therefore, these variants may be risk factors for late-onset hearing loss, and individuals harboring any one of these variants may develop hearing loss during adulthood. Reduced channel activity could be rescued by KCNQ activators, suggesting the possibility of medical intervention. These findings indicate that KCNQ4 variants may contribute more to late-onset NSHL than expected, and therefore, genetic screening for this gene is important for the prevention and treatment of NSHL. A gene associated with hereditary hearing loss may play a greater role than previously recognized in age-related auditory impairment. Many cases of autosomal dominant non-syndromic hearing loss (ADNSHL) arise from defects in KCNQ4, a protein that maintains the cellular ionic conditions needed for normal inner ear function. Researchers led by Heon Yung Gee and Jae Young Choi at Yonsei University College of Medicine, Seoul, South Korea, have now uncovered numerous previously overlooked mutations in the gene encoding KCNQ4 that may also contribute to adult-onset hearing loss. Their survey of human genome databases revealed 23 additional sequence variants that can meaningfully impair function of this protein. The effects of some of these mutations can be at least partially corrected with existing chemical compounds, indicating the potential to protect a subset of ADNSHL patients from future deafness. The KCNQ4 gene was first cloned and characterized by Kubisch et al.3. They mapped it to the DFNA2  locus, and a dominant-negative mutation was identified in the DFNA2 pedigree. DFNA2 is characterized as a late-onset disease, with high-frequency hearing loss that progresses to all frequencies over time4.KCNQ4 (KV7.4), a voltage-gated potassium channel, plays a critical role in the auditory function of the inner ear by regulating KKCNQ4 is one of the most commonly mutated genes in ADNSHL5. Approximately 30 pathologic mutations in KCNQ4 have been identified as the cause of DFNA2 (www.deafnessvariationdatabase.org or www.hgmd.cf.ac.uk/ac/index.php). The KCNQ4 channel consists of six transmembrane domains, a pore region, and two intracellular termini6. The mutation hotspots in KCNQ4 associated with DFNA2 are clustered around the pore region7. The first described missense mutation (p.G285S)3 as well as p.L274S, p.W276S, p.L281S, p.G285C, and p.G296S, which correspond to changes around the pore region of KCNQ4, induced a loss of channel function or decreased membrane expression of the channel protein15.16. However, the contribution of genetic predisposition to ARHL is not clear and, therefore, often underestimated. Several single nucleotide polymorphisms in KCNQ4 are significantly associated with ARHL17, and it is plausible that the hypomorphic mutations in KCNQ4 might contribute to ARHL18. In addition, KCNQ4 variants are also implicated in noise-induced hearing loss19.Age-related hearing loss (ARHL) is the most common sensory deficit among the elderly and progresses slowly, similar to DFNA2. ARHL is a complex disease resulting from an interplay between genetic and environmental factorsKCNQ4 variants in public databases and found several missense variants around the pore region of KCNQ4 that are present at a low frequency in the general population and have not been associated with DFNA2. As the pathogenicity or clinical significance of these variants is unknown, we investigated the effects of these variants on KCNQ4 function and found that they significantly decreased K+ channel activity. These findings suggest that these KCNQ4 variants may contribute to NSHL or ARHL.In this study, we examined KCNQ4 variants were examined in several databases: HGMD Professional (http://www.hgmd.cf.ac.uk/ac/index.php), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), the Deafness Variation Database (http://www.deafnessvariationdatabase.org/), and the Genome Aggregation Database (gnomAD) (http://gnomad.broadinstitute.org/). A whole-genome sequencing dataset of 397 Korean individuals from the National Biobank of Korea of the Centers for Disease Control and Prevention was also used.KCNQ4 were purchased from OriGene Technologies  and subcloned into the pENTR-D-TOPO vector . Expression vectors were constructed using LR clonase (Invitrogen) following the manufacturer’s instructions, and a Myg- or FLAG-tag was inserted in the N-terminus. KCNQ4 variant clones were generated by PCR-based site-directed mutagenesis using the Quick Change II XL Site-Directed Mutagenesis Kit .Complementary DNAs (cDNAs) of human KCNQ4 plasmids using Lipofectamine and PLUS reagent, or Turbofect , according to the manufacturer’s instructions. For electrophysiological experiments, CHO-K1 cells were cotransfected with 0.9 μg of the human KCNQ4 plasmids with 0.1 μg of a green fluorescent protein (GFP) gene-containing expression plasmid to visualize the transfected cells. Experiments were performed within 24–36 h after transfection.Human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells were cultured in Dulbecco’s modified essential medium and RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum and penicillin (50 IU/mL)/streptomycin (50 μg/mL) (Invitrogen). Cells were transfected with wild-type (WT) or mutant 20. Anti-BiP (ab21685), anti-β-actin , anti-Myc (sc-40), anti-aldolase A1 , anti-GOLGB1 (HPA011008), and anti-FLAG  antibodies were purchased from commercial sources. Coimmunoprecipitation was performed using EZview Red Anti-FLAG M2 and anti-c-Myc Affinity Gel (Sigma). Surface biotinylation was performed using 0.3 mg/mL EZ-Link Sulfo-NHS-SS-Biotin and NeutrAvidin (Thermo Fisher Scientific). Immunoblotting was performed using primary antibodies at a 1:1000 dilution, followed by corresponding anti-isotype secondary antibodies (Santa Cruz Biotechnology) at a 1:2000 dilution. Signals were visualized using the SuperSignal West-Pico Kit (Thermo Fisher Scientific). For immunofluorescence, blocking buffer containing 10% donkey serum and 1% bovine serum albumin in phosphate buffered saline was used, and dilutions of primary and fluorophore-tagged secondary antibodies were 1:100 and 1:2000, respectively. Confocal images were obtained with a Carl Zeiss LSM780 instrument; ZEN software was used for image processing.Experiments were performed as described previouslyKCNQ4-expressing CHO-K1 cells. Cells were transferred to a bath mounted on the stage of an IX-71 inverted microscope  equipped with a light source set to green fluorescence excitation wavelengths. Patch clamp experiments were performed at room temperature (23 °C–25 °C). Microglass pipettes  were fabricated using a PC-10 dual-stage glass micropipette puller  and an MF-830 microforge with a resistance of 2.0–2.5 MΩ . Currents were recorded using an Axopatch 200B amplifier and Digidata 1440 A interface, digitized at 10 kHz and low pass-filtered at 5 kHz by pClamp software v. 10.3 . The series resistance was compensated by an offset circuit in Axopatch 200B. All voltage and current trace data were analyzed using Clampfit v. 10.3 and Origin v. 8.0 software . The step pulse protocol used for KCNQ4 channel recordings started from a holding potential of −80 mV, depolarized for 2 s from −80 mV to 20 mV with 20 mV increments, and followed by a tail pulse at −50 mV for 0.5 s. Whole-cell patch clamp analysis was conducted using a bath solution containing 147 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose (adjusted to pH 7.4 with NaOH). The pipette solution contained 130 mM KCl, 10 mM HEPES, 1 mM CaCl2, 10 mM EGTA, and 3 mM Mg-ATP (adjusted to pH 7.2 with KOH).Whole-cell patch clamp techniques were used for measuring channel activity in KCNQ4 variants or empty plasmids. After 48 h, the medium was replaced with 80 μL/well of FluxOR (Invitrogen) loading buffer and incubated for 1 h at 37 °C in the dark. The loading buffer was removed, and 100 µL of assay buffer was added to each well. To activate the KCNQ4 channels, cells were pretreated with 10 μM retigabine for 10 min. FluxOR fluorescence (excitation/emission: 490/525 nm) was recorded 10 s prior to the addition of 20 µL of the stimulus buffer containing a low level of thallium ions, and the fluorescence was monitored for an additional 15 s. FluxOR fluorescence was recorded using a Zyla sCMOS camera (Andor Technology) and a Nikon Eclipse Ti inverted microscope (Nikon Instruments) and analyzed using Metamorph analysis software (Molecular Device). All buffers were prepared according to the manufacturer’s instructions.CHO-K1 cells were transfected in 96-well plates with https://swissmodel.expasy.org/). Amino acid residues surrounding the pore region (residues 270–345) of KCNQ1 of Xenopus laevis (PDB 5vms), which shares 70% sequence identity with human KCNQ4 (resides 256–326), was used as the template structure. The tertiary structure of the domains was generated, and the 3D structures of the normal and mutated proteins were visualized using the UCSF Chimera package (http://www.cgl.ucsf.edu/chimera). Protein stability was predicted using Site-Directed Mutator (http://marid.bioc.cam.ac.uk/sdm2/), I-Mutant Suite (http://gpcr2.biocomp.unibo.it/cgi/predictors/I-Mutant3.0/I-Mutant3.0.cgi), and CUPSAT (http://cupsat.tu-bs.de/).To examine the effects of the variants at the protein level, structural modeling of KCNQ4 was performed using Swiss-Model  that span large genomic regions affecting multiple genes, including KCNQ4. Of the 70 single nucleotide variants, five are pathogenic or likely pathogenic, 35 are benign or likely benign, and 30 are of uncertain significance. Of the 12 CNVs in ClinVar, nine are pathogenic, and three are of uncertain significance. Nine pathogenic CNVs, including KCNQ4, are mostly linked to severe phenotypes, such as global developmental delay, intellectual disability, and cardiac defects. One CNV, a duplication of chr1:849,467-248,224,649 (hg19), is associated with ear anomaly, short stature, cardiovascular anomaly, and polydactyly.In ClinVar, 106 KCNQ4 variants in gnomAD, of which 297 are missense or loss-of-function (LoF) variants. Of the 19 LoF KCNQ4 variants, 15 are nonsense or frameshift, and four affect an obligatory splice site  5 and 6 (aa 259–296) surrounding the pore region (aa 271–292), as most known KCNQ4 variants linked to hearing loss are clustered around this region7. There are six variants in this region of KCNQ4 in gnomAD, and none of them has been reported in either HGMD Professional or ClinVar except p.T278A, which has been reported in ClinVar as of unknown significance  detected in the Korean dataset.We first performed immunofluorescence and surface biotinylation assays to examine the expression levels of the WT and variant KCNQ4 proteins at the plasma membrane in HEK 293 cells. Using immunofluorescence with endoplasmic reticulum and Golgi markers, the WT and all variant KCNQ4 proteins were detected at the cell surface Fig. . Surface10, we examined whether variant KCNQ4 proteins affected heteromeric assembly with the WT protein. Coimmunoprecipitation demonstrated that variant proteins were able to interact with the WT protein, indicating that heterotetramers containing the WT and variant proteins can form , the MAF of which is 0.2584, was also included in the functional study. All variants showed reduced potassium channel activity  to mimic the heterozygous condition, and the results show that all six variants exerted dominant-negative inhibitory effects on WT KCNQ4-mediated currents  openerold Fig. . We examold Fig. . We alsoine Fig. , indicatrug Fig. .Fig. 4EfXenopus KCNQ125. The p.W276S variant was predicted to decrease the stability of KCNQ4 by three prediction programs 10. Although slowly progressive hearing loss is not fully attributable to the degeneration of outer hair cells in the cochlea, the main pathomechanism is the impaired potassium recycling in the endolymph, which consequently changes the nature of its electrolyte milieu and debilitates the endocochlear potential27. In mice, the mechanism of hearing loss is not haploinsufficiency but a dominant-negative effect of the mutant KCNQ4 because heterozygous knockout mice do not show any hearing loss27. Therefore, most mutations identified in humans are thought to have a dominant-negative effect.Pathogenic KCNQ4 gene, which is responsible for the ion selectivity of the channel28. KCNQ4 mediates an M-like potassium current in the outer hair cells and type I vestibular hair cells29. The M-like potassium conductance in these cells is dominant-negatively abolished when the pore mutant p.G285S was transfected into the cells29. These findings indicate that the main pathomechanism of DFNA2 resulting from mutations in amino acid residues around the pore region of KCNQ4 is the dominant-negative loss of potassium current in the outer hair cells. Therefore, people with KCNQ4 variants with impaired potassium current are strong candidates for progressive hearing loss linked to DFNA2.The majority of variants linked to DFNA2 are located around the pore region of the protein encoded by the KCNQ4 from public databases, including gnomAD, and found six missense variants that are present in the general population at low frequency , we can calculate the frequencies of individuals who carry these variants in a homozygous or heterozygous state (2pq+q2). Frequencies of heterozygous or homozygous individuals are estimated to be 0.0000244, 0.0000081, 0.0000569, 0.0000081, 0.0000081, and 0.0000081 for p.N264S, p.S269F, p.S273A, p.T278A, p.L281M, and p.L295P, respectively. Their sum is 0.0001137, which is ~5.69% of the prevalence of hearing loss (1/500 = 0.002). Therefore, KCNQ4 variants may contribute more than expected to hearing loss.Based on MAFs (q) of six missense variants around the pore region of KCNQ4 reported in gnomAD, if we assume that these variants are in Hardy-Weinberg equilibrium (phttp://hereditaryhearingloss.org/). NSHL is very heterogeneously inherited. In particular, autosomal dominant hearing loss is mainly related to late-onset and progressive hearing loss; therefore, it is unclear whether young children or adults are affected by hearing loss. Those with mild or moderate hearing loss are likely considered healthy because high-frequency hearing loss is commonly undetected. Thus, it is plausible that sequencing data of people who have mild-to-moderate hearing loss or who are in a subclinical stage of progressive hearing loss are present in public databases. KCNQ4 variants attributable to ARHL or NIHL but not yet reported as pathogenic may also be included in public databases.More than one hundred genes causing NSHL have been described , such as epilepsy and tinnitus32. There have been many trials to develop therapies for these diseases in which KCNQ channels are activated to obtain neuronal hyperexcitability. In fact, retigabine, a small molecule that activates KCNQ2-5 channels, was approved as an anti-epileptic drug by the US Food and Drug Administration33. Retigabine exerts its therapeutic effect by enhancing KCNQ channel activity through the shift of voltage-dependent activation to more negative voltages, resulting in a reduction in the excessive firing of neuronal cells34.KCNQ channels, particularly heteromeric KCNQ2/3, play a critical role in maintaining neuronal excitability by producing outward K35. In our studies, retigabine rescued the voltage-activated currents mediated by KCNQ4 pore mutants, suggesting that retigabine or other KCNQ activators may be useful in the therapeutics of genetic hearing loss. There is an unmet medical need for the treatment of inherited hearing loss caused by mutations in KCNQ4. However, more in vitro studies and clinical trials are needed to determine the therapeutic effects of KCNQ activators on hearing loss.KCNQ4 channels expressed in the basolateral membrane of outer hair cells play a major role in maintaining the resting membrane potential. Therefore, impaired KCNQ4 channel activity caused by dysfunctional KCNQ4 mutants induces degeneration of outer hair cells by prolonged depolarization of the membrane potential, which further leads to progressive hearing loss36. Similarly, our results showed that the almost null potassium activity for the two mutants was not rescued by retigabine. However, it is noteworthy that four of the six pore variants of KCNQ4 with residual voltage-activated currents were activated by retigabine to almost WT levels. Retigabine increases channel function by stabilizing the open K+ conducting form through binding to a single conserved tryptophan residue in the S5 domain (Supplementary Fig. 38. Our results show that retigabine did not enhance channel activity of variant KCNQ4 proteins with completely impaired channel function, whereas residual currents mediated by other variant KCNQ4 proteins were rescuable. Given that KCNQ activators increase KCNQ4 activity through alteration of channel conductance, it is still unclear how pore mutants are responsive to retigabine39. It is possible that retigabine induces a conformational change in the pore region. Further biophysical analyses are needed to clarify this issue.Pore variants of KCNQ4 are known to be unresponsive to retigabine or zinc pyrithione, which is another KCNQ activatorKCNQ4 variant is present, which are potential therapeutic targets of retigabine if only the WT assembly was responsive to the drug. However, more potential retigabine targets would be available if the drug could also rescue the channel activity of pore mutant KCNQ4 as well as WT proteins.Because KCNQ4 assembles into a homotetramer, 6.25% of tetramers are formed of WT subunits only when a heterozygous 40. To avoid these adverse effects, retigabine could be locally administered in the inner ear. Overall, the clinical value of this drug will be scientifically evaluated after further biophysical analysis is performed, and distinct in vivo study results are available.In this context, we found that it may be possible to rescue channel activity by nonselective KCNQ activators, thereby preventing hearing deterioration in patients with DFNA2. However, retigabine has many systemic side effects, such as drowsiness, vertigo, and slurred speech, and thus has not been successful in the marketKCNQ4 is highly important. Furthermore, we identified several KCNQ4 pore variants potentially treatable with a small molecule, which would enable us to realize precision medicine in genetic hearing loss.In conclusion, we found that there are many unreported variants around the pore regions of KCNQ4, possibly associated with DFNA2, in public databases. Given that DFNA2 is one of the most common causes of ADNSHL, genetic testing of Supplementary Information.


Diving within artisanal fishing is a profession carried out by many men in coastal communities of southern Chile. These shellfish divers use surface supplied air for breathing. Among potential health threats are occupational accidents, decompression sickness and barotrauma. Repeated middle and inner ear barotrauma and decompression sickness of the ear may result in hearing loss.To determine the prevalence of hearing loss and related risk factors in artisanal shellfish divers.A cross–sectional study including 125 male shellfish divers was carried out in a coastal village in southern Chile. Participants were interviewed using a standard Spanish questionnaire adapted for this population. Hearing loss was assessed through audiometry. Any hearing loss, sensorineural hearing loss and other types of hearing loss  were used as the outcomes. Bivariate and multiple logistic regression models were carried out to identify risk factors for hearing loss.Median duration on the job was 25 years (range 1–52), 64% of divers had a low level of schooling and 52% reported not knowing how to use decompression tables. Most (86%) of the divers dove deeper than 30 meters exceeding the 20 meters permitted by law. The majority (80%) reported having experienced several episodes of type II decompression sickness during their working life. The prevalence of any type of hearing loss was 54.4%: 29.0% presented sensorineural hearing loss and 25.6% presented other types of hearing impairment. After adjustment for age and other potential risk factors, diving more than 25 years was the main predictor for all kinds of hearing loss under study.Hearing loss is frequent in artisanal shellfish divers and safety measures are limited. Although based on small numbers and lacking an unexposed comparison group, our results suggest the need for community-based interventions. Fishing is one of the most important economic activities of men living in villages in the southern Chilean coast. Shellfish divers extract seafood using compressed air as respiratory support. In contrast to scuba divers, shellfish divers use surface supplied air, also called Hookah technique. With this technique, compressed air is produced on the boat and piped to an air hose through which the diver inhales. The technique allows for longer time under the surface than Scuba diving. The other advantage of Hookah technique, particularly for Chilean shellfish divers, is the low legal requirements for the use of this technique. At the same time, the technique is only permitted up to a diving depth 20 meters, a depth at which almost no shellfish are available.There are various hazards associated with this professional activity. In addition to fatal occupational accidents, the hyperbaric environment, cold water, sea current, and poor lighting might damage divers’ health 234. Furt23578101110111213Professional divers in Chile might be insured against occupational accidents and diseases. This social insurance offers surveillance programs to protect from noise-induced hearing loss . HoweverTherefore, the aim of this study was to determine the prevalence of risk factors for hearing loss in shellfish divers in a coastal village of southern Chile. Based on this, governmental intervention programs should be established in the future.The cross-sectional study was carried out between November 2012 and January 2013 among male artisanal shellfish divers from a coastal village in southern Chile. Based on estimations by the health authority of the local fishing station, approximately 400 artisanal shellfish divers work in the village; however, due to the precarious working conditions, official registries do not exist. Nevertheless, many of the divers are associated with one of the five fishermen unions in the village. The union leaders of the village were informed about the project, and they provided their membership lists, including 243 shellfish divers. From this list, 75 divers were excluded for three reasons: 1) currently not working as a diver, 2) working as a diver outside the village or 3) death. The remaining 168 divers were visited in their homes or in public places and invited to participate in the study. In order to increase the number of participants, 60 shellfish divers who were not on the lists were invited while the researchers and the fishing station paramedic visited those identified (convenience sample).Prior to participation, divers were informed about the purpose and activities of the project, the voluntary nature of participation and the confidentiality of information. Data collection was done anonymously. The study was approved by the ethical committees of the University Hospital in Munich (LMU) and Instituto de Seguridad del Trabajo in Chile.Overall, 131 of 228 divers agreed to participate (57.5%), of which six were excluded from the hearing evaluation for the following reasons: earwax plug in the ear canal (n = 2), recent middle ear barotrauma (n = 1), perforated eardrum (n = 1), acute respiratory infection (n = 1) and known diagnosis of sudden hearing loss (n = 1). Consequently, 125 shellfish divers were included in the study.The questionnaire and audiometry screening were conducted in the first aid room of the local fishing station. This room was a quiet location for the screening, without traffic or noisy machinery.All divers were interviewed by two of the principal authors  using a questionnaire based on the Spanish version of the European Working Condition and Health Survey . Questio18To evaluate hearing, field screening was performed by a trained technician measuring the frequencies from 500 Hz to 8000 Hz by air conduction. If thresholds were altered, bone conduction was also assessed. The definition of hearing loss was based on the criteria established in the Protocolo de Exposición Ocupacional a Ruido PREXOR of the Chilean Health Ministry , consideFour outcome definitions were used:Any type of hearing loss (yes vs. no): prevalence of any type of altered audiogram as described above.Sensorineural hearing loss (yes vs. no): audiogram suggestive of bilateral sensorineural hearing loss with a simultaneous decrease in the threshold of air and bone conduction. For bivariate and multivariate analysis divers with sensorineural hearing loss also identified with other types of hearing loss (see below) were excluded.Other types of hearing loss (yes vs. no): audiogram altered suggestive of conduction, unilateral and mixed hearing loss, after assessment of bone way and without a curve suggestive of bilateral sensorineural hearing loss. Divers identified with sensorineural hearing loss were excluded from this analysis. In order to assess potential risk factors for hearing loss, the following variables were considered:Age was categorized into ≥40 years vs. <40 years.Level of education was differentiated between those with some form of elementary education and those with higher level of education.Active smoker: Yes, if the diver reported to smoke occasionally or daily.Alcohol consumption: Yes, if the diver reported to drink alcohol occasionally or daily.Alcoholism: Yes, if there were two positive responses on the EBBA scale .Diving years: The duration of work as a shellfish diver was dichotomized using the median (25 diving years) as cut-off.Number of working days per week: only holidays and irregular days, Monday to Friday; Monday to Friday and exceptionally Sunday and/or holidays; Monday to Saturday; Monday to Sunday.Weekly dives: The number of dives per week were divided using 7 dives per week as cut-off.Diving duration: Average duration of the dives was determined by using 1 hour as cut-off.Ability to read decompression tables (yes vs. no).Use of decompression tables (yes vs. no).Deep dives: Dives exceeding depths of 30 meters.Middle ear barotrauma: Yes, if divers reported to have suffered barotrauma of middle ear while diving.Symptoms of type II decompression sickness (yes vs. no): Yes, if unexpected fatigue, headache, numbness/tingling, vertigo, loss of balance, difficulty breathing/choking, muscular weakness/paralysis, loss of vision, loss of consciousness, inability to urinate, or confusion/disorientation/memory loss had occurred after a dive more than once in their working life.2 test) and logistic regression analysis assessing the association between potential predictors and hearing loss.Statistical analysis was carried out using EpiInfo Version 3.5.4 through descriptive analysis, bivariate analysis . Only 34% of shellfish divers had completed elementary education, 28% did not finish elementary education, and two divers had no formal education. The median duration of work as a diver was 25 years (range 1–52 years).No statistically significant differences were found between divers of fishermen unions (random sample) and those forming part of the convenience sample Table . Thirty-The prevalence of any type of hearing loss among shellfish divers was 54.4%. After bilateral sensorineural hearing loss (28.8%), the most common alteration of audiometry was unilateral hearing loss (14.4%). Only a few divers suffered from conduction hearing loss (5.6%) or mixed hearing loss (5.6%).In the bivariate analysis, risk factors for all types of hearing loss were age ≥40 years and having more than 25 diving years. With respect to sensorineural hearing loss, being an active smoker was inversely related. For other types of hearing loss, having a lower level of education and not being able to read decompression tables were risk factors , sensorineural hearing loss (OR 9.2 CI 95% 2.6–32.6) and other types of hearing loss (OR 3.7 CI 95% 1.2–11.7). Age was associated with any type of hearing loss (OR 5.4 95% CI 1.5–19.1) and when stratifying, it was related to other types of hearing loss only (OR 6.1 95% CI 1.1–32.0. Table This study indicates a high prevalence of hearing loss among professional shellfish divers in southern Chile. More than 50% of those with a hearing impairment had an audiometric curve suggestive of sensorineural hearing loss. The main predictor for sensorineural and other types of hearing loss were diving years while age was only associated with other types of hearing impairment.A high prevalence of hearing loss has been described in some other studies among professional divers 222324. I22232113As known from the literature 26 age wa6101127Recurring symptoms of type II decompression sickness were not associated with sensorineural hearing loss. Different symptoms of the disease were combined so that the association still might hold true for decompression sickness of the inner ear. Accordingly, future research should include an assessment focused on the history of inner ear decompression sickness and inner ear barotrauma in order to establish possible associations for cumulative damage.Having a high level of education and the ability to read decompression tables were protective factors for other types of hearing loss in the bivariate analysis. It is plausible that these factors contribute to risk-adverse diving behavior and indicate that noise may not be the main risk factor for hearing loss in the group under evaluation. However, future studies including a larger number of divers are necessary in order to explore this question.Limitations of this study include the absence of an official and updated list of shellfish divers and having a small study group with a response is not a rate as it does not involve time of 55%, which made it difficult to generalize the results to the entire population of shellfish divers. In addition, we could not include an unexposed comparison group. One may question if a selection bias exists: for example, divers who previously experienced a hearing impairment may have been more likely to participate in the study. However, less than half of the divers with altered audiometry reported feeling their hearing impairment, which allows us to assume that no such selection bias was present.Due to poor access to a hearing evaluation center, the only way to assess hearing loss among shellfish divers was through field screening audiometry. These conditions were adequate for an audiometric screening, considering that the purpose of this study was not to calculate the percentage of hearing loss. Other factors not included in this study were possible noise exposure in motorboats and the presence of chronic diseases that can affect the inner ear 122529.1225In conclusion, this study indicates that long-term professional diving is a risk factor for hearing loss in artisanal shellfish divers. Whether this is due to noise exposure or the hyperbaric environment remains a question for future research. Surveillance programs and community-based interventions should be considered to evaluate occupational diseases and risk factors and to determine preventive measures to avoid disability among these workers, the majority of who are not covered by social insurance against accidents and occupational diseases.


We aim to clarify some points that have been criticized about our previous paper “Hearing Impairment in MELAS: new prospective in clinical use of microRNA, a systematic review”.We answered to the criticism of Dr. Finsterer point by point, by citing all literature in support of our previous paper.The point by point answering allows us to clarify doubts and to support the proposal that we exposed in our review, the possibility to use microRNA for detecting the hearing damage in patients affected from mitochondrial disease. It is challenging to answer point by point to your remarks in our article .We clearly explained in our paper that we were investigating the use of microRNA (miRs). Our hypothesis is that microRNAs might be an interesting tool to study mitochondrial disorders and its multisystemic manifestations in Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) and we believe that researcher is fully aware of limitations mostly related to the use of preexisting data collected .We stated that additional clinical and experimental studies are necessary. Ideally, a clinical study should link the results of specific audiological and electrophysiological impairments with MELAS and, to the concentration of specific microRNAs. The necessity of prospective studies is clear due to the unspecificity of microRNAs , as you Our suggested microRNAs are the ones that have been identified in the human temporal bones of patients affected from MELAS ; thanks We focused our attention on the variation of concentration of microRNAs only and not, on their multiple functions since this would need more experimental studies.As far as the influence of aging, we reported that the temporal bones observed came from young subjects (“patients were under 30 years old”) and, in our opinion this is the strength of this research, in fact the “age” may be considered as a confounder if we consider the hearing impairment, that is a common finding in older age. Furthermore ROS that increase with age could be a common pathogenetic link to mitochondrial disorders, since mitochondrial deletions increase with age.We pointed out the heterogeneity of the human temporal bone and, we underlined different severities of hearing loss observed in MELAS patients are due to the stochastic segregation of mitochondria by determining changes of different entity and that involves different inner ear structures – spiral ganglion, vascular stria . This isI disagree with you, when you spoke about conductive form of hearing loss (HL) in patients with MELAS, your speculative statement is not correlated with that observed in human clinical observations, patients with MELAS are affected by a sensorineural form of hearing loss even if they present different severity of hearing impairment .The central auditory pathways have been investigated from several authors in patient affected by MELAS , 8 by AuWe included different type of studies in human temporal bones and clinical observations, that we analyzed for explaining that the biomarkers commonly observed in patients affected from MELAS (i.e. creatine kinase (CK), lactic acid) might add to the reported Magnetic Resonance Imaging (MRI) results in the temporal bone analysis.We stated that hearing loss not only affects patients with MELAS, but also other mitochondrial disorders.We think that microRNAs could be a valid biomarker for investigating the damage in hearing pathways, currently the method needs to be tested in prospective human clinical studies as first and, then if its accuracy will be confirmed microRNAs might be used in the screening of patients with sensory neural hearing loss.


Mycoplasma hyopneumoniae and innovative adjuvants designed to induce cellular immunity could improve vaccine efficacy against this pathogen. The aim of this experimental study was to assess the efficacy of three experimental bacterin formulations based on M. hyopneumoniae field strain F7.2C which were able to induce cellular immunity. The formulations included a cationic liposome formulation with the Mincle receptor ligand trehalose 6,6-dibehenate (Lipo_DDA:TDB), a squalene-in-water emulsion with Toll-like receptor (TLR) ligands targeting TLR1/2, TLR7/8 and TLR9 (SWE_TLR), and a poly(lactic-co-glycolic acid) micro-particle formulation with the same TLR ligands (PLGA_TLR). Four groups of 12 M. hyopneumoniae-free piglets were primo- (day (D) 0; 39 days of age) and booster vaccinated (D14) intramuscularly with either one of the three experimental bacterin formulations or PBS. The pigs were endotracheally inoculated with a highly and low virulent M. hyopneumoniae strain on D28 and D29, respectively, and euthanized on D56. The main efficacy parameters were: respiratory disease score , macroscopic lung lesion score (D56) and log copies M. hyopneumoniae DNA determined with qPCR on bronchoalveolar lavage (BAL) fluid . All formulations were able to reduce clinical symptoms, lung lesions and the M. hyopneumoniae DNA load in the lung, with formulation SWE_TLR being the most effective  −67.28%). Further experiments raised under field conditions are needed to confirm these results and to assess the effect of the vaccines on performance parameters.New vaccine formulations that include novel strains of  Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of enzootic pneumonia (EP) in pigs. This chronic respiratory disease is responsible for major economic losses in pig producing countries all over the world due to reduced performance and increased medication use [tion use , 2.M. hyopneumoniae field strain isolated in the UK in the 1950s [Together with biosecurity measurements, management practices and medication, commercial bacterin vaccines are used worldwide to control EP . They arhe 1950s , and adjhe 1950s . Vaccinahe 1950s . Howeverhe 1950s . Also, the 1950s –10. Morehe 1950s , which mhe 1950s .+ T cell responses, are considered to play a major role in protective immunity against M. hyopneumoniae infections [Mycoplasma infections by activating macrophage killing [+ T cells, on the other hand, might dampen the excessive pro-inflammatory Th responses that induce lung lesions and clinical disease [While serum antibodies are not correlated with protection against EP , the rolfections –16. T he killing , while T killing  and recr killing . CD8+ T  disease .M. hyopneumoniae is constantly ongoing. An overview of the different experimental M. hyopneumoniae vaccines already showed that most of them are based on recombinant proteins of M. hyopneumoniae and were evaluated in mice [Research into novel vaccine formulations that may offer a better protection against  in mice . Merely  in mice , 20.M. hyopneumoniae strain F7.2C, a highly virulent field strain shown to be antigenically different from the J strain [M. hyopneumoniae-specific T cell responses. These included a micro-particle and an oil-in-water formulation to deliver a cocktail of Toll-like receptor (TLR) 1/2, TLR9 and TLR7/8 ligands, and a cationic liposomal formulation to deliver a Mincle ligand. The liposomal formulation was able to induce strong Th1 and CD8+ T cell responses, while the oil-in-water formulation induced a strong Th1 response and a moderate CD8+ T cell response. The micro-particle formulation had the unique ability of inducing a potent Th17 response. Therefore, the aim of this study was to assess the protective efficacy of these three innovative bacterin formulations against experimental infection with two M. hyopneumoniae field strains. The main efficacy parameters were respiratory disease score (RDS), macroscopic lung lesion score and log copies M. hyopneumoniae DNA in bronchoalveolar lavage (BAL) fluid. Additionally, microscopic lung lesions, M. hyopneumoniae-specific local and systemic antibodies, M. hyopneumoniae-specific systemic T cell responses and cytokine responses in BAL fluid were assessed.In a previous study , the safJ strain , 23, andM. hyopneumoniae bacterin formulations and identified promising vaccine candidates for further exploration.This study demonstrated the potential of innovative co-glycolic acid) (PLGA) micro-particles and a squalene-in-water emulsion (SWE)) with different immune stimulators. These included the Mincle agonist trehalose 6,6-dibehenate  and a combination of TLR ligands: TLR1/2 ligand Pam3Cys-SK4 , TLR9 ligand CpG oligodeoxynucleotides SL03  and TLR7/8 ligand resiquimod . The selection of the TLR ligands was based on their ability to activate porcine antigen presenting cells [Three adjuvant formulations were developed based on the association of particle-based delivery systems  micro-particles (combined to ethylaminoethyl-dextran) were produced with the double emulsion (W/O/W) methods [Trehalose 6,6-dibehenate (TDB) was combined with dimethyl dioctadecylammonium (DDA) bromide with the thin lipid film method  and foll methods . Pam3Cys methods ) with thM. hyopneumoniae F7.2C was grown in modified Friis medium [8 colour changing units (CCU)/mL, was inactivated by incubation with 4 mM binary ethyleneimine (BEI) under agitation at 35 ± 2 °C for 24 h. Subsequently, the BEI was neutralised by incubating the inactivated culture with 4 mM sodium thiosulfate under agitation at 35 ± 2 °C for 24 h. Inactivated bacteria were pelleted at 15 000 g for 40 min at 4 °C and washed three times in 50 mL sterile phosphate buffered saline (PBS). The final pellet was resuspended in sterile PBS and mixed with the different adjuvants. The composition of each experimental vaccine is given in Table The vaccine strain s medium  for 5 daM. hyopneumoniae-free Rattlerlow-Seghers piglets  were enrolled in the study. All animals originated from a herd that has been M. hyopneumoniae-free for many years based on repeated serological testing, nested PCR testing on tracheobronchial swabs, and absence of clinical signs and pneumonia lesions at slaughter. The piglets arrived at the experimental facilities of the Faculty of Veterinary Medicine, Ghent University, Belgium at 32 days of age. They were housed in stables with absolute air filters for impending particles (HEPA U15) on both incoming and outgoing ventilation shafts and fed ad libitum with a non-antimicrobial-supplemented diet. On the day of arrival (D-7), the piglets were randomly allocated into three vaccination groups and a PBS-injected control group (PCG) of 12 piglets each. Five pigs were included as a non-challenge control group (NCG). After an acclimatization period of 1 week, the pigs of the vaccination groups were prime-boost vaccinated intramuscularly (IM) at day (D) 0 and D14 of the study with 2 mL of experimental bacterin. The pigs of the PCG and NCG received 2 mL sterile PBS IM at both vaccination days. The pigs of the vaccinated groups and the PCG were experimentally infected by endotracheal inoculation of the highly virulent M. hyopneumoniae strain F7.2C (7 mL culture medium containing 107 CCU/mL) on D28 and the low virulent strain F1.12A (7 mL culture medium containing 107 CCU/mL) on D29 [®  and Xyl-M® 2%  IM. Four weeks after challenge infection (D56), all pigs were euthanized by exsanguination after deep anaesthesia with 0.3 mL/kg of the same mixture injected IM.The study was performed after approval by the Ethical Committee for Animal Experiments of the Faculty of Veterinary Medicine, Ghent University . Fifty-three ) on D29 . The pigThe pigs were observed daily between 8 and 10 a.m. for at least 20 min from D-6 until D56 of the study. For each pig, remarkable clinical findings  were recorded and the severity of coughing was assessed by means of a RDS . The scoOn the day of primo-vaccination (D0), challenge (D28) and euthanasia, the pigs were weighed, and the average daily gain  was calculated from D0–28, D28–56 and D0–56 .Mycoplasma-like lesions according to Hannan et al. [At necropsy (D56), the lungs were removed and scored for macroscopic n et al. . The scoM. hyopneumoniae, while scores 3–5 are suggestive for an infection with M. hyopneumoniae.From each pig, samples from the left apical, cardiac and diaphragmatic lung lobe were collected for histopathological examination. If lesions were present, samples were taken from the border of the lesion. Each sample was scored for the degree of peribronchiolar and perivascular lymphohistiocytic infiltration, as well as nodule formation, using light microscopy. A score system ranging from 1 to 5 was used, with 1 = limited infiltration of macrophages and lymphocytes around bronchioles, with airways and alveolar spaces free of cellular exudates, 2 = light to moderate infiltrates with mild diffuse cellular exudates into airways, 3–4–5 = respectively mild, moderate and severe lesions characteristic of broncho-interstitial pneumonia, centred around bronchioles but extending to the interstitium, with lymphofollicular infiltration and mixed inflammatory cell exudates . Scores The percentage of lung area occupied by air (% air) was assessed using an automated image analysis system  and ImageJ  ).® Dog Catheter with Female Luer Mount, Smiths Medical International Ltd., Kent, UK) in the trachea and flushing the lungs with 20 mL sterile PBS [® Blood & Tissue kit, Qiagen, Venlo, The Netherlands) and a quantitative PCR (qPCR) was performed according to Marois et al. [M. hyopneumoniae organisms. The threshold values were converted to the number of organisms using a tenfold dilution series of M. hyopneumoniae F7.2C DNA. Values below the highest dilution  were considered as negative.Two weeks after challenge infection (D42), bronchoalveolar lavage fluid was collected from each pig by inserting a catheter  with a s streak . The agaM. hyopneumoniae seropositive animals was determined with a commercial blocking ELISA  according to the manufacturer‘s instructions. Samples with optical density (OD) lower than 50% of the average OD of the buffer control were considered positive. Samples with OD-values equal or bigger than 50% of the average OD of the buffer control were considered negative.Before primo-vaccination (D0), on the day of booster vaccination (D14), at challenge (D28), 2 weeks after challenge (D42) and at euthanasia (D56), serum samples were collected and the number of Mycoplasma hyopneumoniae-specific IgG and IgA isotypes were measured in serum  with an in-house indirect ELISA using Tween 20-extracted M. hyopneumoniae antigens according to Matthijs et al. [s et al. . All samMycoplasma hyopneumoniae-specific IgA antibodies in BAL fluid collected 2 weeks after challenge infection (D42) and at euthanasia (D56) were measured with an in-house indirect ELISA as previously described [escribed . The BALM. hyopneumoniae-specific T cell responses according to Matthijs et al. [M. hyopneumoniae F7.2C bacterin. For the last 4 h, Brefeldin A was added to inhibit cytokine release and allow intracellular detection of cytokines. Subsequently, cells were harvested and the percentage of cytokine-producing T cells was measured by flow cytometry (FCM) using a 5-step 6-colour staining protocol. Briefly, following incubation with the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit , cells were incubated with anti-CD4  and anti-CD8β  antibodies and then with their corresponding secondary antibodies anti-mouse IgG2b AlexaFluor 488  and anti-mouse IgG2a PE-Cy7 . After fixation and permeabilization of the cells with the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution kit , intracellular cytokines were stained with directly coupled anti-human TNF-α AlexaFluor 647 , anti-pig IFN-γ PerCP-Cy5.5  and anti-human IL-17A PE . All samples were acquired on a CytoFLEX flow cytometer  and the analysis performed with the FlowJo™ software . For each animal, samples were restimulated in triplicate cultures and analysed separately. To define whether an animal had M. hyopneumoniae-specific circulating cells, a threshold value was calculated as the mean % of cytokine-producing cellsall control animals + 3*SDall control animals for D28, and as the mean % of cytokine-producing cellsNCG + 3*SDNCG for D56. Animals with values above the threshold were identified as positive, while animals with values equal to or below the threshold were classified as negative.Shortly before challenge infection (D28) and at euthanasia (D56), blood samples were collected to assess s et al. . Periphe® ELISA, R&D Systems, Minneapolis, MN, USA), IL-6 , IFN-γ  and TNF-α  using a sandwich ELISA according to the manufacturer‘s instructions. The OD-values were converted to cytokine levels by means of a standard curve.The BAL fluid collected at D42 and D56 was tested undiluted for the presence of IL-1β , IL-1, IL-6, IFN-γ and TNF-α were not normally distributed according to the Shapiro–Wilk’s test, and were analysed using a Kruskal–Wallis ANOVA followed by Dunn’s post hoc test. Adjusted P values were computed to account for multiple comparisons, except for Scheffé’s post hoc test, which is already quite conservative [P ≤ 0.05.The RDS data was averaged for the periods D-6 until D56, D-6 until D27 and D28 until D56, and analysed using a repeated measures ANOVA. Pairwise comparisons between groups were obtained with Scheffé’s post hoc test. The T cell data was analysed using a one-way ANOVA with Tukey–Kramer’s post hoc test for pairwise comparisons. The parameters ADG, macroscopic lung lesions, microscopic lung lesions, % air, log copies ervative . The NCGGeneral health, severity of coughing (daily RDS) and ADG of each piglet were closely monitored during the entire study. One piglet from the PCG died during anaesthesia on D28. This piglet was excluded from the RDS and ADG analyses. On D42, one piglet from the Lipo_DDA:TDB group showed severe abdominal breathing after blood sampling. Therefore, BAL fluid was not collected from that animal on D42.P ≤ 0.05; Table None of the non-challenge control animals coughed, except for one pig on D44 . An increase in the mean RDS was first observed in group Lipo_DDA:TDB from 6 to 8 days post challenge (DPC) onwards, followed by groups SWE_TLR, PCG and PLGA_TLR, respectively from 8, 9 and 10 DPC onwards. After several days, coughing decreased in the vaccinated groups, while it continued at a high level in the PCG until the end of the study .For each group, the mean ADG from D0–56, D0–28 and D28–56 is shown in Table P ≤ 0.05). Formulation SWE_TLR induced the highest reduction in macroscopic lung lesions compared to the PCG (88.38%), while formulations PLGA_TLR and Lipo_DDA:TDB reduced macroscopic lung lesions with 81.11% and 69.88%, respectively. All three formulations significantly reduced microscopic lung lesions (P ≤ 0.05), and the highest reduction was again observed in group SWE_TLR. Groups Lipo_DDA:TDB and SWE_TLR had a significantly higher percentage of lung area occupied by air compared to the PCG (P ≤ 0.05).At euthanasia (D56), lungs were collected and scored for macroscopic and microscopic lung lesions. The percentage of lung area occupied by air was measured using image analysis. The results are presented in Table M. hyopneumoniae DNA in BAL fluid and mean log copies M. hyopneumoniae DNA in BAL fluid are shown for each group in Table M. hyopneumoniae organisms were detected in BAL fluid from groups PLGA_TLR and SWE_TLR compared to the PCG (P ≤ 0.05). The reduction in log copies M. hyopneumoniae DNA was 42.41% and 67.28%, respectively. Formulation Lipo_DDA:TDB reduced the number of M. hyopneumoniae organisms in BAL fluid with 34.55% compared to the PCG, but this reduction was not statistically significant (P > 0.05). At euthanasia (D56), all groups had a lower number of M. hyopneumoniae DNA in BAL fluid compared to D42 and no significant differences were observed between the groups (P > 0.05).The number of animals positive for No other respiratory bacteria were detected after inoculating the BAL samples on Columbia blood agar plates.M. hyopneumoniae-specific antibodies in serum 2 weeks after booster vaccination (D28). In groups PLGA_TLR and SWE_TLR, 5/12 and 11/12 pigs seroconverted, respectively. Two weeks after challenge infection (D42), all pigs from the vaccinated groups were seropositive, together with 7/11 pigs from the PCG. At euthanasia (D56), all pigs from the vaccinated groups and PCG were seropositive. The pigs form the NCG remained serologically negative for M. hyopneumoniae during the entire study.According to the commercial blocking ELISA . Two weeks after challenge infection (D42) and at euthanasia (D56), all vaccinated groups had higher M. hyopneumoniae-specific IgG levels compared to the PCG. This was statistically significant for all vaccinated groups (P ≤ 0.05) except group PLGA_TLR on D42 . Nevertheless, 2 weeks after challenge infection (D42), group Lipo_DDA:TDB showed a significantly higher M. hyopneumoniae-specific IgA response compared to the PCG and group PLGA_TLR (P ≤ 0.05). Also, at euthanasia (D56), group Lipo_DDA:TDB had a significantly higher level of M. hyopneumoniae-specific IgA in serum compared to the PCG  and 2 weeks after booster vaccination (D28), none of the vaccine formulations induced a significant M. hyopneumoniae-specific IgA antibodies in BAL fluid collected 2 weeks after challenge (D42). At euthanasia (D56), all animals from the vaccinated groups and the PCG were positive for M. hyopneumoniae-specific IgA. No IgA antibodies were detected in BAL fluid from the non-challenge control animals on both sampling days . At euthanasia (D56), groups Lipo_DDA:TDB and SWE_TLR had significantly higher M. hyopneumoniae-specific IgA levels in BAL fluid compared to the PCG, and group Lipo_DDA:TDB was also significantly higher than group PLGA_TLR  are presented in Figures + (Th1) cells compared to the PCG and group PLGA_TLR (P ≤ 0.05). However, in this group only five animals were above the threshold. In the Lipo_DDA:TDB group four animals and in group PLGA_TLR one animal were above the cut-off value  cells, one pig from group Lipo_DDA:TDB and two pigs from group SWE_TLR were above the cut-off  cells compared to the PCG and the other two vaccinated groups (P ≤ 0.05), although merely five animals appeared to have such cells in the blood , group PLGA_TLR had a significantly higher percentage CD4d Figure D. In thed Figure E. Only id Figure F.P ≤ 0.05). At euthanasia (D56), group PLGA_TLR had a significantly lower IL-1β concentration compared to the PCG . At euthanasia (D56), group SWE-TLR had a significantly higher IFN-γ concentration compared to group PLGA_TLR . The improvements obtained with formulation SWE_TLR seemed similar to or sometimes even better  than the results obtained with commercial M. hyopneumoniae bacterins under experimental conditions [All three vaccine formulations were able to reduce clinical signs, macroscopic lung lesions and histopathological lung lesions, with formulation SWE_TLR being the most effective , the contraction phase corresponds to a huge decrease in the frequency of antigen-specific T cells before they become memory cells. Even though memory cells are still circulating in the peripheral blood, their frequency is low as most memory cells recirculate between lymphoid tissue and blood, migrate to peripheral sites or the bone marrow for long-term survival [Two weeks after booster vaccination (D28), a significantly higher percentage of s et al. , where fs et al.  showed asurvival , 47. It survival , which msurvival . InteresM. hyopneumoniae-induced pneumonia [Two weeks after challenge infection, group Lipo_DDA:TDB had very high IL-1β levels compared to the other groups. This group also had the highest RDS at that time point. Several studies have associated the excessive production of pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α with the development of neumonia –50. Acconeumonia , vaccinaM. hyopneumoniae DNA load in the lung, with the oil-in-water formulation delivering a cocktail of TLR-ligands being the most effective. As the number of animals is limited in experimental infection studies, further research including more animals and raised under field conditions is needed to confirm the present results, and especially to assess the effects of the different vaccine formulations from this study on the reduction of performance losses  due to M. hyopneumoniae infections.In conclusion, all formulations were able to reduce clinical symptoms, macro- and microscopic lung lesions and the Additional file 1. Results of theM. hyopneumoniae-specific antibodies measured at different time points in serum and in BAL fluid. Pigs were prime-boost vaccinated on D0 and D14 with three different experimental M. hyopneumoniae bacterins , challenge infected on D28–29 and euthanized on D56. M. hyopneumoniae-specific antibodies were determined by the IDEIA™ Mycoplasma hyopneumoniae EIA kit  and by indirect in-house ELISAs. For the in-house ELISAs, NetOD-values were calculated by subtracting the OD-value of the blank from the OD-value of the sample. BAL, bronchoalveolar lavage; NCG, non-challenge control group ; PCG, PBS-injected control group ; OD, optical density.


Chocolate consumption was independently associated with low odds of any hearing loss . Moreover, the severity of hearing loss was inversely correlated with the frequency of chocolate consumption. In contrast to chocolate, there was no association between hearing loss and the consumption of sweet products without cocoa. Chocolate consumption was also not associated with tinnitus or tinnitus-related annoyance. Our results suggest that a chocolate-based diet may protect middle-aged people from hearing loss.Chocolate, which is produced from cocoa, exerts antioxidant and anti-inflammatory effects that ameliorate neurodegenerative diseases. We hypothesized that chocolate consumption would protect against hearing loss and tinnitus. We evaluated the hearing and tinnitus data, as well as the chocolate consumption, of middle-aged participants (40–64 years of age) of the 2012–2013 Korean National Health and Nutrition Examination Survey. All of the subjects underwent a medical interview, physical examination, audiological evaluation, tinnitus questionnaire, and nutrition examination. A total of 3575 subjects 40–64 years of age were enrolled. The rate of any hearing loss  in the subjects who consumed chocolate (26.78% (338/1262)) was significantly lower than that in those who did not (35.97% (832/2313)) ( Hearing loss, a highly prevalent sensorineural disorder, imposes a major economic and social burden . The incTinnitus is a common otologic symptom, often called a “phantom sound” as it is a conscious auditory perception without a corresponding physical source . The prePatients and clinicians have concerns regarding the effect of diet on hearing loss and tinnitus ,14. TherBased on the therapeutic effect of chocolate , we hypoThis cohort study used data, from the Korean National Health and Nutrition Examination Survey (KNHANES), representative of the health and nutritional status of Koreans. We examined the association of chocolate consumption with hearing loss and tinnitus in participants 40 to 65 years of age. All subjects gave their informed consent for inclusion before they participated in the study. This study was approved by the Institutional Review Board of Seoul National University Hospital (1902-046-1008). n = 887), did not provide their chocolate consumption frequency (n = 874), or had missing values for confounders (n = 337). Finally, 3575 participants from the 2012–2013 KNHANES aged 40 to 65 years were enrolled in the study , 2098 were excluded because they did not receive a hearing threshold test or respond to a tinnitus-related questionnaire  in a soundproof room. The mean hearing threshold was calculated as the average of the hearing thresholds at 0.5, 1, 2, and 4 kHz. The mean high-tone hearing threshold was determined using the average of the hearing thresholds at 3, 4, and 6 kHz. To exclude subjects with systemic diseases, blood samples were collected and analyzed at the Neodin Medical Institute in Seoul, Korea.With regard to tinnitus, all subjects were also interviewed about the presence of tinnitus and tinnitus-related annoyance. The severity of tinnitus was classified as follows: “no,” “slightly annoying”, and “very annoying and difficult to sleep”. In this study, a response of “slightly annoying” or “very annoying and difficult to sleep” was defined as tinnitus-related annoyance. The parameters of mean hearing threshold according to frequency, presence of tinnitus, and tinnitus-related annoyance were used to evaluate the association with chocolate.The following potential confounders were adjusted for: sleep duration, stress severity, income, current smoking habits, alcohol consumption, noise exposure, and medical conditions. The information obtained included sleep duration , rate of perceived stress, income , current smoking habits, exposure to indoor second-hand smoke (at work or at home), alcohol consumption , and difficulties in controlling alcohol use. The duration of occupational exposure to noise and earphone and headphone use were also measured. In addition, the health status  of the subjects was evaluated. Finally, we used sweet products without cocoa  as a confounder in the evaluation of the non-specific effect of pleasant sensations.As in previous study , chocolaAll statistical analyses were performed using SAS software . The subjects’ demographic and clinical characteristics are presented as medians (interquartile ranges) or numbers (proportions). The comparison between groups, performed by Fisher exact test (binary covariates), chi-squared test (more than three categories), or Wilcoxon rank sum test (continuous covariates), was deemed appropriate. The association between chocolate consumption and hearing loss and tinnitus was evaluated by means of multivariate logistic regression models with adjustment for the following potential confounders: age; sex; sleep duration; perceived stress; current smoking habits; exposure to indoor second-hand smoke; heavy drinking; drinking-related problem; duration of earphone use; occupational exposure to noise; menopause; and history of hypertension, diabetes mellitus, dyslipidemia, anemia, kidney failure, and thyroid disorder. The Spearman correlation rank order was used to evaluate the correlation between the frequency of chocolate consumption and the severity of hearing loss. In the multivariate models of tinnitus or tinnitus-related annoyance, we controlled for unilateral or bilateral hearing loss as well as the above potential confounders. p < 0.001, p < 0.001) and high-tone hearing loss (51.58% (651/1262) vs. 63.60% , p < 0.001), respectively.The rate of any hearing loss  was significantly lower in the subjects who consumed chocolate (26.78% (338/1262)) than in those who did not (35.97% (832/2313)) (p = 0.03), bilateral hearing loss , and high-tone hearing loss   = 0.83, 95% confidence interval (CI) = 0.70 to 0.98,  = 0.02) . In contrast to chocolate, neither hearing loss nor bilateral hearing loss was associated with the consumption of sweet products without cocoa, according to multivariable logistic regression model. Furthermore, chocolate was significantly associated with a decrease of hearing loss after adjusting for cofounders, including consumption of sweet products without cocoa . ρ = −0.117, 95% CI = −0.15 to −0.08, p < 0.001)   A. In add< 0.001) B.Neither the rate of tinnitus nor the rate of tinnitus-related annoyance differed significantly according to chocolate consumption. In addition, subjects who consumed chocolate had 9% and 11% lower odds of tinnitus  and tinnitus-related annoyance , respectively . HoweverTo our knowledge, this is the first study to explore the effect of chocolate on hearing loss and tinnitus in a large cohort of middle-aged people. The rate of hearing loss was significantly lower in the subjects who consumed chocolate than in those who did not. Additionally, there was an inverse correlation between the severity of hearing loss and the frequency of chocolate consumption. These results support those of previous animal studies, which reported that various compounds in chocolate protect against hearing loss . SpecifiThe cochlea is susceptible to oxidative stress because of its high energy requirements and lack of an adequate collateral blood supply . Thus, tAlthough we could not evaluate the direct effect of chocolate on hearing loss by quantifying cocoa, our results suggest that chocolate may prevent hearing loss by a mechanisms other than the pleasant sensation of chocolate. Those who did not consume chocolate had higher risks of hypertension and dyslipidemia than did those who consumed chocolate. Chocolate exerts antioxidant and anti-inflammatory effects and has been shown to have therapeutic benefits for patients with cardiovascular diseases. Specifically, cocoa, a major ingredient of chocolate, attenuates vascular risks by reducing blood pressure and improving endothelium-dependent vasodilation . In lineChocolate is an important dietary source of flavonoids, a subclass of polyphenols . PolypheUnexpectedly, chocolate intake was not associated with tinnitus or tinnitus-related annoyance. In humans there is a relationship between the hearing-loss frequency and the tinnitus pitch, suggesting a strong relationship between auditory deafferentation and tinnitus. However, the association between hearing loss and tinnitus may not be straightforward. For example, subjective tinnitus was reported by approximately 19% of individuals with normal hearing in a cohort study in Korea , suggestThe lack of an association between chocolate consumption and tinnitus may be attributable to strict adjustment for comorbidities, e.g., hearing loss, stress, and sleep . MoreoveThis study had several limitations that should be noted. First, we could not assess the causality of the relationship between chocolate consumption and hearing loss due to the cross-sectional nature of the study. Moreover, no information on chocolate consumption was available for participants over 65 years of age. Given the protective effect of chocolate on age-related hearing loss, the association might have been significant if older persons had been included. Second, the processing technique used in the production of chocolate affects its total polyphenol content . The datNonetheless, the present study had several strengths. First, a large cohort from a nationally representative database was analyzed. Second, this is the first study of the relationship between chocolate intake and hearing loss. Our results will enhance nutritional support for patients with hearing loss.Our results suggest that chocolate plays an otoprotective role against hearing loss but not tinnitus in middle-aged people.


TCs), formerly known as interstitial Cajal‐like cells (ICLCs), exhibits very particular features which unequivocally distinguish these cells from interstitial cells of Cajal (ICCs) and other interstitial cell types. Current research substantiates the existence of TCs and ICCs in the biliary system . Here, we review the distribution, morphology and ultrastructure of TCs and ICCs in the biliary tree, with emphasis on their presumptive roles in physiological and pathophysiological processes.A novel type of interstitial tissue cells in the biliary tree termed telocytes ( Later on, Sun et al in 2006 found ICCs in the mice gallbladder, and in humans, Ortiz‐Hidalgo et al found in stromal tumour of the gallbladder cells with phenotype of ICCs.There is now overwhelming evidence to support canonical ICCs as the origin of pacemaker potentials in the gastrointestinal tract, playing an important role in contractile activity. The gallbladder smooth muscle, which resembles the gastrointestinal smooth muscle, also exhibits spontaneous rhythmic electrical activity. Therefore, it has been proposed that in certain specific regions of the biliary system, these ICCs are likely involved in modulating the excitability of smooth muscle and tissue motility. This hypothesis was subsequently confirmed by researches conducted in China and abroad. In 2012, Pan et al3In 2007, Lavoie et al3.13.1.1Huang et al3.1.22+ release units”; (vi) branching: dichotomous pattern; (vii) organization in network—labyrinthic system: overlapping cytoplasmic processes; (4) gap junctions: with SMCs or with each other; (5) basal lamina: occasionally present; (6) caveolae: 2%‐3% of cytoplasmic volume; ~0.5 caveolae/μm of cell membrane length; (7) mitochondria: 5%‐10% of cytoplasmic volume; (8) endoplasmic reticulum: about 1%‐2%, either smooth or rough; (9) cytoskeleton: intermediate and thin filaments, as well as microtubules, present; (10) myosin thick filaments: undetectable. The ultrastructure of TC in the gallbladder is quite similar among humans and other mammals.Transmission electron microscopy has revealed the characteristics of TCs in the gallbladder of the guinea pig as elongated, electron‐dense cells with multiple long interconnected processes that are rich in mitochondria, smooth endoplasmic reticulum and caveolae. TCs are intercalated between nerves and SMCs and form direct appositions and/or gap junctions with surrounding cells.3.1.32+ transients indicate Ca2+ influx via L‐type Ca2+ channels during action potentials, which were detected in the guinea pig gallbladder SMCs and TCs as rapidly occurring Ca2+ flashes.2+ flashes in smooth muscle cells were synchronized with the electric activity and Ca2+ transients of TCs in a given bundle and that Ca2+ flashes in TCs exhibited higher intensity and longer duration when compared to gallbladder SMCs. They also found that gap junction uncouplers eliminated or greatly reduced Ca2+ flashes in gallbladder SMCs, but the rhythmic activity persisted in TCs, whereas treatment with a Kit tyrosine kinase inhibitor eliminated or reduced APs and Ca2+ flashes in both cell types, as well as associated tissue contractions. These results pointed to the existence of electrical coupling between TCs and gallbladder SMCs, suggesting that TCs may be involved in the generation and propagation of the spontaneous rhythmicity of gallbladder tunica muscularis. This speculation was further substantiated in a recent study showing that the amplitude and frequency of the slow wave were remarkably reduced; concurrently, the tonic contraction was significantly declined in gallbladder smooth muscle strips with impaired TCs.2+ handling in generating spontaneous rhythmicity of TCs and regulating excitability of gallbladder. Overall, these observations support a role of TCs in the generation and propagation of pacemaker activity and in the regulation of gallbladder motility.The excitation‐contraction coupling that is well known to occur in the gastrointestinal smooth muscle is also present in the gallbladder smooth muscle (GBSM) cells. However, GBSM cells are arranged as interdigitated bundles orientated in multiple directions, which is in contrast with the sheet arrangement of smooth muscle cells observed in the gastrointestinal tract.3.1.4A characteristic feature of interstitial TCs in the gallbladder is expression of the transmembrane tyrosine kinase receptor c‐Kit, which enables their convenient identification by immunohistochemical and molecular methods. However, gallbladder mastocytes are also immunopositive for CD117/Kit receptor tyrosine kinase. It is generally accepted that application of antibodies against mast tryptase or toluidine blue staining can effectively distinguish between mast cells and telocytes. PasternakCholecystokinin is released from mucosal endocrine cells in the proximal small intestine in response to a meal, and it is classically known to stimulate the gallbladder contraction. In an early study, researchers identified cholecystokinin A receptor (CCK‐AR) expressed on gallbladder ICCs of guinea pig.3.2Lavoie et alThe aboral side of the common bile duct, where the ampulla opens into the duodenum, is surrounded by the sphincter of Oddi (SO). It has a rather complex muscular structure because the SMCs mainly run in a circular direction along with several longitudinal or oblique muscle bundles. Within the sphincter of Oddi, Huang et al4Since the discovery of TCs in the biliary tree, increasing evidence suggests a correlation between these cells and biliary systemic motility as highlighted above. Accordingly, their potential role in the pathogenesis of some biliary systemic disorders has now begun to attract substantial attention.4.1Cholelithiasis is one of the most common diseases in gastroenterology that is caused by concrement in the biliary system . Several factors are involved in the pathogenesis of this disease, including cholesterol hypersecretion with supersaturation bile, gallbladder hypomotility, as well as accelerated cholesterol crystallization in the bile. The cholesterol saturation index (CSI) is an accepted parameter relevant to bile lithogenicity.4.2In our most recent study,4.3Gastrointestinal stromal tumours (GISTs) are the most common primary, non‐epithelial, mesenchymal tumours of the tubular GI tract, which originate from or manifest the morphological and immunophenotypic characteristics of ICCs. Gradually, stromal tumours which are similar in immunophenotype to TCs [CD117 (c‐Kit)+/CD34+] have also been sporadically discovered outside of the tubular gut, including pancreas, liver, and gallbladder, which are named as extragastrointestinal stromal tumours (EGISTs). In a recent study, Padhi et al5Telocytes and ICCs have been identified in the biliary tree, including gallbladder, cystic duct, extrahepatic bile duct, common bile duct and sphincter of Oddi. TCs in the biliary system are similar to canonical enteric ICCs in the aspect of morphological characteristics and ultrastructure, and they may have a functional relationship with adjacent SMCs and enteric neurons. TCs can be generally divided into two classes according to their location in the biliary tract, namely subepithelial and intramuscular cells. However, it is still not clear whether TCs in the gallbladder form a cellular network. As gallbladder and sphincter of Oddi exhibit spontaneous rhythmic contraction, it is likely that TCs and ICCs regulate excitability of SMCs and tissue motility in specific areas by modulating the generation and propagation of electrical activity. Moreover, there is ample evidence that TCs and ICCs in the bile tree may be related to the development of cholelithiasis, acute cholecystitis, choledochal cysts and stromal tumours of the gallbladder. Works performed in order to understand the role of gallbladder TCs and ICCs under physiological condition has provided new insights and exciting new hypotheses about the causes of some biliary systemic disorders, which can help to give rise to novel potential therapies for these diseases. Nevertheless, the current knowledge pertaining to TCs and ICCs in the biliary tree remains extremely limited, and thus, more studies are certainly needed in both normal and diseased biliary system to advance this field.The authors confirm that there are no conflicts of interest.


Objectives: to analyze the results of molecular methods applying for the diagnosis of mucormycosis in hematologic patients based on a literature review. Data sources: A systematic search in databases PubMed, Google Scholar for August 2019. Review eligibility criteria: original articles published in English, studies of molecular methods for the diagnosis of mucormycosis in hematologic patients. Results. We analyzed the research data from 116 hematological patients with mucormycosis, including children (6%). Patients with localized forms of mucormycosis prevailed (72%), and lung involvement was diagnosed in 58% of these cases. For molecular verification of the causative agent of mucormycosis, blood serum was most often used, less commonly postoperative and autopsy material, biopsy specimens, formalin-fixed paraffin-embedded samples and bronchoalveolar lavage, pleural fluid and sputum. The sensitivity of molecular diagnostics of mucormycosis in a cohort of hematological patients was 88.2%. Conclusion. The use of molecular techniques along with standard mycological methods will improve the diagnostics of mucormycosis in hematologic patients. However, prospective studies of the effectiveness of molecular methods for the diagnosis of mucormycosis of various etiologies in hematological patients, including children, using bronchoalveolar lavage (BAL) and cerebrospinal fluid (CSF) are needed. Aspergillus spp. and the third most common infection after candidiasis and aspergillosis in recipients of allogeneic hematopoietic stem cell transplants  [In recent decades the incidence of mucormycosis has been increasing due to the growth of the number of severely immunocompromised patients. In developed countries, mucormycosis is mostly seen in hematological patients ,2. Mucorlo-HSCT) ,4. At thlo-HSCT) . Mucormylo-HSCT) ,7,8. Acclo-HSCT) , mucormyAccording to this the role of early diagnosis of mucormycosis is growing, but the disease is often detected late, often postmortem ,9,10. ThMolecular methods are promising directions of modern laboratory diagnosis of fungal infections. The main advantages of these tests are high sensitivity and specificity, as well as the ability to use any substrates containing DNA for analysis .Early diagnosis and timely initiation of mucormycosis treatment can improve the survival of patients and give a permit to continue the treatment of underlying hematologic disease. This will also reduce the time of hospitalization and, consequently, lower the treatment cost . HoweverThe aim of this study was to analyze the results of the application of molecular methods for the diagnosis of mucormycosis in hematological patients.In a systematic search in the PubMed, Google Scholar databases we found 38 articles on molecular methods for the detection and identification of mucormycetes in various substrates. For the review we selected sources that met the following requirements: original articles in English, where oncohematological patients with diagnosed mucormycosis were included in the studies in accordance with the criteria of the European Organization for Research and Treatment of Cancer/Mycoses Study Group  .The analysis did not include review articles, abstracts for conferences, descriptions of clinical cases, as well as articles where there were no characteristics of the background states of patients with mucormycosis, or the research results combined for various cohorts of patients, including non-hematological patients. The final search was carried out in August 2019.n = 110; 95%), there were significantly fewer children . Meanwhile, in our register, children and adolescents accounted for 36% of all patients with mucormycosis [The review included seven studies carried out in 2001–2017 ,19,20 dermycosis . TherefoIn all the works selected for this review, molecular diagnosis of mucormycosis was carried out using the polymerase chain reaction (PCR) method. Four studies used real-time PCR (RT PCR) for 18S rDNA ,15,16,17Fusarium infection (In most cases (84%), molecular methods were used in patients with “proven” mucormycosis, according to the criteria of EORTC/MSG, 2008. The samples of patients with “possible” mycosis were less commonly used. In the study conducted by Springer et al. 2016 were included two oncohematological patients with unidentified mycosis, in whom mucormycetes were subsequently detected by RT-PCR. In addition, ten patients had a combination of mucormycosis with “probable” or “proven” aspergillosis, five patients were diagnosed with “probable” aspergillosis prior to molecular diagnosis of mucormycosis, and one patient was “proven” based on histological examination of nfection . A combin = 84, 72%), and lung involvement was diagnosed in 58% of these cases , rhinocerebral mucormycosis was in 9% of patients (n = 11) and isolated central nervous system damage was observed in one patient. This indicates the representativeness of the examined patients: lungs are the main localization of mucormycosis in hematological patients, rhinocerebral form and disseminated process are common [Patients with localized forms of mucormycosis prevailed , Mucor spp. (19%) and Lichtheimia spp. (19%) were most commonly identified in patients with mucormycosis in Europe [Rhizopus spp. (47%), Rhizomucor spp. (28%) and Lichtheimia corymbifera (17%). According to our data, cultures from bronchoalveolar lavage (BAL) fluid and other samples were positive in 65% hematological patients with mucormycosis [Rhizomucor spp., Mucor spp., Cunninghamella spp. and rare pathogens are needed.Etiology of mucormycosis seemed to vary in different countries. For example, n Europe . In compor spp. 1% and LicVarious materials were used for molecular verification of the causative agent of mucormycosis: most often blood serum, less often postoperative and autopsy material, biopsy specimens, histological paraffin blocks, BAL fluid, pleural fluid and sputum . In the The obtained data indicate that molecular methods for a blood serum study can be an important tool for the diagnosis of mucormycosis in hematological patients. Millon et al. in their study showed that mucormycetes DNA can be detected in at least 36/44 (81%) patients with “probable” or “proven” mucormycosis. Due to the retrospective design of the study, serum sampling and volume were not optimal, and the sensitivity level was 81%. However, with the exception of cases with insufficient serum volume for testing, the sensitivity of the molecular method reached 92% .Lichtheimia and Rhizopus) and confirmed the pathogen identified by cultural methods in four out of five cases. Rhizopus spp. was also identified by this method in the case when the genus was not defined in the culture [Springer et al. 2016 showed a high percentage of positive results when have being used RT-PCR for 18S rDNA to analyze sera from patients with “proven” and “probable” mucormycosis. Further sequencing revealed the DNA of two different pathogens of mucormycosis  study, although rhinocerebral mucormycosis was in 9.5% of the examined patients. Probably, additional studies of the effectiveness of molecular diagnostic methods using CSF are also needed.The results of Hammond et al. 2011’s studies showed that the detection of mucormycetes DNA by PCR in histological preparations (tissue fixed in paraffin was used in this work) could also be a useful tool for the diagnosis of mucormycosis. Of 27 tissue samples with initial histological signs of mucormycosis, PCR was positive in 22 samples. In addition, the researchers found a high level of correlation between the results of PCR diagnostics and culture: of 12 samples with a positive culture, mucormycosis was confirmed by PCR in 10 cases (83%), and results of sequencing corresponded to the result of culture identification to genus in nine samples. In addition, PCR diagnostics revealed molds in substrates of 12 patients in whom a fungal culture was not obtained .Analysis of the summarized data as a result of our review indicates a high sensitivity of the molecular diagnostics of mucormycosis in a cohort of hematological patients. So, a positive PCR result was obtained in 98 cases of the 111 examined samples, thus the sensitivity was 88.2% , while tAspergillus PCR assays by the European Aspergillus PCR Initiative (EAPCRI) working group [As our literature analysis has shown, the spectrum of the methods of molecular diagnostics of mucormycosis used to date was mainly limited to RT-PCR and sequencing of 18S rDNA, as well as sequencing of the ITS region. DNA sequencing allowed us to get a slightly better taxonomic resolution, and RT-PCR met the criteria for a quick diagnostic method. In general, molecular diagnostic methods used in the studies we reviewed and intended for amplification of 18S rDNA allowed us to reach a sensitivity of 85–100%. The use of PCR identification of mucormycetes DNA in serum or BAL, along with standard mycological methods, can increase the effectiveness of early diagnosis of mucormycosis in hematological patients. At the moment an important problem is the lack of standardization of molecular methods. Fungal PCR Initiative (FPCRI)/Mucorales Lab working group of the International Society for Human and animal Mycology (ISHAM) are currently solving the questions about improving standardization and providing recommendations for Mucorales PCR assay, as was previously done for ng group ,24.1. Applying molecular methods along with standard mycological methods will improve the diagnosis of mucormycosis in hematologic patients.2. Prospective studies of the effectiveness of molecular methods for the diagnosis of mucormycosis in hematological patients, including children, are needed.Rhizomucor spp., Mucor spp., Cunninghamella spp. and rare pathogens are needed.3. Further researches on the effectiveness of the molecular diagnosis of mucormycosis of various etiologies, including 4. Since the main localizations of mucormycosis in hematological patients are lungs and the central nervous system, additional studies of the effectiveness of molecular diagnostic methods using BAL and CSF are needed.


GJB2) c.235delC variant in the homozygous state wasdetected in the patient. Both parents were heterozygous for this variant, asdocumented by Sanger sequencing. The known pathogenic GJB2c.235delC variant was not detected in 200 healthy controls. It is predicted tobe a disease-causing alteration by generating a truncated protein p.(L79Cfs*3),disturbing the appropriate folding and/or oligomerization of connexins andleading to defective gap junction channels. This study shows that theassociation of homozygosity of the GJB2 c.235delC variant withARNSHL and ANSD in a patient.Autosomal recessive nonsyndromic hearing loss (ARNSHL) is a geneticallyheterogeneous neurosensory disorder, usually characterized by congenital orprelingual hearing loss. We report a Han Chinese male, born to consanguineousparents, presenting with nonsyndromic sensorineural hearing loss, whose clinicalphenotype was also consistent with auditory neuropathy spectrum disorder (ANSD).After exome sequencing, a gap junction protein beta 2 gene( Autosomal recessive nonsyndromic hearing loss (ARNSHL) is a genetically heterogeneousneurosensory disorder, usually characterized by congenital or prelingual hearing loss,and not accompanied by other clinical features . ARNSHL GJB2) were firstidentified as causative of ARNSHL in 1997 .The extreme genetic heterogeneity of nonsyndromic hearing loss makes the use of regularSanger sequencing to identify its genetic cause very challenging, and exome sequencinghas been recommended as a cost-effective alternative strategy , tympanometry,acoustic reflex (AR) thresholds, auditory brainstem responses (ABR), transient evokedotoacoustic emission (TEOAE), and distortion product otoacoustic emission (DPOAE) wereperformed. Potential inner ear congenital malformations were evaluated with magneticresonance imaging (MRI). Audiometric thresholds were evaluated at frequencies 250, 500,1000, 2000, 4000, and 8000 Hz by PTA. Hearing acuity is considered ‘normal’ at athreshold within 25 decibels (dB), and the degree of hearing loss was classed as mild(26-40 dB), moderate (41-60 dB), severe (61-80 dB), or profound (> 81 dB) , according to the manufacturer’sprotocol. The library with the targeted exome was sequenced using the Illumina HiSeq2000 platform. The mean sequencing depth was 101.78, and 99.43% of the targeted exomewas covered. Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels)were detected. Alleles with a frequency > 0.5% in the following databases werescreened out based on the SNPs database (dbSNP version 138), 1000 genomes project (1000genomes release phase 3), HapMap project (2010-08_phase II + III), Exome SequencingProject 6500 (ESP6500) .Sanger sequencing was performed to identify whether candidate variants co-segregated withARNSHL phenotype in the family, using an ABI3500 sequencer   in the GJB2 gene was confirmed inthe patient by Sanger sequencing . Mutationspectrum and frequency in the GJB2 gene vary with ethnicity  missing important functional segments, including CL, TM3, E2, TM4, andC-terminal segments. The glutamine (p.Q80) in the TM2 segment of connexin 26 interactswith arginine (p.R32) in the TM1 segment, thus the variant may interfere with theinterplay between the two TM domains, disturb the appropriate folding and/oroligomerization of connexins, and lead to defective gap junction channels ,in the synapse between IHC and auditory nerve, or in the auditory nerve itself (GJB2-associated ANSD may be caused by immature IHCs.GJB2 gene with the phenotypes of ARNSHL and ANSD.In conclusion, our study shows a novel association of homozygosity for the c.235delCvariant in the