Supplemental Digital Content is available in the text Pendred syndrome (PDS)/DFNB 4 is a disorder with fluctuating and progressive hearing loss, vertigo, and thyroid goiter. We identified pathophysiology of a neurodegenerative disorder in PDS patient derived cochlear cells that were induced via induced pluripotent stem cells and found sirolimus, an mTOR inhibitor, as an inhibitor of cell death with the minimum effective concentration less than 1/10 of the approved dose for other diseases. Given that there is no rational standard therapy for PDS, we planned a study to examine effects of low dose oral administration of sirolimus for the fluctuating and progressive hearing loss, and the balance disorder of PDS by daily monitor of their audio-vestibular symptoms.SLC26A4 genetic testing aged in between 7 and 50 years old at the time of consent are given either placebo or sirolimus tablet (NPC-12T). In NPC-12T placebo arm, placebo will be given for 36 weeks; in active substance arm, placebo will be given for 12 weeks and the NPC-12T for 24 weeks. Primary endpoints are safety and tolerability. The number of occurrences and types of adverse events and of side effects will be sorted by clinical symptoms and by abnormal change of clinical test results. A 2-sided 95% confidence interval of the incidence rate by respective dosing arms will be calculated using the Clopper–Pearson method. Clinical effects on audio-vestibular tests performed daily and precise physiological test at each visit will also be examined as secondary and expiratory endpoints.This is a phase I/IIa double blind parallel-group single institute trial in patient with PDS/DFNB4. Sixteen of outpatients with fluctuating hearing diagnosed as PDS in JMA-IIA00361; Pre-results. While the SLC26A4 gene, which encodes PENDRIN, an anion exchanger, is known to be responsible for PDS, the mechanism that leads to fluctuation and progressive cochlear disorder had long remained unknown. PDS patients with fluctuating hearing loss experience volatile changes in hearing acuity, and thus they suffer not only inconveniences in their daily life, but also fear of losing their ability to communicate orally with people around them following every acute exacerbation. Symptoms of PDS involve rotating vertigo that may last a few hours to a few days once it occurs, in addition to chronic dizziness, which are grave detriments to the patients’ quality of life.Pendred syndrome (PDS)/DFNB4 is a disorder first described by Vaughan Pendred in 1896, the main symptoms of which include fluctuating and progressive hearing loss, vertigo, and goiter.No medication for hereditary hearing loss has pathophysiologically confirmed nonclinical POC anywhere in the world. For PDS, there is no effective treatment available at this time. The only effective interventions that exist are use of devices to augment hearing, such as hearing aids and cochlear implants. Although appropriate adjustment of a hearing aid requires consultation at a medical institution or with an audiologist, volatility in hearing and sporadic occurrence of fluctuations in a PDS patient impede the full performance of such adjustment. The hearing loss in PDS can progress as it fluctuates, and a PDS patient whose symptoms have progressed to severe hearing loss has an option to have a cochlear implant. Although the device provides a sense of sound, the sound quality is significantly deteriorated compared to what can be perceived with inner hair cells, as the number of electrodes used in a cochlear implant is limited to around twenty to date. With regard to vertigo, no medical intervention exists today, and thus patients have no option but to have bed rest and wait until the episode abates.Slc26a4, the responsible gene, develops severe hearing loss from severe malformation. A knock-in mouse for H723R mutations, the most commonly reported mutations in Japanese population with hearing impairment; however, does not develop deafness. This means that fluctuating and progressive hearing loss is not recapitulated in these mouse models. The lack of animal models is one of the impediments to understanding the pathology and developing treatments, and a reason that PDS treatments continue to depend on hearing devices.As described above, PDS is a rare and intractable disorder with no causal treatment, causing significant loss of patients’ QOL, and thus a new treatment has long been awaited. PDS is a hereditary disorder, and a knockout mouse for the  This suggests that animal models generally used to demonstrate nonclinical POC in drug discovery research are essentially inapplicable to PDS due to species difference. Considering this fact, we have undertaken preclinical studies for induced pluripotent stem cells (iPSC)-based drug discovery; identified pathophysiology of a neurodegenerative disorder that involves protein aggregation as a new pathophysiological mechanism of cochlear hearing loss; and performed drug screening with cell death suppression as metrics. We have discovered in this research that patient-derived iPS cells of inner ear present intracellular aggregations of mutant protein and vulnerability to cellular stress, which shares similarities in physiopathology with Parkinson disease and Alzheimer disease. We further screened existing drugs based on this finding, and identified sirolimus, an mTOR inhibitor, as an inhibitor of cell death. Oral administration of this drug has been used for 2 decades worldwide and lot of information, including adverse reaction and toxicity, has been accumulated. Common adverse effects observed among different diseases where Sirolimus was used include, for example, stomatitis, dermatitis, hypertension, and hypercholesterolemia.,9 The minimum effective concentration required for inhibition of cell death was 0.9 ng/mL, which was less than 1/10 of the approved dose of sirolimus preparation.We have described that this discrepancy is attributable to the species difference between primates and rodents, that is, in primates, a family protein with similar functions and SLC26A4 co-express.The natural history of inner ear disorder in PDS patients presents recurrent transient episodes and indolent progression. As no correlation is observed between inner ear malformation and hearing loss in PDS, hypofunction rather than malformation of the inner ear is assumed to be a therapeutic target for the disorder.We constructed 3 different in vitro cell models that correspond to PDS pathophysiology, using the disease-specific cells generated from patient-derived iPS cells:(1)acute injury from exposure to high-concentration epoxomicin;(2)acute injury from exposure to low-concentration epoxomicin; and(3)chronic injury without exposure to any agents.Sirolimus 0.9 ng/mL significantly reduced cell death in all 3 models.The results suggested low-dose sirolimus for PDS patients suffering from exacerbating and progressive symptoms aggravated by life stressors and head trauma may ameliorate vulnerability of the patient's inner ear cells, and further suggested this may lead to attenuation of hearing loss and vertigo episodes and decelerated progression due to reduced cell death.PDS is a rare disease, with an estimated patient population of around 4000 in Japan. The lack of treatment; however, motivates few patients to consult doctors regularly and willingly. Existing reports are limited to those based on limited data collected from outpatients during office visits. Use of existing historical control data on natural course of changes in hearing in PDS; therefore, is assumed to be insufficient for evaluating the efficacy in clinical research. Newly collecting and accumulating such data would thus contribute to evaluation of efficacy in this trial, and will further serve as basic information in preparing protocols for research to be conducted.This trial will collect a larger amount of information and more detailed data by measuring objective functions of the inner ear at patients’ homes, in addition to regular office visits, and explore what tests would enable the most precise evaluation. The trial is positioned as phase I/IIa clinical research aimed at safety analysis and exploratory assessment of efficacy, including endpoints.To explore safety, tolerability, and efficacy of NPC-12T for PDS/DFNB4, for which treatment has yet to be established.22.1To explore safety, tolerability, and efficacy of NPC-12T, 1 mg tablet of Sirolimus, for PDS/DFNB4, for which treatment has yet to be established.2.2This is a phase I/IIa randomized, double blind parallel-group single institute trial in patient with PDS/DFNB4. The trial was designed and will be independently conducted by the Keio University with approval from the ethics committee of Keio University School of Medicine in accordance with the principals of the Declaration of Helsinki. All analyses will be conducted by Keio University, independent of the sponsor, according to the prespecified statistical analysis plan. As a prospective randomized controlled trial, the study strategy will be constructed and presented in accordance with the recommendations of the Standard Protocol Items: Recommendations for Interventional Trials statement. For the detailed protocol please refer Supplemental Digital Content:.2.3Eligible patients are those who meet all the following inclusion criteria and who do not have any listed exclusion criteria at V0.2.4(1)Aged at least 7 and below fifty years at the time of consent.(2)∗ by genetic testing such as Sanger Sequencing.Confirmed PDS-positive(3)Report of subjective symptoms of fluctuating hearing disorder in a medical interview for case registration.(4)Subjective symptoms of episodes of hearing loss and/or dizziness/vertigo within 1 year before the case registration.(5)Had undergone inner-ear function testing at least 3 times, including standard pure-tone audiometry, performed with an interval of 1 day or longer, within 6 months before the case registration.(6)Voluntary informed consent in writing obtainable from the patient or the patient's legally acceptable representative.∗Any of the pathogenic homozygous or compound-heterozygous variant can be included.2.5(1)Prior use of molecular targeted drugs related to mTOR pathway, such as sirolimus, other mTOR inhibitors  and tyrosine kinase .(2)History of hypersensitivity to sirolimus, including sirolimus derivatives, and its additive agents.(3)Hepatitis B surface (HBs) antigen-positive, HBs antibody-positive, hepatitis B surface (HBc) antibody-positive, or active hepatitis C (hepatitis C virus (HCV) antibody-positive patients with inactive hepatitis and with normal-range values of liver function do not apply.) HBs antigen-positive patients for which the cause is presumed to be due to vaccination against hepatitis B may participate in this clinical trial.(4)3, absolute neutrophil count of below 1000/mm3, total white blood cell count of below 3000/mm3.Severe blood abnormalities or hepatic dysfunction: aspartate transaminase (AST) or alanine transaminase (ALT) of 100 IU/L or higher, hematocrits of below 30%, Platelet count of below 80,000/mm(5)Patients with poorly controlled dyslipidemia: serum triglyceride of 500 mg/dL or higher or low-density lipoprotein (LDL) cholesterol level of 190 mg/dL or higher despite being under treatment for dyslipidemia.(6)2.Severe renal impairment: estimated glomerular filtration rate lower than 30 mL/min/1.73 m(7)Immunodeficiency such as human immunodeficiency viruses or primary immunodeficiency.(8)Inability to take investigational products in tablet form, or gastrointestinal dysfunction with risks of malabsorption of sirolimus(9)History of having surgery within 8 weeks before the registration .(10)Female patients who are pregnant, potentially pregnant, or breastfeeding. Male or female patients who decline to consent to contraception during the trial. Contraceptive methods: combination of condoms and spermicide; combination of condoms and diaphragms with spermicide; oral contraceptive; intrauterine contraceptive device; or other methods.(11)Need to take medications that influence CYP3A4 activation after the administration of investigational products.(12)Pulmonary interstitial opacity.(13)Diagnosis by the attending physician during the screening phase or from previous observations from hearing tests that the hearing loss is fixed rather than fluctuating.(14)Bilateral cochlear implant users. Note that the unilateral users are not excluded.(15)Participation in other clinical research and/or trials within 6 months before the date of consent, with the exception of Analysis of Diseases in Otorhinolaryngology Using iPS Cells .(16)Other conditions that the principal investigator or subinvestigator deems inappropriate.2.6A sample size of 16 subjects is expected to be studied: 12 subjects for the NPC-12T active substance arm, and 4 subjects for the NPC-12T placebo arm.As this clinical trial will focus on a rare disease, and is exploratory, feasibility was the main aspect that was considered in setting the sample size. In terms of safety analysis, provided that clinically significant adverse events exist, the size is sufficient to detect adverse events of 15% incidence with 85% accuracy. In terms of efficacy analysis, when making a comparison of NPC-12T arms concerning hearing test endpoints of periods without treatment and with treatment, a difference can be detected at 80% power (2-sided 5% significance level), if the number of evaluable subjects is 11 and Cohen d for endpoints is 0.85. In estimation of correlation coefficient between evaluation indices, if data of 2 arms are integrated for evaluation, and the number of evaluable subjects having population correlation coefficient of 0.7  is 13, then a difference can be detected at a minimum of 80% power (2-sided 5% significance level).2.72.7.1The randomization manager will implement random assignment in accordance with the randomization procedure. When assigning NPC-12T placebo, the randomization manager will specify 3 types of dosage: reduction, increase, or no change in dosage. In specifying reduction or increase, specific visit(s) (V7–V11) when the change in dosage is to be implemented will be indicated. The randomization manager will generate and store a randomization schedule, which is to be concealed. The randomization manager will generate prescription schedules containing information on assignment for respective subjects and specification for change in dosage of NPC-12T placebo tablets. The information for respective subjects will be sealed separately. The prescription schedules will be stored by an unblinded doctor. The randomization manager will store the randomization schedule in strict concealment until the time when the blind is permitted to be broken. When the unblinded doctor makes judgment on dosage change for a subject based on trough concentration measurements (V7–V11), the doctor will unblind the prescription schedule for the subject in question and check the criteria for dosage change. The prescription schedule will be placed in concealment again immediately after the check. The unblinded doctor will keep a record every time the prescription schedule is unblinded or concealed. The randomization manager will generate 2 sets of emergency key codes when the assignment is made, which will be kept strictly in concealment by the randomization manager and the emergency contact center  until the blind is authorized to be broken.2.7.2The randomization manager will verify the indistinguishability of medications and packages before random assignment and after the end of the trial.2.7.3With regard to the blood concentration of sirolimus, any person involved in the trial, with the exception of the unblinded doctor and unblinded clinical research coordinators, will follow the procedures for prescription of investigational products to ensure blindness to the test results is maintained. The test results will be reported to the Independent Data Monitoring Committee and the principal investigator; however, after the blind is broken or if the principal investigator so directs in case of emergency. The unblinded clinical research coordinators will assist in the work of the unblinded doctor and make sure that the dosage change based on trough concentration measurements is appropriately instructed.2.7.4The randomization manager will conceal and store the randomization schedule (randomized code) after the random assignment, and unblind it after the database is locked and the trial is completed.Two sets of emergency key codes will be generated, which will be concealed by the randomization manager. The randomization manager and the emergency contact center  will keep them in strict concealment until the appropriate time when the blind is permitted to be broken. When a serious adverse event or other situation demands urgent need for the principal investigator or subinvestigator to access information on assigned treatment to determine treatment of the subject, or when discovery of subject's pregnancy requires revelation of assigned treatment, the principal investigator will determine whether the code should be partially unblinded. If the principal investigator deems it necessary to unblind the code assigned to the subject in question, the emergency contact center will be requested to allow partial unblinding of the randomization code (emergency key code). The partial unblinding of the emergency key code will be conducted in accordance with the procedure manual for unblinding.The emergency contact center will communicate the unblinded information to the principal investigator. If partial unblinding is performed, the principal investigator will document justification for unblinding and the extent to which the information is made available.2.8The principal investigator or subinvestigator will obtain written informed consent from patients who are thought to satisfy the inclusion criteria, or their legally acceptable representatives, and register the patients2.9The schedule for the trial visits and data collection is summarized in Figure Phase I (single-blind): NPC-12T placebo tablets for 3 months (12 weeks).Phase II (double-blind): NPC-12T active substance tablets or NPC-12T placebo tablets for 6 months (24 weeks).2.10(1)Schedule for tests, observations, and assessmentThe items to be covered in tests and observations, and the schedule of the trial are shown in Table (2)Subjects’ baseline informationSLC26A4 genetic testing will not be recorded on the electronic case report form.The principal investigator or subinvestigator will collect the baseline information listed below from each subject during the period between informed consent and before administration of the investigational products in V1 (D1), and record the information on the subject's electronic case report form. For the purpose of this protocol, an illness that has not subsided before administration of the investigational products is referred to as a concomitant illness, and one that has subsided as a past medical history. The results of (3)Concomitant medications and therapiesThe principal investigator or subinvestigator will examine concomitant medications and therapies from the screening phase (V0) to V13, and record the results on the electronic case report forms.(4)Clinical questionnaireThe principal investigator or subinvestigator will ask subjects to complete the questionnaire specified below (electronic Patient Reported Outcome (ePRO)) every day, in principle, from the screening phase (V0) to V13, and record the results on the electronic case report forms.Questionnaire on daily conditions: medication adherence, hearing loss episodes, vertigo episodes, tinnitus, ear fullness, concomitant medications, handicaps caused by dizziness/vertigo The principal investigator or subinvestigator will ask subjects to complete the Dizziness Handicap Inventory at every visit from the screening phase (V0) to V13 and at discontinuation, and record the results on the electronic case report forms.(5)Tests using portable testing devicesThe principal investigator or subinvestigator will provide subjects training on the portable devices and make adjustments to the devices during the screening phase (V0). Subjects will be asked to perform tests using the portable devices listed below from V0 to V13 every night, in principle. The test results will be collected at every visit from V1 to V13 and at discontinuation.Portable devices used are below:1.Portable audiometer: Audiometer AA-58 For standard pure-tone audiometry (threshold of 4 different frequencies for left and right ears) and recording data in ePRO. The frequencies include 500, 1000, 2000, and 4000 Hz.2.Wireless Frenzel scope: AirMicro Frenzel Wireless Scope For tests such as measurement of rotational eye movement, and direction and amplitude of nystagmus, the results of which shall be transmitted to an Android terminal for storage. The tests will be performed regularly in the evening and as appropriate when an episode occurs, as far as possible.3.Head stability test: JINS MEME ES_R For tests with open and closed eyes, each lasting around 30 seconds, the results of which shall be transmitted to an Android terminal for storage.(6) In-clinic testing and observationsVital signs, physical findings, general laboratory tests Table , thoraci(7)Exploratory tests and observations1.Collection of blood for generation of iPS cells and in vitro efficacy testing of sirolimus The principal investigator or subinvestigator will collect blood to be used for generation of iPS cells. Generation of iPS cells and in vitro efficacy testing using PDS-specific iPS cells will be performed at the Keio University School of Medicine.2.Thyroid ultrasound3.Magnetic resonance imaging (MRI) MRI scan of the inner ear will be performed during phase II and at discontinuation for subjects aged 16 or older with consent to the test, 4 hours after intravenous injection of contrast media.4.Thyroid cytology test Cytology tests of thyroid for an exhaustive gene expression analysis in phase II (V5 and V12) and at discontinuation for subjects with consent(6) Adverse events1.Definition of adverse eventsAn adverse event in this trial refers to any untoward medical occurrence in a subject from initial administration of investigational products (V1) to follow-up observations (V13). The event may not necessarily have to have a causal relationship with administration of investigational products in this trial.2.Definition of serious adverse eventsSeriousness of adverse events is categorized as follows:1)Nonserious: cases other than 2)2)Serious: events for which any of the items from (a) to (g), below, apply. (a) Death, (b) Cases that might result in death, (c) Any case that requires hospitalization for treatment or prolongs the duration of hospitalization, (d) Disability, (e) Cases that might result in disability, (f) Other medically serious condition for which seriousness is equivalent to (a) to (e), above (g) Any congenital diseases or abnormalities in the next generation2.11Assessment of efficacy in this trial is regarded as a secondary objective. Following will be examined.2.11.1(1)Frequency of hearing loss episodes (number of occurrences per year).(2)Highest measurement of auditory threshold shift at a hearing loss episode (dB) Definition of hearing loss episodes: Hearing loss episodes with subjective symptoms and episodes detected as audiometric data will be counted as hearing loss episodes in this trial. Concerning audiometric data, audiometric testing of 4 different frequencies for right and left ears will be performed, and the average of measurements obtained at 5 recent time points will be defined as the baseline. A change of 10 dB or more observed in the testing period including the screening phase will be identified as an episode. The period from the beginning of the episode until the condition returns to the baseline will be counted as 1 episode. Episodes observed in left and right ears or in different frequencies concurrently will be considered as separate episodes.(3)Frequency of dizzy spells (number of occurrences per year) Dizzy spells in which the subject is conscious are to be counted.(4)Maximum amplitude (°/s) and maximum frequency (cycle/s) of nystagmus in a hearing loss episode(5)Percentage of cases in which increase in hearing threshold was observed during remission in the observation period (comparison of measurements at the end of phase I and the end of phase II).(6)Improvement in threshold during remission.(7)Shorter duration of hearing loss episodes.(8)Alleviation of symptoms of dizziness/vertigo Changes in total scores of the Dizziness Handicap Inventory from phase I to phase II.(9)Percentage of cases in which equilibrium exacerbated during remission in the observation period (comparison of measurements at the end of Phase I and the end of Phase II).2.11.21.Reduction in endolymphatic hydrops (MRI findings).2.Inhibition of goiter or thyroid enlargement.3.Cell biological changes in thyrocytes detected by cytology tests.4.Comparison of results of in vitro efficacy evaluation of inner ear cells derived from PDS-specific iPS cells and of clinical evaluation.5.Deviation from threshold value for frequency of abnormalities of auditory brain stem responses and for pure-tone audiometry.2.12Primary endpoints are safety and tolerability. The number of occurrences and types of adverse events and of side effects will be sorted by clinical symptoms and by abnormal change of clinical test results. A 2-sided 95% confidence interval of the incidence rate by respective dosing arms will be calculated using the Clopper–Pearson method.t test will be used for comparison between actual measurements and baseline values at each point in time for each dosage arm. The 2-sided 95% confidence interval for average change for each dosage arm will then be estimated. Using a 2-sample t test, the change or percent change at each point in time between the 2 arms will be compared. The 2-sided 95% confidence interval for average difference between the 2 arms will be estimated. If the data is found to be non-normal, use of nonparametric methods and statistical methods based on probability distribution other than normal distribution will be considered. With regard to binary endpoints, a contingency table will be compiled for each point in time, and Fisher exact test will be applied in making comparison of the distribution in the 2 dosage arms. The Clopper–Pearson method will be applied to estimate the 2-sided 95% confidence interval for each dosage arm. The difference in the 2-sided 95% confidence interval will be estimated using the normal approximation.Assessment of efficacy in this trial is regarded as a secondary objective. With regard to continuous endpoints, summary statistics of changes or percentage changes based on actual measurements and from baseline at each point in time will be calculated for each dosage arm. A 1-sample 2.13Patients or the public were not directly involved in the research. Nevertheless, the main results will be made available in the public domain.2.14Participant recruitment began in May 2018. The final results will be published in international peer-reviewed medical journals.3PDS is a disorder with fluctuating and progressive hearing loss, vertigo, and thyroid goiter and there is so far no rational treatment. We identified pathophysiology of a neurodegenerative disorder that involves protein aggregation in PDS patient derived cochlear cells that were induced via iPSCs and found an mTOR inhibitor sirolimus as a potential therapeutic target. Also, we identified minimum effective concentration less as an inhibitor of cell death by using iPSCs derived disease cells, with the than 1/10 of the approved dose for other diseases. So far as we searched, this is the first study starting clinical trial with the initial Phase I/II trial defined by iPSCs based preclinical proof of concept study. The result will strengthen the concept of iPSCs based drug development itself if we obtained good outcomes. The optional expiratory trial examining correlation in between in vitro effect and actual patient's’ clinical effect may also support the idea.Daily monitoring for audio-vestibular symptoms will be carried out. The result will offer natural time-course of fluctuation in patient with PDS, which has never been reported. Also, the system itself can be applied for other hearing and balance disorders. In addition, episodes in between the patients’ visits, including traumas, are asked carefully in every visit by the coordinators and investigators. In each visit, first the investigators briefly review the data-history collected at home in between visits. Then they ask patients’ daily life-events, especially those at the specific days when the hearing loss and vertigo was documented. We believe the Internet of Things (IoT)-based data collection system may facilitate medical staffs understand the clinical features of individuals more efficiently and accurately.Limitation of the study is that we will only examine patients above 7-year-old because performing audiological tests would be difficult for the younger ages. PDS is a genetic disorder and hearing loss develops from newborn. Further clinical trials will have to be awaited in the future to know the early stage development of PDS.The authors thank Takayuki Abe, former staff member of Department of Preventive Medicine and Public Health, Keio University School of Medicine, Tokyo, Japan for statistical analyses and Yasuko Ochi a member of the Department of Otorhinolaryngology in Keio University School of Medicine for their help in the preparation of this manuscript.Masato Fujioka wrote the manuscript. Masato Fujioka, Takumi Akiyama, Makoto Hosoya, Kayoko Kikuchi, Yuto Fujiki, Yasuko Saito, Keita Tsukada, Hiroyuki Ozawa, Tomonobu Hasegawa,Kaoru Ogawa planned the trial protocol. Masato Fujioka, Keisuke Yoshihama, Shin-Ya Nishio, Shin-Ichi Usami, Tatsuo Matsunaga, Yasunori Sato evaluated basic information regarding genetic tests and calculate affordable sample size. YS planned statistical analyses.


GJB2 c.235delC homozygotes associated with post-lingual and/or milder hearing loss, and explore the possible mechanism of these unconditional phenotypes.To report the phenotypic heterogeneity of GJB2 was performed on all ascertained members from Family 1006983 and three sporadic patients by polymerase chain reaction (PCR) amplification and Sanger sequencing. Next generation sequencing (NGS) was successively performed on some of the affected members and normal controls from Family 1006983 to explore additional possible genetic codes. Reverse transcriptase–quantitative PCR was conducted to test the expression of Connexin30.Mutation screening of GJB2 c.235delC homozygous mutation, affected members from which had post-lingual moderate to profound hearing impairment, and three sporadic patients with post-lingual moderate hearing impairment, instead of congenital profound hearing loss. NGS showed no other particular variants. Overexpression of Connexin30 in some of these cases was verified.We identified a Chinese autosomal recessive hearing loss family with the GJB2 c.235delC homozygotes are not the most common phenotype, revealing the heterogeneity of GJB2 pathogenic mutations. To determine the possible mechanism that rescues part of the hearing or postpones onset age of these cases, more cases are required to confirm both Connexin30 overexpression and the existence of modifier genes.Post-lingual and/or moderate hearing impairment phenotypes of  GJB2, which encodes the gap junction protein Connexin 26, is the most common cause of genetic non-syndromic hearing loss (NSHL). Among the approximately 200 hearing loss associated GJB2 mutations  in the inner ear cochlea. HCs mainly function in transducing sound waves into the electric signals . Hearingutations , the c.2utations , is the utations . The mututations , but no utations . Most papairment . Howeverat onset . ClinicaGJB2, GJB6 is another gene located in DFNB1. GJB6 encodes the gap junction protein Connexin 30, which is co-assemble with Connexin 26 to form hybrid gap junctions  and association studies (unrelated patients) are two strategies can be adopted to identify modifier genes for human disorders . Howeverier gene .GJB2 c.235delC homozygotes, whose phenotypes were late-onset and/or moderate hearing loss. First, we found a Chinese family (Family 1006983) with the GJB2 c.235delC mutation, which was segregated with all of the recruited members. Unlike other patients with the GJB2 c.235delC homozygous mutation, whose phenotype were pre-lingual severe to profound hearing loss, patients in this family showed post-lingual moderate to profound sensorineural hearing loss. In addition, we further identified three sporadic cases with GJB2 c.235delC homozygotes, the phenotypes of which were post-lingual moderate hearing impairment. To decipher the mechanism of partial or late-onset hearing loss in GJB2 c.235delC homozygotes cases, we performed next generation sequencing (NGS), including targeted genes capture and genome sequencing on some of the affected members and controls from 1006983, but no pathogenic variations or modifier genes were identified. Since Connexin26 and Connexin 30 are co-assembled to form hybrid gap junctions and a previous mouse experiment suggested that up-regulation of Connexin or the slowing of its degradation might be a therapeutic strategy to prevent and treat deafness caused by Connexin 30 mutations  was also conducted on some of these patients to test the expression of Connexin30, which is encoded by GJB6  with 133 members presenting with autosomal recessive non-syndromic sensorineural hearing loss (ARNSHL) was ascertained from the Institute of Otolaryngology, Chinese PLA General Hospital , 2. Threpairment . PersonaGJB2 c.235delC mutation in our institute were chosen as the controls to compare phenotypes (A cohort of two hundred and ninety-five hearing loss cases who had the homozygous enotypes .GJB2-Exon2-F: 5′-TTGGTGTTTGCTCAGGAAGA-3′ and GJB2-Exon2-R: 5′-GGCCTACAGGGGTTTCAAAT-3′).Genomic DNA was extracted from the whole blood samples using a Blood DNA kit according to the standard protocol . PCR and Sanger sequencing were performed on all available members from Family 1006983 and the three sporadic patients and their parents to determine whether the potential mutations in causative genes co-segregated with the disease phenotype in these families. Direct PCR products were sequenced using Bigdye Terminator v3.1 Cycle Sequencing Kits  and analysed using an ABI 3700XL Genetic Analyser . Synthesis of cDNA was carried out with a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Primers were designed as follows by Primer5: GAPDH-H-F: GGAGCGAGATCCCTCCAAAAT; GAPDH-H-R: GGAGCGAGATCCCTCCAAAAT; GJB6-H-F: CAAGAGGACTTCGTCTGCAAC; GJB6-H-R: GTGGTTTCGTGCCTGTAGTAG. RT-qPCR was conducted according to the protocol of the “Power SYBR Green PCR Master Mix and RT-PCR” by Applied Biosystems. The quantitative analysis conditions were shown in In Family 1006983, a total of 22 family members, composed of five clinically affected and 17 unaffected individuals were ascertained in this study. In this family, most of the affected members showed post-lingual, approximately symmetrical, and bilateral non-syndromic sensorineural hearing loss. The average onset age for all the affected cases was 6.2 years old. The hearing loss was presented at all frequencies . For theGJB2 c.235delC mutation, four patients had late-onset hearing loss, an incidence of 1.36% (4/295) (Among the 295 cases with the homozygous  (4/295) . The onsThe c.235delC is a known pathogenic ARNSHL related mutation. Sanger sequencing confirmed the co-segregation of c.235delC with the disease phenotype in Family 1006983, the three sporadic patients and their parents . All of A total of 307 hearing loss related genes were captured, and the targeted high-throughput sequencing data were analysed according to the standard process  and no pGJB2 were analysed emphatically. Third, copy number variants (CNVs) of genes interacting with GJB2 gene were analysed1 by selecting the 20M gene sequence before and after the target genes, with no related CNV were deciphered. Last, related genes located in the GJB2 signal pathway were determined  and had no significant difference from that of normal controls, suggesting that GJB2 c.235delC has no correlation with sudden hearing loss .Case 2 complained of sudden hearing loss of the left ear when he came to our clinic on 14th September, 2015. In recent years, some studies have proposed that genetic deafness mutations may be associated with the pathogenesis of sudden hearing loss. Bora et al. once screened the verified . In the ing loss . In our GJB2 c.235delC is the most common deafness-related pathogenic variant in the Chinese population, and reporting the phenotypic heterogeneity of GJB2 c.235delC homozygous cases is helpful throughout the genetic counselling procedure, and would contribute greatly to clinical practice. Except for providing information about incidence, recurrent risk and prevention strategies, phenotype heterogeneity should be accounted both before and after gene testing. For example, information if provided on the possible gene testing results and relevant phenotype prognosis before gene testing, phenotype heterogeneity should be taken into consideration; for the management of an individual with GJB2 c.235delC homozygous mutation after gene testing, especially for a baby who has passed newborn hearing screening, audiology follow-up seems to be much more critical than performing cochlear implantation directly. Gene therapy as a potential clinical treatment method have been reported to be efficient in inherit hearing loss in animal models  for the publication of any potentially identifiable images or data included in this article.HW and YG conceived and designed the experiments. HW, YG, JG, LL, and JY performed the experiments. HW, JG, WX, LX, and LY analysed the data. JY and CZ contributed reagents, materials, and analysis tools. HW wrote the manuscript. DW and QW performed the critical reading and discussion of manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The intention of this paper is to analyze the properties of coral aggregate concrete (CAC) that is reinforced by alkali-resistant glass fibers (ARGF) and the bond performance with BFRP  bars. Two types of ARGF, denoted by Type A and Type B with different manufacturing technologies and fiber lengths, are used in the test. Tests of compressive strength, splitting tensile strength, and flexural performance were performed on ARGF-CAC with four different contents for the two types of ARGF. It is found that the cubic compressive strength is slightly reduced when the fiber volume fraction exceeds 0.5%, but almost keeps invariable if the fiber content further increases. However, the tensile strength, residual strength retention and flexural toughness are improved as more ARGFs are added into CAC, and even higher with Type B ARGF addition. The optimized volume fraction is 1.5% for both the two types of ARGF based on the evaluation of the workability and mechanical performance. Moreover, central pull-out test was performed to study the bond properties of ARGF-CAC with BFRP bars. It is found that both the maximum average bond stress and residual frictional stress are generally reduced as the bond length is longer. The addition of Type B ARGFs can significantly improve the bond strength; however, the Type A ARGFs seem to have marginal effect. Portland cement, river sands, and crushed stones are essential components in conventional concrete. However, a large amount of COoduction . Naturaloduction ,4. Besidoduction , construoduction  can be roduction . MoreoveIn some islands near tropics, such as South China Sea, there are abundant coral reefs. They can be utilized in a rational manner if no damage is ensured in local ecologies. Subsequently, it is feasible to use the crushed coral reefs as aggregates in concrete production . As the Furthermore, it is found that the failure characteristics of CAC are more brittle than those of ordinary concrete ,23,24,25Although the ARGF is widely used in ordinary concrete, studies on ARGF reinforced CAC are relatively few to our best knowledge. Lu et al.  analyzed3 and 1236 kg/m3 for coral sands, and 1899 kg/m3 and 918 kg/m3 for coral coarse aggregates (CCA). Coral sands have a continuous gradation of 0–5 mm and coral coarse aggregates have two continuous gradations of 5–10 mm and 10–20 mm, respectively. In the present study, ARGF reinforced CAC (ARGF-CAC) is mixed by seawater, cement, ground granulated blast furnace slag (GGBFS), Class F fly ash (FA), metakaolin (MK), coral aggregates, and ARGF according to the literature . All of 3. The two types of ARGF are seen in Two types of ARGF are used in the study and obtained from Tanshan Glass Fiber Limited Company. One of them is Cem-FIL 62 ARGF (denoted by Type A ARGF hereafter) with the length of 18 mm and elastic modulus of 72 GPa. It is bound by 200 fiber filaments with diameters 14 μm. The other one is HP-36 ARGF (denoted by Type B ARGF hereafter), which is bound by 600 fiber filaments with diameters 19 μm. It has the length 36 mm and elastic modulus 72 GPa. The densities of all the ARGFs are 2680 kg/mBFRP bars are adopted to study the bond performance with ARGF-CAC. All of the bars have nominal diameters 8 mm and shallow ribs on the surface. It should be noted that the ribs are formed by detaching a demoulding belt, which is initially wound on the smooth surface of each bar. The detailed sizes of rib width and spacing for BFRP bar are shown in It should be noted that the two types of ARGF that are provided by Taishan Fiberglass Inc. have good dispersion properties in the paste. Therefore, relatively large variation ranges of fiber contents are considered in the present study. For each type of ARGF, four different fiber contents, i.e., 0.5%, 1%, 1.5%, and 2%, in the form of volume fractions, are designed. The CAC with no fiber reinforcement is adopted as the control concrete. Moreover, Małek et al.  pointed All of the CCAs were pre-immersed in seawater for 23 h and left in dry condition for 1 h before they were prepared in mixing concrete. SJD-30 mix machine with the maximum range 33 L was used in order to mix the concrete. The rotation rate of the mixer is 45 r/min. During the mixing process, all of the pre-immersed CCAs, coral sands, and cementing materials were first mixed for 30 s in the machine. The seawater and PS were then poured into the solid components. Moreover, the glass fibers are gradually added after the mixture was mixed for 120 s. The fresh concrete is stirred sufficiently with the fiber addition, so that the fibers can be uniformly dispersed in concrete. The whole mixing time lasts about 6 min. The mixing process in the present study is similar to that by Małek et al. . When thAll of the mix was poured into the moulds for the test specimens, as mentioned in The slump values of fresh concrete were determined using a slump cone with the top and base diameters 100 mm and 200 mm, respectively, and height 300 mm, as per BS EN 12350-2 .The dust samples were extracted from the inner regions of the hardened concrete (about 30 mm from the exposed surface), and then dissolved in distilled water, as per RILEM TC 178-TMC recommendations . The pH 3 are adopted in order to determine the cubic compressive strength fcu and three cubic specimens with sizes of 150 × 150 × 150 mm3 are used to determine splitting tensile strength fts. fcu can be expressed byuF is the maximum applied load at failure and A is the cross-sectional area of the specimen.According to Chinese standard GB/T 50081 , three cuF is reached, the tsf is given byA is equal to the square of side length of the cubic sample. It should be noted that the specimens in the splitting tensile strength test, as per BS EN 12390-6 , are cyl3 and spans 300 mm for Type A ARGF-CAC. The width, depth, length, and span are 150 mm, 150 mm, 550 mm, and 450 mm, respectively, for Type B ARGF-CAC beams. The bending test is performed on an electronic universal testing machine with the maximum range of 100 kN as shown in The test of four-point-bending beams is carried out to evaluate both the flexural tensile strength and toughness of ARGF-CAC according to ASTM C1609/C1609M . The widfr, residual strengths S/600 and S/150 (S is the span length of beam), respectively, beam toughness S/150), and equivalent flexural strength ratio The flexural performance can be comprehensively analyzed by virtue of modulus of rupture b, h, and F1 are the beam width, beam height, and first peak load, respectively. f1 is the first-peak strength corresponding to F1 and equal to fr if no first peak load can be detected in the load-displacement curve.Herein, 3 are performed in order to study the bond properties between the two materials [d, 7.5d, and 10d (d is the diameter of BFRP bar), are designed with five samples for each length. The bond length is controlled by using PVC tubes at two sides of steel mould, as shown in Pull-out tests of BFRP bars from ARGF-CAC with sizes of 150 × 150 × 150 mmaterials . OptimizThe electronic universal testing machine with the maximum range of 100 kN is adopted in order to perform pull-out test of BFRP bar from CAC, as shown in The slump values are 115 ± 5 mm for each type of ARGF reinforced concrete. No segregation and bleeding are found in all the fresh mix. The measured pore solution pH value range is 11.8–12 and the addition of ARGFs has hardly the effect on the pH value.fcu varying with fiber contents indicating the maximum and minimum values for the two types of ARGF. When the fiber content is below 0.5% by volume, the fcu almost keeps invariable for Type A ARGF, but shows a slight increase for Type B ARGF with the increasing of fiber content. However, when the fiber volume fraction exceeds 0.5%, the average values of fcu are slightly reduced, as indicated by Sivakumaret al. [fcu of Type A ARGF-CAC almost keeps constant, but the fcu of Type B ARGF-CAC shows a small increment. In fact, the fibers distribute randomly in the concrete and may not be across the longitudinal cracks that are induced by the uni-axial compression. Moreover, even if some of the fibers just distribute across the cracks, the bridging action is so limited due to the insufficient anchorage length when the fibers are very short. If the fiber is long enough, such as Type B ARGF with the length of 36 mm, the bridging effect becomes stronger as the fiber content increases. Thus, the fcu is slightly increased when the fiber volume fraction is larger than 1.0% for Type B ARGF-CAC. In summary, the maximum reduction (%) of the average fcu is 10.5% and 11.7% for Type A ARGF-CAC and Type B ARGF-CAC, respectively, as compared to plain CAC.aret al. . As the fts increases with the fiber content as shown in fts shows marginal variation with the increasing of fiber content. As the fiber volume fraction further increases, the fts is significantly increased and the maximum increment (%) is 18.4% and 27% for Type A ARGF-CAC and Type B ARGF-CAC, respectively, compared to plain CAC. It demonstrates that both the two types of ARGFs can provide good resistance to crack propagation in the concrete under splitting tension. Moreover, when the fiber volume fractions are the same, the fts is generally improved as the fiber length is longer.The average splitting tensile strength F varying with the net displacement Δ at the mid-span of beam for both the two types of ARGF. It can be seen that the F increases almost linearly up to a critical load with the increasing of Δ where the cracks initiate from the bottom of beam. A small non-linear portion is observed in each curve until the maximum load is reached. No first peak load is found in all of the specimens. Subsequently, the F gradually decreases with the increasing of Δ until the beam fails. Moreover, more than 80% of fibers are found to be fractured finally in the critical cross-section, irrespective of fiber type. It means the two types of ARGF used in the study have good bond performance with the CAC.As the fiber content increases, the peak load is improved due to the stronger bridging effect. Stable descending process can be detected in ARGF-CAC beams. When the fiber volume fraction attains 1.5%, the absolute value of descending slope seems to be the lowest. It demonstrates that the addition of the two types of ARGF can significantly improve the strength and toughness of CAC. However, when the fiber content is larger than 1.5% by volume, the mixing becomes too difficult to be sufficient, as indicated in fr, fr, Moreover, the variations of According to the above analysis in the present study, the optimized fiber volume fraction for the two types of ARGFs should be 1.5% by considering actual mixing process of fibers in fresh concrete. Thus, the following section will be aimed at the bond performance between BFRP bars and ARGF-CAC with fiber volume fractions 1.5%. Moreover, the specimens of BFRP bars that are bonded to plain CAC are used for the control ones.L may be varied, even for the same nominal bond length . Thus, the average bond stress τavg determined by Equation (5) is introduced for the sake of comparison. τavg-δ curves under different conditions. Moreover, the specimens have the failure modes of BFRP bar pull-out from the concrete in general. Only three specimens of plain CAC bonded with BFRP bars having nominal bond lengths 10d fail by concrete fracturing before the bars are pulled out.As discussed above, the present study is only aimed at the bond properties between BFRP bars and ARGF-CAC with volume fractions 1.5%. Control specimens are prepared for the bond behavior of BFRP bars with plain CAC. The intention of this section is to compare the pull-out behaviors of BFRP bars from CAC with different fiber types and different bond lengths. However, it should be noted that the actual bond length τavg-δ curve of each specimen still includes four stages. At the beginning of loading, the F or τavg increases with no slip until the frictional stress τs is exceeded. Subsequently, the τavg is further increased with the increasing of δ, but the ascending slope becomes lower. When the maximum value is reached, the applied load is gradually reduced, but the δ still increases. The final part of each curve is a fluctuation line that is also verified in the bond behavior between FRP bar and alkali-activated slag seawater sea sand concrete [τs is then adopted as the average value between the maximum and minimum values at the frictional stage.Very similar to the pull-out behavior of FRP bar from CAC  and alkaconcrete . In factτavg-max and residual frictional stress τs with the nominal bond length being shown in τavg-max and τs in each group are also included in τavg-max and τs commonly decrease. The reasons can be explained, as follows. First, it is acceptable that the non-uniformity of interfacial stress distribution becomes more significant as the bond length increases. It then results in the reductions of the two parameters with the increasing of L. Second, the BFRP bars used in the test have shallow ribs on the surfaces. As the bar is pulled out from the concrete, the ribs are seriously detached from the BFRP surface, as shown in τavg-max and τs for specimens with L = 5d are larger. Besides, radial compression on the bar-to-concrete interface provided by the ribs is inevitable. It may be so strong that the concrete would be cracked along the radial direction. The longer the bond length is, the larger the crack opens. The concrete would be completely fractured before the bar is pulled out if the L is long enough, such as some of the specimens with nominal bond lengths 10d. The τavg-max and τs are reduced as the radial cracks develop in the concrete. Moreover, the values of τs from different specimens are relatively scattered, because the detachment levels of BFRP surfaces are different along the embedment length.The variations of maximum average bond stress τavg-max shows a certain increment. It means that the ARGFs can provide good bridging action on the crack development in the concrete. However, for Type A ARGF, the increase is marginal. If the Type B ARGF is used, the τavg-max is significantly improved. When the L is increased from 7.5d to 10d, the reduction of τavg-max is apparently lower for ARGF-CAC. It demonstrates again that the development of cracks in the concrete is well restricted with the addition of ARGFs. Moreover, the Type A ARGF has a marginal effect on the τs, but the incorporation of Type B ARGFs apparently increases the τs.When the ARGFs are added into the CAC, the d, 7.5d, and 10d) are considered for each type of concrete. The main conclusions are then drawn, as follows.The present study is mainly aimed at the mechanical properties of ARGF reinforced CAC. Two types of ARGF, denoted by Type A and Type B, are used here. Tests of basic mechanical properties and flexural performance are carried out on both the plain CAC and ARGF-CAC with four contents by volume, i.e., 0.5%, 1.0%, 1.5%, and 2.0%, for the two types of ARGF. The optimized fiber volume fractions for both types of ARGF are determined based on evaluating both the workability and mechanical properties of ARGF-CAC. Moreover, a central pull-out test is performed in order to study the bond performance between BFRP bars and both the plain CAC and ARGF-CAC with the optimized fiber volume fraction. Three nominal bond lengths (5(1)fcu increases as the fiber volume fraction is increased to 0.5%, especially for Type B ARGF. When the fiber volume fraction exceeds 0.5%, however, the average values of fcu are slightly reduced for either Type A or Type B ARGF. As the fiber content further increases, the fcu of Type A ARGF-CAC almost keeps constant, but the fcu of Type B ARGF-CAC shows a small increment. But the fts and fr are generally improved with the increasing of fiber volume fraction.As the fiber content increases, the workability of fresh concrete becomes weaker. The average value of (2)Both the residual strength retention and flexural toughness are improved, as more ARGFs are added into CAC, and higher with Type B ARGF addition when compared to Type A ARGF. The improvement becomes smaller when the volume fraction of ARGF exceeds 1.0% for either Type A or Type B ARGF. The optimized volume fraction is 1.5% for both the two types of ARGF by considering the workability and mechanical performance of ARGF-CAC.(3)d are damaged by concrete fracturing before the bars are pulled out. When the bar is pulled out, the ribs on the surface are seriously detached.More than 90% of the specimens have the failure modes of BFRP bar pull-out from the concrete. Only three specimens of plain CAC bonded with BFRP bars having nominal bond lengths 10(4)τavg-max and τs are reduced if L > 5d. The values of τs show relatively large scatters due to the random detachment of ribs on the BFRP surface. When the Type B ARGFs are added into the CAC, the τavg-max is increased significantly. However, the Type A ARGFs seem to have marginal effect on the bond performance.Both the


Deaf and hard-of-hearing adolescents (DHH) experience more peer problems and lower levels of friendships than their hearing peers. This study used a qualitative approach to identify their experiences of peer problems and factors influencing them. A sample of 30, 13–19 year-old DHH adolescents with a moderate to profound hearing loss, drawn from a population-based cohort study in which their receptive language and social–emotional skills had been assessed, underwent semi-structured interviews. Interviews were analyzed using thematic analysis. Participants reported that, overall, they had developed positive and rewarding relationships with their peers, notwithstanding their earlier experience of being bullied. Conflicts and infrequency of interaction in their friendships were mainly reported by girls. Adolescents with moderate hearing loss were identified as facing the same or even more barriers than adolescents with severe to profound hearing loss in making new friends. Implications for educational practice are discussed. Peer relationships are important predictors of academic and life skills in children and young people . Peer reThe current study examines how a heterogeneous group of DHH adolescents experience peer relationships and friendships. A distinction is made here between peer relationships and friendship, with the latter being a subtype of peer relationships. Peer acceptance and popularity define peer relationships, whereas friendship is defined as a close, mutual, dyadic relationship . The peeIn the context of the global increase in inclusive education, recent research has focused on the interactions of children who are DHH with their hearing peers. One longitudinal study reported that, in spite of being no different from hearing adolescents with regard to social skills and behavior, DHH adolescents felt less secure than their hearing peers and faced more difficulties in making friends . SimilarA recent systematic review and meta-analysis of 45 studies identified higher rates of emotional and behavioral difficulties in DHH children and young people compared to hearing peers . Across A subtype of peer relationships is friendship. Friendships are dyadic and reciprocated relationships with peers characterized by shared enjoyment, mutual liking, commitment to each other and closeness . FriendsStudies on the quality of friendship among DHH adolescents are scarce and report inconsistent results. A small study investigating the effect of quality of friendship on the well-being of 12 Dutch DHH 13–19-year olds, found that, compared to their hearing peers, DHH adolescents scored lower on positive qualities of friendships, such as intimacy and companionship . SimilarTaken together, the few studies that compare the quality of friendship of DHH adolescents with that of their hearing peers suggest that DHH peers tend to report lower levels of positive features in their friendships but that friendship quality in DHH children should be considered in the context of their educational setting and their social-emotional skills.Studies exploring the relationships between gender and peer relationships of DHH adolescents are scarce and show mixed results. For example, a study exploring the effect of gender on peer acceptance and popularity of DHH adolescents using sociometric measures found that DHH girls were more popular and more accepted by their hearing peers compared to DHH boys , whereasIt is expected that with the advances in technology  and early diagnosis, the degree of unaided hearing loss might not have an impact on peer relationships. Two studies  exploredThere is growing evidence on the effect of CIs on the spoken language outcomes of DHH adolescents and on their social-emotional skills . StudiesA recent study  using thFunctional communication skills are dependent on language development and on experiencing models of good communication around the developing person . CompareFurthermore, adolescents in mainstream schools with superior levels of spoken language and those in segregated educational settings with superior skills in sign language demonstrated fewer peer problems than their less skilled DHH peers . A longiOne key factor in peer interaction and the preference of DHH adolescents for DHH or hearing friends is the school placement . EarlierOur review of the literature indicated that DHH adolescents face more difficulties in peer relationships compared to their hearing peers and tend to report lower levels of positive features in their friendships. Using qualitative methodology, the present study explores how adolescents themselves view their relationships with peers and provides insight into the perceptions of peer problems as reported by DHH participants. DHH adolescents are a heterogeneous group. It is perhaps for this reason that previous studies revealed inconsistent results on the effect that individual characteristics  and contextual factors  have on their peer relationships. To better understand the peer relationships and quality of friendship of DHH adolescents in relation to the adolescents’ individual characteristics and educational setting, the present study focused on the views and experiences of DHH adolescents themselves. This study aimed to develop a deeper understanding of the experience of their peer relationships and the quality of those relationships using semi-structured interviews in a representative sample of DHH adolescents. In addition, this study aimed to explore how the participants’ characteristics  contribute to their experience of peer relationships. Identification of the positive and negative experiences of peer relationships and friendships of DHH adolescents has the potential to inform the selection of those interventions more likely to be effective in supporting DHH adolescents’ social relationships and overall well-being. We elicited the DHH adolescents’ views to address three research questions:How do adolescents who are DHH experience their relationships with peers and friends?What are the positive and negative qualities of their friendships?How do the DHH adolescents’ characteristics contribute to their experience of peer relationships?Participants of the current qualitative study were selected from a sample of DHH adolescents who had taken part in a wider quantitative study, which investigated language, reading, emotional, and behavioral well-being, and health economics in 76 DHH adolescents and 36 of their hearing peers drawn from a population-based birth cohort from Wessex and Greater London regions of the United Kingdom . FifteenWe classified participants as adolescents who use spoken language and those who use sign language. Six out of seven British Sign Language (BSL) users in the 2017 study participated in the present study. This coincided with our planned purposive selection of a sufficient number of signers to have a reliable indication of signers’ experiences of peer interaction, which we expected to be distinct from that of spoken language users because of their greater likelihood of self-identification with the Deaf world .Semi-structured interview on peer relations and friendship. Data were collected using semi-structured interviews that enabled participants to provide their own interpretation of peer interactions and to explore issues, which might not have been included in a predevised (structured) interview schedule . Questions that were unclear to the pilot study participants were rephrased. Further questions were added based on themes that emerged during the pilot interviews.SDQ. The SDQ is a quantitative measure of emotional and behavioral health , severe (70–94 dB HL), or profound (≥95 dB HL) according to four-frequency averaging of the pure-tone thresholds at 0.5, 1, 2, and 4 kHz in the better ear.Receptive language. Receptive vocabulary skills were quantified using a Receptive Language Composite standard score (RLC) calculated as the mean of standard scores on the Test of Reception of Grammar (TROG) (r (TROG)  and the r (TROG) . These sDHH adolescents with no additional medical conditions who had participated in the 2017 study were asked at that time if they were interested in being interviewed. The interviews were conducted either by the first author (an experienced teacher of the deaf) or by a research assistant , both of whom were trained in qualitative methods. At each interview, there was always a participant and one interviewer, except with BSL users when a BSL interpreter was present as a third party. Written consent for participation was obtained from the adolescents and, if they were younger than 17 years of age, from their parent or guardian. The study was approved by the Southampton and South West Hampshire Research Ethics Committee. Adolescents were assured of the anonymity of the interview content and informed that they could stop their interview if they felt upset or if they did not wish to continue at any time.Interviews were conducted using each adolescent’s preferred mode of communication (spoken English or BSL) and were audio-recorded (see below for procedure to achieve this for the BSL users). Although video recording was considered, it became clear from conversations held in the course of the 2017 study that potential participants were reluctant to be video-taped. We, therefore, deemed that video recording was not appropriate due to the personal and sensitive nature of the conversations.For the BSL users, a communication protocol was based on the following practices as described by https://www.qsrinternational.com/nvivo/what-is-nvivo) to assist with data handling, organization, and coding. Thematic analysis was used to identify, analyze, and report themes within data following six steps: (a) familiarization with the data, (b) generation of initial codes, (c) search for themes, (d) review of themes, (e) defining and naming themes, and (f) report writing . Transcribed interviews were entered into NVivo10  of the interviews. Two independent raters not involved in the data collection, a psychologist ([JK]), and a speech and language therapist (SW), both experienced with the deaf community as researchers and practitioners, re-coded all (SW) or 20% (JK) of the interviews. Inter-rater reliability was calculated in NVivo10 for each piece of coding (themes) and for each transcript. Inter-rater agreement was assessed in NVivo10 by calculating the percentage agreement  between raters for each individual combination of coding and transcript, as recommended by 2  = 6.5, P = 0.03) than was the case among 2017 study participants that were eligible but did not participate in the present study. There were, however, similar percentages of participants and eligible non-participants with severe/profound hearing loss who used spoken language. Twenty-one (70%) participants wore digital hearing aids and nine (30%) were fitted with unilateral or bilateral CIs (Participants ranged in age from 13.7 years to 19.3 years with a mean (SD) age of 16.6 (1.37) years with equal numbers of males and females . Six outeral CIs .2  = 0.91, ns).Eighteen (60%) participants were educated in mainstream settings , 8 (27%)N = 24) in the present study did not differ significantly from the spoken language users (N = 30) who did not participate with respect to their self-ratings on SDQ scales or subscales  (t (54) = 1.13, ns) with a mean score of 90.5  a standard deviation of 12.54 and range of 62 to 106 (The DHH spoken language users (l cases) . Comparel cases) , the per2 to 106 .The qualitative analysis of peer problems resulted in three major themes: experiences of relationships with peers, positive aspects of friendship, and negative aspects of friendship. At least one of the three identified themes emerged from the interview transcripts of all participants. There were 11 subthemes within these three themes . Table 4Experiences of relationships with peers emerged from the interview transcripts of all participants and could be divided into six subthemes: (a) the experience of feeling accepted, (b) ease of making new friends, (c) barriers in making new friends, (d) preference of friends based on their hearing status, (e) the experience of being the only DHH person in a mainstream class, and (f) the experience of being bullied.Feeling accepted. Feeling accepted was defined as feeling part of the class, being at the same achievement level as their peers and being able to ask for help when needed:Yes, most of my friends, like we were roughly around the same level so we were mostly the same classes. And yeah, everyone just helps each other and stuff. Teachers are also supportive and helpful” .“Feeling accepted in class was not only attributed to the good relationships with peers but also to the supported environment provided by teachers and support staff.Obviously, if I didn’t hear anything that the teachers told us to do, what we need to do, then I will ask my friends to explain what we have to do, that’s one thing. The second thing is making sure that I’m writing the right thing, rather than like knowing what to do, but I’m writing completely off the topic.” .“Good relationships with peers were reported by 21 of 30 (70%) participants, they described their classmates as nice, feeling able to ask them for help with work when needed or to seek clarification from them when they had not heard the teacher’s instructions.unny,” “trustworthy,” “smiley” or “polite”). These statements were provided by 23 (77%) participants who explained that they were liked by their classmates because they possess a number of positive character traits:I’m pretty funny, and I think one of the most important things, I’m trustworthy, because I do have quite a few friends from here who tell me everything, cos she has an interesting life compared to me, but she tells me everything that’s going on with her life, and I’m like, “Oh my God,” and I give her advice, but I don’t tell anyone because she only tells me.” .“In response to the question: “What do you think your classmates like about you”? participants commented that they thought they were liked by peers because of their good behavior in the classroom, good academic achievement, and positive traits of their character  participants reported no difficulty in making friends. The ease of making friends depended on the situation and the environment in which this was taking place, with school being the easiest setting.“At school, I wouldn’t say it is difficult because if you are with your mates and you make a mistake they can back you up. Like I am very confident when it comes [to] the group of people in school and talking to people from school.” .“I have like 250 friends on Facebook. I just add them on Facebook and just start talking. I think you only have to do is ask someone on Facebook and then you can find out everything there is to know about them, you know, you can go through all their photos and say oh, this person’s like that and you can sort of go on that” .In the school environment, adolescents felt confident in talking to their peers and expanding their circle of friends. Participants reported that it was easier to make friends in their school environment compared to other social situations . They also shared their anxiety about making friends beyond compulsory education, and when entering further education. However, those participants stated that it was easier to make new friends and to connect with their friends on social media:Connecting with people and making friends through social media seemed to be easier for adolescents who felt less confident in social situations outside their school setting.Barriers in making new friends. Thirteen out of 30 adolescents (43%) found it hard to make new friends and did not feel confident to speak to new people. Eight of those 13 adolescents had moderate hearing loss, three had severe and two profound hearing loss. Difficulty being understood by others resulting in low confidence was a common reason given by the adolescents to explain their difficulty making new friends.“I do find it difficult to make friends because I’m quite a very shy person and I don’t have that much confidence in speaking up to new people. Sometimes when I meet new friends, sometimes they do not understand me because, obviously, when I was born, I was not meant to have my voice, but I’ve like have a speech therapy to improve my voice, talking .”Yeah, so I did find it a bit scary making friends. But when I meet new... Even now, sometimes when I meet a new group of people, like a mixture of guys and girls, I can be a bit shy because none of them know that I have a hearing loss. It can be a bit... It’s hard work sometimes for me, but it’s fine ….I consider myself as hearing impaired I prefer that term because deaf is like fully. All my friends consider me as hearing” .“Besides communication issues, disclosure of the adolescent’s hearing loss was identified as an additional barrier to establishing new friendships. Some adolescents who disliked being labeled “Deaf” disliked disclosing their hearing loss.In addition, a link was made between not wishing to be identified as Deaf, and hearing loss being perceived as an initial barrier to making friends. Adolescents who experienced problems making new friends because they did not wish to disclose their hearing loss and did not want people to take notice of their hearing loss. Those adolescents identified themselves with the hearing world.DHH friends, hearing friends, or no preference. Participants, irrespective of their educational placement or severity of hearing loss, reported having both DHH and hearing friends. The communication mode and the world with which they identified (Deaf or hearing) were the main reasons given for their preference for hearing or DHH friends. Adolescents who communicated using spoken language tended to prefer to have both DHH and hearing friends but explained that it would be difficult to be friends with Deaf people whose main method of communication is signing.“It depends where you are to be honest, if you’re in the Deaf world it’s like harder for me ‘cos I can’t really sign that well.” .“I tend to hang out with hearing people because there is more a range of hearing people, more mixed, and you have to get used to it because if you go to work you have to be with hearing people so you have to get used to talk to them.” .For some adolescents having hearing friends was a way to be included and accepted in the hearing world. Mixing with hearing people was also considered important for later life:“So the person that is not deaf and is hearing, I find that very very difficult to make friends with that person…. With deaf people is easier because I already share something….the same language. I think I know that a deaf person will understand me” .Three signers had only Deaf friends. Those adolescents embraced their Deaf identity, were involved in Deaf clubs and Deaf culture, and felt that they could only feel connected to other DHH people. Deaf friends were perceived by these participants as sharing a similar self-image; sharing the same language and the experience of being DHH were seen as attributes conducive to friendship:In summary, communication and a wish to be connected to either the hearing or the Deaf world or both were the adolescents’ stated reasons for having DHH or hearing friends.Feeling different from peers. The only situation when adolescents reported feeling excluded from their class and unsupported by their peers was when the student was the only DHH student in the classroom (at a mainstream school). This experience was reported by 5 of 18 (27%) adolescents with a moderate hearing loss who were in a mainstream classroom with no DHH peers.“At times I do wish there was like another person who is hearing impaired at [name of the school] like there isn’t one at the moment. I think I’m like my school’s first hearing impaired so it’s quite new for them.” .I don’t... I actually do not know what it was, it might have been that but I just hated it , I just didn’t like... [pause]I think it’s probably just being different and like having to wear it, you know.” .“The aesthetic appearance of their hearing aid and having to ask teachers to use an FM system (the wireless assistive hearing device that enhances the sound of hearing aids or CIs) were the main reasons most often given by participants for feeling different from their hearing peers.Experience of being bullied. In response to the question “What does bullying mean for you”? the adolescents described bullying as behaviors that can hurt other people either physically or emotionally. Adolescents considered a “nasty” behavior as bullying even if it only happened once. However, a further distinction was made between bullying and teasing. Teasing was described as “making fun” of or “playing pranks” on other people.“I have been teased but not bullied. . . I wouldn’t call it bullying. Just tease names like “Can you hear”? My friends say a lot but I can deal with that because I know they are joking.” .The adolescents described situations of being teased in secondary school but reported this behavior as acceptable:t(24) = 0.69, ns).One or more incidents of being bullied in primary school were reported by 23 of 30 (77%). All participants reported that bullying had now (in secondary school) stopped. Thus, all the quotes presented below relate to experiences of being bullied in primary school. Among spoken language users, the group mean Receptive Language Composite standard scores of participants that reported that they had been bullied in primary school did not differ from that of those who did not report this .“Reasons for being bullied varied: some adolescents were not always able to identify the exact reason for being bullied but others attributed it to their hearing loss, communication difficulties, or reasons unrelated to being DHH, such as having a different religion:It can just, I don’t know, it makes me feel angry. Sometimes it makes me feel, it makes me feel, you know, a bit isolated from them because, obviously, they don’t, they haven’t got any disability or any impairments or anything, so they just, you know, they’re alright, they’re perfectly normal.”.“Being bullied had an effect on DHH adolescents’ self-esteem making some of them feel depressed and isolated, whereas others felt angry:“They called me names.. they grabbed my chair.. they put a broken pen on the chair… they take my.. books so I cannot find them.. and they got me into trouble” .The majority of young people described verbal bullying, including gestures, but physical bullying was also reported:There was a Hearing Impaired Unit there, and the numbers fluctuated over the years so I couldn’t tell you how many people were there…..But I was definitely bullied there, it wasn’t just the hearing children that bullied me, the deaf bullied me as well, and the deaf were the worst culprits…..I thought they were my friends at the time and that did upset me, it made me, it made me feel that I was unimportant at the time.”.“Adolescents had been bullied by hearing peers but one participant also described her experience of being bullied by DHH peers.“Not really, [it was in primary school] and I couldn’t, you know, verbalise back, I just used to sit, I was one Deaf person, they were all hearing there and there was a lot of teasing of me and I couldn’t go back there and I just used to sit there and take it and hence my confidence is very low”. .All six adolescents who used BSL as their preferred method of communication reported that they had been bullied in primary school. Those who received their primary school education in mainstream schools attributed the cessation of bullying to their move to a school for the deaf.Twenty-two (73%) participants reported experiencing positive qualities of friendships .Similarities or common interests. Participants described feeling close to friends with whom they shared hobbies, were involved in the same activities, and shared love for the same things in life. For example, one participant described how love of music brought her and her friend close to each other.I mean (friends’ name) and I have a big thing in common which is music, you know we really like music, he’s really good at playing, I am not so good at playing but we both love, you know, sort of music. .“We actually like going to the (name) Deaf Community because like when we were in like Year 6 we used to go on trips, like all deaf children.” .Attending deaf clubs and having an interest in the life and activities of the Deaf community were particularly important for maintaining a close relationship for deaf adolescents who signed and identified themselves as belonging to the Deaf world:“And her Mum and Dad are divorced so sometimes, even though they still get on, there’s sometimes, you know, like sometimes she’ll be a bit like, oh my Dad’s, you know...So when this happened I comforted her and when my dog died, she obviously comforted me. So, you know.” .Another positive quality of friendship was the feeling of intimacy. This was characterized as experience of situations in which friends shared personal information, feelings, and secrets. Discussions about family issues were indications of feelings of intimacy between adolescents and their friends.Discussion about difficulties that participants and their friends had experienced within intimate relationships and disclosure of conflicts that they had with other people were also indications of the intimacy that they felt with their friends.And she had a problem with her boyfriend, so I’m like... And we’re like, “OK, fine, don’t worry about this guy. He’s some idiot, he’s some something,” you know... there’s more fish in the sea.” .The majority of DHH adolescents (72%) also reported some negative experiences in their friendships. These negative experiences were classified as conflict and as losing contact with friends.Experience of conflict. The DHH adolescents described feelings of anger leading to potential conflicts with their friends. Conflicts were attributed to a variety of causes, such as negative characteristics of a friend, inability to keep secrets, and betrayal of trust. This included situations when their friends thought that they had betrayed their trust:Mainly about 3 years ago, me and my friend who were sharing the room we had a conflict. . . We were always quite close but you know sometimes people hear a rumour and then you don’t really understand what happened and then it became awkward. . . I don’t really know what happened but she talked about her family so I haven’t said anything about her family but that is what she thought. . . Deaf people like talk about others the whole time. .“It is probably... that was before the Easter holidays and me and some of my friends were all going to…. and shared a room pretending that nothing had happened and pretending that everything is fine.. we can do that.. we can retain the friendship.. It wasn’t a horrible experience.. but I think she should say sorry but she didn’t. I think she has to say it because …”.Participants often described their relationships as good but sometimes damaged by a single event that was perceived as a sign of betrayal of friendship. However, in most situations, the adolescents described betrayal of trust as being a temporary conflict:Gender differences were observed with 10 of 15 (67%) females describing one or more incidents of arguments/conflicts as a feature of their long-lasting friendships compared with 5 of 15 (33%) boys.Lack of interaction. Participants described situations when they drifted apart from their friends, not because of conflicts but due to lack of interaction. Lack of interaction was attributed by the adolescents to their friends having fewer opportunities to interact with them. They reported lack of interaction in cases where their friends lived far away or when they attended different classes in school. In instances when their friends had moved schools or moved house (to a more distant place), lack of time to engage with their friends outside school had led to a decrease in opportunities for interaction.The main reason for fewer opportunities for interaction reported by 10 out of the 18 DHH adolescents was the situation in which their friends had built a relationship with another peer. Adolescents described feeling rejected, hurt, and lonely in these instances:Me and [name of friend] are... we didn’t talk... We were really close and we’re drifting apart because she’s got close to another girl, and I was thinking, “Oh God, I’ve got... I miss her.” And we don’t talk when we see each other, we never talked that much, so she’s like... she’s like talking to me as if I was kind of a new girl or something, and like, “Mate...” you know. .And well I think he didn’t want to be friends with me one day, I didn’t know why and then...He went down to college and standing near a group of friends, so I was a little bit surprised and thought what’s going on? I think now he wants to be friends again but... if he starts talking to me I will talk back but I wouldn’t be friends with him again. I don’t trust him with my personal stuff but… .However, none of the 10 adolescents (56%) who reported that the lack of interaction was due to their friend forming a new relationship gave any indication on whether the new relationship that their friends developed was with a DHH or a hearing peer.This is, to our knowledge, the first qualitative exploration of relationships and friendships in a sample of DHH adolescents drawn from a population-based cohort study (in the United Kingdom) in which their receptive language and social–emotional skills had been assessed. This is the first and only report of the qualitative findings. The main findings and contribution of this study to existing knowledge are threefold. Firstly, overall DHH adolescents experience positive relationships with their peers and friends notwithstanding their earlier experience of bullying in primary school. Secondly, the majority of adolescents with moderate hearing loss experienced barriers in making friends, felt different from peers in a mainstream classroom due to the physical appearance of hearing technologies and found it easier to make friends in school settings and through social media than in other social situations. Thirdly, DHH girls were generally found to experience more conflicts in their friendships than boys suggesting that girls might benefit from targeted support on developing their peer relationships.In contrast to a previous study in which adolescents reported feeling worried about peer relationships and struggling with their concept of self , particiParticipants’ reports of being rejected by peers and being bullied in primary school are consistent with the report by Nunes and colleagues that DHH children feel neglected in inclusive settings . The cesAlmost three quarters of participants identified similarities or common interests and intimacy as positive qualities, and conflicts and lack of interaction with their friends as negative qualities of their friendships. The finding that a higher percentage of girls than boys experienced conflicts with friends, arising from issues of mistrust and infrequency of interaction, is likely to reflect the importance that girls place on close and exclusive social relationships and their greater dependence on peer feedback to inform their self-worth .Common interests, hobbies, and identification with the Deaf world as well as sharing of personal thoughts and feelings related to family and intimate relationships were considered to reflect positive qualities whereas betrayal of trust and lack of interaction with friends due to the formation of a new friendship reflected negative qualities of friendships. These more specific and detailed descriptions of positive and negative experiences with peers and friends have not been identified by previous quantitative studies and may help in the design and provision of effective and timely support.Feelings of being different from peers and excluded from their mainstream class  were reported by adolescents with moderate hearing loss. Difficulties in being understood by their peers confirm earlier reports of the impact of atypical speech and expressive language used in conversation with peers . This isThe type of communication preferred by DHH adolescents (speech or sign) was identified as a factor affecting the choice of friends of DHH adolescents. Previous studies have also reported that communication type is a strong predictor of choice of DHH or hearing peers as friends . Deaf cuThe lack of a relationship between the receptive language skills of DHH adolescents and their experience of bullying contradicts earlier findings suggesting that poor receptive language ability accounts for higher levels of emotional and behavioral difficulties . A possiA strength of the study is the fact that those studied are representative of the birth cohort from which they were drawn and this is supported by the similarity of their educational settings to the distribution of educational settings reported for DHH adolescents in England by the Consortium for Research on Deaf Education .The verbatim descriptions and qualitative analysis reported here  complement our report of the reading and language skills , 2017 anBias regarding the interpretation of adolescents’ experiences was minimized by using highly qualified interpreters and independent raters to re-code transcripts. The purposive over-representation of BSL users is also a strength of the study, allowing us to capture the range of experience of both this distinct group and also of the spoken language users who constitute the large majority of all DHH adolescents.The absence of a comparison group of hearing adolescents was a limitation because it would have enabled us to determine whether the percentage of adolescents who reported feeling accepted by their peers differed between hearing and DHH adolescents or whether experiences reported in the present study, such as greater prevalence of peer conflicts among girls than boys, are unique to DHH adolescents. We do, however, know from the related quantitative study that the group of DHH adolescents, of whom participants in the present study were a representative sample, had an elevated rate of borderline/abnormal scores on the peer problems subscale of the SDQ whereas the hearing comparison group did not .The absence of video recording of the interviews is also a limitation but was the only course consistent with the expressed views of the participating adolescents who may, as a result, be a larger, more representative sample who were more willing to talk candidly on sensitive topics.An additional limitation is that no connection was made between the adolescents’ experiences and family variables that might have influenced those experiences. Family characteristics with regard to hearing status , type of communication , and communication quality were recorded, but numbers in most of these subcategories were too small to allow examination of the associations between these variables and experiences with peers. Future studies should address family variables in relation to DHH adolescents’ experiences of relationships with peers.The findings of the present study show that difficulties with peers and friends are important for DHH adolescents and might suggest that proactive promotion of communication and positive peer relationships between DHH and hearing adolescents can be potentially beneficial. The negative long-term legacy of bullying on the self-esteem of DHH adolescents  also sugA clear implication of the present study is that schools and teachers should pay more attention to the subgroup of adolescents who have moderate hearing loss. As found in the current study, this subgroup experience the same levels of difficulties as those with severe or profound hearing loss, but the former is often a “neglected” group as the perception is that they “can hear” . Thus, iPeripatetic teachers of the Deaf (teachers of the Deaf visiting DHH children and adolescents in mainstream schools) can support development of effective school strategies for social inclusion and social functioning of DHH children and they too would benefit from further training to consolidate that supporting role. In mainstream schools, support for DHH children’s social-emotional development can come from a variety of school staff  other than just the teachers of the Deaf or the mainstream teachers, all of whom would benefit from training in deaf awareness and communication tactics.This work was supported by the Wellcome Trust (089251).No conflict of interests were reported.