Many cochlear implant users with binaural residual (acoustic) hearing benefitfrom combining electric and acoustic stimulation (EAS) in the implanted ear withacoustic amplification in the other. These bimodal EAS listeners can potentiallyuse low-frequency binaural cues to localize sounds. However, their hearing isgenerally asymmetric for mid- and high-frequency sounds, perturbing or evenabolishing binaural cues. Here, we investigated the effect of afrequency-dependent binaural asymmetry in hearing thresholds on soundlocalization by seven bimodal EAS listeners. Frequency dependence was probed bypresenting sounds with power in low-, mid-, high-, or mid-to-high-frequencybands. Frequency-dependent hearing asymmetry was present in the bimodal EASlistening condition (when using both devices) but was also induced byindependently switching devices on or off. Using both devices, hearing was nearsymmetric for low frequencies, asymmetric for mid frequencies with betterhearing thresholds in the implanted ear, and monaural for high frequencies withno hearing in the non-implanted ear. Results show that sound-localizationperformance was poor in general. Typically, localization was strongly biasedtoward the better hearing ear. We observed that hearing asymmetry was a goodpredictor for these biases. Notably, even when hearing was symmetric apreferential bias toward the ear using the hearing aid was revealed. We discusshow frequency dependence of any hearing asymmetry may lead to binaural cues thatare spatially inconsistent as the spectrum of a sound changes. We speculate thatthis inconsistency may prevent accurate sound-localization even after long-termexposure to the hearing asymmetry. Criteria for cochlear implantation have been expanded to allow adults with goodlow-frequency hearing thresholds and severe-to-profound high-frequency hearing lossin both ears to receive a single cochlear implant. The combined electric andacoustic stimulation (EAS) in the implanted ear and acoustic stimulation in theother provides these cochlear implant recipients with binaural low-frequencyacoustic hearing, which has been shown to improve horizontal-plane soundlocalization . HoweverThe EAS listeners with low-frequency binaural hearing .HoweverIn this study we investigate how the frequency-dependent binaural hearing asymmetriesaffect sound localization by bimodal EAS users. To that end, we present low-, mid-,mid-to-high, and high-frequency sounds and acutely introduce larger hearingasymmetries by turning on and off the devices. We quantify hearing asymmetry as abinaural difference in hearing thresholds separately for each participant, sound,and listening condition. We hypothesize that hearing asymmetries introducelocalization biases to the better hearing ear  to d. ThFurthermore, we hypothesize that, as hearing asymmetry depends on frequency , soundln = 2), Cochlear , or Med-El EAS  and all had worn a contralateral hearing aid for atleast 2 years up to the time of testing  took part in the study, all having a severe sensorineural high-frequencyhearing loss and low-frequency acoustic hearing in both ears. The participantshad 6 months to 10 years of experience with an Advanced Bionics  had limited peripheral vision caused by Usher type2A syndrome, while the other participants had normal, or corrected to normal,vision. Hearing thresholds  were meaHearing devices improved low frequency audibility (<500 Hz) on average by13 dB in the hearing-aid ear compare  and by 4The hearing aid and the cochlear implant were fitted according to local clinicalguidelines. All participants included in the study are clinically monitored atRadboudUMC clinic and reported their device settings to be satisfactory at thestart of the measurements. Experiments were done with their daily devicesettings without any refitting.Some of the data from one participant (P5), who had the best low-frequencyresidual hearing in both ears in this participant group, have been publishedseparately as a case study .Sound-localization experiments were carried out in a dark sound-treated room witha background noise level below 30 dBA . The rooParticipants sat in the center of the setup and wore a light-weight spectacleframe equipped with a head-fixed laser pointer. This pointer ensured thatparticipants kept their gaze centered during the pointing tasks. The searchcoil was mounted on the nose bridge of spectacle frame and the participantsvisually pointed the laser to each of 24 known LED positions for thecoil-calibration procedure. These responses were used to train twothree-layer back-propagation neural networks that transformed thehead-movement data (in mV) into calibrated azimuth and elevation angles indeg, .Sound stimuli were derived from 150-ms long white-noise bursts with 5-mssine-squared onset and cosine-squared offset ramps. Sounds werebandpass-filtered in one of four ways: 100–400 Hz ,500–1500 Hz , 3000–20000 Hz , and500–20000 Hz . The four sound stimuli wereinterleaved in one block and presented at a level of 65 dB(A) from one of 16locations with four repetitions per speaker location with a total of 64 trialsper listening condition. Five listening conditions were tested and a descriptionof each condition is presented in the section “Listening Conditions.” Speakerlocations were randomly selected between −80 deg and +80 deg in azimuth andbetween −45 and +60 deg in elevation. Participants took part in afamiliarization task of 20 trials before the start of the actual experiment.A trial began with the presentation of a green fixation LED from thestraight-ahead location, at  deg. The participant had to point thehead-fixed laser toward this LED. This ensured that the starting position wasthe same for each trial. The participant was provided with a small hand-heldbutton box to start each presentation. Pressing the button turned off thefixation light within 100 to 300 ms and started the sound stimulus 200 ms afterthat. Participants were instructed to orient their head as quickly and asaccurately as possible and direct the head-fixed pointer in the perceiveddirection of the stimulus. Because reaction times exceeded 150 ms (the durationof the stimulus) and no visual feedback was provided, all responses were madeunder complete open-loop conditions.A-EX. Bimodal stimulation with acoustic stimulation at thenon-implanted side (A), electric (E) stimulation and an open earcanal (X) at the implant side (seven participants).A-EA. Bimodal EAS stimulation with acoustic amplification in thenon-implanted ear and electro-acoustic stimulation in the implantedear (seven participants).X-EX. Electric stimulation only without acoustic amplification; openear canal at both sides (five participants). The implant was notre-fitted after taking the acoustic component of the EAS device outof the ear canal.X-EA. Electro-acoustic stimulation only with the hearing aid at thenon-implanted side taken out, leaving the ear canal open (sixparticipants).A-XA. Bilateral acoustic amplification with electric stimulationturned off. All seven participants performed the localization taskwith this listening condition.We chose to measure with open ear canals (X) to have a large range ofhearing asymmetry across participants due to the natural variation in residualhearing were taken as the participant's response, where positive azimuth angles indicatepositions to the implanted ear while negative azimuth angles indicate responsesto the hearing-aid ear.EASp, as a measure of localization bias. This continuous variable can vary from 0,indicating all responses were directed to the hearing-aid ear, to 1, indicatingthat all responses were directed to the implanted side. AEASp value of 0.5 indicates there is nolocalization bias, with 50% of the responses being directed toward the implantedside, and the other half to the contralateral side.As most responses were toward either the EAS or the hearing-aid ear without aclear linear or continuous stimulus-response relationship (see Results), wequantified the proportion of responses toward the implanted ear,RMSE, in deg) for comparison to otherstudies. For normal-hearing listeners, this error is typically less than 10 deg. Chance level error depends on the listener's responsestrategy. If a listener is unable to perceive sound location and then localizesrandomly within the stimulus range, the error will be 73 deg. If that listenerdoes not respond and keeps on looking straight ahead, then the error will be 46deg.We also calculated the more-commonly used root-mean-squared localization error.Sounds were localized around 0 deg in elevation ,irrespective of target location, sound frequency, listening condition orparticipant. This is expected from listeners that do not have access to spectralpinna cues and we do not discuss this further.H) between ears was obtained bycalculating the audibility of the four sound stimuli for each of the fivelistening conditions with a free-field microphone (type-4191) positioned at the location of theparticipant's head. Then, correction was applied in gammatone filter banks to compensate for the bandwidth of the noise burst. Finally,the corrected dB SPL sound level was converted to dB HL  was obtained bysubtracting the audibility in the hearing-aid ear from the audibility in theimplanted ear . In general,hearing asymmetry favored the implanted ear, which is likely due to theaudibility over a broader range of frequencies in the implanted ear (<8 kHz) in comparison to the hearing-aid ear (< 2 kHz).Finally, the hearing asymmetry :ω (dB),the hearing-asymmetry range for which EASpvaried from 0.1 to 0.9. This is the range of hearing asymmetry that shouldyield extreme bias on either side with EASp of0.1 and 0.9 indicating a strong bias toward the hearing-aid and implantedear respectively.We performed logistic regression to fit the localization bias,ω indicate that hearing asymmetry barelyinfluences sound localization. This equation implies mirror symmetry oflocalization bias, when hearing is symmetric . Thedata, however, indicated that sound localization was biased towards thehearing-aid ear contralateral to the implant ear(EASp<0.5), even when audibility wassymmetric (ΔH = 0). Therefore, we adjusted this model forlistening conditions with the hearing aid turned on by including ahearing-aid preference, θ (in dB):EASp is 0.5,when the hearing aid is turned on. Positive or negative values ofθ indicate that sound localization is biased toward thehearing-aid or EAS ear, respectively, more than expected by hearingasymmetry, ΔH, alone – i.e., responses to either the hearing-aid, or the EASear, are favored.Large values of ωand θ were obtained for each participant separately, butalso hierarchical group-level parameters were obtained. Equations 1 and 2,and individual and group-level parameters were all fittedsimultaneously.The fitting , the bias(EASp) and hearing asymmetry (ΔH). The95%-CIs were obtained through bootstrapping using 1000 samples.We determined the mean and 95% confidence interval (95%-CI) for the error forall sounds. When the hearing aid was turned off (bottom two rows), sounds werejudged exclusively to the implanted side . Both findings are in line with the hearing asymmetry inducedby turning either device off . However, hearing asymmetry typically favored the implant , which was not reflected by thelocalization bias (EASp  = 0.6).Bimodal EAS users had to localize sounds consisting of various frequency bands indifferent listening conditions in which their devices were independently turnedon or off. These two experimental manipulations (frequency bands and listeningcondition) systematically affected a participant's hearing asymmetry. The goalof this study was to see whether this also affected sound localization. We willfirst show that this was indeed the case for one of our participants (P6) withwhom we measured all conditions . For theEASp = 1, blue inner circle; exceptfor one response). As these sounds were not audible by the (unaided or aided)ear on the hearing-aid side (H =  + 37 dB (High-frequency sounds (EASpbetween 0.15 and 0.43) for the three other sounds , but did notlocalize all sounds toward either extreme. For these sounds and these listeningconditions, hearing asymmetry was not as extreme as for the high-frequencysounds; also, intermediate levels of hearing asymmetry were observed or with bimodal EAS (A-EA), the participant hada localization bias toward the hearing-aid ear (ERMS > 30 deg), even whenlistening with all devices turned on (A-EA). Nevertheless, listening conditionand noise-burst frequency systematically affected both hearing asymmetry andsound-localization bias of this participant. This happened in a similar, but notidentical, way for both measures (inner and outer colors match to a largeextent). Hearing asymmetry generally favored the implant more than thelocalization bias .Overall, the participant did not localize sounds accurately in any of thelistening conditions  localized sounds in asimilar fashion with incorrect responses either to the side with hearing aid orto the side with cochlear-implant depending sound frequency and listeningcondition. Only the mid and mid-to-high frequency sounds in the bimodal EAS andbimodal listening conditions were localizable to some extent, with considerableresponse bias and variability.In general, the other participants . We observed no overall benefit ofacoustic amplification by the EAS in the bimodal EAS condition (A-AE), whencompared to the bimodal condition (A-XE). For example, the errors formid-to-high frequency sounds (MHF) were comparable at 50 deg  and 60 deg , respectively. However, localizationerrors varied considerably across participants. For example, in the extreme caseof participant P5, the localization errors for the low-frequency sounds (LF)were close to normal-hearing  when the contralateral hearing aid was turned on . When turned off (X-EX and X-EA), however, the bias switched toward theEAS side (EASp > 0.5). Response bias formid-to-high-frequency noise bursts  shifted from the hearing-aid earto the EAS ear as the listening condition changed from the bilateral-acoustic (A– XA) to the EAS-only (X – EA) listening condition, with little average bias(EASp around 0.5) for the bimodal (A – EX)and bimodal EAS (A – EA) conditions. The high-frequency sounds  werealways perceived on the EAS side except when the implant was turned off (A –XA).Localization of low- and mid-frequency sounds (LF - red and MF - blue) was onaverage biased toward the hearing-aid side , whilehigh-frequency sounds were largely localized toward the EAS side (if turned on).Mid-to-high-frequency sounds were more likely perceived with a smaller bias,especially for the bimodal EAS and bimodal hearing conditions. This findinglargely corresponds to the changes in hearing asymmetry cf. .Hearing asymmetry is a continuous variable, incorporating experimental factorslike listening condition and frequency band and the continuous data descriptorslike hearing thresholds. As such, it may be a more informative variable comparedto the unaided hearing thresholds alone to explain the underlying differences inobserved localization bias resulting from listening condition, sound type andhearing thresholds. We tested this assumption by plotting localization bias as afunction of hearing asymmetry and fitting the logistic curve of Equation. .Figure θ. In θ for eachparticipant for visual purposes. The hearing-aid preference was quitesubstantial . Thissuggests that bimodal EAS users have a large preference to localize soundstoward the hearing-aid ear, even when hearing is symmetric.Thus, hearing asymmetry systematically affected the localization bias, both forparticipant P6  and at tWe investigated how bimodal EAS users localize sounds of varying frequencycontent with or without their hearing devices turned on. Overall, our bimodalEAS users localized sounds poorly . When thrmsE of 62 deg), especially when comparedto previous studies visibility) may introduce significant experimentaldifferences , while two more could alsolocalize mid-frequency (500−1500 Hz) sounds to a reasonable extent .We hypothesized that bimodal EAS users can potentially localize low-frequencysounds accurately . These bThe insensitivity to ITDs for many bimodal EAS users . Furthermore, disabling the acoustic part of the EAS device didnot affect sound localization to a large extent  indicating that our participants did not use fine-structureITDs.Another factor may be that these bimodal EAS users weight ITD cues less than ILDcues when forming a spatial percept. Evidence that supports this notion comesfrom turning off the electrical and acoustic parts of the EAS device. Finestructure ITDs are only provided by the acoustic hearing devices and not by thecochlear implant. Therefore, turning off the electrical part should notinfluence sound localization if the listener fully relies on ITD. However,turning off the implant did influence sound localization for the three “good”performers  that (re)weighting of cross-frequency soundlocalization cues can be acute and adaptive (Sound localization was strongly biased for most listening conditions andfrequency bands of the noise bursts . For theWe observed a preference for listeners to localize sounds toward thenon-implanted ear using the hearing aid. The observed hearing asymmetry had tobe adjusted by about 19 dB in favor of the hearing-aid ear (equation ;Figure The observed preference toward the hearing-aid side is not predicted by ourhearing-asymmetry model . It mighOverall, our conceptual hearing-asymmetry model  could exFor clinical purposes, a hearing instrument test with a coupler and real earmeasurements immediately before the experiments could help to optimize fittingand potentially reduce hearing asymmetry. To further consolidate the correlationbetween hearing asymmetry and localization behavior, it would be useful to mapthe loudness growth function for both ears in response to the experimentalstimuli. This would capture the fact that independently acting automatic gaincontrol across the devices will produce different outputs according to the soundpresentation level. For future experiments, it would also be interesting to usemultiple sound levels as independently acting automatic gain control across thedevices might interact with the hearing asymmetry as a function of presentationlevel.Hearing asymmetry in bimodal EAS users causes a frequency-dependent soundlocalization bias. Furthermore, our bimodal EAS users had a global preference tolocalize toward the hearing-aid side. Overall, our results imply that accuratelocalization of sounds with arbitrary spectra by bimodal EAS users with considerablefrequency-dependent hearing asymmetry is hard to obtain.Click here for additional data file.Supplemental material, sj-docx-1-tia-10.1177_23312165221143907 for HearingAsymmetry Biases Spatial Hearing in Bimodal Cochlear-Implant Users DespiteBilateral Low-Frequency Hearing Preservation by Snandan Sharma, Lucas H.M. Mens,Ad F.M. Snik and A. John van Opstal, Marc M. van Wanrooij in Trends inHearing


Is expanded genomic sequencing combined with hearing screening associated with the detection of hearing loss and the improvement in the clinical management of patients in the neonatal intensive care unit (NICU)?In this cohort study that included 8078 patients in the NICU, expanded genomic sequencing was associated with a 15.6% increase in cases of diagnosed hearing loss that were missed by hearing screening and changed the clinical management strategies of these patients. Of 52 patients with a diagnosis of hearing loss, 39 (75%) had genetic findings and experienced a more severe degree of hearing loss.This study suggests that expanded genomic sequencing combined with hearing screening may be effective for diagnosing hearing loss in the NICU setting. Hearing loss is a global social burden. Early identification of hearing loss missed by newborn hearing screening tests in the neonatal intensive care unit is crucial.To assess the association between expanded genomic sequencing combined with hearing screening and detection of hearing loss as well as improvement in the neonatal intensive care unit.This cohort study was performed between August 8, 2016, and December 31, 2020, among 8078 newborns admitted to the neonatal intensive care unit of the Children’s Hospital of Fudan University in Shanghai, China. Follow-up for hearing status was performed via telephone interviews between September 1 and November 30, 2021.A hearing screening test and the expanded genomic sequencing targeting 2742 genes were administered to each patient. Those who failed the hearing screening test or had positive genetic findings were referred for diagnostic audiometry at a median of 3 months of age.The primary outcome was hearing loss missed by hearing screening test. Secondary outcomes were genetic findings and benefits associated with the expanded genomic sequencing for clinical management of patients in the neonatal intensive care unit.GJB2 and SLC26A4 were the most common genes identified. Patients with genetic findings experienced a more severe degree of hearing loss than those without genetic findings , with more bilateral hearing loss . Clinical management strategies were changed for patients who underwent genomic sequencing combined with hearing screening.Of 8078 patients , 52 of 240 (21.7%) received a diagnosis of hearing loss. Expanded genomic sequencing combined with hearing screening was associated with a 15.6% increase (7 of 45 patients) in cases of diagnosed hearing loss that were missed by hearing screening. Of the 52 patients with hearing loss, genetic factors were identified for 39 patients (75.0%); This study suggests that expanded genomic sequencing combined with hearing screening may be effective at detecting hearing loss among patients in the neonatal intensive care unit. This cohort study assesses the association between expanded genomic sequencing combined with hearing screening and the detection of hearing loss and improvement in the clinical management of patients in the neonatal intensive care unit. We assessed the association between expanded genomic sequencing (EGS) combined with NBHS and the early detection of hearing loss among newborns admitted to the NICU in China for better management strategies of patients with this type of hearing loss.26 between August 8, 2016, and December 31, 2020. The inclusion criteria were as follows: newborns admitted to NICU levels 3 and 4  who underwent NBHS. The exclusion criteria were deceased newborns or newborns with insufficient information in the medical records. Patients who tested positive for hearing loss with the NBHS program or had positive genetic findings through EGS were referred for diagnostic audiometry and follow-up. Approval was obtained from the ethics committee of the Children’s Hospital of Fudan University. The parents of each patient provided written informed consent. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline.This cohort study retrospectively included 8078 newborns who were admitted to the Children’s Hospital of Fudan University in Shanghai, China, and underwent genetic testing in the China Neonatal Genomes Project27 perinatal asphyxia (including intrauterine hypoxia28 or birth asphyxia29), mechanical ventilation, craniofacial malformation, severe hyperbilirubinemia , congenital cytomegalovirus infection , neonatal bacterial meningitis,30 family history of hearing loss, and consanguineous parents.For each patient, clinical information, NBHS findings, and genetic findings from the electronic medical record were independently obtained by 2 clinicians (Y.Z. and L.H.). Risk factors were recorded, which included the duration of NICU stay, oxygen exposure, low birth weight (<2500 g), maternal complications during pregnancy , neonatal sepsis,A hearing screening test was performed for each patient. Otoacoustic emission  and automated auditory brainstem response  were tested successively by trained audiologists when the patients had stable conditions during admission. Infants who tested positive for hearing loss with the NBHS program were defined as those with positive NBHS results after testing for otoacoustic emission, automated auditory brainstem response, or both unilaterally or bilaterally.31 A virtual panel including nonsyndromic and syndromic hearing loss–related genes on the Hereditary Hearing Loss Homepage32 or in the Online Mendelian Inheritance in Man database33 were analyzed. Patients with pathogenic or likely pathogenic variants consistent with inheritance patterns received positive genetic findings based on the guidelines of the American College of Medical Genetics and Genomics.34 Sanger sequencing was performed to confirm the variant.Expanded genomic sequencing was performed for every patient. Genomic DNA was extracted from whole-blood samples using a QIAamp DNA Blood Mini Kit (Qiagen). Fragments of DNA were enriched using the Agilent ClearSeq Inherited Disease panel kit (Agilent Technologies) that targeted 2742 genes. Sequencing was performed on a HiSeq 2000/2500 platform (Illumina Inc). The TruSeq Rapid PE Cluster and SBS Kits (Illumina Inc) were used for sequencing. Data analysis was conducted using an in-house pipeline.20 and treatment .Patients who tested positive for hearing loss with the NBHS program or had positive genetic findings were referred for diagnostic audiometry and audiologic counseling by audiologists. Hearing monitoring was arranged if necessary. Follow-up telephone interviews were performed between September 1 and November 30, 2021. Hearing status at the time of the telephone interview was recorded, including diagnosis of hearing loss, degree of hearing loss , symptoms , risk factors,35Hearing loss diagnosed by trained audiologists was defined as a diagnostic audiology test result with a hearing threshold over 25 dB unilaterally or bilaterally and diagnosis of hearing loss by audiologists according to behavioral audiometry (patients ≥6 months of age). The degree of hearing loss according to the latest diagnostic audiometry result was classified as mild , moderate , severe , or profound .2 test or the Fisher exact test was performed for comparative analysis. All P values were from 2-sided tests, and results were deemed statistically significant at P < .05. Statistical analysis was performed using Stata, version 15 (StataCorp LLC).Continuous variables with a skewed distribution are reported as median values with interquartile ranges (IQRs). Categorical variables are expressed as frequencies and percentages. The Pearson χOf 8078 patients included in the study, the median age at admission was 6.3 days , and 4666 (57.8%) were boys ; Figure.Of 8078 patients, 238 (2.9%) tested positive for hearing loss with the NBHS program, while 7840 (97.1%) tested negative. The top 5 risk factors associated with hearing loss among the 238 patients who did not pass the NBHS test were hospitalization in the NICU for 5 days or more (210 [88.2%]), oxygen exposure (71 [29.8%]), maternal complications during pregnancy (69 [29.0%]), low birth weight (67 [28.2%]), and sepsis (62 [26.1%]) (eTable 1 in the GJB2 (OMIM 121011) was identified in 58 patients (64.4%) and SLC26A4 (OMIM 605646) was identified in 17 patients (18.9%). Of these 90 patients, 70.0% (63 of 90) had positive NBHS test results. The remaining 30.0% patients (27 of 90) had negative NBHS test results and had positive genetic findings. Among those patients, GJB2:c.109G>A was the most prevalent variant  received a diagnosis of a genetic condition associated with 15 genes  in the cohort. The allele frequency of GJB2:c.109G>A was higher among patients with a diagnosis of hearing loss than among those without hearing loss . Among these patients with positive genetic findings, the degree of hearing loss was more severe  compared with patients without genetic findings . More patients with genetic findings than without genetic findings received a diagnosis of bilateral hearing loss . However, the median age at onset  and the median age at diagnosis  were not significantly different between patients with positive genetic findings and those without genetic findings.Of the 265 patients referred, 240 (90.6%) reported their hearing status after the telephone interview, while 25 (9.4%) were lost to follow-up  were confirmed to have mild to profound hearing loss bilaterally with early diagnosis . One patient (14.3%) with GJB2:c.235del received a diagnosis at 6 months of age.Of 27 patients who had negative NBHS test results but positive genetic findings, 26 completed the follow-up, 7 of whom (26.9%) received a diagnosis of severe to profound hearing loss . Of thesP = .49) or in the number of risk factors .Of 175 patients who had positive NBHS test results but did not have genetic findings, 155 completed the follow-up. A total of 13 patients (8.4%) received a diagnosis of mild to severe hearing loss, including 4 patients with unilateral hearing loss (eTable 6 in the GJB2:c.109G>A in 34 patients (73.9%), GJB2:c.235del in 1 patient (2.2%), SLC26A4 variants in 3 patients (6.5%), COL11A1 (OMIM 120280) in 2 patients (4.3%), and variants in COL2A1 (OMIM 120140), CREBBP (OMIM 600140), FLNA (OMIM 300017), KCNQ4 (OMIM 603537), MAF (OMIM 177075), and PTPN11 (OMIM 176876), each of which was seen in 1 patient  did not receive a diagnosis of hearing loss. Of these 188 patients, 46 had positive genetic findings, including 27 patients who tested positive after the NBHS and 19 patients who tested negative. The 46 patients who had positive genetic findings were identified with genetic findings of homozygous or heterozygous  patient . These pExpanded genomic sequencing combined with NBHS had a beneficial association with the referral, diagnosis, and clinical management of the newborns in this study. Of 8078 patients, 238 were referred for diagnostic audiometry if only NBHS was performed. For those who underwent both EGS and NBHS, an additional 27 patients at risk for hearing loss were identified; they had positive genetic findings but had negative NBHS test results. Of 52 patients with a diagnosis of hearing loss, 45 were diagnosed through the NBHS program only. However, EGS combined with NBHS identified an additional 15.6% of patients (7 of 45) with hearing loss who were missed by the NBHS program. The increased diagnosis rate was 0.09% (7 of 8078) among the whole cohort. If only NBHS was performed, 45 patients with a diagnosis of hearing loss were followed up. A total of 98 patients (52 patients with hearing loss and 46 patients who had genetic findings but without hearing loss) who underwent EGS and NBHS were followed up . Of the In this study, we assessed EGS combined with NBHS for 8078 patients in the NICU who were at high risk for hearing loss. We found that EGS combined with NBHS was associated with an increase in the referral rate and in the number of cases of hearing loss diagnosed that were missed by NBHS, as well as changes in the clinical management of patients with the possibility of hearing loss. Of the patients with a diagnosis of hearing loss, 75.0% (39 of 52) clarified the genetic findings presented with a more severe degree of hearing loss bilaterally. Such patients need early treatment to improve their prognosis. Expanded genomic sequencing combined with NBHS may be useful in identifying patients with underlying hearing loss who need proper clinical management and/or changes in clinical management.37 Some studies have shown that the referral rate by NBHS only in the general neonatal population ranges from 0.4% to 3.2%,39 while it is 3.9% among the NICU population.19 Both NBHS and genetic testing were associated with a 0.2% to 0.9% increase in the referral rate among the general population.38 Our study reported a referral rate of 2.9% with NBHS only; however, the combination of EGS and NBHS was associated with an increase of 0.4%. Therefore, EGS combined with NBHS was associated with the identification of these referred patients at possible risk for hearing loss.Newborn hearing screening programs have been shown to have significant benefits worldwide.25 In our study, EGS combined with NBHS was associated with a 15.6% increase in the hearing loss diagnosis rate, which was also associated with an additional 0.09% increase in the hearing loss diagnosis rate among the whole cohort. Our diagnosis rate was higher than in previous studies. In our study, we used EGS with a broader detection range compared with commonly used microarray methods in China, including 9 to 20 variants in several hearing genes.23 Expanded genomic sequencing detected the entire coding region, including variants uncovered by microarray. In addition, patients in NICU levels 3 and 4 had a higher incidence of genetic diseases compared with the general neonatal population. Therefore, EGS combined with NBHS was associated with an increased diagnosis rate among this population.Genetic testing identified cases of underlying hearing loss that were missed by the NBHS. Some studies have demonstrated that genetic tests were associated with an increase in the number of additional cases of hearing loss that were missed by the NBHS by 0.005% to 0.05%.41 and of other genetic disorders.46 However, the high cost and long turnaround time limit the applicability of newborn genetic screening. We performed EGS on an NICU cohort, underscoring its effectiveness. Our turnaround time was 20 days, and the cost of EGS was $250.47 With the shortened duration and the decreasing cost of sequencing, EGS combined with NBHS presents a promising possibility for genetic testing in the NICU setting.Exome sequencing presented an advantage in the early diagnosis of hearing loss49 Of 52 patients with a diagnosis of hearing loss in our study, we noted that 75.0% had confirmed genetic findings and presented with a more severe degree of bilateral hearing loss. Such patients need early treatment for a better prognosis. In addition, all patients with unilateral hearing loss were identified among the group that had positive NBHS test results but did not have genetic findings. More patients are needed to delineate the clinical characteristics of unilateral hearing loss.Expanded genomic sequencing plus NBHS is also associated with benefits for clinical management. It was reported that genetic factors account for 50% to 60% of congenital hearing loss cases.GJB2:c.109G>A was mostly prevalent, together with other variants associated with progressive, late-onset hearing loss  or syndromes partially characterized by hearing loss .Of 188 patients without hearing loss up to the last follow-up, 46 had genetic findings. Among these 46 patients, homozygous or heterozygous GJB2:c.109G>A is a hotspot variant in East Asia, with an allele frequency of 8.4%. Patients with c.109G>A variants may develop progressive hearing loss owing to variable expressivity and incomplete penetrance.52 The ages at onset of homozygous or heterozygous c.109G>A range from infancy to 30 years. Individuals with homozygous c.109G>A lose hearing at a rate of 1 dB per year, with penetrance of 17% by young adulthood in the Chinese population.53 Of 31 patients with homozygous c.109G>A in our cohort, 6 (19.4%) developed hearing loss up to our last follow-up. This finding is consistent with previous data.51 Currently, c.109G>A is not detected in neonatal genetic screening for hearing in China. Underlying hearing loss may be missed among patients with this variant. We think that c.109G>A should be included in genetic screening for hearing among newborns. Patients with c.109G>A should be followed up for the progression of hearing loss among those with a diagnosis of hearing loss and for the occurrence among those without hearing loss.GJB2:c.235del, which is associated with moderate to profound congenital hearing loss, is another frequent variant in the Chinese population. A previous study reported patients with homozygous or heterozygous GJB2:c.235del whose hearing loss was missed by otoacoustic emission or automated auditory brainstem response testing, which could delay a diagnosis and may affect the prognosis.54 Therefore, EGS is helpful for the early diagnosis and management of late-onset hearing loss for this type of patient. Patients with variants in SLC26A4, which may lead to sudden hearing loss after head trauma or barotrauma, were strongly advised to avoid situations that may result in sudden changes in intracranial pressure, to prevent or delay the occurrence of hearing loss.55Expanded genomic sequencing combined with NBHS is useful for diagnosing hearing loss, managing patients, monitoring progression, genetic counseling, and educating patients on the prevention of precipitating factors. It also highlights the importance of follow-up for preventing or delaying the occurrence of hearing loss and of early diagnosis for prompt treatment of late-onset hearing loss if it occurs.This study had several limitations. Our single-center cohort was generated from the China Neonatal Genomes Project, which may have led to selection bias. Moreover, patients with a limited follow-up period needed a longer time for monitoring the occurrence of hearing loss. We also did not analyze the cost-effectiveness of the procedure. In the future, multicenter studies will need to be performed for the development of an effective genetic testing strategy for the NICU population.The findings of this cohort study suggest that EGS combined with NBHS was associated with an increase in the referral rate and in the number of cases of underlying hearing loss missed by NBHS and that the genetic factors associated with hearing loss need to be identified for the proper clinical management of hearing loss in patients. Genetic factors were associated with hearing loss in the patients in the NICU. We recommend EGS combined with NBHS for hearing loss diagnosis in the NICU setting.


The preservation of residual hearing is becoming increasingly important in cochlear implant surgery. Conserving residual hearing is a positive prognostic indicator for improved hearing abilities.The primary aim of the study was to explore the preservation of residual hearing following cochlear implantation in a group of recipients at two major cochlear implant centers.A quantitative paradigm was adopted and exploratory research conducted within a retrospective data review design. The sample consisted of 50 surgical records and 53 audiological records from 60 observations . The records were selected using purposive sampling and consisted of records from participants ranging from six to 59 years of age. The average time of when the postoperative audiograms were performed in the current study was 24.7 months (s.d. = ±9.0). Data were analyzed through both qualitative and inferential statistics and a comparative analysis of unaided pre- and postoperative audiological test results was conducted.Results indicated a high success rate of 92% preservation of residual hearing with half of the sample exhibiting complete preservation in cochlear implant recipients across all frequencies postoperatively. A total postoperative hearing loss was found in only 8% of cochlear implantees across all frequencies. There was no relationship between preoperative hearing thresholds and preservation of hearing postoperatively. The two main surgical techniques used in the current study were the contour on stylet and the advance off-stylet techniques, with the majority of surgeons utilizing a cochleostomy approach. From the findings, it became apparent that the majority of cases did not have any reported intraoperative complications. This is a positive prognostic indicator for the preservation of residual hearing.Findings suggest improved cochlear implant surgical outcomes when compared to previous studies implying progress in surgical techniques. The surgical skill and experience of the surgeon are evidenced by the minimal intraoperative complications and the high success rate of hearing preservation. This is a positive prognostic indicator for individuals with preoperative residual hearing as the preserved residual hearing allows for the potential of electro-acoustic stimulation, which in turn has its own hearing benefits. Cochlear implants have revolutionized the manner in which rehabilitation of patients with severe to profound hearing impairment in restoring speech understanding is approached.Verhaegen et al.In the past, only profoundly hearing-impaired individuals with no residual hearing were implanted,8A review of earlier literature on traditional measures to ensure hearing preservation makes comparisons of studies complex, and indicates that caution should be taken when comparing findings as is the case with the current study where retrospective record reviews were conducted without any active manipulation of variables. As early as 1997 it was reported in a study conducted by Hodges et al.Several authorsPreserved hearing has also become beneficial in one of the most significant advances, which is the introduction of bimodal Electric-Acoustic Stimulation (EAS). EAS involves the preserved low frequencies being amplified by means of acoustic stimulation in the form of hearing aids, while the high-frequency hearing loss is being addressed with electrical stimulation in the same ear by means of a cochlear implant.Describing and performing a comparative analysis of hearing function before and after cochlear implant surgery.Determining to what extent residual hearing had been preserved or lost.Establishing whether a relationship exists between the hearing findings and the surgical technique employed, as well as the incidence of intraoperative complications.The primary objective of the study was to explore the preservation of residual hearing function in a group of cochlear implant recipients by:Within a quantitative paradigm, a retrospective data review design was adopted.Purposive sampling technique was used to recruit participants’ files.The records of participants included audiological testing data and surgical records from cochlear implantees with preoperative residual hearing. Participants consisted of male and female, unilateral and bilateral cochlear implant recipients from two cochlear implant units. The children had to be at least 6 years old (to increase reliability of pure tone test findings) at preoperative hearing testing and the adults not more than 59 years old (to minimize the influence of presbycusis) at postoperative testing. The average time of when the postoperative audiograms included in the current study were performed was 24.7 months (s.d. = ±9.0).A pre-requisite of this study was that the participants had to have some preoperative residual hearing at any or all of the following frequencies: 125 Hz, 250 Hz, 500 Hz, 750 Hz, 1000 Hz, 1500 Hz, 2000 Hz, 3000 Hz, 4000 Hz, 6000 Hz and 8000 Hz.The sample comprised 50 surgical records and 53 audiological participant records. Although there were 53 audiological participant files, seven of these individuals were bilaterally implanted, yielding a total sample size of 60 ear observations.Cochlear implants: The participants must have been fitted with a cochlear implant unilaterally or bilaterally at least one month prior to postoperative pure-tone testing.Hearing status: The participants were required to have had a moderate sloping to profound, a severe to profound or a profound sensorineural hearing loss  in both ears and must have had some degree of benefit, even if only minimal, from hearing aids.Speech discrimination: Due to the fact that the participants underwent cochlear implantation, it was assumed that preoperatively, the participants scored ≤50% for sentence recognition in the ear to be implanted and ≤60% in the non-implanted or contralateral ear or bilaterally for speech discrimination, as this is a criterion for cochlear implantation.Auditory nerve functioning: Given the fact that the participants had cochlear implants, the researcher could make the assumption that the participants’ auditory nerve fibers were intact so that they could receive electrical stimuli inside the cochlea.Medical condition: It was assumed that the participants’ medical conditions and inner ear structures met the requirements for implantation, as these are criteria for candidacy.Age: One of the inclusion criteria was that the participants’ ages had to range between 6 and 59 years to ensure reliability of results and to exclude presbycusis loss.The following are factors considered in the inclusion criteria:The following data was collected from each participant file: cochlear implant unit (Unit A/Unit B); left/right ear; Bilateral/unilateral implant; time (months) between last preoperative hearing test and surgery and time (months) between surgery and first postoperative hearing test.The hearing thresholds (in dB) of the pure-tone air-conduction testing conducted preoperatively and postoperatively at the following frequencies: 125 Hz, 250 Hz, 500 Hz, 750 Hz, 1000 Hz, 1500 Hz, 2000 Hz, 3000 Hz, 4000 Hz, 6000 Hz and 8000 Hz.The change: the post-pre difference between the hearing thresholds (in dB) of the pure-tone testing results, at the above frequencies where these frequencies could be measured.The researcher collected additional data, which included the following: age, gender, etiology, duration of hearing loss prior to implantation (in years), implant type, implant manufacturer, electrode type, electrode array insertion, electrode array depth.The types of cochlear implants, electrode array type, insertion depth and surgical technique used are summarized in Fifty percent of implantees were given the CI24RE implant and 25% the CI512.n = 5).The category “Other” (17%) included inter alia the cochlear implant type – Nucleus CI24R with Contour Advance electrode (A large majority of the participants (83%) had the contour advance electrode array implanted.A total of 94% of the participants underwent complete insertion of the electrode array. Very few patients (a mean of 2%) underwent partial insertion of the electrode array.The two main surgical techniques used in the current study were the contour on stylet and the AOS (advance off-stylet) technique with the AOS technique being the most used (67%).The surgical technique used did not have a negative effect on the postoperative hearing threshold levels. The use of the AOS or contour on stylet or “Other” technique did not change the postoperative outcome in terms of preservation of residual hearing.The majority of surgeons utilized a cochleostomy approach, with the exception of four cases where the round window approach was used.The researcher chose to adopt James et al.’s The researcher chose to assign an artificial or numerical value to represent the total loss of residual hearing at the maximum output of the audiometer plus 5 dB. The researcher also elected to represent the maximum output of the audiometer as 120 dB.In calculating the preoperative and postoperative hearing threshold levels, when the hearing threshold level was “no response” (NR), the researcher assigned a value of 125 dB to NR. This value of 125 dB was assigned to replace NR in order to determine the residual hearing or lack thereof pre- or postoperatively.Data analysis was carried out in STATISTICA (version 10). The results were analyzed using descriptive and inferential statistics.To minimize participant variables from impacting on the reliability and validity of results, the researcher excluded any records where participants were reported to have been “unreliable” during testing or where there was “questionable reliability”. Further, as in Kiefer et al.’sPrior to the commencement of the study, the researcher obtained ethical clearance from the University's Human Research Ethics Committee  (Protocol n° M111037). Thereafter, the researcher obtained permission from all the relevant authorities. Furthermore, the work adhered to the Helsinki Declaration of 1975, as revised in 2008.As depicted in The sample size of the current study is believed to be adequate and had a greater number of observations than some other studies reported in the literature. Gstoettner et al.,Data could only be recorded for 11 participants at 125 Hz. This is an important finding which requires attention by the audiology community within this context as Franks et al.As seen in Overall, as depicted in The change in residual hearing indicated by loss of hearing, which was less in the mid and low frequencies, is clinically significant. This preservation of hearing in the low and mid frequencies assists with speech perception in noise and allows for the consideration of EAS – acoustic amplification with a hearing aid and a cochlear implant for the high frequencies.It was expected that there would be a decrease in hearing threshold levels in the high frequencies due to the placement and the insertion of the electrode in the cochlea and due to the tonotopicity of the cochlea. Additionally, the high frequencies are more susceptible to damage, given their placement in the cochlea, as found in the current study.The preoperative and the postoperative hearing threshold levels were plotted on the same graph in order to better depict the change in residual hearing on unity lines as illustrated in The change in hearing function from preoperative to postoperative hearing threshold levels was classified for each participant according to the scheme which is in line with that by Gstoettner et al.Three different combinations of frequencies were used as inputs for the above classification: Gstoettner et al.The change in hearing function, according to the three classifications is provided in From In summary, current findings indicate that the vast majority of the cochlear implantees experienced preservation of residual hearing – either partial or complete hearing preservation. These are positive findings that have implications for both clinical and surgical management of patients with cochlear implants.As depicted in Hodges et al.’sBalkany et al.Verhaegen et al.p = 0.174). The degree of preoperative hearing loss did not determine the outcome of the postoperative hearing threshold levels. These findings are consistent with those by Cosetti et al.The relationship between the postoperative hearing findings and degree of preoperative hearing loss was also explored in the current study. There was no significant correlation between the preoperative hearing threshold levels and the preservation of postoperative residual hearing  and Balkany et al. (1999) (as cited in Di Nardo et al.In Di Nardo et al.’s.The authors are of the view that the positive outcome in the current study is due to the expertise of the surgeons together with improved electrode designs, as evidenced by the minimal intraoperative complications. Despite the fact that the majority of implantees underwent complete insertion of the electrode array, minimal surgical complications occurred and successful hearing preservation was achieved.Preservation of residual hearing following cochlear implantation was successfully achieved in 92% of participants – 42% partial and 50% complete, with 8% of recipients experiencing a complete loss postoperatively. Similar results were obtained regardless of which type of surgical technique was used – AOS or contour on stylet. The majority of surgeons utilized a cochleostomy approach which proved to be successful. Minimal intraoperative complications were reported, resulting in a positive outcome, indicative of successful surgical techniques and expertise of the surgeons, as well as improved electrode designs. Current findings are of clinical significance, making EAS a reality.With the increasing number cochlear implants, the expanding criteria for implantation,The authors declare no conflicts of interest.


Vestibular migraine (VM) presents mainly with recurrent vestibular symptoms and migraine. A great number of patients with VM have cochlea symptoms such as tinnitus, hearing loss.A cross-sectional study was conducted on patients with definite VM (dVM) and probable VM (pVM) who met the diagnostic criteria. Auditory-vestibular tests and psychological assessments were performed. Logistic regression was used to evaluate the predictive effect of EHF pure tone audiometry (PTA) for standard frequency (SF) hearing loss.Fifteen patients with pVM and 22 patients with dVM were recruited. Overall, the two most vertigo types were vestibulo-visual symptoms (83.78%) and internal vertigo (54.05%). A vertigo attack persisted for <5 min in approximately 57% of patients, compared with 5 min to 72 h in 43%, and lasted longer than 72 h in 8%. Approximately 87% of patients had psychological disorders. Most patients with VM (92%) suffered from some degree of EHF hearing impairment, and 68% had SF hearing loss, which is substantially higher than their complaints (43%). Moreover, the mean EHF hearing threshold cutoff value (57 dB HL) worked well in predicting SF hearing loss , outperforming distortion product optoacoustic emission .VM has a wide range of clinical manifestations. Hearing loss had a considerably higher rate compared to actual complaints. Moreover, patients with VM tended to have bilateral EHF and high-frequency hearing loss. The effectiveness of the mean EHF hearing threshold cutoff value in predicting hearing loss supported its use in the early detection of hearing loss and monitoring disease progression. Vestibular migraine (VM) presents with a recurrence of vestibular symptoms, a history of migraine, and a temporal association between vestibular spells and migraine symptoms  hearing plays an obscure but important role in our work and daily lives, including enhancing speech perception in noise  explore the audiological features of patients with VM between standard frequencies (SFs) and EHFs, (2) determine the use of EHF audiometry as a tool for the early detection of hearing loss in patients with VM, and (3) offer a rationale for the integration of a specific audiological assessment in the differential diagnosis and treatment of VM.A prospective cross-sectional study was conducted in the Department of Otorhinolaryngology of a tertiary hospital. The study was approved by the Ethics Committee of Wuhan Union Hospital under code number 20210873. A detailed medical history was taken, auditory-vestibular tests and psychological assessments on the participants were performed, and these data were analyzed in detail.Patients who had a definite VM (dVM) and probable VM (pVM) and attended the otolaryngology outpatient service of a tertiary medical institute between October 2021 and February 2022 were sequentially included. The diagnosis of VM was established on basis of the criteria formulated by Barany society in 2012 . The study excluded patients who had any external or middle ear diseases, had received ear surgery, had Meniere's disease (MD), had an acoustic neuroma, had chronic exposure to noise, had hereditary deafness, had an inner ear deformity, and took ototoxic drugs .A predetermined set of variables was extracted from medical records, supplemented by follow-up telephone calls when necessary, and then entered into a database. Clinical variables included gender, age, vertigo attacks, hearing loss, headache or migraine features, and other accompanying symptoms , motion sickness, sleep disorders, allergies, autoimmune diseases, cardiovascular risk factors, anxiety, depression, and a family history of vertigo or migraine.Pure-tone air conduction audiometry was performed separately in each ear for SFs  and EHFs  using an Astera audiometer . As ISO 389-5 2006 did not provide an equivalent reference threshold for a frequency of 20 kHz, it was excluded from the analysis. The underdetected frequency threshold was measured at 120 dB HL. The frequency could be divided into low frequency (0.125–0.5 kHz), middle frequency (1–2 kHz), high frequency (4–8 kHz), and EHF (10–16 kHz). If no response was obtained at one frequency, it was recorded as 120 dB HL. The result was taken as EHF hearing loss when the hearing threshold was greater than 20 dB HL at one or more frequencies ranging from 10 to 16 kHz in one or both ears.A chart was prepared by comparing the results of a previous study on ETF PTA in healthy people  was recorded by using a cochlear emission analyzer . The frequency ratio was set to 1.22 . DPOAE data were recorded for different frequency regions at 0.75, 1, 2, 3, 4, 6, and 8 kHz. For all frequencies, DPOAE measurements were considered detectable when the signal-to-noise ratio was greater than 6 dB. Abnormality was defined as a decreased or undetectable reactive amplitude at one or more frequencies.The vestibular autorotation test (VAT) was performed using a VAT device . VAT was considered abnormal when the gain, phase, or asymmetry decreased or increased.All patients were psychometrically assessed to confirm the presence of anxiety, depression, and somatic symptoms.Generalized anxiety disorder (GAD) assessment   , and difference analysis was done using two independent sample To analyze the predictive factors for hearing loss , the mean EHF hearing threshold and DPOAE results  were used to perform logistic regression analysis. AUC was used to evaluate the predictive power.A total of 37 adult patients with VM/pVM  were enrolled in this study. Fifteen patients with pVM having a mean age of 45.13 and 22 patients with VM having a mean age of 46.68 years were recruited. The female-to-male ratio was higher in patients with VM (1.5) than in patients with pVM (6.3), but the difference between the two was not significant. There were no statistically significant differences in vertigo features between patients with VM and their pVM counterparts. For the total cohort, the median disease duration was 16 months, the median vertigo spell lasted for 2 days, and the affected median duration was 4 days per month in the past 6 months. Vertigo types in patients with both VM and pVM varied. Overall, the most common types were vestibulo-visual symptoms (83.78%) and internal vertigo (54.05%), followed by dizziness (43.24%), and the rarest was a posture symptom (10.81%). We found that, in 56.76% of patients, the vertigo attack persisted no longer than 5 min, compared with 5 min to 72 h in 43.24% of the subjects. In three patients with VM, the vertigo episode lingered for more than 72 h.It was found that 26.67% of patients with pVM had migraine currently or earlier with or without aura, with the rate being 100% in patients with VM. Migraine features, such as, phonophobia and photophobia, were more frequent in the VM group than in the pVM group.The cochlear symptom in the interval episode were common in patients with VM and pVM. A total of 16 participants (43.24%) complained of hearing loss in one or both ears, and 20 patients (54.05%) had unilateral or bilateral tinnitus.The prevalence of motion sickness, phonophobia, and photophobia was higher in patients with VM than in patients with pVM. The rates of nausea and vomiting, family history, psychological disorder, autoimmune diseases, cardiovascular risk factors, and sleep disorder were frequent in both the pVM and VM groups, and no statistically significant differences in these accompanying disorders or conditions were observed .A difference in SF and EHF threshold of both ears in patients with pVM and VM was not significant .n = 25) had hearing loss, which was much more than their complaint/counterpart (43.24%). In 60% of patients with pVM (n = 9) and 72.73% of patients with VM (n = 16), the threshold was unilaterally or bilaterally elevated across SFs. The rate of bilateral hearing loss at SF  was higher than that of unilateral hearing loss as SF .Hearing loss at standard frequency  was found to be common in patients with pVM/dVM. Approximately 67.57% of patients (n = 32) had bilateral EHF hearing loss. Patients mainly developed total hearing loss (107.54 ± 34.22) at 16 kHz and moderate to severe hearing loss at 10 and 12.5 kHz (38.21 ± 33.71 and 52.24 ± 37.46).A total of 34 participants (91.89%) suffered from some degree of EHF hearing loss , as defined by the International Organization for Standardization. Of them, 94.12%  by gender, statistically significant differences were found only at 12.5 kHz but not at other frequencies .A large number of patients with pVM/dVM had high-frequency (59.5%) and EHF (91.9%) hearing loss as compared to low- (24.3%) and middle-frequency (24.3%) hearing loss .In comparison to the study by Wang et al. , no diffIn terms of SF hearing loss, subjects in our cohort could be further divided into two subgroups, patients with VM who had normal SF hearing and those who had SF hearing loss. In addition, we then selected the ear with the more severe hearing loss to represent the patient's hearing condition. We found that the subgroup with SF hearing loss had poor SF hearing as well as EHF hearing . MoreoveIn terms of the presence or absence of tinnitus, patients could be classified into VM with tinnitus and VM without tinnitus. The group of VM with tinnitus consisted of 20 patients (54.1%), and the group of VM without tinnitus consisted of 17 patients (45.9%). VM with tinnitus had a slightly higher SF and EHF threshold than VM without tinnitus, but the difference between the two was not significant . PatientSeven patients with pVM and 15 patients with VM both had abnormal DPOAE in one or both ears . In contn = 27), approximately 66.7% (n = 18) had central vestibular disorder, 11.1% (n = 3) had peripheral vestibular dysfunction, and 10.8% (n = 4) had vestibular impairment of unknown origin.Twenty-seven patients had VM and seven patients had an abnormal VAT. In patients with abnormal VAT (n = 32) of patients were found to have much more psychological disorder than their complaint (35.14%). In our cohort, 37.8% (n = 14) were suffering from depression, 32.4% (n = 12) from anxiety, and 70.3% (n = 26) from somatic symptoms. The psychological disorders were mostly mild to moderate in nature  curve analysis revealed that another cutoff value of >57.09 dB HL of the mean EHF hearing threshold had maximal sensitivity and specificity for predicting poor hearing  . Moreove 0.0137] .In terms of EHF mean hearing threshold, our patients were classified into two groups: patients with low mean EHF hearing threshold (LEHF) and those with high mean EHF hearing threshold (HEHF). Statistically significant differences were found in several clinical features between the two groups . PatientFirst, this study showed that VM had a wide variety of clinical manifestations, and some of its clinical features were inconsistent with previously reported characteristics. Secondly, we found that auditory system dysfunction, especially hearing loss, was common but less explicable in pVM/dVM. In patients with dVM/pVM, hearing loss was characterized by bilateral involvement and greater severity, particularly at EHF and high frequency, followed by middle and low frequencies. Moreover, we identified the mean EHF threshold as a better predictor for poor SF hearing. Patients with VM could be classified into two groups, namely the LEHF and HEHF groups, in terms of the cutoff value of 57 dB HL of the mean EHF threshold.Vestibular migraine had clinical manifestations, which varied substantially. Women and people at the age of 40 were preponderant, which coincided with previous findings  and DPOAE (62.9%),. This discrepancy could be explained by central and peripheral auditory system damage in VM: (1) reversible vasospasm of the internal auditory artery or its branches  outperformed DPOAE in predicting poor SF hearing. Therefore, EHF PTA might detect potential hearing damage in VM to allow for early intervention. Our study also showed that EHF PTA could be used to monitor disease progression and evaluate treatment efficacy. Hearing function was related to life quality, drop risk, and cognitive ability  and the clinical history of migraine precursors might present some uncertainties, as these data were only collected during a structured interview of the patient. Finally, our sample size was not sufficient, and there was no control group to allow for a more in-depth analysis and the identification of additional risk factors for hearing loss.Vestibular migraine has a wide range of clinical manifestations, including core symptoms (vestibular symptoms and migraine features), accompanying symptoms (auditory symptoms), and comorbidities . Auditory symptoms, especially hearing loss, were more common than previously thought. The majority of the patients with VM (74.3%) had SF hearing loss (0.125–8.0 kHz) and 97.1% had EHF hearing loss (8.0–20 kHz). Patients with VM tend to have bilateral EHF and high-frequency hearing loss (4.0–8 kHz). Moreover, EHF PTA may be used to predict hearing loss, monitor disease progression, and evaluate treatment efficacy. Patients with a high mean EHF hearing threshold (>57 dB HL) may benefit from positive multimodal migraine prophylaxis therapy and should put on hearing protection.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Wuhan Union Hospital. The patients/participants provided their written informed consent to participate in this study.SZ, ZG, and WK designed the research and directed its implication. ZG drafted and modified the manuscript. ZG, ET, JW, and JC contributed to the medical record collection. All authors have read and agreed to the published version of the manuscript.


OTOF gene. The aim of this study was to investigate the pathophysiological mechanisms behind the auditory dysfunction in five patients (2 adults and 3 children) carrying biallelic mutations in OTOF, who showed an uncommon phenotype of mild hearing impairment associated with severe difficulties in speech perception and delay of language development.Congenital profound hearing loss with preserved cochlear outer hair cell activity (otoacoustic emissions and cochlear microphonic) is the most common phenotype associated with mutations in the OTOF gene. Three patients out of five underwent unilateral cochlear implantation. Speech perception measures and electrically evoked auditory nerve potentials were obtained within 1 year of cochlear implant use.Patients underwent audiometric assessment with pure-tone and speech perception evaluation, and otoacoustic emissions and auditory brainstem response recording. Cochlear potentials were recorded in all subjects through transtympanic electrocochleography in response to clicks delivered in the free field from 120 to 60 dB peak equivalent SPL and were compared to recordings obtained from 20 normally hearing controls and from eight children with profound deafness due to mutations in the OTOF were found in all five patients, and five novel mutations were identified. Hearing thresholds indicated mild hearing loss in four patients and moderate hearing loss in one. Distortion product otoacoustic emissions were recorded in all subjects, whereas auditory brainstem responses were absent in all but two patients, who showed a delayed wave V in one ear. In electrocochleography recordings, cochlear microphonics and summating potentials showed normal latency and peak amplitude, consistently with preservation of both outer and inner hair cell activity. In contrast, the neural compound action potential recorded in normally hearing controls was replaced by a prolonged, low-amplitude negative response. No differences in cochlear potentials were found between OTOF subjects showing mild or profound hearing loss. Electrical stimulation through the cochlear implant improved speech perception and restored synchronized auditory nerve responses in all cochlear implant recipients.Pathogenic mutations in the two alleles of OTOF gene who show a stable phenotype of mild hearing loss. Abnormal nerve synchrony with preservation of hearing sensitivity is consistent with selective impairment of vesicle replenishment at the ribbon synapses with relative preservation of synaptic exocytosis. Cochlear implants are effective in restoring speech perception and synchronous activation of the auditory pathway by directly stimulating auditory fibers.These findings indicate that disordered synchrony in auditory fiber activity underlies the impairment of speech perception in patients carrying biallelic mutations in  Auditory neuropathy is a hearing disorder characterized by disruption of the temporal coding of acoustic signals in the auditory nerve fibers affecting auditory perceptions dependent on the processing of temporal cues, the magnitude of these perceptual changes being independent of the changes in audibility (OTOF gene (DFNB9) encoding otoferlin, a transmembrane protein involved in glutamate neurotransmitter release at the IHCs ribbon synapses. Specifically, otoferlin might have a dual role in synaptic vesicle exocytosis, as a calcium sensor for synaptic vesicle fusion  function as indicated by cochlear microphonic (CM) and otoacoustic emissions (OAEs) recordings.Mutations in ulations . Most paOTOF indicated preservation of both outer and IHC function  in profoundly deaf children carrying two mutant alleles in function , 2015. Ifunction .OTOF have been reported in patients showing a temperature-sensitive phenotype . Details of their genetic and audiological findings are summarized in Table We investigated the hearing function in five subjects carrying biallelic mutations in the Three patients were evaluated as children (#3 to #5). They had passed the newborn hearing screening by OAEs recording and were referred for assessment because of delay in language development. Findings from subject #4 have been partially reported in a previous study .Two subjects were diagnosed in their adult life. Subject #1 was evaluated at another institution at the age of 2 because of delay in speech development, but, since hearing thresholds were normal, she was referred for cognitive assessment. When we saw her, she was able to speak quite fluently, but speech perception relied almost completely on lipreading. Subject #2 had undergone several audiological assessments since childhood, first for delay in language development and thereafter for impairment of speech perception. At the time of evaluation, language was quite good, but verbal production showed several phonological and morphosyntactic errors, while speech perception relied on lipreading. Both adult subjects had tried hearing aids without benefit.None showed the temperature-sensitive phenotype.CT and MRI scans of the head and ear as well as growth and motor development were normal in all.Three patients  received unilateral cochlear implant, whereas two refused the procedure.OTOF, as reported . Sanger sequencing was used to screen for mutations in reported .OTOF isoform. Nomenclature follows current Human Genome Variation Society rules as implemented by the Mutalyzer 2.0.3 program . Software for prediction of mutation pathogenicity is indicated in the footnote to Table Mutation nomenclature is based on GenBank accession number NM_001287489.1, which corresponds to the cDNA sequence of the long cochlear This study was approved by the Ethical Committee of Hospital Universitario Ramón y Cajal (in accordance with the 1964 Declaration of Helsinki). Written informed consent was obtained from all participating subjects, or from their parents in the case of minors.All audiological tests were performed in a sound-attenuating room (Marvinacustica). In cooperative subjects (#1 to #3), air and bone conduction hearing thresholds were obtained in each ear through conventional audiometry (Madsen Astera 2), whereas the two younger children performed visual reinforcement audiometry in the free field. The degree of hearing impairment was defined by the pure-tone average (PTA) at 0.5, 1, 2, and 4 kHz .Acoustic reflex thresholds were measured ipsilaterally and contralaterally to the stimulated ear at frequencies of 0.5, 1, 2, and 4 kHz. (Madsen Zodiac impedance audiometer). They were considered absent when no response was found at intensities higher than 110 dB HL.Articulation gain curves were obtained using disyllabic, phonetically balanced words from 10 Italian wordlists for adults or children, each including 20 items . Ten worDistortion product otoacoustic emissions (DPOAEs) were obtained using the ILO-292 OAE system. Primary tones were presented at 65/55 dB SPL and the f2/f1 ratio was kept at 1.21. The f2 frequency was increased in 1/4 octave steps from 1.0 kHz. The upper f2 frequency was kept at 8.0 kHz in subjects #1, 2 and #4, and at 10.0 and 6.0 kHz in subjects #3 and #5, respectively.Gap detection was measured in the two adult subjects (#1 and #2). Stimuli were presented in the free field through one loudspeaker placed 1 m away from the front of the subject’s head at an intensity of 30 dB above threshold. We used the procedures reported by Psychophysical tuning curves (PTCs) were measured in subject #2 for each ear at frequencies of 1, 2, and 4 kHz by presenting the sinusoidal signal at a fixed sensation level (15 dB SL) together with a narrowband noise used as the masker. The level of masker required to just mask the signal was determined for 19 masker center frequencies. The stimulus paradigm consisted of a two-alternative, forced-choice, two-up and one-down procedure. The masker was presented in two intervals , one of which (chosen at random) contained the signal (duration 200 ms). The masker level was changed with a step size of 4 dB until the level of the masker barely masked the signal. Thereafter, the two-up and one-down procedure was repeated using a step size of 1 dB. The measurements were made using a Tucker–Davis Technologies system controlled by a computer. The masker and signal were generated according to In the two adult subjects, the recognition of disyllabic words and identification of consonants were evaluated by presenting recorded speech stimuli in the free field through one loudspeaker placed 1 m away from the front of the subject’s head. Speech material comprised digital anechoic recordings of a native Italian female speaker. Tests were administered at 70 dB(A) in quiet. In subject #2, the recognition of disyllabic words was also performed in the presence of competing speech noise at three signal-to-noise (S/N) ratios  through two additional loudspeakers placed laterally at an angle of 90° on either side of the subject’s head at a distance of 1 m. Sentence recognition was evaluated through live-voice presentation. The speech material was obtained from the protocol of patient candidacy for cochlear implantation (CI) for the Italian language for adult patients . The recIn consonant identification test, stimuli consisted of confusion matrices compiled from two presentations, each comprising the 16 consonants, b d f g k l m n p r s t v z j t∫ presented in an “a-consonant-a” context (chance 6%).In children, all tests were administered through live-voice presentation. The speech material was obtained from the protocol of evaluation of speech perception for the Italian language . The speAuditory brainstem responses (ABRs) were recorded from scalp electrodes in response to 2000 trials of alternating polarity clicks presented monaurally at a maximum intensity of 125 dB peak equivalent (p.e.) SPL . The filter settings of the amplifier were set between 5 and 4000 Hz.The adult patients and children’s parents signed an informed consent form for submission to ECochG recording, which is part of our standard assessment protocol in patients showing the profile of auditory neuropathy. The ECochG protocol was assessed by the regional body for quality control of clinical and therapeutic procedures .Adults were tested under local anesthesia and children under general anesthesia. ECochG procedures have been reported elsewhere . BrieflyThe stimulus paradigm consisted of an initial click, followed 15 ms later by 10 clicks with an interstimulus interval of 2.9 ms, and the sequence was repeated every 191 ms .The potentials were differentially amplified , filtered (5 to 8000 Hz) and digitized (25 µs) for averaging . The procedure of averaging the responses evoked separately by condensation and rarefaction clicks was applied to cancel the CM and extract the CAP with the superimposed summating potential (SP). The resulting curve was subtracted from the potential evoked by condensation clicks to obtain the CM. Since CM attenuation was often incomplete at a high stimulus intensity and CM spectral energy was at a maximum between 1500 and 3000 Hz, a low-pass digital filter  was used to attenuate the residual CM, where needed .Latency was defined relative to CM onset in milliseconds (ms). Amplitude was computed relative to the period 1 ms before CM onset in microvolt (µV).OTOF-related profound hearing loss whose ECochG findings have been reported in a previous paper  tested for presumed cochlear hearing loss, who showed normal thresholds when evoking neural and receptor potentials , while tus paper .Electrically evoked CAPs (e-CAPs) were obtained through the cochlear implant in response to trains of alternate polarity, biphasic, 25 µs width per phase pulses presented at 80 Hz. The evoked electrical activity was recorded two electrodes apart.p <0.05.Separate two-factor ANOVAs for repeated measures with factors of group and stimulus intensity were used to evaluate ECochG measures. Post-hoc tests for multiple comparisons were conducted using the Tukey–Kramer procedure. The level of significance was OTOF gene. Mutations in the two alleles of OTOF were found in all five patients . All four novel missense mutations affect highly conserved residues in otoferlin across diverse vertebrate species , and mild (#4) or moderate (#5) hearing loss in the two children submitted to visual reinforcement audiometry in the free field  who showed a delayed Wave V in their left ear , SP amplitude , and SP peak latency  between groups, although SP amplitudes tended to be smaller in OTOF subjects compared to normally hearing controls. In contrast, the duration of ECochG potentials as measured from SP onset to return to baseline was significantly prolonged in OTOF patients compared to normally hearing controls . No differences in response duration were found between the two groups of OTOF subjects.Mean and individual values of amplitudes and latencies of ECochG potentials from the five OTOF patients with mild to moderate hearing loss are plotted as a function of signal intensity in Figure us paper . No signGap detection was measured in subjects #1 (difference limen = 80 ms) and #2 (difference limen = 24 ms). Values proved remarkably higher in comparison to the scores obtained in a group of 19 normally hearing individuals .PTCs were obtained in subject #2 at frequencies of 1, 2, and 4 kHz. These are displayed in Figure Speech intelligibility was tested in cooperative subjects (#1 to #3)  Fig. .Articulation gain curves  were recorded in all cochlear implant recipients in response to electrical stimulation through the cochlear implant at each electrode location, consistent with restoration of synchronous activation of auditory fibers Fig. B. e-CAP OTOF cause severe to profound congenital hearing loss in a majority of cases, but there are a few patients whose phenotypes deviate from the general rule. Previous studies have reported mild to moderate hearing loss in patients carrying biallelic mutations in OTOF gene for the temperature-sensitive phenotype  that result from mutations in OTOF gene correlate with the presence of at least one missense variant or one in-frame deletion in their associated genotypes (ECochG recordings carried out in patients affected by enotypes , and so enotypes . Howeverenotypes . Diverseenotypes . Only onAlthough the hearing thresholds were not evaluated in each ear for both subjects #4 and #5, we believe that they were symmetrical. Indeed, the threshold of the prolonged ECochG potential recorded in subject #3 was comparable to the hearing thresholds measured through earphones. Moreover, the threshold of the prolonged ECochG component in subjects #4 and #5 was comparable to the hearing thresholds obtained in sound field for both ears. On the basis of these findings, it seems reasonable to admit that hearing thresholds were symmetrical between ears also in subjects performing sound field audiometry.Interestingly, PTCs measured in subject #2 were distorted and showed a truncation of the tip segment. OTOF-related mild hearing loss. In our previous article, we reported the benefits of CI in a group of eight profoundly deaf children carrying mutations in both alleles of OTOF gene (OTOF (OTOF-related hearing disorders.Another relevant finding of this study consists of the effectiveness of cochlear implants in restoring speech perception in TOF gene . A positne (OTOF  and for ne (OTOF . NeverthOTOF gene may result in alterations of temporal coding of acoustic stimuli with good preservation of hearing sensitivity. Speech perception remarkably improves in response to electrical stimulation through unilateral CI, which remains an effective therapeutic option also in the perspective of future gene therapy possibly targeting the contralateral ear.In conclusion, mutations of both alleles of the We are greatly indebted to Prof. Roberto Bovo and Dr Franco Trabalzini who performed surgery. We are also grateful to engineer Antonio Selmo who supported us in setting up the recording apparatus.


Age-related hearing loss is a common, heterogeneous disease with a strong genetic component. More than 100 loci have been reported to be involved in human hearing impairment to date, but most of the genes underlying human adult-onset hearing loss remain unknown. Most genetic studies have focussed on very rare variants (such as family studies and patient cohort screens) or very common variants (genome-wide association studies). However, the contribution of variants present in the human population at intermediate frequencies is hard to quantify using these methods, and as a result, the landscape of variation associated with adult-onset hearing loss remains largely unknown.Here we present a study based on exome sequencing and self-reported hearing difficulty in the UK Biobank, a large-scale biomedical database. We have carried out variant load analyses using different minor allele frequency and impact filters, and compared the resulting gene lists to a manually curated list of nearly 700 genes known to be involved in hearing in humans and/or mice. An allele frequency cutoff of 0.1, combined with a high predicted variant impact, was found to be the most effective filter setting for our analysis. We also found that separating the participants by sex produced markedly different gene lists. The gene lists obtained were investigated using gene ontology annotation, functional prioritisation and expression analysis, and this identified good candidates for further study.Our results suggest that relatively common as well as rare variants with a high predicted impact contribute to age-related hearing impairment and that the genetic contributions to adult hearing difficulty may differ between the sexes. Our manually curated list of deafness genes is a useful resource for candidate gene prioritisation in hearing loss.The online version contains supplementary material available at 10.1186/s12915-022-01349-5. Hearing impairment is one of the most common sensory deficits in the human population and has a strong genetic component. However, the auditory system is a complex system with many interacting parts, which offers many routes to loss of function. Accordingly, although over 150 genes have been identified as contributing to non-syndromic human hearing loss .As early as 1997, it was noted that single-gene mutations can lead to early postnatal or adult-onset progressive hearing loss . This reAlternative approaches are therefore required to identify novel variants and genes associated with age-related hearing loss. Here we have investigated variants associated with self-reported hearing difficulty in 94,312 UK Biobank participants with available exome sequence data. We have assessed variant load in self-reported hearing difficulty at a range of variant minor allele frequencies, from rare variants  < 0.005) to very common variants, and compared the resulting gene lists to a much larger list of known deafness genes that we have curated and present here, based on work in mice as well as in humans. We found the optimal MAF cutoff to be 0.1, which is an intermediate frequency, neither rare nor common. Our results suggest that intermediate frequency variants with a high predicted impact contribute to hearing difficulty, and also that the genetic contributions to hearing impairment may differ between the sexes.After filtering, in the normal hearing group there were 18,235 male (average age = 62.37 years) and 30,496 female (average age = 62.20 years) participants . In the hearing difficulty group, there were 24,237 male (average age = 63.60 years) and 21,344 female (average age = 62.96 years) participants . It is notable that while the overall group sizes are similar, there are many more female participants than male in the normal hearing group, reflecting the better hearing that women have later in life , althougOutlier analyses of the genomic variant loads per gene in each group of people were carried out. Briefly, for each gene, the number of variants in people with hearing difficulty was compared to the number of variants in people with normal hearing using a linear regression. Each regression analysis resulted in two lists of outlier genes; those with a much higher variant load than expected (high variant load in hearing difficulty) and those with a much lower variant load than expected  , and obtained the lists of outlier genes, those genes with more variants than expected in hearing difficulty or in normal hearing  are indicated with a *.A, Gene counts for high variant load lists when using high-impact variants at different MAF cutoffs. B, Gene counts for high variant load lists when using low impact variants, MAF<0.1. C, Gene counts for high variant load lists with sex segregation, MAF<0.1. D, Gene counts for high variant load lists using the MSC cutoff, showing the counts for all participants and separated by sex, MAF<0.1. The As the MAF limit increased, the number of genes in the outlier lists also increased, as did the number of deafness and variable genes in each outlier list Table , A. HoweThe effect of including variants with a low impact . Twenty-four genes were present in both normal hearing high variant load lists, including three deafness genes   is a gene-specific cutoff value which uses data from HGMD and ClinVar . BecauseThe large numbers of outlier genes in people with normal hearing was unexpected, so we asked if there might be different types of variants common in hearing difficulty compared with normal hearing. We investigated the characteristics of the variants in the high variant load lists, taking the most deleterious consequence for each variant in each gene , but not by impact, since all variants included in this analysis were high impact. After correcting for multiple tests, none of the genes retained significant p values. We therefore ranked the gene list by signed log P value (SLP) [p value, with a positive sign indicating that cases have more variants than controls, and a negative sign indicating the opposite. Thus, ranking the genes by SLP would result in one extreme of the list being genes with more variants in the people who did not report hearing difficulty (n = 270 genes with SLP < −2), and the other extreme being genes with more variants in the people who did report difficulty hearing (n = 362 genes with SLP > 2)  . The SLPUSH1C, USH2A, MYO3A, TMC2, ADGRV1, PDZD7 and PKHD1L1. Most are known human deafness genes, but PKHD1L1 and TMC2 have only been identified as mouse deafness genes to date [MYO3B, MYH14, CDH23, CLIC5, CHRNA10, FBXO11, TMC1, GJB2, NAV2, LOXHD1, SLC26A5 and MYO6. Genes annotated with this term which had a high variant load in all participants with hearing loss were COL11A1, MYH14, CDH23, CLIC5, CHRNA10, FBXO11, TMC1, GJB2, LOXHD1, SLC26A5 and MYO6. Most of these are known human deafness genes, but to date, FBXO11 has only been identified in the mouse, not in humans . In summary, the GO term analysis showed enrichment for terms relating to sensory hair cells or cytoskeletal elements known to be important to hair cell function, and this appears to be driven by the enrichment for known deafness genes in the lists analysed. Many more genes were included in the high variant load lists than were described by GO annotation terms, reflecting the limitations in current GO annotations of many genes.In order to look for any clues to pathological mechanisms, we carried out a gene ontology (GO) enrichment analysis using gProfiler  on the h to date , 19. Farp values; after correction for multiple testing, we obtained eight genes from the hearing difficulty lists  and three from the normal hearing lists  , and we also plotted data for the genes known to underlie deafness in both mice and humans which were not already included  as useful markers of cochlear cell types. After selecting those genes showing high variance in expression across the cell types, there were 234 genes from the high variant load lists and 78 more from the known deafness gene list. From the resulting heatmap, clusters of genes were annotated according to their expression, defining high expression levels (red) as 2–3, middle expression levels (orange) as 1–2 and low expression levels as 0–1 (yellow)  and strial cell types, possibly because there were fewer datasets available in the gEAR database for these cell types compared to hair cells, pillar cells and supporting cells, but cluster 2D did show consistent SGN expression  vs controls (people who did not report difficulty hearing). Burden tests compare the average of individual scores in cases and controls, while our outlier approach is an aggregated one, simply summing all the variants in cases and comparing them to the sum in controls. That has proved to be a useful approach for the UK Biobank cohort, which lacks all but the most basic auditory phenotyping data. In a cohort with more detailed auditory phenotyping, a burden test may prove to be a better approach. For example, Ivarsdottir et al. recently reported identifying the candidate gene c cohort .FSCN2, SYNJ2, FBXO11, NAV2, TMC2, ERCC6 and PKHD1L1. Of the 185 known human deafness genes, 118 are also mouse deafness genes . Three of the ToppGene candidates were included in the gEAR heatmap; TGFBR1 and FLNA, which are mainly expressed in the organ of Corti, and MYLK, which is expressed in fibrocytes . It is also subject to ascertainment bias due to the relative lack of data on inner ear cell types which are not hair cells or supporting cells. We obtained 6 datasets from hair cells and supporting cells at different stages from E16 to P35, but only 2 datasets from cochlear lateral wall cell types at 2 adult ages (P20 and P30), and only one dataset from SGNs (P17-33). This means that any gene expressed during development in the lateral wall or SGNs, but not expressed in adult stages, will have been missed out of our heatmap. Additionally, most of the known deafness genes which we plotted on the heatmap are hair cell or supporting cell genes, and this may have biased the clustering. This may be why there are more outlier genes assigned to clusters with expression in hair cells  showed almost all of them were significantly enriched in deafness genes  . Five further genes from our outlier lists were shared between a subset of the GWAS lists; TMPRSS9, LOXHD1 and TUB were identified by Ivarsdottir et al. and Kalra et al., and SLC26A5 and FSCN2 by Ivarsdottir et al. alone. Those genes identified by multiple studies are obvious candidates for involvement in ARHL, but the differences in loci identified using the GWAS approach suggest there are many more to investigate, and the results of our exome sequencing analysis support that as well as suggesting further candidates  and detailed auditory phenotyping. Self-reported hearing difficulty has been shown to be sufficiently informative for general hearing capacity , 47, butFrom this study, we have established that it is useful to include more common variants when investigating a heterogeneous disease such as adult hearing difficulty. In this case, we found the most useful MAF cutoff to be 0.1, but it is likely that this varies by condition. We also found that the genetic contributions to self-reported hearing difficulty differ between the sexes, suggesting that in future studies, it would be useful to separate study participants by sex, as well as analysing all participants together. Future studies would also benefit from more detailed auditory phenotyping data.While these points are based on a study of adult self-reported hearing difficulties, it is likely that they apply to many other conditions. As the availability of large-scale exome and genome sequencing studies grows, it is important to explore questions which could not be asked using earlier paradigms such as genome-wide association studies. This work highlights several such avenues of exploration.UK Biobank (RRID:SCR_012815) is a large-scale biomedical database and research resource containing genetic, lifestyle and health information from half a million UK participants, aged between 40 and 69 years in 2006–2010, who were recruited from across the UK. Participants have consented to provide their data to approved researchers who are undertaking health-related research that is in the public interest. Participants were selected who were ≥55 years of age who had exome sequencing data available  and could be classified as having normal hearing or hearing difficulty, based on their self-report of hearing difficulty, hearing difficulty in noise, or use of a hearing aid. If people reported no hearing difficulties or hearing aid use at any assessment and had been asked about their hearing at least once when they were ≥55, we included them in the “normal hearing” group. If people reported consistent or worsening hearing impairment, or that they had at any point been a hearing aid user and had been asked at least once about their hearing when they were ≥55, we included them in the “hearing difficulty” group. Participants who reported otologic disorders (e.g. Meniere’s disease) were excluded. People who reported high levels of noise exposure or moderate/severe tinnitus were also excluded from the normal hearing group  [TMC1 have been shown to result in either prelingual profound hearing loss or postlingual progressive hearing loss [We compiled a manually curated list of known deafness genes in humans and mice, including all genes listed in the Hereditary Hearing Loss Homepage (RRID:SCR_006469) , and gen_006158) , 68; ave_006158) ; for fuling loss , 72, anding loss , 43.Fig.TTN), or because they belong to groups of paralogues such as olfactory receptors, which are sufficiently similar to make correct alignment difficult, resulting in incorrect variant calls. Two such lists were compiled by Adams et al. [Some genes are often reported as having a high number of variants in multiple exome sequencing projects. This can be because they are large genes  (RRID:SCR_017467) [Hprt expression; where Hprt was not present in the dataset, or had an expression level of 0, we did not use the data. We then summarised the data, taking the maximum level per cell type without accounting for age. Because the expression levels ranged from 0 to 70.6 , we transformed the data such that levels between 10 and 100 were scaled to between 2 and 3, and levels between 1 and 10 were scaled to between 1 and 2. We used R to plot a heatmap of the genes that showed the most variability between cell types, suggestive of specific expression patterns rather than non-specific expression , and to cluster cell types and genes. We further defined gene clusters first based on the R dendrograms and then on the gene expression levels within specific cell types or groups of cell types.To assess the expression of lists of genes of interest in the inner ear, including the list of known mouse and human deafness genes, we used single-cell RNAseq data from the mouse inner ear, accessed via the gEAR portal (_017467) . We chos_017467) , 77, P15_017467) , 79, P20_017467) , 81, P28_017467) –84, P30 _017467) , 86 and _017467) , 88. ExpAdditional file 1: Fig S1. Population characteristics of chosen participants. Fig S2. Variant types in the outlier gene lists. Fig S3. Outlier and deafness gene heatmap. Fig S4. Participant selection.Additional file 2: Table S1. Self-reported ethnicity in the chosen participants. Table S2. Outlier genes with a high variant loads and genes with extreme SLP values from the burden analysis. Table S3. Genes reported to underlie hearing loss in humans and/or mice. Table S4. Highly variable genes. Table S5. Variant classifications in order of impact (high to low). Table S6. GO term analysis. Table S7. ToppGene analysis. Table S8. Expression and classification of clustered genes from the heatmap. Table S9. Protein-coding genes and microRNAs used in this study.