Sensorineural hearing loss is one of the most common sensory deficiencies. However, the molecular contribution to age-related hearing loss is not fully elucidated.N = 362,396) and selected a high confidence set of ten hearing-associated gene products for staining in human cochlear samples: EYA4, LMX1A, PTK2/FAK, UBE3B, MMP2, SYNJ2, GRM5, TRIOBP, LMO-7, and NOX4.We performed genome-wide association studies (GWAS) for hearing loss-related traits in the UK Biobank (All proteins were found to be expressed in human cochlear structures. Our findings illustrate cochlear structures that mediate mechano-electric transduction of auditory stimuli, neuronal conductance, and neuronal plasticity to be involved in age-related hearing loss.Our results suggest common genetic variation to influence structural resilience to damage as well as cochlear recovery after trauma, which protect against accumulated damage to cochlear structures and the development of hearing loss over time.The online version contains supplementary material available at 10.1186/s12916-021-02169-0. In humans, hearing loss is a potentially debilitating condition that affects more than 1.23 billion people worldwide and constitutes one of the world’s top ten causes of years lived with disability [The molecular mechanisms that underlie the development of hearing loss and individual variation in risk are poorly elucidated. Twin studies have demonstrated a moderate-to-high heritability for age-related hearing loss of 40–70% –6 and seNID2), clarin-2 (CLRN2), and the Rho guanine nucleotide exchange factor 28 (ARHGEF28) [Over the last decade, genome-wide association studies (GWAS) have been able to illuminate the genetic architectures behind a number of human traits and diseases. Recently, genetic data have become available from large cohorts with standardized collection of many phenotypic traits. For example, in the UK Biobank, a cross-sectional cohort of more than half a million United Kingdom (UK) residents, information on hearing-related traits was collected by touchscreen questionnaire as well as speech-in-noise hearing tests. A recent GWAS of self-reported hearing difficulty and hearing aid use in ~ 250,000 UK Biobank participants identified 44 genetic loci that were associated with these traits and were able to demonstrate the utility of self-reported data for identification of hearing loss-associated genetic variants. In addition, immunological staining revealed murine cochlear expression of three novel hearing-associated proteins: nidogen-2 (encoded by RHGEF28) .GJB2) [SLC26A5), the motor protein of the outer hair cells [The cochlea is the critical organ for the sense of hearing and several structural and metabolic components of the human cochlea have been linked to rare forms of syndromic and non-syndromic deafness, such as the gap-junction protein connexin 26 (encoded by GJB2)  and presir cells . ImagingIn this study, we visualize human hearing-associated proteins, identified as candidates by GWAS, in human cochlear samples by immunohistochemical staining combined with super-resolution structured illumination microscopy (SR-SIM). Human cochlear samples were collected during surgical removal of life-threatening petroclival meningioma and directly fixed for optimal preservation and antigenicity of protein structures.P < 5 × 10−8): 35 with hearing difficulty, 22 with hearing aid use, eleven with tinnitus, and two with speech-in-noise ; DFNB28 , which contains TRIOBP (TRIO and F-actin-binding protein); DFNB61, which contains the gene that encodes prestin, SLC26A5; DFNA58, DFNB47, and DFNB79, which contains TPRN (taperin) and CACNA1B , also overlapped with additional non-syndromal deafness-associated loci: DNFB36, which contains ESPN (espin), DFNB42, which contains ILDR1 (immunoglobulin-like domain-containing receptor 1), DFNB12, which contains CDH23 (cadherin 23), DFNA3A, which contains GJB2 (connexin26), DFNA9, which contains COCH (cochlin), DFNA4B, which contains CEACAM16 (carcinoembryonic antigen-related cell adhesion molecule 16), and DFNB8/10, which contains TMPRSS3 , which contains the gene CCR9, FSCN2, TYR, SYNJ2, SLC16A8, KLHDC7B, and HLA-DQB1  to be expressed in the cuticular plate of the human outer and inner hair cells Fig. A–C. LMO-deafness . In vitrdeafness . In the deafness .Fig. 2ETRIOBP splice variant isoforms 5 and 4. Non-syndromic autosomal recessive deafness 28 (DFNB28) has also been mapped to nonsense and frameshift mutations that occur mainly in exon 6 of TRIOBP [TRIOBP expression could be observed in the cuticular plates of the inner and outer hair cells: in the cell bodies and stereocilia, and in the nerve fibers that innervate the inner and outer hair cells, as well as the outer spiral bundle Fig. A, C, D. f TRIOBP , 34.FigTRIOBP. Consequently, the results presented in this study reflect the expression patterns of TRIOBP isoforms 5 and 1, which both include these exons.Both humans and mouse exhibit three protein isoforms of TRIOBP: TRIOBP-5, which is encoded by a transcript that includes exons 1–3, 5–9, and 12–24, TRIOBP-4, which is encoded by a transcript that ends after exon 6, and TRIOBP-1, which is encoded by a transcript that only includes exons 11–24 . Of thesCallithrix jacchus [Eya4 expression was determined in the cochlea of the common marmoset due to the phenotypic discrepancies between Eya4-deficient mice and humans with EYA4 linked non-syndromic autosomal dominant deafness 10 (DFNA10). Eya4-deficient mice exhibit Eustachian tube dysmorphology and otitis media with effusion [Eya4 RNA expression studies in the developing rat cochlea indicate that this protein is important for normal development of the cochlea [EYA4 was observed to be expressed in supporting cells of the membranous labyrinth and organ of Corti: in the interdental cells, inner sulcus cells, phalangeal cells, pillar cells, Deiters cells, Hensen cells, Boetcher cells, and cells of Claudius; as well as in the nuclei of the outer and inner hair cells -triphosphate (PIP3) and phosphatidylinositol -biphosphate, and plays a key role in recovery of secretory vesicles in presynaptic neurons via endocytosis [Synj2 was linked to progressive hearing loss in the Mozart mouse strain, which was generated in a N-ethyl-N-nitrosourea (ENU) mutagenesis program at the Australian Phenomics Facility in Canberra [Synj2 and are born with normal hearing that deteriorates by 8 weeks, leading to complete loss of hearing at 12 weeks. Mozart mice also exhibit stereociliar fusion, loss of hair cell bundles, and subsequent hair cell degeneration [Synj2 mutation in the phosphatase catalytic domain was able to confirm the observation of progressive hearing loss in Synj2-deficient mice [SYNJ2 was observed to be expressed in the outer spiral bundles Fig. A. Co-stair cells  as well ir cells   reported FAK protein expression in the supporting cells of the organ of Corti [FAK-stained positive in the outer spiral bundles beneath the outer hair cell layer Fig. A, B. Basof Corti . Expressof Corti . It is pGRM5 encodes the metabotropic glutamate receptor 5, which is a postsynaptic G protein-coupled receptor that activates downstream signaling via phospholipase C after glutamate-binding to the extracellular portion of the receptor. There is no previous data on GRM5 expression in the cochlea, in humans or any other species. However, metabotropic glutamate receptor 5 signaling has been linked to central auditory processing pathways from the medial geniculate nucleus in the brainstem, to the lateral amygdala in rats [GRM5 was also expressed in the spiral ganglion cells Fig. A. Simila in rats , 90, 124 in rats . This re in rats .Fig. 8ELmx1a expression in a discrete type I spiral ganglion neuron subpopulation in the developing mouse cochlea [LMX1A encodes a transcription factor, LIM homeobox transcription factor 1-alpha, which is involved in neuronal differentiation as well as maintenance of neuronal homeostasis in the adult brain [Lmx1a-/- mutant mice exhibit severe developmental defects of the cochlea along with disruptions of cochlear cellular organization [LMX1A expression was observed in the inner and outer pillar cells and the reticular lamina in the organ of Corti Fig. A. Type I cochlea . These s cochlea  and enab cochlea . LMX1A elt brain . In micenization , 130–133NOX4 encodes the NADPH oxidase 4 enzyme, which catalyzes the reduction of di-oxygen (O2) to super-oxide (2O•−), a precursor of reactive oxygen species. Reactive oxygen species are canonically known to damage protein structures within the cell. However, at low concentrations, reactive oxygen species also act as intracellular signaling molecules [NOX4 by RNA interference led to a reduction in filamentous actin stress fibers and reduced cellular migration [NOX4 was observed in the supporting cell of the organ of Corti with particularly strong expression in Hensen cells, pillar cells, and the cell membrane of the outer hair cells. Interestingly, NOX4 staining in the inner hair cells was negative, while expression was observed in the outer hair cell membrane and cochlear supporting cells Fig. A. NOX4 wolecules . In vitrigration .Fig. 10. vs. 91,080 cases and 257,037 controls in the current analysis), as well as a higher number of cases and controls in the GWAS for hearing aid . Consequently, twenty hearing difficulty associated and 22 hearing aid use-associated loci that are reported in our study were not observed to be significantly associated in the previous GWAS [The results from our study are consistent with the recent GWAS of hearing difficulty and hearing aid use on UK Biobank participants . HoweverSYNJ2, SLC26A5 , LMO7, SPTBN1 (DFNA58), and FSCN2. Conversely, Wells et al. reported an association of a missense variant within CDH23, which encodes a tip-link protein, cadherin 23. This protein forms a link between the tips of the stereocilia, usually from the tallest to the shorter, and mediates mechanotransduction during stereociliar deflection by opening spring-gated ion channels on the lower stereocilia [CDH23 have been found to be causal for the recessive autosomal deafness locus DFNB12 [Among the hearing difficulty associated loci that were not reported by Wells et al. were several highly biologically relevant genes that are unlikely to represent spurious associations, such as reocilia . Mutatios DFNB12 .SLC26A5, which encodes the motor protein of the outer hair cells, prestin. A large number of associated loci also contained genes that have previously been linked to hearing-related traits and cochlear structures in vitro and in vivo in other species. Imaging analyses of GWAS-associated genes in human tissue samples are uniquely informative due to the discrepancies in cochlear morphology and protein expression that occur between species. In addition, we were also able to confirm the cochlear expression patterns of several hearing-associated proteins for the first time in human tissue and generate precise mapping of protein expression even at the subcellular level.GWAS of self-reported hearing loss-related traits in the UK Biobank revealed that a substantial amount of the associated loci, approximately 40%, could be linked to genes that were determined to be biologically relevant for human hearing. Furthermore, associations overlapped with human congenital non-syndromic deafness loci and associations were found that were proximal to genes with determined functional roles in human auditory mechanotransduction, such as DYRK2 , RAB27B (Ras-related protein Rab-27B), SEPT11 (Septin-11), NTRK3 (NT-3 growth factor receptor), ASTN2 (Astrotactin-2), BAIAP3 (BAI1-associated protein 3), and MAST4 (microtubule-associated serine/threonine-protein kinase 4). Similar to the hair cells, spiral ganglion neurons are sensitive to environmental factors such as noise, ototoxic compounds, and inflammation [NTRK3, which encodes the receptor for the neurotrophic factor neurotrophin-3 (NT-3) and has previously been found to be expressed in the spiral ganglion of rat [RAB27B (Ras-related protein Rab-27B), which mediates anterograde axonal transport of the BDNF/NT-3 growth factor receptor, TrkB [MMP2), neuronal migration (ASTN2 and FAK), and cytoskeleton organization and neurite outgrowth .Taken together, our findings strongly implicate spiral ganglion function to be critical for hearing loss. Nine of the proteins that were selected for staining were found to be expressed in human type I spiral ganglion neurons: UBE3B, MMP2, PTK2/FAK, GRM5, SYNJ2, LMX1A, EYA4, NOX4, and TRIOBP. Additional genes with neuronal function were also identified from GWAS: ammation . Damage ammation , which iammation . Howeverammation . The anaammation , and to ammation , 143. Inn of rat –147 and n of rat , as wellor, TrkB . AssociaSPTBN1 (beta-II spectrin) [CDH13 (cadherin-13) [BAIAP2L2 (brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2) [FSCN2 (fascin-2) [TPRN (taperin) [Our findings also suggest that structural resilience of the human hair cells, and perhaps more critically the sterocilia, is an important factor for the risk of human hearing loss that is affected by common genetic variation. Imaging of GWAS-associated genes in human cochlear tissue showed the actin-associated proteins LMO-7, TRIOBP, and NOX4 to all be expressed in the human hair cells, the hair cell cuticulae, and stereocilia. In addition, five additional structural regulators that are expressed in the hair cells, stereocilia, and cuticula in other species were also associated with hearing-related traits in our analyses: pectrin) , CDH13 (erin-13) , BAIAP2Lotein 2) , FSCN2 (ascin-2) , and TPRtaperin) . Age-reltaperin) . Unlike taperin) . SurvivaSYNJ2 and GRM5 point towards the cochlear conductance of auditory signals to play a role in hearing loss risk. Both genes encode components of neuronal signaling: GRM5 encodes the metabolic glutamatergic receptor 5, an excitatory G protein-coupled receptor that is activated by glutamate in the synaptic cleft, and SYNJ2 encodes synaptojanin 2, which mediates presynaptic recovery of secretory vesicles after neurotransmitter release. Both proteins were also expressed in type I spiral ganglion neurons. SYNJ2 was also expressed in the outer spiral bundles, i.e., the synaptic connections between the auditory neurons and the outer hair cells.Associations linked to scala media, and LMX1A in the organ of Corti. This suggests a role for these proteins in maintenance of the cellular state of these structures in the adult human cochlea.Our findings also suggest that genetic variants that affect cochlear development and maintenance of cellular identity also contribute to the risk for age-related hearing loss. LMX1A and EYA4 are both transcriptional regulators that have been linked to the development and maintenance of cochlear structures. Our imaging reveals expression of both proteins in discrete structures of the adult human cochlea: within the spiral ganglion neuronal cells, EYA4 within cells of the epithelial layer that faces the The current study is limited by the lack of audiometric data collected in a standardized manner, which would allow for a more optimal assessment of hearing loss in study participants. Collection of such data require specialized equipment such as a sound-proof room, tone generator, and a trained audiologist for accurate measurements, and thus become difficult to perform on the scale that is commonly required for an adequately powered GWAS for complex traits. Self-report data are valuable proxies for hearing loss as the collection of these data is easily scaled. The large number of biologically relevant findings from GWAS, as determined from literature analyses and immunohistochemical staining of human cochlea, also serve as a validation of the utility of self-report data on hearing-related traits for large-scale studies. However, the type of hearing loss that UK Biobank participants suffer from cannot be determined from questionnaire data alone. Although age-related hearing loss can be considered the most common form of sensorineural hearing loss in the elderly (> 60 years) , other tWe are also unable to elucidate the genomic mechanisms that underlie the associations observed in the GWAS. We have instead used a bioinformatic approach to generate plausible mechanisms for associated loci and to select candidate genes for downstream experiments. For hearing-related traits, many associations were found at genetic loci that contain genes with established links to hearing, e.g., at human autosomal nonsyndromic deafness loci, or near genes that affect cochlear morphology in other species. We consider this type of overlap as strong evidence for these genes to be involved in the causal mechanisms that underlie the association with hearing loss. In vitro and in vivo modeling of the effects of genetic variants would allow for determining the causal variant or variants and provide more exact genomic models for how they affect hearing loss risk.n = 10,175) and ICD10-H90 Conductive and sensorineural hearing loss (n = 2927)). Sex-specific effects may nonetheless be present for hearing loss-related traits, which can potentially be identified in stratified analyses.Our study also does not fully account for genetic effects that may differ between sexes or are specifically expressed only in females or males. A recent study on genetic data from the UK Biobank demonstrated that sex-specific effects in the genetic architecture can lead to issues when analyses are performed on a combined set of male and female participants, such as genetic effects being masked from detection due to differences in effect sizes or directions between sexes . This waIn conclusion, GWAS for hearing loss-related traits reveal a genetic architecture for hearing loss in humans. Imaging of GWAS-identified leads findings provide evidence that implicate processes of the spiral ganglion neuronal cells, such as neuronal plasticity and recovery after trauma, to be critical for hearing loss risk. The results also highlight the importance of structural components of the hair cells and hair cell stereocilia and suggest that structural resilience within the organ of Corti is an important factor in determining the risk for sensorineural hearing loss. Importantly, proteins were selected for imaging based on previous cochlear imaging studies in other species. Consequently, it is essential to consider other aspects related to cochlear function that may also be influenced by common hearing loss-associated genetic variation. These findings provide biological insight of the underlying mechanisms of age-related hearing loss among the general population and also offer interesting leads for the development of potential novel therapeutic targets.This research has been conducted using the UK Biobank resource under application number 26808. The UK Biobank is a large cross-sectional cohort of almost half a million participants from the UK that were recruited between 2006 and 2010 . ParticiHearing aid use, hearing difficulty, and tinnitus were assessed via touch-screen questionnaire. Participants were asked “Do you use a hearing aid most of the time?” and could answer “Yes” or “No” (data field 3393). For hearing difficulty, participants were asked “Do you have any difficulty with your hearing?” and could respond “Yes,” “No,” or “I am completely deaf” (data field 2247). For tinnitus, participants were asked “Do you get or have you had noises (such as ringing or buzzing) in your head or in one or both ears that lasts for more than 5 min at a time?,” and could answer “Yes, now most or all of the time,” “Yes, now a lot of the time,” “Yes, now some of the time,” “Yes, but not now, but have in the past,” or “No, never” (data field 4803). Hearing aid use and hearing difficulty responses were coded as 1 (“Yes”) or 0 (“No”). Participants who declined to answer or answered “I am completely deaf” were set as missing. For tinnitus, “Yes, now most or all of the time,” “Yes, now a lot of the time,” “Yes, now some of the time,” or “Yes, but not now, but have in the past” were coded as 1 and “No, never” was set as 0.Speech-in-noise perception was assessed during the touchscreen questionnaire session. The participant was supplied headphones and assessed for the ability to hear three spoken digits alongside a rushing background noise . ParticiUK Biobank participants were genotyped on two separate microarrays. Initially, approximately 50,000 participants were genotyped on the Affymetrix UK BiLEVE Axiom array as part of the UK BiLEVE  study . ApproxiP < 1 × 10−20). This left approximately 35 million SNPs for association testing. Association analyses were performed on the computational cluster at the Uppsala multidisciplinary center for advanced computational science (UPPMAX) under project sens2017538.Genome-wide association tests were performed with Plink v1.90b3n . Hearing−7” for the lead SNP. We used a lenient threshold for correlation between SNPs, “--clump-r2 0.1”, and “--clump-kb 500,” so that all SNPs within 500 kb with a squared correlation of R2 > 0.1 to the lead SNP were assigned to the clump that was represented by the lead SNP. A secondary significance threshold for clumped SNPs of “--clump-p2 5 × 10−7” was also used to designate genome-wide significant SNPs within the clump. We also used a hg19 gene range list to cross-reference and report the overlap between clumps and genes, which is performed with the “--clump-range” flag.Genome-wide association analyses results in a large number of associations between genetic variants and the interrogated phenotype. Associations are commonly seen for many SNPs in close proximity of each other, due to the linkage disequilibrium between SNPs. To determine the number of independent associations, we perform “clumping,” where associated SNPs at each locus are clumped together, and an independent “lead SNP” is identified. Clumping is included as a function in Plink v1.90b3n under the --clump flag and was performed for all traits. We set a lenient threshold for genome-wide significance in the clumping analyses: “--clump-p1 5 × 10(λGC), mean χ2 statistics, and LD-score regression intercepts for each GWAS. LD scores from a random subset of 5000 UK Biobank participants were used as weights to adjust for correlation between SNPs. LD scores were generated from genotype data in the Plink -bed format with the LDSC software using a 1000-kb window around each SNP. Mean χ2 represents the mean of the χ2 statistics for all variants that were tested for association in the GWAS. One minus the intercept of the χ2 statistics regressed against the LD score (LD score regression) provides an estimate of the mean contribution of the confounding bias in the test statistic. This can be presented as a ratio or the proportion of the χ2 statistic that the LD score regression intercept ascribes to causes other than polygenic heritability (Ratio = LD intercept − 1)/(mean(χ2) − 1). In practice, the ratio is commonly around 10–20% [t-statistic with the genomic inflation factor and recalculating the p values.LD score regression   was usedd 10–20% . The GWAwww.ncbi.nlm.nih.gov/snp) [gtexportal.org) [https://www.ebi.ac.uk/gwas, dataset accessed 1 Mar 2019) [To determine possible causal variants and link the associated loci in our GWAS to candidate genes, we cross-referenced our GWAS results with functional annotations from publicly available databases. Databases used were dbSNP build 150 (gov/snp) , the gental.org) , and thear 2019) . dbSNP wR2 > 0.1) were then identified and annotations for all SNPs were extracted from dbSNP and the GWAS catalog. SIFT and PolyPhen2 scores for nonsense, missense, and frameshift mutations were identified manually from Ensembl (www.ensembl.org). SIFT and PolyPhen are tools for predicting the functional consequences of genetic variants within protein-coding regions based on sequence homology as well as the physicochemical properties between the alternate amino acids [The linkage disequilibrium (LD) pattern for each lead SNP from our GWAS was determined using the “--r2” command with the flags “--ld-snp,” “--ld-window-r2 0.1,” and “--ld-window-kb 2000” in Plink v1.90b3n. All SNPs within a 2-Mb window around each lead SNP that were in linkage disequilibrium with the lead SNP  were considered to overlap.For the GTEx database, we determined the correlation of the lead SNPs from our analyses with expression quantitative trait loci (eQTLs) that were included in GTEx. We used a significance threshold for association of genetic variants with gene expression in GTEx of  studies . The corWe wished to determine if any genes within our GWAS-identified loci had been previously linked to hearing or hearing-related traits in humans or other species. We therefore performed a PubMed search of all genes within the clumps that were determined by Plink combined with the terms “AND Hearing.” The search results were then manually filtered for original peer-reviewed research articles that had reported data on at least one of the genes included in the literature search.The surgical specimens were from patients suffering from life-threatening posterior cranial fossa meningioma compressing the brain stem. The operations were performed at Uppsala University Hospital by a team of neurosurgeons and oto-neurosurgeons. Human cochleae were dissected out using diamond drills of various sizes. Tissues were immediately placed in 4% paraformaldehyde diluted with 0.1 M phosphate-buffered saline  in the operating room. After a 24-h period spent in fixative, specimens were washed in 0.1 M PBS and then placed in 10% Na-EDTA solution at pH 7.2 for decalcification. The Na-EDTA solution was changed every 2 days until the decalcification process was completed, which took approximately 3 to 6 weeks. Decalcified cochleae were rinsed with 0.1 M PBS and placed in 25% sucrose in 0.1 M PBS overnight (4 °C). Cochleae were embedded in Tissue-Tek (OCT Polysciences) for frozen sections. Cochleae were rapidly frozen and sectioned at 8–10 μm using a Leica cryostat microtome. Frozen sections were collected onto gelatin/chrome-alum-coated slides and stored in a freezer of − 70 °C before immunohistochemistry procedures , 169. InImmunohistochemistry procedures on human cochlear sections were described in previous publications , 171. SeX–Y plane and 394 nm in the Z plane. The laser and filter were set up as follows: 405 nm laser of excitation coupled with BP 420–480 + LP 750 filter, 488 nm laser of excitation with BP 495–550 + LP750 filter, 561 nm laser of excitation with BP 570–620 + LP 750 filter, and 647 nm laser of excitation with LP 655 filter. From the SR-SIM dataset, 3D reconstruction was done with Imaris 8.2 . A bright-field channel was able to merge fluorescence channels to visualize cell and tissue borders.Stained sections were first investigated with an inverted fluorescence microscope  equipped with a spot digital camera with three filters . Image-processing software , including image merging and a fluorescence intensity analyzer, was installed on a computer system connected to the microscope. For laser confocal microscopy, we used the same microscope equipped with a three-channel laser emission system. SR-SIM  was perfAdditional file 1: Figures 1-3, Tables 1-8. Fig 1. Manhattan plots that illustrate the results from genome-wide association analyses for hearing difficulty, hearing aid use, speech-in-noise and tinnitus. Fig 2. Quantile-quantile plots for the results from genome wide association analyses on hearing difficulty, hearing aid use, speech-in-noise and tinnitus. Fig 3. Comparison of results from the current study and the recent GWAS for hearing difficulty. Table 1. Characteristics of UK Biobank participants that were included in GWAS for hearing-related traits. Table 2. Results from the LDSC analyses on hearing loss-related trait GWAS in the UK Biobank. Table 3. Brief description of the literature for genes within genetic loci that were associated with hearing-related traits in the UK Biobank. Table 4. Non-synonymous coding variants that were linked to genetic loci associated with hearing-related traits in the UK Biobank. Table 5. Expression quantitative trait loci (eQTLs) that were found to be linked to genetic loci associated with hearing related traits in the UK Biobank. Table 6. Summary of expression patterns of candidate proteins in other mammals. Table 7. Characteristics of tissue donors. Table 8. Antibodies used for immunohistochemical staining.Additional file 2: Excel sheets with the lead SNPs that were associated with hearing difficulty, hearing aid use, speech-in-noise and tinnitus. Columns: CHR = chromosome, SNP = variant ids, either as rsID or in the format CHR_POSITION_ALLELE1_ALLELE2, BP = chromosomal position in basepairs , P = P-value for association tests, P.GC_adjusted = P-values after adjusting for genomic inflation (this was only performed for tinnitus), SIGNIFICANT_ASSOCIATION = denotes whether a genome-wide association could be observed after adjusting for the family-wise error rate with Bonferroni-correction (P < 5*10-8), SNPS_IN_CLUMP (N) = the number of associated SNPs that were in linkage disequilibrium with the lead SNPs, CLUMP_COORDS = coordinates for the first and last associated SNPs that were clumped with the lead SNP, CLUMP_SIZE (bp) = the size of the clumped region in basepairs, GENES_IN_CLUMP = genes that were located within each clumped region.


Introduction: Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is an autoimmune disease associated with the NMDA receptor. This paper describes a patient who presented with bilateral hearing loss as the initial symptom of anti-NMDAR encephalitis.Case Report: We describe a 31-year-old young female with anti-NMDAR encephalitis who presented with bilateral severe hearing loss after brief loss of consciousness and then accompanied by other symptoms, such as generalized tonic–clonic seizures, manic episodes, excessive salivation, severe cognitive impairment, and complex non-convulsive status epilepticus. Great improvement was achieved by a combined treatment of steroid, IVIG, and plasmapheresis, especially after surgical removal of the ovarian teratoma. When she was discharged on hospital day 43, her hearing loss obtained a significant improvement.Conclusion: This case study and literature review investigated the impairment of hearing loss and its subsequent treatment in patients with anti-NMDAR encephalitis. N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis is an autoimmune disease associated with IgG antibodies against the NR1 subunit of the NMDA receptor  with a lymphocytic predominance and normal protein and glucose levels. On hospital day 3, the symptoms of this patient were progressing rapidly, developing manic episode, language impairment, and increased salivation. Her manic symptom was gradually controlled when treated with midazolam or diazepam intravenous infusion and risperidone tablets (1 mg twice daily). Since our patient had such signs, the diagnosis of autoimmune encephalitis was suspected. A 5-day course of IV methylprednisolone (1 g/day) and intravenous immunoglobulin (IVIG) (25 g/day) were administered empirically. On hospital day 4, the patient showed significant cognitive dysfunction and even did not know where she came from. CSF PCR for herpes simplex virus (HSV) 1 and 2 was unremarkable. Serum and CSF anti-NMDAR antibodies returned positive by immunofluorescence , confirming the diagnosis of definite anti-NMDAR encephalitis  showed an increased white blood cell (WBC) count  in the right-side ovary . BecauseIn the 1-month follow-up, her hearing was normal and cognitive function improved significantly, just presenting with mild memory deterioration. During this period, no seizures were observed. The MMSE score was 29 and the MoCA score was 27. Regular follow-up will be carried out for our patient. The clinical course is summarized in Anti-NMDAR encephalitis was first identified in 2007 by Dalmau et al. . The cliParaneoplastic syndrome has also been reported in some literature associated with hearing loss. The most frequent is Kelch-like (KLHL) 11 encephalitis that is associated with testicular germ cell tumors, benign teratomas, small-cell lung cancer, and ovarian cancer . The typThe strongest pathogenic evidence of bilateral fluctuating hearing loss is that of an immune-mediated disease. The inner ear is not an immunologically privileged site and may mount an immune response against foreign and self-antigens damaging sensory structures within it –26. BothIn this case, the patient presented with bilateral severe hearing loss when she visited our hospital. Serum and CSF anti-NMDAR antibodies were positive, confirming the diagnosis of definite anti-NMDAR encephalitis. During the treatment with methylprednisolone, gamma globulin, and plasmapheresis intravenously, the hearing showed a degree of volatility. Occasionally, sound could be heard in the left ear, but it did not sustain. Starting from the second day after the surgical excision of ovarian teratomas, binaural hearing of this patient showed some extent of retrieval, especially after the following three sessions of plasmapheresis. Fortunately, the hearing loss in this young patient recovered substantially after 43 days of treatment. So far, the hearing loss in anti-NMDA receptor encephalitis associated with ovarian teratoma has not been reported. Taraschenko et al.  reportedIn summary, our case, as the first episode of anti-NMDAR encephalitis, initially presented with bilateral hearing loss after brief loss of consciousness and then accompanied by other symptoms, such as generalized tonic–clonic seizures, manic episodes, excessive salivation, severe cognitive impairment, and complex non-convulsive status epilepticus. Great improvement was achieved by a combined treatment of steroid, IVIG, and plasmapheresis, especially after surgical removal of ovarian teratoma. An awareness of diverse and uncommon primary manifestations is conducive to early and accurate diagnosis of anti-NMDAR encephalitis.The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Jincheng people's Hospital, Jincheng, China. The patients/participants provided their written informed consent to participate in this study.LJ: put forward research ideas. FY: took the responsibility of communicating with the patient's family and obtaining the authorization in this paper. HC and JZ: responsible for drafting articles. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Exome sequencing (ES) is an effective method for identifying the genetic cause of hearing loss in infants diagnosed through newborn hearing screening programs. ES has the potential to be integrated into routine clinical care, yet little is known about the experiences of clinicians offering this test to families. To address this gap, clinicians involved in a clinical study using ES to identify the cause of infants’ hearing loss were interviewed to explore their experiences with offering and returning results to parents. Interview transcripts were analysed using inductive content analysis. Twelve clinicians participated: seven genetic counsellors, four clinical geneticists, and one paediatrician. Most clinicians were supportive of offering ES to infants with hearing loss, primarily because results may inform the child’s clinical management. However, some expressed concerns, questioning the utility of this information, particularly for isolated hearing loss. Clinicians had differing views regarding the optimal time to offer ES to families; while some felt that families can manage everything at once, others recommended delaying testing until parents have come to terms with their child’s diagnosis. These findings show the complexity involved in determining how ES should be offered to families following the diagnosis of a child with hearing loss, particularly with regards to when testing is suggested. Approximately 0.1% of Australians are born with unilateral or bilateral hearing loss [Research shows that exome sequencing (where the coding regions of a person’s DNA are sequenced) can be used to identify the genetic cause of hearing loss, with a higher diagnostic yield than standard single gene testing . A cliniThe study by Downie et al. showed that, based on the high diagnostic yield and clinical implications, exome sequencing following the identification of infants with hearing loss has the potential to be integrated into routine clinical care . As suchClinicians involved in the overarching clinical study  were deemed eligible to take part in this qualitative interview study. Eligible clinicians, who were identified by the clinical project lead, were emailed an invitation to participate. In accordance with qualitative research methodology, participant recruitment continued until data saturation was reached  and we had achieved sufficient sample heterogeneity by recruiting at least one clinician from each of the clinical specialties involved in the overarching clinical study  and each of the study sites. Our sample size was not intended to be statistically representative, but rather to generate rich data that addressed our study aims ,10. The Interviews were conducted by Lauren Notini, either in person or by telephone depending on the location and preference of the participant. In-person interviews were conducted at the participant’s workplace, either in a meeting room or in the participant’s office. Each interview was audio-recorded and transcribed. Interview transcripts were analysed using inductive content analysis, a method of qualitative data analysis that involves using content categories generated from the data, rather than predetermined categories ,12. Codin = 1) and >20 years (n = 1) with the majority of participants having from 1–4 years (n = 4) or 5–9 years (n = 3) experience. While clinicians were recruited from both study sites, the majority  worked at one site. This uneven distribution of specialties and worksites among our sample precludes meaningful comparison of participants’ responses. Hence, we do not provide separate analyses of the views expressed by clinicians at different worksites, nor provide a comparative analysis of responses from the different clinical specialties.A total of 12 clinicians participated, including seven (58%) genetic counsellors, four (33%) clinical geneticists, and one (8%) paediatrician; this was more than half of the 21 clinicians eligible to participate in the study. Average interview length was 41 min, with a range between 25–59 min. Clinicians varied in terms of how many years they had worked in their current profession; experience ranged between trainee level (Three main content categories were generated from the interview data: (1) clinicians’ views on offering exome sequencing to parents of children with hearing loss; (2) clinicians’ accounts of parents’ decision making regarding exome sequencing for their child’s hearing loss; (3) clinicians’ experiences returning diagnostic exome sequencing results to parents. We describe these content categories, providing illustrative quotes for each, below. Emphasis in quotes represents participants’ original emphasis.Most clinicians were supportive of offering exome sequencing to parents of children with hearing loss, for several reasons. This was often because the results of exome sequencing may inform the child’s clinical management:“The diagnosis of certain genetic syndromes will lead you down a different treatment pathway and allow you to avoid other investigations.”(Clinician 1)Some clinicians also put forward reasons relating to equity, and felt that exome sequencing should be offered to parents of children with any type of hearing loss, and not just the types that met the study eligibility criteria:“I think definitely the hearing component, everybody should be offered, or given the chance to take it up … even the unilateral losses and the mild losses which are not part of eligibility … I think they should also be offered the same testing because it’s just being equitable, they also deserve to have an answer, if there is one.”(Clinician 2)Other reasons given by participants in support of offering exome sequencing to parents of children with hearing loss included that knowledge is power, the results may be helpful in addressing parents’ concerns, and that exome sequencing for hearing loss was seen as a cost-effective intervention worthy of resources.Some clinicians also expressed concerns about offering exome sequencing for paediatric hearing loss. Several questioned the utility of this information, especially for isolated hearing loss, or what are the appropriate limits of parents’ abilities to make decisions for their children. Others queried whether hearing loss is better understood as a disability or a culture, and raised concerns relating to data storage:“It’s difficult with deafness, because deafness alone, we think of deafhood and people that are deaf live a great, long, healthy life. And there’s a bit of an ethical discussion to be had for testing newborn babies that are found to have hearing loss … to an extent you’re really pathologising this condition they have … these parents are making decisions for them and their genetic information is going to be stored indefinitely… when maybe there was no real indication for doing the test in the first place.”(Clinician 8)Other clinicians raised ethical concerns relating to distributive justice, as some families had the opportunity to receive free exome sequencing via the study but other families who could also benefit from testing did not receive this same opportunity:“My main ethical challenge with this project is that it’s come to an end, and now we have lots of families coming to our clinic, and being referred, expecting this level of testing… this was a temporary solution, which was great, but now it’s stopped, we’re back to square one with a lot of these families and there’s a lot of deaf babies born every year and now I just have to sit there and say ‘it’s (AUD) 3000 dollars and we have no means of funding it whatsoever’ … So it’s a question of distributive justice that I am bothered by a lot.”(Clinician 4)Clinicians had different views regarding the optimal time to offer exome sequencing to parents of children with hearing loss. Some clinicians felt that some families can manage everything at once:“There will be keen families, and they’ve got the psychological and coping resource to deal with everything at the same time. And they could come through early.”(Clinician 10)However, most clinicians felt that exome sequencing should be offered to parents at a later date, often because the children were very young infants and parents were still coming to terms with their child’s hearing loss diagnosis. “It was pretty clear that for some parents it was a bit early to be raising this. And so, if I was doing this more broadly, I would say maybe not to bring in the genetics and the genetic question ‘til six months of age or nine months or something like that. ‘Cause it did seem that in this cohort, keeping in mind they’ve just been diagnosed with deafness, so they were coping with that diagnosis, then to be faced with this offer of more stuff, I think it was probably a bit overwhelming for some people… I would say maybe we’ll just try and get these kids at six months, for example, rather than as early as possible.”(Clinician 1)Families are also being invited to participate in other studies relating to hearing loss at this time, and some clinicians discussed the discomfort they and their colleagues experienced offering the test.“That feeling of, you know, are we bombarding the families with so much, at that particular point in their sort of understanding of what’s happening to their kids? So, I think people (study clinicians) felt a bit uncomfortable about that, that it was all happening at the wrong time … not the genetic testing for the cause for the deafness, but the offer, and the idea that they were being invited for two or three projects … simultaneously at a point when they’re still coming to grips with the diagnosis in the child.”(Clinician 10)Some clinicians felt that the decision regarding exome sequencing has important implications, and, therefore, parents should be given more time to decide. Some clinicians felt that there should be no time limit on the offer, and parents should be able to opt in to receive exome sequencing at any time: “Parents should have the option to opt in at any time to have it done, when they’re ready or when they have thought enough about it. ‘Cause there are huge implications once you’ve taken the test, in terms of knowing the result.”(Clinician 2)According to clinicians, the most common reason parents accepted exome sequencing for their child was to try to find out the cause of their child’s hearing loss, as this may help them to better understand their child’s prognosis and inform clinical management:“They want to know the answer because they want an explanation, or they want treatment.”(Clinician 11)Clinicians reported that parents were particularly interested in establishing whether their child’s hearing loss was isolated or part of a syndrome:“… there’s a lot of information out there on the internet and within the groups as well … ‘now that we’ve found this one thing, could it be that there are other things that are going to be going on?’ And I think that for a lot of parents, they’re quite scared of conditions like Usher syndrome and those sorts of things, so I think that was a big motivation for a lot of parents to have the testing for hearing loss.”(Clinician 9)Some clinicians also noted that obtaining a genetic diagnosis for their child’s hearing loss may help to alleviate any feelings of parental blame or guilt: “Parents might feel guilty about something that’s happened in pregnancy… a genetic diagnosis for them would help take away that kind of guilt.”(Clinician 3)Most clinicians reported that some parents also accepted exome sequencing for their child for reasons relating to reproductive planning. For example, some parents wanted to be better prepared if they had another child with hearing loss as noted by this clinician:“Not necessarily that they would do anything to prevent a pregnancy or terminate a pregnancy based on the result, but just so they had an idea of ‘is this likely to be a one-off thing? Could I have another child with this same hearing loss? Will it be a similar type?’ Just to know for that kind of family planning … a lot of families described finding out about the diagnosis as just such an earth-shattering shock to their system… I think they wanted to maybe be a bit more prepared if it happened again.”(Clinician 9)In contrast, other parents wanted to use the results in order to avoid having another child with hearing loss:“They’re having more children and they wish to avoid having a child with hearing loss.”(Clinician 10)Clinicians reported that some parents agreed to sequencing because they wanted to confirm the hearing loss diagnosis in their child or know more about their child’s health, including one family with a known genetic cause of hearing loss who chose to participate in the project to receive additional findings. Others wanted to participate to help explain the parents’ own hearing loss, or simply agreed because exome sequencing was offered to them as a clinical test.Clinicians reported that parents had two main reasons for declining exome sequencing for their child’s hearing loss. First, clinicians reported that some parents declined participation as they felt that the exome sequencing results would not change anything: “I just remember them saying, ‘I don’t mind whether my baby’s hearing loss is because of a genetic change or not, it’s not going to change anything for us or our family.’”(Clinician 6)Second, other families declined participation as they were already overwhelmed by their child’s hearing loss diagnosis and management:“There’s so much support, programs, intervention related to hearing loss that the families just felt run off their feet and overwhelmed by all the services … they were like, ‘I can’t introduce another health service into the picture.’”(Clinician 11)Clinicians explained that some parents declined exome sequencing because they already knew or suspected the cause of their child’s hearing loss or had other family members with hearing loss who felt they were coping well and, therefore, did not feel the need for further testing. Some parents felt that, given the possibility a diagnosis for their child’s hearing loss would not be identified, accepting testing might lead to disappointment. Others either did not want to be involved in research or had concerns about the potential implications of the findings, such as the impact of exome sequencing results on their child’s future ability to obtain insurance or concern it would reveal the child’s father was not their genetic father.“I think at the moment I’d just say it’s a mixture of different factors, which range from they may already have a genetic diagnosis, they may have decided the condition’s not genetic. They may just not want to be involved in research, or be concerned about insurance or other implications. So we’ve got a list, but there’s no sort of single factor that’s repeating, it’s basically a whole lot of different things.”(Clinician 1)At the time of interview, all clinicians had been involved in discussing exome sequencing results relating to hearing loss to parents. In describing their experiences returning these results, some clinicians reported that they had to remind parents about the testing that had taken place, as receiving the results was not always at the forefront of parents’ minds:“Usually the time between consent and results’ return was a number of months. So often when we were calling families to arrange a result appointment, you’d have to kind of re-introduce yourself and go ‘remember me? We talked about this test, the reason that I’m calling is we’re expecting to have some results for you in the next couple of weeks’. But I think a lot of the time you were kind of reminding families what exactly it was you were calling about ... I guess the sense of urgency (around the time of the diagnosis) was usually off by that stage.”(Clinician 6)Some clinicians also found it challenging to explain variants of uncertain significance  to families:“One thing we’ve found is that there seems to be … lots of variants (of uncertain significance) in hearing loss genes. So, it’s quite common to get a report with three or four variants of unknown significance. Or even one that we’re quite confident is the diagnosis and some variants of unknown significance. And I found that very challenging for some families, to explain a genetic finding and then explain other genetic findings that may be less relevant.”(Clinician 3)Clinicians reported that parents who received an answer for their child’s hearing loss (genetic or otherwise) typically reacted positively, even though this information may not necessarily affect clinical management:“For the deafness results, I’ve been surprised at how many families have been really emotionally affected by giving them a deafness result … quite a few families are really ecstatic to have an answer, which I think maybe we didn’t predict at the start because it often doesn’t affect management or treatment that much … just really relieved to have an answer to explain what’s going on…that sense of, yeah, relief I guess is the best way to describe it. So that stood out.”(Clinician 3)Some clinicians described this sense of relief as particularly strong in cases where parents had received a genetic explanation for their child’s hearing loss:“…often people blame themselves for problems in their children, so finding a genetic diagnosis brings a relief to some families that it’s not something that they did during the pregnancy, particularly when people are worried about an infection or something like that, that’s playing on their mind.”(Clinician 4)According to participants, parents’ reactions to the exome sequencing result also depended on whether their child’s hearing loss was isolated or syndromic. Parents who received a molecular diagnosis of isolated (non-syndromic) hearing loss often expressed gratitude and relief to clinicians:“Relief was a big one. Just being grateful that they have an answer, that it doesn’t mean anything else is going to necessarily go wrong.”(Clinician 9)However, other parents whose results confirmed an isolated hearing loss experienced negative feelings. Some held concerns about stigmatisation of genetic conditions or the impact of the results on future reproductive planning:“A subsample … were expecting it to come back genetic, but when it did it kind of just confirmed like ‘oh, it’s something that’s genetic.’ And I think maybe, like there’s still a stigma around genetic conditions and having something different with you. So, I think for some of them they may have been a bit taken aback, like ‘oh ok, it means there’s a 25% chance it could happen again if it was a recessive cause … not that I’d terminate a pregnancy based on it, but now I’m in a bit of a conflict of, there’s a chance I could have another child with a hearing loss’.”(Clinician 9)For others, the result may have confirmed a hearing loss diagnosis that they were denying:“Humans are unpredictable (laughs). So, you think someone might be very happy that they’ve received an answer because it’s confirmed that it’s isolated hearing loss. The parent absolutely knew the child has hearing loss, and that, yep, they have the answer, they want to use it for reproductive choices, it’s ticking all those boxes for them. But in actual fact maybe they had somewhere in the back of their mind that they hoped it wasn’t really hearing loss and it was something the child will grow out of, and now you’ve just given them this in-writing thing that says ‘no, you’re child definitely has this’. And so for them it’s devastating.”(Clinician 5)Some parents who received a syndromic diagnosis for their child’s hearing loss reacted relatively positively to this news because it allowed them to be proactive in their child’s clinical management:“For the syndromic forms of hearing loss … for some families they found it as a helpful explanation for things and appreciated that there could be other screening or other testing that could be done for their child, and were grateful that this was also picked up early… like ‘we found out they could have a heart condition, so we’re going to send them to have their heart looked at as well. It’s something we wouldn’t necessarily have done’ or ‘their kidneys looked at’. So, I think there was value in that for quite a lot of families.”(Clinician 9)However, some clinicians mentioned parents who had received a syndromic diagnosis and reacted more negatively due to the devastating nature of the diagnosis or because this diagnosis was unexpected and meant extra medical management for their child: “I was personally involved with a family who received a diagnosis of Usher syndrome, and that was just absolutely heartbreaking for them. It was their biggest fear, and it eventuated. So, yeah. It was utterly devastating for them.”(Clinician 9)For parents who did not receive an explanation for their child’s hearing loss, clinicians reported mixed reactions. Many parents who did not receive an explanation reacted quite well and were particularly relieved that no syndromic diagnosis was found:“As much as we didn’t find a diagnosis at all, a lot of families were relieved that we didn’t find a syndromic diagnosis or something like that.”(Clinician 6)Other parents who did not receive an answer for their child’s hearing loss reacted more negatively. This included parents who were wishing to use this result to inform decisions about future medical management and future reproductive planning:“…some families were quite disappointed if they didn’t get an answer, particularly if they were hoping to understand if their child needed any other check-ups moving forward other than just getting their hearing checked every now and again, or some families were keen to use that information in a reproductive setting for future pregnancies.”(Clinician 6)Clinicians reported that future re-analysis of exome sequencing results was offered to these parents, many of whom expressed interest in this: “People are interested in the possibility of re-analysis down the track, so most people expressed an interest in that when we brought it up as a possibility.”(Clinician 4)This is the first published study exploring clinicians’ views and experiences with offering and returning results from exome sequencing for hearing loss in infancy. Most of our participants highlighted the potential benefits of offering exome sequencing for infant hearing loss, such as informing the child’s clinical management. However, concerns about offering such testing were also raised by some clinicians, particularly in the context of isolated (non-syndromic) hearing loss, for which the clinical benefits of offering such testing may be less clear. Those who were in favour of testing cited reasons relating to clinical and personal utility, equity, and cost-effectiveness. Those who raised concerns also mentioned factors relating to utility, yet, in addition, questioned the boundaries of parental autonomy, deafness as a disability, and distributive justice. Interestingly, there was no clear pattern with clinician type, study site, or years of experience as to whether participants were more or less in favour of offering exome sequencing for hearing loss.Despite the general agreement that exome sequencing for paediatric hearing loss should be offered to parents, clinicians had more varied views regarding the most optimal timing to offer such testing. While some clinicians felt some parents coped well with early testing, most felt testing should be offered later, once parents had had more time to come to terms with their child’s hearing loss diagnosis. This finding was supported by participants’ reports that some parents declined the offer because of how overwhelmed they were at the time. There are several advantages to offering exome sequencing to parents soon after the child’s diagnosis of hearing loss. First, exome sequencing increases diagnostic yield, which subsequently improves the quality of care infants receive, either through reducing the need for, or allowing for furthermore targeted further diagnostic investigations . The cosHowever, there are also disadvantages to offering exome sequencing during early infancy. Parents may already be overwhelmed by either the fact they have a newborn or the recent hearing loss diagnosis. Offering exome sequencing at a later time could provide parents with more space to consider the various factors when making their decision regarding exome sequencing for hearing loss. The appropriate timing to offer exome sequencing for hearing loss will likely depend on the particular family and their circumstances. While offering early exome sequencing may be beneficial for one family, it may be considered detrimental for another, as supported by our finding that some families decline testing because they are overwhelmed. The challenge for clinicians will be in determining when the possibility of testing might best be broached with each family. While clinicians reported that some families were overwhelmed by the offer of testing, it is difficult to determine whether this was due to the offer itself or the fact that the offer had an expiry date. The suggestion raised by some clinicians of allowing parents to choose exome sequencing for hearing loss at any time (rather than as a limited time offer) may provide a solution. Our participants described how parents exhibited varying reactions to receiving results from exome sequencing. Most parents generally reacted positively to a genetic cause for the hearing loss, which accords with other studies that have explored health professionals’ reports of parents’ reactions to receiving results for children with other genetic conditions . However, our participants recounted that some parents react more negatively, which may be due to the stigma associated with genetics or because a result provided unwanted confirmation of the hearing loss itself. Health professionals from other studies have suggested other reasons for negative reactions by parents, such as shock that the condition is genetic, frustration the answer did not lead to a treatment or cure, or distress that it confers a progressive course or worse prognosis than anticipated . Similarly, mixed responses were reported by our participants in relation to negative results. While some parents expressed relief, as seen in other studies , other parents were disappointed at not having information to make management and reproductive decisions. This disappointment has been reported by others but seemed to be more pronounced when parents had high expectations that a cause would be identified . These findings suggest that the nature of the genetic condition being tested needs to be taken into account when preparing families to receive results.Some of our participants raised ethical concerns relating to equity of access depending on the type of hearing loss diagnosed in the infant. While the overarching clinical study had strict eligibility criteria  , cliniciHowever, such strict eligibility criteria may be questioned when considering the notion of justice as equity. Applying this notion of justice requires asking what the similarities and differences are between two groups, and whether these justify treating these two groups differently. It could be argued that infants with permanent, bilateral congenital hearing loss of moderate, severe, or profound severity stand to benefit more from receiving an answer for their hearing loss via exome sequencing . It may also be possible that parents of these infants are more affected by their child’s hearing loss diagnosis and management and could, therefore, stand to benefit more from the opportunity to receive an answer for their child’s hearing loss. However, infants with unilateral or mild forms of hearing loss and their parents may also benefit from the opportunity to receive exome sequencing. For example, this group too may appreciate the opportunity to investigate if the cause of the hearing loss is genetic, and the reproductive planning benefits of such testing may apply equally to this group, if a genetic cause is established. On the other hand, the lower probability of finding a diagnosis also means that there is a greater chance of identifying variants of uncertain significance in these infants, which increases the workload for laboratories, the complexity of the counselling process for clinicians, and the uncertainty for families. A lower diagnostic yield also means fewer families would receive results that could be used for reproductive planning purposes. Further research assessing the diagnostic yield, as well as the clinical and personal utility of exome sequencing in infants diagnosed with unilateral and mild hearing loss, would be informative.Another aspect relating to justice raised by some of our participants concerned the ethical justifiability of offering exome sequencing for free to some families (those who were able to sign up for the study in time) but not others (those who missed the project cut-off). This issue is, of course, inherent in all research projects as evidence for clinical benefit often needs to be generated before applications for healthcare funding can be made to include testing in routine clinical care. Nonetheless, the incorporation of exome sequencing into clinical practice will help to ensure that the offer reaches more families of infants with hearing loss who could derive benefit. In addition, health professionals may benefit from more support in helping them navigate some of these ethical challenges.There are several factors that must be considered when drawing inferences from our results. First, although we interviewed over half of the clinicians deemed eligible to take part in this qualitative study, it is important to note that this was a self-selected sample and, therefore, there may be some views that are not represented in the data. For example, the majority of participants were recruited from one study site. Although this is partly because two-thirds of the potential participants were based at one site, it could also be due to our closer research and clinical connections with that site, which may have encouraged participation. We used a qualitative methodology, which aims to generate rich data that addresses study aims, rather than to generate data that is statistically representative of the broader population . As suchOur study provides insights into clinicians’ experiences of clinical exome sequencing in infants with hearing loss. In particular, we have identified that some of the key challenges relate to when is the most appropriate time to offer testing to this cohort and who to offer it to. The findings are particularly important as much of the existing literature exploring parents’ choices and clinicians’ views regarding exome sequencing in children have been focused on older children, rather than infants. Moreover, as clinicians sought consent to exome sequencing from parents in a real-time, clinical context , our findings have important implications for clinical practice and high relevance to health professionals working in this area. However, further research is needed to explore direct accounts of parents’ views and experiences. In particular, it will be important for future qualitative research to explore parents’ views on when such exome sequencing should be offered, as well as which children or types of hearing loss should be deemed eligible for this testing. In particular, it would be interesting to compare parents’ choices and experiences when they are allowed to choose both if and when to proceed with sequencing in this setting. It would also be worthwhile to compare and contrast the experiences of parents with and without a family history of hearing loss . Finally, more insights into how the clinicians frame the testing offer and the potential impact this has on parental decision making could be gained by observing consultations. The rich, empirical data from clinicians generated by our study will serve as a useful start point with which to compare parents’ own views and accounts of decision making regarding exome sequencing for their child’s hearing loss.


This study aimed to compare the perceived hearing difficulties of a community sample of older adults to two clinical samples of older adults, one with no hearing aid experience and the other with hearing aid experience.N = 243) and compared to scores from two clinical samples, one without (N = 342) and one with prior hearing-aid experience (N = 179). General linear model (GLM) analyses were performed to examine the effects of data sample type and other factors on CPHI scale scores. Scores for the Hearing Handicap Inventory for the Elderly (HHIE) were also available for most participants and were analyzed.Scale scores from the Communication Profile for the Hearing Impaired (CPHI) were analyzed for a community sample of older adults (GLM analyses of each of the 20 CPHI scale scores showed significant effects of sample type with hearing-loss severity and age most frequently showing significant effects as well. GLM analyses controlling for hearing-loss severity and age across sample types found significant differences on most CPHI scales between the community sample and each of the two clinical samples. Significant differences between the two clinical samples were also found on several CPHI scales and on the HHIE.Older adults from the community who did not seek help for hearing difficulties self-reported less difficulty and a greater denial or lack of awareness of communication problems than those who sought assistance at an audiology clinic. For those presumed to have sought a hearing evaluation, those acquiring hearing aids perceived greater communication difficulties in all environments, had greater awareness of communication difficulties, were more accepting of their hearing loss, but tended to allocate more responsibility for their difficulties to others, compared to those who sought clinical assistance but did not acquire hearing aids. The prevalence of hearing loss increases with advancing age . WorldwiDue to the poor uptake of hearing aids, millions of older adults with untreated hearing loss continue to suffer broader consequences. It is well known, for example, that the loss of audibility alone causes many difficulties for everyday speech communication, including poor speech perception e.g.,  and incrAn assumption underlying the foregoing is that the audiogram (and better-ear PTA4 derived from it) is the most appropriate way to define candidacy for amplification as well as the potential benefit to be experienced from hearing aids. It has long been known that older adults with identical audiograms can have markedly different perceived hearing difficulties e.g., . If one Among those who do seek help, many do not follow through with recommendations to obtain hearing aids. For example, Perhaps the most detailed assessment of perceived hearing difficulties, and the individual's reaction to those difficulties, is the Communication Profile for the Hearing Impaired CPHI; , 1987. Tn = 26) was compared to two groups of adherents, one group who had accepted hearing aids after the 30-day trial period and was still using them 6 months later (n = 26) and one group who had discontinued using their hearing aids six months later (n = 24), about half of whom returned their hearing aids during the initial 30-day trial. Significant differences in CPHI scale scores were observed on several CPHI scales between adherents and nonadherents in the studies by Two studies have examined differences in CPHI scores between older adults seen at the audiology clinic who were advised to pursue hearing aids and did (adherents) and those who received the same advice but chose not to do so (non adherents). Aside from these two studies of CPHI scores in adherents and nonadherents, there are several large studies of CPHI scores from clinical samples. The CPHI was originally normed, for example, on a large sample of 827 patients at the Walter Reed Army Medical Center . GarstecIt appears, however, that little is known about the communication difficulties and reactions to them for older adults in the broader community, that is, those who have not sought help at an audiology clinic or acquired hearing aids. To our knowledge, detailed analyses of the communication problems experienced and the reactions to those problems via the CPHI have not been provided for such a community-based sample. Rather, to date, the focus of the CPHI has been to obtain profiles of communication difficulties and reactions to those difficulties from individuals, including older adults, who have been seen at an audiology clinic. The CPHI was envisioned as a tool to assess perceived hearing difficulties and reactions to those problems and guide the subsequent rehabilitation of adults with or without measurable hearing loss as determined at the audiologic evaluation.n = 243); (b) a combined clinical sample comprised of those referred to the MUSC longitudinal study after being seen at the MUSC audiology clinic who were not also current hearing aid wearers (n = 77), and older adults from the Bloomington, Indiana community who had responded to ads for participation in hearing-aid research at Indiana University (IU) and had not worn hearing aids previously (n = 265), resulting in a total population size of 342 for this group; and (c) a combined MUSC and IU clinical sample who were either current hearing aid wearers  or had ever worn hearing aids  for a total population size of 179 for this group of older adults. As will be seen, the nonclinical MUSC community sample of older adults includes many older adults with impaired hearing, yet they have opted, it appears, to not pursue audiologic evaluation, albeit they did volunteer to participate in a longitudinal study of hearing. Also, it is not possible to confirm from the information available whether any of these individuals sought hearing evaluations outside of the MUSC clinic. It is assumed here that the vast majority did not do so, consistent with the modeling of This study sought to fill this gap by analyzing CPHI scores from 764 older adults divided into three groups: (a) a convenience sample of the Charleston, South Carolina community recruited for participation in a longitudinal study of hearing over the adult lifespan at the Medical University of South Carolina , those who obtained an audiology evaluation but did not acquire hearing aids (Clinic–No Hearing Aid [Clinic–No HA] sample), and those who obtained an audiology evaluation and acquired hearing aids (Clinic–Hearing Aid [Clinic–HA] sample). Through such evaluation, it is hoped to gain a better understanding of whether and how differences in perceived hearing difficulties underlie the help-seeking behaviors of older adults with impaired hearing.SD = 6.1 years) for the females and a mean age of 68.2 years (SD = 6.5 years) for the males. The Clinic–No HA sample included individuals from the Charleston, SC area recruited for the longitudinal study of hearing (n = 77) and individuals from the Bloomington, IN area recruited for a study of hearing-aid outcomes (n = 265). All individuals in this combined sample (n = 342) were recruited from the affiliated university clinics or were volunteering for a study on hearing-aid outcomes in which they knew hearing-aid purchase was required. At the time of the completion of the CPHI, however, no individuals in this group self-reported current hearing aid use (MUSC data set) or ever having used hearing aids (IU data set). Of the 342 individuals in this sample, 39% were female. Ages ranged from 57 to 89 years with a mean age of 74.5 years (SD = 7.0 years) for the females and 70.8 years (SD = 6.8 years) for the males. The Clinic–HA sample included 127 members of the Bloomington, IN community who had volunteered for the study of hearing-aid outcomes who reported that they had previously worn hearing aids and 52 members of the Charleston, SC community who had volunteered for the longitudinal study of hearing who reported that they were currently using hearing aids. As noted previously, about two thirds of those from the Bloomington community who had reported prior hearing aid use were current hearing aid users. The Clinic–Hearing Aid sample, total n = 179, ranged in age from 52 to 87 years with a mean age of 75.7 years (SD = 6.7 years) for the females and a mean age of 71.8 years (SD = 7.0 years) for the males.There were three samples of older adults included in these analyses. The Community sample was entirely from the Charleston, SC area and responded to ads placed in the community for participation in a longitudinal study of hearing over the adult lifespan. Of the 243 members of this sample, 141 (58%) were female. Ages for this sample ranged from 50 to 88 years with a mean age of 68.6 years  age between 60 and 89 years; (b) hearing loss that was flat or gently sloping ; (c) hearing loss that was of sensorineural origin ; (d) hearing loss that was bilaterally symmetrical ; (e) pure-tone thresholds within the following ranges at frequencies of 250, 500, 1000, 1500, 2000, 3000, 4000, and 6000 Hz, respectively: 5–85, 5–85, 10–90, 20–95, 25–95, 30–120, 30–120, and 30–120 dB HL ; (f) no For the IU data set, from 1997 to 2008, there was a total of 930 individuals who responded to the ads and were screened for study eligibility. For the period 2004–2008, when this information was collected more formally and in detail, approximately 30% of those screened were found to be ineligible. This percentage was applied to the entire sample of 930 screened individuals, leaving 651 eligible individuals. Of the estimated 651 eligible individuals, 392 (60.2%) enrolled in the studies of hearing-aid outcomes. They paid the full purchase price for the devices at the time of enrollment and were paid as research subjects for the completion of other data-collection sessions.As noted above, an audiogram was completed as a part of the eligibility criteria for the IU data set. After completion of the audiological evaluation, the results were explained briefly to the individual and, if eligible for study enrollment, they were informed about the study at this time. As noted, the study was investigating hearing-aid outcomes and they were informed that they would be required to purchase hearing aids for that study. Those interested in participating were consented and enrolled. No other counseling was provided to these individuals until after the hearing-aid fitting, which took place after the completion of all pre-fit measures, including the CPHI.For the MUSC data set, the following inclusion criteria applied: (a) age 18 and over, (b) good general health , (c) no evidence of conductive hearing loss or active otologic/neurologic disease, and (d) ability to provide reliable results for a majority of the protocol measures. The consent form and all procedures used in this study were approved by the MUSC Institutional Review Board. During a screening visit, the study was explained briefly to the individual and, if eligible for study enrollment, they were informed about the study. Those interested in participating were consented and enrolled. No counseling was provided to study participants until after completion of the CPHI or Hearing Handicap Inventory for the Elderly (HHIE).For the MUSC data set, from the start of this longitudinal study in 1987, approximately 90% of individuals screened for eligibility were enrolled in the study. The average attrition rate is approximately 14%; however, a large proportion of participants who withdrew from the study were less than 50 years of age, which is the minimum age of participants whose baseline data were included in the current analyses.Prior to completing the CPHI, each participant in each group received a complete audiological evaluation at study enrollment, including pure-tone and speech audiometry, as well as immittance measures. The CPHI was administered at each site using a paper-and-pencil response format with the participant seated in a quiet room. Booklets were provided to the participant that contained the standardized written instructions for each section of the CPHI. For each item, one of five response choices is selected. Computer software supplied by Marilyn Demorest was used to score the CPHI . As indiIn addition, almost 80% of the participants included in the current CPHI analyses had also completed the full 25-item HHIE . The HHI2 (2) = 45.5, p < .001, and follow-up z-tests of the proportion of females in each group found that all three samples differed from one another (p < .05). The Community sample was 58.0% female, the Clinic–No HA sample was 38.6% female, and the Clinic–HA sample was 26.3% female. Because of these significant differences in male/female proportions for each sample, as well as the mixed findings regarding the influence of this variable on CPHI scores as noted earlier, sex will serve as another independent variable for all comparisons of the three samples.Chi-squared analysis indicated that there were significant differences in the proportion of males and females across the three samples, χF = 24.3, p < .001, and sample type, F = 38.6, p < .001, emerged. The interaction between sex and sample type, however, was also significant, F = 4.7, p < .01. Post hoc paired comparisons revealed that there were no significant differences in age between the males and females for the Community sample (p > .05), but females were significantly older than males (p < .05) for the other two samples. As a result, as well as the prior literature reviewed earlier that documented effects of age on CPHI scores, age will also serve as a covariate when comparing CPHI results across sample types.When the age composition of each of the three samples was analyzed using general linear models (GLMs) with sex as another independent variable, significant main effects of sex, F = 175.8, p < .001, and sex, F = 32.0, p < .001, but the interaction of sample type and sex was also significant, F = 5.9, p < .01. Regarding the effects of test frequency for the right ear, the main effect was significant, F = 1,253.9, p < .001, as were all of the interactions with frequency: Frequency × Sample Type, F = 11.3, p < .001; Frequency × Sex, F = 77.4, p < .001; Frequency × Sample Type × Sex, F = 2.0, p < .05. For the left ear, significant main effects were observed for sample type, F = 170.9, p < .001, and sex, F = 51.0, p < .001, but the interaction of sample type and sex was also significant, F = 5.0, p < .01. Regarding the effects of test frequency for the right ear, the main effect was significant, F = 1,441.6, p < .001, as were all of the interactions with frequency: Frequency × Sample Type, F = 9.7, p < .001; Frequency × Sex, F = 75.1, p < .001; Frequency × Sample Type × Sex, F = 2.2, p < .01. When these GLM analyses of the audiograms in F = 2.5, p > .05.The three data sample types also differed regarding their pure-tone air-conduction hearing thresholds. Mean hearing thresholds for the males and females comprising each sample type are shown in Difference in audiograms between males and females, especially in the higher frequencies, are well known and are not of primary interest here other than to note that examination of the effects of sex on CPHI scale scores must control for differences in hearing loss between males and females. The differences in audiograms among the three sample types, moreover, are also expected. The vertical bars in N = 1,004) derived across five sites by SD of the normative mean, is observed between the norms and the means of the Clinic–HA sample with similarly good agreement between the norms and the means of the Clinic–No HA sample for 18 of the 20 scales. The Clinic–No HA sample shows means slightly higher than the clinic norms for the PA-stress and PA-withdrawal CPHI scales. In general, however, the clinical data reported here are in good agreement with the previously published clinical norms for the CPHI (The filled circles with error bars appearing in both panels of the CPHI .p < .001) of sample type was found, all F > 7.23. Moreover, the partial eta-squared effect sizes for the 19 significant results were large in 11 cases, medium in seven, and small for one  or .0026, revealed that, for 15 of the 19 significant effects of sample type, all three samples differed from one another. For the remaining four significant effects of sample type , the Community sample had higher scale scores than both of the two clinic samples (indicating better outcomes) and the two clinics samples did not differ from one another. Where significant effects of sample type were observed, that is, 19 of the 20 CPHI scales, the Community sample had significantly higher scores for 15 scales, but had significantly lower scores for four CPHI scales, CP-problem awareness, CS-verbal strategies, CS-nonverbal strategies, and the PA-denial scales.Visual inspection of the means in F > 12.2, p < .001, except for CE-physical characteristics, F = 3.3, p > .05. Moreover, the partial eta-squared effect sizes for better-ear PTA4 were large for five scales , small for two , and medium for all other CPHI scales  > 4.2, p < .05. Based on partial eta-squared effect sizes for age, six of the seven had small effect sizes  and one had a medium effect size (CE-communication need).As noted previously, the sample types differed significantly in hearing loss, age, and sex. The significant effects of sample type on CPHI scales appearing in I scales . RegardiF > 4.3, p < .05. The estimated marginal means, at a mean PTA4 of 34.0 dB HL and a mean age of 71.1 years, are shown in the top panel of Regarding the effect of sex on CPHI scale scores, a significant main effect, without interaction with sample type, was observed for four CPHI scales, all F > 3.1, p < .05. The partial eta-squared effect sizes for these three significant interactions between sex and sample type were all small (p < .05) from one another and in the direction reflected in the bottom panel of p < .05). For the CE-physical characteristics scale (middle panel), for females, all three sample types again differed significantly (p < .05) from one another. For males, the Community sample differed significantly (p < .05) from both clinic samples but the two clinic samples did not differ significantly (p > .05) from one another. Finally, for the CS-nonverbal strategies CPHI scale, as shown in the bottom panel of p < .05) higher scale score than each of the other two sample types, but the Community and Clinic–No HA samples did not differ significantly (p > .05) from one another on this scale.For three additional CPHI scales, CP-social, CE-physical characteristics, and CS-nonverbal strategies, there were significant interactions between sex and the other independent variable, sample type, all ll small . The botF > 3.3, p < .05, with 11 of the 12 having small partial eta-squared effect sizes and one (CP-problem awareness) having a medium effect size (p < .05) from the two clinic sample types but the latter did not differ from one another. The two exceptions were CP-work, for which the Community sample only differed significantly (p < .05) from the Clinic–HA sample, and CE-attitudes of others, for which the Community sample differed only from the Clinic–No HA sample. Regarding the six PA scales in the bottom panel of p < .05) from the two clinic samples but the two clinic samples did not differ from one another. For two PA scales, PA-discouragement and PA-withdrawal, the only significant paired comparison was between the Community and Clinic–HA samples. For the remaining PA scale in the bottom panel of p < .05).Finally, the main effect of sample type, free from significant interactions with sex, was significant for 12 of the 20 scales after controlling for age and better-ear PTA4, all ect size . Figure In summary, these GLM analyses found significant main effects of sample type, after controlling for sample differences in age and better-ear PTA4, on 12 of the 20 CPHI scales and the prevailing pattern was for the Community sample to differ significantly from one or both of the clinic samples with those clinic samples not differing from one another. In addition, recall that there were three other CPHI scales for which there were significant interactions between sample type and sex . When the effects of sample type were examined separately for each sex for these three CPHI scales, the Community sample always differed from one or both clinic samples. In total, the Community sample differed from the clinic samples on 15 of the 20 CPHI scales after controlling for age and better-ear PTA4. The direction of the difference was not consistent across the CPHI scales such that sometimes the Community sample had higher scale scores and for other scales that sample had the lowest score.F > 8.1, p < .01, and mostly medium or large effect sizes  were observed. Age was a significant covariate for six of 20 CPHI scales, all F > 4.0, p < .05. When statistically controlling for better-ear PTA4 and age, significant differences were observed between the Clinic–No HA and the Clinic–HA samples on seven of the 20 CPHI scales, all F > 4.1, p < .05. Partial eta-squared effect sizes were small for five of the six scales and medium for the remaining scales with significant effects of sample type. For the most part, however, differences were seldom observed between the two clinic samples, those with and without hearing aids. To examine this more closely and with greater statistical power, another series of GLM analyses was performed comparing only the two clinical samples and removing sex as a second independent variable. As noted earlier, sex was found to have a significant effect somewhat infrequently and these effects were not the primary focus of this evaluation. Age and better-ear PTA4 remained as covariates. PTA4 was a significant covariate for 19 of the 20 CPHI scales, all F = 120.3, p < .001, and post hoc Bonferroni-adjusted paired comparisons revealed that all sample types differed significantly (p < .05) in HHIE score from one another. This was without controlling for age and better-ear PTA4, the two main covariates based the CPHI analyses, which are likely to impact HHIE scores significantly and are known from the prior analyses of CPHI scores to differ significantly across the sample types. The GLM analysis of HHIE scores was repeated with age and better-ear PTA4 as covariates. Age, F = 7.8, p < .01, PTA4, F = 201.4, p < .001, and sample type, F = 10.1, p < .001, all had significant effects on HHIE-total scores. Post hoc Bonferroni-adjusted paired comparisons among the three sample types found that all sample types differed significantly (p < .05) from one another when evaluated at a mean age of 70.4 years and a mean better-ear PTA4 of 32.2 dB HL. These findings from most participants included in the CPHI analyses support the principal findings of those CPHI analyses. First, there are significant differences in age, hearing loss, and perceived hearing difficulties across the three sample types, but the latter may be mediated to some extent by the differences across the sample types in age and hearing loss. Second, when the differences across sample types in age and hearing loss were controlled statistically, residual differences in perceived hearing difficulties remain across the three sample types.In addition to the full CPHI, 596 of the 764 (78%) participants included in the MUSC and IU data sets also had completed the full 25-item HHIE measure. Those with HHIE scores available constituted 100% of the Community sample, 64% of the Clinic–No HA sample, and 75% of the Clinic–HA sample. The HHIE-total scores were used to corroborate the effects of sample type on perceived hearing difficulties reported here for the CPHI. The mean (standard error) HHIE scores for the three sample types were 12.8 (1.1), 27.2 (1.2), and 41.6 (1.5) for the Community, Clinic–No HA, and Clinic–HA samples, respectively (lower scores indicating less perceived hearing handicap). There was a significant effect of sample type on HHIE scores, All told, the clinical data from the present analyses of CPHI scale scores from older adults are consistent with the prior data from large-scale studies of clinical samples . These pWithout controlling for age, hearing loss (PTA4), or sex differences across the three sample types, significant effects of sample type were observed for 19 of the 20 CPHI scales; all except CE-communication need. Subsequent post hoc paired comparisons among the three sample types for these 19 CPHI scales revealed that all three sample types differed significantly most often  with the Community sample differing from both of the clinic samples but the two clinic samples not differing significantly from one another. Recall that the Community sample was younger and had lower better-ear PTA4 values than the two clinic samples whereas the two clinic samples differed only in PTA4 with the hearing-aid adopters having more audiometric hearing loss. This suggests, not surprisingly, that an older adult with more hearing loss and a more advanced age is more likely to have an audiologic evaluation, and further increases in hearing loss increase the likelihood of adhering to recommendations to acquiring hearing aids.A more interesting question, however, is whether differences in older adults' perceived communication difficulties and their reactions to those difficulties among the sample types remain after controlling for hearing loss and age. This was addressed here by a series of GLM analyses examining the effects of sex and sample type while statistically controlling for age and better-ear hearing loss (PTA4). Here, the Community sample still consistently differed significantly from the two clinic samples. In particular, even when controlling for the significant differences between sample types in age and hearing loss, the Community sample reported better communication performance in all settings and had less awareness of communication problems relative to one or both clinical samples . The low, the Community sample differed from one or both clinical samples on 15 of 20 scales  and nonadherents (who did not). p < .05) in HHIE total scores between groups akin to the Clinic–No HA and Clinic–HA sample types investigated here.The results obtained for the HHIE for most participants included in the CPHI analyses support the principal findings on the impact of sample type on the CPHI. The greatest limitation to the study relates to the way in which membership in one of the three samples was determined. For the Community sample, for instance, it is known that the members of that sample, (a) volunteered for the MUSC longitudinal study of hearing, (b) were not referred to the study from the MUSC audiology clinic, and (c) were not current hearing-aid users. As noted previously, this does not preclude them from having had a hearing evaluation previously but at a non-MUSC clinic or from having tried hearing aids previously but discontinued their use prior to enrollment in the longitudinal study. Likewise, for the Clinic–No HA sample, it is known that they received an audiological evaluation at either the MUSC or the IU clinic, but for the MUSC members of that sample, it is only known that they were not currently using hearing aids. That is, it is possible that they had tried hearing aids (adherents) but stopped using them prior to enrollment in the MUSC longitudinal study. Whereas, the MUSC study confirmed current hearing aid usage, inquiries about prior hearing aid usage were not made. For the members of the Clinic–No HA sample from IU, those who had never tried hearing aids comprised the presumed group of nonadherents. Furthermore, for the entire Clinic–No HA sample, it is presumed that hearing aids were recommended for every member of this sample, but they declined to pursue them, thereby making them nonadherents. For the Clinic–HA sample, considered to be adherents, it is again assumed that they adhered to the recommendation which was to acquire hearing aids. It is, of course, unlikely that hearing aids were acquired without a clinical evaluation and recommendation. Nonetheless, because this was not asked directly of each participant at the time of enrollment in either the MUSC or the IU studies, this cannot be confirmed directly.In addition to this main limitation, there are other common limitations on the generalization of these findings. For example, older adults from only two communities, Charleston, SC and Bloomington, IN, participated and the participants were overwhelming White and non-Hispanic. This limits the broad generalization of the findings to other demographic groups. In addition, all members of all three samples were research volunteers and those who volunteer for research studies may not be representative of the broader community from which they were drawn.In conclusion, this study found significant differences in CPHI scale scores (and HHIE scores) across Community, Clinic–No HA, and Clinic–HA sample types. Hearing loss, and to a lesser extent, age and sex, impacted the effects of sample type on these self-report measures. It is not surprising that an individual's likelihood of taking action, whether to get a hearing evaluation or to acquire hearing aids after such an evaluation, increases as with age and the severity of hearing loss. Importantly, even when controlling for hearing loss and age, many significant differences in CPHI scale scores (and HHIE scores) remained across these sample types. Those who sought clinical evaluation of their hearing loss, regardless of hearing-aid decision, differed from those in the community who did not. The major differences were in the degree of perceived communication difficulty, with those not seeking clinical evaluation reporting less perceived difficulty, and a greater denial or lack of awareness of communication problems among those in the community who did not seek clinical assistance. For the two groups who did seek clinical evaluation of their hearing difficulties, one obtaining hearing aids and the other not doing so, those who acquired hearing aids perceived greater difficulty in communicating in all environments, had greater awareness of communication difficulties, were more likely to use nonverbal strategies to compensate for their difficulties, were more accepting of their hearing loss, but tended to allocate more responsibility for difficulties to their communication partners. Thus, beyond the differences in measured audiometric hearing loss and age across the three sample types examined here, there were real differences in their perceived hearing difficulties and their reactions to them. This reinforces the notion that, ultimately, it is the older adult's self-awareness of hearing difficulties that is most critical to seeking help for hearing problems, as well as remediation for those problems. Efforts to promote and make available self-assessment of hearing difficulties by older adults should be advocated and included in opportunities for hearing screening. In that regard,