Background: An early etiological diagnosis of hearing loss positively impacts children’s quality of life including language and cognitive development. Even though hearing loss associates with extremely high genetic and allelic heterogeneity, several studies have proven that Next-Generation Sequencing (NGS)-based gene panel testing significantly reduces the time between onset and diagnosis.Methods: In order to assess the clinical utility of our custom NGS GHELP panel, the prevalence of pathogenic single nucleotide variants, indels or copy number variants was assessed by sequencing 171 nuclear and 8 mitochondrial genes in 155 Spanish individuals with hearing loss.Results: A genetic diagnosis of hearing loss was achieved in 34% (52/155) of the individuals (5 out of 52 were syndromic). Among the diagnosed cases, 87% (45/52) and 12% (6/52) associated with autosomal recessive and dominant inheritance patterns respectively; remarkably, 2% (1/52) associated with mitochondrial inheritance pattern. Although the most frequently mutated genes in this cohort were consistent with those described in the literature , causative variants in less frequent genes such as TMC1, FGF3 or mitCOX1 were also identified. Moreover, 5% of the diagnosed cases (3/52) were associated with pathogenic copy number variants.Conclusion: The clinical utility of NGS panels that allows identification of different types of pathogenic variants–not only single nucleotide variants/indels in both nuclear and mitochondrial genes but also copy number variants–has been demonstrated to reduce the clinical diagnostic odyssey in hearing loss. Thus, clinical implementation of genomic strategies within the regular clinical practice, and, more significantly, within the newborn screening protocols, is warranted. Hearing loss, defined as any degree of loss on the ability to hear sounds at thresholds considered normal, is the most prevalent sensory disorder . FurtherHearing loss profoundly affects children’s quality of life. Since language acquisition is delayed, behavioral problems may arise and poor academic performance is usually related to hearing impairment . An earlInterestingly, new genomic diagnostic tools based on Next-Generation Sequencing (NGS) promise identification of pathogenic sequence variants associated with hearing loss not only as part of newborn screening test, but also for patients with delayed-onset hearing loss. Given nowadays affordable cost of genomic tools, implementation of NGS approaches in hearing loss etiological diagnosis is warranted , yet it The international collaboration project GHELP has engaged eight institutions in three countries  in order to develop an NGS- based custom diagnostic tool that allows the identification of any type of pathogenic sequence variant  in all nuclear and mitochondrial genes previously associated with hereditary hearing loss. The aim of this study was to assess the prevalence of clinically relevant sequence changes in hearing loss individuals in the Spanish population with clinical suspicion of genetic origin and endorse the utility of a custom gene panel in early diagnosis and precision medicine in hearing loss.This research is circumscribed within the GHELP project “Boosting innovation in the early detection of hearing loss in children within SUDOE space: towards a personalized medicine based on genomic diagnostic tools”, co-financed by the Interreg Sudoe Program (abbreviation of Cooperation Program Interreg V-B Southwest Europe) through the European Regional Development Fund (ERDF).: the inclusion criteria were age of onset of the hearing loss between 0 and 18 years , presence of permanent sensorineural hearing loss (SNHL) either in the presence or absence of a clinically defined syndrome and availability of clinical data at presentation and follow-up.- Retrospective cohort (n = 134 patients)- Prospective cohort (n = 21 patients): the inclusion criteria were age of onset between 0 and 3 months, presence of any type SNHL and failure in the second tier of hearing screening with OtoAcoustic Emissions (OAE) and ABR.A total of 155 Spanish individuals with hearing loss were recruited for this study. Two cohorts of patients were defined, with unilateral o bilateral permanent hearing loss, with known clinical features (retrospective cohort) and patients in whom hearing loss has been detected during neonatal screening program and confirmed by auditory brainstem response (ABR) in at least one ear (prospective cohort).For both cohorts, clinical information included demographic data, personal  and family history, alterations in other organs and systems, performance on the hearing screening, results of the hearing tests performed and associated exploratory findings. In addition, for the retrospective cohort, data about the type of treatment for hearing loss and the results obtained following such treatments were recorded.The clinical data was incorporated into a database for statistical analysis using the SPSS Statistics v20 software and consisted of a total of 83 variables for the retrospective cohort and 62 variables for the prospective cohort .GJB2 and the m.1555A>G mitochondrial DNA mutation in MT-RNR1 . The remaining 154 patients were not prescreened for any common hearing loss mutation.Only one patient in this study had been previously tested for common mutations in The study (#2016.034) was approved by the Ethics Clinical and Research Committee of the University Clinic of Navarra.Thorough review of the available literature and commercially available tests for hearing loss allowed for the identification of a total of 179 genes, 171 nuclear and 8 mitochondrial, that were clinically related to any type of hereditary hearing loss (syndromic and non-syndromic) regardless the pattern of inheritance. Every commercially available gene with a strong evidence of relevance for hearing loss was included in the design. Furthermore, other candidate genes were included based on their frequency among our target populations and additional mitochondrial genes were also included in the design and define the list of target genes in our in-house-developed NGS GHELP panel design . Target Briefly, DNA was isolated from peripheral blood or saliva using the Maxwell RSC Instrument (Promega) and characterized by Qubit (ThermoFisher). Target-region capture using Agilent SureSelect custom probes-based enrichment protocol prior to paired-end Illumina MiSeq sequencing (control software 2.6.2.1) was established as the preferred wet-lab methodology at CIMA LAB Diagnostics (Universidad de Navarra).in vitro diagnostic medical devices in accordance with current legislation (Licence Number: 7157-PS). A custom pipeline was used for secondary analysis of raw FASTQ files resulting from sequencing. The pipeline integrates the following steps: data quality control assessment using FastQC software  and clinical related database (ClinVar); variants visualization through an online platform allowing easy filtering and variant prioritization. Sequence variants were named following Human Genome Variation Society (HGVS) recommendations.The secondary and tertiary bioinformatic analyses were performed by Dreamgenics S.L through the Genome One platform, certified with the IVD/CE mark for software ; removalsoftware ; alignmesoftware ; sorted software  and remosoftware ; variantsoftware  and Dreasoftware . It alsoThe genetic variants identified were classified following the 2015 American College of Medical Genetics and Genomics (ACMG) recommendation guidelines . VariantPatients included in either the retrospective or prospective cohorts were classified in three different groups according to the genetic findings identified by analysis with the NGS GHELP panel - Group 1: individuals with a confirmed genetic diagnosis; Group 2: individuals for whom a clear genetic diagnosis was not achieved but a potential genetic origin could still be suspected based on the genetic variants identified, and Group 3: individuals for whom a genetic cause of hearing loss was not found.GJB2 (n = 21), OTOF (n = 7) and MYO7A (n = 4) (A genetic cause for hearing loss was identified in 32% of the patients (49 out of 155) . The mos (n = 4) . SummaryCausative likely pathogenic/pathogenic variants were identified in genes associated with recessive SNHL in 86% of the cases (42 out of 49), in genes associated with autosomal dominant SNHL in 12% of the cases (6 out of 49), and in a mitochondrial gene in 2% of the cases (1 out of 49) in individuals in Group 1. The benefits of using a comprehensive genomic tool for the diagnosis of hearing loss extend beyond the identification of common single nucleotide variations in frequently mutated genes. Some of the advantages of using an NGS panel like ours that allows for the identification of any type of variant in either mitochondrial or nuclear genes for the diagnosis of hearing loss are exemplified by the following families:GJB2 (NM_004004.6): c.35delG p.  and the well-described Spanish founder ∼309-Kb deletion ∆GJB6-D13S1830  presented with neurosensorial bilateral profound hearing loss with a family history of hearing impairment and clinical features of Waardenburg syndrome, including heterochromia iridis and presence of white forelock. In both cases, we identified a pathogenic heterozygous alteration consisting in the deletion of exons 2 to 5 of the mitCOX1 : c.224G>A p. (Arg75Gln) heterozygous pathogenic variant. This variant has already been described to act in a dominant manner causing non-syndromic hearing loss (DFNA3) or hearing loss with palmoplantar keratoderma : c.617A>G p. (Asn206Ser) and c.109G>A p. . These two non-classical variants in GJB2 have already been described as causative of non-syndromic hearing loss, ichthyosis and in some cases keratoderma on the palms and soles  were diagnosed with syndromic hearing loss, being SLC26A4, related with either Pendred syndrome or autosomal recessive deafness-4 (DFNB4) with enlarged vestibular aqueduct, although the clinical signs usually associated with Pendred syndrome as temporal bone abnormalities and euthyroid goiter could not be clinically confirmed. Four additional individuals  recommendations for reporting results of diagnostic genetic testing .Individuals for whom a potential genetic cause could be suspected represents 25% (39 out of 155) of the subjects in the study. SNHL individuals were classified in this group 2 if 1) a pathogenic or likely pathogenic genetic variant and a variant of unknown clinical significance (VOUS) , or two CDH23 (NM_022124.6) gene: a likely pathogenic variant c.4021G>A p.(Asp1341Asn) and two VOUS: c.4780C>G p.(Arg1594Gly) and c.3178C>T p.(Arg1060Trp) , was identified and classified as likely pathogenic: c.65C>A p. (Ser22*). Homozygous (and very rarely heterozygous) loss of function mutations in EDNRB have previously been associated with Waardenburg syndrome type 4A (WS4A) whereas heterozygous mutations have been described in association with isolated Hirschsprung disease, and suggestively with Waardenburg syndrome type 2 (WS2)  in whom a previously not described heterozygous nonsense variant in the endothelin-B receptor gene,  2 (WS2) . A segreFinally, in 43% of the studied SNHL individuals (67 out of 155) no pathogenic or likely pathogenic or suspicious VOUS that could explain the SNHL phenotype were identified. In this group, a single pathogenic or likely pathogenic variant in a gene previously associated with recessive SNHL (carriers) , likely/Undiagnosed and untreated hearing loss gives rise to worse outcomes in language and cognitive abilities besides social, academic and behavioral problems that negatively affects the quality of life of people with hearing loss . In the In this regard, here we contribute to reinforce the utility of transferring NGS target panels to the clinical practice for early genetic diagnosis of hearing loss, which can boost the diagnostic yield and may help revealing a hidden syndrome. Indeed, NGS testing is included in the hearing loss diagnostic algorithm within the newest ACMG guidelines . TherefoIn the light of this need, our NGS GHELP panel has been designed and has shown the capacity to identify different types of sequence variants known to be pathogenic including SNVs, indels and CNVs within both nuclear and mitochondrial genes. This entails some progress compared to other published NGS-based panels that may analyze a lower number of genes, lack mitochondrial genes in their design  or were In this study, a genetic diagnosis was established in 34% of the studied individuals (52/155), with hearing loss as a part of a syndrome in at least five of them. Providing an accurate diagnosis in these patients through genetic testing and the consequent genetic counseling positively may impact children management and outcome, and benefit their families. Besides providing information about etiology regarding hearing loss, contributing to a better understanding of the nature of this trait, likelihood of recurrence might be estimated, several rehabilitation options may be considered, and referral to other specialties in case surveillance is recommended in previously undiagnosed syndromes is feasible. Moreover, although still in preclinical stages, a growing number of gene therapy approaches have arisen in recent years, as a potential treatment for hearing loss.One of the first crucial approaches when implementing NGS in the clinical practice is to define the set of genes that the panel will target. Establishing a strong association between the targeted genes and SNHL was needed for the genes to be included in the design , so inciRegarding different types of potentially causative alteration, not only is hearing loss caused by pathogenic SNV and indels, but CNVs have also been described as common contributors to this trait. Therefore, CNV analysis should be required when genetic testing for hearing loss is considered. Sufficient coverage analysis for the targeted region and bioinformatic analyses should also be guaranteed, and MLPA confirmation is recommended whenever possible. Patients in families #F1 and #F2 highlight the relevance of including not only SNVs and indels, but also CNVs assessment in the design of genomic tools like our NGS GHELP panel.STRC deletion was identified, which is unexpected given the high prevalence of STRC alterations in Europe, accounting for 16% of the autosomal recessive cases in this population  in both nuclear and mitochondrial genes, another cornerstone in NGS diagnostic pipelines is variant classification and clinical interpretation of the genetic findings by geneticists. In an effort to foster standardization and reduce discrepancies across laboratories, guidelines for variant interpretation have been developed. A Hearing Loss Variant Curation Expert Panel has been also created to provide expert guidance in the context of genomic interpretation of variants in hearing loss-related genes . Besidesa priori be concluded. Benefits of this cooperation are brought to light especially in families in Group 2, in whom a follow up was needed in order to conclude with a diagnosis. Access to several members in family #F6 for segregation studies allowing pathogenicity assessment of identified variants was possible thanks to collaboration with clinicians. Further reviewing genotype-phenotype correlation for family #F7 with the clinicians, highlighted the fact that the proband in this family was born before 35 weeks of a twin pregnancy; thus, being a high-risk premature infant for having non-genetic hearing loss, consistent with the fact that no genetic cause could be elucidated.A multidisciplinary approach when it comes to clinical interpretation of genomic results is mandatory. Cooperation between geneticists and physicians facilitated follow-up studies when needed, and eased access to clinical and family history information helping in the clinical interpretation process. In our laboratory at least two variant curators analyzed every case, so discrepancies in variant classification were discussed and agreed prior to final report writing. Periodic interdepartmental sessions with otorhinolaryngologists from Clínica Universidad de Navarra were arranged in order to assess genotype-phenotype correlation for every case. This approach can help increase the diagnostic yield, especially for those cases in which a genetic diagnosis cannot Even though NGS approaches have been proven to reduce diagnostic odyssey in hearing loss, our GHELP panel has failed to identify a genetic cause for hearing loss in 67% of the patients (103 out of 155), even if the panel design allows for the detection of any variant type. The diagnostic yield reached in this study might be slightly lower than in other published works in the field due to several reasons. Firstly, a reason for having missed pathogenic variants responsible for hearing loss may be the panel design itself: causative genes that may not be included in the gene list; deep intronic variants which are not assessed, since only coding sequence ± 10 bp including splice sites are evaluated; and poor coverage due to insufficient read depth or misalignment may prevent variant identification throughout some coding regions. In addition to these technical limitations, it is possible that the criteria for including patients during the recruitment process were not adequately fulfilled. This oversight could have led to negative genetic testing results in patients experiencing hearing loss due to reasons other than genetics, as demonstrated by the aforementioned patient in family #F7. Within this specific group of patients, it is plausible that environmental factors played a significant role in contributing to their hearing impairments. Patients in whom there were other known risk factors for ototoxicity and no suspicion of genetic origin for hearing loss should have not been recruited. Furthermore, patients who had previously tested positive for common variants were not included in the study. Only patients who had tested negative or with not known previous genetic studies were recruited, which may have impacted the diagnostic yield. Further studies in larger unscreened populations are needed in order to have a deeper knowledge of the genetic landscape of hearing loss.All things considered, here we contribute to endorse the clinical utility of comprehensive NGS panels in clinical practice and screening protocols for hearing loss, contributing to precision medicine in this condition. Furthermore, the authors reckon that the clinical data and observed genotype-phenotype correlations collected in this study will be immensely valuable as prognostic information for future newly diagnosed patients. The use of genomic tools that allow identification of causative pathogenic variants–SNVs/indels and CNVs—in both nuclear and mitochondrial genes in a suitable time span lessens the diagnostic odyssey in hearing loss, making its implementation in diagnostic algorithms worthwhile and warranted.


We know that the effects of unmanaged hearing loss are significant. It leads to an increased risk of both social isolation and poor mental health, and is a major driver for leaving the workforce early. Hearing loss has also been identified as the largest potentially modifiable risk factor for dementia. Yet still, people wait an average of 10 years before they seek help for their hearing loss. World Hearing Day 2023 and beginning their hearing health journey. The check takes just 3 minutes and measures how well you can hear speech in background noise. Those whose result shows that they may have hearing loss are signposted to their GP. In line with The National Institute for Health and Care Excellence (NICE) guidance, RNID recommends GPs investigate this potential hearing loss by taking the actions listed in In 2022, the Royal National Institute for Deaf People (RNID), celebrated over 100 000 people taking their free online hearing checkBox 1The following is recommended:otoscopy to exclude impacted wax or infection, thenarrange an audiological assessment, andrefer for additional diagnostic assessment if needed.We know that hearing loss is a growing public health challenge in the UK, and RNID have released research which painted a startling picture of the nation’s hearing health.Despite 91% of adults saying they rate their hearing as good, the research found that:more than a third (34%) admit they’ve pretended to follow conversations they've struggled to hear, for example by smiling and nodding along;the same amount (34%) have found it difficult to follow conversations in noisy surroundings like restaurants or parties;almost a quarter (24%) have found themselves asking people to repeat themselves;more than 1 in 10 (13%) say they have been told by their partner they think they might have a problem with their hearing;however, 83% have never taken any action in relation to their hearing, although 74% said they would be likely to take an online hearing check that is free and takes 3 minutes. There is also a growing body of evidence to suggest that hearing aids slow cognitive decline. This evidence has become particularly pertinent in the light of the 2020 Lancet Commission on dementia, which identified hearing loss as the largest potentially modifiable risk factor for dementia, with unmanaged hearing loss responsible for up to 8.2% of all dementia cases.It is well established that hearing aids have a large beneficial effect on people’s ability to take part in everyday life. NICE recommends hearing aids as the most cost-effective intervention for hearing loss. It is estimated that in England, only 2 million people have hearing aids out of the 6 million who have hearing loss that is significant enough to benefit from them.Yet many people experiencing hearing loss do not seek medical advice and remain undiagnosed. Typically, people who are referred for a hearing assessment are aged in their mid-70s and on average wait 10 years from the initial onset of hearing loss until they seek medical advice.The reasons for delaying seeking medical advice may be due to multiple factors. Individuals may underestimate the serious effects that hearing loss can have; they may fear the stigma associated with hearing loss and hearing aids; they may not realise the impact it is having due to its gradual progression; they may be in denial; or they may simply deprioritise it. We also know that hearing loss becomes a risk factor for dementia in mid-life. Supporting earlier identification and intervention, therefore, could ensure that individuals are supported to manage their hearing loss at an age when they are likely to benefit the most.We know that the ability to adapt to and manage hearing loss becomes increasingly difficult the older people are when they make an appointment for assessment and intervention.For this reason, NICE recommends referring adults with diagnosed or suspected dementia, or mild cognitive impairment for hearing assessment in audiology. NICE also recommends referring these patients and patients with a diagnosed learning disability for hearing assessment every 2 years.GPs have the power to improve their patients’ hearing health with a potentially transformative effect on people’s overall health and quality of life, through preventing social isolation, supporting mental health, enabling work, and potentially slowing cognitive decline. RNID has set out some clear guidance to help GPs identify and support their patients with hearing loss to access the management that they need.If someone has taken the RNID hearing test and had an outcome that suggests that they could have hearing loss, refer them for a full diagnostic test with a specialist as shown in Over 70% of over 70 year olds have hearing loss, with a high proportion undiagnosed. If you notice a patient struggling to hear you, discuss this with them and offer to refer them for a full diagnostic test with a specialist.free online hearing test. It takes three minutes and can be done in the comfort of one's own home or at the GP surgery in a quiet room. It isn’t the same as a full hearing test, but it’s a reliable way to find out if your patient needs one.Share the RNID  to educate GPs on D/deafness and hearing loss. Find the D/deafness and hearing loss toolkit here.RNID collaborated with the Royal College of General Practitioners and NHS England in developing a toolkitPractical tips on accessibility and communicating with patients who are D/deaf or have hearing loss are available via the RNID website.Ensure GP services are accessible to people with hearing loss and comply with the Accessible Information Standard in England. RNID has recently called for ear wax removal services to be brought back into primary care or community settings. If one of your patients is suffering from a build-up of ear wax, refer them to a local NHS removal service.The RNID hearing check is suitable for most adults, however the check should not be recommended if any of the following apply:unilateral or asymmetric hearing loss ;hyperacusis or severe tinnitus;suspected conductive hearing loss;diagnosed or suspected dementia or cognitive impairment;diagnosed learning disabilities; orunder 18 years old.Instead a referral for diagnostic assessment in ENT or audiology should be made, in line with NICE guidelines and local pathways.