Loss-of-function mutations in MYO15A and OTOF cause non-syndromic hearing loss in two Yemeni families.

BACKGROUND: Hearing loss is a rare hereditary deficit that is rather common among consanguineous populations. Autosomal recessive non-syndromic hearing loss is the predominant form of hearing loss worldwide. Although prevalent, hearing loss is extremely heterogeneous and poses a pitfall in terms of diagnosis and screening. Using next-generation sequencing has enabled a rapid increase in the identification rate of genes and variants in heterogeneous conditions, including hearing loss. We aimed to identify the causative variants in two consanguineous Yemeni families affected with hearing loss using targeted next-generation sequencing (clinical exome sequencing). The proband of each family was presented with sensorineural hearing loss as indicated by pure-tone audiometry results. RESULTS: We explored variants obtained from both families, and our analyses collectively revealed the presence and segregation of two novel loss-of-function variants: a frameshift variant, c.6347delA in MYO15A in Family I, and a splice site variant, c.5292-2A > C, in OTOF in Family II. Sanger sequencing and PCR-RFLP of DNA samples from 130 deaf and 50 control individuals confirmed that neither variant was present in our in-house database. In silico analyses predicted that each variant has a pathogenic effect on the corresponding protein. CONCLUSIONS: We describe two novel loss-of-function variants in MYO15A and OTOF that cause autosomal recessive non-syndromic hearing loss in Yemeni families. Our findings are consistent with previously reported pathogenic variants in the MYO15A and OTOF genes in Middle Eastern individuals and suggest their implication in hearing loss.

RNA base editing therapy cures hearing loss induced by OTOF gene mutation.

Otoferlin (OTOF) gene mutations represent the primary cause of hearing impairment and deafness in auditory neuropathy. The c.2485C>T (p. Q829X) mutation variant is responsible for approximately 3% of recessive prelingual deafness cases within the Spanish population. Previous studies have used two recombinant AAV vectors to overexpress OTOF, albeit with limited efficacy. In this study, we introduce an enhanced mini-dCas13X RNA base editor (emxABE) delivered via an AAV9 variant, achieving nearly 100% transfection efficiency in inner hair cells. This approach is aimed at treating OTOFQ829X, resulting in an approximately 80% adenosine-to-inosine conversion efficiency in humanized OtofQ829X/Q829X mice. Following a single scala media injection of emxABE targeting OTOFQ829X (emxABE-T) administered during the postnatal day 0-3 period in OtofQ829X/Q829X mice, we observed OTOF expression restoration in nearly 100% of inner hair cells. Moreover, auditory function was significantly improved, reaching similar levels as in wild-type mice. This enhancement persisted for at least 7 months. We also investigated P5-P7 and P30 OtofQ829X/Q829X mice, achieving auditory function restoration through round window injection of emxABE-T. These findings not only highlight an effective therapeutic strategy for potentially addressing OTOFQ829X-induced hearing loss but also underscore emxABE as a versatile toolkit for treating other monogenic diseases characterized by premature termination codons.

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue.

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

Predicting the Impact of OTOF Gene Missense Variants on Auditory Neuropathy Spectrum Disorder.

Auditory neuropathy spectrum disorder (ANSD) associated with mutations of the OTOF gene is one of the common types of sensorineural hearing loss of a hereditary nature. Due to its high genetic heterogeneity, ANSD is considered one of the most difficult hearing disorders to diagnose. The dataset from 270 known annotated single amino acid substitutions (SAV) related to ANSD was created. It was used to estimate the accuracy of pathogenicity prediction using the known (from dbNSFP4.4) method and a new one. The new method (ConStruct) for the creation of the protein-centric classification model is based on the use of Random Forest for the analysis of missense variants in exons of the OTOF gene. A system of predictor variables was developed based on the modern understanding of the structure and function of the otoferlin protein and reflecting the location of changes in the tertiary structure of the protein due to mutations in the OTOF gene. The conservation values of nucleotide substitutions in genomes of 100 vertebrates and 30 primates were also used as variables. The average prediction of balanced accuracy and the AUC value calculated by the 5-fold cross-validation procedure were 0.866 and 0.903, respectively. The model shows good results for interpreting data from the targeted sequencing of the OTOF gene and can be implemented as an auxiliary tool for the diagnosis of ANSD in the early stages of ontogenesis. The created model, together with the results of the pathogenicity prediction of SAVs via other known accurate methods, were used for the evaluation of a manually created set of 1302 VUS related to ANSD. Based on the analysis of predicted results, 16 SAVs were selected as the new most probable pathogenic variants.

Temperature-Sensitive Auditory Neuropathy: Report of a Novel Variant of OTOF Gene and Review of Current Literature.

Background and objectives: Otoferlin is a multi-C2 domain protein implicated in neurotransmitter-containing vesicle release and replenishment of the cochlear inner hair cell (IHC) synapses. Mutations in the OTOF gene have been associated with two different clinical phenotypes: a prelingual severe-to-profound sensorineural hearing loss (ANSD-DFNB9); and the peculiar temperature-sensitive auditory neuropathy (TS-ANSD), characterized by a baseline mild-to-moderate hearing threshold that worsens to severe-to-profound when the body temperature rises that returns to a baseline a few hours after the temperature has fallen again. The latter clinical phenotype has been described only with a few OTOF variants with an autosomal recessive biallelic pattern of inheritance. Case report: A 7-year-old boy presented a picture compatible with TS-ANSD exacerbated by febrile states or physical exercise with mild-to-moderate hearing loss at low and medium frequencies and a decrease in speech discrimination that worsened with an unfavorable speech-to-noise ratio. Otoacoustic emissions (OAEs) were present whereas auditory brainstem responses (ABRs) evoked by a click or tone-burst were generally absent. No inner ear malformations were described from the CT scan or MRI. Next-generation sequencing (NGS) of the known deafness genes and multi-phasic bioinformatic analyses of the data detected in OTOF a c.2521G>A missense variant and the deletion of 7.4 Kb, which was confirmed by array-comparative genomic hybridization (array-CGH). The proband's parents, who were asymptomatic, were tested by Sanger sequencing and the father presented the c.2521G>A missense variant. Conclusions: The picture presented by the patient was compatible with OTOF-induced TS-ANSD. OTOF has been generally associated with an autosomal recessive biallelic pattern of inheritance; in this clinical report, two pathogenic variants never previously associated with TS-ANSD were described.

Mutational and phenotypic spectrum of OTOF-related auditory neuropathy in Koreans: eliciting reciprocal interaction between bench and clinics.

BACKGROUND: While auditory neuropathy spectrum disorder (ANSD) is a heterogeneous disorder and its management quite varies depending upon the etiology, even including self-resolution, OTOF is an important molecular etiology of prelingual ANSD and has emerged as an attractive target for implementation of precision medicine in terms of timing and prognosis prediction of auditory rehabilitation. However, to date, the literature is lacking in the genotype-phenotype relationship of this gene as well as efficient molecular testing strategy in the clinic in many populations and to make things more complicated in Koreans, the most prevalent variant p.Arg1939Gln among Korean ANSD children frequently evaded detection by next generation sequencing (NGS), resulting in delayed genetic diagnosis and late cochlear implantation (CI). The aims of this study are to document the mutational and phenotypic spectrum of OTOF-related ANSD (DFNB9) in the Korean population, further establishing genotype-phenotype correlation and proposing a set of the most commonly found OTOF variants to be screened first. METHODS: Genetic diagnosis through the NGS-based sequencing was made on patients with ANSD in two tertiary hospitals. Genotype and phenotypes of eleven DFNB9 patients were reviewed. For data analysis, Mann-Whitney test and Fisher's exact test were applied. RESULTS: This study disclosed four prevalent variants in Koreans: p.Arg1939Gln with an allele frequency of 40.9%, p.Glu841Lys (13.6%), p.Leu1011Pro and p.Arg1856Trp (9.1%). Three novel variants (c.4227 + 5G > C, p.Gly1845Glu, and p.Pro1931Thr) were identified. Interestingly, a significant association of p.Arg1939Gln with worse ASSR thresholds was observed despite consistently no ABR response. Ten of 11 DFNB9 patients received CI for auditory rehabilitation, showing favorable outcomes with more rapid improvement on early-CI group (age at CI ≤ 18 mo.) than late-CI group. CONCLUSIONS: This study included the largest Korean DFNB9 cohort to date and proposed a set of the most frequent four OTOF variants, allowing the potential prioritization of exons during Sanger sequencing. Further, a significant association of p.Arg1939Gln homozygotes with poor residual hearing was observed. We may have to suspect p.Arg1939Gln homozygosity in cases of poor auditory thresholds in ANSD children with putative negative OTOF variants solely screened by NGS. Reciprocal feedback between bench and clinics regarding DFNB9 would complement each other.

An OTOF Frameshift Variant Associated with Auditory Neuropathy Spectrum Disorder.

BACKGROUND: Auditory Neuropathy Spectrum Disorder (ANSD) is manifested as impairment of auditory nerve activity but preservation of the outer hair cell function. OBJECTIVE: This study was to detect the disease-causing gene and variant(s) in a Chinese ANSD family. METHODS: A four-generation consanguineous Chinese ANSD family and 200 unrelated healthy controls were enrolled. Exome sequencing and Sanger sequencing were applied to identify the genetic basis for ANSD in this family. RESULTS: Exome sequencing detected a c.1236delC variant of the otoferlin gene in an apparently homozygous state. Sanger sequencing confirmed that the variant co-segregating with the phenotype of hearing impairments in this family. The variant was not detected in 200 healthy controls. The c.1236delC alteration may result in a truncated otoferlin missing the C2C-C2F domains and the C-terminal transmembrane domain, and thus severely damages Ca2+-dependent synaptic vesicle fusion and targeting function of the otoferlin. CONCLUSION: Our study suggested that the c.1236delC alteration in the otoferlin gene may be the disease-causing variant in this family, and also shed new light on genetic counseling to this ANSD family.

Novel compound heterozygous mutations in the OTOF Gene identified by whole-exome sequencing in auditory neuropathy spectrum disorder.

BACKGROUND: Many hearing-loss diseases are demonstrated to have Mendelian inheritance caused by mutations in single gene. However, many deaf individuals have diseases that remain genetically unexplained. Auditory neuropathy is a sensorineural deafness in which sounds are able to be transferred into the inner ear normally but the transmission of the signals from inner ear to auditory nerve and brain is injured, also known as auditory neuropathy spectrum disorder (ANSD). The pathogenic mutations of the genes responsible for the Chinese ANSD population remain poorly understood. METHODS: A total of 127 patients with non-syndromic hearing loss (NSHL) were enrolled in Guangxi Zhuang Autonomous Region. A hereditary deafness gene mutation screening was performed to identify the mutation sites in four deafness-related genes (GJB2, GJB3, 12S rRNA, and SLC26A4). In addition, whole-exome sequencing (WES) was applied to explore unappreciated mutation sites in the cases with the singularity of its phenotype. RESULTS: Well-characterized mutations were found in only 8.7% (11/127) of the patients. Interestingly, two mutations in the OTOF gene were identified in two affected siblings with ANSD from a Chinese family, including one nonsense mutation c.1273C > T (p.R425X) and one missense mutation c.4994 T > C (p.L1665P). Furthermore, we employed Sanger sequencing to confirm the mutations in each subject. Two compound heterozygous mutations in the OTOF gene were observed in the two affected siblings, whereas the two parents and unaffected sister were heterozygous carriers of c.1273C > T (father and sister) and c.4994 T > C (mother). The nonsense mutation p.R425X, contributes to a premature stop codon, may result in a truncated polypeptide, which strongly suggests its pathogenicity for ANSD. The missense mutation p.L1665P results in a single amino acid substitution in a highly conserved region. CONCLUSIONS: Two mutations in the OTOF gene in the Chinese deaf population were recognized for the first time. These findings not only extend the OTOF gene mutation spectrum for ANSD but also indicate that whole-exome sequencing is an effective approach to clarify the genetic characteristics in non-syndromic ANSD patients.

Mutations in OTOF, CLDN14 & SLC26A4 genes as major causes of hearing impairment in Dhadkai village, Jammu & Kashmir, India.

Background & objectives: A high incidence of hearing impairment is reported from the village of Dhadkai in the State of Jammu and Kashmir, India. Prevalence of endogamy in this community suggested a common genetic basis for the disorder. A genetic study was undertaken to ascertain the basis for the high incidence of hearing impairment in this region. Methods: In a two-step approach to identify the causative mutation/s, a whole-genome-based linkage analysis of an extended family of 45 members was carried out, which included 23 affected and 22 unaffected members. Mutational analysis for the candidate deafness genes helped reveal causative mutations in the family. In addition, seven deafness-causing genes, Cx26, SLC26A4, CLDN14, TMPRSS3, TMC1, TMIE and USH1C, were analyzed in smaller families with hearing impairment. Results: In the 45-member extended family, the critical chromosomal region mapped to 2p24-p22.The c.2122C>T (p.R708X) mutation in OTOF in 2p24-p22was identified as being the causal change. Linkage to 2p24-p22 locus was not observed in a particular branch of this extended family. Analysis of seven known deafness-causing genes in this branch revealed a mutation, c.254T>A (p.V85D), in CLDN14. Among seven small families unrelated to the 45-member extended family, hearing loss was attributable to p.R708X in OTOF in three families and to p.V85D in CLDN14 in one family; a new mutation c.1668T>A (p.Y556X) SLC26A4 was identified in two families and the causative change could not be identified in one family. Interpretation & conclusions: This study suggested considerable genetic heterogeneity in the causation of hearing loss in Dhadkai. Recessive mutations were observed in at least three genes causing hearing loss: OTOF (p.R708X), SLC26A4 (p.Y556X) and CLDN14 (p.V85D). Mutation p.R708X appeared to be the major cause of hearing impairment in Dhadkai.

High frequency of OTOF mutations in Chinese infants with congenital auditory neuropathy spectrum disorder.

Auditory neuropathy spectrum disorder (ANSD) is one of the most common diseases leading to hearing and speech communication barriers in infants and young children. The OTOF gene is the first gene identified for autosomal recessive non-syndromic ANSD, and patients with OTOF mutations have shown marked improvement of auditory functions from the cochlear implantation, but the true involvement of OTOF mutations in Chinese ANSD patients is still unknown which precludes the effective management of this disease. Here, we investigated the contribution of OTOF mutations to congenital ANSD patients in China. In all, 37 infants and young Children with ANSD were screened for all the exons of OTOF gene, of them 34 patients had no neonatal risk factors who were considered as congenital ANSD. The clinical manifestation and audiometric features were also investigated and compared in patients with and without OTOF mutations. In all, 14 of these subjects were shown to carry two or three mutant alleles of OTOF with the high frequency of 41.2% in congenital ANSD patients. In total, 15 novel pathogenic mutations and 10 reported mutations were identified. Our results confirmed that mutations in OTOF gene were a major cause of congenital ANSD in China. Identification of OTOF mutations can facilitate diagnosis, clinical intervention and counseling for congenital ANSD.

Further phenotype description, genotype characterization in patients with de novo interstitial deletion on 2p23.2-24.1.

Interstitial deletions of the distal part of chromosome 2p seem to be rarely identified or reported: to date, only nine distinct patients have been published. The last three patients were diagnosed with the use of more recent molecular karyotyping technology (SNP array). We report on the natural history of an 8-year-old boy with dysmorphic features, postnatal overgrowth, microcephaly, generalized hypotonia, and global developmental delay. The diagnosis was accomplished by SNP array investigation that led to the identification of a de novo 7.4 Mb deletion of 2p23.2-p24.1. The present patient also developed a nonsyndromic auditory neuropathy. Since the deletion encompassed the OTOF gene, this haploinsufficiency suggests second allele sequencing as a possible cause (DFNB9). We describe the phenotype of the patient and review reports in patients with del 2p23 subsequent to the advent of the genomic era. At the time of identification of "new" micro- deletion and -duplication syndromes, the present report adds to the description of phenotype in patients with del(2)p(23.2;24.1) and the 2p23.2 region in particular.

Preclinical Efficacy And Safety Evaluation of AAV-OTOF in DFNB9 Mouse Model And Nonhuman Primate.

OTOF mutations are the principal causes of auditory neuropathy. There are reports on Otof-related gene therapy in mice, but there is no preclinical research on the drug evaluations. Here, Anc80L65 and the mouse hair cell-specific Myo15 promoter (mMyo15) are used to selectively and effectively deliver human OTOF to hair cells in mice and nonhuman primates to evaluate the efficacy and safety of OTOF gene therapy drugs. A new dual-AAV-OTOF-hybrid strategy to transfer full-length OTOF is generated, which can stably restore hearing in adult OTOFp.Q939*/Q939* mice with profound deafness, with the longest duration being at least 150 days, and the best therapeutic effect without difference in hearing from wild-type mice. An AAV microinjection method into the cochlea of cynomolgus monkeys without hearing impairment is further established and found the OTOF can be safely and effectively driven by the mMyo15 promoter in hair cells. In addition, the therapeutic dose of AAV drugs has no impact on normal hearing and does not cause significant systemic toxicity both in mouse and nonhuman primates. In summary, this study develops a potential gene therapy strategy for DFNB9 patients in the clinic and provides complete, standardized, and systematic research data for clinical research and application.

AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness.

Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno-associated virus (AAV)-mediated OTOF gene replacement therapy is extensively validated in Otof-deficient mice. However, the clinical safety and efficacy of AAV-OTOF is not reported. Here, AAV-OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non-human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV-OTOF is delivered into one cochlea of a 5-year-old deaf patient and into the bilateral cochleae of an 8-year-old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure-tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5-year-old patient can be restored to the normal range at 1 month after AAV-OTOF injection, while the 8-year-old patient can hear the conversational sounds. Most importantly, the 5-year-old patient can hear and recognize speech only through the AAV-OTOF-injected ear. This study is the first to demonstrate the safety and efficacy of AAV-OTOF in patients, expands and optimizes current OTOF-related gene therapy and provides valuable information for further application of gene therapies for deafness.

A novel mutation in the OTOF gene in a Chinese family with auditory neuropathy.

Gene therapy for monogenic auditory neuropathy (AN) has successfully improved hearing function in target gene-deficient mice. Accurate genetic diagnosis can not only clarify the etiology but also accurately locate the lesion site, providing a basis for gene therapy and guiding patient intervention and management strategies. In this study, we collected data from a family with a pair of sisters with prelingual deafness. According to their auditory tests, subject Ⅱ-1 was diagnosed with profound sensorineural hearing loss (SNHL), Ⅱ-2 was diagnosed with AN, Ⅰ-1 was diagnosed with high-frequency SNHL, and Ⅰ-2 had normal hearing. Using whole-exome sequencing (WES), one nonsense mutation, c.4030C>T (p.R1344X), and one missense mutation, c.5000C>A (p.A1667D), in the OTOF (NM_001287489.1) gene were identified in the two siblings. Their parents were heterozygous carriers of c.5000C>A (father) and c.4030C>T (mother). We hypothesized that c.5000C>A is a novel pathogenic mutation. Thus, subject Ⅱ-1 should also be diagnosed with AN caused by OTOF mutations. These findings not only expand the OTOF gene mutation spectrum for AN but also indicate that WES is an effective approach for accurately diagnosing AN.

Recovery kinetics of dual AAV-mediated human otoferlin expression.

Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3' portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression.

[Study on gene therapy for DPOAE and ABR threshold changes in adult Otof-/- mice].

Objective:This study aims to analyze the threshold changes in distortion product otoacoustic emissions（DPOAE） and auditory brainstem response（ABR） in adult Otof-/- mice before and after gene therapy, evaluating its effectiveness and exploring methods for assessing hearing recovery post-treatment. Methods:At the age of 4 weeks, adult Otof-/- mice received an inner ear injection of a therapeutic agent containing intein-mediated recombination of the OTOF gene, delivered via dual AAV vectors through the round window membrane（RWM）. Immunofluorescence staining assessed the proportion of inner ear hair cells with restored otoferlin expression and the number of synapses.Statistical analysis was performed to compare the DPOAE and ABR thresholds before and after the treatment. Results:AAV-PHP. eB demonstrates high transduction efficiency in inner ear hair cells. The therapeutic regimen corrected hearing loss in adult Otof-/- mice without impacting auditory function in wild-type mice. The changes in DPOAE and ABR thresholds after gene therapy are significantly correlated at 16 kHz. Post-treatment,a slight increase in DPOAE was observeds,followed by a recovery trend at 2 months post-treatment. Conclusion:Gene therapy significantly restored hearing in adult Otof-/- mice, though the surgical delivery may cause transient hearing damage. Precise and gentle surgical techniques are essential to maximize gene therapy's efficacy.

Apoptosis of type I spiral ganglion neuron cells in Otof-mutant mice.

Otof, which encodes otoferlin, knockout mice are considered model mice for auditory neuropathy spectrum disorder, which is characterized by an absent auditory brainstem response (ABR) despite preserved distortion product otoacoustic emission (DPOAE). Although otoferlin-deficient mice lack neurotransmitter release at the inner hair cell (IHC) synapse, it remains unclear how the Otof mutation affects spiral ganglions. Thus, we used Otof-mutant mice carrying the Otoftm1a(KOMP)Wtsi allele (Otoftm1a) and analyzed spiral ganglion neurons (SGNs) in Otoftm1a/tm1a mice by immunolabeling type Ⅰ SGNs (SGN-Ⅰ) and type II SGNs (SGN-II). We also examined apoptotic cells in SGNs. Four-week-old Otoftm1a/tm1a mice had an absent ABR but normal DPOAEs. The number of SGNs was significantly lower in Otoftm1a/tm1a mice on postnatal day 7 (P7), P14, and P28 compared with that of wild-type mice. Moreover, significantly more apoptotic SGNs were observed in Otoftm1a/tm1a mice than in wild-type mice on P7, P14, and P28. SGN-IIs were not significantly reduced in Otoftm1a/tm1a mice on P7, P14, and P28. No apoptotic SGN-IIs were observed under our experimental conditions. In summary, Otoftm1a/tm1a mice showed a reduction in SGNs accompanied by apoptosis of SGN-Ⅰs even before the onset of hearing. We speculate that the reduction in SGNs with apoptosis is a secondary defect caused by a lack of otoferlin in IHCs. Appropriate glutamatergic synaptic inputs may be important for the survival of SGNs.

Membrane Protein OTOF Is a Type I Interferon-Induced Entry Inhibitor of HIV-1 in Macrophages.

In humans, HIV-1 infection induces innate immune responses mediated mainly by type I interferon (IFN). Type I IFN restricts HIV-1 replication by upregulating the expression of IFN-stimulated genes with diverse anti-HIV properties. In this study, we report that the cell membrane protein otoferlin (OTOF) acts as a type I IFN-induced effector, inhibiting HIV-1 entry in myeloid lineage macrophages and dendritic cells (DCs). OTOF is significantly induced by type I IFN in macrophages and DCs but not in CD4+ T lymphocytes. Silencing OTOF abrogates the IFN-mediated suppression of HIV-1 infection in macrophages and DCs. Moreover, OTOF overexpression exhibits anti-HIV activity in macrophages and CD4+ T cells. Further evidence reveals that OTOF inhibits HIV-1 entry into target cells at the cell membrane. Collectively, OTOF is a downstream molecule induced by type I IFN to inhibit HIV-1 entry in macrophages; it is a new potential agent for the treatment of HIV infection. IMPORTANCE In patients with HIV-1 infection, the virus is recognized by innate immune sensors that trigger the production of type I interferons (IFNs), which are well-known cytokines that exert broad antiviral effects by inducing the expression of antiviral genes. By comparing the gene expression profiles of untreated patients and healthy donors, we systematically identified OTOF as a new antiviral gene induced by IFN-α in primary macrophages and dendritic cells (DCs). Additionally, silencing OTOF alleviates IFN-α-induced resistance to HIV-1 infection in both myeloid cell lineage macrophages and DCs. In contrast, OTOF overexpression potently restricts HIV-1 transmission in macrophages. We further explored the molecular mechanism through which OTOF inhibits the HIV-1 virion across the cell membrane. Overall, OTOF is a newly identified type I IFN-induced antiviral factor that inhibits the transmembrane activity of HIV-1 in myeloid cells.

Generation of a Human Deafness Sheep Model Using the CRISPR/Cas System.

CRISPR/Cas9 system is a promising method for the generation of human disease models by genome editing in non-conventional experimental animals. Medium/large-sized animals like sheep have several advantages to study human diseases and medicine. Here, we present a protocol that describes the generation of an otoferlin edited sheep model via CRISPR-assisted single-stranded oligodinucleotide-mediated Homology-Directed Repair (HDR), through direct cytoplasmic microinjection in in vitro produced zygotes.Otoferlin is a protein expressed in the cochlear inner hair cells, with different mutations at the OTOF gene being the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. By using this protocol, we reported for the first time an OTOF KI model in sheep with 17.8% edited lambs showing indel mutations, and 61.5% of them bearing knock-in mutations by HDR . The reported method establishes the bases to produce a deafness model to test novel therapies in human disorders related to OTOF mutations.

Case report: Clinical and genetic analysis of a family with nonsyndromic auditory neuropathy.

Background: Auditory neuropathy (AN) is a hearing disorder caused by the failure of inner hair cells, auditory nerve synapses and/or auditory nerves. With the development of high-throughput sequencing technology, the genetic factors of AN have been revealed, and genetic testing has become an important tool for identifying different types of AN. Case description: To study the genetic cause of nonsyndromic auditory neuropathy in a Chinese family. The family was from Henan Province with three affected individuals. The audiological examinations were performed on the affected individuals, and whole-exome sequencing was carried out on the proband. The suspected pathogenic variants screened by the bioinformatic analysis were validated using Sanger sequencing in the family members. We identified three novel variants c.3277G > A (p.Glu1093Lys), c.4024-4G > T, and c.898-2A > G of the OTOF gene in the three children with AN. The first two variants were inherited from their father, and the third variant was inherited from their mother. A minigene assay was designed to test the effect of c.4024-4G > T on splicing. The variants c.3277G > A, c.4024-4G > T, and c.898-2A > G could be classified as likely pathogenic/pathogenic following the ACMG guidelines, and they are considered as the genetic causes for the patients in the family. Conclusion: New pathogenic/likely pathogenic variants of the OTOF gene were identified in a family with AN, enriching the mutational spectrum of the OTOF gene.