Targeted genome editing restores auditory function in adult mice with progressive hearing loss caused by a human microRNA mutation.

Mutations in microRNA-96 ( MIR96 ) cause dominant delayed onset hearing loss DFNA50 without treatment. Genome editing has shown efficacy in hearing recovery by intervention in neonatal mice, yet editing in the adult inner ear is necessary for clinical applications. Here, we developed an editing therapy for a C>A point mutation in the seed region of the Mir96 gene, Mir96 14C>A associated with hearing loss by screening gRNAs for genome editors and optimizing Cas9 and sgRNA scaffold for efficient and specific mutation editing in vitro. By AAV delivery in pre-symptomatic (3-week-old) and symptomatic (6-week-old) adult Mir96 14C>A mutant mice, hair cell on-target editing significantly improved hearing long-term, with an efficacy inversely correlated with injection age. We achieved transient Cas9 expression without the evidence of AAV genomic integration to significantly reduce the safety concerns associated with editing. We developed an AAV-sgmiR96-master system capable of targeting all known human MIR96 mutations. As mouse and human MIR96 sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lays the foundation for future treatment of DFNA50 caused by MIR96 mutations.

Cytokine Profiling of Cyst Fluid and Tumor-Associated Macrophages in Cystic Vestibular Schwannoma.

BACKGROUND: The vestibular schwannoma (VS) secretome can initiate monocyte recruitment and macrophage polarization to M1 (proinflammatory) and/or M2 (protumorigenic) phenotypes, which in turn secrete additional cytokines that contribute to the tumor microenvironment. Profiling cyst fluid and cerebrospinal fluid (CSF) in cystic VS provides a unique opportunity to understand mechanisms that may contribute to tumor progression and cyst formation. HYPOTHESIS: Cystic VSs secrete high levels of cytokines into cyst fluid and express abundant M1 and M2 macrophages. METHODS: Tumor, CSF, and cyst fluid were prospectively collected from 10 cystic VS patients. Eighty cytokines were measured in fluid samples using cytokine arrays and compared with normal CSF from normal donors. Immunofluorescence was performed for CD80 + M1 and CD163 + M2 macrophage markers. Demographic, audiometric, and radiographic information was obtained through retrospective chart review. RESULTS: Cyst fluid expressed more osteopontin and monocyte chemotactic protein-1 (MCP-1; p < 0.0001), when compared with normal CSF. Cyst fluid also expressed more protein ( p = 0.0020), particularly MCP-1 ( p < 0.0001), than paired CSF from the same subjects. MCP-1 expression in cyst fluid correlated with CD80 + staining in VS tissue ( r = 0.8852; p = 0.0015) but not CD163 + staining. CONCLUSION: Cyst fluid from cystic VS harbored high levels of osteopontin and MCP-1, which are cytokines important in monocyte recruitment and macrophage polarization. MCP-1 may have a significant role in molding the tumor microenvironment, by polarizing monocytes to CD80 + M1 macrophages in cystic VS. Further investigations into the role of cytokines and macrophages in VS may lead to new avenues for therapeutic intervention.

Gene regulation analysis of patient-derived iPSCs and its CRISPR-corrected control provides a new tool for studying perturbations of ELMOD3 c.512A>G mutation during the development of inherited hearing loss.

The ELMOD3 gene is implicated in causing autosomal recessive/dominant non-syndromic hearing loss in humans. However, the etiology has yet to be completely elucidated. In this study, we generated a patient-derived iPSC line carrying ELMOD3 c.512A>G mutation. In addition, the patient-derived iPSC line was corrected by CRISPR/Cas9 genome editing system. Then we applied RNA sequencing profiling to compare the patient-derived iPSC line with different controls, respectively (the healthy sibling-derived iPSCs and the CRISPR/Cas9 corrected iPSCs). Functional enrichment and PPI network analysis revealed that differentially expressed genes (DEGs) were enriched in the gene ontology, such as sensory epithelial development, intermediate filament cytoskeleton organization, and the regulation of ion transmembrane transport. Our current work provided a new tool for studying how disruption of ELMOD3 mechanistically drives hearing loss.

Polystyrene nanoplastics aggravate reproductive system damage in obese male mice by perturbation of the testis redox homeostasis.

The potential impact of the combination of a high-fat diet (HFD) and polystyrene nanoplastics (PS-NPs) on fertility cannot be ignored, especially when the fertility rate is declining. However, it has not attracted considerable attention. In this study, an obese mouse model was established using an HFD, and the reproductive function of male mice was evaluated after intragastric administration of 100 µL of a 10 mg/mL PS-NP suspension for 4 weeks. By determining the morphology and vitality of sperm and related indicators of testosterone production, it was found that PS-NPs aggravated the destruction of sperm mitochondrial structure, decrease sperm activity, and testosterone production in HFD-fed mice. To comprehensively analyze the injury mechanism, the integrity of the blood testicular barrier (BTB) and the function of Leydig and Sertoli cells were further analyzed. It was found that PS-NPs could destroy BTB, promote the degeneration of Leydig cells, reduce the number of Sertoli cells, and decrease lactate secretion in HFD-fed mice. PS-NPs further interfered with redox homeostasis in the testicular tissues of HFD-fed mice. This study found that PS-NPs could aggravate the damage to the reproductive system of obese male mice by further perturbing its redox homeostasis and revealed the potential health risk of PS-NPs exposure under an HFD.

Treatment of monogenic and digenic dominant genetic hearing loss by CRISPR-Cas9 ribonucleoprotein delivery in vivo.

Mutations in Atp2b2, an outer hair cell gene, cause dominant hearing loss in humans. Using a mouse model Atp2b2Obl/+, with a dominant hearing loss mutation (Oblivion), we show that liposome-mediated in vivo delivery of CRISPR-Cas9 ribonucleoprotein complexes leads to specific editing of the Obl allele. Large deletions encompassing the Obl locus and indels were identified as the result of editing. In vivo genome editing promotes outer hair cell survival and restores their function, leading to hearing recovery. We further show that in a double-dominant mutant mouse model, in which the Tmc1 Beethoven mutation and the Atp2b2 Oblivion mutation cause digenic genetic hearing loss, Cas9/sgRNA delivery targeting both mutations leads to partial hearing recovery. These findings suggest that liposome-RNP delivery can be used as a strategy to recover hearing with dominant mutations in OHC genes and with digenic mutations in the auditory hair cells, potentially expanding therapeutics of gene editing to treat hearing loss.

microRNA-92b-3p augments colon cancer development through inhibiting KLF3.

Colon cancer (CC) is a tumor of the large intestine. miR-92b-3p is often deregulated in the tumorigensis. Here, the role of miR-92b-3p in the development of CC was investigated. miR-92b-3p and Kruppel-like factor 3 (KLF3) expression was examined in CC tissues and cells. miR-92b-3p inhibitor or KLF3 overexpression vector was transfected into CC cells, respectively to observe its role in CC cell proliferation, invasion, migration, and apoptosis. The targeting relationship between miR-92b-3p and KLF3 was validated. Meanwhile, rescue experiments were performed by co-transfection of miR-92b-3p inhibitor and KLF3 siRNA, followed by determining CC cell proliferation, invasion, migration, and apoptosis. Higher miR-92b-3p and lower KLF3 expression levels were observed in CC tissues and cells. miR-92b-3p inhibition or KLF3 overexpression reduced proliferation, invasion, and migration whereas induced apoptosis of CC cells. KLF3 was validated to be the target gene of miR-92b-3p. Depletion of KLF3 could reverse the antitumor role of miR-92b-3p inhibition in CC cells. miR-92b-3p augments CC development through inhibiting KLF3, which may confers a novel way to develop future treatment target.

Genotype-Phenotype Correlations in TMPRSS3 (DFNB10/DFNB8) with Emphasis on Natural History.

BACKGROUND: Mutations in TMPRSS3 are an important cause of autosomal recessive non-syndromic hearing loss. The hearing loss associated with mutations in TMPRSS3 is characterized by phenotypic heterogeneity, ranging from mild to profound hearing loss, and is generally progressive. Clinical presentation and natural history of TMPRSS3 mutations vary significantly based on the location and type of mutation in the gene. Understanding these genotype-phenotype relationships and associated natural disease histories is necessary for the successful development and application of gene-based therapies and precision medicine approaches to DFNB8/10. The heterogeneous presentation of TMPRSS3-associated disease makes it difficult to identify patients clinically. As the body of literature on TMPRSS3-associated deafness grows, there is need for better categorization of the hearing phenotypes associated with specific mutations in the gene. SUMMARY: In this review, we summarize TMPRSS3 genotype-phenotype relationships including a thorough description of the natural history of patients with TMPRSS3-associated hearing loss to lay the groundwork for the future of TMPRSS3 treatment using molecular therapy. KEY MESSAGES: TMPRSS3 mutation is a significant cause of genetic hearing loss. All patients with TMPRSS3 mutation display severe-to-profound prelingual (DFNB10) or a postlingual (DFNB8) progressive sensorineural hearing loss. Importantly, TMPRSS3 mutations have not been associated with middle ear or vestibular deficits. The c.916G>A (p.Ala306Thr) missense mutation is the most frequently reported mutation across populations and should be further explored as a target for molecular therapy.

Single Fraction and Hypofractionated Radiation Cause Cochlear Damage, Hearing Loss, and Reduced Viability of Merlin-Deficient Schwann Cells.

BACKGROUND: Vestibular schwannomas (VS) are benign intracranial tumors caused by loss of function of the merlin tumor suppressor. We tested three hypotheses related to radiation, hearing loss (HL), and VS cell survival: (1) radiation causes HL by injuring auditory hair cells (AHC), (2) fractionation reduces radiation-induced HL, and (3) single fraction and equivalent appropriately dosed multi-fractions are equally effective at controlling VS growth. We investigated the effects of single fraction and hypofractionated radiation on hearing thresholds in rats, cell death pathways in rat cochleae, and viability of human merlin-deficient Schwann cells (MD-SC). METHODS: Adult rats received cochlear irradiation with single fraction (0 to 18 Gray [Gy]) or hypofractionated radiation. Auditory brainstem response (ABR) testing was performed for 24 weeks. AHC viabilities were determined using immunohistochemistry. Neonatal rat cochleae were harvested after irradiation, and gene- and cell-based assays were conducted. MD-SCs were irradiated, and viability assays and immunofluorescence for DNA damage and cell cycle markers were performed. RESULTS: Radiation caused dose-dependent and progressive HL in rats and AHC losses by promoting expression of apoptosis-associated genes and proteins. When compared to 12 Gy single fraction, hypofractionation caused smaller ABR threshold and pure tone average shifts and was more effective at reducing MD-SC viability. CONCLUSIONS: Investigations into the mechanisms of radiation ototoxicity and VS radiobiology will help determine optimal radiation regimens and identify potential therapies to mitigate radiation-induced HL and improve VS tumor control.

Rescue of auditory function by a single administration of AAV-TMPRSS3 gene therapy in aged mice of human recessive deafness DFNB8.

Patients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10. For these patients, cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knockin mouse model with a frequent human DFNB8 TMPRSS3 mutation. The Tmprss3A306T/A306T homozygous mice display delayed onset progressive hearing loss similar to human DFNB8 patients. Using AAV2 as a vector to carry a human TMPRSS3 gene, AAV2-hTMPRSS3 injection in the adult knockin mouse inner ear results in TMPRSS3 expression in the hair cells and the spiral ganglion neurons. A single AAV2-hTMPRSS3 injection in Tmprss3A306T/A306T mice of an average age of 18.5 months leads to sustained rescue of the auditory function to a level similar to wild-type mice. AAV2-hTMPRSS3 delivery rescues the hair cells and the spiral ganglions neurons. This study demonstrates successful gene therapy in an aged mouse model of human genetic deafness. It lays the foundation to develop AAV2-hTMPRSS3 gene therapy to treat DFNB8 patients, as a standalone therapy or in combination with cochlear implantation.

Puerarin Prevents Cadmium-Induced Neuronal Injury by Alleviating Autophagic Dysfunction in Rat Cerebral Cortical Neurons.

Autophagic dysfunction is one of the main mechanisms of cadmium (Cd)-induced neurotoxicity. Puerarin (Pue) is a natural antioxidant extracted from the medicinal and edible homologous plant Pueraria lobata. Studies have shown that Pue has neuroprotective effects in a variety of brain injuries, including Cd-induced neuronal injury. However, the role of Pue in the regulation of autophagy to alleviate Cd-induced injury in rat cerebral cortical neurons remains unclear. This study aimed to elucidate the protective mechanism of Pue in alleviating Cd-induced injury in rat cerebral cortical neurons by targeting autophagy. Our results showed that Pue alleviated Cd-induced injury in rat cerebral cortical neurons in vitro and in vivo. Pue activates autophagy and alleviates Cd-induced autophagic blockade in rat cerebral cortical neurons. Further studies have shown that Pue alleviates the Cd-induced inhibition of autophagosome-lysosome fusion, as well as the inhibition of lysosomal degradation. The specific mechanism is related to Pue alleviating the inhibition of Cd on the expression levels of the key proteins Rab7, VPS41, and SNAP29, which regulate autophagosome-lysosome fusion, as well as the lysosome-related proteins LAMP2, CTSB, and CTSD. In summary, these results indicate that Pue alleviates Cd-induced autophagic dysfunction in rat cerebral cortical neurons by alleviating autophagosome-lysosome fusion dysfunction and lysosomal degradation dysfunction, thereby alleviating Cd-induced neuronal injury.

Preimplantation genetic testing for hereditary hearing loss in Chinese population.

PURPOSE: To evaluate the clinical validity of preimplantation genetic testing (PGT) to prevent hereditary hearing loss (HL) in Chinese population. METHODS: A PGT procedure combining multiple annealing and looping-based amplification cycles (MALBAC) and single-nucleotide polymorphisms (SNPs) linkage analyses with a single low-depth next-generation sequencing run was implemented. Forty-three couples carried pathogenic variants in autosomal recessive non-syndromic HL genes, GJB2 and SLC26A4, and four couples carried pathogenic variants in rare HL genes: KCNQ4, PTPN11, PAX3, and USH2A were enrolled. RESULTS: Fifty-four in vitro fertilization (IVF) cycles were implemented, 340 blastocysts were cultured, and 303 (89.1%) of these received a definite diagnosis of a disease-causing variant testing, linkage analysis and chromosome screening. A clinical pregnancy of 38 implanted was achieved, and 34 babies were born with normal hearing. The live birth rate was 61.1%. CONCLUSIONS AND RELEVANCE: In both the HL population and in hearing individuals at risk of giving birth to offspring with HL in China, there is a practical need for PGT. The whole genome amplification combined with NGS can simplify the PGT process, and the efficiency of PGT process can be improved by establishing a universal SNP bank of common disease-causing gene in particular regions and nationalities. This PGT procedure was demonstrated to be effective and lead to satisfactory clinical outcomes.

Rescue of Auditory Function by a Single Administration of AAV- TMPRSS3 Gene Therapy in Aged Mice of Human Recessive Deafness DFNB8.

Patients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10 for whom cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knock-in mouse model with a frequent human DFNB8 TMPRSS3 mutation. The Tmprss3 A306T/A306T homozygous mice display delayed onset progressive hearing loss similar to human DFNB8 patients. Using AAV2 as a vector to carry a human TMPRSS3 gene, AAV2-h TMPRSS3 injection in the adult knock-in mouse inner ears results in TMPRSS3 expression in the hair cells and the spiral ganglion neurons. A single AAV2-h TMPRSS3 injection in aged Tmprss3 A306T/A306T mice leads to sustained rescue of the auditory function, to a level similar to the wildtype mice. AAV2-h TMPRSS3 delivery rescues the hair cells and the spiral ganglions. This is the first study to demonstrate successful gene therapy in an aged mouse model of human genetic deafness. This study lays the foundation to develop AAV2-h TMPRSS3 gene therapy to treat DFNB8 patients, as a standalone therapy or in combination with cochlear implantation.

Cadmium induces mitochondrial dysfunction via SIRT1 suppression-mediated oxidative stress in neuronal cells.

Cadmium is a widespread environmental contaminant and its neurotoxicity has raised serious concerns. Mitochondrial dysfunction is a key event in Cd-induced nervous system disease; however, the exact molecular mechanism involved has not been fully elucidated. Increasing evidences have shown that Sirtuin 1 (SIRT1) is the key target protein impaired in Cd-induced mitochondrial dysfunction. In this study, the role of SIRT1 in Cd-induced mitochondrial dysfunction and cell death and the underlying mechanisms were evaluated in vitro using PC12 cells and primary rat cerebral cortical neurons. The results showed that Cd exposure caused cell death by inhibiting SIRT1 expression, thus inducing oxidative stress and mitochondrial dysfunction in vitro. However, inhibition of oxidative stress by the antioxidant puerarin alleviated Cd-induced mitochondrial dysfunction. Furthermore, activation of SIRT1 using the agonist Srt1720 significantly abolished Cd-induced oxidative stress and mitochondrial dysfunction and ultimately alleviated Cd-induced neuronal cell death. Collectively, our data indicate that Cd induced mitochondrial dysfunction via SIRT1 suppression-mediated oxidative stress, leading to the death of PC12 cells and primary rat cerebral cortical neurons. These findings suggest a novel mechanism for Cd-induced neurotoxicity.

Zearalenone damages the male reproductive system of rats by destroying testicular focal adhesion.

Zearalenone (ZEA), a common mycotoxin in animal feed, is harmful to public health and causes huge economic losses. The potential target proteins of ZEA and its derivatives were screened using the PharmMapper database and the related genes (proteins) of the testis were obtained from Genecards. We obtained 144 potential targets of ZEA and its derivatives related to the testis using Venn diagrams. The PPI analysis showed that ZEA had the most targets in testis, followed by ZAN, α-ZAL, ß-ZEL, α-ZEL, and ß-ZAL. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses evaluated the metabolic and cancer pathways. We further screened four hub genes: RAC3, CCND1, EP300, and CTNNB1. Eight key biological processes were obtained by GO analysis, and four important pathways were identified by KEGG analysis. Animal and cell experimental results confirmed that ZEA could inhibit the expression of four key KEGG pathway protein components and four hub proteins that interfere with cell adhesion by inhibiting the focal adhesion structure of the testis, Leydig cells, and Sertoli cells. Collectively, our findings reveal that the destruction of the focal adhesion structure in the testis is the mechanism through which ZEA damages the male reproductive system.

A non-coding variant in 5' untranslated region drove up-regulation of pseudo-kinase EPHA10 and caused non-syndromic hearing loss in humans.

Hereditary hearing loss has a genetic and phenotypic heterogeneity. However, it is still difficult to explain this heterogeneity perfectly with known deafness genes. Here, we report a novel causative gene EPHA10 as well as its non-coding variant in 5' untranslated region identified in a family with post-lingual autosomal dominant non-syndromic hearing loss from southern China. One affected member of this family had an ideal hearing restoration after cochlear implantation. We speculated that there were probable deafness-causing abnormalities in the cochlea according to clinical imaging and auditory evaluations. A heterozygous variant c.-81_-73delinsAGC was found co-segregating with hearing loss. Epha10 was expressed in mouse cochlea at both transcription and translation levels. The variant caused upregulation of EPHA10 which may result from promoter activity enhancement after sequence change. Overexpression of Eph (the homolog of human EPHA10) exerted effects on the structure and function of chordotonal organ in fly model. In summary, our study linked pseudo-kinase EPHA10 to hearing loss in humans for the first time.

Responsiveness of the electrically stimulated cochlear nerve in patients with a missense variant in ACTG1: Preliminary Results.

This preliminary study identified a missense variant in ACTG1 (NM_001614.5) in a family with autosomal dominant non-syndromic hearing loss (ADNSHL). The responsiveness of the electrically-stimulated cochlear nerve (CN) in two implanted participants with this missense change was also evaluated and reported. Genetic testing was done using a custom capture panel (MiamiOtoGenes) and whole exome sequencing. The responsiveness of the electrically-stimulated CN was evaluated in two members of this family (G1 and G4) using the electrically evoked compound action potential (eCAP). eCAP results from these two participants were compared with those measured three implanted patient populations: children with cochlear nerve deficiency, children with idiopathic hearing loss and normal-sized cochlear nerves, and postligually deafened adults. Sequencing of ACTG1 identified a missense c.737A>T (p. Gln246Leu) variant in ACTG1 (NM_001614.5) which is most likely the genetic cause of ADNSHL in this family. eCAP results measured in these two participants showed substantial variations. The results indicated the missense c.737A>T (p. Gln246Leu) variant in ACTG1 (NM_001614.5) co-segregated with hearing loss in this family. The responsiveness of the electrically-stimulated CN can vary among patients with the same genetic variants, which suggests the importance of evaluating the functional status of the CN for individual CI patients.

Puerarin alleviates cadmium-induced rat neurocyte injury by alleviating Nrf2-mediated oxidative stress and inhibiting mitochondrial unfolded protein response.

Cadmium (Cd) is a highly neurotoxic environmental pollutant. Puerarin (Pur) is a natural antioxidant isolated from Kudzu root that exhibits a powerful neuroprotective effect. Herein, we illustrated the mechanism underlying the protective effect of Pur on Cd-induced rat neurocyte injury in an in vivo rat model as well as in vitro using PC12 cells and primary rat cerebral cortical neurons. First, the results showed that Pur alleviated Cd-induced cerebral cortical pathological damage and decreased the viability of neurocytes. Furthermore, Cd activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, which plays a negative role in Cd-induced rat neurocyte injury. In addition, Pur alleviated Cd-induced oxidative stress by enhancing antioxidant defense, reducing reactive oxygen species (ROS) accumulation and lipid peroxidation, and inhibiting activation of the Nrf2 signaling pathway in rat neurocytes. Moreover, Pur inhibited the Cd-induced mitochondrial unfolded protein response (UPRmt) in rat neurocytes. Overall, Pur alleviated Cd-induced rat neurocyte injury by alleviating Nrf2-mediated oxidative stress and inhibiting UPRmt.

PINK1/Parkin-mediated mitophagy modulates cadmium-induced apoptosis in rat cerebral cortical neurons.

Cadmium is a persistent environmental pollutant whose neurotoxicity is of serious concern. Mitochondrial dysfunction and its mediated mitophagy and apoptosis are considered key events in Cd-induced neurological pathologies, but the exact molecular mechanism has not been fully elucidated. The aim of this study was to investigate the relationship between Cd-induced mitophagy and apoptosis and their role in Cd-induced neuronal death. Using the mitophagy inhibitor cyclosporine A (CsA), we found that the extent of mitophagy mediated by the PTEN-induced putative kinase protein 1 (PINK1)/E3 ubiquitin ligase (Parkin) pathway decreased, whereas the level of apoptosis and cell death increased in rat cerebral cortical neurons in vitro. Consistent with this, the knockdown of PINK1 also exacerbated Cd-induced apoptosis and neuronal death. Furthermore, the results of the in vivo experiments showed that Cd simultaneously activated both mitophagy and apoptosis and that the suppression of mitophagy by CsA aggravated Cd-induced apoptosis. In summary, our results indicate that PINK1/Parkin-mediated mitophagy exerts an important neuroprotective effect by inhibiting Cd-mediated apoptosis in rat cerebral cortical neurons both in vitro and in vivo. This work may allow the development of new therapeutic strategies for Cd-induced central nervous system disorders.

Elucidation of repeat motifs R1- and R2-related TRIOBP variants in autosomal recessive nonsyndromic hearing loss DFNB28 among indigenous South African individuals.

BACKGROUND: DFNB28, a recessively inherited nonsyndromic form of deafness in humans, is caused by mutations in the TRIOBP gene (MIM #609761) on chromosome 22q13. Its protein TRIOBP helps to tightly bundle F-actin filaments, forming a rootlet that penetrates through the cuticular plate into the cochlear hair cell body. Repeat motifs R1 and R2, located in exon 7 of the TRIOBP-5 isoform, are the actin-binding domains. Deletion of both repeat motifs R1 and R2 results in complete disruption of both actin-binding and bundling activities, whereas deletion of the R2 motif alone retains F-actin bundling ability in stereocilia rootlets. METHODS: Target sequencing, using a custom capture panel of 180 known and candidate genes associated with sensorineural hearing loss, bioinformatics processing, and data analysis were performed. Genesis 2.0 was used for variant filtering based on quality/score read depth and minor allele frequency (MAF) thresholds of 0.005 for recessive NSHL, as reported in population-based sequencing databases. All variants were reclassified based on the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines together with other variant interpretation guidelines for genetic hearing loss . Candidate variants were confirmed via Sanger sequencing according to standard protocols, using the ABIPRISM 3730 DNA Analyzer. DNA sequence analysis was performed with DNASTAR Lasergene software. RESULTS: Candidate TRIOBP variants identified among 94 indigenous sub-Saharan African individuals were characterized through segregation analysis. Family TS005 carrying variants c.572delC, p.Pro191Argfs*50, and c.3510_3513dupTGCA, p.Pro1172Cysfs*13, demonstrated perfect cosegregation with the deafness phenotype. On the other hand, variants c.505C > A p.Asp168Glu and c.3636 T > A p.Leu1212Gln in the same family did not segregate with deafness and we have classified these variants as benign. A control family, TS067, carrying variants c.2532G > T p.Leu844Arg, c.2590C > A p.Asn867Lys, c.3484C > T p.Pro1161Leu, and c.3621 T > C p.Phe1187Leu demonstrated no cosegregation allowing us to classify these variants as benign. Together with published TRIOBP variants, the results showed that genotypes combining two truncating TRIOBP variants affecting repeat motifs R1 and R2 or R2 alone lead to a deafness phenotype, while a truncating variant affecting repeat motifs R1 and R2 or R2 alone combined with a missense variant does not. Homozygous truncating variants affecting repeat motif R2 cosegregate with the deafness phenotype. CONCLUSION: While a single intact R1 motif may be adequate for actin-binding and bundling in the stereocilia of cochlear hair cells, our findings indicate that a truncated R2 motif in cis seems to be incompatible with normal hearing, either by interfering with the function of an intact R1 motif or through another as yet unknown mechanism. Our study also suggests that most heterozygous missense variants involving exon 7 are likely to be tolerated.