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.

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.

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.

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.

Review of Genotype-Phenotype Correlations in Usher Syndrome.

Usher syndrome (USH) encompasses a group of clinically and genetically heterogenous disorders defined by the triad of sensorineural hearing loss (SNHL), vestibular dysfunction, and vision loss. USH is the most common cause of deaf blindness. USH is divided clinically into three subtypes-USH1, USH2, and USH3-based on symptom severity, progression, and age of onset. The underlying genetics of these USH forms are, however, significantly more complex, with over a dozen genes linked to the three primary clinical subtypes and other atypical USH phenotypes. Several of these genes are associated with other deaf-blindness syndromes that share significant clinical overlap with USH, pointing to the limits of a clinically based classification system. The genotype-phenotype relationships among USH forms also may vary significantly based on the location and type of mutation in the gene of interest. 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 USH. Currently, the state of knowledge varies widely depending on the gene of interest. Recent studies utilizing next-generation sequencing technology have expanded the list of known pathogenic mutations in USH genes, identified new genes associated with USH-like phenotypes, and proposed algorithms to predict the phenotypic effects of specific categories of allelic variants. Further work is required to validate USH gene causality, and better define USH genotype-phenotype relationships and disease natural histories-particularly for rare mutations-to lay the groundwork for the future of USH treatment.

Screening Strategies for Deafness Genes and Functional Outcomes in Cochlear Implant Patients.

OBJECTIVES: To review the current state of knowledge about the influence of specific genetic mutations that cause sensorineural hearing loss (SNHL) on cochlear implant (CI) functional outcomes, and how this knowledge may be integrated into clinical practice. A multistep and sequential population-based genetic algorithm suitable for the identification of congenital SNHL mutations before CI placement is also examined. DATA SOURCES, STUDY SELECTION: A review was performed of the English literature from 2000 to 2019 using PubMed regarding the influence of specific mutations on CI outcomes and the use of next-generation sequencing for genetic screening of CI patients. CONCLUSION: CI is an effective habilitation option for patients with severe-profound congenital SNHL. However, it is well known that CI outcomes show substantial inter-patient variation. Recent advances in genetic studies have improved our understanding of genotype-phenotype relationships for many of the mutations underlying congenital SNHL, and have explored how these relationships may account for some of the variance seen in CI performance outcomes. A sequential genetic screening strategy utilizing next-generation sequencing-based population-specific gene panels may allow for more efficient mutation identification before CI placement. Understanding the relationships between specific mutations and CI outcomes along with integrating routine comprehensive genetic testing into pre-CI evaluations will allow for more effective patient counseling and open the door for the development of mutation-specific treatment strategies.

Spectrum of MYO7A Mutations in an Indigenous South African Population Further Elucidates the Nonsyndromic Autosomal Recessive Phenotype of DFNB2 to Include Both Homozygous and Compound Heterozygous Mutations.

MYO7A gene encodes unconventional myosin VIIA, which, when mutated, causes a phenotypic spectrum ranging from recessive hearing loss DFNB2 to deaf-blindness, Usher Type 1B (USH1B). MYO7A mutations are reported in nine DFNB2 families to date, none from sub-Saharan Africa.In DNA, from a cohort of 94 individuals representing 92 families from the Limpopo province of South Africa, eight MYO7A variations were detected among 10 individuals. Family studies identified homozygous and compound heterozygous mutations in 17 individuals out of 32 available family members. Four mutations were novel, p.Gly329Asp, p.Arg373His, p.Tyr1780Ser, and p.Pro2126Leufs*5. Two variations, p.Ser617Pro and p.Thr381Met, previously listed as of uncertain significance (ClinVar), were confirmed to be pathogenic. The identified mutations are predicted to interfere with the conformational properties of myosin VIIA through interruption or abrogation of multiple interactions between the mutant and neighbouring residues. Specifically, p.Pro2126Leufs*5, is predicted to abolish the critical site for the interactions between the tail and the motor domain essential for the autoregulation, leaving a non-functional, unregulated protein that causes hearing loss. We have identified MYO7A as a possible key deafness gene among indigenous sub-Saharan Africans. The spectrum of MYO7A mutations in this South African population points to DFNB2 as a specific entity that may occur in a homozygous or in a compound heterozygous state.

Genetics and the Individualized Therapy of Vestibular Disorders.

Background: Vestibular disorders (VDs) are a clinically divergent group of conditions that stem from pathology at the level of the inner ear, vestibulocochlear nerve, or central vestibular pathway. No etiology can be identified in the majority of patients with VDs. Relatively few families have been reported with VD, and so far, no causative genes have been identified despite the fact that more than 100 genes have been identified for inherited hearing loss. Inherited VDs, similar to deafness, are genetically heterogeneous and follow Mendelian inheritance patterns with all modes of transmission, as well as multifactorial inheritance. With advances in genetic sequencing, evidence of familial clustering in VD has begun to highlight the genetic causes of these disorders, potentially opening up new avenues of treatment, particularly in Meniere's disease and disorders with comorbid hearing loss, such as Usher syndrome. In this review, we aim to present recent findings on the genetics of VDs, review the role of genetic sequencing tools, and explore the potential for individualized medicine in the treatment of these disorders. Methods: A search of the PubMed database was performed for English language studies relevant to the genetic basis of and therapies for vestibular disorders, using search terms including but not limited to: "genetics," "genomics," "vestibular disorders," "hearing loss with vestibular dysfunction," "individualized medicine," "genome-wide association studies," "precision medicine," and "Meniere's syndrome." Results: Increasing numbers of studies on vestibular disorder genetics have been published in recent years. Next-generation sequencing and new genetic tools are being utilized to unearth the significance of the genomic findings in terms of understanding disease etiology and clinical utility, with growing research interest being shown for individualized gene therapy for some disorders. Conclusions: The genetic knowledge base for vestibular disorders is still in its infancy. Identifying the genetic causes of balance problems is imperative in our understanding of the biology of normal function of the vestibule and the disease etiology and process. There is an increasing effort to use new and efficient genetic sequencing tools to discover the genetic causes for these diseases, leading to the hope for precise and personalized treatment for these patients.

Electrophysiology and genetic testing in the precision medicine of congenital deafness: A review.

BACKGROUND: Congenital hearing loss is remarkably heterogeneous, with over 130 deafness genes and thousands of variants, making for innumerable genotype/phenotype combinations. Understanding both the pathophysiology of hearing loss and molecular site of lesion along the auditory pathway permits for significantly individualized counseling. Electrophysiologic techniques such as electrocochleography (ECochG) and electrically-evoked compound action potentials (eCAP) are being studied to localize pathology and estimate residual cochlear vs. neural health. This review describes the expanding roles of genetic and electrophysiologic evaluation in the precision medicine of congenital hearing loss.The basics of genetic mutations in hearing loss and electrophysiologic testing (ECochG and eCAP) are reviewed, and how they complement each other in the diagnostics and prognostication of hearing outcomes. Used together, these measures improve the understanding of insults to the auditory system, allowing for individualized counseling for CI candidacy/outcomes or other habilitation strategies. CONCLUSION: Despite tremendous discovery in deafness genes, the effects of individual genes on neural function remain poorly understood. Bridging the understanding between molecular genotype and neural and functional phenotype is paramount to interpreting genetic results in clinical practice. The future hearing healthcare provider must consolidate an ever-increasing amount of genetic and phenotypic information in the precision medicine of hearing loss.

Angioimmunoblastic T-cell Lymphoma: A Mimic for Lupus.

New-onset systemic lupus erythematosus (SLE) is uncommon in elderly patients. We report the case of a 71-year-old woman who was diagnosed with SLE based on clinical manifestations of fever, alopecia, bicytopenia, hepatomegaly, lymphadenopathy, glomerulonephritis, positive antinuclear antibody (ANA) and anti-double stranded DNA (anti-dsDNA) antibody. Renal biopsy was consistent with lupus nephritis and excision biopsy of a right inguinal lymph node was initially reported as having features of reactive hyperplasia. However, a more careful review of the lymph node biopsy subsequently confirmed a concurrent angioimmunoblastic T-cell lymphoma. This case illustrates the importance of investigating secondary causes and possible alternative diagnoses in patients who present with atypical features of connective tissue disease, and the challenges in diagnosing a rare form of lymphoma. LEARNING POINTS: A thorough work-up for secondary causes and careful evaluation to exclude possible alternative diagnoses is important in cases of elderly-onset lupus.The disease presentations of lupus and haematological malignancies such as lymphoma may mimic each other and differentiation between the two can be clinically challenging; lupus can be associated with cytopenias, hepatomegaly and lymphadenopathy, but the degree of severity and the context of the clinical presentation need to be considered carefully before attributing these features to it.As some lymphomas are rare and difficult to diagnose, if there is a high clinical suspicion despite negative histological studies, discussion with the pathologist is important and a review of histology should be sought.

Screening Consanguineous Families for Hearing Loss Using the MiamiOtoGenes Panel.

Background: Hearing loss (HL) is one of the most common and genetically heterogeneous sensory disorders in humans. Genetic causes underlie 50-60% of all HL and the majority of these cases exhibit an autosomal recessive model of inheritance. Methods: In our study, we used our targeted custom MiamiOtoGenes panel of 180 HL-associated genes to screen 23 unrelated consanguineous Iranian families with at least two affected children to identify potential causal variants for HL. Results: We identified pathogenic variants in seven genes (MYO7A, CDH23, GIPC3, USH1C, CAPB2, LOXHD1, and STRC) in nine unrelated families with varying HL profiles. These include five reported and four novel mutations. Conclusion: For small consanguineous families that were unsuitable for conventional linkage analysis the employment of the MiamiOtoGenes panel helped identify the genetic cause of HL in a cost-effective and timely manner. This rapid methodology provides for diagnoses of a significant fraction of HL patients, and identifies those who will need more extensive genetic analyses such as whole exome/genome sequencing.

Recent advancements in understanding the role of epigenetics in the auditory system.

Sensorineural deafness in mammals is most commonly caused by damage to inner ear sensory epithelia, or hair cells, and can be attributed to genetic and environmental causes. After undergoing trauma, many non-mammalian organisms, including reptiles, birds, and zebrafish, are capable of regenerating damaged hair cells. Mammals, however, are not capable of regenerating damaged inner ear sensory epithelia, so that hair cell damage is permanent and can lead to hearing loss. The field of epigenetics, which is the study of various phenotypic changes caused by modification of genetic expression rather than alteration of DNA sequence, has seen numerous developments in uncovering biological mechanisms of gene expression and creating various medical treatments. However, there is a lack of information on the precise contribution of epigenetic modifications in the auditory system, specifically regarding their correlation with development of inner ear (cochlea) and consequent hearing impairment. Current studies have suggested that epigenetic modifications influence differentiation, development, and protection of auditory hair cells in cochlea, and can lead to hair cell degeneration. The objective of this article is to review the existing literature and discuss the advancements made in understanding epigenetic modifications of inner ear sensory epithelial cells. The analysis of the emerging epigenetic mechanisms related to inner ear sensory epithelial cells development, differentiation, protection, and regeneration will pave the way to develop novel therapeutic strategies for hearing loss.

Diagnostic and therapeutic applications of genomic medicine in progressive, late-onset, nonsyndromic sensorineural hearing loss.

The progressive, late-onset, nonsyndromic, sensorineural hearing loss (PNSHL) is the most common cause of sensory impairment globally, with presbycusis affecting greater than a third of individuals over the age of 65. The etiology underlying PNSHL include presbycusis, noise-induced hearing loss, drug ototoxicity, and delayed-onset autosomal dominant hearing loss (AD PNSHL). The objective of this article is to discuss the potential diagnostic and therapeutic applications of genomic medicine in PNSHL. Genomic factors contribute greatly to PNSHL. The heritability of presbycusis ranges from 25 to 75%. Current therapies for PNSHL range from sound amplification to cochlear implantation (CI). PNSHL is an excellent candidate for genomic medicine approaches as it is common, has well-described pathophysiology, has a wide time window for treatment, and is amenable to local gene therapy by currently utilized procedural approaches. AD PNSHL is especially suited to genomic medicine approaches that can disrupt the expression of an aberrant protein product. Gene therapy is emerging as a potential therapeutic strategy for the treatment of PNSHL. Viral gene delivery approaches have demonstrated promising results in human clinical trials for two inherited causes of blindness and are being used for PNSHL in animal models and a human trial. Non-viral gene therapy approaches are useful in situations where a transient biologic effect is needed or for delivery of genome editing reagents (such as CRISPR/Cas9) into the inner ear. Many gene therapy modalities that have proven efficacious in animal trials have potential to delay or prevent PNSHL in humans. The development of new treatment modalities for PNSHL will lead to improved quality of life of many affected individuals and their families.

Genomics, Epigenetics, and Hearing Loss in Neurofibromatosis Type 2.

OBJECTIVES: In this review, we discuss current knowledge about the genetics and epigenetics of vestibular schwannoma (VS) in relation to hearing loss. A multistep and sequential genetic algorithm suitable for the identification of Neurofibromatosis Type 2 (NF2) constitutional and somatic mutations is discussed. DATA SOURCES, STUDY SELECTION: A review was performed of the English literature from 1990 to 2019 using PubMed regarding genetics and epigenetics of vestibular schwannoma and NF2. CONCLUSION: NF2 is a genetic disorder characterized by NF2 mutations that affect the function of a tumor suppressor called merlin. In particular, individuals with NF2 develop bilateral VS that can lead to hearing loss and even deafness. Recent advances in genetic and epigenetic studies have improved our understanding of the genotype-phenotype relationships that affect hearing in NF2 patients. Specific constitutional NF2 mutations including particular truncating, deletion, and missense mutations have been associated with poorer hearing outcomes and more severe clinical manifestations. Epigenetic events, such as DNA methylation and histone modifications, also contribute to the development and progression of hearing loss in NF2 patients. Furthermore, the accumulation of multiple NF2 and non-NF2 genetic and epigenetic abnormalities at the level of the tumor may contribute to worse hearing outcomes. Understanding genetic and epigenetic signatures in individual NF2 patients and particularly in each VS will allow us to develop novel gene therapies and precision medicine algorithms to preserve hearing in NF2 individuals.

Transcriptomic Analyses of Inner Ear Sensory Epithelia in Zebrafish.

Analysis of gene expression has the potential to assist in the understanding of multiple cellular processes including proliferation, cell-fate specification, senesence, and activity in both healthy and disease states. Zebrafish model has been increasingly used to understand the process of hearing and the development of the vertebrate auditory system. Within the zebrafish inner ear, there are three otolith organs, each containing a sensory macula of hair cells. The saccular macula is primarily involved in hearing, the utricular macula is primarily involved in balance and the function of the lagenar macula is not completely understood. The goal of this study is to understand the transcriptional differences in the sensory macula associated with different otolith organs with the intention of understanding the genetic mechanisms responsible for the distinct role each organ plays in sensory perception. The sensory maculae of the saccule, utricle, and lagena were dissected out of adult Et(krt4:GFP)sqet4 zebrafish expressing green fluorescent protein in hair cells for transcriptional analysis. The total RNAs of the maculae were isolated and analyzed by RNA GeneChip microarray. Several of the differentially expressed genes are known to be involved in deafness, otolith development and balance. Gene expression among these otolith organs was very well conserved with less than 10% of genes showing differential expression. Data from this study will help to elucidate which genes are involved in hearing and balance. Furthermore, the findings of this study will assist in the development of the zebrafish model for human hearing and balance disorders. Anat Rec, 303:527-543, 2020. © 2019 American Association for Anatomy.

Zebrafish Model for Nonsyndromic X-Linked Sensorineural Deafness, DFNX1.

Hereditary deafness is often a neurosensory disorder and affects the quality of life of humans. Only three X-linked genes (POU class 3 homeobox 4 (POU3F4), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), and small muscle protein X-linked (SMPX)) are known to be involved in nonsyndromic hearing loss. Four PRPS1 missense mutations have been found to associate with X-linked nonsyndromic sensorineural deafness (DFNX1/DFN2) in humans. However, a causative relationship between PRPS1 mutations and hearing loss in humans has not been well studied in any animal model. Phosphoribosyl pyrophosphate synthetase 1 (PRS-I) is highly conserved in vertebrate taxa. In this study, we used the zebrafish as a model to investigate the auditory role of zebrafish orthologs (prps1a and prps1b) of the human PRPS1 gene with whole mount in situ hybridization, reverse transcription polymerase chain reaction, phenotypic screening, confocal imaging, and electrophysiological methods. We found that both prps1a and prps1b genes were expressed in the inner ear of zebrafish. Splice-blocking antisense morpholino oligonucleotides (MO1 and MO2) caused exon-2 skip and intron-2 retention of prps1a and exon-2 skip and intron-1 retention of prps1b to knock down functions of the genes, respectively. MO1 and MO2 morphants had smaller otic vesicles and otoliths, fewer inner ear hair cells, and lower microphonic response amplitude and sensitivity than control zebrafish. Therefore, knockdown of either prps1a or prps1b resulted in significant sensorineural hearing loss in zebrafish. We conclude that the prps1 genes are essential for hearing in zebrafish, which has the potential to help us understand the biology of human deafness DFNX1/DFN2. Anat Rec, 303:544-555, 2020. © 2019 American Association for Anatomy.

The Generation of Zebrafish Mariner Model Using the CRISPR/Cas9 System.

Targeted genome editing mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has emerged as a powerful tool for gene function studies and has great potential for gene therapy. Although CRISPR/Cas9 has been widely used in many research fields, only a few successful zebrafish models have been established using this technology in hearing research. In this study, we successfully created zebrafish mariner mutants by targeting the motor head domain of Myo7aa using CRISPR/Cas9. The CRISPR/Cas9-generated mutants showed unbalanced swimming behavior and disorganized sterocilia of inner ear hair cells, which resemble the phenotype of the zebrafish mariner mutants. In addition, we found that CRISPR/Cas9-generated mutants have reduced number of stereociliary bundles of inner ear hair cells and have significant hearing loss. Furthermore, phenotypic analysis was performed on F0 larvae within the first week post fertilization, which dramatically shortens data collection period. Therefore, results of this study showed that CRISPR/Cas9 is a quick and effective method to generate zebrafish mutants as a model for studying human genetic deafness. Anat Rec, 303:556-562, 2020. © 2019 American Association for Anatomy.

Progressive Dominant Hearing Loss (Autosomal Dominant Deafness-41) and P2RX2 Gene Mutations: A Phenotype-Genotype Study.

OBJECTIVES/HYPOTHESIS: P2RX2 encoding P2X purinoreceptor 2 has been identified as the gene responsible for autosomal dominant deafness-41 (DFNA41) as well as mediating vulnerability to noise-induced hearing loss (NIHL). The objective of this study was to investigate the audiological and molecular characteristics of P2RX2-related deafness, with emphasis on its role in NIHL by determining the audiological characteristics of a previously reported six-generation DFNA41 family with a 10-year follow-up. We have also summarized phenotype-genotype correlations of P2RX2-related deafness in human and mouse models. STUDY DESIGN: We describe clinical longitudinal follow-up in the DFNA41 family with P2RX2 (p.Val60Leu) mutation and perform a systematic literature search in PubMed and poster presentations on meeting/conference websites to identify current insights into P2RX2-mediated NIHL. METHODS: Clinical and physical examinations of the family members were performed, and audiograms were obtained to assess the hearing thresholds. Clinical follow-up features in this DFNA41 family are presented along with correlation analyses of phenotype-genotype in all reported families with P2RX2-related deafness. RESULTS: Progressive hearing impairment was confirmed by history and by audiological follow-up testing in all the patients. The onset of hearing loss was between age 25 and 35 years. All affected subjects had bilateral sensorineural hearing loss involving all frequencies with some significant gender differences. CONCLUSIONS: Our study and the review of the literature suggest that P2RX2 plays a crucial role in predisposition to noise-induced and age-related hearing loss. A better knowledge about the P2RX2-associated genetic variants can help in developing novel therapeutic strategies. LEVEL OF EVIDENCE: 2b Laryngoscope, 130:1657-1663, 2020.

Recent Perspectives on Gene-Microbe Interactions Determining Predisposition to Otitis Media.

A comprehensive understanding about the pathogenesis of otitis media (OM), one of the most common pediatric diseases, has the potential to alleviate a substantial disease burden across the globe. Advancements in genetic and bioinformatic detection methods, as well as a growing interest in the microbiome, has enhanced the capability of researchers to investigate the interplay between host genes, host microbiome, invading bacteria, and resulting OM susceptibility. Early studies deciphering the role of genetics in OM susceptibility assessed the heritability of the phenotype in twin and triplet studies, followed by linkage studies, candidate gene approaches, and genome-wide association studies that have helped in the identification of specific loci. With the advancements in techniques, various chromosomal regions and genes such as FBXO11, TGIF1, FUT2, FNDC1, and others have been implicated in predisposition to OM, yet questions still remain as to whether these implicated genes truly play a causative role in OM and to what extent. Meanwhile, 16S ribosomal RNA (rRNA) sequencing, microbial quantitative trait loci (mbQTL), and microbial genome-wide association studies (mGWAS) have mapped the microbiome of upper airways sites and therefore helped in enabling a more detailed study of interactions between host polymorphisms and host microbiome composition. Variants of specific genes conferring increased OM susceptibility, such as A2ML1, have also been shown to influence the microbial composition of the outer and middle ear in patients with OM, suggesting their role as mediators of disease. These interactions appear to impact the colonization of known otopathogens (Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis), as well as Neisseria, Gemella, Porphyromonas, Alloprevotella, and Fusobacterium populations that have also been implicated in OM pathogenesis. Meanwhile, studies demonstrating an increased abundance of Dolosigranulum and Corynebacterium in healthy patients compared to those with OM suggest a protective role for these bacteria, thereby introducing potential avenues for future probiotic treatment. Incorporating insights from these genetic, microbiome, and host-pathogen studies will allow for a more robust, comprehensive understanding of OM pathogenesis that can ultimately facilitate in the development of exciting new treatment modalities.