Detection and Functional Verification of Noncanonical Splice Site Mutations in Hereditary Deafness

Splice site mutations contribute to a significant portion of the genetic causes for mendelian disorders including deafness. By next-generation sequencing of 4 multiplex, autosomal dominant families and 2 simplex, autosomal recessive families with hereditary deafness, we identified a variety of candidate pathogenic variants in noncanonical splice sites of known deafness genes, which include c.1616+3A > T and c.580G > A in EYA4, c.322-57_322-8del in PAX3, c.991-15_991-13del in DFNA5, c.6087-3T > G in PTPRQ and c.164+5G > A in USH1G. All six variants were predicted to affect the RNA splicing by at least one of the computational tools Human Splicing Finder, NNSPLICE and NetGene2. Phenotypic segregation of the variants was confirmed in all families and is consistent with previously reported genotype-phenotype correlations of the corresponding genes. Minigene analysis showed that those splicing site variants likely have various negative impact including exon-skipping (c.1616+3A > T and c.580G > A in EYA4, c.991-15_991-13del in DFNA5), intron retention (c.322-57_322-8del in PAX3), exon skipping and intron retention (c.6087-3T > G in PTPRQ) and shortening of exon (c.164+5G > A in USH1G). Our study showed that the cryptic, noncanonical splice site mutations may play an important role in the molecular etiology of hereditary deafness, whose diagnosis can be facilitated by modified filtering criteria for the next-generation sequencing data, functional verification, as well as segregation, bioinformatics, and genotype-phenotype correlation analysis.

Brain auditory evoked response test, the standard method for the diagnosis of the hereditary deafness in dogs

Hearing deficiency is one of the most common hearing impairments that affect humans and other mammalian alike. Hearing loss is not painful or a life-threatening change but can endanger the patient by taking into account a large number of breeds predisposed to hereditary deafness, this short communication aims to synthesize the steps and the method for BAER test. For the affected breeds, the BAER test is recommended starting at the age of two months

Agent-based modeling of DFNB1A prevalence with regard to intensity of selection pressure in isolated human population: will cochlear implantation increase the cases of hereditary deafness?

It was evidenced, that the increase in the prevalence of autosomal recessive deafness 1A (DFNB1A) in populations of European descent was promoted by assortative marriages among deaf people. Assortative marriages become possible with a widespread introduction of sign language resulting in increased the genetic fitness of deaf individuals, thus relaxing selection against deafness. Currently, cochlear implantation is becoming a common method of rehabilitation for deaf patients, restoring their hearing ability and promoting the acquirement of spoken language. Whether the mass cochlear implantation could affect the spread of hereditary deafness is unknown. We have developed an agent-based computer model for analysis of the spread of DFNB1A. Using the model, we tested impact of different intensity of selection pressure on an isolated human population for 400 years. The modeling of the "purifying" selection pressure on deafness resulted in decrease of the proportion of deaf individuals and the pathogenic allele frequency. The modeling of relaxed selection resulted in increase of the proportion of deaf individuals and the decrease of the pathogenic allele frequency. The results of neutral selection pressure modeling showed no significant changes in both the proportion of deaf individuals and the pathogenic allele frequency after 400 years. Thus, initially low genetic fitness of deaf people can be significantly increased in the presence of assortative mating by deafness, resulting in a higher prevalence of DFNB1A. Contrary, frequency of pathogenic allele and the incidence of hereditary hearing loss will not increase in a population where all deaf individuals undergo cochlear implantation.

Deafness-in-a-dish: modeling hereditary deafness with inner ear organoids

Sensorineural hearing loss (SNHL) is a major cause of functional disability in both the developed and developing world. While hearing aids and cochlear implants provide significant benefit to many with SNHL, neither targets the cellular and molecular dysfunction that ultimately underlies SNHL. The successful development of more targeted approaches, such as growth factor, stem cell, and gene therapies, will require a yet deeper understanding of the underlying molecular mechanisms of human hearing and deafness. Unfortunately, the human inner ear cannot be biopsied without causing significant, irreversible damage to the hearing or balance organ. Thus, much of our current understanding of the cellular and molecular biology of human deafness, and of the human auditory system more broadly, has been inferred from observational and experimental studies in animal models, each of which has its own advantages and limitations. In 2013, researchers described a protocol for the generation of inner ear organoids from pluripotent stem cells (PSCs), which could serve as scalable, high-fidelity alternatives to animal models. Here, we discuss the advantages and limitations of conventional models of the human auditory system, describe the generation and characteristics of PSC-derived inner ear organoids, and discuss several strategies and recent attempts to model hereditary deafness in vitro. Finally, we suggest and discuss several focus areas for the further, intensive characterization of inner ear organoids and discuss the translational applications of these novel models of the human inner ear.

Single-molecule force spectroscopy reveals the dynamic strength of the hair-cell tip-link connection

The conversion of auditory and vestibular stimuli into electrical signals is initiated by force transmitted to a mechanotransduction channel through the tip link, a double stranded protein filament held together by two adhesion bonds in the middle. Although thought to form a relatively static structure, the dynamics of the tip-link connection has not been measured. Here, we biophysically characterize the strength of the tip-link connection at single-molecule resolution. We show that a single tip-link bond is more mechanically stable relative to classic cadherins, and our data indicate that the double stranded tip-link connection is stabilized by single strand rebinding facilitated by strong cis-dimerization domains. The measured lifetime of seconds suggests the tip-link is far more dynamic than previously thought. We also show how Ca2+ alters tip-link lifetime through elastic modulation and reveal the mechanical phenotype of a hereditary deafness mutation. Together, these data show how the tip link is likely to function during mechanical stimuli.

Local Macrophage-Related Immune Response Is Involved in Cochlear Epithelial Damage in Distinct Gjb2-Related Hereditary Deafness Models

The macrophage-related immune response is an important component of the cochlear response to different exogenous stresses, including noise, ototoxic antibiotics, toxins, or viral infection. However, the role of the immune response in hereditary deafness caused by genetic mutations is rarely explored. GJB2, encoding connexin 26 (Cx26), is the most common deafness gene of hereditary deafness. In this study, two distinct Cx26-null mouse models were established to investigate the types and underlying mechanisms of immune responses. In a systemic Cx26-null model, macrophage recruitment was observed, associated with extensive cell degeneration of the cochlear epithelium. In a targeted-cell Cx26-null model, knockout of Cx26 was restricted to specific supporting cells (SCs), which led to preferential loss of local outer hair cells (OHCs). This local OHC loss can also induce a macrophage-related immune response. Common inflammatory factors, including TNF-α, IL-1β, Icam-1, Mif, Cx3cr1, Tlr4, Ccl2, and Ccr2, did not change significantly, while mRNA of Cx3cl1 was upregulated. Quantitative immunofluorescence showed that the protein expression of CX3CL1 in Deiters cells, a type of SC coupled with OHCs, increased significantly after OHC death. OHC loss caused the secondary death of spiral ganglion neurons (SGNs), while the remaining SGNs expressed high levels of CX3CL1 with infiltrated macrophages. Taken together, our results indicate that CX3CL1 signaling regulates macrophage recruitment and that enhancement of macrophage antigen-presenting function is associated with cell degeneration in Cx26-null mice.

Molecular etiology of hereditary deafness using next generation sequencing in northwest China

Objective: To analyze the molecular etiology of 92 hereditary deafness families and explore the genetic mechanism of newly identified genes in deafness heritability. Methods: We analyzed the medical history, audiology, imaging, and physical examination data of 92 probands and their family members. Probands were selected from the hereditary deafness database; they did not have any of the common genetic mutation sites. Genomic DNA was extracted from blood samples and next generation sequencing was performed on an Illumina platform, followed by co-segregation analysis of family members. The control group included the clinical data and blood samples from 207 normal hearing people. Results: Among the 92 samples, 30 homozygous variants were identified in 29 autosomal recessive hereditary deafness families, including 6 reported mutations and 26 novel mutations. Among them, MYO15A was the most frequently detected (6/92), followed by mutations in CDH23, OTOF, FGF3 (3/92 each), MYO7A, SLC26A4, MYO6 (2/92 each), BSND, CLDN14, DFNB59, ILDR1, LHFPL5, LRTOMT, TMPRSS3, TPRN, USH1C, and LOXHD1 (1/92 each). Conclusion: In patients with autosomal recessive deafness, the MYO15A, CDH23, OTOF, and FGF3 genes could be used as candidate genes for conventional genetic studies in northwest China.