Comprehensive molecular analysis of 61 Egyptian families with hereditary nonsyndromic hearing loss.

Nonsyndromic hearing loss is an extremely heterogeneous disorder. Thus, clinical diagnostics is challenging, in particular due to differences in the etiology of hearing loss between populations. With this study, we wanted to elucidate the genetic basis of hearing loss in 61 consanguineous Egyptian families. In 25 families, linkage analysis was used as a prescreening to identify regions for targeted sequencing of candidate genes. Initially, the coding regions of 12 and later of 94 genes associated with hearing loss were enriched and subjected to massively parallel sequencing (MPS) with diagnostic yields of 36% and 75%, respectively. Causative variants were identified in 48 families (79%). They were found in 23 different genes with the majority being located in MYO15A (15.3%), SLC26A4 (9.7%), GJB2 (8.3%), and MYO7A (6.4%). As many as 32 variants were novel ones at the time of detection. Five variants were shared by two, three, or even four families. Our study provides a first survey of the mutational spectrum of deaf patients in Egypt revealing less GJB2 variants than in many European populations. It underlines the value of targeted enrichment of well-selected deafness genes in combination with MPS in the diagnostics of this frequent and genetically heterogeneous disorder.

Frequency of mitochondrial m.1555A > G mutation in Syrian patients with non-syndromic hearing impairment.

BACKGROUND: Mitochondrial maternally inherited hearing impairment (HI) appears to be increasing in frequency. The incidence of mitochondrial defects causing HI is estimated to be between 6 and 33% of all hearing deficiencies. Mitochondrial m.1555A > G mutation is the first mtDNA mutation associated with non-syndromic sensorineural deafness and also with aminoglycoside induced HI. Its prevalence varied geographically between different populations. METHODS: We carried out PCR, restriction enzyme based screening, and sequencing of 337 subjects (including 132 patients diagnosed clinically with hereditary deafness) from 54 families from Syria for m.1555A > G mitochondrial mutation. RESULTS: Mitochondrial m.1555A > G mutation was detected in one of fifty-four families (1.85%), six out of the 132 (4.5%) of all patients with NSHI and one propositus of the 205 individuals with normal hearing (0.48%). CONCLUSION: This is the first study to report prelingual deafness causative gene mutations identified by sequencing technology in Syrian families. It is obvious from the results that the testing for the m.1555A > G mutation is useful for diagnosis of hearing loss in Syrian patients and should also be considered prior to treatment with aminoglycosides in predisposed individuals.

Loss of mammal-specific tectorial membrane component carcinoembryonic antigen cell adhesion molecule 16 (CEACAM16) leads to hearing impairment at low and high frequencies.

The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16(-/-) mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16(-/-) mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and ß-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range.

Generation of somatic electromechanical force by outer hair cells may be influenced by prestin-CASK interaction at the basal junction with the Deiter's cell.

The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism. This observation suggests that cochlear amplification is critically dependent on the surface expression and distribution of prestin. Searching for proteins involved in organizing the subcellular localization of prestin to the basolateral plasma membrane, we identified cochlear expression of a novel truncated prestin splice isoform named prestin 9b (Slc26A5d) that contains a putative PDZ domain-binding motif. Using prestin 9b as the bait in a yeast two-hybrid assay, we identified a calcium/calmodulin-dependent serine protein kinase (CASK) as an interaction partner of prestin. Co-immunoprecipitation assays showed that CASK and prestin 9b can interact with full-length prestin. CASK was co-localized with prestin in a membrane domain where prestin-expressing OHC membrane abuts prestin-free OHC membrane, but was absent from this area for thyroid hormone deficiency. These findings suggest that CASK and the truncated prestin splice isoform contribute to confinement of prestin to the basolateral region of the plasma membrane. By means of such an interaction, the basal junction region between the OHC and its Deiter's cell may contribute to efficient generation of somatic electromechanical force.

Ergic2, a brain specific interacting partner of Otoferlin.

BACKGROUND: Otoferlin, a postulated calcium sensor of 230 kDa, was proposed to trigger calcium dependent fusion of vesicles with plasma membrane in the ribbon synapses of cochlear IHCs. Otoferlin's interaction with Rab8b and Myo6, proteins involved in the intracellular membrane trafficking, extended the previous hypothesis assigning Otoferlin an additional role in trans-Golgi trafficking. Here, we present another Otoferlin binding partner, Ergic2, a protein with a still unknown function but presenting sequence homology to other proteins involved in ER/Golgi vesicle trafficking. METHODS: Novel binding partners of Otoferlin were searched by yeast two-hybrid screening in a rodent cochlear cDNA library (P3-P15). RT-PCR, western blot, immunohistochemistry staining and co-immunoprecipitation were applied to analyze and confirm an interaction between Ergic2 and Otoferlin. RESULTS: The Y-2-H screening, using baits covering parts of Otoferlin's C2D domain, identified Ergic2 as an interacting protein for Otoferlin. Both are co-expressed (mRNA and protein level) in rodent cochlea and brain before- and after-onset of hearing. By RT-PCR Ergic2 was detected in cochlear IHCs and OHCs and in brain regions where Otoferlin is known to be present. Co-localization studies revealed an overlap of Ergic2 and Otoferlin signals in IHCs and neurons of cerebral cortical layer I making Ergic2 the promising binding candidate. However, while Ergic2 was co-precipitated by an anti-Otoferlin antibody in protein lysates from murine brain, this specific protein interaction was not detected in cochlea. CONCLUSION: Our new data on Otoferlin's interactome suggest that Otoferlin can form different, tissue-specific protein complexes.

Temporal expression pattern of Fkbp8 in rodent cochlea.

BACKGROUND: FKBP8 is a multifunctional protein involved in many distinct processes like formation of central nervous system, viral RNA replication and inhibition of apoptosis. Fkbp8 expression was reported in different tissues, various cell lines and malignancies, in the latter displaying changes during carcinogenesis. Loss of Fkbp8 leads to substantial neurodegenerations during regular mouse development, thus hearing onset in mice could also potentially depend on Fkbp8 expression. Since Fkbp8 is crucial for patterning of neuronal function, we studied its expression during maturation of the rodent auditory function. METHODS: Fkbp8 gene expression in rodent cochlear samples was studied by RT-PCR, qPCR, and western blot. Localization of Fkbp8 transcripts and protein was analyzed by in-situ hybridization and immunohistochemistry. RESULTS: Studies of auditory organ demonstrate that Fkbp8 gene activity is increasing just before hearing onset and gradually decreasing after onset of hearing. Western blot analysis suggests substantial levels of Fkbp8 protein before hearing onset, and slow degradation after onset of hearing. The Fkbp8 mRNA is localized in spiral ganglion of cochlea but its distribution changes over time to the stria vascularis, a finding supported by immunohistochemistry staining. Additionally, in pre-hearing time Fkbp8-specific signal was also observed in the tectorial membrane, whose α- and ß-Tectorin components show similar time-dependent expression of mRNA as Fkbp8. CONCLUSION: These results indicate a temporal shift in expression of Fkbp8 which correlates with cochlear maturation, strongly suggesting a contribution of Fkbp8 to the onset of the rodent hearing processes.

Deafness in TRbeta mutants is caused by malformation of the tectorial membrane.

Thyroid hormone receptor beta (TRbeta) dysfunction leads to deafness in humans and mice. Deafness in TRbeta(-/-) mutant mice has been attributed to TRbeta-mediated control of voltage- and Ca(2+)-activated K(+) (BK) channel expression in inner hair cells (IHCs). However, normal hearing in young constitutive BKalpha(-/-) mutants contradicts this hypothesis. Here, we show that mice with hair cell-specific deletion of TRbeta after postnatal day 11 (P11) have a delay in BKalpha expression but normal hearing, indicating that the origin of hearing loss in TRbeta(-/-) mutant mice manifested before P11. Analyzing the phenotype of IHCs in constitutive TRbeta(-/-) mice, we found normal Ca(2+) current amplitudes, exocytosis, and shape of compound action potential waveforms. In contrast, reduced distortion product otoacoustic emissions and cochlear microphonics associated with an abnormal structure of the tectorial membrane and enhanced tectorin levels suggest that disturbed mechanical performance is the primary cause of deafness resulting from TRbeta deficiency.

Rab8b GTPase, a protein transport regulator, is an interacting partner of otoferlin, defective in a human autosomal recessive deafness form.

Mutations within OTOF encoding otoferlin lead to a recessive disorder called DFNB9. Several studies have indicated otoferlin's association with ribbon synapses of cochlear sensory hair cells, as well as data showing the protein's presence in neurons, nerve fibers and hair cells, suggesting a more ubiquitous function. Otoferlin's co-localization not only with ribbon synaptic proteins, but also with additional endosomal (EEA1) or Golgi proteins (GM130) were motivation for a search for further binding partners of otoferlin by a yeast two-hybrid screen in a rodent cochlear cDNA library (P3-P15). This screen identified Rab8b GTPase as a novel interacting partner, substantiated by transient co-expression and co-localization in HEK 293 cells and co-immunoprecipitation of the complex using tagged proteins in vitro and native proteins from cochlea. This finding implies that otoferlin could be a part of components contributing to trans-Golgi trafficking.

Functional analysis of a novel I71N mutation in the GJB2 gene among Southern Egyptians causing autosomal recessive hearing loss.

Mutations in GJB2, a gene encoding the gap junction protein connexin 26 (Cx26), are a major cause for inherited and sporadic non-syndromic hearing loss, albeit with highly variable clinical effects. To determine new mutations and their frequencies in a Southern Egyptian population restriction fragment length polymorphism, gene sequencing, and single strand conformational polymorphism revealed only 2 mutations for GJB2: c.35delG and p.I71N. The allelic frequency of the c.35delG mutation was 8.7% (found in 27 out of 310 investigated alleles) resulting in a relatively low carrier frequency (1.6%) in Upper Egypt. The new mutation, a substitution of isoleucin (I) (a non-polar amino acid) by the polar amino acid asparagin (N), was localized within the conserved Cx26 structure. The functional significance of p.I71N was tested by injection of cRNA into Xenopus laevis oocytes. Cx26 hemi-channel activity was measured by depolarization activated conductance in non-coupled oocytes. As a result, the p.I71N mutated channel was non-functional. The study discloses a novel, functionally relevant GJB2 mutation and defines the contribution of Cx26 alterations to the hearing loss in the Southern Egyptian population.

Frequency of c.35delG Mutation in GJB2 Gene (Connexin 26) in Syrian Patients with Nonsyndromic Hearing Impairment.

BACKGROUND: Hearing impairments (HI) are the most common birth defect worldwide. Very large numbers of genes have been identified but the most profound is GJB2. The clinical interest regarding this gene is very pronounced due to its high carrier frequency (0.5-5.4%) across different ethnic groups. This study aimed to determine the prevalence of common GJB2 mutations in Syrian patients with profound sensorineural HI. METHODS: We carried out PCR, restriction enzyme based screening, and sequencing of 132 Syrian patients diagnosed clinically with hereditary deafness for different GJB2 mutations. RESULTS: The result revealed that, in GJB2 gene, c.35delG is the most prevalent among affected studied subjects (13.64%), followed by c.457G>A (2.4%). CONCLUSION: The benefit of this study on the one hand is its first report of prelingual deafness causative gene mutations identified by sequencing technology in the Syrian families. It is obvious from the results that the deployment in biomedical research is highly effective and has a great impact on the ability to uncover the cause of genetic variation in different genetic diseases.

A TMC1 (transmembrane channel-like 1) mutation (p.S320R) in a Polish family with hearing impairment.

After excluding frequent mutations in common genes like GJB2, SLC26A4 and MT-RNR1 by straightforward Sanger sequencing in about 20 Polish families with hearing impairment, new and possibly pathogenic mutations were searched for by next-generation sequencing (NGS) screening using a specialised panel including more than 80 genes connected with hearing disorders. Due to high rates of false-positive pathogen predictions for newly discovered single-nucleotide polymorphisms (SNPs), different prediction models were combined to enhance the prediction power. In one family with a record of over four generations, II,3 and II,4 were suspected of hearing impairment without medical records. A male person (III,2) displayed hearing loss of 40 dB hearing level (HL) and his two sons, IV,1 and IV,2, were both affected; one with 90 dB HL and the other with 40 dB HL. Here, one heterozygous, non-synonymous variant was detected, with the SNP causing an amino acid substitution in TMC1 (transmembrane channel-like 1), a gene reported with many mutations in DFNA36 and DFNB7/11 (OMIM #606705 and #600974, respectively). Until now, the substitution p.S320R has not been described in any database. Instead of the significance of this mutation by bioinformatics tools, we confirmed the genotype-phenotype co-segregation in family members. The involvement of TMC1 in hereditary hearing impairment has not been observed in the Polish population so far.

Autonomous functions of murine thyroid hormone receptor TRα and TRß in cochlear hair cells.

Thyroid hormone acts on gene transcription by binding to its nuclear receptors TRα1 and TRß. Whereas global deletion of TRß causes deafness, global TRα-deficient mice have normal hearing thresholds. Since the individual roles of the two receptors in cochlear hair cells are still unclear, we generated mice with a hair cell-specific mutation of TRα1 or deletion of TRß using the Cre-loxP system. Hair cell-specific TRß mutant mice showed normal hearing thresholds but delayed BK channel expression in inner hair cells, slightly stronger outer hair cell function, and slightly reduced amplitudes of auditory brainstem responses. In contrast, hair cell-specific TRα mutant mice showed normal timing of BK channel expression, slightly reduced outer hair cell function, and slightly enhanced amplitudes of auditory brainstem responses. Our data demonstrate that TRß-related deafness originates outside of hair cells and that TRα and TRß play opposing, non-redundant roles in hair cells. A role for thyroid hormone receptors in controlling key regulators that shape signal transduction during development is discussed. Thyroid hormone may act through different thyroid hormone receptor activities to permanently alter the sensitivity of auditory neurotransmission.

Rare variants in BMP2 and BMP4 found in otosclerosis patients reduce Smad signaling.

HYPOTHESIS: Genetic variation in BMP2 and BMP4 found in otosclerosis patients result in altered Smad signaling. BACKGROUND: Otosclerosis is a common form of adult-onset conductive hearing loss resulting from abnormal bone remodeling of the bony labyrinth that surrounds the inner ear. Both genetic and environmental factors are implicated in the disease, yet very little is known about its pathogenesis. The evidence for a genetic component has been established through family-based linkage and population-based association studies. Previously, members of the TGF-ß superfamily of genes have been associated with otosclerosis. METHODS: Sequencing of BMP2 and BMP4 coding regions was performed to identify common and rare variation in German otosclerosis patients compared with controls. Functional analyses of rare variation in the patient cohort were conducted by exposing an osteosarcoma cell line to conditioned media containing either wild type or variant forms of BMP2 or BMP4 and analyzing Smad1/5/8 phosphorylation. RESULTS: Although no significant association with common variation in these 2 genes was detected, there were 8 singleton variants identified in the German population. Of the 4 coding variants found solely in otosclerosis patients, two--BMP4(N150K) and BMP2(K357-R396del)--were found to decrease Smad1/5/8 signaling. CONCLUSION: Rare variants in BMP2 and BMP4 are not a major genetic component in the otosclerosis population. However, those with functional affect showed decreased Smad signaling. Further analysis of Smad signaling molecules should be performed to determine if these pathways in combination are a major contributor to otosclerosis, which could lead to additional treatment options for otosclerosis patients.

Deficient forward transduction and enhanced reverse transduction in the alpha tectorin C1509G human hearing loss mutation.

Most forms of hearing loss are associated with loss of cochlear outer hair cells (OHCs). OHCs require the tectorial membrane (TM) for stereociliary bundle stimulation (forward transduction) and active feedback (reverse transduction). Alpha tectorin is a protein constituent of the TM and the C1509G mutation in alpha tectorin in humans results in autosomal dominant hearing loss. We engineered and validated this mutation in mice and found that the TM was shortened in heterozygous Tecta(C1509G/+) mice, reaching only the first row of OHCs. Thus, deficient forward transduction renders OHCs within the second and third rows non-functional, producing partial hearing loss. Surprisingly, both Tecta(C1509G/+) and Tecta(C1509G/C1509G) mice were found to have increased reverse transduction as assessed by sound- and electrically-evoked otoacoustic emissions. We show that an increase in prestin, a protein necessary for electromotility, in all three rows of OHCs underlies this phenomenon. This mouse model demonstrates a human hearing loss mutation in which OHC function is altered through a non-cell-autonomous variation in prestin.