Spectrum and frequencies of non GJB2 gene mutations in Czech patients with early non-syndromic hearing loss detected by gene panel NGS and whole-exome sequencing.

Non-syndromic autosomal recessive hearing loss is an extremely heterogeneous disease caused by mutations in more than 80 genes. We examined Czech patients with early/prelingual non-syndromic, presumably genetic hearing loss (NSHL) without known cause after GJB2 gene testing. Four hundred and twenty-one unrelated patients were examined for STRC gene deletions with quantitative comparative fluorescent PCR (QCF PCR), 197 unrelated patients with next-generation sequencing by custom-designed NSHL gene panels and 19 patients with whole-exome sequencing (WES). Combining all methods, we discovered the cause of the disease in 54 patients. The most frequent type of NSHL was DFNB16 (STRC), which was detected in 22 patients, almost half of the clarified patients. Other biallelic pathogenic mutations were detected in the genes: MYO15A, LOXHD1, TMPRSS3 (each gene was responsible for five clarified patients, CDH23 (four clarified patients), OTOG and OTOF (each gene was responsible for two clarified patients). Other genes (AIFM1, CABP2, DIAPH1, PTPRQ, RDX, SLC26A4, TBC1D24, TECTA, TMC1) that explained the cause of hearing impairment were further detected in only one patient for each gene. STRC gene mutations, mainly deletions remain the most frequent NSHL cause after mutations in the GJB2.

A link between planar polarity and staircase-like bundle architecture in hair cells.

Sensory perception in the inner ear relies on the hair bundle, the highly polarized brush of movement detectors that crowns hair cells. We previously showed that, in the mouse cochlea, the edge of the forming bundle is defined by the 'bare zone', a microvilli-free sub-region of apical membrane specified by the Insc-LGN-Gαi protein complex. We now report that LGN and Gαi also occupy the very tip of stereocilia that directly abut the bare zone. We demonstrate that LGN and Gαi are both essential for promoting the elongation and differential identity of stereocilia across rows. Interestingly, we also reveal that total LGN-Gαi protein amounts are actively balanced between the bare zone and stereocilia tips, suggesting that early planar asymmetry of protein enrichment at the bare zone confers adjacent stereocilia their tallest identity. We propose that LGN and Gαi participate in a long-inferred signal that originates outside the bundle to model its staircase-like architecture, a property that is essential for direction sensitivity to mechanical deflection and hearing.

[Screening of common deaf genes in pregnant women and prevention of deafness at birth].

OBJECTIVE: To determine the carrier rate for common mutations causing deafness among pregnant women in order to prevent births of deaf children. METHODS: For 893 pregnant women, 2 mL peripheral venous blood was taken and DNA was extracted. A deafness DNA microarray screening was applied to such samples, and DNA sequencing was applied to husbands of women with positive screening results. RESULTS: A total of 40 carriers were detected, with the overall mutation rate being 4.48%. Among such carriers, GJB2 235delC was the most common heterozygous mutation (18 cases) and the mutation rate was 2.02%. GJB2 299A-T heterozygous mutation was detected in 7 cases with a mutation rate of 0.78%. IVS7-2A to G heterozygous mutation was detected in 9 cases with a mutation rate of 1.02%. There were 2 cases carrying GJB3 heterozygous mutation and 2 cases of mitochondrial 12S rRNA heterozygous mutation, with a mutation rate of 0.22%. IVS7-2A>G with GJB3 538C>T double heterozygous mutation was detected in 1 case, and IVS7-2A>G with GJB2 299A-T double heterozygous mutation was detected in another case, with the mutation rate of each being 0.11%. DNA sequencing has failed to find presence of mutations in the same gene in the husbands. The results of neonatal hearing follow-up were all normal. CONCLUSION: Applications of the deaf genes screening in pregnant women may play prove to be valuable for the early detection for neonatal deafness.

Role of p63 and the Notch pathway in cochlea development and sensorineural deafness.

The ectodermal dysplasias are a group of inherited autosomal dominant syndromes associated with heterozygous mutations in the Tumor Protein p63 (TRP63) gene. Here we show that, in addition to their epidermal pathology, a proportion of these patients have distinct levels of deafness. Accordingly, p63 null mouse embryos show marked cochlea abnormalities, and the transactivating isoform of p63 (TAp63) protein is normally found in the organ of Corti. TAp63 transactivates hairy and enhancer of split 5 (Hes5) and atonal homolog 1 (Atoh1), components of the Notch pathway, known to be involved in cochlear neuroepithelial development. Strikingly, p63 null mice show morphological defects of the organ of Corti, with supernumerary hair cells, as also reported for Hes5 null mice. This phenotype is related to loss of a differentiation property of TAp63 and not to loss of its proapoptotic function, because cochleas in mice lacking the critical Bcl-2 homology domain (BH-3) inducers of p53- and p63-mediated apoptosis--Puma, Noxa, or both--are normal. Collectively, these data demonstrate that TAp63, acting via the Notch pathway, is crucial for the development of the organ of Corti, providing a molecular explanation for the sensorineural deafness in ectodermal dysplasia patients with TRP63 mutations.

Impaired development of melanoblasts in the black-eyed white Mitf(mi-bw) mouse, a model for auditory-pigmentary disorders.

Microphthalmia-associated transcription factor (Mitf) is a regulator for differentiation of melanoblasts that are derived from the neural crest. The mouse homozygous for the black-eyed white (Mitf(mi-bw)) allele is characterized by the white coat color and deafness, with black eye that is associated with the lack of melanocytes in skin and inner ear. The Mitf(mi-bw) mutation is an insertion of the LINE1 retrotransposable element into intron 3 of the Mitf gene that causes the selective deficiency of the melanocyte-specific Mitf isoform, Mitf-M. Here, we show the expression of Mitf-M mRNA in the trunk region of the homozygous Mitf(mi-bw)(bw) mouse at embryonic days (E) 11.5 and E12.5, but Mitf-M mRNA is undetectable at E13.5. In addition, using bw mouse that carries the lacZ transgene under the control of a melanoblast-specific promoter, we show that the number of migrating melanoblasts in bw embryos was less than 10% of that in control embryos at E11.5 and E12.5, and melanoblasts disappear by E13.5. The loss of melanoblasts in bw embryos was probably caused by apoptosis. Finally, forced expression of Mitf-M in the cultured neural tube of bw embryos ensured the differentiation of melanoblasts. Therefore, the correct dose of Mitf-M is required for the normal development of melanoblasts.

Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin.

Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4(-/-), are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO(3) (-) transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4(-/-) mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4(+/-) and Slc26a4(-/-) mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4(-/-) mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4(-/-) mice, and possibly in humans, lacking functional pendrin expression.

Multispectral four-dimensional imaging reveals that evoked activity modulates peripheral arborization and the selection of plane-polarized targets by sensory neurons.

The polarity of apical stereocilia endows hair cells with directional excitability, which in turn enables animals to determine the vectorial component of a sound. Neuromasts of the lateral line of aquatic vertebrates harbor two populations of hair cells that are oriented at 180 degrees relative to each other. The resulting sensory-vectorial ambiguity is solved by lateralis afferent neurons that discriminate between hair cells of opposite polarities to innervate only those with the same orientation. How neurons select identically oriented hair cells remains unknown. To gain insight into the mechanism that underlies this selection, we devised a simple method to gather dynamic morphometric information about axonal terminals in toto by four-dimensional imaging. Applying this strategy to the zebrafish allowed us to correlate hair cell orientation to single afferent neurons at subcellular resolution. Here we show that in zebrafish with absent hair cell mechanoreception, lateralis afferents arborize profusely in the periphery, display less stability, and make improper target selections. Central axons, however, show no dynamic changes and establish normal contacts with the Mauthner cell, a characteristic second-order target in the hindbrain. We propose that the hardwired developmental mechanisms that underlie peripheral arborization and target recognition are modulated by evoked hair cell activity. This interplay between intrinsic and extrinsic cues is essential for plane-polarized target selection by lateralis afferent neurons.

First report of prenatal diagnosis of genetic congenital deafness in a routine prenatal genetic test.

OBJECTIVE: We aimed to screen for connexin26 gene (GJB2) mutations associated with autosomal recessive non-syndromic neurosensory deafness (NSRD) in a general risk population. METHODS: Screening for the most common connexin26 gene mutations was offered to all women undergoing a second-trimester amniocentesis for fetal karyotype analysis in our Center. After rapid DNA extraction from amniotic fluid, PCR amplification was performed and products analysed to detect mutations of GJB2 gene by a sequencing technique. In particular, we searched for the 20 most frequently reported mutations (out of the approximately 90 so far described) and for which there are commercially available tests. RESULTS: From a total of 4819 consecutive amniotic fluids examined, the following five different heterozygous mutations were detected: 35delG in 80 cases, 167delT in 3 cases and 1 occurrence of each of the following mutations: M34T, 35insG and W77R. From these data, a prevalence of 1 : 56 (1.78%) for the heterozygous condition can be estimated in the Mediterranean general risk population. The striking predominance of 35delG mutation is confirmed. In addition, we detected a homozygous 35delG mutation condition in a foetus of no risk parents. In this case, the early diagnosis permitted prompt application of an acoustic prosthesis allowing for cochlear implantation in due time, with significant improvement of the prognosis. CONCLUSIONS: In a general risk population, a carrier status for congenital deafness can be observed in 1 : 56 (1.78%) amniotic fluids; this is mostly due to the presence of a 35delG mutation of the connexin26 gene. Occasional identification of homozygous states, although rare, allows the best therapeutic approach.

Stereocilia defects in waltzer (Cdh23), shaker1 (Myo7a) and double waltzer/shaker1 mutant mice.

Mutations in myosin VIIa (Myo7a) and cadherin 23 (Cdh23) cause deafness in shaker1 (sh1) and waltzer (v) mouse mutants respectively. In humans, mutations in these genes cause Usher's syndrome type 1B and D respectively, as well as certain forms of non-syndromic deafness. Examination of the organ of Corti from shaker1 and waltzer mice has shown that these genes are required for the proper organisation of hair cell stereocilia. Here we show that at embryonic day 18.5, the outer hair cells of Cdh23(v) homozygote mutant mice appear immature, projecting fewer recognisable stereocilia than heterozygote controls, and by post-natal day (P) 4 their stereocilia are arranged in a disorganised pattern rather than in the regular 'V'-shape seen in heterozygotes. Inner hair cell stereocilia are also disorganised in Cdh23(v) mutant homozygotes. Myo7a was expressed normally in the hair cells of P0 Cdh23(v2J) mutants demonstrating that cadherin 23 is not required for Myo7a expression at this stage. No stereocilia defects were observed in P4 Cdh23(v)/Myo7a(4626SB) double heterozygotes (+/Cdh23(v) +/Myo7a(4626SB)) and neither the Cdh23(v) nor Myo7a(4626SB) homozygote phenotypes were affected by the presence of one mutant copy of Myo7a or Cdh23 respectively. The hair cell phenotype of double homozygote mutant mice did not differ from single Myo7a(4626SB) homozygote mutants. Finally, we found no significant correlation between loss of hearing and double heterozygosity for mutations in Cdh23 and Myo7a in mice aged between 7.5 and 10 months. These findings suggest that Cdh23 and Myo7a are both required for establishing and/or maintaining the proper organisation of the stereocilia bundle and that they do not genetically interact to affect this process nor to cause age-related hearing loss.

[Connexin26 and -30 in the Cochlea and their clinical relevance]. / Connexin26 und -30 im Innenohr und ihre klinische Bedeutung.

INTRODUCTION: Gap junction channels consist of different connexin proteins and play an important role in the physiology of hearing. Connexin26 and connexin30 have been demonstrated in the inner ear by immunohistochemistry and Northern Blot analysis. Mutations in the genes for connexin26 and connexin30 have been described to be responsible for non-syndromic hearing loss. METHODS: We investigated the prevalence of connexin26 and connexin30 mutations in patients with profound hearing loss or deafness by SSCP-analysis and sequencing. RESULTS: 30 connexin26 mutations (22 %) were detected among 134 patients with profound hearing loss or deafness. The most frequent connexin26 mutation 30delG was found in 25 patients. In 5 patients other connexin26 mutations were identified. No connexin30 mutation was found. CONCLUSION: Therefore connexin26 mutations also play an important role for non-syndromic hearing loss in Germany. We propose that every patient with suspected hereditary hearing loss should be screened for a connexin26 mutation.

[The early postnatal development of the hamster cochlea]. / Die frühe postnatale Entwicklung der Cochlea des Goldhamsters.

The Early Postnatal Development of the Hamster Cochlea. Inborn deafness can be diagnosed very early. Deafness in early childhood can be caused by: genetic defects (30 % - 40 %), embryopathies, embryonic noxae (60 %) and pre-, peri- and postnatal noxae. Which structures of the cochlea are disturbed, is unknown in most cases. Systematic studies are necessary with directed experimental impairments of the cochlea in the early development to elucidate these mechanisms. An animal model has to be established in which directed impairments can be carried out in single individuals. The cochlea of the hamster is at birth in a very early state of development. In the apical turn the epithelial layer of the cochlear duct is undifferentiated, whereas in the basal turn the papilla basilaris (Kölliker's organ), the lateral wall and Reissner's membrane are visible. Between the base of the cochlea (1st coil lateral) and the apical part (3rd coil lateral) the difference in the development of the hamster cochlea is exactly four days. This is evident for the 1st and the 5th day after birth. The cochlea development of the hamster is very rapid. The development during one day represents one week of development in the human cochlea. The difference in development in one hamster cochlea from the apex to the base at a certain day representing four days of development is comparable with the development time of four weeks in human. The different developmental stages which are present in the hamster cochlea of a certain day represent the development of the human cochlea of a whole month. The hamster cochlea at the 1st day after birth covers the 2nd embryonic month of the human (4th to 8th week) and at the 5th day the 3rd embryonic month (8th to 12th week). The hamster cochlea at the 1st and the 5th day after birth is especially suitable to study experimental disturbances in the early stages of the development of the cochlear duct when the tectorial membrane, the spiral limbus, the stria vascularis, Reissners membrane and the external spiral Sulcus with the root cells are differentiating. The biggest advantage is that the noxae (hypoxia, teratogenic substances, ototoxic antibiotics and intense noise) can be applied to single individuals which offers better control than the treatment of the mother.

Expression of the PAX2 gene in human embryos and exclusion in the CHARGE syndrome.

The CHARGE syndrome comprises ocular coloboma, heart malformation, choanal atresia, retarded growth and development, central nervous system malformations, genital hypoplasia, ear abnormalities, or deafness. The cause of the CHARGE syndrome remains unknown. In the present study, we analyzed the distribution pattern of the PAX2 gene in human embryos and found that PAX2 gene expression occurs in the primordia affected in the CHARGE syndrome. These data prompted us to consider the PAX2 gene a candidate gene in the CHARGE "association." We analyzed the PAX2 gene in 34 patients fulfilling the diagnostic criteria of the CHARGE syndrome for deletion and nucleotidic variations of the coding sequence and identified only polymorphisms. Our data suggest that mutation of the PAX2 gene is not a cause of the CHARGE association. However, the pattern of expression of PAX2 suggests that genes encoding downstream targets effectors could be candidate genes for the CHARGE syndrome.

Isolated congenital internal auditory canal atresia with normal facial nerve function.

The internal auditory canal forms as a result of mesoderm enveloping the eighth cranial nerve in the developing embryo. The mesoderm eventually transforms into cartilage and ultimately ossifies around the nerve, forming the internal auditory canal. It is theorized that atresia or stenosis of the internal auditory canal results from altered cochleovestibular nerve development secondary to faulty chemotactic mechanisms or a lack of end organ targets. Unilateral internal auditory canal anomalies are frequently seen in conjunction with other inner ear anomalies and occasionally with middle or external ear anomalies. Infrequently, it will occur as either an isolated or bilateral finding, but rarely simultaneously. The few citations of isolated, unilateral or bilateral internal auditory canal anomalies that are reported in the literature are usually associated with other systemic developmental anomalies, such as, cardiac septal defects, polycystic kidney disease, skeletal deformities and duodenal atresia. We present a case report of a patient with bilateral, congenital, internal auditory canal atresia and cochleovestibular deficits but, normal facial nerve function. A review of the literature is discussed as well as diagnostic considerations and treatment options including audiologic and communication rehabilitation.

Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development.

The neurally expressed genes Brn-3.1 and Brn-3.2 (refs 1-6) are mammalian orthologues of the Caenorhabditis elegans unc-86 gene that constitute, with Brn-3.0 (refs 1-3,8,9), the class IV POU-domain transcription factors. Brn-3.1 and Brn-3.2 provide a means of exploring the potentially distinct biological functions of expanded gene families in neural development. The highly related members of the Brn-3 family have similar DNA-binding preferences and overlapping expression patterns in the sensory nervous system, midbrain and hindbrain, suggesting functional redundancy. Here we report that Brn-3.1 and Brn-3.2 critically modulate the terminal differentiation of distinct sensorineural cells in which they exhibit selective spatial and temporal expression patterns. Deletion of the Brn-3.2 gene causes the loss of most retinal ganglion cells, defining distinct ganglion cell populations. Mutation of Brn-3.1 results in complete deafness, owing to a failure of hair cells to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia.

Human myosin VIIA responsible for the Usher 1B syndrome: a predicted membrane-associated motor protein expressed in developing sensory epithelia.

The gene encoding human myosin VIIA is responsible for Usher syndrome type III (USH1B), a disease which associates profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. The reconstituted cDNA sequence presented here predicts a 2215 amino acid protein with a typical unconventional myosin structure. This protein is expected to dimerize into a two-headed molecule. The C terminus of its tail shares homology with the membrane-binding domain of the band 4.1 protein superfamily. The gene consists of 48 coding exons. It encodes several alternatively spliced forms. In situ hybridization analysis in human embryos demonstrates that the myosin VIIA gene is expressed in the pigment epithelium and the photoreceptor cells of the retina, thus indicating that both cell types may be involved in the USH1B retinal degenerative process. In addition, the gene is expressed in the human embryonic cochlear and vestibular neuroepithelia. We suggest that deafness and vestibular dysfunction in USH1B patients result from a defect in the morphogenesis of the inner ear sensory cell stereocilia.

The ocular pathology of Norrie disease in a fetus of 11 weeks' gestational age.

The ocular pathology of Norrie disease was studied for the first time in a fetus of 11 weeks' gestation, following prenatal diagnosis using genetic markers for Norrie disease and elective abortion. The eyes were histologically normal, with no evidence of primary neuroectodermal maldevelopment of the retina, previously postulated to be the cause of the ocular changes. We believe that the retinal and other manifestations of Norrie disease are the result of a primary abnormality of vascular proliferation, probably in relation to persistent hyperplastic primary vitreous after approximately 14 weeks' gestation. We postulate that the ocular and otological effects of Norrie disease may be due to a genetically mediated abnormality of secretion of, or sensitivity to, angiogenic growth factors at endodermal-neuroectodermal interfaces during fetal and postnatal development.