DFNA9 is a progressive audiovestibular dysfunction with a microfibrillar deposit in the inner ear.

OBJECTIVES: Several mutations in the COCH gene were recently identified in American and European families with DFNA9, an autosomal dominant progressive sensorineural hearing loss with onset in high frequencies. Our preliminary vestibular studies in one American family indicated progressive vestibular dysfunction. More complete vestibular studies in European families have shown vestibular abnormalities in the affected individuals. Our temporal bone studies on two families with DFNA9 revealed, in addition to neurosensory degeneration, a unique acidophilic deposit in the membranous labyrinths of the affected individuals. The purposes of this study were 1) to further investigate the vestibular abnormalities in members of one American family for the purposes of genotype-phenotype correlation and 2) to investigate the electron microscopic structure of the acidophilic deposit to obtain further insights into the pathogenesis of DFNA9. STUDY DESIGN: Prospective analysis. METHODS: Extensive vestibular testing was performed in some unaffected and affected members of a family with DFNA9. One temporal bone was analyzed by electron microscopy of celloidin-embedded tissue. RESULTS AND CONCLUSIONS: The findings indicate progressive vestibular dysfunction in many of the patients affected with hearing loss. Thus, despite different mutations in the COCH gene, the American and European families manifest auditory and vestibular dysfunction. Electron microscopic analysis shows the spiral ligament to be enriched for a highly branched non-banded microfibrillar substance that is decorated with glycosaminoglycan granules. Additionally, the spiral ligament lacks the 67-nm-thick straight periodically banded bundles of type II collagen that are normally abundant in this structure. A speculative pathogenetic model is proposed for this unique disease and its relationship with other late-onset or adult-onset audiovestibular diseases and Meniere's disease is investigated.

Morphological and neurochemical correlates of vestibular compensation.

Morphometric analysis of the cat's superior vestibulo-ocular neurons (SVON) 8 weeks, and 1 and 2 years following vestibular neurectomy or labyrinthectomy revealed similar changes which indicate that an excitatory mode of input to the denervated SVON is responsible for the behavioral recovery. These changes include an increased proportion of strong asymmetric synapses, somal spines surrounding the SP, increased size of and number of SP at long (> 1 year) survival periods. There is a parallel decrease < 1 year and increase > 1 year of contralateral vestibular nerve SP on SVON which matches in timing and magnitude the number of ipsilateral vestibular nerve SP after surgical ablation. These unexpected SVON are consistent with the hypothesis that neurotrophins regulate symmetry in the adult vestibular system. This hypothesis was tested in a series of 13 heterozygous brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4) knockout mice. Following unilateral surgical labyrinthectomy the BDNF and NT4 knockout mice demonstrated no delay in behavioral recovery compared to their normal littermate controls. However, the NT3 knockout mice required twice the time to recover from balance deficits as their littermate controls. These results indicate that NT3 protein is important for normal vestibular function.

Inner ridge cells may be the main source of tectorial membrane type II collagen: evidence from quantitative mRNA in situ hybridization.

In a previous study we showed that COL2A1 mRNA is expressed in both ectodermally and mesodermally derived structures of second trimester human fetal cochlea, whereas type II collagen is present in mesodermally derived structures and in tectorial and basilar membranes. Because the tectorial membrane is acellular and therefore does not make its own proteins, the source of type II collagen and proteoglycans in this membrane has been of interest. We have attempted to address this issue, at least in part, by performing quantitative cRNA mRNA in situ hybridization on second trimester human fetal cochlear sections using a COL2A1 probe. By counting the number of silver grains cell in the interdental cells, inner sulcus cells and inner ridge Kolliker organ cells and by an analysis of variance of these quantitative data. inner ridge cells were found to have significantly higher levels of COL2A1 mRNA than interdental and inner sulcus cells (p < 0.0001). On the basis of significantly higher COL2A1 mRNA levels in inner ridge cells and their higher numbers than interdental and inner sulcus cells we postulate that type II collagen for human fetal tectorial membrane is derived mostly from inner ridge Kolliker organ cells. The lower COL2A1 mRNA in interdental cells appears to provide type II collagen for the spiral limbus and the tectorial membrane. The inner sulcus cells, hair cells. Deiter's and Hensen's cells also appear to contribute lesser amounts of type II collagen to the tectorial membrane. In analogy to these findings it is possible that other tectorial membrane proteins, including proteoglycans and other collagens, are also largely derived from these cells during human fetal development.

Neurotrophin 3, not brain-derived neurotrophic factor or neurotrophin 4, knockout mice have delay in vestibular compensation after unilateral labyrinthectomy.

On the basis that neurotrophins (NTs) affect neuronal synaptic plasticity, are expressed in various cell types of the vestibular system, and exert a trophic influence on statoacoustic neurons, the authors hypothesized a role for NTs in vestibular compensation. To test this hypothesis, they performed unilateral surgical labyrinthectomy in 11 heterozygous (+/-) neurotrophin 3 (NT3) and brain-derived neurotrophic factor (BDNF) knockout mice and in two neurotrophin 4 (NT4) homozygous (-/-) knockout mice, each with a control (+/+) sibling, for a total of 26 mice. Four BDNF(+/-) and four NT3(+/-) mice with their (+/+) controls each were allowed to recover in a normal lighted room for 3, 7, 14, and 30 days following labyrinthectomy. Two BDNF(+/-) and two NT4(-/-) mice with controls were kept in total darkness for 1- and 16-day survival periods. One NT3(+/-) mouse without a control (which died in surgery) was sacrificed after 16 days in darkness. The behavior of all mice was videorecorded to monitor their recovery. Compared with normal (+/+) littermate controls, NT3(+/-) mice demonstrated a delay in compensation (8 to 10 days) in light surround, whereas NT4(-/-) mice showed only a minor delay in dark surround. Despite a 40% lower vestibular ganglion cell population in BDNF(+/-) mice compared with (+/+) controls, BDNF(+/-) mice did not reveal a detectable delay in recovery following labyrinthectomy. These findings suggest that a 50% loss of NT3 protein significantly affects vestibular recovery in adult mice. Perhaps variations in achieving vestibular compensation in humans may be partly secondary to genetically different NT3 levels in vestibular pathways.

Norrie disease gene mutation in a large Costa Rican kindred with a novel phenotype including venous insufficiency.

A large Costa Rican kindred has been identified with 15 males affected with congenital blindness, progressive bearing loss, and venous insufficiency. Due to ophthalmological and audio-otological findings, including bilateral retinal dysplasia and detachment, progressive bilateral sensorineural hearing loss, and an X-linked pattern of inheritance, a tentative diagnosis of Norrie disease was considered. However, venous insufficiency is a clinical finding not reportedly associated with Norrie disease. Genetic linkage analysis using microsatellite repeat markers demonstrated linkage to Xp11.23-11.4 (z = 2.723 at theta = 0.0). A candidate gene approach using the Norrie disease gene (NDP), which maps to Xp11.3, revealed a point mutation in the third exon resulting in substitution of phenylalanine for leucine at position 61. The precise function of the gene product, norrin, has yet to be elucidated; however, it has been postulated to be involved in the regulation of neural cell differentiation and proliferation, although hypotheses have been considered for its role in vascular development in the eye. The finding of a mutation in NDP in association with peripheral vascular disease may provide valuable insight into the potential role of this gene in cellular processes.

A gene for non-syndromic autosomal dominant progressive postlingual sensorineural hearing loss maps to chromosome 14q12-13.

We report a novel locus responsible for postlingual progressive sensorineural hearing loss (designated DFNA9) that maps to chromosome 14q12-13. A large kindred with autosomal dominant transmission of non-syndromic hearing loss was clinically studied. Hearing in affected individuals deteriorated at approximately 20 years of age and progressed to anacusis in the fifth decade. A random genome-wide search using polymorphic short tandem repeats demonstrated linkage with D14S121 (maximum two point LOD score = 6.19, theta = 0). Haplotype analysis of recombination events defined a 9 cM disease interval, between D14S252 and D14S49.

GNAZ in human fetal cochlea: expression, localization, and potential role in inner ear function.

Dissociation of an activated alpha-subunit from the beta-gamma complex directly regulates secondary messenger proteins. To address the potential role of G proteins expressed in human fetal cochlea, degenerate oligonucleotide primers corresponding to the 3'-end of the conserved region of alpha-subunits were used for polymerase chain reaction amplification of reverse-transcribed total human fetal cochlear mRNAs; GNAZ and GNAQ were isolated. These two G proteins are unique among the G-protein family because they lack a typical pertussis modification site. GNAZ is expressed in high levels in neural tissue while GNAQ is ubiquitously expressed. We characterized GNAZ expression using Northern blots, tissue in-situ hybridization and immunohistochemistry techniques to elucidate the potential role of this protein in inner ear function. Our data suggest that GNAZ may play a role in maintaining the ionic balance of perilymphatic and endolymphatic cochlear fluids.

Expression and localization of COL2A1 mRNA and type II collagen in human fetal cochlea.

The expression and localization of COL2A1 mRNA and protein was examined in human fetal cochlea to study the role of this gene in hearing and to begin to understand the pathogenesis of mutations in COL2A1 in hearing disorders. Northern blot analysis revealed COL2A1 expression in fetal membranous cochlea to be markedly greater than that in fetal skin, kidney, cartilage, eye and brain. In situ hybridization revealed COL2A1 expression in marrow cells, osteoblasts, fibroblasts and some osteocytes, in addition to chondrocytes in otic capsule. In the membranous cochlea, connective tissue elements (spiral ligament, spiral limbus and modiolar connective tissue), neuronal cells, secretory cells (stria vascularis) and organ of Corti cells (sensory hair cells) were found to express COL2A1. Immunohistochemistry was performed to assess distribution of type II collagen and correlation with COL2A1 mRNA in these morphologically and functionally diverse cell populations. In otic capsule, only cartilage was found to stain positively, and in membranous cochlea, only connective tissue structures including spiral ligament, spiral limbus, tectorial and basilar membranes, modiolar and spiral lamina cartilage contained type II collagen. Nonconnective tissue cells, marrow cells and osteoblasts did not contain immunohistochemically identifiable protein. Absence of type II collagen in a subset of cochlear cells may reflect potentially either inability to detect low levels of protein in these cells or posttranscriptional regulation.

Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening.

We used a combination of subtractive hybridization and differential screening strategies to identify genes that may function normally in hearing and, when mutated, result in deafness. A human fetal cochlear (membranous labyrinth) cDNA library was subtracted against total human fetal brain RNAs by an avidin-biotin-based procedure to enrich for cochlear transcripts. Subtracted cochlear clones were differentially screened with 32P-labeled total cochlear and total brain cDNA probes. Sequence analysis of clones that hybridized more intensely with cochlear than with brain cDNA probes revealed some previously characterized genes, including mitochondrial sequences, collagen type I alpha-2 (COL1A2), collagen type II alpha-1 (COL2A1), collagen type III alpha-1 (COL3A1), spermidine/spermine N1-acetyltransferase (SAT), osteonectin (SPARC), and peripheral myelin protein 22 (PMP22). Also identified were clones that are potential novel cochlear genes. Northern blots of cochlear and brain RNAs probed with COL1A2, COL2A1, COL3A1, SAT, SPARC, PMP22, and a novel sequence, designated Coch-5B2, confirm results of the subtractive procedure by showing preferential cochlear expression. A number of these genes serve structural or regulatory functions in extracellular matrix or neural conduction; defects in some of these genes are associated with disorders involving hearing loss. Partial sequence analysis of Coch-5B2 reveals a von Willebrand factor type A-like domain in this cDNA. To assess the cochlear specificity of Coch-5B2, a Northern blot panel of 14 human fetal tissue RNAs was probed with Coch-5B2, showing differential expression of this novel gene in the cochlea.

COL1A2 and COL2A1 expression in temporal bone of lethal osteogenesis imperfecta.

OBJECTIVE: COL1A2 and COL2A1 genes are expressed at high levels in many cochlear cells of 16- to 23-week-old human fetuses. Given these prior observations and the rare opportunity to obtain temporal bones from a deceased neonate with osteogenesis imperfecta (OI) type II, we determined the cellular distribution and level of expression of COL1A2 mRNA in OI type II inner ear compared with the expression in second-trimester human fetal cochlea. Expression of COL2A1 mRNA was assessed for its normal role in OI type II neonatal cochlea and to address potential spatial and temporal changes along with our observations in fetal cochlea. We describe our tissue in situ hybridization protocol and document its usefulness in assessing gene expression in human temporal bone obtained at autopsy. DESIGN: RNA-RNA in situ hybridization was performed in formaldehyde-fixed, decalcified, paraffin-embedded temporal bone sections from a neonate with OI type II. Semi-quantitative assessment of gene expression was performed by visual inspection of grain densities. RESULTS AND CONCLUSIONS: COL1A2 and COL2A1 were expressed at moderate-to-high levels in many membranous cochlear cells, and no dramatic alterations in pattern or level of expression of these genes was noted compared with human fetal cochlea. Consistent with in vitro studies, expression of COL1A2 in osteoblasts lining enchondral and endosteal layers is less than that in identical cells of the fetal otic capsule undergoing osteoid deposition and mineralization. Expression of COL1A2 mRNA in osteoblasts lining the outer periosteum of otic capsule is markedly higher than osteoblasts lining enchondral and endosteal layers, suggesting that differential expression may exist between osteoblasts lining endosteal, enchondral, and periosteal surfaces of bone in OI type II.

Autosomal dominant sensorineural hearing loss. Pedigrees, audiologic findings, and temporal bone findings in two kindreds.

We report the clinical and otopathologic findings in three persons from two kindreds affected with adult-onset autosomal dominant progressive sensorineural hearing loss. The primary pathologic change is a deposit of acid polymucosaccharide ground substance in the cribrose areas; in the spiral ligament, limbus, and spinal lamina of the cochlea; and in the stroma of the maculae and cristae. These deposits obstruct the channels that accommodate the dendritic nerve fibers to the auditory and vestibular sense organs. The end result is strangulation and degeneration of dendrites followed by retrograde neuronal degeneration in association with varying degrees of atrophic change in the sense organs.

Investigations into the cause of vertigo in sudden sensorineural hearing loss.

In this light microscopic study of the temporal bone, an attempt has been made to find a morphologic correlate of vertigo associated with idiopathic sudden sensori-neural hearing loss (ISSHL). Hair cell densities of the three cristae and both maculae, as well as vestibular ganglion cell (neuronal) count estimation, was done in nine ears that had documented histories of ISSHL. There were five ears with vertigo and four without. These quantitative data--i.e., hair cell densities and neuronal counts, of the vertiginous ears (group I) and nonvertiginous ears (group II)--was compared by histograms and statistically. Additionally, in each of the two groups, the data from three opposite normal hearing ears were taken as a control and used for comparison with the ISSHL ears. The differences between the vertiginous, nonvertiginous, and control ears were not significant at the 0.01 level, indicating that the vertigo was not caused by hair cell or neuronal degeneration. Gross morphologic alterations in the vestibular system, such as membrane ruptures, endolymphatic hydrops, etc., were also assessed, but no clear-cut pathology was identified in the vertiginous and nonvertiginous ears. The absence of a light microscopic morphologic correlate for vestibular disturbances associated with ISSHL suggests that the symptoms could result from ultrastructural changes in the hair cells and their synapses or from biochemical alterations in their environment.

Idiopathic sudden sensorineural hearing loss and postnatal viral labyrinthitis: a statistical comparison of temporal bone findings.

Although the cause of idiopathic sudden sensorineural hearing loss remains uncertain, a viral origin has been suggested in many cases on the basis of anamnestic microbiologic and pathologic data. Twenty-two temporal bone specimens from 18 patients who during life suffered a sudden partial or complete sensorineural hearing loss were studied. On the basis of clinical data, these cases were assigned to one of three diagnostic categories, and the temporal bones were studied by light microscopy and serial section analysis. The implications of the histopathologic findings for the pathogenesis of idiopathic sudden sensorineural hearing loss are discussed.

Temporal bone findings in a case of bilateral Menière's disease treated by parenteral streptomycin and endolymphatic shunt.

A patient with bilateral Menière's disease who had progressive hearing loss and intractable vertigo was treated at ages 60 and 62 with parenteral streptomycin to ablate vestibular function, and at age 74 by a left endolymphatic shunt procedure. He was confined to a wheelchair because of ataxia from age 75 until the time of his death at age 81. Both temporal bones show congenitally hypoplastic endolymphatic sacs and severe endolymphatic hydrops consistent with Meniere's disease. There was atrophy of the striae vasculares and loss of cochlear neurons consistent with presbycusis. A loss of hair cells in the cristae and saccules was consistent with streptomycin ototoxicity. In the left ear the Silastic strip that was intended to function as an endolymphatic shunt into the mastoid lies encased in fibrous tissue and failed by 4.5 mm to reach the hypoplastic endolymphatic sac.

In search of pathologic correlates for hearing loss and vertigo in Paget's disease. A clinical and histopathologic study of 26 temporal bones.

Mixed sensorineural and conductive hearing loss is a common clinical manifestation of Paget's disease of the temporal bone, and while there are numerous clinical and pathologic reports on the condition, none have identified a consistent pathologic explanation for the hearing loss. We performed histologic studies on 26 temporal bones exhibiting Paget's disease from 16 persons, of whom 7 had audiometric testing performed. Contrary to common opinion, the conductive hearing loss is not caused by ossicular fixation; in fact, no cause could be found in the seven ears with documented conductive hearing losses. While the sensorineural hearing losses were greater than normal for age, we could not identify cochlear disorders that could be attributed to Paget's disease. It is concluded that the hearing losses in Paget's disease are caused by changes in bone density, mass, and form that serve to dampen the finely tuned motion mechanics of the middle and inner ears.