Dominant modifier DFNM1 suppresses recessive deafness DFNB26.

More than 50% of severe childhood deafness is genetically determined, approximately 70% of which occurs without other abnormalities and is thus termed nonsyndromic. So far, 30 nonsyndromic recessive deafness loci have been mapped and the defective genes at 6 loci, DFNB1, DFNB2, DFNB3, DFNB4, DFNB9 and DNFB21, have been identified, encoding connexin-26 (ref. 3), myosin VIIA (ref. 4), myosin XV (ref. 5), pendrin, otoferlin and alpha-tectorin, respectively. Here we map a new recessive nonsyndromic deafness locus, DFNB26, to a 1.5-cM interval of chromosome 4q31 in a consanguineous Pakistani family. A maximum lod score of 8.10 at theta=0 was obtained with D4S1610 when only the 8 affected individuals in this family were included in the calculation. There are seven unaffected family members who are also homozygous for the DFNB26-linked haplotype and thus are non-penetrant. A dominant modifier, DFNM1, that suppresses deafness in the 7 nonpenetrant individuals was mapped to a 5.6-cM region on chromosome 1q24 with a lod score of 4.31 at theta=0 for D1S2815.

Small molecule delivery across a perforated artificial membrane by thermoreversible hydrogel poloxamer 407.

Microperforations in the round window membrane have been suggested for enhancing the rate and reliability of drug delivery into the cochlea. Intratympanic injection, the most common delivery method, involves injecting therapy into the middle ear to establish a reservoir from which drug diffuses across the round window membrane into the cochlea. This process is highly variable because (i) the reservoir, if liquid, can lose contact with the membrane and (ii) diffusion across the membrane is intrinsically variable even with a stable reservoir. To address these respective sources of variability, we compared the thermoreversible hydrogel poloxamer 407 (P407) to saline as a drug carrier and studied the effect of membrane microperforations on drug diffusion rate. We used Rhodamine B as a drug proxy to measure permeance across an artificial membrane in a horizontal diffusion cell. We found that permeance of Rhodamine B from a saline reservoir was an order of magnitude higher than that from a P407 reservoir across unperforated membranes. Moreover, permeance increased with total perforation cross-sectional area regardless of number of perforations (p < 0.05 for all saline-based experiments), but the same association was not found with P407. Rather, for a P407 reservoir, only a large perforation increased permeance (p < 0.001), while multiple small perforations did not (p = 0.749). These results confirm that for drug dissolved in saline, multiple small perforations can effectively enhance diffusion. However, for drug dissolved in P407, larger perforations are necessary.

The cochlear-carotid interval: anatomic variation and potential clinical implications.

BACKGROUND AND PURPOSE: A temporal bone CT study in a patient with episodic mid-tone sensorineural hearing loss and tinnitus demonstrated absence of bone between the petrous internal carotid artery and the basal turn of the cochlea. The potential implications with respect to increasingly popular cochlear implant surgery compelled us to retrospectively analyze a series of temporal bone CT scans to establish typical measurements for this region, which we termed the "cochlear-carotid interval" (CCI). METHODS: After IRB exemption, 2 observers independently measured the bony interval between the cochlea and the petrous internal carotid artery canal on coronal images from 30 consecutive temporal bone CT studies. The 1-mm thick coronal images were either acquired directly or were reconstructed from an axial dataset acquired at 0.75 or 0.6 mm section thickness. All measurements were performed by using electronic calipers on a Sienet MagicView VE 42 Siemens PACS station. Mixed model analysis of variance was used to evaluate differences between readers and sides with respect to the mean CCI but adjusted for age and accommodating the correlation among observations generated for the same subject. RESULTS: The patient in our case report had a right CCI of 0.2 mm and left CCI of 0.0 mm. In the other 30 patients, the right CCI ranged from 0.2 to 3.8 mm (mean, 1.2 +/- 0.8 mm; median, 0.9) and the left CCI from 0.2 to 5.0 mm (mean, 1.1 +/- 0.9 mm; median, 0.8). The CCI did not exhibit a significant association with subject age (P = .1336), and there were no significant differences between readers (P = .824) or sides (P = .350) in terms of mean CCI. CONCLUSION: The CCI varies widely between patients and may be as small as zero. Analysis of anatomic relationships suggests a potential relationship between small CCI and mid-tone sensorineural hearing loss, as in our reported patient. Preoperative knowledge of thin or absent bone between the cochlea and petrous carotid canal may help prevent inadvertent penetration of the carotid canal during cochlear implant surgery.

The guinea pig cochlear AE2 anion exchanger: cDNA cloning and in situ localization within the cochlea.

This study has characterized the repertoire of the anion exchanger (AE) family members expressed within the guinea pig organ of Corti, the auditory neuroepithelia. Both AE2 and AE3 cDNAs were present, but AE1 cDNA was not detected. The more abundant AE2 was sequenced and its expression characterized in the cochlea. The 3888 base pairs (bp) AE2 sequence, compiled from multiple clones, includes 150 bp of upstream non-coding sequence and 3717 bp of open reading frame encoding a protein of 1238 amino acids. Immunoblot of cochlear homogenate revealed a single AE2-immunoreactive band of Mr 180 kDa. In situ hybridization and immunohistochemical analysis localized AE2 expression to several tissues and cell types within the guinea pig inner ear, including superior half of the spiral ligament and within the interdental cells lining the spiral limbus. However, AE2 was not clearly detected in the outer hair cells (OHC) of the organ of Corti by either immunohistochemistry or in situ hybridization. The results of these studies imply a physiologic role of AE2 in the cochlear homeostasis, but do not support its role as a potential 'motor protein' in mediating the in vitro-observed voltage-gated, ATP-independent OHC motility.

Refinement of the DFNA4 locus to a 1.44 Mb region in 19q13.33.

Many forms of autosomal dominant non-syndromic hearing impairment are known. While the underlying gene defects and causative mutations have been discovered for some forms, the gene responsible for DFNA4 has remained elusive to date. Examination of a German four-generation kindred led to the identification of a 1.44 Mb map segment in contig NT_011109 as being the most likely DFNA4 candidate region in 19q13.33. The recombination breakpoints in this family and the intervals of two previously reported DFNA4 families allowed us to delineate a minimum consensus region between the markers D19S879 and D19S246. In our family, a maximum two-point LOD score of 4.5 was obtained at theta = 0 for the marker D19S867. Within the refined DFNA4 interval the public databases list more than 50 genes, from which several appear to be promising DFNA4 candidates due to similarities with animal models and with other causative genes involved in hearing disability.

Utility of electronystagmography in the prediction of post-operative outcome following cochlear implantation.

OBJECTIVE: To examine the relationship between pre-operative electronystagmography and videonystagmography test results and post-operative outcomes in dizziness, auditory sensitivity and speech recognition. METHODS: A retrospective chart review was performed. Auditory sensitivity and speech perception ability were tested pre- and post-operatively in 37 adult cochlear implant recipients. Auditory sensitivity was evaluated using either pure tones (for testing with earphones) or frequency-modulated warble tones (for sound-field testing). Speech perception ability was evaluated using Northwestern University Auditory Test Number 6. RESULTS: No correlation was found between pre-operative electronystagmography test results and post-operative subjective dizziness. However, pre-operative electronystagmography testing and post-operative hearing sensitivity as measured by warble tone average (dB HL) correlated significantly at six months or later after cochlear implant activation (r  ≥  -0.34, n = 34, p < 0.05). CONCLUSION: This study, which has a level of evidence 4, demonstrates that pre-operative electronystagmography testing has a potential use in predicting post-operative outcomes in hearing sensitivity following cochlear implantation. However, larger studies are needed to confirm this novel finding.

Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.

The utility of lentivirus as a gene delivery vector in the cochlea was evaluated in vitro and in vivo. Lentivirus transduction was assessed through expression analysis of a reporter gene, green fluorescent protein (GFP), integrated within the viral genome. In vitro characterization of lentivirus-GFP was assessed by infection of explants from cochleas of neonatal rat. The lentiviral vector transduced both spiral ganglion neurons (SGNs) and glial cells. In vivo characterization of lentivirus-GFP was assessed by directly infusing the vector into the guinea pig cochlea via an osmotic minipump. Sections of lentivirus-infused cochlea revealed a highly restricted fluorescence pattern limited to the periphery of the perilymphatic space. Transduction of SGNs and glial cells by lentivirus in vitro but not in vivo suggests limited dissemination of the viral vector from the perilymphatic space. The cellular and tissue architecture of the lentivirus-infused cochlea was intact and free of inflammation. Restricted transduction of cell types confined to the periphery of the perilymphatic space by the lentivirus is ideal for stable production of gene products secreted into the perilymph.

Cochlear gene delivery through an intact round window membrane in mouse.

Cochlear gene transfer studies in animal models have utilized mainly two delivery methods: direct injection through the round window membrane (RWM) or intracochlear infusion through a cochleostomy. However, the surgical trauma, inflammation, and hearing loss associated with these methods lead us to investigate a less invasive delivery method. Herein, we studied the feasibility of a vector transgene-soaked gelatin sponge, Gelfoam, for transgene delivery into the mouse cochlea through an intact RWM. The Gelfoam absorbed with liposomes and adenovirus, but not with adeno-associated virus (AAV), was successful in mediating transgene expression across an intact RWM in a variety of cochlear tissues. The Gelfoam technique proved to be an easy, atraumatic, and effective, but vector-dependent, method of delivering transgenes through an intact RWM. Compared with the more invasive gene delivery methods, this technique represents a safer and a more clinically viable route of cochlear gene delivery in humans.

Assessment of denaturing high-performance liquid chromatography (DHPLC) in screening for mutations in connexin 26 (GJB2).

Mutations in the gene GJB2 encoding connexin 26 (Cx26), a gap junction protein, have been shown to be responsible for a majority of recessive nonsyndromic hereditary hearing impairment in children. Over 60 different mutations in Cx26 have been reported. To obviate the need for direct sequencing of each specimen, a variety of screening techniques have been used to detect mutations in Cx26. However, each of these methods has significant shortcomings including expense, time consumption, and limited sensitivity. Denaturing high-performance liquid chromatography (DHPLC) has been recently introduced as a rapid and highly sensitive method of detecting sequence alterations. We have assessed the efficacy of DHPLC as a screening assay for detecting mutation in Cx26 coding region in 154 patients with hereditary hearing impairment. The GJB2 coding exon was amplified in one or two fragments, analyzed by DHPLC, and sequenced. Sequence analysis identified sequence variations in 34 patients concordant with abnormal DHPLC results. Three novel Cx26 mutations were identified: a single base pair substitution 511G>A, a 4 bp insertion 504insAACG, and a 3 bp deletion 358delAGG in three unrelated patients. In 120 patients with normal Cx26 sequence, DHPLC was normal. These results yield sensitivity and specificity of 100% for DHPLC-based detection of Cx26 mutations, and demonstrate that DHPLC is a highly sensitive and specific method of screening for sequence variations in Cx26 that is time and labor efficient. Further, our experience suggests that DHPLC screening alone followed by DNA sequencing only when DHPLC is abnormal may be adequate for identification of all sequence alterations in Cx26.

Point mutation in the MITF gene causing Waardenburg syndrome type II in a three-generation Indian family.

Waardenburg syndrome (WS) is an autosomal-dominant neural crest cell disorder phenotypically characterized by hearing impairment and disturbance of pigmentation. A presence of dystopia canthorum is indicative of WS type 1, caused by loss of function mutation in the PAX3 gene. In contrast, type 2 WS (WS2) is characterized by normally placed medial canthi and is genetically heterogeneous; mutations in MITF (microphthalmia associated transcription factor) associated with WS2 have been identified in some but not all affected families. Here, we report on a three-generation Indian family with a point mutation in the MITF gene causing WS2. This mutation, initially reported in a Northern European family, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking the HLH-Zip or Zip structure necessary for normal interaction with its target DNA motif. Comparison of the phenotype between the two families demonstrates a significant difference in pigmentary disturbance of the eye. This family, with the first documented case of two unrelated WS2 families harboring identical mutations, provides additional evidence for the importance of genetic background on the clinical phenotype.

Isolated metastatic melanoma of the cerebellopontine angle: case report.

Malignant melanoma is a common cause of central nervous system metastases. This report describes an extremely rare case of metastatic melanoma presenting as an isolated cerebellopontine angle tumor. Clinically and radiographically, the lesion mimicked an acoustic neuroma. The patient had neuro-otological symptoms, including tinnitus, vertigo, sensorineural hearing loss, facial nerve dysfunction, and prominent cerebellar dysfunction. Magnetic resonance images showed a lesion of the internal auditory canal and cerebellopontine angle that was hypointense on T1-weighted images, hyperintense on T2-weighted images, and enhanced after the administration of gadolinium. T2-weighted images showed significant cerebellar edema. Subtotal resection of the tumor through a suboccipital craniotomy palliated the symptoms, but the patient died of tumor progression 6 months later. In contrast to other metastatic tumors of the temporal bone, melanoma initially metastasizes to the internal auditory canal and is characterized by early neurovascular infiltration. This report highlights the pathophysiological characteristics, radiological findings, differential diagnosis, and treatment of metastatic melanoma of the internal auditory canal and cerebellopontine angle.

The transsphenoethmoid approach to the sphenoid sinus and clivus.

Surgical access to the sphenoid sinus and clivus for the resection of benign and malignant disease is difficult and is often associated with significant morbidity. The transsphenoethmoid approach, an extension of a familiar otolaryngological procedure, with or without a limited medial maxillectomy, allows access to this region with little morbidity and excellent cosmetic results. Since 1988, the transsphenoethmoid approach has been used in 15 patients at our institution for resection of primary and recurrent chordomas, chondrosarcomas, pituitary macroadenomas, repair of cerebrospinal fluid leaks, and drainage of petroclival cysts. In most instances, an ipsilateral approach is most satisfactory. When necessary, a contralateral transsphenoethmoid approach is used when the tumor is posterolateral to the internal carotid artery and as far lateral as the abducens nerve.

Grading venous restrictive disease in patients with dural arteriovenous fistulas of the transverse/sigmoid sinus.

Dural arteriovenous fistulas of the transverse/sigmoid sinus usually cause pulse-synchronous bruit but may present catastrophically. Current systems for classifying these vascular malformations do not consider obstruction of venous outflow, which increases the risk of intracranial hemorrhage due to retrograde flow via cortical veins. The authors have developed a grading system based on the severity of venous restrictive disease determined by superselective angiography. In a retrospective analysis of 25 patients with dural arteriovenous fistulas of the transverse/sigmoid sinus treated between 1988 and 1990, the grade of venous restrictive disease reflected the clinical presentation. Visual symptoms and central nervous system hemorrhage were more common in patients with cortical venous drainage and more severe distal venous occlusion (Grade 3: 31% and 31%, respectively; Grade 4: 67% and 100%, respectively) than in patients with cortical venous drainage and mild-to-moderate venous restrictive disease (Grade 2: 13% and 0%, respectively) or those without venous outflow (Grade 1: 0% and 0%, respectively). These preliminary results suggest that this grading system may be useful for predicting the risk of catastrophic clinical presentation and for guiding therapeutic decision-making in patients with dural arteriovenous fistulas of the transverse/sigmoid sinus. A prospective study of a larger number of patients is needed to validate the predictive value of this new grading system.

Bilateral temporal bone encephaloceles after cranial irradiation. Case report.

Irradiation of the central nervous system may cause significant morbidity, including endocrine dysfunction and intellectual impairment. The authors report a case of bilateral temporal bone encephaloceles in a 21-year-old man who had received prophylactic central nervous system irradiation for acute lymphocytic leukemia in early childhood. Endaural encephaloceles are uncommon, and most occur as a complication of mastoid surgery. The etiology, clinical features, radiological diagnosis, and surgical treatment of temporal bone encephaloceles are discussed.

A surgical approach appropriate for targeted cochlear gene therapy in the mouse.

Therapeutic manipulations of the mammalian cochlea, including cochlear gene transfer, have been predominantly studied using the guinea pig as the experimental model. With the significant developments in mouse genomics and the availability of mutant strains of mice with well-characterized hearing loss, the mouse justifiably will be the preferred animal model for therapeutic manipulations. However, the potential advantages of the mouse model have not been fully realized due to the surgical difficulty of accessing its small cochlea. This study describes a ventral approach, instead of the routinely used postauricular approach in other rodents, for accessing the mouse middle and inner ear, and its application in cochlear gene transfer. This ventral approach enabled rapid and direct delivery of liposome-transgene complex to the mouse inner ear while avoiding blood loss, facial nerve morbidity, and mortality. Transgene expression at 3 days was detected in Reissner's membrane, spiral limbus, spiral ligament, and spiral ganglion cells, in a pattern similar to that previously described in the guinea pig. The successful access and delivery of material to the mouse cochlea and the replication of gene expression seen in the guinea pig demonstrated in this study should promote the use of the mouse in future studies investigating targeted cochlear therapy.

Green fluorescent protein as a reporter for gene transfer studies in the cochlea.

This study examined the 'humanized, red-shifted' version of the jellyfish Aequorea victoria green fluorescent protein (hrGFP) as a novel reporter for in vivo gene transfer studies in the cochlea using adeno-associated virus (AAV) vectors. Approximately 10(5) AAV vectors containing the hrGFP reporter gene were infused over 2 days or 1 week into the cochlea of the guinea pig via an osmotic minipump. Saline infused, non-infused, as well as AAV-beta-galactosidase infused guinea pigs served as the negative controls. The hrGFP transgene expression was detected as moderate intensity fluorescence easily distinguished from the background. Increased fluorescence was seen in the spiral ganglion, spiral ligament, spiral limbus, organ of Corti, and Reissner's membrane of the AAV-hrGFP infused animals. Control animals showed minimal fluorescence throughout the cochlea. Comparison of the 2 day and 1 week AAV-hrGFP infused animals showed qualitatively increased fluorescence in the 2 day animals. Background autofluorescence in the stria vascularis was noted in both the experimental and the control animals. In addition, fluorescence was detected in the contralateral cochlea of the AAV-hrGFP infused animals. Subsequent PCR analysis confirmed the presence of viral particles in the AAV-hrGFP infused cochlea as well as in the brain and the contralateral cochlea. This finding has important implications for the eventual implementation of cochlear gene therapy. The results not only reinforce the need to assess the introduction and expression of foreign genes in the target cochlea but also consider issues of viral spread, safety, and modes of gene delivery. This study establishes hrGFP as an effective reporter of gene transfer and transgene expression in the cochlea. GFP's small gene size, stability, ease of detection, and potential for diverse biological applications will be invaluable for a variety of future gene transfer and expression studies in the cochlea.

Cationic liposome mediated transgene expression in the guinea pig cochlea.

Sensorineural hearing loss affects nearly 10% of the American population that is refractory to conventional therapy. Gene therapy represents an intervention with potential therapeutic efficacy. We studied the feasibility of cationic liposome mediated gene transfer within the guinea pig cochlea in vivo following direct microinjection into the cochlea. Transgene expression was persistent up to 14 days in the neurosensory epithelia and surrounding tissue without toxicity and inflammation in the target organ. This study represents the first successful use of cationic liposomes for cochlear gene transfer thus providing a safe and rapid alternative to the use of recombinant viral vectors in gene therapy for inner ear disorders.

Sensorineural and conductive hearing loss associated with lateral semicircular canal malformation.

OBJECTIVE: Lateral semicircular canal (LSCC) malformation is one of the most common radiological inner ear malformations. Traditionally, inner ear malformations are thought to be associated with sensorineural hearing loss (SNHL). Recent experience with patients with LSCC malformation suggested that LSCC malformation may be associated with both SNHL and conductive hearing loss (CHL). The auditory phenotype associated with LSCC malformation is not well delineated. The objective of this study is to define the nature of the hearing loss associated with LSCC malformation. STUDY DESIGN: Retrospective review METHODS: Retrospective review of clinical records, audiological evaluation, and imaging studies. RESULTS: Two patients with unilateral and 13 patients with bilateral LSCC malformation were identified. LSCC malformation was associated with CHL in 14% (4 ears), SNHL in 71% (20 ears), normal hearing in 11% (3 ears) and CHL due to atresia in one ear. Hearing loss varied from mild to profound but did not correlate with the severity of LSCC malformation. In bilateral malformation, the hearing loss was asymmetric in half of the cases. Malformation of the posterior limb of the LSCC was always associated with a large vestibular aqueduct. An absent or rudimentary LSCC was invariably associated with a cochlear abnormality. CONCLUSIONS: LSCC malformation, like other inner ear malformations such as large vestibular aqueduct and X-linked mixed deafness with perilymph gusher, can be associated with CHL, SNHL, or normal hearing. Children with unexplained conductive hearing loss often undergo exploratory surgery to improve hearing. Given that inner ear malformations may be associated with a pure CHL, it is critical that children undergo computed tomography scan of the temporal bone prior to undergoing exploratory surgery.

Contemporary management of cervical tuberculosis.

Although excisional biopsy has traditionally been required to diagnose cervical tuberculosis (TB), fine needle aspiration biopsy (FNAB) has also been found to be useful. The presentation and management of 47 patients diagnosed with cervical TB between 1984 and 1988 were retrospectively reviewed. Chest x-rays were normal in 58% of the patients, and purified protein derivative (PPD) skin testing was positive in 96%. When FNAB was used, TB could be suspected in 83% of cases and definitively established in 62%. Open biopsy correctly diagnosed cervical TB in all masses excised. Medical therapy alone resulted in resolution of disease in 94% of patients diagnosed by FNAB, and subsequent excisional biopsy was necessary in only one patient. The results suggest that FNAB is a useful initial procedure in the diagnosis of cervical TB. Excisional biopsy should be reserved for cases where no diagnosis by FNAB can be made, or for persistent cervical disease despite full-course antituberculous chemotherapy.