SDHB, SDHC, and SDHD mutation screen in sporadic and familial head and neck paragangliomas.

Mutations within three genes, SDHB, SDHC, and SDHD, encoding distinct subunits of a hetero-oligomeric protein known as the mitochondrial complex II, a component of the mitochondrial electron transport chain and the Krebs cycle have been implicated in the pathogenesis of hereditary paraganglioma (PGL). This study describes a mutation screen of SDHB, SDHC, and SDHD in blood and tumor samples of 14 sporadic and three familial cases of head and neck PGL (HNP). Germline mutations in SDHB and SDHD were identified in two of the three affected individuals with familial HNP. The SDHB mutation was a novel 3 base pair, in-frame deletion of AGC at nucleotide 583-585 encoding serine (delS195). The SDHD mutation was a C to T transition within codon 81 causing substitution of proline with leucine (P81L). In contrast to familial cases, no germline or somatic mutations were identified in the 14 sporadic cases of HNP. The presence of mutations within SDHB and SDHD in two of the three samples of familial PGLs and absence of mutations in sporadic cases is consistent with the significant contribution of these genes to familial but not sporadic PGL. The etiology of sporadic PGL remains to be elucidated.

Mutation analysis of Connexin 31 (GJB3) in sporadic non-syndromic hearing impairment.

Mutations in GJB3, the gene encoding the gap junction protein Connexin 31 (CX31), have been pathogenically linked to erythrokeratodermia and non-syndromic autosomal dominant (DFNA2) or recessive hereditary hearing impairment (HHI). To determine the contribution of CX31 to sporadic deafness, we assessed 63 individuals with non-syndromic hearing impairment for CX31 mutations. Single coding exon of CX31 was amplified from genomic DNA and then sequenced. Single nucleotide sequence alteration was present in 15 out of 63 patients (24%), all of the positives being heterozygous for the four different single base pair changes that were detected: C94T, C201T, C357T and C798T. Of these, only C94T transition, identified in two patients, results in amino acid change, R32W, while the other three changes are single nucleotide polymorphisms (SNPs). The R32W substitution in CX31 has been previously documented and is speculated to manifest variable penetrance, similar to the polymorphic allele encoding CX26M34T. Over one-third of all samples were also screened with denaturing high-performance liquid chromatography (dHPLC). Seven out of 25 individuals screened were determined to be positive for CX31 sequence variation. Sequence analysis of the 25 individuals screened identified nucleotide alterations in all of the 7 'positives' and in none of the 16 'negatives' yielding a specificity and sensitivity of 100%. Thus, dHPLC represents a highly efficient CX31 screening technique. This study suggests that while sequence alterations are common, pathogenic mutations of CX31 are infrequent in sporadic non-syndromic hearing impairment.

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.

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.

Detection of fungi in the nasal mucosa using polymerase chain reaction.

HYPOTHESIS: Fungi have been increasingly recognized as important pathogens in sinusitis. However, detection of fungus with conventional culture techniques is insensitive and unreliable. Polymerase chain reaction (PCR) is an exquisitely sensitive assay that can detect the DNA of 10 or less fungal elements. The aim of this study was to compare the sensitivity of conventional culture techniques using PCR analysis. METHODS: Nasal swabs and DNA samples were collected from the nasal cavities of control subjects and patients with chronic sinusitis. Fungal-specific PCR analysis and standard cultures were performed on every sample. chi2 analysis was used to test for statistical differences between groups. RESULTS: PCR analysis detected fungal DNA in 42% and 40% of control subjects and patients with chronic sinusitis while standard cultures were positive in 7% and 0%, respectively. There was no statistically significant difference in the prevalence of fungi in the normal volunteers and patients with chronic rhinosinusitis. CONCLUSION: PCR is significantly more sensitive than nasal swab cultures in detecting the presence of fungi in nasal mucosa. In addition, our study suggests that the presence of fungi alone is insufficient to implicate it as the pathogen in chronic sinusitis.

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.

Cochlear microinjection and its effects upon auditory function in the guinea pig.

Microinjection through the round window membrane has been found to represent a method for vector delivery in intracochlear gene transfer in animal models but breaches the round window membrane, making it necessary to evaluate animals for possible postinjection hearing loss. In the present study healthy guinea pigs were evaluated for their baseline click auditory brainstem response (ABR) thresholds. Each animal was then injected with saline via the round window membrane. After 1 week auditory function was evaluated by click ABR. Animals with increased ABR thresholds were retested at 4 weeks. Animals with 1-week postoperative ABRs similar to baseline were not retested. Results showed that postoperative ABR thresholds in five animals (71%) remained unchanged from baseline, while two animals had increases of 20-25 dB in ABRs after 1 week but recovered baseline ABRs after 4 weeks. The mean baseline ABR threshold was 25.7 dB and was 27.9 dB after 1 week after injection. The difference between preoperative and 1-week postoperative averages was not significant (P = 0.707). In this preliminary study saline microinjection through the round window membrane did not cause permanent hearing loss in the guinea pigs tested, and any damage caused by microinjection appeared to be reversible.

Safety of adeno-associated virus as cochlear gene transfer vector: analysis of distant spread beyond injected cochleae.

The adeno-associated virus (AAV), inoculated into the perilymph, has been shown to be an effective vector for mediating intracochlear transgene expression. The unexpected finding of transgene expression in the contralateral cochlea in previous work raised concern about dissemination of the virus from the target tissue. The current study was undertaken to assess the extent of AAV dissemination following its introduction into the inner ear. Adult male guinea pigs were injected with recombinant AAV into their left ears and sacrificed at 2 or 4 weeks. Various organs including the cochleae were harvested to characterize the presence and expression of the viral DNA. Virus DNA was detected via polymerase chain reaction in the infused and contralateral cochlea and in the cerebellum but not in any other organs, including cortex, heart, lung, liver, spleen, and kidney. Although the viral presence was established in the cerebellum, transgene expression in this organ was undetectable with either Western blot or immunohistochemistry. Transgene expression was demonstrated via immunohistochemistry in multinucleated giant cells in the bone marrow spaces adjacent to the infused and contralateral cochleae. Collectively, these results suggest potential routes for AAV dissemination from the infused cochlea via the cochlear aqueduct or by extension through the temporal bone marrow spaces. This study reinforces the need to investigate factors that mitigate viral leakage.

Human nonsyndromic hereditary deafness DFNA17 is due to a mutation in nonmuscle myosin MYH9.

The authors had previously mapped a new locus-DFNA17, for nonsyndromic hereditary hearing impairment-to chromosome 22q12.2-q13. 3. DFNA17 spans a 17- to 23-cM region, and MYH9, a nonmuscle-myosin heavy-chain gene, is located within the linked region. Because of the importance of myosins in hearing, MYH9 was tested as a candidate gene for DFNA17. Expression of MYH9 in the rat cochlea was confirmed using reverse transcriptase-PCR and immunohistochemistry. MYH9 was immunolocalized in the organ of Corti, the subcentral region of the spiral ligament, and the Reissner membrane. Sequence analysis of MYH9 in a family with DFNA17 identified, at nucleotide 2114, a G-->A transposition that cosegregated with the inherited autosomal dominant hearing impairment. This missense mutation changes codon 705 from an invariant arginine (R) to histidine (H), R705H, within a highly conserved SH1 linker region. Previous studies have shown that modification of amino acid residues within the SH1 helix causes dysfunction of the ATPase activity of the motor domain in myosin II. Both the precise role of MYH9 in the cochlea and the mechanism by which the R705H mutation leads to the DFNA17 phenotype (progressive hearing impairment and cochleosaccular degeneration) remain to be elucidated.

Molecular pathogenesis in sporadic head and neck paraganglioma.

HYPOTHESIS: Similar to familial tumors, sporadic head and neck paragangliomas are associated with chromosomal deletions at either 11q13 or 11q22-23. BACKGROUND: Familial paragangliomas are inherited in an autosomal dominant pattern with genomic imprinting of the maternal allele. Genetic studies of familial paragangliomas have localized the causative genetic defect to two separate loci: 11q13.1 and 11q22-23. The molecular pathogenesis of sporadic head and neck paragangliomas has not been studied. METHODS: Blood and tumor samples from patients with sporadic head and neck paragangliomas were screened for deletions on chromosome 11 using DNA microsatellite markers and polymerase chain reaction. Polymerase chain reaction-amplified alleles from tumor specimens were compared with those from the blood of eight patients. A greater than 50% reduction in band intensity (as determined by densitometric analysis) between blood and tumor sample was indicative of a chromosomal deletion. RESULTS: Three of the eight patients were found to have deletions at chromosome 11q: two at chromosome 11q22-23 and one at 11q13. CONCLUSIONS: Sporadic head and neck paragangliomas are associated with deletions at chromosome 11q13 and 11q22-23. It is thus likely that sporadic and familial paragangliomas share a similar molecular pathogenesis.

Identification of aquaporin 5 (AQP5) within the cochlea: cDNA cloning and in situ localization.

Dysfunction of fluid and electrolyte homeostasis is considered to cause variety of inner ear disorders. One group of candidate proteins that may play a critical role in the inner ear fluid homeostasis is the aquaporins, a family of proteins whose members have well defined roles in fluid transport in variety of organs. This study reports the identification of AQP5, a member of the aquaporin family, within the rat inner ear and its in situ localization. AQP5 was initially identified within rat cochlear RNA via RT-PCR and sequence analysis of the amplified fragments. Immunoblot of cochlear homogenate yielded a predominant AQP5-immunoreactive band of M(r) 35 kDa. The anti-AQP5 immunoreactivity, indicating expression of the AQP5 polypeptide, was localized within the cochlea in situ to the cell types that form the lateral wall of the cochlear duct-the external sulcus (ES) cells and the cells of the spiral prominence. Expression of AQP5 was observed in the apical turn but not the basal turn of the cochlea; nor was it observed in the vestibular neuroepithelia or its supporting cells. The restricted expression of AQP5 to the apical turns of the cochlea suggests its potential role in low frequency hearing.

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.

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.

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.

Expression of adeno-associated virus integrated transgene within the mammalian vestibular organs.

HYPOTHESIS: Adeno-associated virus (AAV) is a suitable viral vector for transgene expression within the mammalian vestibular organs. BACKGROUND: In vivo introduction and expression of a foreign gene within the cochlear tissues have been established using a variety of viral vectors and guinea pig as the animal model. However, the vestibular neuroepithelia of the mammalian inner ear as a potential target for transgene expression remain to be investigated. METHODS: Transgene expression was assessed within the vestibular neuroepithelia of guinea pigs after intracochlear infusion of the recombinant AAV vector with the aid of an osmotic minipump. Evaluation of the transgene within the vestibular apparatus focused on its duration of expression from 2-24 weeks after intracochlear AAV infusion using immunohistochemistry. RESULTS: In the AAV-beta-galactosidase (beta-gal)-infused animals, the sensory hair cells as well as the supporting epithelial cells of cristae and maculae were positive for the transgene expression. The relative level of beta-gal expression was noted to decrease progressively over time. Transduction of the vestibular neuroepithelia also was observed in the contralateral ear, a finding that has been documented previously in AAV-integrated transgene expression in the cochlea. CONCLUSION: This study reports the first demonstration of introduction and long-term transgene expression within the vestibular neuroepithelia. The ability to express a foreign gene with the vestibular system allows the possibility of experimental and therapeutic application of gene therapy technology to address vestibular function and dysfunction.

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.

Genotype-phenotype correlations in type 1 Waardenburg syndrome.

Type 1 Waardenburg syndrome (WS1) is an autosomal dominant disorder characterized by dystopia canthorum, sensorineural deafness, and pigmentary disturbances. Previous work has linked the disease to PAX3, a transcription factor with two highly conserved DNA binding motifs: a paired box and a homeobox. Several mutations within the paired box of PAX3 have been reported. We have identified the first two mutations within the homeobox in two different families. Herein, we report the phenotypic expression of WS1 in these two families and explore the implications for possible genotype-phenotype correlations. The phenotypic expression was variable within and between the families. Sensorineural hearing loss was present in 71% of affected individuals. Spectrum of hearing loss included unilateral high frequency (>4000 Hz), profound unilateral hearing loss, and bilateral profound hearing loss. The severity of hearing impairment varied significantly within and among families. Phenotypically, these two WS1 families with mutations within the PAX3 homeobox could not be differentiated from those families with paired box mutations. More precise genotype/phenotype correlation may be possible when additional mutations are described throughout the PAX3 gene.

Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig.

Gene therapy is currently being used to treat many disorders including cancer, viral infection and the degenerative and fatal diseases of the cardiovascular and the central nervous systems. However, the potential use of gene therapy for alleviation of hearing impairment has not been investigated despite the absence of effective therapy for most forms of inherited hearing disorders. The purpose of this study was to assess the feasibility of introducing genetic material directly into the peripheral auditory system using adeno-associated virus (AAV) as the transfection vector and Hartley guinea pigs as the animal model. Approximately 10(5) particles of AAV containing the bacterial beta-galactosidase (beta-gal) sequence with Ad 2 major late promoter were infused into the cochlea of the animal with the aid of an osmotic minipump. Animals were killed after 2 weeks. Two Hartley guinea pigs with intracochlear saline infusion and four unoperated (nonperfused) animals served as negative controls. Both, the infused and the contralateral, non-infused cochleae were harvested from each animal, decalcified, and embedded in paraffin. Sections, 8 microns in width, were cut from the embedded cochleae and assayed for beta-gal expression via immunohistochemistry. Animals perfused with AAV showed intense immunohistochemical reactivity in the spiral limbus, spiral ligament, spiral ganglion cells and the organ of Corti in the perfused cochlea and a much weaker staining but with similar pattern in the contralateral ear. Cochleae from saline-infused and unoperated animals were devoid of the DAB stain. This study demonstrates for the first time in vivo expression of a foreign gene within the mammalian inner ear resulting from its localized, AAV-mediated introduction. The ability to introduce and stably express exogenous genetic material in the peripheral auditory system will have both experimental and therapeutic benefits. These results lay the groundwork for future studies assessing the potential use of gene therapy for alleviation of hearing impairment.