Unique Clinical Language Patterns Among Expert Vestibular Providers Can Predict Vestibular Diagnoses.

OBJECTIVE: To identify novel language usage by expert providers predictive of specific vestibular conditions. STUDY DESIGN: Retrospective chart review and natural language processing. Level IV. SETTING: Tertiary referral center. PATIENTS: Patients seen for vestibular complaint. INTERVENTION(S): Natural language processing and machine learning analyses of semantic and syntactic patterns in clinical documentation from vestibular patients. MAIN OUTCOME MEASURE: Accuracy of Naïve Bayes predictive models correlating language usage with clinical diagnoses. RESULTS: Natural language analyses on 866 physician-generated histories from vestibular patients found 3,286 unique examples of language usage of which 614 were used 10 or greater times. The top 15 semantic types represented only 11% of all Unified Medical Language System semantic types but covered 86% of language used in vestibular patient histories. Naïve Bayes machine learning algorithms on a subset of 255 notes representing benign paroxysmal positional vertigo, vestibular migraine, anxiety-related dizziness and central dizziness generated strong predictive models showing an average sensitivity rate of 93.4% and a specificity rate of 98.2%. A binary model for assessing whether a subject had a specific diagnosis or not had an average AUC for the receiver operating characteristic curves of .995 across all conditions. CONCLUSIONS: These results indicate that expert providers utilize unique language patterns in vestibular notes that are highly conserved. These patterns have strong predictive power toward specific vestibular diagnoses. Such language elements can provide a simple vocabulary to aid nonexpert providers in formulating a differential diagnosis. They can also be incorporated into clinical decision support systems to facilitate accurate vestibular diagnosis in ambulatory settings.

TMTC2 variant associated with sensorineural hearing loss and auditory neuropathy spectrum disorder in a family dyad.

BACKGROUND: Sensorineural hearing loss (SNHL) is a common form of hearing loss that can be inherited or triggered by environmental insults; auditory neuropathy spectrum disorder (ANSD) is a SNHL subtype with unique diagnostic criteria. The genetic factors associated with these impairments are vast and diverse, but causal genetic factors are rarely characterized. METHODS: A family dyad, both cochlear implant recipients, presented with a hearing history of bilateral, progressive SNHL, and ANSD. Whole-exome sequencing was performed to identify coding sequence variants shared by both family members, and screened against genes relevant to hearing loss and variants known to be associated with SNHL and ANSD. RESULTS: Both family members are successful cochlear implant users, demonstrating effective auditory nerve stimulation with their devices. Genetic analyses revealed a mutation (rs35725509) in the TMTC2 gene, which has been reported previously as a likely genetic cause of SNHL in another family of Northern European descent. CONCLUSION: This study represents the first confirmation of the rs35725509 variant in an independent family as a likely cause for the complex hearing loss phenotype (SNHL and ANSD) observed in this family dyad.

Association of TMTC2 With Human Nonsyndromic Sensorineural Hearing Loss.

IMPORTANCE: Sensorineural hearing loss (SNHL) is commonly caused by conditions that affect cochlear structures or the auditory nerve, and the genes identified as causing SNHL to date only explain a fraction of the overall genetic risk for this debilitating disorder. It is likely that other genes and mutations also cause SNHL. OBJECTIVE: To identify a candidate gene that causes bilateral, symmetric, progressive SNHL in a large multigeneration family of Northern European descent. DESIGN, SETTING, AND PARTICIPANTS: In this prospective genotype and phenotype study performed from January 1, 2006, through April 1, 2016, a 6-generation family of Northern European descent with 19 individuals having reported early-onset hearing loss suggestive of an autosomal dominant inheritance were studied at a tertiary academic medical center. In addition, 179 unrelated adult individuals with SNHL and 186 adult individuals reporting nondeafness were examined. MAIN OUTCOMES AND MEASURES: Sensorineural hearing loss. RESULTS: Nine family members (5 women [55.6%]) provided clinical audiometric and medical records that documented hearing loss. The hearing loss is characterized as bilateral, symmetric, progressive SNHL that reached severe to profound loss in childhood. Audiometric configurations demonstrated a characteristic dip at 1000 to 2000 Hz. All affected family members wear hearing aids or have undergone cochlear implantation. Exome sequencing and linkage and association analyses identified a fully penetrant sequence variant (rs35725509) on chromosome 12q21 (logarithm of odds, 3.3) in the TMTC2 gene region that segregates with SNHL in this family. This gene explains the SNHL occurrence in this family. The variant is also associated with SNHL in a cohort of 363 unrelated individuals (179 patients with confirmed SNHL and 184 controls, P = 7 × 10-4). CONCLUSIONS AND RELEVANCE: A previously uncharacterized gene, TMTC2, has been identified as a candidate for causing progressive SNHL in humans. This finding identifies a novel locus that causes autosomal dominant SNHL and therefore a more detailed understanding of the genetic basis of SNHL. Because TMTC2 has not been previously reported to regulate auditory function, the discovery reveals a potentially new, uncharacterized mechanism of hearing loss.

The Chinchilla Research Resource Database: resource for an otolaryngology disease model.

The long-tailed chinchilla (Chinchilla lanigera) is an established animal model for diseases of the inner and middle ear, among others. In particular, chinchilla is commonly used to study diseases involving viral and bacterial pathogens and polymicrobial infections of the upper respiratory tract and the ear, such as otitis media. The value of the chinchilla as a model for human diseases prompted the sequencing of its genome in 2012 and the more recent development of the Chinchilla Research Resource Database (http://crrd.mcw.edu) to provide investigators with easy access to relevant datasets and software tools to enhance their research. The Chinchilla Research Resource Database contains a complete catalog of genes for chinchilla and, for comparative purposes, human. Chinchilla genes can be viewed in the context of their genomic scaffold positions using the JBrowse genome browser. In contrast to the corresponding records at NCBI, individual gene reports at CRRD include functional annotations for Disease, Gene Ontology (GO) Biological Process, GO Molecular Function, GO Cellular Component and Pathway assigned to chinchilla genes based on annotations from the corresponding human orthologs. Data can be retrieved via keyword and gene-specific searches. Lists of genes with similar functional attributes can be assembled by leveraging the hierarchical structure of the Disease, GO and Pathway vocabularies through the Ontology Search and Browser tool. Such lists can then be further analyzed for commonalities using the Gene Annotator (GA) Tool. All data in the Chinchilla Research Resource Database is freely accessible and downloadable via the CRRD FTP site or using the download functions available in the search and analysis tools. The Chinchilla Research Resource Database is a rich resource for researchers using, or considering the use of, chinchilla as a model for human disease.Database URL: http://crrd.mcw.edu.

Development of a Statistical Model for the Prediction of Common Vestibular Diagnoses.

IMPORTANCE: Treatment of patients with vestibular disorders can be complex, requires lengthy clinic visit time, and uses greater clinical resources for diagnosis. A pre-encounter intake questionnaire may predict the most common disorders, allowing for more efficient allocation of resources and use of clinicians. OBJECTIVE: To develop a statistical model for predicting vestibular diagnoses, prior to clinical evaluation, from an intake questionnaire. DESIGN, SETTING, AND PARTICIPANTS: Retrospective review of 414 consecutive new vestibular patient intake questionnaires (September 2012 through January 2014) and associated medical records with performance of logistic regression analyses and development of predictive models (July 2013 through May 2015). INTERVENTIONS: Use of a vestibular intake questionnaire for triaging of new patients with complaints of dizziness. MAIN OUTCOMES AND MEASURES: Predictors for the diagnosis of benign paroxysmal positional vertigo (BPPV), Ménière's disease, and vestibular migraine. RESULTS: Of the 414 questionnaires analyzed, 381 (92%) had clinician information necessary to define a final diagnosis. Patients were 34% male and had a mean (range) age of 57 (19-91) years. Of the diagnoses, 183 (48%) were ear related (including 103 BPPV and 49 Meniere's disease), 141 (37%) neurological (including 109 vestibular migraine), 36 (9%) medical, 8 (2%) of psychological origin, 46 (12%) of unknown etiology, and 33 (9%) other causes. The diagnosis of BPPV could be predicted from 4 variables with a sensitivity of 79% and specificity of 65%. The diagnosis of Ménière's disease could be predicted from 5 variables with a sensitivity of 81% and specificity of 85%. The diagnosis of vestibular migraine could be predicted from 4 variables with a sensitivity of 76% and specificity of 59%. CONCLUSIONS AND RELEVANCE: A pre-encounter history questionnaire can provide useful diagnostic information for common vestibular disorders. This can help direct appointment scheduling to improve clinical efficiency, time to intervention, and use of resources. Further refinement may enable the use of shorter questionnaires or screening algorithms.

Nontypeable Haemophilus influenzae inhibits autolysis and fratricide of Streptococcus pneumoniae in vitro.

Streptococcus pneumoniae (SP) and nontypeable Haemophilus influenzae (NTHi) are common commensals of the human airway and major bacterial pathogens of otitis media (OM) and other upper airway infections. The interaction between them may play an important role in the pathogenesis of polymicrobial infections. Although previous studies suggested NTHi could promote pneumococcal survival and biofilm formation, how NTHi affects pneumococcal activities has not been defined. Our data in the present studies indicated that the outcome of the interaction between SP and NTHi was in a cell-density-dependent manner and the enhancement of pneumococcal survival happened at the later stages of culturing. Using quantitative PCR, we found that the expression of pneumococcal genes regulating autolysis and fratricide, lytA and cbpD, were significantly down-regulated in co-culture with NTHi. We further observed that influence of NTHi was not on direct cell-to-cell contact, but that this contact may contribute to the interaction between these two microorganisms. These results suggest that pneumococcal survival and biofilm formation can be enhanced by down-regulating pneumococcal cell wall hydrolase production thereby inhibiting pneumococcal autolysis and fratricide in the presence of NTHi.

A novel otoferlin splice-site mutation in siblings with auditory neuropathy spectrum disorder.

We characterize a novel otoferlin mutation discovered in a sibling pair diagnosed with auditory neuropathy spectrum disorder and investigate auditory nerve function through their cochlear implants. Genetic sequencing revealed a homozygous mutation at the otoferlin splice donor site of exon 28 (IVS28 + 1G>T) in both siblings. Functional investigation showed that the intronic sequence between exons 28 and 29 was retained in the mutated minigenes that were expressed in 293T cells. Auditory nerve compound action potential recovery functions in the siblings demonstrated different rates of neural recovery, with sibling AN1 showing rapid recovery (1.14 ms) and AN2 showing average recovery (0.78 ms) compared to subjects with sensorineural hearing loss (average: adults 0.71 ms, children 0.85 ms). Differences in neural recovery were consistent with speech perception differences between the siblings. Genotype information may indicate site of lesion in hearing loss; however, additional, as yet, unknown factors may impact clinical outcomes and must be considered.

Cholesteatoma growth and proliferation: posttranscriptional regulation by microRNA-21.

OBJECTIVES: The goal of this study was to identify novel regulatory mechanisms controlling the growth and proliferation of cholesteatoma. Specifically, the potential role of microRNAs, regulators of protein translation, was studied in cholesteatoma. STUDY DESIGN: This study represents a molecular biologic investigation characterizing and comparing microRNA and protein expression in cholesteatoma and normal postauricular skin. METHODS: Cholesteatoma and normal skin were taken from patients at the time of surgery. Tissue was processed for RNA and protein extraction. Real-time reverse-transcriptase-polymerase chain reaction was used to assess levels of human microRNAs, reverse-transcriptase-polymerase chain reaction was used to confirm the presence of upstream regulators, and Western blot analyses were used to assess levels of downstream target proteins. RESULTS: Among the microRNAs investigated, human microRNA-21 (hsa-miR-21) showed a 4.4-fold higher expression in cholesteatoma as compared with normal skin (p = 0.0011). The downstream targets of hsa-miR-21, PTEN and programmed cell death 4, were found to be greatly reduced in 3 of 4 cholesteatoma samples. Proposed upstream regulators of hsa-miR-21 expression (CD14, interleukin 6R, gp130, and signal transducer and activator of transcription 3) were present in all cholesteatoma tissues. CONCLUSION: MicroRNAs represent powerful regulators of protein translation, and their dysregulation has been implicated in many neoplastic diseases. This study specifically identified up-regulation of hsa-miR-21 concurrent with down-regulation of potent tumor suppressor proteins PTEN and programmed cell death 4. These proteins control aspects of apoptosis, proliferation, invasion, and migration. The results of this study were used to develop a model for cholesteatoma proliferation through microRNA dysregulation. This model can serve as a template for further study into potential RNA-based therapies for the treatment of cholesteatoma.

Mucin gene 19 (MUC19) expression and response to inflammatory cytokines in middle ear epithelium.

Mucin gene 19 (MUC19) has been identified as a major gel-forming mucin in the human middle ear (ME). The objectives of this investigation were to characterize the expression and assess the regulation of MUC19 in the ME cell culture models utilized in the study of otitis media (OM). Findings demonstrate that MUC19 is expressed in both human immortalized cell culture (HMEEC) and chinchilla primary epithelial culture (CMEEC). ME exposure to inflammatory cytokines TNF-alpha, IL-1beta, IL-6 and IL-8 up-regulate MUC19 transcription, most robustly after exposure to TNF-alpha. Kinetic experiments suggest a relative early response in MUC19 transcription and a down-regulation after prolonged exposure. Glycoprotein production was increased in response to the increased transcription as well. Similar to other mucin genes in the ME, MUC19 is differentially regulated after exposure to inflammatory cytokines. The large size, gel-forming properties and up-regulation in response to important inflammatory cytokines of MUC19 suggest that it has significant potential to play a role in both physiology and pathophysiology of the ME.

Gene expression differences in infected and noninfected middle ear complementary DNA libraries.

OBJECTIVES: To investigate genetic differences in middle ear mucosa (MEM) with nontypeable Haemophilus influenzae (NTHi) infection. Genetic upregulation and downregulation occurs in MEM during otitis media (OM) pathogenesis. A comprehensive assessment of these genetic differences using the techniques of complementary DNA (cDNA) library creation has not been performed. DESIGN: The cDNA libraries were constructed from NTHi-infected and noninfected chinchilla MEM. Random clones were picked, sequenced bidirectionally, and submitted to the National Center for Biotechnology Information (NCBI) Expressed Sequence Tags database, where they were assigned accession numbers. These numbers were used with the basic local alignment search tool (BLAST) to align clones against the nonredundant nucleotide database at NCBI. RESULTS: Analysis with the Web-based statistical program FatiGO identified several biological processes with significant differences in numbers of represented genes. Processes involved in immune, stress, and wound responses were more prevalent in the NTHi-infected library. S100 calcium-binding protein A9 (S100A9); secretory leukoprotease inhibitor (SLPI); beta(2)-microglobulin (B2M); ferritin, heavy-chain polypeptide 1 (FTH1); and S100 calcium-binding protein A8 (S100A8) were expressed at significantly higher levels in the NTHi-infected library. Calcium-binding proteins S100A9 and S100A8 serve as markers for inflammation and have antibacterial effects. Secretory leukoprotease inhibitor is an antibacterial protein that inhibits stimuli-induced MUC1, MUC2, and MUC5AC production. CONCLUSIONS: A number of genes demonstrate changes during the pathogenesis of OM, including SLPI, which has an impact on mucin gene expression; this expression is known to be an important regulator in OM. The techniques described herein provide a framework for future investigations to more thoroughly understand molecular changes in the middle ear, which will likely be important in developing new therapeutic and intervention strategies.

Distribution of two-pore-domain potassium channels in the adult rat vestibular periphery.

Constitutively active background or "leak" two-pore-domain potassium (K(+)) channels (Kcnk family), as defined by lack of voltage and time dependency are central to electrical excitability of cells by controlling resting membrane potential and membrane resistance. Inhibition of these channels by several neurotransmitters, e.g. glutamate, or acetylcholine, induces membrane depolarization and subsequent action potential firing as well as increases membrane resistance amplifying responses to synaptic inputs. In contrast, their opening contributes to hyperpolarization. Because of their central role in determining cellular excitability and response to synaptic stimulation, these channels likely play a role in the differential effects of vestibular efferent neurons on afferent discharge. Microarray data from previous experiments showed Kcnk 1, 2, 3, 6, 12 and 1 5 mRNA in Scarpa's ganglia. Real-time RT-PCR showed Kcnk 1, 2, 3, 6, 12 and 15 mRNA expression in Scarpa's ganglia and Kcnk 1, 2, 3, 6, 12 but not 15 mRNA expression in the crista ampullaris. We studied the distribution of two-pore-domain potassium channels K(2P)1.1, 2.1, 3.1 and 6.1 like immunoreactivity (corresponding to Kcnk genes 1, 2, 3 and 6) in the vestibular periphery. K(2P)1.1 (TWIK 1) immunoreactivity was detected along nerve terminals, supporting cells and blood vessels of the crista ampullaris and in the cytoplasm of neurons of the Scarpa's ganglia. K(2P)2.1 (TREK 1) immunoreactivity was detected in nerve terminals and transitional cells of the crista ampullaris, in the vestibular dark cells and in neuronal fibers and somata of neurons of Scarpa's ganglia. K(2P)3.1 (TASK 1) immunoreactivity was detected in supporting cells and transitional cells of the crista ampullaris, in vestibular dark cells and in neuron cytoplasm within Scarpa's ganglia. K(2P)6.1 (TWIK 2) immunoreactivity was detected in nerve terminals, blood vessels hair cells and transitional cells of the crista ampullaris and in the somata and neuron fibers of Scarpa's ganglia.

In silico analysis of 2085 clones from a normalized rat vestibular periphery 3' cDNA library.

The inserts from 2400 cDNA clones isolated from a normalized Rattus norvegicus vestibular periphery cDNA library were sequenced and characterized. The Wackym-Soares vestibular 3' cDNA library was constructed from the saccular and utricular maculae, the ampullae of all three semicircular canals and Scarpa's ganglia containing the somata of the primary afferent neurons, microdissected from 104 male and female rats. The inserts from 2400 randomly selected clones were sequenced from the 5' end. Each sequence was analyzed using the BLAST algorithm compared to the Genbank nonredundant, rat genome, mouse genome and human genome databases to search for high homology alignments. Of the initial 2400 clones, 315 (13%) were found to be of poor quality and did not yield useful information, and therefore were eliminated from the analysis. Of the remaining 2085 sequences, 918 (44%) were found to represent 758 unique genes having useful annotations that were identified in databases within the public domain or in the published literature; these sequences were designated as known characterized sequences. 1141 sequences (55%) aligned with 1011 unique sequences had no useful annotations and were designated as known but uncharacterized sequences. Of the remaining 26 sequences (1%), 24 aligned with rat genomic sequences, but none matched previously described rat expressed sequence tags or mRNAs. No significant alignment to the rat or human genomic sequences could be found for the remaining 2 sequences. Of the 2085 sequences analyzed, 86% were singletons. The known, characterized sequences were analyzed with the FatiGO online data-mining tool (http://fatigo.bioinfo.cnio.es/) to identify level 5 biological process gene ontology (GO) terms for each alignment and to group alignments with similar or identical GO terms. Numerous genes were identified that have not been previously shown to be expressed in the vestibular system. Further characterization of the novel cDNA sequences may lead to the identification of genes with vestibular-specific functions. Continued analysis of the rat vestibular periphery transcriptome should provide new insights into vestibular function and generate new hypotheses. Physiological studies are necessary to further elucidate the roles of the identified genes and novel sequences in vestibular function.

Molecular characterization of two novel splice variants of G alphai2 in the rat vestibular periphery.

GTP binding proteins play an important role in mediating signals transduced across the cell membrane by membrane-bound receptors. We previously described a partial sequence, termed Galphai2vest, obtained from rat vestibular tissue that was nearly identical to rat Galphai2. Using an experimental strategy to further characterize Galphai2vest (GenBank accession number AF189020) and identify other possible Galphai2-related transcripts expressed in the rat vestibular periphery, we employed a RecA-based gene enrichment protocol in place of conventional library screening techniques. We identified two novel Galphai2 splice variants, Galphai2(a) (GenBank accession number AY899210) and Galphai2(b) (GenBank accession number AY899211), that have most of exons 8 and 9 deleted, and exons 5 through 9 deleted, respectively. In situ hybridization studies were completed to determine the differential expression of Galphai2 between the vestibular primary afferent neurons and the vestibular end organs. Computer modeling and predicted 3D conformation of the wild type Galphai2 and the two splice variants were completed to evaluate the changes associated with the Gbetagamma and GTP binding sites. These two novel alternatively spliced isoforms of Galphai2 putatively encode truncated proteins that could serve unique roles in the physiology of the vestibular neuroepithelium. Galphai2vest was found to be a processed pseudogene.

G-protein Golfalpha (GNAL) is expressed in the vestibular end organs and primary afferent neurons of Rattus norvegicus.

Guanine nucleotide binding proteins (G-proteins) play an important role in mediating signals transduced across the cell membrane by membrane-bound receptors. The precise role of these proteins and their coupled receptors in the physiology of the vestibular neuroepithelium is poorly understood. Although Golfalpha was originally discovered in the olfactory neuroepithelium and striatum, we recently identified this G-protein alpha subunit in a normalized cDNA library constructed from rat vestibular end organs and vestibular nerves including Scarpa's ganglia. In order to further characterize Golfalpha in the rat vestibular periphery, we used in situ hybridization and reverse transcription polymerase chain reaction to determine the anatomic context of this gene expression. Golfalpha was found in both the end organs and the ganglia and could serve unique roles in the physiology of the vestibular neuroepithelium.

Assessment of differential gene expression in vestibular epithelial cell types using microarray analysis.

Current global gene expression techniques allow the evaluation and comparison of the expression of thousands of genes in a single experiment, providing a tremendous amount of information. However, the data generated by these techniques are context-dependent, and minor differences in the individual biological samples, methodologies for RNA acquisition, amplification, hybridization protocol and gene chip preparation, as well as hardware and analysis software, lead to poor correlation between the results. One of the significant difficulties presently faced is the standardization of the protocols for the meaningful comparison of results. In the inner ear, the acquisition of RNA from individual cell populations remains a challenge due to the high density of the different cell types and the paucity of tissue. Consequently, laser capture microdissection was used to selectively collect individual cells and regions of cells from cristae ampullares followed by extraction of total RNA and amplification to amounts sufficient for high throughput analysis. To demonstrate hair cell-specific gene expression, myosin VIIA, calmodulin and alpha9 nicotinic acetylcholine receptor subunit mRNAs were amplified using reverse transcription-polymerase chain reaction (RT-PCR). To demonstrate supporting cell-specific gene expression, cyclin-dependent kinase inhibitor p27kip1 mRNA was amplified using RT-PCR. Subsequent experiments with alpha9 RT-PCR demonstrated phenotypic differences between type I and type II hair cells, with expression only in type II hair cells. Using the laser capture microdissection technique, microarray expression profiling demonstrated 408 genes with more than a five-fold difference in expression between the hair cells and supporting cells, of these 175 were well annotated. There were 97 annotated genes with greater than a five-fold expression difference in the hair cells relative to the supporting cells, and 78 annotated genes with greater than a five-fold expression difference in the supporting cells relative to the hair cells.

Selective acquisition of individual cell types in the vestibular periphery for molecular biology studies.

OBJECTIVES: To develop a method for characterizing the transcriptome of individual cell types in the inner ear sensory epithelia. STUDY DESIGN: We employed the technique of laser capture microdissection to obtain enriched populations of hair cells and supporting cells. The respective mRNAs were extracted, reverse transcribed, and amplified using PCR. RESULTS: We were able to isolate RNAs with good integrity from enriched cell populations obtained with laser capture microscopy and amplify specific mRNA targets. CONCLUSIONS: We can now investigate the molecular differences between the different cell types in the inner ear sensory epithelia as identified by morphological criteria. SIGNIFICANCE: Analysis of gene expression profiles in the inner ear cell types has been hampered by the small size of this tissue and by the compact histoarchitecture of the sensory epithelia; however, the present technique offers new possibilities for the analysis of transcriptomes in the vestibular periphery using available high-throughput gene expression analysis methods.

Connexin 26 and connexin 30 mutations in children with nonsyndromic hearing loss.

OBJECTIVES/HYPOTHESIS: Mutations in the connexin 26 (Cx26) or gap junction beta 2 gene are the leading cause of hereditary nonsyndromic sensorineural hearing loss in Caucasians. The Cx26 coding region of 68 children with nonsyndromic sensorineural hearing loss was sequenced to determine the frequency and type of Cx26 mutations in this population. Screening was also performed for a common connexin 30 (Cx30) or gap junction beta 6 mutation (del [GJB6-D13S1830]). Children also underwent audiological testing to determine whether any correlation exists between Cx26 mutations and severity of hearing loss. STUDY DESIGN: In all, 68 children with nonsyndromic sensorineural hearing loss were screened for Cx26 and Cx30 mutations by polymerase chain reaction and direct sequencing. METHODS: Genomic DNA was amplified by polymerase chain reaction using primers that flank the entire Cx26 coding region. Screening for the 342-kb Cx30 deletion was performed using primers that amplified the breakpoint junction of the deletion. The amplicons were then sequenced in both directions and analyzed for mutations. Audiometric testing, including pure-tone audiometry and auditory evoked brainstem response, was also performed to determine the degree of hearing loss. RESULTS: Twenty-seven of 68 children tested had mutations in Cx26 with 35delG being the most prevalent. Ten additional Cx26 mutations were detected including a novel compound heterozygote. Two children were heterozygous for the Cx30 del (GJB6-D13S1830) mutation. CONCLUSION: Cx26 and Cx30 mutations were present in 41.2% of children tested in the study population. Audiometric data supported previous studies demonstrating a greater degree of hearing loss in subjects who are homozygous for the 35delG mutation.

Gene discovery using a human vestibular schwannoma cDNA library constructed from a patient with neurofibromatosis type 2 (NF2).

BACKGROUND: Despite a strong association of schwannomin/merlin gene mutations with vestibular schwannoma formation, the regulatory mechanisms and biologic pathways involved are still largely unknown. The hypothesis of this study is that the genesis and growth characteristics of neurofibromatosis type 2 (NF2)-associated vestibular schwannomas are determined by genetic alterations that vary in gene transcript expression; this transcript expression includes oncogenic gene products that may be identified by construction and sequencing of a cDNA library from NF2-associated vestibular schwannoma. METHODS: Approximately 3 mL of fresh tumor was obtained during resection of a 4-cm vestibular schwannoma from a patient with NF2. Poly(A)(+) mRNA was isolated, synthesized into double-stranded cDNA, and unidirectionally inserted into Uni-Zap XR (Stratagene, La Jolla, CA) bacteriophage vectors. Bacteriophage vectors containing cDNA inserts were processed into phagemids according to Uni-Zap XR protocol, and inserted vectors were sequenced and analyzed using BLAST software (National Institutes of Health, Bethesda, MD) with GenBank, EMBL, DDBJ, and PBD databases. RESULTS: The cDNA library contained 2.4 million primary plaques. Inserts averaged 1.8 kilobases (kb) in length, with a range of 0.8 to 3.0 kb. BLAST multidatabase comparison of the sequence data obtained from 50 randomly selected clones yielded identification of 13 sequences representing known human genes and 17 sequences representing cloned sequences with unknown function. Three clones represented sequences not previously described in vestibular schwannomas but strongly implicated in oncogenesis within other tissues. CONCLUSIONS: These data have implications for understanding the molecular mechanisms of vestibular schwannoma tumor biology. Identified genes may provide future diagnostic/prognostic markers and therapeutic targets.

Expression of G-protein alpha subunit genes in the vestibular periphery of Rattus norvegicus and their chromosomal mapping.

OBJECTIVE: Heterotrimeric G-proteins play an important role in mediating signals transduced across the cell membrane by membrane-bound receptors. The precise role of G-proteins and their coupled receptors in the physiology of the vestibular neuroepithelium is not well understood. The purpose of this study was to better define the role of these proteins by examining their expression in the rat vestibular periphery and characterizing their chromosomal location. MATERIAL AND METHODS: To characterize G-protein alpha subunit gene expression in the target tissue of interest, we performed polymerase chain reaction (PCR) using degenerate G-protein primers corresponding to conserved regions in the G-protein alpha subunit coding sequence on a normalized rat vestibular cDNA library. PCR amplicons were cloned and 50 clones were randomly selected and sequenced. Radiation hybrid (RH) mapping was used to determine the chromosomal location of G alpha(olf) and two previously identified G-protein alpha subunits--G alpha(i2) and G alpha(i2(vest))--in the rat genome. RESULTS: The following G-protein alpha subunits were identified in the normalized cDNA library: G alpha(olf), G alpha(s), G alpha(o) and G alpha(s2). G alpha(olf) maps to chromosome 18 between markers D18Mit17b and D18Mgh2. G alpha(i2) maps to chromosome 8 between markers D8Rat65 and D8Mgh2. G alpha(i2(vest)) maps to chromosome 1 between markers D1Rat132 and D1Rat202. These chromosomal locations in the rat genome are syntenic to chromosomal regions in which the homologous G-protein alpha subunit genes have been localized in the human and mouse genomes, further validating RH mapping as an effective and accurate tool. We were unable to RH map the location of G alpha(o) due to its extensive homology with the hamster gene. CONCLUSION: The characterization of G-protein alpha subunit gene expression in the vestibular periphery and the chromosomal localization of these genes in the rat revealed that a diverse group of these second messengers are expressed.

Radiation hybrid mapping of five muscarinic acetylcholine receptor subtype genes in Rattus norvegicus.

Acetylcholine is the main neurotransmitter of the vestibular efferent system and a wide variety of muscarinic and nicotinic acetylcholine receptors are expressed in the vestibular periphery. The role of these receptors and in particular the role of muscarinic acetylcholine receptors in the physiology of the vestibular neuroepithelium is not understood. Congenic and consomic rats are a convenient way to investigate the involvement of candidate genes in the manifestation of defined traits. To use congenic or consomic rats to elucidate the roles of these receptors in vestibular physiology or pathology the chromosomal location of the genes encoding these receptors has to be determined. Using radiation hybrid (RH) mapping and a rat RH map server (www.rgd.mcw.edu/RHMAP SERVER/), we determined the chromosomal locations of the muscarinic acetylcholine receptor genes in the rat (Rattus norvegicus). The m1-m5 muscarinic subtypes mapped to the following chromosomes: Chrm1, chromosome 1; Chrm2, chromosome 4; Chrm3, chromosome 17; Chrm4, chromosome 3; and Chrm5, chromosome 3. With the chromosomal location for each of these muscarinic subtypes known, it is now possible to develop congenic and consomic strains of rats that can be used to study the functions of each of these subtypes.