Christy B. Erbe

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.

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.

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-α, IL-1β, IL-6 and IL-8 up-regulate MUC19 transcription, most robustly after exposure to TNF-α. 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.

Radiation hybrid mapping of 11 alpha and beta nicotinic acetylcholine receptor genes in rattus norvegicus

Acetylcholine is the main neurotransmitter of the vestibular efferents and a wide variety of muscarinic and nicotinic acetylcholine receptors are expressed in the vestibular periphery. To date, 11 nicotinic subunits (alpha and beta) have been reported in mammals. Previously, our group [Brain Res. 778 (1997) 409] reported that these nicotinic acetylcholine receptor alpha and beta subunits were differentially expressed in the vestibular periphery of the rat. To begin an understanding of the molecular genetics of these vestibular efferents, this study examined the chromosomal locations of these nicotinic acetylcholine receptor genes in the rat (Rattus norvegicus). Using radiation hybrid mapping and a rat radiation hybrid map server (www.rgd.mcw.edu/RHMAP SERVER/), we determined the chromosomal position for each of these genes. The alpha2-7, alpha9, alpha10, and beta2-4 nicotinic subunits mapped to the following chromosomes: alpha2, chr. 15; alpha3, chr. 8; alpha4, chr. 3; alpha5, chr. 8; alpha6, chr. 16; alpha7, chr. 1; alpha9, chr. 14; alpha10, chr. 7; beta2, chr. 2; beta3, chr. 16; and beta4, chr. 8. With the location for each of these nicotinic subunits known, it is now possible to develop consomic and/or congenic strains of rats that can be used to study the functional genomics of each of these subunits.

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

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.

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.

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 α 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 α 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 Gαolf and two previously identified G-protein α subunits—Gαi2 and Gαi2(vest)—in the rat genome. Results—The following G-protein α subunits were identified in the normalized cDNA library: Gαolf, Gαs, Gαo and Gαs2. Gαolf maps to chromosome 18 between markers D18Mit17b and D18Mgh2. Gαi2 maps to chromosome 8 between markers D8Rat65 and D8Mgh2. Gαi2(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 α 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 Gαo 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.

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.