African-Americans Are Less Frequently Assessed For Hereditary Colon Cancer.

INTRODUCTION: Colorectal cancer (CRC) is the third most common malignancy in the United States and disproportionately affects African-Americans. Approximately 5-10% of CRC results from hereditary cancer syndromes. A detailed family history is recommended as an initial component of cancer risk assessment to help determine initiation, frequency, screening method and genetic counselling referral. This study evaluated the rate of hereditary CRC risk assessment in African-American and white patients. METHODS: A chart review of all patients referred for CRC screening in a university gastroenterology clinic during a 3 month period was performed. Patient self-described race/ethnicity, gender, age, documentation of multi-generational family medical history (3+ generations) were obtained. Amsterdam II Criteria, Bethesda Criteria and Colorectal Cancer Risk Assessment Tool were used to determine which patients with family histories should receive referrals for genetic counselling. Statistical analysis was performed using Fisher's Exact Test with significance set at p < 0.05. The study was IRB approved. RESULTS: 872 medical records were reviewed, including 452 African-American (276 females, 176 males; mean age 60.2), 263 White (123 females, 140 males; mean age 59.4), 45 Hispanic, and 42 Asian. Multi-generational family history was obtained from 143 (16.4%); 62 African-American (13.7%; 47 females, 15 males), 58 White (22.1%; 37 females, 21 whites), 3 Hispanic (6.7%), and 4 Asian (9.5%). There was a significant difference (p = 0.0050) in the rate of detailed family history in African-Americans and whites. However, African-Americans and Whites similarly qualified for genetic counselling when family history was obtained (p = 0.7915); 58.1% African-Americans (36; 30 females, 6 males) and 50% Whites (29: 19 females, 10 males) qualified for genetic counselling. Overall referral rate to genetic counselling was 16.5% with no significant difference (p = 0.7586) between African-Americans and whites. CONCLUSIONS: CRC risk assessment with detailed family medical history was inconsistently performed in all patients. There was significantly lower rate of obtaining multi-generational family medical histories in African-Americans. Referrals of all patients for genetic counselling and testing were also insufficient. Appropriate identification of individuals at increased risk for hereditary cancer syndromes, particularly African-Americans, is critical to prevention, early detection, and treatment of CRC and improving disparities in care.

A Bariatric Curveball: A Rare Case of Recurrent Lactobezoars after Roux-en-Y Gastric Bypass Surgery.

Bariatric surgery is a major risk factor for bezoar formation secondary to decreased gastric motility, gastric acidity, and pyloric function.1 This case is about a 49-year-old female veteran, 3 weeks status-post Roux-en-Y gastric bypass surgery, who presented with acute abdominal pain and oral intolerance. After being diagnosed with a bezoar and esophagogastroduodenoscopic removal, the patient had immediate relief of symptoms. Unfortunately, over the course of 4 months, this patient experienced three recurrent episodes of bezoar formation (with a possible fourth episode that could not be confirmed secondary to resolution of symptoms after administration of oral contrast load). Based on her dietary history and gross appearance of the bezoar, the patient was determined to have developed recurrent lactobezoars. Lactobezoars are composed of milk and mucous proteins and are commonly found in neonates with immature gastrointestinal tracts.7 This unusual complication demonstrates how current dietary recommendations encouraging dairy consumption to meet daily protein requirements may have increased this patient's risk for lactobezoar formation. This case illustrates the importance of balancing the risks and benefits of macronutrient requirements after bariatric surgery with postsurgical bezoar complications.

Treatment of advanced colorectal cancer in a patient with cardiotoxic reactions to 5-fluorouracil and capecitabine using suboptimal doses.

A 32-year-old female with stage IV colorectal cancer and metastasis to the liver experienced cardiotoxic reactions after treatment with 5-fluorouracil and its oral prodrug capecitabine even at two-thirds the recommended dose. After careful considerations, the decision was made to attempt capecitabine retrial at a further suboptimal dose with combination chemotherapy where she no longer experienced cardiac events. As a result, the liver tumour shrank and rectal mass stabilised, tumour markers dropped and she underwent surgical resection of both masses. Later there was local recurrence of disease near the previous liver tumour, so the suboptimal capecitabine therapy was restarted without complaint. The patient became a candidate for a NanoKnife procedure, offering a potentially curative therapy. This case report summarises a novel treatment strategy for those patients with advanced colorectal cancer who experience cardiotoxic reactions to fluoropyrimidines, the active agent of gold standard treatment.

Progressive Consent and Specimen Accrual Models to Address Sustainability: A Decade's Experience at an Oregon Biorepository.

BACKGROUND: The Legacy Biorepository is a College of American Pathologists-accredited biorepository operating within a seven-hospital healthcare system, with a decade's experience in specimen accrual, storage, and distribution. While standardization of our practices through accreditation remains a priority, we along with others face challenges with regard to sustainability. Purposeful changes in our consent process, which we term "progressive consent," are expected to improve sustainability and operational flexibility while increasing our scientific impact. METHODS: Until 2015, informed consent was performed primarily by biorepository staff at an estimated time of 1 hour per case. After a process improvement exercise, we successfully changed our informed consent process to a modified front-door model, with use of material and data for research as an opt-in or opt-out selection on the institutional patient informed consent form provided to surgery patients in the healthcare system. Successful implementation of this change required the engagement and participation of multiple stakeholders in healthcare system leadership, hospital administration, research, legal, regulatory, and patient care levels. RESULTS: A modified front-door consent enabled us to collect an additional 38 specimens in the first two quarters of 2016, with a time commitment of 15.75 hours, a time savings per specimen increasing in Q2 over Q1. We estimate a potential savings of 43 hours in 2016. This progressive model allowed us to maintain our frozen sample collection while increasing the availability of paraffin-embedded tissue and bodily fluids. Augmenting our tissue collection added little expense per case (approximately half that of each frozen tissue aliquot) and increased the range of biospecimens collected. CONCLUSIONS: Biorepository financial sustainability is a critical issue. Thorough evaluation and modification of existing procedures and collection models, as well as cost recovery initiatives, can translate into savings. Sustainability, process improvement, and scientific impact broadly overlap and continue to require operational critique and implementation of strategic changes.

A comparison of obstetrical outcomes and costs between misoprostol and dinoprostone for induction of labor.

OBJECTIVE: The objective of this study is to compare resource utilization (efficiency) and obstetrical/cost outcomes of single dose misoprostol versus dinoprostone for induction of labor (IOL) at term. METHODS: Retrospective cohort of induced deliveries 37-41 weeks gestation presenting with a Bishop score ≤4 using single-dose-50 mcg vaginal misoprostol or 10 mg-dinoprostone vaginal-inserts. Dinoprostone patients were compared (5:1) with misoprostol patients. The primary outcome variable was length of L&D stay (proxy for resource utilization). Baseline characteristics, clinical outcomes, and costs were compared. RESULTS: Three-hundred thirty-one patients were included, 276 received dinoprostone and 55 received misoprostol. The misoprostol group had statistically significant decreased time to active labor [median 8 h (1.6,24) versus 12(0.8,52)], time-to-delivery [median 11 h (4,31) versus 17(2.8,56)] and L&D stay [median 16 h (13,28) versus 24(18,30)]. Differences remained significant after adjustment for race, method of delivery, birth weight, gravidity/parity, gestational age, and BMI (adjusted p values <0.001, <0.01, and < 0.05, respectively). There were no statistical differences in Apgar scores, tachysystole rate, cesarean section rate, and composite maternal/neonatal morbidity. A policy of using misoprostol would result in annual cost savings of approximately $242 500 at our institution as compared with dinoprostone. CONCLUSION: Single-dose misoprostol is more efficient in IOL at term with respect to L&D utilization and cost and its use is not associated with increased adverse obstetrical outcomes.

MicroRNA-21 overexpression contributes to vestibular schwannoma cell proliferation and survival.

HYPOTHESIS: Elevated levels of hsa-microRNA-21 (miR-21) in vestibular schwannomas (VSs) may contribute to tumor growth by downregulating the tumor suppressor phosphatase and tensin homolog (PTEN) and consequent hyperactivation of protein kinase B (AKT), a key signaling protein in the cellular pathways that lead to tumor growth. BACKGROUND: Vestibular schwannomas are benign tumors that arise from the vestibular nerve. Left untreated, VSs can result in hearing loss, tinnitus, vestibular dysfunction, trigeminal nerve dysfunction, and can even become life threatening. Despite efforts to characterize the VS transcriptome, the molecular pathways that lead to tumorigenesis are not completely understood. MicroRNAs are small RNA molecules that regulate gene expression posttranscriptionally by blocking the production of specific target proteins. METHODS: We examined miR-21 expression in VSs. To determine the functional significance of miR-21 expression in VS cells, we transfected primary human VS cultures with anti-miR-21 or control, scrambled oligonucleotides. RESULTS: We found consistent overexpression of miR-21 when compared with normal vestibular nerve tissue. Furthermore, elevated levels of miR-21 correlated with decreased levels of PTEN, a known molecular target of miR-21. Anti-miR-21 decreased VS cell proliferation in response to platelet-derived growth factor stimulation and increased apoptosis, suggesting that increased miR-21 levels contributes to VS growth. CONCLUSION: Because PTEN regulates signaling through the growth-promoting phosphoinositide 3-kinase/AKT pathway, our findings suggest that miR-21 may be a suitable molecular target for therapies aimed specifically at reducing VS growth.

Partial characterization of normal and Haemophilus influenzae-infected mucosal complementary DNA libraries in chinchilla middle ear mucosa.

OBJECTIVES: We sought to construct and partially characterize complementary DNA (cDNA) libraries prepared from the middle ear mucosa (MEM) of chinchillas to better understand pathogenic aspects of infection and inflammation, particularly with respect to leukotriene biogenesis and response. METHODS: Chinchilla MEM was harvested from controls and after middle ear inoculation with nontypeable Haemophilus influenzae. RNA was extracted to generate cDNA libraries. Randomly selected clones were subjected to sequence analysis to characterize the libraries and to provide DNA sequence for phylogenetic analyses. Reverse transcription-polymerase chain reaction of the RNA pools was used to generate cDNA sequences corresponding to genes associated with leukotriene biosynthesis and metabolism. RESULTS: Sequence analysis of 921 randomly selected clones from the uninfected MEM cDNA library produced approximately 250,000 nucleotides of almost entirely novel sequence data. Searches of the GenBank database with the Basic Local Alignment Search Tool provided for identification of 515 unique genes expressed in the MEM and not previously described in chinchillas. In almost all cases, the chinchilla cDNA sequences displayed much greater homology to human or other primate genes than with rodent species. Genes associated with leukotriene metabolism were present in both normal and infected MEM. CONCLUSIONS: Based on both phylogenetic comparisons and gene expression similarities with humans, chinchilla MEM appears to be an excellent model for the study of middle ear inflammation and infection. The higher degree of sequence similarity between chinchillas and humans compared to chinchillas and rodents was unexpected. The cDNA libraries from normal and infected chinchilla MEM will serve as useful molecular tools in the study of otitis media and should yield important information with respect to middle ear pathogenesis.

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.

Differential expression of cytoskeletal genes in the cochlear nucleus.

The relationship between structure and function is clearly illustrated by emerging evidence demonstrating the role of the neuronal cytoskeleton in physiological processes. For example, alterations in axonal caliber, a feature of the cytoskeleton, have been shown to affect reflex arc latencies and are prominent features of several neuropathological disorders. Even in the nonpathologic situation, however, axonal diameter may be a crucial element for the normal function of specialized auditory neurons. To investigate this relationship, we used serial analysis of gene expression and microarray analyses to characterize the expression of cytoskeletal genes in the central auditory system. These data, confirmed by real-time RT-PCR, identified differential expression of intermediate neurofilament transcripts (i.e., Nefh, Nef3, and Nfl) among the subdivisions of the cochlear nucleus. In situ hybridization was used to identify specific classes of neurons within the cochlear nucleus expressing these neurofilament genes. Robust neurofilament expression was seen in bushy cells and cochlear nerve root neurons, suggesting an association between cytoskeletal structure and rapid conduction velocities. Gene expression data were also identified for other classes of cytoskeletal and structural genes important in neuronal function. These results may help to explain some causes of hearing loss in hereditary neuropathies and provide an anatomic basis for understanding normal neuronal function in the central auditory system.

Signaling pathways in interleukin-1beta-mediated middle ear mucin secretion.

OBJECTIVES: The objectives of this study were to investigate the role of the phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC), and nitric oxide synthase (NOS) pathways during upregulation of mucin secretion by middle ear epithelium after exposure to interleukin-1beta and to examine the ability of a specific interleukin-1 receptor antagonist (IL-1betara) to block this increased secretion. MATERIALS AND METHODS: Primary chinchilla middle ear epithelial cultures were established and exposed to IL-1beta. Specific inhibitors of calmodulin, PC-PLC, PKC, and NOS pathways were used to investigate the potential role of these pathways leading to increased epithelial mucin secretion after exposure to IL-1beta. Mucin secretion was characterized by exclusion chromatography and liquid scintillation. RESULTS: Epithelial cultures exposed to IL-1beta demonstrate an increase in mucin secretion that is blocked by specific inhibitors of PC-PLC, PKC, and NOS, but not by inhibitors of calmodulin. In addition, mucin secretion stimulated by IL-1beta was reversible with use of a specific IL-1betara. CONCLUSIONS: IL-1beta stimulates mucin secretion from middle ear epithelium and its effects can be reversed by IL-1betara. PC-PLC, PKC, and NOS pathways play a role in the increased secretion of mucin in middle ear epithelial cells after exposure to IL-1beta.

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.

Serial analysis of gene expression in neurofibromatosis type 2-associated vestibular schwannoma.

HYPOTHESIS: The genesis, morphology, and growth characteristics of vestibular schwannomas are determined by genetic alterations which vary gene transcript expression and this transcript expression can be qualitatively and quantitatively evaluated using the SAGE technique. By use of such technique, gene products with tumorigenic potential may be identified, providing insight and targets for future study. BACKGROUND: Serial analysis of gene expression (SAGE) is a powerful new technique that allows detailed qualitative and quantitative evaluation of cellular gene transcript expression. Tissue in limited quantity (5 x 10 to 2 x 10 cells) may be analyzed by a modified version of SAGE called microSAGE. Application of SAGE or microSAGE to study vestibular schwannoma gene expression has not been previously reported. METHODS: Fresh, vestibular schwannoma specimen from an individual with the diagnosis of neurofibromatosis type 2 was attained intraoperatively and maintained in a sealed container at -80degreesC until the time of analysis. The tissue was processed according to the microSAGE protocol, using 180 mg of vestibular schwannoma as starting material. RESULTS: The protocol resulted in the generation and sequencing of a tag library involving 458 tags representing 277 different gene products, including many transcripts known to be expressed in vestibular schwannomas. Several gene products with tumorigenic potential were identified. CONCLUSIONS: These data demonstrate that microSAGE is a useful technique to study vestibular schwannoma gene expression. Future studies will include building more comprehensive libraries and comparing libraries from various vestibular schwannoma phenotypes to identify useful diagnostic or prognostic markers, and targets for therapeutic intervention.

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.