Allele-specific Expression of a Bitter Taste Receptor

Study Overview
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Brief Summary
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This single-site, within-subject experimental basic research study is designed to analyze the hypothesis that allele-specific expression of the bitter taste receptor T2R38 in taste tissue of individuals heterozygous for the taste receptor gene TAS2R38 correlates with that in nasal epithelium, and is responsible for differences in acyl-homoserine lactone-induced respiratory defenses. Subjects will include 100 predominantly European adults without chronic rhinosinusitis who will be undergoing a sinonasal procedure for reconstructive purposes. All subjects will provide saliva samples for genotyping, from which 25 subjects heterozygous for TAS2R38 (AVI/PAV) will be identified. These individuals will be asked to complete a beverage frequency questionnaire and taste test prior to the procedure that will evaluate for a number of compounds, among them bitter ligands specific to T2R38. Their tongue will also be photographed to evaluate the anatomy of their fungiform papillae, the mushroom-like structures on the tongue which contain taste buds. Subjects will subsequently provide nasal epithelium and taste tissue, which will be processed to 1) evaluate for allele-specific expression of TAS2R38 mRNA in both the taste and nasal tissue, with the nasal tissue concurrently being cultured in an air-liquid interface system to 2) assess the AHL-induced respiratory defenses of ciliary beat frequency (CBF) and nitric oxide (NO) production. Should subjects require a subsequent sinonasal procedure for clinically-determined reasons, taste and nasal tissue will again be obtained and analyzed for TAS2R38 mRNA, allowing for 3) longitudinal evaluation of mRNA expression level.

Detailed Description
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The Basic Biology of Bitter Taste. The perception of bitter taste is thought to have evolved as a mechanism to protect against the ingestion of toxic materials, and is the result of ligand-activation of one of more than 25 different bitter taste receptors, so-called T2Rs (1). These receptors are found on the tongue in what are called fungiform papillae, mushroom-like structures that contain taste buds with receptors responding to a variety of tastes including sweet, salty, sour, umami, and bitter. A prototypical example of one of these bitter ligands is phenylthiocarbamide (PTC), which actives the T2R38 receptor. While initially identified in type II taste cells, T2R38 is also expressed in nasal epithelium, where it participates in innate immune defense responses to invading bacteria (2-6). TAS2R38: A Model System for Genotype-Phenotype Studies. Prior studies have identified two main forms of T2R38, active and inactive, which are characterized by three genetic variants in the TAS2R38 gene. These variants result in three amino acid changes, proline (P) to alanine (A) at position 49, alanine (A) to valine (V) at position 262, and valine (V) to isoleucine (I) at position 296 in the T2R38 receptor. Individuals who are homozygous for the active (PAV/PAV) form detect bitterness in compounds containing a thiourea (-N-C=S) moiety, including PTC, 6-n-propylthiouracil (PROP), and the plant compound goitrin, common in foods such as green vegetables (7-9). They also respond to acyl-homoserine lactones (AHLs), a class of compounds produced as signaling molecules by certain bacteria, triggering a rapid defense reaction consisting of increased ciliary beat frequency (CBF) to facilitate mucociliary clearance, and generation of nitric oxide (NO), a gas that can diffuse into the airway and kill bacteria (4). In contrast, those who are homozygous for all three variants (AVI/AVI) consume these compounds without perceiving them as bitter and do not appear to respond to AHLs (10). The frequency of both the active and inactive forms of TAS2R38 is at a near balance of 50:50 in many human racial groups, including Americans of European and African descent. The Heterozygote Hypothesis. Interestingly, individuals heterozygous for the active form of the receptor (AVI/PAV) exhibit highly variable phenotypes, with some people very sensitive to bitter compounds, and others needing high concentrations to taste them at all (11). While the investigators know that taste papillae density plays at least some role in this variability , our preliminary taste data suggest that the range of response is tied to how much mRNA is expressed from the active (PAV) form of the receptor, a concept called allele-specific expression (12). For example, this is the case when analyzing caffeine consumption, which strongly correlates with active mRNA expression (12). The investigators therefore hypothesize that the abundance of active TAS2R38 mRNA in heterozygous individuals also predicts the biologically significant change in magnitude of defensive responses in the presence of AHLs (13-15). The proposed study will determine whether this is in fact the case, and whether those people who have high mRNA abundance in taste tissue (fungiform papillae) also have correspondingly high abundance in nasal epithelium, or whether regulation is tissue-dependent. This will allow us to determine whether taste tests could provide a reliable representation of receptor function in other tissues and cell types. Should mRNA abundance prove to be a key factor, the investigators will determine whether high expressers sustain this expression over time. Of note, a study performed by Dr. Reed and collaborators found that the population in Philadelphia contained 18% of individuals in the homozygous nontaster (AVI) group, 17% in the homozygous taster (PAV) group and 37% in the common heterozygous group (AVI/PAV) (16). The remaining 28% were distributed among ten other less common genotypes, which will not be analyzed in this study. Thus, as the majority of the population is heterozygous, a thorough understanding of their ability to fight infection is clinically important. Clinical Significance. As one of the most common chronic conditions in the United States, chronic rhinosinusitis invokes a direct treatment cost of $3.5-5 billion annually. Its incidence is 146 per 1,000 and increasing (17). Our prior studies have shown that individuals with two copies of the active form of T2R38 have nasal epithelium that defends very effectively against certain bacteria, such as Pseudomonas aeruginosa, and are less likely to develop severe chronic rhinosinusitis requiring surgery, while those with two inactive forms cannot defend themselves as effectively, and are more likely to develop severe chronic rhinosinusitis requiring surgical intervention. Because the treatment of chronic rhinosinusitis involves multiple rounds of antibiotics and often surgical management through functional endoscopic sinus surgery (FESS), this research has significant implications for antibiotic stewardship, surgical morbidity and mortality, and health care expenditures.

Official Title
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Bitter Taste and Allele-specific Expression of the Human TAS2R38 Gene

Conditions
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Rhinosinusitis

Intervention / Treatment
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* Other: Observational study


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Key inclusion criteria include age 21-50 years English speaking, and plans to undergo a sinonasal procedure for reconstructive purposes or other reasons. Exclusion Criteria: Key exclusion criteria include a history of chronic rhinosinusitis Plans to undergo a procedure for reasons other than reconstruction Oral disease Pregnancy, or any condition that would prevent psychophysical testing. Subjects showing signs of oral disease, including tongue lesions or xerostomia, would be excluded from tongue sampling, and therefore excluded from the study. Subjects will not be excluded because of economic status, gender, race or ethnicity.

Ages Eligible for Study
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Minimum Age: 21 Years
Maximum Age: 50 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| PAV/PAV Tasters<br>Individuals homozygous for the taster allele of the TAS2R38 gene, PAV/PAV. | |
| AVI/PAV.<br>Individuals heterozygous for the taster allele of the TAS2R38 gene, AVI/PAV. | Other: Observational study<br> <br> |
| AVI/AVI<br>Individuals homozygous for the non-taster allele of the TAS2R38 gene, AVI/AVI. | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| TAS2R38 genotype | A participant's genotype will be identified on enrollment in the study, the results of which could take up to 1 month to be performed. | Up to 1 month after enrollment. | 
| TAS2R38 mRNA expression levels measured by RT-qPCR | Measured approximately 6 weeks after enrollment, based on the date of the participant's procedure. | Approximately 6 weeks after subject enrollment | 
| Ciliary beat frequency | Measured approximately 6 weeks after enrollment, based on the date of the participant's procedure. | Approximately 6 weeks after subject enrollment | 
| Production of nitric oxide by a participant's nasal epithelium culture measured in fold change of 4,5-diaminofluorescence diacetate | The production of nitric oxide (NO) by a patient's nasal epithelium culture will be measured approximately 6 weeks after enrollment, based on the date of his or her procedure. This will be measured by quantifying the fold change in fluorescence by using the NO-sensitive marker 4,5-diaminofluorescence diacetate (DAF-2). | Approximately 6 weeks after subject enrollment | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Bitter taste perception as measured by a visual analog scale | Psychophysical bitter taste perception will be assessed by asking participants to taste a solution and rate its bitterness on a visual analog scale ranging from no intensity at all to extremely intense. This will be assessed at participant's post-procedure follow-up appointment, on average 6 weeks after enrollment. | Approximately 6 weeks after subject enrollment | 
| Caffeine intake measured by number caffeinated beverages consumed per week (normalized to 1 cup = 180 mg caffeine) | The scale will be normalized to 1 cup = 180 mg caffeine. This will be assessed at participant's post-procedure follow-up appointment, on average 6 weeks after enrollment. | Approximately 6 weeks after subject enrollment | 
| Taste papillae density | Papillae density will be assessed at participant's post-procedure follow-up appointment, on average 6 weeks after enrollment. | Approximately 6 weeks after subject enrollment | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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chronic rhinosinusitis, airway physiology, taste receptors, tas2r, epithelial biology, upper respiratory disease