FREQUENCY OF ALUYA5 INSERTION IN A REGULATORY REGION OF MICROSOMAL EPOXIDE HYDROLASE IN HUMAN DISEASED TISSUE

Open Access
Author:
Fay , Emily Kathryn
Area of Honors:
Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Curtis John Omiecinski, Thesis Supervisor
  • Stephen Wade Schaeffer, Honors Advisor
Keywords:
  • detoxification
  • bioactivation
  • carcinogenesis
  • microsomal epoxide hydrolase
  • Alu insertion element
  • Hardy-Weinberg equilibrium
Abstract:
Human microsomal epoxide hydrolase (mEH) is an enzyme involved in both detoxification and bioactivation of endogenous and xenobiotic compounds. mEH catalyzes the trans-addition of water to reactive epoxide intermediates to form less toxic diols; however, mEH is also responsible for bioactivating metabolic intermediates, resulting in potentially toxic metabolites. mEH is required for the activation of polyaromatic hydrocarbons (PAHs) found in cigarette smoke to genotoxic carcinogens. Several common genetic polymorphisms exist in the mEH gene (EPHX1) that may cause differences in epoxide hydrolase expression and activity between individuals. Epidemiological studies have shown that certain genetic variants are correlated with altered disease susceptibilities, suggesting they may serve as markers for predicting disease. This study investigates the population frequencies of a double AluYa5 insertion element that occurs in the far upstream promoter region of EPHX1 that impacts the gene’s transcriptional status. Cell or tissue-derived DNA samples from several hundred unrelated individuals were analyzed in these investigations. A focus of the study was to evaluate genotype status of the EPHX1 upstream promoter region using DNA extracts from normal uninvolved tissues that were derived from a human tissue bank, consisting of lung and liver tissues obtained from otherwise diseased individuals. The DNA samples were analyzed for the presence or absence of the double Alu insertion, as well as Alu (-/-), Alu (+/-), and Alu (+/+) gene frequencies. Using Pearson’s chi-square test, we compared the observed and expected genotype frequencies in a previously studied healthy population to determine whether the insertion was in Hardy-Weinberg equilibrium. We used the allele frequencies of the healthy population to calculate the expected genotype frequencies of the diseased population, and Pearson’s chi-square test was performed to identify any significant difference between observed and expected genotype frequencies. Additionally, the allele frequencies of the healthy population were used to calculate the expected genotype frequencies in the lung and liver samples, male and female samples, and samples from donors age 0-49 and age 50 and older, to determine whether any correlations existed between genotype and tissue type, gender, and age of development. Pearson’s chi-square test was also used to compare observed and expected genotype frequencies in each subpopulation analyzed. These analyses allowed us to determine whether the double Alu insertion was in Hardy-Weinberg equilibrium in the subpopulations. Through these investigations, we determined that the double Alu insertion was indeed in Hardy-Weinberg equilibrium in both the healthy and diseased populations, indicating that the insertion was not under selection pressures and therefore did not correlate with protection from disease nor initiation of disease. The double Alu insertion was also in Hardy-Weinberg equilibrium in both lung and liver samples, female and male donors, and donors younger than and older than age 50, and no significant differences in genotype frequency were identified in any subpopulation analyzed. Our findings suggest that the double Alu insertion does not play a major role in altering disease susceptibility in the samples analyzed and does not appear to be a useful biomarker for disease prediction within the spectrum of diseases assessed.