Function and Regulation of Cytochrome P450 4V2 and the Implications in Bietti’s Crystalline Dystrophy

Thesis (Ph.D.)--University of Washington, 2016-06

Background: Cytochrome P450 4V2 (CYP4V2) is one of 57 functional human isozymes in the cytochrome P450 superfamily of heme-containing monooxygenase enzymes. CYP4V2 is one of thirteen human P450 enzymes classified as ‘orphans’ because their substrate specificity and role in physiological function are not well characterized. CYP4V2 is ubiquitously expressed, with known ω-hydroxylase activity for medium-chain fatty acids, and is believed to play a role in lipid homeostasis. In 2004, researchers at the National Eye Institute identified a mutation in the ‘orphan’ cytochrome P450 4V2 (CYP4V2) gene in patients with Bietti’s crystalline dystrophy (BCD), a rare, degenerative, blinding retinal disease. In genetic studies aside from BCD, a common polymorphism in CYP4V2 has been associated with deep vein thrombosis, and expression of CYP4V2 has been inversely correlated with tumor grade in breast cancer. The research described in this dissertation was conducted to better understand normal physiological function of CYP4V2, to further elucidate the molecular mechanisms behind BCD, and to identify potential therapeutic targets that may lead to treatment options. Methods: Murine model of BCD. With assistance from the University of Washington Transgenic Core Facility, embryonic stem cells with targeted disruption of the Cyp4v3 gene were obtained from the Knockout Mouse Project. The embryonic stem (ES) cell clone was expanded on embryonic feeder cells, evaluated for correct gene targeting by polymerase chain reaction (PCR), and then injected into albino C57Bl/6 blastocysts to generate chimeric mice. Offspring that inherited the targeted Cyp4v3 allele were interbred as heterozygotes for production of Cyp4v3-/- mice. To confirm the null allele in Cyp4v3-/- mice, genotype analysis was performed by PCR. To confirm that the Cyp4v3 gene targeting event is truly null, CYP4V3 protein expression was assessed by Western blot analysis. To screen for changes in ocular appearance, a Micron II small animal retinal imaging system from Phoenix Research Laboratories was used to capture fundus images. Analysis of 20 μL sera from Cyp4v3 knockout and wild-type mice for PUFA content was performed using methanol and methyl-t-butyl ether (MTBE), and total fatty acids were analyzed by GC-EI/MS. For histologic analysis, whole eyes were harvested from mice and immediately fixed in 10% formalin for standard histological processing and hematoxylin & eosin staining. Whole eyes were also immediately frozen in Tissue-Tek optimum temperature cutting compound for histologic processing and Oil Red O staining for lipid content. Additionally, whole eyes were immediately submerged in 1/2x Karnovsky’s fixative, postfixed in osmium tetroxide and processed, sectioned, and examined by transmission electron microscopy. In addition to fundus images additional tests for visual acuity and function were conducted in collaboration with colleagues at Oregon Health and Science University. Electroretinograms were measured on dark-adapted mice anesthetized with ketamine/xylazine. Pupils were dilated, electrodes applied, and the electrical transmission of the retina was recorded from light flash intensities ranging from -4.34 to 3.55 log cd•s/m2. Optical coherence tomography was conducted on mice sedated using 1.5% isofluorane delivered via nose cone, with anesthetized corneas and dilated pupils, using linear horizontal scans in the temporal and nasals quadrants and linear vertical scans in the superior and inferior quadrants. Visual acuity was measured by optokinetic response in which computer monitors form a virtual cylinder of rotating sine-wave vertical gratings, which randomly rotates clockwise or counterclockwise, and mice reflexively respond to the rotating gratings by turning their head in the corresponding direction. A minimum of two to seven days lapsed between anesthesia and testing for each mouse. CYP4V2 mutations in BCD. At regular intervals, the patient underwent ophthalmic examination and testing, which included best-corrected visual acuity (BCVA), fundus color photography, wide-field fundus autofluorescence (AF; Optos), wide-field fluorescein angiography (FA; Optos), semi-automated kinetic and automated static full-field perimetry (Octopus 101/900, Haag-Streit, Inc), spectral-domain optical coherence tomography (OCT; Heidelberg Engineering), and ERG. To conduct the genotype analysis, DNA was extracted from buccal swabs from a BCD patient and a healthy control. Each exon was individually amplified from extracted DNA by standard polymerase chain reaction and validated by gel electrophoresis. Sequencing results were analyzed for homology to known CYP4V2 sequence using the Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov). A homology model of wild-type CYP4V2 was constructed using the I-TASSER server, and all molecular dynamics (MD) simulations were performed in Gromacs 4.6.5. Regulation of CYP4V2. Human liver microsomes, total mRNA, and total RNA was extracted from human liver tissue from the University of Washington liver bank. Whole genome expression was measured from total mRNA, SDS-PAGE and Western blot analysis of prepared human liver microsomes were conducted and quantified, and total RNA was used to screen for miRNA expression by microarray analysis. To validate a regulatory interaction by the candidate miRNA (miR-146b-5p), HepG2 cells were stably transfected with plasmid vectors containing the complete CYP4V2 3’UTR, and either a vector to overexpress miR-146b-5p, or a non-targeting control. Luciferase activity was evaluated using the SecretePair® Dual Luminescence Assay and measured with a Synergy HTX plate reader. ARPE-19 cells were transiently transfected with plasmids to overexpress miR-146b-5p, or a non-targeting control miRNA, fixed in methanol, and incubated with fluorescent-tagged antibodies. The processed samples were analyzed with a Nikon Ti-S microscope, and the ratio of CYP4V2 to GFP signal was determined for each transfection condition. Absolute protein was measured using LC-MS/MS techniques and two unique signature peptides. Results: We demonstrated that Cyp4v3-/- mice recapitulate the most significant elements of the phenotype observed in BCD, particularly the development of glistening yellow-white crystalline deposits in the retinal tissue, a defining characteristic of BCD. In a patient with BCD, we successfully determined her genotype, the first homozygous point mutation that had previously only been reported as one allele in a compound heterozygous patient. We also were the first to develop a sequence homology model describing the functional deficiency caused by this homozygous point mutation. In an effort to elucidate the biochemistry of CYP4V2 in normal physiological function, we showed strong evidence that CYP4V2 is influenced by regulatory pressure of microRNA (miR-146b-5p). Finally, we described rationale for conducting a pilot gene replacement therapy study in our knockout mouse model using an adeno-associated virus (AAV) vector packaged with functional CYP4V2 cDNA. Conclusion: This dissertation presents a collection of studies aimed at better characterizing CYP4V2 in normal physiological function, and to begin to elucidate the aberrant physiology observed in BCD. We developed a knockout mouse model that recapitulates the hallmark phenotype observed in BCD, and is a promising preclinical model for further evaluation of the pathophysiology of BCD, as well as a platform for screening potential therapeutic options, including gene replacement therapy or treatment with small molecule candidates. Finally, we identified miR-146b-5p as a candidate for regulation of expression and function of CYP4V2, which contributes to our knowledge of the role of CYP4V2 in normal physiology and can lead to better understanding of the abnormal pathophysiological mechanism of disease.