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Genetic Determinants of Human Serum Sterol Levels

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Genetic Determinants of Human Serum Sterol Levels GENETIC DETERMINANTS OF HUMAN SERUM STEROL LEVELS APPROVED BY SUPERVISORY COMMITTEE David W. Russell, Ph.D. Ralph J. Deberardinis, M.D., Ph.D. Jonathan C. Cohen, Ph.D. Jay D. Horton, M.D. I dedicate this thesis to my late undergraduate research advisor, David Sterling Marsh, Ph.D., who always showed enthusiasm and excitement for my research projects, taught me how to interpret unexpected results and revealed how new questions and hypotheses can arise from ‘uninterpretable’ data. My life would not have been the same without his endless sarcasm, wit and humor. I thank him for always making time for one of his ‘chilluns’; you will not be forgotten, Dave. ACKNOWLEDGEMENTS First and foremost, I thank my mentor and advisor, David Russell, for his endless support, patience, and guidance and for providing an excellent learning environment for my graduate studies. The knowledge and wisdom that I have acquired from working in his lab is priceless. Technical help of Daphne Rye, Bonne Thompson and Jeff McDonald over the years has been invaluable. I consider myself lucky to have been a student under such a great mentor, and it is an honor to be his last graduate student. I am grateful to have been a student in the Department of Molecular Genetics; the cooperative and collaborative environment is unlike any other. I am also incredibly lucky to have distinguished scientists on my thesis committee. I appreciate the insight, suggestions and discussion on my project and future career. Special thanks to my family. Thanks to my father, Ken Stiles, for instilling in me the value of a good education and hard work. Thanks to my mother, Sharon McNutt, for always supporting my decisions and for teaching me that time is scarce. To my younger siblings, I hope that my experience has instilled in you the importance of pursuing your dreams and achieving your goals, no matter how hard they may be. To the rest of my family, thank you for supporting me in every way and for doing more than your best to help me achieve my goals. Finally, thanks to my best friend, my love, Marcus Long, for being one of my biggest supporters, I am thrilled to have you in my life and look forward to our future together. And to Cooper and Jane, our four-legged kids, thanks for always cheering me up; you bring a smile to my face. GENETIC DETERMINANTS OF HUMAN SERUM STEROL LEVELS by ASHLEE RENEE STILES DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center Dallas, Texas May, 2013 GENETIC DETERMINANTS OF HUMAN SERUM STEROL LEVELS ASHLEE R. STILES, Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2013 DAVID W. RUSSELL, Ph.D. Dozens of different cholesterol metabolites are synthesized by mammalian cells and are known to play important physiological roles in the liver, brain, and immune system. These metabolites are ultimately inactivated by conversion into bile acids in the liver and are thereafter excreted from the body. Mutations in several genes encoding enzymes that metabolize cholesterol have been identified and the clinical consequences of these mutations range from progressive central nervous system neuropathy to spastic paraplegia to liver failure in children. There are several genes in the cholesterol metabolic pathway in which human mutations and their clinical consequences have not yet been identified. For example, the metabolite 24- hydroxycholesterol is produced in the brain by cholesterol 24-hydroxylase (encoded by the CYP46A1 gene). Disruption of this gene in the mouse causes severe learning defects, but it is not known whether mutations in the human gene cause the same phenotype. To overcome the uncertainty inherent in guessing what the phenotype arising from mutations in the human CYP46A1 gene might be and to detect mutations in other genes specifying cholesterol metabolic enzymes, we developed mass-spectrometry based methods to measure >60 different sterol metabolites in small volumes (200 µl) of human serum. We applied these methods to v quantify sterols in 3,230 serum samples derived from clinically well-characterized subjects participating in the Dallas Heart Study. Large intra-individual variation was detected in the serum levels of approximately 22 of the >60 sterols assessed. To identify the genes underlying the observed variation, we took both targeted and untargeted approaches. In the targeted approach, advantage was taken of the fact that the substrates and products of many sterol metabolizing enzymes are known and we thus sequenced the corresponding genes in subjects with very high or low levels of the sterol in question. In the untargeted approach, the 3,230 individuals whose sterol levels were measured were genotyped for 9,229 non-synonymous (amino acid-changing) variations (SNPs) in multiple human genes. For 24-hydroxycholesterol, the metabolite mentioned above, both approaches yielded concordant results. Direct sequencing of the oxysterol 7α-hydroxylase gene (CYP39A1), which uses 24- hydroxycholesterol as a substrate, identified several mutations that decrease enzyme activity and thus lead to increased serum levels of this sterol substrate. In the genetic linkage analysis, a SNP in CYP39A1 was strongly (P=2.1 x 10-27) correlated with serum 24-hydroxycholesterol levels. Expression analysis indicated that this SNP also decreased CYP39A1 enzyme activity. Together, these results illustrate that our approach has the ability to identify genetic determinants of serum sterol levels. Additional correlations were identified between a SNP in EPHX2 and levels of 24,25-epoxycholesterol (P=9.6 x 10-25) and a SNP in SDR42E1 and levels of 8-dehydrocholesterol levels (P=1.5 x 10-15). Biochemical assays were performed to characterize these previously unidentified genetic associations and to reveal their effect on oxysterol and sterol metabolism. Simultaneously, SNP genotypes and sterol levels were correlated with specific clinical phenotypes in an effort to shed light on the role of non- cholesterol sterols in disease. The next step is to apply these methods to the other 19 sterols that are routinely detected in serum and to correlate the analytical and genetic findings with the >100 clinical phenotypes of the 3,230 subjects whose sterol levels we have measured. vi TABLE OF CONTENTS TITLE FLY......................................................................................................................................i DEDICATION................................................................................................................................ii ACKNOWLEDGEMENTS.............................................................................................................iii TITLE PAGE.................................................................................................................................iv ABSTRACT....................................................................................................................................v TABLE OF CONTENTS...............................................................................................................vii PRIOR PUBLICATIONS...............................................................................................................ix LIST OF TABLES AND FIGURES….............................................................................................x LIST OF APPENDICES...............................................................................................................xii LIST OF ABBREVIATIONS.........................................................................................................xiii CHAPTER 1 INTRODUCTION………………………………………………………………………..1 CHAPTER 2 A COMPREHENSIVE METHOD FOR EXTRACTION AND QUANTITATIVE ANALYSIS OF STEROLS AND SECOSTEROIDS FROM HUMAN PLASMA..............................................................................................................18 A. INTRODUCTION B. EXPERIMENTAL SECTION C. RESULTS D. DISCUSSION E. FUTURE DIRECTIONS F. TABLES AND FIGURES CHAPTER 3 CYP7B1: ONE CYTOCHROME P450, TWO HUMAN GENETIC DISEASES, AND MULTIPLE PHYSIOLOGICAL FUNCTIONS…………………………….......48 CHAPTER 4 ENVIRONMENTAL, GENETIC, AND ANATOMIC DETERMINANTS OF SERUM STEROL LEVELS……………………………………………………………………..61 vii A. INTRODUCTION B. MATERIALS AND METHODS C. RESULTS D. DISCUSSION E. TABLES AND FIGURES CHAPTER 5 CONCLUDING REMARKS……………………………………...………………….107 APPENDIX A METHOD DETAILS………………………………………………………………....112 APPENDIX B OXYSTEROL METHOD FILE………………………………………………...…….122 APPENDIX C STEROL METHOD FILE…………………………………………………………….132 APPENDIX D LATHOSTEROL METHOD FILE……………………………………………………141 APPENDIX E STANDARD CONCENTRATIONS, RECIPES, AND DEUTERATED ANALOG PAIRS………………………………………………………………………………...145 BIBLIOGRAPHY........................................................................................................................146 viii PRIOR PUBLICATIONS Stiles AR, McDonald JG, Bauman DR, Russell DW. CYP7B1: one cytochrome P450, two human genetic diseases, and multiple physiological functions. J Biol Chem. 2009 284:28485-9. Stiles AR and Russell DW. SRD5A3: a surprising role in glycosylation. Cell 2010 23:196-8. McDonald JG, Smith DD, Stiles AR, Russell DW. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J Lipid Res. 2012 53:1399-409. ix LIST OF FIGURES AND
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