ABSTRACT Title of Dissertation: RECESSIVE OSTEOGENESIS IMPERFECTA: PREVALENCE AND PATHOPHYSIOLOGY OF COLLAGEN PROLYL 3-HYDROXYLATION COMPLEX DEFECTS Wayne Anthony Cabral, Doctor of Philosophy, 2014 Dissertation directed by: Dr. Joan C. Marini, MD, PhD Bone and Extracellular Matrix Branch, NICHD National Institutes of Health Professor Stephen M. Mount, PhD Department of Cell Biology and Molecular Genetics Osteogenesis Imperfecta (OI) is a clinically and genetically heterogeneous heritable bone dysplasia occurring in 1/15,000-20,000 births. OI is a collagen-related disorder, with the more prevalent dominant forms caused by defects in the genes encoding the α1 and α2 chains of type I collagen (COL1A1 and COL1A2). Rare recessive forms of OI are caused by deficiency of proteins required for collagen post-translational modifications or folding. We identified deficiency of components of the ER-resident collagen prolyl 3- hydroxylase (P3H) complex as a cause of recessive OI. The P3H complex, consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP) and cyclophilin B/peptidyl-prolyl isomerase B (CyPB/PPIB), modifies the α1(I) P986 and α2(I) P707 residues of type I collagen. The most common P3H complex defects occur in LEPRE1, the gene encoding P3H1, and over one-third of these cases are due to a founder mutation we identified among individuals of West African and African American descent. Our screening of contemporary cohorts revealed that 0.4% of African Americans and nearly 1.5% of West Africans are carriers for this mutation, predicting a West African frequency of recessive OI due to homozygosity for this mutation at 1/18,260 births, equal to de novo dominant OI. Furthermore, haplotype analysis of affected families was consistent with a single founder for this mutation, occurring 650-900 years ago (1100-1350 C.E.). Patients deficient in P3H1 and CRTAP have consistent bone phenotypes and collagen biochemistry. However, the rare cases of CyPB deficiency have variable findings distinct from P3H1/CRTAP. To clarify the OI mechanism of CyPB deficiency, we generated a Ppib knock-out mouse. In the absence of CyPB, only residual collagen prolyl 3- hydroxylation is detectable in KO cells and tissues. The delay in collagen folding in KO cells is further increased upon cyclophilin inhibition, supporting CyPB’s role as an isomerase and the presence of redundancy for collagen ER PPIases. Site-specific alterations of collagen post-translational modification, particularly at residues involved in helical crosslinking, suggest that CyPB is critical to the function of collagen hydroxylases, especially LH1. Thus our studies indicate novel roles for CyPB, separate from the P3H complex, which directly and indirectly regulate collagen biosynthesis and bone development. Recessive Osteogenesis Imperfecta: Prevalence and Pathophysiology of Collagen Prolyl 3-Hydroxylation Complex Defects by Wayne Anthony Cabral Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2014 Advisory Committee: Dr. Joan C. Marini, MD, PhD, Co-Chair Professor Stephen M. Mount, PhD, Co-Chair Professor Ian H. Mather, PhD Professor John Moult, PhD Professor Adam H. Hsieh, PhD ©Copyright by Wayne Anthony Cabral 2014 ACKNOWLEDGEMENTS There are a number of people that have had an undeniable, positive impact on my professional development, and to whom I wish to express my sincerest gratitude. Several people have sacrificed their time and efforts to encourage, teach and challenge me, including Dr. Denise Pettit, Dr. Krista Fischer-Stenger and Dr. Antonella Forlino. I am extremely grateful for their generosity during the early years of my career. I would also like to acknowledge my appreciation to Dr. Elisabeth Gantt, Professor Emerita, for supporting my admission to the MOCB Program. I thank members of my thesis committee, Professors Ian Mather, Stephen Mount, John Moult and Adam Hsieh, for offering their time and constructive guidance. In addition, I would like to thank Dr. Stephen Mount and Dr. Ian Mather, Professor Emeritus, for their long-term commitment and support of my non-standard route through graduate school. Most importantly, I wish to express my sincere thanks for the efforts of my advisor, mentor and coach, Joan C. Marini, M.D., Ph.D., for her unwavering support of my personal and professional development, pushing me to reach my potential, and for teaching me to never give up. To my parents, Dr. Guy Cabral and Dr. Francine Marciano-Cabral, the greatest Mom, Dad, and role models that a son could have, I strive every day to make you proud. Finally, I am indebted to my fiancée, Adriana Zingone, M.D., Ph.D., for her patience, love and support. ii LIST OF TABLES Table 1. Primers utilized in haplotype analysis ............................................................ 111 Table 2. LEPRE1 c.1080+1G>T Carrier Frequency in African Americans ................. 114 Table 3. LEPRE1 c.1080+1G>T Carrier Frequency in Contemporary Africans .......... 116 Table 4. LEPRE1 c.1080+1G>T Allele Haplotypes in West Africans ......................... 120 Table 5. LEPRE1 c.1080+1G>T Allele Haplotypes in African Americans ................. 121 Table 6. Type I Collagen Post-Translational Hydroxylation ........................................ 146 Table 7. Post-translational Modification of Type I Collagen Lysine Residues ............ 151 Table 8. Reported Cases of PPIB Deficiency in Type IX Osteogenesis Imperfecta .... 171 iii LIST OF FIGURES Figure 1. The Collagen Family of Proteins ..................................................................... 3 Figure 2. Type I Procollagen Structure ........................................................................... 5 Figure 3. Procollagen Synthesis and Modification ......................................................... 8 Figure 4. The Collagen Prolyl 3-Hydroxylation Complex ............................................ 24 Figure 5. Procollagen Trafficking .................................................................................. 33 Figure 6. Collagen Fibril Structure ................................................................................ 44 Figure 7. Lysyl Oxidase-Mediated Collagen Crosslinking ............................................ 49 Figure 8. Endochondral Bone Formation and Mineralization ....................................... 53 Figure 9. The Cellular Components of Bone ................................................................. 58 Figure 10. Stress-Strain Curve for Cortical Bone .......................................................... 71 Figure 11. Clinical Features of Classical Osteogenesis Imperfecta ............................... 75 Figure 12. Biochemical Determination of Type I Collagen Overmodification ............. 77 Figure 13. West African Origin of LEPRE1 c.1080+1G>T Mutation ........................... 97 Figure 14. Screening of Genomic DNA for the LEPRE1 c.1080+1G>T Mutation ...... 113 Figure 15. Pedigrees of African American (AA) and West African (WA) Families Included in Haplotype Analysis .................................................................. 118 Figure 16. Generation of a Murine Model of Cyclophilin B Deficiency ...................... 142 Figure 17. Absence of Ppib Expression Affects Bone Development ........................... 144 Figure 18. Synthesis of Type I Collagen ...................................................................... 145 Figure 19. Cyclophilin B Catalyzes Folding of Type I Collagen ................................. 147 Figure 20. Post-translational Modification of Type I Collagen from Tissues .............. 149 Figure 21. Altered Post-translational Modification of Specific Type I Collagen Lysine Residues in the Absence of Cyclophilin B ....................................... 150 iv Figure 22. Expression of ER Resident Collagen Lysine Modification Enzymes ......... 154 Figure 23. Dysregulation of Collagen Deposition and Fibril Assembly ...................... 156 Figure 24. Abnormal crosslink profile in Ppib-null mouse bone ................................. 157 v LIST OF ABBREVIATIONS 3Hyp 3-hydroxyproline 4Hyp 4-hydroxyproline AA cohort African American cohort aBMD areal bone mineral density αMEM minimum essential medium ADAMTS a disintegrin and metalloproteinase with thrombospondin motifs BFR bone formation rate BMDD bone mineral density distribution BMP bone morphogenetic protein Brtl Brittle mouse model of dominant osteogenesis imperfecta BS bone surface BV bone volume Ca calcium CNBr cyanogen bromide COL1A1 gene encoding alpha1 chain of type I collagen COL1A2 gene encoding alpha2 chain of type I collagen COP coat protein complex CRTAP cartilage-associated protein Cs cross-section CsA cross-sectional area Ct cortical (bone) cTAGE cutaneous T-cell lymphoma-associated antigen CyP cyclophilin DHLNL dihydroxylysinonorleucine crosslink DMEM Dulbecco’s Modified Eagle Medium DNA deoxyribonucleic acid DSC differential scanning calorimetry DXA dual Xray absorptiometry EDS Ehlers-Danlos Syndrome EDTA ethylenediaminetetraacetic acid ERES endoplasmic reticulum exit sites ERGIC endoplasmic reticulum-golgi intermediate complex FKBP FK506 binding protein GAP GTPase activating protein GAPDH glyceraldehyde 3-phosphate dehydrogenase GEF guanine nucleotide exchange factor GGT glucosylgalactosylhydroxylysine GLT25D1 galactosyltransferase 25 KDa, D1 GLT25D2 galactosyltransferase 25 KDa, D2 GPC Golgi to plasma membrane