IMMUNE FUNCTION IN ATP6V0A2-RELATED CUTIS LAXA by Michelle Morrow BS, The Ohio State University, 1992 PhD, Northwestern University, 2005 Submitted to the Graduate Faculty of the Department of Human Genetics Graduate School of Public Health in partial fulfillment of the requirements for the degree of Master of Science University of Pittsburgh 2017 UNIVERSITY OF PITTSBURGH GRADUATE SCHOOL OF PUBLIC HEALTH This thesis was presented by Michelle Morrow It was defended on April 19, 2017 and approved by Adrea L. Durst, MS, DrPH, LCGC, Assistant Professor, Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh Louise D’Cruz, PhD, Assistant Professor, Department of Immunology, School of Medicine, University of Pittsburgh Thesis Director: Zsolt Urban, PhD, Associate Professor, Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh ii Copyright © by Michelle Morrow 2017 iii Zsolt Urban, PhD IMMUNE FUNCTION IN ATP6V0A2-RELATED CUTIS LAXA Michelle Morrow, MS University of Pittsburgh, 2017 ABSTRACT Cutis laxa (CL) is a genetic disorder of connective tissue. CL has known effects on multiple body systems, but its relationship to immune function has not been characterized. Recently, the laboratory of Jordan Orange, MD, PhD identified a patient with immune deficiency disease who also had a mutation in a known CL-causing gene, ATP6V0A2. This observation prompted us to look for evidence of immune dysfunction among individuals with ATP6V0A2- related CL. We administered an infection history survey to study participants with ATP6V0A2- related CL, their unaffected relatives, and individuals with ELN-related CL. We calculated a novel age weighted infection score for each participant and compared the three groups. We found that individuals with ATP6V0A2-related CL had significantly higher infection scores than unaffected family members (p=0.0004) or individuals with ELN-related cutis laxa (p=0.0016). These data suggest that ATP6V0A2 mutations have a deleterious effect on immune function. Our findings have public health significance because they further characterize the natural history of ATP6V0A2-related CL and thus enhance our ability to assess this condition in the population. The infection history questionnaire has the potential to be used in guiding genetic testing and in guiding medical management, support the public health function of policy development. In addition, research in CL is relevant to aging and to conditions that affect larger populations, such as asthma, chronic obstructive pulmonary disease, and heart disease. iv TABLE OF CONTENTS PREFACE ................................................................................................................................... IX 1.0 INTRODUCTION .............................................................................................................. 10 2.0 LITERATURE REVIEW .................................................................................................. 12 2.1 ELASTIC FIBER FORMATION ............................................................................. 12 2.2 REVIEW OF GENETIC FORMS OF CUTIS LAXA ............................................ 13 2.2.1 ELN-related cutis laxa .................................................................................... 13 2.2.2 FBLN5-related cutis laxa ............................................................................... 14 2.2.3 FBLN4-related cutis laxa ............................................................................... 15 2.2.4 LTBP4-related cutis laxa ................................................................................ 15 2.2.5 PYCR1-related cutis laxa ................................................................................ 16 2.2.6 ALDH18A1-related cutis laxa ........................................................................ 17 2.2.7 ATP7A-related cutis laxa ................................................................................ 17 2.2.8 Geroderma Osteodysplasticum ..................................................................... 19 2.2.9 MACS syndrome ............................................................................................. 19 2.2.10 ATP6V0A2-related cutis laxa ....................................................................... 20 2.2.11 Recently identified ATP6V-related cutis laxa subtypes ............................. 21 2.3 ATP6V0A2 AND IMMUNE FUNCTION ............................................................... 22 2.3.1 Possible roles for ATP6V0A2 in immune function ...................................... 22 v 2.3.2 Vacuolar ATPases: structure, expression pattern, function ....................... 22 2.3.3 Glycoimmunology: potential roles for ATP6V0A2 .................................... 23 2.3.4 ATP6V0A2 regulation of immune response ................................................. 26 3.0 MANUSCRIPT: INCREASED INCIDENCE OF INFECTIONS IN ATP6V0A2- RELATED CUTIS LAXA .......................................................................................................... 31 3.1 BACKGROUND ........................................................................................................ 31 3.2 SUBJECTS AND METHODS .................................................................................. 34 3.2.1 Study Participants .......................................................................................... 34 3.2.2 Methods ........................................................................................................... 35 3.3 RESULTS .................................................................................................................... 40 3.4 DISCUSSION ............................................................................................................. 48 3.5 CONCLUSIONS ........................................................................................................ 53 4.0 RESEARCH SIGNIFICANCE TO GENETIC COUNSELING AND PUBLIC HEALTH ...................................................................................................................................... 55 APPENDIX A: IRB APPROVAL LETTER ............................................................................ 59 APPENDIX B: INFECTION HISTORY QUESTIONNAIRE .............................................. 60 APPENDIX C: CUTIS LAXA CLINICAL QUESTIONNAIRE ............................................ 66 APPENDIX D: CUTIS LAXA STUDY SCREENING SCRIPT ............................................. 76 BIBLIOGRAPHY ........................................................................................................................ 82 vi LIST OF TABLES Table 1. Mutation data .................................................................................................................. 36 Table 2. Infection history data ...................................................................................................... 38 Table 3. Clinical characteristics of individuals with cutis laxa .................................................... 43 Table 4: Statistical test results (all subjects) .................................................................................. 47 Table 5. Statistical test results (adults only) ................................................................................. 48 vii LIST OF FIGURES Figure 1. Age vs. total infection score (unweighted) ................................................................... 39 Figure 2. Location of mutations on ATP6V0A2 protein ............................................................. 41 Figure 3. Familial relationships between individuals with ATP6V0A2-related CL and unaffected relatives ......................................................................................................................................... 42 Figure 4. Age-weighted total infection score for each participant ............................................... 45 Figure 5. Between-group comparison of age-weighted mean infection scores ............................ 46 Figure 6. Between-group comparisons of mean infection scores (adults only) ........................... 47 viii PREFACE I would like to thank my advisor Zsolt Urban, PhD, my assistant program director Andrea Durst, MS, DrPH, LCGC, and my committee member Louise D’Cruz, PhD. I would also like to thank my program director Robin Grubs, MS, PhD, LCGC, my class of 2017 family, my colleagues Emily Spoth and Jennifer Peck the Magnificent, Elizabeth Lawrence and the rest of the Urban lab. I could not have done this without the support of my husband David Fraser, my daughter Milly Fraser, the Huen-Hillers, the Finkels, Anu Hoey, Jennifer Furin, Chelsea Dawn, my parents and siblings, and all the friends who make my life better. You know who you are. ix 1.0 INTRODUCTION Cutis laxa (CL) is a family of connective tissue disorders (Berk, Bentley, Bayliss, Lind, & Urban, 2012) affecting approximately 300 families worldwide. The most prevalent feature in CL is loose, wrinkled skin, giving an aged appearance to individuals affected by this condition. There is variable systemic involvement, although pulmonary and cardiovascular complications are common. The nervous system, musculoskeletal system, and genitourinary system can also be affected. The constellation of symptoms seen in a particular individual is largely dependent on the underlying genetic cause of the condition. CL is usually diagnosed by a combination of clinical exam, abnormal structure and