Towards the Identification of Causal Genes and Contributing
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TOWARDS THE IDENTIFICATION OF CAUSAL GENES AND CONTRIBUTING MOLECULAR PROCESSES UNDERLYING STRABISMUS by Xin Ye B.Sc., The University of British Columbia, 2011 M.Sc., The University of British Columbia, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2019 © Xin Ye, 2019 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Towards the identification of causal genes and contributing molecular processes underlying strabismus submitted by Xin Ye in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Genetics Examining Committee: Wyeth Wasserman Supervisor Angela Brooks-Wilson Supervisory Committee Member Orson Moritz University Examiner Douglas Allan University Examiner Additional Supervisory Committee Members: Supervisory Committee Member Supervisory Committee Member ii Abstract Eye misalignment, or strabismus, has a frequency of up to 4% in a population, and is known to have both environmental and genetic causes. Genes associated with syndromic forms of strabismus (i.e. strabismus concurrent with multiple phenotypes) have emerged, but genes contributing to isolated strabismus remain to be discovered. Only one isolated strabismus locus, STBMS1 on chromosome 7, has been confirmed in more than one family, but the inheritance model of the locus is inconsistent between studied families and no specific causal variant has been reported. The large set of syndromes with strabismus suggests that within the visual system multiple perturbations of an underlying genetic network(s) can have the common output of disrupted eye alignment. Thus, I used a bioinformatic-driven approach to analyze curated genes associated with strabismus to provide insight into the biological mechanisms underlying strabismus, highlighting a link to the Ras-MAPK pathway. During the process, I noticed strabismus presenting within a large number of intellectual disability disorders. Therefore, I studied the co-occurrence of strabismus and other common phenotypes in a series of patients with intellectual disability, which confirmed a significant correlation between eye alignment and intellectual disability. Finally, I resumed efforts from my prior studies to identify the genetic cause in a seven-generation family with isolated strabismus inherited in an autosomal dominant manner. The likely casual gene disruption, altering a likely cis-regulatory region of the FOXG1gene, was identified through the incorporation of linkage analysis, next generation sequencing, and in-depth bioinformatic analyses. This thesis identifies potential roles for genes participating in the Ras-MAPK pathway, emphasizes the role of the central nervous system, and reveals FOXG1 as a causal gene candidate for isolated strabismus. iii Lay Summary Eye misalignment, or strabismus, can affect vision and self-image. Untreated strabismus can lead to blindness in extreme cases. Multiple surgeries may be needed to align the eyes, but the outcome may not be satisfying. Hippocrates observed that strabismus was often presented as a familial condition over 2400 years ago, implying a genetic cause of strabismus. However, we have little understanding of the genetics and mechanism(s) of strabismus today. I combined different methods to study the role of genetics in strabismus, including finding the gene likely causing strabismus in a large family with many afflicted. Strabismus is a complex condition that may involve mechanisms related to eyes, muscles and the brain. This study leads to new directions for research to unravel the causes of strabismus, and the improved understanding may lead to better prevention and treatment. iv Preface Chapter 1 is built upon a published review: Ye XC, Pegado V, Patel MS, Wasserman WW. Strabismus genetics across a spectrum of eye misalignment disorders. Clin Genet. 2014 Aug;86(2):103–11. A version of chapter 2 is published as: Ye XC, van der Lee R, Wasserman WW. Curation and bioinformatic analysis of strabismus genes supports functional heterogeneity and proposes candidate genes with connections to RASopathies. Gene. 2019. doi: 10.1016/j.gene.2019.02.020. WWW helped design the computational studies, reviewed and edited the manuscript, and acquired funding. RvdL reviewed/edited the manuscript and advised on the analyses. I designed and conducted the computational studies, collected and analyzed the data, wrote the manuscript, and made tables and figures. A version of chapter 3 is under review in Pediatric Neurology as: Ye XC, van der Lee R, Wasserman WW, CAUSES Study, Friedman JM, Lehman A. Strabismus in children with intellectual disability: part of a broader motor control phenotype? AL helped design the study, reviewed and edited the manuscript. RvdL, WWW and JMF reviewed/edited the manuscript. I designed and conducted the study, collected and analyzed the data, wrote the manuscript and made tables and figures. CAUSES Study collected the data. v Chapter 4 is in preparation for publication as: Ye XC, Horton J, Lyons C, Pegado V, Ross C, Shyr C, Richmond P, Roslin N, Peterson A, Han X, Higginson M, Giaschi D, Gregory-Evans C, Patel M, Wasserman WW. Linkage analysis identifies an isolated strabismus locus at 14q12 overlapping the FOXG1 syndrome region. WWW and MP helped design the experiments, reviewed and edited the manuscript, and acquired funding. JH, VP, and CL performed ophthalmological exams. NR and AP performed the initial linkage analysis through the CARE4RARE network. MH extracted DNA from blood samples and prepared samples for WES and WGS. CR and XH designed and performed Sanger sequencing. CS and PR maintained and improved the WES and WGS pipelines and performed the analyses. DG was counselled for project development and helped develop the MRI protocol. CGE helped develop the project and provided experimental design support. I designed and conducted the experiments, worked with the family to build the pedigree, extracted DNA from saliva samples, performed the second round of linkage analysis with WGS data, performed WES and WGS analyses, analyzed and integrated relevant data for candidate prioritization, helped develop the MRI protocol, analyzed the data, wrote the manuscript and made tables and figures. Ethics statement: Approval for the study was obtained from University of British Columbia Children’s & Women’s Research Ethics Board (approval number CW10-0317/H10-03215). vi Table of Contents Abstract .................................................................................................................................... iii Lay Summary ........................................................................................................................... iv Preface ....................................................................................................................................... v Table of Contents .................................................................................................................... vii List of Figures ......................................................................................................................... xii List of Supplementary Material ............................................................................................ xiii List of Abbreviations ............................................................................................................. xiv Acknowledgements ................................................................................................................. xv Chapter 1: Introduction ........................................................................................................... 1 1.1 Overview ..................................................................................................................... 1 1.2 Introduction of strabismus ........................................................................................... 2 1.2.1 Classification ....................................................................................................... 2 1.2.2 Epidemiology ...................................................................................................... 2 1.2.3 Pathology............................................................................................................. 3 1.2.4 Animal models..................................................................................................... 4 1.3 Genes of strabismus ..................................................................................................... 5 1.3.1 Family and twin study .......................................................................................... 5 1.3.2 Linkage analysis .................................................................................................. 7 1.3.3 Genome-wide association studies ......................................................................... 8 1.3.4 Next generation sequencing ................................................................................. 9 1.4 Frontiers of strabismus research - genes implicated in strabismus .............................. 10 vii 1.4.1 Duane retraction syndrome ................................................................................ 10 1.4.2 Congenital fibrosis of the extraocular muscles ..................................................