University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Genetics And Retinal Degeneration: Challenges In Optogenetic Therapy And Indentifying Pathogenic Variants Laura Mary Bryant University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Genetics Commons, and the Ophthalmology Commons Recommended Citation Bryant, Laura Mary, "Genetics And Retinal Degeneration: Challenges In Optogenetic Therapy And Indentifying Pathogenic Variants" (2017). Publicly Accessible Penn Dissertations. 2988. https://repository.upenn.edu/edissertations/2988 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2988 For more information, please contact [email protected]. Genetics And Retinal Degeneration: Challenges In Optogenetic Therapy And Indentifying Pathogenic Variants Abstract Inherited retinal degenerative diseases are one of the leading causes of childhood blindness. While over 200 causative genes have been identified, many cases still have an unknown underlying genetic cause. With the advent of next generation sequencing (NGS), it should be possible to identify the genetic cause in almost every case, provided enough relatives are willing to participate. However, the massive amount of data generated by NGS can make identifying the pathogenic variant challenging. It is necessary to filter the data in order to create a manageable candidate list, but overly strict filtering or erroneous assumptions can result in filtering out the pathogenic ariant.v Identifying the genetic cause of retinal degeneration in each patient will allow us to better identify candidate genes for gene therapy and bring us a step closer to precision medicine. Here we developed an efficient screening system to find candidate mutations with minimal assumptions to avoid screening out pathogenic variants and better identify good candidates for novel gene discovery. Out of an initial cohort of 69 patients we identify the pathogenic variant(s) in 44 of them and identified 11 subjects as good candidates for novel gene discovery. We also need a broad treatment for retinal degeneration to help those who have mutations in genes that are poor candidates for gene therapy or who have an unknown genetic cause. We tested the efficacy of using the GRM6 minimal promoter as a bipolar cell specific promoter to express channelrhodopsin in bipolar cells after photoreceptor degeneration to make the bipolar cells directly respond to light. Surprisingly, we found that unlike in the wildtype mouse retina, the GRM6 promoter is not bipolar cell specific in multiple mouse models of retinal degeneration. This suggests that the genetic profiles of the cells in the inner retina change during retinal degeneration. Understanding these fundamental changes in cell specific gene expression during retinal degenerative processes will be critical in order to develop effective therapeutic strategies for late stage retinal degeneration. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Neuroscience First Advisor Jean Bennett Keywords GRM6, PRPF31, Retinal Degeneration, SEMA4A, Whole Exome Sequencing Subject Categories Genetics | Ophthalmology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2988 GENETICS AND RETINAL DEGENERATION: CHALLENGES IN OPTOGENETIC THERAPY AND INDENTIFYING PATHOGENIC VARIANTS Laura M. Bryant A DISSERTATION in Neuroscience Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation ______________________ Jean Bennett M.D., Ph.D. Professor of Ophthalmology Graduate Group Chairperson _______________________ Joshua I. Gold, PhD Professor of Neuroscience Dissertation Committee Kenneth S. Shindler M.D., Ph.D., Associate Professor of Ophthalmology Valder Arruda M.D., Ph.D., Associate Professor of Pediatrics Claire H. Mitchell, Ph.D., Associate Professor of Anatomy & Cell Biology Marcella Devoto Ph.D., Professor of Pediatrics at the Children's Hospital of Philadelphia (external member) ABSTRACT GENES AND RETINAL DEGENERATION: CHALLENGES IN OPTOGENETICS THERAPY AND IDENTIFYING PATHOGENIC VARIANTS Laura M. Bryant Jean Bennett M.D., Ph.D. Inherited retinal degenerative diseases are one of the leading causes of childhood blindness. While over 200 causative genes have been identified, many cases still have an unknown underlying genetic cause. With the advent of next generation sequencing (NGS), it should be possible to identify the genetic cause in almost every case, provided enough relatives are willing to participate. However, the massive amount of data generated by NGS can make identifying the pathogenic variant challenging. It is necessary to filter the data in order to create a manageable candidate list, but overly strict filtering or erroneous assumptions can result in filtering out the pathogenic variant. Identifying the genetic cause of retinal degeneration in each patient will allow us to better identify candidate genes for gene therapy and bring us a step closer to precision medicine. Here we developed an efficient screening system to find candidate mutations with minimal assumptions to avoid screening out pathogenic variants and better identify good candidates for novel gene discovery. Out of an initial cohort of 69 patients we identify the pathogenic variant(s) in 44 of them and identified 11 subjects as good candidates for novel gene discovery. ii We also need a broad treatment for retinal degeneration to help those who have mutations in genes that are poor candidates for gene therapy or who have an unknown genetic cause. We tested the efficacy of using the GRM6 minimal promoter as a bipolar cell specific promoter to express channelrhodopsin in bipolar cells after photoreceptor degeneration to make the bipolar cells directly respond to light. Surprisingly, we found that unlike in the wildtype mouse retina, the GRM6 promoter is not bipolar cell specific in multiple mouse models of retinal degeneration. This suggests that the genetic profiles of the cells in the inner retina change during retinal degeneration. Understanding these fundamental changes in cell specific gene expression during retinal degenerative processes will be critical in order to develop effective therapeutic strategies for late stage retinal degeneration. iii Table of Contents Abstract…………………………………………………………………………...………i List of Tables…………………………………………………………………………..…iv List of Figures…………………………………………………………………………….v Chapter 1: Introduction………………………………………………………….…….…1 Chapter 2: Prescreening Whole Exome Sequencing Results from Patients with Retinal Degeneration for Variants in Genes Associated with Retinal Degeneration…………….25 Chapter 3: Identification of a novel pathogenic missense mutation in PRPF31 using whole exome sequencing ……………………………………………………………..…71 Chapter 4: On variants and disease-causing mutations: Case studies of a SEMA4A variant identified in inherited blindness………………………………………………….87 Chapter 5: Loss of ON-bipolar cell specific expression from the GRM6 and TRPM1 promoters during retinal degeneration………………………………………………...…94 Chapter 6: General Conclusions and Future Directions ……………………………….117 iv List of Tables Table 2-1: Breakdown of diagnoses included in this study……………………………...47 Table 2-2: Pathogenic and probable pathogenic variants in known disease causing genes identified in subjects in this study……………………………………………………48-53 Table 2-3: Heterozygous variants identified in subjects with recessive disease...............54 Table 2-4: Additional heterozygous variants of uncertain significance found in this study………………………………………………………………………………......55-59 Table 2-5: Multiple compound heterozygous mutations were identified in a subject with multiplex retinitis pigmentosa……………………………………………………………60 Table 5-1: Specific mutations and course of retinal degeneration in mouse lines used in this study………………………………………………………………..……………...107 v List of Figures Figure 2-1: Final outcome of initial screening for variants in genes associated with retinal degeneration……………………………………………………………………...46 Figure 3-1: Clinical features and molecular data for PRPF31 family…………………...81 Figure 3-2: Localization of PRPF31WT and PRPF31L197P in ARPE19 cells as measured by immunofluorescence for the HA tag………………………………………………….83 Figure 4-1: Family Pedigrees and clinical findings……………………………………...92 Figure 5-1: Expression profile of the 4xGRM6-SV40-CaTCH-eGFP construct in WT and rd1 mice……………………………………………………………………..………….108 Figure 5-2: Comparison of young and old rd1 mice 6 weeks after injection with the 4xGRM6-SV40- EGFP in AAV8BP2…………………………………………………..109 Figure 5-3: Variable expression is seen in the neural retina in different sections within the same eye……………………………………………………………………………...…110 Figure 5-4: 4xGRM6-SV40- EGFP expression in various mouse models of retinal degeneration…………………………………………………………………………….111 Figure 5-5: TRPM1 promoter driven expression in WT and rd1 mice and crd1 dog retinal explant cultures…………………………………………………………………………112 Figure 5-6: Comparison of tropism of different AAV capsids in rd1 mice…………….113 vi Chapter 1: Introduction Phenotype and genotype variability in inherited retinal degeneration Hereditary retinal degeneration causes blindness independent of environmental factors, with disease onset often occurring during childhood. This includes non-syndromic forms like Stargardt