Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine 1-1-2019 Multimodal Imaging And Asymmetry Of Disease Progression In Rhodopsin-Associated Autosomal Dominant Retinitis Pigmentosa Lawrence Chan Follow this and additional works at: https://elischolar.edu/ymtdl Part of the Medicine and Health Sciences Commons Recommended Citation Chan, Lawrence, "Multimodal Imaging And Asymmetry Of Disease Progression In Rhodopsin-Associated Autosomal Dominant Retinitis Pigmentosa" (2019). Yale Medicine Thesis Digital Library.edu/ymtdl/3480 This Open Access Thesis is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale. For more information, please contact elischolar@yale.
Multimodal Imaging and Asymmetry of Disease Progression in Rhodopsin- associated Autosomal Dominant Retinitis Pigmentosa A Thesis Submitted to the Yale University School of Medicine in Partial Fulfillment of the Requirements for the Degree of Doctor of Medicine by Lawrence Chan 2019 Abstract Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited retinal degenerative diseases with no known cure to date. The recent gene therapy treatment for Leber’s congenital amaurosis and RP caused by mutations in RPE65 have resulted in dramatic improvements in vision, leading to excitement for other potential gene therapies on the horizon. Upcoming clinical trials will be targeting patients with specific mutations, and measurements of disease progression will be needed for each genetic subtype of RP in order to determine whether treatments are successful. In this retrospective cohort study, we examined 27 RP patients with confirmed autosomal dominant mutations in the rhodopsin gene by monitoring rates of progression as measured structurally with ellipsoid zone (EZ) line width on spectral domain optical coherence tomography (SD-OCT), horizontal and vertical hyperautofluorescent ring diameters on short wavelength fundus autofluorescence (SW-FAF), and as measured functionally with 30 Hz flicker amplitudes on electroretinography (ERG).
Each structural parameter was measured twice by the author four weeks apart. The mean rates of progression were -158.4%) for EZ line widths, -122.5%) for horizontal diameters, and -108.9%) for vertical diameters. High test-retest reliability was observed for the parameters (EZ line intraclass coefficient [ICC] = 0.9989, horizontal diameter ICC = 0.9889, vertical diameter ICC = 0. The three parameters were also correlated with each other (r = 0.9325 for EZ line and horizontal diameter; r = 0.9081 for EZ line and vertical diameter; r = 0.9630 for horizontal and vertical diameters).
No significant changes in ERG amplitude were seen. The subjects were classified by rhodopsin mutation class (I, IIa, IIb, III) and morphology of the hyperautofluorescent ring (typical vs. No significant differences in rates of structural progression were observed by rhodopsin mutation class or by ring morphology. Finally, higher rates of asymmetry of progression between the left and right eyes were detected for EZ line width (23% of subjects), horizontal diameter (17%), and vertical diameter (25%), as compared to studies on other forms of RP.
Acknowledgments I would like to thank my mentors and thesis advisors Dr. Stephen Tsang and Dr. Ron Adelman for their mentorship and guidance with this project and my path through medicine and ophthalmology. I would also like to thank members of the Tsang lab for their invaluable assistance, including Dr.
Ronaldo Carvalho for his help with planning the experimental design and Jimmy Duong for his much-needed statistical wizardry and patience with my incompetence. Furthermore, I also thank Dr. Ching-Hwa Sung from Weill Cornell Medicine for her expertise in biochemical characterization of rhodopsin mutations. I would like to express my gratitude to Dr.
Ninani Kombo for her efforts in helping me with the revision process. I also want to show my deepest appreciation to the Yale Department of Ophthalmology and Visual Science, especially to Deana Ralston for her incredible guidance with finalizing the thesis. I thank my wonderful friends, classmates, mentors, and family for their unending support for as long as I can remember. Finally, I want to thank my fiancée and life partner Yue Meng for her unconditional love and guidance at every step of my life these past seven years.
Table of Contents Introduction………………………………………………………………………………1 Statement of Purpose…………………………………………………………………16 Methods…………………………………………………………………………………17 Results………………………………………………………………………………….47 1 Introduction Retinitis pigmentosa (RP), a group of inherited retinal diseases with an incidence of approximately one in 4000 people, is characterized by progressive photoreceptor death and irreversible vision loss (1). Typically, the initial loss of photoreceptors primarily involves the rods, thereby diminishing peripheral and night vision, followed by worsening tunnel vision and eventual loss of central vision mediated by cone photoreceptor death (1). Ophthalmoscopic hallmarks of the disease include retinal arteriolar attenuation, bone-spicule peripheral pigment deposits, and waxy pallor of the optic disc (2). The clinical presentation of retinitis pigmentosa is highly variable.
The severity and pattern of vision loss may be mild or severe. The rate of disease progression can be slow or rapid, and the age of onset can be as early as childhood while some individuals remain asymptomatic until mid-adulthood. Allelic heterogeneity, in which each gene locus may have different mutations that cause the same disease entity, contributes to the diverse genetic etiology of RP; for example, over 300 different RPGR mutations have been identified in families with X-linked RP (3). Even among members of the same family, the same mutation may result in different phenotypic manifestations.
RP is also a genetically heterogeneous disease, with over 50 genes that have been found to be associated with non-syndromic RP. Further complicating the heterogeneity of the disease is that different mutations in the same gene may result in different modes of inheritance. The pattern of inheritance can be autosomal recessive (15-20%), autosomal dominant (20- 2 25%), X-linked recessive (10-15%), or sporadic (30%) (2, 4). RP may also be syndromic, as seen in Bardet-Biedl syndrome, Usher syndrome, abetalipoproteinemia (Bassen-Kornzweig syndrome), and phytanic acid oxidase deficiency (Refsum disease) (2).
Despite the genetic complexity of RP, improvements in the cost and efficiency of molecular techniques that allow for the high-throughput DNA sequencing of patients have resulted in clinicians being able to append a molecular diagnosis to their clinical diagnosis. Specifically, the advent of next-generation sequencing (NGS), which is able to perform massively parallel sequencing runs on the order of millions of DNA fragments using micron-sized beads, has dramatically increased the speed of sequencing many-fold and enabled the capture of a broader spectrum of mutations compared to conventional Sanger sequencing (5). Molecular basis of the visual cycle To understand how mutations in certain genes may cause RP, an outline of the visual cycle will need to be described. The first step in vision occurs when light enters the eye and is focused by the cornea and lens onto the retina (photosensitive tissue located posteriorly within the eye).
In the retina, the light- sensitive photoreceptor cells called rods and cones convert the external light stimuli into electrical impulses that the brain processes to form an image. Rod photoreceptors contain the visual pigment rhodopsin, which is a light-sensitive G- 3 protein coupled receptor that consists of the apoprotein opsin and 11-cis-retinal, a chromophore. When light is absorbed by rhodopsin, the 11-cis-retinal is converted to all-trans-retinal and leads to a series of conformational changes of the opsin that activates the GTP-binding protein transducin, triggering a canonical cyclic guanosine monophosphate (cGMP) second-messenger cascade through the activation of cGMP phosphodiesterase (PDE) (2). PDE hydrolyzes cGMP, leading to closure of the cGMP-dependent cation channels normally responsible for influx of Na+, Ca2+, and Mg2+.
The resulting hyperpolarization of the photoreceptor cell decreases the rate of transmitter release and elicits responses in second-order (bipolar) cells for further neural transmission (6). The all-trans-retinal is converted to all-trans-retinol and is transported to the retinal pigment epithelium (RPE) to be recycled into 11-cis-retinal for transport back into the rods (2). Rods are sensitive to low levels of light, and psychophysical experiments have shown that they can register single photon absorptions (6). Since rods play a crucial role in enabling vision in low-light scenarios and are anatomically located in the periphery of the retina, RP patients usually experience night blindness (nyctalopia) and loss of peripheral vision as their initial symptoms.
The organization of the rod photoreceptor consists of a synaptic body that interfaces with the bipolar/horizontal cells, a cell body, an inner segment (IS) which contains the endoplasmic reticulum, mitochondria, and Golgi apparatus, 4 and an outer segment (OS) which houses membranous discs containing mostly opsin within a plasma membrane. The IS and OS are connected by the connecting cilium, and the OS interfaces with and is phagocytosed by the RPE. a) Illustration showing cell organization within the retina. b) Cross- sectional H&E stain of retina.
Image from Wikimedia Commons. 5 Structure of rhodopsin As previously mentioned, rhodopsin (RHO) is the G-protein coupled receptor (GPCR) that is responsible for the first step in allowing rod photoreceptors to detect light. It is synthesized in the rough endoplasmic reticulum and then transported through the Golgi apparatus where it ultimately functions within the discs of the OS (7). 30% to 40% of all autosomal dominant RP (adRP) is caused by mutations in the RHO gene, and over 120 different mutations in RHO have been identified (2, 8).
One study of 200 families with clinical evidence of adRP found that rhodopsin mutations were the most common cause of disease, representing 26.5% of the total cases of adRP (9). In addition to its role in adRP, rhodopsin was the first GPCR whose crystal structure was elucidated, and it served as a prototype template for understanding the rest of the GPCR superfamily (8). Rhodopsin is a highly conserved protein among vertebrate species, and similar proteins have even been found in the visual systems of invertebrates such as Drosophila melanogaster (10). The structure of rhodopsin consists of four specialized domains that assist in the maintenance of protein structure, trafficking, and phototransduction: 1) cytoplasmic, 2) intradiscal, 3) transmembrane, and 4) ligand-binding domains (11).
The cytoplasmic C-terminal domain of rhodopsin regulates its trafficking and interactions with other proteins in the phototransduction cascade such as transducin (11). The intradiscal domain contains the extracellular loops between transmembrane domains and the N- terminus. Research suggests that mutations in the intradiscal domain result in 6 misfolding of the protein and accumulation of the protein within the secretory system, leading to disease (12). The transmembrane domains have been shown to have several residues that are important for rhodopsin protein stability and function (13).
The ligand-binding domain is where the 11-cis-retinal chromophore binds with the opsin apoprotein (14). Biochemical classification of rhodopsin mutations Mutations in rhodopsin causing adRP have been grouped into three classes (Table 1) based on the phenotypes of the proteins from in vitro studies that transfected human tissue culture cells with wild-type and mutant rhodopsin cDNA clones (8, 11, 12). Class I mutations are located near the C-terminus of the protein or within the first transmembrane segment. The protein resembles wild- type rhodopsin in terms of protein levels, ability to associate with the 11-cis- retinal chromophore, and subcellular localization (15, 16).
However, these mutations cause rhodopsin to activate transducin inefficiently in the presence of light (17). Class II mutations cause decreased binding to 11-cis-retinal and result in accumulation within the endoplasmic reticulum, possibly due to issues with protein folding and stability (15, 17). Within class II, further subclassification can be made for those mutants that predominantly localize intracellularly (class IIa) and those that preferentially localize to the cell surface (class IIb) (16). Finally, class III mutants form rhodopsin poorly and at low levels, are retained in the endoplasmic reticulum, and may form aggresomes, causing targeted degradation 7 by the ubiquitin proteasome system (18).
Studies have suggested that impaired endocytic activity is the primary mechanism by which class III mutations cause RP (19). One common finding among all three classes of mutations is the decreased sensitivity to light and less efficient activation of transducin (17).