MOLECULAR GENETICS OF HUMAN RETINAL DISEASE

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1 ?Annu. Rev. Genet : Copyright c 1999 by Annual Reviews. All rights reserved Amir Rattner, 1,4 Hui Sun, 1,4 and Jeremy Nathans Department of Molecular Biology and Genetics, 2 Department of Neuroscience, 3 Department of Ophthalmology, 4 Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; jnathans@jhmi.edu Key Words macular degeneration, retinitis pigmentosa, photoreceptor, ophthalmic genetics Abstract The past decade has witnessed extraordinary progress in retinal disease gene identification, the analysis of animal and tissue culture models of disease processes, and the integration of this information with clinical observations and with retinal biochemistry and physiology. During this period over twenty retinal disease genes were identified and for many of these genes there are now significant insights into their role in disease. This review presents an overview of the basic and clinical biology of the retina, summarizes recent progress in understanding the molecular mechanisms of inherited retinal diseases, and offers an assessment of the role that genetics will play in the next phase of research in this area. Introduction The Human Retina Summary of Some Common Clinical Phenotypes Mechanisms of Cell Death in Retinitis Pigmentosa Diagnosis of Retinal Disease Specific Gene Defects Phototransduction Structure and Biosynthesis MOLECULAR GENETICS OF HUMAN RETINAL DISEASE CONTENTS The Visual Cycle Retinal Pigment Epithelium Development Metabolism Miscellaneous Future Directions Genetic Analysis Prospects for Therapy /99/ $

2 90 RATTNER SUN NATHANS INTRODUCTION? This review summarizes recent work on the molecular mechanisms of inherited retinal disease. It emphasizes those retinal diseases for which some insight exists into pathogenic mechanisms and gives only cursory coverage to systemic diseases that have retinal manifestations and to retinal diseases for which the responsible genes have not yet been identified. Had this review been written ten years ago, it could have provided comprehensive coverage of this topic in only a few pages. However, the past decade has witnessed extraordinary progress in retinal disease gene identification, the analysis of animal and tissue culture models of disease processes, and the integration of this information with clinical observations and with retinal biochemistry and physiology. As a result, this review can present only a summary of current information. 1 For additional information, the reader may wish to consult the following sources. Retinal physiology with an emphasis on clinical applications is presented in Reference 89 and with an emphasis on fundamental processes in Reference 199. A comprehensive treatment of the retinal pigment epithelium can be found in Reference 146, and descriptions of the natural history and management of retinal diseases can be found in References 92 and 177. An invaluable on-line resource, RetNet ( has an up-to-date compendium of references on inherited retinal disease and is maintained by Dr. Stephen Daiger at the University of Texas. Recent reviews on inherited retinal disease include References 17, 20, 59, 64, and 84. The Human Retina Structure and Cell Biology The retina is an outpouching of the central nervous system (CNS) that covers the back wall of the eye. Three layers of cells comprise 1 Abbreviations: Amino acids are referred to by the single letter code; amino acid substitutions are referred to by the identity of the original amino acid, the codon number, and the identity of the new amino acid, e.g. K296E refers to the substitution of glutamate for lysine at codon 296; ABCR, retina-specific ABC transporter; ADRP, autosomal dominant RP; AMD, age-related macular degeneration; ARRP, autosomal recessive RP; CACNA1F, calcium channel α-subunit; CNCG, cyclic nucleotide gated channel; CNS, central nervous system; CRALBP, cellular retinaldehyde binding protein; CRX, cone-rod homeobox; CSNB, congenital stationary (i.e. nonprogressive) night blindness; ECM, extracellular matrix; EOG, electrooculogram; ERG, electroretinogram; GA, gyrate atrophy; GCAP, guanylate cyclase activating protein; LHON, Leber hereditary optic neuropathy; MMP, matrix metalloproteinase; ND, Norrie Disease; NRL, neural retina leucine zipper; OAT, ornithine amino transferase; PDE, phosphodiesterase; rds, retinal degeneration slow; rd, retinal degeneration; REP-1, rab escort protein-1; RetGC, retinal guanylate cyclase; RGS, regulator of G-protein signaling; ROM-1, rod outer segment membrane protein- 1; ROS, rod outer segment; RP, retinitis pigmentosa; RPE, retinal pigment epithelium; RPGR, retinitis pigmentosa GTPase regulator; SFD, Sorsby fundus dystrophy; TIMP, tissue inhibitor of metalloproteinases; XLRP, X-linked RP; XLRS, X-linked retinoschisis.

3 ?HUMAN RETINAL DISEASE 91 Figure 1 Schematic of the vertebrate retina and the adjacent choroid. The vitreous is at the bottom and the sclera is at the top. Most of the major cell types and structures are shown: a choroidal blood vessel, Bruch s membrane separating the choroid from the RPE, an RPE cell, rods and cones, bipolar cells, a ganglion cell, and a Muller glial cell. Horizontal and amacrine cells that process information in the outer and inner plexiform layers, respectively, are not shown. the neural retina (Figure 1). The outer layer contains the photoreceptor cells, the rods and cones, which mediate dim light (scotopic) and bright light (photopic) vision, respectively. In humans only 5% of photoreceptor cells are cones and 95% are rods. Cones are found throughout the retina but are most concentrated within a small central region, the fovea. A somewhat larger zone, called the macula, is centered on the fovea and also includes the immediately surrounding rod-rich retina. Cones mediate color vision, and in humans come in three types that differ depending upon which one of three light sensing pigments (visual pigments) is present. The phototransduction proteins are located within a modified and greatly enlarged cilium, referred to as an outer segment, that protrudes from the apical face of each photoreceptor. The outer segment is filled with flattened membrane sacs called discs and is constantly renewed by new synthesis and assembly at its base, and by shedding of older material from its tip. The second (inner) layer of cells contains bipolar cells, the second-order neurons onto which photoreceptors synapse, as well as horizontal and amacrine cells, interneurons that process visual information in the outer and inner retina, respectively. The third and innermost

4 92 RATTNER SUN NATHANS? Figure 2 The visual cycle (adapted from Reference 191). cell layer contains ganglion cells, the output units of the retina, as well as some amacrine cells. Ganglion cell axons track along the inner surface of the retina, coming together at the optic disc to form the optic nerve. Adjacent to the retina is the retinal pigment epithelium (RPE), a CNS derivative that separates the retina from the choroidal circulation, the blood supply for the outer retina. The RPE lies in close apposition to the outer segments and each RPE cell contacts approximately 45 outer segments. As one of its functions, the RPE engulfs and digests the distal 10% of each outer segment daily, a total biomass equivalent to several average-size cells. This digestion, which continues throughout life, makes the RPE cell the most active phagocytic cell in the human body. The RPE also participates in a unique exchange with the photoreceptors known as the visual cycle, which plays a critical role in visual pigment regeneration (Figure 2). All vertebrate visual pigments consist of an apoprotein, opsin, linked covalently to a chromophore 11-cis retinal (an aldehyde derivative of vitamin A). Photoactivation isomerizes retinal from 11-cis to all-trans, after which all-trans retinal dissociates from opsin, to be replaced by a new molecule of 11-cis retinal. In rods, the released all-trans retinal chromophore is reduced to all-trans retinol (the corresponding alcohol derivative of vitamin A) and transported to the RPE. Within the RPE it is esterified to a lipid, chemically isomerized to the 11-cis configuration, hydrolyzed from the ester linkage, and finally oxidized to the aldehyde and returned to the photoreceptor. As discussed below, the isomerization cycle for cones may occur within the retina rather than the RPE. The flux through the visual cycle is extremely high: gazing at the blue sky on a sunny day produces 20,000 photoisomerizations

5 ?HUMAN RETINAL DISEASE 93 per rod per second (199). Under these viewing conditions, the visual cycle in each RPE cell therefore processes approximately 1,000,000 chromophores per second. Phototransduction The excitation phase of phototransduction begins with photoactivation of a visual pigment, which induces a conformational change that catalyzes the activation of a photoreceptor G protein, transducin (Figure 3A). Each activated alpha subunit of transducin, now bound to GTP, displaces the inhibitory subunit of a cgmp phosphodiesterase (PDE), which then catalyzes the hydrolysis of cgmp to GMP. A cgmp-gated cation channel located in the outer segment plasma membrane closes in response to lower intracellular cgmp, leading to a decrease in sodium and calcium influx. The resulting membrane hyperpolarization leads to a graded attenuation in neurotransmitter release at the photoreceptor synapse. Within the outer segment, the drop in intracellular calcium triggers a negative feedback loop that mediates light adaptation in the case of a sustained stimulus, or restores the cell to its preactivated state in the case of a transient stimulus. This feedback loop is described in greater detail under Guanylate Cyclase and Guanylate Cyclase Activating Protein (GCAP). The recovery phase of phototransduction involves active turnoff and recycling of the transduction components (Figure 3A). Rhodopsin is inactivated by the combined action of rhodopsin kinase, which specifically phosphorylates photoactivated rhodopsin, and arrestin, which binds phosphorylated rhodopsin (see below under Arrestin and Rhodopsin Kinase ). Transducin hydrolyzes GTP to GDP and P i,a reaction that is accelerated by a member of the regulator of G-protein signaling (RGS) family, RGS-9, thereby allowing PDE to return to its inactive state. The action of guanylate cyclase leads to an increase in the cytosolic cgmp concentration, which in turn leads to an increase in the number of open cgmp-gated channels. The locations within the outer segment of the main structural and phototransduction proteins are shown in Figure 3B. More extensive descriptions of phototransduction can be found in References 115, 188, and 262. In the industrialized world, the most common diseases involving the retina are diabetic retinopathy, glaucoma, and age-related macular degeneration (AMD), which together affect several percent of the population. Each of these diseases has both genetic and nongenetic components. By contrast, the simple Mendelian retinal diseases, which are the focus of this review, affect in aggregate approximately one person in Many of the Mendelian diseases have an earlier onset and some have a more severe clinical course than typically observed for the three more common disorders listed above and, for the most part, they are untreatable. These characteristics, together with the possibility of using genetic approaches to understand disease mechanisms, have focused attention on the Mendelian disorders. Hereditary retinal diseases are characterized by age of onset, severity and topographic pattern of visual loss, rod vs cone involvement, ophthalmoscopic findings, Summary of Some Common Clinical Phenotypes

6 ?(A) The phototransduction cascade. Upper, the excitation phase; lower, the Figure 3 recovery phase. Activated species are shown in italics with an asterisk ( ) appended. Catalytic reactions that amplify the signal are shown as bold arrows. Arr, arrestin; RK, rhodopsin kinase; R, dark rhodopsin, i.e. with 11-cis retinal attached; R, photoactivated rhodopsin, i.e. with all-trans retinal attached; T, transducin; PDE, cgmp phosphodiesterase; GCAP, guanylate cyclase activating proteins; G cyclase, guanylate cyclase; CNCG, cgmp-gated channel; RGS, regulator of G-protein signaling. (B) Subcellular localization of rod outer segment proteins. Each of the four schematic diagrams shows a rod outer segment with a stack of (internal) disc membranes. The indicated proteins are localized to the following regions (left to right): the edge of the discs, the plasma membrane, the disc and plasma membranes, the cytosol. Several proteins indicated as cytosolic are tethered to the cytosolic leaflet of the membrane by a covalently linked lipid.

7 ?HUMAN RETINAL DISEASE 95 and family history. Retinitis pigmentosa (RP), a clinically and genetically heterogeneous group of disorders, is classically characterized by impaired rod function, a progressive degeneration of the retina beginning in the midperiphery, and a characteristic retinal deposit, the appearance of which has given it the name bone spicule pigmentary deposit (91, 176). RP usually spares the central retina, which mediates high-acuity vision, until late in the disease. Eventually, most RP patients lose both rod and cone function. In a minority of patients with RP or RP-like diseases, cone dysfunction occurs early in the disease; this is referred to as conerod dystrophy. RP can have X-linked (XLRP), autosomal recessive (ARRP), or autosomal dominant (ADRP) modes of inheritance, and as described below, each of these forms shows both locus and allelic heterogeneity. Macular degenerations show the converse anatomic pattern, preferentially affecting the central retina and causing a loss of acuity often with minimal impairment in peripheral vision. Leber congenital amaurosis is the name given to nonsyndromic retinal dystrophies that are diagnosed shortly after birth or in infancy. Additional descriptions of clinical phenotypes are presented in the text below. In humans with RP (162) and in mouse models of RP (30, 140, 187), photoreceptor cell death occurs by apoptosis as determined by analysis of DNA fragmentation and by the absence of an inflammatory response. Interestingly, in human RP retinas there is patchy loss of both rod and cone photoreceptors (34), and in chimeric or mosaic mouse models of RP, diseased or dying photoreceptor cells induce cell death in adjacent genetically normal photoreceptor cells (102, 117). The deleterious effect of proximity to defective and/or dying cells is presumably responsible for the eventual loss of cones in those RP patients who carry rodspecific gene defects. Within this group of patients, the progressive loss of conemediated vision has a far greater impact on quality of life than does the absence of rod function. One reasonable therapeutic goal for this group of patients might be to diminish cone loss by preserving rod viability even in the absence of rod function. Genetic analysis depends on accurate disease classification. Retinal diseases represent a favorable group in this respect because, in contrast to most tissues, the appearance and function of the retina can be monitored with high precision using methods that are minimally invasive. The following paragraphs summarize the principal methods used for clinical testing of retinal disease (16, 25, 255). Mechanisms of Cell Death in Retinitis Pigmentosa Diagnosis of Retinal Disease Fundus Imaging The back of the eye, the fundus, can be observed through the ophthalmoscope. The clinician can therefore monitor the appearance of the retina and its vasculature, and can detect gross abnormalities such as defects in the RPE, retinal and subretinal deposits, neovascularization, and retinal detachments.

8 96 RATTNER SUN NATHANS? Psychophysics Fundus photography can be combined with intravenous fluorescein injection to monitor intraocular blood flow, a technique referred to as fluorescein angiography. Fluorescein angiography can detect the neovascularization that often accompanies macular degeneration and diabetic retinopathy. Fluorescein angiography is also useful for identifying defects in the RPE, since fluorescent signals from the choroidal circulation are normally attenuated by melanin within the RPE. Several novel imaging methods show great promise for high-resolution noninvasive monitoring of retinal disease processes. Optical coherence tomography uses laser interferometry to measure optical reflectivity and generates cross-sectional images of the retina with a spatial resolution of 10 µm (95, 101). A related technique, autofluorescent imaging of the fundus, measures the distribution and levels of autofluorescent pigments deposited within the RPE, a characteristic of the aging human eye that appears to be a risk factor for macular degeneration (245). Most recently, adaptive optics, a method developed to enhance the quality of astronomical images, has been used to view the human retina at single-cell resolution (201). By definition, psychophysical testing relies on a behavioral response to a stimulus. Human visual psychophysics enjoys the advantages of an enormous range of stimuli and a highly developed nervous system. Visual stimuli can vary in intensity, wavelength, duration, and spatial extent, and they can be superimposed on adapting stimuli or convey a complex pattern. In practice, the clinical assessment is usually confined to tests of visual acuity, color vision, and the visual field using either static or kinetic stimuli to map out the regions within the retina that show decreased or absent sensitivity (scotomas). Less commonly, dark adaptation is assessed. For this purpose, the subject is placed in a dark room following exposure to a bright light, and at frequent intervals over the ensuing minutes the threshold for detection of a brief test flash of variable intensity is determined. Subjects with retinitis pigmentosa, congenital stationary night blindness (CSNB), or any of a number of diseases that affect scotopic vision can show altered kinetics of dark adaptation. Electrophysiology The retina generates a complex electrical response to illumination that can be recorded by measuring the fluctuating potential between a corneal and a reference electrode. This signal, referred to as the electroretinogram (ERG), consists of sequential waves of activity that originate in the photoreceptors (the a-wave), the inner retina (the b-wave), and the RPE (the c-wave). Ganglion cell and optic nerve activity do not contribute to the ERG response to flashes of light. The ERG can selectively measure rod or cone responses: Rod responses are obtained under scotopic conditions, and cone responses are obtained under photopic conditions or by using a high-frequency stimulus train that the more sluggish rods cannot track. A patient with photoreceptor loss over a fraction of the retina will show a proportional decrease in ERG amplitude under full-field (ganzfeld) illumination. Because of its high information content and because it is an objective

9 ?HUMAN RETINAL DISEASE 97 test, the ERG has become an important clinical tool in diagnosing retinal disease. A second test, the electrooculogram (EOG), measures a standing potential across the eye that originates in the RPE. Its greatest utility is in assessing diffuse disease of the RPE, especially Best vitelliform macular dystrophy (described below). In this section we briefly describe each of the inherited retinal diseases for which the responsible gene has been identified. The normal functions of the protein products of some of these genes are not well understood, and for most of the genes our understanding of the pathophysiological consequences of mutation is incomplete. In particular, we cannot easily connect what is known of the function or abundance of the encoded protein with those aspects of disease that are most important to the patient the rapidity and severity of visual loss. Despite these limitations in our current knowledge base, we have taken the liberty in this review of grouping the genes described below by the known or likely mechanism of action of their protein products or the cell types in which the disease genes are likely to act. Genes listed in the miscellaneous group are ones for which there are insufficient data to assign a mechanistic or cell-type category. As described in the introductory paragraphs, rods and cones utilize a G-protein coupled enzyme cascade to amplify the signal derived from a photoactivated visual pigment. For rod photoreceptors, the major protein components of this cascade have been identified and the corresponding cdnas encoding them have been isolated. Although the possibility exists that additional regulatory proteins remain to be discovered, the broad outlines of the phototransduction cascade are now well established. In general, phototransduction in cones appears to utilize proteins that are homologous to, but distinct from, those in rods. Because cones constitute only a small minority of photoreceptors in most mammalian retinas, including the human retina, cone phototransduction components have been identified principally by using the cloned rod cdnas as hybridization probes. One of the most successful approaches to identifying retinal disease genes has been systematic mutation screening of the approximately 15 rod phototransduction genes using a panel of several hundred unrelated patients who are affected by a diverse set of retinal diseases (53). In its first application the identification of rhodopsin gene mutations in ADRP (60) this effort was focused by information obtained from linkage analysis (157), but for the most part it has been applied without linkage information. Systematic mutation screening is well suited to the study of retinal diseases because of the extreme genetic heterogeneity within this population and the high frequency of recessive disease that together preclude effective linkage analysis for the majority of patients. SPECIFIC GENE DEFECTS Phototransduction

10 98 RATTNER SUN NATHANS Rhodopsin? As described below under Structure and Biosynthesis, most rhodopsin mutations that produce a phenotype in humans appear to affect folding, stability, or intracellular trafficking and cause ADRP. Exceptions to this pattern are seen in two rhodopsin mutations (G90D and A292E) in patients with CSNB (54, 222). In vitro analysis of these proteins indicates that they are active in G-protein signaling in the absence of the retinal chromophore (54, 189). In both cases, the newly acquired carboxylate appears to weaken the salt bridge between lysine 296, the site of covalent attachment of the chromophore in the seventh transmembrane domain, and its counterion, glutamate 113, in the third transmembrane domain. A large body of work shows that this salt bridge is essential for maintaining opsin in an inert state (190). In heterozygotes these mutant proteins greatly reduce the amplitude of the rod ERG, presumably because the small fraction of mutant protein that is unliganded produces a signal that mimics a desensitizing light stimulus. The lack of rod degeneration in carriers of the G90D and A292E mutations most likely reflects the ability of these proteins to bind 11-cis retinal, which stabilizes them and holds them in an inactive state. Two mutant rhodopsins that are unable to bind 11-cis retinal due to substitution at lysine 296 (K296E and K296M) are found in patients with ADRP. In vitro experiments indicate that the lysine 296 mutants can activate transducin (196, 197), reflecting a lack of the inhibitory salt bridge between residues 113 and 296. Transgenic mice expressing low levels of K296E opsin show minimal rod ERG abnormalities early in life, but their rods degenerate over the ensuing several months regardless of whether they are or are not exposed to light (133). Rod outer segments (ROS) from the K296E transgenic mice contain significant quantities of K296E opsin that is constitutively phosphorylated and bound by arrestin. Removal of arrestin (by washing the ROS membranes in urea) and dephosphorylation of opsin and rhodopsin reveals the expected light-independent transducin activation by the K296E opsin. Taken together, the in vivo and in vitro experiments suggest that the K296E opsin exhibits constitutive activity in vivo and is shut off by phosphorylation and arrestin binding. The mechanism by which K296E opsin causes photoreceptor cell death may be related to the considerably lower stability of opsin compared to rhodopsin (circa C; 104). Transducin The single known transducin alpha mutation (G38D) is of special interest because it is responsible for autosomal dominant CSNB in one of the largest and most famous pedigrees in human genetics, the Nougaret family, which traces its ancestry to Jean Nougaret ( ), who along with many of his descendents experienced severely reduced night vision (58). Residue 38 forms a critical part of the active site for GTP hydrolysis as judged by its position within a loop that hydrogen bonds with the beta and gamma phosphates of GTP (124). Mutations in the corresponding position (codon 12) in the small monomeric G-protein p2l ras diminish GTPase activity and are oncogenic as a result of excessive signaling (129). The G38D mutation in the alpha chain of transducin presumably leads to excessive signaling in response to light, thereby elevating the background

11 ?HUMAN RETINAL DISEASE 99 noise within rod photoreceptors. A defect at the level of the outer segment would eliminate the a-wave in the electroretinogram, a finding that has been observed in some CSNB subjects (27, 28). Other CSNB subtypes are associated with a normal a-wave but abnormal or absent b-waves, indicative of defects beyond the outer segment, for example, in neurotransmission (see below under Calcium Channel CACNA1F ). Arrestin and Rhodopsin Kinase These two proteins act together to shut off rhodopsin activity (Figure 3A). Within one or a few hundred milliseconds of photoactivation, rhodopsin kinase phosphorylates photoexcited rhodopsin, which is then capped by arrestin binding. Rhodopsin regeneration following photoactivation, which involves all-trans retinal release, 11-cis retinal binding, rhodopsin dephosphorylation, and arrestin release, occurs on a time scale of minutes. Recessive loss-of-function mutations in either arrestin (72) or rhodopsin kinase (35, 259) produce Oguchi disease, a variant of CSNB. Oguchi disease due to rhodopsin kinase mutation causes slowing of both rod and cone recovery after light exposure, which implicates rhodopsin kinase in cone pigment phosphorylation (35). A diagnostic fundus abnormality in Oguchi disease is the Mizuo-Nakamura phenomenon, a metallic golden-yellow color change that is induced by light. The origin of the Mizuo-Nakamura phenomenon is still uncertain, but it is also seen in patients with X-linked retinoschisis (46) and X-linked recessive cone dystrophy (94). It has been suggested to arise from an abnormal concentration of extracellular potassium (46). Interestingly, the same arrestin gene mutation responsible for most cases of Oguchi disease in Japan (a frame shift at codon 309) is responsible for RP in several percent of Japanese patients with ARRP (171). In this study, one member of a pair of siblings who were homozygous for a codon 309 frameshift mutation exhibited classic RP without the Mizuo-Nakamura phenomenon whereas the other sibling had Oguchi disease. cgmp PDE The alpha and beta chains of PDE are each approximately 90 kda and together form the catalytically active complex. The gamma subunit associates with alpha and beta to form an enzymatically inactive alpha 1 beta 1 gamma 2 heterotetramer. When liganded to GTP, the alpha subunit of transducin induces the dissociation of the gamma subunits, thereby activating the alpha-beta heterodimer. Recessive mutations within the alpha (103) and beta (45, 155, 156, 242) subunits of PDE account for several percent of patients with ARRP. This disease association was predicted by earlier work on the retinal degeneration (rd) mouse, which carries a defective beta PDE gene and exhibits a marked elevation in cgmp and a progressive loss of rod photoreceptors (63). In the rd mouse and in this subset of ARRP patients, increased cgmp presumably leads to an increase in the number of cgmp gated channels that are open, an increase in sodium and calcium influx across the ROS plasma membrane, and a resulting increase in the requirement for high-energy phosphate to extrude intracellular sodium. No disease-causing mutations have yet been identified in the gamma subunit of PDE.

12 100 RATTNER SUN NATHANS? Cyclic Nucleotide Gated Channel (CNCG) In one family, a beta-pde missense mutation (H258D) has been found to cause autosomal dominant CSNB (75). This mutation maps near the PDE gamma binding site in the amino-terminal half of the protein, whereas most of the ARRP mutations affect the carboxy-terminal catalytic domain. The CSNB mutation may act by decreasing the effectiveness of PDE inhibition by the gamma subunit, leading to constitutive hydrolysis of cgmp and rod desensitization. Rod and cone CNCGs consist of heterotetramers of two homologous subunits (264). Mutations in the alpha subunit of the rod CNCG are responsible for 1 2% of ARRP (55). The mutations identified thus far either produce early truncations or destabilize the channel as determined by a decrease in the number of channels in the plasma membrane of transiently transfected cells. Missense mutations within the alpha subunit of the cone CNCG are found in autosomal recessive rod monochromacy, a nonprogressive disorder characterized by a complete absence of cone function (121). The functional properties of the mutant cone CNCGs have not yet been characterized, but several mutations are located within the cgmp-binding domain and therefore may impair gating by cgmp. Guanylate Cyclase and Guanylate Cyclase Activating Protein (GCAP) In the photoreceptor outer segment, cgmp hydrolysis in response to light is balanced by cgmp synthesis (from GTP) by guanylate cyclase (Figure 3A). The photoreceptorspecific guanylate cyclases, RetGC-1 and RetGC-2, are members of a large family of guanylate cyclases that have an extracellular domain, a single membrane span, and a large intracellular domain within which the catalytic domain occupies the extreme carboxy-terminus (51, 141, 260, 261). Guanylate cyclase activity is tightly regulated by calcium it increases when the calcium concentration drops during light exposure and it decreases when calcium levels rise in the dark. This regulation is mediated by two homologous calcium-binding proteins, guanylate cyclase activating protein-1 and -2 (GCAP-1 and -2), which each have four EFhands (186). Intracellular calcium declines following light exposure because closure of the plasma membrane cgmp gated channel blocks calcium entry while intracellular calcium continues to be extruded via the plasma membrane sodium/ calcium/potassium exchanger. Thus, the calcium-dependent modulation of guanylate cyclase activity serves to restore the outer-segment cgmp level to baseline. This feedback loop is the principal mechanism by which photoreceptors adapt to light and damp out fluctuations in membrane potential (262). Recessive missense and frameshift mutations in RETGC-1 are responsible for a subset of cases of Leber congenital amaurosis (184). A naturally occurring null mutation in the chicken orthologue of RETGC-1 produces a recessive photoreceptor degeneration that begins after hatching, thus providing an animal model for this form of Leber congenital amaurosis (217). Two missense mutations in adjacent codons within a putative intracellular dimerization domain in RetGC-1 (127), E837D and R838C, are responsible for one form of autosomal dominant

13 ?HUMAN RETINAL DISEASE 101 cone-rod dystrophy (119). The adjacent locations of these two mutations suggests that they impart a specific gain of function to the mutant protein and that cone-rod dystrophy in these families does not arise from haploinsufficiency, a conclusion that is reinforced by the lack of a phenotype in carriers of the recessive null mutations in RETGC-1 responsible for Leber congenital amaurosis. An unanswered question at present is why the second photoreceptor guanylate cyclase, RetGC-2, cannot compensate for defects in RetGC1. Affected individuals in a single family with dominant cone dystrophy have been found to carry a missense mutation (Y99C) in the third EF hand of guanylate cyclase activating protein-1 (182). In vitro, GCAP-1 (Y99C) resembles wildtype GCAP-1 in stimulating the guanylate cyclase activity of recombinant RetGC-1 and of crude bovine rod outer segments in low (50 nm) calcium (50, 225). However, in high (2 µm) calcium, a concentration at which wild-type GCAP-1 shows no stimulatory activity, GCAP1 (Y99C) exhibits 30 50% of the stimulatory activity seen in low calcium. Thus, GCAP1 (Y99C) would be predicted to inappropriately stimulate cgmp synthesis in dark-adapted photoreceptors and lead to a derangement similar to that observed with loss-of-function mutations in cgmp PDE. The preferential degeneration of cones in patients carrying the GCAP-1 (Y99C) mutation may reflect the high level of expression of GCAP-1 in cone compared to rod photoreceptors (82, 100). Rhodopsin Approximately 30% of cases of ADRP are caused by rhodopsin gene mutations, most of which produce single amino acid substitutions (56, 232). As described below, nearly all of the approximately 35 ADRP mutant rhodopsins that have been studied in transfected cells and/or in transgenic mice have been found to differ from wild-type rhodopsin in one or more biochemical or cell biological properties. One group of rhodopsin mutations, referred to as class I, cluster at the extreme carboxy-terminus (Figure 4). In transfected 293 or COS cells, class I rhodopsin mutants are indistinguishable from wild type in all aspects examined thus far: yield, efficiency of localization to the plasma membrane, 11-cis retinal binding, transducin activation, and phosphorylation by rhodopsin kinase (116, 163, ). In transgenic mice, one class I mutant (Q344ter) shows inefficient outer segment localization of the mutant opsin but not the endogenous wild-type opsin (234), and a second mutant (P347S) induces massive accumulation of vesicles at the base of the outer segment (134a). Experiments in which synthetic peptides from rhodopsin s carboxy-terminus were added to cell-free homogenates of retina reveal a partial inhibition of cell-free vesicular transport of opsin by wild type but not by three class I mutant carboxy-terminal peptides (49). The role of rhodopsin s carboxy-terminus in protein transport and sorting to the outer segment has recently been clarified with the discovery that the wild-type carboxy-terminus binds to the Tctex-1 protein, a widely expressed dynein light chain, whereas various single amino acid Structure and Biosynthesis

14 102 RATTNER SUN NATHANS?(Right) Mutations responsible for ADRP that are near the carboxy-terminus Figure 4 of rhodopsin (44) and (Left) amino acid substitutions near the carboxy-terminus of vertebrate cone and rod pigments. The compendium of vertebrate rod and cone pigments is from human, mouse, goldfish, zebrafish, chicken, gecko, and frog. substitutions of rhodopsin s carboxy-terminus responsible for ADRP fail to bind (237a). Tctex-1 is abundant in photoreceptor inner segments and is likely to guide the transport of rhodopsin-laden post-golgi vesicles to their destination. As seen in Figure 4, a compendium of ADRP mutations near rhodopsin s carboxy-terminus reveals numerous amino acid substitutions at positions 345 and 347, as well as frameshift and stop codon mutations that alter or remove the last few amino acids. Figure 4 also shows that among vertebrate visual pigments, positions 345 and 347 are distinguished from other positions near the carboxyterminus by their high degree of evolutionary conservation. Taken together, these data indicate that valine 345, proline 347, and possibly the free carboxylate following the terminal amino acid at position 348 comprise a protein sorting signal that is not required for ER Golgi plasma membrane movement in cultured cells but that plays a specialized role in outer segment protein localization. The second group of rhodopsin mutants, referred to as class II, show defects in stability and/or protein folding when expressed in cultured cells (76, 116, 137, 163, ). The mutant proteins accumulate to reduced levels relative to the wild type, are localized predominantly to the endoplasmic reticulum, and bind 11-cis retinal variably or not at all. Most class II mutants are single amino acid substitutions and are distributed throughout rhodopsin s transmembrane and intradiscal domains (the latter is topologically equivalent to an extracellular domain), a pattern that was anticipated by earlier mutagenesis studies aimed at identifying domains that contribute to the stability and folding of bovine rhodopsin (52). The asymmetric location of amino acids involved in stability and/or folding may reflect a requirement for flexibility in the cytosolic domain of rhodopsin, which must

15 ?HUMAN RETINAL DISEASE 103 undergo a conformational transition to interact with transducin, rhodopsin kinase, and arrestin. In the analysis of class II mutants, it is noteworthy that different investigators studying the same amino acid substitutions have observed different degrees of functional impairment, as assessed by protein yield, degree of 11-cis retinal binding, and subcellular localization. In general, a greater degree of impairment has been reported with human rhodopsin mutants expressed in 293 cells and reconstituted with 11-cis retinal after membrane purification (233, 235) relative to the corresponding mutants in bovine rhodopsin expressed in COS cells and reconstituted in intact cells (116, 137, 163). In ADRP, the photoreceptor cell demise that results from a mixture of normal and unstable or misfolded opsin may reflect the high steady-state level of rhodopsin ( rhodopsins per cell) and the high rate of rhodopsin synthesis ( rhodopsins per cell per day). In keeping with this hypothesis, in Drosophila the most frequent class of dominant photoreceptor cell degeneration mutants obtained after chemical mutagenesis carry point mutations in the rhodopsin gene that resemble class II rhodopsin mutations in ADRP (36, 123, 249). One class II rhodopsin mutant, T17M, exhibits a significantly increased yield and a conversion from ER to plasma membrane localization if the transfected cells are grown in the dark in the continuous presence of 11-cis retinal (134). In transgenic mice that express T17M rhodopsin, disease progression is slowed by a diet high in vitamin A, as determined by the amplitude of the ERG and by the thickness of the photoreceptor cell layer. A similar slowing of disease progression is not observed in transgenic mice expressing P347S rhodopsin, a class I mutant. These observations probably reflect the C increment in stability that 11-cis retinal binding imparts to opsin (104), and they suggest that rhodopsin mutants that are marginally stable in the absence of 11-cis retinal may achieve a significant degree of stability upon 11-cis retinal binding. If 11-cis retinal binds to newly synthesized opsin in the rod inner segment, as current data suggest (227), that binding may permit the transport of unstable mutants to the outer segment, a pattern that is observed in transgenic mice expressing the class II mutant proteins P23H and T17M (134, 180). Premature termination mutations at codons 64 and 249 in the rhodopsin gene have been characterized in two families and found to cause dominant and recessive RP, respectively (142, 202). Presumably, the codon 64 mutation produces a toxic protein fragment, whereas the codon 249 mutation either produces less protein as a result of mrna instability [often seen in mrnas carrying premature termination codons (154)] or a protein fragment that is less toxic. Mice carrying rhodopsin gene knockouts show rapid retinal degeneration in the homozygous state and a very slow degeneration in the heterozygous state (105, 128). The heterozygous knockout phenotype is in contrast to the rapid degeneration seen in mice expressing various ADRP rhodopsin transgenes, which suggests that this subtype of ADRP is caused by a dominant gain-of-function (i.e. toxic) protein rather than haploinsufficiency.

16 104 RATTNER SUN NATHANS Cone Pigments? Approximately 8 10% of Caucasian males and about 5% of Asian and African males carry rearrangements within the red and green visual pigment gene array on the X-chromosome that produce benign anomalies of color vision (175). Rarely, alterations within this locus either eliminate a locus control region upstream of the array or introduce deleterious missense or stop codon mutations within an array that carries only a single gene (173, 174, 193). These rare alterations produce blue cone monochromacy or one of its variants, disorders associated with nearly complete color blindness and a loss of visual acuity. A subset of individuals with blue cone monochromacy or its variants experience a progressive degeneration of the central retina (68, 173, 174, 193), presumably via mechanisms analogous to those responsible for the progressive degeneration of the peripheral retina in RP patients with rhodopsin gene mutations. A similar RP-like mechanism probably accounts for the loss of blue cone function seen in autosomal dominant blue-blindness (tritanopia), a disorder caused by missense mutations in the transmembrane domains of the blue pigment that are predicted to disrupt its folding and/or stability (253, 254). Peripherin and Rod Outer Segment Membrane Protein-1 (ROM-1) Peripherin/rds and ROM-1 are homologous integral membrane proteins located at the disc rim in rod and cone photoreceptors. Peripherin/rds was identified independently in bovine rod outer segments (38, 167) and by virtue of its mutation in the retinal degeneration slow (rds) mouse (240). ROM-1 was identified by differential hybridization as the product of a retina-specific cdna (9). Peripherin/rds and ROM-1 form heterotetrameric complexes that are linked by a disulfide bond in the intradiscal domain of the proteins to form larger oligomeric complexes (80, 81). The abundance, disc rim location, and oligomeric properties of these proteins strongly suggest that they play an important role in stabilizing the high membrane curvature at the disc rim and possibly in anchoring the discs to the adjacent plasma membrane (166). Over 40 mutations have been identified in the peripherin/rds gene in patients with a wide variety of retinal diseases with dominant modes of inheritance: ADRP, macular dystrophies of various types, and disorders associated with an accumulation of yellow/white deposits in the retina and/or RPE (118). This degree of diversity in disease phenotypes is unusual among retinal disease genes. When produced in transfected cells, many of the mutant peripherin/rds proteins found in human retinopathies fail to correctly fold and multimerize with coexpressed ROM-1 (78). Although the spontaneous murine peripherin/rds mutation, which involves a large rearrangement and appears to be a null, is generally considered a recessive allele (206), heterozygous animals exhibit disorganization and shortening of outer segments (90). Different peripherin/rds alleles do not correlate in a simple way with the various disease phenotypes defined ophthalmoscopically (118). For example, one family carrying a deletion in codons has affected members with RP, pattern dystrophy, and fundus flavimaculatus (256). The effect of genetic background on peripherin/rds phenotypes has been dramatically demonstrated in several families

17 ?HUMAN RETINAL DISEASE 105 in which individuals who are heterozygous for both a Ll85P allele of peripherin/rds and a null allele of ROM-1 have RP. By contrast, family members who are single heterozygotes are not affected, the first clear example in human genetics of a digenic disorder (57, 114). In transfected cells, L185P peripherin/rds does not form homotetramers as does wild-type peripherin/rds, but it can form heterotetramers with ROM-1 (79). Presumably, in double heterozygotes a reduction in ROM-1 concentration unmasks the homomultimerization defect in the 50% of the peripherin/rds protein that is mutant. Despite macular involvement in many peripherin/rds-based retinopathies, no sequence alterations in this gene have yet been associated with AMD. Rab Escort Protein-1 (REP-1) Choroideremia is an X-linked disorder that affects approximately in 1 in 50,000 males and shows clinical symptoms much like RP (40). It is distinguished from RP by a dramatic and early loss of the RPE accompanied by a progressive atrophy of the choroidal vasculature (93, 153). The responsible gene, REP-1, was independently discovered by positional cloning (42) and by purification of geranylgeranyl transferase, a multiprotein complex that adds the prenyl group geranylgeranyl to the carboxy-termini of rab (and possibly other) proteins (213, 214). In the biochemistry literature, the REP-1 protein is also referred to as component A of geranylgeranyl transferase. The identity of REP-1 as the choroideremia gene product has been demonstrated not only by mutation analysis of patient DNA (243) but also by the finding that geranylgeranyl transferase activity is deficient in lymphoblastoid cell lines from choroideremia patients (212). The rab proteins are involved in a variety of intracellular trafficking processes and require prenylation for membrane localization and function. REP-1 is distinct from the catalytic component of the geranylgeranyl transferase complex, and appears to act by delivering newly synthesized rab proteins to the catalytic component and then to their target membranes (1, 6). In Saccharomyces cerevisiae, the REP-1 orthologue is essential for viability, as are the catalytic components of geranylgeranyl transferase. If geranylgeranylation is also essential for viability in mammals, then the restricted phenotype of REP-1 deficient patients might be explained by the existence of REP-2, a REP-1 homologue that has partially overlapping but nonidentical substrate specificity (39, 215). As both REP-1 and REP-2 are widely expressed, they might partially compensate for each other in many tissues. The spectrum of REP-1 mutations in choroideremia patients is distinctive in that it consists overwhelmingly of apparent null mutations (243). This pattern of mutations suggests either that low levels of REP-1 activity might be sufficient for normal ocular development and function, or conversely that partially active alleles interfere with geranylgeranylation and produce a more severe and/or extraocular phenotype. Myosin VIIA and USH2A Usher syndrome is a recessive, genetically heterogeneous group of disorders characterized by the combination of congenital sensorineural hearing loss and retinitis pigmentosa (22, 67, 91, 176). Usher syndrome

18 106 RATTNER SUN NATHANS? The Visual Cycle affects approximately 1 in 25,000 persons. Three subtypes of Usher syndrome are distinguished by the severity of auditory and vestibular dysfunction: Type I patients are profoundly deaf and lack vestibular function, type II patients have mild hearing loss and normal vestibular function, and type III patients have progressive hearing loss with variable vestibular function. At least five loci are responsible for Usher syndrome type I (USH1), and one of these, USH1B, encodes myosin VIIA, an unconventional myosin with a predicted M r of 250 kda (130, 139, 251). A distinct group of recessive mutations in this gene are responsible for isolated (i.e. nonsyndromic) deafness (252), and recessive mutations in the orthologous murine gene cause the deafness and vestibular dysfunction seen in the shaker mouse (77). In one study, myosin VIIA was localized by immunoelectron microscopy of both primate and rodent retinas to the ciliary base of rod and cone outer segments and to the apical microvilli of the RPE, which interdigitate between the outer segments (138). However, a second research group observed myosin VIIA immunoreactivity in primate but not rodent photoreceptors, which led to the suggestion that a species difference in protein distribution accounts for the lack of retinal pathology in the shaker mouse (61). Notwithstanding the partial inconsistency in immunolocalization, the data suggest a role for myosin VIIA in intracellular transport, and specifically in outer segment biogenesis in the human retina. Recently, a second Usher syndrome gene, USH2A, has been shown to be responsible for Usher syndrome IIA (62). USH2A encodes a putative extracellular matrix protein of 1551 amino acids that is expressed in the retina and inner ear, and at lower levels in other tissues. Cellular Retinaldehyde Binding Protein (CRALBP) CRALBP is one of several intra- and extracellular retinoid binding proteins in the retina and RPE that orchestrate the visual cycle (Figure 2). CRALBP preferentially binds 11-cis retinol and is present in the RPE and in Muller glia in the retina. In vitro CRALBP promotes oxidation of 11-cis retinol to 11-cis retinal and inhibits the esterification of 11-cis retinol (205). The oxidation reaction is the final chemical step in the recycling pathway, whereas esterification produces a stored form of retinol. A variety of mutations in CRALBP have been found in recessively inherited progressive retinal degenerations that are variably characterized by night blindness, maculopathy, and yellow/white deposits in the fundus (26, 152, 168). Recombinant CRALBP carrying one of the disease-causing mutations, R150Q, is unable to bind 11-cis retinal, suggesting that this mutation impairs the oxidation of 11-cis retinol to 11-cis retinal in vivo and, by inference, reduces the 11-cis retinal available for regeneration of visual pigment (152). 11-cis Retinol Dehydrogenase As noted above, conversion of 11-cis retinol to 11-cis retinal occurs within the RPE. This reaction is catalyzed by 11-cis retinol dehydrogenase, encoded by the RDH5 gene (223a). Two recessive missense