BBS SPECIAL ISSUE: Controversies in Neuroscience III: Signal Transduction in the Retina and Brain

CORRELATION OF PHENOTYPE WITH GENOTYPE IN INHERITED RETINAL DEGENERATION

Keywords

Abstract

1. INTRODUCTION

Within the past decade rapid progress has been made in identifying genes and mutations causing many forms of inherited retinal degeneration in humans and other animals. However, we are still ignorant of the biological mechanisms that underlie many inherited diseases. The connection between genotype or specific mutations and phenotype, such as retinal degeneration, is often obscure or unknown. Although much has been learned about human genes, less than 2% of the human genome has been sequenced and fewer than 1% of human genes have been fully characterized (Burks et al. 1991). Also, gene products function within complex cellular environments, and the relationship between proteins, their substrates, organelles and other cellular components is poorly understood. Further, we know even less about the function of proteins as they become degraded with time. Finally, proteins may function differently in different tissues or their function may change in the same tissue at different developmental stages.

The vertebrate retina has served as a model system for investigating embryogenesis, cellular ultrastructure, signal transduction, signal transmission and processing, and cellular turnover and death. We believe that the retina, the mammalian retina in particular, also serves as a useful model for investigating genotype-phenotype relationships in inherited degenerative diseases and disorders. Many retinal genes have been cloned, and many specific mutations of photoreceptor-specific genes are known to cause retinal degeneration in humans. Such mutations can contribute to our understanding of both abnormal and normal visual processes.

There are at least 200 inherited diseases that lead to retinal degeneration in humans (probably an underestimate). At least 10% of inherited diseases involve the retina directly or indirectly (McKusick 1992). Over the past 5 years, more than 40 genes causing retinal degeneration in humans have been mapped, mostly by linkage methods. Seven of these genes have been cloned. Thus there are a number of opportunities to correlate genotype with phenotype of the resulting disease. This discussion focuses on the genes for rhodopsin, peripherin/RDS and the rod phosphodiesterase ~-subunit (PDEB), genes that are expressed specifically in photoreceptors. Rhodopsin is the rod photoreceptor visual pigment located in both disc membranes and in the plasma membrane of rod outer segments (ROS). Rhodopsin constitutes approximately 90% of the protein in ROS. Peripherin/RDS is a protein of undetermined function that is expressed in both rods and cones. It is found on the periphery of photoreceptor discs where it may play a structural role. PDEB is a catalytic subunit of phosphodiesterase, the protein in the visual transduction cascade which hydrolyses cGMP to 5'-GMP when disinhibited by activated transducin (the rod-specific G-protein). The phosphodiesterase subunit that maps to 4p appears to be rod-specific, although expression in other retinal cells cannot be excluded. Of primary interest here are mutations in genes for rhodopsin, peripherin/RDS and PDEB that result in phenotypes which cause progressive retinal degeneration such as autosomal dominant retinitis pigmentosa (adRP), autosomal recessive retinitis pigmentosa (arRP) and autosomal dominant macular dystrophy (adMD). Several recent reviews provide additional details (Berson 1993; Heckenlively 1988; Humphries et al. 1992; Humphries et al. 1993; Musarella et al. 1992).

Recently, mutations in the gene for rod cGMP-gated channel (CNCG) have been suggested as another cause of arRP (McGee et al. 1994). This supprots the expectation that many of the 40 or more genes causing inherited retinal degeneration are likely to be photoreceptor-specific.

2. BACKGROUND 2.1. Diseases causing inherited retinal degeneration

Diseases causing inherited retinal degeneration in humans can be classified broadly into those that first affect peripheral vision and the peripheral retina, such as retinitis pigmentosa, and those that primarily affect central vision and the macula, such as macular dystrophy. Although the macula, especially the fovea, has the highest concentration of cones and the peripheral retina is dominated by rods, factors contributing to whether a given degeneration is expressed as a peripheral disease or a macular disease are more complicated than simply the density of a particular type of photoreceptor.

The clinical symptoms of retinitis pigmentosa include night blindness and loss of peripheral vision (Heckenlively 1988). With time visual impairment progresses toward the center of the retina causing "tunnel-vision". The disease is usually accompanied by pigmentary deposition, although the appearance of pigment is thought to be a consequence of the disease and not its cause. Early symptoms usually occur within the first or second decades of life. For many patients visual fields continue to constrict over a period of years or decades, eventually resulting in blindness. Retinal degeneration is usually bilateral and is often proceeded and accompanied by characteristic changes in the electroretinogram (ERG).

Retinitis pigmentosa can be subdivided into several genetic categories: autosomal dominant, autosomal recessive, X-linked or syndromic. The overall incidence of retinitis pigmentosa is about 1/4000 without apparent ethnic or racial distinctions (Fishman 1978; Halloran 1985). Autosomal dominant retinitis pigmentosa (adRP) accounts for about 20% of cases; autosomal recessive (arRP) for 15%; X-linked (xlRP) for 10%; and syndromic forms, such as the recessive disease Usher syndrome (which combines congenital deafness with retinitis pigmentosa), for 20%. The remaining 35% of cases are isolated or sporadic. Thus the mode of inheritance cannot be determined for all patients but it is assumed that all cases have a genetic basis.

There are also a number of clinical categories for retinitis pigmentosa. Limiting discussion to autosomal dominant forms of the disease, adRP families and patients can be classified by a number of clinical criteria such as age of onset of night blindness and visual impairment, rate of progression, whether pigmentary deposits are diffuse or regional or sectorial, and whether ERG responses are diminished (or absent) from rods, from cones or from both. These classes have been condensed into two broad categories. Type 1 retinitis pigmentosa is characterized by rapid progression and diffuse, severe pigmentation; type 2 retinitis pigmentosa has a slower progression and more regional, less severe pigmentation (Massof & Finkelstein 1981). In an alternate system of classification (Lyness et al. 1985), these two types correspond roughly to type D or "diffuse" and type R or "regional" retinitis pigmentosa . Some investigators propose ~sectorial~ retinitis pigmentosa as a third classification, as well as additional types (Fishman et al. 1985; Heckenlively 1988).

Classification of macular degeneration is more tenuous (Fishman 1990). Macular degeneration can have either a genetic basis or it may be an acquired disease. Approximately 10% of Americans over the age of 50 are afflicted with age-related macular degeneration (Bressler et al. 1988), an acquired form of disease. The inherited forms of macular degeneration are much less common but usually more severe. The inherited forms are characterized by early development of macular abnormalities such as yellowish deposits and atrophic or pigmented lesions, followed by progressive loss of central vision. The macular lesions may appear early in life and often precede the loss of vision by many years. Like retinitis pigmentosa, macular degeneration is usually bilateral. Unlike retinitis pigmentosa, only the central retina is affected.

The relative incidence and importance of inherited versus non-inherited factors is unclear. In addition, genetic factors are likely to play a role in acquired forms of macular degeneration. The high frequency of acquired forms makes it very difficult to determine the incidence of inherited forms. However, several distinct, heritable forms of the disease such as autosomal dominant North Carolina macular dystrophy, autosomal dominant Best macular dystrophy, and autosomal recessive Stargardt disease have been mapped. Genes causing the inherited forms of macular degeneration are very promising candidates for genetic factors that predispose to age related-macular degeneration.

There are many other inherited diseases that cause retinal degeneration in humans. Among these are gyrate atrophy, Norrie disease, choroideremia and various cone-rod dystrophies. In addition there are numerous inherited, systemic diseases, such as Bardet-Biedl, Charcot-Marie-Tooth and Refsum disease, which include retinal degeneration among a multiplicity of other symptoms.

2.2. Mapped and cloned genes causing inherited retinal degeneration

Table 1 lists mapped and cloned genes causing retinal degeneration and related diseases in humans. Most of the diseases have been mapped to specific chromosomal sites by linkage testing. For genes listed as "cloned", the causative gene has been isolated, sequenced and characterized, to a limited extent at least. The table is not exhaustive in that most syndromic and systemic causes of degeneration are not included. The list is certain to be outdated soon because new disease genes are being mapped and cloned at a rapid pace. The table lists the GDB-approved symbol, if known, and the McKusick number. The McKusick number is the code assigned in OMIM (see footnote 1). We use the GDB or OMIM symbols because they are the most commonly accepted terms, but we recognize that different symbols may be used with equal validity (McKusick 1992; Pearson et al. 1992). 2.2.1. Mapped genes causing retinitis pigmentosa and macular degeneration

The first mapped gene for retinitis pigmentosa was RP2, a gene for xlRP which was maps to Xp11 (Bhattacharya et al. 1984). Subsequently one form of adRP was mapped to 3q (McWilliams et al. 1989). Shortly thereafter the 3q form of adRP was shown to be caused by mutations in rhodopsin (Dryja et al. 1990; Farrar et al. 1990a). Subsequently additional genes causing adRP have been mapped to 6p, 7p, 7q, 8q, 17p and 19q (Al-Maghtheh et al. 1994; Blanton et al. 1991; Greenberg et al. 1994; Farrar et al. 1991; Inglehearn et al. 1993; Jordan et al. 1993). Of these, the gene on 6p has been identified as the gene for peripherin/RDS; the others have not been cloned as yet. Two additional genes for xlRP have also been reported (Musarella et al. 1990; Ott et al. 1990). Finally, recessive retinitis pigmentosa may be caused by mutations either in genes for rhodopsin, PDEB (which maps to 4p) or CNCG (which also maps to 4p) (McGee et al. 1994; McLaughlin et al. 1993; Rosenfield et al. 1992). Recently, another form of arRP, RP12, was mapped to 1p but the causative gene is not known (van Soest et al. 1994).

The first mapped gene for adMD was VMD1 which was assigned to 8q (Ferrell et al. 1983). Subsequently, the gene for North Carolina macular dystrophy was mapped to 6q (Small et al. 1992) and the gene for Best macular dystrophy was mapped to llq (Stone et al. 1992a). Additional genes causing adMD have been mapped to 6p and 7p (Jordan et al. 1992; Kremer et al. 1994). One of these has been identified as the gene for peripherin/RDS (Jordan et al. 1992; Kajiwara et al. 1991); the others have not been cloned as yet.

In summary, at least 3 genes can cause xlRP (RP2, RP3 and RP6), 7 can cause adRP (rhodopsin, peripherin/RDS, RP1, RP9, RP10, RP11 and RP13), 4 can cause arRP (rhodopsin, PDEB, CNCG and RP12) and 5 can cause adMD (peripherin/RDS, MCDR1, DCMD, VMD1 and VMD2). In addition, genes for Usher syndrome have been mapped to lq, llp, llq and 14q (Kaplan et al. 1992; Kimberling et al. 1990; Kimberling et al. 1992, Lewis et al. 1990; Smith et al. 1992). As indicated in Table 1, many other retinal conditions also have been mapped. For most disease categories it is important to emphasize that there are affected families whose disease gene is excluded from all known loci (e.g., Kumar-Singh et al. 1993). 2.2.2. Cloned genes causing retinal degeneration and related conditions

From Table 1 and the preceding section, we know that mutations in the genes for rhodopsin, peripherin/RDS and PDEB can cause either adRP or arRP. About 30% of adRP cases are caused by rhodopsin gene mutations and 5% by mutations in the peripherin/RDS gene (Dryja 1992; Kajiwara et al. 1991). Mutations in the PDEB gene may account for 1 to 5% of arRP cases (McLaughlin et al. 1993); the percent of arRP cases caused by rhodopsin is unknown. It is known that mutations in the peripherin/RDS gene also can cause adMD. Furthermore, mutations in rhodopsin and, possibly, PDEB can cause dominant congenital stationary night blindness (adCSNB) (Dryja et al. 1993; Rao et al. 1994; Sieving et al. 1992).

There is an additional form of retinitis pigmentosa with a mode of inheritance distinct from dominant or recessive: digenic RP (Kajiwara et al. 1994). Degeneration in these patients is the result of a compound of one mutation in peripherin/RDS and one in rod outer segment membrane protein 1 (ROM1); neither mutation alone in a heterozygote causes disease. One implication of this finding is that both peripherin/RDS and ROM1 should be screened for mutations in patients with retinitis pigmentosa for which the mode of inheritance is unclear.

The genes for peripherin/RDS and PDEB were first found to be the cause of retinal degeneration in mice before their connection to human diseases was demonstrated. The recessive mouse disease retinal degeneration slow (rds) is caused by a 10 kb (kilobase) insertion in the gene for peripherin/RDS (Connell et al. 1991; Travis et al. 1991a). The recessive disease retinal degeneration (rd) is caused by insertion of a retrovirus gene fragment (Bowes et al. 1990; Bowes et al. 1993) or by a nonsense mutation (Pittler & Baehr 1991) in the gene for PDEB. The mouse rd nonsense mutation is identical to the mouse r mutation described more that 70 years ago (Pittler et al. 1993). Finally, the autosomal recessive disease rod/cone dysplasia 1 (rcdl) found in the Irish Setter is also caused by a nonsense mutation in PDEB (Farber et al. 1992; Suber et al. 1993).

What these findings demonstrate most clearly is the exceptional heterogeneity of inherited retinal degeneration. Different gene mutations can cause the same disease (genetic heterogeneity), different mutations within the same gene can cause different diseases and different clinical subtypes (allelic heterogeneity), or the same mutation can have different clinical consequences in different individuals (clinical heterogeneity). Thus it is not possible to deduce the underlying causative gene mutation based on the clinical phenotype of a patient or family. Eventually psychophysical or electrophysiological findings may distinguish one disease gene from another but this is not possible yet.

2.3 Retinal biology relevant to inherited degeneration

Rhodopsin is synthesized in rod inner segments, post-translationally modified by the addition of oligosaccharides and fatty acids, assembled into vesicles, transported to the connecting cilium, and inserted into nascent discs. Rhodopsin has 7 transmembrane domains, and is a member of the G-coupled receptor superfamily (Stryer 1986; Hargrave & McDowell 1992; Hargrave & McDowell 1992a; Nathans 1992). The amino-terminus of rhodopsin is on the intradiscal or lumenal side of disc membranes, and the carboxyl-terminus is on the cytoplasmic side. Dark-adapted rhodopsin contains 11-cis retinal as its photosensitive chromophore, attached by a protonated Schiff's base to a lysine at codon 296. Light isomerizes the chromophore to an all-trans configuration and activates rhodopsin into an equilibrium between metarhodopsin I and metarhodopsin II. Metarhodopsin II binds transiently to transducin. While transducin is bound to rhodopsin, the `-subunit of the G-protein exchanges a bound GDP for GTP. The `-subunit of transducin with bound GTP dissociates from the ~- and k- subunits so that it can bind to one of the two inhibitory k-subunits of phosphodiesterase. The disinhibited membrane-bound `- and ~-subunits are then activated to hydrolyze cGMP to 5'-GMP. Lowering the cytosolic concentration of cGMP reduces the conductance of a cGMP-gated channel in the plasma membrane, which hyperpolarizes the cell and reduces the rate of transmitter release from the rod synapse. Inactivating this cascade requires phosphorylation of metarhodopsin II by rhodopsin kinase, blockage of further interaction of metarhodopsin II with transducin by binding of arrestin (also called S-antigen) to phosphorylated rhodopsin, release and recycling of all-trans retinal, regeneration of cGMP from GTP by guanylate cyclase and other related but poorly defined steps. Therefore, although many of the proteins (and their genes) involved in phototransduction are well characterized, there are certain to be other genes and proteins involved that are not known yet.

Similar mechanisms and homologous proteins are involved in phototransduction within cones, but the details are different and different genes code for these proteins. Specifically, there are distinct cone opsins, transducin subunits, phosphodiesterase subunits, and cGMP-gated channels (Blatt et al. 1988; Levine et al. 1990; Peng et al. 1992). Thus rod and cone degeneration may be caused by functionally similar proteins that are encoded by different genes.

3. GENERAL CONSIDERATIONS

How can mutations in the genes for three photoreceptor proteins, rhodopsin, peripherin/RDS and PDEB account for several different diseases including adRP, arRP, adMD and adCSNB? To address this question, some generalizations are in order.

First, dominant diseases are often the result of positive acting mutations, particularly in structural proteins, whereas recessive mutations are often the result of null-function mutations, usually in enzymes.

Second, degenerative retinal diseases may be related to each other in several ways. Over most of the retina, rods and cones are closely interdigitated in a dense, two-dimensional cellular matrix. They share many metabolites and cofactors and have at least some genes in common. Thus gene mutations responsible for retinitis pigmentosa and macular degeneration may affect common metabolic pathways and result in similar end effects upon photoreceptor physiology. Additionally, adCSNB is part of a continuum of rod dysfunction that may result from either a diminution of rod function or a loss of the rods themselves.

Third, it is important to recognize inherent biases in the available data. The known associations between diseases and genes derive from linkage testing in families and from mutation screening in selected patients. Because many possible genes may cause similar diseases, absence of linkage in a particular family does not mean that the excluded gene cannot cause the disease in another family. Further, some families are too small to establish linkage, and recessive diseases, in particular, present special problems for detecting linkage because of heterogeneity, etc. Moreover, mutation testing has its own bias, namely the selection of patients to be tested. If we have not guessed the correct gene to test then we will not find the underlying mutation. Also, the problem of many genes causing similar disorders means that an uncommon molecular cause may be missed in a modest sample of patients.

Finally, there are technical difficulties with mutation screening. Several laboratories, our own included, have tested collections of patients for mutations in rhodopsin and peripherin/RDS (Dryja et al. 1991; Gannon et al. 1993; Inglehearn et al. 1992; Kajiwara et al. 1991; Rodriguez et al. 1993a; Sheffield et al. 1991). Less extensive surveys of PDEB and other photoreceptor proteins have also been conducted (Jacobson & Bascom 1993; McGee et al. 1993; Ringens et al. 1990). Most mutation screening is usually done using single-strand conformational analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), GC-clamped DGGE or heteroduplex analysis. At best each of these methods is only 90% effective, thus some mutations are missed. In addition, since multiple PCR reactions are involved, the larger the gene the more cumbersome the test. For example, rhodopsin with 5 exons and peripherin/RDS with 3 exons are amendable to PCR analysis, but PDEB with 22 exons is considerably more cumbersome (Kajiwara et al. 1991; McLaughlin et al. 1993; Rodriguez et al. 1993). Thus, because present methods for detecting mutations are very laborious, no current research group has tested a multiplicity of genes in a wide range of patients. Hopefully, as new techniques are realized, such as analysis of illegitimate transcripts and improvements in DNA sequencing, these limitations will be resolved.

3.1 Available data

Despite the inherent limitations of linkage testing and mutation screening, a large number of disease-causing mutations have been reported. At least 73 mutations in the rhodopsin gene have been described. These are listed in Table 2, including unpublished mutations from our laboratory. At least 23 peripherin/RDS mutations and 5 PDEB mutations, plus many benign variants, have been described (Humphries et al. 1993; McLaughlin et al. 1993). These mutations constitute the raw material for beginning the elucidation of the connection between genotype and phenotype. In this review we focus on the rhodopsin mutations because more rhodopsin-related diseases have been reported, because better clinical data are available, and because rhodopsin is better characterized than peripherin/RDS or PDEB.

3.2 Why do these proteins cause these diseases? 3.2.1. Rhodopsin

Rhodopsin is a membrane-spanning protein that comprises the bulk of ROS protein. Thus mutations that change its tertiary structure within the membrane should have a dominant effect since, in this context, rhodopsin has a structural role. Yet rhodopsin also has a catalytic role in phototransduction, so that, in this context, mutations may be recessive. Still other mutations may simply perturb phototransduction without cell damage, resulting in congenital stationary night blindness in which the perturbation may act dominantly or recessively. We will show examples of each of these possibilities.

Do mutations in cone opsins cause cone degeneration? Abnormalities in cone opsins are generally associated with color blindness, not degeneration (Nathans 1992; Nathans et al. 1992a). X-linked deuteranopia is caused by absence of green function, X-linked protanopia is caused by absence of red function and autosomal dominant tritanopia is caused by absence of blue function. X-linked blue cone monochromacy results from absence of both red and green function (Nathans et al. 1993). The molecular basis of these diseases is well established (Nathans 1992). The red and green cone opsins are X-linked; blue cone opsin maps to 7q. Deuteranopia, protanopia and tritanopia can be caused by missense mutations, nonsense mutations, deletions or rearrangements in each applicable gene. Tritanopia can be caused by multiple mutations, by rearrangements of the red and green opsins or by deletion of a locus control region for these genes. However, none of the known mutations causes cone degeneration. Retinal degeneration is occasionally seen with blue cone monochromacy (mutations unknown) but never with dominant tritanopia. It may be that the density of each cone type taken separately is too low to cause recognizable degeneration, especially the blue cones which are absent from the central retina. Also, developmental pathways during embryogenesis may simply exclude production of cones with an aberrant opsin, compensating for the deficit with additional normal cones. In any case, this remains a challenging mystery. 3.2.2. Peripherin/RDS

Peripherin/RDS, like rhodopsin, is a membrane-spanning protein found in photoreceptor discs (Arikawa et al. 1992; Connell et al. 1991; Travis et al. 1991). Unlike rhodopsin it is expressed both in rods and cones. Peripherin/RDS has 4 transmembrane domains so both the amino-terminus and carboxyl-terminus are on one side of the disc, the cytoplasmic side. Very little is known about the function of peripherin/RDS. In the disc membrane it forms a homodimer and is associated with another membrane-spanning protein, ROM1, which is about 30% similar in sequence to peripherin/RDS (Bascom et al. 1992). These two proteins are found predominately around the rim of discs in rods where, it is speculated, they contribute to the formation and maintenance of the disc structure.

Diseases caused by mutations in peripherin/RDS run the gamut of dominant retinal degeneration including adRP, adMD, retinitis punctate albescens, fundus flavimaculatus and various pattern dystrophies (Kajiwara et al. 1993; Keen et al. 1994; Nichols et al. 1993). Furthermore, one mutation, a 3 bp deletion of a lysine at codon 153 or 154, causes retinitis pigmentosa, pattern macular dystrophy and Stargardt dystrophy in different members of the same family (Weleber et al. 1993).

In the absence of a clear understanding of the functional domains of peripherin/RDS it would be naive to try to explain these findings mechanistically. Since the gene for peripherin/RDS is expressed in both rods and cones, some domains may be important for rod function, some for cone function and some for both. ROM1 is not expressed in cones, so one distinguishing domain may be the ROM1 binding site. It is reasonable to speculate that destabilization of the disc membrane would be very damaging to the photoreceptor. Why some mutations are highly variable in their expression is currently unknown, though this suggests that genetic background and environmental factors play important roles. At present, the limited number of known, disease-causing mutations is insufficient to make meaningful correlations. However, the accumulating number of pedigrees with mutations in the peripherin/RDS gene may prove instructive. 3.2.3. Phosphodiesterase ~-subunit (PDEB)

Mutations in PDEB are associated with arRP and adCSNB (Gal et al. 1994; McLaughlin et al. 1993). Reduced or absent PDEB should retard hydrolysis of cGMP and make photoreceptors less sensitive to light. Thus dominant CSNB is a logical outcome. Recessive degeneration, though, is less expected. The reported PDEB mutations causing arRP are compound heterozygotes of 3 possible nonsense mutations, 1 out-of-frame deletion and 1 missense mutation (McLaughlin et al. 1993). These mutations cause typical retinitis pigmentosa with early onset and no detectable rod ERG in adulthood. They are likely to be null-function mutations which, among other consequences, would elevate cGMP levels. 3.2.4. Why do these mutations lead to cell death?

For many photoreceptor mutations the initiating cause of retinal degeneration can be inferred, such as membrane instability, aberrant activation of phototransduction or increased cGMP levels. Studies of retinal degeneration in animals suggest that increased cGMP may be toxic to photoreceptors (Lolley et al. 1977; Ulshafer et al. 1980). The connection between increased cGMP and cell death is not known, but increased cellular permeability, direct cytotoxicity or unbalanced Ca2+/Na+ flows are possibilities (Hargrave & McDowell 1992). Why these various conditions lead to degeneration is unclear. One possibility is a simple necrotic process wherein the cell ceases its metabolic activity, its membranes decompose and phagocytic activity, either by the RPE or by infiltrating macrophages, removes the cellular debris. An alterative is an enhanced, catabolic interaction between the RPE and the ROS, possibly exacerbated by a feedback loop triggered by accumulating disc debris. However, recent evidence implicates apoptosis, a form of programmed cell death, as the mechanism responsible for photoreceptor cell death in rodent models of retinal degeneration. In apoptosis, genetic switches in the cell initiate a process which turns off normal cellular activity and causes condensation of nuclei, DNA degradation and cell death. The cardinal features of apoptosis have been observed in the rd mouse, the rds mouse and a transgenic mouse with the rhodopsin Pro347His mutation (Chang et al. 1993; Lolley et al. 1994). Thus it seems likely that some forms of retinal degeneration in humans may result from apoptosis. However, these observations do not preclude alternative mechanisms in other forms of degeneration. Further, they beg the question of what triggers apoptosis in the first place.

4. CLINICAL CONSEQUENCES OF RHODOPSIN MUTATIONS

Table 2 summarizes data on human rhodopsin mutations from many sources. (References are in Table 2 unless otherwise noted.) The table gives the codon, nucleotide change and amino acid change for each mutation. In addition, functional properties, protein domain and evolutionary conservation of affected amino acids are given (Fryxell & Myerowitz 1991; Hargrave & O~Brien 1991; Nathans et al. 1993.) Also listed is the type of disease observed, clinical features of the disease, if reported, and comments. Included in the comments is the biochemical class (I, IIa or IIb) based on transfection-expression experiments by Sung and colleagues (Sung et al. 1991; Sung et al. 1993).

The table is grouped into disease-causing missense mutations; nonsense mutations; deletions, insertions or splice-site mutations; and benign variants. There are 73 reported disease-causing mutations of the rhodopsin gene, including 63 missense mutations, 3 nonsense mutations and 7 rearrangements. Of these, 70 cause dominant retinal degeneration, 2 cause adCSNB and 1 causes recessive retinal degeneration (only homozygotes or compound heterozygotes are affected). The locations of the missense mutations are diagrammed in Figure lb. This organization emphasizes the missense mutations since simple amino acid substitutions are easiest to interpret in terms of functional consequences. Thus most of the following discussion is based on the 63 missense mutations, though the other mutations are also considered.

Important functional domains in rhodopsin include the intradiscal, transmembrane and cytoplasmic domains. Amino acids with critical functions include the acetylated amino-terminus, glycosylated asparagines at codons 2 and 15, cystines at codons 110 and 187 which form a disulfide bound (Karnik & Khorana 1990), the glutamate at codon 113 which provides a counterion for the Schiff base linkage to codon 296, lipid-binding cystines at codons 322 and 323 which attach to the cytoplasmic side of the membrane, and phosphorylation sites at codons 334, 336, 338, 340, 342 and 343 (Figure la) (Hargrave & O~Brien 1991).

Mutations in the rhodopsin gene have also been classified by the severity of the disease produced, if reported. There are many ways to classify retinitis pigmentosa: by type, by progression, by fundus findings, by ERG findings, etc. Several classification schemes have been proposed (Fishman et al. 1985; Heckenlively 1988; Massof & Finkelstein 1981; Lyness et al. 1985). Unfortunately different patients have been classified in different ways. Lacking uniform classification criteria (an undertaking we strongly advocate), we have adopted the simplest possible clinical categories. Thus in what follows we equate "mild" adRP with type 2 or R or late onset and slow progression, and "severe" adRP with type 1 or D or early onset and rapid progression. "Moderate" is intermediate. We also consider adCSNB and arRP as inherently mild. We do not incorporate ERG profiles or funduscopic findings though, needless to say, they would be relevant to a more sophisticated analysis. Based on these criteria, a total of 40 rhodopsin mutations can be classified, admittedly a small sample.

4.2 General considerations 4.2.1. Population distribution

The majority of mutations in Table 2 are unique, occurring in one patient or one family only. A few mutations occur in multiple, unrelated families. Among the latter are the Thr17Met, Thr58Arg and Pro347Leu mutations found in both Europe and Japan; the Pro23His mutation found in nearly 10% of American Caucasian adRP patients (but not in Europeans) (Farrar et al. 1990); the Thr178Cys and ~Ile255/256 mutations found in the British Isles; and the Glyl06Arg, Glu181Lys, Asp190Asn, His211Arg and Pro347Arg mutations found in European and American families.

Do these multiple pedigrees represent multiple mutational events or descent from a common ancestor (founder effect)? A single chromosomal haplotype is in complete linkage disequilibrium with the Pro23His mutation, providing strong evidence for founder effect (Dryja et al. 1991). In addition, those mutations confined to a limited geographical range, such as the Tyr158Cys and ~Ile255/256 mutations, are probably the result of founder effect. By contrast, molecular evidence suggests that the Thr58Arg and Pro347Leu mutations arose on multiple occasions. Also, it seems likely that the mutations found in both Europe and Japan arose independently. The main effect of this uncertainty is to imply that Table 2 should include multiple entries for some of the mutations. Unfortunately the multi-family mutations are not helpful in population screening because, taken in aggregate, they represent only a small percent of all reported rhodopsin mutations. 4.2.2. General distribution and types of mutations

Based on Table 2, the ratio of transitions to transversions is 43:32, which is not significantly different from the expected ratio of 2:1 in mammalian genes (Nei 1987). Although a few CT transitions in CpG dinucleotides are observed, the number is not exceptional (Barker et al. 1984). Most of the amino acid substitutions change the charge, hydrophobicity or size of the wild-type residue, but this also is not exceptional since most random changes would affect one or more of these properties. However, there are 18 evolutionarily-conserved sites in human rhodopsin (Fryxell & Meyerowitz 1991); 29% of the 63 missense mutations occur at conserved sites whereas only 5% are expected by chance alone. Thus substitutions at conserved sites are significantly more likely to cause retinal degeneration.

There are two other striking anomalies in the distribution of mutations, first, in the number of codons with multiple, independent mutations and, second, in the distribution of mutations within protein domains. For the pedigrees with rhodopsin-based retinal degeneration studied to date, 305 of the 348 amino acids in human rhodopsin have zero mutations, 32 have one mutation each, 6 have two, 3 have three, 1 has five (arginine at codon 135) and 1 has six (proline at codon 347). The expected random distribution would be 290 amino acids with zero mutations, 53 with one, 5 with two, 0.30 with three and less than 10-6 with five or more (Chakraborty 1993). Thus codons 135 and 347 are highly unusual, particularly considering the multiple, independent families with a Pro347Leu mutation. Note that although both codons contain a CpG dimer, this is an insufficient explanation since many other codons contain the same dimer.

At a superficial level the mutations seem uniformly distributed between the intradiscal, transmembrane and cytoplasmic regions of the protein. About 50% of rhodopsin amino acids are within the membrane and 25% are on each side. The observed distribution of mutations is 19:32:12 which is not significantly different from 1:2:1. However, nearly all the cytoplasmic mutations are in the 8 terminal amino acids; there is a paucity of mutations in the four cytoplasmic loops. The cytoplasmic loops include the binding sites for `-subunit of transducin K(nig et al. 1989; Min et al. 1993) and arrestin. We speculate that mutations may be as frequent here as elsewhere in the molecule, but that they do not cause adRP. 4.2.3. General distribution of clinical types

There are no striking anomalies in the distribution of mild, moderate or sever mutations. Six intradiscal mutations are mild and 4 are severe; 2 transmembrane mutations are mild (4 counting adCSNB) and 8 are severe; and 2 cytoplasmic mutations are mild and 2 severe. Thus protein domain alone does not determine severity of disease.

Interestingly, many of the "mild" forms of adRP caused by rhodopsin mutations show a sectorial phenotype in some patients. These include the Thr17Met, Pro23His, Thr58Arg, Gly106Arg, Gly182Ser and Pro267Leu mutations. Given that clinicians will differ in attributing this phenotype to a particular patient, the evidence suggests that sectorial adRP is not a separate genetic category. 4.2.4. Distribution of biochemical classes

Sung and colleagues (Sung et al. 1991; Sung et al. 1993) have used site-directed mutagenesis to distinguish broad classes of rhodopsin mutations. They constructed expression vectors containing the human rhodopsin sequence with specific mutations and used these to transfect human embryonic kidney cells. They then evaluated synthesis, transport, opsin binding capability and immunologic properties of the mutant rhodopsins. In total, they tested 21 human mutant types. Based on their findings they classified the mutants into three classes, class I with wild-type properties, class IIa with aberrant properties and complete failure to leave the site of synthesis, and class IIb with aberrant properties but partial transport to the plasma membrane. Class I mutants clustered within the first transmembrane domain and at the extreme carboxyl-terminus. Class II mutants clustered within the other transmembrane and extracellular domains (Sung et al. 1993). There are clinical data on 18 of the 20 mutations tested. Nine of the mutations produce mild disease, 7 produce severe disease and 2 produce moderate disease. Surprisingly, there is no simple correlation between severity and class: the ratios of mild-to-severe for class I are 1:2, for class IIa are 6:3 and for class IIb are 2:2.

4.3 The connection between phenotype and genotype of specific rhodopsin mutations.

Within the limits of the data cited above, specific rhodopsin genotypes can be correlated with broad phenotypic categories. Different mutations cause retinal degeneration for distinctly different reasons. However, there is an organizing principal: the clinical phenotype is a consequence of where and when the mutation affects the function of rhodopsin. 4.3.1. Mutations producing no protein or unstable protein will be mild (or recessive) in heterozygotes

This may seem counterintuitive, since it is often assumed that a defective protein will accumulate at the point of synthesis and "poison" the cell. However the cellular system for degradation and clearance of aberrant proteins is highly efficient (Klausner & Sitia 1990). Further, a 50% diminution in rhodopsin levels may be mildly deleterious at worst. This category includes mutations producing no message or unstable message, mutations producing truncated or otherwise aberrant proteins, or mutations producing proteins that are not transported to or inserted into the disc membrane because of incorrect folding or incorrect post-translational modification. Examples include early stop mutations, such as Gln64stop and Glu249stop, and substantial deletions, such as ~68-71 and 1231-23del30ins150 that produce mild disease (or recessive disease in the case of Glu249stop). The two splice-site mutations, 1230+1GT and 1231-1GA, produce very mild disease and are highly variable in expression. This may be because the splice-site mutations are spliced differently in different individuals, that is, they are "leaky" mutations. Finally, the Asn15Ser mutation would preclude glycosylation of this highly-conserved site, a potentially major defect. However, the phenotype of this mutation is also mild, suggesting this change may simply interfere with targeting or insertion into the disc (Hargrave & O~Brien 1991). 4.3.2 Heterozygous mutations producing proteins that are inserted into but destabilize the membrane will be severe

Since the discs are geometrically-precise structures with complex functional requirements and rapid turnover, and since rhodopsin constitutes the bulk of the disc protein, destabilizing mutations are expected to be severe. We speculate that this is why the Leu40Arg, Leu46Arg, Arg135Leu, Arg135Trp and Met207Arg mutations are severe. The Arg135 mutations are particularly noteworthy because this is a highly-conserved site at the margin of the membrane. Several mutations at this site cause severe adRP. Additionally, the Arg135Leu and Arg135Trp mutations are class IIb. If results from embryonic kidney cells can be extrapolated to intact photoreceptors, this implies that some protein gets to the disc membrane. Further, this amino acid may have a critical role in rod function beyond simple membrane integrity since Arg135Leu and Arg135Trp mutants fail to activate transducin (Fahmy & Sakmar 1992; Min et al. 1993). However, as we consider subsequently, this effect alone is not likely to cause severe dominant disease.

The Cysl87Tyr mutation eliminates the disulfide bond with Cys110 and produces severe disease, possibly by destabilizing the membrane. The ~255/256Ile deletion, another transmembrane mutation, also produces severe disease. On the other hand, some transmembrane mutations, such as Thr58Arg, only produce mild disease, suggesting that mutations of transmembrane regions may not necessarily perturb the membrane in significant ways. 4.3.3. Heterozygous mutations that fail to incorporate 11-cis retinal or disrupt the Schiff base counterion will constitutively activate transducin and produce severe disease

Artificial mutations that change the lysine at codon 296 or the glutamate at codon 113 result in constitutive activation of transducin (Robinson et al. 1992). No human pedigrees expressing a Glu113 mutation have been identified. However, the Lys296Glu and Lys296Met mutations have been found which produce very severe, dominant degeneration. Opsins with substitutions at the Lys296 position fail to bind 11-cis retinal and are constitutively active. Thus these mutations are likely to induce high levels of cGMP with toxic consequences (Lolley et al. 1977; Ulshafer et al. 1980). 4.3.4. Heterozygous mutations that bind 11-cis retinal normally but also activate transducin in the absence of chromophore will be mild or stationary

The revealing examples in this case are the two mutations that cause adCSNB, Gly90Asp and Ala292Glu. Both mutant opsins bind chromophore, are inactive in the dark and are activated normally by light (Dryja et al. 1993; Rao et al. 1994). However, in contrast to wild-type opsin, these proteins can also activate transducin in the absence of chromophore. Thus, unlike the Lys296 mutations which are permanently activated, these proteins cause inappropriate activation of transducin when all-trans retinal leaves opsin during the regeneration cycle. This minor abnormality is sufficient to diminish sensitivity to dim light but not sufficient to damage the photoreceptor. Significantly, the Lys296, Glu113 and Gly90 sites all fall in close proximity within the three-dimensional structure of rhodopsin (Baldwin 1993) suggesting that the glycine participates in chromophore stabilization. 4.3.5. Heterozygous mutations in amino acids outside of the membrane are likely to be mild or recessive unless indirectly involved in membrane stability or chromophore binding

We speculate that the following general principles apply to such mutations. First, mutations of some of the amino-terminal amino acids will affect targeting of rhodopsin to the disc and will have mild consequences (vide 4.3.1). Second, some of the amino acids in the intradiscal and cytoplasmic loops will be essential for proper protein folding. If mutations in these sites prevent insertion of the protein into the membrane then the consequences will be mild (vide 4.3.1). Third, heterozygous mutations in the intradiscal loops are not likely to affect phototransduction directly unless they disturb chromophore binding or isomerization (vide 4.3.3 and 4.3.4). Fourth, mutations in the cytoplasmic loops will affect transducin binding or activation (Franke et al. 1988;K nig et al. 1989). The consequences of such mutations in a heterozygote may be diminished sensitivity to dim light or adCSNB (or recessive CSNB), but not significant degeneration. Finally, heterozygous mutations affecting phosphorylation sites may affect recovery from photobleaching but not cell survival.

Possible examples of mild mutations that meet these criteria are Thr17Met, Pro23His, Gly106Arg and Glu341Lys. The Thr58Arg mutation is an example of a mild phenotype associated with diminished transducin activation (Min et al. 1993). Such example may help to explain the paucity disease pedigrees caused by dominant mutations within the cytoplasmic loops.

One intriguing observation is the importance of the penultimate proline, Pro347, in the carboxyl-terminal cytoplasmic domain (Humphries et al. 1993). The two terminal amino acids (Pro-Ala) are highly conserved in mammals. This is the most common site of mutations causing adRP, and the disease phenotype is severe. By contrast, two mutations which delete these amino acids, 1313delC and 1515del8, have a mild phenotype. However, by coincidence, the terminal Pro-Ala residues are preserved in each of these mutations. Unfortunately for the argument, this is not the case for the 1289del17 and 1312del24 mutations, which also produce mild disease. 4.3.6. We do not know enough about rhodopsin to guess which mutations might interfere with disc assembly, progression along the ROS, shedding or phagocytosis; nor can we know their clinical consequences

We do not know what role rhodopsin plays, if any, in disc morphogenesis, axial displacement, shedding and phagocytosis by the RPE. We also do not know whether defects in these processes might be related to human retinal degeneration. In any case, we know so little about these processes that guessing may not be productive. It is our expectation that new disease-causing mutations will reveal previously-unrecognized functional sites in rhodopsin that are involved in disc membrane renewal.

ACKNOWLEDGEMENTS

Supported by grants from the RP Foundation Fighting Blindness and the George Gund Foundation, and NIH NRSA Grant EY06467 to L.A.S. and a Fellowship from the Schissler Foundation to J.A.R.

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TABLE 1

Cloned and/or Mapped Human Genes Causing Inherited Retinal Diseases(a)

(In Chromosomal Order)

Symbol(b) McKusick Location Disease(c); Protein

Number

(STDG1) 248200 1p21-p13 recessive juvenile Stargardt's

disease (fundus flavimaculatus)

USH2 276901 1q recessive Usher syndrome, type 2

RP12 1q31-q32.1 recessive RP

RHO 180380 3q21-q24 dominant RP, recessive RP and

others; rhodopsin

PDEB 180072 4p16.3 recessive RP and dominant CSNB;

mouse rd, mouse r and Irish Setter

rcd1; cGMP phosphodiesterase a

CNCG 123825 4p14-q13 recessive RP; rod cGMP-gated

channel

RP7 179605 6p21.2-cen dominant RP, dominant MD, digenic

RP with ROM1 and others; mouse rds;

peripherin-RDS

MCDR1 136550 6q13-q16 dominant North Carolina MD

RCD1 180020 6q25-q26 dominant retinal-cone dystrophy 1

RP9 180104 7p dominant RP

DCMD 153880 7p15-p21 dominant cystoid MD

RP10 180105 7q dominant RP

BCP 190900 7q31.3-32 dominant tritanopia;

blue cone pigment

RP1 180100 8q11-q21 dominant RP

VMD1 153840 8q24 dominant atypical vitelliform MD

OAT 258870 10q26 dominant gyrate atrophy;

ornithine aminotransferase

USH1C 276904 11p15-p13 recessive Usher syndrome, Acadian

ROM1 180721 11q13 "digenic" RP with RDS, possible

dominant RP; rod outer segment

membrane protein 1

VMD2 153700 11q13 dominant Best MD

EVR1 133780 11q13-q23 dominant familial exudative

vitreoretinopathy

VRNI 193235 11q13 dominant neovascular inflammatory

vitreoretinopathy

USH1B 276903 11q14 recessive Usher syndrome, type 1

RMCH 216900 14 recessive rod monochromacy

USH1A 276900 14q32 recessive Usher syndrome, French

(RP13) 600059 17p dominant RP (RP12 in OMIM)

CORD1 120970 18q21- dominant cone-rod dystrophy 1

q21.3

CORD2 19q13.1- dominant cone-rod dystrophy (2)

q13.2

RP11 19q13.4 dominant RP, locus distinct from

CORD2

SFD 136900 22q13-qter dominant Sorby's fundus dystrophy

RS 312700 Xp22.2- X-linked retinoschisis

p22.1

RP6 312612 Xp21.3- X-linked RP

p21.2

DMD 310200 Xp21.3- Oregon eye disease; may be

p21.1 dystrophin

RP3 312610 Xp21.1 X-linked RP

COD1 304020 Xp21.1- X-linked cone dystrophy 1

p11.2

PRD 312500 Xp11.3 primary retinal dysplasia

NDP; 310600 Xp11.4- Norrie disease, familial exudative

XLFEVR p11.3 vitreoretinopathy; Norrie disease

protein

CSNB1 310500 Xp11.4- X-linked CSNB

p11.23

RP2 312600 Xp11.4- X-linked RP

p11.23

AIED 300600 Xp11.4-q21 land island eye disease

CHM 303100 Xq21.1- choroideremia; geranylgeranyl

q21.3 transferase A

RCP 303900 Xq28 protanopia; red cone pigment

GCP 303800 Xq28 deuteranopia and rare retinal

dystrophy; green cone pigment

a References in GDB and Omim or in the text. b Symbols in parentheses are pending. c RP = retinitis pigmentosa; MD = macular dystophy; CSNB = congenital stationary night blindness.

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Table 2

Codon Codon Codon Location or Mutation(b) Clinical Consequences; References No. Change Function; Sequence Clinical Phenotype;

Conservation(a) Comment(c)

A. Disease-causing missense mutations

4 ACA> 1st intradiscal Thr4Lys dominant RP; affects Bunge 93

AAA (T4K) glycosylation of Asn-2

(possibly not

incorporated into

membrane)

15 AAT> 1st intradiscal, Asn15Ser dominant RP; type 2, Kranich 93;

AGT glycosylated; con- (N15S) regional Sullivan 93

served in

vetebrates

17 ACG> 1st intradiscal Thr17Met dominant RP; type 2, Bunge 93;

ATG (T17M) regional, retinal Dryja 91;

neovascularization in Fishman 92a;

Japanese; class IIa, Fujiki 92;

multiple families, C>T Hayakawa 93;

in CpG Sheffield 91

23 CCC> 1st intradiscal; Pro23His dominant RP; type 2, Berson 91;

CAC conserved in (P23H) regional, variable Dryja 90a;

vetebrates severity (some with Heckenl. 90;

normal function); Stone 91

class IIa, 10% of USA

Caucasians

23 CCC> 1st intradiscal; Pro23Leu dominant RP; class IIa Dryja 91

CTC conserved in (P23L)

vetebrates

28 CAG> 1st intradiscal Gln28His dominant RP Bunge 93

CAC (Q28H)

40 CTG> 1st transmembrane Leu40Arg dominant RP; early Al-Maghtheh

CGG (L40R) onset, severe 94; Kim 93

45 TTT> 1st transmembrane Phe45Leu dominant RP; class I Sung 91

CTT (F45L)

46 CTG> 1st transmembrane Leu46Arg dominant RP; type 1, Rodriguez 93

CGG (L46R) rapid progression

51 GGC> 1st transmembrane Gly51Ala probable dominant RP Macke 93

GCC (G51A)

51 GGC> 1st transmembrane Gly51Arg dominant RP Dryja 92

CGC (G51R)

51 GGC> 1st transmembrane Gly51Val dominant RP; class I Dryja 91

GTC (G51V)

53 CCC> 1st transmembrane Pro53Arg dominant RP; class IIb Inglehearn 9

CGC (P53R)

58 ACG> 1st transmembrane Thr58Arg dominant RP; type 2, Bunge 93;

AGG (T58R) regional; class IIb, Dryja 90;

multiple families, re- Fishman 91;

duced transducin Min 93;

activation Richards 91

87 GTC> 2nd transmembrane Val87Asp dominant RP; class IIb Sung 91

GAC (V87D)

89 GGT> 2nd transmembrane Gly89Asp dominant RP; class IIb Dryja 91;

GAT (G89D) Sung 91

90 GGC> 2nd transmembrane Gly90Asp dominant congenital Rao 94;

GAC (G90D) stationary night Sieving 92

blindnes; consti-

tutively activates

transducin w/o

chromophore

106 GGG> 1st intradiscal Gly106Arg dominant RP; mild, Fishman 92;

AGG loop; conserved in (G106R) regional; class IIb, Inglehearn

vetebrates multiple families 92; Macke 93

106 GGG> 1st intradiscal Gly106Trp dominant RP; class IIb Sung 91

TGG loop; conserved in (G106W)

vetebrates

110 TGC> 1st intradiscal Cys110Tyr dominant RP Dryja 92

TAC loop, disulf. (C110Y)

w/187; conserved

in G-coupled

receptors

125 CTG> 3rd transmembrane Leu125Arg dominant RP; class IIb Dryja 91

CGG (L125R)

135 CGG> 3rd transm., Arg135Gly dominant RP; class IIb Bunge 93;

GGG transducin (R135G) Macke 93

activation;

conserved in G-

coupled receptors

135 CGG> 3rd transm., Arg135Leu dominant RP; rapid Andrasson 9

CTG transducin (R135L) progression, severe;

activation; Swedish family,

conserved in G- distinct from

coupled receptors Arg135Leu (CGG>CTT)

below

135 CGG> 3rd transm., Arg135Leu dominant RP; severe; Jacobson 91;

CTT transducin (R135L) class IIb, absent Min 93; Sung

activation; transducin activation; 91

conserved in G- unusual GG>TT mutation

coupled receptors

135 CGG> 3rd transm., Arg135Pro dominant RP Rodriguez 93

CCG transducin (R135P)

activation;

conserved in G-

coupled receptors

140 TGT> 2nd cyto. loop, Cys140Ser dominant RP Macke 93

TCT transducin inter- (C140S)

action; conserved

in vetebrates

167 TGC> 4th transmembrane Cys167Arg dominant RP; class IIb Dryja 91

CGC (C167R)

171 CCA> 4th transmembrane; Pro171Leu dominant RP; class IIa Dryja 91

CTA conserved in (P171L)

vetebrates and

invetebrates

171 CCA> 4th transmembrane; Pro171Ser dominant RP Stone 93;

TCA conserved in (P171S) Vaithinathan

vetebrates and 93

invetebrates

178 TAC> 2nd intradiscal Tyr178Cys dominant RP; type 1, Bell 92;

TGC loop (Y178C) diffuse, early onset; Farrar 91a

class IIa, multiple

families from British

Isles

180 CCC> 2nd intradiscal Pro180Ala dominant RP Rodriguez

GCC loop; conserved in (P180A) unpub.

vetebrates and

invetebrates

181 GAG> 2nd intradiscal Glu181Lys dominant RP; type 1; Bunge 93;

AAG loop (E181K) class IIa, multiple Dryja 91;

families Rodriguez

unpub; Saga

93

182 GGC> 2nd intradiscal Gly182Ser dominant RP; mild, Fishman 92a;

AGC loop; conserved in (G182S) regional; class IIa Sheffield 91

vetebrates and

invetebrates

186 TCG> 2nd intradiscal Ser186Pro dominant RP; class IIa Dryja 91

CCG loop; conserved in (S186P)

vetebrates

187 TGT> 2nd intradiscal Cys187Tyr dominant RP; severe; Nathans 93;

TAT loop, disulfide (C187Y) polymorphic C203R Scott 93

w/110; cons. in mutation at homologous

vet. and invet. site in green opsin

causes inactivation

but not degeneration

188 GGA> 2nd intradiscal Gly188Arg dominant RP; class IIa Bunge 93;

AGA loop; conserved in (G188R) Dryja 91

vetebrates

188 GGA> 2nd interdiscal Gly188Glu dominant RP; class IIa Macke 93

GAA loop; conserved in (G188E)

vetebrates

190 GAC> 2nd intradiscal Asp190Asn dominant RP; mild, Bunge 93;

AAC loop (D190N) regional; class IIa, Dryja 91;

multiple familes Keen 91;

Rodriguez 93

190 GAC> 2nd intradiscal Asp190Gly dominant RP; class IIa Dryja 91;

GGC loop (D190G) Sung 91

190 GAC> 2nd intradiscal Asp190Tyr dominant RP; severe, Fishman 92b

TAC loop (D190Y) diffuse

207 ATG> 5th transmembrane Met207Arg dominant RP; type 1, Farrar 92

AGG (M207R) severe; first Irish

family

209 GTG> 5th transmembrane Val209Met probable dominant RP Macke 93

ATG (V209M)

211 CAC> 5th transmembrane, His211Arg dominant RP; type 1- Reig 94;

CGC strongly stabi- (H211R) type 2; class IIa, Rodriguez

lizes multiple families 93a; Macke

metarhodopsin II 93; Weitz 92

211 CAC> 5th transmembrane, His211Pro dominant RP; class IIa Keen 91;

CCC strongly stabi- (H211P) Weitz 92

lizes

metarhodopsin II

216 ATG> 5th transmembrane; Met216Lys dominant RP Al-Maghtheh

AAG Leu found in all (M216K) 94

other opsins

220 TTT> 5th transmembrane Phe220Cys dominant RP Bunge 93

TGT (F220C)

222 TGC> 5th transmembrane Cys222Arg dominant RP Bunge 93

CGC (C222R)

267 CCC> 6th transmembrane; Pro267Leu dominant RP; mild, Fishman 92b;

CTC conserved in (P267L) regional; class IIa, Sheffield 91

vetebrates P264S mutation in ho- Weitz 92a

mologous site in blue

opsin causes dominant

tritanopia

292 GCG> 7th transmembrane Ala292Glu dominant congenital Dryja 93

GAG (A292E) stationary night

blindness; constituti-

vely activates

transducin w/o chro-

mophore

296 AAG> 7th transmembrane, Lys296Glu dominant RP; type D, Bunge 93;

GAG 11-cis retinal (K296E) severe; constitutively Keen 91

attachment; cons. activates transducin

in vet. & invet.

296 AAG> 7th transmembrane, Lys296Met dominant RP; profound Sullivan 93

ATG 11-cis retinal (K296M) early rod loss,

attachment; cons. central cone involve-

in vet. & invet. ment minimal

328 CTG> last cytoplasmic Leu328Pro dominant RP Rodriguez 93

CCG region (L328P)

341 GAG> last cytoplasmic Glu341Lys dominant RP; mild Scott 93

AAG region; conserved (E341K)

in vetebrates

342 ACG> last cytoplasmic Thr342Met dominant RP; C>T in Stone 93

ATG region, phosphor- (T342M) CpG

ylated

345 GTG> last cytoplasmic Val345Leu dominant RP Vaithinathan

TTG region; conserved (V345L) 93

or in vetebrates

345 GTG> last cytoplasmic Val345Met dominant RP; moderate, Bunge 93;

ATG region; conserved (V345M) variable severity; Berson 91b;

in vetebrates class I Dryja 91

347 CCG> last cyto. region; Pro347Ala dominant RP Stone 93

GCG penultimate (P347A)

proline conserved

in vetebrates

347 CCG> last cyto. region; Pro347Arg dominant RP; type 1, Bunge 93; Ga

CGG penultimate (P347R) moderate; multiple 91; Niemeyer

proline conserved families 92

in vetebrates

347 CCG> last cyto. region; Pro347Gln dominant RP Vaithinathan

CAG penultimate (P347Q) 93

proline conserved

in vetebrates

347 CCG> last cyto. region; Pro347Leu dominant RP; type 1, Apfelstedt-

CTG penultimate (P347L) diffuse, early onset; Sylla 92;

proline conserved class I, multiple Berson 91a;

in vetebrates families including Bunge 93;

European and Japanese; Dryja 90;

C>T in CpG Fujiki 92;

Hotta 92; Na

kazawa 91;

Orth 91;

Shiono 92

347 CCG> last cyto. region; Pro347Ser dominant RP; type 1; Bunge 93;

TCG penultimate (P347S) class I Dryja 90

proline conserved

in vetebrates

347 CCG> last cyto. region; Pro347Thr dominant RP Rodriguez 93

ACG penultimate (P347T)

proline conserved

in vetebrates

B. Disease-causing premature stop mutations

64 CAG> 1st cytoplasmic Gln64stop dominant RP; mild, Jacobson 94;

TAG loop (Q64X) some with normal Macke 93

retina

249 GAG> remainder of Glu249stop recessive RP; Rosenfield 9

TAG protein; conserved (E249X) remainder missing

in vetebrates starting with 6th

transmemb. domain;

heterozygotes at lower

limit of ERG response

to dim blue flash;

early night blindness

344 CAG> last cytoplasmic Gln344stop dominant RP; mild, Jacobson 91;

TAG region through (Q344X) some with normal Jacobson 94;

carboxy-terminus retina; class I; Sung 91

deletes terminal Gln-

Val-Ala-Pro-Ala

C. Disease-causing insertions, deletions and splice-site mutations

68- --- 1st cytoplasmic ~68-71 dominant RP; mild; Keen 91; Min 71 loop (496del12) class IIa, deletes 93

Leu-Arg-Thr-Pro, does

not bind 11-cis

retinal

255 ATC> 6th transmembrane ~Ile255/25 dominant RP; type 1, Artlich 92; / --- 6 severe, diffuse; class Bunge 93; 256 (1057del3) IIa, multiple families Inglehearn 9

264 TGC> 6th transmembrane; ~Cys264 dominant RP Vaithinathan

--- conserved in (1084del3) 93

vetebrates

(312 --- last cytoplasmic 1230+1G>T dominant RP; highly Jacobson 94; -313) region through variable expression Macke 93;

carboxy-terminus (Macke 93), some with Rosenfield 9

normal retina or

unaffected (Rosenfield

92); intron 4 donor

splice site G>T elimi-

nating terminal 36

amino acids

(312 --- last cytoplasmic 1231-1G>A dominant RP; type 2, Rodriguez -313) region through mild; intron 4 unpub.

carboxy-terminus acceptor splice site

G>A, effect on protein

unknown

(312 --- last cytoplasmic 1231-23del dominant RP; type 2, Al-Maghtheh -313) region through 30ins150 regional; 30 bp 94a; Kim 93

carboxy-terminus deletion of 3' intron

4 (23 bp) and 5' exon

5 (7 bp) plus 150 bp

insertion

332 GAG> last cytoplasmic ~332stop dominant RP; type 1- Rodriguez 93

G-- region, phosphor- (1289del17 type 2; 17 bp out of

ylated ) frame deletion, new

stop at codon 346

340 ACG> last cytoplasmic ~340 dominant RP; mild, Horn 92

A-G region, phosphor- (1313delC) late onset, diffuse; 1

ylated bp deletion replaces

terminal 9 AA's with

19 new AA's (preserves

terminal Pro-Ala)

340 --- last cytoplasmic ~340-348 dominant RP; mild, Restagno 93 - region through (1312del24 slow progression; 348 carboxy-terminus, ) deletion of stop codon

phosphorylated may lengthen to 386

amino acids

341 --- last cytoplasmic ~341-343 dominant RP; mild; 8 Horn 92; - region, phosphor- (1515del8) base-pair deletion Bunge 93 343 ylated replaces terminal 8

AA's with 9 new AA's

(preserves terminal

Pro-Ala)

D. Silent substititions, benign variants and polymorphisms

104 GTC> 1st intradiscal Val104Ile normal variant Macke 93

ATC loop; conserved in (V104I)

vetebrates

int- --- --- [CA]n polymorphic Weber 1989 ron 1 microsatellite

variation in intron 1

120 GGC> 3rd transmembrane Gly120Gly normal variant - Bunge 93;

GGT (654C>T) silent substitution Dryja 91;

Macke 93

146 TTC> 2nd cytoplasmic Phe146Phe normal variant - Macke 93

TTT loop, transducin (2515C>T) silent substitution

interaction

160 ACC> 4th transmembrane Thr160Thr normal variant - Reig 94; Sun

ACT (2577C>T) infrequent silent 91; Macke 93

substitution;

ACC>ACA (T160T) found

in a Spanish family

173 GCC> 4th transmembrane Ala173Ala normal variant - Bunge 93;

GCT (2593C>T) silent substitution; Dryja 91

C>T in CpG

186 TCG> 2nd intradiscal Ser186Ser normal variant - Rosenfield 9

TCA loop; conserved in (2840G>A) silent substitution

vetebrates

244 CAG> 3rd cytoplasmic, Gln244Gln normal variant - Rosenfield 9

CAA transducin inter- (4130G>A) silent substitution

action; conserved

in vetebrates

248 AAG> 3rd cytoplasmic, Lys248Lys normal variant - Sung 91

AAA transducin inter- (4142G>A) infrequent silent

action substitution

297 AGC> 7th transmembrane Ser297Ser normal variant - Rosenfield

AGT (4289C>T) silent substitution; 92; Saga 93;

C>T in CpG Macke 93

--- --- 3' noncoding 1384A>G normal variant - Sung 91

region polymorphic with 14%

frequency

--- --- intron 4 1231-23G>A normal variant - Sung 91

infrequent

action substitution

297 AGC> 7th transmembrane Ser297Ser normal variant - Rosenfield

AGT (4289C>T) silent substitution; 92; Saga 93;

C>T in CpG Macke 93

--- --- 3' noncoding 1384A>G normal variant - Sung 91

region polymorphic with 14%

frequency

--- --- intron 4 1231-23G>A normal variant - Sung 91

infrequent

--- --- 5' noncoding 1338C>A normal variant- Sung 91

region polymorphic with 13%

frequency

a Based on Fryxell and Meyerowitz [1991] with 6 vetebrate opsins (human red, green and blue; human, pig ovine and bovine rhodopsin), 2 Drosophila opsins and 10 additional, homologous G-coupled-protein receptors. b Nomenclature based on Beaudet and Tsui [1993] numbering nucleotides from the 5' start of the cDNA. c Class refers to rhodopsin cDNA transfection experiments in human embryonic kidney cells by Sung et al. [1991a; 1993]. Class I = wild type characteristics; class IIa = distinctly abnormal and remains in endoplasmic reticulum; class IIb = distinctly abnormal but some localizes in plasma membrane.

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