Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

protein shows reduced binding to DNA sequence elements, and the R90W mutation also reduces CRX mediated transactivation of the rhodopsin promotor in vitro experiments. The carrier parents with the heterozygous R90W CRX mutations developed a mild CRD, which consisted of photoaversion, dark adaptation difficulties, subtle ERG abnormalities of both rod and cone systems, color vision abnormalities (measured by Ischihara plates), and perifoveal punctate changes of the retina (75). One frameshift CRX mutation was found in both an affected LCA child and her normal-vision father, suggesting mosaicism in the father or recessive inheritance in the affected child (67). CRX is implicated in both dominant LCA (74) and recessive LCA (75), but also in dominant cone-rod dystrophy (CORD) (68), and dominant RP (76). Most authors report a severe phenotype for the CRX genotype (76) with a profound maculopathy, indicating abnormal foveal development, whereas we have reported (77) one patient with LCA with marked improvement in acuity, visual field, and cone ERG, when measured over a period of 11 yr. A patient with LCA with a heterozygous Ala 177 (1bp del) frameshift mutation was diagnosed with LCA at age 6 wk. Repeat ERGs showed a nondetectable rod and cone signal. At age 6, we measured acuities at 20/900 and this improved to 20/150 at age 11. An ERG at age 11 showed an electronegative cone signal with a small a- and b-wave, whereas the visual field was measurable by Goldman perimetry. We provide clinical evidence that in some patients with CRX, postnatal OS lengthening and improved vision may take place after the initial insult of the CRX lesion (77).

In conclusion, the CRX defects lead to a highly variable phenotypic spectrum of disease. CRX mutations may lead to a dominant CORD (68) or a dominant or recessive form of LCA (74). CRX defects appear to be a rare cause of LCA (78,79). Patients affected with LCA have moderately severe visual loss, often associated with a macular lesion, although one report noted improvements in several visual function parameters (61). The obligate carrier parents appear to have a mild cone-rod dysfunction (on ERG) (75). The histological phenotype is very similar to the rd mouse (with a cGMP PDE defect and elevated cGMP levels) (71), and the expression of the γ subunit of cGMP PDE is markedly decreased (72), suggesting that the molecular mechanism of CRX lesions, may be cGMP elevation (Table 1).

CRB1

CRB1 was cloned by den Hollander et al. in the Netherlands (80). The gene resides on 1q31, has 12 exons, and encodes a protein of 1376 amino acids and harbors mutations that have been found in a wide variety of retinal dystrophies (9,80–83). These include AR RP (ARRP) of the original RP12 family, which has a severe phenotype with hyperopia, nystagmus, and preservation of the para-arteriolar RPE (also known as PPRPE type of RP), ARRP without the PPRPE, RP with the Coats-like exudative vasculopathy, and LCA with and without the PPRPE type of retinal changes.

The CRB1 protein shows 35% structural similarity to the Drosophila Crumbs (CRB) gene. The similarity with the CRB protein suggests a role for CRB1 in cell–cell interaction and possibly in the maintainance of epithelial cell polarity of ectodermally derived cells. The human CRB1 protein contain a signal peptide, 19 epidermal growth factor-like domains, three laminin A Globular-like domains, a transmembrane domain, and a highly conserved 37 amino acid cytoplasmic domain with a C-terminal ERL1

motif (82). The cytoplasmic domain of the Drosophila CRB and human CRB1 proteins domains, when overexpressed in Drosophila embryos, rescue the Crumbs phenotype to a large degree, suggesting a critical function for this small domain (82).

The architecture of epithelial cells depends on the distribution of cellular junctions and other membrane associated protein complexes. A core component of these complexes is the transmembrane protein CRB. Human photoreceptors are packed together in the ONL of the retina together with processes of Müller glial cells for structural and metabolic support. The establishment and maintenance of apical–basal polarization and cell adhesion is important for the photoreceptors. The apical part of photoreceptors is the OS (which abuts the RPE), whereas the basal part represents the synapse of the photoreceptor with bipolar cells. At the apical site of photoreceptors an adhesion belt named the outer limiting membrane (OLM) contains multiple adherence junctions which are present between photoreceptors and Müller cells. These adherence junctions consist of multiprotein complexes and are linked to the cytoskeleton of the cell for cell shape. The adhesion belt runs along the photoreceptor cells at the division of the IS and the cellbody and nucleus. The potential space between the OLM and the RPE represents the subretinal space and the photoreceptor IS and OS normally project and “float” in this space. Cells lacking CRB fail to organize a continuous zonular adherens and fail to maintain cell polarity. Pellikka et al. (83) showed that CRB1 localizes to a subdomain of the photoreceptor apical plasma membrane, namely the IS. They proposed that CRB is a central component of a molecular scaffold that controls zonula adherens assembly. The role of mammalian CRB1, however was still unclear.

Clues about the function of CRB1 came initially from a natural CRB1 mutant mouse, called retinal degeneration 8 (rd8). This mouse model produces a secreted truncated CRB1 protein of 1207 amino acids, which lacks both the transmembrane domain and the intracellular domain. The rd8 mouse develops striking retinal abnormalities, including focal photoreceptor degeneration and irregularities of the OLM (84). Mehalow et al. (84), reported that CRB1 localizes to both Müller cells and photoreceptor IS. They found clinically that the rd8 mice developed irregular-shaped large white subretinal spots, more heavily concentrated in the inferonasal quadrant of the retina. These spots correspond histopathologically to regions with retinal folds and pseudorosettes. They also found OLM fragmentation, OS shortening but normal IS. By 5 mo, the OS have virtually disappeared, the IS are swollen, and the Müller cell processes are unusually prominent. The retinal degeneration was focal in appearance, with nearly normal retina present at the edge of a region with severe degeneration (85). The photoreceptor dysplasia and degeneration reported by Mehalow et al. in the rd8 mouse with the CRB1 mutations strongly vary with the genetic background, suggesting modifier effects from other retinal genes.

The rd8 mutation is probably not a null allele, therefore van de Pavert et al. (86) inactivated both CRB1 alleles (CRB1–/–) to produce a complete null and examined the resulting mouse retina. There were marked differences between the CRB1–/– and the rd8 retinal findings. Two-week- nor 2-mo-old CRB1–/– mice had retinal abnormalities. At 3 mo, however, the KO mice developed focal areas of retinal degeneration. The OLM was ruptured and there was protrusion of single or multiple photoreceptors both into the subretinal space and into the inner nuclear layer. One of the most strikng histological

findings were double photoreceptors layers (half rosettes). These rosettes developed normal inner segments and a full OLM, very much unlike the rd8 model. This finding suggests that CRB1 is not essential for the formation of junctional complexes and OLM, but rather for the maintenance of these structures. In 6-mo-old CRB1–/– mice large ectopic photoreceptor layers were identified, which were so large that they resembled a “funnel” abutting the ganglion cell and inner limiting membrane. They suggest that the initial insult of the CRB1 mutation is the loss of the photoreceptor to Müller glial cell adhesion in retinal foci. Light exposure experiments revealed a significant increase in retinal degeneration in the CRB1–/– mouse especially inferotemporally; therefore, light enhances the retinal degeneration in the CRB1–/– retinas (86). This may be a correlate of the thickened retinas that lacked the distinct layers of the normal retina, found by Jacobson et al. (87), who used in vivo high-resolution microscopy (also known as optical coherence tomography [OCT 3]) in patients with LCA with known CRB1 mutations. Comparable retinas from patients with LCA with RPE65 mutations were thinned when examined by the same methodology (87).

Because of the severity of the PPRPE type RP, CRB1 was postulated also to cause LCA. Den Hollander et al. (9) and Lotery et al. (85) indeed found that 13% of cases with LCA can be explained by mutations in the CRB1 gene, making it a common and important gene for LCA. In addition, CRB1 mutations were identified in five of the nine patients with RP and Coats-like exudative vasculopathy, a severe complication of RP (9). Patients with LCA with CRB1 mutations in three of seven cases also showed the PPRPE picture (9). An overview of the CRB1 mutation spectrum can be found in den Hollander et al. (83).

The phenotype of patients with LCA with CRB1 lesions may be distinct as the following constellation of findings may point to a CRB1 defect. Visual acuities range from 20/40 to light perception (61,85). Many patients have hyperopic refractions and the retinal appearance, although variable and overlapping with other LCA phenotypes may be distinct when PPRPE or white dots are found. Many but not all patients with LCA with CRB1 mutations have one of these two features. Thick, abnormally laminated retinas by OCT imaging may be a pathognomonic feature of LCA retinas with degeneration caused by CRB1 mutations (87). Pigmented paravenous chorioretinal atrophy, a dominant form of RP characterized by paravenous pigmentary deposits, was found to be associated with a heterozygous Val162Met CRB1 missense mutation in a large RP family (88). Obligate heterozygous parents of offspring with LCA and CRB1 mutations have an ERG and a multifocal ERG phenotype that may be distinct from the phenotype of obligate heterozygotes with other LCA gene defects (62,89). Cremers hypothesized that LCA may be associated with complete loss of function of CRB1, whereas patients with RP (early-onset RP with and without PPRPE, and RP with Coats-like exudative vasculopathy) in whom the visual loss is more gradual and later in life, may have residual CRB1 function (90). This hypothesis is supported by the facts that 37% of LCA CRB1 alleles are presumed null alleles, whereas only 19% of RP CRB1 alleles appear to be null (p = 0.01 by Fisher’s exact test) (90).

In conclusion, CRB1 defects are an important and frequent cause of LCA (9,85). Both the affected status and obligate heterozygotic parents may be recognizable clinically (61,62). The affected CRB1 phenotype may be relatively mild and distinct,

as some patients have PPRPE and relatively good visual function (61). The partial (84) and complete (86) KO mice models of CRB1 suggest that the pathology involves fragmentation of the adhesion belt, the OLM, and absence of the zonula adherens, which results in the loss of polarity of the photoreceptors, ectopic development of photoreceptors, and initially a focal retinal degeneration.

GUCY2D

The gene for retinal guanylyl cyclase GUCY2D was cloned by Shyjan et al. in 1992 (91) and mapped to 17p13.1 by Oliviera et al. (92). In 1995, Camuzat et al. (93,94) mapped a gene for LCA to this region (17p13.1) by a homozygosity-mapping strategy using consanguineous LCA families of North African origin. Perrault et al. (5) subsequently reported missense and frameshift mutations in GUCY2D in four unrelated LCA1 probands of these same families. The human gene contains 20 exons, and has thus far been implicated in AR LCA and, surprisingly, also the later-onset AD CRD (95) (also known as CORD6). GUCY2D is expressed in the plasma membrane of photoreceptor OS, but at higher levels in cones than in rods (96,97). The protein of GUCY2D is GC and is a transmembrane protein that serves a key function in photoreceptor physiology as it synthesizes the intracellular transmitter of photoexcitation guanosine 3′,5′-cyclic monophosphate (cGMP). A unique feature of GC is its regulation by three small Ca2+-binding proteins called GC activating proteins (GCAPs) (98–101). These regulatory proteins sense changes in the cytoplasmic Ca2+-concentration (Ca2+) during illumination and activate GCs when the (Ca2+) decreases below the value in a dark-adapted cell of 500–600 nM (102). GC is responsible for the production of second messenger cGMP after it has been hydrolysed by cGMP PDE (for a complete review, see ref. 103). cGMP PDE becomes activated after light stimulation and depletes cGMP, resulting in closure of photoreceptor cGMP gated channels and subsequent decreased levels of calcium, which leads to hyperpolarization of the membrane. The drop in calcium stimulates GCAP, to stimulate GC to produce cGMP. GC is an enzyme that is composed of an extracellular ligand-binding N-terminal segment, a transmembrane domain, an internal protein kinase homology region, and a C-terminal catalytic domain. In an attempt to identify the significance of the extracellular domain (ECD), Laura et al. (104) created deletion mutants of GC and removed the ECD and showed normal GCAP stimulated cGMP production by the mutant GC, concluding that GCAP interacts with GC through the intracytoplasmic portion. Duda et al. (105) showed that a Phe514Ser mutation (found in a patient with LCA) from the kinase homology domain of the protein severely compromised the ability to produce cGMP (105), whereas Rozet et al. (106) tested the basal functional capacities of catalytic domain and ECD mutations and found that the catalytic mutations almost completely abolished basal cGMP production. The extracellular domain mutations had no effect on basal cGMP production. Tucker et al. (107) then showed that catalytic domain mutations severely compromise the ability of GCAP to stimulate cGMP production, whereas ECD mutations cause only about 50% decrease in catalytic ability (108). Tucker et al. also showed that recessive GUCY2D mutations in heterozygous state found in humans with LCA cause dominant negative effects on the WT allele (108), and this correlates with significantly abnormal cone electroretinographic responses in parents who are obligate heterozygotes for these same mutations (109), who have children with LCA.

We were puzzled by the observations that mutations in the same gene, GUCY2D, can cause two different diseases with very different onsets and severities; i.e., dominant GUCY2D mutations (in the dimerization domain of the protein) are associated with conerod dystrophy, whereas recessive GUCY2D mutations (found in all other protein domains) are well known causes of the much more severe LCA. These facts prompted us to hypothesize that parents of children with LCA with GUCY2D mutations must be obligate heterozygotes for the same mutations and may therefore exhibit a mild cone-rod disease (109,107). We tested this hypothesis by performing detailed ERGs on parents with known GUCY2D lesions (P858S, L954P) and documented repeatable and significant cone dysfunctions in four subjects (109). To address the question of whether abnormalities in the heterozygous state were caused by haploinsufficiency or dominant negative effects, we tested the same mutations in an in vitro expression system (107). We constructed the mutations in GUCY2D cDNA by site-directed mutagenesis (108) and tested the ability of the mutant protein to produce cGMP. First, we showed (107,110) that mutations in the catalytic domain dramatically compromise the ability of GCAP to stimulate cGMP production (107). Membrane GCs are thought to exist in a dimeric state (111,112) and, therefore, we tested whether the P858S and L954P mutants affect WT RetGC-1 activity when co-expressed. We co-transfected HEK 293 cells with 2.5 µg WT RetGC-1 and 2.5 or 5 µg of either pRc-cytomegalovirus (CMV) (as a control), P858S, or L954P. Western blots showed that the total amount of WT and mutant protein transfected correlates with the amount of protein used in the assays. The addition of increasing amounts of either P858S or L954P reduced GCAP-2 stimulated activity by upto 55%. These results showing a significant decrease in wild-type RetGC-1 activity when L954P or P858S are coexpressed suggest that any heterodimers formed are inactive or poorly active (107). We then expressed mutations from the extracellular domain of RetGC-1 (C105Y and L325P) to investigate their effects on cGMP production and found that these only reduce RetGC1 activity by 50%.

Disruption of the retinal GC gene in mice leads to normal development and normal numbers of rod and cone photoreceptors (113). However, by 5 wk, the number of cones in the mouse KO has been reduced dramatically and rapidly in the null mice compared to age-matched controls (113). The degeneration of cones is not matched by a rod decline. Both numbers and morphology of the rod photoreceptors remains normal. However, the rod responses are also dramatically decreased despite their normal appearance (113). The GC KO in the chicken also leads to rapid retinal degeneration after the photoreceptors appear to have developed normally (114). Light-driven translocation of photoreceptor proteins between inner and outer segment plays an important role in adaptation of photoreceptors to light and this process is disrupted in the mouse GC KO (115). The α cone transducin molecule, which is normally found in the OS, was found in IS and synapses of cones in the GC KO, whereas arrestin, which is normally shifted from the OS to IS during light exposure of the WT mice, was found only in the outer segments of the GC KO (115). This may indicate that the absence of GC may be the biochemical equivalent of light exposure and the sequestration of transducin in the inner segmet may be a protective mechanism in the face of chronic light adaptation and chronic hyperpolarization of the cell membrane (115). The molecular mechanism of photoreceptor cell death in LCA retinas with GUCY2D defects may relate to the abnormally

low cGMP levels, low calcium levels, chronic hyperpolarization, all consequences of chronic light adaptation. The link between the former biochemical changes and apoptosis still needs to clarified.

In some populations, GUCY2D may be the most common LCA gene, with 20% of patients with LCA carrying this genotype (79,116). Others, including our own, found that 8% of LCA is caused by this gene (78). The phenotype of GUCY2D-type LCA is distinct (78,117). Dharmaraj et al. found a severe phenotype for patients with LCA with GUCY2D defects, but the visual course was stable (over a 20-yr period). Marked hyperopia, photo-aversion, and an essentially normal retinal appearance were also noted (78). Obligate heterozygous parents with GUCY2D mutations were found to have mild cone ERG abnormalities (109), and may be recognizable clinically, reducing the time and effort of the molecular diagnostic process (109). Silva et al. determined that a GUCY2D mutation modified the phenotype of a patient with LCA with RPE65 defects (118). Histological studies of a patient with LCA with GUCY2D mutations revealed the surprizing presence of cone photoreceptors (24). In this study, Milam et al. (24) found a Arg660Gln mutation and a deletion adjacent to the GUCY2D gene in an 11-yr-old patient with LCA with light perception vision, +4.00 D hyperopia, and a subtle retinal pigmentary mottling. OCT showed a thinned retina, whereas further histological study showed OS loss, a normal bipolar layer, and a thinned ganglion cell layer (24). Autofluorescence studies (which measure the active lipofuscin deposition in the RPE, and indicates viability of the photoreceptor–RPE complex) showed normal autofluorescence patterns in patients with LCA with GUCY2D mutations (119). These data (24,76,119) suggest the possibility that patients with LCA with the GUCY2D genotype may have viable retinas and may also be amenable to gene therapy.

In conclusion, the LCA disease phenotype associated with GUCY2D is a severe congenital cone-rod dystrophy associated with high hyperopia, a relatively normal retinal aspect and a relatively stable course (76,79,117). The obligate heterozygous parents of LCA offspring develop a recognizable subclinical cone dysfunction (109). Histological and autofluorescence studies of affected patients suggest that some photoreceptors remain intact in patients with the GUCY2D LCA (24,119). The molecular mechanism appears to be a depression of the level of cGMP (Table 1).

RPE65

The RPE65 gene was cloned by Hamel et al. (120) was found to be exclusively expressed by the RPE and plays an important role in the vitamin A cycle. The gene for RPE65 is located on 1p22 (121) and is composed of 14 exons, whereas the protein has 533 amino acids (122). RPE65 mutations are associated with a variety of overlapping severe retinal dystrophies ranging from LCA (most severe), AR childhood-onset severe retinal dystrophy, and to juvenile RP (least severe) (7,123,124). In an attempt to discern the function of RPE65, a RPE65–/– KO mouse was constructed (125), which revealed that OS discs of rod photoreceptors in RPE65–/– mice are disorganized compared to those of RPE65+/– and RPE65+/+ mice. Rod function, as measured by electroretinography, was abolished in RPE65–/– mice, although some cone function remained. The remaining visual function in the RPE65–/– mouse was later determined to be generated by the rods, not cones as shown by Seeliger et al. (126).

RPE65–/– mice lack rhodopsin as a result of an inability to recycle 11-cis retinal, but not opsin apoprotein, whereas all-trans-retinyl esters overaccumulate in the RPE, therefore, RPE65 is necessary for the production of rhodopsin and prevention of the accumulation of retinyl esters (125). The logical conclusion could be that in humans and animals lacking RPE65, the accumulation of the retinyl esters is somehow toxic to the RPE and, therefore, involved in the pathogenesis and death of the photoreceptor cells. However, there is also evidence that persistant opsin or rhodopsin signaling can cause photoreceptor pathology, and Fain et al. have named this phenomenon the equivalent light hypothesis (127,128). In an attempt to distinguish these two possibilities (accumulation of retinyl esters vs light independent signaling), Woodruff et al. compared RPE65 null mice with RPE65 null/transducin (that have a block in the phototransduction cascade) mice and hypothesized that continuous, light independent opsin (unbound to its ligand 11-cis retinal) activity causes photoreceptor degeneration, without retinyl ester accumulation playing a role (129). They postulated that if spontaneous opsin activity simulates the light adapted state of the rod, then there must be a diminished circulating current, a reduced light sensitivity, an accelerated light response kinetics, closure of the cGMP gated channels, and subsequent reduction of the intracellular calcium levels (129). If these predictions are accurate, they argued, then by blocking the transducin-ediated signaling, the photoreceptor degeneration must be prevented. In the RPE65 KO mouse, Woodruff et al. showed that indeed the circulating currents are much lower, the light sensitivity is diminished, and the turn off of the photoresponse was accelerated in the RPE65 KO mouse compared to the WT. To test whether spontaneous-unliganded opsin causes photoreceptor death through transducin-mediated signaling, the authors crossbred mice null for RPE65 with mice null for transducin and examined the retinal histology, by counting the number of photoreceptor nuclei in the ONL. Compared to WT, null/transducin mice, and RPE65 heterozygotes, which maintain 9–11 rows of nuclei, the RPE65 KO mice have 6–7 rows at 40 wk. The crossbred mice (KO for RPE65 and transducin) had 8–10 rows of nuclei at 40 wk and were completely protected from the retinal degeneration. Dark rearing of the RPE65 KO mouse did not protect the retinal cells from degeneration as expected, because the opsin activation is light independent. Retinyl esters accumulations were found to be 14 times higher in the double KO than in the control mice, showing that the retinyl esters are not responsible for the degeneration. Finally, Ca2+ levels were measured and found to be very low as expected in the RPE65 KO, illustrating the consequences of closed cGMP gated channels (129). How aberrant opsin signaling, closure of the rod-cGMP channels, and low Ca2+ leads to death is not currently known, but low Ca2+ is known to trigger apoptosis in neurons and photoreceptors (128,130). In addition to the KO mouse, a natural KO of the RPE65 gene exists in the Briard dog, which harbors a 4-bp deletion in RPE65. Because the retinal histology was essentially normal except for RPE lipid vacuoles, the retinal phenotype of the Briard dog was initially called congenital stationary night blindness (131). Further analysis of the phenotype and retinal architecture revealed that the diagnosis was more a progressive retinal dystrophy (132), and a good model for human RPE65 type retinal dystrophies.

Perrault et al. suggested that, based on the divergence of the underlying molecular defects for patients with GUCY2D- and RPE65-type LCA, the resulting phenotypes may be identifiable and distinguishable clinically (117). The observation of missense

and frameshift GUCY2D mutations suggests that the cGMP production in photoreceptor cells is markedly reduced or abolished in LCA (117). As a consequence, the excitation process of rod and cone photoreceptors would be markedly impaired because of constant closure of cGMP-gated cation channels, with chronic hyperpolarization of the membrane. The cGMP concentration in photoreceptor cells would not be restored to the dark level, leading to a situation equivalent to constant light exposure during photoreceptor development. Thus, in contrast to GUCY2D mutations, RPE65 mutations would decrease the rhodopsin production, leading to a situation equivalent to a retina kept in a constant dark state (117). Although it appears possible to separate patients LCA with GUCY2D defects from those with RPE65 defects (78,117), the results of Woodruff et al. (129) strongly suggest that both GUCY2D and RPE65 defects lead to similar molecular defects, i.e., abnormal lowering of cGMP and subsequent lowering of the intracellular Ca2+ levels, a situation consistent with the equivalent light hypothesis suggested by Fain et al. (127,128).

In the patients harboring GUCY2D mutations, no visual improvement was observed, the pendular nystagmus remained unchanged, and visual acuity was reduced to light perception or ability to count fingers held in the visual field. In addition, the patients complained of severe photophobia and usually preferred half light, and significant hyperopia was observed, although visual fields were not recordable because of profound loss of visual acuity (78,117). However, in the patients with RPE65 mutations, transient improvement in visual function was regularly noted by the parents (78,117, 133). This improvement or adaptation of visual function was later confirmed objectively by Lorenz et al. and Paunescu et al. (133,134). Young children became able to follow light or objects, especially during the daytime. They complained of night blindness and usually preferred bright light. Visual acuity reached 20/100–20/200, mild or no hyperopia was observed, and mild myopia occured occasionally. Finally, the visual field in this group was usually recordable and frequently displayed a peripheric concentric reduction. They concluded that GUCY2D defects lead to a functional outcome that is different and recognizable from the functional outcome of patients with RPE65 defects. Patients with GUCY2D develop a congenital stationary cone-rod dystrophy, whereas patients with RPE65 defects develop a progressive rod-cone dystrophy (78,117,133). Silva et al. found a modification of the RPE65 phenotype by a GUCY2D missense mutation (118).

The relative burden of the RPE65 locus in LCA may be as high as 16% (124), although most large studies found a burden between 6.1% (11 out of 179 patients LCA) and 8.2% (8 out of 98 patients) (79,135). We screened 275 new patients with LCA and found that 25 (9%) carry at least one RPE65 mutation, and 7 of these were novel. The new LCA microchip constructed by the Allikmets group, which contains 81 mutant RPE65 alleles (the largest number of any gene), only identified 2.4% of 200 new patients with LCA with RPE65 defects (136,137). Strict clinical criteria must be maintained for LCA (including poor fixation, wandering nystagmus, amaurotic pupils and nondetectable ERG) to allow exclusion of milder phenotypes, such as juvenile RP and AR childhood-onset severe retinal dystrophy (133). In a large consanguinous family with homozygous Y368H mutations in RPE65, we found a surprising variability in visual evolution, with some patients having stable vision, others declining, whereas yet

others were measured to improve, implying the action of modifier effects from genetic or environmental factors or both (138).

We have thus far analyzed nine obligate RPE65 carriers and found that most carriers have characteristic foveal drusen at a young age and all have normal rod and cone ERG functions (62,89). We found that affected patients with RPE65 mutations have a characteristic retinal aspect, with a transluscent RPE and areas of atrophy. Histological analysis of the KO mouse and Briard dog shows subtle abnormalities of the RPE, but essentially normal retinal architecture (131,132), although one human fetus with RPE65 mutations had attenuation of the photoreceptor layer (139,140). Autofluorescence studies confirm the block in the vitamin A recycling process, as the autofluorescence of patients with mutated RPE65 is very abnormal and low (119). Autofluorescent patterns may provide a clinical flag for patients with the RPE65 genotype (119). Measurable acuities, visual fields, and small cone ERG are possible in patients with RPE65 and this possibly makes their phenotype distinct (78,117,133).

Recently, mechanism-based pharmacological therapy has been shown to arrest photoreceptor death in the RPE65–/– mouse model of LCA, based on the emerging knowledge of the genes and their functions in the retina. Van Hooser et al. (43), using recent knowledge that RPE65 mutations in mice lead to an inability to form 11-cis retinal (which binds to rod opsin to form light sensitive rhodopsin), supplemented the mouse diet with a vitamin A derivative and consequently showed short-term improvements in rod physiology. The long-term consequences of this intervention are not known. Theoretically, adding vitamin A to a system, which is known to have a block as a result of a mutant RPE65 protein, could lead to long-term accumulation of a toxic intermediate, making the disease worse. Acland et al. (34) studied the effects of RPE65 replacement in the Briard dog, which harbors a natural, homozygous 4-bp deletion in the RPE65 gene and is blind at birth. ERG function is nondetectable, despite the normal appearance of the retinas, including the photoreceptor layer in this dog model. Subretinal injections in one eye of three dogs containing the adenoassociated virus with cDNA of dog RPE65, with a CMV promotor, B-actin enhancer and internal ribosome entry sequence were performed at age 4 mo (34). Rod-mediated ERGs, cone-mediated ERGs, visual-evoked potential, pupillometry, and behavioral testing all showed dramatic improvements in visual function at about 8 mo of age. Genomic PCR and RT-PCR demonstrated expression of the WT message in the retina and RPE, whereas immunoblots showed persistent RPE65 protein in RPE cells. This is the first study to demonstrate restoration of visual function in a large-eyed animal model with LCA caused by an RPE gene defect, and this persisted for more than 3 yr after one injection (Jean Bennett, personal communication). It is currently not known whether the retinas of human patients with LCA with RPE65 defects are intact, and whether the photoreceptor layer is present. The time window before the retina undergoes cell death is also not known. Also, it is not known whether photoreceptor gene replacements have similar dramatic effects, which will now have to be evaluated. A human clinical trial involving well-characterized LCA gene defects in babies with LCA may commence in 2005.

In conclusion, the disease phenotype of patients with LCA with RPE65 defects appears to be recognizable based on retinal appearance, relative mildness of the visual

defect, a transient improvement in vision, and an absence of autofluorescence, and has been termed early-onset severe retinal dystrophy (133). The molecular mechanism of disease appears to be very similar to that of patients with GUCY2D defects, i.e., a depression of the level of cGMP (129). RPE65 defects lead to lowered cGMP levels, not through a depressed production, but as a result of constitutively active opsin, which stimulates cGMP PDE to continously hydrolyze cGMP (129).

RPGRIP1

The RPGRIP1 gene was discovered by Roepman et al. (141), Boyle and Wright (142), and Hong et al. (143). RPGRIP1 is expressed both in rod and cone photoreceptors, but surprisingly also in amacrine cells (144). RPGRIP1 is the molecular partner of RPGR and both proteins co-localize to the connecting cilium, which connects the inner to the OS of the photoreceptor cell (141–143). RPGR mutations cause several types of retinal degenerations, including x-linked forms of RP, CRD, and macular dystrophy (145). The RPGRIP1 protein may be a structural component of the ciliary axoneme (146). The genes associated with NPHP, an important cause of end-stage kidney disease (sometimes associated with LCA and/or RP), are also associated with ciliary function, and there is evidence that NPHP proteins interact with the RPGRIP1 protein (147). The exact function of RPGRIP1 is still unknown, however, and investigations are complicated by the multiple RPGRIP1 isoforms, which have distinct cellular, subcellular localizations and biochemical properties in the retina (148,149). To understand RPGR and RPGRIP1 function in the retina, the molecular partners of RPGR were sought in twoyeast hybrid experiments. By screening bovine cDNA retinas with the RDH of RPGR as bait, a novel RPGRIP was subsequently identified by three independent groups and the protein bears no homology to any thus far identified retinal proteins (141–143). This protein was named RPGRIP-1, and the gene RPGRIP1 was mapped to chromosome 14q11 (141–143). RPGRIP1 consists of 3861 bp (i.e., 1287 amino acids) (Gerber et al. [150] divided more than 25 exons).

In 2000, Roepman et al. found that RPGRIP1 contains a C-terminal RPGR interacting domain and a coiled-coil domain, which the authors suggested is homologous to proteins involved in vescicular trafficking (141). In vivo expression experiments with RPGR mutations (from X linked retinitis pigmentosa [XRRP] patients) were observed to impair RPGR–RPGRIP1 interactions. Both RPGR and RPGRIP1 were found in this study to co-localize to the OS of rod and cone photoreceptors. Based on these observations, the authors concluded that the site of pathology for RPGRIP1 associated disease is in mediating vescicular transport-mediated processes. In the same year, Boylan and Wright (142) also isolated RPGRIP1 and showed its expression in retina and testis. RPGR–RPGRIP1 interactions were confirmed by co-immunoprecipitation experiments and RPGR mutation co-expression studies (142). Hong et al. then subsequently confirmed that RPGRIP1 is indeed a molecular partner of RPGR and found that both proteins co-localize in the photoreceptor connecting cilium (143). Their data suggested that RPGRIP1 is a structural component of the ciliary axoneme of both rods and cones and functions to anchor RPGR within the cilium. Unlike the suggestions of Roepman et al., the subcellular localization of RPGRIP1 to the connecting cilium by Hong et al. would suggest that RPGRIP1 could be critically important for the directional transport of