Properties and Therapeutic Implications of an Enigmatic D477G RPE65 Variant Associated with Autosomal Dominant Retinitis Pigmentosa.

RPE65 isomerase, expressed in the retinal pigmented epithelium (RPE), is an enzymatic component of the retinoid cycle, converting all-trans retinyl ester into 11-cis retinol, and it is essential for vision, because it replenishes the photon capturing 11-cis retinal. To date, almost 200 loss-of-function mutations have been identified within the RPE65 gene causing inherited retinal dystrophies, most notably Leber congenital amaurosis (LCA) and autosomal recessive retinitis pigmentosa (arRP), which are both severe and early onset disease entities. We previously reported a mutation, D477G, co-segregating with the disease in a late-onset form of autosomal dominant RP (adRP) with choroidal involvement; uniquely, it is the only RPE65 variant to be described with a dominant component. Families or individuals with this variant have been encountered in five countries, and a number of subsequent studies have been reported in which the molecular biological and physiological properties of the variant have been studied in further detail, including observations of possible novel functions in addition to reduced RPE65 enzymatic activity. With regard to the latter, a human phase 1b proof-of-concept study has recently been reported in which aspects of remaining vision were improved for up to one year in four of five patients with advanced disease receiving a single one-week oral dose of 9-cis retinaldehyde, which is the first report showing efficacy and safety of an oral therapy for a dominant form of RP. Here, we review data accrued from published studies investigating molecular mechanisms of this unique variant and include hitherto unpublished material on the clinical spectrum of disease encountered in patients with the D477G variant, which, in many cases bears striking similarities to choroideremia.

Phenotype-genotype correlation with Sanger sequencing identified retinol dehydrogenase 12 (RDH12) compound heterozygous variants in a Chinese family with Leber congenital amaurosis.

BACKGROUND: Leber congenital amaurosis (LCA) is a group of clinically and genetically heterogeneous retinal dystrophy. To date, 22 genes are known to be responsible for LCA, and some specific phenotypic features could provide significant prognostic information for a potential genetic etiology. This study is to identify gene variants responsible for LCA in a Chinese family using direct Sanger sequencing, with the help of phenotype-genotype correlations. METHODS: A Chinese family with six members including two individuals affected with LCA was studied. All patients underwent a complete ophthalmic examination. Based on phenotype-genotype correlation, direct Sanger sequencing was performed to identify the candidate gene on all family members and normal controls. Targeted next-generation sequencing was used to exclude other known LCA genes. RESULTS: By Sanger sequencing, we identified two novel missense variants in the retinol dehydrogenase 12 (RDH12) gene: a c.164C>A transversion predicting a p.T55K substitution, and a c.535C>G transversion predicting a p.H179D substitution. The two affected subjects carried both RDH12 variants, while their parents and offspring carried only one of heterozygous variants, showing complete cosegregation of the variants. The compound heterozygous variants were not present in 600 normal controls. Besides, the RDH12 variants were confirmed by targeted next-generation sequencing. CONCLUSIONS: The RDH12 compound heterozygous variants might be the cause of the LCA family. Our study adds to the molecular spectrum of RDH12-related retinopathy and offers an effective example of the power of phenotype-genotype correlations in molecular diagnosis of LCA.

Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants.

Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD(+) biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD(+) synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome.

Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis.

RPE65 is a membrane-associated retinoid isomerase involved in the visual cycle responsible for sustaining vision. Many mutations in the human RPE65 gene are associated with distinct forms of retinal degenerative diseases. The pathogenic mechanisms for most of these mutations remain poorly understood. Here, we show that three Leber congenital amaurosis -associated RPE65 mutants (R91W, Y249C and R515W) undergo rapid proteasomal degradation mediated by the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13) in cultured human retinal pigment epithelium (RPE) cells. These mutant proteins formed cytosolic inclusion bodies or high molecular weight complexes via disulfide bonds. The mutations are mapped on non-active sites but severely reduced isomerase activity of RPE65. At 30°C, however, the enzymatic function and membrane-association of the mutant RPE65s are significantly rescued possibly due to proper folding. In addition, PSMD13 displayed a drastically decreased effect on degradation of the mutant proteins in the cells grown at 30°C. These results suggest that PSMD13 plays a critical role in regulating pathogenicity of the mutations and the molecular basis for the PSMD13-mediated rapid degradation and loss of function of the mutants is misfolding of RPE65.

Nature of the visual loss in observers with Leber's congenital amaurosis caused by specific mutations in RPE65.

PURPOSE: To characterize visual losses associated with genetic mutations in the RPE65 gene that cause defects in the RPE-specific isomerase, RPE65. RPE65 is an important component of the retinoid cycle that restores 11-cis-retinal after its photoisomerization to its all-trans form. The defects investigated here cause Leber's congenital amaurosis (LCA2), an autosomal, recessively-inherited, severe, congenital-onset rod-cone dystrophy. METHODS: Vision was assessed in nine patients and 10 normal controls by measuring: (1) long-wavelength sensitive (L-) cone temporal acuity (critical flicker fusion frequency or cff) as a function of target illuminance, and (2) L-cone temporal contrast sensitivity as a function of temporal frequency at a fixed-target illuminance. Measurements were made by modulating either a 650-nm light superimposed on a 480-nm background or the red phosphor of a color monitor on a background produced by the monitor's blue phosphor. RESULTS: RPE65-mutant observers have severely reduced cffs with shallower cff versus log illuminance functions that rise with a mean slope of 4.53 Hz per decade of illuminance compared with 8.69 Hz in normal controls. Consistent with the cff differences, RPE65-mutant observers show losses in temporal contrast sensitivity that increase rapidly with temporal frequency. CONCLUSIONS: All RPE65-mutant observers have consistent and substantial losses in temporal acuity and sensitivity compared with normal observers. The losses can be characterized by the addition of two sluggish filters within the mutant visual pathway, both filters with a time constant of 29.5 ms (i.e., low-pass filters with cut-off frequencies of 5.40 Hz).

Novel compound heterozygous NMNAT1 variants associated with Leber congenital amaurosis.

PURPOSE: The gene encoding nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) was recently found to be mutated in a subset of patients with Leber congenital amaurosis (LCA) with macular atrophy. The aim of this study was to determine the occurrence and frequency of NMNAT1 mutations and associated phenotypes in different types of inherited retinal dystrophies. METHODS: DNA samples of 161 patients with LCA without genetic diagnosis were analyzed for variants in NMNAT1 using Sanger sequencing. Variants in exon 5 of NMNAT1, which harbors the majority of the previously identified mutations, were screened in 532 additional patients with retinal dystrophies. This cohort encompassed 108 persons with isolated or autosomal recessive cone-rod dystrophy (CRD), 271 with isolated or autosomal recessive retinitis pigmentosa (RP), and 49 with autosomal dominant RP, as well as 104 persons with LCA in whom the causative mutation was previously identified. RESULTS: Compound heterozygous alterations were found in six patients with LCA and in one person with early-onset RP. All except one carried the common p.E257K variant on one allele. Macular atrophy was absent in one patient, who carried this variant in combination with a truncating mutation on the other allele. The p.E257K alteration was also found in a heterozygous state in five individuals with LCA and one with RP while no mutation was detected on the other allele. Two individuals with LCA carried other NMNAT1 variants in a heterozygous state, whereas no NMNAT1 variants in exon 5 were identified in individuals with CRD. The p.E257K variant was found to be enriched in a heterozygous state in individuals with LCA (0.94%) compared to Caucasian controls (0.18%), although the difference was statistically insignificant (p=0.12). CONCLUSIONS: Although macular atrophy can occur in LCA and CRD, no NMNAT1 mutations were found in the latter cohort. NMNAT1 variants were also not found in a large group of patients with sporadic or autosomal recessive RP. The enrichment of p.E257K in a heterozygous state in patients with LCA versus controls suggests that this allele could act as a modifier in other genetic subtypes of LCA.

AIPL1, A protein linked to blindness, is essential for the stability of enzymes mediating cGMP metabolism in cone photoreceptor cells.

Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor interacting protein like-1 (AIPL1) are linked to blinding diseases, including Leber congenital amaurosis (LCA) and cone dystrophy. While it is apparent that AIPL1 is needed for rod and cone function, the role of AIPL1 in cones is not clear. In this study, using an all-cone animal model lacking Aipl1, we show a light-independent degeneration of M- and S-opsin containing cones that proceeds in a ventral-to-dorsal gradient. Aipl1 is needed for stability, assembly and membrane association of cone PDE6, an enzyme crucial for photoreceptor function and survival. Furthermore, RetGC1, a protein linked to LCA that is needed for cGMP synthesis, was dramatically reduced in cones lacking Aipl1. A defect in RetGC1 is supported by our finding that cones lacking Aipl1 exhibited reduced levels of cGMP. These findings are in contrast to the role of Aipl1 in rods, where destabilization of rod PDE6 results in an increase in cGMP levels, which drives rapid rod degeneration. Our results illustrate mechanistic differences behind the death of rods and cones in retinal degenerative disease caused by deficiencies in AIPL1.

Intervisit variability of visual parameters in Leber congenital amaurosis caused by RPE65 mutations.

PURPOSE: To determine the intervisit variability of kinetic visual fields and visual acuity in patients with Leber congenital amaurosis (LCA) caused by mutations in the RPE65 (Retinal Pigment Epithelium-specific protein 65kDa) gene. METHODS: RPE65-LCA patients (n = 20; ages 11-40 years) were studied on at least two visits separated by fewer than 120 days using Goldmann visual field (GVF) and ETDRS visual acuity (VA) in a retrospective review. GVFs were quantified by computing the spherical coordinates of their vertices and calculating the solid angle subtended, and reported in normalized solid-angle units (nsu) as a percentage of average normal field extent. Repeatability coefficients were calculated using 95% confidence intervals on log(10)-converted variables. RESULTS: Visual field extents in RPE65-LCA spanned a wide range from 4 to 95 nsu. The repeatability coefficient was 0.248 (log(10)nsu), suggesting cutoffs for significant change (in nsu) of +77% for improvement and -44% for worsening. VA in RPE65-LCA ranged from logMAR = 0.14 to 1.96 (20/40 to 20/1250). The repeatability coefficient was 0.170 (logMAR) (±8.5 ETDRS letters). Comparisons with published studies of ungenotyped retinitis pigmentosa showed that the RPE65-LCA patients had higher variability in kinetic field extent. VA variability in RPE65-LCA fell within reported results for retinitis pigmentosa. CONCLUSIONS: Variability data for GVF and VA are provided to permit interpretation of the significance of increases and decreases of these functional outcomes in ongoing and planned clinical trials of therapy for LCA caused by RPE65 mutations.

Determining consequences of retinal membrane guanylyl cyclase (RetGC1) deficiency in human Leber congenital amaurosis en route to therapy: residual cone-photoreceptor vision correlates with biochemical properties of the mutants.

The GUCY2D gene encodes retinal membrane guanylyl cyclase (RetGC1), a key component of the phototransduction machinery in photoreceptors. Mutations in GUCY2D cause Leber congenital amaurosis type 1 (LCA1), an autosomal recessive human retinal blinding disease. The effects of RetGC1 deficiency on human rod and cone photoreceptor structure and function are currently unknown. To move LCA1 closer to clinical trials, we characterized a cohort of patients (ages 6 months-37 years) with GUCY2D mutations. In vivo analyses of retinal architecture indicated intact rod photoreceptors in all patients but abnormalities in foveal cones. By functional phenotype, there were patients with and those without detectable cone vision. Rod vision could be retained and did not correlate with the extent of cone vision or age. In patients without cone vision, rod vision functioned unsaturated under bright ambient illumination. In vitro analyses of the mutant alleles showed that in addition to the major truncation of the essential catalytic domain in RetGC1, some missense mutations in LCA1 patients result in a severe loss of function by inactivating its catalytic activity and/or ability to interact with the activator proteins, GCAPs. The differences in rod sensitivities among patients were not explained by the biochemical properties of the mutants. However, the RetGC1 mutant alleles with remaining biochemical activity in vitro were associated with retained cone vision in vivo. We postulate a relationship between the level of RetGC1 activity and the degree of cone vision abnormality, and argue for cone function being the efficacy outcome in clinical trials of gene augmentation therapy in LCA1.

The ciliopathy-associated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase.

Recent studies have established ciliary dysfunction as the underlying cause of a broad range of multi-organ phenotypes, known as 'ciliopathies'. Ciliopathy-associated proteins have a common site of action in the cilium, however, their overall importance for ciliary function differs, as implied by the extreme variability in ciliopathy phenotypes. The aim of this study was to gain more insight in the function of two ciliopathy-associated protein homologs, RPGR interacting protein 1 (RPGRIP1) and RPGRIP1-like protein (RPGRIP1L). Mutations in RPGRIP1 lead to the eye-restricted disease Leber congenital amaurosis, while mutations in RPGRIP1L are causative for Joubert and Meckel syndrome, which affect multiple organs and are at the severe end of the ciliopathy spectrum. Using tandem affinity purification in combination with mass spectrometry, we identified Nek4 serine/threonine kinase as a prominent component of both the RPGRIP1- as well as the RPGRIP1L-associated protein complex. In ciliated cells, this kinase localized to basal bodies, while in ciliated organs, the kinase was predominantly detected at the ciliary rootlet. Down-regulation of NEK4 in ciliated cells led to a significant decrease in cilium assembly, pointing to a role for Nek4 in cilium dynamics. We now hypothesize that RPGRIP1 and RPGRIP1L function as cilium-specific scaffolds that recruit a Nek4 signaling network which regulates cilium stability. Our data are in line with previously established roles in the cilium of other members of the Nek protein family and define NEK4 as a ciliopathy candidate gene.

Long-term preservation of cones and improvement in visual function following gene therapy in a mouse model of leber congenital amaurosis caused by guanylate cyclase-1 deficiency.

Leber congenital amaurosis (LCA) is a severe retinal dystrophy manifesting from early infancy as poor vision or blindness. Loss-of-function mutations in GUCY2D cause LCA1 and are one of the most common causes of LCA, accounting for 20% of all cases. Human GUCY2D and mouse Gucy2e genes encode guanylate cyclase-1 (GC1), which is responsible for restoring the dark state in photoreceptors after light exposure. The Gucy2e(-/-) mouse shows partially diminished rod function, but an absence of cone function before degeneration. Although the cones appear morphologically normal, they exhibit mislocalization of proteins involved in phototransduction. In this study we tested the efficacy of an rAAV2/8 vector containing the human rhodopsin kinase promoter and the human GUCY2D gene. Following subretinal delivery of the vector in Gucy2e(-/-) mice, GC1 protein was detected in the rod and cone outer segments, and in transduced areas of retina cone transducin was appropriately localized to cone outer segments. Moreover, we observed a dose-dependent restoration of rod and cone function and an improvement in visual behavior of the treated mice. Most importantly, cone preservation was observed in transduced areas up to 6 months post injection. To date, this is the most effective rescue of the Gucy2e(-/-) mouse model of LCA and we propose that a vector, similar to the one used in this study, could be suitable for use in a clinical trial of gene therapy for LCA1.

Novel mutations in MERTK associated with childhood onset rod-cone dystrophy.

PURPOSE: To report the clinical phenotype in patients with a retinal dystrophy associated with novel mutations in the MER tyrosine kinase (MERTK) gene. METHODS: A consanguineous family of Middle Eastern origin was identified, and affected members underwent a full clinical evaluation. Linkage analysis was performed using the Affymetrix 50K chip. Regions of homozygosity were identified. The positional candidate genes protocadherin 21 (PCDH21), retinal G protein-coupled receptor (RGR), and MERTK were polymerase chain reaction (PCR) amplified and sequenced. Long-range PCR was performed to characterize the deletion. Two hundred and ninety-two probands with autosomal recessive, childhood onset, retinal dystrophies were analyzed using the Asper Ophthalmics Leber congenital amaurosis chip to screen for known MERTK mutations. RESULTS: Analysis of a 50K-Affymetrix whole genome scan identified three regions of homozygosity on chromosomes 2 and 10. Screening of the candidate gene MERTK showed a possible deletion of exon 8. Long-range PCR identified a ~9 kb deletion within MERTK that removes exon 8. Screening of DNA from a panel of Saudi Arabian patients with autosomal recessive retinitis pigmentosa identified a second consanguineous family with the same mutation. One patient with a known MERTK mutation (p.R651X) was identified using the Asper Ophthalmics Leber congenital amaurosis chip. Further screening of the gene identified a second novel splice site mutation in intron 1. The phenotype associated with these identified MERTK mutations is of a childhood onset rod-cone dystrophy with early macular atrophy. The optical coherence tomography (OCT) appearance is distinctive with evidence of debris beneath the sensory retina. CONCLUSIONS: Mutations in MERTK are a rare cause of retinal dystrophy. Non homologous recombination between Alu Y repeats near or within disease genes may be an important cause of retinal dystrophies.

The Leber congenital amaurosis protein, AIPL1, is needed for the viability and functioning of cone photoreceptor cells.

Leber congenital amaurosis (LCA) caused by mutations in Aryl hydrocarbon receptor interacting protein like-1 (Aipl1) is a severe form of childhood blindness. At 4 weeks of age, a mouse model of LCA lacking AIPL1 exhibits complete degeneration of both rod and cone photoreceptors. Rod cell death occurs due to rapid destabilization of rod phosphodiesterase, an enzyme essential for rod survival and function. However, little is understood regarding the role of AIPL1 in cone photoreceptors. Cone degeneration observed in the absence of AIPL1 could be due to an indirect 'bystander effect' caused by rod photoreceptor death or a direct role for AIPL1 in cones. To understand the importance of AIPL1 in cone photoreceptor cells, we transgenically expressed hAIPL1 exclusively in the rod photoreceptors of the Aipl1(-/-) mouse. Transgenic expression of hAIPL1 restored rod morphology and the rod-derived electroretinogram response, but cone photoreceptors were non-functional in the absence of AIPL1. In addition, the cone photoreceptors degenerate, but at a slower rate compared with Aipl1(-/-) mice. This degeneration is linked to the highly reduced levels of cone PDE6 observed in the hAIPL1 transgenic mice. Our studies demonstrate that AIPL1 is needed for the proper functioning and survival of cone photoreceptors. However, rod photoreceptors also provide support that partially preserves cone photoreceptors from rapid death in the absence of AIPL1.

Negative charge of the glutamic acid 417 residue is crucial for isomerohydrolase activity of RPE65.

RPE65 is the isomerohydrolase essential for regeneration of 11-cis retinal, the chromophore of visual pigments. Here we compared the impacts of two mutations in RPE65, E417Q identified in patients with Leber congenital amaurosis (LCA), and E417D on isomerohydrolase activity. Although both mutations decreased the stability of RPE65 and altered its sub-cellular localization, E417Q abolished isomerohydrolase activity whereas the E417D mutant retained partial enzymatic activity suggesting that the negative charge of E417 is important for RPE65 catalytic activity. Loss of charge at this position may represent a mechanism by which the E417Q mutation causes blindness in LCA patients.

Activation of retinal guanylyl cyclase RetGC1 by GCAP1: stoichiometry of binding and effect of new LCA-related mutations.

Retinal membrane guanylyl cyclase (RetGC) and Ca(2+)/Mg(2+) sensor proteins (GCAPs) control the recovery of the photoresponse in vertebrate photoreceptors, through their molecular interactions that remain rather poorly understood and controversial. Here we have determined the main RetGC isozyme (RetGC1):GCAP1 binding stoichiometry at saturation in cyto, using fluorescently labeled RetGC1 and GCAP1 coexpressed in HEK293 cells. In a striking manner, the equimolar binding of RetGC1 with GCAP1 in transfected HEK293 cells typical for wild-type RetGC1 was eliminated by a substitution, D639Y, in the kinase homology domain of RetGC1 found in a patient with a severe form of retinal dystrophy, Leber congenital amaurosis (LCA). A similar effect was observed with another LCA-related mutation, R768W, in the same domain of RetGC1. In contrast to the completely suppressed binding and activation of RetGC1 by Mg(2+)-liganded GCAP1, neither of these two mutations eliminated the GCAP1-independent activity of RetGC stimulated by Mn(2+). These results directly implicate the D639 (and possibly R768)-containing portion of the RetGC1 kinase homology domain in its primary recognition by the Mg(2+)-bound activator form of GCAP1.