Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.     

Retinopathy and attenuated circadian entrainment in Crx-deficient mice

Crx, an Otx-like homeobox gene, is expressed specifically in the photoreceptors of the retina and the pinealocytes of the pineal gland. Crx has been proposed to have a role in the regulation of photoreceptor-specific genes in the eye and of pineal-specific genes in the pineal gland. Mutations in human CRX are associated with the retinal diseases, cone-rod dystrophy-2 (adCRD2; refs 3, 4, 5), retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), which all lead to loss of vision. We generated mice carrying a targeted disruption of Crx. Crx-/- mice do not elaborate photoreceptor outer segments and lacked rod and cone activity as assayed by electroretinogram (ERG). Expression of several photoreceptor- and pineal-specific genes was reduced in Crx mutants. Circadian entrainment was also affected in Crx-/- mice.     

Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53

The relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (RPE) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. The molecular bases for both primary photoreceptor and RPE diseases that cause blindness have been identified. Gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and RPE in a large-animal model of human disease has not been reported. Here we study one of the most clinically severe retinal degenerations, Leber congenital amaurosis (LCA). LCA causes near total blindness in infancy and can result from mutations in RPE65 (LCA, type II; MIM 180069 and 204100). A naturally occurring animal model, the RPE65-/- dog, suffers from early and severe visual impairment similar to that seen in human LCA. We used a recombinant adeno-associated virus (AAV) carrying wild-type RPE65 (AAV-RPE65) to test the efficacy of gene therapy in this model. Our results indicate that visual function was restored in this large animal model of childhood blindness.     

Identification and characterization of rod-derived cone viability factor

Retinitis pigmentosa is an untreatable, inherited retinal disease that leads to blindness. The disease initiates with the loss of night vision due to rod photoreceptor degeneration, followed by irreversible, progressive loss of cone photoreceptor. Cone loss is responsible for the main visual handicap, as cones are essential for day and high-acuity vision. Their loss is indirect, as most genes associated with retinitis pigmentosa are not expressed by these cells. We previously showed that factors secreted from rods are essential for cone viability. Here we identified one such trophic factor by expression cloning and named it rod-derived cone viability factor (RdCVF). RdCVF is a truncated thioredoxin-like protein specifically expressed by photoreceptors. The identification of this protein offers new treatment possibilities for retinitis pigmentosa.     

Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12).

Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of diseases that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. To isolate candidate genes for chorioretinal diseases, we cloned cDNAs specifically or preferentially expressed in the human retina and the retinal pigment epithelium (RPE) through a novel suppression subtractive hybridization (SSH) method. One of these cDNAs (RET3C11) mapped to chromosome 1q31-q32.1, a region harbouring a gene involved in a severe form of autosomal recessive RP characterized by a typical preservation of the para-arteriolar RPE (RP12; ref. 3). The full-length cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A G-like domains and a C-type lectin domain. This protein is homologous to the Drosophila melanogaster protein crumbs (CRB), and denoted CRB1 (crumbs homologue 1). In ten unrelated RP patients with preserved para-arteriolar RPE, we identified a homozygous AluY insertion disrupting the ORF, five homozygous missense mutations and four compound heterozygous mutations in CRB1. The similarity to CRB suggests a role for CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration.     

New views on RPE65 deficiency: the rod system is the source of vision in a mouse model of Leber congenital amaurosis

Leber congenital amaurosis (LCA) is the most serious form of the autosomal recessive childhood-onset retinal dystrophies. Mutations in the gene encoding RPE65, a protein vital for regeneration of the visual pigment rhodopsin in the retinal pigment epithelium, account for 10-15% of LCA cases. Whereas previous studies of RPE65 deficiency in both animal models and patients attributed remaining visual function to cones, we show here that light-evoked retinal responses in fact originate from rods. For this purpose, we selectively impaired either rod or cone function in Rpe65-/- mice by generating double- mutant mice with models of pure cone function (rhodopsin-deficient mice; Rho-/-) and pure rod function (cyclic nucleotide-gated channel alpha3-deficient mice; Cnga3-/-). The electroretinograms (ERGs) of Rpe65-/- and Rpe65-/-Cnga3-/- mice were almost identical, whereas there was no assessable response in Rpe65-/-Rho-/- mice. Thus, we conclude that the rod system is the source of vision in RPE65 deficiency. Furthermore, we found that lack of RPE65 enables rods to mimic cone function by responding under normally cone-isolating lighting conditions. We propose as a mechanism decreased rod sensitivity due to a reduction in rhodopsin content to less than 1%. In general, the dissection of pathophysiological processes in animal models through the introduction of additional, selective mutations is a promising concept in functional genetics.     

Mutation of the gene encoding the ROR2 tyrosine kinase causes autosomal recessive Robinow syndrome

Robinow syndrome is a short-limbed dwarfism characterized by abnormal morphogenesis of the face and external genitalia, and vertebral segmentation. The recessive form of Robinow syndrome (RRS; OMIM 268310), particularly frequent in Turkey, has a high incidence of abnormalities of the vertebral column such as hemivertebrae and rib fusions, which is not seen in the dominant form. Some patients have cardiac malformations or facial clefting. We have mapped a gene for RRS to 9q21-q23 in 11 families. Haplotype sharing was observed between three families from Turkey, which localized the gene to a 4. 9-cM interval. The gene ROR2, which encodes an orphan membrane-bound tyrosine kinase, maps to this region. Heterozygous (presumed gain of function) mutations in ROR2 were previously shown to cause dominant brachydactyly type B (BDB; ref. 7). In contrast, Ror2-/- mice have a short-limbed phenotype that is more reminiscent of the mesomelic shortening observed in RRS. We detected several homozygous ROR2 mutations in our cohort of RRS patients that are located upstream from those previously found in BDB. The ROR2 mutations present in RRS result in premature stop codons and predict nonfunctional proteins.     

Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin

Nephronophthisis (NPHP) is the most frequent genetic cause of chronic renal failure in children. Identification of four genes mutated in NPHP subtypes 1-4 (refs. 4-9) has linked the pathogenesis of NPHP to ciliary functions. Ten percent of affected individuals have retinitis pigmentosa, constituting the renal-retinal Senior-Loken syndrome (SLSN). Here we identify, by positional cloning, mutations in an evolutionarily conserved gene, IQCB1 (also called NPHP5), as the most frequent cause of SLSN. IQCB1 encodes an IQ-domain protein, nephrocystin-5. All individuals with IQCB1 mutations have retinitis pigmentosa. Hence, we examined the interaction of nephrocystin-5 with RPGR (retinitis pigmentosa GTPase regulator), which is expressed in photoreceptor cilia and associated with 10-20% of retinitis pigmentosa. We show that nephrocystin-5, RPGR and calmodulin can be coimmunoprecipitated from retinal extracts, and that these proteins localize to connecting cilia of photoreceptors and to primary cilia of renal epithelial cells. Our studies emphasize the central role of ciliary dysfunction in the pathogenesis of SLSN.     

ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy

Mutations in the gene encoding ABCR are responsible for Stargardt macular dystrophy. Here we show by immunofluorescence microscopy and western-blot analysis that ABCR is present in foveal and peripheral cone, as well as rod, photoreceptors. Our results suggest that the loss in central vision experienced by Stargardt patients arises directly from ABCR-mediated foveal cone degeneration.     

Mutations in NMNAT1 cause Leber congenital amaurosis with early-onset severe macular and optic atrophy

In addition to its activity in nicotinamide adenine dinucleotide (NAD(+)) synthesis, the nuclear nicotinamide mononucleotide adenyltransferase NMNAT1 acts as a chaperone that protects against neuronal activity-induced degeneration. Here we report that compound heterozygous and homozygous NMNAT1 mutations cause severe neonatal neurodegeneration of the central retina and early-onset optic atrophy in 22 unrelated individuals. Their clinical presentation is consistent with Leber congenital amaurosis and suggests that the mutations affect neuroprotection of photoreceptor cells.     

Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans

Remodeling of the cytoskeleton is central to the modulation of cell shape and migration. Filamin A, encoded by the gene FLNA, is a widely expressed protein that regulates re-organization of the actin cytoskeleton by interacting with integrins, transmembrane receptor complexes and second messengers. We identified localized mutations in FLNA that conserve the reading frame and lead to a broad range of congenital malformations, affecting craniofacial structures, skeleton, brain, viscera and urogenital tract, in four X-linked human disorders: otopalatodigital syndrome types 1 (OPD1; OMIM 311300) and 2 (OPD2; OMIM 304120), frontometaphyseal dysplasia (FMD; OMIM 305620) and Melnick-Needles syndrome (MNS; OMIM 309350). Several mutations are recurrent, and all are clustered into four regions of the gene: the actin-binding domain and rod domain repeats 3, 10 and 14/15. Our findings contrast with previous observations that loss of function of FLNA is embryonic lethal in males but manifests in females as a localized neuronal migration disorder, called periventricular nodular heterotopia (PVNH; refs. 3-6). The patterns of mutation, X-chromosome inactivation and phenotypic manifestations in the newly described mutations indicate that they have gain-of-function effects, implicating filamin A in signaling pathways that mediate organogenesis in multiple systems during embryonic development.     

Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa

Mutation of a receptor tyrosine kinase gene, Mertk, in the Royal College of Surgeons (RCS) rat results in defective phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) and retinal degeneration. We screened the human orthologue, MERTK, located at 2q14.1 (ref. 10), in 328 DNA samples from individuals with various retinal dystrophies and found three mutations in three individuals with retinitis pigmentosa (RP). Our findings are the first conclusive evidence implicating the RPE phagocytosis pathway in human retinal disease.     

Genetic basis of total colourblindness among the Pingelapese islanders

Complete achromatopsia is a rare, autosomal recessive disorder characterized by photophobia, low visual acuity, nystagmus and a total inability to distinguish colours. In this disease, cone photoreceptors, the retinal sensory neurons mediating colour vision, seem viable but fail to generate an electrical response to light. Achromatopsia, or rod monochromatism, was first mapped to 2p11-2q12 (MIM 216900; ref. 3), where it is associated with missense mutations in CNGA3 (ref. 4). CNGA3 encodes the alpha-subunit of the cone cyclic nucleotide-gated cation channel, which generates the light-evoked electrical responses of cone photoreceptors. A second locus at 8q21-q22 has been identified among the Pingelapese islanders of Micronesia, who have a high incidence of recessive achromatopsia (MIM 262300). Here we narrow the achromatopsia locus to 1.4 cM and show that Pingelapese achromatopsia segregates with a missense mutation at a highly conserved site in CNGB3, a new gene that encodes the beta-subunit of the cone cyclic nucleotide-gated cation channel. Two independent frameshift deletions establish that achromatopsia is the null phenotype of CNGB3. Combined with earlier findings, our results demonstrate that both alpha- and beta-subunits of the cGMP-gated channel are essential for phototransduction in all three classes of cones.     

Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration

Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with NMNAT1 mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (Wld(s)) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder.     

Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood-onset severe retinal dystrophy

We identified three consanguineous Austrian kindreds with 15 members affected by autosomal recessive childhood-onset severe retinal dystrophy, a genetically heterogeneous group of disorders characterized by degeneration of the photoreceptor cells. A whole-genome scan by microarray analysis of single-nucleotide polymorphisms (ref. 2) identified a founder haplotype and defined a critical interval of 1.53 cM on chromosome 14q23.3-q24.1 that contains the gene associated with this form of retinal dystrophy. RDH12 maps in this region and encodes a retinol dehydrogenase proposed to function in the visual cycle. A homozygous 677A-->G transition (resulting in Y226C) in RDH12 was present in all affected family members studied, as well as in two Austrian individuals with sporadic retinal dystrophy. We identified additional mutations in RDH12 in 3 of 89 non-Austrian individuals with retinal dystrophy: a 5-nucleotide deletion (806delCCCTG) and the transition 565C-->T (resulting in Q189X), each in the homozygous state, and 146C-->T (resulting in T49M) and 184C-->T (resulting in R62X) in compound heterozygosity. When expressed in COS-7 cells, Cys226 and Met49 variants had diminished and aberrant activity, respectively, in interconverting isomers of retinol and retinal. The severe visual impairment of individuals with mutations in RDH12 is in marked contrast to the mild visual deficiency in individuals with fundus albipunctatus caused by mutations in RDH5, encoding another retinal dehydrogenase. Our studies show that RDH12 is associated with retinal dystrophy and encodes an enzyme with a unique, nonredundant role in the photoreceptor cells.     

A null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retinitis pigmentosa.

Mutations within the rhodopsin gene are known to give rise to autosomal dominant retinitis pigmentosa (RP), a common hereditary form of retinal degeneration. We now describe a patient with autosomal recessive RP who is homozygous for a nonsense mutation at codon 249 within exon 4 of the rhodopsin gene. This null mutation, the first gene defect identified in autosomal recessive retinitis pigmentosa, should result in a functionally inactive rhodopsin protein that is missing the sixth and seventh transmembrane domains including the 11-cis-retinal attachment site. We also found a different null mutation carried heterozygously by an unrelated unaffected individual. Heterozygous carriers of either mutation had normal ophthalmologic examinations but their electroretinograms revealed an abnormality in rod photoreceptor function.