Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290‐related phenotypes

Leber Congenital Amaurosis (LCA), the most severe inherited retinal dystrophy, is genetically heterogeneous, with 14 genes accounting for 70% of patients. Here, 91 LCA probands underwent LCA chip analysis and subsequent sequencing of 6 genes (CEP290, CRB1, RPE65, GUCY2D, AIPL1and CRX), revealing mutations in 69% of the cohort, with major involvement of CEP290 (30%). In addition, 11 patients with early‐onset retinal dystrophy (EORD) and 13 patients with Senior‐Loken syndrome (SLS), LCA‐Joubert syndrome (LCA‐JS) or cerebello‐oculo‐renal syndrome (CORS) were included. Exhaustive re‐inspection of the overall phenotypes in our LCA cohort revealed novel insights mainly regarding the CEP290‐related phenotype. The AHI1 gene was screened as a candidate modifier gene in three patients with the same CEP290 genotype but different neurological involvement. Interestingly, a heterozygous novel AHI1 mutation, p.Asn811Lys, was found in the most severely affected patient. Moreover, AHI1 screening in five other patients with CEP290‐related disease and neurological involvement revealed a second novel missense variant, p.His758Pro, in one LCA patient with mild mental retardation and autism. These two AHI1 mutations might thus represent neurological modifiers of CEP290‐related disease. © 2010 Wiley‐Liss, Inc.     

Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis

Mutations in the centrosomal-ciliary gene CEP290/NPHP6 are associated with Joubert syndrome and are the most common cause of the childhood recessive blindness known as Leber congenital amaurosis (LCA). An in-frame deletion in Cep290 shows rapid degeneration in the rod-rich mouse retina. To explore the mechanisms of the human retinal disease, we studied CEP290-LCA in patients of different ages (7-48 years) and compared results to Cep290-mutant mice. Unexpectedly, blind CEP290-mutant human retinas retained photoreceptor and inner laminar architecture in the cone-rich central retina, independent of severity of visual loss. Surrounding the cone-rich island was photoreceptor loss and distorted retina, suggesting neural-glial remodeling. The mutant mouse retina at 4-6 weeks of age showed similar features of retinal remodeling, with altered neural and synaptic laminae and Muller glial activation. The visual brain pathways in CEP290-LCA were anatomically intact. Our findings of preserved foveal cones and visual brain anatomy in LCA with CEP290 mutations, despite severe blindness and rapid rod cell death, suggest an opportunity for visual restoration of central vision in this common form of inherited blindness.     

CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium

Joubert syndrome (JS) is a developmental brain disorder characterized by cerebellar vermis hypoplasia, abnormal eye movement, ataxia and mental retardation. Mutations in CEP290 mutations are responsible for the cerebello-oculo-renal subtype of JS that includes kidney cysts and retinal degeneration, two phenotypes commonly linked to ciliopathies. CEP290 mutations are also associated with Meckel-Gruber syndrome and Bardet-Biedl syndrome (BBS). Here we demonstrate that CEP290 interacts with a centriolar satellite protein PCM-1, which is implicated in BBS4 function. CEP290 binds to PCM-1 and localizes to centriolar satellites in a PCM-1- and microtubule-dependent manner. The depletion of CEP290 disrupts subcellular distribution and protein complex formation of PCM-1. In accord with PCM-1's role in microtubule organization, CEP290 knockdown causes the disorganization of the cytoplasmic microtubule network. Moreover, we show that both CEP290 and PCM-1 are required for ciliogenesis and are involved in the ciliary targeting of Rab8, a small GTPase shown to collaborate with BBS protein complex to promote ciliogenesis. Our results suggest that PCM-1 is a potential mediator that may link CEP290 with BBS proteins in common molecular pathways.     

Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome

Meckel-Gruber syndrome (MKS) is an autosomal recessive, lethal multisystemic disorder characterized by meningooccipital encephalocele, cystic kidney dysplasia, hepatobiliary ductal plate malformation, and postaxial polydactyly. Recently, genes for MKS1 and MKS3 were identified, putting MKS on the list of ciliary disorders (ciliopathies). By positional cloning in a distantly related multiplex family, we mapped a novel locus for MKS to a 3-Mb interval on 12q21. Sequencing of the CEP290 gene located in the minimal critical region showed a homozygous 1-bp deletion supposed to lead to loss of function of the encoded centrosomal protein CEP290/nephrocystin-6. CEP290 is thought to be involved in chromosome segregation and localizes to cilia, centrosomes, and the nucleus. Subsequent analysis of another consanguineous multiplex family revealed homozygous haplotypes and the same frameshift mutation. Our findings add to the increasing body of evidence that ciliopathies can cause a broad spectrum of disease phenotypes, and pleiotropic effects of CEP290 mutations range from single organ involvement with isolated Leber congenital amaurosis to Joubert syndrome and lethal early embryonic multisystemic malformations in Meckel-Gruber syndrome. We compiled clinical and genetic data of all patients with CEP290 mutations described so far. No clear-cut genotype-phenotype correlations were apparent as almost all mutations are nonsense, frameshift, or splice-site changes and scattered throughout the gene irrespective of the patients' phenotypes. Conclusively, other factors than the type and location of CEP290 mutations may underlie phenotypic variability.     

E-cadherin regulates cell movements and tissue formation in early zebrafish embryos.

E-cadherin is maternally expressed in most vertebrate species, but its function during early development of the vertebrate embryo proper is unknown. To directly examine E-cadherin gene (cdh1) function in zebrafish, morpholino oligonucleotides (MOs) that inhibit E-cadherin protein (Cdh1) expression were injected into embryos. Cdh1 knockdown reduced embryo survival. In early cdh1 MO-injected embryos, the cleavage plane orientation between blastomeres was irregular and adhesion defects prevented normal compaction. Cdh1 knockdown inhibited epiboly cell movements. Epiboly delay caused yolk cell lysis and produced embryos with a bifurcated embryonic axis. Cdh1 knockdown inhibited gastrulation cell movements, causing defects in convergence and extension. Additionally, prechordal plate derivatives were absent in Cdh1 knockdown embryos even though presumptive prechordal plate markers were induced normally. E-cadherin mRNA coinjection demonstrated the specificity of cdh1 MO-induced defects. Our experiments illustrate the importance of cdh1 in regulating morphogenetic cell movements and tissue formation in the early embryo.     

Gucy2f zebrafish knockdown--a model for Gucy2d-related leber congenital amaurosis

Mutations in retinal-specific guanylate cyclase (Gucy2d) are associated with Leber congenital amaurosis-1 (LCA1). Zebrafish offer unique advantages relative to rodents, including their excellent color vision, precocious retinal development, robust visual testing strategies, low cost, relatively easy transgenesis and shortened experimental times. In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific GUCY2D-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f knockdown. Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. Its expression pattern was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown using two different morpholino-modified oligos (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay on 6-days-post-fertilization larvae, and by analyzing changes in retinal histology. Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared with uninjected and control MO-injected zebrafish larvae. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments. A zebrafish model of Gucy2f-related LCA1 displays early visual dysfunction and photoreceptor layer dystrophy. This study serves as proof of concept for the use of zebrafish as a simple, inexpensive model with excellent vision on which further study of LCA-related genes is possible.     

Involvement of the centrosomal protein 55 (cep55) gene in zebrafish head formation

Mammalian CEP55 (centrosomal protein 55 kDa) is a coiled‐coil protein localized to the centrosome in interphase cells and is required for cytokinesis. A homozygous non‐sense mutation in human CEP55 has been recently identified in perinatal lethal MARCH (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly) syndrome. We have isolated zebrafish cep55 mutants defective in head morphology. The zebrafish cep55 gene was expressed in the head including the retina and the pectoral fin at 1 day post‐fertilization (dpf), and extensive cell death was widely observed in the head and tail of the cep55 mutant. In the cep55 mutant, the anterior–posterior distance of the ventral pharyngeal arches was short, and retinal lamination was disorganized. Neural cells, such as islet1‐positive cells and pax2‐positive cells, and fli1b‐positive vascular cells were reduced in the head of the cep55 mutant. Thus, we propose that the zebrafish cep55 mutant is a model organism for human MARCH syndrome.     

In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse

Centrosome- and cilia-associated proteins play crucial roles in establishing polarity and regulating intracellular transport in post-mitotic cells. Using genetic mapping and positional candidate strategy, we have identified an in-frame deletion in a novel centrosomal protein CEP290 (also called NPHP6), leading to early-onset retinal degeneration in a newly identified mouse mutant, rd16. We demonstrate that CEP290 localizes primarily to centrosomes of dividing cells and to the connecting cilium of retinal photoreceptors. We show that, in the retina, CEP290 associates with several microtubule-based transport proteins including RPGR, which is mutated in approximately 15% of patients with retinitis pigmentosa. A truncated CEP290 protein (DeltaCEP290) is detected in the rd16 retina, but in considerably reduced amounts; however, the mutant protein exhibits stronger association with specific RPGR isoform(s). Immunogold labeling studies demonstrate the redistribution of RPGR and of phototransduction proteins in the photoreceptors of rd16 retina. Our findings suggest a critical function for CEP290 in ciliary transport and provide insights into the mechanism of early-onset photoreceptor degeneration.     

Cone photoreceptors are the main targets for gene therapy of NPHP5 (IQCB1) or NPHP6 (CEP290) blindness: generation of an all-cone Nphp6 hypomorph mouse that mimics the human retinal ciliopathy

Leber congenital amaurosis (LCA), a severe autosomal recessive childhood blindness, is caused by mutations in at least 15 genes. The most common molecular form is a ciliopathy due to NPHP6 (CEP290) mutations and subjects have profound loss of vision. A similarly severe phenotype occurs in the related ciliopathy NPHP5 (IQCB1)-LCA. Recent success of retinal gene therapy in one form of LCA prompted the question whether we know enough about human NPHP5 and NPHP6 disease to plan such treatment. We determined that there was early-onset rapid degeneration of rod photoreceptors in young subjects with these ciliopathies. Rod outer segment (OS) lamination, when detectable, was disorganized. Retinal pigment epithelium lipofuscin accumulation indicated that rods had existed in the past in most subjects. In contrast to early rod losses, the all-cone human fovea in NPHP5- and NPHP6-LCA of all ages retained cone nuclei, albeit with abnormal inner segments and OS. The rd16 mouse, carrying a hypomorphic Nphp6 allele, was a good model of the rod-dominant human extra-foveal retina. Rd16 mice showed normal genesis of photoreceptors, including the formation of cilia, followed by abnormal elaboration of OS and rapid degeneration. To produce a model of the all-cone human fovea in NPHP6-LCA, we generated rd16;Nrl-/- double-mutant mice. They showed substantially retained cone photoreceptors with disproportionate cone function loss, such as in the human disease. NPHP5- and NPHP6-LCA across a wide age spectrum are thus excellent candidates for cone-directed gene augmentation therapy, and the rd16;Nrl-/- mouse is an appropriate model for pre-clinical proof-of-concept studies.     

Impairment of the tRNA-splicing endonuclease subunit 54 (tsen54) gene causes neurological abnormalities and larval death in zebrafish models of pontocerebellar hypoplasia

Pontocerebellar hypoplasia (PCH) represents a group (PCH1-6) of neurodegenerative autosomal recessive disorders characterized by hypoplasia and/or atrophy of the cerebellum, hypoplasia of the ventral pons, progressive microcephaly and variable neocortical atrophy. The majority of PCH2 and PCH4 cases are caused by mutations in the TSEN54 gene; one of the four subunits comprising the tRNA-splicing endonuclease (TSEN) complex. We hypothesized that TSEN54 mutations act through a loss of function mechanism. At 8 weeks of gestation, human TSEN54 is expressed ubiquitously in the brain, yet strong expression is seen within the telencephalon and metencephalon. Comparable expression patterns for tsen54 are observed in zebrafish embryos. Morpholino (MO) knockdown of tsen54 in zebrafish embryos results in loss of structural definition in the brain. This phenotype was partially rescued by co-injecting the MO with human TSEN54 mRNA. A developmental patterning defect was not associated with tsen54 knockdown; however, an increase in cell death within the brain was observed, thus bearing resemblance to PCH pathophysiology. Additionally, N-methyl-N-nitrosourea mutant zebrafish homozygous for a tsen54 premature stop-codon mutation die within 9 days post-fertilization. To determine whether a common disease pathway exists between TSEN54 and other PCH-related genes, we also monitored the effects of mitochondrial arginyl-tRNA synthetase (rars2; PCH1?and PCH6) knockdown in zebrafish. Comparable brain phenotypes were observed following the inhibition of both genes. These data strongly support the hypothesis that TSEN54 mutations cause PCH through a loss of function mechanism. Also we suggest that a common disease pathway may exist between TSEN54- and RARS2-related PCH, which may involve a tRNA processing-related mechanism.     

OCRL localizes to the primary cilium: a new role for cilia in Lowe syndrome

Oculocerebral renal syndrome of Lowe (OCRL or Lowe syndrome), a severe X-linked congenital disorder characterized by congenital cataracts and glaucoma, mental retardation and kidney dysfunction, is caused by mutations in the OCRL gene. OCRL is a phosphoinositide 5-phosphatase that interacts with small GTPases and is involved in intracellular trafficking. Despite extensive studies, it is unclear how OCRL mutations result in a myriad of phenotypes found in Lowe syndrome. Our results show that OCRL localizes to the primary cilium of retinal pigment epithelial cells, fibroblasts and kidney tubular cells. Lowe syndrome-associated mutations in OCRL result in shortened cilia and this phenotype can be rescued by the introduction of wild-type OCRL; in vivo, knockdown of ocrl in zebrafish embryos results in defective cilia formation in Kupffer vesicles and cilia-dependent phenotypes. Cumulatively, our data provide evidence for a role of OCRL in cilia maintenance and suggest the involvement of ciliary dysfunction in the manifestation of Lowe syndrome.     

Sema3E is required for migration of cranial neural crest cells in zebrafish: Implications for the pathogenesis of CHARGE syndrome

CHARGE syndrome is a congenital disorder with multiple malformations in the craniofacial structures, and cardiovascular and genital systems, which are mainly affected by neural crest defects caused by loss‐of‐function mutations within chromodomain helicase DNA‐binding protein 7 (CHD7). However, many patients with CHARGE syndrome test negative for CHD7. Semaphorin 3E (sema3E) is a gene reported to be mutated in patients with CHARGE syndrome. However, its role in the pathogenesis of CHARGE syndrome has not been verified experimentally. Here, we report that the knockdown of sema3E results in severe craniofacial malformations, including small eyes, defective cartilage and an abnormal number of otoliths in zebrafish embryos, which resemble the major features of CHARGE syndrome. Further analysis reveals that the migratory cranial neural crest cells are scattered in the region of the hindbrain, and the postmigratory neural crest cells are reduced in the pharyngeal arches upon sema3E knockdown. Notably, immunostaining and time‐lapse imaging analyses of a neural crest cell‐labelled transgenic fish line, sox10:EGFP, show that the migration of cranial neural crest cells is severely impaired, and many of these cells are misrouted upon sema3E knockdown. Furthermore, the sox10‐expressing cranial neural crest cells are scattered in chd7 homozygous mutants, which phenocopied the phenotype in sema3E morphants. Overexpression of sema3E rescues the phenotype of scattered cranial neural crest cells in chd7 homozygotes, indicating that chd7 may control the expression of sema3E to regulate cranial neural crest cell migration. Collectively, our data demonstrate that sema3E is involved in the pathogenesis of CHARGE syndrome by modulating cranial neural crest cell migration.     

Genetic and physical interaction between the NPHP5 and NPHP6 gene products

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.     

A meckelin-filamin A interaction mediates ciliogenesis

MKS3, encoding the transmembrane receptor meckelin, is mutated in Meckel-Gruber syndrome (MKS), an autosomal-recessive ciliopathy. Meckelin localizes to the primary cilium, basal body and elsewhere within the cell. Here, we found that the cytoplasmic domain of meckelin directly interacts with the actin-binding protein filamin A, potentially at the apical cell surface associated with the basal body. Mutations in FLNA, the gene for filamin A, cause periventricular heterotopias. We identified a single consanguineous patient with an MKS-like ciliopathy that presented with both MKS and cerebellar heterotopia, caused by an unusual in-frame deletion mutation in the meckelin C-terminus at the region of interaction with filamin A. We modelled this mutation and found it to abrogate the meckelin-filamin A interaction. Furthermore, we found that loss of filamin A by siRNA knockdown, in patient cells, and in tissues from Flna(Dilp2) null mouse embryos results in cellular phenotypes identical to those caused by meckelin loss, namely basal body positioning and ciliogenesis defects. In addition, morpholino knockdown of flna in zebrafish embryos significantly increases the frequency of dysmorphology and severity of ciliopathy developmental defects caused by mks3 knockdown. Our results suggest that meckelin forms a functional complex with filamin A that is disrupted in MKS and causes defects in neuronal migration and Wnt signalling. Furthermore, filamin A has a crucial role in the normal processes of ciliogenesis and basal body positioning. Concurrent with these processes, the meckelin-filamin A signalling axis may be a key regulator in maintaining correct, normal levels of Wnt signalling.     

Safety and efficacy of gene transfer for Leber's congenital amaurosis

Leber's congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477 [ClinicalTrials.gov]). Three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study provides the basis for further gene therapy studies in patients with LCA.     

Expression of VE-cadherin in zebrafish embryos: a new tool to evaluate vascular development.

We have identified the zebrafish homologue of VE-cadherin and documented its expression in the developing vascular system. The zebrafish VE-cadherin gene is specifically expressed in the vascular endothelial cell lineage beginning with the differentiation and migration of angioblasts and persists throughout vasculogenesis, angiogenesis, and endocardium development. Staining zebrafish embryos by whole-mount in situ hybridization with the VE-cadherin probe provides a method to screen embryos for vascular defects. To illustrate this utility, we used VE-cadherin expression to demonstrate a conservation of vascular endothelial growth factor-A (VEGF-A) function. The morpholino antisense oligonucleotide knockdown of VEGF-A function in zebrafish embryos results in a loss of angiogenic blood vessels, as indicated by the lack of VE-cadherin expression in the intersegmental vasculature. This loss can be restored in embryos supplemented with either zebrafish or human VEGF-A, the latter indicating that genes crucial to angiogenesis have highly conserved functional activities in vertebrates.     

Hypomorphic mutations identified in the candidate Leber congenital amaurosis gene CLUAP1

PurposeLeber congenital amaurosis (LCA) is an early-onset form of retinal degeneration. Six of the 22 known LCA genes encode photoreceptor ciliary proteins. Despite the identification of 22 LCA genes, the genetic basis of ~30% of LCA patients remains unknown. We sought to investigate the cause of disease in the remaining 30% by examining cilia-associated genes.MethodsWhole-exome sequencing was performed on an LCA cohort of 212 unsolved probands previously screened for mutations in known retinal-disease genes. Immunohistochemistry using mouse retinas was used to confirm protein localization and zebrafish were used to perform rescue experiments.ResultsA homozygous nonsynonymous mutation was found in a single proband in CLUAP1, a gene required for ciliogenesis and cilia maintenance. Cluap1 knockout zebrafish exhibit photoreceptor cell death as early as 5 days after fertilization, and rescue experiments revealed that our proband’s mutation is significantly hypomorphic.ConclusionConsistent with the knowledge that CLUAP1 plays an important role in cilia function and that cilia are critical to photoreceptor function, our results indicate that hypomorphic mutations in CLUAP1 can result in dysfunctional photoreceptors without systemic abnormalities. This is the first report linking mutations in CLUAP1 to human disease and establishes CLUAP1 as a candidate LCA gene.Genet Med18 10, 1044–1051.