Early-onset Severe Retinal Dystrophy Research Articles

Restoration of rod function following gene therapy in patients with mutations in the gene encoding the RPE65 protein required for recycling all-trans-retinal to 11-cis-retinal

Rod visual function can be confirmed unequivocally by demonstrating that visual performance measured as a function of wavelength has a rod spectral sensitivity. We have used this test to assess retinal function in some of the patients with early onset severe retinal dystrophy enrolled in an ongoing phase-I/II dose-escalation clinical trial of gene therapy for RPE65 deficiency. The therapy involves subretinal delivery of a recombinant adeno-associated viral vector expressing RPE65. The particular visual performance task used was the threshold detection of 1-Hz flicker following 40 minutes of dark adaptation. The flickering target was 3.55-deg in diameter presented 10-deg in the superior retina (close to the treatment site). We made spectral sensitivity measurements prior to gene therapy and then at 2-monthly intervals following gene therapy in the last five patients enrolled in the trial (to date). Prior to gene therapy, no clear evidence of rod function was found in the spectral sensitivity measurements. The flicker spectral sensitivity functions in all patients were cone-like and similar to those found during the cone plateau after an intense bleach. After gene therapy, however, substantial improvements in flicker sensitivity were found in the first two patients tested. One patient showed a 1000-fold improvement in dark-adapted flicker sensitivity at 500 nm, 4 months after treatment and the second a 100-fold improvement, 6 months after treatment. In both cases, the spectral sensitivity became clearly rod-like, indicative of restored rod function. To date, the other patients have not shown an improvement using this test. The trial is ongoing.

A Novel Nonsense Mutation inCEP290Induces Exon Skipping and Leads to a Relatively Mild Retinal Phenotype

PURPOSE. To identify the genetic defect in a family with variable retinal phenotypes. The proband had a diagnosis of Leber congenital amaurosis (LCA), whereas her two cousins had an early-onset severe retinal dystrophy (EOSRD) with useful vision. A distant family member had retinitis pigmentosa (RP). METHODS. DNA samples of the affected family members were genotyped with 250 K genome-wide SNP microarrays. Genetic defects were localized by linkage analysis and homozygosity mapping, and candidate genes were analyzed by sequencing. Patients underwent a full ophthalmic examination. RESULTS. Compound heterozygous mutations in CEP290 were identified in the proband and her two cousins: the frequent c.2991+1655A>G founder mutation and a novel nonsense mutation in exon 7 (c.451C>T, p.Arg151X). The proband had nystagmus, hyperopia, a flat electroretinogram (ERG), and decreased visual acuity (20/250) from birth. The two cousins had minimal scotopic ERG responses at the age of 2. In one of these patients, visual acuity had reached a level of 20/32 at age 5, which is high for patients with CEP290 mutations. Analysis of the CEP290 mRNA in affected individuals revealed altered splice forms in which either exon 7 or exons 7 and 8 were skipped. In both mutant cDNA products, the open reading frame was not disrupted. Furthermore, homozygosity mapping and mutation analysis in the distant family member affected by RP revealed a homozygous mutation in MERTK, but no CEP290 mutations. This MERTK mutation was heterozygously present in the most severely affected (LCA) patient, but was absent in the two more mildly affected cousins. CONCLUSIONS. A novel nonsense mutation in CEP290 results in nonsense-associated altered splicing. That the remaining open reading frame is intact may explain the less severe phenotype observed in the two affected cousins. The additional heterozygous mutation in MERTK may clarify the more severe phenotype in the proband. This study extends the phenotypic spectrum of CEP290-associated diseases at the mild end.

Prospects for gene therapy of inherited retinal disease

Gene-based therapies offer the means to address gene defects responsible for inherited retinal disorders. A number of studies in experimental and preclinical models have demonstrated proof-of-principle that gene replacement therapy can mediate significant quantifiable improvements in ocular morphology and visual function. The first results of clinical trials of gene therapy for early-onset severe retinal dystrophy caused by defects in RPE65 show proof-of-concept for efficacy and short-term safety in humans. The challenges for gene therapy of conditions caused by gain-of-abnormal function are being addressed by strategies to knock down expression of the disease allele. Vector-mediated expression of neuroprotective proteins may offer a generic approach for preserving vision in single-gene and multi-gene retinal degenerations. Gene therapy is likely to be most successful where stable expression of the therapeutic transgene can be achieved at an appropriate level in diseases in which retinal development is unaffected and a significant number of target cells survive at the point of intervention.

An Assessment of the Apex Microarray Technology in Genotyping Patients with Leber Congenital Amaurosis and Early-Onset Severe Retinal Dystrophy

Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EOSRD) are genetically heterogeneous, with 11 genes currently implicated. The LCA chip may be used to interrogate many variants in one hybridization reaction. The purpose of this study was to assess the utility of this technology. One hundred fifty-three patients with LCA and EOSRD were screened using an array (Asper Ophthalmics, Tartu, Estonia) containing 344 published disease-causing variants and polymorphisms in eight genes: AIPL1, GUCY2D, CRB1, CRX, RPGRIP1, RPE65, MERTK, and LRAT. One hundred thirty-six probands underwent bidirectional sequencing of the full coding region of the RPE65 gene. The same technique was also used to confirm CRB1 and AIPL1 mutations initially identified with the Apex chip (Asper Ophthalmics). Single nucleotide polymorphism (SNP) analysis within control populations was performed for two variants, P701S and W21R, on the chip for GUCY2D. Of the possible 109,392 interrogations, 3,346 (3.06%) failed on one strand whereas 259 (0.47%) failed on both. The chip reported mutations in 68 (44%) patients; 26 patients had two alleles identified (17%). Direct sequencing of RPE65 showed no discrepancies, whereas sequencing of AIPL1 and CRB1 revealed seven samples called erroneously. The SNP analysis of both GUCY2D variants revealed equal prevalence in the EOSRD panel and the normal population. Subsequent reanalysis, after excluding these polymorphisms, revealed one (18.3%) or two (11.7%) mutations identified in 46 patients. When evaluated by diagnosis, 46% of patients with LCA had one or two mutations identified, compared with 24% of patients with EOSRD. This approach is a rapid and reasonably low-cost technique for identifying both previously identified mutations and common polymorphisms. The addition of further genes and mutations to the chip will improve its utility, though it is advised that all results be checked by direct sequencing.

Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Fatty acid retinyl esters are the storage form of vitamin A (all-trans-retinol) and serve as metabolic intermediates in the formation of the visual chromophore 11-cis-retinal. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, acts by transferring an acyl group from the sn-1 position of phosphatidylcholine to retinol. To define the membrane association and localization of LRAT, we produced an LRAT-specific monoclonal antibody, which we used to study enzyme partition under different experimental conditions. Furthermore, we examined the membrane topology of LRAT through an N-linked glycosylation scanning approach and protease protection assays. We show that LRAT is localized to the membrane of the endoplasmic reticulum (ER) and assumes a single membrane-spanning topology with an N-terminal cytoplasmic/C-terminal luminal orientation. In eukaryotic cells, the C-terminal transmembrane domain is essential for the activity and ER membrane targeting of LRAT. In contrast, the N-terminal hydrophobic region is not required for ER membrane targeting or enzymatic activity, and its amino acid sequence is not conserved in other species examined. We present experimental evidence of the topology and subcellular localization of LRAT, a critical enzyme in vitamin A metabolism.

Fundus autofluorescence in children and teenagers with hereditary retinal diseases

In adults, evaluation of fundus autofluorescence (AF) plays an important role in the differential diagnosis of retinal diseases. The aim of this study was to evaluate the feasibility of recording AF in children and teenagers and to define typical AF findings of various hereditary retinal diseases during childhood. Fifty patients aged 2 to 16 years with hereditary retinal diseases were analysed using the HRA (Heidelberg Retina Angiograph). To enhance the AF signal, a mean of up to 16 single images was calculated. Twenty healthy children (aged 4-16 years) served as controls. In many children as young as 5 years of age and even in one 2-year-old child good AF images could be obtained. To achieve high quality images, larger image series (about 50 single images) were taken and appropriate single images were chosen manually to calculate the mean. Characteristically, Stargardt disease shows a central oval area of reduced AF, often surrounded by more irregular AF. In patients with Best disease, a central round structure with regular or irregular intense AF is visualised. Some patients with X-linked retinoschisis show central radial structures. In many patients with rod-cone dystrophies, a central oval ring-shaped area of increased AF is present. In early-onset severe retinal dystrophy (EOSRD) with RPE65 mutations AF is completely absent, whereas in other forms of Leber congenital amaurosis, AF is normal. Fundus autofluorescence may visualise disease-specific distributions of lipofuscin in the retinal pigment epithelium, often not (yet) visible on ophthalmoscopy. AF images can be used in children to differentiate hereditary retinal diseases and to facilitate follow-up controls. In many cases, four single images are sufficient to analyse the AF pattern.

Longitudinal and cross-sectional study of patients with early-onset severe retinal dystrophy associated with RPE65 mutations

To quantify retinal function longitudinally and cross-sectionally in patients with autosomal-recessive early-onset severe retinal dystrophy (EOSRD) associated with RPE65 mutations. The ocular phenotype was characterized in four children from three families up to the second decade of life, and in three siblings from one family aged 43-54 years carrying compound heterozygous or homozygous mutations in RPE65. Standard clinical examination included colour vision testing, fundus photography and Goldmann visual fields (GVF). Full-field ERGs (in all) and multifocal ERGs (in two patients) were also recorded. Visual performance and fundus appearance were compared to literature data. In childhood, visual acuity (VA) ranged from 0.1 to 0.3, and GVF for target V4 was well preserved. VA and GVF were measurable in only one of the three adult siblings. Nystagmus was present in two of four children and two of three adults. Photophobia was absent in childhood and developed in adulthood. Funduscopic changes were discrete during the first decade of life in three of four children; one patient had clear macular changes already at age 5 years. All three adult siblings had distinct retinal changes including the macula. Bone spicules were not a feature. Residual colour vision was present in all patients with measurable VA. Rod ERGs were absent at any age; cone ERGs were detectable in early childhood. To date, VA data have been reported in 51 patients, visual fields in 29 patients, and a detailed fundus description in 34 patients. For all three parameters, data were comparable to the results in our patient cohort. In childhood, patients with RPE65 mutations have better visual functions than typically seen in Leber congenital amaurosis. The phenotype shows a common progressive pattern with intrafamilial and interfamilial variation. The data suggest a preserved retinal morphology at young ages, arguing for vision-restoring gene therapy trials in childhood.

Lack of fundus autofluorescence to 488 nanometers from childhood on in patients with early-onset severe retinal dystrophy associated with mutations in RPE65

Purpose Fundus autofluorescence is due to accumulation of lipofuscin in the retinal pigment epithelium (RPE) resulting from incomplete digestion of N-retinylidene-phosphatidyl-ethanolamine from shed photoreceptor outer segment discs. Alteration in autofluorescence reflects changes in lipofuscin content of the RPE. Mutations on both alleles of RPE65 result in absent or largely decreased formation of rhodopsin, due to a defect in all-trans retinol isomerization in the RPE. Autofluorescence could therefore be altered. This study was conducted to evaluate fundus autofluorescence in patients with early-onset severe retinal dystrophy (EOSRD, or early-onset rod–cone dystrophy) associated with mutations on both alleles of RPE65. Design Case series. Participants and controls Ten 10- to 55-year-old patients with EOSRD and compound heterozygous or homozygous mutations in RPE65. For comparison, 6 heterozygous parents and 2 patients with other forms of EOSRD were examined. Methods Participants underwent, in addition to standard clinical and electrophysiological examination, autofluorescence imaging using a confocal scanning laser ophthalmoscope. Three of the patients were also examined by optical coherence tomography (OCT) to evaluate the status of retinal degeneration. Mutations in 7 patients have been reported previously; the other patients were investigated by polymerase chain reaction–single-strand conformation polymorphism and direct sequencing for mutations in RPE65 and lecithin retinol acyltransferase ( LRAT). Main outcome measures Fundus autofluorescence and OCT. Results Absent or minimal autofluorescence was found in all patients with compound heterozygous or homozygous RPE65 mutations. Autofluorescence was normal in the heterozygous parents. Autofluorescence was present in 2 children with EOSRD not associated with mutations in RPE65 or LRAT, another gene involved in retinol recycling. Optical coherence tomography in younger patients revealed an intraretinal appearance similar to that of their healthy, heterozygous parents. Conclusions Lack of autofluorescence in patients with EOSRD associated with mutations in RPE65 is in accordance with the biochemical defect and can be used as a clinical marker of this genotype. Optical coherence tomography results in younger patients would indicate still viable photoreceptors despite the absence of autofluorescence.

Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver.

Lecithin-retinol acyltransferase (LRAT), an enzyme present mainly in the retinal pigmented epithelial cells and liver, converts all-trans-retinol into all-trans-retinyl esters. In the retinal pigmented epithelium, LRAT plays a key role in the retinoid cycle, a two-cell recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. We disrupted mouse Lrat gene expression by targeted recombination and generated a homozygous Lrat knock-out (Lrat-/-) mouse. Despite the expression of LRAT in multiple tissues, the Lrat-/- mouse develops normally. The histological analysis and electron microscopy of the retina for 6-8-week-old Lrat-/- mice revealed that the rod outer segments are approximately 35% shorter than those of Lrat+/+ mice, whereas other neuronal layers appear normal. Lrat-/- mice have trace levels of all-trans-retinyl esters in the liver, lung, eye, and blood, whereas the circulating all-trans-retinol is reduced only slightly. Scotopic and photopic electroretinograms as well as pupillary constriction analyses revealed that rod and cone visual functions are severely attenuated at an early age. We conclude that Lrat-/- mice may serve as an animal model with early onset severe retinal dystrophy and severe retinyl ester deprivation.

Mutations in the gene encoding lecithin retinol acyltransferase are associated with early-onset severe retinal dystrophy.

The chromophore of the visual pigments, 11-cis retinal, is derived from vitamin A (all-trans retinol) through a series of reactions that take place in retinal pigment epithelium (RPE); (ref. 1). The first of these reactions is catalyzed by lecithin retinol acyltransferase (LRAT); (ref. 2). We screened 267 retinal dystrophy patients for mutations in LRAT and identified disease-associated mutations (S175R and 396delAA) in three individuals with severe, early-onset disease. We showed that the S175R mutant has no acyltransferase activity in transfected COS-7 cells. Our findings highlight the importance of genetic defects in vitamin A metabolism as causes of retinal dystrophies and extend prospects for retinoid replacement therapy in this group of diseases.