RPE65 Mutations Research Articles

A dominant mutation in RPE65 identified by whole-exome sequencing causes retinitis pigmentosa with choroidal involvement

Linkage testing using Affymetrix 6.0 SNP Arrays mapped the disease locus in TCD-G, an Irish family with autosomal dominant retinitis pigmentosa (adRP), to an 8.8 Mb region on 1p31. Of 50 known genes in the region, 11 candidates, including RPE65 and PDE4B, were sequenced using di-deoxy capillary electrophoresis. Simultaneously, a subset of family members was analyzed using Agilent SureSelect All Exome capture, followed by sequencing on an Illumina GAIIx platform. Candidate gene and exome sequencing resulted in the identification of an Asp477Gly mutation in exon 13 of the RPE65 gene tracking with the disease in TCD-G. All coding exons of genes not sequenced to sufficient depth by next generation sequencing were sequenced by di-deoxy sequencing. No other potential disease-causing variants were found to segregate with disease in TCD-G. The Asp477Gly mutation was not present in Irish controls, but was found in a second Irish family provisionally diagnosed with choroideremia, bringing the combined maximum two-point LOD score to 5.3. Mutations in RPE65 are a known cause of recessive Leber congenital amaurosis (LCA) and recessive RP, but no dominant mutations have been reported. Protein modeling suggests that the Asp477Gly mutation may destabilize protein folding, and mutant RPE65 protein migrates marginally faster on SDS-PAGE, compared with wild type. Gene therapy for LCA patients with RPE65 mutations has shown great promise, raising the possibility of related therapies for dominant-acting mutations in this gene.

The human visual cortex responds to gene therapy–mediated recovery of retinal function

Leber congenital amaurosis (LCA) is a rare degenerative eye disease, linked to mutations in at least 14 genes. A recent gene therapy trial in patients with LCA2, who have mutations in RPE65, demonstrated that subretinal injection of an adeno-associated virus (AAV) carrying the normal cDNA of that gene (AAV2-hRPE65v2) could markedly improve vision. However, it remains unclear how the visual cortex responds to recovery of retinal function after prolonged sensory deprivation. Here, 3 of the gene therapy trial subjects, treated at ages 8, 9, and 35 years, underwent functional MRI within 2 years of unilateral injection of AAV2-hRPE65v2. All subjects showed increased cortical activation in response to high- and medium-contrast stimuli after exposure to the treated compared with the untreated eye. Furthermore, we observed a correlation between the visual field maps and the distribution of cortical activations for the treated eyes. These data suggest that despite severe and long-term visual impairment, treated LCA2 patients have intact and responsive visual pathways. In addition, these data suggest that gene therapy resulted in not only sustained and improved visual ability, but also enhanced contrast sensitivity.

Cone opsin determines the time course of cone photoreceptor degeneration in Leber congenital amaurosis

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal recycling and cause Leber congenital amaurosis (LCA), the most severe retinal dystrophy in early childhood. We used Lrat(-)(/-), a murine model for LCA, to investigate the mechanism of rapid cone degeneration. Although both M and S cone opsins mistrafficked as reported previously, mislocalized M-opsin was degraded whereas mislocalized S-opsin accumulated in Lrat(-)(/-) cones before the onset of massive ventral/central cone degeneration. As the ventral and central retina express higher levels of S-opsin than the dorsal retina in mice, our results may explain why ventral and central cones degenerate more rapidly than dorsal cones in Rpe65(-)(/-) and Lrat(-)(/-) LCA models. In addition, human blue opsin and mouse S-opsin, but not mouse M-opsin or human red/green opsins, aggregated to form cytoplasmic inclusions in transfected cells, which may explain why blue cone function is lost earlier than red/green-cone function in patients with LCA. The aggregation of short-wavelength opsins likely caused rapid cone degenerations through an endoplasmic reticulum stress pathway, as demonstrated in both the Lrat(-)(/-) retina and transfected cells. Replacing rhodopsin with S-opsin in Lrat(-)(/-) rods resulted in mislocalization and aggregation of S-opsin in the inner segment and the synaptic region of rods, ER stress, and dramatically accelerated rod degeneration. Our results demonstrate that cone opsins play a major role in determining the degeneration rate of photoreceptors in LCA.

600. Safety and Efficacy of Re-Administration of AAV2.hRPE65v2 in Subjects with Leber Congenital Blindness Due to RPE65 Mutations

600. Safety and Efficacy of Re-Administration of AAV2.hRPE65v2 in Subjects with Leber Congenital Blindness Due to RPE65 Mutations

Preclinical Safety Evaluation of AAV2-sFLT01— A Gene Therapy for Age-related Macular Degeneration

AAV2-sFLT01 is a vector that expresses a modified soluble Flt1 receptor designed to neutralize the proangiogenic activities of vascular endothelial growth factor (VEGF) for treatment of age-related macular degeneration (AMD) via an intravitreal injection. Owing to minimal data available for the intravitreal route of administration for adeno-associated virus (AAV), we initiated a 12-month safety study of AAV2-sFLT01 administered intravitreally at doses of 2.4 × 10(9) vector genomes (vg) and 2.4 × 10(10) vg to cynomolgus monkeys. Expression of sFlt01 protein peaked at ~1-month postadministration and remained relatively constant for the remainder of the study. Electroretinograms, fluorescein angiograms, and tonometry were assessed every 3 months, with no test article-related findings observed in any group. Indirect ophthalmoscopy and slit lamp exams performed monthly revealed a mild to moderate but self-resolving vitreal inflammation in the high-dose group only, which follow-up studies suggest was directed against the AAV2 capsid. Histological evaluation revealed no structural changes in any part of the eye and occasional inflammatory cells in the trabecular meshwork, vitreous and retina in the high-dose group. Biodistribution analysis in rats and monkeys found only trace amounts of vector outside the injected eye. In summary, these studies found AAV2-sFLT01 to be well-tolerated, localized, and capable of long-term expression.

Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa

For dominantly inherited disorders development of gene therapies, targeting the primary genetic lesion has been impeded by mutational heterogeneity. An example is rhodopsin-linked autosomal dominant retinitis pigmentosa with over 150 mutations in the rhodopsin gene. Validation of a mutation-independent suppression and replacement gene therapy for this disorder has been undertaken. The therapy provides a means of correcting the genetic defect in a mutation-independent manner thereby circumventing the mutational diversity. Separate adeno-associated virus (AAV) vectors were used to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. Viruses were subretinally coinjected into P347S mice, a model of dominant rhodopsin-linked retinitis pigmentosa. Benefit in retinal function and structure detected by electroretinography (ERG) and histology, respectively, was observed for at least 5 months. Notably, the photoreceptor cell layer, absent in 5-month-old untreated retinas, contained 3–4 layers of nuclei, whereas photoreceptor ultrastructure, assessed by transmission electron microscopy (TEM) improved significantly. The study provides compelling evidence that codelivered suppression and replacement is beneficial, representing a significant step toward the clinic. Additionally, dual-vector delivery of combined therapeutics represents an exciting approach, which is potentially applicable to other inherited disorders.

Fundus Albipunctatus Associated with Compound Heterozygous Mutations in RPE65

To describe a family with an 18-year-old woman with fundus albipunctatus and compound heterozygous mutations in RPE65 whose unaffected parents and 1 female sibling harbored single heterozygous RPE65 mutations. Observational study. Four family members. Clinical examinations included full-field electroretinogram (ffERG) after standard (30-minute) and prolonged (17-hour) dark adaptation, multifocal electroretinogram (mfERG), optical coherence tomography (OCT), and fundus autofluorescence (FAF). Molecular genetic testing included sequencing of RDH5 and RLBP1 and screening for known autosomal-recessive retinitis pigmentosa mutations by a commercially available microarray technique. RPE65 sequencing was performed after the identification of a known heterozygous splice-site mutation by array screening. We recorded ffERG and mfERG amplitudes, OCT characteristics, the FAF intensity index, and the outcomes of DNA sequencing regarding RPE65 mutations. Uniform, yellow-white dots typical of fundus albipunctatus were demonstrated in the proband. These dots corresponded with discrete, hyperreflective formations extending from the Bruch's membrane and retinal pigment epithelium (RPE) into the level of the external limiting membrane, thus spanning along the entire RPE and photoreceptor outer and inner segments. A reduced thickness of the central retina and the RPE-outer segment complex was demonstrated. The intensity of the FAF was severely reduced in the entire fundus. At age 18, ffERG-including prolonged dark adaptation-demonstrated a barely recordable rod response after standard dark adaptation and normalization (increase by more than 700%) of the response after prolonged dark adaptation. The cone 30-Hz flicker response was reduced after standard dark adaptation and increased by >50% after prolonged dark adaptation. In addition, mfERG demonstrated reduced central and peripheral responses. Molecular genetic analysis demonstrated compound heterozygous mutations (IVS1+5G>A and c.344T>C) in RPE65. No mutations were found in RDH5 or RLBP1. No significant abnormalities of retinal structure or function were detected in the parents and sister carrying single heterozygous mutations in RPE65. This is the first reported association between compound heterozygous RPE65 mutations and fundus albipunctatus, indicative of a mutation-specific phenotypic effect in this gene. This finding, together with the reduced FAF, supports that disruption of retinoid recycling in the RPE is essential for the development of fundus albipunctatus.

The Phenotype of Severe Early Childhood Onset Retinal Dystrophy (SECORD) from Mutation ofRPE65and Differentiation from Leber Congenital Amaurosis

To describe in detail the characteristic clinical phenotype and electrophysiological features of Severe Early Childhood Onset Retinal Dystrophy (SECORD) caused by mutation of RPE65. Ophthalmological examination, color fundus photography, visual field testing, detailed electrophysiological assessment, and screening of RPE65 were undertaken in five subjects. Selected patients also had spectral domain optical coherence tomography. All five patients had life-long, extremely poor night vision. Variable degrees of nystagmus were present; three cases lacked nystagmus at the time of assessment. Bilateral disc drusen were evident in three subjects. While case 1 had an undetectable electroretinogram and features supporting a diagnosis of Leber congential amaurosis (LCA) as an infant, her level of acuity and function into the second decade of life was more consistent with SECORD. In two cases, both vision and electrophysiological responses were seen to improve into the second decade of life. The objective demonstration of improved retinal function over time, with electrophysiological testing, has not been previously reported. Cases 4 and 5 had evidence of fine white retinal dots. The authors propose that these represent abnormal accumulations of retinyl esters, as has been demonstrated in animal models, and has also been observed as lipid droplets within the retinal pigment epithelium (RPE). These white dots were seen to fade with time in the patients and were replaced by RPE changes. The identification of patients with mutations in RPE65 has attained greater significance now that gene replacement trials have begun. The features presented in this article assist in the recognition of this form of LCA/SECORD.

Gene therapy rescues cone structure and function in the 3-month-old rd12 mouse: a model for midcourse RPE65 leber congenital amaurosis.

RPE65 function is necessary in the retinal pigment epithelium (RPE) to generate chromophore for all opsins. Its absence results in vision loss and rapid cone degeneration. Recent Leber congenital amaurosis type 2 (LCA with RPE65 mutations) phase I clinical trials demonstrated restoration of vision on RPE65 gene transfer into RPE cells overlying cones. In the rd12 mouse, a naturally occurring model of RPE65-LCA early cone degeneration was observed; however, some peripheral M-cones remained. A prior study showed that AAV-mediated RPE65 expression can prevent early cone degeneration. The present study was conducted to test whether the remaining cones in older rd12 mice can be rescued. Subretinal treatment with the scAAV5-smCBA-hRPE65 vector was initiated at postnatal day (P)14 and P90. After 2 months, electroretinograms were recorded, and cone morphology was analyzed by using cone-specific peanut agglutinin and cone opsin-specific antibodies. Cone degeneration started centrally and spread ventrally, with cells losing cone-opsin staining before that for the PNA-lectin-positive cone sheath. Gene therapy starting at P14 resulted in almost wild-type M- and S-cone function and morphology. Delaying gene-replacement rescued the remaining M-cones, and most important, more M-cone opsin-positive cells were identified than were present at the onset of gene therapy, suggesting that opsin expression could be reinitiated in cells with cone sheaths. The results support and extend those of the previous study that gene therapy can stop early cone degeneration, and, more important, they provide proof that delayed treatment can restore the function and morphology of the remaining cones. These results have important implications for the ongoing LCA2 clinical trials.

Restoration of Cone Vision in the CNGA3−/− Mouse Model of Congenital Complete Lack of Cone Photoreceptor Function

Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce viral gene replacement therapy as a potential treatment for this disease in the CNGA3(-/-) mouse model. We show that such therapy can restore cone-specific visual processing in the central nervous system even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3(-/-) mice were able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular cyclic nucleotide-gated (CNG) channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cyclic guanosine monophosphate (cGMP) levels in cones, delayed cone cell death and reduced the inflammatory response of Müller glia cells that is typical of retinal degenerations. Furthermore, ganglion cells from treated, but not from untreated, CNGA3(-/-) mice displayed cone-driven, light-evoked, spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated, vision-guided behavior.

Molecular Anthropology Meets Genetic Medicine to Treat Blindness in the North African Jewish Population: Human Gene Therapy Initiated in Israel

The history of the North African Jewish community is ancient and complicated with a number of immigration waves and persecutions dramatically affecting its population size. A decade-long process in Israel of clinical-molecular screening of North African Jews with incurable autosomal recessive blindness led to the identification of a homozygous splicing mutation (c.95-2A > T; IVS2-2A > T) in RPE65, the gene encoding the isomerase that catalyzes a key step in the retinoid-visual cycle, in patients from 10 unrelated families. A total of 33 patients (four now deceased) had the severe childhood blindness known as Leber congenital amaurosis (LCA), making it the most common cause of retinal degeneration in this population. Haplotype analysis in seven of the patients revealed a shared homozygous region, indicating a population-specific founder mutation. The age of the RPE65 founder mutation was estimated to have emerged 100-230 (mean, 153) generations ago, suggesting it originated before the establishment of the Jewish community in North Africa. Individuals with this RPE65 mutation were characterized with retinal studies to determine if they were candidates for gene replacement, the recent and only therapy to date for this otherwise incurable blindness. The step from molecular anthropological studies to application of genetic medicine was then taken, and a representative of this patient subgroup was treated with subretinal rAAV2-RPE65 gene therapy. An increase in vision was present in the treated area as early as 15 days after the intervention. This process of genetically analyzing affected isolated populations as a screen for gene-based therapy suggests a new paradigm for disease diagnosis and treatment.

Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy

Leber congenital amaurosis (LCA) is a rare hereditary retinal degeneration caused by mutations in more than a dozen genes. RPE65, one of these mutated genes, is highly expressed in the retinal pigment epithelium where it encodes the retinoid isomerase enzyme essential for the production of chromophore which forms the visual pigment in rod and cone photoreceptors of the retina. Congenital loss of chromophore production due to RPE65-deficiency together with progressive photoreceptor degeneration cause severe and progressive loss of vision. RPE65-associated LCA recently gained recognition outside of specialty ophthalmic circles due to early success achieved by three clinical trials of gene therapy using recombinant adeno-associated virus (AAV) vectors. The trials were built on multitude of basic, pre-clinical and clinical research defining the pathophysiology of the disease in human subjects and animal models, and demonstrating the proof-of-concept of gene (augmentation) therapy. Substantial gains in visual function of clinical trial participants provided evidence for physiologically relevant biological activity resulting from a newly introduced gene. This article reviews the current knowledge on retinal degeneration and visual dysfunction in animal models and human patients with RPE65 disease, and examines the consequences of gene therapy in terms of improvement of vision reported.

AAV Provides an Alternative for Gene Therapy of the Peripheral Sensory Nervous System

Recombinant adeno-associated virus (rAAV) is a promising vector for applications in the central nervous system (CNS). Clinical phase I/II studies have demonstrated safety of rAAV-expressing therapeutic transgenes for a variety of benign (i.e., noncancer) CNS disorders, namely, Canavan disease,1 Parkinson’s disease,2,3,4 and Batten disease.5 Human studies were also recently reported targeting the retina.6,8,9,9 From the early stages of preclinical development, rAAV was appealing because it was found to be neurotrophic and capable of long-term activity without immune interference or toxicity when injected into the CNS.10

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A(2) treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.

Visual Acuity in Patients with Leber's Congenital Amaurosis and Early Childhood-Onset Retinitis Pigmentosa

To correlate visual acuity of patients with Leber's congenital amaurosis (LCA) and early childhood-onset retinitis pigmentosa (RP) with mutations in underlying LCA genes. Multicentered retrospective observational study. After exclusion of 28 subjects, 169 patients with the diagnosis of LCA and 27 patients with early childhood-onset RP were included in the study because the underlying mutations in AIPL1, GUCY2D, RDH12, RPE65, CRX, CRB1, RPGRIP1, CEP290, LCA5, and TULP1 genes could be identified in this cohort of patients. We collected data on best-corrected visual acuity as recorded at the time of the patient's most recent visit to one of the participating ophthalmology departments. The median and range of visual acuities for each genetic subtype were calculated separately for the LCA and early childhood-onset RP groups. The range and median best-corrected visual acuities for each genetic subtype and age-related mean visual acuities for each genetic subtype. A wide variation in visual acuity was observed in patients with LCA and RPE65, RDH12, and CRB1 mutations, whereas AIPL1, GUCY2D, CRX, and RPGRIP1 gene mutations were associated with severely decreased visual acuities beginning within the first year of life. It was also noted that patients with either an RPE65 or CRB1 mutation have progressive visual loss with advancing age. Onset of visual symptoms after infancy was associated with a relatively better visual prognosis. The data obtained from this study will help clinicians provide counseling on visual prognosis to patients with known mutations in LCA genes and be of value in future studies aimed at the treatment of LCA and early childhood-onset RP.

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.

Differential Macular Morphology in Patients withRPE65-,CEP290-,GUCY2D-, andAIPL1-Related Leber Congenital Amaurosis

To evaluate genotypic and macular morphologic correlations in patients with RPE65-, CEP290-, GUCY2D-, or AIPL1-related Leber congenital amaurosis (LCA) using spectral-domain optical coherence tomography (SD-OCT). SD-OCT macular scans were performed in 21 patients, including 10 with RPE65, 7 with CEP290, 3 with GUCY2D, and 1 with AIPL1 mutations. An image processing software was used to manually draw segmentation lines by three observers. Lamellar structure was evaluated based on the number of retinal layers on segmented images. Total retinal thickness was measured at the central macular and perifoveal areas by using an automated algorithm. All three patients with GUCY2D mutations (age range, 20-53 years) retained six retinal layers with visible photoreceptor inner/outer segment juncture (PSJ). However, the preservation of lamellar structures did not parallel better visual acuity. Patients with other mutations had poorly defined PSJ and disorganized retinal lamellar structures, where only one to three retinal layers could be observed. Patients with CEP290 mutations trended to have retention of the outer nuclear layer at the fovea and macular thickening, especially at younger ages. In patients with RPE65 (age range, 20-71 years) and AIPL1 mutations (age, 22 years), macular thickness was markedly decreased. Disorganization of retinal lamellar structures in the RPE65 group trended toward a worsening with increasing age. Variations of macular microstructures were observed among LCA patients with different genotypes. Disorganization of retinal lamellar structure was generally age related. Preservation of retinal microanatomic structures may not be associated with better visual acuity.

The Digest

The Digest

Novel Lipofuscin Bisretinoids Prominent in Human Retina and in a Model of Recessive Stargardt Disease

Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve all-trans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-electrospray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE). The second pigment, A2-dihydropyridine-ethanolamine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at approximately 490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidylethanolamine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.

CRB1Gene Mutations Are Associated with Keratoconus in Patients with Leber Congenital Amaurosis

To present an association of mutations in the CRB1 gene with keratoconus in patients with Leber congenital amaurosis (LCA). Sixteen patients with genotyped LCA (having the CRB1, CRX, RetGC, RPE65, and AIPL1 mutations) were recruited from one ophthalmology practice and examined for the presence of keratoconus. Corneal topography, visual acuity, and slit lamp biomicroscopic examination were performed in all cases. The mean age of the patients was 34.5 years (range, 13-74). Visual acuities ranged from 20/40 to light perception. Corneal topography was successfully collected in 15 of the cases. Five of the 16 cases had slit lamp and/or topographic features consistent with keratoconus. One patient had a clinical picture that was keratoglobus-like. Of these six cases, four had a CRB1 mutation and two had a CRX mutation. Of the three subjects with the CRX mutation, one had keratoconus, one had the keratoglobus-like presentation, and one was normal. Our cohort represents 14 separate, unrelated families. Only one family comprised multiple members with LCA. These were three affected brothers, one with keratoconus, all with CRB1 mutations. Although the results cannot exclude other gene mutations, they suggest that LCA patients with a CRB1 mutation may have a particular susceptibility to keratoconus.