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Purpose: To determine the human retinal phenotype caused by mutations in the gene encoding AIPL1 (Aryl hydrocarbon receptor-interacting protein-like 1) now that there are proof-of-concept results for gene therapy success in Aipl1-deficient mice., Methods: Leber congenital amaurosis (LCA) patients (n = 10) and one patient with a later-onset retinal degeneration (RD) and AIPL1 mutations were studied by ocular examination, retinal imaging, perimetry, full-field sensitivity testing, and pupillometry., Results: The LCA patients had severe visual acuity loss early in life, nondetectable electroretinograms (ERGs), and little or no detectable visual fields. Hallmarks of retinal degeneration were present in a wide region, including the macula and midperiphery; there was some apparent peripheral retinal sparing. Cross-sectional imaging showed foveal cone photoreceptor loss with a ring of minimally preserved paracentral photoreceptor nuclear layer. Features of retinal remodeling were present eccentric to the region of detectable photoreceptors. Full-field sensitivity was reduced by at least 2 log units, and chromatic stimuli, by psychophysics and pupillometry, revealed retained but impaired rod function. The RD patient, examined serially over two decades (ages, 45-67 years), retained an ERG in the fifth decade of life with abnormal rod and cone signals; and there was progressive loss of central and peripheral function., Conclusions: AIPL1-LCA, unlike some other forms of LCA with equally severe visual disturbance, shows profound loss of foveal as well as extrafoveal photoreceptors. The more unusual late-onset and slower form of AIPL1 disease may be better suited to gene augmentation therapy and is worthy of detection and further study.

Purpose: To define the phenotype of the retinal degeneration associated with mutations in the CERKL gene., Methods: Six patients (ages, 26-54 years) from three unrelated families with CERKL mutations were studied clinically and by electroretinography, kinetic, and chromatic static perimetry, autofluorescence (AF) imaging, and optical coherence tomography (OCT)., Results: Three siblings were homozygotes for p.R257X mutation; two siblings were compound heterozygotes for p.R257X and a novel p.C362X mutation; and one patient had only p.R257X mutation identified to date. There was a spectrum of severity: from mild visual acuity loss to light perception; from full kinetic fields with relative central scotomas to remnant peripheral islands; from reduced ERGs (some with negative waveforms) to nondetectable signals. Maculopathy showed residual foveal islands or extensive central rod and cone scotomas. With AF imaging, there was evidence of hyperautofluorescence at earlier and hypoautofluorescence at later disease stages. Peripheral function was generally less affected than central function. With OCT there were small foveal islands of outer nuclear layer (ONL) in those with preserved acuity. Eccentric to an annular region with no discernible ONL, there could be ONL in the midperiphery. At early disease stages, ganglion cell layer thickness was less affected than ONL. Later disease stages were accompanied by inner nuclear layer and nerve fiber layer abnormalities., Conclusions: CERKL mutations are associated with widespread retinal degeneration with prominent maculopathy. The clinical presentation is that of an autosomal recessive cone-rod dystrophy. Photoreceptor loss appears at all stages of disease and inner laminopathy complicates the phenotype at later stages.

Human gene therapy with rAAV2-vector was performed for the RPE65 form of childhood blindness called Leber congenital amaurosis. In three contemporaneous studies by independent groups, the procedure was deemed safe and there was evidence of visual gain in the short term. At 12 months after treatment, our young adult subjects remained healthy and without vector-related serious adverse events. Results of immunological assays to identify reaction to AAV serotype 2 capsid were unchanged from baseline measurements. Results of clinical eye examinations of study and control eyes, including visual acuities and central retinal structure by in vivo microscopy, were not different from those at the 3-month time point. The remarkable improvements in visual sensitivity we reported by 3 months were unchanged at 12 months. The retinal extent and magnitude of rod and cone components of the visual sensitivity between 3 and 12 months were also the same. The safety and efficacy of human retinal gene transfer with rAAV2-RPE65 vector extends to at least 1 year posttreatment.

Leber congenital amaurosis (LCA) is a group of autosomal recessive blinding retinal diseases that are incurable. One molecular form is caused by mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene. A recombinant adeno-associated virus serotype 2 (rAAV2) vector, altered to carry the human RPE65 gene (rAAV2-CBSB-hRPE65), restored vision in animal models with RPE65 deficiency. A clinical trial was designed to assess the safety of rAAV2-CBSB-hRPE65 in subjects with RPE65-LCA. Three young adults (ages 21-24 years) with RPE65-LCA received a uniocular subretinal injection of 5.96 x 10(10) vector genomes in 150 microl and were studied with follow-up examinations for 90 days. Ocular safety, the primary outcome, was assessed by clinical eye examination. Visual function was measured by visual acuity and dark-adapted full-field sensitivity testing (FST); central retinal structure was monitored by optical coherence tomography (OCT). Neither vector-related serious adverse events nor systemic toxicities were detected. Visual acuity was not significantly different from baseline; one patient showed retinal thinning at the fovea by OCT. All patients self-reported increased visual sensitivity in the study eye compared with their control eye, especially noticeable under reduced ambient light conditions. The dark-adapted FST results were compared between baseline and 30-90 days after treatment. For study eyes, sensitivity increases from mean baseline were highly significant (p < 0.001); whereas, for control eyes, sensitivity changes were not significant (p = 0.99). Comparisons are drawn between the present work and two other studies of ocular gene therapy for RPE65-LCA that were carried out contemporaneously and reported.

Purpose: To determine the retinal phenotype of Usher syndrome type III (USH3A) caused by clarin-1 (CLRN1) gene mutations in a non-Finnish population., Methods: Patients with USH3A (n = 13; age range, 24-69) representing 11 different families were studied and the results compared with those from patients with USH2A (n = 24; age range, 17-66). The patients were evaluated by ocular examination, kinetic and static perimetry, near-infrared autofluorescence, and optical coherence tomography (OCT)., Results: Ten of 11 families had Ashkenazi Jewish origins and the N48K CLRN1 mutation. Rod function was lost in the peripheral field in the first two decades of life, but central rod function could be retained for another decade. Peripheral cone function was detectable into the third decade of life. Central cone function had a slower decline that extended for decades. Photoreceptor layer loss and features of retinal remodeling were present in retinal regions with severe visual dysfunction, even at the youngest ages tested. Central retinal structure could be normal in younger patients but structural integrity was lost in older patients. RPE disease generally paralleled photoreceptor degeneration. Comparisons between USH3A and USH2A suggested a common rod and cone phenotype but a more accelerated time course of rod loss in USH3A., Conclusions: USH3A and USH2A share patterns of rod and cone dysfunction and retinal structural abnormalities. Peripheral function measurements showed USH3A to be more rapidly progressive than USH2A.

Purpose: To determine the underlying retinal micropathology in subclasses of autosomal dominant retinitis pigmentosa (ADRP) caused by rhodopsin (RHO) mutations., Methods: Patients with RHO-ADRP (n = 17, ages 6-73 years), representing class A (R135W and P347L) and class B (P23H, T58R, and G106R) functional phenotypes, were studied with optical coherence tomography (OCT), and colocalized visual thresholds were determined by dark- and light-adapted chromatic perimetry. Autofluorescence imaging was performed with near-infrared light. Retinal histology in hT17M-rhodopsin mice was compared with the human results., Results: Class A patients had only cone-mediated vision. The outer nuclear layer (ONL) thinned with eccentricity and was not detectable within 3 to 4 mm of the fovea. Scotomatous extracentral retina showed loss of ONL, thickening of the inner retina, and demelanization of RPE. Class B patients had superior-inferior asymmetry in function and structure. The superior retina could have normal rod and cone vision, normal lamination (including ONL) and autofluorescence of the RPE melanin; laminopathy was found in the scotomas. With Fourier-domain-OCT, there was apparent inner nuclear layer (INL) thickening in regions with ONL thinning. Retinal regions without ONL had a thick hyporeflective layer that was continuous with the INL from neighboring regions with normal lamination. Transgenic mice had many of the laminar abnormalities found in patients., Conclusions: Retinal laminar abnormalities were present in both classes of RHO-ADRP and were related to the severity of colocalized vision loss. The results in human class B and the transgenic mice support the following disease sequence: ONL diminution with INL thickening; amalgamation of residual ONL with the thickened INL; and progressive retinal remodeling with eventual thinning.

Purpose: To determine macular pigment (MP) optical density (OD) in patients with ABCA4-associated retinal degenerations (ABCA4-RD) and the response of MP and vision to supplementation with lutein., Methods: Patients with Stargardt disease or cone-rod dystrophy and known or suspected disease-causing mutations in the ABCA4 gene were included. All patients had foveal fixation. MPOD profiles were measured with heterochromatic flicker photometry. Serum carotenoids, visual acuity, foveal sensitivity, and retinal thickness were quantified. Changes in MPOD and central vision were determined in a subset of patients receiving oral supplementation with lutein for 6 months., Results: MPOD in patients ranged from normal to markedly abnormal. As a group, patients with ABCA4-RD had reduced foveal MPOD, and there was a strong correlation with retinal thickness. Average foveal tissue concentration of MP, estimated by dividing MPOD by retinal thickness, was normal in patients, whereas serum concentration of lutein and zeaxanthin was significantly lower than normal. After oral lutein supplementation for 6 months, 91% of the patients showed significant increases in serum lutein, and 63% of the patients' eyes showed a significant augmentation in MPOD. The retinal responders tended to be female and to have lower serum lutein and zeaxanthin, lower MPOD, and greater retinal thickness at baseline. Responding eyes had significantly lower baseline MP concentration than did nonresponding eyes. Central vision was unchanged after the period of supplementation., Conclusions: MP is strongly affected by the stage of ABCA4 disease leading to abnormal foveal architecture. MP could be augmented by supplemental lutein in some patients. There was no change in central vision after 6 months of lutein supplementation. Long-term influences of this supplement on the natural history of these macular degenerations require further study.

Purpose: Human blindness caused by mutation of visual cycle genes has been discussed as potentially treatable by retinoid replacement either through gene transfer or pharmacological bypass. Mutations in the RDH12 gene disrupt the visual cycle in vitro, but little is known of the in vivo effects of mutant RDH12, other than the association with severe early-onset autosomal recessive retinal disease. The relationship of retinal organization and visual function in patients with RDH12 mutations was determined and comparisons made with the disease from mutations in another visual cycle gene, RPE65., Methods: Young patients with RDH12 mutations were studied with optical coherence tomography (OCT) and colocalized measures of vision with dark-adapted absolute thresholds. Results were compared to those in patients with RPE65 mutations., Results: Retinal architecture of patients with RDH12 mutations was appreciably distorted, precluding identification of the normal laminae. Some RDH12-mutant retinas were remarkably thick and others were thin, but all had the same dysplastic pattern. A comparison with the structural and functional consequences in patients with mutations in RPE65 indicated that the pathogenesis of retinal degeneration in RDH12 mutations was distinctly different., Conclusions: The results demand critical consideration of the human disease mechanism and the therapeutic approach in patients with mutations in the putative visual cycle gene RDH12.

Mutations in RPE65, a gene essential to normal operation of the visual (retinoid) cycle, cause the childhood blindness known as Leber congenital amaurosis (LCA). Retinal gene therapy restores vision to blind canine and murine models of LCA. Gene therapy in blind humans with LCA from RPE65 mutations may also have potential for success but only if the retinal photoreceptor layer is intact, as in the early-disease stage-treated animals. Here, we use high-resolution in vivo microscopy to quantify photoreceptor layer thickness in the human disease to define the relationship of retinal structure to vision and determine the potential for gene therapy success. The normally cone photoreceptor-rich central retina and rod-rich regions were studied. Despite severely reduced cone vision, many RPE65-mutant retinas had near-normal central microstructure. Absent rod vision was associated with a detectable but thinned photoreceptor layer. We asked whether abnormally thinned RPE65-mutant retina with photoreceptor loss would respond to treatment. Gene therapy in Rpe65(-/-) mice at advanced-disease stages, a more faithful mimic of the humans we studied, showed success but only in animals with better-preserved photoreceptor structure. The results indicate that identifying and then targeting retinal locations with retained photoreceptors will be a prerequisite for successful gene therapy in humans with RPE65 mutations and in other retinal degenerative disorders now moving from proof-of-concept studies toward clinical trials.

Purpose: To investigate the retinal disease expression in USH2C, the subtype of Usher syndrome type 2 recently shown to be caused by mutation in the VLGR1 gene, and compare results with those from USH2A, a more common cause of Usher syndrome., Methods: Three siblings with USH2C and 14 patients with USH2A were studied. Visual function was measured by kinetic perimetry, static chromatic perimetry, and electroretinography (ERG). Central retinal microstructure was studied with optical coherence tomography (OCT)., Results: The siblings with VLGR1 mutation showed abnormal photoreceptor-mediated function in all retinal regions, and there was greater rod than cone dysfunction. USH2A had a wider spectrum of disease expression and included patients with normal function in some retinal regions. When abnormalities were detected, there was more rod than cone dysfunction. Retinal microstructure in both USH2C and USH2A shared the abnormality of loss of outer nuclear layer thickness. Central retinal structure in both genotypes was complicated by cystic macular lesions. A coincidental finding in an USH2C patient was that oral intake of antihistamines was associated with temporary resolution of the macular cystic change., Conclusions: USH2C and USH2A manifest photoreceptor disease with rod- and cone-mediated visual losses and thinning of the outer nuclear layer. An orderly progression through disease stages was estimated from cross-sectional and limited longitudinal data. Intrafamilial and interfamilial variation in retinal severity in USH2A, however, suggests that genetic or nongenetic modifiers may be involved in the disease expression.

Purpose: The Arg677ter mutation in the RP1 gene is one of the most common causes of autosomal dominant retinitis pigmentosa (RP). In the current study, a de novo Arg677ter RP1 gene mutation was identified in a patient with RP., Methods: RP1 gene mutation screening was performed in probands with simplex RP. In one proband with the RP1 mutation, paternity was established by analyzing 24 short tandem repeat polymorphisms. Additional candidate RP genes, including rhodopsin, RDS/peripherin, RP2, and RPGR, were also examined in this proband. Phenotype was characterized with psychophysics, electroretinography, and optical coherence tomography., Results: An RP1 (Arg677ter) mutation was identified in one of the patients with simplex RP, but the sequence change was not detected in his parents. Parentage was confirmed, and other candidate genes were negative for mutations. Retinal function and cross-sectional imaging studies in the patient indicated greater rod than cone dysfunction with a photoreceptor basis for the abnormalities., Conclusions: The de novo origin of an RP1 (Arg677ter) mutation in a patient with simplex RP suggests that this common autosomal dominant RP mutation can arise independently in the population and supports the hypothesis of a mutational hotspot in the RP1 gene.