Inverse correlation between fatty acid transport protein 4 and vision in Leber congenital amaurosis associated with RPE65 mutation.

Fatty acid transport protein 4 (FATP4), a transmembrane protein in the endoplasmic reticulum (ER), is a recently identified negative regulator of the ER-associated retinal pigment epithelium (RPE)65 isomerase necessary for recycling 11-cis-retinal, the light-sensitive chromophore of both rod and cone opsin visual pigments. The role of FATP4 in the disease progression of retinal dystrophies associated with RPE65 mutations is completely unknown. Here we show that FATP4-deficiency in the RPE results in 2.8-fold and 1.7-fold increase of 11-cis- and 9-cis-retinals, respectively, improving dark-adaptation rates as well as survival and function of rods in the Rpe65 R91W knockin (KI) mouse model of Leber congenital amaurosis (LCA). Degradation of S-opsin in the proteasomes, but not in the lysosomes, was remarkably reduced in the KI mouse retinas lacking FATP4. FATP4-deficiency also significantly rescued S-opsin trafficking and M-opsin solubility in the KI retinas. The number of S-cones in the inferior retinas of 4- or 6-mo-old KI;Fatp4-/- mice was 7.6- or 13.5-fold greater than those in age-matched KI mice. Degeneration rates of S- and M-cones are negatively correlated with expression levels of FATP4 in the RPE of the KI, KI;Fatp4+/- , and KI;Fatp4-/- mice. Moreover, the visual function of S- and M-cones is markedly preserved in the KI;Fatp4-/- mice, displaying an inverse correlation with the FATP4 expression levels in the RPE of the three mutant lines. These findings establish FATP4 as a promising therapeutic target to improve the visual cycle, as well as survival and function of cones and rods in patients with RPE65 mutations.

Loss of CRB2 in Müller glial cells modifies a CRB1-associated retinitis pigmentosa phenotype into a Leber congenital amaurosis phenotype.

Variations in the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Müller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.

Ciliopathies and the Kidney: A Review.

Primary cilia are specialized sensory organelles that protrude from the apical surface of most cell types. During the past 2 decades, they have been found to play important roles in tissue development and signal transduction, with mutations in ciliary-associated proteins resulting in a group of diseases collectively known as ciliopathies. Many of these mutations manifest as renal ciliopathies, characterized by kidney dysfunction resulting from aberrant cilia or ciliary functions. This group of overlapping and genetically heterogeneous diseases includes polycystic kidney disease, nephronophthisis, and Bardet-Biedl syndrome as the main focus of this review. Renal ciliopathies are characterized by the presence of kidney cysts that develop due to uncontrolled epithelial cell proliferation, growth, and polarity, downstream of dysregulated ciliary-dependent signaling. Due to cystic-associated kidney injury and systemic inflammation, cases result in kidney failure requiring dialysis and transplantation. Of the handful of pharmacologic treatments available, none are curative. It is important to determine the molecular mechanisms that underlie the involvement of the primary cilium in cyst initiation, expansion, and progression for the development of novel and efficacious treatments. This review updates research progress in defining key genes and molecules central to ciliogenesis and renal ciliopathies.

SENIOR-LØKEN SYNDROME: A Case Series and Review of the Renoretinal Phenotype and Advances of Molecular Diagnosis.

PURPOSE: To report genetic and clinical findings in a case series of 10 patients from eight unrelated families diagnosed with Senior-Løken syndrome. METHODS: A retrospective study of patients with Senior-Løken syndrome. Data collected included clinical findings electroretinography and ocular imaging. Genetic analysis was based on molecular inversion probes, whole-exome sequencing (WES), and Sanger sequencing. RESULTS: All patients who underwent electrophysiology (8/10) had widespread photoreceptor degeneration. Genetic analysis revealed two mutations in NPHP1, two mutations in NPHP4, and two mutations in IQCB1 (NPHP5). Five of the six mutations identified in the current study were found in a single family each in our cohort. The IQCB1-p.R461* mutation has been identified in 3 families. Patients harboring mutations in IQCB1 were diagnosed with Leber congenital amaurosis, while patients with NPHP4 and NPHP1 mutations showed early and sector retinitis pigmentosa, respectively. Full-field electroretinography was extinct for 6 of 10 patients, moderately decreased for two, and unavailable for another 2 subjects. Renal involvement was evident in 7/10 patients at the time of diagnosis. Kidney function was normal (based on serum creatinine) in patients younger than 10 years. Mutations in IQCB1 were associated with high hypermetropia, whereas mutations in NPHP4 were associated with high myopia. CONCLUSION: Patients presenting with infantile inherited retinal degeneration are not universally screened for renal dysfunction. Modern genetic tests can provide molecular diagnosis at an early age and therefore facilitate early diagnosis of renal disease with recommended periodic screening beyond childhood and family planning.

Leber Congenital Amaurosis Due to GUCY2D Mutations: Longitudinal Analysis of Retinal Structure and Visual Function.

Gene augmentation therapy is being planned for GUCY2D-associated Leber congenital amaurosis (LCA). To increase our understanding of the natural history of GUCY2D-LCA, patients were evaluated twice with an interval of 4 to 7 years between visits using safety and efficacy outcome measures previously determined to be useful for monitoring this disorder. In this group of molecularly-identified LCA patients (n = 10; ages 7-37 years at first visit), optical coherence tomography (OCT) was used to measure foveal cone outer nuclear layer (ONL) thickness and rod ONL at a superior retinal locus. Full-field stimulus testing (FST) with chromatic stimuli in dark- and light-adapted states was used to assay rod and cone vision. Changes in OCT and FST over the interval were mostly attributable to inter-visit variability. There were no major negative changes in structure or function across the cohort and over the intervals studied. Variation in severity of disease expression between patients occurs; however, despite difficulties in quantifying structure and function in such seriously visually impaired individuals with nystagmus, the present work supports the use of OCT as a safety outcome and FST as an efficacy outcome in a clinical trial of GUCY2D-LCA. A wide age spectrum for therapy was confirmed, and there was relative stability of structure and function during a typical time interval for clinical trials.

CRB2 in immature photoreceptors determines the superior-inferior symmetry of the developing retina to maintain retinal structure and function.

The mammalian apical-basal determinant Crumbs homolog-1 (CRB1) plays a crucial role in retinal structure and function by the maintenance of adherens junctions between photoreceptors and Müller glial cells. Patients with mutations in the CRB1 gene develop retinal dystrophies, including early-onset retinitis pigmentosa and Leber congenital amaurosis. Previously, we showed that Crb1 knockout mice developed a slow-progressing retinal phenotype at foci in the inferior retina, although specific ablation of Crb2 in immature photoreceptors leads to an early-onset phenotype throughout the retina. Here, we conditionally disrupted one or both alleles of Crb2 in immature photoreceptors, on a genetic background lacking Crb1, and studied the retinal dystrophies thereof. Our data showed that disruption of one allele of Crb2 in immature photoreceptors caused a substantial aggravation of the Crb1 phenotype in the entire inferior retina. The photoreceptor layer showed early-onset progressive thinning limited to the inferior retina, although the superior retina maintained intact. Surprisingly, disruption of both alleles of Crb2 in immature photoreceptors further aggravated the phenotype. Throughout the retina, photoreceptor synapses were disrupted and photoreceptor nuclei intermingled with nuclei of the inner nuclear layer. In the superior retina, the ganglion cell layer appeared thicker because of ectopic nuclei of photoreceptors. In conclusion, the data suggest that CRB2 is required to maintain retinal progenitor and photoreceptor cell adhesion and prevent photoreceptor ingression into the immature inner retina. We hypothesize, from these animal models, that decreased levels of CRB2 in immature photoreceptors adjust retinitis pigmentosa because of the loss of CRB1 into Leber congenital amaurosis phenotype.

Cone-rod homeobox CRX controls presynaptic active zone formation in photoreceptors of mammalian retina.

In the mammalian retina, rod and cone photoreceptors transmit the visual information to bipolar neurons through highly specialized ribbon synapses. We have limited understanding of regulatory pathways that guide morphogenesis and organization of photoreceptor presynaptic architecture in the developing retina. While neural retina leucine zipper (NRL) transcription factor determines rod cell fate and function, cone-rod homeobox (CRX) controls the expression of both rod- and cone-specific genes and is critical for terminal differentiation of photoreceptors. A comprehensive immunohistochemical evaluation of Crx-/- (null), CrxRip/+ and CrxRip/Rip (models of dominant congenital blindness) mouse retinas revealed abnormal photoreceptor synapses, with atypical ribbon shape, number and length. Integrated analysis of retinal transcriptomes of Crx-mutants with CRX- and NRL-ChIP-Seq data identified a subset of differentially expressed CRX target genes that encode presynaptic proteins associated with the cytomatrix active zone (CAZ) and synaptic vesicles. Immunohistochemistry of Crx-mutant retina validated aberrant expression of REEP6, PSD95, MPP4, UNC119, UNC13, RGS7 and RGS11, with some reduction in Ribeye and no significant change in immunostaining of RIMS1, RIMS2, Bassoon and Pikachurin. Our studies demonstrate that CRX controls the establishment of CAZ and anchoring of ribbons, but not the formation of ribbon itself, in photoreceptor presynaptic terminals.

Overlap of abnormal photoreceptor development and progressive degeneration in Leber congenital amaurosis caused by NPHP5 mutation.

Ciliary defects can result in severe disorders called ciliopathies. Mutations in NPHP5 cause a ciliopathy characterized by severe childhood onset retinal blindness, Leber congenital amaurosis (LCA), and renal disease. Using the canine NPHP5-LCA model we compared human and canine retinal phenotypes, and examined the early stages of photoreceptor development and degeneration, the kinetics of photoreceptor loss, the progression of degeneration and the expression profiles of selected genes. NPHP5-mutant dogs recapitulate the human phenotype of very early loss of rods, and relative retention of the central retinal cone photoreceptors that lack function. In mutant dogs, rod and cone photoreceptors have a sensory cilium, but develop and function abnormally and then rapidly degenerate; L/M cones are more severely affected than S-cones. The lack of outer segments in mutant cones indicates a ciliary dysfunction. Genes expressed in mutant rod or both rod and cone photoreceptors show significant downregulation, while those expressed only in cones are unchanged. Many genes in cell-death and -survival pathways also are downregulated. The canine disease is a non-syndromic LCA-ciliopathy, with normal renal structures and no CNS abnormalities. Our results identify the critical time points in the pathogenesis of the photoreceptor disease, and bring us closer to defining a potential time window for testing novel therapies for translation to patients.

Retinal gene therapy.

Introduction: Inherited retinal diseases are the leading cause of sight impairment in people of working age in England and Wales, and the second commonest in childhood. Gene therapy offers the potential for benefit. Sources of data: Pubmed and clinicaltrials.gov. Areas of agreement: Gene therapy can improve vision in RPE65-associated Leber Congenital Amaurosis (RPE65-LCA). Potential benefit depends on efficient gene transfer and is limited by the extent of retinal degeneration. Areas of controversy: The magnitude of vision improvement from RPE65-LCA gene therapy is suboptimal, and its durability may be limited by progressive retinal degeneration. Growing points: The safety and potential benefit of gene therapy for inherited and acquired retinal diseases is being explored in a rapidly expanding number of trials. Areas timely for developing research: Developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Optimization of trial design will accelerate reliable assessment of outcomes.

Leber Congenital Amaurosis Associated with Mutations in CEP290, Clinical Phenotype, and Natural History in Preparation for Trials of Novel Therapies.

PURPOSE: To investigate and describe in detail the demographics, functional and anatomic characteristics, and clinical course of Leber congenital amaurosis (LCA) associated with mutations in the CEP290 gene (LCA-CEP290) in a large cohort of adults and children. DESIGN: Retrospective case series. PARTICIPANTS: Patients with mutations in CEP290 identified at a single UK referral center. METHODS: Review of case notes and results of retinal imaging (color fundus photography, fundus autofluorescence [FAF] imaging, OCT), electrophysiologic assessment, and molecular genetic testing. MAIN OUTCOME MEASURES: Molecular genetic testing, clinical findings including visual acuity and retinal imaging, and electrophysiologic assessment. RESULTS: Forty patients with LCA-CEP290 were identified. The deep intronic mutation c.2991+1655 A>G was the most common disease-causing variant (23/40 patients) identified in the compound heterozygous state in 20 patients (50%) and homozygous in 2 patients (5%). Visual acuity (VA) varied from 6/9 to no perception of light, and only 2 of 12 patients with longitudinal VA data showed deterioration in VA in their better-seeing eye over time. A normal fundus was found at diagnosis in younger patients (mean age, 1.9 years), with older patients showing white flecks (mean age, 5.9 years) or pigmentary retinopathy (mean age, 21.7 years). Eleven of 12 patients (92%) with OCT imaging had preservation of foveal architecture. Ten of 12 patients (83%) with FAF imaging had a perifoveal hyperautofluorescent ring. Having 2 nonsense CEP290 mutations was associated with worse final VA and the presence of nonocular features. CONCLUSIONS: Detailed analysis of the clinical phenotype of LCA-CEP290 in a large cohort confirms that there is a window of opportunity in childhood for therapeutic intervention based on relative structural preservation in the central cone-rich retina in a significant proportion of patients, with the majority harboring the deep intronic variant potentially tractable to several planned gene editing approaches.

Genetic profile and mutation spectrum of Leber congenital amaurosis in a larger Indian cohort using high throughput targeted re-sequencing.

To provide a comprehensive data on the prevalence of mutations in Leber congenital amaurosis (LCA) candidate genes from a larger Indian cohort. Ninety-two unrelated subjects were recruited after complete ophthalmic examination and informed consent. Targeted re-sequencing of 20 candidate genes was performed using Agilent HaloPlex target enrichment assay and sequenced on Illumina MiSeq platform. The data were analyzed using standard bioinformatics pipeline, variants annotated, validated and segregated. Genotype-phenotype correlation was performed for the mutation-positive cases. Targeted next generation sequencing (NGS) for the 20 candidate genes generated data with an average sequence coverage and depth of 99.03% and 134X, respectively. Mutations were identified in 61% (56/92) of the cases, which were validated, segregated in the families and absent in 200 control chromosomes. These mutations were observed in 14/20 candidate genes and 39% (21/53) were novel. Distinct phenotypes were observed with respect to genotypes. To our knowledge, this study presents the first comprehensive mutation spectrum of LCA in a large Indian cohort. The mutation-negative cases indicate scope for finding novel candidate gene(s) although mutations in deep intronic and regulatory regions cannot be ruled out.

Leber Congenital Amaurosis.

Leber congenital amaurosis (LCA) is a part of the spectrum of early-onset retinal dystrophy (EORD). It usually presents in the first few years of life, most often before the age of 1 year. The prevalence is about 1:80,000. Also known as congenital retinitis pigmentosa (RP), patients have wandering nystagmus, with reduced vision from birth. The fundus is almost normal to start with. Later, pigmentary disturbances develop. The best corrected visual acuity (BCVA) ranges from no light perception (in nearly one third of cases) to no better than 20/400, often with a hyperopic refraction (≥5.0D). Patients have absent pupillary reflex and some of them have keratoconus (CRB1 and AIPL1). Oculodigital reflex (eye rubbing or poking) is a common association. Electroretinography (ERG) responses are almost undetectable. Patients may have normal intelligence, but some studies suggest that as many as 20% of children with LCA without associated anomalies develop intellectual disability. Patients with GUCY2D mutations have a stable clinical course; those with RPE65 mutations show a period of limited improvement followed by progressive deterioration; and those with AIPL1, CRB1, CEP290, and NMNAT1 mutations show gradual progression over several decades.

Ciliopathy: Senior-Løken Syndrome.

Senior-Løken syndrome is a rare autosomal recessive disease with a prevalence of 1:1,000,000. Retinopathy may progress as Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), or sector RP (Figs. 34.1 and 34.2). Onset of photophobia, nystagmus, and hyperopia can occur in the first few years of life or later in childhood. Patients experience nephronophthisis, characterized by cystic kidney disease (medullary cystic kidney disease), reduced concentrating ability, and chronic tubulointerstitial nephritis, which progresses to end-stage renal disease. Hypertension is common.

[Ciliopathies]. / Retinale Ziliopathien.

Ciliopathies are disorders caused by ciliary dysfunction and can affect an organ system or tissues. Isolated or syndromic retinal dystrophies are the most common ocular manifestation of ciliopathies. The photoreceptor connecting cilium plays a leading role in these ciliopathy-related retinal dystrophies. Dysfunctional photoreceptor cilia cause the most severe type of retinal dystrophy: Leber's congenital amaurosis (LCA). The most common syndromic ciliopathies with an ocular manifestation are Bardet-Biedl syndrome (BBS) and Usher syndrome. Molecular-genetic analysis revealed a large number of cilia genes with a high phenotype heterogeneity. Diagnosis of ciliopathies require a multi-disciplinary approach. Causative treatment of ciliopathies is not yet available; therefore, rehabilitative and supportive treatment is mandatory.

Mouse Models of NMNAT1-Leber Congenital Amaurosis (LCA9) Recapitulate Key Features of the Human Disease.

The nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD(+) in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea-generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1(V9M) mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD(+) metabolism.

The Role of the Human Visual Cortex in Assessment of the Long-Term Durability of Retinal Gene Therapy in Follow-on RPE65 Clinical Trial Patients.

PURPOSE: Gene therapy (GT) has offered immense hope to individuals who are visually impaired because of RPE65 mutations. Although GT has shown great success in clinical trials enrolling these individuals, evidence for stability and durability of this treatment over time is still unknown. Herein we explored the value of functional magnetic resonance imaging (fMRI) as an objective measure to assess independently the longevity of retinal GT. DESIGN: Individuals with RPE65 mutations who underwent GT in their worse-seeing eye in a phase 1 clinical trial received a second subretinal injection in their contralateral eye in a follow-on clinical trial. Functional magnetic resonance imaging (MRI) was performed longitudinally to assess brain responses of patients with RPE65 mutations after stimulation of their most recently treated eye before and 1 to 3 years after GT. PARTICIPANTS: Seven participants with RPE65 mutations who were part of the follow-on clinical trial gave informed consent to participate in a longitudinal neuroimaging fMRI study. METHODS: All participants underwent fMRI using a 3-Tesla MRI system and a 32-channel head coil. Participants' cortical activations were assessed using a block design paradigm of contrast reversing checkerboard stimuli delivered using an MRI-compatible video system. MAIN OUTCOME MEASURES: The primary parameters being measured in this study were the qualitative and quantitative fMRI cortical activations produced by our population in response to the visual task. RESULTS: Functional MRI results showed minimal or no cortical responses before GT. Significant increase in cortical activation lasting at least 3 years after GT was observed for all participants. Repeated measures analysis showed significant associations between cortical activations and clinical measures such as full-field light sensitivity threshold for white, red, and blue colors; visual field; and pupillary light reflex. CONCLUSIONS: Participants with RPE65 mutations showed intact visual pathways, which became responsive and strengthened after treatment. Functional MRI results independently revealed the efficacy and durability of a 1-time subretinal injection. The fMRI results paralleled those recently reported during the long-term clinical evaluations of the same patients. Results from this study demonstrated that fMRI may play an important role in providing complementary information to patients' ophthalmic clinical evaluation and has usefulness as an outcome measure for future retinal intervention studies.

Genotypic and Phenotypic Characteristics of CRB1-Associated Retinal Dystrophies: A Long-Term Follow-up Study.

PURPOSE: To describe the phenotype, long-term clinical course, clinical variability, and genotype of patients with CRB1-associated retinal dystrophies. DESIGN: Retrospective cohort study. PARTICIPANTS: Fifty-five patients with CRB1-associated retinal dystrophies from 16 families. METHODS: A medical record review of 55 patients for age at onset, medical history, initial symptoms, best-corrected visual acuity, ophthalmoscopy, fundus photography, full-field electroretinography (ffERG), Goldmann visual fields (VFs), and spectral-domain optical coherence tomography. MAIN OUTCOME MEASURES: Age at onset, visual acuity survival time, visual acuity decline rate, and electroretinography and imaging findings. RESULTS: A retinitis pigmentosa (RP) phenotype was present in 50 patients, 34 of whom were from a Dutch genetic isolate (GI), and 5 patients had a Leber congenital amaurosis phenotype. The mean follow-up time was 15.4 years (range, 0-55.5 years). For the RP patients, the median age at symptom onset was 4.0 years. In the RP group, median ages for reaching low vision, severe visual impairment, and blindness were 18, 32, and 44 years, respectively, with a visual acuity decline rate of 0.03 logarithm of the minimum angle of resolution per year. The presence of a truncating mutation did not alter the annual decline rate significantly (P = 0.75). Asymmetry in visual acuity was found in 31% of patients. The annual VF decline rate was 5% in patients from the genetic isolate, which was significantly faster than in non-GI patients (P < 0.05). Full-field electroretinography responses were extinguished in 50% of patients, were pathologically attenuated without a documented rod or cone predominance in 30% of patients, and showed a rod-cone dysfunction pattern in 20% of RP patients. Cystoid fluid collections in the macula were found in 50% of RP patients. CONCLUSIONS: Mutations in the CRB1 gene are associated with a spectrum of progressive retinal degeneration. Visual acuity survival analyses indicate that the optimal intervention window for subretinal gene therapy is within the first 2 to 3 decades of life.

"Visual" Cortex of Congenitally Blind Adults Responds to Syntactic Movement.

Human cortex is comprised of specialized networks that support functions, such as visual motion perception and language processing. How do genes and experience contribute to this specialization? Studies of plasticity offer unique insights into this question. In congenitally blind individuals, "visual" cortex responds to auditory and tactile stimuli. Remarkably, recent evidence suggests that occipital areas participate in language processing. We asked whether in blindness, occipital cortices: (1) develop domain-specific responses to language and (2) respond to a highly specialized aspect of language-syntactic movement. Nineteen congenitally blind and 18 sighted participants took part in two fMRI experiments. We report that in congenitally blind individuals, but not in sighted controls, "visual" cortex is more active during sentence comprehension than during a sequence memory task with nonwords, or a symbolic math task. This suggests that areas of occipital cortex become selective for language, relative to other similar higher-cognitive tasks. Crucially, we find that these occipital areas respond more to sentences with syntactic movement but do not respond to the difficulty of math equations. We conclude that regions within the visual cortex of blind adults are involved in syntactic processing. Our findings suggest that the cognitive function of human cortical areas is largely determined by input during development. SIGNIFICANCE STATEMENT: Human cortex is made up of specialized regions that perform different functions, such as visual motion perception and language processing. How do genes and experience contribute to this specialization? Studies of plasticity show that cortical areas can change function from one sensory modality to another. Here we demonstrate that input during development can alter cortical function even more dramatically. In blindness a subset of "visual" areas becomes specialized for language processing. Crucially, we find that the same "visual" areas respond to a highly specialized and uniquely human aspect of language-syntactic movement. These data suggest that human cortex has broad functional capacity during development, and input plays a major role in determining functional specialization.

Results at 2 Years after Gene Therapy for RPE65-Deficient Leber Congenital Amaurosis and Severe Early-Childhood-Onset Retinal Dystrophy.

PURPOSE: To provide an initial assessment of the safety of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in adults and children with retinal degeneration caused by RPE65 mutations. DESIGN: Nonrandomized, multicenter clinical trial. PARTICIPANTS: Eight adults and 4 children, 6 to 39 years of age, with Leber congenital amaurosis (LCA) or severe early-childhood-onset retinal degeneration (SECORD). METHODS: Patients received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of 2 dose levels, and were followed up for 2 years after treatment. MAIN OUTCOME MEASURES: The primary safety measures were ocular and nonocular adverse events. Exploratory efficacy measures included changes in best-corrected visual acuity (BCVA), static perimetry central 30° visual field hill of vision (V30) and total visual field hill of vision (VTOT), kinetic perimetry visual field area, and responses to a quality-of-life questionnaire. RESULTS: All patients tolerated subretinal injections and there were no treatment-related serious adverse events. Common adverse events were those associated with the surgical procedure and included subconjunctival hemorrhage in 8 patients and ocular hyperemia in 5 patients. In the treated eye, BCVA increased in 5 patients, V30 increased in 6 patients, VTOT increased in 5 patients, and kinetic visual field area improved in 3 patients. One subject showed a decrease in BCVA and 2 patients showed a decrease in kinetic visual field area. CONCLUSIONS: Treatment with rAAV2-CB-hRPE65 was not associated with serious adverse events, and improvement in 1 or more measures of visual function was observed in 9 of 12 patients. The greatest improvements in visual acuity were observed in younger patients with better baseline visual acuity. Evaluation of more patients and a longer duration of follow-up will be needed to determine the rate of uncommon or rare side effects or safety concerns.

Variability and Errors of Manually Digitized Goldmann Visual Fields.

PURPOSE: Goldmann visual fields (GVFs) are useful for tracking changes in areas of functional retina, including the periphery, in inherited retinal degeneration patients. Quantitative GVF analysis requires digitization of the chart coordinates for the main axes and isopter points marked by the GVF operator during testing. This study investigated inter- and intra-digitizer variability among users of a manual GVF digitization program. METHODS: Ten digitizers were trained for 1 hour, then digitized 23 different GVFs from inherited retinal degeneration patients in each of three testing blocks. Digitizers labeled each isopter as seeing or non-seeing, and its target size. Isopters with the same test target within each GVF were grouped to create isopter groups. RESULTS: The standard deviation of isopter group area showed an approximate square-root relationship with total isopter group area. Accordingly, the coefficient of variation for isopter group area decreased from 68% to 0.2% with increasing isopter group area. A bootstrap version of ANOVA did not reveal a significant effect of digitizers on isopter group area. Simulations involving random sampling of digitizers showed that five to seven digitizers would be required to catch 95% to 99% of labeling errors and isopter misses, on the basis of data discrepancies, with 99% probability. CONCLUSIONS: These data suggest that any minimally trained digitizer would be capable of reliably determining any isopter area, regardless of size. Studies using this software could either use five to seven minimally trained digitizers for each GVF, three digitizers who demonstrate low frequencies of errors on a practice set of GVFs, or two digitizers with an expert reader to adjudicate discrepancies and catch errors.