Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics.

The RPE65 gene encodes the isomerase of the retinoid cycle, the enzymatic pathway that underlies mammalian vision. Mutations in RPE65 disrupt the retinoid cycle and cause a congenital human blindness known as Leber congenital amaurosis (LCA). We used adeno-associated virus-2-based RPE65 gene replacement therapy to treat three young adults with RPE65-LCA and measured their vision before and up to 90 days after the intervention. All three patients showed a statistically significant increase in visual sensitivity at 30 days after treatment localized to retinal areas that had received the vector. There were no changes in the effect between 30 and 90 days. Both cone- and rod-photoreceptor-based vision could be demonstrated in treated areas. For cones, there were increases of up to 1.7 log units (i.e., 50 fold); and for rods, there were gains of up to 4.8 log units (i.e., 63,000 fold). To assess what fraction of full vision potential was restored by gene therapy, we related the degree of light sensitivity to the level of remaining photoreceptors within the treatment area. We found that the intervention could overcome nearly all of the loss of light sensitivity resulting from the biochemical blockade. However, this reconstituted retinoid cycle was not completely normal. Resensitization kinetics of the newly treated rods were remarkably slow and required 8 h or more for the attainment of full sensitivity, compared with <1 h in normal eyes. Cone-sensitivity recovery time was rapid. These results demonstrate dramatic, albeit imperfect, recovery of rod- and cone-photoreceptor-based vision after RPE65 gene therapy.

Cone and cone-rod dystrophy segregating in the same pedigree due to the same novel CRX gene mutation.

AIM: To describe the detailed phenotypes of a multi-generation family affected by autosomal dominant cone-rod dystrophy (adCRD) and characterised by marked intrafamilial heterogeneity, due to a novel frameshift mutation in the CRX gene. METHODS: Six affected and two unaffected family members underwent detailed ophthalmological examination as well as psychophysical and electrophysiological testing. Mutation screening of the CRX gene and segregation analysis were performed in 14 family members from three generations. RESULTS: Clinical examination of six available mutation carriers showed marked phenotypic heterogeneity, presenting with a reduced cone electroretinogram (ERG) and normal rod ERG in one family branch and a negative ERG in the other as the most striking feature. Genetic screening identified a novel mutation in the CRX gene, c.636delC, that independently segregates with the disease in both branches of the family. CONCLUSION: The authors identified a novel disease causing mutation in the CRX gene associated with adCRD. Furthermore, we show here for the first time the coexistence of a reduced cone and a negative ERG component in different individuals of the same family, all affected by the same mutation.

Maturation of rod function in preterm infants with and without retinopathy of prematurity.

OBJECTIVES: To establish normal development of rod electroretinograms in preterm infants and to assess the effects of retinopathy of prematurity (ROP). STUDY DESIGN: We measured 88 Naka-Rushton functions from 41 preterm infants at maturities from 30 to 72 weeks postmenstrual age (PMA). Outcomes (log sigma, retinal sensitivity and V(max), retinal responsivity) were compared between control (no ROP), untreated ROP, and treated ROP. RESULTS: In control infants, sensitivity increased by 1.5 log units from 30 to 40 weeks PMA and by a further 0.5 log units by 50 weeks PMA but was 0.5 log units less than in similarly-mature, healthy, term-born infants. Average retinal responsivity increased from 23 microV to 90 microV between 30 and 40 weeks PMA and was 35 muV greater at 40 weeks PMA than in similarly-mature term-born infants. At around 36 weeks PMA, (when onset of ROP peaks), infants with untreated ROP had average retinal sensitivity 0.2 log units lower than control infants; sensitivity was reduced further in infants treated for ROP. Retinal responsiveness did not differ between control subjects and untreated infants with ROP but was greatly reduced in infants treated for ROP. CONCLUSIONS: Maturation of rod sensitivity appears to be slowed by preterm birth whereas maturation of rod responsivity is accelerated. ROP reduces retinal sensitivity, and treated ROP reduces both sensitivity and responsivity.

Isolated ocular disease is associated with decreased mucolipin-1 channel conductance.

PURPOSE: To evaluate a 15-year-old boy with MLIV (mucolipidosis type IV) and clinical abnormalities restricted to the eye who also had achlorhydria with elevated blood gastrin levels. METHODS: In addition to a detailed neuro-ophthalmic and electrophysiological assessment, his mutant mucolipin-1 was experimentally expressed in liposomes and its channel properties studied in vitro. RESULTS: The patient was a compound heterzygote for c.920delT and c.1615delG. Detailed neuro-ophthalmic examination including electroretinography showed him to have a typical retinal dystrophy predominantly affecting rod and bipolar cell function. In vitro expression of MCOLN1 in liposomes showed that the c.1615delG mutated channel had significantly reduced conductance compared with wild-type mucolipin-1, whereas the inhibitory effect of low pH and amiloride remained intact. CONCLUSIONS: These findings suggest that reduced channel conductance is relatively well tolerated by the brain during development, whereas retinal cells and stomach parietal cells require normal protein function. MLIV should be considered in patients with retinal dystrophy of unknown cause and screened for using blood gastrin levels.

The status of cones in the rhodopsin mutant P23H-3 retina: light-regulated damage and repair in parallel with rods.

PURPOSE: This study tests whether cones in the rhodopsin-mutant transgenic P23H-3 retina are damaged by ambient light and whether subsequent light restriction allows repair of damaged cones. METHODS: P23H-3 rats were raised in scotopic cyclic (12 hours of 5 lux, 12 hours of dark) ambient light. At postnatal day 90 to 130, some were transferred to photopic conditions (12 hours of 300 lux, 12 hours of dark) for 1 week and then returned to scotopic conditions for up to 5 weeks. Photoreceptor function was assessed by the dark-adapted flash-evoked electroretinogram, using a two-flash paradigm to isolate the cone response. Outer-segment structure was demonstrated by immunohistochemistry for cone and rod opsins and by electron microscopy. RESULTS: Exposure for 1 week to photopic ambient light reduced the cone b-wave, the rod b-wave, and the rod a-wave by 40% to 60% and caused shortening and disorganization of cone and rod outer segments. Restoration of scotopic conditions for 2 to 5 weeks allowed partial recovery of the cone b-wave and the rod a- and b-waves, and regrowth of outer segments. CONCLUSIONS: Modest increases in ambient light cause rapid and significantly reversible loss of cone and rod function in the P23H-3 retina. The reduction and recovery of cone function are associated with shortening and regrowth of outer segments. Because the P23H mutation affects a protein expressed specifically in rods, this study emphasizes the close dependence of cones on rod function. It also demonstrates the capacity of cones and rods to repair their structure and regain function.

Cone dystrophy with supernormal rod response is strictly associated with mutations in KCNV2.

PURPOSE: Cone dystrophy with supernormal rod response (CDSRR) is a retinal disorder characterized by reduced visual acuity, color vision defects, and specific alterations of ERG responses that feature elevated scotopic b-wave amplitudes at high luminance intensities. Mutations in PDE6H and in KCNV2 have been described in CDSRR. A combined clinical and genetic study was conducted in a cohort of patients with CDSRR, to substantiate these prior METHODS: Seventeen patients from 13 families underwent a detailed ophthalmic examination including color vision testing, Goldmann visual fields, fundus photography, Ganzfeld and multifocal ERGs, and optical coherence tomography. The coding sequences and flanking intron/UTR sequences of PDE6C and KCNV2 were screened for mutations by means of DHPLC and direct DNA sequencing of PCR-amplified genomic DNA. results. Whereas no mutations were detected in the PDE6H gene, mutations in KCNV2 were identified in all patients, in either the homozygous or compound heterozygous state. Ten of the 11 identified mutations were novel, including three missense and six truncating mutations and one gross deletion. The mutations concordantly segregate in all available families according a recessive mode of inheritance. The CDSRR phenotype was associated with reduced visual acuity of variable degree and color vision defects. Macular defects ranging from mild pigmentary changes to distinct foveal atrophy were present in nine patients. Progression of the disease was observed in only three of seven patients with follow-up data. CONCLUSIONS: The phenotype of cone dystrophy with supernormal rod response is tightly linked with mutations in KCNV2.

Measuring rod and cone dynamics in age-related maculopathy.

PURPOSE: A cathode-ray-tube (CRT) monitor-based technique was used to isolate clinically significant components of dark adaptation. The utility of the technique in identifying adaptation abnormalities in eyes with age-related maculopathy (ARM) is described. METHODS: A CRT dark adaptometer was developed to assess cone and rod recovery after photopigment bleach. The following measures were obtained: cone recovery rate (R(c); in decades per minute) and absolute threshold (Tf(c); log candelas per square meter), rod recovery rate (R(r); decades per minute), and rod-cone transition (rod-cone break [RCB], in minutes). These components were isolated by appropriately selecting stimulus size, stimulus location, pigment bleach, and test duration and by coupling the CRT with judiciously selected neutral-density (ND) filters. The protocol was developed by using 5 young observers and was tested on 27 subjects with ARM in the study eye and 22 age-matched control subjects. RESULTS: The parameters necessary for effective isolation of cone and early phase rod dark adaptation were a 2.6 ND filter (for a standard CRT monitor, 0.08-80 cd . m(-2) luminance output); a 4 degrees foveated, 200-ms, achromatic spot; approximately 30% pigment bleaching; and a 30-minute test duration. These settings returned obvious rod and cone recovery curves in control and ARM eyes that were compatible with conventional test methods and identified 93% of participants with ARM as having delayed dynamics in at least one of the parameters. Cone recovery dynamics were significantly slower in the ARM group when compared with age-matched control subjects (R(c), 0.99 +/- 0.35 vs. 2.63 +/- 0.61 decades . min(-1), P < 0.0001). Three of the 27 eyes with ARM did not achieve RCB during the allowed duration (30 minutes). The remaining eyes with ARM (n = 24) exhibited a significant delay in rod recovery (R(r)(,) ARM, 0.16 +/- 0.03 vs. controls, 0.22 +/- 0.02 decades . min(-1), P < 0.0001) and the average time to RCB (+/-SD) in the ARM group was significantly longer than in the control subjects (19.12 +/- 5.17 minutes vs. 10.40 +/- 2.49 minutes, P < 0.0001). CONCLUSIONS: The CRT dark-adaptation technique described in this article is an effective test for identifying abnormalities in cone and rod recovery. Slowed cone and rod recovery and a delayed RCB were evident in the eyes with ARM. The test method is potentially useful for clinical intervention trials in which ARM progression is monitored.

Dissociation of rod and cone sensitivity by acute localized retinal pigment epithelium loss.

PURPOSE: To assess the impact of acute retinal pigment epithelium (RPE) loss on photopic and scotopic sensitivity. METHODS: A 68-year-old woman who had been followed for drusenoid RPE detachment in age-related macular degeneration presented with an acute spontaneous retinal pigment epithelium tear. Three months later, she was seen for routine follow-up and was examined by manual photopic and scotopic threshold perimetry (a static 0.46-degree-diameter 660 nm stimulus under photopic conditions; then, following 25 min of dark adaptation, a static 0.46-degree-diameter 532 nm stimulus under scotopic conditions). The stimuli were applied over the RPE defect and at reference points of similar eccentricity in the opposite vertical haemifield of the same eye where the RPE remained present. RESULTS: Acute RPE loss was associated with only a marginal reduction of photopic sensitivity (-1.5 dB) but a pronounced loss of scotopic sensitivity (-19.5 dB). CONCLUSION: Our observations show that RPE is essential for scotopic but not for photopic retinal function, supporting the theory that cone photopigment regeneration occurs within the human neurosensory retina independently of the RPE.

Structural and functional remodeling in the retina of a mouse with a photoreceptor synaptopathy: plasticity in the rod and degeneration in the cone system.

Knowledge about the plastic and regenerative capacity of the retina is of key importance for therapeutic approaches to restore vision in patients who suffer from degenerative retinal diseases. In the retinae of mice, mutant for the presynaptic scaffolding protein Bassoon, signal transfer at photoreceptor ribbon synapses is disturbed due to impaired ribbon attachment to the active zone. In a long-term study we observed, with light and electron microscopic immunocytochemistry and electroretinographic recordings, two overlapping events in the Bassoon mutant retina, i.e. loss of photoreceptor synapses in the outer plexiform layer, and structural remodeling and formation of ectopic photoreceptor synapses in the outer nuclear layer, a region usually devoid of synapses. Formation of ectopic synaptic sites starts around the time when photoreceptor synaptogenesis is completed in wild-type mice and progresses throughout life. The result is a dense plexus of ectopic photoreceptor synapses with significantly altered but considerable synaptic transmission. Ectopic synapse formation is led by the sprouting of horizontal cells followed by the extension of rod bipolar cell neurites that fasciculate with and grow along the horizontal cell processes. Although only the rod photoreceptors and their postsynaptic partners show structural and functional remodeling, our study demonstrates the potential of the retina for long-lasting plastic changes.

Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction.

Age-related macular degeneration is the most common form of legal blindness in westernized societies, and polymorphisms in the gene encoding complement factor H (CFH) are associated with susceptibility to age-related macular degeneration in more than half of affected individuals. To investigate the relationship between complement factor H (CFH) and retinal disease, we performed functional and anatomical analysis in 2-year-old CFH-deficient (cfh(-/-)) mice. cfh(-/-) animals exhibited significantly reduced visual acuity and rod response amplitudes on electroretinography compared with age-matched controls. Retinal imaging by confocal scanning laser ophthalmoscopy revealed an increase in autofluorescent subretinal deposits in the cfh(-/-) mice, whereas the fundus and vasculature appeared normal. Examination of tissue sections showed an accumulation of complement C3 in the neural retina of the cfh(-/-) mice, together with a decrease in electron-dense material, thinning of Bruch's membrane, changes in the cellular distribution of retinal pigment epithelial cell organelles, and disorganization of rod photoreceptor outer segments. Collectively, these data show that, in the absence of any specific exogenous challenge to the innate immune system, CFH is critically required for the long-term functional health of the retina.

Cone- and rod-mediated dark adaptation impairment in age-related maculopathy.

OBJECTIVE: To examine impairment in cone- versus rod-mediated dark adaptation in the parafovea of persons with age-related maculopathy (ARM). DESIGN: Cross-sectional. PARTICIPANTS: Older adults with ARM at various severity levels from early to advanced (n = 83) and in good retinal health (n = 43), as determined by stereo fundus photographs evaluated with the Age-Related Eye Disease Study severity scale. METHODS: Dark adaptation, both cone- and rod-mediated components, was measured with a modified Humphrey Field Analyzer using a target located 12 degrees in the inferior visual field on the vertical meridian, after exposure to a 98% bleach. Information was collected on self-reported problems for activities at night or under dim illumination (Low Luminance Questionnaire [LLQ]) and for activities during daytime conditions (modified National Eye Institute Visual Function Questionnaire [NEI VFQ]). MAIN OUTCOME MEASURES: Cone- and rod-mediated parameters of dark adaptation. RESULTS: Compared with older adults in normal retinal health, ARM patients had significant impairments in rod-mediated parameters of dark adaptation (rod-cone break, rod slope, rod sensitivity) (P<0.0001), which were increasingly abnormal as disease severity increased. Cone-mediated parameters (cone time constant and cone sensitivity) were not impaired. Low Luminance Questionnaire scores and NEI VFQ scores decreased with increased ARM severity (P = 0.0004 and P = 0.0005, respectively); the percent decrease in LLQ scores as a function of disease severity was larger in magnitude than the percent decrease in NEI VFQ scores. CONCLUSIONS: Disturbances in rod-mediated but not cone-mediated dark adaptation in the parafovea at 12 degrees in the inferior field on the vertical meridian are characteristic of ARM even in its early phases.

An adaptive ERG technique to measure normal and altered dark adaptation in the mouse.

The time-course of dark adaptation provides valuable insights into the function and interactions between the rod and cone pathways in the retina. Here we describe a technique that uses the flash electroretinogram (ERG) response to probe the functional integrity of the cone and rod pathways during the dynamic process of dark adaptation in the mouse. Retinal sensitivity was estimated from the stimulus intensity required to maintain a 30 microV criterion b-wave response during a 40 min period of dark adaptation. When tracked in this manner, dark adaptation functions in WT mice depended upon the bleaching effects of initial background adaptation conditions. Altered dark adaptation functions, commensurate with the functional deficit were recorded in pigmented mice that lacked cone function (Gnat2 ( cplf3 )) and in WT mice injected with a toxin, sodium iodate (NaIO(3)), which targets the retinal pigment epithelium and also has downstream effects on photoreceptors. These data demonstrate that this adaptive tracking procedure measures retinal sensitivity and the contributions of the rod and/or cone pathways during dark adaptation in both WT control and mutant mice.

Novel mutations in the KCNV2 gene in patients with cone dystrophy and a supernormal rod electroretinogram.

PURPOSE: To identify mutations in KCNV2 in patients with a form of cone dystrophy characterized by a supernormal rod electroretinogram (ERG). METHODS: The 2 exons and flanking intron DNA of KCNV2 from 8 unrelated patients were PCR amplified and sequenced. RESULTS: We found 1 frameshift, 2 nonsense, 1 non-stop, and 6 missense mutations. Every patient had one or two mutations identified. Of the missense mutations, 4 affected residues were in the amino terminal region of the protein, and two in the pore region. CONCLUSIONS: KCNV2 mutations account for most if not all cases of cone dystrophy with a supernormal rod ERG.

Rod photoreceptor function predicts blood vessel abnormality in retinopathy of prematurity.

PURPOSE: To test the hypothesis that early rod dysfunction predicts the blood vessel abnormalities that are the clinical hallmark of retinopathy of prematurity (ROP). METHODS: Two rat models of ROP, induced by exposure to alternating 50%/10% oxygen (50/10 model) from postnatal day (P) 0 to P14, or exposure to 75% oxygen (75 model) from P7 to P14, and controls reared in room air were studied. In a longitudinal design, electroretinographic (ERG) records and digital fundus images were obtained at P20 +/- 1, P30 +/- 1, and P60 +/- 1. Rod sensitivity was derived from the ERG a-wave. Integrated curvature for the arterioles was calculated using Retinal Image multi-Scale Analysis (RISA) software. RESULTS: In both ROP models, rod sensitivity was low at P20. Sensitivity improved by P60 in the 50/10 model, but remained low in the 75 model. Integrated curvature was high at P20 in both ROP models, decreased nearly to normal by P30 in the 50/10 model, but remained high in the 75 model, even at P60. At P20, rod sensitivity correlated with integrated vessel curvature. Furthermore, low rod sensitivity at P20 predicted abnormal retinal vasculature--that is, high integrated curvature--at P30 and P60. In contrast, vessel curvature at P20 did not predict sensitivity at P30 or P60. CONCLUSIONS: The rods may instigate the vascular abnormalities that are the clinical hallmark of ROP.

Constitutive excitation by Gly90Asp rhodopsin rescues rods from degeneration caused by elevated production of cGMP in the dark.

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca(2+) sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca(2+) in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca(2+) measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca(2+) sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca(2+) concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca(2+) can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

Dark rearing rescues P23H rhodopsin-induced retinal degeneration in a transgenic Xenopus laevis model of retinitis pigmentosa: a chromophore-dependent mechanism characterized by production of N-terminally truncated mutant rhodopsin.

To elucidate the molecular mechanisms underlying the light-sensitive retinal degeneration caused by the rhodopsin mutation P23H, which causes retinitis pigmentosa (RP) in humans, we expressed Xenopus laevis, bovine, human, and murine forms of P23H rhodopsin in transgenic X. laevis rod photoreceptors. All P23H rhodopsins caused aggressive retinal degeneration associated with low expression levels and retention of P23H rhodopsin in the endoplasmic reticulum (ER), suggesting involvement of protein misfolding and ER stress. However, light sensitivity varied dramatically between these RP models, with complete or partial rescue by dark rearing in the case of bovine and human P23H rhodopsin, and no rescue for X. laevis P23H rhodopsin. Rescue by dark rearing required an intact 11-cis-retinal chromophore binding site within the mutant protein and was associated with truncation of the P23H rhodopsin N terminus. This yielded an abundant nontoxic approximately 27 kDa form that escaped the ER and was transported to the rod outer segment. The truncated protein was produced in the greatest quantities in dark-reared retinas expressing bovine P23H rhodopsin and was not observed with X. laevis P23H rhodopsin. These results are consistent with a mechanism involving enhanced protein folding in the presence of 11-cis-retinal chromophore, with ER exit assisted by proteolytic truncation of the N terminus. This study provides a molecular mechanism for light sensitivity observed in other transgenic models of RP and for phenotypic variation among RP patients.

Flicker assessment of rod and cone function in a model of retinal degeneration.

Critical flicker frequency (CFF) is the lowest frequency for which a flickering light is indistinguishable from a non-flickering light of the same mean luminance. CFF is related to light intensity, with cone photoreceptors capable of achieving higher CFF than rods. A contemporaneous measure of rod and cone function can facilitate characterization of a retinal degeneration. We used sinusoidal flicker ERG to obtain CFF values, over a wide range of light intensities, in RCS dystrophic (RCS-p(+)) and wild type rats. Recordings were made at PN23, PN44, and PN64. The CFF curve in control animals increased in proportion to the log of stimulus intensity, with a gentle slope over the lowest 4 log-unit intensity range. The slope of the CFF curve dramatically increased for higher intensities, indicating a rod-cone break. In the RCS rats the rod driven CFF was significantly lower in amplitude compared to normal rats at the earliest age tested (PN23). By PN64 the rod driven CFF was immeasurable in the RCS rats. The amplitude of the cone driven CFF approached normal values at PN23, but was greatly reduced by PN44. By PN64 the entire CFF function was greatly depressed and there was no longer a discernable rod-cone break. These CFF/ERG data show that RCS rats exhibit significant early degeneration of the rods, followed soon after by degeneration of the cones. Using this approach, rod and cone function can be independently accessed using flicker ERG by testing at a few select intensities.

Functional study in NSE-Hu-Bcl-2 transgenic mice: a model for retinal diseases starting in Müller cells.

In NSE-Hu-Bcl-2 transgenic mice, line 71, retina undergoes early postnatal degeneration linked to the prior death of Müller cells. The purpose of this study was to complete the characterization of this retinal dysfunction by using electroretinographic (ERG) recordings in both scotopic and photopic conditions. Here, we showed that both rod and cone systems were profoundly affected in NSE-Hu-Bcl-2 transgenic mice as soon as 15 postnatal days in accordance with histological study performed previously.

Attenuation of oscillatory potentials in nob2 mice.

PURPOSE: To examine changes in inner retinal function of nob2 mice, expressing a null mutation in Cacna1f encoding the Ca(V)1.4 subunit of voltage-dependent calcium channels. CACNA1F mutations underlie one form of incomplete X-linked congenital stationary night blindness (CSNB2). In addition to a loss of dark-adapted (rod-driven) visual sensitivity, electroretinogram (ERG) b-waves and oscillatory potentials (OPs) are decreased in CSNB2 patients. METHODS: ERGs were recorded under dark-and light-adapted conditions from the corneal surface of nob2 mice, WT littermates and nob4 mice. ERG frequency spectra were calculated by fast Fourier transform (FFT). A FFT-based high-pass filter was used to derive OP waveforms. RESULTS: Under dark-adapted conditions, the dominant frequency of the OPs varied between 90 to 120 Hz in WT mice. In WT mice, OP frequency first increased with flash intensity and then decreased at the highest flash levels while overall OP amplitude increased monotonically with increasing flash intensity. In response to low stimulus flashes, reliable OPs were not obtained from nob2 mice. OPs were only seen at stimulus intensities at or above -1.8 log cd s/m(2), where they occurred at a lower frequency range (70-90 Hz) than for WT mice. When flash stimuli were superimposed against a steady rod-desensitizing adapting field, the amplitude and frequency of WT OPs increased with flash intensity above 0.4 log cd s/m(2). In comparison to WT results, cone-mediated OPs obtained from nob2 mice were smaller in amplitude, of lower frequency and had delayed implicit times. We compared the extent to which OPs and the b-wave were reduced in nob2 mice, by normalizing to the results obtained from WT mice. In comparison to the b-wave, the OPs were relatively spared, under both dark- and light-adapted conditions. CONCLUSIONS: In nob2 mice, rod- and cone-driven OPs are reduced in amplitude and occur at a lower frequency range. Since Ca(V)1.4 is expressed in both the inner and outer plexiform layers, these changes are likely to reflect reduced transmission from photoreceptors to bipolar cells as well as alterations in inner retinal function. That the OPs were better preserved than b-waves suggests that inner retinal pathways may be reorganized in response to the decreased bipolar cell response in nob2 mice.

Genotype-phenotype correlation in a German family with a novel complex CRX mutation extending the open reading frame.

PURPOSE: To describe the genotype-phenotype correlation in a German family with a novel CRX mutation and to perform a comparative analysis of published cases. DESIGN: Retrospective observational case series, systematic review, and comparative analysis of the literature. PARTICIPANTS: Four related patients with progressive retinal degeneration. METHODS: Mutation screening by single-strand polymorphism analysis and direct sequencing. Clinical examination included kinetic visual fields (VFs), 2-color threshold perimetry (2CTP), full-field electroretinography, fundus photography, optical coherence tomography, and fundus autofluorescence (FA) recording. MAIN OUTCOME MEASURES: Visual fields, subjective and objective cone- and rod-specific function, fundus aspect, retinal stratification, and FA. RESULTS: A novel heterozygous complex mutation (c.816delCACinsAA) in CRX predicting the substitution of 27 C-terminal amino acids by 44 novel amino acids, thus abolishing the OTX tail, was identified in a 2-generation family finally diagnosed with cone-rod dystrophy (CRD), which was confirmed by 2CTP. Patients presented with variability in progression, nystagmus, and nyctalopia. Most of the patients were hyperopic. Electroretinography recordings showed residual rod and mixed cone-rod responses in 2 of the subjects. Age-dependent VF losses followed funduscopic changes of progressive atrophy of the retinal pigment epithelium and neuroretina in the macula and midperiphery marked by disturbed FA. Optical coherence tomography showed decreased central retinal thickness. Comparative analysis of the 131 published data sets revealed 2 groups: patients with early and late onset. CONCLUSIONS: We described a 2-generation family with a novel mutation in CRX. The resulting phenotype is that of CRD with variable age at onset and progression. The phenotype description of previously published cases is conclusive only for CRD.