Caterina Ripamonti

X-Linked Cone Dystrophy Caused by Mutation of the Red and Green Cone Opsins

X-linked cone and cone-rod dystrophies (XLCOD and XLCORD) are a heterogeneous group of progressive disorders that solely or primarily affect cone photoreceptors. Mutations in exon ORF15 of the RPGR gene are the most common underlying cause. In a previous study, we excluded RPGR exon ORF15 in some families with XLCOD. Here, we report genetic mapping of XLCOD to Xq26.1-qter. A significant LOD score was detected with marker DXS8045 (Z(max) = 2.41 [theta = 0.0]). The disease locus encompasses the cone opsin gene array on Xq28. Analysis of the array revealed a missense mutation (c. 529T>C [p. W177R]) in exon 3 of both the long-wavelength-sensitive (LW, red) and medium-wavelength-sensitive (MW, green) cone opsin genes that segregated with disease. Both exon 3 sequences were identical and were derived from the MW gene as a result of gene conversion. The amino acid W177 is highly conserved in visual and nonvisual opsins across species. We show that W177R in MW opsin and the equivalent W161R mutation in rod opsin result in protein misfolding and retention in the endoplasmic reticulum. We also demonstrate that W177R misfolding, unlike the P23H mutation in rod opsin that causes retinitis pigmentosa, is not rescued by treatment with the pharmacological chaperone 9-cis-retinal. Mutations in the LW/MW cone opsin gene array can, therefore, lead to a spectrum of disease, ranging from color blindness to progressive cone dystrophy (XLCOD5).

Early onset retinal dystrophy due to mutations in LRAT: Molecular analysis and detailed phenotypic study

PURPOSEnTo report novel variants and characterize the phenotype associated with the autosomal recessive retinal dystrophy caused by mutations in the lecithin retinol acyltransferase (LRAT) gene.nnnMETHODSnA total of 149 patients with Lebers congenital amaurosis (LCA) or early onset retinal dystrophy were screened for mutations in LCA-associated genes using an arrayed-primer extension (APEX) genotyping microarray (Asper Ophthalmics). LRAT sequencing was subsequently performed in this 148-patient panel. Patients identified with mutations underwent further detailed phenotyping.nnnRESULTSnAPEX analysis identified one patient with a previously reported homozygous LRAT mutation. Sequencing of the panel identified three additional patients with novel homozygous LRAT mutations in exon 2. All four patients had severe progressive nyctalopia, visual field constriction, and photophilia in childhood. Visual acuity ranged from 0.22 logMAR to hand motion. Funduscopy revealed severe retinal pigment epithelial atrophy and minimal retinal pigmentation. Asteroid hyalosis and macular epiretinal fibrosis were frequent. All demonstrated reduced fundus autofluorescence. Optical coherence tomography identified disrupted retinal lamination, outer-retinal debris, and an unidentifiable photoreceptor layer in two cases. Full-field electroretinograms were undetectable or showed severe rod-cone dysfunction. Photopic perimetry revealed severe visual field constriction. Dark-adapted perimetry demonstrated markedly reduced photoreceptor sensitivity. Dark-adapted spectral sensitivity measurements identified functioning rods in two of three patients. All three had severely reduced L- and M-cone sensitivity and poor color discrimination.nnnCONCLUSIONSnLRAT mutations cause a severe, early childhood onset, progressive retinal dystrophy. Phenotypic similarities to the retinal dysfunction associated with RPE-specific protein 65 kDa mutations, another visual cycle gene, suggest that LRAT deficiency may show a good response to novel therapies.

Nature of the Visual Loss in Observers With Leber's Congenital Amaurosis Caused by Specific Mutations in RPE65

PURPOSEnTo characterize visual losses associated with genetic mutations in the RPE65 gene that cause defects in the RPE-specific isomerase, RPE65. RPE65 is an important component of the retinoid cycle that restores 11-cis-retinal after its photoisomerization to its all-trans form. The defects investigated here cause Lebers congenital amaurosis (LCA2), an autosomal, recessively-inherited, severe, congenital-onset rod-cone dystrophy.nnnMETHODSnVision was assessed in nine patients and 10 normal controls by measuring: (1) long-wavelength sensitive (L-) cone temporal acuity (critical flicker fusion frequency or cff) as a function of target illuminance, and (2) L-cone temporal contrast sensitivity as a function of temporal frequency at a fixed-target illuminance. Measurements were made by modulating either a 650-nm light superimposed on a 480-nm background or the red phosphor of a color monitor on a background produced by the monitors blue phosphor.nnnRESULTSnRPE65-mutant observers have severely reduced cffs with shallower cff versus log illuminance functions that rise with a mean slope of 4.53 Hz per decade of illuminance compared with 8.69 Hz in normal controls. Consistent with the cff differences, RPE65-mutant observers show losses in temporal contrast sensitivity that increase rapidly with temporal frequency.nnnCONCLUSIONSnAll RPE65-mutant observers have consistent and substantial losses in temporal acuity and sensitivity compared with normal observers. The losses can be characterized by the addition of two sluggish filters within the mutant visual pathway, both filters with a time constant of 29.5 ms (i.e., low-pass filters with cut-off frequencies of 5.40 Hz).

Cone Dystrophy With ''Supernormal'' Rod ERG: Psychophysical Testing Shows Comparable Rod and Cone Temporal Sensitivity Losses With No Gain in Rod Function

PURPOSEnWe report a psychophysical investigation of 5 observers with the retinal disorder cone dystrophy with supernormal rod ERG, caused by mutations in the gene KCNV2 that encodes a voltage-gated potassium channel found in rod and cone photoreceptors. We compared losses for rod- and for cone-mediated vision to further investigate the disorder and to assess whether the supernormal ERG is associated with any visual benefit.nnnMETHODSnL-cone, S-cone, and rod temporal acuity (critical flicker fusion frequency) were measured as a function of target irradiance; L-cone temporal contrast sensitivity was measured as a function of temporal frequency.nnnRESULTSnTemporal acuity measures revealed that losses for vision mediated by rods, S-cones, and L-cones are roughly equivalent. Further, the gain in rod function implied by the supernormal ERG provides no apparent benefit to near-threshold rod-mediated visual performance. The L-cone temporal contrast sensitivity function in affected observers was similar in shape to the mean normal function but only after the mean function was compressed by halving the logarithmic sensitivities.nnnCONCLUSIONSnThe name of this disorder is potentially misleading because the comparable losses found across rod and cone vision suggest that the disorder is a generalized cone-rod dystrophy. Temporal acuity and temporal contrast sensitivity measures are broadly consistent with the defect in the voltage-gated potassium channel producing a nonlinear distortion of the photoreceptor response but after otherwise normal transduction processes.

Spectral sensitivity measurements reveal partial success in restoring missing rod function with gene therapy

Restored rod visual function after gene therapy can be established unequivocally by demonstrating that, after dark adaptation, spectral sensitivity has the shape characteristic of rods and that this shape collapses to a cone-like shape before rods have recovered after an intense bleach. We used these tests to assess retinal function in eight young adults and children with early-onset severe retinal dystrophy from Phase II of a clinical gene-therapy trial for RPE65 deficiency that involved the subretinal delivery of a recombinant adeno-associated viral vector carrying RPE65. We found substantial improvements in rod sensitivity in two participants: dark-adapted spectral sensitivity was rod-like after treatment and was cone-like before rods had recovered after a bleach. After 40 min of dark adaptation, one participant showed up to 1,000-fold sensitivity improvements 4 months after treatment and the second up to 100-fold improvements 6 months after treatment. The dark-adapted spectral sensitivities of the other six participants remained cone-like and showed little improvement in sensitivity.

Visual consequences of molecular changes in the guanylate cyclase-activating protein.

PURPOSEnWe characterized and modeled changes in visual performance associated with a Tyr99Cys mutation in guanylate cyclase-activating protein-1 (GCAP1) in four family members aged between 39 and 55 years old. Guanylate cyclase and its activating protein are molecules in the visual transduction pathway that restore cyclic GMP (cGMP) following its light-activated hydrolysis. The mutation causes an excess of cGMP in the dark and results in progressive photoreceptor loss.nnnMETHODSnL-cone temporal acuity was measured as a function of target irradiance, and L-cone temporal contrast sensitivity was measured as a function of temporal frequency.nnnRESULTSnAll four mutant GCAP1 family members showed sensitivity or acuity losses relative to normal observers. The data for the youngest family member are consistent with an abnormal speeding up of the visual response relative to that in normals, but those for the older members showed a progressively higher-frequency sensitivity loss consistent with a slowing down of their response.nnnCONCLUSIONSnThe speeding up of the visual response in the youngest observer is consistent with the Tyr99Cys mutation that results in the more rapid replacement of cGMP after light exposure and, thus, in a reduction of temporal integration and relative improvement in high-frequency sensitivity compared to normals. The high-frequency losses in the older observers are consistent with their vision being limited by the interposition of some sluggish process. This might result from some residual or malfunctioning molecular process limiting transduction within damaged photoreceptors or from an active or passive postreceptoral reorganization caused by the paucity of functioning photoreceptors.

Hue shifts produced by temporal asymmetries in chromatic signals depend on the alignment of the first and second harmonics

When M- or L-cone-isolating sawtooth waveforms flicker at frequencies between 4 and 13.3 Hz, there is a mean hue shift in the direction of the shallower sawtooth slope. Here, we investigate how this shift depends on the alignment of the first and second harmonics of sawtooth-like waveforms. Below 4 Hz, observers can follow hue variations caused by both harmonics, and reliably match reddish and greenish excursions. At higher frequencies, however, the hue variations appear as chromatic flicker superimposed on a steady light, the mean hue of which varies with second-harmonic alignment. Observers can match this mean hue against a variable-duty-cycle rectangular waveform and, separately, set the alignment at which the mean hue flips between reddish and greenish. The maximum hue shifts were approximately frequency independent and occurred when the peaks or troughs of the first and second harmonics roughly aligned at the visual input-consistent with the hue shifts being caused by an early instantaneous nonlinearity that saturates larger hue excursions. These predictions, however, ignore phase delays introduced within the chromatic pathway between its input and the nonlinearity that produces the hue shifts. If the nonlinearity follows the substantial filtering implied by the chromatic temporal contrast-sensitivity function, phase delays will alter the alignment of the first and second harmonics such that at the nonlinearity, the waveforms that produce the maximum hue shifts might well be those with the largest differences in rising and falling slopes-consistent with the hue shifts being caused by a central nonlinearity that limits the rate of hue change.

Hue shifts produced by temporal asymmetries in chromatic signals

Observers viewed M- or L-cone-isolating stimuli and compared slowly-on and slowly-off sawtooth waveforms of the same mean chromaticity and luminance. Between 6 and 13 Hz, the mean hue of slowly-on L-cone and slowly-off M-cone sawtooth flicker appeared redder, and the mean hue of slowly-off L-cone and slowly-on M-cone sawtooth stimuli appeared greener-despite all the waveforms having the same mean, near-yellow-appearing chromaticity. We measured the effect of the modulation depth and the slope of the sawtooth on the mean hue shifts as a function of temporal frequency. The results are complex but show that discriminability depended mainly on the second harmonic of the waveforms. We considered several models with combinations of linear and nonlinear stages. First, we considered models in which a nonlinear stage limits the rate of change of hue and restricts the steep slope of the sawtooth waveform more than its shallow slope, thus shifting the mean hue in the direction of the shallower slope (such a nonlinearity is also known as a slew-rate limit). Second, we considered saturation models in which the nonlinear stage compresses hue signals and thus shifts the mean of asymmetrical waveforms with or without differentiation before the nonlinearity. Overall, our modeling and results suggest that the hue shift occurs at some nonlinear mechanism in the chromatic pathway; and that, in terms of the Fourier components of the various waveforms, the effect of the nonlinearity depends crucially on the timing of the second harmonic relative to the first.

Vision in observers with enhanced S-cone syndrome: an excess of s-cones but connected mainly to conventional s-cone pathways.

PURPOSEnThe effect of increased numbers of S-cone photoreceptors in enhanced S-cone syndrome (ESCS) was investigated psychophysically in six ESCS observers to understand more about relative cone sensitivities and postreceptoral organization.nnnMETHODSnMeasures of temporal sensitivity or delay were made: S- and L-cone temporal acuity (critical flicker fusion, or CFF), S-cone temporal contrast sensitivity, and S-cone delay.nnnRESULTSnESCS observers showed uniform enhancements of S-cone CFF of between 0.85 and 6.25 Hz, but reductions in L-cone CFF. They also showed higher S-cone temporal contrast sensitivities at medium and high S-cone adaptation levels, with sensitivity functions that peaked near 7.5 Hz but fell off at lower and higher frequencies. In contrast, the mean normal function was flat at low frequencies and fell off only at high frequencies. The S-cone signal, as in the normal, is subject to large phase delays.nnnCONCLUSIONSnWe interpret the enhancements in CFF as increases in S-cone number in ESCS of between 1.39 and 11.32 times normal density (with a mean of 3.48). The peaked ESCS contrast-sensitivity functions are consistent with S-cone signal interactions that increase sensitivity at intermediate frequencies through constructive interference but decrease it at lower and higher frequencies through destructive interference. Measurements of S-cone delays relative to L- and M-cone signals show that the predominant S-cone signals in ESCS are negative and delayed as in normal observers, but reveal another faster, positive S-cone signal. This signal is also likely to be the cause of constructive and destructive interference in the contrast-sensitivity data of ESCS observers.

A novel missense mutation in both OPN1LW and OPN1MW cone opsin genes causes X-linked cone dystrophy (XLCOD5).

X-linked cone and cone-rod dystrophies (XLCOD and XLCORD) are an inherited group of retinal disorders primarily involving cone photoreceptors. The most common cause is mutation of RPGR. In a British family with XLCOD, we mapped the disorder to Xq26.1-qter, excluding RPGR and other known retinal degeneration genes. The cone opsin gene array on Xq28 was a positional candidate locus. A novel missense mutation (c.529T > C; p.W177R) was identified in exon 3 of both the long wavelength-sensitive (OPN1LW; LW, red) and medium wavelength-sensitive (OPN1MW; MW, green) cone opsin genes, which segregated with disease. Exon 3 sequences of both genes were identical, derived from the OPN1MW gene by partial gene conversion. The amino acid W177 is conserved in all opsins across species. We have shown that W177R in MW opsin results in protein misfolding and retention in the endoplasmic reticulum (ER). Mutations in the OPN1LW /OPN1MW cone opsin gene array can therefore cause a spectrum of phenotypes, from colour blindness to progressive cone dystrophy (XLCOD5).