In this report, we describe a male subject who presents with a complex phenotype of myopia associated with cone dysfunction and a protan vision deficiency. Retinal imaging demonstrates extensive cone disruption, including the presence of non-waveguiding cones, an overall thinning of the retina, and an irregular mottled appearance of the hyper-reflective band associated with the inner segment ellipsoid portion of the photoreceptor. Mutation screening revealed a novel p.Glu41Lys missense mutation in a hybrid L/M opsin gene. Spectral analysis shows that the mutant opsin fails to form a pigment in vitro and fails to be trafficked to the cell membrane in transfected Neuro2a cells. Extensive sequence and quantitative PCR analysis identifies this mutant gene as the only gene present in the affected subject's L/M opsin gene array, yet the presence of protanopia indicates that the mutant opsin must retain some activity in vivo. To account for this apparent contradiction, we propose that a limited amount of functional pigment is formed within the normal cellular environment of the intact photoreceptor, and that this requires the presence of chaperone proteins that promote stability and normal folding of the mutant protein.

We report the first observations of the three-dimensional morphology of cone photoreceptors in the living human retina. Images were acquired with a high-speed adaptive optics (AO) spectral-domain optical coherence tomography (SD-OCT) camera. The AO system consisted of a Shack-Hartmann wavefront sensor and bimorph mirror (AOptix) that measured and corrected the ocular and system aberrations at a closed-loop rate of 12 Hz. The bimorph mirror was positioned between the XY mechanical scanners and the subject's eye. The SD-OCT system consisted of a superluminescent diode and a 512 pixel line scan charge-coupled device (CCD) that acquired 75,000 A-scans/s. This rate is more than two times faster than that previously reported. Retinal motion artifacts were minimized by quickly acquiring small volume images of the retina with and without AO compensation. Camera sensitivity was sufficient to detect reflections from all major retinal layers. The regular distribution of bright spots observed within C-scans at the inner segment / outer segment (IS/OS) junctions and at the posterior tips of the OS were found to be highly correlated with one another and with the expected cone spacing. No correlation was found between the posterior tips of the OS and the other retinal layers examined, including the retinal pigment epithelium.

We report first observations of the three-dimensional morphology of cone photoreceptors in the living human retina. Images were acquired with a high-speed adaptive optics (AO) spectral domain optical coherence tomography (SD-OCT) camera. The AO system consists of a Shack-Hartmann wavefront sensor and bimorph deformable mirror (AOptix) that measure and correct the ocular and system aberrations at a closed-loop rate of 12 Hz. Unlike previous AO-OCT and AO-SLO instruments, the bimorph mirror was strategically positioned between the XY mechanical scanners and the subject's eye so as to avoid beam distortion at the pupil plane, which is created when the mirror compensates for the refractive error of the eye. This new configuration is evaluated empirically and with commercial ray tracing software. The SD-OCT system consists of a superluminescent diode and a 512 pixel line scan charge-coupled device (CCD) that acquires 75,000 A-scans/sec. This rate is more than two times faster than that previously reported. Retina motion artifiacts were minimized by quickly acquiring small volume images of the retina with and without AO compensation. Camera sensitivity was sufficient to detect reflections from all major retinal layers. The distribution of bright spots observed within C-scans at the inner segment / outer segment (IS/OS) junction and at the posterior tips of the OS were found to be highly correlated with one another and with the expected cone spacing. No correlation was found between the IS/OS junction and either the plexiform layers or the layers immediately behind the OS posterior tips.

Purpose

To study retinal structure in choroideremia patients and carriers using high-resolution imaging techniques.

Methods

Subjects from four families (six female carriers and five affected males) with choroideremia (CHM) were characterized with best-corrected visual acuity (BCVA), kinetic and static perimetry, full-field electroretinography, and fundus autofluorescence (FAF). High-resolution macular images were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography (SD-OCT). Coding regions of the CHM gene were sequenced.

Results

Molecular analysis of the CHM gene identified a deletion of exons 9 to 15 in family A, a splice site mutation at position 79+1 of exon 1 in family B, deletion of exons 6 to 8 in family C, and a substitution at position 106 causing a premature stop in family D. BCVA ranged from 20/16 to 20/63 in carriers and from 20/25 to 5/63 in affected males. FAF showed abnormalities in all subjects. SD-OCT showed outer retinal layer loss, outer retinal tubulations at the margin of outer retinal loss, and inner retinal microcysts. Patchy cone loss was present in two symptomatic carriers. In two affected males, cone mosaics were disrupted with increased cone spacing near the fovea but more normal cone spacing near the edge of atrophy.

Conclusions

High-resolution retinal images in CHM carriers and affected males demonstrated RPE and photoreceptor cell degeneration. As both RPE and photoreceptor cells were affected, these cell types may degenerate simultaneously in CHM. These findings provide insight into the effect of CHM mutations on macular retinal structure, with implications for the development of treatments for CHM. (ClinicalTrials.gov number, NCT00254605.).