Subjects

This prospective cross-sectional study included 14 eyes of 14 patients with a clinical diagnosis of RP and hyperautofluorescent rings of different diameters on FAF where expansion, constriction, or no change of the ring diameter was documented at follow-up. The patients (6 female, 8 male) ranged in age from 10 to 68 years. The clinical findings of the study patients were evaluated by 1 retina specialist and the full-field scotopic and photopic electroretinograms (ERGs) were performed according to the International Society for Clinical Electrophysiology of Vision standards. Both clinical features and ERG tracings were consistent with the diagnosis of RP in all patients. All eyes in the study had clear media facilitating FAF and SD-OCT imaging. Eyes were excluded if there was a refractive error greater than ±6.0 diopters spherical or ±2.0 diopters cylindrical, evidence of cystoid macular edema, an epiretinal membrane, and evidence or a history of other ocular diseases (eg, glaucoma, diabetes).

All patients were screened for genetic mutations. RPar genotyping microarrays (Asper Ophthalmics, Tartu, Estonia) were used to screen for 585 mutations in CERKL , CNGA1 , CNGB1 , MERTK , PDE6A , PDE6B , PNR , RDH12 , RGR , RLBP1 , SAG , TULP1 , CRB , RPE65 , USH2A , USH3A , LRAT , and PROML1 genes; RPad genotyping microarrays (Asper Ophthalmics) were used to screen for 370 mutations in CA4 , FSCN2 , IMPDH1 , NRL , PRPF3 , PRPF31 , PRPF8 , RDS , RHO , ROM1 , RP1 , RP9 , CRX , TOPORS , and PNR ; early-onset retinal dystrophy array was used to screen for 495 mutations in AIPL1 , CRB1 , CRX , GUCY2D , LRAT , TULP1 , MERTK , CEP290 , RDH12 , RPGRIP1 , LCA5 , and RPE65 genes. Usher array and Bardet-Biedl array (Asper Ophthalmics) were applied to selected cases.

Fundus Autofluorescence

FAF imaging was performed using a confocal scanning laser ophthalmoscope Heidelberg Retina Angiograph (HRA) 2 or Spectralis HRA+OCT (Heidelberg Engineering, Dossenheim, Germany) after pupil dilation with topical 0.5% tropicamide and 2.5% phenylephrine eye drops. FAF imaging was performed using a 30-degree field of view at a resolution of 1536 × 1536 pixels. An optically pumped solid-state laser (488 nm) was used for excitation and a 495-nm barrier filter was used to modulate the blue argon excitation light. A standard procedure was followed for the acquisition of FAF images, including focus of the retinal image in the infrared reflection mode at 820 nm, sensitivity adjustment at 488 nm, and acquisition of 9 single 30 × 30-degree FAF images encompassing the entire macular area with at least a portion of the optic disc. The 9 single images were computationally averaged to produce a single frame with improved signal-to-noise ratio.

The external and internal boundaries of the hyperautofluorescent ring in both horizontal and vertical axes were defined as the visible limits seen on FAF. The external and internal diameters of the hyperautofluorescent ring were measured along the horizontal and vertical axes that passed through the fovea. The baseline and follow-up FAF images were registered using commercial software (MatLab R2006; The MathWorks, Inc, Natick, Massachusetts, USA). We tested the accuracy of each registration by superimposing the baseline and follow-up image as a layered image in Photoshop CS2 (Adobe Systems Inc, San Jose, California, USA) and flickering the superficial layer on and off. The requirements for accuracy were that constant features (ie, the retinal vasculature) and corresponding vessels should remain stationary. If registration was inaccurate, a new registration was performed until the result was satisfactory. Because they were precisely superimposed, both image scales were identical and exact spatial relations between FAF abnormalities in the initial and follow-up images could be determined. The external and internal boundaries for each hyperautofluorescent ring along both the horizontal and vertical axes were measured at baseline and at 12-, 24-, 36-, and 48-month follow-up examinations. Distances were measured using the measuring tool contained in Photoshop. A fovea-to-optic-disc-margin distance of 3000 μm was used as a reference. Of the 14 eyes studied, 12 eyes were examined at 12 months, 7 eyes at 24 months, 5 eyes at 36 months, and 1 eye at 48 months follow-up. The percentage change in diameter of the ring on FAF at each follow-up examination was compared to the baseline measurement in all subjects and calculated.

The decrease in the diameter of the hyperautofluorescent ring was defined as the value at the baseline subtracted from the value obtained at 12-, 24-, and 36-month follow-up visits. The 95% confidence interval was calculated as the mean of the differences ± 1.96 multiplied by the standard deviation of the difference. The general linear model (GLM) with t tests was used to determine the significance of the changes in the hyperautofluorescent ring diameter in both horizontal and vertical axes at follow-up examinations. The Bonferroni test was also applied since there was difference in follow-up examination time among the study patients.

Spectral-Domain Optical Coherence Tomography

SD-OCT was obtained at baseline examination in 8 eyes. Of these, 6 had follow-up examinations at 12 months and 4 at 24 months. SD-OCT was performed with the Cirrus SD-OCT (Carl Zeiss Meditec Inc, Dublin, California, USA). The acquisition protocol consisted of a 5-line raster scan and a macular cube 512 × 128-scan pattern in which a 6 × 6-mm region of the retina was scanned (a total of 65 536 sampled points) within a scan time of 2.4 seconds. After image acquisition, those with a signal strength ≤8 were excluded. Horizontal line scans through the center of the foveal region were repeated 3 times.

The measurements obtained using the Cirrus SD-OCT were confirmed with the Spectralis HRA+OCT in 3 eyes. The simultaneous acquisition of OCT and FAF images facilitates point-to-point correlation between the en-face and cross-sectional images. Spectralis SD-OCT imaging was acquired by a broadband 870-nm superluminescent diode that scanned the retina at 40 000 A-scans per second with an optical depth resolution of 7 μm. The standard protocol included 25 OCT scans averaged to improve the signal-to-noise ratio.

The length of the intact IS/OS junction was measured at baseline and follow-up examinations using the horizontal foveal scan at the same precise landmark. The length of the IS/OS junction was defined as the distance between the nasal and temporal limits of the hyperreflective band that represents the IS/OS junction layer on SD-OCT. The horizontal length of the intact IS/OS junction lamina was measured using the caliper available on the SD-OCT software. The horizontal length of the IS/OS junction at each follow-up examination was compared to that at baseline, and the percentage change in measurement was calculated.

The diameter of the hyperautofluorescent ring and the length of the IS/OS junction lamina were measured independently by 2 of the authors (L.L. and T.B.), who were masked to the other findings and data of the patients. In the event of disagreement, a third investigator (S.T.) was consulted for the final determination. The FAF and OCT data were analyzed at the baseline and follow-up visits.

Pearson correlation was performed to correlate the length of the intact IS/OS junction on SD-OCT with both external and internal boundaries of the hyperautofluorescent ring along the horizontal axis at baseline. A P value <.05 was considered statistically significant. All analyses were performed using SPSS software version 3.1(SPSS Inc, Chicago, Illinois, USA).