Delineating the Clinical Phenotype of Patients With the c.629C>G, p.Pro210Arg Mutation in Peripherin-2

Abstract

Purpose: More than 200 different mutations in peripherin-2 (PRPH2) are associated with multiple subtypes of inherited retinal diseases (IRDs), including retinitis pigmentosa and cone or macular diseases. Our goal was to understand how the poorly characterized PRPH2 mutation p.Pro210Arg (P210R) affects visual function and retinal structure as well as gain insight into the mechanism driving the clinical pathology.

Methods: Eleven patients had clinical assessments including best-corrected visual acuity (BCVA), full field and multifocal electroretinography (ERG), static (spot size V) and kinetic perimetry (Octopus 900), and dark-adapted chromatic (DAC; Medmont; spot size V) perimetry. Images were acquired with the Optos ultra-wide field camera and spectral-domain optical coherence tomography (SD-OCT). Molecular characteristics of the P210R mutant protein were evaluated in vitro.

Results: Patients with the P210R mutation had BCVA (Snellen) ranging from 20/15 to 20/80. Perimetry showed a reduction in sensitivity, while ERG findings suggested that cone function was more impaired than rod function. Scotomas were identified corresponding to atrophic retinal lesions. Imaging revealed heterogeneous outer retinal changes such as hyperfluorescent flecks, hypo-autofluorescence (AF) regions of atrophy, and thinning of the photoreceptor layer on SD-OCT. In vitro findings suggested that P210R-Prph2 retains the ability to interact with binding partner Rom1 but abnormally accumulates in the endoplasmic reticulum (ER), suggesting the protein does not fold properly.

Conclusions: Rod and cone sensitivities were decreased in subjects with the P210R mutation in PRPH2. There was scotomatous vision loss that occurred within the macula, likely due to atrophy that occurs after drusen have formed and have begun to resolve. This suggests that although rod and cone photoreceptors are dependent on PRPH2, preventing blindness in this specific subgroup of patients could involve therapeutics that impede the formation or lifecycle of drusen.

Figure 1.

Representative electroretinography responses. (A) The ffERG responses from the left eye (OS) of patient C1 rod response (top) amplitude within normal limits (WNL) and decreased amplitude for the cone response (bottom; solid black line) compared to normal (gray dashed line). (B) The mfERG of the right eye (OD) from patient G1 revealed reduced amplitudes compared to the (C) normal reference response densities. N, nasal; T, temporal.

Figure 2.

Dark-adapted chromatic perimetry results showing rod sensitivity measured throughout the visual field. Sensitivity (dB) of loci tested in the left eyes (OS) of patients A1 (A), A2 (B), A4 (C), and C1 (D). Sensitivity of loci tested in the right eyes (OD) of patients B1 (E), D1 (F), and E1 (G), and a 43-year-old normal-sighted control. Blue colors indicate better rod function whereas red areas specify loci that were not detected with the brightest intensity of the stimulus. Eccentricity 0, 0 was the position of the fovea. The blind spot in the field is the red spot slightly below the horizontal midline at −10 degrees to −15 degrees OS or 10 degrees to 15 degrees OD. No (red), or 0 dB means that the patient did not see the stimulus at the brightest intensity.

Figure 3.

Left eye fundus images from patients with the P210R mutation in PRPH2. (A) Wide-field pseudo-colored and (B) autofluorescent (AF) images from patients A1, A2, and C1. (C) Blue-wavelength AF images from patients A1 (age 60 years), A2, E1, and D1. Patient age when the image was acquired is in parentheses.

Figure 4.

Right eye spectral-domain optical coherence tomography (SD-OCT) images from patients with the P210R mutation in PRPH2. NIR images (left) showing the horizontal axis (green line) of the scan position of the SD-OCT images (right) from patients A1 (A), A2 (B), A4 (C), B1 (D), C1 (E), D1 (F), D2 (G), E1, (H), F1 (I), and G1 (J). Note that SD-OCT was not obtained for patient A3. White boxes, foveal sparing; red arrows, foveal drusen; yellow arrows, parafoveal drusen; black arrows, breaks in the photoreceptor inner/outer segment (ellipsoid) junction; blue arrows, migrating retinal pigment epithelium (RPE). Outer nuclear layer (ONL), and external limiting membrane (ELM).

Figure 5.

P210R Prph2 retains the ability to bind Rom1 but accumulates abnormally in the ER. COS-7 cells were single transfected with P210R-Prph2 (A) or WT-Prph2 (B), and cells were labeled for Prph2 (red) and the ER marker calreticulin (green); and nuclei are counterstained with DAPI (blue). P210R accumulated in the ER (yellow), in contrast to WT-Prph2 (red) which was distributed in punctae throughout the cell. (C-D) COS-7 cells were double transfected with P210R-Prph2 (C) or WT-Prph2 (D) and Rom1 and labeled for Prph2 (red), Rom1 (blue), and calreticulin (green); and the nuclei are counterstained with DAPI (grey). WT-Prph2 and Rom1 co-localized (magenta) with very little found in the ER (green). P210R-Prph2 and Rom1 exhibited some co-localization in the ER (white signal, orange arrow), and the small amount of P210R that exited the ER (red, red arrows highlight examples) did not appear to co-localize with Rom1 (blue, blue arrows highlight examples). Images captured at 100x, scale bar is 10 µm for all images, A and C show two different representative cells. (E-F) HEK 293T cells were co-transfected with P210R-Prph2 and Rom1 (E) or WT-Prph2 and Rom1 (F). Prph2 antibodies (RDS-CT) were used to immunoprecipitated (IP) Prph2 complexes from cell lysates. Blots were immunoblotted (IB) for Prph2 or Rom1. Input, starting lysate; Pre., preclear; beads + IgG; Bound, fraction bound to Prph2 antibody; Un., unbound flow through.