Cellular Changes in Retinas From Patients With BEST1 Mutations

Abstract

Best disease (BD), also known as vitelliform macular dystrophy, is an inherited disease of the central retina caused by more than 300 pathogenic variants in the BEST1 gene. The phenotype of BD is variable, and there are just a few reports on the histopathology of eyes from donors with BD. Here, we describe the histopathological comparison of donor's eyes from two patients with BD. Eyes obtained from 85-year-old (donor 1) and 65-year-old (donor 2) donors were fixed within 25 h postmortem. Perifoveal and peripheral retinal regions were processed for histology and immunocytochemistry using retinal-specific and retinal pigment epithelium (RPE)-specific antibodies. Three age-matched normal eyes were used as controls. DNA was obtained from donor blood samples. Sequence analysis of the entire BEST1 coding region was performed and identified a c.886A > C (p.Asn296His) variant in donor 1 and a c.602T > C (p.Ile201Thr) variant in donor 2; both mutations were heterozygous. Fundus examination showed that donor 1 displayed a macular lesion with considerable scarring while donor 2 displayed close to normal macular morphology. Our studies of histology and molecular pathology in the perifovea and periphery of these two BD donor eyes revealed panretinal abnormalities in both photoreceptors and RPE cellular levels in the periphery; donor 1 also displayed macular lesion. Our findings confirm the phenotypic variability of BD associated with BEST1 variants.

Keywords: BEST1 gene; best disease; histopathology; photoreceptors; retinal pigment epithelium.

FIGURE 1

Impact of BEST1 pathogenic variants on gross pathology and retinal morphology compared to an unaffected, age-matched control eye. (A) The macroscopic fundus image shows a control eye free of any pathology. Donor 1 (c.886A > C, p.Asn296His, an 85-year-old donor) displayed a visible macular lesion (white arrows) while donor 2 (c.602T > C, p.Ile201Thr, a 65-year-old donor) did not display any obvious retinal lesion; however, this donor displayed significant postmortem fixation artifacts (areas of retinal detachment). Visible fovea is indicated by white arrowhead, optic nerve head = ON. (B) Histology of a control retina (a 95-year-old donor) in the perifovea and periphery displayed typical characteristics including structured lamina consisting of retinal cells. Donor 1 perifovea shows a fibrovascular scar present between the Bruch’s membrane and the retina (star), accompanied by thin patchy RPE (black arrowhead) and inter photoreceptor matrix edema (black arrow); asterisk = drusen. GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; POS = photoreceptor outer segments; RPE = retinal pigment epithelium, choroid (Ch). In the periphery, both donors 1 and 2 displayed a distinct GCL, INL, ONL, RPE, and Ch. Scale bar: A = 2 mm (all low-magnification images) and Scale bar B = 50 μm (all images).

FIGURE 2

Impact of BEST1 pathogenic variants in RPE autofluorescent granules. Cryosections obtained from the BD donors and a 95-year-old control were observed using the green channel (FITC filter) and red channel (TRITC filter). Autofluorescence was overlaid on differential interference contrast (DIC) images. Bruch’s membrane is indicated by hashed white line, asterisk = drusen. Scale bar = 40 μm (all images).

FIGURE 3

Impact of BEST1 pathogenic variants in photoreceptors. (A) Cryosections obtained from the BD donors and a 65-year-old control were labeled with antibodies specific to rhodopsin (green), while cell nuclei were labeled with TO-PRO-3 (blue). (B) Cryosections were also labeled with antibodies specific to red/green cone opsin (green) and GFAP (red), while cell nuclei have been labeled with TO-PRO-3 (blue). Arrow = abnormal distribution of rhodopsin into cell body; arrowheads = abnormal distribution of red/green cone opsin into the cell body. Bruch’s membrane is indicated by the hashed white line. Scale bar = 40 μm (all images).

FIGURE 4

Impact of BEST1 pathogenic variants in gross pathology and RPE. (A) Cryosections obtained from the BD donors and an 88-year-old control were labeled with antibodies specific to EBP50 (green), while cell nuclei were labeled with TO-PRO-3 (blue). (B) Cryosections were also labeled with antibodies specific to MCT3 (green), while cell nuclei have been labeled with TO-PRO-3 (blue). Bruch’s membrane is indicated by the hashed white line. Arrow = abnormal RPE apical microvilli; arrowheads = mislocalized apical RPE distribution of MCT3; double arrowheads = Muller cell apical processes. Scale bar = 40 μm (all images).

FIGURE 5

Impact of BEST1 pathogenic variants in bestrophin-1 RPE localization. Cryosections obtained from the BD donors and an 88-year-old control were labeled with antibodies specific to bestrophin-1 (green), while cell nuclei have been labeled with TO-PRO-3 (blue). Bruch’s membrane is indicated by the hashed white line. Arrow = mislocalized apical RPE distribution of bestrophin-1; arrowheads = basolateral RPE distribution of bestrophin-1; double arrowheads = intracellular bestrophin-1. Scale bar = 40 μm (all images).