Histopathological comparison of eyes from patients with autosomal recessive retinitis pigmentosa caused by novel EYS mutations

Abstract

To evaluate the retinal histopathology in donor eyes from patients with autosomal recessive retinitis pigmentosa (arRP) caused by EYS mutations. Eyes from a 72-year-old female (donor 1, family 1), a 91-year-old female (donor 2, family 2), and her 97-year-old sister (donor 3, family 2) were evaluated with macroscopic, scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) imaging. Age-similar normal eyes and an eye donated by donor 1's asymptomatic mother (donor 4, family 1) were used as controls. The perifovea and peripheral retina were processed for microscopy and immunocytochemistry with markers for cone and rod photoreceptor cells. DNA analysis revealed EYS mutations c.2259 + 1G > A and c.2620C > T (p.Q874X) in family 1, and c.4350_4356del (p.I1451Pfs*3) and c.2739-?_3244 + ?del in family 2. Imaging studies revealed the presence of bone spicule pigment in arRP donor retinas. Histology of all three affected donor eyes showed very thin retinas with little evidence of stratified nuclear layers in the periphery. In contrast, the perifovea displayed a prominent inner nuclear layer. Immunocytochemistry analysis demonstrated advanced retinal degenerative changes in all eyes, with near-total absence of rod photoreceptors. In addition, we found that the perifoveal cones were more preserved in retinas from the donor with the midsize genomic rearrangement (c.4350_4356del (p.I1451Pfs*3) and c.2739-?_3244 + ?del) than in retinas from the donors with the truncating (c.2259 + 1G > A and c.2620C > T (p.Q874X) mutations. Advanced retinal degenerative changes with near-total absence of rods and preservation of some perifoveal cones are observed in arRP donor retinas with EYS mutations.

Fig. 1. Mutational Analysis of individuals with arRP due to EYS mutations

a Pedigree of family 1. Slashed symbols reflect deceased family members. Affected family members are shown with filled symbols, unaffected family members are shown with unfilled symbols and unaffected carrier family members are shown with unfilled symbols with a black dot inside. Postmortem analysis was done on affected member II-4 referred to as donor 1 (*) and in their unaffected mother (I-2) referred to as donor 4 (*). b Pedigree of family 2. Postmortem analysis was done on affected members II-1 and II-2 referred to as donors 2 (*) and 3 (*), respectively. c Sequence analysis of family 1 identified two heterozygous EYS mutations, c.2259+1G>A and p.Q874X. DNA analysis was performed on all three affected members (II-4, II-5 and II-6), their two unaffected living sisters (II-7 and II-8), and their mother (I-2). d Sequence analysis of family 2 identified a heterozygous 7 base pair deletion in EYS, p.I1451Pfs*3. Comparative genomic hybridization identified a heterozygous deletion of exons 15 – 18, c.2739-?_3244+?del. DNA analysis was performed in two affected family members, II-1 and II-2. e Predicted domain structure and distribution of the four EYS mutations identified in this study.

Fig. 2. Ex-vivo imaging of arRP donor eyes with EYS mutations

Fundus images (a-d) and SLO images (e-l) were collected from donor 1, 2 and 3 and an age-similar control. In the control eye, detached retinas are apparent with all imaging modalities (a, e, i, *). In all three arRP eyes, fundus images reveal bone spicule pigment in mid-peripheral and peripheral areas to varying degrees (b-d). SLO-IR imaging identified degeneration in the entire posterior pole region including the macula, perimacula and areas surrounding the optic nerve due to focal loss of RPE in donors 2 (g) and 3 (h) as compared to an age-matched control eye (e). SLO-AF imaging identified hypofluorescence in one contiguous region involving the macula and area surrounding the optic disk of donor 3 (l) as opposed to the individually demarcated and isolated regions seen with both donors 1 (j) and 2 (k) and the control eye (i). Scale bars in fundus image = 0.5 cm.

Fig. 3. Ex-vivo OCT imaging of arRP donor eyes with EYS mutations

OCT images were collected from donors 1, 2 and 3 and an age-matched control. En face images reveal the location (dashed lines) of the in-depth, B-scan images of control (a) and arRP donors 1 (b), 2 (c) and 3 (d). The fovea (arrowhead) and optic nerve (ON) were identified in all donor eyes. In-depth B-scans from the control eye (e) revealed a normal appearing retina with clearly defined fovea, some evidence of laminar architecture, and no appreciable evidence of retinal thinning or degeneration. Images from the arRP donors (f-h) revealed retinas of appreciable thickness but with less organized architecture and integrity in the macular region (horizontal bracket). In contrast, the perimacular regions showed some evidence of thinned retina relative to the control retina suggesting degeneration. Donor’s 2 (g) and 3 (h) had macular (*) and choroidal (arrow) detachments as indicated in the B-scans. B-scan scale is 0.5mm.

Fig. 4. Histology of arRP donor eyes with EYS mutations

A fundus image of the studied eye (OS) with a schematic drawing of the regions cut and processed for cryosectioning and immunolabeling is shown (a). Toluidine blue-stained plastic sections (1μm) of retinas from donors 1, 2 and 3 and an age-matched control. Morphology of the control retina in the periphery (b) and perifoveal (f) regions displayed typical retinal lamina. Histology of the periphery of all three donors revealed a highly degenerated retina with disorganization of the lamina and cellular layers and gliosis in all areas analyzed (c-e). Photoreceptor outer segments were also absent in all areas analyzed. Intraretinal bone spicule pigments were visible in the retinas of donors 2 (d, arrow) and 3 (e, arrow). In contrast, the perifovea of all arRP donors displayed a prominent inner nuclear layer. Donors 2 (h) and 3 (i) displayed localized areas of RPE atrophy whereas the RPE was thin in the perifovea of donor 1 (g) as compared to the control donor (f). Patchy, disorganized cone remnants were observed on top of the RPE in the perifovea of donors 2 (h, arrowhead) and 3 (i, arrowhead). GCL= ganglion cell layer; INL= inner nuclear layer; ONL= outer nuclear layer; POS = photoreceptor outer segments; RPE= retinal pigment epithelium; Ch = Choroid. Scale bar =50μm.

Fig. 5. Immunocytochemistry of arRP retinal sections with EYS mutations stained with rhodopsin antibodies

Immunofluorescence of arRP retinal sections labeled with antibodies to rhodopsin (green) showed significantly decreased staining when compared to control. The control retina showed that rhodopsin was restricted to the rod outer segments in both the periphery (a) and perifovea (e). Rhodopsin-labeled cells were essentially absent from the periphery of donor 1 (e). However, rhodopsin-labeled cells were still present in the periphery of donors 2 (c) and 3 (d). Of interest, some rhodopsin-labeled cells were observed in the choroid of these eyes (arrows). In the perifovea, donors 1 (f) and 2 (g) displayed no rods while donor 3 (h) displayed a few disorganized rods. Bruch’s membrane is indicated by hashed white line. Nuclei were labeled with TO-PRO-3. GCL= ganglion cell layer; INL= inner nuclear layer; ONL= outer nuclear layer; POS = photoreceptor outer segments; RPE= retinal pigment epithelium. Scale bar = 40μm.

Fig. 6. Immunocytochemistry of arRP retinal sections with EYS mutations stained with cone-specific antibodies

Immunofluorescence of arRP retinal sections labeled with antibodies to cone arrestin (green) and red/green cone opsin (red) showed significantly decreased staining in the periphery when compared to control. In the peripheral (a) and perifoveal (e) regions of the control retina, cone arrestin was distributed along the entire plasma membrane, from the tip of the outer segment to the synaptic base, while the red/green cone opsin was restricted to the outer segments. Cone-specific labeled cells were mostly absent from the periphery of all three arRP donor retinas (b-d). In contrast, cone-specific labeled cells were present but highly disorganized in the perifovea of donor 1 (f). Interestingly, the perifovea from both donor 2 (g) and 3 (h) displayed disorganized cone-specific labeled cells concentrated in the areas that still maintained RPE. Bruch’s membrane is indicated by hashed white line. Nuclei were labeled with TO-PRO-3. GCL= ganglion cell layer; INL= inner nuclear layer; ONL= outer nuclear layer; POS = photoreceptor outer segments. Scale bar = 40μm.

Fig. 7. Immunocytochemistry of retinal sections of an unaffected carrier with a heterozygous EYS mutation

Immunofluorescence of peripheral retina sections of the unaffected mother of donor 1 (donor 4) labeled with antibodies to rhodopsin, cone arrestin and red/green cone opsin showed no significant changes in staining of these proteins as compared to control. The unaffected carrier donor retina displayed both rhodopsin (b, green) and red/green cone opsin (d, red) restricted to the photoreceptor outer segments as observed in control retinas (a, c). Cone arrestin labeling in the unaffected carrier donor retina (d, green) displayed similar distribution when compared to the control retina (c). Nuclei were labeled with TO-PRO-3. GCL= ganglion cell layer; INL= inner nuclear layer; ONL= outer nuclear layer; POS = photoreceptor outer segments. Scale bar = 40μm.