X-linked juvenile retinoschisis: clinical diagnosis, genetic analysis, and molecular mechanisms

Abstract

X-linked juvenile retinoschisis (XLRS, MIM 312700) is a common early onset macular degeneration in males characterized by mild to severe loss in visual acuity, splitting of retinal layers, and a reduction in the b-wave of the electroretinogram (ERG). The RS1 gene (MIM 300839) associated with the disease encodes retinoschisin, a 224 amino acid protein containing a discoidin domain as the major structural unit, an N-terminal cleavable signal sequence, and regions responsible for subunit oligomerization. Retinoschisin is secreted from retinal cells as a disulphide-linked homo-octameric complex which binds to the surface of photoreceptors and bipolar cells to help maintain the integrity of the retina. Over 190 disease-causing mutations in the RS1 gene are known with most mutations occurring as non-synonymous changes in the discoidin domain. Cell expression studies have shown that disease-associated missense mutations in the discoidin domain cause severe protein misfolding and retention in the endoplasmic reticulum, mutations in the signal sequence result in aberrant protein synthesis, and mutations in regions flanking the discoidin domain cause defective disulphide-linked subunit assembly, all of which produce a non-functional protein. Knockout mice deficient in retinoschisin have been generated and shown to display most of the characteristic features found in XLRS patients. Recombinant adeno-associated virus (rAAV) mediated delivery of the normal RS1 gene to the retina of young knockout mice result in long-term retinoschisin expression and rescue of retinal structure and function providing a 'proof of concept' that gene therapy may be an effective treatment for XLRS.

Copyright © 2012 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Retina imaging of individuals with XLRS

(a) Severe retinoschisis involving almost the complete retina observed in a boy 3 months of age carrying a RS1-gene p.R213W mutation. (b) Fundus autofluorescence indicating a spoke-wheel pattern due to overlying foveal retinoschisis in a 12 year old boy carrying a RS1-gene p.R102W mutation. (c) Near-infrared image of foveal retinoschisis in a 21 year old patient carrying a RS1-gene p.D126_L127delinsE mutation. (d) Spectral domain optical coherence tomography in a 22 year old male carrying a RS1-gene p.W96G mutation. The upper scan shows marked retinoschisis in different retinal layers. The lower scan after 3 months of local application of dorzolamide shows a smaller foveal retinoschisis cavity and absence of perifoveal retinoschisis cavities. (e) Color image of peripheral retinoschisis in the lower temporal quadrant of the right eye in a 20 year old male carrying a RS1-gene p.R209H mutation.

Figure 2. Light micrographs of an adult mouse retina immunolabeled for retinoschisin

Left: Differential interference contrast (DIC) image of a mouse retina stained with DAPI to show the nuclear layers. Right: Immunofluorescence image of the same section showing retinoschisin distribution (green) in the retinal cell layers. Intense immunoflurorescence staining is observed in the inner segment layer with more moderate staining in the outer nuclear, outer plexiform, inner nuclear and inner plexiform layers. OS, outer segment; IS, inner segment; ONL, outer nuclear layer, OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bar −20 μm.

Figure 3. Sequence alignment and structural homology model of retinoschisin discoidin domain

(a) Alignment of the discoidin domain sequences of human (Hu-RS1) and mouse (Mo-RS1) retinoschisin with D2 discoidin domain sequences of human Factor V (Hu-FV) and human Factor VIII (Hu-FVIII). Numbering is for retinoschisin; yellow shows amino acid identity; βstrands and spike regions are shown below sequences; cysteine residues involved in disulphide bonds are outlined. (b) Homology model of the discoidin domain of retinoschisin obtained using Factor V as a template. Cysteine residues including those involved in intramolecular disulphide bonds are shown in blue. Modified from (Wu and Molday, 2003).

Figure 4. Oligomeric structure of retinoschisin

(a) SDS gels of wild-type (WT) and mutant retinoschisin proteins run under nonreducing conditions. WT and C40S mutant run as a 186 kDa octamer; C59S/C223S mutant runs as a dimer; and C40S/C59S/C223S runs as a monomer. (b) Working model for the organization of retinoschisin subunits into an octameric complex via disulphide bonding. The octameric structure is maintained by C59–C223 disulphide bonds. Within the octamer, four dimers are maintained by disulphide bonding involving C40. Rs1 domain and discoidin domain (DS) and carboxyl terminal segment (Ct) - are indicated. Modified from (Wu et al., 2005).

Figure 5. Diagram depicting the effect of various disease-causing missense mutations on retinoschisin synthesis, protein folding/ER retention, and subunit oligomerization

(a) Wild-type retinoschisin synthesized off of ribosomes associated with the ER membrane is threaded through the translocon and the signal sequence is cleaved by a signal peptidase in the ER lumen to produce the mature folded retinoschisin (RS1) polypeptide. RS1 assembles into a disulphide-linked octameric complex which is exported from cells via the secretory pathway (not shown). (b) Mutations in the signal sequence (L12H and L13P) prevent the insertion of the nascent polypeptide chain into the translocon of the ER resulting in a misfolded polypeptide localized to the cytoplasm where it is rapid degraded by the proteosome. Mutations in the discoidin (DS) domain enable the nascent polypeptide chain to be transported into the ER lumen, but the protein fails to fold into a native conformation and as a result is retained in the ER. Cysteine mutations (C59S and C223R) in the regions flanking the DS domain result in relatively normal protein synthesis, folding, and disulphide-linked dimerization, but fail to further oligomerize into an octameric complex. As a result the retinoschisin dimers are secreted from cells, but are nonfunctional due to their failure to form disulphide-linked octamers.

Figure 6. Location of selected missense mutations within retinoschisin structure

(a) A linear diagram showing the organization of retinoschisin into it various domains along with selected disease-associated missense mutations (SS – cleavable signal sequence; Rs1 domain; discoidin domain; and Ct - C-terminal segment). (b) Selected missense mutations are shown within a model of the mature retinoschisin subunit. The structure of the Rs1 domain and C-terminal segment is not known and drawn as lines within a dashed ellipse with mutations shown in green spheres. The discoidin domain structure is based on the homology modeling shown in Figure 3b with mutations shown in blue spheres with the exception of R141H which is shown as a red sphere.

Figure 7. Retinoschisin expression and retinal structure in a Rs1h knockout mouse treated with rAAV containing the human RS1 cDNA under control of an upstream mouse opsin promoter (rAAV-mOps-RS1)

The right eye of a 14 day old Rs1h knockout mouse was injected subretinally with rAAV-mOps-RS1 (Treated); the left eye was not injected and served as a contra lateral control (Untreated). One year post-treatment, the mouse was sacrificed and the both the untreated and treated retinas were immunolabeled for retinoschisin and visualized by confocal scanning microscopy. The untreated eye showing no retinoschisin expression was highly disorganized with a merging of the inner and outer nuclear layers and disrupted outer plexiform layer. The treated eye showed retinoschisin expression and distribution comparable to that of a wild-type retina (see Figure 2) and significant improvement in retinal structure and photoreceptor survival as determined by the thickness of the outer nuclear layer (ONL). Bar − 20 micrometers.