Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

Fig. 1. Variation of retinal abnormalities in patients with RS. (A) Macular area with clearly visible spoke-wheel pattern surrounding the fovea in a 16-yr-old patient with RS. (B) Severe RS in a 3-mo-old infant. The RS involves nearly the complete retina forming two bullous schisis cavities with the retina visible behind the lens and only a single small central horizontal area with nonschitic retina.

males are misdiagnosed as amblyopic. Usually, the underlying cause is not detected until a thorough retinal investigation including electroretinography is conducted.

CLINICAL MANIFESTATION

The penetrance of RS is almost complete with the vast majority of male mutation carriers presenting with at least one sign of RS pathology, e.g., foveal changes (3). In contrast, expressivity is highly variable. In our series of 86 patients with RS, manifestations ranged from nearly complete RS in both eyes at the age of 3 mo (Fig. 1B) to normal visual acuity with mild pigmentary macular abnormalities and a negative electroretinogram (ERG) in a 57-year-old male from a single RS family (4). In the majority of cases, the expression of the disease is symmetrical in both eyes; however, a marked asymmetry of visual function can be present, especially in cases where additional complications occur (5). Visual acuity is reduced to 20/100 in most patients, although it may vary greatly. Although macular abnormalities, such as the spoke-wheel pattern in younger patients and pigmentary changes in older patients, are present in nearly all affected males, peripheral retinal abnormalities are less common (in about 40–50% of patients). They are most frequent in the lower temporal quadrant of the retina (4). In these cases, a sharply delineated RS usually is limited to the periphery or mid-periphery, but may extend from the periphery to the macula, including the fovea in some cases. If the inner sheet of the RS degenerates, the retinal vessels may remain running free through the vitreous cavity (presenting as so called vitreous veils). If additional breaks occur in the outer sheet of the RS, a retinal detachment may occur.

In the majority of patients, the disease either shows no or minimal progression (6). Around the age of 30 yr, the macular alterations may change from the characteristic spoke-wheel pattern to unspecific mild retinal pigment abnormalities. In some cases, severe visual loss with increased age has been described (7), although incidence data are not available as a result of the lack of long-term follow-up studies. In contrast to other X-linked disorders, female carriers have rarely been reported with retinal abnormalities or visual loss. In two cases of female RS, unilateral retinal cysts (8,9) and an abnormal

Fig. 2. Full-field ERG. Three responses at dark adaptation (DA) at low-stimulus intensity (first row), high-stimulus intensity (second row), and with special filtering for oscillatory potentials are shown for a normal individual (left column) and a patient with RS (right column). In addition, a single-flash response at light adaptation (LA) and a response to 30 Hz flicker is depicted. Note, the response in the second row reveals a severe reduction in the second part of the response (i.e., the b-wave) in the patient with RS (the so-called “negative” ERG). The conedependent responses at LA and 30 Hz flicker are reduced as well.

electroretinogram were noted (9). In addition, two affected woman were homozygous carriers of disease mutations (10,11).

CLINICAL DIAGNOSIS OF RS

Diagnosis of RS is unambiguous when a typical spoke-wheel pattern or even peripheral RS are present. In boys with bilateral reduced visual acuity, a thorough examination of the fovea with specific high-magnification lenses is necessary to detect retinal abnormalities. If a foveal RS cannot be detected or excluded, other techniques can be used to demonstrate the macular alterations. With optical coherent tomography (OCT), reflectance of retinal structures can be measured facilitating the detection of even small RS cavities (12). Measurement of the fundus autofluorescence will reveal an increased foveal autofluorescence in most cases of RS, most likely because of the altered light transmission in the area of RS. Fluorescein angiography may reveal RPE alterations in older males; however, this is of limited value in children.

The major functional test for the diagnosis of RS is the full-field ERG. Typically, a bright flash of light will elicit a “negative” response from the diseased retina, in which the a-wave is larger than the b-wave in contrast to the normal findings (Fig. 2). Usually, the light adapted responses show an amplitude reduction as well. The origin of the retinal dysfunction is an abnormality in the ONand OFF-pathways on the level of the bipolar cells (13). A “negative” ERG can be associated with various retinal disorders; however, in young males, the only major differential diagnosis is congenital stationary night blindness (CSNB). The combination of macular alteration and a “negative” ERG indicates RS. Recently, detailed evaluation of macular dysfunction with the multifocal ERG has demonstrated a widespread cone dysfunction (14). Other functional tests are of limited value. As in most disorders affecting the macula, color vision may be abnormal to

a variable degree without any value for differential diagnosis. If peripheral RS is present, visual field testing will reveal an absolute scotoma in the corresponding area, but otherwise the visual fields will be normal.

COMPLICATIONS IN RS

Complications include vitreous hemorrhages or retinal detachment. Vitreous hemorrhage mostly clears spontaneously and only rarely requires vitreous surgery. Results of retinal detachment surgery are of limited benefit even with advanced surgical techniques (15). Prophylactic treatment of RS either by laser or vitreoretinal surgery cannot be recommended owing to possible severe complications (4,16). Overall, the frequency of secondary problems is approx 5% in all affected males, with complications most frequently developing in the first decade of life.

Differential Diagnosis in RS

Differential diagnosis of RS includes disorders with congenital or juvenile retinal detachment, other forms of inherited RS, and inherited disorders affecting macular function in the first decade of life.

RS is more frequently compared to disorders with retinal detachment. These include the X-linked Norrie syndrome (NS), in which complete retinal detachment is present at birth and visual function is nearly absent. NS is associated with mutations in the Norrie disease gene on Xp11.4 (17,18). A less severe variant of NS is the X-linked familial exudative vitreoretinopathy (FEVR), also associated with mutations in the Norrie disease gene. Peripheral vascular retinal abnormalities are present in a variable degree, which may lead to retinal detachment. Similar alterations can be observed in autosomal dominant FEVR (Criswick-Schepens syndrome) and can be associated with mutations in the frizzled-4 (FZD4) gene on 11q14.2 (19) or the LRP5 gene on 11q13.2 (20). The ophthalmoscopic features in both forms of FEVR are distinct from RS. Incontinentia pigmentii (BlochSulzberger syndrome) can present with early-onset retinal detachment; however, as the condition is lethal in males, it is not a differential diagnosis for RS. Other forms of congenital or juvenile retinal detachment, e.g., following trauma or retinopathy of prematurity, can usually be excluded by the patient’s history.

Foveal RS has rarely been reported as an apparent autosomal recessive trait in families with predominantly affected females (21–23). In Goldmann-Favre syndrome (alias enhanced S-cone syndrome), which is associated with mutations in the NR2E3 gene on 15q23 (24), a foveal RS may be present. The ERG is quite different from RS and distinguishes the two disorders.

Other inherited conditions affecting macular function are the macular dystrophies and CSNB. The macular dystrophies, e.g., Stargardt disease, usually show a markedly progressive course within 1 or 2 yr, which is not typical for RS. However, as expression in both disorders varies, an ERG can easily distinguish RS from other macular dystrophies because of the presence of the “negative” ERG response (Fig. 2). CSNB, most frequently inherited as an X-linked trait, presents with a similar reduction of visual function and shows a “negative” ERG similar to RS (25). In contrast to RS, the retina is normal on ophthalmoscopy.

TREATMENT OPTIONS

In most cases, treatment of RS is limited to the prescription of low-vision aids. Surgical interventions are indicated in vitreous hemorrhage without spontaneous resolution or when retinal detachment occurs.

ISOLATION OF THE RS1 GENE AND STRUCTURAL FEATURES OF ITS GENE PRODUCT

In 1969, a first tentative association of RS with Xg blood group markers was reported suggesting a localization of the genetic defect to the distal short arm of the X chromosome (26). Later, a number of polymorphic DNA markers derived from Xp became available and mapped the RS locus more precisely to chromosomal region Xp22.1-p22.2 (27–29). In the following years, the DNA marker map of the X chromosome grew increasingly dense, eventually facilitating the refinement of the RS1 gene locus to an approx 1000-kb interval on Xp22.13 flanked by markers DXS418 on the distal side and DXS999 on the telomeric side (30,31). Following the initial localization, the minimal RS region on Xp22.13 was subsequently cloned and searched for disease gene candidates. From a total of 14 genes positioned within the DXS418-DXS999 interval (32), one transcript, later termed RS1 (alias XLRS1), was found to be abundantly expressed exclusively in the retina (33). Mutational analysis of RS1 in a number of multigeneration RS families revealed distinct mutations segregating with the disease, thus providing strong and convincing evidence for a causal role of the gene in the etiology of RS (33).

The RS1 gene locus spans 32.4 kb of genomic DNA and is organized in six exons coding for retinoschisin, a 224-amino acid protein of which the N-terminal 23 amino acids reveal a signature characteristic for proteins destined for cellular secretion (33). Significant homology exists between a large portion of the predicted retinoschisin sequence (157 amino acid residues including codon 63 to codon 219) and the so-called discoidin domain, which was first identified in the discoidin I protein of the slime mold Dictyostelium discoidium (34). This motif is highly conserved in a number of secreted and transmembrane proteins from many eukaryotic species and is known to be involved in functions such as neuronal development and cellular adhesion (35–37). Two minor portions of the RS1 protein are unique with no homologies to other known proteins. N terminal to the discoidin domain is a 38-amino acid RS1-specific sequence, whereas 5 unique amino acid residues flank the discoidin domain on the C-terminal side. Functional properties of the two RS1specific protein domains have not been identified so far, but may be confined to structural aspects of subunit assembly in protein complex formation (38,39).

SPECTRUM AND MOLECULAR PATHOLOGY

OF RS-ASSOCIATED MUTATIONS

As of January 2005, 127 distinct disease-associated mutations have been reported from patients with RS of various genealogical ancestry, such as British (40), Chinese (41), Colombian (42), Danish (43), Finnish (44), French (45), German (33,45), Greek (40), Icelandic (46), Italian (47), Japanese (48), North American (33,49), Swedish (50), and Taiwanese (51). These findings further confirm the initial disease-association of the

Fig. 3. RS-associated mutations in the RS1 gene. The RS1 locus is drawn schematically with six coding exons represented by bars and intronic sequences by lines. Bar colors indicate the functionally distinct domains of the RS1 protein encoded by the respective exonic sequences (yellow = N-terminal 23-amino acid signal peptide; gray = RS1-specific domain; orange = discoidin domain). Distinct mutations reported in the RS mutation database (http://www.dmd.nl/) are divided in three categories including missense mutations, mutations resulting in protein truncation (nonsense, splice site, frame shift, in-frame deletion), and larger genomic rearrangement causing the loss of partial or complete exons.

RS1 gene and demonstrate locus homogeneity within the studied ethnic backgrounds (although with considerable allelic heterogeneity). Of the identified mutations, 75 (59.1%) are missense, 18 (14.2%) cause a shift in the reading frame consequently resulting in a truncation of the protein, 13 (10.2%) represent a nonsense mutation, 11 (8.7%) affect the correct splicing of the pre-messenger RNA (mRNA), and 1 (0.8%) results in an in-frame deletion of a single amino acid (Asn85del) (Fig. 3). Nine mutations involve genomic deletions of entire or partial exons and range in size from 8 bp (321_326+1del) to at least 15 kb (1-?-184+?del). Three of the smaller genomic deletions (173-184+21del, 181_184+10del, 321_326+1del) affect splice donor sequences and are likely to interfere with correct splicing of the pre-mRNA sequence. With the exception of the missense mutations and the single in-frame deletion, the other sequence changes represent true null alleles and thus should produce no protein or truncated, non-functional, versions of the RS1 protein.

Although some families segregate unique mutations (e.g. Gly70Arg or Asn104Lys), a number of sequence alterations are recurrent (for a comprehensive listing of mutations, see RS1 mutation database at http://www.dmd.nl/). Most notably, the latter type of sequence changes are exclusively missense mutations with the most frequent disease alleles Glu72Lys and Arg102Trp reported 50 and 25 times, respectively. The missense mutations Gly70Ser, Trp96Arg, Arg102Gln, Gly109Arg, Arg141Cys, Pro192Ser,

Arg200Cys were found in eight or more apparently unrelated families. In addition, the Glu-72 and the Gly-109 residues have been affected by four different amino acid substitutions, codons Arg-197 and Arg-209 were affected three times each (Fig. 3). These commonly altered amino acid residues could be indicative of mutational hotspots and/or functionally important moieties of the RS1 protein.

The distribution of missense mutations along the coding sequence of the RS1 gene is nonrandom with significant clustering of this type of alterations in exons 4 to 6 (Fig. 3). This uneven distribution coincides with the extent of the discoidin domain encoded by codons 63 through 219. Although the 157 amino acid residues of the discoidin domain are affected by 68 missense mutations (0.43 mutations per codon), there are only 7 missense mutations reported in the remaining 67 amino acids of RS1 (0.11 mutations per codon). This amounts to an approximately fourfold excess of missense mutations within the discoidin domain compared to the rest of the protein. In contrast, other types of mutations combined (i.e., frame shift, nonsense, splice site, and in-frame deletion mutations) are randomly distributed within the coding region. Compared to 23 such mutations within the discoidin domain (0.15 mutations per codon), 8 mutations are localized outside of the conserved motif (0.12 mutations per codon). Together, these data suggest that absence of RS1 or loss of protein function may be the main molecular mechanism underlying RS pathology. In particular, the discoidin domain appears to be most crucial for RS1 function with strong constraints on the proper amino acid sequence.

To assess the pathological mechanisms of RS1 missense mutations, Wang and associates (52) have conducted in vitro studies expressing mutant RS1 protein (Leu12His, Cys59Ser, Gly70Ser, Arg102Trp, Gly109Arg, Arg141Gly, Arg213Trp). Their findings have led to the suggestion that in the majority of cases failure of cellular secretion (either partial or complete) may underlie disease pathology. Based on an extended series of elegant biochemical experiments, Wu and Molday (38) and Wu and associates (39) have developed a structural model of the RS1 discoidin domain that identifies particular cysteine residues as crucial components for intraand intermolecular disulfide bond formation and thus proper protein folding and subunit assembly (Fig. 4A,B). Essentially, intramolecular disulfide bonds are formed between residues Cys-63 and Cys-219 and between Cys-110 and Cys-142 thus playing a central role in proper subunit folding (Fig. 4A). Eight such subunits are then joined together by disulfide bonds between the discoidin-flanking cysteine residues Cys-59 and Cys-223, which are crucial for subunit oligomerization. Within this complex, the homomeric subunits are further organized into dimers by Cys-40-Cys40 disulfide bond formation with dimerization and octamerization processes evidently independent of each other (39) (Fig. 4B).

Based on this model, Wu and Molday (38) have pointed out three specific disease mechanisms underlying RS. First, pathological missense mutations in the discoidin domain (e.g., Glu72Lys, Gly109Glu, Cys110Tyr, Arg141Cys, Cys142Trp, Asp143Val, Arg182Cys, Pro203Leu, Cys219Arg) cause protein misfolding and consequently retention of the mutant protein in the endoplasmatic reticulum (ER). Second, missense mutations affecting the disulfide-linked subunit assembly (e.g., Cys59Ser, Cys223Arg) do not significantly interfere with cellular secretion but cause a failure to assemble a functional oligomeric RS1 protein complex. Third, missense mutations in the 23-amino acid leader sequence of RS1 (e.g., Leu13Pro) prevent proper insertion of the protein into the

Fig. 4. Topology and subunit organization of the retinoschisin discoidin domain. (A) Ribbon diagram of the retinoschisin-discoidin domain modeled after the F5/8-type C-domain fold. The eight core β strands are labeled β1–β8. The three loops (“spikes”) demonstrating amino acid sequence-dependent ligand affinities are marked. The intramolecular disulfide bonds C63-C219 and C110-C142 as well as the C-terminal (C223) and N-terminal (C59) cysteine residues participating in intermolecular disulfide bond formation are shown (38). (B) Schematic illustrating the formation of the homo-octameric RS1 complex from intermolecular disulfide-linked monomers. Subunits within the octamer are further organized into dimers mediated by C40–C40 disulfide bonds (39).

ER membrane resulting in cellular mislocalization and thus defective secretion. Together, these studies suggest that the known disease-associated RS1 mutations (i.e., missense mutations and protein-truncating mutations) represent loss-of-function mutations. This is also consistent with the observation that disease severity in RS does not appear to be correlated with the specific type of mutational change.

FUNCTIONAL PROPERTIES OF RETINOSCHISIN

The Discoidin Domain and Its Putative Role in Protein Function

Retinoschisin belongs to a family of proteins that contain one or two phylogenetically highly conserved discoidin domains (53). This motif, also known as the F5/8 type C-domain fold, has been found in a variety of proteins such as coagulation factors V and VIII (F5, F8), milk fat globule-epidermal growth factor (EGF) factor 8 (MFGE8), EGFlike repeatsand discoidin I-like domains-containing protein-3 (EDIL3), neurexin IV (NRXN4), neuropilin-1 and -2 (NRP1 and NRP2, respectively), discoidin domain receptors-1 and -2 (DDR1 and DDR2, respectively), or aortic carboxypeptidase-like protein. Many of these proteins are expressed only transiently, in response to specific stimuli or during development and are associated with cellular adhesion, migration, or

Fig. 5. Expression of murine Rs1h. (A) Reverse-transcriptase polymerase chain reaction analysis of Rs1h, the murine orthologe of human RS1, in comparison to the cone-rod homeoboxcontaining gene (Crx) and β-actin. Rs1h mRNA expression is detectable starting at P0 with increasing expression until postnatal day P7. This level of expression is then maintained throughout life. This is in contrast to the Crx transcript level where peak expression around P11 decreases in development and only low levels of expression remain during adulthood. β-actin serves as a control for cDNA integrity. (B) RS1 immunolabelling of outer and inner murine retina. Arrows point to the labeling of bipolar cell surfaces.

aggregation via membrane surfaces. High-resolution crystal structures of human F5 and F8 C2 domains have been reported (54,55) and provide a framework for the homology-based modeling of the three-dimensional structure of the RS1 discoidin domain (Fig. 4A). Its central feature is an eight-stranded antiparallel β-barrel consisting of tightly packed five-stranded (β1, β2, β4, β5, β7) and three-stranded (β3, β6, β8) β-sheets. This conserved β-barrel scaffold exhibits three protruding loops (“spikes”) (Fig. 4A) that have been shown to display specific amino acid-dependent affinities (56). A number of ligands have been identified for discoidin-containing proteins and include negatively charged membrane surfaces with defined phospholipids (F5 [57], F8 [58], MFGE8 [59]), integrin receptors that bind to the classic integrin-binding RGD sequence (discoidin I protein of D. discoideum [60]; EDIL3 [61]), as well as collagen (DDR1, DDR2 [62,63]). Modelling of the RS1 loops indicate that several hydrophobic amino acid residues may be exposed at their spike apexes. This could suggest that similar to coagulation factors F5 or F8, the discoidin domain of RS1 could also interact with phospholipid membranes.

Expression of RS1 and Localization of the Protein in the Mammalian Retina

Expression of RS1 is restricted to the retina as shown by Northern blot hybridization to a number of human (33) and mouse (64) tissues. Furthermore, in situ hybridization experiments revealed RS1 mRNA transcripts in rod and cone photoreceptor inner segments (65,66) and also in other cell bodies of the retinal layers namely in bipolar cells, amacrine cells, and retinal ganglion cells (67). In postnatal eye development, measurable levels of RS1 expression resume around P1 and reach a maximum between P5 and P7 (Fig. 5A). This level of expression is then maintained throughout adult live indicating that continued de novo synthesis of RS1 is required and is essential for the maintenance of retinal integrity.

As suggested by the presence of an N-terminal signal peptide sequence, retinoschisin was shown experimentally to be secreted from the cell after removal of the first 23-amino acid residues (38). To localize the protein within the mammalian retina, a number of monoand polyclonal antibodies were raised against the RS1-specific N-terminus of the mature protein (66–69). In the adult retina, prominent immunolabeling is consistently observed at the extracellular surfaces of the inner segments of both the rod and cone photoreceptors, most bipolar cells, as well as the two plexiform layers (66–68,70) (Fig. 5B). There is still some controversy with regard to the immunoreactivity of ganglion cells (66,67) and Müller cells (70).

Developmental Expression of Retinoschisin

Immunostaining of the developing rat retina revealed weak RS1 labeling at P6 within the neuroblastic zone (68). The staining intensity of the outer retina increased over time with intense staining of the newly formed inner segment layer at P10. Adult pattern labeling was reached around P12. Developmental expression of RS1 was also investigated in the mouse retina (67). Similar to the rat, inner segment labeling of the photoreceptors became evident by P7 and more prominent by P10 to P14 when finally an adult pattern of immunostaining developed. Interestingly, Takada and associates (67) observed a transient pattern of expression of RS1 in retinal ganglion cells in murine stages E16.5, P1, and P3, but not at later time points of development. With subsequent layer formation, this expression was found to move posteriorily through the retinal layers as additional types of neurons became differentiated. If confirmed, this would suggest that RS1 is produced locally at several defined neuronal cell populations (67), arguing against the need for a trans-retinal transport system of photoreceptoror bipolar-secreted retinoschisin as suggested by Reid and associates (70).

Mouse Model for X-Linked Juvenile Retinoschisis

The ability to develop mouse strains with targeted mutations in a defined protein has tremendously expanded our spectrum of experimental tools allowing us to gain insight into physiological mechanisms controlled by these proteins. In particular, because photoreceptor cells cannot be maintained in vitro, the study of animal models with a mutant photoreceptor protein is instrumental in understanding pathological processes and in testing therapeutic approaches. With these objectives in mind, a mouse line deficient in Rs1h, the murine orthologe of the human RS1 gene (64), was generated and the clinical phenotype characterized by electroretinography, histology, and immunohistochemistry (69). The Rs1h−/Y mouse shares several diagnostic features with human RS, including the typical “negative” ERG response and the development of cystic structures In addition, the diseased murine retina shows a general disorganization and a disruption of the synapses between the photoreceptors and bipolar cells accompanied by a dramatic and progressive loss of photoreceptor cells (Fig. 6). Starting at postnatal day 14, minor and major gaps within the inner nuclear layer become evident. Some of the schisis cavities are filled with fragmented nerve cell terminals containing synaptic vesicles. In accordance with the observed lack of immunoreactivity of Müller cells (67,68), glial cells are relatively unaffected in the Rs1h−/Y retina (69).

Fig. 6. Retinal cryosections of 12-mo-old wild-type and retinoschisin-deficient mice (Rs1h–/Y) stained with hematoxylin and eosin. In the Rs1h–/Y retina, note the striking reduction of photoreceptor cell bodies of the outer nuclear layer (ONL) and the layer disorganization/cell displacement greatly affecting the outer plexiform layer (OPL) and the inner nuclear layer (INL). The outer (OS) and inner segments (IS) of the photoreceptor are markedly shortened in the knockout mouse, whereas histology of the inner plexiform layer (IPL) and ganglion cell layer (GCL) appears normal.

Evidently, the site of schisis in the retinoschisin-deficient mouse retina within the inner nuclear layer is not consistent with the classic view of human RS pathology formerly described as a splitting between the inner limiting membrane and the nerve fiber layer (2,71). However, recent histopathological data from a 19-year-old patient with RS, have emphasized a more general disorganization of the retinal layers with splitting readily apparent in the inner and outer plexiform layers (IPL and OPL, respectively) (72). Similarly, cross sectional views of younger patients with RS by OCT revealed schisis cavities in multiple layers, both superficial and deeper in the retina including the outer and inner nuclear layers (ONL and INL, respectively) (5,12,73).

Gene Therapy in RS

Because RS pathology arises as a consequence of a missing functional RS1 protein (38,66), delivery of the normal RS1 gene product to the retina may be an amenable treatment option for RS. To test this, we have delivered an adeno-associated virus (AAV) serotype 5 vector containing the human RS1 cDNA under the control of the mouse opsin promoter (AAV5-mOPs-RS1) into the subretinal space of the right eyes of Rs1h-deficient 15-day-old mice (74,75). At 2 and 3 mo after treatment, ERG recordings revealed a significant improvement of a- and b-waveform characteristics in injected over noninjected left control eyes. At 5-mo, five treated animals were sacrificed and retinal cryosections analyzed by immunohistochemistry (Fig. 7). Retinal tissues from treated eyes showed an RS1 immunostaining pattern similar to that of wild-type with intense RS1 labeling present in the inner segment layer while more moderate staining was seen in the ONL and