Retinal Pathology of Canine X-linked Progressive Retinal Atrophy, the Locus Homologue of RP3

Abstract

purpose. To describe the course of photoreceptor disease in canine X-linked retinal degeneration.

methods. Retinas from 55 dogs (44 males, 8 carrier females, 3 homozygous females) were obtained by enucleation under general anesthesia. After fixation and dehydration, tissues were embedded in epoxy resin, sectioned at 1 μm for light microscopy and stained with azure II/methylene blue and a paraphenylenediamine counterstain. For electron microscopy, regions identified by light microscopy were selected and cut at 60 nm. Sections were stained with uranyl acetate-lead citrate. Electroretinography from an additional group of normal males, affected males, and carrier females was performed and the rod and cone responses evaluated.

results. The earliest lesion detectable by electron microscopy was vesiculation of rod discs, followed by disruption of outer segments and death of rods. Loss of cones and progressive atrophy of inner retinal layers followed. Lesions were most severe in the peripheral retina and advanced toward the optic disc with disease progression. Significant variation in disease severity was present in males despite the presence of the same disease allele in all affected dogs. Carrier females displayed generalized reduction in photoreceptor density as well as multifocal areas of complete rod loss. The electroretinogram (ERG) findings were compatible with the histopathologic abnormalities. Homozygous females had lesions similar to those seen in affected males.

conclusions. X-linked retinal degeneration is characterized by initial degeneration of rod photoreceptors, followed by loss of cones and progressive atrophy of the inner retina. Carrier females display a phenotype consistent with random X-chromosome inactivation. Variation in genetic background may alter expression of the disease allele in affected animals, thus accounting for variation in phenotypic expression of the disease.

Retinitis pigmentosa (RP) describes a group of genetically heterogeneous inherited retinal disorders characterized by progressive photoreceptor disease, degeneration, and cell death. RP may be inherited in autosomal recessive, dominant or X-linked patterns. Of these forms, the X-linked form is the least prevalent (16%–33% of all RP cases) ^{1 2} but the most severe in early age of onset and rate of progression. ³ Approximately 70% of X-linked retinitis pigmentosa (XLRP) cases map to one of two loci on the X chromosome: RP2 or RP3. ⁴ RP3 is the predominant form of XLRP and has been localized to a 1-cM region at Xp21. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for 20% to 30% of RP3 cases. ^{5 6 7} More recently, mutations in a gene with homology to human cofactor C, a protein involved in the ultimate step of β-tubulin folding, have been demonstrated in patients with RP2. ⁸  

Before the discovery of RPGR, there were only two reported histopathologic studies of X-linked RP. ^{9 10} However, it is not known to which linkage group the subjects in these studies belonged; thus the influence of genetic heterogeneity on the development of retinal lesions could not be assessed. Several descriptions of clinical and histopathologic findings in patients with RPGR mutations have recently been published. ^{11 12 13 14} Clinically, disease severity appears to correspond to the site of the mutation in RPGR. Mutations in the RCC1-homologous regions appear to create a more severe phenotype than molecular defects in the 3′ half of the gene. Retinal disease in a carrier is described in one of these studies, ¹² but the advanced degree of retinal degeneration in this case only provides a histologic picture dominated by nonspecific end-stage degenerative changes. 

Assessment of early retinal disease in XLRP is generally dependent on electroretinographic (ERG) studies. ^{12 15} From these data, the target cell affected and the sequence of disease and degeneration of rods and cones, respectively, are unclear. Additionally, it is unclear whether the broad phenotypic variability present in carriers is due to locus, genetic, or allelic heterogeneity, or to skewed X-inactivation patterns. ¹³ Consequently, the sequence of degenerative events in XLRP, the age of onset, the spatiotemporal characteristics, and the influence of genetic variability on disease development remain poorly defined. 

There is a paucity of spontaneous animal models forX-linked retinal disease in general and XLRP in particular. Two genetically altered mouse models, the choroideremia knockout mouse ¹⁶ and the mosaic rds mutant mouse ¹⁷ have been produced. The choroideremia model does not accurately reproduce the human disease because the mutation is lethal in males and can thus be assessed only in heterozygous females. The rds mosaic mouse was generated after insertion of a rescue transgene for rds/peripherin into the X chromosome of rds ^−/− mice. In heterozygous females, random X chromosome inactivation resulted in patchy photoreceptor rescue similar to the general pattern observed in carriers of XLRP. To date, the Siberian husky with X-linked progressive retinal atrophy (XLPRA) represents the only spontaneous animal model of X-linked retinal disease. ¹⁸ Hemizygous males show ophthalmoscopic evidence of generalized retinal degeneration (hyperreflectivity due to retinal thinning, vascular attenuation, optic disc pallor) and ERG evidence of progressive photoreceptor dysfunction, predominantly affecting rods. These abnormalities typically become apparent after 1 year of age, and progress to severe visual impairment by 2 to 3 years. Obligate heterozygous females show clinical evidence of random X inactivation that is apparent by the presence of multifocal patches of atrophy surrounded by ophthalmoscopically normal retina. 

Our recent studies have indicated that XLPRA is the locus homologue of RP3. ¹⁹ We have found that the disease is tightly linked to the RPGR gene with a lod score of 11.7, and zero recombinants. Cloning of the canine RPGR cDNA, and examination of the coding sequence for disease-causing mutations have shown no sequence differences between normal and affected animals. Moreover, examination of several of the retinal-expressed transcripts also has shown no abnormalities. Thus, XLPRA is similar to XLRP in most human patients whose disease is linked to the RP3 locus, but in whom a mutation has not yet been identified. Because the RPGR gene is large and complex, and has multiple transcripts that are tissue specific, ²⁰ it is possible that other mutations, in either humans or dogs, are present in the promoter region or in the splice sites. Alternatively, there may be mutations in regions that are some distance away from the RPGR gene but that alter expression of the gene because of a positional effect. 

The purpose of our study was to define the sequence of cytologic changes characteristic of XLPRA. We also have described the topographic distribution and temporal characteristics of the disease. Although this study focused on the changes seen in hemizygous males, preliminary data on heterozygous and homozygous affected females are presented as well. The dogs in this study are from a colony generated by outbreeding a single affected male dog to normal laboratory beagles. This has resulted in the propagation of a single mutant allele throughout the colony, thus eliminating the effect of genetic and allelic heterogeneity on the disease process. Because the XLPRA phenotype segregates with zero recombination with an intragenic canine RPGR polymorphism, XLPRA represents a genotypically defined model for RP3. 

Materials and Methods

Animals

We developed the XLPRA colony from one affected male Siberian husky by outbreeding to laboratory beagles known to be free from other gene loci causally associated with inherited retinal degeneration. Details of the breeding used to derive the colony have been presented previously. ¹⁸ The colony is housed in the Retinal Disease Studies (RDS) Facility (Kennett Square, PA) and is supported by the National Institutes of Health (Grant EY06855) and the Foundation Fighting Blindness. 

The purpose of this colony is to produce enough animals of known genotype to use for linkage and positional cloning studies of the XLPRA gene, and identification of the disease-causing mutation. To this end, a series of breedings were performed that are detailed in the pedigree (Fig. 1) . Because histologic assessment of disease is the benchmark criterion used for assignment of phenotype in the linkage studies, we selected 55 dogs for detailed morphologic examination. From histologic examination of retinas in the initial study describing XLPRA, ¹⁸ it was apparent that the disease process in affected males spanned the age bracket from approximately 8 months to 2 years of age. Before 8 to 12 months, the retina appears histologically normal, and after 2 years, the retina is diffusely affected with the nonspecific changes characteristic of end-stage retinal atrophy. Consequently, to evaluate the earlier and more specific stages of the disease process, most of the male dogs in this study were examined during the period of active retinal degeneration. Of 44 male dogs studied, 16 were normal males (age range, 11 months to 1.5 years), and 22 were affected males (21 dogs between 11 months and 2 years of age and 1 [dog 1, propositus] 7 years of age). Six male dogs were less than 11 months of age (indicated by stars in the pedigree [Fig. 1 ] and in Table 1 ) and could not be assigned a status on the basis of histopathology because their retinas all appeared normal at this age. However, by typing with the RPGR marker, ²¹ two dogs segregated with the disease allele, and four dogs segregated with the normal allele and were classified accordingly. Of the remaining 11 dogs, 8 were carrier females (age range, 1.4–7.8 years), and three were homozygous affected females (age range, 3.7–4.9 years; Table 1 ). Females were considered carriers if they had produced both normal and affected male offspring. Carriers have funduscopically evident multifocal retinal thinning, but do not develop clinical blindness. Homozygous affected females were the progeny of carrier female–affected male matings, and showed severe diffuse retinal degeneration at an age comparable to the age of affected males. In addition, when typed with the RPGR polymorphism, all affected females were homozygous for the disease-associated allele. 

Tissue Processing and Evaluation

Eyes were obtained by enucleation of dogs deeply anesthetized with intravenous sodium pentobarbital, and the dogs were euthanatized after enucleation. All procedures involving animals were performed in adherence to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Tissues were fixed using a triple-fixative protocol (3% glutaraldehyde-2% formaldehyde, 2% glutaraldehyde-1% osmium tetroxide, and 2% osmium tetroxide), as previously described. ²² After fixation, the posterior segment was trimmed into four quadrants extending from the optic disc to the ora serrata, taking care to preserve their topographic orientation. After dehydration, tissues were embedded in an epoxy resin (PolyBed 812, Polysciences, Warrington, PA), sectioned at 1 μm for light microscopy on a microtome (Supercut 2065; Leica , Deerfield, IL), and stained with azure II-methylene blue and a paraphenylenediamine counterstain. 

For each dog, 1-μm sections extending continuously from the optic disc to the ora serrata of superior, inferior, and temporal meridians were evaluated using a light microscope (Axioplan; Carl Zeiss, Thornwood, NY). For electron microscopy selected regions identified by light microscopy were cut at 60 nm, stained with uranyl acetate-lead citrate and examined with a transmission electron microscope (model 109; Zeiss). Eyes from one of the affected females (dog 17) was fixed in Bouin’s solution, embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin-eosin. Eyes from the remaining two affected females were embedded in plastic as described. In addition to establishing the cytologic characteristics of the photoreceptor disease from the retinal sections, we determined the topographic distribution of the disease. For this, each retinal section was sequentially examined from optic nerve to ora serrata, using the× 40 objective. For each field, several indicators of retinal degeneration were noted (rod and cone outer segment morphology, rod and cone inner segment morphology, rod-to-cone ratio, number of nuclei in the outer nuclear layer [ONL], and width of outer plexiform layer[ OPL]) and used to establish the topographic pattern of disease severity. In addition, the number of photoreceptor nuclei in the ONL was counted at three locations of the superior and inferior quadrants: 350 μm distal to the optic nerve, 350 μm proximal to the ora serrata, and midway between these two points. At each location, the number of nuclei in three adjacent photoreceptor columns was counted and an average taken to provide the final value. 

ERG

ERGs were recorded from a selected group of halothane-anesthetized dogs in response to stimuli and under conditions designed to enable separate evaluation of rod- and cone-mediated response components. ²² In brief, the ERG was recorded during dark adaptation in response to stimulation with low-intensity red light. Once dark adapted, the retina was stimulated with scotopically balanced red and blue light flashes and then with white-light stimuli of increasing intensity. Finally, to isolate the rod and cone components of the ERG better, a series of responses were recorded to repetitive (flicker) stimuli. Rod flicker responses were recorded to 5- and 12-Hz flashes of dim white light (2.0 log foot lamberts), and cone flicker responses to 5-, 12-, and 30-Hz bright white light (4.0 log foot lamberts). 

Results

Retinal Morphology

Hemizygous Males.

Photoreceptors undergo an ordered sequence of cellular changes culminating in degeneration and death of rods initially, followed by cone degeneration. These cytologic changes are stereotypic and consistent within and between subjects and are described in the next section regarding the sequence of degenerative events. However, despite presence of the same disease allele in all affected animals, phenotypic variability is seen in the spatial distribution of disease and in its temporal expression. These changes are described in the later section regarding distribution of disease in affected males. 

Sequence of Degenerative Events in Photoreceptors.

A distinct sequence of cellular transformations affecting the photoreceptors and outer retinal layers was evident in most sections examined. To define the disease process better, we categorized the light microscopic appearance of this progression into stages (stage 0 through stage 7). This staging system is described below, summarized in Table 2 , and illustrated in Figure 2 . Because most affected males were examined at an age when retinal degeneration is most active, all stages were present in most eyes. Normal retina (stage 0; Fig. 2A ) was present in the regions adjacent to the optic nerve in most eyes. The severity of the lesions in individual eyes was variable and topographically defined (see following section). In the normal canine retina, photoreceptor outer and inner segments are sharply defined and are aligned parallel to one another. There are approximately 8 to 10 photoreceptor nuclei per column in the ONL. Subsequent stages (stage 1 is the least severe and stage 7 the most severe) were present as a continuum that worsened in severity from the posterior pole to the periphery. For ultrastructural examination, retinal sections from affected males were selected to obtain the ultrastructural correlates of the first four stages of XLPRA, defined by light microscopy. Emphasis was placed on stage 1 in an effort to detect the earliest typical changes characteristic of the disease. Sections from comparable regions of the retina from normal dogs were used as control samples. 

In the earliest stages of the disease (stage 1), rod outer segments (ROSs) were mildly disorganized and had slightly irregular profiles (Fig. 2B) . A faint banding pattern was seen in many ROSs, which was due to separation of packets of discs (Figs. 2B 3 A). Otherwise, the retina was normal. Ultrastructurally, approximately half the ROSs exhibited several changes affecting discs. The earliest detectable was an apparent fragmentation of individual discs seen as rows of small vesicles located between intact discs (Fig. 3C) . These rows of vesicles were located at all levels of the ROS, and were seen in approximately 50% of these structures. This change was presumably the cause of the ROS banding pattern observed by light microscopy. As these vesicles became larger and coalesced, variably sized vacuoles were formed that disrupted the architecture of adjacent normal discs (Figs. 3D 3E) . Groups of discs were disorganized and disoriented, resulting in irregular outer segment contours. Phagosomes, indicative of normal phagocytosis of shed photoreceptor discs, were present, and the retinal pigment epithelial (RPE) cells themselves, as well as Bruch’s membrane, were normal at this stage and remained so throughout the disease process. 

Distinct rod degeneration became apparent in stage 2 (Fig. 2C) . ROSs were distorted and fragmented. There was moderate loss of rods as evidenced by visible spaces between remaining rod inner segments (RISs), and the ONL was reduced to approximately 70% of its original thickness. Cones had widened inner segments in regions of rod loss but were otherwise normal. Ultrastructurally, all ROSs were disrupted to variable degrees by the accumulation of membrane-bound vesicles (Fig. 3B) . These were generally contained within the cell membrane of the outer segment. Coalescence of vesicles to form large vacuoles resulted in disruption of the cell membrane, fracture of outer segments, and location of vesicular profiles in the interphotoreceptor space (Fig. 3F) . In some cases, individual outer segments had completely disintegrated and were reduced to an amorphous mass of vesicular material. Associated with this degeneration, small numbers of macrophages were present in the interphotoreceptor space. RISs were shorter and broader than normal but were otherwise ultrastructurally normal. The only other abnormality at this stage was a subjective increase in the number of myelin-like figures located in photoreceptor inner segments and cell bodies (Fig. 4C ). These structures consisted of whorls of electron-dense membranous material usually located in the cytoplasm, although, in some cases, they were found within lysosomal inclusions. Cells containing myelin-like figures increased in number as the disease progressed and extended from the ONL to involve the OPL and finally the inner plexiform layers. 

By stage 3 (Fig. 2D) , rod degeneration was more advanced. ROSs were reduced to short stacks of discs interspersed among photoreceptor debris in the subretinal space, and RISs were shortened to half their original length. Phagocytic cells appeared in the subretinal space, together with occasional extruded rod nuclei. The ONL was reduced to approximately 50% of its original width, and the cone-to-rod ratio was increased because of preferential rod loss. Cone inner segments were widened, but cone outer segments were still present. Ultrastructurally, ROSs were completely fragmented and could be seen as profiles of short disc packets floating in the subretinal space or enclosed in RPE microvilli (Fig. 4A) . The subretinal space was filled with vesicular debris from disintegrated outer segments and abundant fine granular material. RISs were markedly reduced in number. They were separated by wide gaps in which the microvillous processes of Müller cells were now clearly visible. These remaining inner segments were wider and shorter, ended bluntly at their apices, and extended irregular pseudopodia from their lateral surfaces. Photoreceptor nuclei were reduced to approximately half the original number (Fig. 2D) . This was reflected in the large cytoplasmic spaces that could be seen between the photoreceptor nuclei beneath the external limiting membrane. Degeneration also was apparent in a few cones by stage 3. 

A spectrum of degenerative changes was seen more proximally in the photoreceptor cell bodies. These ranged from intracytoplasmic myelin figures (Fig. 4C) to multiple membrane bound intracytoplasmic vacuoles (Fig. 4D) . These degenerative changes were seen predominantly in rod cell bodies, although they were present occasionally in cone cell bodies as well. However, at no time were the characteristic outer segment changes observed in rods present in cones. Very few dead cells were apparent; when present, these had a shrunken, pyknotic nucleus surrounded by a contracted dense cytoplasm (Fig. 4E) . 

Complete rod degeneration was apparent in stage 4 (Fig. 2E) , and cone disease was now distinct. Cone inner segments were shorter and wider and often showed cytoplasmic vesiculation. When present, cone outer segments were distorted and shortened, and the subretinal space was narrowed. The ONL was two to three nuclei thick and contained many pyknotic nuclei. The OPL was narrowed, but the remaining inner retinal layers were normal. Outer segment material was rarely visible, but, when present, it appeared as disorganized and disoriented profiles. Most remaining inner segments at this stage were cones whose apices were almost apposed to the RPE as a result of marked narrowing of the subretinal space (Fig. 4B) . Apart from widening and shortening, many cone inner segments appeared to be relatively well preserved. Occasional inner segments had abnormalities ranging from those described in stage 3 to marked cytoplasmic membrane blebbing and complete disintegration. Although macrophages were still seen in the subretinal space (Fig. 4B) , the interphotoreceptor layer appeared almost clear, with minimal amounts of the granular debris so evident in the previous two stages. The OPL was markedly narrowed as a result of severe photoreceptor loss. Degenerative changes (vacuolation, membranous vesiculation) were seen within remaining photoreceptor cell bodies and the rod and cone synaptic terminals as well. Spaces previously seen between photoreceptor nuclei were now filled by hypertrophic Müller cell cytoplasm. 

By stage 5 (Fig. 2F) , a few residual inner segment structures and scattered membranous debris remained in the interphotoreceptor space. There was multifocal collapse of the interphotoreceptor space with apposition of the RPE apical surface to the external limiting membrane. The ONL contained a single row of photoreceptor nuclei, and these were almost exclusively cones. In the terminal stages of retinal atrophy (stages 6 [Fig. 2G ] and 7 [Fig. 2H ]), there was progressive loss of all photoreceptor structures with collapse of the subretinal space. The ONL contained scattered remaining nuclei that eventually disappeared. Consequently, there was loss of the OPL and moderate atrophy of the inner nuclear layer. Small numbers of melanin-containing cells could be found in the peripheral retina. Finally, all retinal cells were lost, and the neuroretina was replaced by a thin glial cord. 

Cone disease develops once rod disease is advanced. Cones initially develop broad inner segments (stages 2 and 3) presumably due to loss of the lateral support formerly provided by RISs. They remain structurally normal until stage 4, at which point inner and outer segment degeneration is present. During the period of active photoreceptor degeneration (stages 2 to 5), phagocytic cells and extruded rod and cone nuclei were consistently seen in the subretinal space. In addition, pyknotic and shrunken nuclei (presumably apoptotic photoreceptor nuclei) were prominent in the ONL during these stages. In the terminal stages of disease, occasional hypertrophic RPE cells could be seen in the peripheral retina, but otherwise the RPE and choroid remained normal. 

Six male dogs less than 11 months of age were examined and found to have normal retinal histology. When these six dogs were typed with the RPGR polymorphism, two segregated with the disease-associated allele and could thus be assigned XLPRA-affected status. Thus it appears that in the dogs in this study the disease is not detectable histologically before 11 months of age. 

Temporal and Topographic Distribution of Disease in Affected Males.

A consistent spatial pattern of photoreceptor degeneration was present in all eyes examined. The most severe stages of the disease in affected dogs were always present in the peripheral retina, with disease decreasing in severity toward the central retina. Thus it appears that the disease process affects the peripheral retina first and extends centrally over time. This pattern was consistent in superior, inferior, and temporal meridians. The transition between stages was usually gradual, but, in a minority of animals, transition from early (stage 1) to advanced stages (stages 3, 4, and 5) was abrupt. Within the same eye, disease in the inferior retina was approximately one stage more advanced than that in the superior retina. 

The extent of severity of these retinal changes varied among affected males. Even in animals of approximately the same age, the disease severity varied. In the least affected ones, mild disease, equivalent to stage 1, was limited to the portion of the retina peripheral to the termination of the tapetum lucidum in superior and temporal meridians. (Fig. 5A ). Early to advanced stages of the disease were still present but were contracted so that only the peripheral retina was involved. In the far periphery, adjacent to the ora serrata, disease was always advanced (stages 5 to 6). Normal dogs, particularly those that are older than 5 to 6 years, may have an acute diminution in the number of photoreceptors in the terminal portion of the retina adjacent to the ora serrata. However, such changes seen in normal dogs are not present early in life, and when present in older dogs, do not extend centrally more than 1000 μm, whereas in dogs affected with the least severe form of XLPRA, degenerative changes extended over 2500 μm or more of the peripheral retina. In moderately affected dogs, most of the retina was affected with the disease process. The central retina was either normal or affected with stage 1 disease, and disease severity worsened gradually toward the periphery (Fig. 5B) . In the most severe form, the entire retina was affected with stage 2 disease or worse (Fig. 5C) . 

With advancing age, the disease process extended from the peripheral retina to include the central retina. In most animals, at 11 to 18 months of age, all stages of retinal degeneration were present in the retina—that is, from stage 1 in the central retina to stage 4 or 5 in the periphery. Stages 5 and 6 were seen only in dogs older than 18 months. In the 7-year-old propositus, the entire retina consisted of regions with stages 5, 6, or 7 of the disease. Although older dogs had more severe disease than younger dogs, when age-matched males of similar parentage were examined, a spectrum of disease severity was also evident, i.e., some dogs had mild peripheral degeneration, whereas their siblings were affected with the more severe form. Thus, mild peripheral degeneration characterizes young dogs in early stages of XLPRA or older dogs that express a less severe form of the disease. With time, peripheral degeneration worsens to affect the entire retina. 

Another way of assessing the topographic variation in disease severity is to plot the changes in the ONL with age in a quadrant- and region-specific manner. To this end, we counted the number of photoreceptor nuclei in three regions of the superior and inferior meridians: centrally (350 μm from the disc), peripherally (350 μm from the ora serrata), and equatorially (midway between these two points). These observations for the superior quadrant are illustrated in Figure 6 . As expected, we found an earlier and more severe loss of photoreceptor nuclei in the periphery, and this change gradually progressed to the equatorial region. With time, the posterior pole region was affected. Note that affected animals have a large range of photoreceptor nuclear counts. This is a reflection of the great interanimal variation that we found in the severity of the retinal disease phenotype, even in animals of approximately the same age. 

Affected Females.

Cellular characteristics of disease in affected (homozygous) females resembled those seen in affected males—that is, diffuse photoreceptor degeneration. Because these dogs ranged from 3.7 to 4.9 years of age, their spectrum of disease was more advanced than that seen in the younger males. Photoreceptor degeneration in the superior retina was advanced centrally; the ONL was reduced to two rows of nuclei, and stubs of remaining photoreceptor inner segments occupied the narrowed subretinal space. The inferior retina was markedly more affected than the superior retina. Broad patches of complete retinal atrophy were interspersed with less affected regions where a single row of photoreceptor nuclei remained in the ONL (data not shown). Because of the relatively advanced age of the retinas, degeneration was advanced throughout (i.e., stages 5 through 7). This was comparable to the retinal disease seen in older affected males, but differed greatly from the disease present in carrier females of similar age. There was minimal variation in disease severity among the three affected females, probably a result of their relatively advanced age and of progression of degeneration to its terminal stages. 

Carrier Females.

Carriers exhibited two distinct cellular changes: 1) There was uniform rod photoreceptor loss resulting in reduction of the ONL to approximately 50% of its original width (Figs. 7A , 7C ). Remaining photoreceptors in these areas, or in areas where there was more severe rod loss, were ultrastructurally normal (Fig. 7E) . ² ) There were scattered foci of complete rod loss, presumably resulting from random X inactivation (lyonization; Figs. 7B 7C , and 7D ). In these foci, remaining cones were tightly clustered in a rod-free region, and the ONL consisted entirely of a cluster of cone nuclei flanked by a more normal, although thinner, layer of rod nuclei. The foci of retinal degeneration in the central retina were smaller, measuring 150 to 300μ m, whereas those in the peripheral retina were larger and the surrounding disease more severe (stages 4 through 6). In these regions, cones had degenerated to leave an empty patch devoid of photoreceptors flanked by more normal retina (Fig. 7D) . Compared with males, all the carriers examined were older and exhibited disease of comparable levels of severity. In contrast to males, which have progressive disease resulting in complete retinal atrophy over time, degeneration in carriers appears to be slowly progressive after the initial pattern of disease is established. Thus carriers of 7 to 8 years of age have normal vision, whereas affected males of the same age are completely blind. In contrast to affected males, all the carriers examined were approximately similar in the nature and severity of retinal disease. 

ERG

ERG studies were performed in a selected group of study animals. Dogs younger than 6 months of age (n = 6) showed no detectable abnormalities in the rod- and cone-mediated responses. Between 6 months and 1 year of age (n = 3), there was a decrease in the amplitude of the rod-mediated b-wave response, which was prominent at the highest stimulus intensities (Fig. 8A ). Dogs older than 1 year of age (n = 4) showed decreased ERG amplitudes and elevation of the dark-adapted b-wave threshold (Fig. 8B) . Distinct cone flicker responses were recordable and were initially normal (data not shown). Older heterozygous females showed decreased amplitude for the rod- and cone-mediated responses, and these findings were similar to those obtained in very young affected male dogs (Fig. 8A) . Unlike hemizygous males whose ERG responses progressively deteriorated, older heterozygous females had no disease-associated worsening of the ERG responses with time. 

Discussion

X-linked progressive retinal atrophy is a close phenotypic correlate of XLRP. Both are characterized by primary photoreceptor outer segment degeneration, and both affect hemizygous males in early adulthood and progress to complete blindness by middle age. Our previous work has shown that XLPRA is tightly linked to an intragenic marker within the canine RPGR gene, making XLPRA the locus homologue of RP3. ¹⁹ This study thus provided the opportunity to describe in detail the sequence of degenerative changes in an animal model for one form of XLRP in humans, RP3. 

Our studies confirm that XLPRA in dogs is a primary photoreceptor disease that begins as an ROS disorder. Disruption and disintegration of the ROS precede inner segment abnormalities and culminate in complete rod loss. The staging system described in this article is based on the sequence of degenerative events seen in rods. Cones follow a course of degenerative events that differ temporally from that of rods, in that it begins after significant rod loss has already occurred. Cone morphology is normal until relatively advanced retinal degeneration (i.e., stage 4). At this stage, cone disease is characterized by short, wide inner segments, loss of outer segments, and extrusion of cone nuclei into the subretinal space. The outer segment abnormalities (disc vesiculation and vacuolization) characterizing rod degeneration are not seen in cones. A series of secondary changes—for example, degeneration of synaptic terminals, narrowing of the plexiform layers, and Müller cell hypertrophy—are seen as late developments. The absence of histologically detectable disease in young males (less than 11 months of age) that possess the disease-associated allele of RPGR indicates that XLPRA begins well after retinal development is complete. ERG studies performed in a selected sample of study animals support these observations. 

The XLPRA pedigree is derived from a single mutant X-chromosome, because the affected male propositus was bred to homozygous normal beagles. Subsequent progeny were crossed to normal animals of different breeds. ¹⁸ We can conclude, therefore, that all affected animals shared the same mutation and consequently should have displayed a uniform clinical and histologic disease phenotype. In regard to the sequence of degenerative events in the retina, this was true. All affected dogs showed the same cytologic and topographic abnormalities. Degeneration affected the peripheral retina first and progressed centrally until the entire retina was affected with disease. By the time the central retina was affected with stage 1 disease, the peripheral retina had advanced to the terminal stages of degeneration. This pattern of disease was present in all affected retinas, but the extent to which the retinas of individual animals was affected differed—that is, the extent of disease in XLPRA was qualitatively similar, but quantitatively variable. 

Young affected males had disease limited to the peripheral half of the retina, and with age, disease extended to affect the central retina as well. However, affected male littermates showed variable degrees of disease severity as well, indicating that factors other than age affected the extent of disease expression. Examples of identical mutations resulting in variable phenotypes are widespread, but the mechanisms underlying this variability are poorly understood. ²³ Phenotypic variability is most commonly ascribed to the effects of modifier genes that provide a variable genetic background against which the mutant gene is expressed. ²⁴ Because the mutant XLPRA allele was propagated in a variety of breeds, this hypothesis seems plausible. Polymorphisms within the disease gene itself may modify disease expression. For example, this mechanism has been described in the cystic fibrosis gene. ²⁵ However, in an X-linked disease such as XLPRA, in which a single mutant allele derived from a founder male is present, it is unlikely that an intragenic polymorphism accounts for disease severity. Other mechanisms such as variable methylation of C residues in CpG islands at the disease locus ²⁶ or the amplification of stochastic developmental events as described by Kurnit et al. ²⁷ may also play a role in modifying the disease phenotype. 

Preferential degeneration of rods in XLPRA was demonstrated most clearly in carrier females that had multiple foci of lyonization characterized by complete loss of rods with retention of cones. Subsequent loss of cones suggests that cone degeneration is secondary to an altered interphotoreceptor environment due to rod death, and that the specific genetic defect affects rods only. This topographic gradient of disease severity in carrier females was similar to that seen in affected males—that is, disease was more advanced peripherally than centrally. The topographic expression of XLPRA may be influenced by molecular gradients inherent to the normal composition of the retina. Common pathologic findings are seen between carriers of XLPRA and two previously described elderly human carriers of XLRP. ^{10 13} In the younger patient, multiple patches of photoreceptor degeneration were present in the mid- and far periphery. In the older patient, there was retention of cones in the central retina only. These changes were more severe than those seen in XLPRA carriers, probably a reflection of the advanced age of the human subjects, and as well, the variability in the extent of random X inactivation in each carrier female. In contrast to carriers of XLRP, which exhibit broad phenotypic variability, ¹³ carriers of XLPRA had retinal disease of comparable severity, even between animals of different ages. 

Description of the cellular changes in a young man with XLRP in whom a disease locus was not defined ⁹ differs from disease characteristics seen in XLPRA in several aspects. In that study, although both rods and cones were affected, cone disease predominated. Cones in the far periphery were best preserved and had outer segment vesiculation similar to that seen in XLPRA. Rods in the region had shortened outer segments but were otherwise normal. These changes differ from those seen in XLPRA, in which rod degeneration clearly precedes cone degeneration, and retinal disease is always more severe peripherally than centrally. The later stages of retinal disease in XLPRA are not unique, in that they conform to a stereotypic sequence of events seen in many genetically diverse forms of retinal degeneration. ^{9 10 28 29 30} The one difference between the canine and human diseases is that prominent intraretinal migration of retinal pigment epithelial cells into perivascular sites in the retina does not occur in the dog. This difference is not specific for XLPRA, however, as it is a characteristic of the dog retina in all inherited photoreceptor degenerations. 

Among animals, XLPRA may be appropriately compared with those forms of inherited retinal degeneration that begin after retinal development is complete. These include autosomal recessive retinal degeneration in the Abyssinian cat, ³¹ Purkinje cell degeneration (pcd) in the mouse, ³² and progressive rod–cone degeneration (prcd) in the dog. ³³ XLPRA is most similar clinically and histologically to prcd, which is inherited as an autosomal recessive trait and differs slightly from XLPRA in its spatiotemporal disease expression. In prcd, degeneration begins several months earlier and is markedly more severe in the inferior than in the superior retina. The earliest ultrastructural lesion in prcd consists of disintegration of the outer segment to form many small vesicles that are distributed throughout the interphotoreceptor space. The vesicular blebbing and scrolling of disc membranes within intact outer segment, as seen in stage 1 of XLPRA, is not evident in prcd. Disintegration of outer segments to form free-floating vesicles occurs in XLPRA, but only in stage 2. The earliest detectable change in affected Abyssinian cats is lamellar disorganization and vesiculation of the outer segments that appears similar in severity to that seen in stage 2 of XLPRA. However, in the Abyssinian cat, mitochondrial degeneration, which may occur as a fixation artifact, appears prominent. ³¹ This change is never seen in XLPRA. In the pcd mouse, the first detectable abnormalities appear at postnatal day 18, approximately 4 days after retinal development is completed, and are characterized by the appearance of distinct membranous blebs between photoreceptor inner segments. ³² Thus, the pathologic origin of pcd appears to be in the inner segments and involves outer segments secondarily. Individual cells in XPLRA-affected retinas demonstrated changes consistent with death by apoptosis. Apoptosis appears to be the predominant means of cell death in several hereditary degenerations and in light-induced retinal damage ³⁴ and is probably a prevalent mechanism in XLPRA as well. 

XLPRA as a model system for XLRP has several advantages. The sequence of degenerative events can be systematically described throughout the course of the disease. Thus, the most informative (i.e., early) stages of the disease can be selected for further studies (e.g., immunohistochemistry, in situ hybridization, reverse transcription–polymerase chain reaction, and northern blot analysis) to characterize the molecular events associated with the disease. Once the molecular defect is defined in this locus homologue of RP3, XLPRA-affected animals will provide an excellent model in which to assess therapeutic strategies that may be applicable in humans. 

 Supported in part by the Foundation Fighting Blindness; Grant EY06855 from the National Eye Institute, National Institutes of Health; and the Baker Institute Progressive Retinal Atrophy Research Fund.

 Submitted for publication March 17, 1999; revised June 3, 1999; accepted June 21, 1999.

 Commercial relationships policy: N.

 

Figure 1.

 

Canine X-linked progressive retinal atrophy pedigree. The propositus from which the XLPRA colony was developed, dog 1, is indicated with an arrow. Dog 6 is indicated twice for the purposes of drawing the pedigree. Gender and disease status of dogs are depicted by the following symbols: normal male, open square; affected male, filled square; normal female, open circle; carrier female, open circle containing black dot; affected female, filled circle. Dogs labeled by number are Siberian husky–derived dogs, and dogs labeled with letters are nonhusky dogs that are normal at the XLPRA and other retinal disease loci. ∗Dogs less than 11 months of age whose retinal histology appears normal, but whose status is predicted after genotyping with the RPGR polymorphism.

Table 1.

 

Status, Gender, and Age of Dogs Included in the Morphologic Study

Normal Males			Affected Males			Carrier Females			Affected Females
Dog	Age	Dog	Age	Dog	Age	Dog	Age
56*	5 mo	41*	7 mo	2	7.8	30	4.9
57*	5 mo	43*	8 mo	3	3.5	31	3.7
58*	6 mo	33	11 mo	4	6	17	4
42*	7 mo	34	11 mo	8	4.9
49	11 mo	28	1	7	5.7
16	1.1	15	1.1	18	3.8
9	1.1	55	1.1	40	1.4
11	1.1	10	1.1	, †	3
12	1.1	21	1.3
44	1.1	22	1.3
20	1.3	23	1.3
45	1.3	24	1.3
46	1.3	25	1.3
29	1.3	47	1.3
26	1.3	27	1.3
19	1.3	35	1.4
54	1.5	36	1.4
51	1.5	52	1.5
50	1.5	53	1.5
		48	1.7
		37	1.7
		39	2
		14	2
		1	7

 Age is expressed in years, unless marked otherwise.

*  Status predicted after genotyping with the RPGR polymorphism.

†  Carrier female examined histologically at a previous date and not included in the pedigree.

Table 2.

 

Staging System for Retinal Disease of XLPRA in Affected Males

Stage	Cellular changes
0	Normal retina
1	Mild disorganization of ROS; retina otherwise normal
2	ROS distorted, fragmented, and shortened; loss of rod nuclei, and ONL approximately 70% of normal thickness; mild broadening of cone inner segments
3	Marked ROS disintegration and rod loss; ONL approximately 50% of normal thickness; phagocytic cells and extruded photoreceptor nuclei appear in subretinal space
4	Complete rod degeneration; ONL only two to three nuclei thick; narrow OPL; distinct cone disease showing short, wide, vacuolated cone inner segments and short cone outer segments
5	Most outer segment material is absent; remaining inner segments belong predominantly to cones; ONL is 1 nucleus thick
6	Loss of retinal layer organization; intact RPE
7	Retina mostly devoid of cells and reduced to a glial cord; multifocal disruption of RPE with adhesion of neuroretina to choroid

Figure 2.

 

Stages of retinal degeneration in males affected with XLPRA, as seen by light microscopy. (A) Normal retina. Arrow, external limiting membrane. (B) Stage 1: outer segments have uneven borders and are faintly banded. (C) Stage 2: outer segments are fragmented and disorganized. Spaces between inner segments (IS) are due to photoreceptor loss, and cone inner segments are broader than normal. Apoptotic nuclei are present. (D) Stage 3: cone inner segments are broad, but cone outer segments are intact. ONL is reduced to 50% of its original width and the subretinal space is narrowed. (E) Stage 4: most outer segment material is absent. Remaining inner segments belong to cones. The ONL is two to three nuclei thick with prominent pyknotic nuclei, and the OPL is atrophic. (F) Stage 5: short stubs of inner segments are apposed to the RPE. The ONL is reduced to a single layer of nuclei and is close to the inner nuclear layer due to atrophy of the OPL. An extruded photoreceptor nucleus is present in the subretinal space (arrow). (G) Stage 6: all inner and outer segment material is absent, and there is disorganization of retinal architecture. (H) Stage 7: the neuroretina is reduced to a glial cord infiltrated with clusters of hypertrophic RPE cells. Note that the RPE is nonpigmented in tapetal regions in (A, C, and D), and pigmented in nontapetal regions in (B and E through H). OS, outer segment; IS, inner segment; INL, inner nuclear layer; IPL inner plexiform layer. Bar, 30 μm.

Figure 3.

 

Ultrastructural changes in stages 1 and 2 of XLPRA in hemizygous males (A, B) and spectrum of ROS disease (C through F). (A) Stage 1: ROSs are vesiculated, disorganized, and occasionally disintegrated (arrow). Cone (C) inner and outer segments are normal. All photoreceptor inner segments are normal. (B) Stage 2: ROSs are markedly distorted, shortened, and fragmented. Loss of rods is reflected by the increase in space between inner segments and an increased proportion of cone inner segments; cone inner segments are widened but otherwise normal. (C) A large phagocytic cell process is present in the interphotoreceptor space (∗). Visible spaces are seen between outer fibers of photoreceptor cell bodies beneath the external limiting membrane. (C through F) Spectrum of disease seen in ROSs. Individual changes are not stage specific; however, less severe changes (C, D, and E) predominate in earlier stages of disease, whereas more severe changes (F) predominate in later stages. (C) Rows of vesicles located between ROS discs are the earliest detectable ultrastructural change. (D) Packets of ROS discs are malaligned and disoriented. (E) Small vesicles coalesce to form large vesicles and vacuoles that begin to distort the ROS. (F) A completely disintegrated ROS is flanked by two intact ones. Magnification, (A, B)× 2,300; (C through F) ×27,700.

Figure 4.

 

(A, B) Ultrastructural changes in stages 3 and 4 of XLPRA in hemizygous males. (A) Stage 3: outer segments are uniformly disrupted and consist largely of isolated packets of discs. The interphotoreceptor space is narrowed. Remaining RISs are shorter and wider and contain a moderately distended endoplasmic reticulum. (B) Stage 4. Almost no outer segments remain. Surviving inner segments are short and wide. An intact cone inner segment is present (C), but its outer segment is absent. A macrophage is present in the subretinal space (M). Müller cell processes in the interphotoreceptor space are prominent and spaces between inner fibers of photoreceptors are now occupied by hypertrophic Müller cells. (C through E) Spectrum of disease seen in photoreceptor cell bodies. (C) Rod cell body. A myelin figure is present within the cytoplasm (arrow). (D) Rod cell body. Multiple intracytoplasmic vacuoles are present adjacent to the nucleus. (E) Rod cell body. A shrunken pyknotic nucleus is surrounded by contracted cytoplasm. Magnification, (A, B)× 2300; (C through E) ×5400.

Figure 5.

 

Topographic variation in disease severity in males affected with XLPRA. Superior, inferior, and temporal quadrants extending from optic nerve (white circle) to ora serrata (semicircle) are depicted. Severity of disease (normal, stages 1 through 5) is indicated by shaded rectangles, with darker shading denoting more severe disease. The tapetum lucidum occupies most of the superior quadrant and is shaded with a brick pattern. The RPE becomes pigmented in regions where the tapetum lucidum is absent. These regions are indicated with diagonal lines. (A) XLPRA: peripheral disease. All stages of XLPRA are present, and their topographic pattern is maintained; however, they are contracted to mainly affect the periphery of the retina only. (B) XLPRA: moderate disease. All stages of XLPRA are present. The entire retina is affected with the disease process, which is least severe near the optic nerve and progresses in severity toward the periphery. (C) XLPRA: severe disease. The topographic distribution of disease illustrated in Figures 3 and 4 is maintained, but the entire retina is affected with severe stages of disease (stages 2 and higher).

Figure 6.

 

Histograms comparing numbers of photoreceptor nuclei in normal and XLPRA-affected males in three regions of the superior retina: central (350 μm distal to the optic nerve), peripheral (350 μm proximal to the ora serrata), and equatorial (midway between these two points). The mean and range (the vertical line associated with each bar) for normal and affected males in five different age groups is shown. These are: group 1: 7 to 8 months (n = 2); group 2: 11 months to 1.2 years (n = 6); group 3: 1.3 to 1.5 years (n = 11); group 4: 1.7 to 2 years (n= 4); and group 5: 7 years (n = 1). Disease severity increases with age, but there is wide variation in severity within age groups (as seen by the large range of ONL nuclear counts).

Figure 7.

 

Light and ultrastructural findings in carriers of XLPRA. (A, B) Three-year-old carrier (Table 1 , dog†); (C through E) 4-year-old carrier. (A) The ONL is reduced to approximately half its normal width. The subretinal space is slightly narrowed because of shortening of outer segments, but the remaining cells are normal. (B) A cluster of residual cones in a lyonized focus is illustrated (arrow). (C) A lyonized focus in the central retina is depicted (arrow). (D) In the periphery, foci of lyonization expand to become large focal regions of generalized photoreceptor loss accompanied by focal retinal atrophy. (E) Ultrastructural appearance of a lyonized focus. A cluster of residual cones in a lyonized focus is illustrated. Outer segments of both cones (C) and adjacent rods (R) are normal. Magnification, (E)× 2300. Bar, (A, B and C, D) 30 μm.

Figure 8.

 

(A) Selected ERG responses recorded from a normal male (1 year; left), an affected male (11 months; center), and a carrier female (8 years; right) in response to 50-msec single-light stimuli of different colors (first three rows), or flickering-light stimuli of different intensities (bottom two rows). The affected dog at 11 months of age (center) showed decreased rod-mediated responses under all stimulus conditions. Based on the slight reduction in cone b-wave amplitude at this age, the extent of functional rod loss was greater. The 8-year-old carrier female (right) showed a reduction in the ERG amplitude that primarily affected the rod system; at this advanced age, the ERG of affected male dogs was nonrecordable. (B) Mean dark-adapted rod b-wave responses to 50-msec white-light stimuli of increasing intensity recorded from nine normal male dogs and affected males of two age groups: 6 months to 1 year (n = 3) and 1 to 1.5 year (n = 4). The mean rod b-wave amplitude was decreased in affected males 6 month to 1 year of age at the higher stimulus intensities. In the older male dogs, the ERG b-wave amplitude was markedly reduced, and the dark-adapted threshold was elevated.

The authors thank Sue Pearce–Kelling for preparing all sections for ultrastructural analysis, Julie Alling and Sue Pearce–Kelling for their assistance in preparation of tissues for thick sections, Vicki Baldwin for her assistance in the molecular biology laboratory, Jill Czarnecki and Jennifer Johnson for graphics, Kunal Ray for many helpful discussions and for participating in the molecular studies that are part of a separate study, and the staff of the RDS Facility for assistance with breeding and rearing of the research dogs.