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From the James A. Baker Institute, College of Veterinary Medicine, Cornell University, Ithaca, New York.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 mouse16 and the mosaic rds mutant mouse17 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.
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Materials and Methods |
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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.
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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 x40 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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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) .
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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.
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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 300
µm, 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 locus26 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 defined9 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 damage34 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.
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Acknowledgements |
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Footnotes |
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Submitted for publication March 17, 1999; revised June 3, 1999; accepted June 21, 1999.
Commercial relationships policy: N.
Corresponding author: Gustavo D. Aguirre, James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. E-mail: gda1{at}cornell.edu
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); (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)
x2300. Bar, (A, B and C, D) 30 µm.