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Hereditary X-Linked Juvenile Retinoschisis: A Review of the Role of Müller Cells
Arch Ophthalmol. 2002;120:979-984.
Hereditary X-linked retinoschisis (RS) is the most common cause of juvenile
macular degeneration in males1-2
and may lead to vitreoretinal degeneration characterized by cystic spoke-wheel
maculopathy, peripheral retinoschisis, alterations of the vitreous body, and
a reduced b wave on the electroretinogram. Its prevalence ranges from 1:5000
to 1:25 000.3-4 The
condition is usually bilateral and affects males only. Males with juvenile
RS usually seek treatment because of diminished vision at school age, followed
by progressive visual deterioration later in life. Peripheral retinoschisis
is found in 50% of patients and may be limited to the inferior temporal quadrant.
Breaking of the inner schisis layer may lead to unsupported retinal vessels
in the vitreous cavity, called a "congenital vascular veil."5
There have been few reports on the histopathologic characteristics of RS.6-11
The principal feature in all these cases was a large schisis cavity originating
from the nerve fiber layer (NFL). Several theories concerning the pathogenesis
of RS have been postulated. First, findings on fluorescein angiography led
to a vascular theory of RS development12
because of delayed development of the retinal and choroidal vasculature in
which the retina outgrows its blood supply infratemporally. Vascular changes
might play a role in the evolution of the schisis,13
and RS may be complicated by neovascular glaucoma.14
Second, Schepens15 believed that the primary
abnormality was vitreous traction on the inner retinal surface caused by inadequate
growth or shrinkage of the cortical vitreous. The histologic characteristics
of RS in a male infant with congenital retinal detachment and splitting in
the inner retina but no schisis16 and in
2 male infants with congenital hereditary RS17
supported the theory of a vitreoretinal developmental anomaly. Third, based
on pathological findings, several authors postulated that juvenile RS arises
from a basic inherited defect in probably the innermost portion of the cytoplasm
of Müller cells.7, 10-11
Current molecular genetic and immunohistochemical findings contradict the
theory of a primary defect in the Müller cells18
and suggest an abnormality that interacts with a Müller cell receptor
or components of the extracellular matrix.19
Based on immunohistochemical analysis with a RS1-specific antibody applied
to the enucleated eye of a relatively young patient with RS, we support the
theory that photoreceptors appear to be the cells primarily involved in the
pathologic characteristics of RS.
Patient, Materials, and Methods
At age 5 months, our patient was diagnosed as having X-linked juvenile
RS. At age 19 years, his right eye was enucleated. The enucleated eye was
fixed in 4% formaldehyde solution in a 0.1M phosphate buffer. After horizontal
sectioning, the eye was embedded in paraffin. Sections (5-µm) were incubated
with polyclonal antibody glial fibrillary acid protein (DAKO, Glostrup, Denmark)
(dilution, 1:1200; incubation, 30 min at room temperature; peroxidase-antiperoxidase
method). The monoclonal antibodies vimentin (BioGenex, San Ramon, Calif),
and fibronectin (DAKO) and neurofilaments (Sanbio, Uden, the Netherlands)
were applied using the avidin-biotin complex method (dilution, 1:3200, 1:1200,
and 1:300, respectively; incubation, 10 min). Prior to incubation with vimentin
and neurofilaments, slides were pretreated for 15 minutes in citrate buffer
(microwave); prior to fibronectin incubation, slides were pretreated with
pronase for 10 minutes. The RS1 antibody was provided by one of us (B.H.F.W.)
and is identical to the RS1 antibody described in Molday et al.18
Raising the RS1 antibody and the specificity have been described previously.18 In short, the amino peptide LSSTEDEGEDPWYQKAC,
corresponding to aa22-39 of the human RS1 precursor protein,20
was conjugated to keyhole limpet hemocyanin and used to immunize New Zealand
White rabbits. For immunolabeling, a 1:1000 dilution of rabbit serum was used.
Prior to incubation, slides were pretreated for 10 minutes in citrate buffer
(microwave). A formalin-fixed paraffin-embedded eye with a healthy human retina
was used as a control.
Material was sampled from the formalin-fixed retina and embedded in
epoxy resin after dehydration with grading acetone. Semithin sections (1 µm)
for light microscopy were made with a glass knife and stained with toluidine
blue (1% weight-volume ratio). Ultrathin sections (70-80 nm) were cut with
a diamond knife and mounted on unfilmed 300-mesh copper grids. After staining
for 30 minutes with uranyl acetate and 2 minutes with lead citrate, the ultrathin
sections were examined with a Zeiss EM 902 transmission electron microscope
(Carl Zeiss, Oberkochen, Germany) with an acceleration voltage of 80 kV.
Our patient was also enrolled in a large study by the Retinoschisis
Consortium21 on screening for mutations
of the gene involved in RS (RS1).
Results
The family pedigree revealed an X-linked mode of RS inheritance with
several males affected (Figure 1).
In our patient, pursuit movements, a convergent strabismus of his right eye,
and remnants of persistent pupillary membranes were recorded on early examination.
At age 5 years, a cataract developed in his right eye. Visual acuity was light
perception OD and 20/200 OS. At age 8 years, his right eye showed a mature
cataract with posterior synechiae. Recurrent granulomatous uveitis with large
iris nodules occurred in the right eye from age 18 years onward (Figure 1A) and initially responded to topical
steroids and cycloplegia. Laboratory testing did not reveal a cause for the
uveitis. The patient was treated with 200 mg hydroxychloroquine sulfate per
day. Electroretinography and visual evoked potential were almost nonrecordable
in the right eye. At age 19 years, iris neovascularization developed in the
right eye with secondary glaucoma; it was treated with acetazolamide and local
therapy. Eventually, the right eye was enucleated. The visual acuity of the
left eye was counting fingers at the most recent examination (Figure 2B).
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Figure 1. The family pedigree reveals an
X-linked mode of inheritance with several affected males. Open square indicates
male; shaded square, male with juvenile retinoschisis; shaded square with
slash, male with juvenile retinoschisis who died; open circle, female; circle
with dot, carrier female; and asterisk, the index patient.
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Figure 2. Cataract and large iris nodules
in the right eye (A). Funduscopy of the left eye (B) of a patient with juvenile
retinoschisis shows delicate retinal cysts. In the enucleated eye, a depigmented
area is noted macroscopically in the posterior pole (C) with some vascular
veils extending anteriorly. Microscopically, the cysts originated from schisis
in the nerve fiber layer in the inferotemporal part of the retina (asterisks)
(D). The underlying retina and the nasal retina were detached (arrows), and
the retina was folded at the base of the schisis cavities (hematoxylin-eosin,
original magnification x1.7). In the nasal-posterior part of the retina
(E), there is splitting in the inner and outer plexiform layers (arrows) (hematoxylin-eosin,
original magnification x100). In the central retina (F) multiple PAS
(periodic acid–Schiff)–positive globules are present in all retinal
layers (original magnification x400). In the anterior segment (G), occlusion
of the pupil is present. The lens shows a hypermature cataract with posterior
synechiae and rupture of the anterior lens capsule with a reactive inflammatory
infiltrate (hematoxylin-eosin, original magnification x25). Glial fibrillary
acid protein stains strongly positive throughout the retina (H) (original
magnification x400). The nerve fiber layer (I) stains strongly positive
with S100 (original magnification x250). A healthy human retina (J)
with strong RS1 antibody immunostaining in the inner segments of the photoreceptors,
strong membranous staining in the outer nuclear layer, moderate immunostaining
in the inner nuclear layer and the plexiform layers, and negative staining
in the ganglion cell layer and the nerve fiber layer (original magnification
x250). The retinoschisis-affected eye with negative RS1 antibody staining
in the atrophic central retina (K) and markedly reduced staining in the relatively
well-preserved peripheral retina (L) (original magnification x250).
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The eye was fixed in formalin and transported to the pathology department.
Macroscopically, occlusion of the pupil, a mature cataract, and posterior
synechiae were noted in the anterior segment. In the posterior pole, a depigmented
area was found, with some vascular veils extending anteriorly (Figure 2C). The retina was partly detached, with delicate cysts
inferiorly in the eye. On microscopic examination, the cysts were seen to
have originated from schisis in the NFL in the inferotemporal part of the
retina and were covered by a glial membrane. The underlying retina and the
nasal retina were detached (Figure 2D).
There was marked splitting in the NFL of the nasal retina along the plane
of the ganglion cell layer and detachment of the inner limiting membrane (ILM).
Alcian blue/hyaluronidase staining was negative. The retina was folded at
the base of the schisis cavities with marked hyalinization of intraretinal
vessels and degenerative calcification. In the nasal-posterior part of the
retina, there was splitting in the inner and outer plexiform layers (Figure 2E). In the depigmented posterior
pole, the retinal pigment epithelium showed proliferative and degenerative
changes with atrophy of the photoreceptors and the outer nuclear layer. In
the pupil-optic block, the retina was partly detached without obvious schisis
cavities. The inner retina showed splitting in the NFL and detachment of the
ILM. In the central retina, multiple PAS (periodic acid-Schiff)–positive
globules were present in all retinal layers, sometimes with lumens (Figure 2F). In the macular area, degenerative
changes were found in the outer plexiform and nuclear layers. In the anterior
segment iris, neovascularization, occlusion of the pupil, and iris bombé
were present. The lens showed a hypermature cataract with posterior synechiae
and rupture of the anterior lens capsule (Figure 2G). A reactive mixed inflammatory infiltrate was present,
with histiocytes and giant cells within the lens capsule. Foamy cells surrounded
the lens and were present in the anterior chamber. Granulomas were noted along
the pigment epithelium of the iris and ciliary body and focally at the retinal
pigment epithelium, with associated uveitis.
On immunohistochemical examination, glial fibrillary acid protein (Figure 2H) and vimentin stained strongly
positive throughout the retina and the inner and outer layer of the schisis
cavities. The NFL (Figure 2I) and
the inner and outer layers of the schisis cavities stained strongly positive
with S100. The roof of the schisis cavity and the NFL stained focally positive
with neurofilaments. The PAS-positive globules stained strongly positive with
fibronectin. In the healthy human retina, immunostaining with the RS1 antibody
revealed intense staining of the inner segments of the photoreceptors, strong
membranous staining in the outer nuclear layer, moderate staining in the inner
nuclear layer and the plexiform layers, and negative staining in the ganglion
cell layer and the NFL (Figure 2J).
The RS-affected eye showed negative staining in the atrophic central retina
(Figure 2K) and markedly reduced
staining in the relatively well-preserved peripheral retina (Figure 2L).
On electron microscopic examination, splitting had occurred in the NFL
in the semithin sections. Intraretinal globules were present in the inner
nuclear layer and the inner part of the outer plexiform layer and were composed
of basement membrane–like material in the ultrathin sections (Figure 3A). A glial membrane was present
at the vitreal side of the ILM. The retinal surface of the ILM was attached
to footplates of degenerated Müller cells. The plasma membrane of some
Müller cells was focally deficient with intraretinal deposits of intermediate
filaments (Figure 3B).
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Figure 3. Electron microscopic examination
shows intraretinal globules composed of basement membrane–like material
(A). The plasma membrane of some Müller cells was focally deficient with
intraretinal deposits of intermediate filaments (B) (original magnification
x7000).
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In our patient and his family, the missense mutation Arg102Trp was found
in exon 4 containing part of the conserved discoidin domain of the RS1 gene.20
Comment
The histological findings in our patient are characteristic of juvenile
RS with an unusual complication of phacoantigenic endophthalmitis, which explains
the clinical findings of granulomatous anterior uveitis. Immunostaining with
the RS1-specific antibody18 was markedly
reduced in the RS-affected eye. The healthy human retina stained strongly
positive in the inner segments of the photoreceptors and the outer nuclear
layer, moderately positive throughout the inner nuclear layer and the plexiform
layers, and negative in the inner retina. This is consistent with findings
for the same antibody applied in mouse and monkey retinas and a normal human
retina.18 Similarly, a retina-specific polyclonal
antibody, designated retinoschisin, has been described in mouse and human
retinas.19 Although messenger RNA of the
causative RS1 gene was detected only in the photoreceptor
layer, the protein product of the gene (retinoschisin) was present both in
the photoreceptors and within the inner portions of the peripheral human retina,
and there was patchy immunoreactivity in the inner and outer nuclear layers
at the macula.
By genetic linkage analysis, RS was first mapped to the distal region
of Xp, and subsequent refinement eventually localized the RS gene in Xp22.2.22 Sauer et al20
identified a candidate gene for RS, designated RS1
(alias XRLS1). The RS1 gene
has 6 exons and encodes a 224 amino acid protein, which contains a highly
conserved discoidin domain. The RS1 mRNA encodes
a secretable adhesion protein.20, 23
Its role is implicated in cell-cell adhesion and phospholipid binding, indicating
that RS1 is important in cell adhesion processes
during retinal development.20-21
It was postulated that the protein product RS1 is expressed and assembled
in photoreceptors of the outer retina and bipolar cells of the inner retina
as a disulfide-linked oligomeric protein complex.18-19
Recently, it has been demonstrated in vitro that retinoschisin is selectively
taken up and transported by Müller cells into the inner retina in a direction-specific
manner.24 Juvenile RS may therefore be caused
by abnormalities in the secreted photoreceptor protein at some distance from
the site of RS pathologic characteristics.19
Discoidin domains are present in extracellular or transmembrane proteins in
cell adhesion or cell-cell interactions.25
The interaction of RS1 protein with a Müller cell surface receptor or
the extracellular matrix would be in keeping with its discoidin domain.19
We found no expression of RS1 protein in the central atrophic retina
and markedly reduced staining in the relatively well-preserved peripheral
retina in the RS-affected eye. This is consistent with a recent study showing
reduced antibody staining in chimera mice with a targeted RS1 knockout.26 The reduced staining
in the human RS-affected eye may be explained by the missense DNA mutation
found in our patient, which may have resulted in a dysfunctional protein with
a reduced half-life and defective cellular adhesive function. Many missense
and protein-truncated mutations of the causative RS1
gene have now been identified and are thought to be inactivating.19 Such a defective adhesive protein may still be
transported by the Müller cells into the inner retina, eventually leading
to schisis formation. The basement membrane of the Müller cells forms
part of the ILM. The Müller cell is the principal glial cell of the retina
and is in intimate contact with the inner segments of the photoreceptors and
the cells of the middle retinal layers, surrounding large areas of retinal
vessels. The dysfunctional protein or abnormalities in the interaction of
the protein with a Müller cell receptor or extracellular matrix may therefore
be expected to affect the middle and inner retinal layers and to produce structural
defects in the ILM and the NFL. This could account for the schisis, which
was present not only in the inner retinal layers but also nasal-posteriorly
in the inner and outer plexiform layers. The cone-shaped zone of Müller
cells in the central and inner part of the fovea centralis plays an important
role in the structural integrity of the macula, and defective cell-cell interaction
may explain the characteristic foveo-macular schisis, later replaced by atrophic
changes.27 Similarly, Müller cells
may also be involved in the extracellular deposits of amorphous PAS-positive
dots in the retina and, possibly, walls of small vessels. The PAS-positive
deposits were noted in all retinal layers in the atrophic central retina and
were not restricted to the schisis cavities.10-11
In our patient, the immunohistochemical (glial fibrillary acid protein, S100,
and neurofilament) and electron microscopic findings (presence of degenerative
Müller cells and deposits of intermediate filaments) are consistent with
earlier findings. However, glial fibrillary acid protein and S100 positivity
were not restricted to the retina adjacent to the schisis.10-11
These differences may be explained by the age at the time of enucleation (age,
19 years vs 55, 53, and 83 years10-11);
our case probably represents an earlier stage of the disease. We support the
hypothesis that the basement membrane–like material and filaments that
accumulate extracellularly within the atrophic central retina may be caused
by abnormalities in the interaction of the (defective) RS protein and a Müller
cell receptor or extracellular matrix.19
In summary, earlier studies18-19
have established through immunohistochemical analysis the cellular distribution
localization of RS protein in mammalian and healthy human retinas. The photoreceptors
and bipolar cells appeared to be the cell types primarily involved in maintaining
the integrity of the central and peripheral retina, secreting a cell adhesion
protein taken up and transported by Müller cells into the inner retina.18-19 In our study of an RS-affected
human eye, a mutation of the RS1 gene appears to
give rise to a dysfunctional adhesive protein, resulting in defective cellular
retinal adhesion that eventually leads to schisis formation.
AUTHOR INFORMATION
This study was presented in part at the annual meeting of the Verhoeff-Zimmerman
Society, Portland, Ore, April 24, 1999.
Cornelia M. Mooy, MD, PhD
Dordrecht, the Netherlands
L. Ingeborgh van den Born, MD;
Seerp Baarsma, MD;
Dion A. Paridaens, MD
Rotterdam, the Netherlands
Thea Kraaijenbrink, MSc;
Arthur Bergen, PhD
Amsterdam, the Netherlands
Bernhard H. F. Weber, PhD
Würzburg, Germany
Corresponding author and reprints: Cornelia M. Mooy, MD, PhD, Pathology
Laboratory Dordrecht, Jkvr Van den Santheuvelweg 2A, 3317NL Dordrecht, the
Netherlands (e-mail: cmooy{at}paldordt.com).
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SECTION EDITOR: W. RICHARD GREEN, MD
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