Clinicopathologic Effects of Mutant
GUCY2D in Leber Congenital Amaurosis
Ann H. Milam, PhD,1,2Mark R. Barakat, AB,1Nisha Gupta, PhD,1Linda Rose, MD, PhD,1
Tomas S. Aleman, MD,1Michael J. Pianta, PhD,1Artur V. Cideciyan, PhD,1Val C. Sheffield, MD, PhD,3
Edwin M. Stone, MD, PhD,4Samuel G. Jacobson, MD, PhD1
(LCA) caused by mutation in GUCY2D.
Two subjects with LCA; postmortem eye from one LCA patient and three normal donors.
Clinical and visual function studies were performed between the ages of 6 and 10 years in the LCA
eye donor and at age 6 in an affected sibling. Genomic DNA was screened for mutations in known LCA genes.
The retina of the 111⁄2-year-old subject with LCA was compared with normal retinas from donors age 3 days, 18
years, and 53 years. The tissues were processed for histopathologic studies and immunofluorescence with retinal
Vision in both siblings at the ages examined was limited to severely impaired cone function.
Mutation in the GUCY2D gene was identified in both siblings. Histopathologic study revealed rods and cones
without outer segments in the macula and far periphery. The cones formed a monolayer of cell bodies, but the
rods were clustered and had sprouted neurites in the periphery. Rods and cones were not identified in the
midperipheral retina. The inner nuclear layer appeared normal in thickness throughout the retina, but ganglion
cells were reduced in number.
An 111⁄2-year-old subject with LCA caused by mutant GUCY2D had only light perception but
retained substantial numbers of cones and rods in the macula and far periphery. The finding of numerous
photoreceptors at this age may portend well for therapies designed to restore vision at the photoreceptor
level. Ophthalmology 2003;110:549–558 © 2003 by the American Academy of Ophthalmology.
To study the retinal degeneration in an 111⁄2-year-old patient with Leber congenital amaurosis
Comparative human tissue study.
Leber congenital amaurosis (LCA) is a retinal degeneration
with blindness or severe vision loss at birth or shortly
thereafter.1–3LCA accounts for 5% of all inherited retinal
diseases but is higher in countries with high rates of con-
sanguineous unions.4,5Recent studies have demonstrated
that LCA is genetically heterogeneous.6,7To date, at least
10 LCA genes have been identified or localized: GUCY2D
(retinal guanylate cyclase 1, also known as RETGC, and
AIPL121,26,27; RPGRIP128,29; LCA330; LCA531; CRB132,33;
There are relatively few histopathologic studies of hu-
man LCA eyes, usually of retinas with advanced disease and
none with a known gene defect. Most published reports on
LCA retinas have revealed death of photoreceptors, the rods
before the cones. Loss of inner retinal neurons, including
ganglion cells, and intraretinal migration of retinal pigment
We studied the postmortem eye of a young (111⁄2-year-
old) LCA subject whose vision was carefully documented in
life. Her younger sister also has LCA. The molecular defect
in the family is mutation in the GUCY2D gene. The post-
mortem donor retina was well preserved and offered the
opportunity to address three questions:
● The subject had only light perception at last examina-
tion. What is the microscopic correlate of the func-
● How does mutation in the GUCY2D gene affect the
rods and cones?
Originally received: March 29, 2002.
Accepted: July 26, 2002.
1Department of Ophthalmology, Scheie Eye Institute, University of Penn-
sylvania, Philadelphia, Pennsylvania.
2F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute,
University of Pennsylvania, Philadelphia, Pennsylvania.
3Department of Pediatrics, University of Iowa Hospital and Clinics, Iowa
4Department of Ophthalmology, University of Iowa Hospital and Clinics,
Iowa City, Iowa.
Supported by NIH grants EY05627, EY13385, EY13203, and EY13729,
Bethesda, Maryland; Foundation Fighting Blindness, Owings Mills, Mary-
land; Pennsylvania Lions Sight Conservation and Eye Research Founda-
tion, Feasterville, Pennsylvania; Macula Vision Research Foundation,
West Conshohocken, Pennsylvania; Paul and Evanina Mackall Trust, New
York, New York; Fight for Sight Research Division of Prevent Blindness
America, Schaumburg, Illinois; The Grousbeck Family Foundation, San
Francisco, California; and the F. M. Kirby Foundation, Morristown, New
Jersey. VCS is supported by the Howard Hughes Medical Institute; SGJ is
an RPB Senior Scientific Investigator; AVC is an RPB Special Scholar.
Reprint requests to Ann H. Milam, PhD, Scheie Eye Institute, University of
Pennsylvania, 51 North 39th St., Philadelphia, PA 19104.
Manuscript no. 220247.
© 2003 by the American Academy of Ophthalmology
Published by Elsevier Science Inc.
ISSN 0161-6420/03/$–see front matter
● Would a retina at the stage of disease of the eye donor
be amenable to any form of therapy?
With these questions in mind, we present detailed clini-
cal, genetic, and histopathologic observations on the retina
of this young LCA subject.
Material and Methods
Subjects and Clinical Studies
A North American family of German and Italian ancestry had 2
female children affected with LCA and 2 unaffected female chil-
dren (Fig 1A). The two LCA subjects had complete clinical ocular
examinations and visual function tests, including Goldmann ki-
netic perimetry, full-field electroretinography (ERG), and optical
coherence tomography (OCT). Details of our perimetry, full-field
ERG, and OCT methods and data analyses are published.22,40–43
All subjects gave informed consent; institutional review board
approval was obtained, and the tenets of the Declaration of Hel-
sinki were followed.
DNA was extracted from peripheral blood using a previously
described protocol.44The proband was screened for mutations in
the coding sequences of the AIPL1, CRB1, CRX, GUCY2D, and
RPE65 genes using single-strand conformational polymorphism
(SSCP) analysis.45,46The primer sequences used for SSCP screen-
ing of the coding regions are published.9,13,32,47–49
The polymerase chain reaction amplification products were
denatured for 3 minutes at 94° C, electrophoresed on 6% poly-
acrylamide-5% glycerol gels at 25 W for approximately 3 hours,
and stained with silver nitrate.50Polymerase chain reaction prod-
ucts from samples with aberrant electrophoretic patterns were
sequenced bidirectionally with fluorescent dideoxynucleotides on
an ABI model 377 automated sequencer (Applied Biosystems,
Foster City, CA).
Detection of only a single amino acid–altering sequence change
in the proband led to complete bidirectional sequence analysis of
the coding sequences of the GUCY2D locus. In addition, a geno-
typic survey of the GUCY2D locus was performed using 20
(D17S974, D17S1879, D17S786, D17S720, D17S938, D17S1881,
D17S804, D17S960, D17S919, D17S796, D17S1854, D17S1149,
D17S1852, D17S731, and D17S1159).
Oligonucleotide primers complementary to sequences flanking
these markers were obtained from Research Genetics (Huntsville,
AL). The polymerase chain reaction conditions used for short-
tandem repeat polymorphism genotyping were the same as for
SSCP analysis. However, the amplification products were dena-
tured, electrophoresed on gels consisting of 6% polyacrylamide-7
M urea at 65 W for approximately 3 hours, and stained with silver
Postmortem human eyes were obtained through the donor program
of the Foundation Fighting Blindness (FFB, Owings Mills, MD)
and the University of Washington Lions’ Eye Bank (Seattle, WA).
The eye from an 111⁄2-year-old girl with LCA (FFB #648) was
fixed 13.5 hours postmortem in a mixture of 4% paraformaldehyde
and 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. After
4 days in this fixative, the globe was stored in 2% paraformalde-
hyde in the same buffer. As controls, normal eyes from three
donors (#0849-94, 3-day-old, 2.5 hours postmortem; #0966-91,
18-year-old, 10 hours postmortem; and #11-23-99, 53-year-old,
surgical specimen) were processed in the same way.
As reported previously,51retinal samples were treated with 1%
sodium borohydride, infiltrated overnight at 4° C with 30% su-
crose in 0.1 M phosphate buffer, pH 7.3, and cryosectioned at 12
?m. Sections and retinal flat mounts were processed for immuno-
fluorescence.51The following retinal cell-specific antibodies were
used: mouse monoclonal antibody (mAb) 7G6 specific for cone
cytoplasm (1:250, from Dr. P. MacLeish, Morehouse School of
Medicine, Atlanta, GA); mouse mAb 4D2 anti-rhodopsin specific
for rod outer segments (1:40, from Dr. R. Molday, University of
British Columbia, Vancouver, B. C., Canada); sheep polyclonal
antibody (pAb) anti-rhodopsin (1:1000, from Dr. D. Papermaster,
University of Connecticut Health Center, Farmington, CT); rabbit
pAb JH492 anti-red/green cone opsin (1:5000, from Dr. J.
Nathans, Johns Hopkins University, Baltimore, MD); rabbit pAb
anti-red/green cone opsin (1:200, from Dr. J. Saari, University of
Washington, Seattle, WA); rabbit pAb JH455 anti-blue cone opsin
(1:5000, from Dr. Nathans); mouse mAb anti-blue cone opsin
(1:10,000 from Dr. A. Szel, Semmelweis University Medical
School, Budapest, Hungary); mouse mAb 3B6 anti-rds/peripherin,
specific for rod and cone outer segments (undiluted, Dr. Molday);
rabbit pAb anti-recoverin, specific for rods, cones, and cone bipo-
lar cells52(1:1000, from Dr. A. Dizhoor, Kresge Eye Institute,
Detroit, MI); mouse mAb anti-SV2, specific for synaptic vesicles
(1:400, from Dr. K. Buckley, Harvard University, Boston, MA);
mouse mAb anti-parvalbumin, specific for horizontal cells (P-
3099, 1:10,000, from Sigma. St. Louis, MO); mouse mAb anti-
calbindin, specific for red/green cone cytoplasm, horizontal cells
and inner retinal neurons (C8666, 1:200, from Sigma); rabbit pAb
anti-GABA and anti-glycine, specific for amacrine cells (1:100,
from Dr. R. Marc, University of Utah, Salt Lake City, UT); guinea
pig pAb anti-GABA (1:1000, from Incstar Co., Stillwater, MN);
rabbit pAb anti-glial fibrillary acidic protein (GFAP), specific for
astrocytes and reactive Mu ¨ller cells (1:750, Dako Corporation,
Carpinteria, CA); and rhodamine-conjugated peanut agglutinin and
wheat germ agglutinin lectins (1:1000, Vector Laboratories, Bur-
lingame, CA). The secondary antibodies (goat anti-rabbit, anti-
mouse, anti-sheep, or anti-guinea pig immunoglobulin, 1:50) were
labeled with Alexa Fluor 488 (green; Molecular Probes, Eugene,
OR), Cy-2 (green), or Cy-3 (red) (Jackson ImmunoResearch Lab-
oratories, Inc., West Grove, PA). Cell nuclei were stained (blue)
with 4?,6?-diamidino-2-phenylindole (1 ?g/ml; Molecular Probes).
Control sections were treated in the same way with omission of
The immunolabeled retinal sections were examined with a
microscope equipped for epifluorescence (Leica DMR, Deerfield,
IL) and photographed with Kodak EliteCHROME ASA 400 film
(Rochester, NY). Images were digitized with a flatbed scanner
(Saphir HiRes, Heidelberg CPS GmbH, Bad Hamburg, Germany)
using LinoColor Elite 5.1 software (Heidelberg CPS GmbH),
imported into a graphics program (Photoshop 5.0, Adobe, San
Jose, CA), and dye-sublimation prints were generated.
Eye Donor and Family Members: Clinical Studies
The family (Fig 1A) had two female children (II-2, II-3) with LCA
and two unaffected female children (II-1, 11-4). There was no
known parental consanguinity.
Subject II-2 (Eye Donor). The proband as an infant had
abnormal eye movements and visual disturbances. On examination
Volume 110, Number 3, March 2003
Figure 1. A, Pedigree of the family with Leber congenital amaurosis (LCA). Circle, female; square, male; filled circle, affected; open symbol, unaffected.
Arrow indicates deceased proband. B, Kinetic visual fields using two targets (V-4e and I-4e) in the two subjects with LCA. C, Standard electroretinograms
(ERGs) in the two LCA subjects. Rod, mixed cone-rod, and cone ERGs from the two subjects are compared with those of a young normal subject. D,
In vivo cross-sectional retinal images obtained with optical coherence tomography in the two LCA patients and representative normal subjects (N). Images
are displayed with the logarithm of reflectivity mapped to a gray scale that corresponds with the more commonly used pseudocolor displays (1, white; 2,
red; 3, yellow; 4, green; 5, blue; and 6, black). Images shown are from 3 different locations: a scan from the fovea to 15° S, a section superior and temporal
to the fovea (5°S, 5°T), and an area superior and nasal to the optic nerve (5°S, 25°N). Scale bars on the images indicate 0.3 mm. Longitudinal reflectivity
profiles (LRPs), averaged from 1° sections, are shown to the right of the images. All LRPs and images are aligned by matching the depth of the most sclerad
peak of the outer retina-choroidal complex, ORCC (*).
Milam et al ? Leber Congenital Amaurosis
at age 6 years, she had nystagmus and exotropia; visual acuities
were 1/200; and cycloplegic retinoscopy was ?4.00 in both eyes.
Ophthalmoscopy showed attenuated retinal vessels and a granular
appearance to the fundus, most obvious in the midperiphery.
Kinetic perimetry (V-4e test target) suggested that the visual field
was limited to a central island of function (Fig 1B). The ERGs had
no detectable rod b-waves to a dim blue flash, dark adapted, but
there were abnormally reduced waveforms to a maximal mixed
cone-rod stimulus and to cone stimuli at 1 Hz and 29 Hz (Fig 1C).
A clinical diagnosis of LCA was made. At ages 9 and 10 years,
visual acuities had declined to light perception, and there was no
measurable visual field by kinetic perimetry. Ophthalmoscopic
appearance was unchanged, except for faint bone spicule pigment
in the midperiphery. The macula did not seem to be atrophic.
Cross-sectional retinal images with OCT were obtained at extrafo-
veal locations (Fig 1D). Retinal thickness was reduced in the LCA
proband compared with normal at the locations sampled. A normal
OCT longitudinal reflectivity profile has 2 prominent peaks, one at
the vitreoretinal surface and the other in the outer retina, known as
the outer retina-choroidal complex (ORCC). Unlike the normal
double-banded ORCC, the proband’s ORCC contained only a
single peak (Fig 1D). At age 111⁄2 years, the proband died from
complications after tonsillectomy.
Subject II-3. A sibling (5 years younger) of the proband was
noted at age 4 years to have some visual inattention and night
vision disturbances. On examination at age 6 years, there was no
nystagmus or strabismus; visual acuities were 20/200; and cyclo-
plegic retinoscopy was ?3.00 ? 1.50 ? 180 in both eyes. Oph-
thalmoscopy showed attenuated retinal vessels and some granu-
larity to the fundus appearance. Kinetic visual fields to the V-4e
test target had slight generalized constriction but were full in
peripheral extent; response to a I-4e target was mainly in the
peripheral field with a relative central and midperipheral scotomas
(Fig 1B). The ERGs had no detectable rod b-waves; responses to
a mixed cone-rod stimulus and to cone stimuli were abnormally
reduced (Fig 1C). With OCT, a more complete series of cross-
sectional retinal images was obtained, because this patient did not
have nystagmus. A scan through the fovea showed a relatively
normal contour, but, as in the proband, there was retinal thinning
and the normally double-banded ORCC had only a single peak at
the locations examined (Fig 1D).
A total of 74 amplimers (collectively containing the coding sequences
of five LCA-causing genes: AIPL1, CRB1, CRX, GUCY2D, and
RPE65) was screened with SSCP analysis for coding sequence mu-
region of the GUCY2D gene of the proband and her affected sister
was directly sequenced twice with automated DNA sequencing. Only
two sequence variations (both in GUCY2D and both heterozygous)
were found in this entire experiment. One of these changes is a fairly
common T3C polymorphism at position 703, 143 base pairs 3? to
the last nucleotide of the GUCY2D stop codon. The other is a
variation that would be expected to change the basic arginine residue
not been observed in another LCA proband or in any of 143 control
Detection of a single plausible disease-causing allele in the two
affected sisters led to further scrutiny of the GUCY2D locus using
short-tandem repeat polymorphism. In all, 20 polymorphisms at
the GUCY2D locus were genotyped in this nuclear family, and 6
markers were informative for both parents. All 6 markers revealed
that the 2 affected sisters share a genotype and, in addition, that
this shared genotype is distinct from that of an unaffected sister
(Fig 2). One marker (D17S974) seems to be hemizygous in the
father, and no normal paternal allele was detectable in either
affected daughter. This marker is 1.14 cM away from the coding
sequences of the GUCY2D gene.
Normal 3-day-old Macula. Cones and rod cytoplasm and
outer segments were strongly labeled with anti-recoverin
Figure 2. Haplotype analysis. Each vertical line represents a portion of
chromosome 17 containing the GUCY2D gene. The alleles of various
polymorphisms are grouped into deduced haplotypes on either side of these
lines. The pedigree symbols above each vertical line indicate the gender
and affection status of the individuals harboring the indicated haplotypes
(circle, female; square, male; closed symbol, affected; diagonal line, de-
ceased proband). The parents are shown at the top of the figure and their
three children below. The GUCY2D gene is shown as a box superimposed
on each haplotype, and the presence of three intragenic variations (at
codons 660, 703, and in the 3?UTR) are depicted using these numbers,
whereas an “N” is used to depict the wild-type sequence at these positions.
The observed alleles of 6 informative short-tandem repeat polymorphisms are
shown numerically. The order of the polymorphisms from top to bottom in
GUCY2D, D17S974, D17S1879. An X indicates a putative deletion involv-
ing D17S974. Although the figure is drawn with the 5? end of the GUCY2D
gene pointing toward the telomere, this orientation has not been definitively
established. The map order shown in this figure was obtained from the
Genatlas database by way of www.infobiogen.fr.
Volume 110, Number 3, March 2003
(Fig 3A). The cone cytoplasm was positive throughout with
mAb 7G6 (Fig 3B), and the cone outer segments were short
and positive for either blue cone (Fig 3B) or red/green cone
opsin. The short cone and rod outer segments were positive
for rds/peripherin (Fig 3C). The short cone matrix sheaths
were peanut agglutinin positive (Fig 3D), and the rod
sheaths were wheat germ agglutinin positive. The short rod
outer segments were rhodopsin positive (Fig 3E), and
GFAP reactivity was restricted to the astrocytes (Fig 3E).
The synapses of the cones and rods were positive for the
synaptic vesicle protein, SV2 (Fig 3A). Inner retinal cells
were well labeled with the following markers: horizontal
cells, calbindin and parvalbumin; amacrine cells, gamma-
aminobutyric acid (GABA), glycine, and calbindin. The
outer and inner plexiform layers were uniform in thickness
and well labeled with anti-SV2 (Fig 3A). The macular
ganglion cells were 6 to 8 cells deep, as revealed by 4?,6?-
diamidino-2-phenylindole staining of their nuclei.
Normal 18-year-old and 53-year-old Maculas. Cones
and rods were labeled as at 3 days, except the outer seg-
ments were normal in length, shown in cones with mAb
7G6 (Fig 3F), including blue cones (Figs 3F and G) and
red/green cones (Fig 3H). Rod and cone outer segments
were positive for rds/peripherin (Fig 3I), and rod and cone
matrix sheaths (Fig 3J) had normal length. Rod outer seg-
ments were rhodopsin positive (Fig 3K), and GFAP was
restricted to the astrocytes (Fig 3K). The inner retina was
labeled as at 3 days, including SV2-positive outer and inner
plexiform layers (Fig 3L) and recoverin-positive cone bipo-
lar cells (Fig 3L). Horizontal, bipolar, and amacrine cells
were labeled normally as in the 3-day retina.
Gross Pathology. The dimensions of the cornea were 12 ?
11 mm, and the globe was 21 ? 23 ? 20 mm. The cornea
appeared normal with no keratoconus; the lens and vitreous
were clear. The optic nerve head appeared pale and waxy.
The retina showed postmortem edema and a fold that in-
cluded the macula; yellow macular pigment was visible.
The retina contained scattered small intraretinal hemor-
rhages. Faint bone spicule pigment was present in the mid-
Macula. All cones and rods lacked outer segments, evi-
denced by absence of labeling with anti-opsins and anti-rds/
peripherin (Fig 3M). The cones formed a monolayer of
7G6-positive cell bodies (Fig 3N). Some cones were posi-
tive for blue cone opsin (Fig 3O) but no cones were red/
green cone opsin positive (Fig 3P), as tested with two
different antibodies. The more degenerate cones at the edge
of the macula had variable intensity of cytoplasmic staining
for calbindin, recoverin, and 7G6 (Fig 3Q). The cytoplasm
of the cones was weakly peanut agglutinin positive (Fig
3R), but cone sheaths could not be identified. Sections
treated with secondary antibodies only had weak autofluo-
rescence of lipofuscin granules in the retinal pigment epi-
The rods were present in clusters, as demonstrated in
sections (Figs 3O, S, and X) and retinal flat mounts (Fig
3T). The rod cell bodies were rhodopsin positive but lacked
outer segments, confirmed by lack of rds/peripherin reac-
tivity. The rods were irregular in shape but did not show
neurite sprouting in the macula. No wheat germ agglutinin-
positive rod sheaths were identified.
A foveal pit could not be identified, because the central
macula was edematous and folded. The foveal cones were
identified by their uniform columnar shape and lack of
labeling with anti-calbindin.53The foveal cones lacked
outer segments and a few were stained throughout the
cytoplasm with anti-blue cone opsin, but the rest of the
foveal cones were red/green cone opsin negative. The bases
of the foveal cones were SV2 positive but the outer plexi-
form layer was thinned (Fig 3U). The cytoplasm of the
foveal cones was intensely labeled with anti-recoverin (Fig
3U) and mAb 7G6.
Peripheral Retina. The midperipheral retina lacked
photoreceptors, corresponding to the area of faint bone
spicule pigment noted grossly. The far peripheral retina
contained numerous photoreceptors that lacked outer seg-
ments. There was a nearly continuous monolayer of cone
cell bodies (Fig 3V) and rods that showed neurite sprouting
Inner Retina. There was weak labeling of horizontal
cells with anti-parvalbumin and anti-calbindin. Normal
numbers of cells were present in the inner nuclear layer,
where scattered cone bipolar cells were positive for recov-
erin (Fig 3U) or calbindin. Amacrine cells were positive
with anticalbindin, but few were positive for GABA or
glycine. The inner plexiform layer, identified by labeling
with anti-SV2 and anti-GABA, was thinned and irregular in
the periphery but uniform in thickness and SV2-labeling in
the macula (Fig 3U). Ganglion cells, identified by 4?,6?-
diamidino-2-phenylindole staining of their round nuclei,
were three to five cells deep in the macula (normal, six to
eight) but reduced to a few, scattered cells in the periphery.
The Mu ¨ller cells had undergone reactive gliosis throughout
the retina (Fig 3X), and their hypertrophied processes were
GFAP positive, particularly in the nerve fiber layer where
ganglion cell axons had been lost.
Photoreceptor guanylyl cyclases are outer segment mem-
brane proteins that catalyze conversion of guanosine
triphosphate to cyclic guanosine monophosphate; this opens
gated cation channels and restores the dark state in photo-
receptors after light exposure.7The GUCY2D gene, on
chromosome 17p, was the first gene identified with muta-
tions causing LCA,9and subsequent reports indicate that
disease-causing changes can be present in extracellular,
kinaselike, dimerization and catalytic domains of the mol-
ecule.10–12This is in contrast to the heterozygous dimeriza-
tion domain GUCY2D mutations that cause autosomal dom-
inant cone-rod dystrophy.54–56
The Arg-660-Gln variation observed in the GUCY2D
gene of the proband and affected sister in this study is likely
Milam et al ? Leber Congenital Amaurosis
Volume 110, Number 3, March 2003
to be a disease-causing mutation for the following reasons:
it alters the predicted amino acid sequence of the protein in
a way that would alter the charge of the molecule at neutral
pH; this change has not been observed in any other LCA
patients (more than 400 chromosomes) or in any control
subjects (more than 280 chromosomes); the mutation is
present in both affected sisters, and no plausible disease-
causing mutations were found in 4 other LCA-associated
genes. Moreover, haplotype analysis of the GUCY2D locus
clearly reveals that the 2 affected sisters received the same
GUCY2D genes from both parents, whereas their unaffected
sister received a different paternal allele. Finally, a marker
suggests that the father’s putative disease-causing allele
harbors a deletion of unknown size adjacent to the GUCY2D
gene. It will require further study of this region to confirm
the existence of this deletion and to assess the likelihood
that it could be responsible for inactivation of the adjacent
Among the goals of this study were to determine the
effect of mutant GUCY2D on the human retina and to
provide clinicopathologic correlations in this 111⁄2-year-old
patient donor with LCA who had been followed clinically
for 51⁄2 years before her death. The retinal disease of the
proband-donor progressed in the period between age 6 and
101⁄2 years. About 11⁄2 years before death, vision had de-
clined to light perception only; there was no measurable
kinetic visual field with conventional targets, and the ERG
had been nondetectable years previously. Correlating with
the severe retinal dysfunction in the subject was the lack of
photoreceptor outer segments. The absence of outer seg-
ments was evidenced by lack of labeling with anti-rds/
peripherin, a marker specific for rod and cone outer seg-
ments,57,58and lack of outer segment labeling with antiblue
or anti-red/green cone opsin, or with anti-rhodopsin. We
found that many rods and cones, however, had survived in
the macula and periphery, although in decreased numbers.
In vivo cross-sectional images with OCT predicted an ab-
normally thinned retina, and the histopathology confirmed
The macular cones were reduced to cell bodies that
closely resembled those in newborn human retinas (this
study and59) and in retinas with advanced retinitis pigmen-
tosa (RP).60,61However, the LCA cones lacked outer seg-
ments, which are present but very short in newborn and RP
macular cones. In the LCA retina, some cone cell bodies
were well labeled with anti-blue cone opsin, but no cones
were labeled with either antibody against red/green cone
opsin. Both antibody preparations strongly labeled red/
Figure 3. Immunofluorescence images of normal and Leber congenital amaurosis (LCA) human retinas processed with cell-specific antibodies. Nuclei are
stained (blue) with 4?,6?;-diamidino-2-phenylindole. Bar ? 100 ?m in all panels. R, autofluorescent retinal pigment epithelium layer; P, photoreceptor
layer; O, outer plexiform layer; N, inner nuclear layer; I, inner plexiform layer; G, ganglion cell layer. A, Normal 3-day-old human retina labeled with
anti-recoverin (green) and anti-synaptic vesicle protein (SV2) (red). Note recoverin-positive rods and cones (P) with very short outer segments (*).
Synapses in the outer plexiform layer (O) and inner plexiform layer (I) are strongly positive (red) for SV2. The outer plexiform layer is yellow gold because
it is positive for both recoverin and SV2. B, Normal 3-day old human retina labeled with monoclonal antibody (mAb) 7G6 (red) and anti-blue cone opsin
(green). Note 7G6-positive cone outer segments and cytoplasm and two blue cone opsin-positive outer segments (arrowheads). C, Normal 3-day old
human retina labeled with anti-rds/peripherin (red). Note short outer segments (*) of the rods and cones. D, Normal 3-day old human retina labeled with
peanut agglutinin (PNA) (red). Note heavy labeling of the short cone matrix sheaths (*). E, Normal 3-day old human retina labeled with anti-rhodopsin
(red) and anti-glial fibrillary acidic protein (GFAP) (green). Note rhodopsin-positive rod outer segments (arrowhead) and GFAP in astrocytes (A) in the
inner retina. F, Normal 53-year-old human retina labeled with mAb 7G6 (red) and anti-blue cone opsin (green). The cones are positive throughout their
outer segments and cytoplasm with mAb 7G6. Note single blue cone outer segment (arrowhead). The RPE cells (R) across the bottom of the image contain
autofluorescent lipofuscin granules. G, Normal 53-year-old human retina labeled with anti-blue cone opsin to demonstrate a blue cone cell body and outer
segment. H, Normal 53-year-old human retina labeled with anti-red/green cone opsin (green). Note that most of the cone outer segments (arrowheads)
contain red/green cone opsin. I, Normal 53-year-old human retina labeled with anti-rds/peripherin (red). Note positive staining of rod and cone outer
segments (*). J, Normal 53-year-old human retina labeled (red) with PNA. Note long cone matrix sheaths (*). K, Normal 53-year-old human retina
labeled with anti-rhodopsin (red) and anti-GFAP (green). Note rhodopsin-positive rod outer segments (*) and GFAP-positive astrocytes (A) in the nerve
fiber layer and around a blood vessel. L, Normal 53-year-old human retina labeled with anti-recoverin (green) and anti-SV2 (red). Note recoverin-positive
rods and cones (P) and scattered recoverin-positive cone bipolar cells (arrowheads) in the inner nuclear layer (N). Synapses in the outer plexiform layer
(O) and inner plexiform layer (I) are strongly positive for SV2. The outer plexiform layer is yellow gold, because it is positive for both recoverin and SV2.
M, LCA retina labeled with anti-rds/peripherin. Note lack of specific labeling because of absence of photoreceptor outer segments. N, LCA retina labeled
with mAb 7G6. Note intense labeling of cone cytoplasm (*). R, retinal pigment epithelium. O, LCA retina labeled with anti-blue cone opsin (green)
and anti-rhodopsin (red). Note lack of cone outer segments and presence of two blue cone cell bodies (arrowheads) and a cluster of rod cell bodies (arrow).
P, LCA retina labeled with anti-red/green cone opsin. Note lack of cone labeling. Q, Edge of macula of LCA retina labeled (red) with mAb 7G6. Note
variable intensity of cytoplasmic labeling of individual cones (arrowheads). R, Labeling of LCA retina with PNA (green). Note very weak labeling of cone
inner segments (arrowheads) and lack of cone matrix sheaths. S, LCA retina labeled with anti-rhodopsin (red). Note absence of rod outer segments.
Rhodopsin is labeled in a cluster of rod cell bodies (arrowhead). T, Flat mount of edge of macula in the LCA retina labeled with anti-rhodopsin (red).
Note patchy loss of rods across the retina. A normal retina labeled in the same manner shows uniform distribution of rod cells (see67.) U, LCA retina
labeled with anti-recoverin (green) and anti-SV2 (red). Note intense labeling of recoverin in the cone cell bodies (*) and the thin outer plexiform layer
(O). Recoverin-positive cone bipolar cells (arrowheads) are present in the inner nuclear layer (N). The inner plexiform layer (I) has normal thickness
and is intensely positive for SV2. V, Far periphery of LCA retina labeled (red) with mAb 7G6. Note monolayer of cone cell bodies (arrowheads) that
stops abruptly at the ora serrata (*). W, Far periphery of LCA retina labeled (red) with anti-rhodopsin. Note row of rod cell bodies adjacent to the ora
serrata. Some rods have sprouted long neurites (arrowheads) that pass through the inner nuclear layer (N). X, LCA retina labeled with anti-rhodopsin
(red) and anti-GFAP (green). Note cluster of rhodopsin-positive rod cell bodies (arrowheads) that lack outer segments. Mu ¨ller cells have undergone
reactive gliosis in response to photoreceptor cell death, and their cytoplasm is filled with GFAP-positive filaments (green). Note heavy labeling with
anti-GFAP of the nerve fiber layer (F), reflecting loss of some ganglion cells.
Milam et al ? Leber Congenital Amaurosis
green cone outer segments in the normal 3-day-old, 18-year-
old, and 53-year-old human retinas, and the basis of this
deficit in the LCA retina is unknown.
We also noted that, unlike normal macula cones, some
LCA cones, identified by their characteristic shape, had
decreased immunoreactivity for certain cytoplasmic pro-
teins involved in visual transduction (recoverin) and other
cellular functions (calbindin and 7G6). Similar loss of re-
activity for cytoplasmic proteins in degenerate cones has
been noted in human RP retinas with Rho mutations62and
in detached retinas of cats.63,64This apparent loss of cyto-
plasmic proteins may indicate dedifferentiation of the cones
and contribute to loss of cone-mediated vision, even though
the cone cell bodies are present.62
In addition to abnormal cones, the LCA macula also
contained rods that were strongly positive for rhodopsin in
their cell bodies and axons. We did not find evidence of rod
neurite sprouting in the macula, but rod neurites were abun-
dant in the periphery, in agreement with studies of RP
retinas, where rod neurites were limited to this region.60,65
Another unusual feature of the LCA retina was that the
remaining rods were in patches rather than spread uniformly
across the macula and periphery like the cones. The basis of
this pattern is unknown but may be consistent with sugges-
tions that rods secrete factors trophic for other photorecep-
tors,66perhaps including fellow rods as well as cones.
Similar patchy loss of rods was found in retinas from
patients with RP caused by Rho mutations.67
What do we know about the effect on other retinas of
mutations in the gene encoding photoreceptor guanylyl cy-
clase? To date, no other retinas from LCA human patients
with this genetic defect have been reported. There is infor-
mation from two animals, the rd chicken, which has a null
mutation in retGC1,68and a mouse with targeted disruption
of the Gucy2e gene.69The photoreceptors in newborn rd
chick retinas are fully differentiated and ultrastructurally
normal, but they do not have recordable ERGs. After ap-
proximately 1 week, the photoreceptor outer segments and
then their cell bodies and nuclei degenerate.70The rd
chicken cones downregulate expression of Gcap1, and
Gcap1 protein levels are reduced by more than 90%.71This
is reminiscent of the loss of cone cytoplasmic proteins in
human RP retinas62and in the LCA retina studied here.
Previous studies72comparing histopathology and OCT
of rd chicken retinas (ages 10–15 months) showed a pattern
of OCT thinning with a single-peaked ORCC that is remi-
niscent of the OCT in the human LCA patients in this study.
The rd chicken retinas examined morphologically had some
variation in severity of retinal degeneration.72The most
severe phenotype lacked photoreceptors but in less severe
disease, outer nuclear and outer plexiform layers were re-
tained, albeit thinned. No rod photoreceptors were identi-
fied, but some cone inner segments with tiny outer segments
were retained,72confirming earlier observations of surviv-
ing cones.73Mice with disrupted Gucy2e also show ERG
abnormalities that precede morphologic changes. The dis-
ease expression in mice has been described as affecting
cones more than rods.69The species differences in disease
expression are not understood.
From a therapeutic standpoint, even though the LCA
patient had only light perception at the time of death, her
retina still contained numerous macular and peripheral rods
and cones, although they lacked outer segments, and some
cones had lost reactivity for certain cytoplasmic proteins.
This should be a source of tempered encouragement for
those developing treatments for severe early-onset retinal
degenerations such as LCA.74Successful treatment might
cause the rods and cones to form outer segments and lead to
expression of all cone cytoplasmic proteins needed for
A second critical consideration for any photoreceptor-
based therapy is the status of the inner retina, because the
inner retinal circuitry must remain intact for vision to be
restored once the photoreceptor defect is corrected. On the
positive side, the inner nuclear layer seemed intact with
normal numbers of cells, some of which were identified as
cone bipolar and amacrine cells. However, we failed to find
normal parvalbumin or calbindin reactivity in horizontal
cells, and GABA and glycine labeling of amacrine cells was
greatly reduced. In addition, ganglion cell numbers were
reduced in both the macula and periphery. Yet, the patient
retained light perception, suggesting that sufficient inner
retinal circuitry may have remained for improved visual
function if the photoreceptor defects could be corrected.
Acknowledgments. The authors thank the scientists who gen-
erously provided antibodies for this study, Ms. J. Fisher for assis-
tance with donor tissue, Dr. N. Syed for help with gross pathology,
and Ms. H. Hanes, Ms. C. Taylor, Ms. P. Rothenberg, Drs. J.
Huang, and W.-Y. Tang for technical assistance. Clinical coordi-
nation was provided by Ms. L. Gardner and Ms. J. Emmons.
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