Clinicopathologic Effects of MutantGUCY2D in Leber Congenital Amaurosis
Ann H. Milam, PhD,1,2 Mark R. Barakat, AB,1 Nisha Gupta, PhD,1 Linda Rose, MD, PhD,1
Tomas S. Aleman, MD,1 Michael J. Pianta, PhD,1 Artur V. Cideciyan, PhD,1 Val C. Sheffield, MD, PhD,3
Edwin M. Stone, MD, PhD,4 Samuel G. Jacobson, MD, PhD1
Purpose: To study the retinal degeneration in an 111⁄2-year-old patient with Leber congenital amaurosis(LCA) caused by mutation in GUCY2D.
Study Design: Comparative human tissue study.Participants: Two subjects with LCA; postmortem eye from one LCA patient and three normal donors.Methods: 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, 18years, and 53 years. The tissues were processed for histopathologic studies and immunofluorescence with retinalcell-specific antibodies.
Results: 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 coneswithout outer segments in the macula and far periphery. The cones formed a monolayer of cell bodies, but therods were clustered and had sprouted neurites in the periphery. Rods and cones were not identified in themidperipheral retina. The inner nuclear layer appeared normal in thickness throughout the retina, but ganglioncells were reduced in number.
Leber congenital amaurosis (LCA) is a retinal degenerationwith blindness or severe vision loss at birth or shortlythereafter.1–3 LCA accounts for 5% of all inherited retinaldiseases but is higher in countries with high rates of con-sanguineous unions.4,5 Recent studies have demonstrated
that LCA is genetically heterogeneous.6,7 To date, at least10 LCA genes have been identified or localized:GUCY2D(retinal guanylate cyclase 1, also known asRETGC, andRETGC1)8–12; RPE65 13–19; CRX20–24; TULP125;AIPL121,26,27; RPGRIP128,29; LCA3 30; LCA531; CRB1 32,33;andLRAT.34
There are relatively few histopathologic studies of hu-man LCA eyes, usually of retinas with advanced disease andnone with a known gene defect. Most published reports onLCA retinas have revealed death of photoreceptors, the rodsbefore the cones. Loss of inner retinal neurons, includingganglion cells, and intraretinal migration of retinal pigmentepithelium cells occur secondary to photoreceptordeath.35–39
We studied the postmortem eye of a young (111⁄2-year-old) LCA subject whose vision was carefully documented inlife. Her younger sister also has LCA. The molecular defectin the family is mutation in theGUCY2D gene. The post-mortem donor retina was well preserved and offered theopportunity to address three questions:
● The subject had only light perception at last examina-tion. What is the microscopic correlate of the func-tional defect?
● How does mutation in theGUCY2D gene affect therods and cones?
Originally received: March 29, 2002.Accepted: July 26, 2002. Manuscript no. 220247.1 Department of Ophthalmology, Scheie Eye Institute, University of Penn-sylvania, Philadelphia, Pennsylvania.2 F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute,University of Pennsylvania, Philadelphia, Pennsylvania.3 Department of Pediatrics, University of Iowa Hospital and Clinics, IowaCity, Iowa.4 Department 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, NewYork, New York; Fight for Sight Research Division of Prevent BlindnessAmerica, Schaumburg, Illinois; The Grousbeck Family Foundation, SanFrancisco, California; and the F. M. Kirby Foundation, Morristown, NewJersey. VCS is supported by the Howard Hughes Medical Institute; SGJ isan RPB Senior Scientific Investigator; AVC is an RPB Special Scholar.
Reprint requests to Ann H. Milam, PhD, Scheie Eye Institute, University ofPennsylvania, 51 North 39th St., Philadelphia, PA 19104.
● Would a retina at the stage of disease of the eye donorbe amenable to any form of therapy?
With these questions in mind, we present detailed clini-cal, genetic, and histopathologic observations on the retinaof this young LCA subject.
Material and Methods
Subjects and Clinical StudiesA North American family of German and Italian ancestry had 2female children affected with LCA and 2 unaffected female chil-dren (Fig 1A). The two LCA subjects had complete clinical ocularexaminations and visual function tests, including Goldmann ki-netic perimetry, full-field electroretinography (ERG), and opticalcoherence tomography (OCT). Details of our perimetry, full-fieldERG, and OCT methods and data analyses are published.22,40–43
All subjects gave informed consent; institutional review boardapproval was obtained, and the tenets of the Declaration of Hel-sinki were followed.
Molecular StudiesDNA was extracted from peripheral blood using a previouslydescribed protocol.44 The proband was screened for mutations inthe coding sequences of the AIPL1, CRB1, CRX, GUCY2D, andRPE65 genes using single-strand conformational polymorphism(SSCP) analysis.45,46 The primer sequences used for SSCP screen-ing of the coding regions are published.9,13,32,47–49
The polymerase chain reaction amplification products weredenatured 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.50 Polymerase chain reaction prod-ucts from samples with aberrant electrophoretic patterns weresequenced bidirectionally with fluorescent dideoxynucleotides onan ABI model 377 automated sequencer (Applied Biosystems,Foster City, CA).
Detection of only a single amino acid–altering sequence changein the proband led to complete bidirectional sequence analysis ofthe coding sequences of the GUCY2D locus. In addition, a geno-typic survey of the GUCY2D locus was performed using 20short-tandem repeat polymorphisms that map to 17p13.1(D17S974, D17S1879, D17S786, D17S720, D17S938, D17S1881,D17S804, D17S960, D17S919, D17S796, D17S1854, D17S1149,D17S1353, D17S1805, D17S1796, D17S945, D17S954,D17S1852, D17S731, and D17S1159).
Oligonucleotide primers complementary to sequences flankingthese markers were obtained from Research Genetics (Huntsville,AL). The polymerase chain reaction conditions used for short-tandem repeat polymorphism genotyping were the same as forSSCP analysis. However, the amplification products were dena-tured, electrophoresed on gels consisting of 6% polyacrylamide-7M urea at 65 W for approximately 3 hours, and stained with silvernitrate.46,50
HistopathologyPostmortem human eyes were obtained through the donor programof 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) wasfixed 13.5 hours postmortem in a mixture of 4% paraformaldehydeand 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. After4 days in this fixative, the globe was stored in 2% paraformalde-
hyde in the same buffer. As controls, normal eyes from threedonors (#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,51 retinal 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.51 The following retinal cell-specific antibodies wereused: mouse monoclonal antibody (mAb) 7G6 specific for conecytoplasm (1:250, from Dr. P. MacLeish, Morehouse School ofMedicine, Atlanta, GA); mouse mAb 4D2 anti-rhodopsin specificfor rod outer segments (1:40, from Dr. R. Molday, University ofBritish Columbia, Vancouver, B. C., Canada); sheep polyclonalantibody (pAb) anti-rhodopsin (1:1000, from Dr. D. Papermaster,University of Connecticut Health Center, Farmington, CT); rabbitpAb JH492 anti-red/green cone opsin (1:5000, from Dr. J.Nathans, Johns Hopkins University, Baltimore, MD); rabbit pAbanti-red/green cone opsin (1:200, from Dr. J. Saari, University ofWashington, 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 MedicalSchool, 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 cellsand inner retinal neurons (C8666, 1:200, from Sigma); rabbit pAbanti-GABA and anti-glycine, specific for amacrine cells (1:100,from Dr. R. Marc, University of Utah, Salt Lake City, UT); guineapig pAb anti-GABA (1:1000, from Incstar Co., Stillwater, MN);rabbit pAb anti-glial fibrillary acidic protein (GFAP), specific forastrocytes and reactive Muller cells (1:750, Dako Corporation,Carpinteria, CA); and rhodamine-conjugated peanut agglutinin andwheat 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) werelabeled 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 ofprimary antibody.
The immunolabeled retinal sections were examined with amicroscope 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, SanJose, CA), and dye-sublimation prints were generated.
Results
Eye Donor and Family Members: Clinical StudiesThe family (Fig 1A) had two female children (II-2, II-3) with LCAand two unaffected female children (II-1, 11-4). There was noknown parental consanguinity.
Subject II-2 (Eye Donor). The proband as an infant hadabnormal eye movements and visual disturbances. On examination
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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). Imagesare 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 temporalto 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 reflectivityprofiles (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 scleradpeak of the outer retina-choroidal complex, ORCC (*).
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at age 6 years, she had nystagmus and exotropia; visual acuitieswere 1/200; and cycloplegic retinoscopy was �4.00 in both eyes.Ophthalmoscopy showed attenuated retinal vessels and a granularappearance to the fundus, most obvious in the midperiphery.Kinetic perimetry (V-4e test target) suggested that the visual fieldwas limited to a central island of function (Fig 1B). The ERGs hadno detectable rod b-waves to a dim blue flash, dark adapted, butthere were abnormally reduced waveforms to a maximal mixedcone-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 nomeasurable visual field by kinetic perimetry. Ophthalmoscopicappearance was unchanged, except for faint bone spicule pigmentin 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 LCAproband compared with normal at the locations sampled. A normalOCT longitudinal reflectivity profile has 2 prominent peaks, one atthe vitreoretinal surface and the other in the outer retina, known asthe outer retina-choroidal complex (ORCC). Unlike the normaldouble-banded ORCC, the proband’s ORCC contained only asingle peak (Fig 1D). At age 111⁄2 years, the proband died fromcomplications after tonsillectomy.
Subject II-3. A sibling (5 years younger) of the proband wasnoted at age 4 years to have some visual inattention and nightvision disturbances. On examination at age 6 years, there was nonystagmus 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-4etest target had slight generalized constriction but were full inperipheral extent; response to a I-4e target was mainly in theperipheral field with a relative central and midperipheral scotomas(Fig 1B). The ERGs had no detectable rod b-waves; responses toa mixed cone-rod stimulus and to cone stimuli were abnormallyreduced (Fig 1C). With OCT, a more complete series of cross-sectional retinal images was obtained, because this patient did nothave nystagmus. A scan through the fovea showed a relativelynormal contour, but, as in the proband, there was retinal thinningand the normally double-banded ORCC had only a single peak atthe locations examined (Fig 1D).
Molecular StudiesA total of 74 amplimers (collectively containing the coding sequencesof five LCA-causing genes: AIPL1, CRB1, CRX, GUCY2D, andRPE65) was screened with SSCP analysis for coding sequence mu-tations in the proband and affected sister. In addition, the entire codingregion of the GUCY2D gene of the proband and her affected sisterwas directly sequenced twice with automated DNA sequencing. Onlytwo sequence variations (both in GUCY2D and both heterozygous)were found in this entire experiment. One of these changes is a fairlycommon T3C polymorphism at position 703, 143 base pairs 3� tothe last nucleotide of the GUCY2D stop codon. The other is avariation that would be expected to change the basic arginine residueat codon 660 to an uncharged glutamine residue. The latter change hasnot been observed in another LCA proband or in any of 143 controlsubjects.
Detection of a single plausible disease-causing allele in the twoaffected sisters led to further scrutiny of the GUCY2D locus usingshort-tandem repeat polymorphism. In all, 20 polymorphisms atthe GUCY2D locus were genotyped in this nuclear family, and 6markers were informative for both parents. All 6 markers revealedthat the 2 affected sisters share a genotype and, in addition, thatthis 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 eitheraffected daughter. This marker is 1.14 cM away from the codingsequences of the GUCY2D gene.
Retinal Histopathology
Controls
Normal 3-day-old Macula. Cones and rod cytoplasm andouter segments were strongly labeled with anti-recoverin
Figure 2. Haplotype analysis. Each vertical line represents a portion ofchromosome 17 containing the GUCY2D gene. The alleles of variouspolymorphisms are grouped into deduced haplotypes on either side of theselines. The pedigree symbols above each vertical line indicate the genderand 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 theirthree children below. The GUCY2D gene is shown as a box superimposedon each haplotype, and the presence of three intragenic variations (atcodons 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 areshown numerically. The order of the polymorphisms from top to bottom inthe figure is as follows: centromere-D17S720, D17S938, D17S1881, D17S786,GUCY2D, D17S974, D17S1879. An X indicates a putative deletion involv-ing D17S974. Although the figure is drawn with the 5� end of the GUCY2Dgene pointing toward the telomere, this orientation has not been definitivelyestablished. The map order shown in this figure was obtained from theGenatlas database by way of www.infobiogen.fr.
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(Fig 3A). The cone cytoplasm was positive throughout withmAb 7G6 (Fig 3B), and the cone outer segments were shortand positive for either blue cone (Fig 3B) or red/green coneopsin. The short cone and rod outer segments were positivefor rds/peripherin (Fig 3C). The short cone matrix sheathswere peanut agglutinin positive (Fig 3D), and the rodsheaths were wheat germ agglutinin positive. The short rodouter segments were rhodopsin positive (Fig 3E), andGFAP reactivity was restricted to the astrocytes (Fig 3E).The synapses of the cones and rods were positive for thesynaptic vesicle protein, SV2 (Fig 3A). Inner retinal cellswere well labeled with the following markers: horizontalcells, calbindin and parvalbumin; amacrine cells, gamma-aminobutyric acid (GABA), glycine, and calbindin. Theouter and inner plexiform layers were uniform in thicknessand well labeled with anti-SV2 (Fig 3A). The macularganglion 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. Conesand rods were labeled as at 3 days, except the outer seg-ments were normal in length, shown in cones with mAb7G6 (Fig 3F), including blue cones (Figs 3F and G) andred/green cones (Fig 3H). Rod and cone outer segmentswere positive for rds/peripherin (Fig 3I), and rod and conematrix sheaths (Fig 3J) had normal length. Rod outer seg-ments were rhodopsin positive (Fig 3K), and GFAP wasrestricted to the astrocytes (Fig 3K). The inner retina waslabeled as at 3 days, including SV2-positive outer and innerplexiform layers (Fig 3L) and recoverin-positive cone bipo-lar cells (Fig 3L). Horizontal, bipolar, and amacrine cellswere labeled normally as in the 3-day retina.
LCA Retina
Gross Pathology. The dimensions of the cornea were 12 �11 mm, and the globe was 21 � 23 � 20 mm. The corneaappeared normal with no keratoconus; the lens and vitreouswere 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-periphery.
Histopathology/Immunocytochemistry
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 of7G6-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 twodifferent antibodies. The more degenerate cones at the edgeof the macula had variable intensity of cytoplasmic stainingfor calbindin, recoverin, and 7G6 (Fig 3Q). The cytoplasmof the cones was weakly peanut agglutinin positive (Fig3R), but cone sheaths could not be identified. Sectionstreated with secondary antibodies only had weak autofluo-rescence of lipofuscin granules in the retinal pigment epi-thelium.
The rods were present in clusters, as demonstrated insections (Figs 3O, S, and X) and retinal flat mounts (Fig3T). The rod cell bodies were rhodopsin positive but lackedouter segments, confirmed by lack of rds/peripherin reac-tivity. The rods were irregular in shape but did not showneurite sprouting in the macula. No wheat germ agglutinin-positive rod sheaths were identified.
A foveal pit could not be identified, because the centralmacula was edematous and folded. The foveal cones wereidentified by their uniform columnar shape and lack oflabeling with anti-calbindin.53 The foveal cones lackedouter segments and a few were stained throughout thecytoplasm with anti-blue cone opsin, but the rest of thefoveal cones were red/green cone opsin negative. The basesof the foveal cones were SV2 positive but the outer plexi-form layer was thinned (Fig 3U). The cytoplasm of thefoveal cones was intensely labeled with anti-recoverin (Fig3U) and mAb 7G6.
Peripheral Retina. The midperipheral retina lackedphotoreceptors, corresponding to the area of faint bonespicule pigment noted grossly. The far peripheral retinacontained numerous photoreceptors that lacked outer seg-ments. There was a nearly continuous monolayer of conecell bodies (Fig 3V) and rods that showed neurite sprouting(Fig 3W).
Inner Retina. There was weak labeling of horizontalcells with anti-parvalbumin and anti-calbindin. Normalnumbers 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 positivewith anticalbindin, but few were positive for GABA orglycine. The inner plexiform layer, identified by labelingwith anti-SV2 and anti-GABA, was thinned and irregular inthe periphery but uniform in thickness and SV2-labeling inthe 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 toeight) but reduced to a few, scattered cells in the periphery.The Muller cells had undergone reactive gliosis throughoutthe retina (Fig 3X), and their hypertrophied processes wereGFAP positive, particularly in the nerve fiber layer whereganglion cell axons had been lost.
Discussion
Photoreceptor guanylyl cyclases are outer segment mem-brane proteins that catalyze conversion of guanosinetriphosphate to cyclic guanosine monophosphate; this opensgated cation channels and restores the dark state in photo-receptors after light exposure.7 The GUCY2D gene, onchromosome 17p, was the first gene identified with muta-tions causing LCA,9 and subsequent reports indicate thatdisease-causing changes can be present in extracellular,kinaselike, dimerization and catalytic domains of the mol-ecule.10–12 This 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 GUCY2Dgene of the proband and affected sister in this study is likely
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to be a disease-causing mutation for the following reasons:it alters the predicted amino acid sequence of the protein ina way that would alter the charge of the molecule at neutralpH; this change has not been observed in any other LCApatients (more than 400 chromosomes) or in any controlsubjects (more than 280 chromosomes); the mutation ispresent in both affected sisters, and no plausible disease-causing mutations were found in 4 other LCA-associatedgenes. Moreover, haplotype analysis of the GUCY2D locusclearly reveals that the 2 affected sisters received the sameGUCY2D genes from both parents, whereas their unaffectedsister received a different paternal allele. Finally, a markersuggests that the father’s putative disease-causing alleleharbors a deletion of unknown size adjacent to the GUCY2Dgene. It will require further study of this region to confirmthe existence of this deletion and to assess the likelihoodthat it could be responsible for inactivation of the adjacentGUCY2D gene.
Among the goals of this study were to determine theeffect of mutant GUCY2D on the human retina and toprovide clinicopathologic correlations in this 111⁄2-year-oldpatient donor with LCA who had been followed clinicallyfor 51⁄2 years before her death. The retinal disease of theproband-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 measurablekinetic visual field with conventional targets, and the ERGhad been nondetectable years previously. Correlating withthe severe retinal dysfunction in the subject was the lack ofphotoreceptor 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,57,58 and lack of outer segment labeling with antiblueor anti-red/green cone opsin, or with anti-rhodopsin. Wefound that many rods and cones, however, had survived inthe macula and periphery, although in decreased numbers.In vivo cross-sectional images with OCT predicted an ab-normally thinned retina, and the histopathology confirmedthis observation.
The macular cones were reduced to cell bodies thatclosely resembled those in newborn human retinas (thisstudy and59) and in retinas with advanced retinitis pigmen-tosa (RP).60,61 However, the LCA cones lacked outer seg-ments, which are present but very short in newborn and RPmacular cones. In the LCA retina, some cone cell bodieswere well labeled with anti-blue cone opsin, but no coneswere labeled with either antibody against red/green coneopsin. 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 arestained (blue) with 4�,6�;-diamidino-2-phenylindole. Bar � 100 �m in all panels. R, autofluorescent retinal pigment epithelium layer; P, photoreceptorlayer; O, outer plexiform layer; N, inner nuclear layer; I, inner plexiform layer; G, ganglion cell layer. A, Normal 3-day-old human retina labeled withanti-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 becauseit 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 oldhuman retina labeled with anti-rds/peripherin (red). Note short outer segments (*) of the rods and cones. D, Normal 3-day old human retina labeled withpeanut 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 theinner retina. F, Normal 53-year-old human retina labeled with mAb 7G6 (red) and anti-blue cone opsin (green). The cones are positive throughout theirouter segments and cytoplasm with mAb 7G6. Note single blue cone outer segment (arrowhead). The RPE cells (R) across the bottom of the image containautofluorescent lipofuscin granules. G, Normal 53-year-old human retina labeled with anti-blue cone opsin to demonstrate a blue cone cell body and outersegment. 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 outersegments (*). J, Normal 53-year-old human retina labeled (red) with PNA. Note long cone matrix sheaths (*). K, Normal 53-year-old human retinalabeled with anti-rhodopsin (red) and anti-GFAP (green). Note rhodopsin-positive rod outer segments (*) and GFAP-positive astrocytes (A) in the nervefiber layer and around a blood vessel. L, Normal 53-year-old human retina labeled with anti-recoverin (green) and anti-SV2 (red). Note recoverin-positiverods 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 labeledwith 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. Notevariable intensity of cytoplasmic labeling of individual cones (arrowheads). R, Labeling of LCA retina with PNA (green). Note very weak labeling of coneinner 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 retinalabeled 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 thicknessand is intensely positive for SV2. V, Far periphery of LCA retina labeled (red) with mAb 7G6. Note monolayer of cone cell bodies (arrowheads) thatstops 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 oraserrata. 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. Muller cells have undergonereactive gliosis in response to photoreceptor cell death, and their cytoplasm is filled with GFAP-positive filaments (green). Note heavy labeling withanti-GFAP of the nerve fiber layer (F), reflecting loss of some ganglion cells.4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
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green cone outer segments in the normal 3-day-old, 18-year-old, and 53-year-old human retinas, and the basis of thisdeficit in the LCA retina is unknown.
We also noted that, unlike normal macula cones, someLCA cones, identified by their characteristic shape, haddecreased immunoreactivity for certain cytoplasmic pro-teins involved in visual transduction (recoverin) and othercellular functions (calbindin and 7G6). Similar loss of re-activity for cytoplasmic proteins in degenerate cones hasbeen noted in human RP retinas with Rho mutations62 andin detached retinas of cats.63,64 This apparent loss of cyto-plasmic proteins may indicate dedifferentiation of the conesand contribute to loss of cone-mediated vision, even thoughthe cone cell bodies are present.62
In addition to abnormal cones, the LCA macula alsocontained rods that were strongly positive for rhodopsin intheir cell bodies and axons. We did not find evidence of rodneurite sprouting in the macula, but rod neurites were abun-dant in the periphery, in agreement with studies of RPretinas, where rod neurites were limited to this region.60,65
Another unusual feature of the LCA retina was that theremaining rods were in patches rather than spread uniformlyacross the macula and periphery like the cones. The basis ofthis pattern is unknown but may be consistent with sugges-tions that rods secrete factors trophic for other photorecep-tors,66 perhaps including fellow rods as well as cones.Similar patchy loss of rods was found in retinas frompatients with RP caused by Rho mutations.67
What do we know about the effect on other retinas ofmutations in the gene encoding photoreceptor guanylyl cy-clase? To date, no other retinas from LCA human patientswith this genetic defect have been reported. There is infor-mation from two animals, the rd chicken, which has a nullmutation in retGC1,68 and a mouse with targeted disruptionof the Gucy2e gene.69 The photoreceptors in newborn rdchick retinas are fully differentiated and ultrastructurallynormal, but they do not have recordable ERGs. After ap-proximately 1 week, the photoreceptor outer segments andthen their cell bodies and nuclei degenerate.70 The rdchicken cones downregulate expression of Gcap1, andGcap1 protein levels are reduced by more than 90%.71 Thisis reminiscent of the loss of cone cytoplasmic proteins inhuman RP retinas62 and in the LCA retina studied here.
Previous studies72 comparing histopathology and OCTof rd chicken retinas (ages 10–15 months) showed a patternof 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 somevariation in severity of retinal degeneration.72 The mostsevere phenotype lacked photoreceptors but in less severedisease, 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 segmentswere retained,72 confirming earlier observations of surviv-ing cones.73 Mice with disrupted Gucy2e also show ERGabnormalities that precede morphologic changes. The dis-ease expression in mice has been described as affectingcones more than rods.69 The species differences in diseaseexpression are not understood.
From a therapeutic standpoint, even though the LCA
patient had only light perception at the time of death, herretina still contained numerous macular and peripheral rodsand cones, although they lacked outer segments, and somecones had lost reactivity for certain cytoplasmic proteins.This should be a source of tempered encouragement forthose developing treatments for severe early-onset retinaldegenerations such as LCA.74 Successful treatment mightcause the rods and cones to form outer segments and lead toexpression of all cone cytoplasmic proteins needed fornormal function.
A second critical consideration for any photoreceptor-based therapy is the status of the inner retina, because theinner retinal circuitry must remain intact for vision to berestored once the photoreceptor defect is corrected. On thepositive side, the inner nuclear layer seemed intact withnormal numbers of cells, some of which were identified ascone bipolar and amacrine cells. However, we failed to findnormal parvalbumin or calbindin reactivity in horizontalcells, and GABA and glycine labeling of amacrine cells wasgreatly reduced. In addition, ganglion cell numbers werereduced in both the macula and periphery. Yet, the patientretained light perception, suggesting that sufficient innerretinal circuitry may have remained for improved visualfunction 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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