Histopathology of the Retina from a Three Year-Old Suspected to Have Joubert Syndrome

Research Article

Austin J Clin Ophthalmol. 2015; 2(4): 1057.

Histopathology of the Retina from a Three Year-Old Suspected to Have Joubert Syndrome

Bonilha VL1,2*, Rayborn ME¹, Bell BA¹, Marino MJ¹, Traboulsi EI¹, Hagstrom SA1,2 and Hollyfield JG1,2

¹Cole Eye Institute, Cleveland Clinic, Cleveland, USA

²Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, USA

*Corresponding author: Bonilha VL, Cole Eye Institute, Cleveland Clinic, Ophthalmic Research - i3, 19500 Euclid Avenue, Cleveland, OH 44195, USA

Received: June 23, 2015; Accepted: August 25, 2015; Published: September 21, 2015


Purpose: To define the retinal pathology in a 3 year-old eye donor who died from complications of an undiagnosed genetic syndrome.

Methods: Eyes were fixed and analyzed using macroscopic fundus photography (MF), confocal scanning laser ophthalmoscopy (cSLO) and spectral-domain optical coherence tomography (SD-OCT). Small areas from the perifovea and periphery were processed for histology and indirect immunofluorescence, using antibodies specific to retinal proteins such as rhodopsin, cone arrestin, RPE65 and others. Available medical records were also reviewed.

Results: With all three imaging modalities, the affected donor’s eyes lacked the distinct morphological detail typically observed with these techniques in postmortem control eyes. MF images showed a “photonegative effect” due to a hypopigmented macula relative to a hyperpigmented retinal background. cSLO imaging demonstrated a weak autofluorescence signal that was largely devoid of the usual retinal structures compared to the control. SD-OCT suggested disorganization of the affected retina, absence of a photoreceptor layer, and degeneration of the choroid in the macular area. Histologic findings indicated a highly disorganized photoreceptor layer in the macula and periphery. The RPE layer displayed thinning in some regions of the periphery and decreased pigmentation in most areas. Rods and cones were significantly reduced in the affected retina but a few cones were detected in the perifovea. Centrin-2 labeling was mostly absent from the connecting cilium of the photoreceptor cells. Medical record review pointed to a possible clinical diagnosis of Joubert syndrome.

Conclusions: The retinal degenerative findings, and absence of centrin-2 labeling are compatible with the expected retinal phenotype in patients with Joubert syndrome.

Keywords: Genetic abnormalities; Retina; Histology; Disorganized photoreceptors; Immunohistochemistry


Genetic testing has allowed the identification of the underlying cause of a large number of inherited eye diseases. Eye conditions such as coloboma, microphthalmia, and the retinal dystrophies can be isolated, or can be part of a clinical syndrome [1]. It has been well established that there is significant genetic, allelic, and phenotypic heterogeneity amongst genetic eye diseases, especially the inherited retinal dystrophies. While clinical presentation and examination can often provide a clinical diagnosis, genetic testing and the demonstration of the underlying molecular pathology establish the cause of disease.

Here we report the analysis of eyes of a male donor with profound visual impairment. The patient had received a clinical diagnosis of Leber Congenital Amaurosis (LCA). Genetic testing was carried out but failed to identify the disease-causing mutation(s). A medical record review indicated a wider range of clinical signs and symptoms and suggested the possibility of a syndromic form of retinal dystrophy.

Materials and Methods

Patient information

The organ donation was coordinated through The Foundation Fighting Blindness Rare Eye Donor Program (#852). The review of medical records and tissue analysis were performed with the approval of the Cleveland Clinic Institutional Review Board (IRB #14-057). This male child was the product of a consanguineous relationship between his mother and her maternal uncle. He was removed from his mother and placed in foster care. The donor died at the age of 3 years from respiratory distress. When his globes were donated to this program, he carried a clinical diagnosis of Leber congenital amaurosis (LCA).

Genetic analysis

Peripheral blood was collected from the donor. DNA was extracted and purified from leukocytes in the blood by means of the Gentra Systems PUREGENE DNA Purification Kit (Qiagen). Since the presenting diagnosis was LCA, DNA was evaluated by Asper Ophthalmics for 641 mutations in 13 genes known to cause LCA (AIPL1, CRB1, CRX, GUCY2D, LRAT, MERTK, CEP290, RDH12, RPGRIP1, RPE65, TULP1, LCA5, SPATA7). However, due to insufficient DNA quality, some amplicons were missed and the analysis failed to identify any specific genetic mutation.

Medical Record Review

Approximately 5 years after obtaining this eye donation, the donor’s medical records were obtained through consent and coordination with the Washington State Department of Social and Health Services. Both the genetic counselor and ophthalmic geneticist analyzed all records. Based on the review of the donor’s clinical findings, a differential diagnosis was developed.

Ex-vivo Imaging of globes

Macroscopic fundus images of the posterior globes were acquired as previously described [2]. Briefly, the globes were bisected along the equator, and the posterior poles were transferred to a custommade plexiglass chamber filled with PBS solution to a level above the cut surface of the globe, and the posterior poles were imaged using a Zeiss AxioCam MRC5 camera and an angled illumination from a bifurcated fiber optic light source.

Scanning Laser Ophthalmoscopy (SLO)

SLO images of the fixed posterior poles were collected using a model HRA2 confocal scanning laser ophthalmoscope (Heidelberg Engineering, Inc.) as previously described [2]. The HRA2 was rotated 90o and the system was operated in high-resolution mode, which provides an image pixel format of 1536 x 1536 when used with a 55° wide-field objective lens. SLO images of the posterior pole were collected using Infrared Reflectance (SLO-IR), and Autofluorescence (SLO-AF) imaging modes at field of view (FOV) settings of 55°, 35°, and 25°.

Spectral Domain Optical Coherence Tomography (SD-OCT)

SD-OCT images of the posterior pole were collected using an SDOCT system (Model SDOIS, Bioptigen, Inc.), as previously described [2]. SD-OCT imaging was performed using the following scan parameters: (1) 5mm linear scan of the horizontal meridian through the optic nerve and fovea @ 1000 A-scans/B-scan, (2) 10mm linear scan of the horizontal meridian through the optic nerve and fovea @ 1000 A-scans/B-scan, (3) 5mm² Volume Scan of the posterior pole @ 500 B-Scans/Volume x 250 A-scans/B-scan, and (4) 10mm² volume scan of the posterior pole @ 500 B-Scans/Volume x 250 A-scans/Bscan. Post-acquisition images were averaged with a line and frame filter settings of 3 and 3, respectively using the Bioptigen InVivoVue SDOIS Software version


The globes were fixed 11 hours post-mortem in a mixture of 4% paraformaldehyde and 0.5% glutaraldehyde made in PBS. After 1 month in fixative, the globes were transferred and stored in 2% paraformaldehyde prepared in the same buffer. Eyes from a 3 year-old female with no history of retinal disease, who died from asphyxiation, were used as controls and were fixed 2 hours post-mortem. A small area of the retina/RPE/choroid tissue from the perifovea and periphery of the affected donor and age-matched control were cut and further processed as previously described [2]. Cryosections (8μm) were cut and labeled with the following antibodies: B630N to rhodopsin (1:50, from Dr. G. Adamus, Oregon Health and Science University, Portland, OR), 7G6 to cone arrestin (1:100, from Dr. P. MacLeish, Morehouse School of Medicine, Atlanta, GA), ab10062 to GFAP (1:400, Abcam), PETLET to RPE65 (1:500, from Dr. R. Crouch, University of South Carolina, Charleston, SC), AB1778 to calbindin D (1:500, Chemicon) and sc-27794 to centrin-2 (1:50, Santa Cruz). Cell nuclei were labeled with TO-PRO®-3 iodide (1mg/ml, Molecular Probes, Eugene, OR). Secondary antibodies (goat anti-mouse or anti-rabbit IgG; 1:1000) were labeled with Alexa Fluor 488 (green; Molecular Probes) and Alexa Fluor 594 (red; Molecular Probes). Sections were analyzed using the same acquisition parameters in a Leica laser scanning confocal microscope (TCS-SP2, Leica, Exton, PA). A series of 1 μm xy en face sections were collected through the whole section and processed into a three-dimensional projection of the entire cryosection (sum of all images in the stack). Microscopic panels were composed using AdobePhotoshop CS3 (Adobe, San Jose, CA).


When first examined at the age of 1.5 years, the donor was found to have profound visual impairment and was diagnosed with LCA. He had failure to thrive (less than 3rd percentile for weight), hypotonia, developmental delay (one assessment showed 2 - 5 months developmental level at chronological age of 16 months), microcephaly (less than 3rd percentile), plagiocephaly, hyperextensible joints, pectus excavatum, and an undescended left testicle. An MRI of the brain showed mild to moderate cerebellar atrophy, with a hypoplastic inferior cerebellar vermis, and dilation of the fourth ventricle. A hearing test was normal. Additional exams included normal chromosomal analysis, negative for fragile X syndrome, negative for Smith- Lemli- Opitz syndrome, normal very long chain fatty acids, and normal extended newborn screen. The donor sustained several episodes of aspiration pneumonia as well as numerous other medical problems such as respiratory obstruction, hypertension, hypotension, and bradycardia. He underwent surgical interventions including tonsillectomy and adenoidectomy, fundoplication, gastrojejunostomy, and right orchiopexy.

As we characterized the retinal histopathology of this donor we intended to determine the specific mutation affecting this donor. However, there was insufficient DNA to perform additional genetic testing.

Fundus imaging of the affected donor eye showed a “photonegative effect” in the macula due to a hypopigmented macula relative to a hyperpigmented, dark retinal background (Figure 1B, arrow) when compared to the control eye (Figure 1A). SLO-IR imaging identified the optic disk (Figure 1D, asterisk) and the hypopigmented macula identified by fundus image (Figure 1D, arrow). SLO-AF imaging identified a weak AF signal (Figure 1F) that was devoid of any structural detail compared to the control, which clearly showed retinal vasculature and lipofuscin AF background (Figure 1E). All imaging modalities identified precipitated crystals from the buffer in the fixative solution in the control eye (Figures 1A, 1C, 1E, arrowheads).