Approval from the institutional review boards of the University of Utah, Moorfields Eye Hospital, and Institut de Recherche en Ophtalmologie was obtained for this study, and informed consent was obtained from all patients. Participating in the study were 27 individuals at risk for inheriting STGD4 in the first kindred, from the United States, a black family; 9 individuals at risk for inheriting MCDR2 in the second kindred, from England, a family of mixed European descent; and 15 individuals at risk for inheriting cone-rod dystrophy in the third kindred, from Switzerland.
Genetic linkage and mutation screening.
DNA was extracted from blood samples, and genetic linkage was assessed using microsatellite markers D4S1582
, and GATA158G03
(tightly linked to the STGD4
loci) using previously established methods (6
). We performed 2-point linkage analysis using the FASTLINK (37
) version of MLINK from the LINKAGE Program Package (38
). An autosomal-dominant mode of inheritance with full penetrance was used for LOD score computation. Disease allele frequency was set at 0.0001 (39
mutation screening was performed by denaturing HPLC (dHPLC) analysis followed by direct sequencing of PCR-amplified DNA fragments for all 23 PROM1
exons using previously established methods (6
). PCR primers were designed to include flanking intronic sequences of each exon according to published protocols (6
). Amplified products were purified using the QIAquik Gel Extraction Kit (Qiagen) and sequenced with forward and reverse primers by the Taq Dyedeoxy Terminator Cycle Sequencing Kit (Beckman-Coulter) according to the manufacturer’s instructions. dHPLC analysis was used according to the manufacturer’s instructions (Transgenomics Inc.) to screen for changes in PROM1
sequence and to determine the presence or absence of the mutation in the controls.
Generation of expression constructs.
Forward and reverse primers (5′-CCGCTCGAGCGTTGCTAGCTATGGCCCTC-3′ and 5′-ATAGTTTAGCGGCCGCATTCTTATTCAATGTTGTGATGGGCTTGTC-3′, respectively) containing Xho
l and Not
l restriction sites were designed for amplification of WT human PROM1
cDNA. Following Xho
l and Not
l restriction digest, the amplified cDNA was cloned into the Xho
l and Not
l sites of the pcDNA3.1(–) vector (Invitrogen). The 1117 C>T (R373C
) mutation was introduced into the WT PROM1
pcDNA3 construct by PCR-based site-directed mutagenesis. The recombinant plasmids were purified using a Qiagen plasmid isolation kit (Qiagen). Mouse PCDH21 expression constructs were generously provided by A. Rattner and J. Nathans (Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; ref. 21
). All constructs were verified by restriction digestion and DNA sequencing.
Generation of PROM1 transgenic mice.
WT or mutant human PROM1
cDNAs, generated as described above, were subcloned into the pRHO4.4 plasmid, containing the 4.4-kb human rhodopsin
promoter — directing expression to rod and cone photoreceptors (15
) — and a bovine poly A site. All constructs were verified by restriction enzyme digestion and direct DNA sequencing. Inserts were isolated by Not
l and Kpn
l digestion, microinjected into C57BL/6 mouse embryos, and implanted into pseudopregnant foster female mice. Founder mice were identified by PCR. Human PROM1
primers (forward, 5′-CCGCTCGAGCGTTGCTAGCTATGGCCCTC-3′; reverse, 5′-CGGGATCCCGCTATCAATGTTGTGATGGGC-3′) were used for PCR analysis of genomic DNA extracted from mouse tail biopsies. WT and mutant transgenic lines were established and propagated in the C57BL/6 mouse strain. All procedures were approved by the IACUC of the University of Utah and carried out according to NIH guidelines.
PROM1 expression in transgenic mice.
After removal of the lens, retinas from 1-month-old mice were manually separated from the RPE and lysed in 150 mM NaCl; 50 mM Tris, pH 7.5; 1 mM EDTA; 1% Triton X-100; 0.5% SDS; and protease inhibitor mixture (Roche Applied Science). Protein from each retinal lysate (10 μg) was separated by SDS-PAGE, transferred to PVDF filters, and probed with either human PROM1– or mouse Prom1–specific antibodies, followed by incubation with HRP-conjugated secondary antibodies and standard ECL detection.
Fundus photography and fluorescein angiography.
Fundus photographs and fluorescein angiographs for C57BL/6 and WT and mutant PROM1 transgenic mice were recorded at 6 and 9 months of age using a Kowa RC-2 handheld fundus camera (Kowa Genesis). The eyes were dilated with scopolamine (0.25%; Isopto Hyoscine; Alcon) 1 h prior to photography. Fluorescein angiographs were recorded with negative black and white film after intraperitoneal injection of 0.2 ml 25% sodium fluorescein diluted 1:1 with sterile PBS.
Histology and light microscopy.
Mice were maintained in a continuous 12-h light/12-h dark cycle and sacrificed 8–12 h after the onset of the light phase. Anesthetized mice were perfused with 0.1 M PBS and then with 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) by intracardiac injection. The superior sclera of each eye was marked for orientation. Eyecups were processed for embedding in Epon. Sections of 0.5 μm, oriented along the dorsoventral axis of the retina and containing the optic nerve head, were used for measuring photoreceptor ONL width. Photoreceptor nuclear counts were measured within 200–300 μm dorsal and ventral regions flanking the optic nerve head. Five separate counts per side (total 10 counts) were averaged, and ONL width was expressed as average number of nuclei.
Electroretinograms were obtained from 42 mice between the ages of 3 and 15 months: 19 PMT14, 8 PWT20, and 15 C57BL/6. Several mice were tested at more than one age. Mice were dilated with 0.25% scopolamine, dark-adapted overnight, and prepared for recording under a dim red light while the dilation was reinforced with scopolamine. Mice were anesthetized with intraperitoneal injection of 0.008 ml/g of a mixture of ketamine (20%), xylazine (0.5%), and sodium chloride solution (79.5%). The corneal electrode was a Burian-Allen mouse electrode referenced to a needle electrode in the scalp. A second needle electrode in the tail served as ground. Mice were placed between 2 heating pads to stabilize body temperature, which was continuously monitored with a digital probe. The eye was numbed with proparacaine hydrochloride (Alcaine; Alcon) and Refresh Celluvisc (Allergan Inc.) was added to the contact lens electrode to protect the eye. Signals were amplified 10,000× and filtered (8-pole Butterworth 60-Hz notch filter) to remove line noise before averaging (n
= 20–200) by computer. A ganzfeld dome and the incorporated Grass photostimulator, similar to systems used in human testing, was used to produce flashes comparable to the International Society for the Clinical Electrophysiology of Vision standard (40
). Rod a-waves were elicited by a high-intensity Xenon flash (Novatron). The rod phototransduction model was fit to the leading edges of a-waves generated in response to high-intensity stimuli, and the dashed curves were the best fitting curves (41
). Cone b-wave responses were obtained in the presence of a rod-saturating background (3.2 log photopic trolands).
Eyes from WT and mutant PROM1 transgenic mice were fixed in 2% paraformaldehyde plus 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and embedded in Epon 812 resin. Ultrathin sections were mounted on copper grids and stained with uranyl acetate and lead citrate and examined with a Philips electron microscope (model 208).
Antibodies used in these studies included rabbit polyclonal anti-human PROM1 (42
); rat monoclonal anti-mouse Prom1 (11
); rabbit polyclonal anti-mouse PCDH21 (diluted 1:500; gift from A. Rattner and J. Nathans; ref. 16
); mouse monoclonal anti-mouse CNGCA1 (diluted 1:100; gift from R. Molday, University of British Columbia, Vancouver, British Columbia, Canada; ref. 19
); mouse monoclonal anti-mouse ROM1 (diluted 1:100; gift from R. Molday; ref. 43
); and rabbit polyclonal anti-mouse Na+
-ATPase (diluted 1:100; obtained from the Developmental Studies Hybridoma Bank, University of Iowa).
Cryostat sections (12 μm) were washed with 1× PBS, blocked in immunohistochemical buffer (0.2% Triton X-100 in PBS) containing 10% goat serum at room temperature for 1 h. Sections were incubated in primary antibody overnight at 4°C. After washing, sections were incubated in FITC-conjugated goat anti-rabbit or Texas Red–conjugated goat anti-mouse secondary antibodies (diluted 1:100; Invitrogen) at room temperature for 1 h. Immunofluorescence was examined, and images were captured on a Zeiss 510 confocal microscope.
Cell transfection and immunoprecipitation reactions of PROM1 and PCDH21.
Cultured cells were propagated in DMEM (Gibco; Invitrogen) supplemented with 10% FBS (Gibco; Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were transfected with recombinant human WT or mutant PROM1 and mouse recombinant PCDH21 (containing a C-terminal Myc epitope tag) with FuGENE 6 Transfection Reagent (Roche Applied Science) according to the manufacturer’s recommended protocol. At 36 h after transfection, cells were lysed in 150 mM NaCl; 50 mM Tris, pH 7.5; 1 mM EDTA; 1% Triton X-100; 0.5% SDS; and a protease inhibitor mixture (Roche Applied Science). Cell lysates were centrifuged at 1,600 g for 5 min at 4°C to remove nuclei and insoluble material. The anti-PROM1 antibody was used to immunoprecipitate PROM1-binding proteins. The immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF filters, and probed with the Myc antibody on Western blot. The reciprocal experiment was performed using the PCDH21 antibody for immunoprecipitations followed by Western blot with anti-PROM1. For retinal coimmunoprecipitation studies, retinas were isolated and pooled from 10 WT or mutant PROM1 transgenic mice, and protein was extracted as described above.
Western blots of PROM1 and PCDH21 in transgenic mouse retinas.
Dissected retinas were briefly homogenized in buffer containing 150 mM NaCl; 50 mM Tris, pH 7.5; 1 mM EDTA; 1% Triton X-100; 0.5% SDS; and complete protease inhibitor mixture (Roche Applied Science). Following a 20-min incubation on ice, lysate was cleared of nuclei and insoluble material by centrifugation at 1,600 g for 5 min at 4°C. Soluble proteins were separated by SDS-PAGE, after which Western blot analysis was performed. The polyclonal PCDH21 C-terminal antibody was used to detect full-length PCDH21 and the C-terminal proteolytic fragment in C57BL/6 and PROM1 transgenic mice. A polyclonal antibody specific for human PROM1 without cross-reactivity to the mouse Prom1 was used to detect WT and mutant PROM1 transgene expression. An anti-mouse β-actin antibody (Sigma-Aldrich) was used as a loading control. For visualization, blots were probed with a HRP-conjugated anti-rabbit or anti-mouse secondary antibody (diluted 1:4,000; GE Healthcare) and developed with an ECL kit according to the manufacturers’ protocol (GE Healthcare).
Immunoprecipitation of PROM1 and actin.
HEK293 cells transfected with WT or mutant PROM1 were lysed in the wash buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1% NP-40; and 0.5% sodium deoxycholate) supplemented with complete protease inhibitors (Roche Applied Science), centrifuged at 10,000 g for 5 min to obtain postnuclear supernatant, and immunoprecipitated using affinity-purified polyclonal PROM1 antibodies. Immunocomplexes were precipitated by Protein A-Agarose (Pierce Biotechnologies Inc.) and washed 4 times with wash buffer. Immunoprecipitated protein samples were eluted with Laemmli buffer with 100 mM DTT, resolved by 10% SDS-PAGE, and immunoblotted with β-actin monoclonal antibodies (Sigma-Aldrich).
Dissected retinas from 1-month-old PWT20 mice were homogenized in buffer containing 150 mM NaCl; 50 mM Tris, pH 7.5; 1 mM EDTA; 1% Triton X-100; 0.5% SDS; and complete protease inhibitor mixture (Roche Applied Science). Lysate was centrifuged at 10,000 g for 5 min to obtain postnuclear supernatant and then immunoprecipitated using affinity-purified polyclonal PROM1 antibody. Immunocomplexes were precipitated by Protein A-Agarose, resolved by 10% SDS-PAGE, and immunoblotted with a β-actin monoclonal antibody.
Immunostaining and fluorescence microscopy of PROM1 and actin.
HEK293 cells were transfected with WT or mutant PROM1. Immunostaining was performed essentially as described previously (44
). Slides were incubated for 1 h at room temperature in anti-PROM1 (diluted 1:500) or rhodamine-conjugated phalloidin for F-actin (diluted 1:2,000). Laser-scanning confocal microscopy was performed on a Zeiss LSM510 microscope with krypton-argon and helium-neon lasers.
Cryosections of PWT20 and PM3 retinas were labeled with FITC-conjugated phalloidin (Invitrogen) to visualize actin filaments and with tubulin antibody. Immunofluorescence was examined, and images were captured on a Zeiss 510 confocal microscope.
Significance of differences was determined using SPSS software. Comparisons were made by 2-tailed Student’s t test. A P value less than 0.05 was considered significant.