Human Molecular Genetics, 2002, Vol. 11, No. 11 1333-1342
© 2002 Oxford University Press
Protein localization in the human eye and genetic screen of opticin
1Department of Ophthalmology and 2Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada T6G-2H7, 3Molecular Endocrinology and Oncology, Laval University Hospital (CHUL) Research Center, Québec City, QC, Canada G1V-4G2, 4Unit of Ophthalmology, University Clinical Departments, Liverpool, UK L69 3GA, and 5Department of Ophthalmology, CHUL and Laval University, Québec City, QC, Canada G1V-4G2
Received February 4, 2002; Accepted March 15, 2002
DDBJ/EMBL/GenBank accession nos
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The opticin (OPTC) gene encodes a protein that is a member of the small leucine-rich repeat protein (SLRP) family. OPTC is located on chromosome 1q31–q32 within an age-related macular degeneration (AMD) susceptibility locus. We have developed an affinity-purified N-terminal anti-opticin antibody and used it to examine opticin expression in human eye tissues. The antibody was also used for opticin protein localization in human eye sections. Immunoblots of human eye tissues detected a predominant band of approximately 62 kDa in size in iris, trabecular meshwork/ciliary body, retina, vitreous, and optic nerve. Immunohistochemical experiments revealed that opticin is specifically localized in human cornea, iris, ciliary body, vitreous, choroid and retina. Due to opticin's protein profile in the eye, we have also screened OPTC for mutations in individuals with primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG) or AMD. We identified four sequence variations, all of which were observed in normal controls except for the Arg229Cys change. Three amino acid substitutions (Ile182Thr, Arg229Cys and Arg325Trp) were in residues conserved in dog, mouse, pig and human. The Arg229Cys alteration was present in a homozygous state in one individual with neovascular AMD. Examination of the other AMD afflicted family members showed that the OPTC Arg229Cys variant did not segregate with the disorder within the family. The protein localization pattern of opticin and our preliminary screen of AMD patients suggest that a larger AMD patient screen may be warranted.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Age-related macular degeneration (AMD) and glaucoma are two major causes of blindness worldwide. AMD is the single largest cause of blindness in the Western world, while glaucoma is predicted to affect least 67 million people and cause nearly 6.7 million cases of bilateral blindness worldwide (1–5). One method to identify genes involved in these disorders is to use the candidate gene approach, whereby genes are examined based on their expression profile or known function. We have used this approach to test Opticin as a candidate gene for AMD and glaucoma.
Opticin (OPTC) has been previously identified by our laboratory and other groups (6–8). We isolated ‘oculoglycan’ (opticin) from a screen identifying genes highly expressed in a human adult iris cDNA library. OPTC cDNA has also been isolated from another iris cDNA library and retinal cDNA (7,8). OPTC is localized to lq31–q32 and encodes a 1.6 kb transcript in the eye containing a 996 bp open reading frame (6,7). The Opticin transcript has been examined by others and us, in both northern blot and RT–PCR experiments. Northern analysis determined that Opticin is expressed in the human iris and fetal liver and in a pool of RNA containing retina, ciliary body and vitreous (6,8). OPTC was found not to be expressed in a wide variety of tissues, including heart, brain, lung, skeletal muscle and kidney (6). Additional transcripts were observed by RT–PCR in retina, skin and ligament (6,8).
The opticin protein is predicted to be 332 amino acids in length and has homology to the Class III small leucine-rich repeat proteins (SLRPs) epiphycan and osteoglycin. Members of the SLRP family contain leucine-rich repeats bounded by conserved cysteine residues and are associated with the extracellular matrix (9,10). Opticin protein has been isolated from bovine vitreous extracts containing collagen fibrils and has been reported to be glycosylated with sialylated O-linked oligosaccharides in bovine vitreous (8). In situ hybridization analysis of opticin in mice showed expression in ciliary body during development from at least 15.5 dpc to adulthood (11). The canine ortholog was recently examined and excluded as a candidate for canine oculo-skeletal dysplasia (12). A recent study reporting susceptibility loci for age-related macular degeneration (AMD) included one locus whose region encompassed the OPTC gene (13).
Two small leucine-rich repeat proteins, keratocan and nyctalopin, have recently been shown to be involved in ocular disease. Keratocan, a class II SLRP, is mutated in individuals with a recessive form of cornea plana, a disorder in which the curvature of the cornea is flattened (14,15). Thirty-five Finnish families and a Chinese-American individual with cornea plana and, recently, a family from Bangladesh with cornea plana and micropthalmia had either homozygous amino acid substitutions or a homozygous stop mutation in the keratocan gene. These alterations may either eliminate keratocan activity or possibly alter the binding of keratocan to collagen (14,15). Nyctalopin, a glycosylphosphatidyl (GPI)-anchored protein, is mutated in an X-linked form of congenital stationary night blindness in a variety of families (16,17). Changes reported included missense mutations, insertions and deletions in the nyctalopin gene. The mutations observed are thought to affect retinal cell-to-cell interconnections (16,17).
We report here the protein localization of opticin in the human eye using an N-terminal affinity purified antibody as well as the results of the mutational screening of primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG) and AMD patients in a French-Canadian population.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Immunoblot analysis
To test the specificity of our affinity-purified anti-opticin antibody, we first performed immunoblot experiments with a recombinant opticin–green fluorescent protein (GFP) protein. Immunoblot analysis of COS-7 cell extracts transfected with and without the OPTC cDNA in the pEGFPN1 vector demonstrated two prominent bands. One band, of approximately 47.5 kDa, was observed in both transfected and untransfected lanes. The second larger band of about 60 kDa, consistent with the expected size of unmodified opticin (
37 kDa) with GFP (26 kDa), was seen in transfected lysates (Fig. 1A). To verify that the 60 kDa band was indeed the opticin–GFP fusion protein, we performed an immunoblot experiment with anti-GFP antibody and detected a single 60 kDa band (Fig. 1B), confirming that our affinity-purified anti-opticin antibody is able to detect human opticin in mammalian cells.
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After these preliminary tests showed that the anti-opticin antibody was able to specifically detect opticin, we expanded our analysis to include human eye tissues. Immunoblot experiments on human eye tissue protein extracts showed a prominent band of approximately 62 kDa in iris, trabecular meshwork/ciliary body, retina, optic nerve and vitreous. Weaker bands were observed at 45 and 38 kDa. The 45 kDa band was seen in iris, trabecular meshwork/ciliary body and optic nerve, while the 38 kDa band was present in iris and retina (Fig. 1C).
Immunohistochemical analysis
To further delineate the location of opticin protein in the human eye, we performed immunohistochemical analysis on eye sections from a human male donor (Fig. 2). Several components of the eye were labeled with the anti-opticin antibody, with the most intense staining in the vitreous base and cortex regions (Fig. 2H). The basal layer of the corneal epithelium (Fig. 2B) exhibited immunoreactivity at a level similar to that seen in the vitreous. The anterior corneal stroma (Fig. 2B) was diffusely stained at a level above background but less than the vitreous base. In the iris, vessel walls and surrounding stroma labeled as intensely as the vitreous, but the sphincter and dilator muscles appeared to stain only at background levels (Fig. 2D). Likewise, the staining in the ciliary body was not in the muscle itself but in the stroma around the muscle (Fig. 2F). Staining was not seen in the non-pigmented ciliary epithelium, although it was not possible to exclude staining in the pigment epithelial layer because of its heavy pigmentation (Fig. 2F). The sclera and trabecular meshwork (Fig. 2F) showed patchy labeling similar to that seen in the anterior corneal stroma. The uveal tract also stained with the anti-opticin antibody (Fig. 2F). Choroidal stromal staining was as intense as that in the vitreous (Fig. 2H). Retinal vessel walls were immunoreactive (Fig. 2J). Except where vitreous tufts appeared to be inserted into the retina at the vitreous base, the peripheral neuroretina did not stain (Fig. 2H). However, there was a gradually increasing level of focal stain as the posterior inner retina approached the optic nerve head, and the level of staining in the optic nerve head approximated that of the vitreous (data not shown). It was not possible to exclude retinal pigment epithelial labeling because of the dense pigmentation in that monolayer. Bruch's membrane was mostly negative, with some patchy staining. Drusen did not stain, while the choriocapillaris was strongly stained. Extraocular blood vessel adventitia and extraocular nerve peri/epineurium also stained at a level less than the vitreous but greater than background. Intraluminal blood vessel labeling was observed at a level similar to that of the adventitia, but red cells did not label.
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Opticin ortholog cDNA isolation
We had previously isolated the human opticin cDNA. In order to identify potentially important residues in the predicted opticin protein, we additionally isolated the mouse and pig orthologs of opticin. The dog ortholog of opticin was also recently determined (12). Residues completely conserved among all orthologs can be considered important for opticin's protein function. A four-species amino acid alignment is shown in Figure 3A. TIGR database and NCBI BLAST searches were performed and identified one mouse EST sequence (AA832880) and one mouse tentative consensus sequence (TC207501) with homology to human OPTC. PCR was used to amplify the internal Optc cDNA from mouse eye cDNA, and the fragment was sequenced. A single-base-pair difference was observed in the coding region between our mouse Optc cDNA sequence and the cDNA sequence recently reported (GenBank AF333980) (11). The base-pair change is in the first codon position of the encoded amino acid 168, which we predict to be aspartate instead of tyrosine. Porcine OPTC cDNA was isolated using PCR and RACE methods. The pig OPTC cDNA is at least 1364 bp in length and encodes a putative protein 333 amino acids in length. The human and dog putative proteins are 76.5% identical and 87.5% similar. The human and pig putative proteins are 74.2% identical and 88.6% similar, while the human and mouse encoded proteins are 73.5% identical and 88.3% similar.
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Opticin genetic screen
We decided to perform a genetic screen of OPTC in AMD and glaucoma patients, as opticin protein is present in the eye and because of OPTC's location within an AMD locus. Glaucoma patients were also tested, since opticin protein was present in tissues associated with this condition, namely the ciliary body and the optic nerve.
The OPTC gene was screened for mutations in a total of 197 individuals. A schematic of the OPTC gene with the relative positions of PCR primer pairs used and base-pair changes seen is shown in Figure 3B. Individuals affected by AMD and/or glaucoma were separated into three ‘affected’ subgroups according to their phenotypes. The affected sporadic group comprised 45 individuals diagnosed with AMD, 10 individuals with AMD plus POAG or NTG, and 87 individuals with POAG or NTG alone. The control group consisted of 55 clinically investigated individuals asymptomatic for AMD or glaucoma.
A total of five coding and one non-coding sequence variations were found among all the participating subjects (Table 1). Four of the five coding sequence variations resulted in amino acid changes. These changes were Ile182Thr, Arg229Cys, Leu268Pro and Arg325Trp. Non-synonymous coding changes are shown in a four-species (human, dog, mouse and pig) amino acid alignment in Figure 3A. Non-synonymous amino acid alterations Ile182Thr, Arg229Cys and Arg325Trp were at positions completely conserved among human, dog, mouse and pig. The Leu268Pro alteration was at a conserved residue in humans, pigs and mice. However, in canine the orthologous position is a proline. The Ile182Thr substitution and Leu270Leu synonymous codon variation were respectively identified in one and two clinically investigated normal subjects. The Leu268Pro variation was detected in four sporadic individuals with AMD and in six sporadic cases of POAG for a total frequency of 7.0% (10/142) among the affected phenotype groups. The same variation was identified in eight clinically investigated normal subjects for a total frequency of 14.5% (8/55) in the control population.
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The Arg325Trp variation was detected in three sporadic individuals with POAG, but not in individuals from the other two affected groups. This same variation was detected in three investigated normal subjects. The frequency of the Arg325Trp substitution among sporadic individuals with NTG or POAG was 3.4% (3/87), similar to the 5.5% (3/55) seen in the control population. A variation in the non-coding sequence near an exon–intron junction, causing a cytosine-to-thymine change at the third base of intron 2, was detected in a sporadic individual affected by AMD. As the orthologous position of Leu268 in canine is proline, it is likely that the Leu268Pro alteration is a polymorphism. Although the Ile182Thr and Arg325Trp alterations were at amino acid positions that were conserved in human, mouse, dog and pig, there is no current evidence that they are associated with AMD.
Interestingly, the Arg229Cys variation, observed in one of the sporadic patients affected by AMD, was detected as a homozygous change. This individual, BN007, was diagnosed at age 71 years with advanced macular degeneration with choroidal neovascularization in his left eye (exudative AMD). The right eye, however, only displayed some rare drusen. Since the Arg229Cys variation was not present in any other subjects from the initial mutational screen, additional individuals were tested for this variation. One group of 83 unrelated individuals with POAG and a second cohort of 48 non-clinically investigated spouses from glaucoma families were tested. None of these individuals harbored the Arg229Cys variation. The close relatives of individual BN007 were recruited to test for co-segregation of the variant with AMD (Fig. 4). Six siblings were investigated and the OPTC gene was screened for mutations (Table 2). The Arg229Cys change was found in two siblings (BN003 and BN009) in a heterozygous state. BN003 and BN009 both have normal retinas and optic disks at ages 78 and 68 years, respectively. On the other hand, two other family members (BN004 and BN008) harboring the wild-type arginine at position 229 were diagnosed with ocular diseases. One member (BN004) was affected by foveomacular dystrophy, while her sister (BN008) was diagnosed with early AMD. Neither sibling displayed as severe a form of retinal disease as the BN007 proband.
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Using Fisher's exact test, we compared the presence of the homozygous Arg229Cys variation in our AMD population with our control population using the program created by David Nash at http://www.zi.ku.dk/personal/drnash/Pages/program.htm. Using individuals having only AMD (n=45), and clinically investigated normal subjects (n=55), we determined a non-statistically significant P-value of 0.45. This demonstrates that the association between the homozygous Arg229Cys and AMD is not statistically significant. However, a wider screen of AMD patients may reveal additional patients with OPTC alterations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We report here the protein localization of opticin in the human eye and the results of a mutation screen of OPTC in patients with POAG, NTG or AMD. The N-terminal affinity-purified antibody used in the immunoblot and immunohistochemistry experiments was initially tested against a recombinant opticin–GFP fusion protein. We determined that the antibody was able to specifically identify the opticin protein, since immunoblot experiments identified a band of the expected size in only the transfected COS-7 cells. This finding was confirmed through immunoblot experiments using anti-GFP antibody. The opticin recombinant immunoblot experiment additionally detected an approximate 47.5 kDa band in both the transfected and untransfected lanes. The size observed corresponds to the previously observed size of bovine opticin (8), and suggests that COS-7 cells endogenously express opticin.
In human tissues, we observed, through immunoblot blot analysis, a prominent band at 62 kDa in iris, trabecular meshwork/ciliary body, retina, optic nerve and vitreous and weaker bands at 45 and 38 kDa. Previous work using antisera raised against a C-terminal peptide of opticin, in immunoblot experiments, showed major bands in human iris, ciliary body and retina at 48 kDa and other, smaller, heavy bands in retina (7). In rat tissues, the same antisera generated bands in trabecular meshwork/iris, vitreous, retina, optic nerve head and brain (7). In dog, opticin was reported to be present in skeletal muscle, testes, skin, ligament and rib chondrocyte (12). In dog eye, opticin was observed in retinal pigment epithelium, retina, iris and vitreous (12). It is possible that the observed differences in the major protein size of human opticin are due to differences in protein treatment, or differing levels of post-translational modification between the individuals examined. The differing sizes of opticin protein seen between species by others and us (data not shown) suggest that levels of post-translational modifications of opticin may vary both between and within species.
Interestingly, we also observed opticin protein in the optic nerve. As the optic nerve tissue was extracted prior to eye disruption, there was no possibility for this tissue to be contaminated by other opticin-containing tissues. As a secreted extracellular matrix protein, it is possible that opticin protein traveled from the eye to the optic nerve. Alternatively, OPTC mRNA may be expressed at some low level in the human optic nerve. Further work will be required to delineate the presence of OPTC mRNA in the optic nerve.
AMD is the leading cause of blindness in North America. AMD incidence ranges, depending on age, from 0.2% among those aged 55–64 years to 13% in those over 85 years of age (18). Strongly associated with AMD is the presence of drusen, located between Bruch's membrane and the basal lamina of the retinal pigment epithelium. Drusen may be described as being ‘hard’ or ‘soft’, with ‘soft’ drusen being more strongly associated with AMD. Work using electron microscopy has shown that cell processes from the choroid pass through Bruch's membrane into drusen (19). Studies of carbohydrates within drusen identified a drusen ‘core’ with O-linked oligosaccharides (20). The presence of O-linked oligosaccharides was observed after neuraminidase digestion of the eye sections used (20). Opticin was previously reported to contain sialylated O-linked oligosaccharides (8). The physical location of opticin in the choroid, the observation that drusen may be choroidally based and opticins previously described post-translational modifications make it tempting to hypothesize that opticin protein may be located within the core regions of drusen. Although we did not observe opticin protein within drusen in our study, we did not treat our tissue in the same manner as the aforementioned study did (20).
Recently, a whole-genome scan for susceptibility loci for AMD was carried out (13). In this paper, the three groups of patients (models A–C) studied had incidences of neovascularization ranging from 65% to 72%, depending on the clinical stringency applied. In our study, the incidence of neovascularization in our AMD patient pool was at least 70%. In our AMD-with-glaucoma group, no patients were reported to have neovascularization. The authors identified a region between markers D1S1660 and D1S1647 with a LOD score of 2.46. Interestingly, these markers overlap with our RH localization of OPTC (6). When examined using the NCBI map viewer (http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/hum_srch), the D1S1660-to-D1S1647 region was observed to contain the opticin gene.
We observed a single proband with severe AMD who had a homozygous arginine-to-cysteine alteration at the encoded amino acid 229 of the opticin protein. The change observed was in an amino acid completely conserved among human, dog, mouse and pig opticin orthologs. The Arg229Cys change is of note because opticin and other SLRPs contain six conserved cysteine residues, of which at least two are known to form internal disulfide bonds (10). The addition of another cysteine residue to the opticin protein could conceivably cause problems in protein folding. Mutations involving cysteine residues have been observed in nyctalopin. One observed mutation in nyctalopin eliminated a cysteine through alteration to a serine at amino acid 31 (17). A second change inserted the three amino acids ‘CysLeuArg’, while a third mutation removed eight amino acids, including two cysteine residues (16). The opticin Arg229Cys alteration was only seen in two other individuals. Both were siblings of the proband with the alteration in heterozygous form and each had a normal phenotype. These findings are consistent with individuals heterozygous with keratocan mutations having no disease phenotype (14,15). The presence of the more severe neovascular form of AMD in our homozygous Arg229Cys proband is consistent with the finding that all keratocan or nyctalopin allele(s) are mutated in their respective diseases. This finding raises the possibility that opticin could be a modifier of the AMD phenotype. However, our statistical analysis has determined that the association between the Arg229Cys variation and AMD is not significant. With our current patient pool, we estimate that we would have to screen over 860 control individuals to achieve a significant result. Therefore any association between opticin and AMD should currently be considered speculative. We conclude then, that larger studies would have to be performed to more fully examine the relationship, if any, between opticin and AMD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibody generation
A polyclonal chicken antibody was raised against a peptide based on amino acid positions 24–42 of human opticin. The polypeptide sequence chosen did not have homology to any other protein besides opticin using BLAST searches. In addition, the region selected included a homology gap of seven amino acids between opticin and its nearest human homologs, epiphycan and osteoglycin (6). Chicken egg IgY was isolated and affinity-purified against the peptide (Research Genetics, Huntsville, AL).
Opticin constructs
The OPTC open reading frame was cloned into a pEGFP vector. Maxipreps (Qiagen, Mississauga, ON) of each construct were carried out. Construct DNA (8 µg) was transfected into mammalian COS-7 cells [2x106 cells per 100 mm plate in 16 ml of Dulbecco's modified Eagle's medium (DMEM)+10% fetal bovine serum-(FBS)] with the FuGENE 6 transfection reagent (24 µl; Roche Molecular Biochemicals, Laval QC). Cells were harvested by scraping 48 hours after transfection and sonicated in lysis buffer (20 mM HEPES pH 7.6, 25% glycerol, 150 mM NaCl and 0.5 mM dithiothreitol, with protease inhibitors: aprotinin 5 µg/ml, pepstatin 5 µg/ml, leupeptin 5 µg/ml and phenylmethanesulfonyl fluoride 1 mmol). Soluble and insoluble fractions were collected after a 17 000g centrifugation for 10 minutes at 4°C. Fractions were then mixed into 2x SDS sample buffer and boiled for 5 minutes prior to storage. Samples were boiled for 5 minutes prior to loading onto 9% SDS–PAGE gels.
Immunoblot analysis
One pair of normal donor eyes (female, age 87 years) was obtained post mortem from the comprehensive tissue centre at the University of Alberta Hospital, Edmonton. Different ocular tissues (iris, trabecular meshwork/ciliary body, vitreous, retina and optic nerve) were subsequently isolated and frozen at -80°C. All human eye proteins, except for vitreous, were homogenized and extracted in 4 mM guanidine–HCl. Human vitreous was extracted in a 50 mM Tris–HCl pH 7.4 buffer cocktail containing 1% NP-40, 0.2% SDS and 1% Triton X, with protease inhibitors: aprotinin 5 µg/ml, pepstatin 5 µg/ml, leupeptin 5 µg/ml and phenylmethanesulfonyl fluoride 1 mmol.
Human protein samples of approximately l00 µg were size-separated using a 3% stacking and an 11% separating gel. The gels were then transferred to Trans-Blot Transfer Medium (Biorad, Mississauga, ON). The blots were blocked for at least 1 hour prior to a 1-hour incubation with the anti-opticin antibody (1 : 95 dilution). The blots were then washed five times in 5-minute intervals with TBST (100 mM Tris, 150 mM NaCl, 0.05% Tween-20) before incubation in rabbit anti-chick antibody (1 : 2000 dilution) conjugated to horseradish peroxidase (HRP) (Amersham-Pharmacia, Baie d'Urfé, QC) for 1 hour. Blots were washed again in TBST before detection using chemiluminescent substrate (Pierce, Rockford, IL) and exposure to Biomax film (Kodak, Rochester, NY). Anti-GFP antibody was kindly provided by Dr Luc Berthiaume (Department of Cell Biology, University of Alberta) and was used at 1 : 3333 dilution. The anti-GFP antibody was detected using an anti-rabbit antibody (1 : 3333 dilution) conjugated to HRP (Amersham-Pharmacia, Baie d'Urfé, QC).
Immunohistochemical experiments
Paraffin-embedded eye sections of a normal male eye used in immunohistochemical analysis were obtained as a generous gift from Dr Ian MacDonald (Department of Ophthalmology, University of Alberta). Slides were first dewaxed in xylene and rehydrated in gradual changes of ethanol. The slides were further rehydrated in PBS for 30 minutes before blocking with 10% FBS. Endogenous peroxidase activity was then removed by placing the cells in a 3% hydrogen peroxide/methanol solution for 20 minutes. Slides were washed in PBS prior to antibody incubation at 37°C in a humidity chamber. The anti-opticin antibody (1 : 5 dilution) was used as the primary antibody and incubated overnight at 4°C. An HRP-labeled anti-chick antibody (1:100 dilution) was used an the secondary antibody and incubated for 1.5 hours at 37°C. The tertiary antibody was incubated for 1.5 hours at 37°C using an HRP-labeled donkey anti-rabbit antibody (1 : 100 dilution). Antibody staining was observed using the red chromagen 9-amino-3-ethylcarbamazole (AEC) in acetate buffer with hydrogen peroxide.
Mouse sequence cDNA
TIGR database (http://www.tigr.org.tdb/) searches using the human OPTC cDNA revealed one mouse tentative consensus sequence (TC207501) and one mouse EST (AA832880), corresponding to the 5' and 3' sections of Optc respectively. Primers (5'-TTAAGTGAAGAAATGAGATTAGGG-3', and 5'-CTTGGGCTACAGATAGACTTTC-3') were designed from each EST and used to amplify the internal mouse Optc sequence from mouse eye cDNA generated in a manner previously described (6).
Pig sequence cDNA
Pig trabecular meshwork/ciliary body RNA collection and RACE cDNA generation was performed in the same manner as previously described (6). Primers specific to human OPTC (5'-TCCTGGCTTTCCTGAGTCTG-3', 5'-TCCAGGAACTCAATGCCAC-3') were selected for amplification from pig cDNA. The annealing temperature used was 55°C. A band of expected size (approximately 660 bp) was obtained and cloned into the pGEM-T Easy vector (Promega, Nepean, ON) prior to manual sequencing. 5' and 3'-RACE primers were then chosen, and RACE was performed to isolate the remaining cDNA sequence. The 5'- and 3'-RACE fragments were cloned and sequenced as above.
Mutational screening of OPTC
This research has been approved by the Centre Hospitalier de l'Université Laval (CHUL) Research Center Ethics Committee. All participants, affected or not, signed an informed-consent form before entering the study. A total of 197 individuals were clinically investigated. Clinical assessment comprised a complete ophthalmologic assessment, including fluorescein angiography when indicated.
Individuals recruited for the AMD study were investigated at the CHUL or at the Clinique d'ophtalmologie de la Cité, both in Québec City, Canada. Diagnostic criteria for early AMD were presence of large soft drusen and/or pigmentary abnormalities of the retinal pigment epithelium. Diagnostic criteria for advanced AMD were photocoagulation or other treatment for choroidal neovascularization, geographic atrophy involving the center of the macula, non-drusenoid retinal pigment epithelial detachment, serous or hemorrhagic retinal detachment, hemorrhage under the retina or the retinal pigment epithelium, and/or subretinal fibrosis.
Individuals recruited for the familial and the sporadic glaucoma studies were investigated by members of the Québec Glaucoma Network, comprising 103 ophthalmologists from different regions of the Province of Québec. Diagnostic criteria for POAG were intra-ocular pressures 22 mmHg or more in one or both eyes, characteristic optic disk damage and/or visual field impairment, grade III or IV (open-angle) gonioscopy, and exclusion of secondary causes (e.g. uveitis, steroid-induced glaucoma or trauma). Persons with intraocular pressures less than 22 mmHg and with visual field impairment as well as characteristic optic disk damage were diagnosed as NTG. Fifty-five other subjects selected at random had a complete normal ophthalmologic assessment, and served as investigated normal individuals. Forty-eight spouses of the individuals investigated in the glaucoma study were considered as randomly selected individuals from the general population.
The ages of the asymptomatic ‘normal’ investigated individuals in this screen ranged from 43 to 83 years, with one subject 43 years old, 14 subjects between 50 and 59, 29 subjects between 60 and 69, 10 subjects between 70 and 79 and one subject 83 years in age. The mean age of the investigated group was 63.2 years. The average age of the 45 patients with only AMD was 72.3 years. All individuals examined in this study were ethnically matched Caucasians of French–Canadian ancestry.
Polymerase chain reaction and DNA sequencing
Genomic DNA was obtained from 28 ml of whole blood drawn by venipuncture in four 7 ml EDTA tubes. DNA was extracted using the Puregene DNA Isolation Protocol for Whole Blood. The OPTC gene was screened for mutations by amplifying the six coding exons using PCR before sequencing. The amplicons used for sequencing were obtained by using six primer pairs, each amplifying one exon of the gene. The primer pairs used are as follows:
Pair A: forward 5'-TCTCAGTCCCATCTGACTCC-3',
reverse 5'-AGGGAATGTAGTTGGTCTGC-3';
Pair B: forward 5'-CCAGAGTCCAAAGTTAAGTCC-3',
reverse 5'-CCTATGACCTAGGGATATTGC-3;
Pair C: forward 5-CTCCCTTTGTTCTGTCTTCC-3',
reverse 5'-GTTGGTGACTGTCCTAGTGG-3';
Pair D: forward 5'-CTGGTTTCTCTCTTTGTTCTCC-3',
reverse 5'-TGGTGGAGGTGATAGATAGTGG-3';
Pair E: forward 5'-CAGCCTCCTACACTCTTTGC-3',
reverse 5'-GTTTATCACCCTTGCTCTGG-3';
Pair F: forward 5'-CAGCTGATGTGAGCCTTTGG-3',
reverse 5'-AGATGACCTGGGAGGAGTGG-3'.
The initial PCRs were performed on a Hybaid Omnigene Temperature Cycling System in a total volume of 50 µl containing 100 ng of genomic DNA, 20 pmol of each primer, 200 µM dNTPs, 50 mM KCl, 10 mM Tris (pH 9), 1.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100 and 1 U Taq polymerase (Invitrogen, Burlington, ON). Amplifications were carried out using a ‘hot-start’ procedure. Taq polymerase was added after a 5-minute denaturation step at 95°C. Samples were then processed through 35 cycles of denaturation (95°C for 30 seconds) and annealing (55–60°C for 30 seconds), followed by one last step of elongation (30 seconds at 72°C). PCR products were diluted in five volumes of Qiagen PB buffer, transferred on a Whatman GF/C filter plate, washed twice with a 80% ethanol 20 mM Tris pH 7.5 solution and eluted in 50 µl of water. Samples were quantified by the PicoGreen reagent protocol. A second PCR was performed on an Applied Biosystem Gene_Amp PCR System 9700 (96 wells) or 9700 Viper (384 wells) to incorporate the sequencing dyes using a protocol of 25 cycles of denaturation (95°C for 10 seconds) and annealing (55°C for 5 seconds), followed by one last step of elongation (2 minutes at 59°C). PCR products were then purified by the ABI ethanol–EDTA precipitation protocol, collected in a Beckman-Coulter Allegra 6R centrifuge and resuspended in a 50% HiDi–formamide solution. Samples were then run on an Applied Biosystems Prism 3700 DNA Analyser automated sequencer. Sequence data was analysed with the Staden pregGap4 and Gap4 programs.
AciI restriction enzyme test
A cytosine-to-thymine transition was detected at base pair 685 of the OPTC coding sequence. This transition introduced an Arg229Cys missense variation and destroyed an AciI restriction site. Screening for additional carriers of the Arg229Cys missense variation was thus performed using an AciI restriction enzyme test on the OPTC PCR-amplified fragment of exon 4. The wild-type sequence of exon 4 contained three restriction fragments of 89, 93 and 99 bp, whereas the altered sequence contained one fragment of 99 bp and one of 182 bp. PCR was conducted according to the conditions previously described for exon 4. The PCR products were purified in a QIAquick PCR Purification Kit spin column from Qiagen. In a total reaction volume of 20 µl, 10 µl of the purified PCR fragments were added to 7 µl of water, 1 µl (5 U/µl) of AciI (New England Biolabs, Mississauga, ON) and 2 µl of buffer NEB3 (100 mM NaCl, 50 mM Tris–HCl, 10 mM MgCl2 and 1 mM dithiothreitol). The reaction was incubated at 37°C for 2 hours, after which digested DNA samples were separated by electrophoresis on a 2.5% agarose gel. Presence of the Arg229Cys alteration in a heterozygous or homozygous state was confirmed by automated sequencing of the amplified fragment.
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ACKNOWLEDGEMENTS |
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We would first like to thank all of the individuals who took part or donated tissue for our study. We also thank Rose Arseneault for helping to recruit and collect data on the BN family. We thank the Québec Glaucoma Network for clinically investigating those involved in the study. We appreciate the critical comments from the Ocular Genetics Laboratory, Dr Ian MacDonald, Dr Rachel Wevrick Dr Roseline Godbout, Kerry McTaggart and Fiona Punter. We thank Dr Laurie Russell for technical assistance and Dr Bruce Rannala for his helpful comments. Acknowledgments also go to Annie Duchesne and Marc-André Rodrigue for their expert technical assistance in the DNA extraction and sequencing laboratory. J.S.F. is a CIHR Doctoral Fellow. V.R. is a Fonds de la Recherche en Santé du Québec (FRSQ) National Investigator. M.A.W. is a CIHR investigator and an AHFMR senior scholar. Funding for this work was received from the Canadian Genetic Diseases Network, the Canadian Foundation for Innovation (Grant no. 548) and the FRSQ Health Vision Research Network and from a Sigma Xi Grant in Aid.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Ocular Genetics Laboratory, Room 8-32, Medical Sciences Building, University of Alberta, Edmonton, AB, Canada T6G-2H7, Tel: +1 780 492 9805; Fax: +1 780 492 6934; Email: mwater{at}ualberta.ca
AY077681, AY077682 and AY077683
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