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1.  Methodologies for analysis of patterning in the mouse RPE sheet 
Molecular Vision  2015;21:40-60.
Purpose
Our goal was to optimize procedures for assessing shapes, sizes, and other quantitative metrics of retinal pigment epithelium (RPE) cells and contact- and noncontact-mediated cell-to-cell interactions across a large series of flatmount RPE images.
Methods
The two principal methodological advances of this study were optimization of a mouse RPE flatmount preparation and refinement of open-access software to rapidly analyze large numbers of flatmount images. Mouse eyes were harvested, and extra-orbital fat and muscles were removed. Eyes were fixed for 10 min, and dissected by puncturing the cornea with a sharp needle or a stab knife. Four radial cuts were made with iridectomy scissors from the puncture to near the optic nerve head. The lens, iris, and the neural retina were removed, leaving the RPE sheet exposed. The dissection and outcomes were monitored and evaluated by video recording. The RPE sheet was imaged under fluorescence confocal microscopy after staining for ZO-1 to identify RPE cell boundaries. Photoshop, Java, Perl, and Matlab scripts, as well as CellProfiler, were used to quantify selected parameters. Data were exported into Excel spreadsheets for further analysis.
Results
A simplified dissection procedure afforded a consistent source of images that could be processed by computer. The dissection and flatmounting techniques were illustrated in a video recording. Almost all of the sheet could be routinely imaged, and substantial fractions of the RPE sheet (usually 20–50% of the sheet) could be analyzed. Several common technical problems were noted and workarounds developed. The software-based analysis merged 25 to 36 images into one and adjusted settings to record an image suitable for large-scale identification of cell-to-cell boundaries, and then obtained quantitative descriptors of the shape of each cell, its neighbors, and interactions beyond direct cell–cell contact in the sheet. To validate the software, human- and computer-analyzed results were compared. Whether tallied manually or automatically with software, the resulting cell measurements were in close agreement. We compared normal with diseased RPE cells during aging with quantitative cell size and shape metrics. Subtle differences between the RPE sheet characteristics of young and old mice were identified. The IRBP−/− mouse RPE sheet did not differ from C57BL/6J (wild type, WT), suggesting that IRBP does not play a direct role in maintaining the health of the RPE cell, while the slow loss of photoreceptor (PhR) cells previously established in this knockout does support a role in the maintenance of PhR cells. Rd8 mice exhibited several measurable changes in patterns of RPE cells compared to WT, suggesting a slow degeneration of the RPE sheet that had not been previously noticed in rd8.
Conclusions
An optimized dissection method and a series of programs were used to establish a rapid and hands-off analysis. The software-aided, high-sampling-size approach performed as well as trained human scorers, but was considerably faster and easier. This method allows tens to hundreds of thousands of cells to be analyzed, each with 23 metrics. With this combination of dissection and image analysis of the RPE sheet, we can now analyze cell-to-cell interactions of immediate neighbors. In the future, we may be able to observe interactions of second, third, or higher ring neighbors and analyze tension in sheets, which might be expected to deviate from normal near large bumps in the RPE sheet caused by druse or when large frank holes in the RPE sheet are observed in geographic atrophy. This method and software can be readily applied to other aspects of vision science, neuroscience, and epithelial biology where patterns may exist in a sheet or surface of cells.
PMCID: PMC4301600
2.  Gene expression changes during retinal development and rod specification 
Molecular Vision  2015;21:61-87.
Purpose
Retinitis pigmentosa (RP) typically results from individual mutations in any one of >70 genes that cause rod photoreceptor cells to degenerate prematurely, eventually resulting in blindness. Gene therapies targeting individual RP genes have shown efficacy at clinical trial; however, these therapies require the surviving photoreceptor cells to be viable and functional, and may be economically feasible for only the more commonly mutated genes. An alternative potential treatment strategy, particularly for late stage disease, may involve stem cell transplants into the photoreceptor layer of the retina. Rod progenitors from postnatal mouse retinas can be transplanted and can form photoreceptors in recipient adult retinas; optimal numbers of transplantable cells are obtained from postnatal day 3–5 (P3–5) retinas. These cells can also be expanded in culture; however, this results in the loss of photoreceptor potential. Gene expression differences between postnatal retinas, cultured retinal progenitor cells (RPCs), and rod photoreceptor precursors were investigated to identify gene expression patterns involved in the specification of rod photoreceptors.
Methods
Microarrays were used to investigate differences in gene expression between cultured RPCs that have lost photoreceptor potential, P1 retinas, and fresh P5 retinas that contain significant numbers of transplantable photoreceptors. Additionally, fluorescence-activated cell sorting (FACS) sorted Rho-eGFP-expressing rod photoreceptor precursors were compared with Rho-eGFP-negative cells from the same P5 retinas. Differential expression was confirmed with quantitative polymerase chain reaction (q-PCR).
Results
Analysis of the microarray data sets, including the use of t-distributed stochastic neighbor embedding (t-SNE) to identify expression pattern neighbors of key photoreceptor specific genes, resulted in the identification of 636 genes differentially regulated during rod specification. Forty-four of these genes when mutated have previously been found to cause retinal disease. Although gene function in other tissues may be known, the retinal function of approximately 61% of the gene list is as yet undetermined. Many of these genes’ promoters contain binding sites for the key photoreceptor transcription factors Crx and Nr2e3; moreover, the genomic clustering of differentially regulated genes appears to be non-random.
Conclusions
This study aids in understanding gene expression differences between rod photoreceptor progenitors versus cultured RPCs that have lost photoreceptor potential. The results provide insights into rod photoreceptor development and should expedite the development of cell-based treatments for RP. Furthermore, the data set includes a large number of retinopathy genes; less-well-characterized genes within this data set are a resource for those seeking to identify novel retinopathy genes in patients with RP (GEO accession: GSE59201).
PMCID: PMC4300221
3.  Gene expression changes during retinal development and rod specification 
Molecular Vision  2015;21:61-87.
Purpose
Retinitis pigmentosa (RP) typically results from individual mutations in any one of >70 genes that cause rod photoreceptor cells to degenerate prematurely, eventually resulting in blindness. Gene therapies targeting individual RP genes have shown efficacy at clinical trial; however, these therapies require the surviving photoreceptor cells to be viable and functional, and may be economically feasible for only the more commonly mutated genes. An alternative potential treatment strategy, particularly for late stage disease, may involve stem cell transplants into the photoreceptor layer of the retina. Rod progenitors from postnatal mouse retinas can be transplanted and can form photoreceptors in recipient adult retinas; optimal numbers of transplantable cells are obtained from postnatal day 3–5 (P3–5) retinas. These cells can also be expanded in culture; however, this results in the loss of photoreceptor potential. Gene expression differences between postnatal retinas, cultured retinal progenitor cells (RPCs), and rod photoreceptor precursors were investigated to identify gene expression patterns involved in the specification of rod photoreceptors.
Methods
Microarrays were used to investigate differences in gene expression between cultured RPCs that have lost photoreceptor potential, P1 retinas, and fresh P5 retinas that contain significant numbers of transplantable photoreceptors. Additionally, fluorescence-activated cell sorting (FACS) sorted Rho-eGFP-expressing rod photoreceptor precursors were compared with Rho-eGFP-negative cells from the same P5 retinas. Differential expression was confirmed with quantitative polymerase chain reaction (q-PCR).
Results
Analysis of the microarray data sets, including the use of t-distributed stochastic neighbor embedding (t-SNE) to identify expression pattern neighbors of key photoreceptor specific genes, resulted in the identification of 636 genes differentially regulated during rod specification. Forty-four of these genes when mutated have previously been found to cause retinal disease. Although gene function in other tissues may be known, the retinal function of approximately 61% of the gene list is as yet undetermined. Many of these genes’ promoters contain binding sites for the key photoreceptor transcription factors Crx and Nr2e3; moreover, the genomic clustering of differentially regulated genes appears to be non-random.
Conclusions
This study aids in understanding gene expression differences between rod photoreceptor progenitors versus cultured RPCs that have lost photoreceptor potential. The results provide insights into rod photoreceptor development and should expedite the development of cell-based treatments for RP. Furthermore, the data set includes a large number of retinopathy genes; less-well-characterized genes within this data set are a resource for those seeking to identify novel retinopathy genes in patients with RP (GEO accession: GSE59201).
PMCID: PMC4301594
4.  Transcriptional regulation of crystallin, redox, and apoptotic genes by C-Phycocyanin in the selenite-induced cataractogenic rat model 
Molecular Vision  2015;21:26-39.
Purpose
This study was designed to examine the constrictive potential of C-Phycocyanin (C-PC) in regulating changes imposed on gene expression in the selenite-induced cataract model.
Methods
Wistar rat pups were divided into three groups of eight each. On P10, Group I received an intraperitoneal injection of normal saline. Groups II and III received a subcutaneous injection of sodium selenite (19 μmol/kg bodyweight); Group III also received an intraperitoneal injection of C-PC (200 mg/kg bodyweight) on P9–14. Total RNA was isolated on P16, and the relative abundance of mRNA of the crystallin structural genes, redox components, and apoptotic cascade were ascertained with real-time PCR with reference to the internal control β-actin.
Results
Real-time PCR analysis showed the crystallin genes (αA-, βB1-, γD-) and redox cycle components (Cat, SOD-1, Gpx) were downregulated, the apoptotic components were upregulated, and antiapoptotic Bcl-2 was downregulated in Group II. Treatment with 200 mg/kg bodyweight C-PC (Group III) transcriptionally regulated the instability of the expression of these genes, thus ensuring C-PC is a prospective anticataractogenic agent that probably delays the onset and progression of cataractogenesis induced by sodium selenite.
Conclusions
C-PC treatment possibly prevented cataractogenesis triggered by sodium selenite, by regulating the lens crystallin, redox genes, and apoptotic cascade mRNA expression and thus maintains lens transparency. C-PC may be developed as a potential antioxidant compound applied in the future to prevent and treat age-related cataract.
PMCID: PMC4301595
5.  Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity 
Molecular Vision  2015;21:12-25.
Purpose
Keratoconus (KC) is characterized by progressive vision loss due to corneal thinning and structural abnormalities. It is hypothesized that KC is caused by deregulated collagen levels and collagen fibril-maturating enzyme lysyl oxidase (LOX). Further, it is currently not understood whether the gene expression deregulated by the corneal epithelium influences KC pathogenesis. We studied (i) the expressions of the LOX, collagen I (COL IA1), collagen IV (COL IVA1), MMP9, and IL6 genes in KC corneal epithelia, (ii) validated their expression levels in patient tissues, and (iii) correlated expression levels with KC disease severity. The primary goal of this study was to evaluate the importance of these genes in the progression of KC.
Methods
We analyzed the gene expression levels of the key proteins LOX, collagens (COL IA1 and COL IVA1), MMP9, and IL6 in debrided corneal epithelia from a large cohort of KC patients (90 eyes) and compared them to control patients (52 eyes) without KC. We measured the total LOX activity in the tears of KC patients compared to controls. We also correlated the protein expression levels of LOX and collagens by immunohistochemistry (IHC) in primary tissues from KC patients (27 eyes) undergoing keratoplasty compared to healthy donor corneas (15 eyes).
Results
We observed a significant reduction in LOX transcript levels in KC corneal epithelia, and LOX activity in KC tears correlated with disease severity. Collagen transcripts were also reduced in KC while MMP9 transcript levels were upregulated and correlated with disease severity. IL6 was moderately increased in KC patients. IHC demonstrated a reduction in the protein expression levels of LOX in the epithelium and collagen IV in the basement membrane of KC patients compared to healthy donor corneas.
Conclusions
The data demonstrates that the structural deformity of the KC cornea may be dependent on reduced expressions of collagens and LOX, as well as on MMP9 elevated by the corneal epithelium.
PMCID: PMC4301596
6.  Mutational analysis and genotype-phenotype correlations in southern Indian patients with sporadic and familial aniridia 
Molecular Vision  2015;21:88-97.
Purpose
Aniridia is a rare panocular disorder characterized by iris hypoplasia and other associated eye anomalies. Heterozygous null mutations in paired box gene 6 (PAX6) are the major cause of the classic aniridia phenotype. This study aims to detect the mutational spectrum of PAX6 and associated phenotypes in southern Indian patients with sporadic and familial aniridia.
Methods
Genomic DNA was isolated from peripheral blood from all participants. The coding regions and flanking intronic sequences of PAX6 were screened with Sanger sequencing in 30 probands with aniridia. The identified variations were further evaluated in available family members and 150 healthy controls. The pathogenic potential of the mutations were assessed using bioinformatics tools.
Results
Thirteen different mutations were detected in eight sporadic and five familial cases. Eleven novel mutations, including five insertions (c.7_10dupAACA, c.567dupC, c.704dupC, c.868dupA and c.753_754insTA), two deletions (c.242delC and c.249delT), and four splicing variants (c.10+1G>A, c.141G>A, c.141+4A>G and c.764A>G) were identified in this study. Clinical findings of the patients revealed phenotypic heterogeneity with the same or different mutations.
Conclusions
This study reported 11 novel mutations and thus expanded the spectrum of PAX6 mutations. Interestingly, all mutations reported in this study were truncations, which confirms the hypothesis that haploinsufficiency of PAX6 causes the aniridia phenotype. Our observations revealed inter- and intrafamilial phenotypic variability with PAX6 mutations. The common ocular findings associated with PAX6 mutations were iris hypoplasia, nystagmus, and foveal hypoplasia reported in almost all cases, with cataract, glaucoma, and keratopathy reported in approximately 50% of the patients.
PMCID: PMC4309133
7.  Cluster analysis of multiplex ligation-dependent probe amplification data in choroidal melanoma 
Molecular Vision  2015;21:1-11.
Purpose
To determine underlying correlations in multiplex ligation-dependent probe amplification (MLPA) data and their significance regarding survival following treatment of choroidal melanoma (CM).
Methods
MLPA data were available for 31 loci across four chromosomes (1p, 3, 6, and 8) in tumor material obtained from 602 patients with CM treated at the Liverpool Ocular Oncology Center (LOOC) between 1993 and 2012. Data representing chromosomes 3 and 8q were analyzed in depth since their association with CM patient survival is well-known. Unsupervised k-means cluster analysis was performed to detect latent structure in the data set. Principal component analysis (PCA) was also performed to determine the intrinsic dimensionality of the data. Survival analyses of the identified clusters were performed using Kaplan–Meier (KM) and log-rank statistical tests. Correlation with largest basal tumor diameter (LTD) was investigated.
Results
Chromosome 3: A two-cluster (bimodal) solution was found in chromosome 3, characterized by centroids at unilaterally normal probe values and unilateral deletion. There was a large, significant difference in the survival characteristics of the two clusters (log-rank, p<0.001; 5-year survival: 80% versus 40%). Both clusters had a broad distribution in LTD, although larger tumors were characteristically in the poorer outcome group (Mann–Whitney, p<0.001). Threshold values of 0.85 for deletion and 1.15 for gain optimized the classification of the clusters. PCA showed that the first principal component (PC1) contained more than 80% of the data set variance and all of the bimodality, with uniform coefficients (0.28±0.03). Chromosome 8q: No clusters were found in chromosome 8q. Using a conventional threshold-based definition of 8q gain, and in conjunction with the chromosome 3 clusters, three prognostic groups were identified: chromosomes 3 and 8q both normal, either chromosome 3 or 8q abnormal, and both chromosomes 3 and 8q abnormal. KM analysis showed 5-year survival figures of approximately 97%, 80%, and 30% for these prognostic groups, respectively (log-rank, p<0.001). All MLPA probes within both chromosomes were significantly correlated with each other (Spearman, p<0.001).
Conclusions
Within chromosome 3, the strong correlation between the MLPA variables and the uniform coefficients from the PCA indicates a lack of evidence for a signature gene that might account for the bimodality we observed. We hypothesize that the two clusters we found correspond to binary underlying states of complete monosomy or disomy 3 and that these states are sampled by the complete ensemble of probes. Consequently, we would expect a similar pattern to emerge in higher-resolution MLPA data sets. LTD may be a significant confounding factor. Considering chromosome 8q, we found that chromosome 3 cluster membership and 8q gain as traditionally defined have an indistinguishable impact on patient outcome.
PMCID: PMC4316690
8.  Mutational analysis and genotype-phenotype correlations in southern Indian patients with sporadic and familial aniridia 
Molecular Vision  2015;21:88-97.
Purpose
Aniridia is a rare panocular disorder characterized by iris hypoplasia and other associated eye anomalies. Heterozygous null mutations in paired box gene 6 (PAX6) are the major cause of the classic aniridia phenotype. This study aims to detect the mutational spectrum of PAX6 and associated phenotypes in southern Indian patients with sporadic and familial aniridia.
Methods
Genomic DNA was isolated from peripheral blood from all participants. The coding regions and flanking intronic sequences of PAX6 were screened with Sanger sequencing in 30 probands with aniridia. The identified variations were further evaluated in available family members and 150 healthy controls. The pathogenic potential of the mutations were assessed using bioinformatics tools.
Results
Thirteen different mutations were detected in eight sporadic and five familial cases. Eleven novel mutations, including five insertions (c.7_10dupAACA, c.567dupC, c.704dupC, c.868dupA and c.753_754insTA), two deletions (c.242delC and c.249delT), and four splicing variants (c.10+1G>A, c.141G>A, c.141+4A>G and c.764A>G) were identified in this study. Clinical findings of the patients revealed phenotypic heterogeneity with the same or different mutations.
Conclusions
This study reported 11 novel mutations and thus expanded the spectrum of PAX6 mutations. Interestingly, all mutations reported in this study were truncations, which confirms the hypothesis that haploinsufficiency of PAX6 causes the aniridia phenotype. Our observations revealed inter- and intrafamilial phenotypic variability with PAX6 mutations. The common ocular findings associated with PAX6 mutations were iris hypoplasia, nystagmus, and foveal hypoplasia reported in almost all cases, with cataract, glaucoma, and keratopathy reported in approximately 50% of the patients.
PMCID: PMC4316699
9.  The effects of actomyosin disruptors on the mechanical integrity of the avian crystalline lens 
Molecular Vision  2015;21:98-109.
Purpose: Actin and myosin within the crystalline lens maintain the structural integrity of lens fiber cells and form a hexagonal lattice cradling the posterior surface of the lens. The actomyosin network was pharmacologically disrupted to examine the effects on lenticular biomechanics and optical quality.
Methods: One lens of 7-day-old White Leghorn chickens was treated with 10 µM of a disruptor and the other with 0.01% dimethyl sulfoxide (vehicle). Actin, myosin, and myosin light chain kinase (MLCK) disruptors were used. The stiffness and the optical quality of the control and treated lenses were measured. Western blotting and confocal imaging were used to confirm that treatment led to a disruption of the actomyosin network. The times for the lenses to recover stiffness to match the control values were also measured.
Results: Disruptor-treated lenses were significantly less stiff than their controls (p≤0.0274 for all disruptors). The disruptors led to changes in the relative protein amounts as well as the distributions of proteins within the lattice. However, the disruptors did not affect the clarity of the lenses (p≥0.4696 for all disruptors), nor did they affect spherical aberration (p = 0.02245). The effects of all three disruptors were reversible, with lenses recovering from treatment with actin, myosin, and MLCK disruptors after 4 h, 1 h, and 8 min, respectively.
Conclusions: Cytoskeletal protein disruptors led to a decreased stiffness of the lens, and the effects were reversible. Optical quality was mostly unaffected, but the long-term consequences remain unclear. Our results raise the possibility that the mechanical properties of the avian lens may be actively regulated in vivo via adjustments to the actomyosin lattice.
PMCID: PMC4316703
10.  Localization of complement factor H gene expression and protein distribution in the mouse outer retina 
Molecular Vision  2015;21:110-123.
Purpose
To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.
Methods
Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.
Results
Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results.
Conclusions
Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.
PMCID: PMC4323684
11.  Ophthalmological phenotype associated with homozygous null mutation in the NEUROD1 gene 
Molecular Vision  2015;21:124-130.
Purpose
NEUROD1 is a tissue-specific basic helix loop helix (bHLH) protein involved in the development and maintenance of the endocrine pancreas and neuronal elements. Loss of NEUROD1 causes ataxia, cerebellar hypoplasia, sensorineural deafness, and severe retinal dystrophy in mice. Heterozygous loss-of-function mutations in NEUROD1 have previously been described as a cause of maturity-onset diabetes of the young (MODY) and late-onset diabetes. To date, homozygous loss-of-function NEUROD1 mutations have only been detected in two patients. Both mutations caused permanent neonatal diabetes and severe neurologic defects, including visual impairment. However, a detailed ophthalmological phenotype of this novel syndrome has not yet been reported. Our aim was to characterize the ophthalmological phenotype associated with the previously reported homozygous c.427_428CT mutation in the NEUROD1 gene.
Methods
The female patient was investigated on multiple occasions between 2009 (age 14) and 2014 (age 19), including visual acuity testing, automated perimetry, funduscopy, anterior-segment imaging, optical coherence tomography of the posterior pole, standard full-field electroretinography, and fundus-autofluorescence imaging.
Results
The patient had nyctalopia, blurry vision, and visual field constriction from early childhood. Her best corrected visual acuity ranged between 20/25 and 15/25 during the investigation period. Perimetry showed concentric constriction of the visual field, sparing only the central 30 degrees in both eyes. The anterior segment did not show any morphological changes. Optical coherence tomography revealed total absence of the photoreceptor layer of the retina outside the fovea, where a discoid remnant of cone photoreceptors could be detected. Neither setting of the standard full-field electroretinography could detect any electrical response from the retina. Color fundus photos presented peripheral chorioretinal atrophy and central RPE mottling. A hyperreflective parafoveal ring was detected on fundus autofluorescent photos, a characteristic sign of hereditary retinal dystrophies.
Conclusions
To the best of our knowledge, this is the first report on the ophthalmological phenotype associating with a homozygous NEUROD1 null mutation in humans. Our results indicate that the loss of NEUROD1 has similar functional and anatomic consequences in the human retina as those described in mice. The present description can help the diagnosis of future cases and provide clues on the rate of disease progression.
PMCID: PMC4323689
12.  Ocular Inflammation in HLA-B27 Transgenic Mice Reveals a Potential Role for MHC Class I in Corneal Immune Privilege 
Molecular Vision  2015;21:131-137.
Purpose
HLA-B27 is a major histocompatibility complex class I (MHCI) allele that has been closely associated with the development of ankylosing spondylitis and acute anterior uveitis (AAU), the most common form of uveitis worldwide. We have been characterizing the phenotypes of transgenic mice carrying a human HLA-B27 allele, but that are deficient in endogenous mouse MHCI genes (H-2K−/− and H-2D−/− double knockout, or DKO) to create the HLA-B27/DKO line. In maintaining and expanding this colony, we observed a rare sporadic severe central keratitis that developed in transgenic animals, but that was not present in wild-type (WT) animals.
Methods
The corneas of affected HLA-B27/DKO and DKO mice were compared to their WT counterparts by staining with standard histological methods for markers of inflammation and neovascularization. A model of experimental corneal inflammation was subsequently used to test the responses of each genotype to insult.
Results
We identified a previously unreported corneal pathology in naïve HLA-B27/DKO mice, and we describe significantly prolonged CD4+- and CD8+-associated inflammation in these animals following an experimentally induced corneal injury.
Conclusions
These results demonstrate an increased T-cell response in B27/DKO corneas due to the expression of the HLA-B27 allele, suggesting that low MHCI expression in WT corneas is an important contributor to immune privilege.
PMCID: PMC4323692
13.  Chondrogenesis in scleral stem/progenitor cells and its association with form-deprived myopia in mice 
Molecular Vision  2015;21:138-147.
Purpose: Previously, we demonstrated that scleral stem/progenitor cells (SSPCs) from mice have a chondrogenic differentiation potential, which is stimulated by transforming growth factor-β (TGF-β). In the present study, we hypothesized that chondrogenesis in the sclera could be a possible mechanism in myopia development. Therefore, we investigated the association of form-deprivation myopia (FDM) with expressions in mice sclera representing the chondrogenic phenotype: collagen type II (Col2) and α-smooth muscle actin (α-SMA).
Methods: The mRNA levels of α-SMA and Col2 in cultured murine SSPCs during chondrogenesis stimulated by TGF-β2 were determined by real-time quantitative RT–PCR (qRT-PCR). The expression patterns of α-SMA and Col2 were assessed by immunohistochemistry in a three dimensional pellet culture. In an FDM mouse model, a western blot analysis and immunofluorescence study were used to detect the changes in the α-SMA and Col2 protein expressions in the sclera. In the RPE-choroid complex, qRT-PCR was used to detect any changes in the TGF-β mRNA expression.
Results: The treatment of SSPCs in vitro with TGF-β2 for 24 h at 1 or 10 ng/ml led to increased levels of both the α-SMA and Col2 expressions. In addition, we observed the formation of cartilage-like pellets from TGF-β2-treated SSPCs. Both α-SMA and Col2 were expressed in the pellet. In an in-vivo study, the α-SMA and Col2 protein expressions were significantly increased in the sclera of FDM eyes in comparison to contralateral control eyes. Similarly, the levels of TGF-β in the RPE-choroid complex of an FDM eye were also significantly elevated.
Conclusion: Based on the concept of stem cells possessing multipotent differentiation potentials, scleral chondrogenesis induced by SSPCs may play a role in myopia development. The increased expressions of the cartilage-associated proteins Col2 and α-SMA during scleral chondrogenesis may be potential markers for myopia development. In addition, the increased levels of TGF-β mRNA in the RPE-choroid complex might induce the chondrogenic change in the sclera during myopia development.
PMCID: PMC4323720
14.  Ablation of Kcnj10 expression in retinal explants revealed pivotal roles for Kcnj10 in the proliferation and development of Müller glia 
Molecular Vision  2015;21:148-159.
Purpose
We previously found that Kcnj10, an inwardly-rectifying potassium channel, is a gene expressed in c-kit-positive retinal progenitor cells on P1. The shRNA-mediated screening of the functions of the genes for retinal development in retinal explant culture suggested a role for Kcnj10 in the differentiation of 23Müller glia. In the present study, we extended the work and focused on analyzing the role of Kcnj10 in retinal development.
Methods
shRNA-mediated downregulation of Kcnj10 in retinal explants and the in vivo mouse retina at the P1 stage was performed. Differentiation and proliferation of the retina were examined with immunohistochemistry. The effect of barium (Ba2+) treatment, which inhibits potassium currents by blocking potassium channels, on retinal development was examined.
Results
When Kcnj10 was downregulated at E18, cellular proliferation and morphological differentiation were perturbed; in particular, a decreased number of Müller glial cells with abnormal morphological maturation was observed. The overexpression of Kcnj10 in retinal progenitors did not result in gross abnormality during retinal development, but rescued the abnormal differentiation induced with sh-Kcnj10. The presence of Ba2+ in the retinal explant medium led to a phenotype similar to that seen with sh-Kcnj10. Ba2+ exerts an effect mainly during late retinal development, and sh-Kcnj10 in the P1 retina affected Müller glia maturation, suggesting that Kcnj10 plays a pivotal role in the maturation of retinal cell subsets. A previous study of Kcnj10-knockout mice showed no obvious abnormality in retinal differentiation, especially of Müller glia. We examined the effects of the downregulation of Kcnj10 with in vivo electroporation of sh-Kcnj10 in the P1 retina. Retinal differentiation was perturbed, as seen following the in vitro downregulation of Kcnj10, suggesting that compensatory gene expression and/or signaling occurred in the Kcnj10-knockout mice in the retina, leading to normal eye development.
Conclusion
Kcnj10 plays a role in Müller glia maturation during retinal development probably through ionic channel activities.
PMCID: PMC4323724
15.  Fibroblast growth factor receptor 1 (Fgfr1) is not required for lens fiber differentiation in mice 
Molecular vision  2006;12:15-25.
Purpose
The developing lens expresses at least three different FGF receptor genes (Fgfr1-3) and FGFs have been shown to induce lens epithelial cells to differentiate into fiber cells both in vitro and in vivo. While the loss of Fgfr2 alone does not prevent fiber differentiation and the loss of Fgfr3 alone does not appear to affect lens development the independent role of Fgfr1 in lens development has not been reported. These experiments were conducted to determine if Fgfr1 plays an independent essential role in lens development.
Methods
To address this question, we took two complementary approaches. First, we employed the aphakia (ak) lens complementation system to show that Fgfr1-deficient embryonic stem (ES) cells were able to form a normal embryonic lens that maintains a normal pattern of crystallin gene expression. Second, we employed the Cre-loxP system to achieve lens-specific inactivation of Fgfr1.
Results
Fgfr1-null embryonic stem cells were able to rescue normal embryonic lens development in chimeric combination with aphakia mutant embryos. In addition, conditional deletion of Fgfr1 does not compromise lens development either before or after birth.
Conclusions
The results of both approaches suggest that lens epithelial cell integrity, cell cycle regulation and lens fiber differentiation are intact in the Fgfr1-deficient lens. Overall, our results demonstrate that Fgfr1 is not cell autonomously essential for lens development and suggests functional redundancy among different FGFRs with respect to lens fiber differentiation.
PMCID: PMC4280232  PMID: 16446698
16.  Differential effects of hepatocyte growth factor and keratinocyte growth factor on corneal epithelial cell cycle protein expression, cell survival, and growth 
Molecular Vision  2014;20:24-37.
Purpose
Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are secreted in the cornea in response to injury. In this study, we investigated the HGF- and KGF-mediated effect on the expression of cell cycle and apoptosis controlling proteins, cell survival, and growth in the corneal epithelium to better understand the possible role of their signaling mechanisms in repairing epithelial injuries.
Methods
The cell survival capability of HGF and KGF in epithelial primary cultures was evaluated by using a staurosporine-induced apoptosis model. Apoptosis was quantified with image analysis following nuclear staining with Hoechst fluorescent dye and DNA laddering. Western immunoblotting was used to study the effect of growth factors on the expression of cell cycle- and apoptosis-regulating proteins.
Results
HGF and KGF protected cells from apoptosis for a short duration (10 h), but only KGF exhibited cell survival capability and maintained cell growth for a longer period (24 h). The onset of apoptosis was accompanied by a significant increase in cell cycle inhibitor p27kip. HGF and KGF suppressed p27kip levels in the apoptosis environment; however, KGF- but not HGF-dependent downregulation in p27kip expression was sustained for a longer duration. Inhibition of phosphatidylinositol 3-kinase/Akt activation blocked HGF- and KGF-mediated control of p27kip expression. Further, when compared to HGF, the presence of KGF produced significant downregulation of p53 and poly(adenosine diphosphate-ribose) polymerase, the key proteins involved in apoptosis and blocked the degradation of G1/S cell cycle progression checkpoint protein retinoblastoma. HGF and KGF upregulated the levels of p21cip, cyclins A, D, and E and cyclin-dependent kinases (CDK2 and CDK4) as well, but the KGF-mediated effect on the expression of these molecules lasted longer.
Conclusions
Sustained effect of KGF on cell survival and proliferation could be attributed to its ability to inhibit p53, retinoblastoma, caspases, and p27kip functions in apoptosis and cell cycle arrest and promote the expression of cell cycle progressing molecules for longer duration. Designing therapeutic strategies targeting cell cycle control through KGF may be beneficial for repairing difficult-to-heal corneal epithelial injuries that require sustained growth and cell survival promoting signals.
PMCID: PMC3888494  PMID: 24426773
17.  Expression profiling of the RPE in zebrafish smarca4 mutant revealed altered signals that potentially affect RPE and retinal differentiation 
Molecular Vision  2014;20:56-72.
Purpose
The purpose of this study was to develop a framework for analyzing retinal pigment epithelium (RPE) expression profiles from zebrafish eye mutants.
Methods
The fish model we used was SWI/SNF-related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (smarca4), a retinal dystrophic mutant with a previously described retinal phenotype and expression profiles. Histological and Affymetrix GeneChip analyses were conducted to characterize the RPE defects and underlying differential expression, respectively.
Results
Histological analysis revealed that smarca4 RPE was formed, but its differentiation was abnormal. In particular, ultrastructural analysis of smarca4 RPE by transmission electron microscopy demonstrated several defects in melanogenesis. The nature of these defects also suggests that the cytoskeletal dynamics, which are tightly linked with melanogenesis, were impaired in smarca4 RPE. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, the gene expression profiles of microdissected retinas and RPE-attached retinas were measured with Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples by subtracting the retinal expression values from the expression values of the RPE-attached retinas. A factorial analysis was conducted using the expression values of the RPE, retinal, and whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted models to select for three groups of genes: 1) smarca4-regulated RPE genes, 2) smarca4-regulated retinal genes, and 3) smarca4-regulated RPE genes that are not differentially expressed in the retina. Interestingly, the third group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, and paracrine and intracellular signal transduction.
Conclusions
Our analytical framework provides an experimental approach to identify differentially-regulated genes in the retina and the RPE of zebrafish mutants in which both of these tissues are affected by the underlying mutation. Specifically, we have used the method to identify a group of 39 genes that can potentially explain the melanogenesis defect in the smarca4 RPE. In addition, several genes in this group are secreted signaling molecules. Thus, this observation further implicates that the smarca4 RPE might play a role in the retinal dystrophic phenotype in smarca4.
PMCID: PMC3888495  PMID: 24426776
18.  AIPL1 implicated in the pathogenesis of two cases of autosomal recessive retinal degeneration 
Molecular Vision  2014;20:1-14.
Purpose
To localize and identify the gene and mutations causing autosomal recessive retinal dystrophy in two consanguineous Pakistani families.
Methods
Consanguineous families from Pakistan were ascertained to be affected with autosomal recessive retinal degeneration. All affected individuals underwent thorough ophthalmologic examinations. Blood samples were collected, and genomic DNA was extracted using a salting out procedure. Genotyping was performed using microsatellite markers spaced at approximately 10 cM intervals. Two-point linkage analysis was performed with the lod score method. Direct DNA sequencing of amplified genomic DNA was performed for mutation screening of candidate genes.
Results
Genome-wide linkage scans yielded a lod score of 3.05 at θ=0 for D17S1832 and 3.82 at θ=0 for D17S938, localizing the disease gene to a 12.22 cM (6.64 Mb) region flanked by D17S1828 and D17S1852 for family 61032 and family 61227, which contains aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), a gene previously implicated in recessive Leber congenital amaurosis and autosomal dominant cone-rod dystrophy. Sequencing of AIPL1 showed a homozygous c.773G>C (p.Arg258Pro) sequence change in all affected individuals of family 61032 and a homozygous c.465G>T (p.(H93_Q155del)) change in all affected members of family 61227.
Conclusions
The results strongly suggest that the c.773G>C (p.R258P) and c.465G>T (p.(H93_Q155del)) mutations in AIPL1 cause autosomal recessive retinal degeneration in these consanguineous Pakistani families.
PMCID: PMC3888496  PMID: 24426771
19.  Maternal germline mosaicism of kinesin family member 21A (KIF21A) mutation causes complex phenotypes in a Chinese family with congenital fibrosis of the extraocular muscles 
Molecular Vision  2014;20:15-23.
Purpose
To identify the causative mutation with its possible origin in a Chinese family with congenital fibrosis of extraocular muscles type 1 (CFEOM1) and to characterize the ocular phenotypes and lesions in the corresponding intracranial nerves.
Methods
Three affected siblings and their asymptomatic parents underwent comprehensive ophthalmic examinations and neuropathologic analysis involving magnetic resonance imaging (MRI). KIF21A, PHOX2A, and TUBB3 genes were sequenced on the leukocyte-derived DNA to detect variants. The disease-linked haplotype was analyzed using four microsatellite markers across the KIF21A locus.
Results
All three affected individuals displayed typical CFEOM1. MRI revealed complicated but consistent neuromuscular abnormalities in the two patients examined, including hypoplastic oculomotor nerves, complete absence of bilateral superior rectus muscles, and unilateral absence of the abducens nerve with marked atrophy of the corresponding lateral rectus muscle. A heterozygous hotspot mutation KIF21A c.2860C>T was identified in all patients, but it was absent in both parents. Haplotype analysis of the disease locus showed the likely maternal inheritance of the disease-associated haplotype to all three affected offspring, strongly suggesting maternal germline mosaicism of the mutation.
Conclusions
Germline mosaicism of KIF21A c.2860C>T is likely to cause the high occurrence of this mutation in the population. This information may be useful for genetic counseling. KIF21A mutations can affect the abducens nerve and cause complete absence of the bilateral superior rectus muscles. MRI characterization of new CFEOM1 phenotypes would assist clinical management.
PMCID: PMC3888497  PMID: 24426772
20.  Age-related macular degeneration: Beyond anti-angiogenesis 
Molecular Vision  2014;20:46-55.
Recently, anti-vascular endothelial growth factor therapies for neovascular age-related macular degeneration have been developed. These agents, originally developed for their anti-angiogenic mechanism of action, probably also work through an anti-permeability effect in preventing or reducing the amount of leakage from submacular neovascular tissue. Other treatment modalities include laser photocoagulation, photodynamic therapy with verteporfin, and submacular surgery. In reality, these latter treatments can be similarly categorized as anti-angiogenic because their sole aim is destroying or removing choroidal neovascularization (CNV). At the cellular level, CNV resembles stereotypical tissue repair that consists of several matricellular components in addition to neovascularization. In the retina, the clinical term CNV is a misnomer since the term may more appropriately be referred to as aberrant submacular repair. Furthermore, CNV raises a therapeutic conundrum: To complete or correct any reparative process in the body, angiogenesis becomes an essential component. Anti-angiogenic therapy, in all its guises, arrests repair and causes the hypoxic environment to persist, thus fueling pro-angiogenesis and further development of CNV as a component of aberrant repair. However, we realize that anti-vascular endothelial growth factor therapy preserves vision in patients with age-related macular degeneration, albeit temporarily and therefore, repeated treatment is needed. More importantly, however, anti-angiogenic therapy demonstrates that we can at the very least tolerate neovascular tissue beneath the macula and preserve vision in contrast to our historical approach of total vascular destruction. In this clinical scenario, it may be possible to look beyond anti-angiogenesis if our goal is facilitating submacular repair without destroying the neurosensory retina. Thus, in this situation of neovascular tolerance, it may be timely to consider treatments that facilitate vascular maturation, rather than its arrest or destruction. This would neutralize hypoxia, thus removing the stimulus that drives neovascularization and in turn the need for repeated lifelong intravitreal therapy. A pro-angiogenic approach would eliminate neovascular leakage and ultimately complete repair and preserve the neurosensory retina.
PMCID: PMC3888498  PMID: 24426775
21.  Activation of the lectin pathway of complement in experimental human keratitis with Pseudomonas aeruginosa 
Molecular Vision  2014;20:38-45.
Purpose
Pseudomonas aeruginosa (P. aeruginosa) microbial keratitis (MK) is a sight-threatening disease. Previous animal studies have identified an important contribution of the complement system to the clearance of P. aeruginosa infection of the cornea. Mannose-binding lectin (MBL), a pattern recognition receptor of the lectin pathway of complement, has been implicated in the host defense against P. aeruginosa. However, studies addressing the role of the lectin pathway in P. aeruginosa MK are lacking. Hence, we sought to determine the activity of the lectin pathway in human MK caused by P. aeruginosa.
Methods
Primary human corneal epithelial cells (HCECs) from cadaveric donors were exposed to two different P. aeruginosa strains. Gene expression of interleukin (IL)-6, IL-8, MBL, and other complement proteins was determined by reverse transcription-polymerase chain reaction (RT–PCR) and MBL synthesis by enzyme-linked immunosorbent assay and intracellular flow cytometry.
Results
MBL gene expression was not detected in unchallenged HCECs. Exposure of HCECs to P. aeruginosa resulted in rapid induction of the transcriptional expression of MBL, IL-6, and IL-8. In addition, expression of several complement proteins of the classical and lectin pathways, but not the alternative pathway, were upregulated after 5 h of challenge, including MBL-associated serine protease 1. However, MBL protein secretion was not detectable 18 h after challenge with P. aeruginosa.
Conclusions
MK due to P. aeruginosa triggers activation of MBL and the lectin pathway of complement. However, the physiologic relevance of this finding is unclear, as corresponding MBL oligomer production was not observed.
PMCID: PMC3888499  PMID: 24426774
22.  L-2-oxothiazolidine-4-carboxylic acid attenuates oxidative stress and inflammation in retinal pigment epithelium 
Molecular Vision  2014;20:73-88.
Purpose
Oxidant- and inflammation-induced damage to retinal pigment epithelial (RPE) cells is central to the pathogenesis of age-related macular degeneration (AMD). Thus, developing novel strategies to protect these cells is important. We reported previously on the robust antioxidant and therefore cell-protective effects of the cysteine pro-drug L-2-oxothiazolidine-4-carboxylic acid (OTC) in cultured human RPE cells. New reports citing a novel anti-inflammatory role for OTC in addition to the known glutathione-stimulating and antioxidant properties emerged recently; however, this role has not been evaluated in RPE cells or in intact retina. Given the crucial causative roles of oxidative stress and inflammation in AMD pathogenesis, knowing whether OTC might exhibit a similar benefit in this cell and tissue type has high clinical relevance; thus, we evaluated OTC in the present study.
Methods
ARPE-19 and primary RPE cells isolated from wild-type, Gpr109a−/−, or Slc5a8−/− mouse eyes were exposed to TNF-α in the presence or absence of OTC, followed by analysis of IL-6 and Ccl2 expression with real-time quantitative polymerase chain reaction or enzyme-linked immunosorbent assay. Cellular and molecular markers of inflammation and oxidative stress (i.e., IL-1β, TGF-β, ABCG1, ABCA1, reduced glutathione, and dihydroethidium) were evaluated in Ccl2−/−/Cx3cr1−/− double knockout mice on rd8 background (DKO rd8) treated with OTC (10 mg/ml) in drinking water for a period of 5 months.
Results
OTC treatment significantly inhibited the expression and secretion of IL-6 and Ccl2 in TNF-α-stimulated ARPE-19 cells. Studies conducted using DKO rd8 animals treated with OTC in drinking water confirmed these findings. Cellular and molecular markers of inflammation were significantly suppressed in the retinas of the OTC-treated DKO rd8 animals. Subsequent in vitro and in vivo studies of the possible mechanism(s) to explain these actions revealed that although OTC is an agonist of the anti-inflammatory G-protein coupled receptor GPR109A and a transportable substrate of the sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8), these properties may play a role but do not explain entirely the anti-inflammatory effects this compound elicits in cultured RPE cells and the intact mouse retina.
Conclusions
This study represents, to our knowledge, the first report of the suppressive effects of OTC on inflammation in cultured RPE cells and on inflammation and oxidative stress in the retina in vivo.
PMCID: PMC3888500  PMID: 24426777
23.  CFH haplotypes and ARMS2, C2, C3, and CFB alleles show association with susceptibility to age-related macular degeneration in Mexicans 
Molecular Vision  2014;20:105-116.
Purpose
To evaluate the contribution of genetic variants of complement factor H (CFH), complement component 2 and 3 (C2 and C3), complement factor B (CFB), and age-related maculopathy susceptibility 2 (ARMS2) to age-related macular degeneration (AMD) risk in the Mexican Mestizo population.
Methods
Analysis included 282 unrelated Mexican patients with advanced AMD, 205 healthy controls, and 280 population controls. Stereoscopic fundus images were graded on the Clinical Age-Related Maculopathy System (CARMS). We designed a resequencing strategy using primers with M13 adaptor for the 23 exons of the CFH gene in a subgroup of 96 individuals clinically evaluated: 48 AMD cases and 48 age- and sex-matched healthy controls. Single nucleotide polymorphisms (SNPs) in C3 (Arg80Gly and Pro292Leu), C2 (rs547154), CFB (Leu9His), and ARMS2 (Ala69Ser) were genotyped in all patients, healthy and population controls using TaqMan assay.
Results
All evaluated individuals were Mexican Mestizos, and their genetic ancestry was validated using 224 ancestry informative markers and calculating Fst values. The CFH resequencing revealed 19 SNPs and a common variant in the intron 2 splice acceptor site; three CFH haplotypes inferred from individual genotypes, showed significant differences between cases and controls. The risk alleles in C3 (rs1047286, odds ratio [OR]=2.48, 95% confidence interval [CI]=1.64–3.75, p=1.59E-05; rs2230199, OR=2.15, 95% CI=1.48–3.13, p=6.28E-05) and in ARMS2 (rs10490924, OR=3.09, 95% CI=2.48–3.86, p=5.42E-23) were strongly associated with risk of AMD. The protective effect of alleles in C2 (rs547154) and CFB (rs4151667) showed a trend but was not significantly associated after correction for multiple testing.
Conclusions
Our results show that ARMS2 and C3 are major contributors to advanced AMD in Mexican patients, while the contributions of CFH, C2, and CFB are minor to those of other populations, reveling significant ethnic differences in minor allele frequencies. We provide evidence that two specific common haplotypes in the CFH gene predispose individuals to AMD, while another may confer reduced risk of disease in this admixed population.
PMCID: PMC3891434  PMID: 24453474
24.  Association between genotype and phenotype in families with mutations in the ABCA4 gene 
Molecular Vision  2014;20:89-104.
Purpose
To investigate the genotype and phenotype in families with adenosine triphosphate–binding cassette, sub-family A, member 4 (ABCA4)–associated retinal degeneration.
Methods
Three families with at least one family member with known homozygous or compound heterozygote mutations in the ABCA4 gene were studied. The investigations included full field electroretinography (ff-ERG), multifocal ERG (mERG), Goldmann visual fields, optical coherence tomography (OCT), and standard ophthalmological examination. Microarray (Asper) was used for ABCA4 genotyping.
Results
In family 1, the proband (age 23) was homozygote for the c768 G>T mutation. She was diagnosed with cone rod dystrophy (CRD) while her aunt (age 69) was compound heterozygote for the c768 G>T and c2894 A>G mutations and had autosomal recessive retinitis pigmentosa (arRP). The father (age 61) and the mother (age 60) of the proband were asymptomatic carriers of the c768 G>T mutation. In family 2, the proband (age 25) was homozygote for the c5917del. She was diagnosed with CRD. Her father and two sisters were compound heterozygote for the c5917del and c5882 G>A mutations. The eldest sister (age 23) suffered from Stargardt disease (STGD) while the youngest sister (age 12) and their father (age 48) had no visual complaints. Anyhow, their ERG measurements indicated changes corresponding to STGD. The mother (age 42), (heterozygote for the c5917 delG mutation) and the youngest child (age 9; heterozygote for the c5882 G>A mutation) had a normal phenotype. In family 3, the proband (age 43) was compound heterozygote for c768 G>T and c3113 C>T and had been diagnosed with STGD. Her son (age 12), who was homozygote for the c768 G>T mutation, had wider scotomas with earlier onset (age 6), ff-ERG cone responses in the lower range of normality, and reduced mERG. At the moment, he is classified as having STGD but may progress to CRD. The father (age 45) was asymptomatic and heterozygote for the c768 G>T mutation. The patients with progressive disorders (CRD or arRP) had prolonged implicit times for the 30 Hz flicker ff-ERG and the mERG. All patients with two mutations in the ABCA4 gene demonstrated attenuation of retinal thickness on the OCT macular map.
Conclusions
This study confirms that ABCA4 mutations lead to a spectrum of retinal degenerations ranging from STGD to CRD or arRP. At the time of diagnosis, it is not possible to predict the severity of the condition only from genotyping. Our results suggest that prolongation of implicit times for the ff-ERG and/or mERG seem to be associated with progressive conditions such as CRD and arRP. Since ABCA4 mutations are common in the general population, different family members can harbor various combinations of mutations resulting in diverse phenotype and prognosis in the same family, further emphasizing the importance of a combination of genetic and electrophysiological tests at the first visit and follow-up.
PMCID: PMC3892680  PMID: 24453473
25.  Identification of proteins that interact with alpha A-crystallin using a human proteome microarray 
Molecular Vision  2014;20:117-124.
Purpose
To identify proteins interacting with alpha A-crystallin (CRYAA) and to investigate the potential role that these protein interactions play in the function of CRYAA using a human proteome (HuProt) microarray.
Methods
The active full-length CRYAA protein corresponding to amino acids 1–173 of CRYAA was recombined. A HuProt microarray composed of 17,225 human full-length proteins with N-terminal glutathione S-transferase (GST) tags was used to identify protein–protein interactions. The probes were considered detectable when the signal to noise ratio (SNR) was over 1.2. The identified proteins were subjected to subsequent bioinformatics analysis using the DAVID database.
Results
The HuProt microarray results showed that the signals of 343 proteins were higher in the recombinant CRYAA group than in the control group. The SNR of 127 proteins was ≥ 1.2. The SNR of the following eight proteins was > 3.0: hematopoietic cell-specific Lyn substrate 1 (HCLS1), Kelch domain-containing 6 (KLHDC6), sarcoglycan delta (SGCD), KIAA1706 protein (KIAA1706), RNA guanylyltransferase and 5′-phosphatase (RNGTT), chromosome 10 open reading frame 57 (C10orf57), chromosome 9 open reading frame 52 (C9orf52), and plasminogen activator, urokinase receptor (PLAUR). The bioinformatics analysis revealed 127 proteins associated with phosphoproteins, alternative splicing, acetylation, DNA binding, the nuclear lumen, ribonucleotide binding, the cell cycle, WD40 repeats, protein transport, transcription factor activity, GTP binding, and cellular response to stress. Functional annotation clustering showed that they belong to cell cycle, organelle or nuclear lumen, protein transport, and DNA binding and repair clusters. CRYAA interacted with these proteins to maintain their solubility and decrease the accumulation of denatured target proteins. The protein–protein interactions may help CRYAA carry out multifaceted functions.
Conclusions
One-hundred and twenty-seven of 17,225 human full-length proteins were identified that interact with CRYAA. The advent of microarray analysis enables a better understanding of the functions of CRYAA as a molecular chaperone.
PMCID: PMC3893783  PMID: 24453475

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