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1.  Anterior Keratocyte Depletion in Fuchs’ Endothelial Dystrophy 
Archives of ophthalmology  2011;129(5):555-561.
After endothelial keratoplasty for Fuchs’ endothelial dystrophy, vision is degraded by scattered light because of structural changes in the retained host stroma. In this study, we determined if keratocyte populations were different in corneas with Fuchs’ dystrophy compared to normal corneas.
Eleven corneas excised at penetrating keratoplasty for Fuchs’ dystrophy, and five normal corneas of eyes enucleated for choroidal melanoma, were examined by using light microscopy. Twenty normal corneas, age-matched to the corneas with Fuchs’ dystrophy, were examined by using confocal microscopy in vivo. The number of keratocytes in a full thickness column of central stroma with frontal area of 1 mm2 (referred to as a “column”), determined by using histologic and confocal methods, was compared between Fuchs’ dystrophy and normal.
By histology, the number of cells in a full-thickness column of stroma in Fuchs’ dystrophy (12,215 ± 1,394, [mean ± standard deviation]) was less than in normal corneas (15,628 ± 710, p<0.001). The number of keratocytes in the anterior 10% of the stroma of corneas with Fuchs’ dystrophy (682 ± 274 cells) was less than that in the normal corneas measured by using histology (1,858 ± 404 cells, p<0.001) and by using confocal microscopy (1,481 ± 397 cells, p<0.001).
Keratocytes are depleted by 54–63% in the anterior 10% of the stroma of corneas requiring penetrating keratoplasty for Fuchs’ dystrophy. Keratocyte loss might contribute to anterior stromal changes that persist and degrade vision after endothelial keratoplasty.
PMCID: PMC3902089  PMID: 21220622
2.  Genetic control of the alternative pathway of complement in humans and age-related macular degeneration 
Human Molecular Genetics  2009;19(1):209-215.
Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.
PMCID: PMC2792151  PMID: 19825847
3.  Copy number variation in the complement factor H-related genes and age-related macular degeneration 
Molecular Vision  2011;17:2080-2092.
To determine the contribution of copy number variation (CNV) in the regulation of complement activation (RCA) locus to the development of age-related macular degeneration (AMD).
A multiplex ligation-dependent probe amplification assay was developed to quantify the number of copies of CFH, CFHR3, CFHR1, CFHR4, CFHR2, and CFHR5 in humans. Subjects with (451) and without (362) AMD were genotyped using the assay, and the impact on AMD risk was evaluated.
Eight unique combinations of copy number variation were observed in the 813 subjects. Combined deletion of CFHR3 and CFHR1 was protective (OR=0.47, 95% confidence interval 0.36–0.62) against AMD and was observed in 88 (82 [18.6%] with one deletion, 6 [1.4%] with two deletions) subjects with AMD and 127 (108 [30.7%] with one deletion, 19 [5.4%] with two deletions) subjects without AMD. Other deletions were much less common: CFH intron 1 (n=2), CFH exon 18 (n=2), combined CFH exon 18 and CFHR3 (n=1), CFHR3 (n=2), CFHR1 (n=1), combined CFHR1 and CFHR4 (n=15), and CFHR2 deletion (n=7, 0.9%). The combined CFHR3 and CFHR1 deletion was observed on a common protective haplotype, while the others appeared to have arisen on multiple different haplotypes.
We found copy number variations of CFHR3, CFHR1, CFHR4, and CFHR2. Combined deletion of CFHR3 and CFHR1 was associated with a decreased risk of developing AMD. Other deletions were not sufficiently common to have a statistically detectable impact on the risk of AMD, and duplications were not observed.
PMCID: PMC3156785  PMID: 21850184
4.  Characterization of the retinal proteome during rod photoreceptor genesis 
BMC Research Notes  2010;3:25.
The process of rod photoreceptor genesis, cell fate determination and differentiation is complex and multi-factorial. Previous studies have defined a model of photoreceptor differentiation that relies on intrinsic changes within the presumptive photoreceptor cells as well as changes in surrounding tissue that are extrinsic to the cell. We have used a proteomics approach to identify proteins that are dynamically expressed in the mouse retina during rod genesis and differentiation.
A series of six developmental ages from E13 to P5 were used to define changes in retinal protein expression during rod photoreceptor genesis and early differentiation. Retinal proteins were separated by isoelectric focus point and molecular weight. Gels were analyzed for changes in protein spot intensity across developmental time. Protein spots that peaked in expression at E17, P0 and P5 were picked from gels for identification. There were 239 spots that were picked for identification based on their dynamic expression during the developmental period of maximal rod photoreceptor genesis and differentiation. Of the 239 spots, 60 of them were reliably identified and represented a single protein. Ten proteins were represented by multiple spots, suggesting they were post-translationally modified. Of the 42 unique dynamically expressed proteins identified, 16 had been previously reported to be associated with the developing retina.
Our results represent the first proteomics study of the developing mouse retina that includes prenatal development. We identified 26 dynamically expressed proteins in the developing mouse retina whose expression had not been previously associated with retinal development.
PMCID: PMC2843734  PMID: 20181029
5.  Using a Seed-Network to Query Multiple Large-Scale Gene Expression Datasets from the Developing Retina in Order to Identify and Prioritize Experimental Targets 
Understanding the gene networks that orchestrate the differentiation of retinal progenitors into photoreceptors in the developing retina is important not only due to its therapeutic applications in treating retinal degeneration but also because the developing retina provides an excellent model for studying CNS development. Although several studies have profiled changes in gene expression during normal retinal development, these studies offer at best only a starting point for functional studies focused on a smaller subset of genes. The large number of genes profiled at comparatively few time points makes it extremely difficult to reliably infer gene networks from a gene expression dataset. We describe a novel approach to identify and prioritize from multiple gene expression datasets, a small subset of the genes that are likely to be good candidates for further experimental investigation. We report progress on addressing this problem using a novel approach to querying multiple large-scale expression datasets using a ‘seed network’ consisting of a small set of genes that are implicated by published studies in rod photoreceptor differentiation. We use the seed network to identify and sort a list of genes whose expression levels are highly correlated with those of multiple seed network genes in at least two of the five gene expression datasets. The fact that several of the genes in this list have been demonstrated, through experimental studies reported in the literature, to be important in rod photoreceptor function provides support for the utility of this approach in prioritizing experimental targets for further experimental investigation. Based on Gene Ontology and KEGG pathway annotations for the list of genes obtained in the context of other information available in the literature, we identified seven genes or groups of genes for possible inclusion in the gene network involved in differentiation of retinal progenitor cells into rod photoreceptors. Our approach to querying multiple gene expression datasets using a seed network constructed from known interactions between specific genes of interest provides a promising strategy for focusing hypothesis-driven experiments using large-scale ‘omics’ data.
PMCID: PMC2735966  PMID: 19812791
gene expression; gene network; cell fate determination; retina; photoreceptor

Results 1-5 (5)