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Focus on Fellows 2013 Abstracts


Mapping the Receptor-binding Site on PEDF: Implications for a Neurotrophic Role of PEDF-R

Jason Kenealey1, Preeti Subramanian1, David Hoover2, S. Patricia Becerra1

1Protein Structure & Function Section, Laboratory of Retinal Cell & Molecular Biology, National Eye Institute; 2Center for Information Technology, National Institutes of Health

Purpose: Pigment epithelium-derived factor (PEDF) protects the retina by blocking pathogenic angiogenesis, and by protecting the neural retina against degeneration. Our lab has identified a neurotrophic PEDF receptor, PEDF-R and mapped its ligand-binding domain to amino acid positions 203-232 of the human PEDF-R. However, the region on PEDF that binds PEDF-R has not been identified. Therefore, this study is designed to map the region on PEDF that binds PEDF-R to exert biological activity.

Methods: Binding was determined by ligand blotting, pull-down, surface plasmon resonance (SPR), and fluorescence anisotropy. The structure of P1 was predicted in an ab initio model, and the resultant structure was then used in a global docking search using the Rosetta software. Retina survival assays were performed with rat retina precursor R28 cells induced to cell death by serum starvation, and measuring apoptosis by TUNEL staining.

Results: The 44mer peptide bound specifically to P1 peptide by ligand blot, SPR, and anisotropy assays; whereas, the 34mer did not bind. The 44mer, and not the 34mer, bound to full-length PEDF-R in a pull-down assay. The affinity of the 44mer:P1 interaction calculated from ligand blot data was KD=70±7.7 nM compared to 9.1±1.1 nM for that of PEDF:P1. The 17mer bound P1 with a KD=410±80 nM. Alanine scanning of the 17mer demonstrated that R99 of 17mer was critical for P1 binding, and that 17mer[H105A] variant bound P1 more tightly than the unmodified peptide. Molecular modeling revealed that P1 can fold into three alpha helices and dock in the 44mer region of the PEDF protein. Like PEDF, the 44mer and 17mer peptide, protected R28 cells from apoptosis induced by serum deprivation, but the 34mer had no neuroprotective effect.

Conclusions: Our results identify a novel PEDF-R binding site on PEDF in its neurotrophic region. Further, these data support the role of PEDF-R as a neurotrophic receptor for PEDF.

Gene Therapy for X-linked Retinitis Pigmentosa Due to RP2 Mutation

Suddhasil Mookherjee1, Suja Hiriyanna1, Kayleigh Kaneshiro1, Haohua Qian2, Tiansen Li3, Hemant Khanna4, Peter Colosi1, Anand Swaroop5, Zhijian Wu1

1Unit on Ocular Gene Therapy; 2Visual Function Core; 3Retinal Cell Biology Degeneration Section, Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health; 4Department of Ophthalmology, University of Massachusetts Medical School; 5Retinal Development, Genetics, & Therapy Section, Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health

Retinitis pigmentosa (RP) is a degenerating eye disorder associated with early night blindness, progressive loss of peripheral vision and eventual loss of central vision. The disease can be inherited as autosomal dominant, autosomal recessive or X-linked form. A mutation in RP2 gene causes relatively severe form of the disease and accounts for 10-20% of total X-linked RP cases. Here we report the efficacy of AAV (adeno-associated virus) vector mediated gene therapy in an Rp2 knockout mouse model, generated by crossing an Rp2flox mouse line with a Cre expressing line. The mice showed early and severe cone photoreceptor degeneration and moderate rod degeneration later in life. These knockout mice were treated unilaterally with subretinal injections of an AAV (serotype 8) vector carrying a human RP2 expression cassette at 4-6 weeks of age with 1e8, 3e8, or 1e9 vector genomes (v.g.)/eye. The fellow eyes were injected with saline as a control. The AAV vector genome has a self-complementary structure and is composed of the rhodopsin kinase promoter, the CMV/beta-globin intron, the human RP2 cDNA and the beta globin polyadenylation site. Immunohistochemistry showed AAV-mediated RP2 expression in the inner segments and cell bodies of the photoreceptors. The treated mice were followed periodically for visual function change by electroretinogram (ERG). A significantly better cone ERG in vector-treated eyes was observed as early as 4 months of age, which sustained up to 12 months, advocating its efficacy in maintaining cone viability. Due to the slow rod degeneration, no obvious difference in the rod function was observed between the vector-treated and control eyes in the two lower doses whereas a decline in rod ERG in 1e9 v.g. treated group indicated toxicity of the vector at this dose. Studies are in progress to further evaluate efficacy of the gene therapy strategy. Our study provides a framework for gene therapy for retinitis pigmentosa due to RP2 mutation.

Dissecting Human Photoreceptor Development Using Retinal Organoids

Rossukon Kaewkhaw, Kohei Homma, Anand Swaroop

Retinal Development, Genetics and Therapy Section, Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health

A better understanding of human photoreceptor development is critical for designing new treatments for retinal degenerative diseases, as animal models do not fully recapitulate human diseases. Organoids can be generated from floating aggregates of human embryonic stem cells (hESCs), giving a three-dimension (3D) tissue structure that would be useful for elucidating mechanism of retinal neurogenesis.

Our aim is to identify intrinsic genetic network and extrinsic factors directing photoreceptor differentiation using human retinal organoids. We generated a hESC line carrying green fluorescent protein (GFP) driven by promoter of cone-rod homeobox (CRX) gene into a safe harbor AAVS1 locus. The hESCs were differentiated into stratified neural retina by floating aggregates, which formed a highly ordered optic vesicle-like structure expanding with culture time. Co-immunostaining experiments validated that the CRXp-GFP construct recapitulated the endogenous CRX expression in differentiating cells. Furthermore, optic vesicle-like structures established apical-basal polarity and contained at least five types of retinal neurons and Müller glia at different culture states between days 37, 47, 67 and 90. Upon differentiation, GFP-positive cells on days 67 and 90 expressed cone arrestin, neural retina-specific leucine zipper and opsin proteins. The percentage of GFP-positive cells were 10.9 ± 2.8 on day 67 (mean ± SD, n = 4). We purified GFP-positive cells from each culture state by fluorescence activated cell sorter and are conducting global gene expression analysis using RNA-seq to identify the mechanism(s) underlying human photoreceptor development. Our studies demonstrate that retinal organoids from hESCs can be used as a model for elucidating early development of retinal neurons and designing therapeutic paradigms.

Functional Characterization of Prickle 2, a Core Planar Cell Polarity Protein, in Mouse Retina

Samelia Okpodu1, Chunqiao Liu1, Alexander Bassuk2, Helen May-Simera1, Vinit Mahajan3, Werner Graf4, Anand Swaroop5, Tiansen Li1

1Retinal Cell Biology Degeneration Section, Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health; 2Department of Pediatrics; 3Ophthalmology, University of Iowa Carver College of Medicine; 4Department of Physiology and Biophysics, Howard University; 5Retinal Development, Genetics & Therapy Section, Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health

Purpose: Planar cell polarity (PCP) refers to an asymmetry along the plane of tissue that is perpendicular to the baso-apical axis. PCP is critical for many aspects of development such as convergent extension of cell movement during gastrulation and cochlea duct formation in the inner ear. To understand functions of PCP pathway in retina we focused on functional characterization of Prickle 2, a core player of PCP pathway.

Methods: In situ hybridization and qRT-PCR were used to study Prickle 2 gene expression during retinal development. Polyclonal antibodies against Prickle2 were generated to study Prickle 2 protein expression by immunoblotting and Prickle 2 subcellular localization by immunohistochemistry. Cochlea hair cell formation was examined by phalloidin staining for actin filament in wildtype and prickle2 null mice at postnatal day 1. Retinal functions of Prickle 2 knockout mice were assessed by electroretinography (ERG).

Results: Prickle 2 expression in retina is detected as early as at embryonic day 13.5 (E13.5) by in situ hybridization and is present throughout the developing retina. In adult retina, it is moderately expressed in the inner nuclear layer, weakly expressed in ganglion cells, and minimally expressed in photoreceptor layer. Immunoblotting shows Prickle2 to be a single band of 95 kDa which is absent in the KO retina. Prickle 2 protein is upregulated in NRL knockout mouse retina both by immunoblotting and by qRT-PCR. ERG at one month shows that loss of PK2 protein does not lead to a major deficit in photoreceptor development or function. No gross abnormality in stereocilia bundle formation and orientation was detected in the knockout mice. Further work is in progress, which will investigate the inner retinal neurons and circuitry in addition to the PK2 subcellular localization.

Conclusions: Sensory neurons (photoreceptors and cochlear hair cells) appeared to develop normally in mice lacking Prickle 2. Functional redundancy with Prickle1 may offer an explanation. PK2 expression appears to be influenced by NRL, the significance of which is under investigation.

Resident Myeloid Cells in the Mouse Choroid Show Diverse Associations with Choroidal Vessels and Display Prominent Dynamic Process Motility

Anil Kumar1, Lian Zhao1, Robert Fariss2, Wai Wong1

1Unit on Neuron-Glia Interactions in Retinal Disease, 2Biological Imaging Core, National Eye institute, National Institutes of Health

Choroidal macrophages are resident ocular myeloid cells capable of influencing the inflammatory environment of the outer retina and play a role in AMD pathogenesis. However, choroidal macrophages have not been characterized in detail in terms of their anatomy, distribution, association, behavior, and possible endogenous functions. Here we identify that resident dendritiform myeloid cells in the choroid demonstrate variations in the morphologies and associations with choroidal vessels that vary according to their position in the choroidal vascular tree. These dendritiform cells also exhibit a marked dynamism in the processes that is capable of providing extensive perivascular coverage without overt cellular migration in a function consistent with immunosurveillance. This description of the morphology, distribution, and behavior of these resident cells provide a foundation for understanding immune function in the young healthy choroid and a basis for interpreting age-related and pathological changes in eye disease in future studies.

Tertiary Lymphoid Tissue Formation in the Chronically Inflamed Eye

Jennifer Kielczewski, Reiko Horai, Yingyos Jittayasothorn, Rachel Caspi

Immunoregulation Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health

Tertiary lymphoid tissues (TLT) are germinal center-like lymphoid structures with distinct T and B cell zones that form outside of lymphoid organs in inflamed tissues. TLT can be pathogenic hot spots for autoantibody production and antigen presentation, since they contain antigen-presenting cells, T follicular helper cells and B cells, enabling them to produce inflammatory cell and antibody responses. We developed a transgenic mouse (line R161H) that expresses a retina specific T cell receptor and develops spontaneous autoimmune uveitis of a chronic-progressive nature. These mice develop distinct retinal lymphoid aggregates in their retina, which resemble TLT. We asked whether these lymphoid aggregates fit the criteria of TLT and possess the necessary lymphoid cells and structures that could facilitate inflammation and antigen presentation to occur locally within the eye. Mouse retinas were immunohistochemically stained for various immune cell types and germinal center markers and imaged via confocal microscopy. Selected areas from frozen retinal cross sections were isolated by laser capture microdissection and analyzed for expression of genes of interest. Immunohistochemical analysis of the retinal lymphoid aggregates revealed markers for all the essential immune cells, including antibody-forming cells, which typically compose lymphoid germinal centers. Also, the retinal aggregates stained positive for stromal cell networks, which provide a scaffold for immune cell migration, organization, and interaction. Importantly, we observed evidence of antigen presentation by dendritic cells to T cells, which would tend to perpetuate inflammation at that site. We conclude that the lymphoid aggregates in uveitic retina of R161H mice resemble organized TLT, in which lymphoid cells accumulate to create an environment conducive to immune cell activation, contributing to chronicity of their ocular disease.

Distinct Basal Ganglia Circuits for Controlled and Automatic Behaviors Guided by Flexible and Stable Values

Hyoung Kim, Okihide Hikosaka

Neuronal Networks Section, Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health

Objects around us have values: we are likely to orient to, approach, and interact with more valuable objects. However, the values of some objects change flexibly, or the values of the other objects remain unchanged. Therefore, animals should be able to update the object values flexibly by recent experiences and retain them stably by long-term experiences. Since the flexible and stable values are mutually conflicting, it would be difficult for a single neural circuit to process the potentially conflicting values. We found that distinct circuits of the primate caudate nucleus control behavior selectively in the flexible and stable value conditions. Single caudate neurons encoded the values of visual objects in a regionally distinct manner: flexible value coding in the caudate head and stable value coding in the caudate tail. Monkeys adapted in both flexible and stable conditions by controlled and automatic saccade to the objects with high values. Notably, inactivation of each caudate subregion disrupted the value discrimination selectively in each behavior; caudate head and tail inactivation respectively impaired the controlled and automatic value discrimination. This parallel complementary mechanism enables animals to efficiently choose valuable objects in both flexible and stable conditions.

Altered CD8+ T Cell Function in Human Non-infectious Uveitis

Sima Hirani1, Lai Wei1, Baoying Liu1, Zhiyu Li1, Shayma Jawad1, Ian Thompson1, Ping Chen1, H. Nida Sen1, Richard W. J. Lee2, & Robert Nussenblatt1

1Clincial Immunology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health; 2Inflammation & Immunotherapy Theme, National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust & UCL Institute of Ophthalmology, University Hospitals Bristol NHS Foundation Trust & University of Bristol

Purpose: Although CD8+ T cells are primarily implicated in the clearance of viral infections, they have also recently been shown to be a key determinant of clinical prognosis in patients with non-ocular autoimmune diseases. This challenges the conventional paradigm of CD4+ T cell driven autoimmunity, and raises new questions about the role of CD8+ cells in non-infectious inflammation. We therefore interrogated the phenotype and function of CD8+ cells in patients with sight threatening autoimmune uveitis.

Methods: Fresh whole blood from uveitis patients (n=90) and healthy controls (HCs, n=65) was analyzed using flow cytometry. Four subsets of CD8+ cells were distinguished based on surface expression of CD45RA, and CCR7, representing naÔve (CCR7+CD45RA+), central memory (CCR7+CD45RA-), effector memory (CCR7-CD45RA-), and effector memory RA+ (CCR7-CD45RA+) cells. Flow cytometry was used to assess intracellular expression of cytotoxic markers, CD107a, granzyme B and the transcription factors T-bet and Eomes, which are master regulators of CD8+ effector differentiation.

Results: There was significantly higher percentage of effector memory CD8+ cells in uveitis patients compared to age matched healthy controls. Effector memory CD8+ cells from uveitis patients contained higher percentage of CD107a+, and higher expression of granzyme B. Furthermore, CD8+ cells from uveitis patients had a significantly higher T-bet:Eomes ratio in comparison with healthy controls.

Conclusions: The increased expression of CD107a and granzyme B in effector memory CD8+ T cells from uveitis patients indicates a greater capacity for cytotoxicity. This is the first report of altered CD8+ cell function in patients with sight-threatening non-infectious uveitis, and further investigations are now needed to determine whether this contributes to the pathogenesis of intraocular inflammation.

Prohibitin is Required for Ocular Development in Zebrafish

Mariana Rius, Sunit Dutta, Brian Brooks

Pediatric, Developmental, & Genetic Ophthalmology Unit, Ophthalmic Genetics & Visual Function Branch, National Eye Institute, National Institutes of Health

Optic fissure closure is a highly conserved and complex biological process in ocular morphogenesis. Failure of the optic fissure to close results in a potentially blinding ocular malformation known as uveal coloboma. We previously used developmental profiling to identify a zinc-finger-containing gene, nlz1, as an important regulator of optic fissure closure. NLZ1 was independently shown to interact with prohibitin (PHB/phb), another gene suggested by our developmental screen. phb is thought to regulate DNA replication and cell division and may have a role in mitochondrial function. This study evaluates phb as a candidate gene for coloboma using a zebrafish model. Whole mount in-situ hybridization confirmed phb1 and phb2 expression in the eye at relevant developmental time points. RT PCR indicated maternal and zygotic expression of phb1 and phb2, and morpholino knockdown revealed a uveal coloboma phenotype in zebrafish. The identification of prohibitinís involvement in the fusion of the optic fissure provides insight into the various types of genes that may participate in such a conserved and poorly understood mechanism.

OKR Arena - an Open Source System to Quantify Optokinetic Responses

Friedrich Kretschmer, Tudor C. Badea

Retinal Circuit Development & Genetics Unit, Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health

The measurement of the optokinetic reflex (OKR) is a widely established method to assess various parameters of the visual system in several animal species. When a global movement of the world within the visual field happens involuntary compensatory body, head and/or eye movements occur. We have built a novel setup to measure all of these in mice during visual stimulation. While the measurement of eye movements requires a fixation of the animal to keep the eye in the focus of a camera, head and body movements are measured without the need to restrain the animal. Latter are recorded by video tracking the snout and body axis of the animal in real-time. This data can also be used to objectively quantify optokinetic responses.

Stimuli are presented on four computer screens surrounding the animal covering the whole field of view in form of a texture on the surface of a virtual sphere. The sphere is placed in such a way that the animal is always located in the center. Hence, the distance to all areas of the stimulus is held constant. This is an important aspect to measure parameters like spatial acuity which are dependent on the perceived size and therefore on the distance between the head and the presented stimulus.

In the unrestrained condition, the position of the virtual sphere is continuously readjusted according to the location of the animal head determined by our video tracking algorithm. The building plans and software of this setup are going to be released under an open source license in near future.

We are currently using the setup to measure spatial acuity and contrast thresholds under scotopic and photopic light conditions. We compare the thresholds measured by analyzing eye movements to the thresholds determined by solely looking at head/body movements.

CRISPR/Cas9: A New Tool in Genomic Engineering and its Implementation at the NEI GEC

Hazel Lozano, Pinghu Liu, Jingqi Lei, Carl Haugen, Yan Li, Lijin Dong

Genetic Engineering Facility, National Eye Institute, National Institutes of Health

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins have quickly gained prominence as a cost- and time-efficient tool in genetic engineering. Initially discovered as an adaptive immune defense in bacteria, CRISPR-Cas is now valued for its potential in sequence-specific gene editing and modification of gene expression. After foreign invasion, a small (~20bp) piece of the invading plasmid or viral DNA is copied and inserted into the bacterial genome, archiving the insult. This archive is transcribed into a small non-coding RNA pool, the CRISPR RNAs. Upon invasion by the same foreign DNA, a Cas nuclease forms a complex with target-specific CRISPR RNA. Cas is delivered to the target DNA via base pairing, and creates a double-strand break. The system can be analogized to RNA interference by microRNA: microRNA (miRNA) is a small non-coding RNA molecule (~22bp) found in plants and animals, which functions in post-transcriptional regulation of gene expression. Encoded by eukaryotic DNA, miRNAs function within an active RNA-induced silencing complex (RISC) by base pairing with complementary sequences in the 3í UTR of mRNA molecules, usually resulting in gene silencing through translational repression. Coupled with effector domains, a modified Cas protein under the guidance of a target-specific CRISPR RNA can turn genes on or off in a reversible fashion, enabling transcriptional repression or activation. CRISPR-Cas can also be compared to zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs): CRISPR-Cas can target multiple loci simultaneously, with high efficiency and specificity. The CRISPR-Cas system promises much for genetic engineering, and has delivered much in the past year alone. The NEI Genetic Engineering Core has implemented the system for the purpose of mouse genetic engineering in either mouse embryonic stem cells (ESCs) or mouse zygotes; the results of those experiments will be discussed, as well as future avenues of research.

The Role of Macf1 During Ciliated Cell Development

Helen May-Simera, Tiansen Li

Retinal Cell Biology Degeneration Section, National Eye Institute, National Institutes of Health

Ciliopathies comprise a growing class of syndromic disorders caused by disrupted development and function of the primary cilium, a microtubule-based, membrane-bound appendage required for a range of cellular functions. After exit from mitosis the basal body, derived from the mother centriole, migrates to the apical domain of a polarized cell and initiates the process of ciliogenesis. Virtually all ciliopathies are accompanied by retinal degeneration, as the mammalian photoreceptors contain a highly specialized sensory cilium. This makes the photoreceptor a good model system in which to study ciliary function. Molecular trafficking associated with the ciliary axoneme is crucial during ciliogenesis and function, and is highly regulated by the basal body and transition zone at the base of the cilium. We identified an interaction between the spectraplakin Microtubule actin cross-linking factor 1 (Macf1) with the basal body protein Mkks (Bbs6). Spectraplakins are cytoskeletal cross-linkers, which associate with F-actin and microtubules to integrate cytoskeletal networks and have also been shown to play a role in intracellular vesicle trafficking. Mice lacking Macf1 in developing ciliated tissues (retina, cochlea, lateral ventricles) exhibit a complete loss of visual function due to severely disrupted retinal lamination primarily affecting photoreceptors. Loss of apical basal polarity of the outer nuclear layer is seen as early as P5, which is preceded by failure of the basal body to dock at the apical surface of the cell. Ciliary defects are also observed in mutant cochlea sensory neurons and ciliated ventricular neuroepithelia. Immunolocalization using antibodies against the actin-binding domain suggests that Macf1 localizes along microtubules and concentrates at regions where microtubules interact with actin, particularly around the ciliary pocket in ciliated cells. These findings suggest a critical role for Macf1 in ciliogenesis and highlight novel functions for microtubule and actin interactions required for ciliary trafficking. These insights may provide the cellular basis for clinical phenotypes underlying ciliopathies.

Correlations Between Visual Cortical Activity and Perception: What Do They Mean?

Adrian Bondy, Bruce Cumming

Vision Section, Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health

Neurons in visual cortex are activated by particular features in particular regions of the visual field. How the pattern of neural responses in visual cortex is used to recover the nature of the visual stimulus is not well understood. One approach has been to monitor the electrical activity of visual cortical neurons in non-human primates while they report what they see on experimentally controlled displays. Such experiments have shown that seemingly random fluctuations in the activity of neurons correlate with the perceptual reports, even when the stimulus is fixed. This phenomenon, called choice probability (CP), is widely thought to indicate that the animals base their reports on the activity of the recorded neurons, even if the relevant visual feature is absent. However, so many neurons in visual cortex show CP that the influence of any one neuron on perceptual choice must be much smaller than CP. This indicates that correlations in the random fluctuations in activity of neighboring neurons determine CP, regardless of the role of individual neurons in choice. So to correctly interpret CP, we need to understand the origin of these widespread correlations.

A possible source of these correlations is some common cognitive signal related to the impending choice that reaches visual cortical neurons through a feedback pathway, correlating activity between them and with choice. This would be strong evidence against the notion that CP reflects neuronsí causal effect on choice, but conversely, would suggest that CP reflects the effect of choice on visual cortex. To test for this possibility, we recorded simultaneously from many single neurons in macaque primary visual cortex (V1) using multi-electrode arrays, while the animals were engaged in a visual discrimination task. We found that correlations changed dynamically with task instruction — a signature of exactly the top-down scenario described above. These results imply that when we "decide what we see," we change our brainsí representation of the outside world to match.

Global Gene Expression Patterns During Rod Photoreceptor Development

Jung-Woong Kim, Anand Swaroop

Retinal Development, Genetics, & Therapy Section, Neurobiology Neurodegeneration & Repair Laboratory, National Eye Institute, National Institute of Health

Mammalian photoreceptors are highly organized for specialized capture of visual information. Rod photoreceptors respond to the dim light conditions, and the cones mediate day light vision and color perception. Even though their physiological functions are definite, underlying molecular mechanisms are not well elucidated during photoreceptor development. To study epigenetic regulation of rod development, we used Nrlp-GFP transgenic mouse line that expresses green fluorescence only in rod photoreceptors. GFP positive rod cells were collected by fluorescence-activated cell sorter (FACS) at different stages of differentiation, and then, its global mRNA expression levels and DNA methylation patterns were analyzed by RNA-sequencing and reduced-representation bisulfite sequencing (RRBS) using next-generation sequencing techniques. We demonstrate that there are global anti-correlation patterns between DNA methylation and mRNA expression in rods. Rod-specific gene expressions are gradually increased by reduced DNA methylation on promoters and gene body regions. However, expression of cone genes in newborn rods is rapidly decreased after P6, and gene body regions of cone genes were highly methylated compare to rod-specific genes. Our studies provide further evidence in favor of S-cone being the "default" pathway for post-mitotic photoreceptor precursors and suggest how epigenetic modifications can establish gene expression patterns.

Ocular Adnexal Lymphoma: Assessment of a Tumor-Node-Metastasis Staging System

Mary Aronow1,2, Craig Portell3, Lisa Rybicki4, John Sweetenham3, Arun Singh2

1Clinical Trials Branch, Division of Epidemiology & Clinical Applications, National Eye Institute, National Institutes of Health; 2Department of Ophthalmic Oncology, Cole Eye Institute, Cleveland Clinic; 3Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic; 4Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic

Purpose: To assess distribution, correlations, and prognostic effect of T, N, and M staging on relapse and survival.

Methods: 63 patients with primary ocular adnexal lymphoma (OAL) were staged according to the American Joint Committee on Cancer tumor-node-metastasis system.

Results: There were 40 (63.5%) males. Median age was 65 years. The conjunctiva was affected in 27 (42.9%), orbit in 38 (60.3%), and eyelid in 3 (4.8%) patients. Extra-nodal marginal zone lymphoma accounted for 51 (81.0%) cases. There were 14 (23.3%) T1, 42 (70.0%) T2, 1 (1.7%) T3, 3 (5.0%) T4, 48 (82.8%) N0, 10 (17.2%) N1-4, 47 (81.0%) M0, and 11 (19.0%) M1 cases. There were 18 (28.6%) relapses and 6 (9.5%) deaths (median follow-up: 27.9 months). Relapse was not associated with T stage (hazard ratio (HR) 1.14 per 1 level increase, p=0.71), N stage (HR 1.47, p=0.51 N1-4 vs. N0), or M stage (HR 1.22, p=0.76 M1 vs. M0). T stage was not associated with survival (HR 0.86, p=0.81). N1-4 had worse survival than N0 (HR 5.35, p=0.07). M1 had worse survival than M0 (HR 9.27, p=0.008).

Conclusions: T stage does not predict relapse or survival, however N1-4 and M1 stages indicated less favorable survival.

Department of Health and Human Services NIH, the National Institutes of Health