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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyophthalmoscopy, SD-OCT, and high resolution Magnetic ResonanceImaging of the orbits.2) Five patients had choroidal lesions which were visible withophthalmoscopy and hence typical of choroidal nevi 3) Three patientshad choroidal lesions that were invisible to ophthalmoscopy andhence not traditional nevi or other known choroidal lesions. One ofthese 3 patients had skin lesions highly suggestive of NF1.Conclusions: Multi-spectral imaging (MSI) is a novel way toexamine the retina and choroid. This technique revealed choroidallesions associated with Neurofibromatosis 1 which are invisible toophthalmoscopy and many other imaging devices. Such lesions havebeen rarely, if ever, documented in the world’s literature. Wehypothesize that these choroidal lesions revealed by MSI, that wereclinically invisible on SD-OCT and high resolution MRI, mayrepresent a new biomarker for Neurofibromatosis 1. There are at leasta dozen NFI -1 interventional drug studies presently underway.Whether these lesions change with treatment with any of theseexperimental drugs remains to be determined.were above or below the mean ≥ 1 SD and p values ≤ 0.05.Results: About 284 proteins were quantified per mouse strain. Moreproteins were altered in the choroid fraction (n = 44) than in theretina (n = 8), with the majority detected in TIMP3 KO mice. Moreproteins were elevated in KO mice than decreased while very fewproteins were elevated in KI mice and none were common to thoseelevated in KO mice. All TIMP3 KO and KI mouse strains exhibitedsignificantly reduced amounts of crystallins in both the retina andchoroid fraction, including α-crystallin B, β-crystallin B2 and α-crystallin A. Homozygous KI mice also exhibited significantlyreduced levels of several collagens, fibrilin-1 and TGFβ-induced igh3in the choroid fraction.Conclusions: Protein changes observed in both TIMP3 KO and KImice are more likely due to TIMP3 dysfunction than collateralgenetic alterations. Our results suggest lower crystallin levels are aconsequence of TIMP3 dysfunction. Crystallins are inhibitors ofapoptosis. Reduced crystallins may facilitate inflammatory responsesand cellular degeneration in TIMP3 dysfunctional animals, includingheterozygous TIMP3 KI mice which exhibit an SFD-like phenotype.Other proteins decreased in homozygous KI mice implicatedysregulation of extracellular matrix homeostasis.Commercial Relationships: Geeng-Fu Jang, None; Alecia Cutler,None; Lei Zhang, None; John S. Crabb, None; Heidi Stoehr,None; John W. Crabb, SKS Ocular (P), Allergan (C); Bela Anand-Apte, 7 183 256 B2 (P)Support: EY021840, EY022134, EY016490, CA10641505, BIMR1104, FFB Center Grant, RPB Challenge GrantAnnidis-RHA Multi-spectral ImagingCommercial Relationships: Dorothy Hitchmoth, Annidis, Corp.(C); Jerome Sherman, Optos, Inc. (F), Optos, Inc. (C), Optos, Inc.(R), Annidis (C), Annidis (R), Zeiss (R)Support: no supportProgram Number: 1575 Poster Board Number: D0002Presentation Time: 8:30 AM - 10:15 AMQuantitative Proteomic Analysis of TIMP3 DysfunctionGeeng-Fu Jang 1 , Alecia Cutler 1 , Lei Zhang 1 , John S. Crabb 1 , HeidiStoehr 2 , John W. Crabb 1 , Bela Anand-Apte 1 . 1 Cole Eye Institute &Lerner Research Institute, Cleveland Clinic, Cleveland, OH; 2 Instituteof Human Genetics, University of Regensburg, Regensburg,Germany.Purpose: Polymorphisms in tissue inhibitor of metalloproteinase 3(TIMP3) have been associated with Sorsby fundus dystrophy (SFD)and age-related macular degeneration. Toward a molecularunderstanding of TIMP3 dysfunction, we pursued quantitativeproteomic analyses of the retina and choroid from TIMP-3 knockout(KO) mice and knockin (KI) mice expressing the TIMP3 S156Cmutation that causes SFD.Methods: Soluble proteins from isolated retinas and choroidcontainingposterior globes from TIMP3 KO mice (n = 5 mice), KIhomozygotes (n = 5), KI heterozygotes (n = 4), and wild-type mice (n= 7) were quantified by iTRAQ technology. Protein was digestedwith trypsin, peptides labeled with iTRAQ tags, fractionated bystrong cation exchange chromatography, and peptides were analyzedby LC MS/MS. Proteins were identified using the Swiss-Proteindatabase and quantified using code written in R. Proteins quantifiedwith ≥ 2 unique peptides/protein in ≥ 3 mice/strain were consideredsignificantly altered if average ratios relative to wild-type tissuesProgram Number: 1576 Poster Board Number: D0003Presentation Time: 8:30 AM - 10:15 AMAssociation of the polymorphism of the 5' UTR region of theHBD1 gene in patients with infectious blepharitisHector J. Perez-Cano 1 , Ingrid V. Gonzalez Leon 2 , Oscar Fernandez-Vizcaya 2 , Justine Nolasco-Lopez 1 , Jean R. Clemenceau 1 , AtzinRobles-Contreras 1 . 1 Biomedical Research Center, HospitalFoundation "Nuestra Senora de la Luz", Mexico, Mexico; 2 cornea,hospital foundation "Nuestra Señora de la Luz", Mexico, Mexico.Purpose: Recently, there is a particular interest in the participation ofsingle nucleotide polymorphisms (SNPs) in DEFB1 gene andinvolvement in susceptibility to infections. Two polymorphisms havebeen associated in the 5'UTR region of HBD1 gene,-52G/A and -44C/G, and have been reported -44C and -55G alleles associated withincreased risk of HIV infection and respiratory infections withCandida in diabetic patients. However, little is known about theinvolvement of these polymorphisms in ocular infections. Wepropose that polymorphisms in the 5'UTR region in HBD1 gene isassociated with infectious blepharitisThere are not information about infectious blepharitis and itsrelationship to the 5'UTR polymorphism DEFB1 gene for that reasonafter conducting an epidemiological study to identify the mostcommon pathogens that occur in infectious blepharitis in patientsattending at Hospital Foundation Nuestra Senora de la Luz wasperformed an analysis looking association between infectiousblepharitis and 5'UTR DEFB1 gene polymorphism.Methods: One hundred patients with infectious blepharitis, and thirtyone not infected subjects, as controls, were studied. We obtainedsamples from scraping palpebral for DNA extraction. The 5'UTRregion of HBD1 gene was amplified by PCR, and then, the PCRproducts were purified and analyzed the presence of SNPs -52G/Aand -44C/G by direct sequencing.Results: The allelic frequency of -44C was 60% and -52G was 50%of the 5'UTR region of HBD1 gene in patients, and in controls 50%and 60%, respectively. Our results not showed significantly©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologydifferences between patients and control subjects (Chi-square 2.02,p=0.077; Chi-square 2.02, p=0.155 respectively).Conclusions: In this study of Mexican population on infectiousblepharitis and polymorphism of the gene 5'UTR DEFB1 noassociation was found, however, it is necessary to conduct severalstudies to find the risk factors for developing infectious blepharitiswhich might be involved systemic diseases affecting the immunemechanisms.Commercial Relationships: Hector J. Perez-Cano, None; IngridV. Gonzalez Leon, None; Oscar Fernandez-Vizcaya, None;Justine Nolasco-Lopez, None; Jean R. Clemenceau, None; AtzinRobles-Contreras, NoneProgram Number: 1577 Poster Board Number: D0004Presentation Time: 8:30 AM - 10:15 AMComparative Analysis of Donor Medical History and DiseaseAttributesKieron Torres, Verity F. Oliver, Raymond Enke, Jin Song, ShannathL. Merbs. Ophthalmology, Johns Hopkins Wilmer Eye Institute,Baltimore, MD.Purpose: Age-related macular degeneration (AMD) and primaryopen angle glaucoma (POAG) are among the leading causes ofblindness worldwide. We aim to identify DNA methylation patternsassociated with AMD and POAG through a genome-wide analysis ofaffected cells being collected from control or diseased human eyes.This purpose of this study was to determine the accuracy of the tissuebank diagnosis by photographic and histologic methods.Methods: Adult human eyes were obtained from NDRI and theMaryland Anatomy Board. Donor eyes were labeled as either control(no history of retinal disease, POAG or AMD), POAG or AMDbased on what was listed under diagnoses in the provided donormedical history. POAG or AMD eyes that had additional informationincluding duration of disease, type of AMD, or treatment details wereconsidered as confirmed, and eyes without as unconfirmed. Diagnosisof POAG was verified by grading optic nerve cross-sections todetermine the presence of axonal loss. AMD was verified by gradingretinal images taken during eye dissection for posterior funduspathology.Results: 24 control, 13 POAG and 14 AMD donors were identified.23 of 24 controls had normal fundus photos and no axonal loss in atleast 1 eye. The majority of the POAG (11) and AMD (10) donorswere confirmed by additional history from NDRI. 6 of 11 confirmedPOAG individuals were verified by axonal loss. 6 of 10 confirmedAMD donors were verified by photo grading, and of these, 5 had atleast one eye without axonal loss. Additional confirmation of AMDby histologic section is ongoing.Conclusions: To facilitate short preservation times, tissue banksoften do not have access to comprehensive donor medical histories.Even donor eyes with an accurate ocular history may not havedisease-associated pathology if obtained within early stages of thedisease or if treatment had slowed progression. Additionally, controleyes may have undiagnosed or unrecorded POAG or AMD, makingthem inappropriate as controls. Our study shows that diagnoses needto be verified by histologic and photographic methods.Commercial Relationships: Kieron Torres, None; Verity F.Oliver, None; Raymond Enke, None; Jin Song, None; Shannath L.Merbs, NoneSupport: NEI R01EY020406Program Number: 1578 Poster Board Number: D0005Presentation Time: 8:30 AM - 10:15 AMTreatment of Usher syndrome with antisense oligonucleotidesJennifer J. Lentz 1 , Mette Flaat 1 , Francine M. Jodelka 2 , AnthonyHinrich 2 , Yongdong Zhou 1 , Kate McCaffery 2 , Dominik M. Duelli 3 ,Nicolas G. Bazan 1 , Frank Rigo 4 , Michelle L. Hastings 2 .1 Neuroscience Center, LSUHSC, New Orleans, LA; 2 Cell Biologyand Anatomy, Rosalind Franklin University, North Chicago, IL;3 Cellular and Molecular Pharmacology, Rosalind FranklinUniversity, North Chicago, IL; 4 Isis Pharmaceuticals, Carlsbad, CA.Purpose: Usher syndrome (Usher) is the leading genetic cause ofcombined deafness and blindness. Type 1 Usher (Usher 1) ischaracterized by profound hearing impairment and vestibulardysfunction at birth, and the development of retinitis pigmentosa(RP) in early adolescence. The 216G>A (216A) mutation in USH1Ccreates a cryptic splice site that is used preferentially over the correctsite and results in a truncated harmonin protein. We created a mousemodel for Usher 1C by knocking in the 216A mutation. 216AA miceexhibit circling behavior indicative of severe vestibular dysfunctionand deafness, have retinal dysfunction by 1 month of age and beginto lose photoreceptors after 6 months of age. This mouse modelprovides an opportunity to test the feasibility of correcting thedisease-associated genetic defect using antisense oligonucleotides(ASOs).Methods: Antisense oligonucleotides (ASOs) were used to targetUsh1c mutations in vitro and in vivo by systemic or local injection.Correction of splicing was evaluated by RT-PCR and western blot.Immunofluorescence was used to analyze Harmonin expression andASO localization. Hearing and vestibular function were evaluated byauditory-evoked brain stem response (ABR) and open-field chamberanalysis, respectively. Visual function was evaluated byelectroretinogram (ERG) analyses and optical coherence tomography(OCT) imaging was used to examine ocular structures.Results: ASOs effectively corrected splicing of 216A RNA in anUsher patient cell line, and in the cochleae and retinas of 216AAmice when delivered systemically or locally. Cell-free and cellularassays also demonstrated that ASOs targeted to the Ush1c.238dupCmutation result in in-frame skipping of exon 3. Treatment with 216AtargetedASOs to neonate mice corrected protein expression with animprovement in harmonin localization in the hair cells andphotoreceptors; rescued vestibular and hearing function, anddemonstrated a small improvement in visual function.Conclusions: Our results show that ASOs can effectively targetUsh1c mutations both in vitro and in vivo. These results suggest thetherapeutic potential of ASOs in Usher syndrome and other diseasesof the ear and eye.Commercial Relationships: Jennifer J. Lentz, None; Mette Flaat,None; Francine M. Jodelka, None; Anthony Hinrich, None;Yongdong Zhou, None; Kate McCaffery, None; Dominik M.Duelli, None; Nicolas G. Bazan, None; Frank Rigo, None;Michelle L. Hastings, Isis Pharmaceuticals (P)Support: NIH P30 GM103340, NIH/NEI EY005121, Research toPrevent BlindnessProgram Number: 1579 Poster Board Number: D0006Presentation Time: 8:30 AM - 10:15 AMGenetic Heterogeneity Among Patients with Pericentral RetinitisPigmentosaJason Comander 1, 2 , Aliete Langsdorf 1 , Shyana Harper 2 , CarolWeigel-DiFranco 2 , Mark B. Consugar 1 , Michael Sandberg 2 , XiaowuGai 3 , Joseph A. White 1 , Eliot L. Berson 2 , Eric A. Pierce 1, 2 . 1 OcularGenomics Institute, Massachusetts Eye and Ear Infirmary, HarvardMedical School, Boston, MA; 2 Berman-Gund Laboratory for theStudy of Retinal Degenerations, Massachusetts Eye and EarInfirmary, Harvard Medical School, Boston, MA; 3 Center forBiomedical Informatics, Department of Molecular Pharmacology and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
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