Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyConclusions: In a Turkish subpopulation, the LOXL1 genepolymorphism of PES and PEG were different from healtyindividuals, and genetic variation was similar to European andAmerican populations.Commercial Relationships: Nilgun Yildirim, None; Yetkin Yaz,None; Oguz Cilingir, None; Fez an Sahin, None; Zafer Yüksel,NoneSupport: Eskisehir Osmangaz UnversityProgram Number: 1597 Poster Board Number: D0024Presentation Time: 8:30 AM - 10:15 AMElevated TGFβ1 concentration in the plasma of glaucomapatientsJohn Kuchtey, L. Goodwin Burgess, Megan B. Parks, Jessica Kunkel,Milam A. Brantley, Rachel W. Kuchtey. Vanderbilt Eye Institute,Vanderbilt University, Nashville, TN.Purpose: Previously, we formed the hypothesis that microfibrildefects may contribute to glaucoma pathogenesis, based on ouridentification of the microfibril-associated gene, ADAMTS10, as thedisease gene in a colony of dogs with inherited primary open angleglaucoma (POAG). Since microfibrils are a major reservoir forTGFβ, and because other diseases caused by defective microfibrilsare associated with elevated plasma TGFβ, a corollary to themicrofibril hypothesis is that glaucoma patients may have a systemicelevation of TGFβ. The purpose of this study was to test thehypothesis that patients with POAG have elevated plasmaconcentrations of TGFβ.Methods: Blood samples from POAG patients and controls werecollected in lithium heparin tubes, centrifuged at 4 o C for 10 minutesat 1,500 x g and plasma aliquoted and stored at -80 o C.Concentrations of total TGFβ1 and total TGFβ2 were determined byELISA assay of acid-activated plasma samples from POAG patientsand controls matched for age, sex and ancestry.Results: In an initial set of 55 POAG patients and 55 controls, totalTGFβ1 was higher in the plasma of POAG patients compared tocontrols (median concentrations 3.12 vs. 2.54 ng/ml, respectively,p2SD above the mean of controls.Plasma total TGFβ2 concentrations were below limits of detection.Conclusions: Total TGFβ1 is higher in the plasma of POAG patientsas compared to controls, with some patients having pronouncedelevation. This finding is consistent with our hypothesis thatdefective microfibrils contribute to glaucoma pathogenesis andsuggest a mechanism to explain elevated TGFβ in the aqueous humorof glaucoma patients. Elevated plasma TGFβ1 may be used as abiomarker for glaucoma associated with defective microfibrils.Commercial Relationships: John Kuchtey, None; L. GoodwinBurgess, None; Megan B. Parks, None; Jessica Kunkel, None;Milam A. Brantley, None; Rachel W. Kuchtey, NoneSupport: NIH Grant EY020894Program Number: 1598 Poster Board Number: D0025Presentation Time: 8:30 AM - 10:15 AMAssociation of the T allele of rs41435250 of LOXL1 with elevatedrisk of exfoliation syndrome and exfoliation glaucoma in aMexican populationDalia C. Guadarrama 1 , Juan C. Zenteno 1, 2 , Antonio Miranda 3 , CeliaElizondo 1 . 1 GENETICS, INSTITUTE OF OPHTHALMOLOGYCONDE DE VALENCIANA, MEXICO, Mexico;2 BIOCHEMISTRY FACULTAD DE MEDICINA, NATIONALAUTONOMOUS UNIVERSITY OF MEXICO (UNAM), MEXICO,Mexico; 3 GENETICS, NATIONAL REHABILITATIONINSTITUTE, MEXICO, Mexico.Purpose: Pseudoexfoliation syndrome (XFS) is a major risk factorfor developing pseudoexfoliation glaucoma (XFG). The disease hasbeen associated with 3 high risk SNPs (rs3825942, rs1048661 andrs2165241) in the lysil oxidasa-like1 (LOXL1) gene. Particularly, theG allele of rs3825942 is considered the “universal” risk allele as it isconsistently associated to XFS/XFG in distinct ethnic groups.Previous studies in Mexican population showed that the T allele ofrs2165241 SNP confers a greater risk for XFS/XFG than the G alleleof rs3825942 in that population. The purpose of this study was toinvestigate the association of an additional LOXL1 SNP withXFS/XFG risk in Mexican patients.Methods: A total 102 subjects with PEX/PEG and 130 healthy adultcontrols were included. Genomic DNA was extracted fromleukocytes, and the LOXL1 exon 1 coding sequence of LOXL1 wasamplified by PCR and directly sequenced for genotyping the allelesof the rs41435250 SNP. STATA ver.10.0 statistical software packagewas utilized for calculations. Allele frequencies, HardyeWeinbergequilibrium (HWE) and haplotype association analyses were assessedwith Haplo View 4.0 software.Results: The mean age in the patient group was 75 years and of 72years in the control group. The rs41435250 TT genotype wasassociated with an elevated risk as it was observed in 3.92% ofpatients and in 1.54% controls (OR[95% CI]= 2.56 [0.44-14.58]; p=0.004500); accordingly, the GG genotype for this SNP had afrequency of 85.38% in controls and 70.59% in patients and wasassociated with a protective effect: OR[95% CI]= 0.43 [0.22-0.83];p= 0.013. Allele frequencies showed the same tendencies asmentioned for genotypes.Conclusions: Our results indicate that the T allele of rs41435250confers an elevated risk for the development of XFS/XFG in Mexicanpopulation.Commercial Relationships: Dalia C. Guadarrama, None; Juan C.Zenteno, None; Antonio Miranda, None; Celia Elizondo, NoneSupport: YesProgram Number: 1599 Poster Board Number: D0026Presentation Time: 8:30 AM - 10:15 AMRegulation of rhodopsin gene expression by DNA methylationJin Song, Tomohiro Masuda, Donald J. Zack, Shannath L. Merbs.Department of Ophthalmology, Johns Hopkins University, Baltimore,MD.Purpose: We have previously shown that Rhodopsin (RHO) has acell-specific DNA methylation pattern within the retina and ismethylated in non-expressing cells. We aimed to determine if DNAmethylation contributes directly to the suppression of RHOexpression.Methods: Cells expressing low levels of RHO (HEK 293, WERI andY79) were treated with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dc; 72 h treatment) to inducehypomethylation. Global DNA methylation was measured by LUMA(luminometric methylation assay). RHO methylation was measuredby bisulfite pyrosequencing, and the expression level determined byquantitative real-time PCR (qPCR). To explore whether the additionof transcription factors could further increase endogenous RHOexpression in 5-aza-dc-treated HEK 293 cells, a bovine Rho promoterreporter (Gluc luciferase) was co-transfected with CRX and NRLexpression vectors. Endogenous RHO methylation and expressionwas examined.Results: Treatment of HEK 293, WERI, and Y79 cells with 5-aza-dcinduced a drug-dependent global DNA hypomethylation in all threecell lines. At 17 CpG sites of RHO, DNA methylation was reduced in©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.