Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMYO7A mutations, was high-throughput sequenced for targetedexons of genes associated with inherited retinal degenerations.Genetically unsolved samples were subsequently screened fordeletions using a custom design comparative genomic hybridization(CGH) array. When possible, the likely pathogenic variants wereconfirmed by co-segregation in available family members withSanger sequencing.Results: Selective exon capture and Illumina sequencing providedexcellent coverage of the targeted exons, with >95% of exons having>10X sequence depth. With this sequencing approach, we were ableto solve 35% of 49 cases, where mutations in MYO7A were the mostfrequent (16%), followed by CDH23 (10%) and USH1C (6%). 37%of the samples contained one heterozygous likely pathogenic changein a USH1 gene. The remaining, 28% did not carry mutations in anyknown USH1 gene. CGH array analyses for unsolved cases is inprogress and will be reported.Conclusions: Targeted exon sequencing provides an effectiveapproach for genetic diagnostic testing of patients with Ushersyndrome. The results of this study show that there is a considerablenumber of USH1 patients with an unknown genetic cause of thedisease, indicating additional genetic loci for this condition.Acknowledgements:This work was supported by grants from the National Eye Institute(EY012910) and the Foundation Fighting Blindness.Commercial Relationships: Kinga M. Bujakowska, None; EmilyPlace, None; Mark B. Consugar, Agilent Technologies, Inc. (R);Daniel G. Taub, None; Aliete Langsdorf, None; Carol Weigel-DiFranco, None; Shyana Harper, None; Xiaowu Gai, None; EliotL. Berson, None; Eric A. Pierce, NoneSupport: National Eye Institute (EY012910); Foundation FightingBlindnessProgram Number: 1582 Poster Board Number: D0009Presentation Time: 8:30 AM - 10:15 AMEpigenetic factors in the pathogenesis of corneal dystrophyXiaohua Li, Xiaohua Li, Min Yuan, Ruijie Yin. Henan Eye Institute,Henan Provincial Eye Hospital, Zhengzhou, China.Purpose: Tumor suppressor gene Hypermethylated In Cancer 1(HIC1) regulates the expression of SIRT1 and in turn SIRT1deacetylates of P53 (AC-p53) leading to the response to DNA repair,stress and senesces, therefore, a HIC1, SIRT 1 and P53 loop havebeen implied in the pathogenesis of cancer, ageing and degenerativediseases. The current research sought to investigate the roles of HIC1,SIRT1 and P53 in the pathogenesis of corneal dystrophy and itsassociation with CTGF and TGF-beta expression.Methods: Formalin-fixed paraffin embedded cornea section from 25patients (ages 5-68; 17 male, 8 female) with corneal dystrophy wereprepared for the study.Five corneal specimens from normal adultwere included as control. H&E, PAS, Congo red, Masson andcolloidal iron staining were performed to confirm the diagnosis ofcorneal dystrophy. Sections were analyzed for the expressions ofHIC1, SIRT1, acetylated -P53, CTGF, TGF-beta, and α-SMA byimmunohistochemistry. The red chromagen color was developedusing amino ethyl carbazole. The Slides were examined using a digitmicroscope.Results: 14 cases were diagnosed with macular corneal dystrophyand 11 cases were diagnosed with lattice corneal dystrophy accordingto H&E and histochemical staining. In cases of macular dystrophy,the deposits were positive with colloidal iron staining, while in casesof lattice dystrophy, the deposits stained positively with Massontrichrome, PAS and Congo red staining. In corneal specimen bothmacular corneal and lattice corneal dystrophy, HIC1 was highlyexpressed compared with control especially in corneal epithelial cell .The expression of SIRT1 was much weaker compared with the HIC1and normal corneal specimens staining. Notably, Acetylated P53immnunoreactivity is abundant in the degenerated areas of thecornea. In addition, the increased expression of fibrotic inducer suchas CTGF and TGF-beta were seen in the corneal fibrosis lesion,striking, the expression of CTGF was much stronger than TGF-beta.α-SMA was also unregulated in the area where CTGF was detected.Conclusions: The high HIC1 expression is associated with increasedexpression of Ac-p53 and CTGF and the down regulation of SIRT1in the corneal tissue; the distinct expression of the epigenetic factorsHIC1, SIRT1, AC-p53 and their interaction with CTGF may play anessential role in pathogenesis of the corneal dystrophy.Commercial Relationships: Xiaohua Li, None; Xiaohua Li, None;Min Yuan, None; Ruijie Yin, NoneSupport: Grants 81100650, National Natural Science Foundation ofChinaProgram Number: 1583 Poster Board Number: D0010Presentation Time: 8:30 AM - 10:15 AMBiomarkers for Neuronal Injury following Blast Trauma to theEyeSteven G. Hart 1 , XiangDi Wang 1 , Tonia S. Rex 2 , Eldon E. Geisert 1 .1 Ophthalmology, University of Tennessee Health Science Center,Memphis, TN; 2 Ophthalmology, Vanderbilt University, Nashville,TN.Purpose: The pathogenic pathways triggered by blast injury to theeye and biomarkers that reflect the activation of these pathways arelargely unknown. The present study is the initial attempt to definepotential biomarkers that reflect the severity of the retinal injury.Methods: Blast injuries to the eye were produced by a paintball gunfitted with a shortened and narrowed barrel and an integrated pressureregulator. The mice were deeply anesthetized and secured in a PVCpipe. A 45-psi overpressure wave was delivered selectively to the eyeof C57BL/6 mice and DBA/2J mice. The animals were thensacrificed at 2 or 5 days after the blast injury. To aid in our initialsurvey of potential biomarkers of retinal injury, we examined ourOptic Nerve Crush Microarray Dataset and compared it to ourNormal Retinal Microarray Dataset in GeneNetwork.org. This workled to six potential biomarkers for blast injury, Gfap, Iba1, C1q,Aqp4, Sox11 and Hsp25. One set of retinas were removed and stainedby indirect immunohistochemical methods to assess the distributionand intensity of the staining compared to uninjured control retinas.For a second set of retinas, the animals were anesthetized; the retinaswere removed and placed in sample buffer. The level of proteinexpression was determined by semi-quantitative immunoblotmethods.Results: Immunostaining sections of retina revealed that two of themarkers, SOX11 and HSP25, were upregulated in the neurons of theinner retina following blast. Both SOX11 and HSP25 labeled cells inthe ganglion cell layer and the inner nuclear layer. In the ganglioncell layer SOX11 labeled approximately 90% of the cells, indicatingthat it was labeling most ganglion cells and displaced amacrine cells.Furthermore, amacrine cells in the inner nuclear layer were labeledby SOX11. The intensity of staining increased dramatically after blastinjury and on immunoblots there was approximately a 2-fold increasein the intensity of the SOX11 band. A similar pattern of staining wasobserved with HSP25. The increased staining after blast injury didnot appear to be as dramatic as it was for SOX11. On immunoblots,there was also an observed increase in the intensity of the HSP25band following injury.Conclusions: SOX11 and HSP25 are markers for blast injury to theretina, labeling both retinal ganglion cells and amacrine cells. Thebetter of the two markers appears to be SOX11.©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.