Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamutations in individuals without ZEB1 coding region mutations inposterior polymorphous corneal dystrophy (PPCD).Methods: Slit lamp examination and DNA collection was performedfor individuals diagnosed with PPCD, and when available, affectedand unaffected family members. Genomic DNA prepared fromperipheral blood leukocytes and buccal epithelial cells underwentPCR amplification and automated sequencing of the ZEB1 gene andpromoter region.Results: Ten unrelated individuals with PPCD were identified andgenomic DNA was collected from each. ZEB1 mutations wereidentified in 5 of the 10 probands, four of which were novel:p.Ala150fsX36 (spontaneous), p.Arg230fsX7, p.Cys638fsX5 andp.Gly1039fsX6. Screening of the ZEB1 promoter region in 28 PPCDprobands without a ZEB1 coding region mutation identified only twoknown SNPs whose frequency in the affected probands did not differsignificantly from that in the general population.Conclusions: We report four novel frame-shift mutations, oneconfirmed to be spontaneous, in the ZEB1 gene associated withPPCD, bringing the total number of pathogenic mutations to 23, andthe percentage of PPCD associated with ZEB1 mutations to 32%.The absence of ZEB1 promoter region mutations in probands withouta ZEB1 coding region mutation indicates that other genetic loci, suchas PPCD1, are responsible for the majority of cases of PPCD.Commercial Relationships: Pejman Bakhtiari, None; Ricardo F.Frausto, None; Ashley N. Roldan, None; Cynthia Wang, None;Anthony J. Aldave, Alcon (R), Allergan (R), NIH (F), Bausch +Lomb (C), Allergan (C)Program Number: 4733 Poster Board Number: C0116Presentation Time: 11:00 AM - 12:45 PMIdentification by whole-exome next-generation sequencing ofcoding region mutations as candidates for posterior amorphouscorneal dystrophyJonathan Han, Ricardo F. Frausto, Michelle J. Kim, Anthony J.Aldave. Doris Stein, Cornea Division, Jules Stein Eye Institute, LosAngeles, CA.Purpose: To identify the causative coding region variant of posterioramorphous corneal dystrophy (PACD) by whole-exome sequencingof the 12q21.33-q23.1 PACD locus.Methods: Slit-lamp examination was performed on 54 members of amultigenerational pedigree with PACD to determine their affectedstatus. Peripheral blood samples were collected from each familymember, and extracted genomic DNA from selected affected andunaffected individuals were used for whole-exome sequencing. Boththe alignment of the paired-end reads and the variant discovery wereconducted within the Partek Flow software. A list of candidatevariants comprising those identified within the PACD locus (boundedby the D12S1812 and D12S1051 genetic markers) was generatedusing the Partek Genomic Suite software. Twenty-eight variants werethen compared to previous Sanger sequencing results using aspreadsheet application to determine the number of false positivesand false negatives at two different coverage thresholds.Results: Fifteen individuals were diagnosed as affected based oncharacteristic clinical features. Whole-exome sequencing wasperformed on genomic DNA from 5 affected and 1 unaffectedindividuals. Analysis of the sequencing data revealed 12 singlenucleotide variants (SNV) within 9 genes that were present in each ofthe affected samples but not in the unaffected sample. Six of the 12SNVs were coding region variants. In addition, two indels wereidentified that were present only in the affected individuals: a 3’UTRinsertion and a coding region deletion. By comparing variantspreviously identified in the same 5 affected and 1 unaffectedindividuals using Sanger sequencing to those identified with wholeexomesequencing, 23/30 variants (23.3% false negative rate) wereidentified at a coverage threshold 10 and 27/30 variants (10.0% falsenegative rate) were identified at a coverage threshold of 5. No falsepositives occurred at either threshold.Conclusions: We report one of the initial applications of nextgenerationresequencing technology to resequence a candidate regionpreviously associated with a corneal dystrophy. The relatively lownumber of identified variants presents a reasonable cohort ofcandidate variants that will be confirmed by Sanger sequencing,followed by determination of segregation in the remaining 10affected and 38 unaffected individuals.Commercial Relationships: Jonathan Han, None; Ricardo F.Frausto, None; Michelle J. Kim, None; Anthony J. Aldave, Alcon(R), Allergan (R), NIH (F), Bausch + Lomb (C), Allergan (C)Support: RPBProgram Number: 4734 Poster Board Number: C0117Presentation Time: 11:00 AM - 12:45 PMLattice corneal dystrophy, type 1 (LCD1): an epithelial orstromal entity ?Walter Lisch 1 , Berthold Seitz 2 . 1 Ophthalmology, Johannes GutenbergUniversity Mainz, Mainz, Germany; 2 Ophthalmology, SaarlandUniversity, Homburg/Saar, Germany.Purpose: There are controversial reports that lattice cornealdystrophy, type 1 (LCD1) is of epithelial or stromal origin. The aimof this study is to evaluate this question.Methods: We observed ten eyes of five LCD1 patients afterpenetrating keratoplasty (PKP) on both eyes with a follow-up of 8-14years post-op. A slit-lamp photo-documentation of all patients indirect and indirect illumination was performed with dilated pupil.Results: All ten corneal transplants of the LCD1-patients showedsubepithelial diffuse opacities of different severity, beginning after 3-4 years postoperatively, that were often combined with cornealerosions and consecutive pain. In none of our patients, we were ableto disclose any signs of lattice formation in form of gray lines whichrun obliquely from the surface to the midstroma in directillumination. In retroillumination, no lattice opacity units in form oftranslucent and refractile lines were visible.Conclusions: The transforming growth factor beta-induced (TGFBI)gene, that is also responsible for LCD1, is expressed above all by thecorneal epithelial cells but also by the keratocytes. We interpret thesuperficial, diffuse LCD1 opacities on the graft as the product of theepithelial cells, whereas the non-occurrence of lattice lines as long as14 years postoperatively as an indirect sign that the lattice lines arethe product of the keratocytes. We know, that stromal cornealdystrophies such as macular corneal dystrophy may not recur beforedecades on the graft due to the very slow transformation of transplantkeratocytes into pathological host keratocytes. Thus, LCD1 seems torepresent an epithelial-stromal entity, because both, the epithelialcells and keratocytes are pathophysiologically involved.Commercial Relationships: Walter Lisch, None; Berthold Seitz,NoneProgram Number: 4735 Poster Board Number: C0118Presentation Time: 11:00 AM - 12:45 PMProteolytic processing in lattice corneal dystrophyEbbe Toftgaard Poulsen 1 , Kasper Runager 1 , Michael W. Risør 1 , IdaB. Thøgersen 1 , Thomas Dyrlund 1 , Line R. Thomsen 1 , Gordon K.Klintworth 2 , Jan J. Enghild 1 . 1 Department of Molecular Biology,Aarhus University, Aarhus C, Denmark; 2 Departments of Pathologyand Ophthalmology, Duke University, Durham, NC.Purpose: Transforming growth factor beta-induced protein(TGFBIp) is a major component of the human corneal proteome.©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. 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