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Title: Expert Network to study of the genetic origin and molecular pathogenesis of corneal dystrophies Integrated international research to study molecular pathogenesis of corneal dystrophies and its genetic origin ACRONYM: CORNEAGEN Quality of Life ID= 1155582 UNAME= NJ1155582N1 PASSWORD= NJ1155582N1 Memorandum of the 1st meeting in Budapest January 12, 2002 Participating Organizations: CS 01 University of Debrecen, Faculty of Medicine Department of Ophthalmology, Hungary Short name: UDFMDO Scientific coordination Prof. Andras Berta, M.D. Dept. of Ophthalmol., Medical and Health Sciences Center, University of Debrecen, Nagyerdei krt. 98., PO box: 30. H-4012 DEBRECEN, HUNGARY Tel.: +36 30 229 5889 Tel./Fax.: +36 52 415 816 Berta, Andra [email protected] Modis, Laszlo [email protected] User Name is : CORNEAGEN2 Password is : shu407 CO 02 VARIMED Ltd., Hungary (manager) Short name: VARIMED Project and database management Vari, Sandor [email protected] VARIMED Ltd. Budapest, Szalanci u 5, H-1124 Telephone: +36-1-487-0429 Fax: +36-1-487-0430 AC03 VITAMIB SARL. France Short name: VITAMIB eForms, electronic database Xavier Fabre [email protected] User Name is : CORNEAGEN10 Password is : dze530 CR04 University of Erlangen, Department of Ophthalmology, Germany Short name: UEDO Naumann G.O.H., MD Professor and Chairman, Department of Ophthalmology Friedrich-Alexander-Universitat, Erlangen-Nürnberg, Schwabachanlage 6 (Kopfklinikum) D-91054 ERLANGEN/GERMANY Tel. (09131) 8534477 FAX (09131) 8536435 [email protected] Dr. Berthold Seitz Fax (49) 9131 853 4436 [email protected] User Name is : CORNEAGEN4 Password is : mnae459 CR05 Community Hospital Århus, Department of Ophthalmology, Denmark Short name: COHADOP Professor Dr. Niels Ehlers Community Hospital Århus Norrebrogade 44 DK-8000 Arhus C Phone: (45) 8949 3237 Fax: (45) 8949 3230 [email protected] User Name is : CORNEAGEN5 Password is : khe378 CR06 University of Helsinki , Central Hospital, Finland Short Name: UHCH Genome typing Haartmaninkatu 4C, FIN-00029, HYKS, Finland. Tervo, Timo, [email protected] Juha Holopainen [email protected] Tiina Alitalo [email protected] User Name is : CORNEAGEN7 Password is : plo259 CR07 Hadassah University Hospital, Department of Ophthalmology, Israel Short name: HUHDO Professor Dr. J. Frucht-Pery Hadassah University Hospital, Department of Ophthalmology, Israel P.O. Box 12000, 91120 Jerusalem, Israel, Fax: (972) 2 6428896 [email protected] User Name is : CORNEAGEN8 Password is : hmo2001 CR08 Lublin University School Of Medicine (Medical Academy), Ophthalmology & 1st Eye Hospital Short name: LUSMOEH Professor Zbigniew Zagórski, M.D., Professor and Chairman Tadeusz Krwawicz Chair of Ophthalmology & 1st Eye Hospital Lublin University School Of Medicine (Medical Academy) ul. Chmielna 1, 20-079 Lublin, Poland, Tel./Fax: (48) 81-5324827 [email protected] User Name is: CORNEAGEN11 Password is: shou434 CR09 Slovak Postgradual Academy of Medicine, Ophthalmic Department Short name: SPAOP Prof Dr. Andrej Cernak Ophthalmic Department Slovak Postgradual Academy of Medicine St. Cyril and Method Hospital Antolská 11, 851 07 Bratislava, Slovakia Tel: (421) 76867 2039 Fax: (421) 76381 2145 [email protected] User Name is : CORNEAGEN12 Password is : shou721 CR10 Charles University, Department of Ophthalmology, Lexum Laser Excimer Centrum Short name: CUDO-LEXUM Doc. MUDr. Martin Filipec CSc. Lexum Laser Excimer Centrum Valčikova 1950/3 Praha 8 Liben Tel/Fax: (420) 026884240 Czech Republic [email protected] User Name is : CORNEAGEN13 Password is : kle232 CR10 Cedars-Sinai Medical Center, Ophthalmology Research Department of Surgery Existing NIH Grant is the matching activities and fund Short name: CSMCORD Burns & Allen Research Institute 8700 Beverly Blvd. Los Angeles, CA 90048-750 Kenney, Cristina [email protected] Nesburn, Antony [email protected] Cc: Daniel Oshiro [email protected] User Name is : CORNEAGEN9 Password is : bli165 List of the representatives attended of the meeting: Prof. Andras Berta, MD Dr. Laszlo Modis, MD Dr. Tiina Alitalo, PhD Dr. Sandor G. Vari, MD Agenda; FORMATION OF CONSORTIUM 1. Participants of the meeting agreed to form the CORNEAGEN Consortium, simce the Scientific Coordinator of the project received confirmation of interest from the partners listed above. 2. Partners agreed to make a registration on the CORDIS site as a potential EU project (see summary for CORDIS) 3. Partners agreed to cancel the submission of a proposal on February 8, 2002 QoL 14. Support for research infrastructures. 4. Partners agreed to focus on the EU Sixth Framework Expert Networks and organize such a network until November 2002 5. Next meeting will be organized in April or May, 2002 to finalize the WPs, the role and contribution of the partners. Summary for Cordis WEB: The scientific co-ordinator of the project began to investigate the molecular pathogenesis of type I lattice (Haab-Dimmer) dystrophy, which is one of the 5q31 related corneal dystrophies in 1995. The purpose of their former studies was the identification of the amyloid precursor in LCDI and its biochemical characterization. They also carried out immunohistochemical investigations in scarring and keratoconus corneas to better understand the role of mutations in the cornea. In their biochemical investigations, they showed a special protein in the LCDI corneas, the N-terminal sequence of which showed 100% identity with the mutated protein. They also showed the presence of mutated protein in the corneal deposits by immunohistochemistry. In scarring corneas, they found increased beta IG-H3 (kerato-epithelin) level at the sites of scarring. In keratoconus corneas the immunohistochemically detectable beta IG-H3 amount was less than normal, however, it increased when scarring was also present in the specimens. Partners plan to bring together a collaborative group or called thematic network of investigators to further study the molecular genetic basis, immunohistological and biochemical characteristics of 5q31 related (and possibly other) corneal dystrophies. 1. Investigation of the DNA mutations occurring in the subjected region 2. Investigation of the effect of these mutations on the turnover of beta IG-H3 (keratoepithelin) protein and the development of the protein deposits. 3. Definition of potential treatment modalities. Possible inhibition of enzymes involved into the pathological degradation products. Possible replenishment of missing enzymes for dissolution of pathological deposits. Key words: 0150 Databases, Database Management, Data Mining 0152 Decision Support Tools 0217 European Integration 0263 Genetics 0264 Genomes, Genomics Project Goals: The scientific co-ordinator of the project in 1995 began to investigate the molecular pathogenesis of type I lattice (Haab-Dimmer) dystrophy, which is one of the BIGH3 (5q31) related corneal dystrophies. The purpose of their former studies was the identification of the amyloid precursor in LCDI and its biochemical characterization. They also carried out immunohistochemical investigations in scarring and keratoconus corneas to better understand the role of BIGH3 (kerato-epithelin) protein in the cornea. In the biochemical investigations, they showed a 42 kD protein in the LCDI corneas, the Nterminal sequence of which showed 100% identity with BIGH3. They also showed the presence of BIGH3 protein in the corneal deposits by immunohistochemistry. In scarring corneas, they found increased BIGH3 level at the sites of scarring. The immunohistochemically detectable BIGH3 amount was less than normal in keratoconus corneas, however, it increased when scarring was also present in the specimens. In the future, they would like to continue their investigations in two main directions: 1. Investigation of the DNA mutations occurring in Central Europe 3. Investigation of the effect of these mutations on the turnover of BIGH3 protein and the development of amyloid mutations. Ad 1.: At present, at least eighteen different mutations are known in the BIGH3 gene, and lattice type IIIA and IV dystrophies were shown to be BIGH3 related dystrophies as well. The mutations occurring in Central-Eastern Europe have not been studied yet. First, we would like to analyse the mutations occurring in Hungary, which are most probably similar to those occurring in other parts of Europe. We began to Collect DNA samples from the dystrophic patients and their family members. We plan to analyse these samples by PCR amplification, followed by direct automated DNA sequencing. After finding the specific mutations, simpler tests, such as PCR-RFLP or PCR-SSCP may be used. Ad 2.: Investigation of the changes in protein metabolism caused by the mutations is a much more complicated task. The corneal button excised during keratoplasty is too small for extensive biochemical studies and does not allow in vivo investigations. To overcome this problem, we plan to establish immortalized epithelial cell lines from dystrophic and normal corneas. The immortalization is done with viral or plasmid vectors, containing SV40 viral sequences. The establishment of such immortalized cell lines is going on at present in our laboratory. We expect to establish immortalized epithelial cell lines from both normal and dystrophic corneas in one or two years. These immortalized cell lines seem to be suitable model systems for the investigation of the pathogenesis of these dystrophies. They will be helpful in determining the proteolytic steps involved in the metabolism of BIGH3 and identifying the site and the enzymes of proteolysis. On the long term, these studies may help the development of drugs being able to inhibit the accumulation of abnormal protein end-products. Partners plan to bring together a collaborative group called “thematic network” of investigators to further study the molecular genetic basis, immunohistological and biochemical characteristics of corneal dystrophies, with special emphasis on the beta IGH3 (kerato-epithelin) related dystrophies and keratoconus. This project description may be fitted to the study of other corneal dystrophies (macular, Meesmann, etc.) as well, if other partners have more intention or experience in the investigation of these diseases Workplan: WP01 Project Coordination and Management Project Management: The participating partners agreed that the time until February 08, 2002 is too short, the Consortium could not meet the challenges of the “Quality of Life and Management of Living Resources Programme, Support for Research Infrastructures”. Partners will go for the next meeting if we have positive answers from most of the partners no later then February 15, 2002, and based on the information provided by the partners of the Consortium we are planning the next meeting in April or May 2002. WP02 Sample collection Blood all patients diagnosed with dystrophy and available family members Do you have in-house or institutional collaborators to extract DNA from these samples? . Workplan: The investigation of DNA mutations in the 5q31 chromosome region Aim: to better understand the role of BIGH-3 in the following corneal dystrophies: 1. 2. 3. 4. 5. lattice types I, III, IIIa and IV Groenouw I Avellino Reis-Bucklers since some earlier data showed that beta IG-H3 and related proteins may be involved in the pathogenesis of keratoconus we plan to further investigate the relationship of keratoconus and beta IG-H3. Cornea all patients who went through keratoplasty The samples should be cut into two parts: one for tissue culture one for frozen section histopathology (Deep frozen –70C to section for immunohistochemical staining) and/or biochemical investigations Mutation detection: Automated DNA sequencing for the patient. Can you have the DNA extracted from the blood sample sequenced with an automated DNA sequencer? Simplified tests, such as PCR-RFLP or PCR-SSCP for mapping the family (Benefit to investigate penetrance and expressivety of gene.) Are these simple tests available to study of family members in your institution? Tissue culture of dystrophic corneal cells: Can you establish immortalized epithelial cell lines from dystrophic and normal corneas? A. Phenotype characterization by complex biochemical methods: Identification of enzymes involved in the degradation process and detection of defects in the process B. Biochemical analyses of the pathological deposits (amyloid, hyalin ect.): Identification of abnormal molecular products using protein sequencing and linking the abnormality to the morphological and functional defect Development of a European electronic database for genotypes and phenotypes from eForm for mutation (HTML and XML) eForm for clinical and histological data Correlation analysis of European Database (genotypes and phenotypes): Potential treatment modalities: Possible inhibition of enzymes involved in the pathological degradation products. Possible replenishment of missing enzymes for dissolution of pathological deposits.