Genome-wide identification and characterization of well-defined genes involved in glaucoma and pterygium corneae Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Gabriela Chavarría Soley aus San José Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 27.03.2008 Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Georg Fey Zweitberichterstatter: Prof. Dr. Andreas Winterpacht ii Gedruckt mit Unterstützung des Deuschen Akademischen Austauschdienstes iii iv Index 1 Introduction....................................................................................................................... 1 1.1 Glaucoma, general aspects .................................................................................................. 1 1.1.1 Classification of glaucoma .............................................................................................................. 2 1.2 Genetics of POAG................................................................................................................3 1.2.1 Inheritance and implicated loci ...................................................................................................... 3 1.2.2 Known glaucoma genes .................................................................................................................. 6 1.2.2.1 Myocilin (MYOC) ................................................................................................................. 6 1.2.2.2 OPTN.................................................................................................................................... 7 1.2.2.3 WDR36 ................................................................................................................................. 8 1.3 Primary congenital glaucoma (PCG) ................................................................................. 8 1.3.1 CYP1B1 gene and protein............................................................................................................... 9 1.4 Pterygium corneae ............................................................................................................. 11 1.4.1 Genetics of Pterygium corneae...................................................................................................... 12 1.4.2 Factors implicated in the pathogenesis of pterygium corneae....................................................... 12 2 Methods............................................................................................................................ 14 2.1 Patients................................................................................................................................ 14 2.2 DNA standard methods ..................................................................................................... 14 2.2.1 DNA isolation ............................................................................................................................... 14 2.2.1.1 Salting out procedure for DNA extraction...................................................................... 14 2.2.1.2 Automated DNA isolation................................................................................................. 15 2.2.2 Agarose gel electrophoresis .......................................................................................................... 15 2.2.3 Gel extraction of PCR products..................................................................................................... 15 2.2.4 Quantification of dsDNA .............................................................................................................. 15 2.3 RNA standard methods ..................................................................................................... 15 2.3.1 RNA isolation................................................................................................................................ 15 2.3.2 Quantification of RNA with absorbance at 260nm ....................................................................... 16 2.3.3 Evaluation of RNA quality............................................................................................................ 16 2.4 PCR (polymerase chain reaction), microsatellite analysis, and sequencing ................. 16 2.4.1 Polymerase chain reaction (PCR).................................................................................................. 16 2.4.2 Microsatellite Analysis.................................................................................................................. 16 2.4.3 Purification of PCR products ........................................................................................................ 17 2.4.3.1 Enzymatic purification of PCR products ........................................................................ 17 2.4.3.2 Purification of PCR-products magnetic beads.............................................................. 17 2.4.4 Sequencing of purified PCR products with the Sanger method .................................................... 17 2.4.5 Purification of sequencing products with magnetic beads ............................................................ 18 2.5 Plasmid procedures............................................................................................................ 18 2.5.1 Site-directed mutagenesis.............................................................................................................. 18 2.5.2 Midi Plasmid-DNA-Preparation....................................................................................................19 2.6 Yeast methods..................................................................................................................... 19 2.6.1 Yeast Stocks.................................................................................................................................. 19 2.6.2 Competent yeast cells.................................................................................................................... 19 2.6.3 Yeast transformation ..................................................................................................................... 19 2.6.4 Induction of expression and microsome isolation ......................................................................... 20 2.6.5 Microsome Isolation...................................................................................................................... 20 2.6.6 Determination of enzymatic activity .............................................................................................20 2.7 Standard protein methods................................................................................................. 20 2.7.1 Determination of total protein concentration................................................................................. 20 2.7.2 Western Blot.................................................................................................................................. 20 2.8 Bioinformatics tools ........................................................................................................... 21 2.8.1 PCR primer design ........................................................................................................................ 21 2.8.2 Sequencing analysis ...................................................................................................................... 21 v 2.8.3 Microsatellite Analysis.................................................................................................................. 21 2.8.4 Genome Browsers ......................................................................................................................... 21 2.8.5 Single nucleotide polymorphisms (SNPs) databases..................................................................... 21 2.8.6 Linkage disequilibrium visualization ............................................................................................22 2.8.7 Haplotype reconstruction .............................................................................................................. 22 2.8.8 Multiple sequence alignment......................................................................................................... 22 2.8.9 Expression data analysis................................................................................................................ 22 2.8.10 Gene Ontology ......................................................................................................................... 22 2.9 Nomenclature ..................................................................................................................... 22 2.10 Reagents and Materials ..................................................................................................... 23 2.10.1 Kits ........................................................................................................................................... 23 2.10.2 Instruments ............................................................................................................................... 23 2.10.3 Enzymes ................................................................................................................................... 23 2.10.4 Plattes and other consumables.................................................................................................