Systematic mutation analysis and functional characterization of candidate genes for primary open angle glaucoma [Elektronische Ressource] / vorgelegt von Lorena Fernández Martínez

Lfernandez

Sujets

• Gesundheit

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Aus dem Humangenetischen Institut
der
Friedrich-Alexander-Universität Erlangen-Nürnberg
Direktor: Prof. Dr. med. André Reis






Systematic mutation analysis and functional characterization of candidate genes
for primary open angle glaucoma








Inaugural-Dissertation
zur Erlangung der Doktorwürde
der Medizinischen Fakultät
der
Friedrich-Alexander-Universität
Erlangen-Nürnberg










vorgelegt von
Lorena Fernández Martínez
aus
Oviedo

Gedruckt mit Erlaubnis der
Medizinischen Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg














Dekan: Prof. Dr. J. Schüttler

Referent: Prof. Dr. A. Reis

Korreferenten: Prof. Dr. J. Brandstätter
Prof. Dr. A. Winterpacht

Tag der mündlichen Prüfung: 08. Dezember 2009

ii a mi padre,
a mi hermano,
im memoriam





























There is one thing that gives radiance to everything. It is the idea of finding
something around the corner.
- Gilbert Keith Chesterton


When you've got it, you've got it. When you haven't, you begin again. All the rest
is humbug.
-Edouard Manet


iii iv Index
 
1. Summary ......................................................................................................... 1  
2. Zusammenfassung .......................................................................................... 3  
3. Introduction.... 5  
3.1. Genetics of complex diseases.......................................................... 5  
3.1.1. Monogenic versus complex diseases........................................................................ 5  
3.1.2. Methods for genetic dissection of complex diseases............... 6  
3.1.2.1. Linkage analysis................................ 6  
3.1.2.2. Association studies............................................................ 6  
3.1.2.3. Candidate-gene approaches............................................... 7  
3.2. Glaucoma, general aspects .............................................................. 7  
3.2.1. Diagnostics............................................................................... 8  
3.2.2. Classification, prevalence and risk factors............................................................... 9  
3.2.3. Pathogenesis........... 11  
3.2.4. Treatment ............................................................................................................... 12  
3.2.4.1. Classical treatments......................... 12  
3.2.4.2. Investigational Glaucoma Treatments............................................................. 13  
3.2.5. Animal models ....................................................................... 13  
3.3. Genetics of POAG......................................... 14  
3.3.1. Inheritance and implicated loci.............. 15  
3.3.2. Known glaucoma genes ......................................................................................... 16  
3.3.2.1. Myocilin (MYOC)........................... 16  
3.3.2.2. Optineurin (OPTN)......................... 17  
3.3.2.3. WD40-Repeat 36 (WDR36)........................................................................... 18  
3.3.3. Glaucoma candidate genes..................... 19  
3.4. Loci investigated ........................................... 20  
3.4.1. ADCY4 (adenylate cyclase type IV)...................................................................... 21  
3.4.2. BCL2L2 (B-cell/lymphoma 2- like 2).... 22  
3.4.3. DAD1 (defender against cell death 1).... 22  
3.4.4. ISGF3G (interferon-stimulated transcription factor 3 gamma).............................. 22  
3.4.5. MMP14 (matrix metalloproteinase 14).................................................................. 23  
3.4.6. NRL (neural retina leucine zipper)......... 23  
3.4.7. OXA1L (oxidase cytochrome c assembly 1-like).................................................. 23  
v 3.4.8. SALL2 (salivary protein-like 2)............................................................................. 24  
3.4.9. ZNF219 (zinc finger protein 219).......... 24  
3.4.10. RPGRIP1 (retinitis pigmentosa GTPase regulator-interacting protein 1)............ 24  
3.5. The aims of this thesis................................................................................................... 25  
4. Materials and methods................. 26  
4.1. Subjects ......................................................................................................................... 26  
4.1.1. Patients................... 26  
4.1.1.1. Recruitment ..................................................................................................... 26  
4.1.1.2. Diagnosis......... 26  
4.1.2. Controls .................................................................................................................. 27  
4.2. DNA standard methods ................................................................................................. 27  
4.2.1. Genomic DNA isolation......................... 27  
4.2.1.1. Automated DNA isolation............................................................................... 27  
4.2.1.2. DNA isolation from COS-1 cells.... 27  
4.2.2. Quantification of dsDNA....................... 28  
4.3. PCR (polymerase chain reaction) and sequencing........................................................ 28  
4.3.1. Polymerase chain reaction (PCR) .......................................... 28  
4.3.2. Agarose gel electrophoresis................... 28  
4.3.3. Gel extraction of PCR products............................................. 29  
4.3.4. Purification of Pts ................................................. 29  
4.3.4.1. Purification of PCR-products with magnetic beads........ 29  
4.3.4.2. Purification of PCR-products with Millipore Cleanup Kit ............................. 29  
4.3.4.3. Purification of PCR-products with QIAquick PCR Purification Kit .............. 29  
4.3.4.4. Enzymatic purification of PCR-products ........................................................ 30  
4.3.5. Sequencing of purified PCR products with the Sanger method............................. 30  
4.3.6. Purification of sequencing products with magnetic beads..................................... 30  
4.3.7. RT-PCR (reverse transcription polymerase chain reaction).. 31  
4.3.8. Microsatellite analysis............................................................................................ 31  
4.4. Cloning and plasmid procedures in bacteria................................. 31  
4.4.1. Cloning of plasmids and PCR products in a cloning vector.. 31  
4.4.2. Miniprep plasmid preparation ................................................................................ 32  
4.4.3. Midiprep plasmid preparation 32  
4.4.4. Site-directed mutagenesis....................... 32  
4.4.5. Gateway cloning..................................................................................................... 33  
vi 4.5. Yeast two-hybrid experiments ...................................................................................... 34  
4.5.1. Yeast cotransformation.......................... 34  
4.5.1.1. Lithium acetate (LiAC)-mediated cotransformation of fresh growing cells... 34  
4.5.1.2.1. Preparation of frozen competent cells...................................................... 35  
4.5.1.2.2. Transformation of frozen competent cells............... 35  
4.5.2. X-α-Galactosidase assay ........................................................ 35  
4.5.3. β-Galactosidase assays........................................................... 35  
4.5.3.1. β-Galactosidase liquid assay ........................................................................... 36  
4.5.3.2. β-Galactosidase colony-lift filter assay........................... 36  
4.6. Assays in mammalian cells ................................................................ 36  
4.6.1. Culture conditions.................................. 36  
4.6.2. Stock preparation.................................................................... 37  
4.6.3. Cotransfection methods.......................... 37  
4.6.3.1. Nucleofection.................................. 37  
4.6.3.2. Cationic lipid transfection using Lipofectamine and PLUS reagents ............. 37  
4.6.4. Coimmunoprecipitation.......................................................................................... 37  
4.6.5. Immunofluorescence............