Genetic heterogeneity and molecular genetic diagnostics in primary and secondary laminopathies [Elektronische Ressource] / vorgelegt von: Nguyen, Thuy Duong

Giang Son

Sujets

• Gesundheit

Informations

• Publié par: ernst-moritz-arndt-universitat_greifswald
• Publié le: 01 janvier 2008
• Nombre de lectures: 31
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Aus dem Funktionsbereich für Molekulare Gencharakterisierung
(Leiter Univ. - Prof. Dr. M. Wehnert)
Institut für Humangenetik
der Medizinischen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald





Thema:
Genetic heterogeneity and molecular genetic diagnostics
in primary and secondary laminopathies


Inaugural-Dissertation

zur

Erlangung des akademischen Grades

Doktor der Medizin

der

Medizinischen Fakultät

der

Ernst-Moritz-Arndt-Universität

Greifswald

2008

vorgelegt von:
Nguyen, Thuy Duong
geboren am 13. Mai 1978
in Hanoi, Vietnam











Dekan: Prof. Dr. rer. nat. H. K. Kroemer










1. Gutachter: Herr Prof. Dr. med. Manfred Wehnert, Greifswald
2. Gutachter:
3. Gutachter:










Tag der Promotion:


























Content
1. Introduction .......................................................................................................................... 1
2. Materials, methods and patients ....................................................................................... 14
2.1. Materials ........................................................................................................................ 14
2.1.1. Reagents ................................................................................................................ 14
2.1.2. Enzymes ................................................................................................................ 14
2.1.3. Extraction kits ....................................................................................................... 15
2.1.4. Solutions and media .............................................................................................. 15
2.1.4.1. Solutions ............................................................................................................. 15
2.1.4.2. Bacterial culture medium .................................................................................... 16
2.1.5. Vector .................................................................................................................... 16
2.1.6. Oligonucleotides (primers).................................................................................... 17
2.1.7. Equipment ............................................................................................................. 17
2.2. Methods ......................................................................................................................... 19
2.2.1. Determination of DNA content ............................................................................. 19
2.2.2. Agarose gel electrophoresis .................................................................................. 19
2.2.3. Mildly denaturing polyacrylamide gel electrophoresis (PAGE) ........................... 19
2.2.4. DNA extraction ..................................................................................................... 19
2.2.4.1. DNA preparation from blood .............................................................................. 19
2.2.4.2. DNA preparation from cultured cells ................................................................. 20
2.2.4.3. DNA extraction from paraffin blocks ................................................................. 20
2.2.5. Total RNA purification from cultured cells .......................................................... 21
2.2.6. Primer design for polymerase chain reaction ........................................................ 21
2.2.7. Polymerase chain reaction (PCR) ......................................................................... 22
2.2.7.1. Regular PCR ....................................................................................................... 22
2.2.7.1.1. Regular PCR from genomic DNA ........................................................... 22
2.2.7.1.2. Regular PCR from bacterial colonies ..................................................... 23
2.2.7.2. Nested PCR ......................................................................................................... 23
2.2.7.3. RT-PCR ............................................................................................................... 23
2.2.8. Heteroduplex analysis ........................................................................................... 24
2.2.9. Sequencing ............................................................................................................ 26
2.2.9.1. Sequencing procedure ......................................................................................... 26
2.2.9.2. Computer-aided sequence evaluation ................................................................. 27
2.2.9.3. Nomenclature of sequence variations ................................................................. 27
2.2.10. Restriction digestion ............................................................................................. 27
2.2.11. Cloning .................................................................................................................. 28
2.2.11.1. Cloning of genomic DNA ................................................................................. 28
2.2.11.1.1. Processing of PCR products ................................................................. 28
2.2.11.1.2. Preparation of LB-ampicillin plates ..................................................... 29
2.2.11.1.3. Transformation ..................................................................................... 29
2.2.11.2. Cloning of cDNA .............................................................................................. 30
2.2.12. Electronic databases used ...................................................................................... 30
2.3. Patients ........................................................................................................................... 31
3. Results ................................................................................................................................. 32
3.1. Primary laminopathies (LMNA mutations) .................................................................... 32
3.1.1. Emery-Dreifuss muscular dystrophy (EMD) ........................................................ 32
3.1.1.1. Mutational analysis in EDMD patients ............................................................... 32
3.1.1.2. Mutational analysis in EDMD families .............................................................. 35
3.1.2. Dilated cardiomyopathy with conduction disturbances (CMD1A) ...................... 46
3.1.3. Familial partial lipodystrophy (FPLD) and madibuloacral dysplasia (MAD) ...... 51
3.1.4. Progeroid syndromes ............................................................................................. 51
3.1.4.1. Mutational analysis in Hutchinson-Gilford progeria (HGPS) ............................ 51
3.1.4.2. Mutational analysis in a progeroid family .......................................................... 51
3.1.5. Restrictive dermopathy (RD) ................................................................................ 52
3.1.6. Hallermann-Streiff syndrome................................................................................ 52
3.2. Secondary laminopathies ............................................................................................... 54
3.2.1. Restrictive dermopathy (RD) ................................................................................ 54
3.2.1.1. Mutational analysis in RD patients ..................................................................... 54
3.2.1.2. Prenatal diagnosis in RD families ....................................................................... 61
3.2.1.3. Genetic instability in ZMPSTE24 ....................................................................... 62
3.2.2. Mandibuloacral dysplasia (MAD)......................................................................... 64
3.2.3. Emery-Dreifuss muscular dystrophy (EDMD) ZMPSTE24 ................................. 64
3.2.4. Candidate gene testing for secondary laminopathies ............................................ 64
3.2.4.1. NARF .................................................................................................................. 64
3.2.4.2. SREBF1 .............................................................................................................. 64
4. Discussion ............................................................................................................................ 65
5. Summary ............................................................................................................................. 82
6. References ........................................................................................................................... 86
7. Annex ............................................................................