Molecular genetic studies in hereditary laminopathies of man [Elektronische Ressource] / vorgelegt von Le Thi Thanh Huong

Minh Ha-Duong

Sujets

• Gesundheit

Informations

• Publié par: ernst-moritz-arndt-universitat_greifswald
• Publié le: 01 janvier 2010
• Nombre de lectures: 34
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Aus dem Institut für Humangenetik
(Direktorin Univ. - Prof. Dr. Ute Felbor)
der Medizinischen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald





Thema:
Molecular genetic studies in hereditary laminopathies of man


Inaugural-Dissertation

zur

Erlangung des akademischen Grades

Doktor der Naturwissenschaften in der Medizin
(Dr. rer. med.)

der

Medizinischen Fakultät

der

Ernst-Moritz-Arndt-Universität

Greifswald

2010


vorgelegt von:
Le Thi Thanh Huong
geboren am 15. November 1979
in Haiphong, Vietnam








I







































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

1. Gutachter: Prof. Dr. rer. nat. Manfred Wehnert, Greifswald
2. Gutachter: Prof. Dr. med. Peter Wieacker, Münster
(3. Gutachter:)

Ort, Raum: Greifswald, Hörsaal der Klinik für Hals-, Nasen-, Ohrenheilkunde
Tag der Disputation: 22. Juli 2010

II
Content


1. Introduction............................................................................................. 01

2. Materials, methods and patients............................................................ 12
2.1. Materials............................................................................ 12
2.1.1. Reagents............................................................................. 12
2.1.2. Enzymes.................................... 12
2.1.3. Extraction kits.................................................................... 13
2.1.4. Solutions and media................................................................................... 13
2.1.4.1. Solutions............................................................................. 13
2.1.4.2. Bacterial culture medium........................................................................... 14
2.1.5. Vector.......................................................... 14
2.1.6. Oligonucleotides (primers)......................................................................... 15
2.1.7. Equipment........................................................................... 15
2.2. Methods....................................................... 17
2.2.1. Determination of DNA content........................................... 17
2.2.2. Agarose gel electrophoresis........................................................................ 17
2.2.3. Mildly denaturing polyacrylamide gel electrophoresis (PAGE)................ 17
2.2.4. DNA extraction........................................................................................... 17
2.2.4.1. DNA preparation from blood...................................................................... 17
2.2.4.2. DNA preparation from parafin blocks........................................................ 18
2.2.5. Total RNA extraction from parafin blocks................................................. 18
2.2.6. Primer design for polymerase chain reaction.............................................. 18
2.2.7. Polymerase chain reaction (PCR)............................................................... 19
2.2.7.1. Regular PCR............................................................................................... 19
2.2.7.1.1 Regular PCR from genomic DNA...................................... 19
2.2.7.1.2 Regulm bacterial colonies......................................................... 20
2.2.7.2. Nested PCR......................................................................... 20
2.2.7.3. RT-PCR.............................................................................. 20
2.2.8. Heteroduplex analysis......................................................... 21
2.2.9. High resolution melting...................................................... 22
2.2.10. Sequencing.......................................................................... 23
III
2.2.10.1. Sequencing procedure................................................................................. 23
2.2.10.2. Sequencing procedure for ABI-Prism......................................................... 23
TM 2.2.10.3. Sequencing r MegaBACE 1000.......................................... 24
2.2.10.4. Computer-aided sequence evaluation................................. 25
2.2.10.5. Nomenclature of sequence variations................................. 26
2.2.11. Restriction digestion................................................................................... 26
2.2.12. Cloning and expression gene.............................................. 26
2.2.12.1. Cloning of genomic DNA................................................... 26
2.2.12.1.1. Processing of PCR products................................................ 26
2.2.12.1.2. Preparation of LB-ampicillin plates............................................................ 28
2.2.12.1.3. Transformation..................................................................... 28
2.2.12.1.4. Prepararion of plasmid for transfection....................................................... 29
2.2.12.1.5. Preparation of cell for transfection...................................... 29
2.2.12.2. Transfection................................................................................................. 29
2.2.12.3. Total RNA extraction from HEK cell.................................. 29
2.2.12.4. Clonning of c.DNA...................................................................................... 30
2.2.13. Electronic databases used............................ 30
2.3. Patients................................................................................. 32

3. Results......................................................................................................... 33
3.1. Candidate gene testing for laminopathies.................................................... 33
3.1.1. The LAP2 gene............................................................................................ 33
3.1.2. The LEMD2 gene................................................................ 41
3.1.3. The NARF and LMNB2 gene...................................................................... 43
3.2. Mutational analysis in primary laminopathies..................... 44
3.2.1. Mutational anaylsis in muscular phenotypes............................................... 44
3.2.2. Mutational analysis in progeroid phenotypes...................... 47
3.3. Secondary laminopathies............................................................................. 49
3.3.1. Mutationl analysis in RD patients................ 49
3.3.2. Mutational analysis in MAD....................................................................... 57
3.4. Influence of SNP LMNA c.1698C>T (p.H566H)on alternative splicing
efficiency...................................................................................................... 59


IV
4. Discussion.................................................................................................... 62

5. Summary..................................................................................................... 74

6. References................................................................................................... 76

7. Annex.......................................................................................................... 91

Acknowledgements

Eidesstattliche Erklärung

Curriculum vitae

Own publications




























V
Abbreviations

µg microgram/-s
µl microlitre/-s
A adenine
aa amino acid
ADLD autosomal dominant leukodystrophy
AICD automated cardioverter/defibrillator
APL aquired partial lipodystrophy
ATP adenosine triphosphate
aWRN atypical Werner syndrome
AV atrio-ventricular
BAF barrier-to-autointegration factor
bp base pair/-s
BSA bovine serum albumin
C cytosine
cDNA complementary DNA
CK creatine kinase
CMD1A dilated cardiomyopathy type 1A
CMT Charcot-Marie-Tooth neuropathy
CNS central nervous system
CSD conduction system disease
DCM dilateted cardiomyopathy
DES Desmin gene
ddH O double distilled water 2
DMSO dimethyl sulphoxide
DNA deoxyribonucleic acid
Dnase deoxyribonuclease
VI
dNTP deoxynucleotide triphosphates
ECG electrocardiography
EDTA ethylene diamine tetra acetic acid
EDMD Emery-Dreifuss muscular dystrophy
EMG electromyogram
ER endoplasmaticreticulum
FPLD familial partial lipodystrophy
G guanine
membrane protein associated with chromatin,
HA95 h