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

De
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 .................................................................
Publié le : mardi 1 janvier 2008
Lecture(s) : 30
Tags :
Source : UB-ED.UB.UNI-GREIFSWALD.DE/OPUS/VOLLTEXTE/2008/500/PDF/FINAL_THESIS5.PDF
Nombre de pages : 129
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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 ................................................................................................................................... 99
Acknowledgements
Eidesstattliche Erklärung
Curriculum vitae
Own publications








































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
dNTP deoxynucleotide triphosphates
ECG electrocardiography
EDTA ethylene diamine tetra acetic acid
EDMD Emery-Dreifuss muscular dystrophy
EMG electromyogram
ER endoplasmatic reticulum
FPLD familial partial lipodystrophy
G guanine
membrane protein associated with chromatin,
HA95
homologous with AKAP95
HEM Greenberg dysplasia
HGPS Hutchinson-Gilford progeria syndrome
IF intermediate filament
kb kilo base/-s
kDa kilo dalton/-s
l litre
LAP2 lamin associated polypeptide 2 gene
LB luria broth
LBR lamin B receptor
LDHCP hepatic steatosis
LEM Lamin, Emerin, MAN1
LGMD1B limb girdle muscular dystrophy type 1B
LMNA lamine A/C gene
LMNB1 lamin B1 gene
LMNB2 lamin B2 gene
M mole
MAD mandibuloacral dysplasia
MADA mandibularacral dysplasia type A
MADB mandibularacral dysplasia type B
min minute
ml millilitre
mM millimole
ng nanogram
nm nanometer
NLS nuclear localization signal
OD optic density
OMIM online mendelian inheritance in man
PBS phosphate buffered solution
PCR polymerase chain reaction
pg picogram
potential of hydrogen, negative logarithm of the
pH
hydrogen-ion activity
PHA Pelger-Huet abomaly
pmol picomole
PM pace maker
RD restrictive dermopathy
RNA ribonucleic acid
Rnase ribonuclease
rpm rotations per minute
SDS sodium dodecyl sulfate
sec second
Seq Ed sequence editor
SNP single nucleotide polymorphism
SREBF sterol regulating element binding transcription factor
STA emerin gene
T thymine
TAE tris-acetate EDTA buffer
TBE tris-borate EDTA buffer
TE tris-EDTA
U unit
UV ultraviolet
V volt
vol volume
ZMPSTE24 zinc metallo-protease STE24 gene
































Introduction Dissertation
1. Introduction
Laminopathies are a group of rare genetic disorders caused by mutations in genes
encoding proteins of the nuclear lamina. One can distinguish laminopathies into two types:
primary and secondary laminopathies. Primary laminopathies representing at least fourteen
disease phenotypes arise through pleiotropic mutations in LMNA - the gene that codes for the
A-type lamins A and C, mutations in LMNB1 encoding lamin B1 and mutations in LMNB2
encoding lamin B2. Secondary laminopathies including disease phenotypes also observed in
primary laminopathies are caused by genes encoding proteins related to the nuclear lamina
like ZMPSTE24 (FACE1), LAP2, LBR and thus reflecting genetic heterogeneity in
laminopathies.
The nuclear lamina is a scaffolding structure near the inner nuclear membrane and the
peripheral chromatin and is required for maintenance of nuclear shape. It is composed of
lamins, which are also present in the nuclear interior, and lamin-associated proteins (Fig. 1.1).
It is an essential component of metazoan cells and involved in most nuclear activities
including DNA replication, RNA transcription, nuclear and chromatin organization, cell cycle
regulation, cell development and differentiation, nuclear migration and apoptosis (Gruenbaum
et al., 2003).










Figure 1.1: Inner nuclear membrane protein and nuclear lamina (from Foisner et al., 2001)
AKAP149: a kinase-anchoring protein; BAF: barrier to autointegration factor; E2F: transcription
factor; Gp210: integral membrane protein; HP1: Heterochromatin protein 1; INM: inner nuclear
membrane; La A: lamin A; La B: lamin B; LAP1: lamina-associated polypeptide 1; LAP2: lamina-
associated polypeptide 2; LBR: lamin B receptor; LEM: LAP2, emerin and MAN1; MAN1: LEM
domain-containing integral membrane protein; mGCL: mouse germ cell less; ONM: outer nuclear
membrane; P18: intergral membrane protein; P32/34: a low-molecular-weight protein; POM121:
integral membrane protein; PP1: phosphatase; pRb: retinoblastoma protein; RFBP: ring finger
binding protein; RUSH: member of the SWI/SNF family of transcription factors
1

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