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

De
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..................................
Publié le : vendredi 1 janvier 2010
Lecture(s) : 34
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Source : D-NB.INFO/1008137804/34
Nombre de pages : 124
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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 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 Breceptor
LDHCP hepatic steatosis
LEM Lamin, Emerin, MAN1
LEMD2 LEM domain containing 2 gene
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
VII
MADA mandibularacral dysplasia type A
MADB mandsplasia type B
min minute
ml millilitre
mMillimole
NARF nuclear prelamin A recognition factor gene
ng nanogram
nm nanometer
NLS nuclear localization signal
OD optic density
OMIM online mendelianinheritance in man
PBS phosphate buffered solution
PCR polymerasechain 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
STA(EMD) emerin gene
VIII
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
IXIntroduction 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 represent at least fourteen
disease phenotypes including various types of muscular dystrophies, dilated
cardiomyopathies, lipodystrophies, mandibuloacral dysplasia, progeroid syndromes, and
restrictive dermopathy. They arise mainly through pleiotropic mutations in LMNA - the gene
that codes for the A-type lamins A and C. Few primary laminopathies are caused by
mutations in LMNB1 - encoding lamin B1 and 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 or inner nuclear membrane
like STA (EMD), ZMPSTE24 (FACE1) or LAP2 (Bione et al., 1994; Navarro et al., 2004 and
2005; Taylor et al., 2005) and thus reflecting genetic heterogeneity in laminopathies. Most of
the symptoms develop after birth, typically during childhood or adolescence. Some
laminopathies however may lead to early death.
The nuclear lamina, or the inner nuclear membrane (INM), is a dense (~30 to 100 nm
thick) fibrilar network inside the nucleus of a eukaryotic cell. It is a scaffolding structure near
the inner nuclear membrane and the peripheral chromatin. It is required for maintenance of
nuclear shape. This scaffold is made of mostly type V intermediate filament proteins, lamin
A/C, B1 and B2, which together form a complex meshwork underneath the INM (reviewed in
Foisner, 2001; and Wilson et al., 2001). It has been demonstrated that nuclear lamins are
important proteins in maintaining cellular as well as nuclear integrity, and chromatin
organization in the nucleus. Besides providing mechanical support, the nuclear lamins are
involved in a number of essential nuclear functions, including nuclear envelope assembly and
disassembly during cell division, DNA synthesis, transcription, and apoptosis. Additionally, it
participates in chromatin organization and anchors the nuclear pore complexes embedded in
the nuclear envelope (Fig. 1.1). The nuclear lamina is associated with the inner face of the
bilayer nuclear envelope whereas the outer face stays continuous with the endoplasmic
reticulum. The nuclear lamina consists of two components, lamins and nuclear lamin-
associated membrane proteins.




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