New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2015
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| Beschreibung: | XI, 116 Seiten Illustrationen 21 cm |
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| 245 | 1 | 0 | |a New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes |c vorgelegt von Yicheng Liu |
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| adam_text | Titel: New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff an
Autor: Liu, Yicheng
Jahr: 2015
Table of content i
Abbreviation v
Summary viii
Zusammenfassung x
1. Introduction ..............................................................................................................................l
1.1 Aging and aging associated diseases ...............................................................................1
1.2 Progeria syndromes: genomic instability and premature aging .....................................2
1.3 Hutchinson-Gilford progeria syndrome: an example for early aging research .............5
1.4 Hallermann-Streiff syndrome (HSS) and Bloom syndrome: two congenital disease used
as models to study aging processes ....................................................................................6
1.4.1 Hallermann-Streiff syndrome (HSS) ....................................................................................6
1.4.1.1 Molecular studies on Hallerman-Streiff syndrome ..................................................7
1.4.1.2 CHD6: a putative disease causing gene for HSS ...........................................................8
1.4.1.3 Condensin and cohesin .............................................................................................10
1.4.1.3.1 Condensin ..............................................................:..................................................10
1.4.1.3.2 Cohesin ..........................................................................................................13
1.5 Bloom syndrome and DNA-PKs function .........................................................................15
1.5.1 Clinical characteristics in patient s with Bloom syndrome ...........................................15
1.5.2 The causative gene for Bloom syndrome .........................................................................15
1.5.3 DNA-PKs and the classical non-homologous end joining pathway ...............................16
1.5.3.1 Features and functions of DNA-PKs in DNA damage response ................................16
1.5.3.2 The role of DNA-PKcs in the NHEJ pathway ...............................................................18
1.6 Next generation sequencing ............................................................................................18
1.7 An example of NGS application in research on Mendelian disorders ...........................20
2. Study aims ...........................................................................................................................22
3. Materials and methods ..................................................................................................23
3.1 Materials ......................................................................................................................23
3.1.1 Subject DNA samples and patient-derived cells .............................................................23
3.1.2 Chemicals and reagents ....................................................................................................23
3.1.3 Enzymes .............................................................................................................................25
3.1.4 Kits ......................................26
3.1.5 Solutions and buffers ..........................................................................27
3.1.6 Antibodies ................................................................................................................28
3.1.7 Bacteria ......................................................29
3.1.8 Vectors ............................................29
3.1.9 Equipment .......................................................................................................................30
3.1.10 Online databases and software ...................................................................................30
3.1.10.1 Online databases .........................................................................................................30
3.1.10.2 Software ...................................................................................................................32
3.2 Methods ..............................................................................................................................32
3.2.1 Molecular-biological methods .........................................................................................32
3.2.1.1 DNA Isolation from fibroblasts ...................................................................................32
3.2.1.2 Polymerase chain reaction (PCR) .............................................................................33
3.2.1.3 Agarose gel electrophoresis .....................................................................................33
3.2.1.4 DNA sequencing .........................................................................................................34
3.2.1.5 Isolation of RNA ...........................................................................................................34
3.2.1.6 Synthesis of cDNA .......................................................................................................34
3.2.1.7 Quantitative real-time PCR (qRT-PCR) ......................................................................35
3.2.2 Protein-biochemical methods ........................................................................................36
3.2.2.1 Total cell lysates .........................................................................................................36
3.2.2.2 Measuring total protein concentration ....................................................................36
3.2.2.3 SDS polyacrylamide gel electrophoresis (SDS-PAGE) ...............................................36
3.2.2.4 Western blot ...................................................................................................................37
3.2.2.5 Immunofluoresence ...................................................................................................38
3.2.3 Cell culture .........................................................................................................................38
3.2.3.1 Cell culture ...................................................................................................................38
3.2.3.2 Freezing and thawing of cells .....................................................................................38
3.2.3.3 Cell transfection ............................................................................................................
3.2.3.4 Flow cytometry intracelluar protein expression ......................................................39
3.2.3.5 Cell cycle analysis ........................................................................................................................................................................39
3.2.3.5.1 Propidium iodide (PI) DNA staining .............................................................. 39
3.2.3.5.2 Bromdesoxyuridin (BrdU)/PI DNA staining ................................................... 40
3.2.3.5.3 Phosphorylated-Histone3 (pH3)/PI staining ..................................................40
3.2.4 CRISPR/Cas9 Nuclease RNA-guided Genome Editing ..................................................40
3.2.4.1 Sequence targeted oligonucleotide design .........................................................40
3.2.4.2 Bacterial Transformation .........................................................................................42
3.2.4.3 Plasmid isolation ..........................................................................................................42
3.2.4.4 Fluorescence activated cell sorting .........................................................................42
3.2.5 Neutral comet assay ........................................................................................................43
3.2.6 Micro-homologous end joining assay (MMEJ) .............................................................44
4. Results ..................................................................................................................................45
4.1 Molecular and functional analysis of Hallermann-Streiff syndrome (HSS) ..............45
4.1.1 Molecular analysis of Hallermann-Streiff syndrome (HSS) .........................................45
4.1.1.1 Molecular analysis of Hallermann-Streiff syndrome (HSS) ......................................45
4.1.1.2 A de novo mutation in SMC2 in a patient with HSS ...................................................46
4.1.1.3 Molecular analysis of HSS patients K611, K3016, and K3251 ...................................47
4.1.1.3.1 Molecular genetic study of patient K611 ................................................................47
4.1.1.3.2 Molecular genetic study of patient K3016 ...............................................................48
4.1.1.3.3 Molecular genetic study of patient K3251 ................................................................50
4.1.1.3.4 Identification of promising candidate genes in HSS patient K611, K3016, and K3251
............................................................................................................................................50
4.1.1.4 Large-scale targeted genome sequencing in the HSS patient cohort.......................51
4.1.2 Functional analysis of HSS patient fibroblasts....................................................................54
4.1.2.1 DNA damage response in HSS patient fibroblasts ....................................................54
4.1.2.2 Cell cycle distribution of HSS patient fibroblasts ........................................................56
4.1.2.2.1 General cell cycle distribution of HSS patient fibroblasts .........................................56
4.1.2.2.2 Cell cycle distribution of HSS patient fibroblasts after DNA damage induction.......59
4.1.2.3 Analysis of chromosome compaction in HSS patient fibroblasts ..........................59
4.1.2.4 Analysis of the chromosome architecture in HSS patient fibroblasts........................61
4.1.2.5 SMC2 and CHD6 expression in HSS patient fibroblasts ............................................62
4.1.3 Cellular phenotype ofSMC2 heterozygous knockout HEK293 cells (HEKSA4C2+/)........63
4.1.3.1 Generation of a HEK SMC2*1cell line ........................................................................63
4.1.3.2 Analysis of DNA damage response in HEKSMC2+/cells ............................................65
4.1.3.3 Cell cycle distribution and cell proliferation in HEK5MC2+/ cells..............68
4.1.3.4 qPCR analysis of CHD6 expression in HEK SMC2+/~ cells .............69
4.1.3.5 Chromosome architecture in HEK SMC2+/cells .........................................................70
4.2 Molecular and functional analysis of Bloom syndrome ....................................................71
4.2.1 Identification of a variant in PRKDC m a patient with Bloom syndrome ........................71
4.2.2 Functional analysis of Bloom syndrome ...........................................................................74
4.2.2.1 Analysis of the DNA damage response in Bloom syndrome patient fibroblasts ...74
4.2.2.2 Analysis of DNA double-strand break repair in patient fibroblasts ........................77
4.2.2.3 Analysis of microhomology-mediated end joining (MMEJ) in patient fibroblasts 78
4.2.2.4 Analysis of total and phosphorylated DN A-PKcs in patient fibroblasts.....................81
5. Discussion ...............................................................................................................................82
5.1 Identification of the disease asociated genes in patient with Hallermann-Streiff
syndrome (HSS) .....................................................................................................................82
5.2 Identification of additional mutation in SMC2 and CHD6 ...............................................84
5.3 SMC2 and CHD6 expression in HSS patient fibroblasts ...................................................84
5.4 DNA damage response in HSS patient fibroblasts and HEK SMC2*1cells........................86
5.5 Cell cycle distribution in HSS patient fibroblasts and HEK SMC2*1 cells..........................87
5.6 Chromosome compaction in HSS patient fibroblasts .......................................................88
5.7 Anaphase segregation in in HSS patient fibroblasts and HEK SMCT1 cells.....................89
5.8 Hypothesized pathogenic mechanism for HSS...................................................................90
5.9 Homozygous missense mutation in PRKDCin Bloom syndrome .....................................91
5.10 Total and activated DNA-PKcs expression..........................................................................93
5.11 The p.P3174L mutation in DNA-PKcs does not abolish NHEJ............................................94
6. Reference ........................................................................................................................95
7. Ackonwledgement .........................................................................................................115
8. Erklarung ......................................116
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| author | Liu, Yicheng |
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| spelling | Liu, Yicheng Verfasser (DE-588)108068056X aut New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes vorgelegt von Yicheng Liu 2015 XI, 116 Seiten Illustrationen 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität zu Köln 2015 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1084345064/04 Inhaltsverzeichnis HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028975794&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Liu, Yicheng New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes |
| subject_GND | (DE-588)4113937-9 |
| title | New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes |
| title_auth | New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes |
| title_exact_search | New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes |
| title_full | New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes vorgelegt von Yicheng Liu |
| title_fullStr | New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes vorgelegt von Yicheng Liu |
| title_full_unstemmed | New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes vorgelegt von Yicheng Liu |
| title_short | New insights into the molecularpathogenesis of accelerated aging phenotypes in Hallermann-Streiff and Bloom syndromes |
| title_sort | new insights into the molecularpathogenesis of accelerated aging phenotypes in hallermann streiff and bloom syndromes |
| topic_facet | Hochschulschrift |
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