Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease:
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| Format: | Thesis Book |
| Language: | English |
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2015
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| Online Access: | Inhaltsverzeichnis |
| Item Description: | Zsfassung in dt. und engl. Sprache |
| Physical Description: | 156 S. graph. Darst. |
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| adam_text | Titel: Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease
Autor: Pannes, Andrea
Jahr: 2015
Table of contents
Table of contents
List of abbreviations.................................................................................................
1 Introduction...................................................................................................................1
1.1 Autosomal dominant tubulointerstitial kidney disease - history and nomenclature... 2
1.2 Clinical characteristics.............................................................................................3
1.3 Subcategories of ADTKD.........................................................................................5
1.3.1 ADTKD-REN.....................................................................................................5
1.3.1.1 REN expression by juxtaglomerular cells in the kidney...................................5
1.3.1.2 Renin-angiotensin system (RAS)....................................................................7
1.3.1.3 Prorenin and renin protein structure................................................................8
1.3.1.4 Pathogenic variants causing ADTKD-REN......................................................9
1.3.2 ADTKD-MUC1................................................................................................10
1.3.2.1 Mucin family..................................................................................................10
1.3.2.2 MUC1 gene..................................................................................................12
1.3.2.3 MUC1: protein structure and function............................................................14
1.3.2.4 Pathogenic variant causing ADTKD-ML/Cf...................................................16
2 Study aims..................................................................................................................18
3 Material and Methods.................................................................................................19
3.1 Materials, solutions and reagents..........................................................................19
3.1.1 Human subjects and DNA...............................................................................19
3.1.1.1 Substances for the analysis of nucleic acids.................................................19
3.1.1.2 Buffers and solutions for the analysis of nucleic acids...................................20
3.1.1.3 Kits................................................................................................................23
3.1.1.4 Primers.........................................................................................................23
3.1.1.5 Enzymes.......................................................................................................23
3.1.1.6 Vectors..........................................................................................................24
3.1.2 Cell culture.....................................................................................................24
3.1.2.1 Reagents for cell culture...............................................................................24
3.1.2.2 Solutions and buffers for cell culture.............................................................25
3.1.3 Bacteria..........................................................................................................25
3.1.4 Protein analysis..............................................................................................26
3.1.4.1 Reagents for protein analysis........................................................................26
3.1.4.2 Buffers and solutions for protein analysis......................................................27
3.1.4.3 Kits................................................................................................................28
3.1.4.4 Enzymes.......................................................................................................28
3.1.4.5 Antibodies.....................................................................................................28
3.1.5 Equipment......................................................................................................29
3.1.6 Computer programs........................................................................................32
3.1.7 Data bases.....................................................................................................32
3.2 Methods.................................................................................................................
3.2.1 Analysis of nucleic acids.................................................................................
3.2.1.1 Polymerase chain reaction (PGR).................................................................
3.2.1.2 DNA sequencing...........................................................................................
3.2.1.3 Genome-wide linkage analysis......................................................................
3.2.1.4 Microsatellite marker analysis.......................................................................
3.2.1.5 Targeted NGS...............................................................................................
3.2.1.6 Whole-exome sequencing.............................................................................
3.2.1.7 Detection of the MUC1 VNTR InsC by SNaPshot minisequencing................
3.2.1.8 Detection of the MUC1 VNTR InsC by targeted NGS....................................
3.2.1.9 Generation of extra-long reads with PacBio SMRT sequencing
of MUC1 VNTR.............................................................................................
3.2.1.10 Bioinformatic analysis tool for reconstruction of the MUC1 VNTR...............
3.2.1.11 Determination of MUC1 allele sizes by Southern Blot analysis....................
3.2.2 Generation of REN cDNA expression constructs............................................
3.2.3 Transient expression of REN..........................................................................
3.2.4 Determination of transfection efficiency..........................................................
3.2.5 Endoplasmic reticulum stress.........................................................................
3.2.6 Cell culture.....................................................................................................
3.2.7 Analysis of proteins.........................................................................................
3.2.7.1 Standard Western blot analysis.....................................................................
3.2.7.2 Secretion analysis of renin............................................................................
3.2.7.3 Deglycosylation experiments.........................................................................
3.2.7.4 Trypsin digestion...........................................................................................
3.2.7.5 Inhibition of protein biosynthesis by CHX chase experiments........................
3.2.7.6 Suppression of translation by emetine and inhibition of the 26S proteasome
- ubiquitin pathway.......................................................................................
3.2.7.7 Subcellular fractionation................................................................................
3.2.7.8 Ren activity measurement.............................................................................
3.2.7.9 Immunohistochemical staining of kidney biopsies.........................................
3.2.7.10 Immunofluorescence staining of kidney biopsies.........................................
3.2.7.11 Preparation of urinary exovesicles..............................................................
Results........................................................................................................................
4.1.1 ADTKD-RBV...................................................................................................
4.1.1.1 Identification of a novel mutation (c.28T C, p.WIOR) in the signal peptide
of REN......................................................... ........................ ...... .............
4.1.1.2 Identification of a novel mutation (c.77C T, p.T26l) in the propeptide
domain of REN..............................................................................................
4.1.2 Molecular and functional analysis of novel identified pathogenic variants
in REN........................................................................
33
33
33
35
37
38
39
39
40
43
44
45
47
48
48
.49
.49
.50
.50
.50
.51
.51
.52
.52
.52
.52
.53
.53
.54
.54
.56
.57
.57
.59
.60
4.1.2.1 Prorenin and renin expression and secretion in transiently transfected
HEK293T cells..............................................................................................61
4.1.2.2 Glycosylation and prorenin activation studies................................................63
4.1.2.3 Stability of WT and mutant renin proteins......................................................64
4.1.2.4 Inhibition of translation and proteasomal degradation...................................65
4.1.2.5 Subcellular fractionation................................................................................66
4.1.2.6 Investigation of endoplasmic reticulum stress...............................................67
4.1.2.7 Measurement of renin activity (preliminary data)...........................................69
4.2 ADTKD-MUC1.......................................................................................................71
4.2.1 Identification of ADTKD-MUC1 candidate cases.............................................71
4.2.1.1 ADTKD-MUC1 locus linkage analysis...........................................................71
4.2.2 Genetic studies...............................................................................................76
4.2.2.1 SNaPshot minisequencing approach............................................................76
4.2.2.2 Confirmation of the MUC1 VNTR InsC by targeted NGS...............................79
4.2.2.3 Determination of MUC1 VNTR length via Southern blot................................83
4.2.2.4 Sequencing of the MUC1 VNTR region by single molecule, real time
(SMRT) sequencing......................................................................................83
4.2.3 Immunohistochemical and immunofluorescence studies in kidney biopsies.. . 91
4.2.4 MUC1-fs studies in a cell system....................................................................92
4.2.5 Analysis of urinary samples of affected individuals.........................................95
5 Discussion..................................................................................................................97
5.1 Novel identified REN variants causing ADTKD......................................................97
5.2 Pathophysiology of the novel identified REN variants..........................................100
5.2.1 Impaired ER translocation and prorenin and renin biosynthesis....................100
5.2.2 Prorenin activation - impact of the variants..................................................102
5.2.3 Instability of mutant renin precursors............................................................102
5.2.4 Subcellular localization of mutant proteins....................................................103
5.2.5 Rescue effects on prorenin biosynthesis and protein misfolding...................104
5.2.6 The novel signal peptide variant (c.28T C, p.W10R)
- a comparison with known variants.............................................................105
5.2.7 The novel identified propeptide mutation (c.78C T, p.T26l)
- classification within the ADTKD-REN mutation spectrum...........................106
5.2.8 Conclusion and Outlook................................................................................107
5.3 ADTKD-MUC1.....................................................................................................109
5.3.1 Identification of families showing linkage to the ADTKD-MUCf locus...........110
5.3.2 Detection of the MUC1 InsC by SNaPshot minisequencing
- a valid tool for diagnostics purposes?........................................................113
5.3.3 Development of novel techniques for detection of mutations in the
MUC1 VNTR region - gaining insight into VNTR assembly..........................116
120
121
121
123
125
127
129
132
137
154
155
5.3.4 Detection of MUC1-fs in vitro.........................................
5.3.5 MUC1-fs protein as a urinary biomarker........................
5.3.6 Incomplete penetrance, anticipation and ADTKD...........
5.3.7 Tandem repeat mutations associated to other diseases
5.3.8 Conclusion and outlook.................................................
Summary....................................................................................
Zusammenfassung....................................................................
Supplement................................................................................
References.................................................................................
Publications...............................................................................
Appendix....................................................................................
|
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| discipline | Medizin |
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| spelling | Pannes, Andrea Verfasser (DE-588)1074262557 aut Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease vorgelegt von Andrea Pannes 2015 156 S. graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache Köln, Univ., Diss., 2015 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028202888&sequence=000004&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Pannes, Andrea Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease |
| subject_GND | (DE-588)4113937-9 |
| title | Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease |
| title_auth | Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease |
| title_exact_search | Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease |
| title_full | Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease vorgelegt von Andrea Pannes |
| title_fullStr | Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease vorgelegt von Andrea Pannes |
| title_full_unstemmed | Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease vorgelegt von Andrea Pannes |
| title_short | Novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease |
| title_sort | novel functional and genetic findings in autosomal dominant tubulointerstitial kidney disease |
| topic_facet | Hochschulschrift |
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