Evaluation of susceptibility genes for infammatory bowel disease by association study and candidate gene analyses [Elektronische Ressource] / vorgelegt von Weiyue Zheng

W.Zheng

Sujets

• Gesundheit

Informations

• Publié par: christian-albrechts-universitat_zu_kiel
• Publié le: 01 janvier 2006
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Evaluation of susceptibility genes for inflammatory
bowel disease by association study and
candidate gene analyses






Dissertation
zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Christian-Albrechts-Universität
zu Kiel




vorgelegt von


Weiyue Zheng M.sc
















Referent: Prof. Dr. h.c. Thomas C. G. Bosch

Koreferent/in: ……………………………………


Tag der mündlichen Prüfung: .............................................

Zum Druck genehmigt: Kiel, ..............................................


Der Dekan

Abbreviations and symbols ..............................................................................................................5
1. Introduction ..............................................................................................................................8
1.1 Inflammatory bowel disease...............................................................................................8
1.1.1 Basic concept on inflammatory disease ......................................................................8
1.1.1.1 Barrier organs.......................................................................................................8
1.1.1.2 Inflammation as a common response to environmental triggers..........................9
1.1.1.3 Bacterial flora on body surfaces as a disease factor...........................................10
1.1.2 Inflammatory bowel disease......................................................................................11
1.1.2.1 Pathogenesis and pathophysiology of IBD ........................................................11
1.1.2.2 Genetic background of inflammatory bowel disease .........................................16
1.1.2.3 Linkage region on chromosome 12....................................................................17
1.1.2.4 Linkage region on Chromosome 7 .....................................................................18
1.2 Globlet cell function.........................................................................................................18
1.3 Mouse model ....................................................................................................................19
1.4 AGR2 function..................................................................................................................21
1.5 Disease gene detection in complex disease......................................................................23
1.5.1 Linkage analysis........................................................................................................23
1.5.2 Association mapping analysis ...................................................................................25
1.5.3 Candidate gene analysis ............................................................................................27
1.5.4 Pathway-mapping to establish the link between genetic variants and
pathophysiology .....................................................................................................................30
1.6 Aims of this study ..........................................................................................................31
2. Materials and methods ...........................................................................................................32
2.1 Materials...........................................................................................................................32
2.2 Electronic database34
2.3 Participants and study design ...........................................................................................35
2.3.1 Investigated patient sample of AGR gene.................................................................35
2.3.2 Association study population on chromosome 12.....................................................36
2.3.3 Sequencing samples ..................................................................................................37
2.4 Handling of samples.........................................................................................................37
2.4.1 DNA isolation38
2.4.2 Plate design ...............................................................................................................40
2.4.3 Whole Genome Amplification (WGA) plate preparation .........................................41
2.5 Diallelic genotyping42
2.5.1 Taqman assays...........................................................................................................42
2.5.2 SNPlex assays45
2.6 Mutation detection in candidate genes.............................................................................48
2.6.1 PCR optimisation ......................................................................................................48
2.6.2 Sequence analysis49
2.6.3 Mutation detection in candidate gene........................................................................50
2.6.3.1 AGR2 and AGR3 gene on chromosome 7 .........................................................50
2.6.3.2 Candidate genes on chromosome 12...................................................................52
2.7 Internal database...............................................................................................................55
2.8 Statistical analysis ............................................................................................................56
2.9 cDNA amplification .........................................................................................................57
2.9.1 Amplification of AGR2 cDNA .................................................................................57
2.9.2 Amplification of cDNA from candidate genes on chromosome 12..........................58
2.10 Rapid Amplification of cDNA Ends (RACE)................................................................59
2.11 Cell culture, reporter gene constructs and dual luciferase reporter gene assay..............61
2.12 Real-time PCR................................................................................................................63
3. Results ....................................................................................................................................64
3.1 AGR2 and AGR3 genes .....................................................................................................64
3.1.1 Mutation detection results .........................................................................................64
3.1.2 Results of Data Analyses...........................................................................................66
3.1.3 cDNA amplification results.......................................................................................72
3.1.4 Real-time PCR results ...............................................................................................74
3.1.5 Expression of Reporter gene construct......................................................................75
3.2 Association mapping on chromosome 12 ........................................................................76
3.2.1 Identification of the association lead on chromosome 12.........................................76
3.2.2 High density SNP mapping in the association region...............................................77
3.2.3 Analyses of LD in the association region..................................................................80
3.2.4 Candidate gene analyses in association region .........................................................82
3.2.4.1 Mutation detection results ..................................................................................82
3.2.4.2 Statistical analyses results83
3.2.4.3 cDNA amplification results................................................................................84
3.2.4.4 Rapid amplification of cDNA ends (RACE) results ..........................................86
4. Discussion ..............................................................................................................................87
4.1 AGR2 gene........................................................................................................................87
4.2 Chromosome 12 ...............................................................................................................89
4.2.1 Association mapping.................................................................................................89
4.2.2 Candidate genes on chromosome 12.........................................................................94
5. Conclusions ............................................................................................................................99
6. Summary101
7. Zusammenfassung................................................................................................................102
8. References104
9. Index of figures and tables ......................