Genome-wide association study to search for SNPs affecting gene expression in a general population [Elektronische Ressource] / Divya Deepak Mehta

O

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 16
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Experimentelle Genetik


Genome-wide association study to search for SNPs
affecting gene expression in a general population

Divya Deepak Mehta

Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. A. Gierl
Prüfer der Dissertation:
1. Univ.-Prof. Dr. Th. Meitinger
2. apl. Prof. Dr. J. Adamski
3. Univ.-Prof. Dr. H -R. Fries

Die Dissertation wurde am 19.12.2008 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt am 10.10.2009 angenommen.
Table of Contents

ZUSAMMENFASSUNG.............................................................................................................................. 1
1.0 SUMMARY............................................................................................................................................. 2
2.0 INTRODUCTION... 3
2.1 RNAISSANCE AND GENE REGULATION ................................................................................................. 5
2.2 VARIATION IN HUMAN GENE EXPRESSION............................................................................................. 8
2.2.1 HERITABILITY OF GENE EXPRESSION VARIATION ............................................................................... 8
2.2.2 CIS AND TRANS EFFECTS.................................................................................................................... 9
2.2.2.1 CIS-ACTING ELEMENTS ................................................................................................................. 10
2.2.2.2 TRANS-ACTING FACTORS .............................................................................................................. 11
2.2.3 GENE EXPRESSION VARIATION AT THE LEVEL OF ISOFORMS ............................................................ 11
2.3 GENETIC MAPPING OF GENE EXPRESSION VARIATION ......................................................................... 12
2.3.1 LINKAGE STUDIES............................................................................................................................ 12
2.3.2 ASSOCIATION STUDIES..................................................................................................................... 13
2.3.2.1 POPULATION-BASED ASSOCIATION STUDIES ................................................................................. 14
2.3.2.1.1 POPULATION STRATIFICATION: LOOKOUT FOR “SUSHI” GENES ................................................ 14
2.3.2.2 GENOME-WIDE ASSOCIATION STUDIES.......................................................................................... 15
3.0 AIMS OF THE INVESTIGATION .................................................................................................... 17
4.0 MATERIALS AND METHODS......................................................................................................... 19
4.1 MATERIALS ........................................................................................................................................ 19
4.1.1 RNA RESOURCES............................................................................................................................. 19
4.1.1.1 THE KORA F3/S3 POPULATION 19
4.2 METHODS............ 20
4.2.1 RNA ISOLATION .............................................................................................................................. 21
4.2.2 RNA QUALITY CHECK USING AGILENT BIOANALYZER NANO 6000 KIT........................................... 22
4.2.3 THE RIN (RNA INTEGRITY NUMBER) ............................................................................................. 23
4.2.4 RNA QUANTIFICATION USING THE INVITROGEN RIBOGREEN KIT..................................................... 23
4.2.5 GLOBIN REDUCTION EXPERIMENTAL PROCEDURE............................................................................ 24
4.2.6 RNA AMPLIFICATION, REVERSE TRANSCRIPTION AND LABELING .................................................... 26
4.2.7 ILLUMINA MICROARRAY PROCEDURES 29
4.2.7.1 WHOLE GENOME GENE EXPRESSION WITH SENTRIX BEAD CHIP 29
4.2.7.2 MICROARRAY LOADING................................................................................................................ 29
4.2.8 ILLUMINA BEAD STUDIO CONTROL SUMMARY REPORT.................................................................... 32
4.2.9 GENOTYPING ................................................................................................................................... 33
4.2.10 STATISTICAL ANALYSIS ................................................................................................................. 33
5.0 RESULTS............... 35
5.1 DYNAMIC RANGE OF DETECTION ........................................................................................................ 35
5.2 NORMALIZATION OF GENE EXPRESSION DATA .................................................................................... 36
5.3 FILTERING OF EXPRESSION DATA 37
5.4 TECHNICAL AND BIOLOGICAL REPLICATES ......................................................................................... 39
5.5 VARIABILITY IN GENE EXPRESSION LEVELS ........................................................................................ 40
5.6 GENES EXPRESSED IN WHOLE BLOOD.................................................................................................. 43
5.7 CELL-SPECIFIC GENE EXPRESSION PATTERNS...................................................................................... 45
5.8 GLOBIN – TO REDUCE OR NOT REDUCE?.............................................................................................. 47
5.9 GENDER-SPECIFIC DIFFERENCES IN GENE EXPRESSION........................................................................ 50
5.10 AGE-RELATED GENE EXPRESSION PATTERNS .................................................................................... 53
5.11 CIS AND TRANS REGULATORS OF GENE EXPRESSION ......................................................................... 54
5.12 FUNCTIONAL VALIDATION OF GWAS CANDIDATE SNPS USING EXPRESSION PROFILES.................... 63
5.12.1 CONFIRMATION OF KNOWN ESNPS AND IDENTIFICATION OF NOVEL ESNPS.................................. 64
5.12.2 AN EXAMPLE WHERE EXPRESSION PROFILES ALLOWED PRIORITIZATION OF A CANDIDATE GENE... 66
5.12.3 TESTING FOR EFFECTS OF CIS AND TRANS SNPS IN THE CANDIDATE GENES................................... 66
5.13 USE OF GENE EXPRESSION TO FUNCTIONALLY VALIDATE GWAS CANDIDATE GENES....................... 69
5.13.1 FUNCTIONAL VALIDATION OF SLC2A9 INFLUENCING URIC ACID CONCENTRATIONS..................... 69
5.13.2 F VALIDATION OF WDR66 ASSOCIATED WITH MPV IN A GWAS ............................... 71
5.14 IDENTIFICATION OF NOVEL REGULATORY PATHWAY ........................................................................ 72
5.14.1 USE OF EXPRESSION PROFILES TO IDENTIFY IGE REGULATION PATHWAY ...................................... 72
6.0 DISCUSSION AND CONCLUSIONS................................................................................................ 74
6.1 ADVANTAGES AND DISADVANTAGES OF USING WHOLE BLOOD IN TRANSCRIPTOMICS ........................ 74
6.2 ESTABLISHMENT OF THE KORA GENE EXPRESSION DATASET ............................................................ 75
6.2.1 USE OF THE KORA DATASET TO MEASURE VARIABILITY OF GENE EXPRESSION .............................. 76
6.2.2 GENDER-SPECIFIC GENE EXPRESSION SIGNATURES IN THE KORA DATASET.................................... 77
6.2.2.1 ESTABLISHMENT OF A GENDER PREDICTOR................................................................................... 77
6.3 AGE -SPECIFIC GENE EXPRESSION SIGNATURES IN THE KORA DATASET ............................................ 78
6.4 IDENTIFICATION OF CIS AND TRANS EQTLS........................................................................................ 80
6.5 USE OF THE KORA GENE EXPRESSION RESOURCE TO IDENTIFY NOVEL ESNPS .................................. 82
6.6 FUNCTIONAL VALIDATION OF SLC2A9 .............................................................................................. 83
6.7 GENOME-WIDE ASSOCIATION STUDIES - CAVEATS AND FUTURE PERSPECTIVES 85
6.8 VALUE OF GENE EXPRESSION DATA .................................................................................................... 86
7.0 BIBLIOGRAPHY................................................................................................................................. 88
9.0 LIST OF ABBREVIATIONS............................................................................................................ 100
10.0 ACKNOWLEDGEMENTS ............................................................................................................. 102

Zusammenfassung
Die quantitative Erfassung von Gentranskription liefert wertvolle Hinweise bei der
Untersuchung genetischer Risikofaktoren von haüfigen Erkrankungen. Ziel dieser Arbeit
war