Genetic analysis of candidate genes for the metabolic syndrome and type 2 diabetes [Elektronische Ressource] / Harald H. Grallert

Harald.Grallert

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 36
• Langue: English
• Poids de l'ouvrage: 2 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN
Institut für Experimentelle Genetik


Genetic Analysis of Candidate Genes for the Metabolic Syndrome
and Type 2 Diabetes

Harald H. Grallert

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
genehmigte Dissertation.

Vorsitzender: Univ.-Prof. Dr. H.-R Fries

Prüfer der Dissertation:
1. apl. Prof. Dr. J. Adamski
2. Univ.-Prof. Dr. H. Daniel
3. Priv.-Doz. Dr. Th. Illig,
Ludwig-Maximilians-Universität München


Die Dissertation wurde am 31.10.2007 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 04.02.2008 angenommen.
Acknowledgement
The research work and resulting Ph.D. thesis has been accomplished at the Institute of
Epidemiology. I am greatful to Prof. Dr. Dr. H.-Erich Wichmann, head of the institute, for the
possibility to realize this work.

Special thanks go to apl. Prof. Dr. Jerzy Adamski, who supervised this thesis at the
Technische Universität München.

Equally, I would like to thank PD Dr. Thomas Illig for suggesting this theme and his
supervision and support during my assignment at the GSF.

I want to thank Prof. Dr. Hannelore Daniel for evaluating this work as second examiner.

I thank my colleagues Dr. Caren Vollmert, Dr. Henning Gohlke, Melanie Kolz, Dr. Norman
Klopp, Dr. Wiebke Sauter and the rest of the EPIBIOGEN group for supporting me with
words and deeds.

I am grateful to Cornelia Huth for inducting me into the world of statistics as well as Guido
Fischer who taught me SAS programming and data management, providing the basis for the
statistical analysis.

Moreover, I want to thank all cooperation partners who supported my publications with fruitful
discussion.

I further want to thank my former Master students Eva-Maria Sedlmaier and Christina
Holzapfel, who supported this work with additional data and proofreading this thesis.

Although they have not contributed scientifically my family played an important role behind
the story. Thus, I am very grateful to my parents Herbert and Hannelore, my sister Astrid and
my girlfriend Pamela Roith, without whose constant encouragement, support, assistance and
tolerance this dissertation would not have been possible.

Certainly, my thanks are extended to all who are not mentioned above that have been in
contact with me scientifically or amicable.


Table of contents I
Table of contents
Table of contents.......................................................................................................................I
Summary................................................................................................................................ IV
Zusammenfassung.................................................................................................................. V
Abbreviations.......................................................................................................................... VI
1. Introduction .......................................................................................................................1
1.1 Metabolic syndrome........................................................................................................1
1.1.1 Pathophysiology of the metabolic syndrome............................................................3
1.2 Diabetes mellitus ............................................................................................................5
1.3 Inflammation in type 2 diabetes mellitus and metabolic syndrome.................................6
1.4 Epidemiology of type 2 diabetes mellitus and metabolic syndrome ...............................7
1.4.1 Type 2 diabetes mellitus ..........................................................................................7
1.4.2 Metabolic syndrome.................................................................................................7
1.5 Genetic susceptibility in type 2 diabetes mellitus and metabolic syndrome ...................9
1.5.1 Approaches for the identification of genetic susceptibilities.....................................9
1.5.1.1 Linkage..............................................................................................................9
1.5.1.2 Candidate gene approach .................................................................................9
1.5.1.3 Genome wide association analysis ...................................................................9
1.5.2 Genetics of metabolic syndrome..............................................................................9
1.5.2.1 Heritability..........................................................................................................9
1.5.2.2 Results from linkage studies............................................................................10
1.5.2.3 Results from candidate gene studies ..............................................................10
1.5.3 Genetics of Type 2 diabetes ..................................................................................13
1.5.3.1 Heritability........................................................................................................13
1.5.3.2 Results from linkage studies13
1.5.3.3 Results from candidate studies .......................................................................14
1.5.3.4 Results from genome wide association studies...............................................14
1.6 Aims..............................................................................................................................17
2. Methods ..........................................................................................................................18
2.1 SNP selection ...............................................................................................................18
2.2 DNA extraction..............................................................................................................19
2.2.1 DNA extraction from 9 ml EDTA blood...................................................................19
2.2.2 DNA extraction from 1 ml buffy coats.....................................................................19
2.3 DNA quantification........................................................................................................19
2.3.1 Spectrophotometry.................................................................................................19
2.3.2 Nanodrop ...............................................................................................................20
2.4 Agarose gel electrophoresis .........................................................................................20
Table of contents II
2.5 Polymerase Chain Reaction (PCR) ..............................................................................20
2.6 SNP detection via MALDI-TOF mass spectrometry .....................................................21
TM2.6.1 Homogeneous MassExtend Assay.....................................................................21
2.6.2 iPLEX and iPLEX Gold Assay................................................................................22
2.6.3 PCR amplification in scope of hME and iPLEX......................................................22
2.6.4 Shrimp alkaline phosphatase (SAP) reaction.........................................................23
2.6.5 Primer adjustment..................................................................................................23
2.6.5.1 hME Assay ......................................................................................................23
2.6.5.2 iPLEX Assay....................................................................................................24
2.6.6 Primer extension reaction ......................................................................................24
2.6.7 MALDI-TOF MS .....................................................................................................26
2.7 Allelic discrimination with TaqMan................................................................................27
2.7.1 PCR Reaction and allelic discrimination ....................................................................27
2.8 Quality assurance during genotyping ...........................................................................28
2.9 Statistical methods........................................................................................................28
2.9.1 Hardy-Weinberg-Equilibrium..................................................................................28
2.9.2 Linkage disequilibrium............................................................................................29
2.9.3 Descriptive statistics and association analysis.......................................................29
2.9.4 Haplotype analysis.................................................................................................30
3. Material ..