Wang, Guan (2013) Genetic studies of elite athlete status. PhD thesis. http://theses.gla.ac.uk/4594/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Genetic Studies of Elite Athlete Status by Guan Wang, M.Res. A Doctoral Thesis Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy August 2013 Institute of Cardiovascular and Medical Sciences College of Medical, Veterinary, and Life Sciences University of Glasgow © G. Wang 2013 2 Author’s Declaration I declare that this thesis is the result of my own work, except that subject recruitment in each country was orchestrated by the relevant PIs as follows: Dr. Yannis Pitsiladis (Jamaican sprint cohort), Dr. Krista Austin (USA sprint cohort), Dr. Rob Lee (Caucasian swim cohort), Dr. Noriyuki Fuku (Japanese swim and track-and-field athlete cohorts), and Dr. Sandy Hsieh (Taiwanese swim cohort); genotyping of Japanese and Taiwanese swim cohort was conducted by Dr. Eri Mikami and Dr. Li-Ling Chiu; genome-wide genotyping of Jamaican and USA sprint cohorts as well as Japanese track-and-field athlete cohort was overseen by Dr. Noriyuki Fuku and carried out in Japan; raw genotype data of additional 350 African-American controls was received from Dr. Braxton Mitchell at University of Maryland, USA. This work has not been submitted previously for any other degree at the University of Glasgow or any other institution. Guan Wang 3 Acknowledgement I would like to express my sincere thanks to my supervisors Dr. Yannis Pitsiladis and Dr. Sandosh Padmanabhan for their continuous support and guidance. Dr. Pitsiladis inspired me on the path to the completion of my PhD study with his enthusiasm to the field, guided and reminded me to reflect on my weaknesses, and provided me opportunities to be involved in diverse projects to improve my knowledge and skills in the field I am interested in. Dr. Padmanabhan provided me with his immense knowledge and persistent help on the GWAS project. Without the training from Dr. Padmanabhan on GWAS analysis, the completion of my PhD and this thesis would have not been possible. Also, I am truly thankful to Dr. Mark Bailey for his patience and time in helping me understand the essence of genetics, and his insightful comments and questions kept me thinking about the right way through a genetic research. Besides, I would also like to send “many thanks” to Dr. Claire Hastie for her generous help during the initial setting up of the GWAS analysis. Now, it is a great pleasure to thank everyone who provided their help and kindness to assist me at certain stage of my PhD: Dr. Noriyuki Fuku, Dr. Alessandra de Perini, Dr. Eri Mikami, Dr. Wai Kwong Lee, Dr. Jim McCulloch, Ms. Morvern Campbell, Mr. Michael Deason, Dr. Carlos Celis, Dr. Anna Christina Koni, Mr. David Hughes, Mrs. Anne Keenan. I would also like to thank the Great Britain Sasakawa Foundation for providing an award to me for visiting the lab in Japan on genome-wide genotyping technology. Finally, it is the “thanks” that beyond words that can adequately convey. These are to my family, my parents and uncle, for the roles they play in my life and their encouragement to 4 me at every step, and to my boyfriend, his sense of humour, tolerance and kindness helped me through every difficult moment over the past few years. 5 Table of Contents Author’s Declaration .............................................................................................................. 2 Acknowledgement.................................................................................................................. 3 List of Figures ........................................................................................................................ 8 List of Tables........................................................................................................................ 10 List of Abbreviations............................................................................................................ 11 Publications, Awards and Presentations .............................................................................. 16 Abstract ................................................................................................................................ 19 1 Introduction ................................................................................................................... 22 1.1 Elite human performance ......................................................................................... 22 1.2 Genetic variations in elite human performance ....................................................... 23 1.2.1 Family-based studies: genetic evidence ......................................................... 24 1.2.2 Association studies ......................................................................................... 26 1.2.3 Candidate gene association studies ................................................................ 28 1.3 Genome-wide association studies in complex traits ................................................ 30 1.3.1 Sample size and statistical power ................................................................... 32 1.3.2 Statistical significance thresholds .................................................................. 35 1.3.3 Population stratification and its solution ........................................................ 35 1.3.3.1 Genomic control ..................................................................................... 36 1.3.3.2 Structure assessment ............................................................................... 37 1.3.3.3 Principal component analysis ................................................................. 37 1.3.3.4 Family-based controlling approach ........................................................ 38 1.3.4 Genetic architecture of complex traits: CDCV hypothesis, infinitesimal model, rare allele model and the broad sense heritability model.................................. 39 1.3.5 GWAS of exercise-/performance-related traits .............................................. 40 1.3.5.1 GWAS of skeletal muscle trait ............................................................... 45 1.3.5.2 o 2max trainability .................................................................. 47 1.3.6 Replication ..................................................................................................... 48 1.4 Extreme phenotype................................................................................................... 50 1.5 Aims ......................................................................................................................... 51 2 Materials and methods .................................................................................................. 53 2.1 Study samples .......................................................................................................... 53 6 2.1.1 Elite athlete cohorts ........................................................................................ 53 2.1.2 DNA collection, extraction, quantification, storage and transportation......... 55 2.2 Genotyping ............................................................................................................... 57 2.2.1 Taqman® SNP genotyping ............................................................................. 57 2.2.1.1 Taqman® assay ........................................................................................ 57 2.2.1.2 PCR conditions and endpoint reading using StepOneTM software v2.1 . 58 2.2.2 Illumina whole-genome genotyping .............................................................. 59 2.2.2.1 Illumina Infinium DNA analysis BeadChips .......................................... 59 2.2.2.2 Illumina GenomeStudio v2010.3/v1.8: genotyping module ................... 60 2.3 Software ................................................................................................................... 61 2.3.1 PLINK ............................................................................................................ 61 2.3.2 Haploview ...................................................................................................... 62 2.3.3 EIGENSTRAT ............................................................................................... 63 2.4 Statistical analysis .................................................................................................... 65 2.4.1 Candidate gene association analysis .............................................................. 65 2.4.2 Analysis of GWAS ......................................................................................... 66 2.4.2.1 Power calculations .................................................................................. 66 2.4.2.2 Formats converting from Illumina output files to PLINK formats ......... 67 2.4.2.3 Quality control .......................................................................................