Human population history and genetic adaptation in the Himalayan region University of Cambridge Corpus Christi College A thesis submitted for the degree of Doctor of Philosophy Elena Arciero The Wellcome Sanger Institute Wellcome Genome Campus Hinxton, Cambridge CB10 1SA, UK September 2018 2 3 To my parents, Cristina and Virgilio 4 5 Declaration I hereby declare that except where specific reference is made to the work of others, the contents of this dissertation are original and have not been submitted in whole or in part for consideration for any other degree or qualification at the University of Cambridge or any other University or similar institution. This dissertation is my own work carried out under the supervision of Prof. Chris Tyler- Smith at the Wellcome Sanger Institute, while a member of Corpus Christi College, University of Cambridge, and contains nothing which is the outcome of work done in collaboration with others, except as specified in the text and Acknowledgements. This dissertation does not exceed the prescribed word limit of 60,000 words excluding bibliography, figures, tables, equations and appendices. Elena Arciero September 2018 6 7 Acknowledgements First and foremost I would like to express my sincere gratitude to my supervisor Dr Chris Tyler-Smith for giving me the opportunity to carry out this project and for his continuous support, advice and mentorship during my PhD. I would like to thank Dr Yali Xue and Dr Qasim Ayub for their guidance and for their critical and constructive assessment of my work. I would also like to thank Dr Marc Haber for his help in the day to day work. Many thanks to the rest of my thesis committee, Dr Toomas Kivisild and Dr Daniel Gaffney for their insightful comments and ideas on my work. I am also very grateful to the Wellcome Sanger Institute Graduate Programme and the Committee of Graduate Studies, as well as the Wellcome Trust for my PhD studentship. My sincere thanks to all my collaborators including Dr Thirsa Kraaijenbrink, Prof. Peter de Knijff, Dr Asan, Prof. Mark Jobling and Prof. George van Driem for providing their expertise and useful insight on Himalayan populations. Thank you to other people who contributed to this project including Dr Michał Szpak, Dr Massimo Mezzavilla, Dr Laurits Skov, Dr Shane McCarthy and Dr Yuan Chen for the fruitful discussions and their contribution with some of the analyses of this project. Many thanks also to Dr Andrew Knights, Dr Sebastian Lukasiak and Dr Irene Gallego- Romero for their expertise and advice in the design of the functional work on EPAS1. Thank you also to Dr Nikolaos Panousis for his assistance with the RNA-seq data and to Dr Fengtang Yang and the Cytogenetic Core Facility for the karyotyping and support with the cell lines. I would also like to thank all current and former Human Evolution team members, in particular Dr Pille Hallast, Mohamed Almarri, Dr Javi Prado Martinez and Dr Michał Szpak for their support, stimulating discussions over lunch break and all the fun we had together over the years. Thank you to my fellow PhD students, especially Mari Niemi for all the days we spent together with endless hours of work before deadlines. Also thank you to all other friends at campus for the coffee breaks and interesting chats and the gym clubbers for the good time training together. Many thanks to my all my friends in Cambridge, especially María for her positivity and encouragement over the years and the Italian community, including Claudia, Cinzia, Luca, Annalisa, Giuseppe and Gianni for their support, love and for making me always feel at home. I also thank my best friends Francesca, Martina, Chiara, Marina and Chiara for being always with me regardless of our physical distance and for believing 8 in me every time. Thanks to my friend Simone for making me smile every time even in very stressful situations. Many thanks to my boyfriend Chris for his love, support and patience in a difficult moment of my life. Last but not the least, I would like to thank all my family: my parents Cristina and Virgilio, my grandparents Clara e Mario, my grandmother Giovanna that is no longer with us, my cousin Silvia and my aunt Anna, my uncle Arturo and his wife Barbara, and all the others for supporting me throughout my life. 9 Abstract The Himalayan mountain range contains the highest peaks on Earth and has provided a diversity of environments for humans, some of which have required substantial genetic adaptation. I have used a combination of SNP-chip data, genome sequences and functional studies to explore the demographic history, genetic structure and signatures of adaptation in Himalayan populations. Eight hundred and eighty three individuals from 49 different autochthonous groups from Nepal, Bhutan, North India, and the Tibetan Plateau in China were genotyped for ~600,000 genome- wide SNPs. High-coverage whole-genome sequences of 87 individuals from a subset of these populations plus three additional ones were also generated. Himalayan populations share a common genetic component derived from a single ancestral population, followed by the development of local fine structure correlating with language and geographical distribution. I find higher genetic diversification within the Himalayan populations than in the surrounding regions which correlates with the distribution of Indo-European and Tibeto-Burman speakers, suggesting that both language and geography have influenced the genetic structure of these populations. I refined Himalayan population demographic history, using both autosomal and uniparental sequences. Himalayan populations display different proportions of gene flow with neighbouring populations and diverse effective population sizes and split times. The Y-chromosome lineages identified are common in South and East Asia and Tibet, but mostly form distinct clusters in the Himalayas. High altitude adaptation seems to have originated in a single ancestral population and then spread widely in the Himalayan region in the last 5,000 years. Genetic signatures of adaptation to high altitude are observed in the Endothelial PAS Domain Protein 1 gene (EPAS1) and several other known and novel candidates. EPAS1 has previously been reported to be under selection and involved in adaptation to living at high altitudes and was suggested to result from introgression of DNA from an extinct hominin species (Denisovans) into Tibetans. However, functional studies of EPAS1 variants have not been systematically carried out and it is still unknown which variant(s) are responsible for high altitude adaptation and their mechanism of action. I used both in silico and in vitro studies to explore these topics and validate EPAS1 candidate regulatory variants. The introgressed haplotype extends for over 300 kb spanning six 10 genes (EPAS1, TMEM247, ATP6V1E2, RHOQ, CRIPT, PIGF). I optimised a protocol to induce hypoxia in cell lines with and without the Denisovan introgressed haplotype. Preliminary results show that in cell lines without the introgressed haplotype, EPAS1 expression increases under hypoxic conditions, whereas in the cell lines with the introgressed haplotype the expression of EPAS1 remains constant. The most likely functional candidate variants fall in a ~32.7kb region within EPAS1. High altitude adaptation thus seems to be driven by EPAS1 as well as coordinated by other genes involved in the hypoxic response. 11 Table of Contents List of figures .................................................................................................................... 13 List of tables ...................................................................................................................... 17 1. Introduction .................................................................................................................. 19 1.1 Human origins, ancient migrations and mixing ....................................................... 19 1.2 Genetic variation and positive selection in human populations............................... 22 1.2.1 Human genetic variation ................................................................................... 22 1.2.2 Natural selection ............................................................................................... 29 1.3 The Himalayan region: an overview ........................................................................ 32 1.4 High altitude adaptation and the hypoxia molecular pathway ................................. 35 1.4.1 Adaptation at high altitude ................................................................................ 35 1.4.2 Hypoxia molecular pathway ............................................................................. 38 1.5 Aims and objectives ................................................................................................. 42 1.6 Outline of the thesis ................................................................................................. 43 2. Population genetic analyses of Himalayan samples using SNP-genotype data ...... 45 2.1 Introduction .............................................................................................................. 45 2.2 Materials and Methods ............................................................................................. 49 2.2.1 Samples and dataset .......................................................................................... 49 2.2.2 Methods used for the analyses .......................................................................... 52 2.3 Results