Adygei (n = 20), CEU (n = 20) and 1 chimpanzee sample were used as controls.

5

I. ABSTRACT

The proposed Master Thesis refers to the results of the research work carried out at the Wellcome Trust Sanger Institute, Hinxton, UK, under the direct supervision of Dr. Chris Tyler-Smith, Head of the Human Evolution Team.

The aim of the study is to ascertain signals of positive selection in human populations living at high altitude using targeted next-generation DNA re-sequencing of candidate genes and control regions in 96 individuals. For this purpose 55 Daghestani from three ethnic groups were selected as case populations living at high altitudes (>2,000 msl).

Adygei (n = 20), CEU (n = 20) and 1 chimpanzee sample were used as controls.

The Daghestani populations have a long history of permanence (>10.000 years) at high altitudes and their peculiar demographic history make them suitable for hypoxia adaptation studies. In order to disentangle selective and demographic effects on the genome, fifteen candidate genes involved in oxygen metabolism (HIF1α; PHD1; PHD2;

PHD3; VHL; EPO; EPOr; VEGF; EDN1; NOS3; ACE; α,β,γ,δ-Hb) were sequenced together with twenty-seven “control regions” selected among those used in the Hominid Project (Wall, Cox et al. 2008) and the ENCODE3 Project

(http://www.genome.gov/10005107).

DNA samples obtained from whole blood or cell cultures were used to amplify the targeted genomic regions by long-template PCR. The amplimers were visualized by gel electrophoresis and those belonging to the same individuals were pooled together and subsequently purifiedusing the QIAquick PCR Purification Kit. Each pooled sample was indexed by adding an eight nucleotides tag, and 8 samples were combined and sequenced on each lane of an Illumina flow-cel. The sequenced DNA was sorted and aligned to the reference sequence using the MAQ algorithm (Li, Ruan et al. 2008). Parameters checking the goodness of the sequencing outputs as well as summary statistics and neutrality tests (Tajima’s D, Fu and Li’s D and F, Fay and Wu’s H) were calculated on “PHASED”

haplotype data. Candidate targets of positive selection (re-sequenced genes) and neutral

regions were analyzed for putative signals of adaptation to hypoxia and several novel

SNPs characterized. Particularly, four non synonymous mutations were found responsible

for altering the functionality of HIF1α, ACE, EPOr and NOS3 and could be considered as

putative candidate for hypoxia adaptation.

6 II. TABLE OF ABBREVIATIONS

Aa Amino acid

ACE Angiotensin Converting Enzyme AGT Angiotensin

Blast Best Local Alignment Search Tool (software acronym) CEU Caucasian Europeans Chr Chromosome

cM centi-Morgan

dd,d,NTP di-deoxy, deoxy, Tri- phospate Nucleotide DNA Deoxyrybonucleic Acid EBI The European BioInformatics

Institute EDN1 Endoteline

eFN Encode False Negative rate ENCODE Encyclopedia of Coding

Regions EPO Erythropoietin

EPOr Erythropoietin Receptor EST Expressed Sequenced Tag FN False Negative rate FP False positive Rate Fst Fixation index

G,M,K,bp 10

, 10

, 10

, base pairs Glu Glutammate

HapMap Haplotype Map of the human genome Hb Haemoglobin

HGDP Human Genome Diversity Project HIF1α Hypoxia Inducible Factor (alpha

subunit)

IRAS Integrated Research Application System

Indel Insertion/Deletion in a sequence Kybp 1000 years before present LD Linkage Disequilibrium Lys Lysine

MAQ Mapping and Assembly with Quality (software acronym) MDS Multi-Dimensional Scale Msl Meters above sea level SEQ Re-sequencing data from the

present study

NOS3 Nitric Oxide Synthetase 3 NR Non Reference

NS Non Synonymous

PCR Polymerase Chain Reaction PHD1,2,3 Prolyl Hidroxylase

Domain-Containing Protein

REC Research Ethical Committee rFP Raw False Positive rate

SNP Single Nucleotide Polymorphism STR Short Tandem Repeat

UTR Untranslated Region

VEGF Vascular Endothelium Growth Factor

VHL Von Hippel Landau Tumour Suppressor Gene

WGA Whole Genome Amplification WTSI The Wellcome Trust Sanger

Institute