ANALYSIS OF MITOCHONDRIAL DNA VARIATION IN THE EGYPTIAN POPULATION AND ITS IMPLICATIONS FOR FORENSIC DNA ANALYSIS RANIA GOMAA A thesis submitted in partial fulfilment of the requirements of Nottingham Trent University for the degree of Doctor of Philosophy July 2010 ABSTRACT The genetic sequence of human mitochondrial DNA (mtDNA) is of particular interest to forensic investigations involving human identification, as well as population genetics. The current mtDNA database is lacking sufficient representatives of African mitochondrial DNA sequences compared to European and Asian sequences. The present study was concerned with the analysis of mtDNA in the Egyptian population. FTA cards were used for blood sample collection, storage, and shipment and DNA extraction. An optimised laboratory protocol for rapid PCR amplification of the mitochondrial hypervariable regions was developed. A database of 261 mitochondrial hypervariable region I (HVI) sequences and 78 hypervariable region II (HVII) sequences was established from 261 adult Egyptians. A total of 113 polymorphic sites were reported in the HVI region (nt16024-16365) which identified a total of 187 different haplotypes, of which 151 were unique to single individuals. The most commonly observed HVI haplotype was identical to the Cambridge Reference Sequence (CRS). Analysis of 78 HVII sequences (nt73-340) revealed a total of 42 polymorphic sites that identified 62 different haplotypes, of which 51 were unique to single individuals. Sites that showed the highest variability in the HVI and HVII regions agreed with the previously reported mutational hotspots. Combination of the HVI and HVII data resulted in identification of 207 different mitochondrial haplotypes, of which 183 (~88%) were unique to single individuals. Such a large number of unique mitochondrial haplotypes indicates a high diversity of mtDNA in the Egyptian population, which has a direct impact on forensic applications, since the significance of a match between an evidence sample and a reference sample depends on the population frequency of a profile. The random match probabilities in the HVI and HVII datasets were 2.34% and 3.22%, respectively. Combination of the two datasets reduced the random match probability to 1.28%. The genetic diversities in the HVI and HVII sequences were estimated to be 0.9804 and 0.9803, respectively, whereas, the genetic diversity in the combined dataset was 0.9911. A new strategy was developed to facilitate Restriction Fragment Length Polymorphism (RFLP) analysis of the whole mitochondrial genome for the purpose of haplogroup assignment. All the Egyptian individuals were assigned to well known mitochondrial haplogroups and the frequency distribution of different lineages was estimated. A mixed maternal ancestry of the Egyptian population was reported via detection of a mixture of mitochondrial DNA lineages with varying racial backgrounds. The most frequently reported mitochondrial i lineages were haplogroup T (13.8%) followed by haplogroups L3 (12.6%), H (12.3%), U (~10%) and M (8.4%). The genetic relationship between Egypt and its neighbours was revealed, and it was found that present day Egyptians were the closest African population to the Middle East and Europe, with an overall 62.5% European, 25% African, and 12.5% Asian mitochondrial lineages. The data presented here will enrich the limited Egyptian mitochondrial DNA reference data currently available for forensic applications. ii To the memory of my late Mother-in-law, Mrs Fawkiya Who was looking after my family while I was doing my PhD. Without her kind help, this thesis could never have been completed. iii ACKNOWLEGEMENTS I would like to thank my director of study Dr David C Hughes for guiding me into this project and giving me the opportunity to benefit from his valuable expertise in Molecular Biology and Bioinformatics. He was always supportive and sympathetic. I would also like to acknowledge my second supervisor Prof. Ellen Billet for her interest and support. I wish to thank my past supervisor Prof. Mark Darlison who is really missed by leaving NTU. I would also like to acknowledge Mr David Butler for his continual support and encouragement, for informing me about Forensic Science Events at NTU and elsewhere and for nominating me to represent NTU at the International Networking Event in Forensic Science, in Thailand. I would like to thank Dr Christian Thode for being my internal assessor for three years and for providing me with valuable comments and advice. I would also like to thank all my friends at NTU, past and present, especially Dr Sara Croft, Amy Pole, Tina Schwabe, Noha Juma, Fathiya Asteal, Vinaya Phatak, and Muralitharan Ponniah. They have always been supportive and helpful. I have greatly enjoyed my time at NTU. I wish to thank the Egyptian government for the generous fund for this research as a part of a PhD scholarship. I would also like to thank my Professors at Alexandria University who nominated me for this scholarship. I would like to thank my family, especially my Mum and my Dad for giving me all kinds of support and encouragement. I would definitely like to acknowledge my son Seif for being a sensible young man. I wish to thank my lovely daughter Hana for being good. Last but certainly not least, my biggest heartfelt gratitude must go to my husband Mohamed. I could not have completed this project without his indefinite support, love, sympathy and encouragement. iv TABLE OF CONTENTS ABSTRACT ..................................................................................................................... I ACKNOWLEGEMENTS............................................................................................ IV TABLE OF CONTENTS ............................................................................................... V LIST OF FIGURES…………………………………………………………………XIII LIST OF TABLES……………………………………………………………… XVIII ABBREVIATIONS ..................................................................................................... XX 1. GENERAL INTRODUCTION ...................................................................... 1 BIOLOGICAL ISSUES ....................................................................................................... 1 1.1 DNA and human identification ........................................................................ 1 1.2 Mitochondrial genome ..................................................................................... 3 1.2.1 Mitochondria ................................................................................................. 3 1.2.2 Structure and gene content of the mitochondrial DNA ................................. 3 1.2.3 Cambridge Reference Sequence (CRS)…………………………………….5 1.2.4 Mitochondrial inheritance and recombination .............................................. 6 1.2.4.1 Paternal inheritance of mtDNA ............................................................ 7 1.2.4.2 Recombination characteristics of mtDNA ........................................... 8 1.2.5 Heteroplasmy within the mitochondrial genome .......................................... 9 1.3 Modern human populations ............................................................................ 12 1.3.1 Population genetic variation ........................................................................ 13 1.3.2 Mitochondrial Eve ....................................................................................... 13 1.3.3 Pattern of modern human migration ........................................................... 13 1.4 Mitochondrial DNA haplogroups ................................................................... 16 1.4.1 Definition of mitochondrial DNA haplogroups .......................................... 16 1.4.2 Nomenclature of the mitochondrial DNA haplogroups .............................. 18 1.4.3 Distribution of the mitochondrial DNA haplogroups around the world ..... 19 1.4.3.1 African specific mitochondrial haplogroups ..................................... 19 1.4.3.2 Asian specific mitochondrial haplogroups ........................................ 19 1.4.3.3 European Caucasian mitochondrial haplogroups .............................. 20 1.4.3.4 Native American mitochondrial haplogroups .................................... 20 v 1.4.4 Definitive markers for the identification of mitochondrial DNA haplogroups ..................................................................................................................... 21 1.5 Impact of mitochondrial DNA on the forensic domain .................................. 25 1.5.1 Medico legal peculiarities of the mitochondrial genetic system .............. 25 1.5.2 Mitochondrial DNA as a useful tool in human identification (case studies) ..................................................................................................................... 26 1.5.2.1 Identification of historical cases by mitochondrial DNA analysis..... 26 1.5.2.2 Genetic identification of human skeletal remains from mass graves .. 27 1.5.2.3 Utility of mitochondrial DNA analysis in mass disasters .................. 28 1.5.2.4 A case of exchanged body remains solved by mtDNA analysis ........ 30 1.5.2.5 Identification of burned human remains using mtDNA analysis ....... 30 1.5.2.6 A rape case solved by mtDNA mixture analysis ............................... 31 1.6 The association of mitochondrial DNA haplogroups with the health status ... 32 1.7 Amelogenin ....................................................................................................