GENETIC POLYMORPHISM OF MITOCHONDRIAL CONTROL REGION AS A TOOL FOR FORENSIC DISCRIMINATION AND COMPARISON OF ITS DIVERSITY AMONG VARIOUS ETHNIC GROUPS OF PAKISTAN A THESIS SUBMITTED TO UNIVERSITY OF HEALTH SCIENCES IN FULFILMENT OF THE DOCTOR OF PHILOSOPHY IN DISCIPLINE OF HUMAN GENETICS AND MOLECULAR BIOLOGY By Dr. Shahzad Bhatti JULY, 2015 UNIVERSITY OF HEALTH SCIENCE LAHORE- PAKISTAN i CERTIFICATE It is hereby certified that this thesis is based on the results of experiments carried out by Mr. Shahzad Bhatti and that it has not been previously presented for PhD. Degree. Mr. Shahzad Bhatti has done his research work under my supervision. He has fulfilled all the requirements and is qualified to submit the accompanying thesis for the degree of Doctor in Philosophy. PROF.DR. MUHAMMAD ASLAMKHAN M.Sc. (Punjab), D.Sc. (Maize, Germany), FFBSP, FRSTM & FPAMS, Professor& Supervisor Department of Human genetics and Molecular biology University of Health Sciences Lahore, Pakistan. ii ACKNOWLEDGMENT All praises and thanks to the grace of ALLAH ALMIGHTY, WHO is the ultimate source of all knowledge to mankind. He bestowed man with intellectual power and understanding and gave him spiritual insight enabling him to discover his “Self” know his Creator through His wonders and conquer nature. Bow in obscene, I before my Lord, WHO bestows me to fortitude and impetus to accomplish this task and elucidate a drop of already existing ocean of knowledge. WHO made me reach at present pedestal of knowledge with quality of doing something adventurous, novel, thrilling, sensational, and path bearing. Next to all His Messenger HAZRAT MUHAMMAD (Peace Be upon Him) Who is an eternal torch of guidance and fountain of knowledge for humanity, Who made mankind to get out of depths of evil & darkness. It is indeed my honor and pleasure due Dr Muhammad Aslamkhan, Prof, University of the Health Sciences Lahore, for providing me this opportunity to work in Human genetics Molecular Biology and supervise me in solving problems His kind behavior coupled with friendly attitude towards science was a source of inspiration for me. The work presented in this manuscript was accomplished under his enthusiastic guidance, sympathetic attitude and intellectual supervision. I express sincere appreciation to Dr. MH Qazi, Vice Chancellor, The University of Lahore, from the core of my heart for his guidance, assistance, encouragement and providing research facilities in IMBB Lab during my research work. Without the help of him I was unable to complete my research work. I have no words of thanks for his sincere attitude. With profound gratitude and deep sense of devotion and obligation, I wish to especially recognize the guidance and encouragement extended by my wife Miss SANA Abbas their continuous support pulled me through the tough hours of my research. Last but not the least, I am enormously grateful to all those who taught me even a single word of knowledge. I am greatly thankful to Mr. Shahid (lab Attendant) for his assistance and continuous encouragement during the span of my study. SHAHZAD BHATTI iii ACRONYMS Abbreviation Details ATP Adenosine triphosphate bp base pair C-stretch cytosine stretch ddNTP dideoxynucleotides ddNTP’s dideoxynucleotides D-loop Displacement loop DNA Deoxyribonucleic acid GD Genetic Diversity HVS-I Hypervariable segment I HVS-II Hypervariable segment II HVS-III hypervariable segment III mRNA Messenger RNA mtDNA Mitochondrial DNA OXPHOS Oxidative phosphorylation P Probability value PCR Polymerase chain reaction rCRS Revised Cambridge refrence sequence RFLP Restriction fragment length polymorphism RMP random match probability RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid SNP Single nucleotide polymorphism STR Short tandem repeats tRNA Transfer Ribonucleic acid MYA Million years ago KYA Kilo years AMHs Anatomical modern Humans ybp years before present kya Kilo years ago iv TABLE OF CONTENTS Page No. CERTIFICATE ii ACKNOWLEDGMENT iii ACRONYMS iv LIST OF FIGURES vi LIST OF TABLES X ABSTRACT xii INTRODUCTION 1 REVIEW ABOUT PAKISTAN 27 SUBJECTS AND METHODS 65 RESULTS 75 DISCUSSION 155 REFERENCES 182 v LIST OF FIGURES No. Title Page No. 1.1 Map of Lake Turkana in Kennya 2 1.2 Multiregional and out of Africa model 4 1.3 Route Trajectory followed by the Migrant of Africa 6 1.4 Demonstrating Out of Africa Route 7 Mitochondrial DNA showing different Coding and Non coding 1.5 9 regions Updated comprehensive phylogenetic tree of global human 1.6 11 mitochondrial DNA The deep embedded autocanthus clads of M, N and R, 1.7 predominantly inhabit in South Asia and other lineages shared 13 with East Asians and West Eurasians 1.8 Map of Pakistan 27 1.9 Indo-Gangetic Plain 29 1.10 Map of Sistan or Helmand River Drainage Basin 30 Shows the Indus civilization. The archeological sites are marked 1.11 32 with Red dots. 1.12 Excavated sites of Harappa (A) and Moenjodaro (B) 34 1.13 Scanned Rout of Indo-Aryan in Indian Subcontinent 36 Alexandria on Indus situated at the adjunction of Indus and 1.14 39 Acesines Illustration of Map of Pakistan showing an administrative 1.15 43 divisions and Provinces Illustration of Map of Pakistan showing a population per square 1.16 44 meter in the Provinces. 1.17 Social structuring of South Asian population 53 3.1 DNA quantification from blood samples 81 PCR amplification of HVI & HVII segment of mitochondrial 3.2 82 control region vi PCR amplification of HVIII segment of mitochondrial control 3.3 83 region Sequence of HVI segment of mitochondrial control region and 3.4 85 its dendogram Sequence of HVII segment of mitochondrial control region and 3.5 86 its dendogram Sequence of HVIII segment of mitochondrial control region and 3.6 87 its dendogram Dendograms of HVI segments of mtDNA in Pakistani 3.7 90 population Dendograms of HVII segments of mtDNA in Pakistani 3.8 93 population Dendograms of HVIII segments of mtDNA in Pakistani 3.9 96 population Most common transition was c → t at position 16223bp in HVI 3.10 97 region Most common transition was a → g at position 263bp in HVIII 3.11 98 segment Most common transition was t →c at position 489bp of HVIII 3.12 99 region 3.13 C-stretch heteroplasmy in HVII region 100 3.14 Deletion of “C” in control region of mtDNA 102 A network relating haplogroup M of Pakistani caste and tribal populations. Circle areas are proportional to the haplotypes 3.15 116 frequencies and variant nucleotides are numbered and shown along the links between haplotypes. Reduced median networks of haplogroup N based on Pakistani caste and tribal population. The number along the branched 3.16 denotes mutations with reference to CRS (andereson et al 1981). 117 While circle areas are proportional to the haplotypes frequency and reticulations indicate parallel mutational pathways. A phylogenetic network of haplogroup R of Pakistani caste and tribal populations estimated by reduced median method. The size 3.17 of node constitutes frequency distribution of each haplotype and 119 the identities of mutations that define major haplogroup subset are portrayed along selected internodes. vii Major subset of haplogroup U in Pakistani caste and tribal population are exhibited in reduced median networks. Frequency 3.18 121 of each haplotype in term of nodal size and variant nucleotides are numerated and displayed along the links between haplotypes. A reduced median networks of haplogroup J in Pakistani caste and tribal populations. Nodal size acquainted the haplotype 3.19 frequency and reticulation indicated multiple mutations and 123 variant nucleotides are listed as indicated along the links between haplotypes. Phylogeny of haplogroups W AND T are estimated by constructed reduced median networks of Pakistani caste and 3.20 tribal populations. The size of each node directly proportion to 125 the frequency of haplotypes and variant nucleotides were enumerated and shown along the links between haplotypes. A network relating haplogroup H in Pakistani caste and tribal populations. Circle areas are proportional to the haplotypes 3.21 127 frequencies and variant nucleotides are numbered and revealed along the links between haplotypes. An un-rooted neighbor joining network of 22 populations based 3.22 on genetic distances among caste communities and inset 129 indicates the relationship for the major population groups. Graphical presentation of major haplogroups percentage in 22 3.23 130 populations of Pakistan MDS plot of 22 caste of Pakistani population, based on Fst 3.24 131 distances. Electropherograms of multiplex A & B for individual of 3.25 132 Haplogroup M Primer extension assay of multiplex A and B that exhibited 3.26 133 Haplogroup R Electropherograms of multiplex A and B for individual of 3.27 134 Haplogroup N Electropherograms of Multiplex A and B for Individual of 3.28 135 Haplogroup H Electropherograms of multiplex A and B for individual of 3.29 136 haplogroup J Electropherograms of multiplex A and B for individual of 3.30 137 Haplogroup U viii Electropherograms of multiplex A and B for individual of 3.31 138 haplogroup T 3.32 Mismatch distribution in Rajput population 144 Haplotype distance matrix between/within the 22 ethnic groups 3.33 145 of Pakistani population. 3.34 Matrix of pairwise Fst 146 Average number of pairwise differences within and 3.35 147 between population 3.36 Haplotype frequencies in 22 isonym Population of Pakistan 148 3.37 Expected heterozygosity in 22 ethnic groups of Pakistan 149 Number of alleles at different loci in 22 isonym groups of 3.38 150 Pakistan Molecular diversity indexes of 22 isonym population of 3.39 151 Pakistan. 3.40 Co-ancestory coefficient matrix 152 3.41 Slatkin’s linearized Fst’S 153 Detection of loci under selection from genome scans based on 3.42 154 Fst ix LIST OF TABLES No Title Page.