A STUDY OF CERTAIN ASPECTS OF HUMAN GENETICS INCLUDING CONSANGUINITY AND GENETIC DISORDERS IN HUMAN POPULATION OF DG KHAN By MUHAMMAD AMIN UD DIN THESIS SUBMITTED TO BAHAUDDIN ZAKARIYA UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 2007 INSTITUTE OF PURE AND APPLIED BIOLOGY BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN, PAKISTAN In the name of Allah The most compassionate The most Merciful, the most Beneficent Dedicated To My parents Contents S.NO CONTENTS Page NO 1 Acknowledgments I 2 List of Acronyms III 3 List of Tables V 4 List of Figures VII 5 Abstract XV 6 Chapter 1: Introduction • Preamble 1 • Consanguinity 2 • Genetic Disorders 6 9 • Dera Ghazi Khan 12 • Objectives of the Study 7 Chapter 2: Materials And Methods 15 • Data Collection 15 • Ethnicity 16 • Couple’s Relationship and Marriage Types 16 • Educational Status 16 • Socio-economic Status of Husband 17 • Occupational Status 17 • Pregnancy, Mortality, Morbidity 17 • Consanguinity and Genetic Disorders 17 • Families Studied 18 • Clinical Study 18 • Blood Sampling --------------------------------------------------------------------------------------------------------------------------------------------------------------- A study of certain aspects of human genetics including consanguinity and genetic disorders in the human population of DGK Contents 19 • Genomic DNA Extraction 19 • Polymerase Chain Reaction (PCR) 20 • Horizontal Gel Electrophoresis 20 • Vertical Gel Electrophoresis 20 • Genotyping 21 • Linkage Studies 21 • Mutation analysis 22 • Amplification Refractory Mutation System-PCR 22 • Multiplex ARMS- PCR 23 • Data Analyses 8 Chapter 3: Consanguinity 31 • Introduction 32 • Materials and Methods 32 • Results 32 1. Data Collection 32 2. Marriages 32 2.1. Endogamous and Exogamous Marriages 33 2.2. Distribution of Marriage Types 33 2.3. First Cousin Marriages, Sub-Types 33 2.4. Consanguineous Marriages 34 2.5. Trend of Consanguinity 34 2.6. Mean Inbreeding Coefficient (F) 34 3. Factors Effecting Rate of Consanguinity 35 3.1. Male Education 35 3.1.1. Marriage Types and Male Education --------------------------------------------------------------------------------------------------------------------------------------------------------------- A study of certain aspects of human genetics including consanguinity and genetic disorders in the human population of DGK Contents 36 3.1.2. Consanguinity and Male Education 35 3.2. Female Education 37 3.2.1. Marriage Types and Female Education 37 3.2.2. Consanguinity and Female Education 38 3.3. Male Socioeconomic Status 38 3.3.1. Marriage Types and male Socioeconomic Status 39 3.3.2. Consanguinity and male Socioeconomic Status 40 3.4. Male Occupation 40 3.4.1. Marriage Types and Male Occupation 40 3.4.2. Consanguinity and Male Occupation 41 4. Effects of Consanguinity 41 4.1. Reproductive Outputs 41 4.1.1. Pregnancies 42 4.1.2. Pregnancy loss 43 4.1. 3. Perinatal deaths 44 4.1.4. Infant deaths 44 4.2. Genetic Disorders 46 • Discussion 9 Chapter 4: Skin Disorders • Introduction 70 • Ectodermal dysplasia 71 • Alopecia 71 • Families Studied 73 • Linkage Studies 77 • Mutation Screening 78 --------------------------------------------------------------------------------------------------------------------------------------------------------------- A study of certain aspects of human genetics including consanguinity and genetic disorders in the human population of DGK Contents • Discussion 78 10 Chapter 5: Non-syndromic Deafness • Introduction 98 • Families Studied 100 • Linkage Studies 102 • Mutation Screening 103 • Discussion 103 11 Chapter 6: β-Thalassemia • Introduction 115 • Subjects Studied 116 • Molecular Analysis 117 • Ethnic Distribution of β-Thalassemia Mutations 117 • Distribution of β-Thalassemia mutations in various 118 castes • Regional Distribution of β- Thalassemia Mutations 118 • Discussion 119 12 Chapter 7: References 129 • Electronic Database Information 151 • Government Documents --------------------------------------------------------------------------------------------------------------------------------------------------------------- A study of certain aspects of human genetics including consanguinity and genetic disorders in the human population of DGK Acknowledgments ACKNOWLEDGMENTS It is a pleasure to thank the many people who made this thesis possible. Firstly, I take this opportunity to express my gratitude to my supervisor, Professor Dr. Abdus Salam, Institute of Pure and Applied Biology, Bahauddin Zakariya University Multan, under whose benign supervision and able guidance, this research work was done. His affectionate attitude and great devotion encouraged me at different moments of my studies. I offer my humble gratefulness to my Co-supervisor Professor Dr. Wasim Ahmad, Chairman Department of Biochemistry, Quaid-i-Azam University Islamabad.Throughout my research and thesis-writing period, he provided sound advice, affable company, lots of good ideas, and provided the laboratory facilities. His continuous encouragement enables me to achieve my goals. I wish to extend my heartiest thanks to Professor Dr. Javed Iqbal Mirza, Director, Institute of Pure and Applied Biology, Bahauddin Zakariya University Multan, for providence of facilities during present work. I am highly grateful to venerable Dr, Shahid Mahmood Baig (Principal Scientist), NIBGE, Faisalabad for his unconditional academic and technical support especially in molecular studies of thalassemia. I am deeply indebted to Prof Dr Saqlain Naqvi (Chairman Dept of Biochem, UAAR), Dr Arshad Rafiq (Assist Prof. Comsat IBD), Dr Ansar (Assist Prof.), Peter John (PhD scholar) and Jawad Hassan (PhD scholar) QAU Islamabad, for their great help, valuable suggestions, cooperation and collaboration during this study. I am thankful to Dr Sajid Malik, Dr Najeeb Haider, Mr. Rab Nawaz Khan Jalabani, and Ghulam Hussain Khosa who took pains in rechecking statistical results and proof reading the thesis. I am also grateful to my research fellows, Mr. Imran Khaliq, Mr. A B Gulshan, Mr.Shazad Jhangir, Mr. Qazi Abdul Ghafoor, Mr.M. Sharif and Mr. Wajahat Ali for their unforgettable cooperation in every event of this study like traveling, collection of families and photography. I’m also obliged to all the people on whom this research was conducted for their participation and cooperation. I -------------------------------------------------------------------------------------------------- A study of certain aspects of human genetics including consanguinity and genetic disorders in the human population of DGK Acknowledgments I convey my heartiest and sincerest acknowledgements to my friends, Late Dr. Shaheen Akhter (IBD), Prof. Dr Sardar Nasim Khan (AJKU), Dr Naeem (BZU), Mr. Sayed-ul-Haque (T&R Assistant, QAU), Mr. Abdul Wahab Khan (NWFP) and college colleagues Prof. Fayyaz Ahmad (Principal), Haji Akber Chandia, Mr. M. Anwar, Mr Mustafa Leighari, Mr.Iqbal Shah, Mr. Nasim chandia, Dr Rab Nawaz Monis, Mr. G M A Ghani, and Mr. Khalil Akbar for their pleasant company and valuable help. I owe my gratitude to my wife, children, and brothers who gave me their maximum cooperation and moral support during this study. Finally, I wish to thank ‘The Higher Education Commission (HEC)’, Islamabad Pakistan, for providing funds for research work presented in the thesis. Muhammad Amin ud Din September 2007 II -------------------------------------------------------------------------------------------------- A study of certain aspects of human genetics including consanguinity and genetic disorders in the human population of DGK List of Acronyms List of Acronyms AA Alopecia Areata APL Atrichia with Papular Lesions APMR Alopecia with Mental Retardation ARMS-PCR Amplification Refractory Mutation System-PCR bp Base Pair cM CentiMorgans CM Consanguineous Marriage DFC Double First Cousin DGKhan Dera Ghazi Khan dNTP Deoxynucleoside triphosphate DP Dermal Papilla DR Distantly Related DSC Desmocollin DSG Desmoglein EDAR Ectodysplasin 1 Anhidrotic Receptor EDTA Ethylendiamintetraacetic acid EVPL Envoplakin FBD Father’s Brother Daughter FC First Cousin FCOR First Cousin Once Removed FSD Father’s Sister Daughter GJB6 Gap Junction Beta-6 HJMD Hypotrichosis Congenital with Juvenile Macular Dystrophy HR Hairless HTTS Hypotrichosis Simplex of Scalp kb Kilo base LAH Localized Autosomal Recessive Hypotrichosis MBD Mother’s Brother Daughter mg Milligram --------------------------------------------------------------------------------------------------- III A study of certain aspects of human genetics including consanguinity and genetic disorders in the human population of DGK List of Acronyms MSD Mother’s Sister Daughter NCM Non Consanguineous Marriage NETH Netherton Syndrome ng Nano Gram NR Non Related rpm Revolution Per Minute SC Second Cousin SCOR Second Cousin Once Removed Taq Thermophillus aquaticus TBE Tris-Borate EDTA TGM Transglutaminase TE Tris-EDTA UV Ultra violet μl Micro liter --------------------------------------------------------------------------------------------------- IV A study of certain aspects of human genetics including consanguinity and genetic disorders in the human population of DGK List of Tables List of Tables Page Table No Title No 1.1 Population Stratification of Dera Ghazi Khan District 14 2.1 List of microsatellite markers used to test linkage to candidate 24 gene loci in families F and G 2.2 List of autosomal recessive nonsyndromic deafness loci tested for 26 linkage in the present study 2.3 Sequences of the primers used for PCR amplification of exons 2- 28 19 of the human