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Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly

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Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly by Rizwana Kousar Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2014 Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly A thesis submitted in the partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry/ Molecular Biology by Rizwana Kousar Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2014 DECLARATION I hereby declare that the work presented in this thesis is my own effort, except where otherwise acknowledged, and that the thesis is my own composition. No part of this thesis has been previously published or presented for any other degree or certificate. Rizwana Kousar Contents CONTENTS ACKNOWLEDGEMENTS i LIST OF FIGURES ii LIST OF TABLES v LIST OF ABBREVIATIONS vi ABSTRACT ix Chapter 1 1. INTRODUCTION 1 1.1: Microcephaly 2 1.1.1: Etiology of Microcephaly 3 1.1.2: Diagnostic Criteria 3 1.2: MCPH Genetic Heterogeneity 4 1.2.1: Microcephalin/Mcph1/BRITI at MCPH1 5 1.2.2: WD repeat domain 62 (WDR62) at MCPH2 6 1.2.3: CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) at MCPH3 7 1.2.4: 152 KDa Centrosomal protein (CEP152) at MCPH4 9 1.2.5: Abnormal spindle-like microcephaly-associated (ASPM) at MCPH5 9 1.2.6: Centromeric protein J (CENPJ) at MCPH6 11 1.2.7: SCL/TAL1 interrupting locus (STIL) at MCPH7 12 1.2.8: 135 KDa centrosomal protein (CEP135) at MCPH8 13 1.2.9: 63 KDa centrosomal protein (CEP63) at MCPH9 13 1.2.10: MCPH10 14 1.2.11: Cancer susceptibility candidate 5 (CASC5) at MCPH11 14 1.3: Neurogenic Etiology of MCPH 15 1.4: Approaches to MCPH Gene Identification 19 1.5: MCPH spectrum in Pakistan 20 1.6: Objective of the Study 25 Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly Contents Chapter 2 2. MATERIALS AND METHODS 26 2.1: Families Studied 26 2.2: Blood sampling 26 2.3: Extraction of Human Genomic DNA from whole Blood 27 2.3.1: Phenol-chloroform method 27 2.3.2: Commercially designed kit method 28 2.4: Mapping of genes responsible for MCPH 28 2.4.1: Exclusion Mapping 28 2.4.1.1: Polymerase Chain Reaction (PCR) 29 2.4.1.2: Agarose Gel Electrophoresis 30 2.4.1.3: Polyacrylamide Gel Electrophoresis (PAGE) 30 2.4.2: SNP based Genotyping by Microarray 31 2.4.3: Statistical Packages for Linkage Analysis 31 2.4.4: Bioinformatics Tools 32 2.5: DNA sequencing of candidate genes 32 2.5.1: Primary PCR 32 2.5.2: Post primary PCR purification 33 2.5.3: Sequence PCR 33 2.5.4: Post sequence PCR purification 33 2.5.5: Mutation analysis 34 2.6: Amplified fragment length polymorphism (AFLP) analysis 34 Chapter 3 3. AUTOSOMAL RECESSIVE PRIMARY MICROCEPHALY 12 46 3.1: Clinical Description of Family A 46 3.2: Genotyping and linkage analysis 47 3.3: Screening of candidate genes 49 3.4: Discussion 50 Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly Contents Chapter 4 4. AUTOSOMAL RECESSIVE PRIMARY MICROCEPHALY 2 58 4.1: Clinical description 58 4.1.1: Family B 59 4.1.2: Family C 59 4.1.3: Family D 59 4.1.4: Family E 60 4.2: Linkage Analysis of MCPH families 60 4.3: Sequencing of WDR62 gene 62 4.4: Discussion 63 Chapter 5 5. 5. AUTOSOMAL RECESSIVE PRIMARY MICROCEPHALY 5 81 5.1: Clinical description 81 5.1.1: Family F 82 5.1.2: Family G 82 5.1.3: Family H 82 5.1.4: Family I 83 5.1.5: Family J 83 5.1.6: Family K 84 5.2: Linkage analysis 84 5.3: Sequencing ASPM gene 85 5.4: Amplified fragment length polymorphism (AFLP) analysis 87 5.5: Discussion 87 6. CONCLUSION 112 7. REFERENCES 114 Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Thanks to Almighty ALLAH, The most Gracious and most Merciful who blessed me more than I wished. I offer my praises to Hazrat MUHAMMAD (PBUH) who taught us to probe into the secrets of nature. I would like to express my special appreciation and thanks to my supervisor Dr. Muhammad Ansar, for his dynamic supervision, keen interest, valuable suggestions, insightful discussions and encouragement throughout my research work. I owe my deep gratitude to Prof. Dr. Wasim Ahmed. He is an icon whose research expertise, excellent teaching skills and professional devotion has enabled me to develop craze for the subject. I obliged to Chairperson Department of Biochemistry, Prof. Dr. Bushra Mirza, for providing research facilities and student friendly environment in the department. I submit special thanks to the authorities of Higher Education Commision (HEC) for providing me research grant under indigenous scholarship scheme and International Research Support Initiative Program (IRSIP) to support this project. Most importantly, I thank all the affected patients and their families that took part in this project and made this research possible. I would like to appreciate Dr. Jawad Hassan, Dr. Muhammad Salman Chishti, Dr. Musharraf Jelani, and Dr. Sulman Basit for being a valuable source of discussion and ideas as well. I wish to thank every lab fellow especially Dr. Umm-e-Kalsoom, Dr. Gul Naz, Dr. Bushra Khan, Dr. Mariam Khurshid, Dr. James J Cox, Hina Mir, Dr. Muzammil Ahmed Khan, Dr. Rabia Habib and Dr. Saad Ullah Khan Wazir for their kind help, moral support and continuous motivation during my research work. Words cannot express my feelings for the love, practical support and sacrifices of my parents, Father-in-law (Late), brothers, cousins, husband, and my cute Hania and Shaheer throughout the long journey to achieve this goal. I would also like to thank all of my colleagues at the Department of Biology, AIOU for their cooperation and encouragement to peruse Ph. D studies. Last but definitely not the least, I offer my regards and blessings to all of those who supported me in any respect during the completion of this project. Rizwana Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly I List of Figures LIST OF FIGURES Figure Title Page No. No. 1.1 A proposed model representing how mutant MCPH proteins could 18 affect neurogenesis. 3.1 (a) Pedigree of family A showing segregation of MCPH. 52 (b) Pictures of affected individuals. 52 (c) Image of Computed Tomography (CT) scan. 52 3.2 Image of SNP data analyzed by homozygosity mapper 53 3.3 Image of dChip data analysis in family A. 54 3.4 Haplotype of family A shows HBD region flanked by markers 55 D16S3082 (3.05Mb) and D16S675 (7.9Mb) on chromosome 16p13.3-13.2. 3.5 Diagram of chromosome 16 depicts the 4.85 Mb homozygous region 57 on 16p13.2-p13.3 locus identified in family A. 4.1 (a): Pedigree of family B showing segregation of MCPH. 66 (b): Pictures of affected individuals. 66 4.2 (a): Pedigree of family C showing segregation of MCPH. 68 (b): Computerized tomographic (CT) scan. 68 4.3 Pedigrees of families (a) D, (b) E showing segregation of MCPH. 69 4.4 Haplotype of family B shows homozygosity with WDR62 flanking 70 markers on chromosome 19. 4.5 Haplotype of family C depicts homozygosity with WDR62 flanking 72 markers on chromosome 19. 4.6 Haplotype of family D demonstrate homozygosity with WDR62 73 flanking markers on chromosome 19. 4.7 Haplotype of family E shows homozygosity with WDR62 flanking 74 markers on chromosome 19. 4.8 Mutation analysis of the WDR62 gene in family B. 75 4.9 Graphical representation ofWDR62 gene and protein. 77 Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly ii List of Figures 4.10 Mutation analysis of the WDR62 gene in family C. 78 4.11 Mutation analysis of the WDR62 gene in family D. 79 4.12 Mutation analysis of the WDR62 gene in family E. 80 5.1 Pedigrees of families (a) F, (b) G showing autosomal recessive 90 MCPH. 5.2 (a) Pedigree of family H showing autosomal recessive MCPH. 92 (b) Picture of affected individual (V-8) showing reduced head 92 circumference. 5.3 a) Pedigree of family I showing autosomal recessive MCPH. 93 (b) Pictures of Affected individuals (III-3) and (IV-1) presenting 93 characteristic sloping forehead. 5.4 Pedigrees of families (a) J and (b) K showing autosomal recessive 94 MCPH. 5.5 Haplotype of the family F shows homozygosity with ASPM flanking 95 markers on chromosome 1. 5.6 Haplotype of family G shows homozygosity with ASPM flanking 96 markers on chromosome 1. 5.7 Haplotype of the family H shows homozygosity with ASPM flanking 97 markers on chromosome 1. 5.8 Haplotype analysis of the family I shows homozygosity with ASPM 98 flanking markers on chromosome 1. 5.9 Haplotype of the family J shows homozygosity with ASPM flanking 99 markers on chromosome 1. 5.10 Haplotype of the family K shows homozygosity with ASPM flanking 100 markers on chromosome 1. 5.11 (a) ASPM gene; The structure of the ASPM gene 101 (b) ASPM protein 101 5.12 Mutation analysis of the ASPM gene in Family F. 102 5.13 Mutation analysis of the ASPM gene in Family G. 103 5.14 Mutation analysis of the ASPM gene in Family H. 104 5.15 Mutation analysis of the ASPM gene in Family I. 105 Mapping of Genes Responsible for Autosomal Recessive Primary Microcephaly iii List of Figures 5.16 Mutation analysis of the ASPM gene in Family J.
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