Molecular Genetic Studies of Psoriasis in Pakistani Population By Saeeda Munir Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2015 Molecular Genetic Studies of Psoriasis in Pakistani population A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biochemistry/Molecular Biology By Saeeda Munir Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan Dedicated To My beloved Parents Mr. and Mrs. Muhammad Munir Malik Whose best wishes, prayers and guidance at every step made me achieve such a success in life 2015 Table of Contents Acknowledgements……………………………………………………………………………………………………………. i List of Figures…………………………………………………………………………………………………………………….iii List of Tables.......................................................................................................................... iv List of Abbreviations.............................................................................................................. vi Abstract ................................................................................................................................. ix Chapter 1 Introduction ............................................................................................................................ 1 Epidemiology .......................................................................................................................... 1 The Skin .................................................................................................................................. 2 The Psoriasis Skin .................................................................................................................... 3 Clinical Features ...................................................................................................................... 4 Types of Psoriasis .................................................................................................................... 6 Type I Psoriasis........................................................................................................................ 6 Type II Psoriasis ....................................................................................................................... 6 Triggering Factors ................................................................................................................... 6 Diagnosis ................................................................................................................................ 7 Treatment ............................................................................................................................... 7 Topical Therapy....................................................................................................................... 7 Phototherapy .......................................................................................................................... 8 Systemic Treatments ............................................................................................................... 8 Biological Agents ..................................................................................................................... 9 Psoriasis as an Autoimmune Disease ....................................................................................... 9 Psoriasis Immunology ........................................................................................................... 10 Genetics of Psoriasis ............................................................................................................. 12 Family and Twin Studies ........................................................................................................ 13 Inheritance Models ............................................................................................................... 13 Identified Psoriasis Susceptibility Locus (PSORS) .................................................................... 14 Genetic Approaches to find associated genes in monogenic or complex disease…………………..33 Positional cloning…………………………………………………………………………………………………………………..33 Candidate gene approach………………………………………………………………………………………………………35 Aims of the Study .................................................................................................................. 37 Chapter 2 Materials and Methods ......................................................................................................... 40 Study Subjects....................................................................................................................... 40 Extraction of Genomic DNA from Venous Blood Samples ...................................................... 43 DNA Quantification ............................................................................................................... 44 HLA Typing ............................................................................................................................ 44 Polymerase Chain Reaction with Sequence Specific Primers for HLA Class and Class II Typing 46 Agarose Gel Electrophoresis for HLA ..................................................................................... 46 Meta-Analysis ....................................................................................................................... 48 Genotyping of ACE I/D Polymorphism ................................................................................... 48 SNP Genotyping .................................................................................................................... 50 KASP Genotyping .................................................................................................................. 50 KASP genotyping Assay ......................................................................................................... 52 Statistical Analysis ................................................................................................................. 54 Chapter 3 Role of HLA allelic and haplotype polymorphism ................................................................... 56 Introduction .......................................................................................................................... 56 HLA Association with Psoriasis .............................................................................................. 58 Results .................................................................................................................................. 65 Association Analysis of HLA Class I and Class II Alleles............................................................ 65 Haplotype Analysis of HLA Class I and Class II Alleles ............................................................. 67 Meta-Analysis of HLA Class I and Class II Alleles ..................................................................... 67 Discussion ............................................................................................................................. 81 HLA Class I And Class II Association with Psoriasis in Cases and Controls:............................... 81 Association of HLA Class I and Class II Alleles with Type I and II Psoriasis ............................... 82 Haplotype Analysis of HLA Class I and Class II Alleles ............................................................. 84 Meta-Analysis of HLA Class I and Class II Alleles ..................................................................... 85 Chapter 4 ACE gene polymorphism study .............................................................................................. 88 Introduction .......................................................................................................................... 88 Results .................................................................................................................................. 90 Discussion ............................................................................................................................. 94 Chapter 5 Exploring the candidate and GWAS SNPs for association ..................................................... 101 Introduction ........................................................................................................................ 101 Selected genes for the Study ............................................................................................... 102 Aims and Objectives of the Study: ....................................................................................... 120 Results ................................................................................................................................ 123 Discussion ........................................................................................................................... 132 Chapter 6 Conclusion .......................................................................................................................... 141 References.......................................................................................................................... 145 Appendix ............................................................................................................................ 194 ACKNOWLEDGEMENTS All praise for Almighty Allah (SWT) the most compassionate, the most beneficent and ever merciful, who gives me the power to do, the sight to observe and mind to think and judge.