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Characterization of the Alpha Defensin Copy Number Variation in Humans

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Characterization of the Alpha Defensin Copy Number Variation in Humans Fayeza Fatima Khan Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy March 2012 1 Acknowledgements Foremost I would like to thank John for giving me the opportunity to work on this project. My success is down to his infallible mentoring. It has been a very educational and enjoyable time in JALA lab, a place I have looked forward to going to each day these last three years. Equally responsible for the great atmosphere and good times are my friends in the lab: Jess, Raquel, Suhaili, Somwang, Dannie, Tamsin, Sugandha, Holly, Dibo, Omniah, Laura and Ivan. I am especially thankful to Jess for helping me start up when I began, and continuous subsequent support, and to the alpha defensin team for sharing all things ‘alpha’. For the Cystic Fibrosis analysis, I am grateful to Dr. Jane Davies for her collaboration. I am indebted to the University of Karachi for their generous financial support for my PhD studies under HEC’s scholarship program. I must also thank all past teachers, but especially my Biology teacher at PAF College Lahore, Asim Butt. My base in biology (and love for it!) would certainly have been weaker without his direction. Lastly, I will say thank you to all friends and family for their encouragement and love, especially my parents, Dilawer S. Khan and Arifa Khan, and my grandmother, Bilquees Jan. 2 Contents ABSTRACT........................................................................................................6 1. INTRODUCTION........................................................................................7 1.1. Copy Number Variation in the Human Genome.................................7 1.2. Copy Number Assays .......................................................................12 1.3. The Defensin Peptides .....................................................................17 1.4. Human Alpha Defensin Genes and their CNV...................................23 1.5. Alpha Defensins and Disease: Effect of CNV.....................................27 1.6. Aims of This Thesis...........................................................................29 2. METHODS AND MATERIALS....................................................................31 2.1. Copy Number Measurement Assays ................................................31 2.2. Copy Number Validating Assays (Allele Ratio Assays).......................34 2.3. Capillary Electrophoresis .................................................................37 2.4. Assigning Copy Numbers .................................................................38 2.5. DEFA1A3 del Assay ..........................................................................39 2.6. Correlation with microarray data.....................................................39 2.7. Segregation Analysis........................................................................41 2.8. SNP Association with Copy Number Haplotypes ..............................43 2.9. rs4300027 Genotyping Assay...........................................................43 2.10. DEFA4 Deletion Assay ..................................................................45 2.11. PRT DEFTP1 and usDEL Assay .......................................................48 2.12. Upstream Sequence Replacement Assay......................................50 2.13. Protein Methods ..........................................................................52 2.14. Samples, General Reagents and Procedures.................................56 3 3. DEFA1A3 COPY NUMBERS......................................................................59 3.1. Total Measured Copy Numbers .......................................................60 3.2. Copy Number Measurement Quality ...............................................64 3.3. DefHae3 and Indel-5 Assay ..............................................................76 3.4. Conclusion and Discussion ...............................................................79 4. DEFA1A3 HAPLOTYPES ...........................................................................81 4.1. Segregation Analysis of CEPH families..............................................81 4.2. Copy Number Haplotypes in Europeans...........................................86 4.3. Copy Number Haplotypes and SNPs in Europeans ...........................92 4.4. Copy Numbers and SNP Genotypes in Non-European HapMaps ......98 4.5. Upstream Replacement Polymorphism..........................................102 4.6. Conclusion and Discussion .............................................................108 5. INTERROGATING GENOME WIDE ASSOCIATION STUDIES (GWAS)........110 5.1. SNPs as CNV Surrogates.................................................................111 5.2. rs4300027 as a Surrogate for DEFA1A3 Copy Number ...................112 5.3. Investigating GWAS .......................................................................113 5.4. Conclusions and Discussion ...........................................................117 6. EXPRESSION OF DEFA1A3 GENES .........................................................118 6.1. Synthesis of DEFA1/2/3 peptides...................................................118 6.2. Neutrophil variability.....................................................................119 6.3. Determining Alpha Defensin Peptides in Neutrophil Extracts.........121 6.4. Acid Urea PAGE .............................................................................125 6.5. Quantifying Alpha Defensin Peptides (DEFA1/2/3).........................127 6.6. Investigating a Role for Alpha Defensins in Cystic Fibrosis..............128 6.7. Conclusion and Discussion .............................................................137 4 7. DISCUSSION .........................................................................................139 7.1. Measurement of multiallelic CNVs.................................................139 7.2. Evolution of DEFA1A3 Haplotypes .................................................141 7.3. DEFA1A3 CNV and Clinical Phenotypes ..........................................143 8. Bibliography.........................................................................................146 5 ABSTRACT Copy number variation (CNV) has been acknowledged as an important contributor to variation in the human genome, and copy-variable regions harbouring genes are interesting subjects of research for their potential phenotypic effects. However, despite the extensive and continuing discovery of CNVs, multiallelic loci remain technically challenging to measure and study. In this thesis, a PCR-based measurement system for the tandemly repeated CNV that harbours the DEFA1A3 locus has been developed. This locus can either have the DEFA1 or the DEFA3 gene in any given copy of the repeat; the two genes differ by a single nucleotide difference in the coding sequence. This CNV has previously been shown to vary from 4 to 11 copies in a sample of the UK population. The use of this measuring system has allowed a usefully accurate measure and some characterization of this CNV in Europeans, Asians (Chinese and Japanese) and African (Ibadan, Nigeria) samples from the HapMap project, agreeing with the previously found copy number range in Europeans and showing more variability in non-Europeans. Typing of three- generation CEPH families has allowed the inference of haplotype copy numbers from segregation analysis. Combining haplotype data for some CEPH HapMap samples that were part of these families with their SNP genotypes in the same LD block has shown copy number lineages that are surprisingly well- tagged by SNPs. SNP rs4300027 allows the division of copy number haplotypes into low (2 and 3-copy) and high (4 and 5-copy) groups in European HapMap samples, a result that has been corroborated in an independent set of European samples. The Asian and African HapMap samples have failed to show this particular association. However, Japanese samples show copy number lineages tagged by other SNPs, an association not observed in other populations. In the second part of the study, an attempt has been made to explore the phenotypic effects of this variable locus. Copy number-tagging SNPs have been used to investigate published GWAS for indirect signals of association with DEFA1A3 copy number. Preliminary experiments for studying protein expression levels of DEFA1 and DEFA3 have been carried out and the possible role of this CNV as a modifier locus in Cystic Fibrosis disease severity has been investigated through direct typing of CF samples with clinical data, and indirectly through interrogating a CF GWAS. 6 1. INTRODUCTION 1.1. Copy Number Variation in the Human Genome Human Genomes Vary The human genome is a dynamic entity. In a diploid cell, the nuclear genome is about 6 billion base pairs, packaged with the help of proteins into 23 pairs of chromosomes. It is subject to changes in its sequence and structure by cell processes, mainly replication of the genome prior to cell division and by extrinsic factors including chemical, physical and biological agents that are collectively labelled as mutagens. Most changes are thought to occur during replication. Although replication of the genome is a high-fidelity process, it is not always perfectly accurate and can result in errors. These errors can be of various kinds, but the mutation rate for the most common variation, single base substitution, is on average 3x10-8 mutations/nucleotide/generation (Xue et al.,
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