The identification of mutations in immunoglobulin sequences and the application of mutation analysis to the study of antigen selection in parasitized individuals

Analysis of mutations and the inference of antigen selection from patterns of mutations in IGHV genes can provide a better understanding of the immune system. Accurate knowledge of the germline repertoire is, however, required for these analyses. The fact that there could be errors and omissions in the existing, reported IGHV germline repertoire led to the primary objective of this thesis, which was to build an error-free IGHV germline gene database. A new IGHV germline repertoire was built based on the bioinformatic analysis of the existing repertoire, and of rearranged antibody genes in public sequence databases. A five level classification system was developed, reflecting the likelihood that each gene exists. The results of this analysis showed that nearly half of the reported, functional IGHV repertoire was generated from error-prone studies. In addition, twelve previously unreported alleles were identified and three of them were confirmed by genomic sequencing.The finding of twelve previously unreported polymorphisms led to a questioning of the completeness of the existing IGHV repertoire. In order to find other previously unreported IGHV genes and allelic variants, 454 high throughput sequencing was conducted using samples from Papua New Guinea (PNG) donors. A stringent filter was applied to ensure the sequences identified did not include sequencing errors. Strikingly, a new functional IGHV gene (IGHV3-NL*01), as well as 16 previously unreported IGHV germline alleles and 14 immunoglobulin-like sequences, were amplified. After developing a more accurate and complete IGHV repertoire, studies were conducted in PNG into the IgE response. The natural function of IgE involves in the immune response against parasite. Surprisingly, no study has examined IgE sequences in anti-parasite responses. The IgE response in parasitized individuals was therefore investigated. Analysis of the mutation patterns showed that IgE sequences from parasitized individuals are highly mutated, but lack evidence of antigen selection. This study also showed that the mutation level of the IgG subclasses sequences can be related to sequential class-switching. The functionality of IGHV3-NL*01 and of the 12 new alleles identified in the PNG population were also confirmed through the identification of their presence in isotype-linked VDJ rearrangements