Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 1999. / Typescript. Bibliography: leaves 113-124.

No description available.

Understanding the genetic basis of fitness differences has been a major goal for evolutionary biologists over the last two decades. Although there are many studies investigating how natural selection can promote local adaptation, few have succeeded to find the link between genotype and fitness of the phenotype. Polymorphic genes of the major histocompatibility complex (Mhc) are excellent candidates for such associations as they are a central component of the vertebrate immune system, playing an important role in parasite resistance, and hence can have direct effects on survival of their bearers. Although associations between Mhc and disease resistance are frequently documented, the epidemiological basis of the host-parasite interaction is often lacking and few studies have investigated the role that Mhc genes play in individual variation in fitness; thus comparatively little is known about the fitness consequences of Mhc in wild populations. Furthermore, the majority of work to date has involved testing associations between Mhc genotypes and disease. However, the mechanism by which any direct selection on the Mhc acts, depends on how genotypes map to the functional properties of Mhc molecules. The aim of this thesis was to characterize Mhc alleles in terms of their predicted functional properties and to investigate whether and how selection operates on Mhc class I functional variation using the great tit (Parus major) population at Wytham Woods as a model host species. Through a comprehensive characterization effort and the use of 454 pyrosequencing platform, I performed a detailed analysis of genetic variation at Mhc class I exon 3 and grouped alleles with similar antigen-binding affinities into supertypes to classify functionally distinct Mhc types. There was extreme complexity at the Mhc class I of the great tit both in terms of allelic diversity and gene number. A total of 862 alleles were detected from 857 individuals; the highest number yet characterized in a wild bird species. The functional alleles were clustered into 17 supertypes; there was clear evidence that functional alleles were under strong balancing selection. To understand the role of Mhc in disease resistance, I examined the linkage between Mhc supertypes, Plasmodium infection and great tit survival, and showed that certain functional variants of Mhc confer resistance to two divergent Plasmodium parasite species that are common in the environment. I further investigated the fitness consequences of functional variation at Mhc, using mark-recapture methods and long-term breeding data; and tested the hypotheses that selection: (i) maximizes Mhc diversity; (ii) optimizes Mhc diversity, or (iii) favours specific functional variants. I found that the presence of three different supertypes was associated with three different components of individual fitness: adult survival, annual recruitment probabilities and lifetime reproductive success. In contrast, there was no evidence for a selective advantage of Mhc functional diversity, either in terms of maximal or optimal supertype diversity. Finally, I explored the role that Mhc plays in female mate choice decisions and examined the reproductive fitness consequences of Mhc-dependent mating patterns. There was little evidence to suggest that functional dissimilarity at Mhc has any influence on female mate choice decisions or that dissimilarity at Mhc affects the reproductive output of the social pair. Overall, this thesis provides strong support for the suggestion that selection favours specific functional variants of Mhc, possibly as a result of supertype-specific resistance or susceptibility to parasites that exert strong selective pressures on their hosts; whereas there is no support for selection favouring maximal or optimal Mhc diversity. More importantly it demonstrates that functional variants of Mhc class I loci are an important determinant of individual fitness in natural populations.

The Human Leukocyte Antigen (HLA)-B27 is a Major Histocompability Complex (MHC) class I antigen that is strongly associated with development of a group of closely related arthritic diseases, collectively known as the spondyloarthropathies (SpA). However, the mechanism by which HLA-B27 confers this susceptibility is unclear. Studies have shown that HLA-B27 heavy chains can form classical heterotrimers associated with peptide and β2-microglobulin (B27HT), and also non-classical heavy chain homodimers (B27₂). B27₂ assemble intracellularly during maturation and are also expressed at the cell surface following endosomal recycling of B27HT. A pathogenic role for B27₂ has been proposed in two of the current theories of pathogenesis: the B27 homodimer theory and the B27 misfolding and UPR theory. Yet, determinations of the extent, distribution, and triggers of B27₂ expression, as well as the functional consequences of its receptor interactions in AS pathogenesis, have been hampered by the lack of a specific detection reagent. Therefore, to investigate the role of B27₂ in AS, we generated a novel antibody to B27₂ – HD6 – using phage display technology, which binds to in vitro refolded B27₂ but not B27HT complexes by ELISA. This thesis provides evidence that HD6-reactive molecules, which include B27₂, are expressed at the cell surface in both cell lines and in the context of a disease setting. Recognition is B27-specific and strongly correlated with the magnitude of B27 expression, which could account for the lack of staining in some cell subsets. Moreover, staining was comparable in cell lines expressing the disease-associated B*27:05 and the less disease-associated subtype B*27:09. In addition, I have shown cells expressing physiologic levels of B27, including EBV-transformed BCLs and AS patient PBMCs, are capable of expressing the HD6 epitope upon low pH treatment. Interestingly, these ‘acid-inducible HD6’ molecules were absent from cells lacking a functional PLC. Finally, I have shown that HD6-reactive molecules can derive from pre-existing folding B27 molecules at the cell surface, which may be inhibited by the addition of exogenous B27-binding peptides. These findings are consistent with a mechanism of pathogenesis involving the surface expression and recognition of B27₂ and/or other aberrantly folded forms of B27, as proposed in the homodimer theory. HD6 will be a powerful tool to address the potential pathogenic role of B27₂ in SpA and may additionally have therapeutic potential.

The major histocompatibility complex (MHC) is one of the most variable regions in the vertebrate genome. Many genes within the MHC play important roles in the development of an immune response, including the response to pathogens, by presenting pathogen fragments to T cells. Pathogen-mediated balancing selection is thought to be important in maintaining the high levels of allelic variation at these loci, though the precise mechanism remains unclear. The number of studies of MHC diversity in non-model organisms has increased dramatically in recent years as genotype data have become cheaper and easier to generate; however, key limitations in many such studies remain a lack of high quality MHC genotypes and associated phenotype data. Many studies focus on a single MHC locus, assuming that one locus will represent the full range of variation within each MHC haplotype. Alternatively, the products of different loci may co-amplify, preventing locus-specific genotypes and hence heterozygosity being accurately determined. Non-model systems are also often limited by small sample sizes and limited recording of associated host and pathogen measures, which, combined with high levels of allelic variation at MHC loci, can limit statistical power. Finally, few MHC studies control for the general effect of relatedness in explaining host traits before testing for MHC effects. With so many methodological impediments, it is challenging to identify robust associations between MHC variation and host phenotypes, such as parasite burden or fitness, and to draw conclusions about the mechanisms underpinning the maintenance of diversity at MHC loci. In this thesis, I address these problems by developing a SNP-based haplotyping system for a population of unmanaged Soay sheep (Ovis aries) on Hirta, St. Kilda, for which data is available on pedigree, phenotypic traits and fitness and its components over a 30- year study period. The ovine MHC consists of four classes of loci, within which loci are tightly clustered and show reduced recombination rates compared to the genome average. Although the mammalian MHC is usually highly variable, one would expect that the number of haplotypes within an MHC class in an island population of sheep with no immigration to be limited. The class IIa region of the ovine MHC includes the classical class II loci which are typically thought to be involved in the presentation of peptides derived from extracellular pathogens, including gastrointestinal helminths, in sheep and other mammals. In chapters 2 to 4, I describe the characterisation of class IIa haplotypic diversity in the Soay sheep using direct Sanger sequencing of PCR amplified fragments, which, in combination with cloning, revealed eight distinct haplotypes. With this knowledge of haplotypic diversity, and genotypes for a sample of Soay sheep typed on the Ovine Infinium HD chip (approximately 600K SNPs), I developed a panel of 13 SNPs which could be used to impute the class IIa haplotypes. This panel was genotyped by KASP (Kompetitive Allele Specific PCR) in 6034 samples and used to impute the class IIa haplotypes. After quality control measures, class IIa haplotypes were successfully imputed for 5349 individuals. Evidence of balancing selection was identified using the Ewens-Watterson test at different life history stages and within the standing population each year between 1985 and 2012, showing that allele frequencies were more even than would be expected under neutrality. However, there was no evidence of deviation from Hardy-Weinberg equilibrium identified at different life stages or in the standing population in any year. In chapter 5, I investigate associations between the MHC class IIa haplotypes and individual-level data on host phenotypes - body weight, plasma immunoglobulin levels (measured as anti-Teladorsagia circumcincta third larval stage IgA, IgE and IgG) and strongyle faecal egg counts (FEC). Associations were tested within mixed effects models which were used to account for repeated measures and control for fixed effects known to affect the response variables, as well as within an animal model framework to account for relatedness between individuals. Haplotype heterozygosity was unrelated to any of the traits investigated, suggesting a general heterozygote advantage is unlikely to be operating within the Soay sheep. Six of the eight class IIa haplotypes were associated with multiple traits in different age-sex classes, although many of these associations were removed after inclusion within animal models. The evidence of balancing selection and associations between class IIa haplotypes and phenotypes related to health offers a promising glimpse into the evolutionary mechanisms which may be operating to maintain diversity within this region.

Abstract not available

At the centre of the vertebrate immune system is a group of proteins called MHC (major histocompatibility complex) molecules. These function in self – non self recognition and activation of the immune defence against intruding parasites and pathogens. In this thesis I have investigated individual variation in MHC class II genes and antibody producing ability in relation to ecology and behaviour in the great snipe (Gallinago media), a lekking bird, breeding in northern Europe. There was much variation in the MHC genes of the great snipe and the sequence data show that balancing selection has been acting on these genes. I found genetic differentiation in the MHC between two separate geographic regions of the great snipe distribution. Furthermore, this structure was more pronounced than that previously found in neutral genetic markers, suggesting that different selection pressures (possibly resulting from variation in parasitic fauna) are acting in these different regions. The birds produced specific antibodies following injection with two novel antigens. Males that were chosen as mates, had higher antibody titers than their neighbouring males, suggesting that this ability may be important in female mate choice. Such choice could give the offspring an enhanced immune system or could favour females directly by avoidance of sexually transmitted diseases. Females choosing to mate with a male having a different set of MHC genes than their own could give the offspring immune system the ability to react to a wide range of parasites. No such mate choice could, however, be found in the great snipe. Instead, females preferred males with certain MHC alleles, irrespective of their own MHC type. If those alleles confer resistance to parasites currently prevailing in the population, such resistance would be inherited by the offspring, thereby enhancing their fitness.

Class I major histocompatibility complex molecules present antigenic peptides to cytotoxic T lymphocytes, which leads to the elimination of virus infected cells. Class I molecules are heterotrimers consisting of a heavy chain, a light chain termed beta2-microglobulin, and a peptide ligand. Assembly of class I molecules begins in the endoplasmic reticulum where the heavy chain associates with beta2-microglobulin, and the heavy chain-beta2-microglobulin heterodimers enter a peptide loading complex where class I molecules acquire peptides. During the biogenesis of class I molecules, ERp57, a thiol oxidoreductase, associates with free class I heavy chains and, at a later stage, with the peptide loading complex. In this thesis, I show for the first time that ERp57 participates in oxidative folding of the heavy chain. Depletion of ERp57 by RNAi delayed heavy chain disulfide bond formation and slowed folding of the heavy chain alpha3 domain. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no such effect. Since ERp57 associates with the lectin-chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, I examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells, suggesting that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin-chaperones. ERp57 also forms a mixed disulfide with tapasin within the peptide loading complex and I found that the formation of this mixed disulfide is independent of its interaction with calnexin and calreticulin. I also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide-binding site in substrate recognition. Finally, by inactivating the redox active sites of ERp57, I demonstrate that its enzymatic activity is dispensable in stabilizing the loading complex and in supporting efficient peptide loading. Thus, ERp57 plays a structural rather than catalytic role within the peptide loading complex.

Genomic organization of the major histocompatibility complex (MHC) can profoundly influence gene function and multigene family evolution. Situated at the interface of individual genetic variation and the adaptive immune response, MHC class I and II loci are intensively studied for disease associations and used as markers of adaptive genetic variation in evolutionary ecology research. Genomic sequence of MHC-containing cosmid clones from the red-billed gull (Larus scopulinus, Charadriiformes: shorebirds, gulls, and allies) was obtained for comparative analysis of avian MHC evolution. MHCI polymorphism was further investigated using cDNA library screening and locus-specific genotyping protocols. This first information regarding MHC organization and MHCI variation in charadriiforms suggests a complex evolutionary history to MHC architecture in birds. Duplication of MHCIIα loci in tandem MHCIIα/β pairs and their proximity to MHC-region gene COL11A2 are similar to arrangements in nonavian vertebrates, and contrast with the “minimal essential” MHC of the chicken (Gallus gallus, Galliformes: gamebirds). MHCI–TAP2 organization is shared with Galloanserae (gamebirds + waterfowl), as is a proposed major classical function for this MHCI gene. In contrast, the placement of MHCI genes adjacent to sequence from chromosomes 3, 5, and 22 of the chicken and zebra finch (Taeniopygia guttata, Passeriformes: perching birds) indicates interchromosomal rearrangements in birds and the possible genomic dispersal of nonclassical MHCI genes in the red-billed gull. Screening for avian malaria, genetic parentage tests, and field data from red-billed gulls at Kaikoura Peninsula, New Zealand were combined with MHCI genotypes to investigate relationships with disease and reproduction. Plasmodium infection was confirmed in red-billed gulls, and breeding condition was negatively associated with malarial infection and positively related to variation at the putative major MHCI locus. A low rate of extrapair paternity was identified across thirteen breeding seasons. Partners without extrapair young (EPY) had greater MHCI dissimilarity than was expected by chance, whereas lower individual MHCI variation and elevated hatching failure existed for pairs with EPY. In addition to contributing to studies of MHC evolution, sexual selection, and disease dynamics in the New Zealand avifauna, this research will facilitate studies of MHC genes in related charadriiforms, many of which are of conservation concern.

Genomic organization of the major histocompatibility complex (MHC) can profoundly influence gene function and multigene family evolution. Situated at the interface of individual genetic variation and the adaptive immune response, MHC class I and II loci are intensively studied for disease associations and used as markers of adaptive genetic variation in evolutionary ecology research. Genomic sequence of MHC-containing cosmid clones from the red-billed gull (Larus scopulinus, Charadriiformes: shorebirds, gulls, and allies) was obtained for comparative analysis of avian MHC evolution. MHCI polymorphism was further investigated using cDNA library screening and locus-specific genotyping protocols. This first information regarding MHC organization and MHCI variation in charadriiforms suggests a complex evolutionary history to MHC architecture in birds. Duplication of MHCIIα loci in tandem MHCIIα/β pairs and their proximity to MHC-region gene COL11A2 are similar to arrangements in nonavian vertebrates, and contrast with the “minimal essential” MHC of the chicken (Gallus gallus, Galliformes: gamebirds). MHCI–TAP2 organization is shared with Galloanserae (gamebirds + waterfowl), as is a proposed major classical function for this MHCI gene. In contrast, the placement of MHCI genes adjacent to sequence from chromosomes 3, 5, and 22 of the chicken and zebra finch (Taeniopygia guttata, Passeriformes: perching birds) indicates interchromosomal rearrangements in birds and the possible genomic dispersal of nonclassical MHCI genes in the red-billed gull. Screening for avian malaria, genetic parentage tests, and field data from red-billed gulls at Kaikoura Peninsula, New Zealand were combined with MHCI genotypes to investigate relationships with disease and reproduction. Plasmodium infection was confirmed in red-billed gulls, and breeding condition was negatively associated with malarial infection and positively related to variation at the putative major MHCI locus. A low rate of extrapair paternity was identified across thirteen breeding seasons. Partners without extrapair young (EPY) had greater MHCI dissimilarity than was expected by chance, whereas lower individual MHCI variation and elevated hatching failure existed for pairs with EPY. In addition to contributing to studies of MHC evolution, sexual selection, and disease dynamics in the New Zealand avifauna, this research will facilitate studies of MHC genes in related charadriiforms, many of which are of conservation concern.