[Truncated abstract] Sporadic inclusion body myositis (sIBM) is a chronic inflammatory disease that is the most common myopathy in individuals above the age of 50 in the Caucasian population. sIBM is characterised by cytotoxic immune infiltration of skeletal muscle, consisting primarily of CD8+ T-cells and macrophages, as well as a degenerative process, with muscle fibre vacuolation and intracellular filamentous inclusions. The pathogenesis of sIBM is likely to involve a complex interaction between genetic and environmental factors. Whilst the physiological and pathological characteristics of sIBM have been clearly identified, the exact origin and genetic basis of the disease remains unknown. A number of studies show that sIBM is associated with alleles of the major histocompatibility complex (MHC) on chromosome 6p21.3 and specifically with two ancestral haplotypes (AH) in Caucasians – the 8.1AH, defined by HLA-B*0801, HLA-DRB1*0301 and the 35.2AH, defined by HLA-B*3501, HLA-DRB1*0101. Mapping studies subsequently showed that sIBM susceptibility likely originates from a 389kb region of the MHC, spanning from centromeric of PBX2 to telomeric of HLA-DRB1. The central hypothesis of this thesis was that susceptibility to sIBM is conferred by a single allele found within a region defined using the 8.1AH, which is also carried by other haplotypes associated with sIBM. Three patient cohorts from Australia, the U.S.A and Japan were studied. ... Of the 32 alleles genotyped, none were found in all susceptibility haplotypes and one was common, but not unique, to the 8.1AH, 7.2AH and 52.1AH. Five SNPs were also found in two of the three haplotypes, although none were specific to the sIBM susceptibility haplotypes. These data suggest that the 8.1AH is likely to carry an sIBM susceptibility allele independent of the 35.2AH, 7.2AH and 52.1AH. Based on the possible mechanism of action in cellular differentiation and its location within the 8.1AH-defined sIBM susceptibility region reported in 2004, NOTCH4 was a strong candidate for conferring sIBM susceptibility. NOTCH4 coding region polymorphisms were thus investigated in a Caucasian patient cohort to assess any possible role in sIBM susceptibility. While the frequency of some alleles were increased in sIBM patients, the strong linkage disequilibrium throughout the MHC prevented confirmation of any alleles as playing a direct role in sIBM. The 8.1AH-derived sIBM susceptibility region was further refined using recombination mapping. This approach used markers characterised against multiple haplotypes to genotype patients carrying part of the 8.1AH to locate a common, overlapping susceptibility region. Recombination mapping of patients revealed a common overlapping region of the 8.1AH, extending from BTNL2 to HLA-DRB3. The results of the study indicate that 8.1AH-derived susceptibility for sIBM is likely to originate from a 172kb region encompassing HLA-DRA, HLA-DRB3 and part of BTNL2. These genes warrant further investigation in future studies.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xii, 239 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

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.

Este trabalho teve como objetivo principal descrever a diversidade genética e identificar a estrutura populacional de populações de tamanduá-mirim distribuídas ao longo dos biomas brasileiros através do uso de ferramentas de genética de populações e do acesso a regiões neutras e adaptativas do genoma da espécie. A amostragem de indivíduos de tamanduá-mirim é complicada pela difícil detecção do animal em trabalhos de campo, pela sua complicada captura e manipulação. Assim, fez-se necessário o uso de espécimes de museu e de amostras não-invasivas. A fim de validar o uso das mesmas para a genotipagem confiável de oito locos de microssatélites desenvolvidos para a espécie foi utilizado um método de padronização da qualidade das amostras (Índice de Qualidade, QI) e estimativa dos erros de genotipagem. Foi observada uma qualidade superior das amostras não invasivas (N=19) em relação às peles de museu (N=138). Foi possível também eliminar amostras com desempenho ruim (QI<0.7 e sucesso de amplificação maior que 75%), e garantir a confiabilidade dos resultados de microssatélites das amostras que permaneceram no estudo para análises posteriores. Devido à grande área de distribuição da espécie de forma contínua, se tornou complicado traçar populações pré-definidas. Assim, a abordagem da genética da paisagem foi a ferramenta mais apropriada para o estudo da estrutura de populações em T. tetradactyla (N=176). Comparativamente, duas abordagens foram usadas: agrupamento de indivíduos pelo critério de proximidade geográfica, designado aqui como a priori (20 populações), e análises baseadas no indivíduo, sem informação prévia de agrupamento populacional, referida no capítulo como a posteriori (quatro transectos testados). Foram encontrados níveis de diversidade moderados (média de 11.38 alelos por lóco) e poucas evidências de estruturação entre populações das localidades amostradas (K=2 na maioria dos testes). Os indivíduos que mostraram maior diferenciação foram originados da Floresta Amazônica. Esta região também demonstrou maior diversidade genética (riqueza alélica e heterozigosidade esperada) que as outras. Por outro lado, populações distribuídas ao longo da Mata Atlântica e regiões adjacentes demonstraram um padrão de isolamento por distância. Populações do Brasil central (Cerrado e Pantanal) não demonstraram diferenciação em relação às demais. Finalmente, foi estudada a variação genética adaptativa da espécie através da diversidade do gene DRB do complexo MHC. Esta família gênica codifica proteínas envolvidas no reconhecimento de antígeno e ativação da resposta imune adaptativa, e são regulados por seleção natural, especialmente por pressão seletiva dirigida por patógenos. É esperado que a diversidade de patógenos seja distinta nos biomas brasileiros, sendo os ambientes florestais mais biodiversos neste quesito do que ambientes da Diagonal Seca, o que representa pressões seletivas diferentes. Assim, foi investigada a diversidade do gene DRB éxon 2 em indivíduos (N=65) dos diferentes biomas brasileiros através de sequenciamento de nova geração (454 GS Junior), e os resultados de distribuição dos alelos foram comparados com os microssatélites. Foi encontrada uma alta diversidade (60 alelos no nível de aminoácido e 70 alelos no nível de nucleotídeos) e assinaturas claras de seleção positiva no gene (dN/dS=2.94). Maior riqueza alélica e proporção de alelos privados foram encontradas em biomas florestados, especialmente na Floresta Amazônica. Além disso, os marcadores neutros (microssatélites), demonstraram padrões similares ao DRB, revelando a força de eventos demográficos e deriva genética que também moldaram os padrões de diversidade deste gene do MHC / This work aimed to describe the genetic diversity and population structure of populations of the lesser anteater distributed along Brazilian biomes through population genetic tools, accessing neutral and adaptive genomic regions of the species. Sampling lesser anteater individuals is complicated due to infrequent detection of the animal in field works, its complicated capture and manipulation. Thus, it was necessary to use museum specimens and noninvasive samples. To validate these samples for the reliable genotyping of eight microsatellite loci developed for the species, a standardization method of the quality of samples (Quality Index, QI) and genotyping errors was used. A superior quality of noninvasive samples (N=19) compared to study skins (N=138) was observed. It was also possible to eliminate samples with a bad performance (QI<0.7 and amplification success higher than 75%), and thus guarantee the reliability of microsatellites results for samples kept in further analysis. Due to the great and continuous distribution area of the species, it becomes difficult to delineate predefined populations. Thereby, landscape genetics approach was a better suited tool for studying the population structure of T. tetradactyla individuals (N=176). Comparatively, two approaches were used: grouping of individuals by a geographical proximity criterion, designated here as a priori (20 populations), and analysis based on individuals, without previous information of population grouping, referred here as a posteriori (four transects tested). Moderate levels of diversity were found (average of 11.38 alleles per locus), and few evidences for structuring between populations of the sampled localities (K=2 in most tests). The individuals showing the major differentiation were originated from the Amazon Forest. This region also demonstrated higher genetic diversity (allelic richness and expected heterozygosity) than others. By the other hand, populations distributed in Atlantic Forest and adjacent regions demonstrated a pattern of isolation by distance. Populations from central Brazil (Cerrado and Pantanal) did not show distinction from the others. Finally, the adaptive genetic variation of the species was studied through DRB gene diversity, from MHC. This gene family codes for proteins involved in the antigen recognition and activation of the adaptive immune response, and are regulated by natural selection, especially by selective pressure driven by pathogens. It is expected that the pathogen diversity is distinct in Brazilian biomes, being florested biomes more diverse than dry central Brazil environments, which represents different selective pressures. Therefore, the diversity of DRB exon 2 gene in individuals (N=65) from different Brazilian biomes was investigated through Next Generation Sequencing (454 GS Junior), and the results of allele distributions were compared with microsatellites. A high diversity was found (60 alleles in amino acid level and 70 in nucleotide level) and clear signatures of positive selection in DRB (dN/dS=2.94). Greater allelic richness and private allele number were found in forested biomes, especially in the Amazon Florest. Besides, neutral markers (microsatellites) demonstrated similar patterns to DRB, revealing the strength of demographic events and genetic drift in shaping the diversity patterns in MHC

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.