Characterisation of the ovine major histocompatibility complex class II region

Abstract

The main aim of this project has been to provide insights into the genomic organization of the class II region of the ovine major histocompatibility complex (MHC), a chromosomal region containing genes that control adaptive immune responses in vertebrate species. Assessment of the potential of measuring resting serum IgA levels as a predictor of the resistance of sheep to gastrointestinal parasites was also undertaken.Loci within the ovine MHC class II region were identified and sequenced either by direct sequencing of known class II genes using ovine specific primers or from sub-clones constructed from bacterial artificial chromosome (BAC) clones containing the class II region. The loci were apportioned into two groups (IIa and IIb) based on analogy with the class II region in the cattle MHC where it is known that a chromosomal inversion has divided and separated the region into two subregions ≈15 cM apart. All sequences obtained have been deposited in GenBank. A relative map of the ovine class IIa and IIb subregions was constructed based on comparative information from the human and cattle MHCs. Additionally, complete nucleotide sequences were obtained for the protein folding chaperone gene PFDN6; phylogenetic analysis confirmed its homology and impressive conservation with orthologues from other vertebrate. Partial sequences were also generated for several other loci including WDR46 and DRA.Single nucleotide polymorphisms (SNPs) were identified by analysing homologous sequences from a minimum of five individual sheep. In total, 103 SNPs were discovered that were distributed over 20 distinct loci spanning both the putative class IIa and IIb regions. Genotypes were typed for panels of 10 SNPs across each of the ovine class IIa, IIb subregions, plus 10 published SNP loci for the class III region, in a total of 261 sheep. The sheep comprised 68 unrelated parental animals and 193 sheep from nine different family groups for which worm egg count (WEC) data and estimated breeding values (EBV) were available. Allele frequencies for each SNP locus were estimated and determined to be in Hardy Weinberg proportions. Analysis of SNP heterozygosity in sheep revealed a region within the class IIa subregion where heterozygosity was reduced. This region is similar to a corresponding region within the human MHC manifesting a reduced frequency of SNPs. Analysis of linkage disequilibrium (LD) across the panel of 30 SNP loci showed regions of high and low LD. The region of low heterozygosity within the putative sheep class IIa subregion was characterised by low LD.The availability of independently confirmed pedigrees permitted identification of 54 Mendelian inherited haplotypes across the class IIa, IIb and III subregions for all 30 loci. Haplotypes for other sheep were inferred using the EM algorithm as implemented in the SNPstats software package. Analysis of the deduced haplotypes showed evidence for the presence of conserved allele groupings or ‘blocks’ corresponding to each subregion. These ‘blocks’ seemed to occur more frequently than expected despite some SNP loci being homozygous (especially in the sires). Similar blocks were predicted by haplotype function in SNPstats. This result suggests that conserved subregion haplotypes may be present in the sheep MHC as are known to exist in the human MHC. Further analysis of the sheep pedigrees showed four haplotypic crossovers between the class IIb and class IIa subregions. Four sire derived crossovers in 21 meioses is consistent with a physical separation between these two subregions similar to that observed in cattle. No crossovers were observed between the class IIa and class III subregions.Total and parasite-specific IgA levels were measured in serum collected post weaning from 171 unchallenged sheep for which WEC data was available. There were no significant correlations between either total or parasite specific IgA serum levels and post natal WEC or WEC at weaning Hence, this parameter is not a useful predictor of worm immunity and cannot replace the more difficult to measure WEC values. Multiple regression analysis of the data did suggest however that IgA synthesis may have a role in the humoral immune responses of sheep to gastrointestinal parasites.The discoveries reported in this thesis provide a more detailed description of the ovine MHC class II region, evidence of its division into two separated subregions, and the description of haplotypes based on newly discovered SNPs. Haplotypic associations with breeding values based on WEC were identified, but these did not permit speculation concerning the identity of candidate loci for parasite immunity probably due to the relatively small populations studied. These findings will contribute to association and linkage studies in larger sheep populations and between breeds in the search for genetic determinants of immunity to gastrointestinal worms. It is expected that such determinants in turn will facilitate breeding for more disease-resistant sheep by marker-assisted selection with consequent increases in industry productivity.