Resolution of complex feline leukocyte antigen DRB loci by reference strand‐mediated conformational analysis (RSCA)

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Abstract
The DRB genes of the domestic cat are highly polymorphic. Studies based on clonal sequence analysis have suggested the existence of two distinct loci within individual animals and good evidence for 24 distinct FLA-DRB alleles. This variability, the complexity of clonal sequence analysis and its susceptibility to PCR-induced artefacts has represented a bottleneck to further progress. In this study we have applied reference strand-mediated conformational analysis (RSCA) to FLA-DRB. This protocol has been shown to be highly reproducible. Using five reference strands including two derived from non-domestic felines, we could distinguish 23 FLA-DRB alleles. We used RSCA to explore genetic polymorphism of FLA-DRB in 71 cats including 31 for which clonal sequence analysis was also available. On average, RSCA identified 0.9 more alleles within cats than clonal sequence analysis. Reference strand-mediated conformational analysis was also able to identify animals containing new alleles that could be targeted for sequence analysis. Analysis of allele patterns showed clear evidence for different allele distributions between breeds of cats, and suggested the Burmese breed may have highly restricted FLA-DRB polymorphism. Results from two families provided clear evidence for variation in the number of DRB genes on different haplotypes, with some haplotypes carrying two genes and some containing three. This study highlights the utility of RSCA for the resolution of complex amplicons containing up to six distinct alleles. A simple, rapid method for characterizing FLA-DRB makes possible studies on vaccine response and susceptibility/resistance to viral infections, which are a significant clinical problem in cats.