SEQUENCE-BASED TYPING OF HLA CLASS I A, B AND C LOCI.
          CE Garey, P Chretien, PA Nordstrom, D Nuesca, Visible Genetics, Inc., Toronto, Ont.
          As sequence-based-typing (SBT) has the ability to detect existing as well as new HLA polymorphisms, it is recognized as the current gold standard for high resolution HLA analysis. We have therefore developed a SBT-based methodology to provide for the rapid and precise typing of HLA-A, B and C loci using the Visible Genetics, Inc. OpenGene system. In order to assess the performance of SBT in routine typing of samples for Class I alleles, a reference panel of samples providing a broad coverage of polymorphic positions in both heterozgyous and homozygous combinations was obtained from The Toronto Hospital/Canadian Red Cross. These samples were of known HLA types, determined by serology and/or DNA typing methods; however the SBT was conducted as a blind study. For SBT, all samples werePCR-amplified with A, B and C gene-specific primers followed by sequencing with fluorescent dye-labelled primers. The sequence data was generated by a 30 minute run on the VGI MIcroGene Blaster and analysed with the GeneLibrarian HLA typing software. The SBT results were evaluated and compared to results from serology and DNA typing. The concordancy between serology and SBT was 96%, while concordancy between DNA typing and SBT was 99%. Therefore, in almost all cases the results were consistent with serology and DNA typing, with the major advantage being SBT yields a high resolution type. This robust class I SBT methodology can be routinely utilized for matching donor-recipient pairs prior to transplantation and in disease association or immune response studies where precise knowledge of class I sequence variation is demanded.