DETECTION OF HLA CLASS I EXPRESSION VARIANTS BY SEQUENCE-SPECIFIC PRIMERS.
          M Bunce, J Procter and K I Welsh. Oxford Transplant Centre, Oxford, UK.
          Molecular typing is now the mainstay of many HLA laboratories. A potential problem is that rare null, or low expressed alleles are not discriminated from expressed variants, a possibility that might have implications for transplants. We classify expression variants into two main categories: unique mutations and potentially repeatable mutations. For A*0303N, A*2409N, and B*1526N the mutations giving rise to these alleles appear to be unique. In contrast, repeatable mutations may occur frequently at points where unusual nucleotide sequences make accurate DNA replication by DNA polymerases difficult. One example is at nucleotide positions 621-627 where normal alleles exhibit between three and seven consecutive cytosine residues. Incorrect insertion of an extra cytosine in this region is the cause of expression failure in A*2411N and A*0104N alleles. We hypothesise that the same mutation will occur in other HLA-A alleles as well as in HLA-B and HLA-C. A second candidate site is the point mutation in the enhancer B inverted CAT box reported in A*02 in two different families and in A*01 in another family. We have devised an "Expressions" SSP set to a) monospecifically detect unique mutations such as A*0104N, A*2402102L and B*1526 b) screen for the presence or absence of the T-C mutation in the enhancer B inverted CAT box and c) screen for presence or absence of an extra cytosine anywhere between nucleotides 621-627 in any HLA-A, B or C allele. The SSP reactions for screening the cytosine insertion utilise locus-specific sense primers to identify the locus of a potential null allele. The "Expressions" set has been validated wherever possible by using known examples of expression variants but in a preliminary screen of over 300 consecutive individuals previously typed by both serology and SSP we have not yet detected any expression variants. We conclude that the "Expressions" SSP set is a useful tool for laboratories solely using molecular methods but it does appear that such variants are rare.