DISCRIMINATION OF HLA-B27 ALLELES BY GROUP-SPECIFIC AMPLIFICATION FOLLOWED BY SOLID-PHASE SEQUENCING

HLA-B21 is known to be highly associated with ankylosing spondylitis. Until now, nine B27 subtypes have been sequenced and may contribute in different fashions to ankylosing spondylitis. Additionally, the diver gent subtypes may be of clinical importance in bone marrow transplanta tion with alternative donors. The purpose of this study was to determi ne the different subtypes of HLA-B27 by a direct: sequencing approach. The typing strategy is based on a group-specific amplification of the second and third exon followed by automated fluorescence sequencing o f the polymorphic regions. The extensive sharing of sequence motifs be tween the different B alleles made it impossible to specifically ampli fy the B27 group under the precondition of including all sequence vari ations necessary for a postamplification specificity step. Therefore, for setting up a direct sequencing approach of B27, co-amplified B all eles had to be taken into account. In order to get unambiguous sequenc ing chromatograms without any heterozygous positions, nested sequencin g primers were used which selectively matched sequence motifs only pre sent in the second and third exon of the amplified B27 alleles. This s trategy allowed in all cases investigated a clear separation of the ha plotypes, revealing unequivocal sequencing results. Using this method, we have investigated 93 B27-positive individuals. Sequencing identifi ed the alleles B2702, 2703, 2704, 2705, and 2707. B*2701, 2706, 2708, and 2709 were not represented in the population studied.