Sequencing of HLA class II genes based on the conserved diversity of the noncoding regions: sequencing based typing of HLA-DRB genes

In this paper, we present a novel sequencing based typing strategy for the HLA-DRB1, 3, 4 and 5 loci. The new approach is based on a group-specific am plification from intron 1 to intron 2 according to the serologically-define d antigens. For this purpose, we have determined the 3' 500 bp-fragment of intron 1 and the 5' 340 bp-fragment of intron 2 of all serological antigens and their most frequent subtypes. We discovered a remarkably conserved div ersity characterized by lineage-specific sequence motifs This lineage-speci ficity of non-coding motifs in the 1st and 2nd intron offered the possibili ty to establish a clear serology-related amplification strategy, The method allows the complete analysis of the 2nd exon and the definition of the cis /trans linkage of sequence motifs by intron-mediated polymerase chain react ion (PCR)-based separation of the haplotypes in nearly all serologically he terozygous samples. In particular, the non-coding variabilities between the DR52-associated DRB1 groups made the independent amplification possible. T hus, compared to the standard procedures using exon-based amplification pri mers, the groups DR3, DR12, some DR13 alleles (1301, 1302) and the DR14 gro up could be amplified by specific primer mixes. The DR8 could be amplified with an individual primes mix not co-amplifying the DR12. The DR11 and DR13 did not show any individual motif in intron 1 or intron 2. In order to ach ieve a separate amplification, they had to be amplified by multispecific pr imer mixes (DR3/11/13/14; DR3/11/13 or DR11/13/14) excluding the other hapl otype. Thus, exclusively the alleles in rare DR11,13 heterozygosities witho ut a DRB1*1301 or 1302 could not be amplified separately. Fourteen primer m ixes are used to amplify the DR1-14, and 6 primer mixes for the specificiti es DR51-53. The sequence homology of the 3' end of intron 1 facilitated the application of only three different sequencing primers for all DRB alleles .