CURRENT HLA CLASS II GENERIC PRIMER SETS UNABLE TO IDENTIFY THREE NEW DRB3 ALLELES.
          G Coquillard, TF Tang, J Ng, R Hartzman, CK Hurley, CW Bill Young/ DoD Marrow Donor Program, Kensington, Maryland.
          New HLA alleles are often identified initially from observing uncommon patterns found in low resolution typing performed via PCR-SSOP. The most common situation is found within the DRB1 locus, where allelic subtypes are discerned by combining sequence motifs found in the three variable regions of exon 2. Within these three variable regions, the DRB3 locus also shows variability though not as extensive as DRB1. Recently the HLA-DR oligotyping analysis of three Caucasian bone marrow donors from our registry resulted in the identification of three novel DRB3 alleles. It was the absence of a DRB3 allele within these three samples that initiated our investigation. Direct sequencing of the new alleles was accomplished using amplified genomic DNA with allele specific primer pair sets. The first sample was originally characterized as DRB1*0101, 03011;DRB3*(-). A 5' intron-derived primer was neccessary to identify the sequence variation found at codon 8 (TCG instead of TTG). It was realized that the forward generic primer DRBAMP-A (ITR(-3)-EX8) masked the point mutation, causing negative amplification of the novel DRB3*0104 allele. The second sample was characterized DRB1*0401, 1301; DRB3*(-). As seen for the previous sample, our generic primer set from Lifecodes Class II DRB typing kit (ITR(-7)-EX6) failed to amplify the DRB3 allele of this sample. It was later realized that the forward primer differed by one nucleotide from this new allele (intron(-13)). The exon 2 sequence of this new DRB3 was identical to the DRB3*0101 allele. The third sample was identified as DRB1*0301, 04HS, also without a DRB3 type. Many attempts to amplify the DRB3 allele failed whenever the primer pair set contained a primer located within exon 2 suggesting a possible exon 2 deletion. This study has demonstrated lack of conservation at the junction of intron 1 and exon 2 of the DRB3 gene. In light of the first two novel alleles, the current generic primer sets may need to be reviewed and/or modified.