The ideal high-resolution typing strategy for polymorphic genes is sequence-based typing. SBT of genomic DNA has been developed for the HLA class II genes DRB1, DRB3/4/5 and DPB1. For the DQB1 gene the sequence-based typing method was shown to cause a number of problems. To resolve those problems, different primers to amplify and sequence exon 2 of DQB1 were designed and tested. With several primer combinations, preferential amplification was observed in individuals heterozygous for DQB1*02/*03 and DQB1*02/*04. The preference was for DQB1*02 in many instances but could also be demonstrated for DQB1*03 or *04 and resulted occasionally in allelic drop-out. The best primer combination was selected and successfully used to type individuals heterozygous for DQB1*02, *03 and *04. To distinguish DQB1*0201 and *0202, primers for amplification and sequencing of exon 3 were developed and correct subtyping was obtained. The ambiguous typing DQB1*0301/*0302 and DQB1*0303/*0304 was resolved by allele-specific amplification and sequencing. A total of 258 individuals were fully typed for their DQB1 subtypes. All samples had been previously typed by PCR-SSP and serology. Concordant typing results were obtained for all individuals tested. The DQB1 alleles detected included *0501, *0502, *0503, *0601, *0602, *0603, *0604, *0609, *0201, *0202, *0301, *0302, *0303, *0304, *0401 and *0402. Sequence-based typing of the DQB1 gene proved a reliable typing strategy for assignment of the different DQB1 alleles after intensive selection of primers and test conditions.
The Bw4 and Bw6 epitopes were the first HLA-B differences to be recognized by serological methods. Since then 44 serological groups have been identified and more than 250 alleles assigned by molecular typing methods. In general each serological HLA-B group is associated with the presence of either the Bw4 or the Bw6 epitope. There are several exceptions to this rule. Four alleles, B*4601, *7301, *5503 and *1806, show no serological reactivity with either Bw4 or Bw6. Although the Bw6 motif at residues 77-83 is present in these alleles the Bw6 epitope is modified by a valine at residue 76. One or more alleles from the B8, B40 and B62 groups are identified as Bw4 positive, whereas all others are Bw6 positive. In the groups B27, B44 and B47 several alleles are found to be Bw6 positive, while the majority is Bw4 positive. Histocompatibility testing of dialysis patients and their families revealed the serological presence of an unexpected Bw4 epitope associated with B18 in one patient and B56 in another. Allele-specific amplification and sequencing of exons 2 and 3 of these HLA-B alleles revealed the presence of the Bw4 sequence motif for both. The new alleles were assigned B*1809 and B*5607, respectively. In 2 other patients the presence of a new B*07 allele was determined by sequence based typing. Although the new allele, B*0715, showed the Bw6 sequence motif at positions 77 to 83, a substitution of amino acid 76 from glutamic acid to valine was identified. This change resulted in an aberrant Bw6 serological reaction pattern.
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