Sequence-based typing (SBT) has become an important tool in the identification of HLA alleles. In this study a comparison was made between SBT of DRB1/3/4/5 alleles performed in two laboratories each using a different strategy for SBT. The laboratories in Utrecht and in Maastricht performed direct sequencing of PCR amplified genomic DNA from 30 selected samples. Primers and conditions for PCR amplification were different. Sequencing was either performed with T7 polymerase, using internal sequencing primers, or with cycle sequencing using an M13 tailed system. Two different automated DNA sequencers were used; the ALFexpress from Pharmacia and Applied Biosystems 373A. We concluded that nor the method of sequencing nor the sequencing machine influences typing results. However the PCR reaction used for generating template DNA is the most critical step. Different primers and different conditions can lead to false negative reactions. The fact that these false negative reactions can occur with different alleles in different combinations but not in all, implicates that extensive quality control is needed to assure correct typing results.

The high degree of polymorphism of the HLA genes at the nucleotide sequence level has proven sequence-based typing a major typing strategy. For DRB1 the allelic variability is predominantly present in the second exon and by DNA sequencing of exon 2 all hitherto known DRB1 alleles can be detected. For the associated genes DRB3, DRB4 and DRB5 the situation is slightly different. Allelic differences are not limited to exon 2 and the sequence of exon 3 and sometimes exon 4 is needed for complete subtyping. Oligonucleotides to amplify the exons needed for subtyping of DRB3, DRB4 and DRB5 were designed. Gene-specific products were generated to make simultaneous detection of alleles in heterozygous combinations possible. In this way 238 individuals were fully typed for their DRB3, 4 and 5 subtypes. Additional samples were typed for only one of the genes. All samples had been previously typed by PCR-SSP. Concordant typing results were obtained for all individuals tested. The DRB3 alleles typed for included *0101, *0201, *0202 and *0301, for DRB4 they were *01011, *0102 and *0103 and for DRB5 *0101, *0102, *0103, *0105, *0201, *0202 and *0203. All alleles were easily detected by the protocol described except for DRB5*0201. Sequencing of exon 3 and 4 of the DRB5*0201 allele showed this allele to be a sequencing error and the sequences obtained were identical to the exon 2, 3 and 4 sequences of DRB5*0202. Two new alleles were identified in the samples studied, DRB4*0105 and DRB3*0207. Sequence based typing has been recognized as a valuable tool for HLA typing of DRB1, DQB1 and DPB1 since several years. It is shown to be a superior typing method as well in the detection of the different DRB3, 4 and 5 subtypes.

No abstract

No abstract