P063 : IDENTIFICATION OF A NEW DQB1 AND DPB1 ALLELE WITH SBT AND GENE CLONING

Abstract

Aim The objective of this study is to characterize two new HLA class II alleles that we came across during routine clinical testing. Methods HLA typing was performed by medium resolution Luminex SSO (Thermofisher), high-resolution SSP (Olerup), and SBT (Life Technologies). Long-range PCR, followed by DNA cloning and sequencing was also conducted. Results Medium resolution SSO typing revealed the MUD sample is DRB1 ∗ 13:XX, DRB3 ∗ 01:XX and DQB1 ∗ 06:XX homozygous. SBT analysis for DQB1 loci indicates it is likely DQB1 ∗ 06:03 and DQB1 ∗ 06:04 with a G/A mutation at position 599 in Exon 3, which cause an amino acid change from glycine to glutamic acid at codon 168. The confirmation of this new allele has been assured by long-range PCR covering the whole genomic region of DQB1, followed by DNA cloning and Sanger Sequencing. SBT test, covering exon 2, 3, and 4 of DPB1 region, was applied for the referred sample of a problematic DPB1 typing derived from a renal patient. SBT analysis suggested that it is DPB1 ∗ 01:01:01 and a new allele likely joined from Exon 2 of DPB1 ∗ 28:01 with Exon3 of DPB1 ∗ 05:01:02 (or similar, b/c most of the DPB1 Exon3 info are not available in IMGT). We speculate that the new DPB1 allele is very likely generated from a cross-over/conversion event within intron 2 region between DPB1 ∗ 28:01 and DPB1 ∗ 05:01:02 (or similar allele). A long-range PCR and gene cloning covering DPB1 exon 2 to 3′ UTR region has confirmed the identity of this new DPB1 allele. Conclusions We have identified a new DQB1 ∗ 06 allele with an amino acid mutation (G168E) outside the antigen recognition site and a new DPB1 ∗ 28 allele that share the same antigen recognition site as DPB1 ∗ 28:01 but with different backbones. Although these new alleles are not likely going to alter the peptide repertoire being presented to CD4+ T cells, they could be candidate allo-reaction targets in both cellular and humoral immune responses in allotransplantation.