23-OR

Abstract

Aim Our aim was to identify a novel DRB1 ∗ 09 allele not detected by PCR-SSO in an NMDP donor. Methods Luminex-based SSO, SSP, and SBT were used to determine the HLA typing. Results The following typing was established using Luminex based technology: HLA-A ∗ 02:xx, 02:xx; B ∗ 07:xx, 45:xx; C ∗ 07:xx, 16:xx; DRB1 ∗ 13:xx, - ;DQB1 ∗ 03:xx, 05:xx. To investigate the unusual DR-DQ associations, high resolution PCR-SBT was performed (SeCore, Life Technologies). Low resolution results were confirmed and high resolution Class II results were: HLA-DRB1 ∗ 13:02, -; DQB1 ∗ 03:03, 05:01. Further testing included DRB1 low resolution PCR-SSP (Life Technologies and Olerup). The results obtained from both kits indicated the presence of an allele from the DRB1 ∗ 09 group. Next, PCR-SBT was performed using AlleleSeqr (Celera). This typing yielded HLA-DRB1 ∗ 09:xx, 13:02. A one nucleotide difference in exon 2 at position 112 was detected. This resulted in an A to C substitution. To confirm the DRB1 ∗ 09 allele as potentially novel, PCR-SBT was set up using SBTexcellerator (Qiagen). This approach allowed for the separate sequencing of the DRB1 ∗ 09 and DRB1 ∗ 13 alleles. The mutation occurred in the DRB1 ∗ 09 allele. This base substitution resulted in an amino acid change from a lysine to a glutamine at codon 9. The substitution is found on the pleated floor of the HLA protein, affecting its structure and potentially influencing peptide binding and presentation [Fig. 1]. Download full-size image Conclusions The sequence of this new allele is in the process of being submitted to the IMGT/HLA database for an official allele assignment. This demonstrates the need for HLA labs to utilize multiple methods and typing kits to achieve the most comprehensive results.