OR18 Full gene sequencing reveals very limited variation in the regions outside of the antigen recognition domains (ARD) of 360 unrelated hematopoietic stem cell transplant donor-recipient pairs matched for 10/10 at high resolution

Abstract

Aim Traditional DNA-based typing focuses primarily on interrogating the exons of HLA genes that form the ARD (peptide-binding region that interacts with T cell and killer immunoglobulin-like receptors). The relevance of mismatching outside the ARD on hematopoietic stem cell transplantation (HCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in well matched transplant pairs. Methods 360 HLA-A, B, C, DRB1, and DQB1 high resolution matched donor (D)-recipient (R) pairs were selected based on availability of retrospective Sanger-based sequencing and DNA from the CIBMTR Repository. The majority of the population was self-identified Caucasian (80%). Next generation DNA sequencing was performed on the Illumina MiSeq platform and interpreted with Connexio Assign MPS. Class I gene sequences covered 5 ′ UTR-3 ′ UTR; DRB1, intron 1-intron 3; DQA1 5 ′ UTR-exon 4; DQB1, intron 1-3 ′ UTR. DQ noncoding regions were not evaluated. Results The majority (98.1%) of the HLA-A, B, C, DRB1 D-R allele pairs are matched for sequences outside the ARD exons: 0.5% differ in non-ARD exons, 1.9% differ in noncoding regions. A small number (0.2%) differ within ARD exons. Mismatches in non-ARD exons varies from 0.7% for HLA-C and DQA1 to 0% DQB1; noncoding variation ranges from 2.8% for HLA-C to 1.3%, HLA-B and DRB1. Within non-ARD exons, both nonsynonymous (16 allele pairs) and silent (2) variation is present. Intron variation is minor, usually a single nucleotide. Conclusions This is the first study to evaluate the genetic variation and characterize mismatching outside of the ARD in a cohort of HLA-matched donor-recipient pairs. The paucity of exonic mismatches outside of the ARD is striking. Intronic variation is more common but would not contribute to an alloreactive mismatch. At present, it does not appear to be necessary to increase the resolution of HLA typing beyond the ARD in selecting a matched donor except in cases of common non-expressed variants within G-group assignments. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D–R pairs will be difficult to assess in HCT outcome studies since it does not occur very frequently. Further study is warranted to confirm these findings in larger and more diverse cohorts.