P067A simple sequencing based method for identification of DPB1 resident SNP rs9277534 defining TH level of DPB1 expression

Abstract

Aim Hematopoietic cell transplantation from unrelated donors carries the risk of acute graft-versus-host disease (GvHD). HLA-DPB1 matching has recently emerged as an important part of donor selection. HLA-DPB1 classification by T-cell epitope is currently used to identify permissive and non-permissive donor-recipient combinations. In addition, DPB1-linked SNP rs9277534 has been recently implicated in the severity of GvHD following transplantation with non-permissive DPB1 mismatching. Variant rs9277534A was associated in low expression, whereas rs9277534G was associated with high expression of DPB1. Recipients with the high expression variant who received transplant from DPB1-mismatched donors with low expression variant had high risk of GvHD. A simple and low-cost SNP rs9277534A/G test can assist optimal donor selection when multiple HLA suitable donors are available. Methods A simple Sanger sequencing-based method for identification of rs9277534 variants was developed. M13-tagged PCR primers were designed by applying NCBI Primer-BLAST tool to the fragment of DPB1 3’UTR containing the targeted SNP. Amplified fragments were sequenced with M13 sequencing primers. The targeted nucleotide position, n150 from the sequence start, can be visualized using any available sequencing analysis software and clearly shows the variant of SNP (“A”, “G” or “A/G”). Results Using the published list of A/G variants and a large set of samples with different combinations of DPB1 alleles, we defined a number of additional low- and high-expression DPB1 alleles. The published association of individual DPB1 alleles and rs9277534 variants was confirmed. Conclusions The extended list of low- and high-expression DPB1 alleles will provide a basis for assessing the suitability of DPB1-mismatched donors in the situation when a DPB1-matched donor is not available. The developed Sanger sequencing-based method for identification of rs9277534 variants can be used to identify the level of expression of DPB1 alleles previously not assessed.