Genome Wide Single Nucleotide Polymorphism Typing for Identification of Putative Minor Histocompatibility Antigens in Graft Versus Host Disease.

Abstract

Previous studies have demonstrated that disparity across minor histocompatibility antigens (mHA) can cause graft versus host disease (GVHD) in patients who receive hematopoietic stem cell grafts from HLA-identical donors. mHA are peptide epitopes derived from normal cellular proteins presented by self MHC. Most autosomal mHA are generated as a result of non-synonymous coding single nucleotide polymorphisms (cSNPs), which lead to differences in the amino acid sequences of homologous proteins between donor and recipient cells. Although it is estimated that several hundred mHA exist in humans, only 16 have been definitively characterized to date. Using the Affymetrix 20K cSNP array we performed SNP typing on 97, HLA-A2+ hematopoietic stem cell transplant (HSCT) recipients and their sibling donors. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMC) obtained from patients and their donors. All patients were in remission at the time of sampling, all had undergone HSCT at the Dana-Farber Cancer Institute between 1998 and 2005 and all samples were drawn prior to transplantation. The transplants included myeloablative and non-myeloablative conditioning regimens, T cell depleted and non-T cell depleted grafts and sex matched and sex mis-matched donors. Using dChip software, we evaluated each of the 20,000 non-synonymous cSNPs on the array for mismatch between sibling pairs and for an association between mismatch in the GVHD direction and the development of acute or chronic GVHD. Mismatch in the GVHD direction was defined as a homozygous donor (AA or BB) and a heterozygote recipient (AB) or a homozygous donor (AA or BB) and a homozygously mismatched recipient (BB or AA). We ranked the cSNPs on the array in order of the strength of the association between mismatch in the GVHD direction and the development of either acute or chronic GVHD. There was no overlap between the 40 mismatched cSNPs most strongly associated with acute GVHD and the 40 most tightly associated with chronic GVHD. Mismatch at the SNP rs12407003 in the OMA1 gene was most highly associated with acute GVHD with mismatch in the GVHD direction occurring in 13 of 41 pairs with acute GVHD and 2 of 56 without (p=0.0003 by Fisher's Exact Test). OMA1 encodes a mitochondrial membrane-bound metallopeptidase. 65 sibling pairs were assessable for chronic GVHD. Mismatch at the SNP rs2740349 in the GEMIN4 gene was most strongly associated with chronic GVHD with mismatch in the GVHD direction occurring in 10 of 26 pairs with chronic GVHD and 1 of 39 without (p=0.0002). GEMIN4 is part of a cytoplasmic multiprotein complex. This study demonstrates a novel, genome-wide method of identifying putative mHA using a cSNP array. It reveals that mismatch of non-synonymous cSNPs in the GVHD direction occurs at an appreciable frequency in sibling pairs consistent with the hypothesis that the number of mHA in humans is large. Interestingly, the pattern of mismatch differs between acute and chronic GVHD. The study also identifies individual non-synonymous cSNPs for which mismatch in the GVHD direction is highly associated with the development of GVHD. Further evaluation of these cSNPs in larger independent cohorts will be undertaken to validate this association and targeted immunologic analysis of peptides derived from these cSNPs will examine their role as putative mHA.