Abstract
BackgroundThe minor histocompatibility antigens (mHags) are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules. Disparities in mHags are a potential risk for the development of graft-versus-host disease (GvHD) in the recipients of bone marrow from HLA-identical donors. Two alleles have been identified in the mHag HA-1. The correlation between mismatches of the mHag HA-1 and GvHD has been suggested and methods to facilitate large-scale testing were afterwards developed.MethodsWe used sequence specific primer (SSP) PCR and direct sequencing to detect HA-1 gene polymorphisms in a sample of 131 unrelated Italian subjects. We then set up a novel melting temperature (Tm) assay that may help identification of HA-1 alleles without oligonucleotide probes.ResultsWe report the frequencies of HA-1 alleles in the Italian population and the presence of an intronic 5 base-pair deletion associated with the immunogeneic allele HA-1H. We also detected novel variable sites with respect to the consensus sequence of HA-1 locus. Even though recombination/gene conversion events are documented, there is considerable linkage disequilibrium in the data. The gametic associations between HA-1R/H alleles and the intronic 5-bp ins/del polymorphism prompted us to try the Tm analysis with SYBR® Green I. We show that the addition of dimethylsulfoxide (DMSO) during the assay yields distinct patterns when amplicons from HA-1H homozygotes, HA-1R homozygotes, and heterozygotes are analysed.ConclusionThe possibility to use SYBR® Green I to detect Tm differences between allelic variants is attractive but requires great caution. We succeeded in allele discrimination of the HA-1 locus using a relatively short (101 bp) amplicon, only in the presence of DMSO. We believe that, at least in certain assets, Tm assays may benefit by the addition of DMSO or other agents affecting DNA strand conformation and stability.
Highlights
The minor histocompatibility antigens are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules
We report our experience with melting temperature (Tm) analysis, sequence specific primer (SSP)-PCR, and direct sequencing in detecting the HA-1 polymorphism in a cohort of Italian subjects, which allowed us to disclose further polymorphic variations surrounding the two HA-1 alleles and, in particular, an intronic 5-bp deletion, strongly associated with HA-1H allele
In comparison with the sequence retrieved from GenBank under accession number AF092537, which corresponds to the HA-1R, we identified an intronic 5-bp deletion in the first case examined
Summary
The minor histocompatibility antigens (mHags) are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules. Disparities in mHags are a potential risk for the development of graft-versus-host disease (GvHD) in the recipients of bone marrow from HLA-identical donors. Acute graft-versus-host disease (aGvHD) is still a major marrow transplantation, occurring in 10–60% of patients (page number not for citation purposes). A(1) is the forward primer common to site-specific primers A(2) and A(3) designed with the 3'OH-end at c.500C/T. B(4) is the reverse primer common to site-specific primers B(5) and B(6) designed with the 3'OH-end at c.504A/G. Receiving matched sibling allograft, depending on prophylaxis regimen. These figures turned out to be even higher in the case of unrelated matched allograft [1]. Recent studies emphasize the involvement of mHags disparities in the development of aGvHD [2,3,4]
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