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Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes
© Graziano et al; licensee BioMed Central Ltd. 2005
Received: 28 February 2005
Accepted: 04 October 2005
Published: 04 October 2005
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. 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.
We 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.
We 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.
The 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.
Acute graft-versus-host disease (aGvHD) is still a major cause of morbidity after allogeneic HLA-identical bone marrow transplantation, occurring in 10–60% of patients receiving matched sibling allograft, depending on prophylaxis regimen. These figures turned out to be even higher in the case of unrelated matched allograft . Recent studies emphasize the involvement of mHags disparities in the development of aGvHD [2–4].
Among known autosomal mHags, only HA-1 has been implicated as a cause of aGvHD in humans . HA-1 is a nonapeptide from a protein encoded by a gene termed KIAA0223 (GenBank accession no. D86976), a polymorphic gene that has two known alleles differing at positions 500 and 504 of the cDNA sequence, resulting in a single aminoacid change. The HA-1H allele encodes histidine at position 3 of the peptide, is recognized by HLA-A*0201-restricted cytotoxic T cells and is only expressed by cells of haematopoietic origin . Its allelic counterpart, HA-1R, encodes arginine at position 3. HLA-A*0201 molecules have low affinity for the HA-1R peptide and the complex does not generate a detectable immune response . HA-1 disparity can thus be defined as the presence of HA-1H in the recipient but not in the donor, because in such cases T cells in the transplanted donor marrow respond to mHags in the recipient.
Four different DNA-based strategies have been described so far to perform HA-1 allelotyping. They rely on sequence specific primer (SSP) PCR , restriction fragment length polymorphism (RFLP) PCR , reference strand mediated conformation analysis (RSCA) , and allele specific fluorescence-labelled probes .
The two common primers were used to generate a fragment of an expected length of 486 bp containing the polymorphic sites. Relative positions of primer pair sets A and B designed to perform allele-specific PCR typing at the HA-1 locus are reported in figure 1. Forty ng of genomic DNA was used in 50 μL of a reaction mixture containing Applied Biosystems PCR Buffer with 1.5 mmol/L MgCl2, 15 pmol of each primer, 0.8 mmol/L dNTPs, and 2 units of AmpliTaq polymerase (Applied Biosystems). Cycling conditions were according to Wilke et al . Amplicons were purified with the QIAquick Purification Kit (Qiagen). Sequencing was performed on the ABI PRISM 310 Genetic Analyzer according to the protocol provided by the manufacturer.
Melting curve analysis
The primers used to amplify a 101-bp fragment were: forward, 5'-CTTCGCTGAGGGCCTTGAG-3' and reverse, 5'-CCTTGGGTCTGGCTCTGTCTT-3'. The reactions were performed in a total volume of 100 μL containing 50 μL of SYBR® Green PCR Master Mix (Applied Biosystems), a 300/300 mmol/L forward/reverse primer combination and 50 ng of genomic DNA. The cycling conditions were: 94°C for 45 sec, 60°C for 45 sec and 72°C for 45 sec in 33 cycles. After PCR, DMSO at 5% or 10% was added to the reaction tubes and the total volume was divided in 4 wells of an optical reaction plate. The dissociation protocol was performed in accordance to the default thermal profile of the ABI 5700 Sequence Detection System (Applied Biosystems), which included, after a 15 sec hold at 95°C and a 20 sec hold at 60°C, a slow ramp (20 min) from 60°C to 95°C. The fluorescence was monitored in real time (every 3 sec) for each sample. The melting peaks were calculated by the ABI 5700 software as the negative derivative of fluorescence with respect to the temperature (-dF/dT vs T).
Results and discussion
We report our experience with melting temperature (Tm) analysis, 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.
We sampled 131 unrelated subjects from the Italian population, obtaining a genotypic distribution of 23 HA-1H homozygotes, 41 HA-1R homozygotes, and 67 heterozygotes. The resulting allelic frequencies were 0.43 for allele HA-1H and 0.57 for allele HA-1R. The observed genotypes conformed to the Hardy-Weinberg law (X 2 = 0.23, not significant). Furthermore, our allele frequency estimates are associated with a standard error of 0.03 and are in line with those reported by Tseng et al  for North American Caucasians.
HA-1 haplotypes found in 72 random chromosomes of the Italian population.
c.509+213 ins/del TTTAT
In order to make HA-1 typing easier and faster, we tested the Tm approach to define HA-1 alleles. As reported, the ability of Tm analysis to discriminate different PCR products mainly depends on the length of the amplicon and the GC content [11, 12]. Both amplification and Tm analysis are automated processes that can be performed on fluorescence-detection equipped thermal cyclers. Double-stranded DNA products are detected by the SYBR® Green I nucleic acid dye in real-time during PCR. At the end of the reaction the temperature at which the transition double/single strand DNA occurs is revealed by following SYBR® Green I emission decay. Different PCR products may determine specific dissociation profiles [11, 12].
As a matter of fact, knowledge of the gametic associations between HA-1R/H alleles and the intronic 5-bp ins/del polymorphism might allow a straightforward Tm analysis combining H/R allele specific primer and the presence of the 5-bp deletion in a single amplicon. However, the fragment would be too large to allow correct typing (data not shown). In this respect, our results are in keeping with the criticisms raised by von Ahsen et al  towards Tm assays based on SYBR® Green I melting curves [14, 15].
We detected mutations in novel sites relative to a consensus sequence of HA-1 locus. We showed that these variations are arranged onto few rare haplotypes possibly generated through recombination/gene conversion events from two major ancestral haplotypes. The observed sequence diversity, besides HA-1R/H, involves either a synonymous codon or intronic changes making them unlikely direct determinants of allogeneic recognition by cytotoxic T cells.
Probe-free, Tm assays most often require confirmation by other DNA mutation detection assays. However, we propose that Tm-based methods may be successfully applied to find experimental conditions, such as the use of DMSO described here, for genotyping ins/del and/or single nucleotide polymorphisms.
This work was supported by Fondazione Rulfo, Parma and by funds ex MURST 60%. C.G. and M.G. are the recipients of post-doc fellowships of the University of Florence.
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