Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Graziano
Claudio
C
Human Genetics Unit, Department of Clinical Physiopathology, University of Florence, Viale Gaetano Pieraccini 6, 50139 Firenze, Italy. drgraziano@katamail.com
Giorgi
Massimo
M
Malentacchi
Cecilia
C
Mattiuz
Pier Luigi
PL
Porfirio
Berardino
B
D000483:Alleles; D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D006801:Humans; D015778:Minor Histocompatibility Antigens / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes; D009842:Oligopeptides / Q000235:genetics*; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011804:Quinolines; D017422:Sequence Analysis, DNA / Q000379:methods*; D017384:Sequence Deletion; D013696:Temperature
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; C110615:HA-1 antigen; D015778:Minor Histocompatibility Antigens; D015345:Oligonucleotide Probes; D009842:Oligopeptides; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I