Genomic typing of minor histocompatibility antigen HA-1 by reference strand mediated conformation analysis (RSCA).
Disparities in minor histocompatibility antigen (mHAg) HA-1 are involved in the development of acute graft-versus-host disease (GvHD) in adult recipient after HLA-identical sibling donor hematopoietic stem cell transplantation. The mHAg HA-1 is an HLA-A*0201-restricted nonapeptide, which derives from the cleavage of a protein encoded at chromosome 19. The sequence analysis of HA-1 cDNA identified two alleles, termed HA-1H and HA-1R, which differ in only two nucleotides at 3' end of exon A, at positions 500 and 504. DNA-based methods for HA-1 typing were developed in 1998, using polymerase chain reaction with sequence-specific primers (PCR-SSP) and restriction fragment length polymorphism (PCR-RFLP). Here, we report the usefulness of reference strand mediated conformation analysis (RSCA), which was developed for mutation detection and typing of polymorphic loci, to discriminate between the two HA-1 alleles. We performed genomic typing of HA-1 locus in 203 HLA-A*0201-positive samples using RSCA and we confirmed these results by PCR-SSP. The results demonstrate the high reproducibility of this method and their strong correlation with the results obtained by PCR-SSP (99%). Only two samples showed disparity between the RSCA typing and the PCR-SSP. Direct sequencing of these samples confirmed that the correct allele assignment was that obtained by the RSCA typing. Furthermore, HA-1- RSCA-based typing provides additional information about the intronic structure of both alleles. With this approach, we describe the almost constant presence (99.2%) of a 5-bp deletion at intronic position 214-218 associated to the HA-1H allele, previously unidentified. We conclude that HA-1 genomic typing by RSCA is easy to perform and that could be used as a routine typing method.
Aróstegui
J I
JI
Clinical Hematology Department, Institut Català d'Oncologia, Hospital Durán i Reynals, Barcelona, Spain. 30864jag@comb.es
Gallardo
D
D
Rodríguez-Luaces
M
M
Querol
S
S
Madrigal
J A
JA
García-López
J
J
Grañena
A
A
D000483:Alleles; D017931:DNA Primers; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D015778:Minor Histocompatibility Antigens / Q000235:genetics*; D009842:Oligopeptides / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational
D017931:DNA Primers; C110615:HA-1 antigen; D015778:Minor Histocompatibility Antigens; D009842:Oligopeptides