HLA matching of unrelated bone marrow transplant pairs: direct sequencing of in vitro amplified HLA-DRB1 and -DQB1 genes using magnetic beads as solid support.
Sequencing of HLA genes can offer complete information on the HLA class II genes relevant for the outcome of bone marrow transplantation (BMT). Genomic HLA matching of unrelated BMT patient/donor pairs is often based on PCR-SSO typing of HLA class II alleles. Typing a small number of samples by this approach is both expensive and time-consuming, due to the large number of SSO probes required to perform a complete class II typing. Moreover, only polymorphisms explicitly tested for will be found. We now provide the first report of the use of direct sequencing of HLA-DRB1 and -DQB1 genes, using PCR-amplified genomic DNA attached to magnetic beads, for clinical routine HLA matching. Sequencing ladders obtained by this procedure are easily readable, the patterns can be interpreted in HLA homozygous as well as heterozygous individuals, and sequence differences or similarities between the BMT donor and recipient can be directly identified. This genomic typing method is informative, relatively fast and therefore well-suited for the small number of samples usually analyzed in matching of BMT pairs. Furthermore, this technique has the potential for automation.
Spurkland
A
A
Institute of Transplantation Immunology, National Hospital, Oslo, Norway.
Knutsen
I
I
Markussen
G
G
Vartdal
F
F
Egeland
T
T
Thorsby
E
E
D001483:Base Sequence; D016026:Bone Marrow Transplantation / Q000276:immunology*; D015343:DNA Probes, HLA; D004277:DNA, Single-Stranded / Q000235:genetics; D005802:Genes, MHC Class II; D006683:HLA-DQ Antigens / Q000235:genetics*; D059866:HLA-DQ beta-Chains; D006684:HLA-DR Antigens / Q000235:genetics*; D059811:HLA-DRB1 Chains; D006579:Heterozygote; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006650:Histocompatibility Testing / Q000191:economics / Q000379:methods*; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014019:Tissue Donors
D015343:DNA Probes, HLA; D004277:DNA, Single-Stranded; D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C069051:HLA-DQB1 antigen; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II