Comparison of HLA-DRB1 typing by DNA-RFLP, PCR-SSO and PCR-SSP methods and their application in providing matched unrelated donors for bone marrow transplantation.
The aim of the study was to devise a strategy for large batch analysis to determine HLA Class II alleles exhibited by candidate bone marrow transplant donors and prospective recipients using previously published DNA-based typing techniques. Special attention was directed towards the technical aspects of procedures, the level of typing resolution and the speed of data analysis. 200 blood samples from volunteer bone marrow transplant donors typed serologically for HLA-DR and DQ were further investigated using three DNA-based typing methods: (i) restriction fragment length polymorphism (RFLP) analysis, (ii) polymerase chain reaction (PCR) amplification and subsequent hybridisation with sequence specific oligonucleotide probes (PCR-SSO), and (iii) PCR amplification with sequence specific primers (PCR-SSP) to resolve the DRB1* specificity of each individual. In general, the HLA-DR results obtained using PCR-SSO and PCR-SSP correlated well with each other. However, discordant results were obtained between PCR and RFLP based typing in 21 cases, especially in relation to DRB3* alleles associated with the DRB1 gene. These differences were due to three problems pertaining to RFLP analysis: i) alleles with identical DRB, DQA and DQB fragment sizes, ii) reliance on DQA and DQB results to assign the DRB genotype, and iii) a "new polymorphism" of DR7, in a DR7 homozygous, exhibiting a fragment similar in size to DR8. Our findings suggested a strategy requiring PCR-SSO analysis for initial low resolution class II typing involving large numbers of samples, while the use of PCR-SSP is reserved for small numbers of samples, for urgent samples or for situations where higher resolution is required.(ABSTRACT TRUNCATED AT 250 WORDS)
Jordan
F
F
Anthony Nolan Research Centre, Royal Free Hospital, London, England.
McWhinnie
A J
AJ
Turner
S
S
Gavira
N
N
Calvert
A A
AA
Cleaver
S A
SA
Holman
R H
RH
Goldman
J M
JM
Madrigal
J A
JA
D001483:Base Sequence; D016026:Bone Marrow Transplantation; D017931:DNA Primers; D005069:Evaluation Studies as Topic; D005802:Genes, MHC Class II; D006684:HLA-DR Antigens / Q000032:analysis* / Q000235:genetics; D059811:HLA-DRB1 Chains; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008403:Mass Screening; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D014019:Tissue Donors; D009927:Tissue and Organ Procurement / Q000379:methods*
D017931:DNA Primers; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D015345:Oligonucleotide Probes