Selection of unrelated donors for bone marrow transplantation is improved by HLA class II genotyping with oligonucleotide hybridization.

Abstract

As the demand for donors for bone marrow ranplantation increases, the use of HLA-matched, gently unrelated donors represents a promising strategy. It is well documented that the clinical outcome of bone marrow transplantation is directly dependent on optimal matching for HLA class I and clss H specificities. Molecular studies have revealed the existence of a much larger number of HLA class II alleles than was antipated, many of which cannot be recognized by routine serolgical typing. Bone marrow transplantation (BMT) represents a major therapeutic option in the treatment of patients with leukemia and bone marrow failure syndromes (1). Unfortunately, 60-70% ofthese patients lack an HLA-matched related donor (2). Clinical studies have demonstrated the feasibility of using partially mismatched family members or partially or fully matched unrelated volunteers as marrow donors (3-8). Although the availability of unrelated donors is potentially very large, one important limitation is the accuracy of HLA matching of such donors, in particular for the large number of allelic differences that remain undetected by standard serological typing (9). Indeed, clinical experience with patients receiving a marrow graft from unrelated donors or haplo-identical family donors has shown that graft failure and acute graft-versus-host disease increase as the extent of HLA mismatch increases (5-8).The cloning and sequencing of HLA class II genes have revealed an unsuspected degree of diversity of HLA class II molecules. This approach has established in particular that polymorphism at several class II loci (i.e., DRBJ, DRB3, DQBJ, DQAJ, and DPBI) is much greater than was identified from current HLA class II typing by serology (9). A large number of allelic subtypes thus represent "hidden alleles," undetectable by conventional methods. Fortunately, the availability of cloned HLA class II genes makes it possible to analyze the polymorphism directly at the level of DNA (genotyping). We first proposed such an analysis based on restriction fragment length polymorphism (RFLP) (10). More recently we have presented another approach to genotyping, based on the direct recognition of allelic sequences by hybridization with locus-and allelespecific synthetic oligonucleotides ("HLA oligotyping") (11) and have shown that class II micropolymorphism can be accurately analyzed down to single amino acid differences. This HLA oligotyping procedure can now be simplified by the use of DNA previously amplified by the polymerase chain reaction (PCR) (12, 13).Based on the availability of DNA sequence information for all known alleles of DRB, DQB, and DPB loci, we have developed conditions allowing the identification of all DRBI, DRB3, DRBS, DQBJ, and DPBJ alleles in heterozygous individuals with an appropriate choice of informative sequence-specific oligonucleotides (14-18). Our oligotyping system involves two successive steps. In step 1 (HLA-DR generic typing), a battery of 14 sequence-specific probes is used to identify all major DR specificities (DR...

High-resolution HLA-DRB1 and DQB1 genotyping in Japanese patients with testicular germ cell carcinoma

“…The inconsistency in the reports on the association of some of HLA-B antigens with TGC tumours may stem from the strong linkage between the HLA-B and HLA-DRB I regions. The weaker antigenicity of HLA-A than HLA-B reported in organ transplants, despite the antigens' structural similarities, is attributed mainly to the weak association of HLA-A with HLA-DR, whereas HLA-B has a strong association with HLA-DR (Tiercy et al, 1991;Ichikawa et al, 1993).…”

“…The association of cervical carcinoma and MHC class II genes is still a matter of dispute, mainly owing to typing difficulties as stated (Glew et al, 1992). In addition, because a greater number of class II genes have been detected by genomic analysis than by serology, new standards for bone marrow and other organ transplants have been established (Tiercy et al, 1991;Ichikawa et al, 1993).…”

HLA class II DNA polymorphism in a Moroccan population from the Souss, Agadir area

“…The typing of genomic DNA for HLA-DRB1, -DQA1 and -DQBl loci was performed as previously described (15). The second exon of the DRBl, DQAl and DQBl genes was amplified by the polymerase chain reaction (PCR) using Taq polymerase and specific flanking primers (16). Amplified DNA was blotted onto nylon membranes, and alleles were identified following hybridization with digoxigenin-11-dUTPlabeled sequence-specific oligonucleotides (SSO) and detection with a chemoluminescence substrate (AMPPD, beringher Mannheim Biochemicals).…”

Bone marrow transplantation with unrelated donors: what is the probability of identifying an HLA-A/B/Cw/DRB1/B3/B5/DQB1-matched donor?

“…2 After applying DRB1 high resolution testing an HLA-ABDRB1-matched donor can be found for 40-50% of the patients. 2,[5][6][7] Because of the predominance of Caucasoids in most of the national registries, donor searches are more successful for Caucasoid patients while success rates for patients from other ethnic groups are much lower.…”