Sequencing of two HLA-A blank alleles: implications in unrelated bone marrow donor matching.

BACKGROUND

DNA-based methods can type for HLA antigens with no or very low cell surface expression. The role of such serological blank antigens in bone marrow donor selection is unknown.

METHODS

HLA-A serological blank antigens detected in two leukemic patients were cloned and sequenced from genomic DNA. mRNA expression and exon 3 splicing were determined by reverse transcriptase-polymerase chain reaction (PCR).

RESULTS

Two patients typed A2/x and A3/x by serology were revealed to be A01/A0201 and A03/A24 by DNA typing. The HLA-AO1 blank antigen was identified as the A0104N allele with a C insertion in exon 4, which results in a stop codon. The HLA-A24 blank antigen was identified as the A2402102L allele characterized by a mutation in intron 2 leading to impaired splicing of mainly exon 3. As a consequence, functional A24 mRNA was reduced to less than 5% compared with the other HLA-A allele. For both patients, a search for an unrelated donor was initiated on the basis of the functional absence of the serologically blank allele. The second patient with the A24 blank antigen could undergo transplantation with marrow from a "fully A/B/C/DRB1/DRB5/DQB1-compatible" donor, homozygous for the A antigen. Donor T cells did not react against the patient in a pretransplantation cytotoxic T lymphocyte precursor frequency test known to detect all class I incompatibilities. However, despite this functional pretransplantation compatibility, the patient died 44 days after bone marrow transplantation, suffering from graft-versus-host disease grade IV. The presence of potentially functional A2402 mRNA could be demonstrated by reverse transcriptase-PCR and sequencing: 50% of the A24 mRNA molecules were estimated to contain exon 3 with a correct splice site.

CONCLUSIONS

These findings show that serological blank antigens with low mRNA expression might still be recognized after bone marrow transplantation. The determination of such alleles by molecular methods and the assessment of mRNA expression should therefore be included in the unrelated bone marrow donor search protocol.