Zanone-Ramseier R, Gratwohl A, Gmür J, Roosnek E, Tiercy J M
Transplantation Immunology Unit, Division of Immunology and Allergology, Hôpital Cantonal, Geneva, Switerzland.
Transplantation. 1999 May 27;67(10):1336-41. doi: 10.1097/00007890-199905270-00008.
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.
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).
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.
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.
基于DNA的方法可对细胞表面表达缺失或极低的HLA抗原进行分型。此类血清学空白抗原在骨髓供体选择中的作用尚不清楚。
从两名白血病患者中检测到的HLA - A血清学空白抗原，从基因组DNA中进行克隆和测序。通过逆转录聚合酶链反应（PCR）测定mRNA表达和外显子3剪接情况。
两名血清学分型为A2/x和A3/x的患者经DNA分型显示为A01/A0201和A03/A24。HLA - AO1空白抗原被鉴定为A0104N等位基因，其外显子4中有一个C插入，导致产生一个终止密码子。HLA - A24空白抗原被鉴定为A2402102L等位基因，其特征是内含子2发生突变，主要导致外显子3剪接受损。因此，与其他HLA - A等位基因相比，功能性A24 mRNA减少至不到5%。对于这两名患者，基于血清学空白等位基因功能缺失的情况，开始寻找无关供体。第二名携带A24空白抗原的患者能够接受来自一名“A/B/C/DRB1/DRB5/DQB1完全匹配”且A抗原纯合的供体的骨髓移植。在已知可检测所有I类不相容性的移植前细胞毒性T淋巴细胞前体频率试验中，供体T细胞未对患者产生反应。然而，尽管移植前在功能上是相容的，但该患者在骨髓移植后44天死于IV级移植物抗宿主病。通过逆转录PCR和测序可证明存在潜在功能性的A2402 mRNA：估计50%的A24 mRNA分子包含具有正确剪接位点的外显子3。
这些发现表明，mRNA表达低的血清学空白抗原在骨髓移植后仍可能被识别。因此，分子方法对此类等位基因的测定以及mRNA表达的评估应纳入无关骨髓供体搜索方案中。
