The use of molecular techniques for identification of genetic divergence in transplantation : with special reference to MHC genes and HLA typing

Detta är en avhandling från Stockholm : Karolinska Institutet, Biosciences and Nutrition

Sammanfattning: Transplantation immunology basically deals with the immune mechanisms that lead to rejection of transplanted tissues. Lymphocytes are the keymediators of graft rejection. At the time for this observation, the genetic loci responsible for histocompatibility between individuals of the same species were already identified in the mouse. It was through combination of these two major areas in transplantation immunology; cell biology and immunogenetics, that the relationship between T lymphocytes and human leukocyte antigens (HLA), derived from major histocompatibility complex (MHC) genes, was established. MHC genes in humans encode the transplantation antigens; HLA class I and HLA class 11 molecules, which are the main immunological barriers to successful transplantation. Each individual has several MHC gene loci with a remarkable degree of allelic variability. This inter- individual allelic variability in turn derives from DNA sequence variability localised to a number of discrete polymorphic regions in the exon(s) encoding the aminoterminal domains of HLA molecules. Although one individual only express around 1020 different HLA molecules, the potential number of MHC allele-combinations largely exceeds the world's population. However, strings of certain alleles at adjacent loci on the same chromosome (haplotypes) tend to be coherently inherited, and consequently enables HLA matching even between unrelated individuals in transplantation. Serological techniques were earlier the best way to define histocompatibility, i.e. the degree of HLA class I and class II molecular similarity between transplanted tissue and the transplant recipient. However, the use of anti-HLA antibodies (normally present in sera of multiparous women) is inadequate to characterize the extensive molecular polymorphism of the transplantation antigens. We have developed a polymerase chain reaction-based technique, using sequence- specific primers (PCR-SSP) for HLA typing, starting with the HLA class II genes. Typing responses are clearly visible as the mere absence or presence of a number of PCR products. By eliminating tedious post PCR amplification processing, we have managed to perform highly sensitive and specific HLA genomic typing in 2 hours, which makes the PCR-SSP technique possible to use in a routine clinical cadaveric organtransplant situation. Finally, we have retrospectively studied the presence of donor- and recipient- derived hematopoetic cells by an alternative application of PCR amplification with minisatellitespecific primers, in patients with pre-B-cell-acute lymphoblastic leukemia (pre-B-ALL), after bone marrow transplantation (BMT). The chimeric status obtained in the potentially affected cell lineage (B- cells), was compared to the patient-specific clonal origin of leukemic cells, by sequencing of clonally rearranged immunoglobulin (IgH) genes, and T-cell receptor (TCR[delta]) genes. A correlation between the sensitivity of the two assays, as well as a clinical correlation to relapse seen after BMT for patients with mixed chimerism (MC) in the B-cell lineage, suggested that routinely performed mixed chimerism analysis of a potentially affected cell lineage, may facilitate BMT monitoring and rapid therapeutic decisions in monitoring of many transplanted patients.

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