MHC Genotyping Using Next Generation Sequencing (NGS)
Dr. med. Kaimo Hirv
The determination of HLA types is the foundation for assessing the compatibility of donors and recipients in regard to blood stem cell or organ transplantation. Depending to the case, currently only 3-6 different HLA genes are being analyzed; the typing of the different genes is not complete either. The biologically relevant exons 2 and 3 of the HLA class I genes (HLA-A, -B, -C) or exon 2 of the HLA class II genes (HLA-DRB1, -DRB3/4/5, -DQB1) are analyzed.
Even if the analyzed HLA markers match completely, graft-versus-host disease (GVHD) or transplant rejection is a frequent complication of transplantations. The reason is that the success of a transplantation does not only depend on the compatibility of HLA types; other genes influence the course of the disease as well. The major histocompatibility complex, MHC, comprises a group of more than 400 genes on chromosome 6, on which not only HLA markers but a variety of proteins that are involved in the immune response are encoded. This gene cluster (approx. 4 mio bp) shows the highest variability in the human genome. Full sequencing of this region can provide crucial information for research and soon also for clinical use. This method enables characterizing additional genes that lead to the development of GVHD or transplant rejection. Furthermore, donor-recipient differences that contribute to the intensification of graft-versus-leukemia effect can be detected. More detailed information on the genetic variability of the MHC could optimize the selection of donors and therefore lead to better transplantation outcomes. The conventional sequencing methods do not allow sequencing of such a large gene region within one approach. With NGS it is possible to sequence DNA fragments of this scale quickly and at reasonable financial cost. Depending on the case, various MHC regions (MHC class I, II or III) can be analyzed and extended sequencing of HLA markers (including 5' and 3' UTR) can be requested. The diagnosis of autoimmune diseases represents another application of the complete sequencing of the MHC region. Numerous associations of HLA alleles with autoimmune phenomena are described in the literature; however, HLA typing alone does not provide a clear diagnosis. It is assumed that gene variants in proximity of HLA markers play a role in causing these diseases or that several genetic variants acting together lead to the development of autoimmunity.