CASyM winter school of Systems Medicine took place between March 29th and April 1st 2017 in Ljubljana, Slovenia and is entitled »The 3rd SysBioMed hands-on tutorial: Systems Medicine Approaches in Personalized Medicine«
Immunogenetics in Transplantation of Haematopoietic Stem Cells
Sendi Montanič1, Blanka Vidan-Jeras1
1Blood Transfusion Centre of Slovenia
Introduction: HLA system is one of the most intensively studied genetic systems. Its complexity is due to many HLA alleles and their possible combinations. The HLA plays important role in transplantation of organs, tissues and hematopoietic stem cells (HSC), resistance or susceptibility to autoimmune and infectious diseases and reproductive medicine. Specific HLA alleles are more common in certain races and ethnic groups than in others . First methods for molecular typing of HLA have emerged already in the early 80s of the last century. This was followed by expansion of methods based on polymerase chain reaction, such as hybridization with sequence-specific oligonucleotides and amplification with sequence-specific primers. Lately, most laboratories serving HSCT use methods based on the classical Sanger sequencing. Due to the growing number of newly discovered HLA alleles such approaches to HLA typing are no longer sufficient. First alternative is monoallelic sequencing, where each allele is amplified separately. In our laboratory the typing is performed by commercial reagents which amplify exons 2, 3 and 4 for loci HLA-A, B, C and exons 2 and 3 for loci HLA-DR and DQ. This allows us to obtain results almost without ambiguities, with exception of null alleles, where the differences are outside the amplified exons. Second alternative is generation of extended HLA allele sequences by full length hemizygous Sanger sequencing. The method was developed in Tissue Typing Laboratory in Maastricht by Prof. Tilanus and provides unambiguous typing results at allelic level of resolution . Many HLA laboratories are currently introducing various methods of next-generation sequencing, although they were successfully introduced only in high throughput laboratories as they are time-consuming and too expensive for smaller laboratories. Oxford Nanopore has recently developed a small and low cost sequencer MinION® which gives the possibility of sequencing long DNA fragments, up to 13 kilobases . The technology is based on an array of nanopores embedded on a chip. Electrical current flows through the pores, creating tension in the membrane. The transition of an analyte through a pore leads to significant changes in the current and the measurement of these changes enables identification of the molecule. In the case of DNA sequencing the DNA molecule passes through the pore and the current varies depending on which of the bases G, A, T or C passes through the pore. Results: In order to explore the utility value of this new method in our routine laboratory, we compared the results obtained by MinION with the results of classical Sanger sequencing and those obtained with Roche reagents for NGS on Roche GS Junior454 system. We typed 18 samples for loci HLA-A, B and C. Typing resolution, time consuming, applicability for the routine work, availability on the market and prices were analyzed. For samples typed with MinION we obtained results without ambiguity in the exons, some remained in introns where sequences of most HLA alleles are not yet known and computer softwares for the interpretation of the results cannot compare them to a reference sequence. Conclusion: The method represents a significant improvement in the technology, which is necessary due to the growing number of HLA alleles. It provides high coverage, unambiguous results; it is low cost, fast and useful in various branches of diagnostics.
2006 - University of Ljubljana, Faculty of Medicine, Center for Functional Genomics and Bio-chips.