Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high-throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high-throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V- and J-segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T-lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Genomics of Adaptive Immunity Shemyakin Ovchinnikov Institute of Bioorganic Chemistry Moscow Russia

Department of Internal Medicine Haematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

Department of Molecular Technologies Pirogov Russian National Research Medical University Moscow Russia

Laboratory of Cytogenetics and Molecular Genetics Dmitry Rogachev National Medical and Research Centre of Paediatric Haematology Oncology and Immunology Moscow Russia

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Novel bimodal TRBD1-TRBD2 rearrangements with dual or absent D-region contribute to TRB V-(D)-J combinatorial diversity