Sequence conservation of RAG-1 and RAG-2 genes in hematologic malignancies

Abstract

The diversity of the immune repertoire is due to a physiological process of somatic gene rearrangement directed by the V(D)J recombinase. The products of the RAG-1 and RAG-2 genes drive the molecular process of assembly of immunoglobulin and T cell antigen receptor genes. The expression of RAG genes is not restricted to immature lymphocyte populations in the thymus and bone marrow but they are also functional in germinal center B cells. [1][2][3][4] It has been postulated that certain chromosomal translocations could be attributed to an illegitimate recombinase activity of this enzymatic complex. Earlier studies have demonstrated that RAG-1 is expressed in a stage-specific manner among acute lymphoblastic leukemia(ALL)/lymphoblastic lymphoma (LBL) cells. 5 Schwarz et al 6 demonstrated the absence of mature B and T cells owing to truncations of the RAG-1 or RAG-2 genes by missense mutations in the active core of the proteins that eliminate their function and lead to an immunodeficiency.A mutational analysis of the entire coding region of the RAG-1 and RAG-2 genes was performed in a series of different types of hematologic malignancies. Cell lines tested and purchased from DSMZ were: NALM-6 (human B cell precursor leukemia), HEL (human erythroleukemia), RAJI (human Burkitt lymphoma), MV-4,11(human acute monocytic leukemia which carries a 4;11 translocation), SU-DHL-1 (human anaplastic large cell lymphoma which carries the t(2;5)(p23;q35) NPM-ALK fusion gene), DAUDI (human Burkitt lymphoma) and NB-4 (human acute promyelocytic leukemia which carries the t(15;17) PML-RARA fusion gene). Clinical samples harboring well characterized molecular lesions were also analyzed: six acute lymphoblastic leukemias with bcr/abl rearrangements, five anaplastic large cell lymphomas with NPM/ALK rearrangements, five follicular lymphomas carrying the bcl2/J H rearrangement, 14 acute leukemias with MLL rearrangements, nine acute lymphoblastic leukemias and follicular lymphomas with p53 mutations and 11 acute lymphoblastic leukemias with TEL/AML-1 rearrangements. Mutational analysis of RAG-1 gene was performed following the protocol described in an earlier work 7 and RAG-2 gene was studied using seven sets of overlapping oligonucleotide primers covering the coding sequence (primers and PCR conditions are provided on request).The multiple PCR-SSCP analysis of the RAG-1 gene detected shifted bands in four cases with the following diagnosis: two bcr/abl+ ALL, one MLL+ AML and one case diagnosed with FL. These samples were sequenced and a single nucleotide polymorphism from A to G was found at position 2571, which has been described previously. 8 Furthermore, in our study we detected another single transitional base change localized at position 301 in the sequence of the RAJI cell line (human Burkitt lymphoma). However, this abnormal band did not correspond to an amino acid change since both codons codified a Proline. This sequence change was present in a heterozygous state (Figure 1).We were unable to detect sequence changes in the RAG-2 g...

Characterizing disease states from topological properties of transcriptional regulatory networks

“…For example, this graph suggests that the regulation of RAG1 (recombination activating gene 1) by RUNX1/CBFB (which comprises the subunits of the heterodimeric transcription factor, core binding factor) is a crucial link differentiating AML and ALL. RAG1 is known to be expressed in a stage-specific manner in various types of ALL (both T and pre-B) [ 46 ]. Yannoutsos et al recently demonstrated that an intergenic silencer which suppresses expression of RAG1 in developing lymphocytes is dependent on occupation of the RUNX binding sites [ 47 ].…”