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Multiplex PCR reaction for the detection and identification of immunoglobulin kappa deleting element rearrangements in B‐lineage leukaemias
Author(s) -
Stolz Frank,
Panzer Simon,
Renate PanzerGrümayer E.
Publication year - 1999
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1046/j.1365-2141.1999.01557.x
Subject(s) - gene rearrangement , minimal residual disease , biology , microbiology and biotechnology , primer (cosmetics) , clone (java method) , immunoglobulin heavy chain , multiplex polymerase chain reaction , chronic lymphocytic leukemia , multiplex , polymerase chain reaction , kappa , antibody , gene , genetics , leukemia , chemistry , linguistics , philosophy , organic chemistry
Immunoglobulin kappa (Igκ) gene recombinations can be used — similarly to IgH rearrangements — as clonal markers in B‐lineage leukaemias. Based on the extensive junctional diversity, these rearrangements represent valuable targets for the analysis of minimal residual disease (MRD). In order to provide a simple method for the rapid detection of leukaemia clone‐specific kappa deleting element (Kde) mediated rearrangements, we developed a multiplex PCR reaction that is able to amplify the five most frequent rearrangements in one tube. Position of the amplimers were chosen to enable identification of the involved segments according to the size of the PCR product. This method was tested on 101 B‐lineage leukaemias (71 childhood B‐cell precursor acute lymphoblastic leukaemias (BCP ALL) and 30 chronic lymphocytic leukaemias (CLL)). 39 and 22 Kde rearrangements could be readily detected in 30 (44%) BCP ALL and 22 (56%) CLL, respectively. 36% of the Kde rearrangements in BCP ALL and 45% in CLL were intron recombination signal sequence (RSS)‐Kde rearrangements. The other Kde rearrangements involved the Vκ families: VκI in 36% and 50%, VκII in 32% and 16.7%, VκIII in 24% and 25%, and VκIV in 8% and 8.3% in BCP ALL and CLL, respectively. The sensitivity of the multiplex system was 10 −2 –10 −3 . We compared this multiplex PCR assay with multiple single PCR reactions using different sets of primer combinations. Thereby the number and types of rearrangements were confirmed in all cases. Clonality of rearrangements was proven by sequence analysis. Our data show that by this method clonal Kde rearrangements were rapidly detected and precisely identified.