Immunoglobulin Gene Analysis in Chronic Lymphocytic Leukemia

  • Andreas Agathangelidis
  • Richard Rosenquist
  • Frederic Davi
  • Paolo Ghia
  • Chrysoula Belessi
  • Anastasia Hadzidimitriou
  • Kostas Stamatopoulos
Part of the Methods in Molecular Biology book series (MIMB, volume 1881)


The formation of B-cell receptor immunoglobulin (BcR IG) is the result of a multi-step process that starts at the pro-B cell stage with the VDJ gene recombination of IG genes of the heavy chain, followed by VJ recombination of the light chain genes at the pre-B II cell stage. As a result, a fully functional BcR IG is expressed on the surface of any given naive B cell. After antigen encounter, somatic hypermutation (SHM) and class-switch recombination (CSR) act on the rearranged IG genes within the context of affinity maturation, leading to the expression of a BcR IG with unique immunogenetic and functional characteristics. Since B-cell neoplasms arise from the transformation of a single B cell, this renders IG gene rearrangements ideal clonal markers as they will be identical in all neoplastic cells of each individual clone. Furthermore, the rearranged IG sequence can also serve as a cell development/maturation marker, given that its configuration is tightly linked to specific B-cell developmental stages. Finally, in certain instances, as in the case of chronic lymphocytic leukemia (CLL), the clonotypic IG sequence and, more specifically, the load of somatic hypermutations within the rearranged IG heavy variable (IGHV) gene, holds prognostic and potentially predictive value. However, in order to take full advantage of the information provided from the analysis of the clonotypic IG gene rearrangement sequences, robust methods and tools need to be applied. Here, we provide details regarding the methodologies necessary to ensure reliable IG sequence analysis based on the recognized expertise of the European Research initiative on CLL (ERIC). All methodological and analytical steps are described below, starting from the isolation of blood mononuclear cells (PBMC), moving to the identification of the clonotypic IG rearrangement and ending with the accurate interpretation of the SHM status.

Key words

Immunoglobulin gene CDR3 Somatic hypermutation Stereotypy 


Financial Support

Supported in part by H2020 “AEGLE, An analytics framework for integrated and personalized healthcare services in Europe,” by the EU; “MEDGENET, Medical Genomics and Epigenomics Network” (No.692298) by the EU; “TRANSCAN-179” NOVEL JTC 2016; “ESPA for young researchers 2017, Somatic hypermutation in Chronic Lymphocytic Leukemia: ontogenetic and clinical implications from the analysis of NGS immunogenetic data” (No. 5006864).


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Andreas Agathangelidis
    • 1
  • Richard Rosenquist
    • 2
  • Frederic Davi
    • 3
  • Paolo Ghia
    • 4
  • Chrysoula Belessi
    • 5
  • Anastasia Hadzidimitriou
    • 1
  • Kostas Stamatopoulos
    • 1
    • 2
    • 6
  1. 1.Institute of Applied Biosciences, Center for Research and TechnologyThessalonikiGreece
  2. 2.Department of Molecular Medicine and SurgeryKarolinska InstituteStockholmSweden
  3. 3.Hôpital Pitié-SalpêtrièreUniversité Pierre et Marie CurieParisFrance
  4. 4.IRCCS Istituto Scientifico San RaffaeleUniversità Vita-Salute San RaffaeleMilanItaly
  5. 5.Nikea G. HospitalAthensGreece
  6. 6.Department of Immunology, Genetics and PathologyUppsala UniversityUppsalaSweden

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