Immunoglobulin Gene Analysis in Chronic Lymphocytic Leukemia

Methods Mol Biol. 2019;1881:51-62. doi: 10.1007/978-1-4939-8876-1_5.


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.

Keywords: CDR3; Immunoglobulin gene; Somatic hypermutation; Stereotypy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / genetics
  • DNA / isolation & purification
  • DNA Mutational Analysis / instrumentation
  • DNA Mutational Analysis / methods*
  • Genes, Immunoglobulin / genetics*
  • Humans
  • Immunoglobulin Class Switching / genetics*
  • Leukemia, Lymphocytic, Chronic, B-Cell / blood
  • Leukemia, Lymphocytic, Chronic, B-Cell / genetics*
  • Leukocytes, Mononuclear
  • RNA / genetics
  • RNA / isolation & purification
  • Receptors, Antigen, B-Cell / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction / instrumentation
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Somatic Hypermutation, Immunoglobulin


  • Receptors, Antigen, B-Cell
  • RNA
  • DNA