Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation

Abstract

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a 'molecular timeline' since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.

Morphologic and molecular analysis of Richter syndrome in chronic lymphocytic leukaemia patients treated with ibrutinib or venetoclax

“…The analysis of IGHV-IGHD-IGHJ rearrangements was performed as described by Agathangelidis et al 34 DNA amplification with leader primers was followed by bidirectional Sanger sequencing and results were obtained by immunoinformatic analysis using IMGT V-QUEST and ARResT/ AssignSubsets software in concordance with the updated ERIC recommendations. 35 The whole coding region of TP53 gene including the splice sites was analysed using the SureMASTR TP53 library preparation kit (Agilent, USA) and MiSeq sequencing with a minimum mean coverage of 10,000×.…”

Expression of the Chemokine Receptor CCR1 in Burkitt Lymphoma Cell Lines Is Linked to the CD10-Negative Cell Phenotype and Co-Expression of the EBV Latent Genes EBNA2, LMP1, and LMP2

“…The analysis of the immunoglobulin heavy-chain variable region (IGHV) gene mutations was carried out according to the described protocol [ 37 ] using six 5′ IGHV leader primers and four 3′ IGHJ primers. The percentage (%) of the identity shared with the top germline genes was determined by applying the NCBI IGBLAST Tool ( , accessed on 1 January 2021).…”

Chromatin Interaction Neural Network (ChINN): A machine learning-based method for predicting chromatin interactions from DNA sequences

“…IGHV mutation test was performed following the method in Agathangelidis et al 34 . Briefly, IGHV-IGHD-IGHJ gene rearrangements were amplified by 5' IGHV leader primers and 3' IGHJ primers (primer sequences are provided in Supplementary Table 6) using genomic DNA (gDNA) from CLL patient samples.…”