NGS analysis of BCR IGVH regions in wild type (WT) and TCL1 transgenic mice with intact (TCL1 Nfat2+/+) and deleted (TCL1 Nfat2-/-) NFAT2 expression

Chronic lymphocytic leukemia (CLL) can be defined as a clonal expansion of B cells with stereotypic B cell receptors (BCR). In up to 10% of CLL cases, a transformation to an aggressive B cell lymphoma (Richter´s syndrome) with a dismal prognosis can be observed over time. NFAT proteins are transcription factors originally identified in T cells, which also play an important role in B cells. The TCL1 transgenic mouse is a well accepted model of CLL. Upon B cell-specific deletion of NFAT2, TCL1 transgenic mice develop a disease resembling human Richter´s syndrome. Here, we analyzed BCR usage in wild type and TCL1 transgenic mice with and without NFAT2 deletion employing conventional molecular biology techniques and next generation sequencing (NGS). Splenocytes of 7-month-old animals (n=2 each genotype) were isolated and multiplex PCRs were applied for the amplification of BCR heavy chain hypervariable regions (IGVH) IgM. PCR products were cloned into the plasmid pCR4-TOPO and subsequently analysed by Sanger sequencing. Alternatively, PCR products were analyzed by next generation sequencing (NGS). We demonstrate that the loss of NFAT2 in CLL precipitates the selection of unmutated BCRs and the preferential usage of certain VDJ recombinations which subsequently results in the accelerated development of oligoclonal disease.