Genome-wide detection of genes targeted by non-Ig somatic hypermutation in lymphoma

Abstract

The processes of somatic hypermutation (SHM) and class switch recombination introduced by activation-induced cytosine deaminase (AICDA) at the Immunoglobulin (Ig) loci are key steps for creating a pool of diversified antibodies in germinal center B cells (GCBs). Unfortunately, AICDA can also accidentally introduce mutations at bystander loci, particularly within the 5' regulatory regions of proto-oncogenes relevant to diffuse large B cell lymphomas (DLBCL). Since current methods for genomewide sequencing such as Exon Capture and RNAseq only target mutations in coding regions, to date non-Ig promoter SHMs have been studied only in a handful genes. We designed a novel approach integrating bioinformatics tools with next generation sequencing technology to identify regulatory loci targeted by SHM genome-wide. We observed increased numbers of SHM associated sequence variant hotspots in lymphoma cells as compared to primary normal germinal center B cells. Many of these SHM hotspots map to genes that have not been reported before as mutated, including BACH2, BTG2, CXCR4, CIITA, EBF1, PIM2, and TCL1A, etc., all of which have potential roles in B cell survival, differentiation, and malignant transformation. In addition, using BCL6 and BACH2 as examples, we demonstrated that SHM sites identified in these 5' regulatory regions greatly altered their transcription activities in a reporter assay. Our approach provides a first cost-efficient, genome-wide method to identify regulatory mutations and non-Ig SHM hotspots.

Figure 1. Approach to detect aberrant SHM target genes genome-wide.

Figure 2. Novel promoter SNVs target important B cell genes in DLBCL.

iPAGE analysis using Gene Ontology database (#) or a lymphoid specific gene set database (*) showing strong enrichment of genes that are important for germinal center B cells and DLBCLs contain novel promoter SNVs in OCI-Ly1 and OCI-Ly8 cells.

Figure 3. Detection of SHM.

(A). A snapshot of UCSC genome browser showing H3K4me3 ChIP-seq reads density at BACH2 promoter. The top three tracks represent SHMs, novel SNVs, and known SNVs (SNP132) detected in OCI-Ly1 by applying SHMseeqer to OCI-Ly1 H3K4me3 ChIP-seq short reads. (B). OCI-Ly1 H3K4me3 ChIP-seq short reads spanning chr6 position 91062534 (BACH2 intron 1) where a SHM was detected (shaded). (C). Sanger sequencing trace showing detection of both G (wild-type) and A (mutation) at chr6 position 91062534 in OCI-Ly1. (D). Overall validation rates of selected SNVs/SHMs within BACH2, BCL2, BCL6, BTG2, and MYO1E loci. N indicates number of SNVs/SHMs validated by Sanger sequencing in each locus.

Figure 4. Aberrant SHMs affect promoter activity.

The effects of selected SHMs on BCL6 or BACH2 promoter activities were tested by dual luciferase assay in OCI-Ly1. Reporter activity of promoter bearing individual SHM was normalized to reporter activity of the wild-type promoter. Error bars indicate standard errors of three independent experiments.