Activation-induced cytidine deaminase causes recurrent splicing mutations in diffuse large B-cell lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma. A major mutagenic process in DLBCL is aberrant somatic hypermutation (aSHM) by activation-induced cytidine deaminase (AID), which occurs preferentially at RCH/TW sequence motifs proximal to transcription start sites. Splice sequences are highly conserved, rich in RCH/TW motifs, and recurrently mutated in DLBCL. Therefore, we hypothesized that aSHM may cause recurrent splicing mutations in DLBCL. In a meta-cohort of > 1,800 DLBCLs, we found that 77.5% of splicing mutations in 29 recurrently mutated genes followed aSHM patterns. In addition, in whole-genome sequencing (WGS) data from 153 DLBCLs, proximal mutations in splice sequences, especially in donors, were significantly enriched in RCH/TW motifs (p < 0.01). We validated this enrichment in two additional DLBCL cohorts (N > 2,000; p < 0.0001) and confirmed its absence in 12 cancer types without aSHM (N > 6,300). Comparing sequencing data from mouse models with and without AID activity showed that the splice donor sequences were the top genomic feature enriched in AID-induced mutations (p < 0.0001). Finally, we observed that most AID-related splice site mutations are clonal within a sample, indicating that aSHM may cause early loss-of-function events in lymphomagenesis. Overall, these findings support that AID causes an overrepresentation of clonal splicing mutations in DLBCL.

Keywords: B-cell lymphoma; Somatic hypermutation; Splicing mutations.

Fig. 1

Splice mutations in DLBCL and their relationship with aSHM. A RCH (R: A or G; H: not G) and TW (W: A or T) motifs within splicing consensus sequences. Splice sequences can be divided into splice sites (positions ± 1 and ± 2) and splice regions (positions ± 3 to ± 8). C: conserved; NC: non-conserved. B Proportion of RCH and TW motifs across the human genome for each splice sequence position. C Recurrent splice site mutations in DLBCL from Andrades et al. [15] and their distance to the nearest TSS. Circle color represents the nucleotide context and size indicates mutation frequency. Grey lines show transcript length, with transcripts exceeding the plot limits represented by arrowed lines. The chosen threshold to classify mutations into proximal (< 3 kb) or distal (> 3 kb) is marked with a red dashed line. 4 out of the 29 genes described by Andrades (FAS, KMT2D, TBL1XR1 and TNFAIP3) have been omitted for visualization purposes as their splice site mutations far exceed the 4 kb plot limit. The heatmap shows the association of each gene to AID mutagenesis. > 50% RCH/TW splicing mutations, the splice site mutations are mostly in RCH or TW contexts; AID target, the gene has been reported as an AID target by Schmitz et al. [1], Alkodsi et al. [4] or Álvarez-Prado et al. [16]. D Proportion of proximal RCH and TW intronic mis-splicing mutations (in positions ± 1 to ± 8) per cancer type described by Jung et al. [12]. Sample sizes are indicated in parentheses, number of mis-splicing mutations with each sequence motif are indicated in the bars. CNS: central nervous system

Fig. 2

Proximal splice mutations are enriched in aSHM motifs in DLBCL. A Proximal splice site and splice region mutations in DLBCL are significantly enriched in AID motifs compared with the motif distribution of all splice sites and regions annotated in the human genome. B Enrichment analysis of proximal RCH and TW mutations in each genomic feature compared with proximal intronic mutations. C Pan-cancer enrichment analysis of splice site mutations at RCH or TW motifs compared with the motif distribution of all splice sites annotated in the reference genome. Color indicates whether AID-related mutational signatures have been found in a cancer type. “Partial” indicates that the AID activity was present in less than 50% of the samples analyzed [21]. FL: follicular lymphoma; CLL: chronic lymphocytic leukemia; CNS: central nervous system; SCC: squamous cell carcinoma. D Enrichment in G/C transition frequency per genomic feature in Ung^−/−Msh2^−/− mice (N = 2) compared with Aicda^−/− mouse (N = 1). C: conserved; NC: non-conserved; CDS: coding sequence; UTR: untranslated region; OR: odds-ratio. In all panels, Fisher’s exact test FDR-corrected p values are shown (ns: non-significant; *: p < 0.05; **: p < 0.01; *** p < 0.001; **** p < 0.0001). E Estimated cancer cell fraction (CCF) distributions of splice site mutations from Chapuy et al. DLBCL cohort [6]. Mutations are divided into four categories regarding their nucleotide context and their distance to the nearest TSS. A variant is considered clonal when its CCF ≥ 0.9 (dashed line), the proportion of clonal and subclonal mutations in each category is showed in the bar plot