Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients

Abstract

Next-generation sequencing studies on diffuse large B-cell lymphomas (DLBCLs) have revealed novel targets of genetic aberrations but also high intercohort heterogeneity. Previous studies have suggested that the prevalence of disease subgroups and cytogenetic profiles differ between Western and Asian patients. To characterize the coding genome of Chinese DLBCL, we performed whole-exome sequencing of DNA derived from 31 tumors and respective peripheral blood samples. The mutation prevalence of B2M, CD70, DTX1, LYN, TMSB4X, and UBE2A was investigated in an additional 105 tumor samples. We discovered 11 novel targets of recurrent mutations in DLBCL that included functionally relevant genes such as LYN and TMSB4X. Additional genes were found mutated at high frequency (≥10%) in the Chinese cohort including DTX1, which was the most prevalent mutation target in the Notch pathway. We furthermore demonstrated that mutations in DTX1 impair its function as a negative regulator of Notch. Novel and previous unappreciated targets of somatic mutations in DLBCL identified in this study support the existence of additional/alternative tumorigenic pathways in these tumors. The observed differences with previous reports might be explained by the genetic heterogeneity of DLBCL, the germline genetic makeup of Chinese individuals, and/or exposure to distinct etiological agents.

Figure 1

Median number of somatic mutations identified by whole-exome sequencing. Comparison of mutation load between primary and relapse DLBCL samples (A), or between tumors with and without mutations in MMR genes (B-C). MMR-mutated tumors presented a significantly higher median of mutations (**P < .01, Mann-Whitney U test) than nonmutated cases. (B) All mutations. (C) InDels.

Figure 2

Top list of somatic mutation targets in Chinese DLBCL. Genes affected by nonsilent mutations in at least 3 DLBCL samples (10% of cases) and considered to be significantly mutated by cancer gene prediction are displayed.

Figure 3

Pathways most frequently affected by somatic mutations in Chinese DLBCL. Mutation rates were calculated by dividing the total number of mutations encountered in a pathway by the size of coding regions of all genes belonging to that gene set and the number of tumors sequenced (n = 31).

Figure 4

Prevalence of nonsilent mutations in CD70, TMSB4X, B2M, DTX1, UBE2A, and LYN in an extended cohort of DLBCL patients (n = 136). CD70 was the most frequent mutation target in the present cohort. TMSB4X and LYN are novel mutation targets in DLBCL.

Figure 5

Diagrams summarizing the mutations identified in CD70 and LYN. Mutations affecting the CD70 gene were often protein truncating or located at the donor splice site of the exon 2 (A). Mutations in LYN were most often located in the kinase domain of the protein, thus potentially affecting signal transduction through this molecule (B). Wild-type and mutant LYN constructs were transfected into U2OS cells and protein expression detected by immunoblotting. Mutations tested affected LYN’s expression to variable levels (C). neg, negative control (without transfection).

Figure 6

Analysis of DTX1 mutations. (A) Diagram displaying mutations identified in DTX1. Mutations were restricted to the first exon of the gene that encodes for most of the WWE1 domain of DTX1. (B) Distribution of DTX1 mutations by AID targeting hotspot. Distribution of all and mutated cytosines and guanines by WRC/GYW domains in the first exon of DTX1. In spite of the majority of cytosines and guanines being located outside WRC/GYW domains, most mutations were encountered at this AID target motif. (C) Functional characterization of DTX1 mutants. Functional characterization of mutations identified in DTX1. Cotransfection of constructs containing the NCID, DTX1 wild-type, or mutant constructs, and a CSL-responsive luciferase reporter, was performed in U2OS cells. A control pRL-TK plasmid was also cotransfected to allow internal normalization of luciferase activity. The capacity to inhibit an NCID-triggered response by DTX1 was diminished in 4 mutants (p.P11fs*2, p.W30*, p.E49D, and p.D75A). Mann-Whitney U tests were performed to compare the firefly luciferase intensity in wild-type and mutant DTX1 transfected cells (***P < .001). The means and standard deviations of 3 independent experiments are displayed, each containing 3 replicates. (D) Multispecies alignment of the N-terminal portion of DTX1, encoded by the first exon of the gene. The mutations tested in the Notch reporter assay are displayed. Multiple sequence alignment was performed with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and visualized with Jalview.