Genome sequencing analysis identifies Epstein-Barr virus subtypes associated with high risk of nasopharyngeal carcinoma

Abstract

Epstein-Barr virus (EBV) infection is ubiquitous worldwide and is associated with multiple cancers, including nasopharyngeal carcinoma (NPC). The importance of EBV viral genomic variation in NPC development and its striking epidemic in southern China has been poorly explored. Through large-scale genome sequencing of 270 EBV isolates and two-stage association study of EBV isolates from China, we identify two non-synonymous EBV variants within BALF2 that are strongly associated with the risk of NPC (odds ratio (OR) = 8.69, P = 9.69 × 10^-25 for SNP 162476_C; OR = 6.14, P = 2.40 × 10^-32 for SNP 163364_T). The cumulative effects of these variants contribute to 83% of the overall risk of NPC in southern China. Phylogenetic analysis of the risk variants reveals a unique origin in Asia, followed by clonal expansion in NPC-endemic regions. Our results provide novel insights into the NPC endemic in southern China and also enable the identification of high-risk individuals for NPC prevention.

Figure 1. Principal component and phylogenetic analyses of EBV genomes.

(a) Principal component analysis of 270 EBV isolates sequenced in the current study and 97 published isolates. The first two principal-component scores (PC1 and PC2) are plotted. Explaining 26.9% of the total genomic variance, PC1 discriminates between East Asian and Western/African strains; PC2 explains 15.3% of the total variance. (b) Phylogeny of 230 EBV single strains sequenced in the current study and 97 published strains. Macacine herpesvirus 4 genome sequence (NC_006146) was used as the outgroup to root the tree. Type 1 and Type 2 EBV lineages are indicated. The red dot on the phylogeny indicates the lineage of the NPC-dominant EBV strains, where 22 of 37 strains from healthy controls in NPC-endemic regions in southern China were located. Dashed lines in (a) and (b) indicate the separation between East Asian and Western/African strains. (c) Geographical origins and phenotypes of samples from which EBV strains were sequenced are shown with colors as indicated. (d) The normalized values of PC1 and PC2 scores are shown from blue to red. (e) The genotypes of SNPs 162215C>A, 162476T>C, and 163364C>T in each isolate.

Figure 2. Genome-wide association analysis of EBV variants in 156 NPC cases and 47 controls.

(a) Manhattan plot of genome-wide P values from the association analysis using a generalized-linear mixed model. The −log₁₀-transformed P values (y axis) of 1545 variants in 156 NPC cases and 47 controls are presented according to their positions in the EBV genome. The minimum P value (SNP 162507C>T) is 9.17×10⁻⁵. The red line is the suggestive genome-wide significance P value threshold of 4.07×10⁻⁴. The three SNPs 162507C>T, 162852G>T and 162215C>A reaching genome-wide significance are labeled as green. (b) The regional plot of the posterior probabilities of association. The EBV genome was partitioned into overlapping 20-variant bins with 10-variant overlap between adjacent bins. The sum of the posterior probabilities for variants was assigned to each region. The one region from position 160971 to 163629 with strong evidence (> 0.85) for association with NPC risk is shown in green. (c) Schematic of EBV genes. Repetitive regions in EBV genomes are masked by light blue.