Fig. 1
Multiplex EBV BALF2 genotyping qPCR design, validation, and association studies with nasopharyngeal carcinoma in endemic and non-endemic populations. A Analytical sensitivity for each of the four BALF2 qPCR targets. The 95% lower limit of detection with 95% confidence interval is reported for each target in units of EBV copies/mL plasma. In conjunction with the LLODs, the corresponding plasma viral load for 34 screen-detected preclinical NPC cases is presented to indicate likelihood of genotyping success. B Analytical linearity for each of the four BALF2 qPCR targets, plotting cycle threshold (Ct) against nominal dsDNA control concentration in units of log10 copies/μL template. C Mixing studies at fixed total template concentration (100 copies/μL template) combining high-risk and low-risk dsDNA controls, demonstrating detection of minor allele fractions as low as 10% for each of the four targets. Measured concentration is plotted against nominal concentration. In the presence of mixed alleles, the assay is approximately linear as allele fraction decreases. D Study overview and experimental workflow. First, the multiplex BALF2 genotyping assay was analytically validated using synthetic dsDNA controls and wild-type B95–8 whole virus control. Next, our non-endemic cohort of 24 NPC cases and 155 non-NPC controls contributed to BALF2 qPCR/NGS validation, longitudinal BALF2 genotyping, and BALF2-NPC association. Finally, our non-endemic cohort and three predominantly endemic cohorts contributed to a meta-analysis of 755 EBV+ NPC cases and 981 non-NPC controls. This validated the association between BALF2 haplotypes and NPC in multiple cohorts, further defined regional EBV genomic diversity, and was used to develop a variant-informed screening model. E Prevalence of I613V and V317M between EBV+ NPC cases and non-NPC controls in the present study and in the three prior EBV GWAS cohorts. F Log-transformed odds ratios with 95% confidence intervals for association between BALF2 high-risk haplotypes (C-C-T, C-C-C, or both) and EBV+ NPC or other EBV-associated diseases in the current cohort and in the three prior EBV GWAS cohorts. G Individual patient characteristics from current study of 24 NPC cases and 155 non-NPC controls. BALF2 haplotypes are defined by presence or absence of V700L, I613V, and/or V317M, which are associated with clinical phenotype. H Plasma EBV viral load (log10 IU/mL plasma) across phenotypes of 155 patients included in current study, demonstrating no significant difference between plasma viral load and phenotype. I and J Association between NPC and other BALF2 single nucleotide variants identified by next-generation sequencing. Log-transformed P-value from association test and log-transformed odds ratios with 95% confidence intervals are presented for three variants of interest (V700L = 162215C > A, I613V = 162476C > T, V317M = 163364C > T) and 13 additional variants differentially associated with NPC. V700L is mutually exclusive with I613V and V317M, was rare in this population, and was not associated with NPC risk. Only one other variant (163287G > A, synonymous) exceeded the Bonferroni-corrected P-value threshold