Fig. 3

Mutations affecting the NFKBIZ locus and functional effects on mRNA and protein levels. a NFKBIZ mutations were predominantly found within a highly conserved region of the 3′ UTR and were significantly enriched in ABC cases (blue) relative to GCB cases (orange). b A detailed view of the mutated region including the location predicted to have conserved structure (in grey). The pattern of mutations is similar in both the internal validation cohort (322 cases) and the external validation cohort (984 cases). c Mutations in NFKBIZ and MYD88 within ABC and GCB cases in the larger external validation cohort. The same trend of mutual exclusivity was observed in both validation cohorts. d Comparison of mutant variant allele fractions (VAFs) from DNA sequencing and RNA-seq of patient samples with NFKBIZ mutations. VAFs higher in RNA relative to the corresponding DNA indicates allelic imbalance favouring the mutant allele. Significant differences are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, Wilcoxon rank-sum test). e We applied a custom ddPCR assay to eight DLBCL cell lines to determine NFKBIZ mRNA expression levels. Mutant cell lines consistently showed increased NFKBIZ mRNA, and we could attribute this to the mutant allele in lines with 3′ UTR mutations (green). Cell line IκB-ζ expression was assessed by western blot. Only mutant cell lines (green and blue) showed increased protein. f Luciferase reporter assay results show reduced protein expression in the presence of wild-type UTR with restored expression in mutant constructs. Luciferase expression is normalised to a construct containing a latter portion of the UTR. Error bars represent s.d. from three replicates