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TIRAP, a novel familial lymphoma risk gene
in other unaffected family members (Figure 5A). This vari- ant resulted in a substitution of the arginine at amino acid residue 81 to a cysteine (p.R81C) which is highly con- served among species and located in close proximity to the functional TIR domain (Online Supplementary Figure S2B and C). The TIRAP p.R81C variant was predicted to be deleterious by five out of five applied algorithms (Online Supplementary Figure S2D), has a dbSNP identifier (rs138228187) and is reported in COSMIC. It has a global minor allele frequency of 0.00006 and 0.0006 in ExAC and 1000 Genomes, respectively, and 0.0005 in the Japanese population, and is therefore not a polymorphism.36,37 Sanger sequencing of cDNA derived from fresh PBMCs of the family members confirmed the TIRAP p.R81C status and expression of the variant allele in sister 2 and her mother (Figure 5A and Online Supplementary Figure S3). Of note, the p.R81C variant was also identified in the lym- phomas of both siblings (data not shown).
To investigate the functional consequence of TIRAP p.R81C, we assessed the expression of pIRAK1 and total IRAK4, two downstream kinases and activators of the NF- kB signaling pathway. For IRAK4, our analysis was con- fined to the total protein, as an antibody to reliably deter- mine its phosphorylated form on FFPE tissue was not available. GC B-cells of healthy controls showed little to
no pIRAK1 and IRAK4 expression whereas p.R81C TIRAP carrying malignant B-cells of both sisters were clearly pos- itive for these markers (Figure 5B and C). This suggests that the TIRAP downstream signaling is predominantly active in malignant B-cells with the p.R81C mutation. The relevance of this pathway is underlined by the analysis of 36 primary ABC-DLBCLs that revealed that 63% and 17% expressed pIRAK1 and IRAK4, respectively (Figure 5C). GCB-DLBCLs, however, generally lacked the expression of both kinases.
To assess TIRAP/NF-kB pathway activity in non-malig- nant cells, we determined the gene expression of TIRAP as well as genes involved in cell proliferation and survival, among them several targets of NF-kB in PBMCs of living family members. Unsupervised hierarchical clustering analysis revealed two clusters, separating TIRAP p.R81C and wild-type (WT) samples based on the expression sig- nature of selected genes (Figure 5D). PBMCs carrying the TIRAP p.R81C mutation expressed higher levels of TIRAP as well as genes involved in cell survival, cell cycle and proliferation. In contrast, TIRAP WT PBMCs showed higher expression of CASP9, which is implicated in intrin- sic apoptosis.
Next, we studied the impact of TIRAP p.R81C on pri- mary B-cells. An increase in proliferating B-cells as deter-
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Figure 4. Genetic alterations related to the different clinical outcome. (A) Comparison of the somatic landscape in the two lymphomas implementing the lesions identified by whole exome sequencing, array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Only alterations in genes which are present in more than 10% of diffuse large B-cell lymphoma (DLBCL) cases and/or with a pathogenic significance in lymphoid malignancies were considered for this comparison.4-10 Mutations (M), copy number alterations (CN) and translocations (Tx) are sorted according to whether they were found to be mutated in both tumors or restricted to one lymphoma only. Numbers indicate the total amount of identified somatic mutations. In red, genes which have been associated with worse clinical outcome.9,26-29 (B) (Left) Representative FISH signal patterns using MYC and CIITA break apart assay in the primary mediastinal B-cell lymphoma (PMBL) of sister 1. Arrows indicate examples of cells with MYC (multiple FISH signals) gains and CIITA break apart (split red and green FISH signals), respectively. Scale bars: 10 mm. (Right) Immunohistochemistry analysis of PDL1 protein expression in the lymphoma of sister 1. Scale bar: 50 mm.