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TIRAP, a novel familial lymphoma risk gene
mined by Ki-67 was observed in TIRAP p.R81C com- pared to TIRAP WT family members (Figure 5E). This was a surprising finding since circulating B-cells are gen- erally quiescent,38,39 which we observed in age- and gen- der-matched healthy individuals as well as TIRAP WT family members. Sanger sequencing confirmed the absence of TIRAP p.R81C variant in healthy donors (Online Supplementary Figure S3). Activation of TLR
through lipopolysaccharide (LPS) further enhanced B-cell proliferation, preferentially in TIRAP p.R81C individuals (Figure 5E). The higher level of assessed B-cell prolifera- tion in TIRAP p.R81C cases was positively correlated with the increased gene expression levels of TIRAP, as well as BCL2L1 and NFKB1, as main regulators of NF-kB signaling in PBMCs of all family members (Figure 5F). Collectively, our data indicate a potential role of p.R81C
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Figure 6. NF-kB signaling mediated by TIRAP is important for B-cell survival. (A) Peripheral blood mononuclear cells (PBMCs) were isolated from family members and transfected with control (CTRL) or TIRAP-directed siRNA. Twenty-four hours following transfection, cell viability of CD20+ B-cells was assessed by Annexin V/DAPI staining and flow cytometry analysis. A representative flow cytometry dot plot and quantification of living B-cells are shown in (A) and (B), respectively. Student t-test, *P<0.05. (C) Heatmap showing hierarchical clustering of mRNA levels of genes involved in NF-kB pathway, cell survival and proliferation in PBMCs of mother (M), sister 2 (S2), brother (B) and father (F) treated as described in (A). Data were clustered using standard Euclidean’s method based on the average linkage. (D) Bar chart showing the relative expression levels of NFKB1, CASP9, IL6 and MYC genes normalized to ACTb in family members with wild-type (WT) (brother and father) and p.R81C (sister 2 and mother) TIRAP. Dots represent values of each individual of the investigated family. (E) The difference of gene expression in cells transfected with TIRAP or CTRL siRNA was calculated from the mean values shown (D).
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