Figure 3. Attenuated transcriptional activity of PAX5 p.Gly183Ser.

a. Transcriptional activity of PAX5 variants compared to wild-type using a Pax5-dependent reporter gene assay in 293T cells. Bars show mean (± s.e.m.) luciferase activity of six individual experiments with triplicate measurements (PAX5 p.Gly183Val and PAX5 d2-6 four experiments with triplicate measurements). Asterisks indicate significant difference calculated by Dunnett's test (P<0.0001). MIR, MSCV-IRES-mRFP empty vector. b. Transcriptional activity of PAX5 variants using CD79A-dependent sIgM expression in the murine J558LμM plastocytoma cell line. Percentages indicate proportion of mRFP positive cells that show sIgM expression. Bars show mean (± s.e.m.) sIgM expression in two individual experiments with triplicate replicates each. Asterisks indicate significant difference calculated by Dunnett's test (P<0.0001). MIR, MSCV-IRES-mRFP empty vector. c. PAX5-dependent reporter gene assay of PAX5 wild-type and PAX5 p.Gly183Ser run in triplicate as above, with or without co-transfection of 0.05μg of Grg4 as indicated. A PAX5 p.Tyr179Glu (Y179E) mutant that is deficient in binding to Grg4 and empty vector were used as controls. Asterisks indicate significant differences by two-tailed t-test (p<0.0001). d. Heatmap of PAX5 activated genes in mature B cells. Four samples from family 2 (diagnosis and relapse samples from individuals IV1 and IV2) show differential expression of PAX5 activated genes when compared to a group of 139 sporadic B-ALL cases. This indicates an effect of the pGly183Ser mutation on PAX5 function. Red indicates high expression, blue represents low expression. PAX5 mutation status is indicated by the colors above the samples. Green indicates wild type PAX5, yellow indicates heterozygosity for a PAX5 mutation, and pink indicates biallelic PAX5 mutations.