A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

Abstract

Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.

Figure 1. Familial Pre-B cell ALL associated with i(9)(q10) and dic(9;v) in two families harboring a novel, recurrent germline p.Gly183Ser variant

a. Family 1 of Puerto Rican ancestry. The proband is denoted by an arrow. Exome sequencing was undertaken in germline DNA from all available affected (IV1, IV5, IV6, III5) and unaffected (IV9, III3, III4) individuals as well as the diagnostic leukemic sample from IV6. p.Gly183Ser variant status denoted by (+/−). b. Family 2 of African-American ancestry. The proband is denoted by an arrow. Exome sequencing was undertaken in diagnostic, remission and relapse leukemic samples from individuals III4, IV1, and IV2. p.Gly183Ser variant status denoted by (+/−). c. Chromosome 9 copy number heat map for SNP6.0 microarray data of germline and tumor samples from three members of Family 2. These data demonstrate the common feature of loss of 9p in the tumor specimens. Note the focal dark blue band denoting homozygous loss of CDKN2A/B in all samples. Blue indicates deletions and red indicates gains. G, germline; D, diagnosis; R, relapse sample. d. The haplotype flanking the p.Gly183Ser mutation. A five SNP haplotype rs7850825 to rs7020413 (Chr9:36.997-37.002 Mb) proximal to the mutation was concordant in both family 1 and family 2. However, the distal end flanking the mutation rs6476606 was discordant.

Figure 2. Recurrent PAX5 mutations in ALL

a. Gene schematic of PAX5 showing the exons (upper grey numbers), amino acid residues (lower grey numbers), protein domains (as denoted by colored legend) and position of the germline p.Gly183Ser variant (in red) in relation to the somatic PAX5 mutations described in this study (n=13, arrows) and somatic mutations described previously in B-ALL^,,. Primary leukemic samples with confirmed retention of the germline p.Gly183Ser variant denoted by the square shape (Family 1) and diamond shape (Family 2). In one case of i(9)/dic(9) ALL, we found both a heterozygous Val26Gly and a heterozygous Gln350fs mutation, indicating polyclonality of the tumor. b. Conservation of the octapeptide domain in selected PAX family members.

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.