Frequent Down Regulation of the Tumor Suppressor Gene A20 in Multiple Myeloma

<div><p>Multiple myeloma (MM) is a malignant clonal expansion of plasma cells in the bone marrow and belongs to the mature B-cell neoplams. The pathogenesis of MM is associated with constitutive NF-κB activation. However, genetic alterations causing constitutive NF-κB activation are still incompletely understood. Since A20 (<i>TNFAIP3</i>) is a suppressor of the NF-κB pathway and is frequently inactivated in various lymphoid malignancies, we investigated the genetic and epigenetic properties of A20 in MM. In total, of 46 patient specimens analyzed, 3 single base pair exchanges, 2 synonymous mutations and one missense mutation were detected by direct sequencing. Gene copy number analysis revealed a reduced A20 gene copy number in 8 of 45 (17.7%) patients. Furthermore, immunohistochemical staining confirmed that A20 expression correlates with the reduction of A20 gene copy number. These data suggest that A20 contributes to tumor formation in a significant fraction of myeloma patients.</p></div

Genetic aberrations of A20 in multiple myeloma.

<p><b>a: Electropherogram of rs368271377 in exon 7:</b> Arrows indicate the single base pair substitution. <b>b: Electropherogram of rs143002189 in exon 9:</b> The arrow indicates the single base pair substitution. <b>c: Gene copy number analysis of A20 of selected cases:</b> For the gene copy number assays two technical replicates of each samples were used. The blue bar represents the data for exon 4 and the red one for exon 6. Each bar represents the mean values of expression levels ± standard deviation (SD). Cut off for deletion—depicted as red line—was set at 0.7 through the fact that samples exhibited up to 40% non-neoplastic surrounding tissue. <b>d: Representative immunohistochemical A20 staining of multiple myeloma samples.</b> i and ii: multiple myeloma samples with reduced A20 gene copy number. iii and iv: multiple myeloma samples with normal A20 gene copy number.</p

mRNA expression analysis of A20 and 7 NF-κB target genes (BCL2, Cyclin D1, CCR7, CD44, CXCR2, cFlip, IRF4) of MM cases with (n = 6) and without (n = 14) monoallelic A20 deletions and of non-neoplastic bone marrow biopsies (BM; n = 6).

<p>mRNA expression levels were calculated as relative expression in comparison with peripheral mononucleated cells serving as a calibrator. Each bar represents the mean values of expression levels ± standard deviation (SD). The comparison of the expression levels was performed by using the Mann-Whitney U test; all significant associations were corrected for multiple testing by applying a Bonferroni correction.</p