Identification Genetic Variations (GVs) Causing Splicing of TNF Family Members and Adaptor Proteins That Modulate NFkB Pathways in Waldenstrom’s Maroglobulinemia (WM).

The TNF ligand-receptor superfamily and their adaptor proteins regulate important B-cell signaling pathways, including CD40L-CD40 and APRIL/BLYS-TACI through adaptor protein TRAF2. These pathways promote B-cell differentiation and immunoglobulin heavy chain class switching. Defects in immunoglobulin heavy chain class switching and presence of constitutive IgA and IgG hypogammaglobulinemia in patients with WM have previously been reported by us (Hunter et al, ASH2006). In WM patients we identified several novel splice variants of TNF-family members CD40 and BLYS, and their adaptor protein TRAF2. Cloning, sequencing and alignment analysis document that aberrant splice transcripts of CD40, CD40-Va, Vb and Vc, and BLYS, BLYS-Va and Vb, result from partial exon skipping (CD40Va and Vc) and entire or partial intron retention (CD40-Vb, BLYS-Va and Vb), while the TRAF2 variant is a result of exon skipping only. Using RT-PCR DNA fragment analysis, malignant and normal B-cells from the bone marrow of 25 WM patients and 6 healthy donors (HDs) were screened for the expression of CD40, BLYS and TRAF2 splice transcripts. This analysis identified overexpression of CD40-Vb (15/25WM vs. 0/6HD; P=0.01), CD40-Vc (21/25WM vs. 0/6HD; P=0.0003), BLYS-Vb (18/25WM vs. 0/6HD; P=0.002) and TRAF2-V (21/25WM vs. 1/6HD; P=0.004) in WM patients. The expression of other splice variants CD40-Va (18/25WM vs.2/4HD; P=0.09) and BLYS-Va (23/25WM vs. 4/6HD P=0.2) in the same group of WM patients were not significant. We hypothesized that these aberrations are consequences of genetic variations (GVs) distributed in the vicinity of splicing elements of these genes, as well as, alterations may have occurred in the repertoire of splicing factors (SFs) with respect of their expression levels. To address these issues, we started sequencing CD40, BLYS, and TRAF2 gene segments that are subjected to aberrant splicing. Sequencing analysis of the CD40 gene from WM B cells revealed 6 recurrent genetic variations (GVs-defined as mutations occurring more than one patients) that include 2 missense (on exon 3) and 3 silent substitutions (on exons 3-4-5), and 1 frame-shift deletion on exon 5. These substitutions lead to amino acid changes on CD40 gene, while the frame-shift deletion may cause truncation of wild-type CD40 protein. We also identified recurrent GVs on introns 4 and 5. All these GVs detected on exons and introns are distributed in the vicinity of key splicing elements that create and/or activate a new splice site in precisely the position required for the splicing events to create CD40 variants. Also, using TaqMan low density array (TaqMan-LDA) we evaluated levels of major SFs and other TNF family members involved in CD40 and BLYS signaling. These analyses showed that patients expressing aberrant splice variants of CD40 and BLYS overexpress not only other members of TNF family but also major SFs: SF2/ASF (a proto-oncogene), U2, hNRPA1 and SRP55. TaqMan-LDA analysis suggests that these SFs may play a significant role in CD40, BLYS and TRAF2 splicing in WM patients since these transcripts were upregulated (1.2-2.2 fold higher) only in those patients which expressed CD40, BLYS and TRAF2 variants. In conclusion, presence of GVs in the vicinity of splicing elements and upregulation of SFs collectively may promote aberrant CD40, BLYS and TRAF2 splicing and thus modulate TNF family pathways supportive of B-cell differentiation and immunoglobulin heavy chain class switching in patients with WM.