Investigations on hyperphosphorylation of paratarg-7, a significant risk factor and frequent antigenic target of paraproteins in MGUS and multiple myeloma.

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In previous studies we had identified and characterized hyperphosphorylated paratarg-7 (pP-7) as the antigenic target of 15% of all paraproteins of the IgA and IgG type and of 11% of all IgM paraproteins. Moreover, we had shown that pP-7 carriership is the strongest risk factor to develop MGUS/MM/WM known to date and is inherited as an autosomal-dominant trait. During the first three years of DFG support for this project we showed that pP-7 is the target of >1/3 (!) of paraproteins in African-American MGUS/MM patients underlining the importance of pP-7 in the pathogenesis of these diseases. Moreover, we succeeded in identifying additional autoantigenic targets of paraproteins and demonstrated that all these autoantigenic targets were hyperphosphorylated in the respective patients compared to healthy controls (and also inherited autosomal-dominantly), demonstrating that hyperphosphorylation of autoantigenic paraprotein targets is frequent in MGUS/MM/WM patients. Investigating the mechanisms underlying the hyerphosphorylation of the paraprotein targets we demonstrated that in healthy controls P-7 is hyerphosphorylated at serine 17 after stimulation by PKCzeta, and dephosphorylated by protein phosphatase 2A, as are all other autoantigenic paratargs. In pP-7 carriers and carriers of all other hyperphosphorylated autoantigenic paraproteins targets, dephosphorylation of pP-7 is defective due to an exchange of the regulatory subunit B55delta by B56gamma3 in the trimeric PP2A enzyme complex. Sequencing of paratargs and of the for the phosphorylation state responsible PKCzeta as well as the expressed subunits of PP2A, did not reveal SNPs or mutations that were associated with the hyperphosphorylation of P-7. Similarly, whole-exome sequencing which was performed twice in cooperation with two independent cooperating partners did not identify a genetic variant responsible for P-7 hyperphosphorylation, nor did whole-genome methylation analysis. However, a single nucleotide polymorphisms and microsatellite-based genome-wide linkage analysis identified a 4.3 Mb long region of chromosome 4q35 to locate the genetic variant responsible for the hyperphosphorylation of P-7 with a LOD score of 6 (!). In the suggested second period of DFG support, we will narrow-down the DNA region using a functional approach with wtP-7 and pP-7 LCLS and investigate the pathways leading from the respective genetic variant of interest to the hyperphosphorylation of autoantigenic targets of paraproteins. Our investigations will yield important and completely novel information on the pathogenesis of MGUS/MM/WM that will go far beyond their role in paratarg-7 hyperphosphorylation.