• 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • Recently a reciprocal fusion between YPEL yippee like and PP


    Recently, a reciprocal fusion between YPEL5 (yippee-like 5) and PPP1CB (serine/threonine protein phosphatase-1 beta catalytic subunit) was described to be exclusively expressed in more than 90% of primary CLL samples [5] and not in control samples or samples from patients with other hematological malignancies. The two chimeric RNA transcripts appeared be the result of trans-splicing as no sign of genomic reorganization could be detected. The discovery of a common, CLL-specific molecular defect is interesting as it provides a unique target for drug discovery and molecular monitoring.
    Materials and methods
    Results and discussion The apparent frequent and exclusive occurrence of the trans-splicing of YPEL5 and PPP1CB in CLL [5] could be a good target for directed therapy and molecular monitoring of this disease. To maximize the sensitivity and specificity in the detection of this fusion transcript, we used the recently introduced NuPCR technique [6]. The combination of two specific primers and a specific MNAzyme ensures a high specificity of the PCR in combination with a sensitive detection method (Fig. 1). Initially, we tested four samples of CLL patients for the sphingosine 1-phosphate receptor modulator of both YPEL5–PPP1CB and PPP1CB–YPEL5 transcripts. In all samples, we observed low expression of the YPEL5–PPP1CB transcript. In contrast, the PPP1CB–YPEL5 transcript was not detected in any sample (data not shown). Therefore, we focused on the YPEL5–PPP1CB transcript in our further analyses. The panel of primary diagnostic CLL samples was expanded and in total, we detected variable expression of the YPEL5–PPP1CB transcript in 17/18 CLL samples (94%) (Fig. 2A – black bars), confirming the recurrent expression of the YPEL5–PPP1CB transcript in CLL [5]. To test the specificity of the expression of YPEL5–PPP1CB, we tested diagnostic samples of patients with AML (n=3), ALL (n=4), MBL (n=7), CML (n=3), different hematological cell lines (n=10; data not shown) and samples from healthy individuals (n=3) with the NuPCR for YPEL5–PPP1CB. All patient samples and three cell lines (ME-1, Kasumi-1, and MonoMac-6) tested positive, albeit at different levels (Fig. 3A – black bars), showing that the presence of YPEL5–PPP1CB is not specific for CLL. The expression levels in the different sample types were highly variable, but correspond to the levels observed in primary CLL samples. To investigate the discrepancy between the NuPCR and the results obtained by Velusamy et al. [5], we repeated the analysis on the same samples with the primers from the published SYBR Green assay [5] (Fig. 1; further referred to as EvaGreen method). After the PCR, one melting peak was obtained and sequence analysis confirmed the identity of the YPEL5–PPP1CB fusion (Supplementary Fig. S1). The patient samples showed similar expression with the EvaGreen method as obtained by the NuPCR (Figs. 2A and 3A – white bars). This confirmed the wide expression in non-CLL samples, disproving the statement that the YPEL5–PPP1CB is specific for CLL. Globally, the expression level was comparable regardless of the PCR technique used (Figs. 2B and 3B). A report on high-throughput mRNA sequencing in CLL patients [7] also described a low-level expression of the YPEL5–PPP1CB transcript, both in normal and cancer samples. Although the expression level was below the threshold set by the authors, this finding is concordant with our results. The discrepancy between our results and the results from Velusamy et al. [5] is potentially explained by the recent finding that incubation-induced gene dysregulation affects the expression level and splicing of numerous genes in leukemic samples [8]. As the expression level of YPEL5–PPP1CB is very low, even small differences in splicing efficiency, as a result of different sample processing times, could make the difference between being able to detect the transcript or not.
    Authors’ contributions
    Acknowledgments This work was supported by a grant from Ghent University Hospital (Grant no. KW/1294/LBK/003/001) (Klinisch Onderzoeksfonds).