Validation of a molecular diagnostic assay for CALR exon 9 indels in myeloproliferative neoplasms: identification of coexisting JAK2 and CALR mutations and a novel 9 bp deletion in CALR.

INTRODUCTION: The 2008 WHO criteria for the diagnosis and classification of myeloproliferative neoplasms (MPN) rely in part upon the assessment of mutations in JAK2 and MPL genes. Recently, mutations in calreticulin (CALR) have been identified in MPN lacking JAK2 and MPL mutations. We have validated a sensitive fragment analysis assay to detect CALR mutations. METHODS: Genomic DNA from peripheral blood, bone marrow, and FFPE bone marrow clot preparations from 52 MPN specimens with known JAK2 and MPL mutation status and 29 non-MPN specimens was analyzed. CALR mutation testing was performed by fragment length analysis, and the results were confirmed by sequencing. Accuracy, precision, sensitivity, specificity, and robustness of the assay were determined. RESULTS: Forty specimens (32 JAK2+, 2 JAK2-/MPL+, and 6 JAK2-/MPL-) were negative for CALR mutations. Twelve specimens had CALR mutations including 52 bp deletion (5), 5 bp insertion (6), and a novel 9 bp deletion (1). This 9 bp inframe deletion occurring at an allelic burden of 50% would delete three amino acids. One specimen with a 52 bp deletion also had JAK2 V617F mutation. All 29 non-MPN specimens were negative for CALR mutations. The assay accurately identified the mutation status of all 52 MPN specimens and had a coefficient of variation <3% for the fragment size and mutant peaks with a sensitivity of 5% for indels. CONCLUSIONS: Fragment analysis is an accurate and sensitive method for the detection of CALR indels. The novel 9 bp deletion is likely a germline variant. Consequence of coexisting JAK2 V617F and CALR mutations requires careful interpretation.

Efficient packaging of a specific VL30 retroelement by psi 2 cells which produce MoMLV recombinant retroviruses.

FTO-2B rat hepatoma cells acquired mouse VL30 retrotransposon(s) when infected with Moloney murine leukemia virus (MoMLV) recombinant retroviruses produced from psi 2 cells. The VL30 provirus was integrated into the rat genome, expressed at high levels, and its transcription induced 40-fold by dexamethasone, VL30 RNA was detected in hepatoma cells even without selection for the expression of the amino-3'-glycosyl phosphotransferase (neo) gene, which was co-transferred with a MoMLV retrovirus. However, the extent of transfer of the VL30 RNA was inversely related to the titer of the MoMLV recombinant retrovirus. The restriction map analysis of the transferred VL30 provirus was identical to the mouse VL30s of the NVL subfamily which is known to be a significant fraction of the transcriptionally active VL30 subset. Additionally, the regenerating liver from an adult rat, which was infected with a defective MoMLV-derived retrovirus, expressed VL30 RNA. These results indicate that great care should be given to the transfer of unwanted passengers, like VL30, present in retroviral packaging cell lines like the psi 2 cells, which are currently being used for gene therapy.

Receptor-mediated gene transfer into macrophages.

Gene transfer systems targeting various receptors have been developed to introduce functional genes into cells in culture and into intact animals. A synthetic molecular conjugate, consisting of mannosylated polylysine that exploits endocytosis via the macrophage mannose receptor, was constructed and complexed to expression plasmids containing either the Photinus pyralis luciferase or Escherichia coli beta-galactosidase (lacZ) reporter genes. The DNA complexes were used to transfect murine macrophages isolated from peritoneal exudates in vitro. Luciferase and beta-galactosidase activity was found in transfected cells in culture, whereas complexes consisting of an irrelevant plasmid bound to mannosylated polylysine or the expression plasmid bound to galactosylated polylysine resulted in no detectable transgene expression. Gene transfer was inhibited by the addition of excess mannosylated bovine serum albumin to the culture medium before transfection. Reporter genes were also transferred into macrophages residing in the spleen and liver of adult animals using this system. Luciferase activity was maximal at 4 days after transfection and decreased to lower levels by 16 days. Transgene expression conformed to the distribution of cells that had nonspecific esterase, a cytochemical marker for macrophages. Thus, this system can be used to introduce functional genes into macrophages and may be an approach to the treatment of storage diseases that affect the reticuloendothelial system.

Transfer of the human Alpha1-antitrypsin gene into pulmonary macrophages in vivo.

Several viral and nonviral methods have introduced functional genes into the lungs. An alternative strategy, receptor-mediated gene transfer, exploits the ability of receptors on the surface of cells to bind and internalize DNA complexes and could potentially be used to deliver genes to specific cells in the lung. The gene encoding human alpha1-antitrypsin (A1AT) was delivered to macrophages in vitro and in vivo by targeting the mannose receptor with mannose-terminal molecular conjugates. The human A1AT transcript was detected 2 d after transfection of macrophages in culture, but transgene expression was transient. Human A1AT protein was secreted into the culture medium, and Western blot hybridization revealed the mature human antiprotease. In addition, Sprague-Dawley rats underwent intravenous injections of increasing doses of plasmid DNA (0.2 mg, 1.0 mg, and 2.0 mg) complexed to the molecular conjugate. Four days after transfection, human A1AT mRNA was found in lungs from six of the 13 rats (46%) that received the higher doses of plasmid. Transgene expression was limited to cells in perivascular and alveolar regions, which conformed to the distribution of pulmonary macrophages. Human A1AT was measured in the epithelial lining fluid of rats treated with transfection complexes. Animals that received 1.0 mg of plasmid had human A1AT levels of 7.4 +/- 3.4 pM, which was significantly different from nontransfected and mock-transfected controls. Thus the mannose receptor permitted direct delivery of genes to pulmonary macrophages, though transgene expression was detected in the lung only at low levels.