| Objective:To identify the novel MPLL391-V392ins12 spliceosome and confirm that it is a true spliceosome of MPL gene. Then analyze its mutation occurrence in patients with Myeloproliferative neoplasms(MPN), and discuss its clinical significance by compare with JAK2V617F、MPL exon10 and CALR mutated patients with MPN.Methods:1. Identification of MPLL391-V392ins12 spliceosomeThe CDS region of MPL gene is amplified by reverse transcription polymerase chain reaction(RT-PCR), and recyle PCR products.To identify the sequence of the spliceosome,those process were needed listed below: restriction enzyme digestion 、 ligation 、transformation 、 pick clone and extract plasmid. The PCR products are derived from differernt primers, that were designed on the differert position of the spliceosome sequence, and conduct eletrophoresis in polyacrylamide gel and agarose gel. The results were analysised by gel imager.2. The mutation occurrence of MPLL391-V392ins12 in MPN paitentThe specimens of bone marrow or peripheral blood were collected from 248 MPN patients and 200 normal people. Genomic RNA were extracted, and RNA was reverse transcribed into c DNA. Then c DNA were amplified by PCR and conducted eletrophoresis in agarose gel to detect The mutation frequency of MPLL391-V392ins12 in paitent with PV、ET and PMF. The larboratory examination data of ET and PMF patients, who only carry a single mutation of MPLL391-V392ins12 、 JAK2V617 F 、 MPLexon10 or CALR. The relationships between these mutations were analyzed further by statistics.Results:1. A novel aberrant spliceosome of MPL gene(MPLL391-V392ins12) was identified by clone sequencing. It was 36 bp intron was retained between exon7 and exon8, and there were 12 amino acids(EGLKLLPADIPV) inserted. The facticity of MPLL391-V392ins12 was confirmed by PCR, which were conducted with different primers that designed by its sequence.2. MPLL391-V392ins12 mutation was detected in 19 of the 248 patients with MPN(total mutation rate was 7.66%), including 1 PV, mutation rate was 1.92%(1/52); 14 ET,mutation rate was 9.66%(14/145); 4 PMF, mutation rate was 7.84%(4/51). And the mutation was not found in the group of 200 normal people. 2 CML patients were positive,and the other tumours were all negative.3. ET patients with MPLL391-V392ins12 mutation had no statistical significance of gender, age, white blood cell count, blood platelet count and hemoglobin compared to patients with MPL exon10 mutation(P>0.05). ET patients with MPLL391-V392ins12 mutation had lower level of age and white blood cell count than patients with JAK2V617 F mutation(P < 0.05), and lower level of blood platelet count than patients with CALR mutation(P < 0.05). ET patients with MPLexon10 mutation had lower level of age, white blood cell count and hemoglobin than patients with JAK2V617 F mutation(P < 0.05), and lower level of hemoglobin than patients with CALR mutation(P < 0.05). ET patients with JAK2V617 F mutation had higher level of white blood cell count and hemoglobin than patients with CALR mutation(P < 0.05). PMF patients with MPLL391-V392ins12 mutation had lower level of age than patients with JAK2V617 F mutation(P < 0.05). PMF patients with JAK2V617 F mutation had higher level of white blood cell count than patients with CALR mutation(P < 0.05).Conclusions:1. This research found a novel abnormal spliceosome of MPL gene(MPLL391-V392ins12), which was not reported ever. MPLL391-V392ins12 spliceosome was further confirmed by caeses of screening. It can be detected in the diseases of classical MPN, and more occur in ET and PMF. This mutation may play an important role in the process of MPN.2. In ET patients, the group of MPLL391-V392ins12 mutation had different laboratory characteristics compared with those of JAK2V617 F mutation and CALR mutation, while it is semblable to those of MPL exon10 mutation. That meansMPLL391-V392ins12 mutation in the process of MPN may play a role similar to that of the MPLexon10 mutations. |
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