Study On SRSF2 And GATA2 Mutations In The Molecular Pathogenesis Of Myeloid Tumors

Objective:Chronic myelomonocytic leukemia(CMML)is a clonal hematopoietic stem cell disorder and its molecular mechanism is unclear.Recently,the identification of spliceosome gene mutationshas made a major breakthrough in the study of molecular mechanism about CMML.SRSF2 is the most frequently mutated spliceosome gene in CMML.We valuatethe frequency of SRSF2 mutation in patients with CMML and analysis clinical features of these patients,to investigate the contribution in the diagnosis and prognosis of CMML.Methods:We collected bone marrow sample from 29 patients with CMML.After isolation of genomic DNA,SRSF2 mutation was detected by Allele-specific polymerase chain reaction(AS-PCR)followed by direct sequencing.The clinical data was compared between the mutant group and wild type group.Results:Among 29 patients,7(24.1%)patients were found harboring SRSF2 mutations,allaffecting P95,including 4 P95L,2 P95H and 1 P95R point mutation.SRSF2 mutations were correlated with higher age,less pronounced anemia,and normal karyotype.No significantly statistical differences were observed with regard to other clinical characteristics between mutant and wildtype group.2 cases who had follow-up data were recorded leukemic transformationsin the early process.One had died after many of chemotherapy and another had got better after allogeneic hematopoietic stem cell transplantation.Conclusion:We conclude that mutational frequencies were 24.1%for SRSF2 in CMML and all weremissense mutations on P95.SRSF2 mutations were correlated with higher age and less pronounced anemia and might have been associated with a poor prognosis and a higher risk of leukemic transformations in CMML.It might become a new diagnosis maker and therapeutic target in CMML in the future.Objective:Construction of eukaryotic expression vectorsof four different GATA2 genes,including three point mutations and a wild type,and estabilishment of GATA2 transgenic mice investigate the contribution of GATA2 mutations in the pathogenesis of acute myeloid leukemia.Motheds:To construct four specific expression vectors of Vavl-GATA2-P304,Vavl-GATA2-G320D,Vav1-GATA2-R362Q and Vavl-GATA2-WT,we recombine GATA2 CDs and poly A behind of ATG in Vavl gene by homologous recombination technology.These vectors were injected into oocytes to establish transgenie mice.Transgenic positive mice were detected by PCR.GATA2 genes expression was measured by RT-PCR and Q-PCR.We examine the blood routine,biochemical and smear of high expression mice to compare whether there is a difference between mutant and wild group.Results:There are 39 positive founder mice by PCR,including 13 Vavl-GATA2-G320D mice(5 female and 8 male);7 Vavl-GATA2-P304H mice(3 female and 4 male);8 Vavl-GATA2-R362Q mice(2 female and 6 male);11 Vavl-GATA2-WT mice(2 female and 9 male).51 F1 mice were proved positive transgenic mice.33 F1 transgenic mice had been examined GATA2 genes expression by RT-PCR and Q-PCR and only 10 F1 mice had a high level.Now,the vital signs of mice are normal and without AML.Blood routine,biochemical and smear had not be seen obviouslyabnormal.there is no statistical difference between mutant and wild group.Conclution:Our experiment successful established four groups of transgenic mice model about GATA2-P304?GATA2-G320D?GATA2-R362Q and GATA2-WT and provided animal models for the study of the molecular pathogenesis of AML.