Genetic Analysis of A Case of Congenital Dysfibrinogenemia Caused by Arg16His Mutation in Exon 2 of FGA / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype and genotype of a family with congenital dysfibrinogenemia.</p><p><b>METHODS</b>Assays of coagulation, including activated partial thromboplastin time(APTT), pro-thrombin time(PT)and thrombin time(TT) were carried out with Sysmex CA-7000 in the proband and his family members. The quality and quantity of fibrinogen in plasma were determined by Clauss and electrophoresis, respectively. Fibrinogen and inconstituent were analyzed by Native-PAGE. All exon and exon intron boundaries of fibringen genes were analyzed by direct sequencing.</p><p><b>RESULTS</b>The proband had normal APTT, but prolonged PT and TT. The activity of fibrinogen in plasma was decreased while its quantity was normal. These abnormalities were also found in his sisters and daughter, while his wife was normal. Genetic analysis revealed heterozygous G1233A in the exon 2 of FGA which resulted in Arg16His missense mutation.</p><p><b>CONCLUSION</b>Inherited dysfibrinogenemia is caused by Arg16His mutation in exon 2 of FGA.</p>

Signal Patterns of Dual Color Dual Fusion Fluorescence in Situ Hybridization for Detection of Genetic Abnormality in Adult Patients with ALL and Their Clinical Application / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the signal patterns of dual color dual fusion fluorescence in situ hybridization (DCDF-FISH) for detection of genetic abnormality in adult acute lymphoblastic leukemia (ALL) patients and their diagnostic value and clinical application.</p><p><b>METHODS</b>The clinical data of 68 ALL patients confirmed in our hospital were analyzed retrospectively; The bone marrow samples were detected by DCDF-FISH, flow cytometry, conventional cytogenetics (CCG), reverse transcriptase polymerase chain reaction (RT-PCR), and the correlation of these results was compared. And the reaction of patients to treatment was dynamically observed by DCDF-FISH.</p><p><b>RESULTS</b>Sixteen signal patterns were found in DCDF-FISH, including 14 kinds of atypical signal patterns (signal patterns of 1R2G, 2R3G, 2R4G and 3R3G as abnormal signal patterns without BCR/ABL fusion gene. Signal patterns of 1R1G1F, 1R1G3F, 1R1G4F, 1R2G1F, 1R2G2F, 1R2G3F, 1RnG2F (n ≥ 3), 2R2G1F, 1G4F, 1R4F corresponded to t (9;22) karyotype). Ph(+) ALL patients accounted for 17. All cases with Ph chromosome or BCR/ABL positive were B-ALL or My(+)-B-ALL. The Ph chromosome was detected in 12 cases (positive rate was 18%) by CCG. The positive rate was 25% (17/68) by DCDF-FISH and RT-PCR. The DCDF-FISH fluorescence pattern change before and after chemotherapy of the patients showed that the quantity and form of the signal pattern was changed after chemotherapy, and the common characteristics was the Ph chromosome in patients.</p><p><b>CONCLUSION</b>The DCDF-FISH is a sensitive and reliable method for the detection of BCR/ABL rearrangement. Analyzing the dynamical change of DCDF-FISH signal patterns has been comfirmed to have a important guiding significance in the diagnosis, and anlysis of response to therapy, drug resistance and the prognosis of ALL patients.</p>

Expression of CSN Complex in ATRA-induced APL Cell Differentiation and Its Clinical Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of CSN complex (COP9 signal some subunits) in the patients with acute promyelocytic leukemia (APL) and its significance in the ATRA-induced APL differentiation.</p><p><b>METHODS</b>Using the NB4 cells as a model, morphologic observation and myeloid differentiation marker CD11b detection were used to monitor ATRA-induced APL differentiation, the expression of CSN complex in cell differentiation was detected by Western blot and reverse transcription real time fluorescent quantitative PCR (RT-qPCR) method. RT-qPCR was also used to detect the relative expression level of COP9 signalosome subunits in the APL patients and remission after treatment.</p><p><b>RESULTS</b>ATRA could obviously enhance CD11b expression; the cell morphology showed obvious differentiation characteristics. During the differentiation, the expression of COP9 signalosome subunits was down-regulated by ATRA. Meanwhile, the CSN expression level in newly diagnosed APL patients was much higher than that in controls (non-leukemia) (P < 0.05). The level of CSN expression was obviously down-regulated when APL patients achieved complete remission.</p><p><b>CONCLUSION</b>The high CSN expression level in APL patients can be down-regulated by ATRA. CSN complex may have a significant effect on the pathogenesis and therapy of APL.</p>

Early monitoring drug-resistance of patients with BCR/ABL(+) ALL by DCDF-FISH / 中国实验血液学杂志

This study was aimed to investigate the application value of the dual color dual fusion fluorescence in situ hybridization (DCDF-FISH) in BCR/ABL (+) acute lymphoblastic leukemia patients with complex chromosomal translocation. The clinical presentations of a patient with ALL were monitored regularly by bone marrow cell morphology test, chromosome analysis, flow cytometry and DCDF-FISH technique, and the reaction of patients to treatment and disease progression were dynamically observed by DCDF-FISH. The results indicated that the patient showed the typical presentation of B lineage acute lymphoblastic leukemia (B-ALL) with expression of CD10, CD19 and CD34; the chromosome analysis showed 46,XY, i(8), ider(9)t (9; 22) [23]/47, idem, +der(22) t (9;22) [7] karyotype in the bone marrow cells, FISH showed that 83% cells contained BCR/ABL fusion gene in the patient's bone marrow, among which 5% cells showed 1R1G2F signalling model, 14% cells showed 1R1G3F, and 64% cells showed 1R1G4F. The patient got complete remission when the imatinib chemotherapy combined with VTLP was carried out, and the tumor cells decreased to 19%, but the cells with 1R1G2F signal model increased to 18%. The 1R1G2F cell signal model increased up to 38% when patient relapsed. The patient died of the drug-resistance. It is concluded that the BCR/ABL (+) leukemia patient with complex translocation has multiple tumor cell subsets, and the responses of different cell subsets to the treatment are different, therefore the response to therapy and drug resistance of patient can be monitored early by the signal model of DCDF-FISH and the observation of dynamical changes of different cell subset.

ARMS-PCR method for detecting multiple NPM1 mutations / 中国实验血液学杂志

This study was aimed to establish a simple, sensitive detection method for multiple NPM1 mutations, so as to reduce the omission ratio of NMP1 mutant detection. Recombinant plasmids containing wide-type NPM1 and the most common mutations (A, B, C, D) were constructed as the detection objects. The ARMS-PCR for detecting multiple NPM1 mutations was established through designing a pair of specific primers whose 3' end base matched with four mutants (A,B,C,D), but did not matched with wild type NPM1 according to the different base sequence of NPM1 mutants. The feasibility of the ARMS-PCR method was evaluated by assessing the detection range and the sensitivity and comparing with direct sequencing. The results showed that the recombinant plasmids were constructed successfully by restriction analysis and DNA sequencing. The four mutants but not wild type NPM1 were detected by using ARMS-PCR, the detection range of the method was 10(3) copies/ml -10(9) copies/ml and the sensitivity was 0.01%, while the direct sequencing method could not detect the mutations if mutation was less than 10%. It is concluded that the high sensitive ARMS-PCR is established for detecting the four mutations of NPM1 and more than 95% mutants can be detected by this method, providing a new detection method for clinical NPM1 gene mutant.

Effect of curcumin combined with ATRA on differentiation of ATRA-resistant acute promyelocytic leukemia cells / 中国实验血液学杂志

In order to investigate the effect of curcumin combined with all-trans retinoid acid (ATRA) on differentiation of ATRA-resistant acute promyelocytic leukemia (APL) cells and its molecular mechanism, the NB4-R1, an ATRA-resistant APL cells, was used as a model, counting of NB4-R1 and cell morphologic observation were performed, the effect of curcumin alone or combined with ATRA on proliferation, differentiation of NB4-R1 cells was detected by flow cytometry (FCM), the change of AKT phosphorylation in cell differentiation was detected by Western blot. The results showed that ATRA had no influence on NB4-R1 cell proliferation, but enhanced the inhibitory effect of curcumin on NB4-R1 cell growth; the curcumin or ATRA alone did not affect NB4-R1 differentiation; curcumin combined with ATRA could obviously induce CD11b expression; the cell morphology showed obvious differentiation characteristics. ATRA could promote phosphorylation of AKT in NB4 cells at short time, but not had effect on phosphorylation of AKT in NB4-R1 cells; the curcumin could enhance the phosphorylation of AKT in NB4-1R cells, the curcumin combined with ATRA could further enhance the phosphorylation of AKT. It is concluded that PI3K/AKT pathway inactivation may be one of the factors of drug resistance in APL and curcumin promotes differentiation of NB4-R1 through activating PI3K/AKT pathway.

Apoptosis of retinoic acid resistant NB4-R1 cells induced with curcumin and its mechanism / 中国实验血液学杂志

This study was purposed to explore the inhibitory effect of Curcumin on growth of retinoic acid-resistant acute promyelocytic leukemia (APL) cells and its mechanism. The NB4-R1, an APL cell line resistant to retinoic acid, was used as a model. The growth level of NB4-R1 was detected by MTT assay, the morphologic features of cells were observed by light microscopy, the mitochondrial transmembrane potential was determined by flow cytometry, the expressions of apoptosis-related proteins procaspase 3, caspase 3, PARP and BCL-XL were measured by Western blot. The results indicated that the sensitivity of NB4-R1 to Curcumin was consistent with NB4 though NB4-R1 was resistant to retinoic acid, Curcumin displayed inhibitory effect on growth of NB4-R1 in time-and concentration-dependent manners. The morphologic observation showed existence of apoptotic bodies in NB4-R1 cells treated with 20 micromol/L of Curcumin. The flow cytometry indicated that the mitochondrial transmembrane potential in NB4-R1 cells treated with 20 micromol/L of Curcumin obviously decreased. The Western blot detection revealed that expressions of pro-caspase 3 and BCL-XL were down-regulated, expressions of caspase 3 and sheared PAPP were up-regulated in NB4-R1 cells treated with 20 micromol/L of Curcumin. It is concluded that the Curcumin can inhibit the growth and induce the apoptosis of NB4-R1.

A novel molecular mechanism of interferon alpha-regulated expression of retinoic acid-induced gene G / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms by which IFN-alpha regulated retinoic acid-induced gene G (RIG-G) expression.</p><p><b>METHODS</b>The expression of STAT1, p-STAT1 and RIG-G in IFN-alpha-treated NB4 cells was detected by Western blot. The roles of STAT1, STAT2 and IRF-9 in IFN-alpha-induced RIG-G expression were analyzed in STAT1-null U3A cells by cell transfection, reporter gene assay, co-immunoprecipitation and chromatin immunoprecipitaion.</p><p><b>RESULTS</b>In U3A cells, only when STAT2 and IRF-9 were co-transfected, the luciferase activities of RIG-G promoter-containing reporter gene could be highly increased about 8-fold compared with that in the control group. Moreover, in the absence of IFN-alpha, similar effects were observed in either IRF-9 co-transfected with wild type or mutant form of STAT2, whereas IFN-alpha could increase the transactivation activity of wild type STAT2 and IRF-9 by 6-fold compared with that without IFN-alpha, but had no effect on mutant STAT2. In addition, STAT2 could interact with IRF-9 and bind to the RIG-G promoter.</p><p><b>CONCLUSION</b>STAT2 may interact with IRF-9 in a STAT1-independent manner. The complex STAT2/IRF-9 is the key factor mediating the expression of RIG-G gene regulated by IFN-alpha. This is a novel signal transduction cascade for IFN which is different from the classical JAK-STAT pathway.</p>

Expression of ifi56 gene in ATRA-induced APL cell differentiation and construction of ifi56 gene eukaryotic expression plasmid / 中国实验血液学杂志

This study was purposed to investigate the expression of ifi56 gene in the ATRA-induced acute promyelocytic leukemia (APL) NB4 cell differentiation and to construct the eukaryotic expression plasmid of ifi56 gene. RT-PCR was used to detect the expression of ifi56 in NB4 cells treated with ATRA for different time. Human ifi56 cDNA was amplified by RT-PCR and cloned into pEGFP-C1 vector, then was transfected into 293T cells. The expression of the recombinant protein in 293T cells was detected by Western blot. The localization of IFI56 protein was observed by fluorescence microscopy. The results showed that the ifi56 mRNA was almost undetectable in untreated NB4 cells, but it significantly increased after ATRA treatment for 72 hours. The cDNA fragment of ifi56 was inserted into the expressing plasmid pEGFP-C1 successfully. The expression of EGFP-IFI56 fusion protein with a molecular weight about 83 kD was detected by Western blot. The EGFP-IFI56 protein was localized in cytoplasm mainly. It is concluded that the expression of ifi56 is enhanced significantly when the differentiation of APL cells was induced by ATRA. Gene ifi56 is successfully cloned into eukaryotic expression vector and the fusion protein is expressed in the cytoplasm mainly.

Regulation mechanism for rig-g gene expression induced by all-trans retinoic acid / 中国实验血液学杂志

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.

Role of the interferon-stimulated response elements I/II in expression regulation of the retinoic acid induced gene G / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the regulatory role of interferon-stimulated response elements (ISREs) located on the retinoic acid-induced gene G (RIG-G) promoter in RIG-G expression.</p><p><b>METHODS</b>By using point mutation technique, the authors constructed the wide type and site mutant reporter gene plasmids according to the ISRE sequence on RIG-G promoter, and detected the functional activities by luciferase reporter assay.</p><p><b>RESULTS</b>Mutation in ISRE II alone had no obvious effect on the expression of the reporter gene, whereas mutation in ISRE I dramatically inhibited the transactivity of RIG-G promoter. Mutation in both ISRE I and ISRE II resulted in complete loss of its response to the transcription factors for the reporter gene.</p><p><b>CONCLUSION</b>Both ISRE I and ISRE II on the RIG-G promoter are the binding sites for the complex of transcription factors. They are required for RIG-G expression, and ISRE I has a preferential role over ISRE II.</p>