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  1. Aziee, S., Haiyuni, MY, Shafini, MY, Johan, MF, Al-Jamal, HAN, Abdul Wahab, R., et al.
    MyJurnal
    The aims of the study were to investigate the anti-cancer effects of 5-
    Aza and TSA in two leukemic cell lines (CCRF-CEM and HL-60). Inhibition
    concentration of 5-Aza and TSA were measured using trypan blue exclusion
    assay. 5-Aza and TSA at IC50 were treated to both CCRF-CEM and HL-60 cell
    lines for 4-6 days. To confirm the inhibition effects of these agents, Annexin-V
    stained cells were analyzed using flow cytometry to evaluate the apoptotic
    induction. The IC50 values of CCRF-CEM were 2.01±0.1µM and 2.65±0.3µM for
    5-Aza- and TSA-treated, respectively. Whereas, the IC50 values of HL-60 were
    1.98±0.2µM and 2.35±0.2µM for 5-Aza- and TSA-treated, respectively. To
    further substantiate the findings, the time-dependent exposure of both drugs was
    studied. CCRF-CEM cells were reduced to 49.4%±5.0, 49.4%±2.5 and
    41.5%±5.6 by 5-Aza; 56.5%±7.0, 45.3%±4.2 and 40.2%±4.2 by TSA treatment
    at first, third and sixth day. HL-60 cells were reduced to 72.0%±4.5, 51.0%±1.5
    and 40.6%±2.6 by 5-Aza at first, third and sixth day. Meanwhile, HL-60 cells
    reduced to 55.6%±4.5, 45.2%±4.0 and 36.3%±2.9 by TSA at first, second and
    fourth day. Both cell lines were significantly inhibited (p
  2. Zakaria NA, Rosle NA, Siti Asmaa MJ, Aziee S, Haiyuni MY, Samat NA, et al.
    Int J Lab Hematol, 2021 Dec;43(6):1451-1457.
    PMID: 34125992 DOI: 10.1111/ijlh.13628
    INTRODUCTION: Calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) have been reported to be key markers in the molecular diagnosis, particularly in patients lacking JAK2 V617F mutation. In most current reports, CALR mutations were analysed by either allele-specific PCR (AS-PCR), or the more expensive quantitative real-time PCR, pyrosequencing and next-generation sequencing. Hence, we report the use of an alternative method, the conformation sensitive gel electrophoresis (CSGE) for the detection of CALR mutations in BCR-ABL1-negative MPN patients.

    METHODS: Forty BCR-ABL1-negative MPN patients' DNA: 19 polycythemia vera (PV), 7 essential thrombocytosis (ET) and 14 primary myelofibrosis (PMF), were screened for CALR mutations by CSGE. PCR primers were designed to amplify sequences spanning between exons 8 and 9 to target the mutation hotspots in CALR. Amplicons displaying abnormal CSGE profiles by electrophoresis were directly sequenced, and results were analysed by BioEdit Sequence Alignment Editor v7.2.6. CSGE results were compared with AS-PCR and confirmed by Sanger sequencing.

    RESULTS: CSGE identified 4 types of mutations; 2 PMF patients with either CALR type 1 (c.1099_1150del52) or type 2 (c.1155_1156insTTGTC), 1 ET patient with nucleotide deletion (c.1121delA) and insertion (c.1190insA) and 1 PV patient with p.K368del (c.1102_1104delAAG) and insertion (c.1135insA) inframe mutations. Three patients have an altered KDEL motif at the C-terminal of CALR protein. In comparison, AS-PCR only able to detect two PMF patients with mutations, either type 1 and type 2.

    CONCLUSION: CSGE is inexpensive, sensitive and reliable alternative method for the detection of CALR mutations in BCR-ABL1-negative MPN patients.

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