Thermal stability of G-rich anti-parallel DNA triplexes upon insertion of LNA and α-L-LNA.

G-rich anti-parallel DNA triplexes were modified with LNA or α-L-LNA in their Watson-Crick and TFO strands. The triplexes were formed by targeting a pyrimidine strand to a putative hairpin formed by Hoogsteen base pairing in order to use the UV melting method to evaluate the stability of the triplexes. Their thermal stability was reduced when the TFO strand was modified with LNA or α-L-LNA. The same trend was observed when the TFO strand and the purine Watson-Crick strand both were modified with LNA. When all triad components were modified with α-L-LNA and LNA in the middle of the triplex, the thermal melting was increased. When the pyrimidine sequence was modified with a single insertion of LNA or α-L-LNA the ΔTm increased. Moreover, increasing the number of α-L-LNA in the pyrimidine target sequence to six insertions, leads to a high increase in the thermal stability. The conformational S-type structure of α-L-LNA in anti-parallel triplexes is preferable for triplex stability.

Anti-parallel triplexes: Synthesis of 8-aza-7-deazaadenine nucleosides with a 3-aminopropynyl side-chain and its corresponding LNA analog.

The phosphoramidites of DNA monomers of 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine (Y) and 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine LNA (Z) are synthesized, and the thermal stability at pH 7.2 and 8.2 of anti-parallel triplexes modified with these two monomers is determined. When, the anti-parallel TFO strand was modified with Y with one or two insertions at the end of the TFO strand, the thermal stability was increased 1.2°C and 3°C at pH 7.2, respectively, whereas one insertion in the middle of the TFO strand decreased the thermal stability 1.4°C compared to the wild type oligonucleotide. In order to be sure that the 3-aminopropyn-1-yl chain was contributing to the stability of the triplex, the nucleobase X without the aminopropynyl group was inserted in the same positions. In all cases the thermal stability was lower than the corresponding oligonucleotides carrying the 3-aminopropyn-1-yl chain, especially at the end of the TFO strand. On the other hand, the thermal stability of the anti-parallel triplex was dramatically decreased when the TFO strand was modified with the LNA monomer analog Z in the middle of the TFO strand (ΔTm=-9.1°C). Also the thermal stability decreased about 6.1°C when the TFO strand was modified with Z and the Watson-Crick strand with adenine-LNA (A(L)). The molecular modeling results showed that, in case of nucleobases Y and Z a hydrogen bond (1.69 and 1.72Ǻ, respectively) was formed between the protonated 3-aminopropyn-1-yl chain and one of the phosphate groups in Watson-Crick strand. Also, it was shown that the nucleobase Y made a good stacking and binding with the other nucleobases in the TFO and Watson-Crick duplex, respectively. In contrast, the nucleobase Z with LNA moiety was forced to twist out of plane of Watson-Crick base pair which is weakening the stacking interactions with the TFO nucleobases and the binding with the duplex part.

NPM1 gene mutation in Egyptian patients with cytogenetically normal acute myeloid leukemia.

BACKGROUND: Nucleophosmin1 (NPM1) protein encoded from the NPM1 gene is a ubiquitously expressed nucleolar phoshoprotein which shuttles continuously between the nucleus and cytoplasm. NPM1 protein plays an important role in cell proliferation and apoptosis. NPM1 gene mutations at exon 12 represent the hallmark of a large sub-group of cytogenetically normal acute myeloid leukemia (CN-AML) patients worldwide. METHODS: Genomic DNA from 53 CN-AML patients were amplified by PCR and followed by fragment analysis of post-PCR products using GeneMapper software for detection of NPM1 mutations. RESULTS: NPM1 exon 12 mutations were found are 15/53 CN-AML patients (28.3%) including 3 of M1, 3 of M2, 5 of M4, 3 of M5, and 1 of M6 FAB subtypes. The NPM1 mutation was significantly associated with lower relapse rate (p < 0.05). The complete remission (CR) rate was significantly higher in the patients with high NPM1 mutation load (> 50%) than low NPM1 mutation load (< 50%) (87.5% vs. 28.6%; p = 0.02). CONCLUSIONS: The aim of this study was to evaluate the NPM1 gene exon 12 mutation in Egyptian patients with CN-AML and its relation to clinical characteristics and patient outcome and survival.

Prognostic implication of BAALC gene expression in adult acute myeloid leukemia.

BACKGROUND: Recently various molecular markers and global gene expression profiling have been investigated to improve risk profile characterization of AML with normal cytogenetics. Our main objective is to investigate the prognostic impact of brain and acute leukemia, cytoplasmic (BAALC) expression in AML with normal karyotype. METHODS: BAALC expression was analysed using quantitative real time (QRT) PCR. RESULTS: High expression was detected in 22 of 45 patients (48.9%) and its expression did not correlate with the clinical parameters of patients. High BAALC expressers had significantly lower incidence of CR (22.7% vs. 73.9%; p = 0.001), higher mortality rate (72.1% vs. 39.1%; p = 0.023), showed significantly shorter DFS (mean 4.5 vs. 13.21 months, p < 0.001), and inferior overall survival (7.02 vs. 15.02 months, p < 0.001). Multivariable analysis confirmed high BAALC expression as an independent risk factor for DFS and OS. CONCLUSIONS: BAALC expression is an important prognostic factor in AML patients with normal karyotype and its incorporation into novel risk-adapted therapeutic strategies will improve the currently disappointing cure rate of this group of patients.