Gene is Lower Expressed in the Myocardium of Patients with Tetralogy of Fallot / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The most typical cardiac abnormality is conotruncal defects (CTDs) in patients with 22q11 deletion syndrome (22q11DS). HIRA (histone cell cycle regulator) gene, as one of the candidate genes located at the critical region of 22q11DS, was reported as possibly relevant to CTD in animal models. This study aimed to analyze the level of expression of the HIRA gene in tetralogy of Fallot (TOF) patients and the potential DNA sequence variations in the promoter region.</p><p><b>METHODS</b>The messenger RNA (mRNA) expression was examined with quantitative real-time polymerase chain reaction in 39 myocardial tissues of the right ventricular outflow tract (RVOT) from TOF patients and 4 myocardial tissues of RVOT from noncardiac death children. The protein expression was detected using immunohistochemistry in 12 TOF patients and 4 controls. A total of 100 TOF cases and 200 healthy controls were recruited for DNA sequencing.</p><p><b>RESULTS</b>The mRNA and protein expressions of the HIRA gene in the myocardium of the TOF patients were both significantly lower as compared to the controls (P < 0.05). Five single nucleotide polymorphisms (SNPs), including g.4111A>G (rs1128399), g.4265C>A (rs4585115), g.4369T>G (rs2277837), g.4371C>A (rs148516780), and g.4543T>C (rs111802956), were found in the promoter region of the HIRA gene. There were no significant differences of frequencies in these SNPs between the TOF patients and the controls (P > 0.05).</p><p><b>CONCLUSION</b>The abnormal lower expression of the HIRA gene in the myocardium may participate in the pathogenesis of TOF.</p>

Effect of Silencing SET Gene on Acute Promyelocytic Leukemia NB4-R1 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of silencing SET gene on the biological characteristics of acute promyelocytic leukemia NB4-R1 cells.</p><p><b>METHODS</b>The expression vector of pGCSIL containing SET-shRNA were transfected into 293T cells by using other packaging plasmids. The supernatant of the 293T cells was harvested for lentivirus. The SET-shRNA lentiviral vector was transfected into acute promyelocytic leukemia NB4-R1 cells and a stably transfected cell line was established. Real-time quantitative PCR and Western blot were used to assay the silencing efficiency on SET gene and the expression of PP2A. The cell cycle distribution was tested by flow cytometry.</p><p><b>RESULTS</b>The expression of SET in experimental group statistically decreased as compared with that of the control group. The expression of PP2A was obviously raised at the level of mRNA and protein. The percentage of NB4-R1 cells in G0/G1 phase significantly increased, while the percentage of cells in S phase significantly decreased.</p><p><b>CONCLUSION</b>The silencing gene in acute promyelocytic leukemia NB4-R1 cells using SET-shRNA lentiviral vector can increase the expression of PP2A and interfere of the cell cycle in NB4-R1 cells. This study has laid a experimental base for targed therapy of patients with acute promyelocytic leukemia.</p>

Influence of I2PP2A gene silencing by RNA interference on proliferation and apoptosis of human acute promyelocytic leukemia cell line NB4-R1 / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of RNA interference of human I2PP2A gene on the proliferation and apoptosis of retinoic acid-resistant human acute promyelocytic leukemia (APL) cell line NB4-R1.</p><p><b>METHODS</b>Designed and constructed a RNA interference lentiviral vector I2PP2A-shRNA which targeted against I2PP2A gene, then transfected it into NB4-R1 via polybrene mediation. The I2PP2A expression levels before and after transfection were detected by qRT-PCR and Western blot, respectively. Meanwhile, the proliferation and apoptosis rates of each group were determined by CCK-8 and flow cytometry assay. The protein expressions of caspase-8 and PARP were detected by Western blot.</p><p><b>RESULTS</b>Both qRT-PCR and Western blot data showed the I2PP2A expression level was significantly downregulated in the transfection group. The I2PP2A mRNA expression level decreased by (70.0 ± 9.6)% and (64.0 ± 6.2)% respectively, compared with blank control and negative control group, and the I2PP2A protein expression level showed a consistent trend. CCK-8 proliferation assay indicated the NB4-R1 cell proliferation rate in I2PP2A-shRNA transfection group significantly reduced compared to blank control group (P<0.05). Flow cytometry results showed that NB4-R1 apoptosis rate in I2PP2A-shRNA transfection group increased by (6.30 ± 0.67) times and (6.04 ± 0.56) times, respectively (P<0.01). After inhibition of I2PP2A, the total caspase-8 and total PARP expressions decreased by (44.0 ± 3.1)% and (57.0 ± 4.0)%, respectively; Meanwhile, the cleaved caspase-8 (p43) and cleaved PARP (p89) increased by (36.0 ± 2.5)% and (45.0 ± 4.8)%, respectively compared with blank control group (P<0.05).</p><p><b>CONCLUSION</b>I2PP2A gene silenced by RNA interference could inhibit the proliferation and promote the apoptosis of NB4-R1, which may be regulated through caspase-8-induced exogenous apoptosis pathway.</p>

Expression level of SET gene in acute myeloid leukemia and its clinical significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression level of SET gene in patients with acute myeloid leukemia (AML) and evaluate its significance.</p><p><b>METHODS</b>The expression level of SET gene in 141 de novo AML patients was determined by real time quantitative PCR (RQ-PCR), and its relationship with the clinical features and outcomes of these patients were analyzed.</p><p><b>RESULTS</b>SET gene transcript level was detected in 141 AML patients with the median expression level of 0.86(range 0.02-15.69). AML patients with higher SET gene expression had a higher level of white blood cell (WBC ≥ 100 × 10⁹/L) count than of lower SET gene expression ones (31.0% vs 11.4%, P=0.005). In the 136 patients who received treatment after diagnosis, higher SET gene expression group had lower complete remission rate (50.0%) than of lower expression cohort (73.5%) after two cycles of chemotherapy (P=0.005). Survival analysis showed that patients with higher SET gene expression had significantly shorter overall survival(OS) (10 months vs 22 months, P=0.001) and event-free survival (EFS) (2 months vs 14 months, P=0.005) than of lower SET gene expression ones. Multivariate COX regression analysis showed SET overexpression was an independent prognostic factor for OS. In the patients with the normal karyotype, higher SET expression group also had significantly shorter OS (12 months vs 35 months, P=0.010) and EFS (4 months vs 14 months, P=0.026) than of lower SET expression ones.</p><p><b>CONCLUSION</b>High expression of SET gene was associated with poor prognosis and might be a prognostic molecular marker of AML.</p>

Characterization of anti-silencing factor 1 in Leishmania major

Anti-silencing factor 1 (ASF1) is a histone chaperone that contributes to the histone deposition during nucleosome assembly in newly replicated DNA. It is involved in chromatin disassembly, transcription activation and in the cellular response to DNA damage. In Leishmania major the ASF1 gene (LmASF1) is located in chromosome 20 and codes for a protein showing 67% of identity with the Trypanosoma brucei TbASF1a. Compared to orthologous proteins, LmASF1 conserves the main residues relevant for its various biological functions. To study ASF1 in Leishmania we generated a mutant overexpressing LmASF1 in L. major. We observed that the excess of LmASF1 impaired promastigotes growth rates and had no impact on cell cycle progress. Differently from yeast, ASF1 overproduction in Leishmania did not affect expression levels of genes located on telomeres, but led to an upregulation of proteins involved in chromatin remodelling and physiological stress, such as heat shock proteins, oxidoreductase activity and proteolysis. In addition, we observed that LmASF1 mutant is more susceptible to the DNA damaging agent, methyl methane sulphonate, than the control line. Therefore, our study suggests that ASF1 from Leishmania pertains to the chromatin remodelling machinery of the parasite and acts on its response to DNA damage.

Expression of SET-NUP214 fusion gene in patients with T-cell acute lymphoblastic leukemia and its clinical significance / 中国实验血液学杂志

This study was aimed to investigate the occurrence and clinical significance of the SET-NUP214 fusion gene in patients with T-cell acute lymphoblastic leukemia (T-ALL), analyse clinical and biological characteristics in this disease. RT-PCR was used to detect the expression of SET-NUP214 fusion gene in 58 T-ALL cases. Interphase FISH and Array-CGH were used to detect the deletion of 9q34. Direct sequencing was applied to detect mutations of PHF6 and NOTCH1. The results showed that 6 out of 58 T-ALL cases (10.3%) were detected to have the SET-NUP214 fusion gene by RT-PCR. Besides T-lineage antigens, expression of CD13 and(or) CD33 were detected in all the 6 cases. Deletions of 9q34 were detected in 4 out of the 6 patients by FISH. Array-CGH results of 3 SET-NUP214 positive T-ALL patients confirmed that this fusion gene was resulted from a cryptic deletion of 9q34.11q34.13. PHF6 and NOTCH1 gene mutations were found in 4 and 5 out of 6 SET-NUP214 positive T-ALL patients, respectively. It is concluded that SET-NUP214 fusion gene is often resulted from del(9)(q34). PHF6 and NOTCH1 mutations may be potential leukemogenic event in SET-NUP214 fusion gene.

Gene rearrangement pattern of immunoglobulin and T-cell receptor (Ig/TR) and its clinical characteristics in children with SET-NUP214 fusion gene-positive leukemia/lymphoma / 中国实验血液学杂志

The purpose of this study was to analyze the gene rearrangement pattern of immunoglobulin and T-cell receptor (Ig/TR) and its clinical characteristics in three children with SET-NUP214 fusion gene positive leukemia/lymphoma. The transcript of SET-NUP214 fusion gene was detected by RT-nested PCR. The pattern of Ig/TR gene rearrangement was analyzed by using the BIOMED-2 multiplex PCR assays. Allelic-specific primers were designed for further monitoring the minimal residual disease (MRD). The results indicated that the fusion site located between exon 7 of SET and exon 18 of NUP214 at mRNA level in the three patients. The diagnoses were made as the mixed phenotype of acute leukemia (MPAL) for patients 1, acute T-lymphoblastic leukemia (T-ALL) for patients 2, and stage IV T-lymphoblastic lymphoma (T-LBL) for patients 3, respectively. Patient 1 responded to chemotherapy very poorly and relapsed at month 6 after hematopoietic stem cell transplantation. Patient 2 had high MRD (> 10(-2)) at the end of inducing remission therapy (day 33) which implied poor outcome, and died of toxic epidermal necrolysis and sequent serious infection. Patient 3 achieved hematological complete remission (CR) and MRD negative at day 15 and day 33 respectively. The duration of CR lasted for 30 months. Clonal TR gene rearrangements were detected in all the three patients. The rearrangements of TRD, TRG and TRB were found in patient 1 and 3. The rearrangements of TRD, TRB, IgH and IgK Kde were detected in patient 2. All the 6 TRB rearrangements detected were incomplete rearrangements, whereas 85.7% and 14.3% of the TRD, and TRG rearrangements were complete and incomplete, respectively. It is concluded that the transformation of SET-NUP214(+) leukemia/lymphoma cells may occur after the rearrangements of TRD and TRG and shortly after TRB rearrangement. The leukemia/lymphoma cells of patient 1 and 2 are more immature which may be related with poor outcome or response to chemotherapy.

Histones, histone chaperones and nucleosome assembly

Chromatin structure governs a number of cellular processes including DNA replication, transcription, and DNA repair. During DNA replication, chromatin structure including the basic repeating unit of chromatin, the nucleosome, is temporarily disrupted, and then reformed immediately after the passage of the replication fork. This coordinated process of nucleosome assembly during DNA replication is termed replication-coupled nucleosome assembly. Disruption of this process can lead to genome instability, a hallmark of cancer cells. Therefore, addressing how replication-coupled nucleosome assembly is regulated has been of great interest. Here, we review the current status of this growing field of interest, highlighting recent advances in understanding the regulation of this important process by the dynamic interplay of histone chaperones and histone modifications.

Use of amniocytes for prenatal diagnosis of 22q11.2 microdeletion syndrome: a feasibility study / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>A study of prenatal genetic diagnosis for 22q11.2 microdeletion, which has a wide phenotypic spectrum that involves almost all organs, is rarely reported in China. This study aimed to explore the prevalence of 22q11.2 microdeletion in congenitally malformed fetuses via the fluorescent in situ hybridization (FISH) technique and to investigate the feasibility of use of amniocytes to diagnose 22q11.2 microdeletion syndrome prenatally.</p><p><b>METHODS</b>The study enrolled 23 cases of fetal cardiac malformation, as indicated by ultrasound in Beijing Anzhen Hospital and 14 cases of non-cardiac malformation, as determined by type-B ultrasound in Beijing Anzhen Hospital and other hospitals. Amniotic fluid was obtained by amniocentesis before odinopoeia, and the stillborn fetuses of the induced labor were preceded to autopsy. The amniotic fluid of 20 cesarean deliveries during the same period of time was used as a control. The TUPLE1 gene in the amniotic fluid of malformed and normal fetuses was assessed by the FISH method.</p><p><b>RESULTS</b>The prevalence rates of the TUPLE1 gene deletion in the amniotic fluid cells from fetuses with cardiac deformations and fetuses without such malformations were 43.5% and 57.1%, respectively. The deletion of TUPLE1 was significantly associated with fetal malformation.</p><p><b>CONCLUSION</b>Chromosome 22q11.2 microdeletion is one of the major factors leading to fetal congenital malformations, and prenatal FISH screening for 22q11.2 microdeletion syndrome is technically feasible using amniocytes.</p>