Upregulation of SPINK2 in acute myeloid leukemia.

Objectives: Acute myeloid leukemia (AML) is a highly heterogeneous disease. Although patients can be classified into risk groups based on their genetic changes, the prognosis of disease within these categories varies widely. This situation raises the need to search for new molecular markers related to AML. Serine peptidase inhibitor Kazal type 2 (SPINK2) has recently been reported to be upregulated in AML and associated with poor outcomes by meta-analysis and in a limited number of AML patients. Methods: We analyzed SPINK2 mRNA expression in 62 patients (45 adult and 17 pediatric) with AML and 11 cell lines using quantitative Real-Time PCR (qRT-PCR). SPINK2 protein level was determined using ELISA in cell lines. Results: We found that the expression of SPINK2 mRNA and protein levels in AML cell lines (HL60 and NB4) have increased compared to other cell lines (K562, Jurkat and NALM6, MCF7, HeLa, HUVEC, hFOB, 293T, U87). SPINK2 mRNA expression was upregulated in patients with AML compared to controls (p=0.004) and significantly lower in t(8;21)-positive patients compared to negative patients (p=0.0006). Conclusions: Our results suggest that SPINK2 serves an important role in AML development. Further studies are needed to evaluate SPINK2 expression in AML patients with t(8.21) and investigate to clarify its prognostic value in various subgroups of AML.

Age-Related Co-Expression of BCOR and BCORL1 mRNA in Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematological malignancy caused by a variety of genetic abnormalities and epigenetic dysregulation. The incidence of AML is strongly related to age, with the highest incidence rates being in older adults. The loss of function mutations in BCOR and BCORL1 genes have been identified in AML. BCL6 corepressor (BCOR) and BCL6 corepressor like 1 (BCORL1) are important epigenetic regu-lators as a member of Polycomb repressive complex 1 (PRC1.1), involved in histone modification processes. METHODS: We analyzed the BCOR and BCORL1 mRNA expression in 74 adult and 22 pediatric patients with AML by Real-Time quantitative PCR in this study. RESULTS: Our results indicated that both BCOR and BCORL1 mRNA expressions decrease with age (p = 0.009 and p = 0.008, respectively) and there is a positive correlation between BCOR and BCORL1 mRNA expression (p < 0.001). BCOR and BCORL1 mRNA expressions were not significantly different in both adult and pediatric patients with AML compared to control (p > 0.05). CONCLUSIONS: Our findings indicate that expression of BCOR and BCORL1 mRNA are down-regulated with age. The increase in AML incidence with age suggests that age-associated BCOR and BCORL1 down-regulation might potentially contribute to age-related epigenetic alterations and form a predisposing condition for the development of elderly AML.

LEF1 Induces DHRS2 Gene Expression in Human Acute Leukemia Jurkat T-Cells

Objective: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease resulting from the accumulation of genetic changes that affect the development of T-cells. The precise role of lymphoid enhancer-binding factor 1 (LEF1) in T-ALL has been controversial since both overexpression and inactivating LEF1 mutations have been reported to date. Here, we investigate the potential gene targets of LEF1 in the Jurkat human T-cell leukemia cell line. Materials and Methods: We used small interfering RNA (siRNA) technology to knock down LEF1 in Jurkat cells and then compared the gene expression levels in the LEF1 knockdown cells with non-targeting siRNA-transfected and non-transfected cells by employing microarray analysis. Results: We identified DHRS2, a tumor suppressor gene, as the most significantly downregulated gene in LEF1 knockdown cells, and we further confirmed its downregulation by real-time quantitative polymerase chain reaction (qRT-PCR) in mRNA and at protein level by western blotting. Conclusion: Our results revealed that DHRS2 is positively regulated by LEF1 in Jurkat cells, which indicates the capability of LEF1 as a tumor suppressor and, together with previous reports, suggests that LEF1 exhibits a regulatory role in T-ALL via not only its oncogenic targets but also tumor suppressor genes.

Phenotypic variability including Behçet's disease-like manifestations in DADA2 patients due to a homozygous c.973-2A>G splice site mutation.

OBJECTIVES: To describe phenotypic and functional characteristics of patients with the homozygous c.973-2A>G splice site mutation in the adenosine deaminase 2 (ADA2) gene (rs139750129), resulting in deficiency of ADA2 (DADA2). METHODS: We present case synopses of six patients from three unrelated families. Clinical data were analysed and next-generation sequencing (NGS) was performed. We also tested for aberrant RNA splicing and measured ADA2 enzyme activity. RESULTS: One family had common DADA2 symptoms, whereas Behçet's disease-like manifestations were observed in the other two families. We detected the homozygous c.973-2A>G splice site mutation in ADA2 in all patients tested. ADA2 enzyme activity was significantly lower in patients than in healthy controls, but no correlation between ADA2 activity levels and disease severity was observed. Aberrant splicing was detected in a minority of mRNA transcripts, but the formation of other, undetected, aberrant splicing products could not be excluded. Patients were treated with TNF-α inhibitors to prevent recurrence of inflammatory findings including cerebral vasculitis-associated stroke. CONCLUSIONS: We describe three families with the same homozygous splice site mutation in ADA2 and observed a novel combination of manifestations resembling Behçet's disease. This further expands the range of phenotypes caused by ADA2 mutations, although no complete genotype-phenotype association could be determined. Even without active disease, the risk of stroke should be addressed in making decisions regarding treatment of DADA2 patients.

Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis

Background: Multiple myeloma is a plasma cell dyscrasia characterized by transformation of B cells into malignant cells. Although there are data regarding the molecular pathology of multiple myeloma, the molecular mechanisms of the disease have not been fully elucidated. Aims: To investigate the gene expression profiles in bone marrow myeloma cells via RNA-sequencing technology. Study Design: Cell study. Methods: Myeloma cells from four patients with untreated multiple myeloma and B cells from the bone marrow of four healthy donors were sorted using a FACSAria II flow cytometer. The patient pool of myeloma cells and the control pool of B cells were the two comparative groups. A transcriptome analysis was performed and the results were analyzed using bioinformatics tools. Results: In total, 18.806 transcripts (94.4%) were detected in the pooled multiple myeloma patient cells. A total of 992 regions were detected as new exon candidates or alternative splicing regions. In addition, 490 mutations (deletions or insertions), 1.397 single nucleotide variations, 415 fusion transcripts, 132 frameshift mutations, and 983 fusions, which were reported before in the National Center for Biotechnology Information, were detected with unknown functions in patients. A total of 35.268 transcripts were obtained (71%) (25.355 transcripts were defined previously) in the control pool. In this preliminary study, the first 50 genes were analyzed with the MSigDB, Enrichr, and Panther gene set enrichment analysis programs. The molecular functions, cellular components, pathways, and biological processes of the genes were obtained and statistical values were determined using bioinformatics tools and are presented as a supplemental file. Conclusion: EEF1G, ITM2C, FTL, CLPTM1L, and CYBA are identified as possible candidate genes associated with myelomagenesis.

Association of Pro-apoptotic Bad Gene Expression Changes with Benign Thyroid Nodules.

BACKGROUND/AIM: This study aimed to investigate the role of the mitochondrial apoptotic pathway in benign thyroid nodules. MATERIALS AND METHODS: Paired samples of nodular and normal tissues were collected from 26 patients with nodular goiters undergoing thyroidectomy. Variable expression of Bcl-2, Bax and Bad genes were evaluated by quantitative PCR. RESULTS: Expression level of Bad gene in nodules was found to be significantly decreased compared to normal tissues (p=0.049). A positive correlation was observed between nodule size and Bad expression levels (correlation coefficient=0.563, p=0.004); and this correlation was stronger in hot nodules (n=18, correlation coefficient=0.689, p=0.003). No significant difference was observed between nodular and normal tissue expressions of Bax and Bcl-2. CONCLUSION: These results suggest that Bad expression correlates with the size of benign thyroid nodules and also its relatively lower expression in nodules, warrant further investigation.

Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines.

ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.

Low dose monoethyl phthalate (MEP) exposure triggers proliferation by activating PDX-1 at 1.1B4 human pancreatic beta cells.

Phthalate plasticizers used in a wide range of common plastic products are released into the environment and may pose a risk of increased incidence of type 2 diabetes. In this work, we studied the effects of monoethyl phthalate (MEP), the metabolite of diethyl phthalate, exposure on 1.1B4 human pancreatic beta cells at low doses (1-1000 nM). We showed that MEP treatment induced proliferation in 1.1B4 cells. Also PCNA protein expression levels were increased related to proliferation induction. It has been noted that phthalates can exert estrogen mediated response by interacting with ER. In our study 24 h MEP treatment decreased ERα protein expression level conversely it increased the same protein expression level after 72 h treatment. Also MEP treatment decreased ERß expression after 72 h at 1.1B4 cells. Our results further show that insulin content of 1.1B4 cells were increased with low dose MEP treatment. Along with our insulin content results, PDX- 1 expression levels were also increased at 1.1B4 cells with MEP treatment. These findings suggest that MEP acts as an estrogenic compound and PPARγ agonist at lower concentrations. Also it should be noted that PDX-1 may be a critical regulator of 1.1B4 cells treated with MEP.

Mitochondrial mutations in patients with congenital heart defects by next generation sequencing technology.

It has been shown that mitochondrial deoxyribo nucleic acid mutations may play an important role in the development of cardiomyopathy, and various types of cardiomyopathy can be attributed to disturbed mitochondrial oxidative energy metabolism. Several studies have described many mutations in mitochondrial genes encoding for subunits of respiratory chain complexes. Thus, recent studies confirm that pathologic mitochondrial deoxyribo nucleic acid mutations are a major reason of diseases and determining them by next-generation sequencing will improve our understanding of dysregulation of heart development. To analyse mitochondrial deoxyribo nucleic acid mutations, the entire mitochondrial deoxyribo nucleic acid was amplified in two overlapping polymerase chain reaction fragments from the cardiac tissue of the 22 patients with congenital heart disease, undergoing cardiac surgery. Mitochondrial deoxyribo nucleic acid was deep sequenced by next-generation sequencing. A total of 13 novel mitochondrial deoxyribo nucleic acid mutations were identified in nine patients. Of the patients, three have novel mutations together with reported cardiomyopathy mutations. In all, 65 mutations were found, and 13 of them were unreported. This study represents the most comprehensive mitochondrial deoxyribo nucleic acid mutational analysis in patients with congenital heart disease.

Association between endothelial nitric oxide synthase intron 4a/b polymorphism and aortic dissection.

OBJECTIVES: The genetic risk factors that contribute to the risk of developing aortic dissection (AD) have been studied. We assessed the association of endothelial nitric oxide synthase (eNOS) gene polymorphism with AD. STUDY DESIGN: Patients who underwent surgery with the diagnosis of AD and survived after the operation in our center between May 2007 and June 2011 were recruited retrospectively. The eNOS intron 4a/b polymorphism was determined by polymerase chain reaction (PCR) using oligonucleotide primers (sense: 5'-AGGCCCTATGGTAGTGCCTTT-3'; antisense: 5'-TCTCTTAGTGCTGTGGTCAC-3') that flank the region of the 27 bp VNTR in intron 4. RESULTS: Thirty-nine patients (88%) had type A AD, while the remainder (12%) had type B AD. The distribution of eNOS4 a/b gene polymorphism differed significantly from the control group, with higher frequencies of eNOS 4a/a and 4a/b genotypes in the AD group (x(2)=7.16, p=0.03). CONCLUSION: In this study, the distribution of eNOS genotypes differed between the AD and control groups; however, this polymorphism was not found to be an independent factor for the development of AD.

SET oncogene is upregulated in pediatric acute lymphoblastic leukemia.

AIMS AND BACKGROUND: The SET gene is a target of chromosomal translocations in acute leukemia and encodes a widely expressed multifunctional phosphoprotein. It has been shown that SET is upregulated in BCR-ABL1-positive cell lines, patient-derived chronic myeloid leukemia CD34-positive cells, and some solid tumors. METHODS AND STUDY DESIGN: We determined the expression level of SET in 59 pediatric acute lymphoblastic leukemia patients who were BCR-ABL-negative using quantitative real-time reverse-transcriptase-polymerase chain reaction. Results. We showed that SET expression was significantly upregulated in 96.5% of B-acute lymphoblastic leukemia (28 of 29; 16.6 fold) and 93% of T-acute lymphoblastic leukemia (28 of 30; 47.6 fold) patients. This upregulation was not associated with any clinical features or overall and relapse-free survival. CONCLUSIONS: Our results showed that SET is significantly overexpressed in pediatric acute lymphoblastic leukemia samples, and an increased level of SET might contribute to leukemic process.