Effect of DNMT3A R882H Hot Spot Mutations on DDX43 Promoter Methylation in Acute Myeloid Leukemia.

Epigenetic alterations have been observed in many hematological malignancies, including acute myeloid leukemia (AML). Many of these alterations result from mutations in DNA methyl transferase (DNMT) enzymes, disabling them to methylate target genes in a proper way. In this case-control study, we investigated the association between R882H mutation in DNMT3A gene and DDX43 gene methylation in patients with AML. 47 AML patients and 6 controls were included in this study. After DNA extraction, amplification refractory mutation system (ARMS)-PCR was used to evaluate R882H mutations in DNMT3A gene. The high-resolution melting (HRM) method was used to determine the methylation changes of the DDX43 gene promoter. R882H mutation was only found in 10.6% (5 out of 47) of AML patients. The frequency of DDX43 gene methylation was significantly higher in patients without R882H mutations compared to patients with R882H mutations (P < 0.05). The DNMT3A R882H mutation is typically present in a minority of AML patients. Nevertheless, this mutation is associated with a reduced frequency of methylation in the DDX43 promoter region.

Lower Levels of TET2 Gene Expression, with a Higher Level of TET2 Promoter Methylation in Patients with AML; Evidence for the Role of Aberrant Methylation in AML Pathogenesis.

DNA methylation is a key epigenetic mechanism that is dysregulated in leukemia and plays a significant role in leukemogenesis. Ten-eleven translocation 2 (TET2) is one of the most frequently mutated genes among the DNA methylation regulators in hematologic malignancies, indicating its tumor-suppressor function. In this study, we investigated the expression and methylation status of TET2 in patients with AML. Quantitative RT-PCR was used to evaluate TET2 expression in peripheral blood mononuclear cells (PBMCs) from 51 newly diagnosed AML patients and 50 healthy controls. The methylation-sensitive high-resolution melting (MS-HRM) method was used in 45 patients with AML and 15 healthy controls to evaluate the promoter methylation of TET2. TET2 expression was significantly downregulated (P < 0.0001) in patients with AML compared to that in healthy controls. Furthermore, the methylation level of the TET2 promoter was significantly different between patients and controls. Aberrant methylation of the TET2 promoter was observed in 53.3% of the patients. Interestingly, a negative (- 0.3138) and significant (P = 0.0358) correlation between TET2 methylation and expression was found. The survival of patients with downregulated TET2 was poorer than that of other patients. TET2 gene expression was significantly downregulated while the promoter methylation was higher in patients, indicating that TET2 may be a tumor suppressor gene and a prognostic factor in AML and that transcriptional silencing of the TET2 gene may play a role in AML pathogenesis. Since epigenetic mechanisms are reversible, abnormal TET2 methylation could become a therapeutic target in the future.

Interleukin 10, but not tumor necrosis factor-alpha, gene variations are associated with factor VII inhibitor development.

OBJECTIVE: Development of alloantibodies against coagulation factor VII (FVII) is the main therapeutic challenge in severe congenital FVII deficiency. About 7% of patients with severe congenital FVII deficiency develop an inhibitor against FVII. In this research, the relationship between interleukin (IL)-10 and tumor necrosis factor-alpha (TNF)-α gene variants and inhibitor development was evaluated for a group of Iranian patients with severe congenital factor VII deficiency. METHODS: Patients with FVII deficiency were divided into 2 groups: 6 cases and 15 controls. Genotyping was performed using the amplification-refractory mutation system polymerase chain reaction. RESULTS: We found that IL-10 rs1800896 A>G gene variant is associated with the risk of FVII inhibitor development (OR = 0.077, 95% CI = 0.016-0.380, P = .001), whereas the TNFα-rs1800629G>A variant has no relation with inhibitor development in severe FVII deficiency. CONCLUSION: The results show that the IL-10 rs1800896 A>G variant increases the risk of developing an inhibitor in patients with severe congenital FVII deficiency.

Effect of siRNA-mediated silencing of p53R2 gene on sensitivity of T-ALL cellsto Daunorubicin.

INTRODUCTION: p53R2 is a p53-inducible protein that, as one of the subunits of ribonucleotide reductase, plays an important role in providing dNTPs for DNA repair. Although p53R2 is associated with cancer progression, its role in T-cell acute lymphoblastic leukemia (T-ALL) cells is unknown. Therefore, in this study, we evaluated the effect of p53R2 silencing on double-stranded DNA breaks, apoptosis and cell cycle of T-ALL cells treated with Daunorubicin. METHODS: Transfection was performed using Polyethyleneimine (PEI). Gene expression was measured using real-time PCR and protein expression was evaluated using Western blotting. Cell metabolic activity and IC50 were calculated using MTT assay, formation of double-stranded DNA breaks was checked using immunohistochemistry for γH2AX, and cell cycle and apoptosis were evaluated using flow cytometry. RESULTS: We found that p53 silencing synergistically inhibited the growth of T-ALL cells by Daunorubicin. p53R2 siRNA in combination with Daunorubicin but not alone increases the rate of DNA double-strand breaks in T-ALL cells. In addition, p53R2 siRNA significantly increased Daunorubicin-induced apoptosis. p53R2 siRNA also caused a non-significant increase in cells in G2 phase. CONCLUSION: The results of the present study showed that silencing of p53R2 using siRNA can significantly increase the antitumor effects of Daunorubicin on T-ALL cells. Therefore, p53R2 siRNA has the potential to be used as an adjuvant therapy in combination with Daunorubicin in T-ALL.

Ultrasensitive quantitation of FLT3-ITD mutation in patients with acute myeloid leukemia using ddPCR.

BACKGROUND: FLT3-ITD mutations occur in 45-50% of cytogenetically normal AML patients. Conventional fragment analysis using capillary electrophoresis is routinely used to quantitate FLT3-ITD mutations. Fragment analysis however has limited sensitivity. METHODS AND RESULTS: Here, FLT3-ITD was quantified in AML patients using an in-house developed ultra-sensitive droplet digital polymerase chain reaction assay (ddPCR). The allelic ratio of FLT3-ITD was also absolutely measured by both Fragment analysis and ddPCR. The sensitivity of ddPCR in quantitation of FLT3-ITD mutation was superior to Fragment analysis. CONCLUSION: This study demonstrates the feasibility of using the described in-house ddPCR method to quantify the FLT3-ITD mutation and measure FLT3-ITD AR in AML patients.

Dual Effects of Resveratrol on the Expression and Secretion of Angiogenic Factors.

Angiogenesis is an essential process in the growth, development, and transition of tumors from dormancy to proliferating state. Resveratrol (RSV), as a natural polyphenolic compound, is claimed to be effective in regulating angiogenesis. This study aimed to evaluate the impact of RSV onthe angiogenesis process in HUVECs (human umbilical vein endothelial cells) alone and co-cultured with Jurkat cells. The effects of RSV on HUVECs and Jurkat cell viability and apoptosis were measured by MTT and Annexin-V/PI methods. HUVECs were co-cultured with pre-treated Jurkat cells and incubated for 24 h, 48 h and 72 h. The angiogenesis process in HUVECs and Jurkat cells alone and in co-culture models was investigated by analyzing the expression of VEGF, VEGFR-2, and Interleukin-8 (IL-8) employing qPCR and ELISA. RSV at low concentration (40 µM) had no significant effects on apoptosis rate of HUVECs, but higher concentrations (80-160 µM) increased apoptosis in co-culture method and HUVECs alone. RSV significantly reduced VEGFR2 and IL-8 gene expression also, IL-8 protein concentration in HUVECs, but the effects of this drug in the HUVECs-Jurkats co-culture were different. Expression of VEGF in Jurkat cells increased following treatment with RSV. RSV had direct anti-angiogenic effects on HUVECs. Unexpectedly its indirect effects were not significant on HUVECs-Jurkats co-culture. Results of our study showed, RSV may be effective in anti-angiogenesis therapy, but in some situations, it may induce angiogenesis. So, appropriate concentrations should achieve to minimize the unpredicted effects of RSV.

Studying the potential of upregulated PTGS2 and VEGF-C besides hyper-methylation of PTGS2 promoter as biomarkers of Acute myeloid leukemia.

Hereby, we aimed to investigate the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and Vascular Endothelial Factor-C (VEGF-C) besides the methylation of PTGS2 in AML patients. VEGF-C and PTGS2 expression analysis were evaluated in newly diagnosed AML patients and healthy controls by quantitative Reverse Transcriptase PCR method. Also, PTGS2 methylation status was evaluated by Methylation-Sensitive High-Resolution Melting Curve Analysis (MS-HRM). While 34% of patients were female, the mean age of the patients was 43.41 ± 17.60 years suffering mostly from M4 (48.21%) type of AML. Although methylation level between patients and controls was not significantly different, none of the normal controls showed methylation in the PTGS2 promoter. PTGS2 and VEGF-C levels were elevated in AML cases and correlated with WBC, Platelet, and Hemoglobin levels. The survival of patients with overexpressed VEGF-C and PTGS2 was poorer than others. It can be concluded that PTGS2 and especially VEGF-C expression but not PTGS2 methylation can be considered as diagnostic biomarkers for AML.

The most common disease-causing mutation of factor XIII deficiency is corrected by CRISPR/CAS9 gene editing system.

Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.

Inhibition of Skp2 enhances doxorubicin-induced cell death in B cell precursor acute lymphoblastic leukemia.

S-phase kinase-associated protein 2 (Skp2) is a well-defined component of the Skp2-Culin1-F-box (SCF) E3 ubiquitin ligase complex, which is involved in cell cycle progression and considered a prognostic marker in cancers. Overexpression of Skp2 is frequently observed in patients with acute lymphoblastic leukemia (ALL). Inhibition of this protein may be a valuable strategy to induce apoptosis in malignant cells. Less well known is the effect of Skp2 inhibition on the potentiation of the chemotherapeutic-induced cell death in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Our results demonstrated that inhibition of the Skp2 using SZL P1-41, not only resulted in caspase-mediated apoptosis but also potentiated doxorubicin-induced apoptosis in BCP-ALL cell lines (NALM-6 and SUP-B15). SZL P1-41 in combination with doxorubicin altered cell cycle distribution and the level of cyclins and cyclin-dependent kinases in BCP-ALL cells. DNA damage response genes were also upregulated in presence of the doxorubicin and SZL P1-41 in both cell lines. In conclusion, our results indicated that inhibition of Skp2 either alone or in a combination with doxorubicin may hold promise in the future treatment of BCP-ALL.

Noninvasive prenatal diagnosis of congenital factor XIII deficiency in Iran.

Congenital factor (F) XIII deficiency is a rare coagulation factor deficiency that is inherited in an autosomal recessive manner. FXIII deficiency presents various clinical manifestations, such as intracranial hemorrhage (ICH), which is the most common cause of morbidity and mortality. As ICH can occur in the neonatal period, prenatal diagnosis (PND) is an effective way to reduce neonatal ICH and its associated fatal consequences. In this study, we investigated a noninvasive prenatal diagnosis (NIPD) method, cell-free fetal DNA (cffDNA), for PND in FXIII deficiency. This study was conducted on seven pregnant women in the first trimester. After extraction of cffDNA from maternal plasma, PCR-restriction fragment length polymorphism (PCR-RFLP) was performed to find the underlying F13A gene mutations previously identified in the family members. PCR-RFLP was also performed on postnatal DNA samples. Sanger sequencing was performed to confirm the results. Four cases were heterozygous for F13A gene mutations, whereas three were unaffected. PCR- RFLP results for cffDNA and postnatal DNA samples were identical, and Sanger sequencing confirmed the results. cffDNA is a noninvasive and effective method for PND in congenital FXIII deficiency.

Use of antioxidant nanoparticles to reduce oxidative stress in blood storage.

Oxidative damage by free radicals has a negative effect on blood quality during storage. Antioxidant nanoparticles can prevent oxidative stress. We use SOD-CAT-Alb-PEG-PLGA- nanoparticles to reduce the effects of oxidative stress in blood storage. Electrospray was employed to prepare nanoparticles. Nanoparticles entered the test bags and were kept for 35 days from the time of donation under standard conditions. On target days, experiments were performed on the samples taken. The examination included blood smear, red blood cells count, hemoglobin, hematocrit, K, Fe, glutathione peroxidase, glutathion reductase, glucose-6-phosphate dehydrogenase, prooxidant-antioxidant balance, malondialdehyde, and flow cytometric assay for phosphatidylserine. The repeated measures analysis was performed on samples every week. Morphological changes were less in the test group compared to the control. The quantitative hemolysis profile test showed significant changes in the test and control groups (p < 0.05) in consecutive weeks except for K and Fe. Oxidative stress parameters too showed a significant change during the target days of the examination (p < 0.05). Also, the phosphatidylserine expression was increased in control groups more than test in consecutive weeks (p < 0.05). It seems that the use of antioxidant nanoparticles improves the quality of stored red blood cells and can prevent posttransfusion complications and blood loss by reducing oxidative stress.

Dose-dependent efficacy of antioxidant nanoparticles on red blood cells storage.

BACKGROUND: Transfusion of healthy red blood cells (RBCs) after storage is important. One of the storage lesions on blood bags is oxidative stress. One way to prevent increased oxidative stress is to use antioxidant nanoparticles (NPs). Superoxide dismutase (SOD) and catalase (CAT) play an important role in antioxidant defense on RBC. poly lactic-co-glycolic acid (PLGA) is a nontoxic biodegradable polymer that is approved by the Food and Drug Administration for drug delivery. This study aimed to assess dose-dependent efficacy of SOD-CAT-polyethylene glycol -PLGA on RBCs storage. MATERIALS AND METHODS: Using a descriptive study, during 1 month, twenty donors from Bojnourd Blood Donation Center were selected. NPs with different concentrations were injected into the satellite bags after directing blood to them. On target days, experiments were performed on the samples taken. Electrospray was employed to prepare SOD-CAT-PLGA NPs. Twenty packed RBCs were isolated from the whole blood bags by the mechanical method, and certain amount of product was transferred to the satellite bags. On days 1, 7, 14, 21, 28, and 35, bags were sampled. Malondialdehyde (MDA), prooxidant-antioxidant balance (PAB), and Annexin V were performed on the samples taken. The repeated measures analysis with the help of SPSS software version 20 was performed on samples. RESULTS: MDA increased in both groups. The maximum increase in test group was seen in concentration 12 mg (MDA Day 14, test [1.93 ± 0.3], [P MDA < 0.001]). Maximum increase in PAB was seen in concentration 12 mg (from 444 ± 1.7 to 563 ± 2.5) (P PAB = 0.000). Furthermore, PS expression increased in the concentration of 12 mg greater than other concentration in consecutive (from 5.00 ± 0.8 to 22.26 ± 1.7, [P < 0.001]). CONCLUSION: Evaluation of dose dependency showed that different concentrations of antioxidant NPs affect RBC. This effect can be changed oxidative stress and apoptosis. Using both changes to evaluate functional and toxicity can be helpful.

Pathogenic and therapeutic roles of cytokines in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease with high mortality that accounts for the most common acute leukemia in adults. Despite all progress in the therapeutic strategies and increased rate of complete remission, many patients will eventually relapse and die from the disease. Cytokines as molecular messengers play a pivotal role in the immune system. The imbalance release of cytokine has been shown to exert a significant influence on the progression of hematopoietic malignancies including acute myeloid leukemia. This article aimed to summarize current knowledge about cytokines and their critical roles in the pathogenesis, treatment, and survival of AML patients.

Inhibitor in Congenital Factor VII Deficiency; a Rare but Serious Therapeutic Challenge-A Systematic Literature Review.

BACKGROUND: Congenital factor (F) VII deficiency is a rare coagulation factor deficiency with an estimated incidence of 1 per 500,000 individuals. Patients with severe FVII deficiency present a broad range of clinical presentations. Alloimmunization against exogenous FVII, as the main challenge of replacement therapy, is an extremely rare phenomenon that is accompanied by a high rate of life-threatening bleeding, that renders replacement therapy less effective. Due to the importance of the issue, we performed a systematic literature review in order to assess incidence, molecular basis, clinical presentations, and therapeutic challenge and management of inhibitor in congenital FVII deficiency. Strategy of search: This systematic review was performed in accordance with PRISMA guidelines. We performed an English-language literature review in the PubMed, EMBASE, Scopus, and Google Scholar databases, using the following keywords: "factor VII inhibitor", "factor VII inhibitors", "FVII inhibitors", "congenital FVII deficiency", "recombinant factor VII", "anti rFVIIa", "replacement therapy", and "alloantibody". RESULTS: Out of 380 patients in the 13 studies, 27 had inhibitor against FVII; 18 were male, 7 were female, while the sex of 2 was not stated. The majority (92%) developed a high-titer inhibitor (Bethesda Unit > 5). All patients had severe FVII deficiency (FVII:C < 10%), and the majority received recombinant FVII prior to inhibitor development (N: 24, 89%). Among ten patients with a detected mutation, three subjects had a common non-sense (30%), and two had a deletion (20%). CONCLUSIONS: Inhibitor development is a relatively rare phenomenon seen only in severe FVII deficiency, where it is associated with severe and life-threatening presentations, treatment challenge, and economic burden on the patients and their families.

Polymorphisms within methotrexate pathway genes: Relationship between plasma methotrexate levels, toxicity experienced and outcome in pediatric acute lymphoblastic leukemia.

OBJECTIVES: The current study aimed to investigate the relationship of genetic polymorphism and plasma methotrexate (MTX) levels, toxicity experience and event free survival (EFS) in pediatric acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: The study included 74 ALL patients. Polymerase chain reaction and genotyping of methylene tetrahydrofolate reductase (MTHFR) rs1801133, MTHFR rs1801131, ATP-binding cassette superfamily B1 (ABCB1) rs1045642, ATP-binding cassette superfamily G2 (ABCG2) rs2231142 and solute carrier 19A1 (SLC19A1) rs1051266 genetic variations were performed. The plasma MTX levels were investigated at 48 hr after the first dose of MTX infusion. RESULTS: MTHFR rs1801133 TT genotype, ABCBa1 rs1045642 CT genotype and ABCG2 rs2231142 CA genotype revealed a statistically significant association with the MTX plasma levels (P<0.01, P<0.05, P<0.05, respectively). The MTHFR rs1801133 TT genotype had a statistically significant association with hematopoietic toxicity (P<0.01) and interventions (P<0.05). The MTHFR rs1801131 AC genotype was related to the decreased hepatic toxicity (P<0.05). The SLC19A1 rs 1051266 GA genotype was related to the increased hepatic toxicity (P<0.05). Only the ABCB1 rs1045642 CT and TT genotypes had a statistically significant correlation with EFS (P<0.05, P<0.05, respectively). CONCLUSION: Our findings showed that genetic polymorphism could be associated with plasma MTX levels, toxicity experienced and EFS in Iranian pediatric ALL.

Molecular and clinical profile of congenital fibrinogen disorders in Iran, identification of two novel mutations.

INTRODUCTION: Congenital fibrinogen disorders (CFDs) comprise the quantitative and qualitative fibrinogen molecule abnormalities that are caused by fibrinogen gene mutations. The objective of this cohort research was to study the molecular and clinical profiles of patients with CFDs. MATERIALS AND METHODS: Genomic DNA Sanger sequencing of 14 Iranian patients was performed to determine CFDs-causing mutations. The disorders were diagnosed by routine and specific (fibrinogen antigen and functional assay) coagulation tests, and clinical data were obtained from medical records. Molecular dynamics (MD) simulations were performed to investigate the effect of missense mutation on the protein structure. RESULTS: Thirteen out of 14 patients had afibrinogenemia while the remaining patient had dysfibrinogenemia. Umbilical cord bleeding was the most common clinical presentation (n: 9, ~70%) which led to the diagnosis of afibrinogenemia, while menorrhagia led to the diagnosis of dysfibrinogenemia. Six homozygous mutations were identified in afibrinogenemia: three previously described variants in FGA (p.Trp52Ter, p.Ser312AlafsTer109 and p.Gly316GlufsTer105), one in FBG (p.Gly430Asp), and two novel mutations in FGB (p.Gly430Arg) and FGG (p.His366ThrfsTer40), while the FGA (p.Arg38Thr) heterozygous mutation was identified in dysfibrinogenemia. MD simulation indicated that the FGA p. Arg38Thr mutation probably interferes with polymerization of fibrin monomers. CONCLUSIONS: In Iran, with its high rate of consanguinity, autosomal recessive afibrinogenemia with severe clinical presentations is relatively common due to heterogeneous molecular defects.

Effects of Reduced Mir-24 Expression on Plasma Methotrexate Levels, Therapy-Related Toxicities, and Patient Outcomes in Pediatric Acute Lymphoblastic Leukemia.

BACKGROUND: The current study aims to investigate the relationship of miR-24 expression with plasma methotrexate (MTX) levels, therapy-related toxicities, and event-free survival (EFS) in Iranian pediatric acute lymphoblastic leukemia (ALL) patients. METHODS: The study included 74 ALL patients in consolidation phase and 41 healthy children. RNA was extracted from plasma, polyadenylated, and reverse transcribed. miR-24 expression was determined by quantitative polymerase chain reaction (qPCR). Plasma MTX concentrations were measured by high performance liquid chromatography (HPLC) 48 h after high-dose methotrexate (HD-MTX) injection. The diagnosis of ALL was further subclassified as B-ALL or T-ALL via flow cytometry. RESULTS: miR-24 expression was less in pediatric ALL patients than in the control group (p = 0.0038). Furthermore, downregulation of miR-24 was correlated with intermediate- to high-grade HD-MTX therapy toxicities (p = 0.025). Nevertheless, no statistically significant associations were seen between miR-24 levels and plasma MTX levels 48 h after HD-MTX administration (p > 0.05) or EFS in pediatric ALL patients (p > 0.05). CONCLUSION: miR-24 expression may contribute to interindividual variability in response to intermediate- to highgrade HD-MTX therapy toxicities under Berlin Frankfurt Munster (BFM) treatment.

Epigenetic changes in FOXO3 and CHEK2 genes and their correlation with clinicopathological findings in myelodysplastic syndromes.

OBJECTIVES/BACKGROUND: Myelodysplastic syndromes (MDSs) are a heterogeneous disease in terms of clinical course and response to therapy. Epigenetic changes are the primary mechanism of MDS pathogenesis. FOXO3 and CHEK2 genes play significant roles in normal cellular mechanisms and are also known as tumor suppressor genes. We aimed to clarify the correlation of epigenetic changes in these genes with clinicopathologic findings in MDS. METHODS: A total of 54 newly diagnosed MDS patients referred to Shariati and Firouzgar Hospitals (Tehran, Iran) were included in the study from 2013 to 2015, comprising the following cases: 26 with refractory cytopenia with unilineage dysplasia, 10 with refractory cytopenia with multilineage dysplasia, four refractory anemia with excess blasts-1 (RAEB-1), 11 refractory anemia with excess blasts-2 (RAEB-2), and three MDS associated with isolated deletion (5q-). Risk groups were determined according to the Revised International Prognostic Scoring System (IPSS-R). The methylation status of CHEK2 and FOXO3 promoters were determined by methylation-sensitive high-resolution melting analysis of sodium bisulfite-converted DNA. Expressions of CHEK2, FOXO3, and GAPDH were measured by quantitative real-time polymerase chain reaction and fold changes were calculated using the ΔΔCT method. RESULTS: Statistical analysis revealed no promoter methylation of CHEK2 and FOXO3 in healthy control specimens. FOXO3 promoter methylation was associated with high-risk World Health Organization subgroups (p = .017), high-risk IPSS-R (p = .007), high-risk cytogenetics (p = .045), and more than 5% blasts in bone marrow (p = .001). CHEK2 promoter methylation was correlated with more than 5% blasts in bone marrow (p = .009). CONCLUSIONS: Promoter methylation of CHEK2 and especially FOXO3 is associated with adverse clinicopathological findings and disease progression in MDS.

How to Assess Founder Effect in Patients with Congenital Factor XIII Deficiency.

Congenital factor XIII (FXIII) deficiency is an extremely rare bleeding disorder (RBD) with estimated prevalence of one per 2 million in the general population. The disorder causes different clinical manifestations such as intracranial hemorrhage (ICH), recurrent miscarriage, umbilical cord bleeding, etc. High incidence of the disorder might be due to founder effect. To assess founder effect, haplotype analysis is an important step. For this purpose, suitable and reliable genetic markers such as microsatellites (Hum FXIIIA01 and HumFXIIIA02) and single nucleotide polymorphisms (SNP) are suggested. In the present study we tried to describe evaluation of founder effect in patients with congenital FXIII deficiency via haplotype analysis using suitable genetic markers.

Relationship Between Single-Nucleotide Polymorphisms of Tumor Necrosis Factor Alpha, Interleukin-10, Factor II and Factor V with Risk of Inhibitor Development in Patients with Severe Hemophilia A.

BACKGROUND: About one-fourth of patients with hemophilia A (HA) develop alloantibodies against factor (F) VIII, as the main treatment challenge. Here, we assessed the relationship between interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), FII and FV polymorphisms and risk of inhibitor formation in patients with severe HA. METHODS: We divided 39 patients with severe HA in two groups of case (n: 19) and control (n: 20). Genotyping was performed by multiplex amplification tetra arms refractory mutation systempolymerase chain reaction (ARMS-PCR) and PCR-restriction fragment-length polymorphism (PCR-RFLP). RESULTS: TNFα rs1800629 G>A polymorphism decreased the risk of inhibitor development in codominant and dominant inheritance pattern. Moreover, TNFα rs1800629 A allele, decrease the risk of inhibitor formation, while IL10 rs1800896 A>G, FV rs6025 G>A, and FII rs1799963 G>A polymorphisms were not associated with risk of inhibitor development. CONCLUSION: It seems that TNFα rs1800629 G>A polymorphism decreased the risk of inhibitor formation in Iranian patients with HA.