Screening of polymorphisms in the folate pathway in Turkish pediatric acute lymphoblastic leukemia patients

Background and aim: Folate metabolic pathway plays a significant role in leukemogenesis because of its necessity for nucleotide synthesis and DNA methylation. Folate deficiency causes DNA damage. Thus polymorphisms of folate-related genes may affect the susceptibility to childhood Acute Lymphoblastic Leukemia [ALL]. MTHFR [Methylenetetrahydrofolate Reductase], DHFR [Dihydrofolate reductase], CBS [Cystathionine ?-synthase] and TYMS [Thymidylate Synthase] have an important role in folate pathway because their activated variants modulate synthesis of DNA and levels of folate. In this study, we aimed to investigate whether polymorphisms in genes related to folate metabolic pathway influence the risk to childhood ALL

Subject and methods: The patient groups who were diagnosed with childhood ALL at Losante Pediatric Hematology-Oncology Hospital and healthy control groups were included in the study. MTHFR 677 C-T, MTHFR 1298 A-C, CBS 844ins68, DHFR 19-bp and TYMS 1494del6 polymorphisms were screened. Genotyping of these polymorphisms was performed by Restriction Fragment Length Polymorphism [RFLP] analysis and Real Time Polymerase chain Reaction [Real Time-PCR]

Results: In total, we have screened 5 polymorphisms in the studied genes. The results were compared between childhood ALL patients and healthy groups. Genotype frequencies of MTHFR 677 C-T, MTHFR 1298 A-C, CBS 844ins68 and DHFR 19-bp del were similar for childhood ALL patients and healthy groups. However, statistical results showed that TYMS 1494del6 may be associated with ALL pathogenesis [p < 0.001]

Conclusion: We showed that TYMS polymorphism [rs2853542] may be associated with ALL pathogenesis. In addition, our results demonstrated that MTHFR, DHFR and CBS do not affect development of leukemia. Our study displays also importance as it is the first screening results to identify association with the studied polymorphisms in Turkish patients with childhood ALL and determination of the frequency in Turkish population

Detection of TET2, KRAS and CBL variants by Next Generation Sequencing and analysis of their correlation with JAK2 and FLT3 in childhood AML

Background: Acute myeloid leukemia [AML] is a heterogeneous clonal disorder in terms of cytogenetic and molecular aberrations. Ten-Eleven-Translocation 2 [TET2], Kirsten rat sarcoma viral oncogene homolog [KRAS], and Casitas B-cell lymphoma [CBL] have an important role pathogenesis of acute myeloid leukemia [AML] and their activated mutations confer proliferative and survival signals

Aim: In this study, we aimed to find possible genetic markers for molecular analysis in childhood AML by screening hot-spot exons of TET2, KRAS, and CBL using Next Generation Sequencing [NGS] analysis. In addition, association between found variants and mutations of Januse Kinase-2 [JAK2] and Fms-Related Tyrosine Kinase [FLT3] were analyzed which are important prognostic risk factors for AML

Methods: Eight patients who were diagnosed with pediatric AML at Losante Pediatric Hematology- Oncology Hospital were included to the study. Hot-spot exons of TET2, KRAS and CBL genes were screened using the NGS method. Furthermore, FLT3-Internal Tandem Duplicate [FLT3-ITD] and JAK2-V617F were analyzed by Real Time Polymerase chain Reaction [Real Time-PCR]

Results: In total, we identified 20 variants in studied genes by NGS. In our patient group, 16 variants in the TET2 [seven novel, seven missense and two silent], two variants in the KRAS [one missense and one intronic] and two variants in the CBL [two novel] were found. All of AML patients were found negative for JAK V617 F. Three of the eight patients [37.5%] showed mutations of both FLT3-ITD and TET2, KRAS, CBL

Conclusion: We found novel mutations forTET2, KRAS, and CBL. The detected variants in this article seem to be the first screening results of genes studied by NGS in childhood AML patients. Our results also showed some degree of association between FLT3-ITD and TET2, KRAS, CBL mutations

Ankaferd Blood Stopper induces apoptosis and regulates PAR1 and EPCR expression in human leukemia cells

Ankaferd Blood Stopper [ABS] is a preparation of plant extracts originally used as a hemostatic agent. It has pleiotropic effects in many cellular processes such as cell cycle regulation, apoptosis, angiogenesis, signal transduction, inflammation, immunologic processes and metabolic pathways as well as hemostatic activity. This unique preparation has been widely investigated for its properties. However there are no studies investigating its action on leukemic cells. Aim of the study was to examine the ABS action on PAR1 and EPCR in leukemia cells. However, during the experiments, we observed the apoptotic effect of ABS on leukemic cells, particularly Jurkat cells. As a result the mechanism of apoptosis induced by ABS treatment was also explored in the study. Two leukemia cell lines, K-562 and Jurkat, were utilized for the study. Expression analyses of PAR 1, EPCR and p21 upon ABS treatment were performed by quantitative real time PCR. Annexin V method was used for apoptosis detection. Our results demonstrated that ABS alters PAR1 and EPCR expression in K-562 and Jurkat cells in a time and dose dependent manner. Additionally it was found that ABS treatment induces apoptosis in leukemia cells. Possible involvement of PAR 1 and p21 in this apoptotic process was observed in Jurkat cells. This study concludes that depending on the concentration and duration of the application, ABS causes apoptosis by regulating PAR1 and p53-independent p21 involvement in apoptosis stimulation in leukemia cells. The composition of ABS plant extracts might be responsible from the apoptotic effect that was observed. We think that our results could contribute to the development of new treatment for leukemia therapy

novel MEFV gene mutation [A511V] in a Chilean FMF patient

Familial Mediterranean fever [FMF] is an autosomal recessive disease which is characterized by recurrent fever and inflammation of serous membranes. A Chilean FMF patient was investigated for MEFV mutations. After DNA extraction, exons 3, 5, 10 and 30UTR region of MEFV gene were analyzed by DNA sequencing while E148Q and R202Q mutations of exon 2 were detected by RFLP. A novel missense mutation, A511V [c.1532C>T, p.Ala511Val], was found in a heterozygous state in exon 5 of MEFV gene. Also, R202Q [c.605G>A, p.Arg202Gln] was detected in heterozygous state. R202Q was of clinical value in the diagnosis of FMF when combined with a disease causing mutation. In this patient, A511V was detected in compound heterozygous state with R202Q and this association may play an important role in FMF

Hemoglobin alpha 2 gene +861 G>A polymorphism in Turkish population

Thalassemia is an inherited blood disorder which is divided into two groups: alpha and beta. HBA1 and HBA2 are the two genes associated with alpha thalassemia. The aim of this study is to investigate abnormal hemoglobin variants of alpha globin gene in healthy abnormal hemoglobin carrying individuals with intact beta globin gene. DNA was extracted from peripheral blood samples of seven healthy carrier individuals who have abnormal hemoglobin variants and 16 control individuals from Turkey. Complete coding and intronic sequences of HBA1 and HBA2 genes were amplified by polymerase chain reaction [PCR] and PCR products of HBA1 and HBA2 were sequenced. We were unable to find any base change in our carrier group in the HBA1 gene. We have observed an A/G polymorphism in the downstream untranslated region [+861 G>A] of the HBA2 gene. Our study showed that 14.29% [1/7 carriers] of the carrier group and 37.50% [6/16 controls] of the control group were heterozygous for the +861 G>A polymorphism. The distribution of allele frequencies and genotypes of HBA2 between carrier and control samples were analyzed and it is seen that the distribution of allele frequencies and that of genotypes were not statistically significant between carrier and control samples [P-value = 0.4131, P-value= 0.366, respectively]. HBA2 +861 G>A nucleotide substitution is a neutral polymorphism previously reported in other populations. This is the first report in Turkish population

Role of ZIP14 [SLC39A14] gene histidine rich regions in neural tube defects

Neural tube defects [NTDs] comprise a group of congenital malformations that includes spina bifida, anencephaly, meningomyelocele and encephalocele. Reports have implicated zinc deficiency as one of the causative factors of NTDs. Both environmental and genetic factors are involved in the etiology of NTDs. Inadequate folate intake and nutritional deficiency are important environmental risk factors. The aim of this study was to determine the relation of a zinc related gene ZRT and IRT like protein 14 [ZIP14] and neural tube defects in Turkish patients. The case control study included seventy Turkish mothers who gave birth to NTD infants. Two hundred and thirty-nine healthy controls were consecutively selected without any congenital defects or familial NTD history. Following DNA extraction, PCR, SSCP and DNA sequencing analysis of exons of the ZIP14 gene were performed. Our data revealed that no relation of neural tube defects and ZIP14 was detected in Turkish NTD patients. Zinc deficiency have been reported as a risk factor for Turkish population and other possible zinc related gene defects may have importance