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Aim:Thiopurine S-methyltransferase (TPMT) is an essential enzyme in the metabolism of thiopurine drugs, and its activity may change due to different polymorphisms in the TPMT gene. The TPMT gene has different genetic polymorphisms in different ethnic groups. This study aimed to determine the frequency of TPMT polymorphisms in children with acute leukemia/non-Hodgkin lymphoma (AL/NHL) and healthy children and to evaluate their association with severe toxicities in the study population.Materials and Methods:Sixty-seven pediatric AL/NHL patients and 84 healthy children were evaluated. Genotyping for the TPMT*2, TPMT*3A, TPMT*3B, TPMT*3C, TPMT*4, TPMT*5, TPMT*6, and TPMT*7 alleles were performed by the real-time polymerase chain reaction technique. The number of grade 3 or higher hematologic and hepatic toxicities were recorded from the patient charts.Results:In the AL/NHL patients, we found that the patients had generally wild-type TPMT*1 allele in 80.6%, whereas TPMT*2 (238G>C) was seen in 1.5%, TPMT*3A (c.460G>A and c.719A>G) in 0%, and TPMT*3B polymorphisms (460G>A) in 17.9%. We found wild-type TPMT*1 allele in 98.8% and TPMT*3B polymorphisms (460G>A) in 1.2% of the healthy volunteers. Grade ≥3 myelosuppression developed in 22/54 patients with the wild type allele, and it developed in 5/12 patients with TPMT*3B allele. Six (8.9%) patients had grade ≥3 aspartate aminotransferase elevations, 17 (25%) patients had grade ≥3 alanine transaminase elevations (1-5 times), and 42 patients had (62.6%) grade ≥3 total bilirubin elevations.Conclusion:TPMT*3B polymorphism was the most common allele detected in our study group. This allele frequency is very high in comparison to other studies from our country and it was over-represented in comparison to the healthy volunteers. We did not find any relationship between severe hematologic/hepatic toxicities and TPMT gene polymorphisms.

We aimed to determine the genotype distribution, allele frequency, and prognostic impact of IDH1/2, TET2, and ASXL1 single nucleotide polymorphisms (SNPs) in myeloproliferative neoplasms (MPNs). TET2 (rs763480), ASXL1 (rs2208131), and IDH1 (rs11554137) variant homozygous genotype frequencies were found at rates of 1.5%, 9.2%, and 2.3%, respectively. No IDH2 SNP was identified. IDH1 and TET2 frequencies were 5% in essential thrombocythemia (ET) and 1.7% in ET and 5% in primary myelofibrosis (PMF), respectively. ASXL1 frequencies were 8.3%-10% in MPN subgroups. The TET2 mutant allele T and ASXL1 mutant allele G had the highest frequencies with 0.272 in the PMF and 0.322 in the polycythemia vera (PV) group, respectively. There was no impact of the SNPs on prognosis. IDH1 frequency in MPNs was found similar to the literature. ASXL1 frequencies were similar between ET, PV, and PMF patients. The ASXL1 and TET2 allele frequencies of the Turkish population are similar to those of the European population. The role of SNPs in MPNs might be further evaluated in larger multicenter studies.

OBJECTIVE: Follicular lymphoma (FL) is one of the most common lymphomas, and is characterized by t(14;18) (q32;q21) in more than 80% of patients. The aim of this study was to determine the rate of t(14;18) positivity based on the detection of mbr or mcr in paraffin-embedded tissue samples. METHODS: The study included 32 paraffin-embedded tissue samples collected from 32 consecutive FL patients that were diagnosed and followed-up at our hospital between 1999 and 2006. The MBR breakpoint was identified based on real-time PCR using a LightCycler v.2.0 t(14;18) Quantification Kit (MBR), multiplex PCR, and seminested PCR. To identify the mcr breakpoint, real-time PCR was performed using specific primers and the FastStart DNA Master SYBR Green I Kit. To detect t(14;18) via fluorescence in situ hybridization (FISH) nuclei from paraffin-embedded tissue sections were extracted and used together with LSI IgH (immunoglobulin heavy chain) (spectrum green)/bcl-2 (B-cell leukemia-lymphoma 2) (spectrum orange) probes. RESULTS: The DNA and nuclei isolation success rate for B5 formalin-fixed, paraffin-embedded tissue sections (n = 12) was 42% and 33%, respectively, versus 95% and 60%, respectively, for 20 tissue sections fixed in formalin only. In all, 24 paraffin-embedded tissue sections were analyzed and mbr positivity was observed in the DNA of 82.14% via seminested PCR, in 53.57% via multiplex PCR, and in 28.57% via real-time PCR. We did not detect mcr rearrangement in any of the samples. In all, 15 of 16 patients (93.75%) whose nuclei were successfully isolated were observed to be t(14;18) positive via the FISH method. CONCLUSION: Semi-nested PCR and FISH facilitated the genetic characterization of FL tumors. As such, FISH and PCR complement each other and are both essential for detecting t(14;18) translocation.

OBJECTIVE: The aim of this study was to examine Factor V G1691A (Leiden) (FVL) and prothrombin G20210A (PT) gene mutation status, and their relationship with thrombosis in patients with chronic myeloproliferative disorders (CMPDs). METHODS: The study included 160 patients with a CMPD that were regularly followed-up between 1993 and 2009. FVL and PT mutation status was established based on blood samples analyzed via PCR using specific primers. RESULTS: The frequency of FVL and PT mutation was 12.5% and 4.4%, respectively. In total, 27 episodes of thrombosis occurred in 24 (15%) of the patients, and there wasn't an association between the observed thrombotic events, and FVL or PT mutations. Hepatic vein thrombosis was noted in 3 patients that had FVL mutation, of which 1 also had PT mutation. CONCLUSION: We did not observe a relationship between thrombosis, and FVL or PT mutations in CMPD patients; however, 3 of the patients that had hepatic vein thrombosis also had FVL mutation. Larger studies are needed to more clearly determine if all CMPD patients with hepatic vein thrombosis need be investigated for FVL and PT mutation.

Objective: The aim of this study was to examine Factor V G1691A (Leiden) (FVL) and prothrombin G20210A (PT) gene mutation status, and their relationship with thrombosis in patients with chronic myeloproliferative disorders (CMPDs).Materials and Methods: The study included 160 patients with a CMPD that were regularly followed-up between 1993 and 2009. FVL and PT mutation status was established based on blood samples analyzed via PCR using specific primers.Results: The frequency of FVL and PT mutation was 12.5% and 4.4%, respectively. In total, 27 episodes of thrombosis occurred in 24 (15%) of the patients, and there wasn’t an association between the observed thrombotic events, and FVL or PT mutations. Hepatic vein thrombosis was noted in 3 patients that had FVL mutation, of which 1 also had PT mutation.Conclusion: We did not observe a relationship between thrombosis, and FVL or PT mutations in CMPD patients; however, 3 of the patients that had hepatic vein thrombosis also had FVL mutation. Larger studies are needed to more clearly determine if all CMPD patients with hepatic vein thrombosis need be investigated for FVL and PT mutation.

The aim of this study was to investigate the effects of experimentally induced diabetes on (a) germ cells, (b) in vitro fertilization (IVF) success rate, and (c) gap junction and cell adhesion molecule gene and protein expressions during the early blastocyst period. Germ cells were obtained from healthy and diabetic rats, analyzed for number, motility, and morphology, and used for IVF. After reaching the early blastocyst stage, the expressions of genes encoding gap junction proteins and cell adhesion molecules were analyzed by quantitative RT-PCR. Histomorphologically and immunohistochemically analyses were also performed. Diabetes significantly affected sperm number and motility and the development of oocytes. Gene expressions of β-catenin and connexin family members and protein expressions of E-cadherin and connexin-43 significantly decreased in groups including germ cells isolated from diabetic rats. Connective tissue growth factor expression increased in groups that included sperm cells isolated from diabetic male rats, whereas mucin-1 expression increased in the group that included oocytes isolated from diabetic female rats paired with sperm cells isolated from healthy male rats. In summary, experimentally induced diabetes was found to influence gap junctions, cell adhesion molecules, and associated proteins which all have important roles in germ cell maturation, fertilization, and development.

Objective: Studies to reduce the heterogeneity of attention-deficit/hyperactivity disorder (ADHD) have increased interest in the concept of sluggish cognitive tempo (SCT). The aim of this study was to investigate if the prevalence of two variable-number tandem repeats (VNTRs) located within the 3′-untranslated region of the DAT1 gene and in exon 3 of the dopamine D4 receptor (DRD4) gene differ among four groups (31 subjects with SCT but no ADHD, 146 individuals with ADHD but no SCT, 67 subjects with SCT + ADHD, and 92 healthy controls). Methods: We compared the sociodemographic profiles, neurocognitive domains, and prevalence of two VNTRs in SCT and ADHD subjects versus typically developing (TD) controls. Results: The SCT without ADHD group had a higher proportion of females and lower parental educational attainment. Subjects in this group performed worse on neuropsychological tests, except for psychomotor speed and commission errors, compared to controls. However, the ADHD without SCT group performed significantly worse on all neuropsychological domains than controls. We found that 4R homozygosity for the DRD4 gene was most prevalent in the ADHD without SCT group. The SCT without ADHD group had the highest 7R allele frequency, differing significantly from the ADHD without SCT group. Conclusion: The 7R allele of DRD4 gene was found to be significantly more prevalent in SCT cases than in ADHD cases. No substantial neuropsychological differences were found between SCT and ADHD subjects.

Objective: Tyrosine kinase inhibitors (TKIs) which efficiently inhibit BCR-ABL are highly effective for clinical treatment of chronic myeloid leukemia (CML), but development of resistance to TKIs is a big challenge to treatment. Sunitinib is a multitargeted TKI targeting vascular endothelial growth factor receptor and is defined a safe and effective candidate target, but its effect on other signaling pathways is unknown. To investigate the cytotoxic and apoptotic effect of sunitinib in CML cell model K-562 on JAK-STAT signaling pathway components, suppressor genes and oncogenes, hematopoiesis-related genes, cell cycle and VEGF pathway components, and mRNA level expression changes was aimed.Materials and Methods: Sunitinib's effective dose cytotoxic IC50 was determined by trypan blue and WST-1 cell proliferation assay tests. Expression levels of target genes were determined by quantitative reverse transcriptase polymerase chain reaction simultaneously after sunitinib application. Protein expression analysis was determined by "WesternBreeze Chromogenic Kit-Anti-Rabbit" based on the principles of the application kit by Western blot analysis.Results: Assessing the cytotoxicity of K-562 cells following sunitinib treatment revealed that sunitinib decreased cell proliferation in a time- and dose-dependent manner. According to the sunitinib inhibition curve, IC50 dose was calculated as 3.5 μM at 48th h for K-562 cells and apoptosis assays pointed that sunitinib induces apoptotic cell death of leukemic cells at moderate levels.Conclusion: Our study supports that sunitinib might be used as a novel therapeutic target to trigger apoptosis in CML cells which in turn might accelerate therapeutic response in regard to inhibiting oncogenes and enhancing tumor suppressors in cooperation with cell cycle regulatory genes. [ABSTRACT FROM AUTHOR]

Aim and Objective: Surface Plasmon Resonance (SPR) based biosensor system was developed for the detection of Delta F508 (ΔF508del) Cystic Fibrosis (CF) mutation in both synthetic and real samples., Material and Method: In order to provide an effective hybridization between probe and the Polymerase Chain Reaction (PCR) amplicons (target), streptavidin was bound to the surface and biotin-tag probe was sent to the streptavidin-coated surface. For the target preparation, blood samples were collected from the patients who suffer from CF. Following the DNA isolation; samples were amplified with PCR with biotin-tag. Before sending the biotin-tag PCR amplicons onto the modified surface, amplicons were also interacted with the helper oligonucleotides to prevent re-annealing of the denatured DNA strands. This kind of 'multiple surface binding' method helps increasing the sensitivity of the detection., Results: The limit of detection (S/N= 3) was calculated as 12.24 pico-mole/ml for PCR-like synthetic long target sequence and 13x105 molecules for real samples in less than half an hour., Conclusion: Using the both biotin-tag probe and the helper oligonucleotides together, hybridization was achieved much more efficiently than traditional denaturation protocols for real samples and biotinfree hybridization detection. To the best of our knowledge, the procedure described in this study is one of the simplest, rapid and sensitive methods for CF mutation detection with SPR based biosensor system in real samples., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)