Nikola Kotur

6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner

AIM TPMT activity is characterized by a trimodal distribution, namely low, intermediate and high methylator. TPMT gene promoter contains a variable number of GC-rich tandem repeats (VNTRs), namely A, B and C, ranging from three to nine repeats in length in an A(n)B(m)C architecture. We have previously shown that the VNTR architecture in the TPMT gene promoter affects TPMT gene transcription. MATERIALS, METHODS & RESULTS: Here we demonstrate, using reporter assays, that 6-mercaptopurine (6-MP) treatment results in a VNTR architecture-dependent decrease of TPMT gene transcription, mediated by the binding of newly recruited protein complexes to the TPMT gene promoter, upon 6-MP treatment. We also show that acute lymphoblastic leukemia patients undergoing 6-MP treatment display a VNTR architecture-dependent response to 6-MP. CONCLUSION These data suggest that the TPMT gene promoter VNTR architecture can be potentially used as a pharmacogenomic marker to predict toxicity due to 6-MP treatment in acute lymphoblastic leukemia patients.

Functional analysis of a novel KLF1 gene promoter variation associated with hereditary persistence of fetal hemoglobin

Hereditary persistence of fetal hemoglobin (HPFH) is a rare hereditary condition resulting in elevated levels of fetal hemoglobin (HbF) in adults. Typical HPFH is associated with promoter mutations or large deletions affecting the human fetal globin (HBG1 and HBG2) genes, while genetic defects in other genes involved in human erythropoiesis, e.g. KLF1, also result in atypical HPFH. Here, we report the first KLF1 gene promoter mutation (KLF1:g.-148G > A) that is associated with increased HbF level. This mutation was shown to result in drastically reduced CAT reporter gene expression in K562 cells, compared to the wild-type sequence (p = 0.009) and also in reduced KLF1 gene expression in vivo. Furthermore, consistent with in silico analysis, electrophoretic mobility shift analysis showed that the KLF1:g.-148G > A mutation resides in a Sp1 binding site and further that this mutation leads to the ablation of Sp1 binding in vitro. These data suggest that the KLF1:g-148G > A mutation could play a role in increasing HbF levels in adults and further underlines the role of KLF1 as one of the key transcription factors involved in human fetal globin gene switching.

HLA genotyping in pediatric celiac disease patients

Celiac disease (CD) is a chronic inflammatory disease in the small intestine triggered by gluten uptake that occurs in genetically susceptible individuals. HLA-DQ2 protein encoded by HLA-DQA1*05 and DQB1*02 alleles is found in 90-95% of CD patients. All of the remaining patients carry HLA-DQ8 protein encoded by HLA-DQA1*03 and DQB1*03:02 alleles. Specific HLA-DQ genotypes define different risk for CD incidence. Presence of susceptible HLA-DQ genotypes does not predict certain disease development, but their absence makes CD very unlikely, close to 100%. Here we presented for the first time the distribution of HLA-DQ genotypes in the group of pediatric celiac patients from the University Childrens Hospital, Belgrade, Serbia and estimated risk for CD development that these genotypes confer. Seventy three celiac disease patients and 62 healthy individuals underwent genotyping for DQA1, DQB alleles and DRB1 allele. 94.5% of patients carried alleles that encode DQ2 protein variant and 2.7% carried alleles that encode DQ8 protein variant. Two patients carried single DQB1*02 allele. No patients were negative for all the alleles predisposing to CD. The highest HLA-DQ genotype risk for CD development was found in group of patients homozygous for DQ2.5 haplotype, followed by the group of heterozygous carriers of DQ2.5 haplotype in combination with DQB1*02 allele within the other haplotype. The lowest risk was observed in carriers of a single copy of DQB1*02 or DQA1*05 allele or other non-predisposing alleles. HLA genotyping, more informative than serological testing commonly used, proved to be a useful diagnostic tool for excluding CD development.

TPMT gene expression is increased during maintenance therapy in childhood acute lymphoblastic leukemia patients in a TPMT gene promoter variable number of tandem repeat-dependent manner

AIMS 6-mercaptopurine influences in vitro TPMT gene expression in a TPMT promoter variable number of tandem repeats (VNTR)-dependent manner. We studied TPMT expression following 6-mercaptopurine and methotrexate administration in childhood acute lymphoblastic leukemia (ALL) patients and the pharmacogenomic potential of the VNTR architecture. MATERIALS & METHODS TPMT gene expression was determined in childhood ALL patients at diagnosis (n = 57) and during the maintenance therapy (n = 27). RESULTS A threefold increase of TPMT gene expression was obtained during maintenance therapy, modulated by the architecture of the VNTR region. CONCLUSION The TPMT promoter genetic variants need to be considered at the very beginning of the maintenance therapy for childhood ALL patients. The TPMT promoter VNTR region may serve as a pharmacogenomic biomarker when introducing thiopurine therapy.

Variations in inflammatory genes as molecular markers for prediction of inflammatory bowel disease occurrence.

Research on inflammatory bowel disease (IBD) has highlighted genes involved in the regulation of inflammatory responses as contributors to disease pathogenesis. This study aimed to evaluate the associations between IBD and variations in NOD2, TLR4, TNF‐α, IL‐6, IL‐1β and IL‐1RN genes, and to use the genetic data obtained in predictive modeling.

The in vitro antioxidative and cytotoxic effects of selected Salvia species water extracts

The current paper presents antioxidant and cytotoxic activities and total phenolic and flavonoid content of the selected species of genus Salvia (Lamiaceae) growing wild in Macedonia ( S. jurisicii Kosanin, S. amplexicaulis Lam . , S. ringens Sibth. & Sm.) and Libya ( S. fruticosa Mill. and S. lanigera Poir.). Crude water extracts, obtained from aerial parts, were yielded from 6.50 to 14.32%. Total phenolic content was the highest in water extracts of S. amplexicaulis and S. ringens (226.30 and 189.01 mg GAE/g, respectively), while the flavonoids were the most abundant in S. jurisicii extract (32.36 mg QE/g). Antioxidant activities of extracts were measured using DPPH, ABTS and FRAP assays. S. amplexicaulis and S. ringens extracts showed the strongest antioxidant activity, measured using DPPH (14.21 and 23.44 μg/mL, respectively) and ABTS assays (2.91 and 2.42 mg AAE/g, respectively). In FRAP assay, S. amplexicaulis and S. fruticosa extracts exhibited strongest activity (1406.73 and 1191.51 µmol Fe(II)/g). Water extract of S. amplexicaulis and S. ringens performed the strongest cytotoxic activity against K562 cells (151.07 and 173.06 μg/mL, respectively). Based on these findings, it can be concluded that S. amplexicaulis and S. ringens water extracts could be considered as possible source of antioxidant and cytotoxic agents.

Influence of Promoter Polymorphisms of the TNF-α (-308G/A) And IL-6 (-174G/C) Genes On Therapeutic Response To Etanercept In Rheumatoid Arthritis

Summary Background: The study was undertaken to assess the influence of functional -308G/A TNF-α (rs 1800629) and -174G/C IL-6 (rs1800795) promoter polymorphisms on the therapeutic response to etanercept, a TNF-α blocker, in patients with rheumatoid arthritis (RA). Methods: Seventy-three patients suffering from active RA were studied, at baseline and 6 and 12 months after therapy. The therapeutic response was estimated according to the European League Against Rheumatism response criteria. Patients were genotyped for -308G/A TNF-α and -174G/C IL-6 polymorphisms by the PCR-RFLP method, and the influence of genotype on etanercept response was assessed. Results: No difference in the percentage of responders (patients who had DAS28 improvement > 1.2) between patients with the TNF-α-308GG and GA and AA genotype was detected after 6 and 12 months of treatment. After 12 months of treatment the percentage of responders was significantly increased in patients with the IL-6 -174GG genotype compared with those with the GC or CC genotype (p=0.006 by Chi-square test). Evaluation of the patients according to their combined IL-6/TNF-α genotypes showed that patients with the IL-6 -174GG / TNF-α -308GG genotype were more frequent among the responders compared to those with other combined genotypes (p=0.022 by Chisquare test). More precisely, all patients with the combined IL-6 -174GG / TNF-α -308GG genotype were responders after 12 months of etanercept treatment. Conclusions: The study suggests that patients who are genetically low TNF-α and IL-6 producers are the best responders to etanercept therapy.

Genetic predictors of celiac disease, lactose intolerance, and vitamin D function and presence of peptide morphins in urine of children with neurodevelopmental disorders

Gastrointestinal disturbances, nutritional deficiencies, and food intolerances are frequently observed in children with neurodevelopmental disorders (NDD). To reveal possible association of celiac disease risk variants (HLA-DQ), lactose intolerance associated variant (LCT-13910C>T) as well as variant associated with vitamin D function (VDR FokI) with NDD, polymerase chain reaction-based methodology was used. Additionally, intestinal peptide permeability was estimated in NDD patients and healthy children by measuring the level of peptides in urine using high-performance liquid chromatography. Levels of opioid peptides, casomorphin 8, and gluten exorphin C were significantly elevated in urine samples of NDD patients (P = 0.004 and P = 0.005, respectively), but no association of genetic risk variants for celiac disease and lactose intolerance with NDD was found. Our results indicate that increased intestinal peptide permeability observed in analyzed NDD patients is not associated with genetic predictors of celiac disease or lactose intolerance. We have also found that FF genotype of VDR FokI and lower serum levels of vitamin D (25-OH) showed association with childhood autism (CHA), a subgroup of NDD. We hypothesize that vitamin D might be important for the development of CHA.

Genetic and environmental factors significant for the presentation and development of inflammatory bowel disease

Objectives The aim of the study was to evaluate associations between inflammatory bowel disease (IBD) presentation and variants in NOD2, TLR4, TNF-&agr;, IL-6, IL-1&bgr;, and IL-RN genes in order to identify possible environmental factors that may affect IBD occurrence, investigate potential predictors for surgical treatment of IBD, and correlate the presence of granulomas in biopsy specimens with clinical characteristics of Crohn’s disease (CD) patients. Patients and methods We genotyped 167 IBD patients using PCR-based methodology and tested for disease genotype–phenotype associations. Results In CD patients ileal localization of disease was more frequent in NOD2 variant carriers. Ileal CD was associated with IL-6 GC+CC genotypes, identifying C allele as a possible marker of increased risk for ileal CD. In CD patients a positive family history for IBD was related to earlier onset of disease, higher risk for CD-related surgery, and appendectomy. CD patients who are TLR4 299Gly carriers are at higher risk for surgery at onset of the disease compared with TLR4 299Asp variant carriers. The presence of granuloma in biopsy specimens was more frequent in patients in whom a diagnosis of CD was made during emergency surgery. Multivariate analysis showed that CD carriers of the 299Gly allele had a 4.6-fold higher risk for emergency surgery before CD diagnosis is established compared with noncarriers, suggesting an aggressive disease course. Granuloma in endoscopic biopsies is detected 5.4-fold more frequently in patients treated surgically at the time of diagnosis. Conclusion Genetic variants together with epidemiological and clinical data of IBD patients could potentially be used as predictors of the disease course.

Pharmacogenomic markers of glucocorticoid response in the initial phase of remission induction therapy in childhood acute lymphoblastic leukemia

Abstract Background Response to glucocorticoid (GC) monotherapy in the initial phase of remission induction treatment in childhood acute lymphoblastic leukemia (ALL) represents important biomarker of prognosis and outcome. We aimed to study variants in several pharmacogenes (NR3C1, GSTs and ABCB1) that could contribute to improvement of GC response through personalization of GC therapy. Methods Retrospective study enrolling 122 ALL patients was carried out to analyze variants of NR3C1 (rs33389, rs33388 and rs6198), GSTT1 (null genotype), GSTM1 (null genotype), GSTP1 (rs1695 and rs1138272) and ABCB1 (rs1128503, rs2032582 and rs1045642) genes using PCR-based methodology. The marker of GC response was blast count per microliter of peripheral blood on treatment day 8. We carried out analysis in which cut-off value for GC response was 1000 (according to Berlin-Frankfurt-Munster [BFM] protocol), as well as 100 or 0 blasts per microliter. Results Carriers of rare NR3C1 rs6198 GG genotype were more likely to have blast count over 1000, than the non-carriers (p = 0.030). NR3C1 CAA (rs33389-rs33388-rs6198) haplotype was associated with blast number below 1000 (p = 0.030). GSTP1 GC haplotype carriers were more likely to have blast number below 1000 (p = 0.036), below 100 (p = 0.028) and to be blast negative (p = 0.054), while GSTP1 GT haplotype and rs1138272 T allele carriers were more likely to be blasts positive (p = 0.034 and p = 0.024, respectively). ABCB1 CGT (rs1128503-rs2032582-rs1045642) haplotype carriers were more likely to be blast positive (p = 0.018). Conclusions Our results have shown that NR3C1 rs6198 variant and GSTP1 rs1695-rs1138272 haplotype are the most promising pharmacogenomic markers of GC response in ALL patients.