Biljana Stankovic

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.

Thalassemia Syndromes in Serbia: An update

Thalassemia syndromes constitute a group of genetic disorders, widespread throughout the world. The present study contains data on thalassemia syndromes and their chromosomal environment obtained in Serbia over a period of 10 years. Ten different β-thalassemia (β-thal) mutations and two hemoglobin (Hb) variants were detected in 127 members of 68 families. Hb Lepore-Boston-Washington (Lepore-BW) (δ87Gln-β-IVS-II-8), a thalassemic Hb variant, was shown to be the most common cause of thalassemia in Serbia. Haplotype analyses of the β-globin gene clusters of healthy individuals as well as of individuals affected with β-thal showed that haplotype I was the most frequent haplotype in the Serbian population, followed by haplotypes II and IX. Two novel haplotypes were detected. Haplotype analyses showed the association between certain haplotypes and the most common thalassemic mutations. Results presented in this paper will update the Serbian national mutation database and contribute to a better understanding of genographic history of South European and Balkan populations.

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.

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.

Women's Experiences of Childbirth in Serbian Public Healthcare Institutions: a Qualitative Study

PurposeThe aim of the present study was to explore how women, users of public maternity healthcare services in Serbia, experience birth and what the most problematic relational aspects of institutional context associated with negative or even traumatic aspects of birth experiences are, as described by the women themselves.MethodAn exploratory qualitative study was undertaken using semi-structured interviews with 15 primiparous women aged 26 to 49 who have recently given birth in different public healthcare institutions in Belgrade. The basic framework for the analysis of interview transcripts was the interpretative phenomenological analysis (IPA).ResultsThrough the analysis, four relatively broad and partly interrelated themes emerged: (a) feelings of isolation and abandonment, (b) lack of communication, (c) lack of a caring relationship, and (d) lack of control and agency. The aspects of institutional environment that were considered particularly distressing in most of the childbirth experiences are related to distant and cold relationship with healthcare providers which adds to the feelings of isolation and abandonment, in addition to the lack of insight into and control over the process of birth that is managed in the hospital context without relying on women’s subjective involvement in any relevant way.ConclusionThe present study emphasized a supportive and caring relationship with medical practitioners, as well as allowing women to be more involved into their birthing process, as crucial for positive experience of birth, which might have profound and long-lasting psychosocial consequences. Recommendations for policy makers and future research are offered.

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.