Aim: β-Type hemoglobinopathies are characterized by vast phenotypic diversity as far as disease severity is concerned, while differences have also been observed in hydroxyurea (HU) treatment efficacy. These differences are partly attributed to the residual expression of fetal hemoglobin (HbF) in adulthood. The Krüppel-like family of transcription factors (KLFs) are a set of zinc finger DNA-binding proteins which play a major role in HbF regulation. Here, we explored the possible association of variants in KLF gene family members with response to HU treatment efficacy and disease severity in β-hemoglobinopathies patients. Materials & methods: Six tag single nucleotide polymorphisms, located in four KLF genes, namely KLF3, KLF4, KLF9 and KLF10, were analyzed in 110 β-thalassemia major patients (TDT), 18 nontransfusion dependent β-thalassemia patients (NTDT), 82 sickle cell disease/β-thalassemia compound heterozygous patients and 85 healthy individuals as controls. Results: Our findings show that a KLF4 genomic variant (rs2236599) is associated with HU treatment efficacy in sickle cell disease/β-thalassemia compound heterozygous patients and two KLF10 genomic variants (rs980112, rs3191333) are associated with persistent HbF levels in NTDT patients. Conclusion: Our findings provide evidence that genomic variants located in KLF10 gene may be considered as potential prognostic biomarkers of β-thalassemia clinical severity and an additional variant in KLF4 gene as a pharmacogenomic biomarker, predicting response to HU treatment.
β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPβ-globin for the physiological β-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the β-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the β-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce β-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPβ-globin provides the basis for the development of therapeutic EV for gene therapy of β-thalassemias.
Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components underlying NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background holds a great potential for the identification of epi-biomarkers for treatment response however, it is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin. We engaged an in-depth characterization of non-coding (nc)RNAs prevalent in human
The mechanisms by which mutations within the 5' untranslated region (UTR) of the human beta-globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four 'silent' mutations in the human beta-globin 5'UTR, namely: +10(-T), +22(G --> A), +33(C --> G) and +(40-43)(-AAAC), which are present in patients with beta-thalassaemia intermedia. Expression of these genes under the control of the beta-globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61.6%, 68%, 85.2% and 70.6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3' end processing or mRNA stability. Nuclear run-on experiments demonstrated that mutations +10(-T) and +33(C --> G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G --> A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post-transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment.
Sickle cell disease (SCD), although a monogenic disease, exhibits a complex clinical phenotype that hampers optimum patient stratification and disease management, especially on hydroxyurea treatment. Moreover, theranostics, the combination of diagnostics to individualize and optimize therapeutic interventions, has not been firmly on the forefront of SCD research and clinical management to date. We suggest that if tailor-made theranostics in SCD is envisaged, pharmacogenomics is anticipated to be the way forward. Herein, we present the current key pharmacogenomic opportunities and challenges in SCD, considering population variation, ethics, and socioeconomic aspects. We focus on pharmacogenomics and pain management, genethics, and cost-effectiveness in SCD. We searched for and synthesized data from PubMed and Google Scholar, and the references from relevant articles, using the keywords "pharmacogenomics," "sickle cell disease," "hydroxyurea," "ethics," "pain management," "morphine metabolism," "opioids," "pharmacogenomics and chronic pain," "cost-effectiveness," and "economic evaluation." Only articles published in English were included. So far, when pharmacogenomics in SCD has been considered, interindividual variability in hydroxyurea response/toxicity has been of primary interest. We underscore the need to extend pharmacogenomic considerations on other therapeutic interventions currently present using a holistic patient-centric approach, and taking disease complications into account as well. Furthermore, we raise awareness toward socioeconomic, ethical, and population differences in the way sickle cell pharmacogenomics might unfold in the future. If pharmacogenomics in SCD is to be used in the clinic in an evidence-based manner, cost-effectiveness and population-specific empirical ethics data are urgently needed.
summary We present here our overall experience after 27 months of performance of the Procleix Ultrio [HIV‐1, hepatitis C virus (HCV), hepatitis B virus (HBV)] transcription‐mediated amplification (TMA) assay. The aim of this report is to assess the impact of nucleic acid testing (NAT) implementation in blood screening of south‐western Greek blood donors. We processed 38 264 units of blood as neat samples with the Procleix Ultrio TMA assay (Chiron/GenProbe, Emeryville/San Diego, CA, USA) between 1 January 2005 and 31 March 2007. NAT results were compared to those obtained from routine serology tests and quantitative polymerase chain reaction (PCR) assays. Overall, 52 units of blood tested positive for HBV (1·4‰), 8 for HCV (0·2‰) and none for HIV or multiple infections. The yield of TMA was 0·183‰ for HBV (7/38 264) and 0 for HCV. The TMA HBV‐positive donations were tested for HBV DNA by a quantitative PCR assay and were found negative (below the detection limit of the method, 200 copies/mL). Follow‐up testing showed that the TMA HBV‐positive donations were positive for anti‐hepatitis B core antigen immunoglobulin G antibodies. Implementation of the TMA assay in the individual donation configuration increased HBV detection compared to serological screening or a commonly used quantitative PCR assay. Follow‐up studies will determine the impact of NAT implementation in HBV transmission in countries with an intermediate HBV incidence.
The current trends in education for distance and personalized learning, calls for further investigation on the educational benefits of a Virtual Reality (VR) software, regarding laboratory science courses. In this study, we investigated the teaching effectiveness of a VR-oriented innovative method vs a more traditional pedagogical approach, by measuring the Pre-Post change, commonly called as Learning Gain (LG). An Item Response Theory model, The Rasch Model (RM), was used to assess the LG as the difference between the students’ ability before and after the educational treatment. The participants, (N= 54) graduate students enrolled in the Department of Primary Education, at the University of Patras, in Greece, were divided into two groups and followed two different scenarios to be educated on the topic of microscopy. Our findings provided evidence in favor of using simulations as a supplementary tool to the learning procedure. According to the LG analysis, the students that interacted with the VR software showed a higher change in ability, compared to their fellow students, who followed a more classic teaching methodology and obtained no LG between the Pre and Posttesting situation.
Abstract Background The aim of this study was to determine the genotype distribution and allelic frequencies of ACE (I/D), AGTR1 (A +1166 C), BDKRB2 (+9/−9) and LEP (G–2548A) genomic variations in 175 Greek athletes who excelled at a national and/or international level and 169 healthy Greek adults to identify whether some particular combinations of these loci might serve as predictive markers for superior physical condition. Results The D/D genotype of the ACE gene ( p = 0.034) combined with the simultaneous existence of BDKRB2 (+9/−9) ( p = 0.001) or LEP (G/A) ( p = 0.021) genotypes was the most prevalent among female athletes compared to female controls. A statistical trend was also observed in BDKRB2 (+9/−9) and LEP (G–2548A) heterozygous genotypes among male and female Greek athletes, and in ACE (I/D) only in male athletes. Finally, both male and female athletes showed the highest rates in the AGTR1 (A/A) genotype. Conclusions Our results suggest that the co-existence of ACE (D/D), BDKRB2 (+9/−9) or LEP (G/A) genotypes in female athletes might be correlated with a superior level of physical performance.
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