Anthony G. Fenech

Mutation screening of the muscarinic M2 and M3 receptor genes in normal and asthmatic subjects

Muscarinic receptors are important in the development of airway hyperresponsiveness, and dysfunction of these receptors has been suggested to be present in asthma. The human muscarinic M2 and M3 receptor genes were screened for polymorphic variation using single‐stranded conformation polymorphism (SSCP) analysis, complemented by direct fluorescent sequencing. Forty‐six random DNA samples and 46 respiratory physician diagnosed asthmatic samples were used as a template for analysis. Within the muscarinic M2 receptor gene, we identified two degenerate single base substitutions (1197T→C, Thr→Thr and 976A→C, Arg→Arg) in one random and one asthmatic sample respectively. Analysis of the 3′ UTR region revealed an additional ‘A’ at bp 1793 (c.f. ATG). This was present in all of 49 samples analysed by sequencing or BsmI digest, suggesting that the published sequence (GenBank Accession No. M16404) is incorrect. A common 3′ UTR polymorphism (T→A) was found at bp 1696 (c.f. ATG) (allelic frequency=65%, n=60), but this does not alter transcription factor recognition sites. We were unable to identify any polymorphic variation within the muscarinic M3 coding region or the flanking regions investigated, using the methods described. The coding regions for the human muscarinic M2 and M3 receptor genes are both highly conserved. These data suggest that polymorphic variation within these coding sequences is unlikely to account for inter‐individual variability in response to methacholine or anticholinergic therapy. The potential functional significance of the muscarinic M2 receptor 3′ UTR polymorphism (bp 1696) remains to be determined.

Deregulation of the phosphatase, PP2A is a common event in breast cancer, predicting sensitivity to FTY720

BackgroundThe most commonly used biomarkers to predict the response of breast cancer patients to therapy are the oestrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Patients positive for these biomarkers are eligible for specific therapies such as endocrine treatment in the event of ER and PgR positivity, and the monoclonal antibody, trastuzumab, in the case of HER2-positive patients. Patients who are negative for these three biomarkers, the so-called triple negatives, however, derive little benefit from such therapies and are associated with a worse prognosis. Deregulation of the protein serine/threonine phosphatase type 2A (PP2A) and its regulatory subunits is a common event in breast cancer, providing a possible target for therapy.MethodsThe data portal, cBioPortal for Cancer Genomics was used to investigate the incidence of conditions that are associated with low phosphatase activity. Four (4) adherent human breast cancer cell lines, MDA-MB-468, MDA-MB-436, Hs578T and BT-20 were cultured to assess their viability when exposed to various dosages of rapamycin or FTY720. In addition, RNA was extracted and cDNA was synthesised to amplify the coding sequence of PPP2CA. Amplification was followed by high-resolution melting to identify variations.Results and conclusionThe sequence of PPP2CA was found to be conserved across a diverse panel of solid tumour and haematological cell lines, suggesting that low expression of PPP2CA and differential binding of inhibitory PPP2CA regulators are the main mechanisms of PP2A deregulation. Interestingly, the cBioPortal for Cancer Genomics shows that PP2A is deregulated in 59.6% of basal breast tumours. Viability assays performed to determine the sensitivity of a panel of breast cancer cell lines to FTY720, a PP2A activator, indicated that cell lines associated with ER loss are sensitive to lower doses of FTY720. The subset of patients with suppressed PP2A activity is potentially eligible for treatment using therapies which target the PI3K/AKT/mTOR pathway, such as phosphatase activators.

A European spectrum of pharmacogenomic biomarkers: Implications for clinical pharmacogenomics

Pharmacogenomics aims to correlate inter-individual differences of drug efficacy and/or toxicity with the underlying genetic composition, particularly in genes encoding for protein factors and enzymes involved in drug metabolism and transport. In several European populations, particularly in countries with lower income, information related to the prevalence of pharmacogenomic biomarkers is incomplete or lacking. Here, we have implemented the microattribution approach to assess the pharmacogenomic biomarkers allelic spectrum in 18 European populations, mostly from developing European countries, by analyzing 1,931 pharmacogenomics biomarkers in 231 genes. Our data show significant inter-population pharmacogenomic biomarker allele frequency differences, particularly in 7 clinically actionable pharmacogenomic biomarkers in 7 European populations, affecting drug efficacy and/or toxicity of 51 medication treatment modalities. These data also reflect on the differences observed in the prevalence of high-risk genotypes in these populations, as far as common markers in the CYP2C9, CYP2C19, CYP3A5, VKORC1, SLCO1B1 and TPMT pharmacogenes are concerned. Also, our data demonstrate notable differences in predicted genotype-based warfarin dosing among these populations. Our findings can be exploited not only to develop guidelines for medical prioritization, but most importantly to facilitate integration of pharmacogenomics and to support pre-emptive pharmacogenomic testing. This may subsequently contribute towards significant cost-savings in the overall healthcare expenditure in the participating countries, where pharmacogenomics implementation proves to be cost-effective.

Deregulation of the protein phosphatase 2A, PP2A in cancer: complexity and therapeutic options.

The complexity of the phosphatase, PP2A, is being unravelled and current research is increasingly providing information on the association of deregulated PP2A function with cancer initiation and progression. It has been reported that decreased activity of PP2A is a recurrent observation in many types of cancer, including colorectal and breast cancer (Baldacchino et al. EPMA J. 5:3, 2014; Cristobal et al. Mol Cancer Ther. 13:938–947, 2014). Since deregulation of PP2A and its regulatory subunits is a common event in cancer, PP2A is a potential target for therapy (Baldacchino et al. EPMA J. 5:3, 2014). In this review, the structural components of the PP2A complex are described, giving an in depth overview of the diversity of regulatory subunits. Regulation of the active PP2A trimeric complex, through phosphorylation and methylation, can be targeted using known compounds, to reactivate the complex. The endogenous inhibitors of the PP2A complex are highly deregulated in cancer, representing cases that are eligible to PP2A-activating drugs. Pharmacological opportunities to target low PP2A activity are available and preclinical data support the efficacy of these drugs, but clinical trials are lacking. We highlight the importance of PP2A deregulation in cancer and the current trends in targeting the phosphatase.

Anxiety and the management of asthma in an adult outpatient population

Background: Review of the literature suggests that anxiety is more common among patients with asthma than among the general population, yet it does not appear to be given the attention it deserves as part of the overall management of asthma. The aim of this study was to investigate the relationship between anxiety and asthma management, in terms of Global Initiative for Asthma steps, lung function and medication. Methods: A total of 201 consecutive patients with respiratory physician-diagnosed asthma were recruited from an adult outpatient asthma clinic. Participants underwent a sociodemographic review, and a medical interview which included a detailed drug history. Forced expiratory volume in 1 second (FEV1) and peak expiratory flow (PEF) values were recorded using a Micro Medical® portable spirometer. The level of anxiety was assessed using the Beck Anxiety Inventory (BAI). Results: A total of 51.5% of participants registered clinically significant levels of anxiety. Of these only 21% had already been diagnosed and were receiving treatment. Females reported significantly higher BAI scores than males (p < 0.01). More females (66.3%) registered clinically significant levels of anxiety as compared with males (33.7%) (p < 0.05). There was a positive correlation between the BAI score and the prescribed dose of inhaled glucocorticoids (r s = 0.150, p < 0.05) and between anxiety and GINA treatment step (r s = 0.139, p < 0.05). There was also a positive correlation between anxiety and the number of medicines taken by patients (r s = 0.259, p < 0.001). Conclusions: Physicians treating patients with asthma should be sensitised to the association between asthma and anxiety, and should also consider assessing patients for the possibility of anxiety disorders as part of asthma management plans.

The effect of turmeric (Curcumin) supplementation on cytokine and inflammatory marker responses following 2 hours of endurance cycling

BackgroundEndurance exercise induces IL-6 production from myocytes that is thought to impair intracellular defence mechanisms. Curcumin inhibits NF-κB and activator protein 1, responsible for cytokine transcription, in cell lines. The aim of this study was to investigate the effect of curcumin supplementation on the cytokine and stress responses following 2 h of cycling.MethodsEleven male recreational athletes (35.5 ± 5.7 years; Wmax 275 ± 6 W; 87.2 ± 10.3 kg) consuming a low carbohydrate diet of 2.3 ± 0.2 g/kg/day underwent three double blind trials with curcumin supplementation, placebo supplementation, and no supplementation (control) to observe the response of serum interleukins (IL-6, IL1-RA, IL-10), cortisol, c-reactive protein (CRP), and subjective assessment of training stress. Exercise was set at 95% lactate threshold (54 ± 7% Wmax) to ensure that all athletes completed the trial protocol.ResultsThe trial protocol elicted a rise in IL-6 and IL1-RA, but not IL-10. The supplementation regimen failed to produce statistically significant results when compared to placebo and control. IL-6 serum concentrations one hour following exercise were (Median (IQR): 2.0 (1.8-3.6) Curcumin; 4.8 (2.1-7.3) Placebo; 3.5 (1.9-7.7) Control). Differences between supplementation and placebo failed to reach statistical significance (p = 0.18) with the median test. Repeated measures ANOVA time-trial interaction was at p = 0.06 between curcumin supplementation and placebo. A positive correlation (p = 0.02) between absolute exercise intensity and 1 h post-exercise for IL-6 concentration was observed. Participants reported “better than usual” scores in the subjective assessment of psychological stress when supplementing with curcumin, indicating that they felt less stressed during training days (p = 0.04) compared to placebo even though there was no difference in RPE during any of the training days or trials.ConclusionThe limitations of the current regimen and trial involved a number of factors including sample size, mode of exercise, intensity of exercise, and dose of curcumin. Nevertheless these results provide insight for future studies with larger samples, and multiple curcumin dosages to investigate if different curcumin regimens can lead to statistically different interleukin levels when compared to a control and placebo.

Knowledge and sources of information about medicines among adolescents in Malta

Objective To investigate knowledge of medicines and sources of information about medicines among adolescents in Malta. Methods A self-administered questionnaire was used to survey knowledge of medicines and information sources among adolescents attending secondary schools in Malta. A random stratified sample design generated a sample size of 514 students. Knowledge of medicines was investigated by questions that included topics about the efficacy of medicines, proper communication during a medical consultation, the safety of medicines, antibiotic use and pictograms. Results The analytical sample, of which 53.8% were girls, was made up of 474 students, aged 1416 years. The students obtained a mean score of 22.92 points (SD = 4.31) out of a maximum of 32 points for medicine knowledge. More than 30.0% of the respondents did not mark the correct answer for 40.6% of the questions that investigated knowledge of medicines. The family physician, community pharmacist and parents were the sources of information that were mostly cited. A proportion of 4.4% had obtained information from the teacher. A quarter of the respondents (24.7%) had obtained information from friends/schoolmates, young relatives or the media, generally television (17.3%). Conclusion Although the level of knowledge about medicines among this study sample of Maltese students was good, there appeared to be particular misconceptions with regard to important aspects associated with the proper use of medicines. These findings highlight the need to educate adolescents about the proper use of medicines. The most important information providers about medicines appeared to be the physician, pharmacist and parents. It is being suggested that education campaigns should be organized in order to help parents guide adolescents on how to use medicines appropriately. In addition, adolescents would benefit if more information about the proper use of medicines would be disseminated by means of television programs and school-based health education curricula.

Health complaints and use of medicines among adolescents in Malta

Objective To investigate self-reported health complaints and the use of medicines among adolescents in Malta. Methods A self-administered questionnaire was used to survey self-reported health complaints, the use and the sources of medicines that had been accessed, during the preceding 3 months among adolescents attending secondary schools in Malta. A stratified random sample design generated a sample size of 514 students. The health complaints and use of medicines that were investigated included ear problems/hay fever/cold/cough, headache, skin problems, sport injuries, indigestion/diarrhoea/constipation, eye problems and menstrual pain (for girls). The use of vitamins and antibiotics was also investigated. Results A total of 477 students participated in the final data collection. Correct information was submitted by 474 students, (aged 14-16 years), who formed the analytical sample, of which 53.8% were girls. The students reported a mean number of 2.70 (SD = 1.39) out of a total of 7 health complaints and 90.3% reported using at least 1 medicine during the preceding 3 months. The community pharmacy was cited as the most commonly accessed source for most of the medicines that were investigated. A proportion of 24.3% of the students had taken at least 1 medicine without adult guidance during the preceding 3 months. Almost 10% of those who had taken antibiotics, had accessed them from the home medicine cabinet. Conclusion A high proportion of adolescents in Malta reported the use of medicines to alleviate the symptoms of common health complaints. This result is concordant with previous research carried out in the United Kingdom, Germany, Slovakia and Kuwait. A considerable proportion of students in this study had obtained medicines without adult guidance and accessed antibiotics from the home medicine cabinet. This highlights the importance of carefully designed education programs for adolescents that will integrate information about the proper use of medicines.

Functional Polymorphism and Differential Regulation of CYSLTR1 Transcription in Human Airway Smooth Muscle and Monocytes

Cysteinyl leukotrienes play an important role in the pathophysiology of many inflammatory disorders, including asthma. The aim of this study was to characterize the mechanisms underlying transcriptional regulation of the human cysteinyl leukotriene receptor 1 (hCYSLTR1) gene. 5′RACE was performed on human airway smooth muscle (HASM) and peripheral blood mononuclear cells. A 1128-bp region of the hCYSLTR1 main putative promoter was screened for polymorphisms by sequencing of 48 individuals. Luciferase reporter gene assays were performed using fragments of the core promoter (232 bp to 1128 bp) in HASM and THP1 cells. Three hCYSLTR1 transcripts were found, one representing 90% of all messenger RNA identified. The genomic location of the transcription start sites suggested there are two putative hCYSLTR1 promoters. The majority of the transcriptional activity of the main putative promoter was detected between −232 and −679 bp. Four single-nucleotide polymorphisms in strong linkage disequilibrium were found in the region studied: −561 (rs7066737), −642 (rs2806489), −781 (rs2637204), and −940 (rs321029), with three haplotypes observed. In THP1 cells, the G allele (−642) caused a twofold decrease in luciferase expression compared to the A allele. These data suggest that the majority of hCYSLTR1 transcripts in HASM and monocytes arise from a single promoter located immediately upstream of the 5′ untranslated region, although rarer transcripts can also occur. This study also raises the possibility that cell-type-dependent differences in transcriptional activity caused by the presence of specific haplotypes within the main CYSLTR1 promoter may be a predictor of disease risk or treatment response.

Conference Scene: Golden Helix Pharmacogenomics Days: educational activities on pharmacogenomics and personalized medicine

The Golden Helix Pharmacogenomics Days are high-profile international educational scientific meetings discussing pharmacogenomics and personalized medicine. Here, we provide an overview of the scientific lectures and the topics discussed during the 4th Golden Helix Pharmacogenomics Day, held in Cagliari, Italy, on 7 October 2011, and the 5th Golden Helix Pharmacogenomics Day, that was held in Msida, Malta, on 3 December 2011. The scientific programs of both events included scientific and company lectures on pharmacogenomics, bioinformatics and personalized medicine by local and international speakers from Europe and the USA.