A Novel Method to Identify Autoantibodies against Putative Target Proteins in Serum from beta-Thalassemia Major: A Pilot Study.

Abnormal immune reactivity in patients with beta-thalassemia (beta-thal) major can be associated with poor prognosis. Immunome protein-array analysis represents a powerful approach to identify novel biomarkers. The Sengenics Immunome Protein Array platform was used for high-throughput quantification of autoantibodies in 12 serum samples collected from nine beta-thal major patients and three non-thalassemia controls, which were run together with two pooled normal sera (Sengenics Internal QC samples). To obtain more accurate and reliable results, the evaluation of the biological relevance of the shortlisted biomarkers was analyzed using an Open Target Platform online database. Elevated autoantibodies directed against 23 autoantigens on the immunome array were identified and analyzed using a penetrance fold change-based bioinformatics method. Understanding the autoantibody profile of beta-thal major patients would help to further understand the pathogenesis of the disease. The identified autoantigens may serve as potential biomarkers for the prognosis of beta-thal major.

Genetic variations in influx transporter gene SLC22A1 are associated with clinical responses to imatinib mesylate among Malaysian chronic myeloid leukaemia patients.

Imatinib mesylate (IM), a well-established gold standard drug in the treatment of chronic myeloid leukaemia (CML), is a synthetic tyrosine kinase inhibitor. Despite excellent efficacy, a significant number of patients on IM therapy develop resistance to IM. Currently, great focus has been laid on the effect of interindividual pharmacogenetic variability on IM treatment responses. IM uptake is mediated by the hOCT1 protein encoded by the solute carrier 22 gene (SLC22A1). The current study investigated the impact of few single-nucleotide polymorphisms (SNPs) of SLC22A1 on mediating resistance and/or good response to IM among 278 Malaysian CML patients (146 IM-resistant group and 132 IM good response group) undergoing IM therapy on 400 mg daily. Our results showed that the allelic frequencies of heterozygous (CG) and homozygous variant (GG) genotypes of SLC22A1 C480G were significantly higher in the IM-resistant group compared with the IM good response group (41.8% versus 30.3% and 10.9% versus 4.5% with P values of 0.047 and 0.048, respectively). On evaluating the association of genotypes with risk of IM resistance development, heterozygous (CG) and homozygous (GG) variant genotypes showed significantly higher risk for developing resistance to IM treatment with odds ratio (OR): 1.901 (95% confidence interval (CI): 1.142-3.163, P = 0.013) and 3.324 (95% CI: 1.235-8.947, P = 0.017), respectively. Two SNPs and two insertions/deletions were detected in exon 7 of SLC22A1. For exon 7, 1222AA carriers together with the presence of both the 8-bp insertion and 3-bp deletion, and M420del alleles showed higher possibility of developing resistance towards IMtreatment. Our results warrant the need of genotyping this SNP in terms of modulating IM treatment in CML patients.

Aberrant DNA Methylation of SOCS1 Gene is Not Associated with Resistance to Imatinib Mesylate among Chronic Myeloid Leukemia Patients.

BACKGROUND: In exploring the cause of Imatinib Mesylate (IM) resistance among Chronic Myeloid Leukemia (CML) patients who do not harbor BCR-ABL dependent mechanism, BCR-ABL independent pathways are the most probable pathways that should be explored. In BCR-ABL independent pathway, SOCS1 plays an important role as it helps in regulating optimal JAK/STAT activity. OBJECTIVE: To identify the association of SOCS1 gene hypermethylation in mediating IM Resistance. METHOD: The SOCS1 promoter methylation level of 92 BCR-ABL non mutated IM resistant CML patients, 83 IM good response CML patients and 5 normal samples from healthy individuals were measured using Methylation Specific-High Resolution Melt (MS-HRM) analysis. RESULTS: Both primers used to amplify promoter region from -333 to -223 and from -332 to -188 showed less than 10% methylation in all CML and normal samples. Consequently, there was no significant difference in SOCS1 promoter methylation level between IM resistant and IM good response patients. CONCLUSION: SOCS1 promoter methylation level is not suitable to be used as one of the biomarkers for predicting the possibility of acquiring resistance among CML patients treated with IM.

Pharmacogenetics and the treatment of chronic myeloid leukemia: how relevant clinically? An update.

Despite the excellent efficacy and improved clinical responses obtained with imatinib mesylate (IM), development of resistance in a significant proportion of chronic myeloid leukemia (CML) patients on IM therapy have emerged as a challenging problem in clinical practice. Resistance to imatinib can be due to heterogeneous array of factors involving BCR/ABL-dependent and BCR/ABL-independent pathways. Although BCR/ABL mutation is the major contributory factor for IM resistance, reduced bio-availability of IM in leukemic cells is also an important pharmacokinetic factor that contributes to development of resistance to IM in CML patients. The contribution of polymorphisms of the pharmacogenes in relation to IM disposition and treatment outcomes have been studied by various research groups in numerous population cohorts. However, the conclusions arising from these studies have been highly inconsistent. This review encompasses an updated insight into the impact of pharmacogenetic variability on treatment response of IM in CML patients.

Aberrant DNA methylation at HOXA4 and HOXA5 genes are associated with resistance to imatinib mesylate among chronic myeloid leukemia patients.

BACKGROUND: Imatinib mesylate is a molecularly targeted tyrosine kinase inhibitor drug. It is effectively used in the treatment of chronic myeloid leukemia (CML) patients. However, development of resistance to imatinib mesylate as a result of BCR-ABL dependent and BCR-ABL independent mechanisms has emerged as a daunting problem in the management of CML patients. Between these mechanisms, BCR-ABL independent mechanisms are still not robustly understood. AIM: To investigate the correlation of HOXA4 and HOXA5 promoter DNA hypermethylation with imatinib resistance among CML patients. METHODS AND RESULTS: Samples from 175 Philadelphia positive CML patients (83 good response and 92 BCR-ABL non-mutated imatinib resistant patients) were subjected to Methylation Specific High Resolution Melt Analysis for methylation levels quantification of the HOXA4 and HOXA5 promoter regions. Receiver operating characteristic curve analysis was done to elucidate the optimal methylation cut-off point followed by multiple logistic regression analysis. Log-Rank analysis was done to measure the overall survival difference between CML groups. The optimal methylation cut-off point was found to be at 62.5% for both HOXA4 and HOXA5. Chronic myeloid leukemia patients with ≥63% HOXA4 and HOXA5 methylation level were shown to have 3.78 and 3.95 times the odds, respectively, to acquire resistance to imatinib. However, overall survival of CML patients that have ≤62% and ≥ 63% methylation levels of HOXA4 and HOXA5 genes were found to be not significant (P-value = 0.126 for HOXA4; P-value = 0.217 for HOXA5). CONCLUSION: Hypermethylation of the HOXA4 and HOXA5 promoter is correlated with imatinib resistance and with further investigation, it could be a potential epigenetic biomarker in supplement to the BCR-ABL gene mutation in predicting imatinib treatment response among CML patients but could not be considered as a prognostic marker.

Association of GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms with clinical response to imatinib mesylate treatment among Malaysian chronic myeloid leukaemia patients.

The detoxifying activity of glutathione S-transferases (GST) enzymes not only protect cells from the adverse effects of xenobiotics, but also alters the effectiveness of drugs in cancer cells, resulting in toxicity or drug resistance. In this study, we aimed to evaluate the association of GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms with treatment response among Malaysian chronic myeloid leukaemia (CML) patients who everyday undergo 400 mg of imatinib mesylate (IM) therapy. Multiplex polymerase chain reaction (multiplex-PCR) was performed to detect GSTM1 and GSTT1 polymorphisms simultaneously and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was conducted to detect the GSTP1 Ile195Val polymorphism. On evaluating the association of the variant genotype with treatment outcome, heterozygous variant (AG) and homozygous variant (GG) of GSTP1 Ile105Val showed significantly a higher risk for the development of resistance to IM with OR: 1.951 (95% CI: 1.186-3.209, P = 0.009) and OR: 3.540 (95% CI: 1.305-9.606, P = 0.013), respectively. Likewise, GSTT1 null genotype was also associated with a significantly higher risk for the development of resistance to IM with OR = 1.664 (95% CI: 1.011-2.739, P = 0.045). Our results indicate the potential usefulness of GST polymorphism genotyping in predicting the IM treatment response among CML patients.

Review: Beta-thalassemia and molecular chaperones.

Thalassemia is known as a diverse single gene disorder, which is prevalent worldwide. The molecular chaperones are set of proteins that help in two important processes while protein synthesis and degradation include folding or unfolding and assembly or disassembly, thereby helping in cell homeostasis. This review recaps current knowledge regarding the role of molecular chaperones in thalassemia, with a focus on beta thalassemia.

BCR-ABL kinase domain mutations, including 2 novel mutations in imatinib resistant Malaysian chronic myeloid leukemia patients-Frequency and clinical outcome.

Discovery of imatinib mesylate (IM) as the targeted BCR-ABL protein tyrosine kinase inhibitor (TKI) has resulted in its use as the frontline therapy for chronic myeloid leukemia (CML) across the world. Although high response rates are observed in CML patients who receive IM treatment, a significant number of patients develop resistance to IM. Resistance to IM in patients has been associated with a heterogeneous array of mechanisms of which point mutations within the ABL tyrosine kinase domain (TKD) are the frequently documented. The types and frequencies of mutations reported in different population studies have shown wide variability. We screened 125 Malaysian CML patients on IM therapy who showed either TKI refractory or resistance to IM to investigate the frequency and pattern of BCR-ABL kinase domain mutations among Malaysian CML patients undergoing IM therapy and to determine the clinical significance. Mutational screening using denaturing high performance liquid chromatography (dHPLC) followed by DNA sequencing was performed on 125 IM resistant Malaysian CML patients. Mutations were detected in 28 patients (22.4%). Fifteen different types of mutations (T315I, E255K, G250E, M351T, F359C, G251E, Y253H, V289F, E355G, N368S, L387M, H369R, A397P, E355A, D276G), including 2 novel mutations were identified, with T315I as the predominant type of mutation. The data generated from clinical and molecular parameters studied were correlated with the survival of CML patients. Patients with Y253H, M351T and E355G TKD mutations showed poorer prognosis compared to those without mutation. Interestingly, when the prognostic impact of the observed mutations was compared inter-individually, E355G and Y253H mutations were associated with more adverse prognosis and shorter survival (P=0.025 and 0.005 respectively) than T315I mutation. Results suggest that apart from those mutations occurring in the three crucial regions (catalytic domain, P-loop and activation-loop), other rare mutations also may have high impact in the development of resistance and adverse prognosis. Presence of mutations in different regions of BCR-ABL TKD leads to different levels of resistance and early detection of emerging mutant clones may help in decision making for alternative treatment. Serial monitoring of BCR-ABL1 transcripts in CML patients allows appropriate selection of CML patients for BCR-ABL1 KD mutation analysis associated with acquired TKI resistance. Identification of these KD mutations is essential in order to direct alternative treatments in such CML patients.

Prevalence and associated factors of stress, anxiety and depression among prospective medical students.

Many studies have reported that the prevalence of psychological distress among medical students during medical training was high. However, there are very few studies exploring on the psychological health of prospective medical students. This study aimed to determine the prevalence and associated factors for stress, anxiety and depression symptoms among the prospective medical students. A cross-sectional study was done on two cohorts of applicants to a public medical school. A total of 839 applicants were invited to participate in the study. The 21-item Depression Anxiety Stress Scale was administered to the applicants after they completed interviews. A total of 743 (92.2%) applicants took part in the study. The prevalence of moderate to extremely severe level of stress, anxiety and depression were 3.6%, 54.5% and 1.9%, respectively. Stress was significantly associated with extra-curricular activity (p<0.001) and race (p<0.001). Anxiety was associated with extra-curricular activity (p<0.001), race (p<0.001), mother education level (p=0.002) and CGPA group (p=0.034). Depression was associated with academic performance in class (p<0.001) and race (p=0.004). Prevalence of stress and depression among entering medical students was low; however prevalence of anxiety was high which could be due to worry about the interviews to enter medical course. The associated factors of psychological distress among prospective medical students were related to academic, non-academic, parent education and cultural backgrounds.

HOXA4 gene promoter hypermethylation as an epigenetic mechanism mediating resistance to imatinib mesylate in chronic myeloid leukemia patients.

Development of resistance to imatinib mesylate (IM) in chronic myeloid leukemia (CML) patients has emerged as a significant clinical problem. The observation that increased epigenetic silencing of potential tumor suppressor genes correlates with disease progression in some CML patients treated with IM suggests a relationship between epigenetic silencing and resistance development. We hypothesize that promoter hypermethylation of HOXA4 could be an epigenetic mechanism mediating IM resistance in CML patients. Thus a study was undertaken to investigate the promoter hypermethylation status of HOXA4 in CML patients on IM treatment and to determine its role in mediating resistance to IM. Genomic DNA was extracted from peripheral blood samples of 95 CML patients (38 good responders and 57 resistant) and 12 normal controls. All samples were bisulfite treated and analysed by methylation-specific high-resolution melt analysis. Compared to the good responders, the HOXA4 hypermethylation level was significantly higher (P = 0.002) in IM-resistant CML patients. On comparing the risk, HOXA4 hypermethylation was associated with a higher risk for IM resistance (OR 4.658; 95% CI, 1.673-12.971; P = 0.003). Thus, it is reasonable to suggest that promoter hypermethylation of HOXA4 gene could be an epigenetic mechanism mediating IM resistance in CML patients.

The impact of medical education on psychological health of students: a cohort study.

Many studies have shown that the prevalence of psychological distress among medical students during medical training is higher than that in general population. A few studies have shown that the prevalence of psychological distress among medical students before the onset of medical training was similar to general population. This study aimed to investigate psychological health of medical students before and during medical training. A one-year prospective study was done on successful applicants who undergo the first year of medical training for 2010/2011 academic session. The stress, anxiety and depression were measured by the DASS-21 at five intervals; during interview (Time 0), two months (Time 1), four months (Time 2), six months (Time 3) and final examination (Time 4) of the first year medical training. The prevalence of unfavourable stress, anxiety and depression before the onset of medical training was 4.1%, 55.6% and 1.8%, respectively. The prevalence of unfavourable stress during medical training ranged between 11.8% and 19.9%. The prevalence of anxiety during medical training ranged between 41.1% and 56.7%. The prevalence of depression during medical training ranged between 12% and 30%. Mean scores of stress and depression before (Time 0) and during medical training (Time 1-4) were significantly different (p < 0.001). The prevalence and level of unfavourable stress and depression during medical training were significantly higher than before the onset medical training. This study supports views that medical training is not an optimal environment to psychological health of medical students.

A Study of Psychological Distress in Two Cohorts of First-Year Medical Students that Underwent Different Admission Selection Processes.

BACKGROUND: Medical training is often regarded as a stressful period. Studies have previously found that 21.6%-50% of medical students experience significant psychological distress. The present study compared the prevalence and levels of psychological distress between 2 cohorts of first-year medical students that underwent different admission selection processes. METHODS: A comparative cross-sectional study was conducted by comparing 2 cohorts of first-year medical students; 1 group (cohort 1) was selected based purely on academic merit (2008/2009 cohort) and the other group (cohort 2) was selected based on academic merit, psychometric assessment, and interview performance (2009/2010 cohort). Their distress levels were measured by the General Health Questionnaire, and scores higher than 3 were considered indicative of significant psychological distress. RESULTS: The prevalence (P = 0.003) and levels (P = 0.001) of psychological distress were significantly different between the 2 cohorts. Cohort 1 had 1.2-3.3 times higher risk of developing psychological distress compared to cohort 2 (P = 0.007). CONCLUSION: Cohort 2 had better psychological health than cohort 1 and was less likely to develop psychological distress. This study provided evidence of a potential benefit of multimodal student selection based on academic merit, psychometric assessment, and interview performance. This selection process might identify medical students who will maintain better psychological health.

Contribution of BCR-ABL kinase domain mutations to imatinib mesylate resistance in Philadelphia chromosome positive Malaysian chronic myeloid leukemia patients.

Development of resistance to imatinib mesylate (IM) in chronic myeloid leukemia (CML) patients is mediated by different mechanisms that can be classified as BCR-ABL dependent or BCR-ABL independent pathways. BCR-ABL dependent mechanisms are most frequently associated with point mutations in tyrosine kinase domain (TKD) of BCR-ABL1 and also with BCR-ABL gene amplification. Many different types and frequencies of mutations have been reported in different studies, probably due to the different composition of study cohorts. Since no reports are available from Malaysia, this study was undertaken to investigate the frequency and pattern of BCR-ABL kinase domain mutations using dHPLC followed by sequencing, and also status of BCR-ABL gene amplification using fluorescence in situ hybridization (FISH) on 40 IM resistant Malaysian CML patients. Mutations were detected in 13 patients (32.5%). Five different types of mutations (T315I, E255K, Y253H, M351T, V289F) were identified in these patients. In the remaining 27 IM resistant CML patients, we investigated the contribution made by BCR-ABL gene amplification, but none of these patients showed amplification. It is presumed that the mechanisms of resistance in these 27 patients might be due to BCR-ABL independent pathways. Different mutations confer different levels of resistance and, therefore, detection and characterization of TKD mutations is highly important in order to guide therapy in CML patients.

A comparison between infrared tympanic thermometry, oral and axilla with rectal thermometry in neutropenic adults.

BACKGROUND: This study assessed the agreement between infrared tympanic membrane (TM), axillary, corrected axillary (+0.5 degrees C), oral, and corrected oral (+0.3 degrees C) to rectal thermometry as reference standard in neutropenic adults. The sensitivity and specificity of the mentioned thermometries in detecting rectal fever (> or =38 degrees C) were also analysed. METHOD: This is a comparative diagnostic test study. A total of 400 sets of blinded simultaneous temperature readings were measured from 21 haemato-oncology in-patients with neutropenia following chemotherapy. Three-hundred sets were then randomly sampled. Agreements were analysed using random two-way intraclass correlation (ICC). Sensitivity and specificity were analysed using contingency 2x2 table. FINDINGS: Both right and left TM thermometry have good correlation with rectal thermometry; 0.810 (95% CI, 0.748-0.855) and 0.770 (95% CI, 0.713-0.815) respectively. Axilla thermometry has weak agreement (ICC 0.486 (95% CI, 0.118-0.689)) with rectal thermometry. The sensitivity (sn) and specificity (sp) in detecting rectal fever (> or =38 degrees C) were: right TM (sn) 0.712 (95% CI, 0.586-0.814), (sp) 0.957 (95% CI, 0.920-0.978); oral (sn) 0.561 (95% CI, 0.433-0.681), (sp) 0.983 (95% CI, 0.954-0.995); and axilla (sn) 0.348 (95% CI, 0.238-0.477), (sp) 0.996 (95% CI, 0.973-0.999). INTERPRETATION: Single tympanic membrane thermometry is in good agreement with rectal thermometry. It is more sensitive than oral or axillary thermometry in detecting rectal fever.

The absence of factor V Leiden mutation in Malays with recurrent spontaneous abortions.

OBJECTIVES: The objectives of this study were to investigate the prevalence of factor V Leiden mutation in Malay women with recurrent spontaneous abortion and to clarify the contribution of the factor V Leiden mutation to recurrent miscarriages in these women. DESIGN: A prospective case control study between June 1999 and April 2000. SETTING: Hospital University Science of Malaysia, Kubang Kerian, Kelantan, and Maternal and Child Health Clinic, Pasir Mas, Kelantan, Malaysia. SAMPLES: A total of 46 Malay women with a history of three or more first or second trimester miscarriages were studied. The control group consisted of 46 parous women without obstetric complications. METHODS: Diagnosis of factor V Leiden mutation was made by examination of factor V Leiden allele product following Mnl I digestion of factor V Leiden alleles amplified by polymerase chain reaction. RESULTS: None of the 46 women with recurrent spontaneous abortion carried the mutation. Also, we found no subject carrying the factor V Leiden alleles in the control group. CONCLUSION: These results suggest that that there is no association between the factor V Leiden mutation and recurrent spontaneous abortion in the Malay population.