A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies

Paediatric leukaemia DNA methylation profiling using MBD enrichment and SOLiD sequencing on archival bone marrow smears

BACKGROUND: Acute Lymphoblastic Leukaemia (ALL) is the most common cancer in children. Over the past four decades, research has advanced the treatment of this cancer from a less than 60% chance of survival to over 85% today. The causal molecular mechanisms remain unclear. Here, we performed sequencing-based genomic DNA methylation profiling of eight paediatric ALL patients using archived bone marrow smear microscope slides. FINDINGS: SOLiD™ sequencing data was collected from Methyl-Binding Domain (MBD) enriched fractions of genomic DNA. The primary tumour and remission bone marrow sample was analysed from eight patients. Four patients relapsed and the relapsed tumour was analysed. Input and MBD-enriched DNA from each sample was sequenced, aligned to the hg19 reference genome and analysed for enrichment peaks using MACS (Model-based Analysis for ChIP-Seq) and HOMER (Hypergeometric Optimization of Motif EnRichment). In total, 3.67 gigabases (Gb) were sequenced, 2.74 Gb were aligned to the reference genome (average 74.66% alignment efficiency). This dataset enables the interrogation of differential DNA methylation associated with paediatric ALL. Preliminary results reveal concordant regions of enrichment indicative of a DNA methylation signature. CONCLUSION: Our dataset represents one of the first SOLiD™MBD-Seq studies performed on paediatric ALL and is the first to utilise archival bone marrow smears. Differential DNA methylation between cancer and equivalent disease-free tissue can be identified and correlated with existing and published genomic studies. Given the rarity of paediatric haematopoietic malignancies, relative to adult counterparts, our demonstration of the utility of archived bone marrow smear samples to high-throughput methylation sequencing approaches offers tremendous potential to explore the role of DNA methylation in the aetiology of cancer

DNA methylation patterns in paediatric acute lymphoblastic leukaemia

Publication included in thesis:Wong, N. C., Ashley, D., Chatterton, Z., Parkinson-Bates, M., Ng, H. K., Halemba, M. S., et al. (2012). A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia. Epigenetics, 7(6), 535-541. DOI: 10.4161/epi.20193© 2014 Dr. Zac ChattertonIntroduction: Disruption of DNA methylation is the most common molecular alteration in human cancers. Paediatric Acute Lymphoblastic Leukaemia (ALL) is the most prevalent childhood cancer and strong evidence indicates that DNA methylation alterations exist within this disease. Several genetic mutations have been described that contribute to the malignant transformation within the B-cell subtypes of ALL (B-ALL), however many of the malignant phenotypes are unexplained by genetic mutations alone. DNA methylation has the ability to alter gene expression and thus DNA methylation alterations may contribute to observed malignant phenotypes, potentially activating oncogenes or inactivating tumour suppressor genes analogous to genetic mutations. Furthermore, DNA methylation alterations represent viable clinical biomarkers for disease diagnosis, prognosis and disease tracking. At the start of this project, preliminary genome-scale DNA methylation profiling had been performed on paediatric B-ALL with appropriate B-cell controls to identify contributing DNA methylation alterations and only limited studies had investigated techniques, thresholds and assays for the clinical implementation of DNA methylation biomarkers. Materials and Methods: Two approaches were used to characterise genome-scale DNA methylation alterations in 69 paediatric B-ALL patients; the Illumina Infinium HumanMethylation BeadChip arrays HM27 and HM450. Validation of B-ALL DNA methylation alterations was conducted using the SEQUENOM MassARRAY EpiTYPER. Genome-scale analysis of gene expression (Affymetrix microarray) was also performed in 17 B-ALL cases and integrated with B-ALL methylome data. The study also developed novel techniques for the analysis of DNA methylation using MALDI-TOF Mass Spectrometry (SEQUENOM). Results: Genome-scale disruptions in DNA methylation were characterised in paediatric B-ALL, validating a number of previous small scale experiments and identifying hundreds of genes with associated DNA methylation disruption. DNA methylation alterations were found to be prevalent in all paediatric B-ALL subtypes and stable biomarkers of disease. Two highly differentially methylated sites in the gene promoters of FOXE3 and TLX3 were used as targets to establish new MALDI-TOF Mass Spectrometry techniques that could 1) analyse multiple DNA methylation regions in single reaction and 2) sensitively detect rare DNA methylation events. The techniques were applied to patient samples and enabled high sensitivity and specificity measurements for disease diagnosis. Furthermore, these techniques enabled sensitive disease tracking and insights into the detection of minimal residual disease by DNA methylation analysis. Integration of genome-scale DNA methylation and gene expression data identified common and subtype-specific epigenetic disruption in paediatric B-ALL effecting known tumour suppressors and genes implicated in apoptosis, cellular proliferation and cell signalling. Furthermore, this study uncovered prognostic DNA methylation signatures associated with B-ALL relapse, present across several B-ALL subtypes. Conclusions: The findings of this study have revealed common alterations to DNA methylation across the genomes of paediatric B-ALL that establish a mechanism for clonal inheritance of gene deregulation integral to malignant phenotype. Additionally, the study establishes targets, techniques and thresholds for clinical implementation of DNA methylation loci as biomarkers for disease diagnosis, prognosis and tracking

An Exploration of Dispositions for Success among First-Year Teachers

Universities and colleges are charged with the broad task of providing their students with the skills that will allow them to be successful in their chosen fields. The perception that teacher preparation programs hold the key to the knowledge and skills necessary to have a successful career is being unclear from the consumer’s perspective. While investing in education will always be beneficial, it does not come with any guarantees of success in the workplace. The research provides the first year teachers’ perceptions of success. The content knowledge, pedagogical development and field experiences are traditionally aligned for all pre-service teachers. Their success in the classroom is not always a given in spite of all pre-service teachers having been exposed to the edTPA process, state teacher licenses assessments, and in-service evaluation that is aligned to the Danielson framework. Neither the edTPA nor the Danielson framework evaluate disposition directly. An educator needs to be reflective in his/her practice in order to develop his/her disposition along with content knowledge and instructional strategies. This study provides insight to pre-service training, first year in-service experiences, the evaluation processes (edTPA and Danielson framework), and their dispositional perspective. Major themes pertaining to classroom management, time commitment, reflective practices, emotional connection to students, and being self-driven were explored through a dispositional lens. Dispositional characteristics are present throughout the pre-service training and become increasing prevalent in first year teachers. This phenomenological study follows a contextual framework of disconnect between teacher preparation programs and in-service realities resulting in a deeper understanding of first year teachers’ perceptions of success

Reducing the risk of false discovery enabling identification of biologically significant genome-wide methylation status using the HumanMethylation450 array

BACKGROUND: The Illumina HumanMethylation450 BeadChip (HM450K) measures the DNA methylation of 485,512 CpGs in the human genome. The technology relies on hybridization of genomic fragments to probes on the chip. However, certain genomic factors may compromise the ability to measure methylation using the array such as single nucleotide polymorphisms (SNPs), small insertions and deletions (INDELs), repetitive DNA, and regions with reduced genomic complexity. Currently, there is no clear method or pipeline for determining which of the probes on the HM450K bead array should be retained for subsequent analysis in light of these issues. RESULTS: We comprehensively assessed the effects of SNPs, INDELs, repeats and bisulfite induced reduced genomic complexity by comparing HM450K bead array results with whole genome bisulfite sequencing. We determined which CpG probes provided accurate or noisy signals. From this, we derived a set of high-quality probes that provide unadulterated measurements of DNA methylation. CONCLUSIONS: Our method significantly reduces the risk of false discoveries when using the HM450K bead array, while maximising the power of the array to detect methylation status genome-wide. Additionally, we demonstrate the utility of our method through extraction of biologically relevant epigenetic changes in prostate cancer

Hypermethylation and down-regulation of DLEU2 in paediatric acute myeloid leukaemia independent of embedded tumour suppressor miR-15a/16-1

BACKGROUND: Acute Myeloid Leukaemia (AML) is a highly heterogeneous disease. Studies in adult AML have identified epigenetic changes, specifically DNA methylation, associated with leukaemia subtype, age of onset and patient survival which highlights this heterogeneity. However, only limited DNA methylation studies have elucidated any associations in paediatric AML. METHODS: We interrogated DNA methylation on a cohort of paediatric AML FAB subtype M5 patients using the Illumina HumanMethylation450 (HM450) BeadChip, identifying a number of target genes with p 0.4 between leukaemic and matched remission (n = 20 primary leukaemic, n = 13 matched remission). Amongst those genes identified, we interrogate DLEU2 methylation using locus-specific SEQUENOM MassARRAY® EpiTYPER® and an increased validation cohort (n = 28 primary leukaemic, n = 14 matched remission, n = 17 additional non-leukaemic and cell lines). Following methylation analysis, expression studies were undertaken utilising the same patient samples for singleplex TaqMan gene and miRNA assays and relative expression comparisons. RESULTS: We identified differential DNA methylation at the DLEU2 locus, encompassing the tumour suppressor microRNA miR-15a/16-1 cluster. A number of HM450 probes spanning the DLEU2/Alt1 Transcriptional Start Site showed increased levels of methylation in leukaemia (average over all probes >60%) compared to disease-free haematopoietic cells and patient remission samples (<24%) (p < 0.001). Interestingly, DLEU2 mRNA down-regulation in leukaemic patients (p < 0.05) was independent of the embedded mature miR-15a/16-1 expression. To assess prognostic significance of DLEU2 DNA methylation, we stratified paediatric AML patients by their methylation status. A subset of patients recorded methylation values for DLEU2 akin to non-leukaemic specimens, specifically patients with sole trisomy 8 and/or chromosome 11 abnormalities. These patients also showed similar miR-15a/16-1 expression to non-leukaemic samples, and potential improved disease prognosis. CONCLUSIONS: The DLEU2 locus and embedded miRNA cluster miR-15a/16-1 is commonly deleted in adult cancers and shown to induce leukaemogenesis, however in paediatric AML we found the region to be transcriptionally repressed. In combination, our data highlights the utility of interrogating DNA methylation and microRNA in combination with underlying genetic status to provide novel insights into AML biology

RESEARCH Open Access

in paediatric acute myeloid leukaemia independent of embedded tumour suppressor miR-15a/16-

Epigenetic deregulation in pediatric acute lymphoblastic leukemia

Similar to most cancers, genome-wide DNA methylation profiles are commonly altered in pediatric acute lymphoblastic leukemia (ALL); however, recent observations highlight that a large portion of malignancy-associated DNA methylation alterations are not accompanied by related gene expression changes. By analyzing and integrating the methylome and transcriptome profiles of pediatric B-cell ALL cases and primary tissue controls, we report 325 genes hypermethylated and downregulated and 45 genes hypomethylated and upregulated in pediatric B-cell ALL, irrespective of subtype. Repressed cation channel subunits and cAMP signaling activators and transducers are overrepresented, potentially indicating a reduced cellular potential to receive and propagate apoptotic signals. Furthermore, we report specific DNA methylation alterations with concurrent gene expression changes within individual ALL subtypes. The ETV6-RUNX1 translocation was associated with downregulation of ASNS and upregulation of the EPO-receptor, while Hyperdiploid patients (&gt; 50 chr) displayed upregulation of B-cell lymphoma (BCL) members and repression of PTPRG and FHIT. In combination, these data indicate genetically distinct B-cell ALL subtypes contain cooperative epimutations and genome-wide epigenetic deregulation is common across all B-cell ALL subtypes

The importance of acoustic quality in classroom

An effective learning process in a classroom with good speech intelligibility requires good acoustic quality. Low acoustic quality may cause frequent speech repetition and consequently leads to several emotional disorders among the students. The purpose of this study is to improve the acoustic quality of the classroom for a better learning process. Two classrooms at Faculty of Civil Engineering, Universiti Teknologi Malaysia have been selected for this study. Reverberation time is the most important factor in acoustics, which was determined using theoretical calculations and simulations. A Dass-21 of self-report questionnaires was used to measure the levels of depression, anxiety, and stress among the students. The results showed that reverberation times of the classrooms were more than 1 second and 34% of the students have suffered from severe and extreme anxiety. The acoustic quality can be improved through the replacement of a painted concrete wall with high sound absorption material made of kenaf fiber in order to ensure reverberation time does not exceed the limit