Publications by authors named "David I Rodenhiser"

28 Publications

  • Page 1 of 1

Evaluation of DNA Methylation Episignatures for Diagnosis and Phenotype Correlations in 42 Mendelian Neurodevelopmental Disorders.

Am J Hum Genet 2020 03 27;106(3):356-370. Epub 2020 Feb 27.

Université de Paris, Epigénétique et Destin Cellulaire, CNRS, 75013 Paris, France.

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called "episignatures"). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders.
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http://dx.doi.org/10.1016/j.ajhg.2020.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058829PMC
March 2020

Diagnostic Utility of Genome-wide DNA Methylation Testing in Genetically Unsolved Individuals with Suspected Hereditary Conditions.

Am J Hum Genet 2019 04 28;104(4):685-700. Epub 2019 Mar 28.

Department of Pathology and Laboratory Medicine, Western University, London, ON N6A 3K7, Canada; Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, London, ON N6A 5W9, Canada. Electronic address:

Conventional genetic testing of individuals with neurodevelopmental presentations and congenital anomalies (ND/CAs), i.e., the analysis of sequence and copy number variants, leaves a substantial proportion of them unexplained. Some of these cases have been shown to result from DNA methylation defects at a single locus (epi-variants), while others can exhibit syndrome-specific DNA methylation changes across multiple loci (epi-signatures). Here, we investigate the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved ND/CAs. We generate a computational model enabling concurrent detection of 14 syndromes using DNA methylation data with full accuracy. We demonstrate the ability of this model in resolving 67 individuals with uncertain clinical diagnoses, some of whom had variants of unknown clinical significance (VUS) in the related genes. We show that the provisional diagnoses can be ruled out in many of the case subjects, some of whom are shown by our model to have other diseases initially not considered. By applying this model to a cohort of 965 ND/CA-affected subjects without a previous diagnostic assumption and a separate assessment of rare epi-variants in this cohort, we identify 15 case subjects with syndromic Mendelian disorders, 12 case subjects with imprinting and trinucleotide repeat expansion disorders, as well as 106 case subjects with rare epi-variants, a portion of which involved genes clinically or functionally linked to the subjects' phenotypes. This study demonstrates that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of epigenomic testing in the routine clinical assessment of ND/CAs.
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http://dx.doi.org/10.1016/j.ajhg.2019.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451739PMC
April 2019

BAFopathies' DNA methylation epi-signatures demonstrate diagnostic utility and functional continuum of Coffin-Siris and Nicolaides-Baraitser syndromes.

Nat Commun 2018 11 20;9(1):4885. Epub 2018 Nov 20.

Department of Pathology and Laboratory Medicine, Western University, London, N6A 5W9, ON, Canada.

Coffin-Siris and Nicolaides-Baraitser syndromes (CSS and NCBRS) are Mendelian disorders caused by mutations in subunits of the BAF chromatin remodeling complex. We report overlapping peripheral blood DNA methylation epi-signatures in individuals with various subtypes of CSS (ARID1B, SMARCB1, and SMARCA4) and NCBRS (SMARCA2). We demonstrate that the degree of similarity in the epi-signatures of some CSS subtypes and NCBRS can be greater than that within CSS, indicating a link in the functional basis of the two syndromes. We show that chromosome 6q25 microdeletion syndrome, harboring ARID1B deletions, exhibits a similar CSS/NCBRS methylation profile. Specificity of this epi-signature was confirmed across a wide range of neurodevelopmental conditions including other chromatin remodeling and epigenetic machinery disorders. We demonstrate that a machine-learning model trained on this DNA methylation profile can resolve ambiguous clinical cases, reclassify those with variants of unknown significance, and identify previously undiagnosed subjects through targeted population screening.
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http://dx.doi.org/10.1038/s41467-018-07193-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244416PMC
November 2018

Genomic DNA Methylation-Derived Algorithm Enables Accurate Detection of Malignant Prostate Tissues.

Front Oncol 2018 23;8:100. Epub 2018 Apr 23.

Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada.

Introduction: The current methodology involving diagnosis of prostate cancer (PCa) relies on the pathology examination of prostate needle biopsies, a method with high false negative rates partly due to temporospatial, molecular, and morphological heterogeneity of prostate adenocarcinoma. It is postulated that molecular markers have a potential to assign diagnosis to a considerable portion of undetected prostate tumors. This study examines the genome-wide DNA methylation changes in PCa in search of genomic markers for the development of a diagnostic algorithm for PCa screening.

Methods: Archival PCa and normal tissues were assessed using genomic DNA methylation arrays. Differentially methylated sites and regions (DMRs) were used for functional assessment, gene-set enrichment and protein interaction analyses, and examination of transcription factor-binding patterns. Raw signal intensity data were used for identification of recurrent copy number variations (CNVs). Non-redundant fully differentiating cytosine-phosphate-guanine sites (CpGs), which did not overlap CNV segments, were used in an L1 regularized logistic regression model (LASSO) to train a classification algorithm. Validation of this algorithm was performed using a large external cohort of benign and tumor prostate arrays.

Results: Approximately 6,000 probes and 600 genomic regions showed significant DNA methylation changes, primarily involving hypermethylation. Gene-set enrichment and protein interaction analyses found an overrepresentation of genes related to cell communications, neurogenesis, and proliferation. Motif enrichment analysis demonstrated enrichment of tumor suppressor-binding sites nearby DMRs. Several of these regions were also found to contain copy number amplifications. Using four non-redundant fully differentiating CpGs, we trained a classification model with 100% accuracy in discriminating tumors from benign samples. Validation of this algorithm using an external cohort of 234 tumors and 92 benign samples yielded 96% sensitivity and 98% specificity. The model was found to be highly sensitive to detect metastatic lesions in bone, lymph node, and soft tissue, while being specific enough to differentiate the benign hyperplasia of prostate from tumor.

Conclusion: A considerable component of PCa DNA methylation profile represent driver events potentially established/maintained by disruption of tumor suppressor activity. As few as four CpGs from this profile can be used for screening of PCa.
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http://dx.doi.org/10.3389/fonc.2018.00100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925605PMC
April 2018

Peripheral blood epi-signature of Claes-Jensen syndrome enables sensitive and specific identification of patients and healthy carriers with pathogenic mutations in .

Clin Epigenetics 2018 14;10:21. Epub 2018 Feb 14.

1Department of Pathology and Laboratory Medicine, Western University, London, Ontario Canada.

Background: Claes-Jensen syndrome is an X-linked inherited intellectual disability caused by mutations in the gene. Kdm5c is a histone lysine demethylase involved in histone modifications and chromatin remodeling. Males with hemizygous mutations in present with intellectual disability and facial dysmorphism, while most heterozygous female carriers are asymptomatic. We hypothesized that loss of Kdm5c function may influence other components of the epigenomic machinery including DNA methylation in affected patients.

Results: Genome-wide DNA methylation analysis of 7 male patients affected with Claes-Jensen syndrome and 56 age- and sex-matched controls identified a specific DNA methylation defect (epi-signature) in the peripheral blood of these patients, including 1769 individual CpGs and 9 genomic regions. Six healthy female carriers showed less pronounced but distinctive changes in the same regions enabling their differentiation from both patients and controls. Highly specific computational model using the most significant methylation changes demonstrated 100% accuracy in differentiating patients, carriers, and controls in the training cohort, which was confirmed on a separate cohort of patients and carriers. The 100% specificity of this unique epi-signature was further confirmed on additional 500 unaffected controls and 600 patients with intellectual disability and developmental delay, including other patient cohorts with previously described epi-signatures.

Conclusion: Peripheral blood epi-signature in Claes-Jensen syndrome can be used for molecular diagnosis and carrier identification and assist with interpretation of genetic variants of unknown clinical significance in the gene.
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http://dx.doi.org/10.1186/s13148-018-0453-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813334PMC
February 2019

Genomic DNA Methylation Signatures Enable Concurrent Diagnosis and Clinical Genetic Variant Classification in Neurodevelopmental Syndromes.

Am J Hum Genet 2018 01;102(1):156-174

Department of Pathology and Laboratory Medicine, Western University, London, ON N6A5C1, Canada; Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, London, ON N6A5W9, Canada. Electronic address:

Pediatric developmental syndromes present with systemic, complex, and often overlapping clinical features that are not infrequently a consequence of Mendelian inheritance of mutations in genes involved in DNA methylation, establishment of histone modifications, and chromatin remodeling (the "epigenetic machinery"). The mechanistic cross-talk between histone modification and DNA methylation suggests that these syndromes might be expected to display specific DNA methylation signatures that are a reflection of those primary errors associated with chromatin dysregulation. Given the interrelated functions of these chromatin regulatory proteins, we sought to identify DNA methylation epi-signatures that could provide syndrome-specific biomarkers to complement standard clinical diagnostics. In the present study, we examined peripheral blood samples from a large cohort of individuals encompassing 14 Mendelian disorders displaying mutations in the genes encoding proteins of the epigenetic machinery. We demonstrated that specific but partially overlapping DNA methylation signatures are associated with many of these conditions. The degree of overlap among these epi-signatures is minimal, further suggesting that, consistent with the initial event, the downstream changes are unique to every syndrome. In addition, by combining these epi-signatures, we have demonstrated that a machine learning tool can be built to concurrently screen for multiple syndromes with high sensitivity and specificity, and we highlight the utility of this tool in solving ambiguous case subjects presenting with variants of unknown significance, along with its ability to generate accurate predictions for subjects presenting with the overlapping clinical and molecular features associated with the disruption of the epigenetic machinery.
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http://dx.doi.org/10.1016/j.ajhg.2017.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777983PMC
January 2018

Identification of epigenetic signature associated with alpha thalassemia/mental retardation X-linked syndrome.

Epigenetics Chromatin 2017 10;10:10. Epub 2017 Mar 10.

Department of Pathology and Lab Medicine, Western University, London, ON Canada.

Background: Alpha thalassemia/mental retardation X-linked syndrome (ATR-X) is caused by a mutation at the chromatin regulator gene . The mechanisms involved in the ATR-X pathology are not completely understood, but may involve epigenetic modifications. ATRX has been linked to the regulation of histone H3 and DNA methylation, while mutations in the gene may lead to the downstream epigenetic and transcriptional effects. Elucidating the underlying epigenetic mechanisms altered in ATR-X will provide a better understanding about the pathobiology of this disease, as well as provide novel diagnostic biomarkers.

Results: We performed genome-wide DNA methylation assessment of the peripheral blood samples from 18 patients with ATR-X and compared it to 210 controls. We demonstrated the evidence of a unique and highly specific DNA methylation "epi-signature" in the peripheral blood of ATRX patients, which was corroborated by targeted bisulfite sequencing experiments. Although genomically represented, differentially methylated regions showed evidence of preferential clustering in pericentromeric and telometric chromosomal regions, areas where ATRX has multiple functions related to maintenance of heterochromatin and genomic integrity.

Conclusion: Most significant methylation changes in the 14 genomic loci provide a unique epigenetic signature for this syndrome that may be used as a highly sensitive and specific diagnostic biomarker to support the diagnosis of ATR-X, particularly in patients with phenotypic complexity and in patients with gene sequence variants of unknown significance.
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http://dx.doi.org/10.1186/s13072-017-0118-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345252PMC
January 2018

Clinical Validation of Fragile X Syndrome Screening by DNA Methylation Array.

J Mol Diagn 2016 11 29;18(6):834-841. Epub 2016 Aug 29.

Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada; London Regional Cancer Program, London Health Sciences Center, London, Ontario, Canada; Children's Health Research Institute, London Health Sciences Center, London, Ontario, Canada; Molecular Genetics Laboratory, London Health Sciences Center, London, Ontario, Canada. Electronic address:

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. It is most frequently caused by an abnormal expansion of the CGG trinucleotide repeat (>200 repeats) located in the promoter of the fragile X mental retardation gene (FMR1), resulting in promoter DNA hypermethylation and gene silencing. Current clinical tests for FXS are technically challenging and labor intensive, and may involve use of hazardous chemicals or radioisotopes. We clinically validated the Illumina Infinium HumanMethylation450 DNA methylation array for FXS screening. We assessed genome-wide and FMR1-specific DNA methylation in 32 males previously diagnosed with FXS, including nine with mosaicism, as well as five females with full mutation, and premutation carrier males (n = 11) and females (n = 11), who were compared to 300 normal control DNA samples. Our findings demonstrate 100% sensitivity and specificity for detection of FXS in male patients, as well as the ability to differentiate patients with mosaic methylation defects. Full mutation and premutation carrier females did not show FMR1 methylation changes. We have clinically validated this genome-wide DNA methylation assay as a cost- and labor-effective alternative for sensitive and specific screening for FXS, while ruling out the most common differential diagnoses of FXS, Prader-Willi syndrome, and Sotos syndrome in the same assay.
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http://dx.doi.org/10.1016/j.jmoldx.2016.06.005DOI Listing
November 2016

DNA methylation analysis in constitutional disorders: Clinical implications of the epigenome.

Crit Rev Clin Lab Sci 2016 12;53(3):147-65. Epub 2016 Jan 12.

a Departments of Pathology and Laboratory Medicine .

Genomic, chromosomal, and gene-specific changes in the DNA sequence underpin both phenotypic variations in populations as well as disease associations, and the application of genomic technologies for the identification of constitutional (inherited) or somatic (acquired) alterations in DNA sequence forms a cornerstone of clinical and molecular genetics. In addition to the disruption of primary DNA sequence, the modulation of DNA function by epigenetic phenomena, in particular by DNA methylation, has long been known to play a role in the regulation of gene expression and consequent pathogenesis. However, these epigenetic factors have been identified only in a handful of pediatric conditions, including imprinting disorders. Technological advances in the past decade that have revolutionized clinical genomics are now rapidly being applied to the emerging discipline of clinical epigenomics. Here, we present an overview of epigenetic mechanisms with a focus on DNA modifications, including the molecular mechanisms of DNA methylation and subtypes of DNA modifications, and we describe the classic and emerging genomic technologies that are being applied to this study. This review focuses primarily on constitutional epigenomic conditions associated with a spectrum of developmental and intellectual disabilities. Epigenomic disorders are discussed in the context of global genomic disorders, imprinting disorders, and single gene disorders. We include a section focused on integration of genetic and epigenetic mechanisms together with their effect on clinical phenotypes. Finally, we summarize emerging epigenomic technologies and their impact on diagnostic aspects of constitutional genetic and epigenetic disorders.
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http://dx.doi.org/10.3109/10408363.2015.1113496DOI Listing
November 2016

Genome-wide analysis in human colorectal cancer cells reveals ischemia-mediated expression of motility genes via DNA hypomethylation.

PLoS One 2014 31;9(7):e103243. Epub 2014 Jul 31.

Department of Biomedical Sciences; Ontario Veterinary College; University of Guelph; Guelph, ON, Canada.

DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability. We previously demonstrated that hypoxia and hypoglycaemia (ischemia), two common micro-environmental changes in solid tumours, decrease DNA methylation through the downregulation of DNMTs in human colorectal cancer cells. Here, we utilized a genome-wide cross-platform approach to identify genes hypomethylated and upregulated by ischemia. Following exposure to hypoxia or hypoglycaemia, methylated DNA from human colorectal cancer cells (HCT116) was immunoprecipitated and analysed with an Affymetrix promoter array. Additionally, RNA was isolated and analysed in parallel with an Affymetrix expression array. Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. A Matrigel invasion assay revealed that indeed HCT116 cells grown in hypoxic or hypoglycaemic conditions have increased mobility capabilities. Confirmation of upregulated expression of cellular movement genes was performed with qPCR. The correlation between ischemia and metastasis is well established in cancer progression, but the molecular mechanisms responsible for this common observation have not been clearly identified. Our novel data suggests that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs. This is the first report to our knowledge that provides an explanation for the increased metastatic potential seen in ischemic cells; i.e. that ischemia could be driving DNA hypomethylation and increasing expression of cellular movement genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103243PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117527PMC
November 2015

Epigenetics, eh! A meeting summary of the Canadian Conference on Epigenetics.

Epigenetics 2011 Oct 1;6(10):1265-71. Epub 2011 Oct 1.

EpiGenwestern Research Group, Children's Health Research Institute, and Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.

In May 2011, the Canadian Conference on Epigenetics: Epigenetics Eh! was held in London, Canada. The objectives of this conference were to showcase the breadth of epigenetic research on environment and health across Canada and to provide the catalyst to develop collaborative Canadian epigenetic research opportunities, similar to existing international epigenetic initiatives in the US and Europe. With ten platform sessions and two sessions with over 100 poster presentations, this conference featured cutting-edge epigenetic research, presented by Canadian and international principal investigators and their trainees in the field of epigenetics and chromatin dynamics. An EpigenART competition included ten artists, creating a unique opportunity for artists and scientists to interact and explore their individual interpretations of this scientific discipline. The conference provided a unique venue for a significant cross-section of Canadian epigenetic researchers from diverse disciplines to meet, interact, collaborate and strategize at the national level.
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http://dx.doi.org/10.4161/epi.6.10.17491DOI Listing
October 2011

Gene signatures of breast cancer progression and metastasis.

Breast Cancer Res 2011 Jan 24;13(1):201. Epub 2011 Jan 24.

London Regional Cancer Program, Cancer Research Laboratories, London Health Sciences Centre, 790 Commissioners Road East, London, Ontario, N6A 4L6, Canada.

Breast cancer is a heterogeneous disease. Patient outcome varies significantly, depending on prognostic features of patients and their tumors, including patient age, menopausal status, tumor size and histology, nodal status, and so on. Response to treatment also depends on a series of predictive factors, such as hormone receptor and HER2 status. Current treatment guidelines use these features to determine treatment. However, these guidelines are imperfect, and do not always predict response to treatment or survival. Evolving technologies are permitting increasingly large amounts of molecular data to be obtained from tumors, which may enable more personalized treatment decisions to be made. The challenge is to learn what information leads to improved prognostic accuracy and treatment outcome for individual patients.
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http://dx.doi.org/10.1186/bcr2791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109557PMC
January 2011

Human 21T breast epithelial cell lines mimic breast cancer progression in vivo and in vitro and show stage-specific gene expression patterns.

Lab Invest 2010 Aug 10;90(8):1247-58. Epub 2010 May 10.

London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada.

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth.
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http://dx.doi.org/10.1038/labinvest.2010.97DOI Listing
August 2010

Multi-platform whole-genome microarray analyses refine the epigenetic signature of breast cancer metastasis with gene expression and copy number.

PLoS One 2010 Jan 13;5(1):e8665. Epub 2010 Jan 13.

London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada.

Background: We have previously identified genome-wide DNA methylation changes in a cell line model of breast cancer metastasis. These complex epigenetic changes that we observed, along with concurrent karyotype analyses, have led us to hypothesize that complex genomic alterations in cancer cells (deletions, translocations and ploidy) are superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes observed in breast cancer metastasis.

Methodology/principal Findings: We undertook simultaneous high-resolution, whole-genome analyses of MDA-MB-468GFP and MDA-MB-468GFP-LN human breast cancer cell lines (an isogenic, paired lymphatic metastasis cell line model) using Affymetrix gene expression (U133), promoter (1.0R), and SNP/CNV (SNP 6.0) microarray platforms to correlate data from gene expression, epigenetic (DNA methylation), and combination copy number variant/single nucleotide polymorphism microarrays. Using Partek Software and Ingenuity Pathway Analysis we integrated datasets from these three platforms and detected multiple hypomethylation and hypermethylation events. Many of these epigenetic alterations correlated with gene expression changes. In addition, gene dosage events correlated with the karyotypic differences observed between the cell lines and were reflected in specific promoter methylation patterns. Gene subsets were identified that correlated hyper (and hypo) methylation with the loss (or gain) of gene expression and in parallel, with gene dosage losses and gains, respectively. Individual gene targets from these subsets were also validated for their methylation, expression and copy number status, and susceptible gene pathways were identified that may indicate how selective advantage drives the processes of tumourigenesis and metastasis.

Conclusions/significance: Our approach allows more precisely profiling of functionally relevant epigenetic signatures that are associated with cancer progression and metastasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008665PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801616PMC
January 2010

Lymphatic metastasis of breast cancer cells is associated with differential gene expression profiles that predict cancer stem cell-like properties and the ability to survive, establish and grow in a foreign environment.

Int J Oncol 2009 Aug;35(2):297-308

London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada.

Although lymphatic dissemination is a major route for breast cancer metastasis, there has been little work to determine what factors control the ability of tumor cells to survive, establish and show progressive growth in a lymph node environment. This information is of particular relevance now, in the era of sentinel lymph node biopsy, where smaller intranodal tumor deposits are being detected earlier in the course of disease, the clinical relevance of which is uncertain. In this study, we compared differentially expressed genes in cell lines of high (468LN) vs. low (468GFP) lymphatic metastatic ability, and related these to clinical literature on genes associated with lymphatic metastatic ability and prognosis, to identify genes of potential clinical relevance. This approach revealed differential expression of a set of genes associated with 'cancer stem cell-like' properties, as well as networks of genes potentially associated with survival and autonomous growth. We explored these differences functionally and found that 468LN cells have a higher proportion of cells with a cancer stem cell-like (CD44+/CD24-) phenotype, have a higher clonogenic potential and a greater ability to survive, establish and grow in a foreign (lymph node and 3D Matrigel) microenvironment, relative to 468GFP cells. Differentially expressed genes which reflect these functions provide candidates for investigation as potential targets for therapy directed against early lymphatic metastasis.
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August 2009

New clinical and experimental approaches for studying tumor dormancy: does tumor dormancy offer a therapeutic target?

APMIS 2008 Jul-Aug;116(7-8):552-68

Massachusetts General Hospital Cancer Center, and Department of Medicine, Harvard Medical School, Boston, MA, USA.

Tumor dormancy is a significant clinical problem. Primary treatment of a cancer may be apparently successful, and yet the tumor may recur either locally or as distant metastases years or even decades later. The ability to predict which patients are likely to develop recurrences is imprecise, relying on probabilities of recurrence based on features of the primary cancer. This uncertainty presents clinical challenges regarding who to treat and how, in order to prevent recurrence after periods of dormancy. Recent clinical trials in breast cancer support the idea that some patients may harbor tumor cells that are capable of forming late-developing metastases years after removal of the primary tumor, and that these dormant cancer cells may in some cases be effectively treated with long-term therapy. Advances in experimental studies of tumor dormancy are shedding light on the nature of dormancy, and are providing both new technologies and conceptual approaches for studying tumor dormancy. A better understanding of mechanisms responsible for tumor dormancy and recurrence will be important for improving care of patients at risk for late-developing metastases.
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http://dx.doi.org/10.1111/j.1600-0463.2008.001059.xDOI Listing
November 2008

Distinct karyotypes in three breast cancer cell lines --21PTCi, 21NTCi, and 21MT-1 --derived from the same patient and representing different stages of tumor progression.

Cancer Genet Cytogenet 2008 Oct;186(1):33-40

Cytogenetics Laboratory, London Health Sciences Centre, University of Western Ontario, London, Ontario, Canada.

Three breast carcinoma cell lines --21PTCi, 21NTCi, and 21MT-1 --all originating from a 36-year-old woman with metastatic breast cancer, have been characterized previously as stably representing different stages of progression: (nontumorigenic [21PTCi]; tumorigenic, nonmetastatic [21NTCi]; and tumorigenic, weakly metastatic [21MT-1]). These cell lines were investigated for cytogenetic characteristics using G-banding and spectral karyotyping. All three cell lines have multiple chromosome aberrations, but they differ in the types of rearrangements and breakpoints. 21PTCi cells have a modal number (mn) of 56, with 55 types of aberrations, including 16 numeric and 39 structural. 21NTCi cells have a mn of 56, with 70 types of aberrations, including 19 numerical and 51 structural. Finally, 21MT-1 cells have a mn of 54, with 43 types of aberrations, including 14 numerical and 29 structural. The most common rearrangements differ in each cell line [i.e., der(X)t(X;3), der(4)t(1;4), del(6q) and der(19)t(17;19)(q11.2;q13.4) in 21PTCi; der(4)t(1;4), der(12)t(12;15) and -16 in 21NTCi; and der(1)t(1;10), +5, der(6)t(6;7), der(11)t(11;13), -20, and der(20)t(20;21) in 21MT-1]. This cytogenetic result is consistent with previous findings in that the three cell lines represent different stages of tumor progression. We hypothesize that the cytogenetic changes in these cell lines may be related to their distinct biologic characteristics. These three cell lines, with their different karyotypes and biologic characteristics, provide a vital tool for further study of the genetic and epigenetic events involved in transitions between premalignant and malignant phenotypes.
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http://dx.doi.org/10.1016/j.cancergencyto.2008.05.012DOI Listing
October 2008

Epigenetic mapping and functional analysis in a breast cancer metastasis model using whole-genome promoter tiling microarrays.

Breast Cancer Res 2008 18;10(4):R62. Epub 2008 Jul 18.

London Regional Cancer Program, Victoria Research Laboratories, London Health Sciences Centre, 790 Commissioners Road East, London, Ontario, N6A 4L6, Canada.

Introduction: Breast cancer metastasis is a complex, multi-step biological process. Genetic mutations along with epigenetic alterations in the form of DNA methylation patterns and histone modifications contribute to metastasis-related gene expression changes and genomic instability. So far, these epigenetic contributions to breast cancer metastasis have not been well characterized, and there is only a limited understanding of the functional mechanisms affected by such epigenetic alterations. Furthermore, no genome-wide assessments have been undertaken to identify altered DNA methylation patterns in the context of metastasis and their effects on specific functional pathways or gene networks.

Methods: We have used a human gene promoter tiling microarray platform to analyze a cell line model of metastasis to lymph nodes composed of a poorly metastatic MDA-MB-468GFP human breast adenocarcinoma cell line and its highly metastatic variant (468LN). Gene networks and pathways associated with metastasis were identified, and target genes associated with epithelial-mesenchymal transition were validated with respect to DNA methylation effects on gene expression.

Results: We integrated data from the tiling microarrays with targets identified by Ingenuity Pathways Analysis software and observed epigenetic variations in genes implicated in epithelial-mesenchymal transition and with tumor cell migration. We identified widespread genomic hypermethylation and hypomethylation events in these cells and we confirmed functional associations between methylation status and expression of the CDH1, CST6, EGFR, SNAI2 and ZEB2 genes by quantitative real-time PCR. Our data also suggest that the complex genomic reorganization present in cancer cells may be superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes.

Conclusion: This is the first whole-genome approach to identify genome-wide and gene-specific epigenetic alterations, and the functional consequences of these changes, in the context of breast cancer metastasis to lymph nodes. This approach allows the development of epigenetic signatures of metastasis to be used concurrently with genomic signatures to improve mapping of the evolving molecular landscape of metastasis and to permit translational approaches to target epigenetically regulated molecular pathways related to metastatic progression.
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http://dx.doi.org/10.1186/bcr2121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575535PMC
April 2009

Epigenetic contributions to cancer metastasis.

Clin Exp Metastasis 2009 2;26(1):5-18. Epub 2008 Apr 2.

Departments of Biochemistry, Oncology and Paediatrics, University of Western Ontario, London, Ontario, Canada.

The molecular basis of cancer encompasses both genetic and epigenetic alterations. These epigenetic changes primarily involve global DNA methylation changes in the form of widespread loss of methylation along with concurrent hypermethylation events in gene regulatory regions that can repress tissue-specific gene expression. Increasingly, the importance of these epigenetic changes to the metastatic process is being realized. Cells may acquire an epi-genotype that permits their dissemination from the primary tumour mass or the ability to survive and proliferate at a secondary tissue site. These epigenetic changes may be cancer-type specific, or in some cases may involve a common target gene providing a selective advantage to multiple metastatic cell types. In this review, I examine the growing volume of literature related to the epigenetic contributions to cancer metastasis. I discuss the functional importance of these epigenetic phenomena and how new epigenetic biomarkers may permit the identification of diagnostic signatures of metastasis and the development of new cancer therapies.
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http://dx.doi.org/10.1007/s10585-008-9166-2DOI Listing
February 2009

Molecular cytogenetic characterization of human breast cancer cell line MDA-MB-468 and its variant 468LN, which displays aggressive lymphatic metastasis.

Cancer Genet Cytogenet 2008 Feb;181(1):1-7

Cytogenetics Laboratory, London Health Sciences Centre, University of Western Ontario, 375 South Street, Room N258, London, Ontario N6A 4G5, Canada.

Two human breast carcinoma cell lines, MDA-MB-468 and its variant MDA-MB-468LN, which displays aggressive lymphatic metastasis, were investigated for molecular cytogenetic characteristics using G-banding, spectral karyotyping, and fluorescence in situ hybridization (FISH). Both cell lines have multiple chromosome aberrations, but differ in the types of rearrangements and their breakpoints. The MDA-MB-468 karyotype identified in the present study differs from that previously reported, which suggests that this cell line is unstable. Neither cell line exhibited amplification of ERBB2 (alias HER-2) by FISH. MDA-MB-468 cells have a modal number of 60, with 42 types of aberrations: 11 numerical and 31 structural. 468LN cells have a modal number of 55, with 37 types of aberrations: 10 numerical and 27 structural. The most common aberrations in MDA-MB-468 cells were der(5)t(5;16), i(7)(p10), i(18)(p10), der(19)t(2;19), del(6)(q23), and der(10)t(1;10). The most common aberrations in 468LN cells were der(18)t(10;18), +5, der(1;7)(q10;q10), der(19)t(2;18;19), and der(20)t(20;21). This cytogenetic result is consistent with previous findings that showed differences in tumorigenicity and metastatic capability of these two cell lines and indicates that 468LN is a new cell line distinctive from MDA-MB-468. We hypothesize that the cytogenetic changes in 468LN may be related to its new biological characteristics. Knowledge of the chromosome aberrations and breakpoints identified could be useful for further genetic and epigenetic studies of breast cancer.
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http://dx.doi.org/10.1016/j.cancergencyto.2007.05.030DOI Listing
February 2008

Genome-wide H3K9 histone acetylation profiles are altered in benzopyrene-treated MCF7 breast cancer cells.

J Biol Chem 2008 Feb 7;283(7):4051-60. Epub 2007 Dec 7.

London Regional Cancer Program, London Health Sciences Centre, University of Western Ontario, London, Ontario, Canada.

Current toxicogenomic approaches generate transcriptional profiles that can identify functional gene expression signatures of environmental toxicants. However, the intricate processes governing transcription are overlaid with a complex set of molecular instructions involving epigenetic modifications. These commands regulate both gene expression and chromatin organization through coordinated sets of histone modifications and heritable DNA methylation patterns. Although the effects of specific environmental toxicants on gene expression are the subject of much study, the epigenetic effects of such compounds are poorly understood. Here we have used human promoter tiling arrays along with chromatin immunoprecipitation to identify changes in histone acetylation profiles because of chemical exposure. Chromatin from cells exposed to the polyaromatic hydrocarbon benzo(a)pyrene was immunoprecipitated with antibodies against acetylated histones. Affymetrix promoter tiling microarrays were probed to generate epigenomic profiles of hypo- and hyperacetylated chromatin localized to gene promoter regions. Statistical analyses, data mining, and expression studies revealed that treated cells possessed differentially acetylated gene promoter regions and gene-specific expression changes. This chromatin immunoprecipitation-on-chip approach permits genome-wide profiling of histone acetylation patterns that can identify chromatin-related signatures of environmental toxicants and potentially determine the molecular pathways these changes target. This approach also has potential applications for profiling histone modifications and DNA methylation changes during embryonic development, in cancer biology, and in the development and assessment of cancer therapeutics.
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http://dx.doi.org/10.1074/jbc.M707506200DOI Listing
February 2008

DNA methylation analysis using CpG microarrays is impaired in benzopyrene exposed cells.

Toxicol Appl Pharmacol 2007 Dec 23;225(3):300-9. Epub 2007 Aug 23.

Department of Biochemistry, University of Western Ontario, Canada.

Epigenetic alterations have emerged as a key mechanism involved in tumorigenesis. These disruptions are partly due to environmental factors that change normal DNA methylation patterns necessary for transcriptional regulation and chromatin compaction. Microarray technologies are allowing environmentally susceptible epigenetic patterns to be mapped and the precise targets of environmentally induced alterations to be identified. Previously, we observed BaP-induced epigenetic events and cell cycle disruptions in breast cancer cell lines that included time- and concentration-dependent loss of proliferation as well as sequence-specific hypo- and hypermethylation events. In this present report, we further characterized epigenetic changes in BaP-exposed MCF-7 cells. We analyzed DNA methylation on a CpG island microarray platform with over 5400 unique genomic regions. Depleted and enriched microarray targets, representative of putative DNA methylation changes, were identified across the genome; however, subsequent sodium bisulfite analyses revealed no changes in DNA methylation at a number of these loci. Instead, we found that the identification of DNA methylation changes using this restriction enzyme-based microarray approach corresponded with the regions of DNA bound by the BaP derived DNA adducts. This DNA adduct formation occurs at both methylated and unmethylated CpG dinucleotides and affects PCR amplification during sample preparation. Our data suggest that caution should be exercised when interpreting data from comparative microarray experiments that rely on enzymatic reactions. These results are relevant to genome screening approaches involving environmental exposures in which DNA adduct formation at specific nucleotide sites may bias target acquisition and compromise the correct identification of epigenetically responsive genes.
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http://dx.doi.org/10.1016/j.taap.2007.08.013DOI Listing
December 2007

Epigenetic inactivation of BRCA1 is associated with aberrant expression of CTCF and DNA methyltransferase (DNMT3B) in some sporadic breast tumours.

Eur J Cancer 2007 Jan 27;43(1):210-9. Epub 2006 Oct 27.

The University of Western Ontario and the London Regional Cancer Program, London Health Sciences Centre, Room A4-134, 790 Commissioners Rd. East, London, Ont., Canada N6A 4L6.

We assessed expression of the BRCA1, CTCF and DNMT3b methyltransferase genes along with BRCA1 promoter methylation to better define the epigenetic events involved in BRCA1 inactivation in sporadic breast cancer. These gene expression patterns were determined in 54 sporadic breast tumours by immunohistochemistry and the methylation status of the BRCA1 promoter was evaluated using methylation-specific PCR. We observed significant DNMT3b expression in 80% of the tumours and that 43% of tumours exhibited novel cytoplasmic CTCF expression. Pairwise analyses of gene expression patterns showed that 28/32 tumours lacked BRCA1 expression and also exhibited cytoplasmic CTCF staining, while 24/32 of these tumours also overexpressed DNMT3b. Furthermore, 86% of the BRCA1 low-expressing tumours were methylated at the BRCA1 promoter and a subset of these tumours displayed both cytoplasmic CTCF and increased DNMT3b expression. Thus, tumour subsets exist that display concurrent decreased BRCA1 expression, BRCA1 promoter methylation, cytoplasmic CTCF expression and with DNMT3b over-expression. We suggest that these altered CTCF and DNMT3b expression patterns represent (a) critical events responsible for the epigenetic inactivation of BRCA1 and (b) a diagnostic signature for epigenetic inactivation of other tumour suppressor genes in sporadic breast tumours.
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http://dx.doi.org/10.1016/j.ejca.2006.09.002DOI Listing
January 2007

Benzopyrene exposure disrupts DNA methylation and growth dynamics in breast cancer cells.

Toxicol Appl Pharmacol 2006 Nov 30;216(3):458-68. Epub 2006 Jun 30.

Department of Biochemistry, University of Western Ontario, Canada.

Exposures to environmental carcinogens and unhealthy lifestyle choices increase the incidence of breast cancer. One such compound, benzo(a)pyrene (BaP), leads to covalent DNA modifications and the deregulation of gene expression. To date, these mechanisms of BaP-induced carcinogenesis are poorly understood, particularly in the case of breast cancer. We tested the effects of BaP exposure on cellular growth dynamics and DNA methylation in four breast cancer cell lines since disruptions in DNA methylation lead to deregulated gene expression and the loss of genomic integrity. We observed robust time- and concentration-dependent loss of proliferation, S phase and G2M accumulation and apoptosis in p53 positive MCF-7 and T47-D cells. We observed minimal responses in p53 negative HCC-1086 and MDA MB 231 cells. Furthermore, BaP increased p53 levels in both p53 positive cell lines, as well as p21 levels in MCF-7 cells, an effect that was prevented by the p53-specific inhibitor pifithrin-alpha. No changes in global levels of DNA methylation levels induced by BaP were detected by the methyl acceptor assay (MAA) in any cell line, however, methylation profiling by AIMS (amplification of intermethylated sites) analysis showed dynamic, sequence-specific hypo- and hypermethylation events in all cell lines. We also identified BaP-induced hypomethylation events at a number of genomic repeats. Our data confirm the p53-specific disruption of the cell cycle as well as the disruption of DNA methylation as a consequence of BaP treatment, thus reinforcing the link between environmental exposures, DNA methylation and breast cancer.
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http://dx.doi.org/10.1016/j.taap.2006.06.012DOI Listing
November 2006

DNA binding sites for putative methylation boundaries in the unmethylated region of the BRCA1 promoter.

Int J Cancer 2004 Sep;111(5):669-78

London Regional Cancer Centre, Child Health Research Institute, London, Ontario, Canada.

Changes in DNA methylation patterns are frequently observed in human cancers and are associated with a decrease in tumor suppressor gene expression. Hypermethylation of the BRCA1 promoter has been reported in a portion of sporadic breast tumours that correspond to a reduction in BRCA1 transcription and expression. Questions remain concerning the maintenance of methylation free zones in promoter regions of tumor suppressor genes in normal tissues. Sodium bisulfite based analysis of the BRCA1 promoter defines a methylation free zone in normal breast tissue that starts 650 bp upstream of the transcription start site and extends for 1.4 kb through most of the BRCA1 CpG island. We provide data implicating 2 proteins, Sp1 and CTCF, in the maintenance of this methylation-free zone. Both of these proteins have been shown to function as methylation boundaries in other genes. Four Sp1 sites have been identified in the BRCA1 promoter by gel shift assays. In vivo binding of Sp1 has been confirmed at 2 of these sites in the BRCA1 promoter and at 2 CTCF sites that flank the unmethylated region. Our data suggest that these DNA binding sites for Sp1 and CTCF may act as boundary elements that are important in maintaining genomic integrity by delineating the normal methylation free BRCA1 promoter. Inactivation or disruption of these boundaries may facilitate an epigenetic "hit", in this case DNA methylation, leading to BRCA1 downregulation and contributing to tumorigenesis, particularly the genesis of sporadic breast tumours.
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http://dx.doi.org/10.1002/ijc.20324DOI Listing
September 2004

Chemically induced DNA hypomethylation in breast carcinoma cells detected by the amplification of intermethylated sites.

Breast Cancer Res 2004 30;6(4):R329-37. Epub 2004 Apr 30.

The London Regional Cancer Centre, Child Health Research Institute, and the Department of Biochemistry, Paediatrics and Oncology, at the University of Western Ontario, London, Ontario, Canada.

Introduction: Compromised patterns of gene expression result in genomic instability, altered patterns of gene expression and tumour formation. Specifically, aberrant DNA hypermethylation in gene promoter regions leads to gene silencing, whereas global hypomethylation events can result in chromosomal instability and oncogene activation. Potential links exist between environmental agents and DNA methylation, but the destabilizing effects of environmental exposures on the DNA methylation machinery are not understood within the context of breast cancer aetiology.

Methods: We assessed genome-wide changes in methylation patterns using a unique methylation profiling technique called amplification of intermethylated sites (AIMS). This method generates easily readable fingerprints that represent the investigated cell line's methylation profile, based on the differential cleavage of DNA with methylation-specific isoschisomeric restriction endonucleases.

Results: We validated this approach by demonstrating both unique and reoccurring sites of genomic hypomethylation in four breast carcinoma cell lines treated with the cytosine analogue 5-azacytidine. Comparison of treated with control samples revealed individual bands that exhibited methylation changes, and these bands were excized and cloned, and the precise genomic location individually identified. In most cases, these regions of hypomethylation coincided with susceptible target regions previously associated with chromosome breakage, rearrangement and gene amplification. Similarly, we observed that acute benzopyrene exposure is associated with altered methylation patterns in these cell lines.

Conclusion: These results reinforce the link between environmental exposures, DNA methylation and breast cancer, and support a role for AIMS as a rapid, affordable screening method to identify environmentally induced DNA methylation changes that occur in tumourigenesis.
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http://dx.doi.org/10.1186/bcr799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC468641PMC
November 2004

Characterization of functional elements in the neurofibromatosis (NF1) proximal promoter region.

Oncogene 2004 Jan;23(2):330-9

London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada.

An essential requirement to understand how genes contribute to genetic disease is the thorough knowledge of the transcriptional regulation of gene expression. Here, we have characterized transcription factor binding sites within the type 1 neurofibromatosis (NF1) proximal regulatory region, and addressed the molecular mechanisms that regulate NF1 transcription. Overlapping regions of the NF1 proximal promoter have been cloned and characterized for use in luciferase reporter assays. These assays have identified a 500 bp region displaying activities up to 80-fold higher than control reporter levels. Mutations at putative CRE and SP1-binding sites immediately 5' to the transcription start site have dramatic effects that lead to a 70-90% decrease in reporter activity in all cell lines tested. Gelshift assays confirm binding of CREB and SP1/KLF family members to their putative recognition sequences, and provide the first evidence identifying functional sites likely involved in regulating NF1 transcription. These assays have also revealed a putative repressor region within the NF1 promoter region corresponding to CCCTC-rich sequences between the transcription and translation start sites. This work provides new information concerning the transcriptional regulation of the NF1 gene, and is the most thorough attempt to date to map functionally relevant regions within the NF1 proximal promoter region.
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http://dx.doi.org/10.1038/sj.onc.1207053DOI Listing
January 2004
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