ABSTRACT
The purpose of this document is to provide pre-analytical, analytical and post-analytical considerations and recommendations to Canadian clinical laboratories developing, validating and offering next-generation sequencing (NGS)-based BRCA1 and BRCA2 (BRCA1/2) tumour testing in ovarian cancers. This document was drafted by the members of the Canadian College of Medical Geneticists (CCMG) somatic BRCA Ad Hoc Working Group, and representatives from the Canadian Association of Pathologists. The document was circulated to the CCMG members for comment. Following incorporation of feedback, this document has been approved by the CCMG board of directors. The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. The current CCMG Practice Guidelines were developed as a resource for clinical laboratories in Canada; however, they are not inclusive of all information laboratories should consider in the validation and use of NGS for BRCA1/2 tumour testing in ovarian cancers.
Subject(s)
Clinical Laboratory Services , Ovarian Neoplasms , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Canada , Carcinoma, Ovarian Epithelial , Female , Genetic Testing , Germ-Line Mutation , High-Throughput Nucleotide Sequencing , Humans , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/geneticsABSTRACT
BACKGROUND: With high survival rates for pediatric acute lymphoblastic leukemia (ALL), long-term quality of life is a prominent consideration in treatment. We concurrently evaluated cognition, behavior, and quality of life in child and adolescent ALL survivors and determined associations between them. METHODS: The sample included 83 controls (mean age: 12.5 years) and 71 ALL survivors (mean age: 11.9 years, mean age at diagnosis: 3.8 years). Participants completed measures of general intellectual abilities, math achievement, and fine motor skills. Parents and teachers completed a survey assessing child participants' cognitive, behavioral, and emotional function. Parents additionally completed a survey about their child's quality of life. RESULTS: Survivors had lower scores on measures of working memory, processing speed, timed math, and fine motor skills (effect size 0.5-1, p < 0.001). Parents identified more problems with executive function and learning in survivors than controls (effect size > 0.7, p < 0.001), and indicated a lower quality of life in all categories evaluated (effect size > 0.7, p < 10-4). Reduced quality of life was associated with lower math achievement scores and with inattention and executive function problems. CONCLUSIONS: ALL survivors experience diffuse cognitive, behavioral, and motor impairments, which are associated with reduced quality of life. These findings underscore the need to address these challenges in ALL survivors. IMPACT: Compared with cancer-free peers, parents of childhood acute lymphoblastic leukemia survivors treated with chemotherapy only reported reduced quality of life. Math difficulties and behavioral problems increased the risk for reduced quality of life. Reduced quality of life is associated with mild cognitive and behavioral difficulties, suggesting that even relatively mild impairments have broad implications for ALL survivors. Screening and early intervention targeting cognitive and behavioral function may enhance quality of life for ALL survivors.
Subject(s)
Adolescent Behavior , Adolescent Development , Cancer Survivors/psychology , Child Behavior , Child Development , Cognition , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Quality of Life , Adolescent , Age Factors , Case-Control Studies , Child , Child, Preschool , Emotions , Executive Function , Female , Humans , Intelligence , Male , Memory, Short-Term , Motor Skills , Risk Assessment , Risk FactorsABSTRACT
Epigenetic dysregulation has emerged as a recurring mechanism in the etiology of neurodevelopmental disorders. Two such disorders, CHARGE and Kabuki syndromes, result from loss of function mutations in chromodomain helicase DNA-binding protein 7 (CHD7LOF) and lysine (K) methyltransferase 2D (KMT2DLOF), respectively. Although these two syndromes are clinically distinct, there is significant phenotypic overlap. We therefore expected that epigenetically driven developmental pathways regulated by CHD7 and KMT2D would overlap and that DNA methylation (DNAm) alterations downstream of the mutations in these genes would identify common target genes, elucidating a mechanistic link between these two conditions, as well as specific target genes for each disorder. Genome-wide DNAm profiles in individuals with CHARGE and Kabuki syndromes with CHD7LOF or KMT2DLOF identified distinct sets of DNAm differences in each of the disorders, which were used to generate two unique, highly specific and sensitive DNAm signatures. These DNAm signatures were able to differentiate pathogenic mutations in these two genes from controls and from each other. Analysis of the DNAm targets in each gene-specific signature identified both common gene targets, including homeobox A5 (HOXA5), which could account for some of the clinical overlap in CHARGE and Kabuki syndromes, as well as distinct gene targets. Our findings demonstrate how characterization of the epigenome can contribute to our understanding of disease pathophysiology for epigenetic disorders, paving the way for explorations of novel therapeutics.
Subject(s)
Abnormalities, Multiple/genetics , CHARGE Syndrome/genetics , DNA Methylation , Epigenesis, Genetic , Face/abnormalities , Hematologic Diseases/genetics , Vestibular Diseases/genetics , Abnormalities, Multiple/diagnosis , CHARGE Syndrome/diagnosis , Cell Line , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Genome, Human , Hematologic Diseases/diagnosis , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Mutation , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Reproducibility of Results , Sensitivity and Specificity , Vestibular Diseases/diagnosisABSTRACT
Epigenetic dysfunction has been implicated in a growing list of disorders that include cancer, neurodevelopmental disorders, and neurodegeneration. Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7) are rare neurodevelopmental disorders with broad phenotypic spectra caused by deletion and duplication, respectively, of a 1.5-Mb region that includes several genes with a role in epigenetic regulation. We have identified striking differences in DNA methylation across the genome between blood cells from children with WS or Dup7 and blood cells from typically developing (TD) children. Notably, regions that were differentially methylated in both WS and Dup7 displayed a significant and symmetrical gene-dose-dependent effect, such that WS typically showed increased and Dup7 showed decreased DNA methylation. Differentially methylated genes were significantly enriched with genes in pathways involved in neurodevelopment, autism spectrum disorder (ASD) candidate genes, and imprinted genes. Using alignment with ENCODE data, we also found the differentially methylated regions to be enriched with CCCTC-binding factor (CTCF) binding sites. These findings suggest that gene(s) within 7q11.23 alter DNA methylation at specific sites across the genome and result in dose-dependent DNA-methylation profiles in WS and Dup7. Given the extent of DNA-methylation changes and the potential impact on CTCF binding and chromatin regulation, epigenetic mechanisms most likely contribute to the complex neurological phenotypes of WS and Dup7. Our findings highlight the importance of DNA methylation in the pathogenesis of WS and Dup7 and provide molecular mechanisms that are potentially shared by WS, Dup7, and ASD.
Subject(s)
DNA Methylation/genetics , Epigenesis, Genetic/genetics , Gene Dosage/genetics , DNA Primers/genetics , Gene Frequency , Humans , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA , Statistics, Nonparametric , Williams SyndromeABSTRACT
The multidrug resistance efflux transporter ATP-binding cassette subfamily G member 2 (ABCG2) is not only overexpressed in certain drug-resistant cancers but is also highly expressed in the mammary gland during lactation, carrying xenobiotics and nutrients into milk. We sought to investigate the molecular mechanisms involved in the upregulation of ABCG2 during lactation. Expression profiling of different mouse Abcg2 mRNA isoforms (E1a, E1b, and E1c) revealed that E1b is predominantly expressed and induced in the lactating mouse mammary gland. Despite this induction, analyses of CpG methylation status and published ChIP-seq datasets reveal that E1b promoter sequences in the virgin gland are already hypomethylated and marked with the open chromatin histone mark H3K4me2. Using a forced-weaning model to shut down lactation, we found that within 24 h there was a significant reduction in Abcg2 mRNA expression and a loss of signal transducer and activator of transcription-5 (STAT5) occupancy at the mouse Abcg2 gene. Luciferase reporter assays further showed that some of these STAT5-binding regions that contained interferon-γ-activated sequence (GAS) motifs function as an enhancer after prolactin treatment. We conclude that Abcg2 is already poised for expression in the virgin mammary gland and that STAT5 plays an important role in Abcg2 expression during lactation.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Epigenesis, Genetic , Epithelial Cells/metabolism , Lactation/genetics , Mammary Glands, Animal/metabolism , RNA Isoforms/genetics , RNA, Messenger/genetics , STAT5 Transcription Factor/physiology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/metabolism , Animals , CpG Islands , DNA Methylation , Female , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Promoter Regions, Genetic , Signal Transduction , Up-RegulationABSTRACT
Imprinted genes are critical for normal human growth and neurodevelopment. They are characterized by differentially methylated regions (DMRs) of DNA that confer parent of origin-specific transcription. We developed a new strategy to identify imprinted gene-associated DMRs. Using genome-wide methylation profiling of sodium bisulfite modified DNA from normal human tissues of biparental origin, candidate DMRs were identified by selecting CpGs with methylation levels consistent with putative allelic differential methylation. In parallel, the methylation profiles of tissues of uniparental origin, i.e., paternally-derived androgenetic complete hydatidiform moles (AnCHMs), and maternally-derived mature cystic ovarian teratoma (MCT), were examined and then used to identify CpGs with parent of origin-specific DNA methylation. With this approach, we found known DMRs associated with imprinted genomic regions as well as new DMRs for known imprinted genes, NAP1L5 and ZNF597, and novel candidate imprinted genes. The paternally methylated DMR for one candidate, AXL, a receptor tyrosine kinase, was also validated in experiments with mouse embryos that demonstrated Axl was expressed preferentially from the maternal allele in a DNA methylation-dependent manner.
Subject(s)
DNA/genetics , Genomic Imprinting , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Alleles , Animals , Base Sequence , CpG Islands/genetics , DNA/chemistry , DNA Methylation , Embryo, Mammalian , Female , Genetic Variation , Genome , Humans , Hydatidiform Mole/genetics , Hydatidiform Mole/metabolism , Mice , Microarray Analysis/methods , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Pregnancy , Pregnancy Complications/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Sex Factors , Sulfites/chemistry , Teratoma/genetics , Teratoma/metabolism , Axl Receptor Tyrosine KinaseABSTRACT
A distinct subset of acute myeloid leukemia (AML) is characterized by the presence of the Philadelphia chromosome (Ph+), due to reciprocal translocation t(9;22)(q34;q11.2). This chromosomal rearrangement leads to the fusion of the breakpoint cluster region (BCR) gene on chromosome 22 with the ABL1 gene on chromosome 9, generating the BCR::ABL1 fusion gene. The Ph+ AML subtype is associated with poor prognosis and resistance to conventional chemotherapy. Beyond the well-established BCR::ABL1 fusion, recent studies have shed light on additional genetic abnormalities in Ph+ AML, including associations with rearrangements involving core binding factor beta (CBFB). We describe a case of de novo AML with concurrent BCR::ABL1 and CBFB::MYH11 rearrangements.
ABSTRACT
The Illumina Infinium HumanMethylation27 BeadChip (Illumina 27k) microarray is a high-throughput platform capable of interrogating the human DNA methylome. In a search for autosomal sex-specific DNA methylation using this microarray, we discovered autosomal CpG loci showing significant methylation differences between the sexes. However, we found that the majority of these probes cross-reacted with sequences from sex chromosomes. Moreover, we determined that 6-10% of the microarray probes are non-specific and map to highly homologous genomic sequences. Using probes targeting different CpGs that are exact duplicates of each other, we investigated the precision of these repeat measurements and concluded that the overall precision of this microarray is excellent. In addition, we identified a small number of probes targeting CpGs that include single-nucleotide polymorphisms. Overall, our findings address several technical issues associated with the Illumina 27k microarray that, once considered, will enhance the analysis and interpretation of data generated from this platform.
Subject(s)
Chromosomes, Human, X/genetics , Chromosomes, Human/genetics , DNA Methylation/genetics , DNA Probes/genetics , Oligonucleotide Array Sequence Analysis/statistics & numerical data , Computational Biology , CpG Islands/genetics , DNA Probes/chemistry , Data Interpretation, Statistical , Humans , Polymorphism, Genetic/genetics , Sequence HomologyABSTRACT
BACKGROUND: Fetal exposure to maternal GDM increases offspring risk for adult-onset metabolic syndromes. Epigenetic modifications such as DNA methylation are modulators for fetal metabolic programming and susceptibility to adult-onset disease. This study investigates genome-wide DNA methylation in GDM exposed cord blood and placenta. METHODS: Oral glucose tolerance testing between 24 and 28 weeks of pregnancy was used to determine severity of glucose intolerance. We measured DNA methylation (DNAm) using the Illumina Infinium 450 K array in 42 fetal cord blood and 36 placenta samples. RESULTS: We identified 662 and 99 CpG sites in GDM placenta and cord blood, respectively at p-value <0.01 and a methylation difference >5% after adjustment for confounders. Annotated sites for AHRR and PTPRN2 were common to cord blood and placenta. Adding published GDM cord blood DNAm data we segregated patients based on treatment (Diet Only vs. +Insulin) and identified altered CpG sites to be unique to each GDM treatment group. CONCLUSION: Consistency in findings with other studies provides evidence for the role of DNAm in placental and fetal responses to hyperglycemia. However, segregating DNAm analysis of GDM samples based on treatment may help delineate whether observed DNAm alterations are reflective of adaptive responses or treatment effects in utero.
Subject(s)
DNA Methylation/genetics , Diabetes, Gestational/blood , Fetal Blood/metabolism , Placenta/metabolism , Adult , Diabetes, Gestational/genetics , Female , Humans , PregnancyABSTRACT
Many neurodevelopmental disorders (NDDs) share common learning and behavioural impairments, as well as features such as dysregulation of the oxytocin hormone. Here, we examined DNA methylation (DNAm) in the 1st intron of the oxytocin receptor gene, OXTR, in patients with autism spectrum (ASD), attention deficit and hyperactivity (ADHD) and obsessive compulsive (OCD) disorders. DNAm of OXTR was assessed for cohorts of ASD (blood), ADHD (saliva), OCD (saliva), which uncovered sex-specific DNAm differences compared to neurotypical, tissue-matched controls. Individuals with ASD or ADHD exhibiting extreme DNAm values had lower IQ and more social problems, respectively, than those with DNAm within normative ranges. This suggests that OXTR DNAm patterns are altered across NDDs and may be correlated with common clinical outcomes.
Subject(s)
Attention Deficit Disorder with Hyperactivity , Autism Spectrum Disorder , Obsessive-Compulsive Disorder , Attention Deficit Disorder with Hyperactivity/genetics , Autism Spectrum Disorder/genetics , DNA Methylation , Female , Humans , Male , Oxytocin/metabolism , Receptors, Oxytocin/geneticsSubject(s)
Aging/genetics , DNA Methylation , Prefrontal Cortex/growth & development , Prefrontal Cortex/metabolism , Female , Humans , MaleABSTRACT
INTRODUCTION: Cure rates for pediatric acute lymphoblastic leukemia (ALL) have reached an all-time high (>90%); however, neurocognitive difficulties continue to affect quality of life in at least a subset of survivors. There are relatively few quantitative neuroimaging studies in child and adolescent ALL survivors treated with chemotherapy only. Use of different outcome measures or limited sample sizes restrict our ability to make inferences about patterns of brain development following chemotherapy treatment. In this study, we used magnetic resonance imaging (MRI) to evaluate brain outcomes in ALL survivors, comparing against a group of typically developing, cancer free peers. MATERIALS AND METHODS: Participants included 71 ALL survivors, on average 8 years after diagnosis and 8-18 years of age, and 83 typically developing controls. Anatomical MRI was performed to evaluate brain structure; diffusion and magnetization transfer MRI were used to examine brain tissue microstructure. RESULTS: Successful MRI scans were acquired in 67 survivors (94%) and 82 controls (99%). Structurally, ALL survivors exhibited widespread reductions in brain volume, with 6% less white matter and 5% less gray matter than controls (p = 0.003 and 0.0006 respectively). Much of the brain appeared affected - 71 of 90 evaluated structures showed smaller volume - with the most notable exception being the occipital lobe, where no significant differences were observed. Average full-scale IQ in the survivor and control groups were 95 (CI 92-99) and 110 (CI 107-113), respectively. Using data from the NIH Pediatric MRI Data Repository, we evaluated the extent to which elevated IQ in the control group might affect the structural differences observed. We estimated that two thirds of the observed brain differences were attributable to ALL and its treatment. In addition to the structural changes, survivors showed, on average, globally lower white matter fractional anisotropy (-3%) and higher radial diffusivity (+5%) (p < 10-6), but no differences in magnetization transfer ratio. CONCLUSIONS: Neuroanatomical alterations in late childhood and adolescent ALL survivors treated with chemotherapy-only protocols are widespread, with white matter being somewhat more affected than gray matter. These MRI results indicate brain development is altered in ALL survivors and highlight the need to examine how these alterations emerge.
Subject(s)
White Matter , Adolescent , Aged, 80 and over , Brain/diagnostic imaging , Child , Gray Matter/diagnostic imaging , Humans , Magnetic Resonance Imaging , Quality of Life , Survivors , White Matter/diagnostic imagingABSTRACT
BACKGROUND: A growing body of research has demonstrated associations between specific neurodevelopmental disorders and variation in DNA methylation (DNAm), implicating this molecular mark as a possible contributor to the molecular etiology of these disorders and/or as a novel disease biomarker. Furthermore, genetic risk variants of neurodevelopmental disorders have been found to be enriched at loci associated with DNAm patterns, referred to as methylation quantitative trait loci (mQTLs). METHODS: We conducted two epigenome-wide association studies in individuals with attention-deficit/hyperactivity disorder (ADHD) or obsessive-compulsive disorder (OCD) (aged 4-18 years) using DNA extracted from saliva. DNAm data generated on the Illumina Human Methylation 450 K array were used to examine the interaction between genetic variation and DNAm patterns associated with these disorders. RESULTS: Using linear regression followed by principal component analysis, individuals with the most endorsed symptoms of ADHD or OCD were found to have significantly more distinct DNAm patterns from controls, as compared to all cases. This suggested that the phenotypic heterogeneity of these disorders is reflected in altered DNAm at specific sites. Further investigations of the DNAm sites associated with each disorder revealed that despite little overlap of these DNAm sites across the two disorders, both disorders were significantly enriched for mQTLs within our sample. CONCLUSIONS: Our DNAm data provide insights into the regulatory changes associated with genetic variation, highlighting their potential utility both in directing GWAS and in elucidating the pathophysiology of neurodevelopmental disorders.
Subject(s)
Attention Deficit Disorder with Hyperactivity , Obsessive-Compulsive Disorder , Attention Deficit Disorder with Hyperactivity/genetics , DNA Methylation/genetics , Genetic Variation/genetics , Humans , Obsessive-Compulsive Disorder/geneticsABSTRACT
Buccal epithelial cells are among the most clinically accessible tissues and are increasingly being used to identify epigenetic disease patterns. However, substantial variation in buccal DNA methylation patterns indicates heterogeneity of cell types within and between samples, raising questions of data quality. We systematically estimated cell-type composition for a large collection of buccal and saliva samples from 11 published studies of DNA methylation. In these we identified numerous cases of buccal samples with questionable purity, which may be affected by sampling from individuals with neurodevelopmental disorders, and by the brushes used for sample collection. Further challenges are involved in comparisons with tissues such as saliva, in which buccal component varies widely. We propose a reference-based method of correcting for buccal purity that reduces unwanted variation while preserving cross-tissue differences. Our work demonstrates the wide variation of buccal quality in epigenetic studies and suggests a possible approach to overcome this issue.
Subject(s)
DNA Methylation , Mouth Mucosa/chemistry , Specimen Handling/methods , CpG Islands , Epigenesis, Genetic , Epithelial Cells/chemistry , Epithelial Cells/cytology , Female , Humans , Male , Mouth Mucosa/cytology , Saliva/chemistryABSTRACT
PURPOSE: Chemotherapy for childhood acute lymphoblastic leukemia (ALL) can cause late-appearing side effects in survivors that affect multiple organs, including the heart and brain. However, the complex ALL treatment regimen makes it difficult to isolate the causes of these side effects and impossible to separate the contributions of individual chemotherapy agents by clinical observation. Using a mouse model, we therefore assessed each of eight representative, systemically-administered ALL chemotherapy agents for their impact on postnatal brain development and heart function. EXPERIMENTAL DESIGN: Mice were treated systemically with a single chemotherapy agent at an infant equivalent age, then allowed to age to early adulthood (9 weeks). Cardiac structure and function were assessed using in vivo high-frequency ultrasound, and brain anatomy was assessed using high-resolution volumetric ex vivo MRI. In addition, longitudinal in vivo MRI was used to determine the time course of developmental change after vincristine treatment. RESULTS: Vincristine, doxorubicin, and methotrexate were observed to produce the greatest deficiencies in brain development as determined by volumes measured on MRI, whereas doxorubicin, methotrexate, and l-asparaginase altered heart structure or function. Longitudinal studies of vincristine revealed widespread volume loss immediately following treatment and impaired growth over time in several brain regions. CONCLUSIONS: Multiple ALL chemotherapy agents can affect postnatal brain development or heart function. This study provides a ranking of agents based on potential toxicity, and thus highlights a subset likely to cause side effects in early adulthood for further study.
Subject(s)
Antineoplastic Agents/adverse effects , Brain Injuries/etiology , Brain/drug effects , Brain/growth & development , Heart Diseases/etiology , Leukemia/complications , Animals , Antineoplastic Agents/administration & dosage , Brain Injuries/diagnostic imaging , Brain Injuries/physiopathology , Child , Disease Models, Animal , Echocardiography , Female , Heart Diseases/diagnostic imaging , Heart Diseases/physiopathology , Heart Function Tests , Humans , Infant , Leukemia/drug therapy , Magnetic Resonance Imaging , Male , MiceABSTRACT
Knowledge about cognitive late effects in survivors of childhood acute lymphoblastic leukemia (ALL) is largely based on standardized neuropsychological measures and parent reports. To examine whether cognitive neuroscience paradigms provided additional insights into neurocognitive and behavioral late effects in ALL survivors, we assessed cognition and behavior using a selection of cognitive neuroscience tasks and standardized measures probing domains previously demonstrated to be affected by chemotherapy. 130 ALL survivors and 158 control subjects, between 8 and 18 years old at time of testing, completed the n-back (working memory) and stop-signal (response inhibition) tasks. ALL survivors also completed standardized measures of intelligence (Wechsler Intelligence Scales [WISC-IV]), motor skills (Grooved Pegboard), math abilities (WIAT-III), and executive functions (Delis-Kaplan Executive Function System). Parents completed behavioral measures of executive functions (Behavior Rating Inventory of Executive Function [BRIEF]) and attention (Conners-3). ALL survivors exhibited deficiencies in working memory and response inhibition compared with controls. ALL survivors also exhibited deficits on WISC-IV working memory and processing speed, Grooved Pegboard, WIAT-III addition and subtraction fluency, and numerical operations, as well as DKEFS number-letter switching. Parent reports suggested more attention deficits (Conners-3) and behavioral difficulties (BRIEF) in ALL survivors compared with referenced norms. Low correspondence between standardized and experimental measures of working memory and response inhibition was noted. The use of cognitive neuroscience paradigms complements our understanding of the cognitive deficits evident after treatment of ALL. These measures could further delineate cognitive processes involved in neurocognitive late effects, providing opportunities to explore their underlying mechanisms.
Subject(s)
Cognition/physiology , Cognitive Dysfunction/psychology , Neuropsychological Tests , Precursor Cell Lymphoblastic Leukemia-Lymphoma/psychology , Survivors/psychology , Adolescent , Child , Cognitive Dysfunction/diagnosis , Cognitive Dysfunction/epidemiology , Cognitive Neuroscience , Executive Function/physiology , Female , Humans , Intelligence Tests , Male , Memory, Short-Term/physiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/epidemiology , Problem Solving/physiologyABSTRACT
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.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Lobular/metabolism , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA-Binding Proteins/metabolism , Genes, BRCA1 , Repressor Proteins/metabolism , Aged , BRCA1 Protein/metabolism , Breast Neoplasms/genetics , CCCTC-Binding Factor , Carcinoma, Ductal, Breast/genetics , Carcinoma, Lobular/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , DNA-Binding Proteins/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Immunohistochemistry , Ki-67 Antigen/metabolism , Middle Aged , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Polymerase Chain Reaction , Repressor Proteins/genetics , DNA Methyltransferase 3BABSTRACT
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.
Subject(s)
Genes, Neurofibromatosis 1 , Neurofibromatoses/genetics , Promoter Regions, Genetic/genetics , Response Elements/genetics , Cell Line , DNA Methylation , Electrophoretic Mobility Shift Assay , Genes, Reporter/genetics , Humans , Luciferases/genetics , Luciferases/metabolism , Sequence DeletionABSTRACT
Childhood cancer survivors frequently experience long-lasting consequences of chemotherapy on health outcomes. Neurocognitive late effects of chemotherapy occur in 40 - 60% of acute lymphoblastic leukemia (ALL) survivors. These deficits affect mental health, school performance, job success, and are associated with poor quality of life, therefore presenting a clinical challenge for psychiatrists. However, not all cancer survivors are impacted by treatment in the same manner and emerging evidence suggests that genetic variation may modulate neurocognitive outcomes. Much like other complex psychopathologies, neurocognitive deficits in cancer survivors are the result of complex interactions between genetic and environmental variables. This review describes adverse neurocognitive outcomes observed in survivors of acute lymphoblastic leukemia (ALL) and discusses genetic variability in biochemical pathways targeted by chemotherapeutic agents as a possible mechanism contributing to psychopathology in ALL survivors.
Les survivants d'un cancer pédiatrique éprouvent souvent des conséquences durables de la chimiothérapie sur leurs résultats de santé. Les effets tardifs neurocognitifs de la chimiothérapie surviennent chez 40% à 60% des survivants de la leucémie lymphoblastique aiguë (LLA). Ces déficits touchent la santé mentale, le rendement scolaire, la réussite professionnelle et sont associés à une piètre qualité de vie, et présentent donc un défi clinique pour les psychiatres. Cependant, les survivants du cancer ne sont pas tous affectés de la même manière par le traitement et de nouvelles données probantes suggèrent que la variation génétique puisse moduler les résultats neurocognitifs. À l'instar d'autres psychopathologies complexes, les déficits neurocognitifs chez les survivants du cancer sont le résultat d'interactions complexes entre les variables génétiques et environnementales. Cette revue décrit les résultats neurocognitifs indésirables observés chez les survivants de la leucémie lymphoblastique aiguë (LLA) et discute de la variabilité génétique des trajectoires biochimiques ciblées par les agents chimiothérapeutiques comme mécanisme possible contribuant à la psychopathologie chez les survivants de la LLA.
ABSTRACT
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.