DNA methylation is an important epigenetic mark and dysregulation of DNA methylation is associated with many diseases including cancer. Advances in next-generation sequencing now allow unbiased methylome profiling of entire patient cohorts, greatly facilitating biomarker discovery and presenting new opportunities to understand the biological mechanisms by which changes in methylation contribute to disease. Enrichment-based sequencing assays such as MethylCap-seq are a cost effective solution for genome-wide determination of methylation status, but the technical reliability of methylation reconstruction from raw sequencing data has not been well characterized.
We analyze three MethylCap-seq data sets and perform two different analyses to assess data quality. First, we investigate how data quality is affected by excluding samples that do not meet quality control cutoff requirements. Second, we consider the effect of additional reads on enrichment score, saturation, and coverage. Lastly, we verify a method for the determination of the global amount of methylation from MethylCap-seq data by comparing to a spiked-in control DNA of known methylation status.
We show that rejection of samples based on our quality control parameters leads to a significant improvement of methylation calling. Additional reads beyond ~13 million unique aligned reads improved coverage, modestly improved saturation, and did not impact enrichment score. Lastly, we find that a global methylation indicator calculated from MethylCap-seq data correlates well with the global methylation level of a sample as obtained from a spike-in DNA of known methylation level.
We show that with appropriate quality control MethylCap-seq is a reliable tool, suitable for cohorts of hundreds of patients, that provides reproducible methylation information on a feature by feature basis as well as information about the global level of methylation.
Advances in whole genome profiling have revolutionized the cancer research field, but at the same time have raised new bioinformatics challenges. For next generation sequencing (NGS), these include data storage, computational costs, sequence processing and alignment, delineating appropriate statistical measures, and data visualization. Currently there is a lack of workflows for efficient analysis of large, MethylCap-seq datasets containing multiple sample groups.
The NGS application MethylCap-seq involves the in vitro capture of methylated DNA and subsequent analysis of enriched fragments by massively parallel sequencing. The workflow we describe performs MethylCap-seq experimental Quality Control (QC), sequence file processing and alignment, differential methylation analysis of multiple biological groups, hierarchical clustering, assessment of genome-wide methylation patterns, and preparation of files for data visualization.
Here, we present a scalable, flexible workflow for MethylCap-seq QC, secondary data analysis, tertiary analysis of multiple experimental groups, and data visualization. We demonstrate the experimental QC procedure with results from a large ovarian cancer study dataset and propose parameters which can identify problematic experiments. Promoter methylation profiling and hierarchical clustering analyses are demonstrated for four groups of acute myeloid leukemia (AML) patients. We propose a Global Methylation Indicator (GMI) function to assess genome-wide changes in methylation patterns between experimental groups. We also show how the workflow facilitates data visualization in a web browser with the application Anno-J.
This workflow and its suite of features will assist biologists in conducting methylation profiling projects and facilitate meaningful biological interpretation.
The transcriptional response driven by Hypoxia-inducible factor (HIF) is central to the adaptation to oxygen restriction. Despite recent characterization of genome-wide HIF DNA binding locations and hypoxia-regulated transcripts in different cell types, the molecular bases of HIF target selection remain unresolved. Herein, we combined multi-level experimental data and computational predictions to identify sequence motifs that may contribute to HIF target selectivity. We obtained a core set of bona fide HIF binding regions by integrating multiple HIF1 DNA binding and hypoxia expression profiling datasets. This core set exhibits evolutionarily conserved binding regions and is enriched in functional responses to hypoxia. Computational prediction of enriched transcription factor binding sites identified sequence motifs corresponding to several stress-responsive transcription factors, such as activator protein 1 (AP1), cAMP response element-binding (CREB), or CCAAT-enhancer binding protein (CEBP). Experimental validations on HIF-regulated promoters suggest a functional role of the identified motifs in modulating HIF-mediated transcription. Accordingly, transcriptional targets of these factors are over-represented in a sorted list of hypoxia-regulated genes. Altogether, our results implicate cooperativity among stress-responsive transcription factors in fine-tuning the HIF transcriptional response.
The Liebowitz Social Anxiety Scale (LSAS) is a widely used measure of social anxiety. However, no study has examined the psychometric properties of the LSAS in an African American sample. The current study examined the LSAS characteristics in 97 African Americans diagnosed with an anxiety disorder. Overall, the original LSAS subscales showed excellent internal consistency and temporal stability. Similar to previous reports, fear and avoidance subscales were so highly correlated that they yielded redundant information. Confirmatory factor analyses for three previously proposed models failed to demonstrate an excellent fit to our data. However, a four-factor model showed minimally acceptable fit. Overall, the LSAS performed similarly in our African American sample as in previous European American samples. Exploratory factor analyses are warranted to determine whether a better factor structure exists for African Americans.
LSAS; African American; psychometric; confirmatory factor analyses; anxiety
While tumor suppressor genes frequently undergo epigenetic silencing in cancer, how the instructions directing this transcriptional repression are transmitted in cancer cells remain largely unclear. Expression of cyclin-dependent kinase inhibitor 1C (CDKN1C), an imprinted gene on chromosomal band 11 p15.5, is reduced or lost in the majority of breast cancers. Here, we report that CDKN1C is suppressed by estrogen through epigenetic mechanisms involving the chromatin-interacting noncoding RNA KCNQ1OT1 and CCCTC-binding factor (CTCF). Activation of estrogen signaling reduced CDKN1C expression 3-fold (P < 0.001) and established repressive histone modifications at the 5′ regulatory region of the locus. These events were concomitant with induction of KCNQ1OT1 expression as well as increased recruitment of CTCF to both the distal KCNQ1OT1 promoter-associated imprinting control region (ICR) and the CDKN1C locus. Transient depletion of CTCF by small interfering RNA increased CDKN1C expression and significantly reduced the estrogen-mediated repression of CDKN1C. Further studies in breast cancer cell lines indicated that the epigenetic silencing of CDKN1C occurs in part as the result of genetic loss of the inactive methylated 11p15.5 ICR allele (R2 = 0.612, P < 0.001). We also found a novel cis-encoded antisense transcript, CDKN1C-AS, which is induced by estrogen signaling following pharmacologic inhibition of DNA methyltransferase and histone deacetylase activity. Forced expression of CDKN1C-AS was capable of repressing endogenous CDKN1C in vivo. Our findings suggest that in addition to promoter hypermethylation, epigenetic repression of tumor suppressor genes by CTCF and noncoding RNA transcripts could be more common and important than previously understood.
Advances in whole genome profiling have revolutionized the cancer research field, but at the same time have raised new bioinformatics challenges. For next generation sequencing (NGS), these include data storage, computational costs, sequence processing and alignment, delineating appropriate statistical measures, and data visualization. The NGS application MethylCap-seq involves the in vitro capture of methylated DNA and subsequent analysis of enriched fragments by massively parallel sequencing. Here, we present a scalable, flexible workflow for MethylCap-seq Quality Control, secondary data analysis, tertiary analysis of multiple experimental groups, and data visualization. This workflow and its suite of features will assist biologists in conducting methylation profiling projects and facilitate meaningful biological interpretation.
next generation sequencing; DNA methylation; epigenetics; cancer; data analysis; data visualization
The current study examined the naturalistic course of generalized anxiety disorder (GAD) in a sample of 113 primary care patients across a 2-year period. Initial diagnoses were established using structured clinical interviews according to DSM-IV diagnostic criteria. Results indicated that the majority of patients meeting DSM-IV diagnostic criteria for GAD were still symptomatic to some degree after 2 years of follow-up. Rates of full and partial recovery from GAD, however, were found to be higher than those reported for previous studies of GAD in psychiatric patients. Diagnostic comorbidity, severity of psychosocial impairment, and gender were found to be significantly associated with achieving full or partial recovery from GAD. Psychiatric treatment was not found to be associated with time to full or partial recovery from GAD symptoms, likely due to a treatment-biasing effect. These results underscore that GAD is a chronic and persistent illness in primary care patients.
GAD; psychiatric treatment; GAD course; anxiety; primary care
Posttraumatic stress disorder (PTSD) is receiving growing attention as a pervasive and impairing disorder but is still under-treated. Our purpose was to describe the characteristics of mental health treatment received by primary care patients diagnosed with PTSD.
4383 patients from 15 primary care, family practice, or internal medicine clinics were screened for anxiety symptoms using a self-report questionnaire developed for the study. Those found positive for anxiety symptoms (N = 539) were interviewed with the Structured Clinical Interview for DSM-IV. Of these patients, 197 met diagnostic criteria for PTSD and were examined in the present study regarding the rates and types of mental health treatment they were currently receiving. Data were gathered from July 1997 to May 2001.
Nearly half (48%) of the patients in general medical practice with PTSD were receiving no mental health treatment at the time of intake to the study. Of those receiving treatment, psychopharmacologic interventions were most common. Few patients were receiving empirically supported psychosocial interventions. Current comorbid major depressive disorder and current comorbid panic disorder with agoraphobia were significantly associated with receiving mental health treatment (major depressive disorder, p < .10; panic disorder with agoraphobia, p < .05). The most common reason patients gave for not receiving medication was the failure of physicians to recommend such treatment, which was also among the most common reasons for not receiving psychosocial treatment.
Despite the morbidity, psychosocial impairment, and distress associated with PTSD, substantial proportions of primary care patients with the disorder are going untreated or are receiving inadequate treatment. Results suggest a need for better identification and treatment of PTSD in the primary care setting.
The current report examines the rates of psychiatric comorbidity in a sample of 539 primary care patients diagnosed with anxiety disorders using the Structured Clinical Interview for DSM-IV (SCID-IV). Though not a typical psychiatric sample, rates of comorbidity were found to be as high or higher than those reported in studies conducted in traditional mental health settings. Multiple anxiety disorders were diagnosed in over 60% of participants and over 70% of participants had more than one current axis I diagnosis. Rates of current and lifetime comorbid major depression were also very high. Patterns of diagnostic comorbidity were also examined, with significantly elevated risks for the co-occurrence of several specific pairings of disorders being found. The study results are discussed in context of a recently published, large-scale study of anxiety disorder comorbidity in psychiatric patients (Brown et al., 2001). Implications of these results for both the mental health and primary care fields are also discussed.
The past decade witnessed considerable debate over the factor structure of the Anxiety Sensitivity Index (ASI), with an eventual consensus emerging that supported a hierarchically organized factor structure. The present study attempted to replicate and examine the overall stability and utility of the hierarchical ASI factor pattern using a large sample of outpatients participating in an ongoing longitudinal study of anxiety disorders. Results supported a hierarchical factor structure for the ASI consisting of three lower-order factors measuring physical concerns, mental incapacitation concerns, and social concerns, all of which loaded significantly on a single second-order factor. Correlational analyses show good test–retest reliability and consistent patterns of intercorrelation for these factor-derived subscales across a 10-month time frame. Additional analyses provide support for the discriminant validity of the ASI subscales with regard to individuals with specific anxiety disorders. The theoretical implications of these findings for future evaluations of anxiety sensitivity are discussed.
Anxiety sensitivity; Anxiety disorders
Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.
Bisphenol A; Estrogen; DNA methylation; Epigenetics; Breast cancer
Early exposure to xenoestrogens may predispose to breast cancer risk later in adult life. It is likely that long-lived, self-regenerating epithelial progenitor cells are more susceptible to these exposure injuries over time and transmit the injured memory through epigenetic mechanisms to their differentiated progeny. Here, we used progenitor-containing mammospheres as an in vitro exposure model to study this epigenetic effect. Expression profiling identified that, relative to control cells, 9.1% of microRNAs (82 of 898 loci) were altered in epithelial progeny derived from mammospheres exposed to a synthetic estrogen, diethylstilbestrol. Repressive chromatin marks, trimethyl Lys27 of histone H3 (H3K27me3) and dimethyl Lys9 of histone H3 (H3K9me2), were found at a down-regulated locus, miR-9-3, in epithelial cells preexposed to diethylstilbestrol. This was accompanied by recruitment of DNA methyltransferase 1 that caused an aberrant increase in DNA methylation of its promoter CpG island in mammosphere-derived epithelial cells on diethylstilbestrol preexposure. Functional analyses suggest that miR-9-3 plays a role in the p53-related apoptotic pathway. Epigenetic silencing of this gene, therefore, reduces this cellular function and promotes the proliferation of breast cancer cells. Promoter hypermethylation of this microRNA may be a hallmark for early breast cancer development, and restoration of its expression by epigenetic and microRNA-based therapies is another viable option for future treatment of this disease.
Several studies have reported that a high expression ratio of HOXB13 to IL17BR predicts tumor recurrence in node-negative, estrogen receptor (ER) α-positive breast cancer patients treated with tamoxifen. The molecular mechanisms underlying this dysregulation of gene expression remain to be explored. Our epigenetic analysis has found that increased promoter methylation of one of these genes, HOXB13, correlate with the decreased expression of its transcript in breast cancer cell lines (P < 0.005). Transcriptional silencing of this gene can be reversed by a demethylation treatment. HOXB13 is suppressed by the activation of estrogen signaling in ERα-positive breast cancer cells. However, treatment with 4-hydroxytamoxifen (4-OHT), an antiestrogen, abrogates the ERα-mediated suppression in cancer cells. The notion that this transcriptional induction of HOXB13 occurs in vitro with simultaneous exposure to both estrogen and 4-OHT may provide a biological explanation for its aberrant expression in many node-negative patients undergoing tamoxifen therapy. Interestingly, promoter hypermethylation of HOXB13 is more frequently observed in ERα-positive patients with increased lymph node metastasis (P = 0.031) and large tumor sizes (>5 cm) (P = 0.008). In addition, this aberrant epigenetic event is associated with shorter disease-free survival (P = 0.029) in cancer patients. These results suggest that hypermethylation of HOXB13 is a late event of breast tumorigenesis and a poor prognostic indicator of node-positive cancer patients.
Psychometric characteristics of the Mobility Inventory (MI) were examined in 216 outpatients diagnosed with panic disorder with agoraphobia participating in a longitudinal study of anxiety disorders. An exploratory principal components analysis replicated a three-component solution for the MI reported in prior studies, with components corresponding to avoidance of public spaces, avoidance of enclosed spaces, and avoidance of open spaces. Correlational analyses suggested that the components tap unique but related areas of avoidance that were remarkably stable across periods of 1,3, and 5 years between administrations. Implications of these results for future studies of agoraphobia are discussed.
Agoraphobia; Panic disorder; Mobility inventory; Factor analysis
Although the only widely accepted role for benzodiazepines in alcohol dependence is the treatment of withdrawal syndromes, they are frequently prescribed outside of this clinical setting. There is little empirical evidence to guide the rational use of benzodiazepines in the common clinical situation where anxiety disorders are comorbid with alcohol use disorders (AUD). Since January 1989, the Harvard Anxiety Research Program has naturalistically monitored the prospective clinical course of people with anxiety disorders, some of whom had a history of AUD. Earlier research showed that the use of benzodiazepines was not significantly associated with the presence or absence of a history of an AUD over the first year of follow-up. This report extends that investigation.
Using standard parametric analytic methods, patterns of benzodiazepine use (routinely prescribed medication and as-needed [PRN] use) among participants receiving benzodiazepine treatment was prospectively examined over the course of 12 years. Differences in benzodiazepine usage patterns were examined in each year of follow-up between participants who did (n = 120) and did not (n = 425) have a new episode of AUD. Using proportional hazards regression analysis, benzodiazepine usage levels were examined as predictors of recovery and recurrence of AUD. Additionally, random-effects regression analyses were used to examine the patterns of benzodiazepine use before and after the onset of a prospectively observed episode of AUD.
Benzodiazepine usage levels remained stable for the full sample over the course of the 12 years. Benzodiazepine use did not distinguish participants who had a new AUD from those who did not. Over the 12 years of follow-up, participants who had an AUD used more PRN medication in years five to eight. This difference reached statistical significance but was not clinically significant. Benzodiazepine usage levels did not predict recovery or recurrence in AUD subjects. Neither the total dose nor the PRN usage of benzodiazepines was significantly associated with the onset of AUD, but when combined into a measure of any benzodiazepine use, a relationship between increased use and the onset of AUD emerged.
For participants in the Harvard Anxiety Research Program with comorbid alcohol dependence and anxiety disorders, there was little association between the use of benzodiazepines and the occurrence of a new AUD. Neither was there a temporal relationship between the use of benzodiazepines and the onset of a new AUD. Whether or not this finding extends to a broader patient population or a group of people who present to addictions treatment awaits further investigation.
Benzodiazepines; Alcohol Use Disorders; Harvard Anxiety Research Program; Anxiety; Comorbid Alcohol Dependence; Anxiety Disorders
Hypermethylation of CpG islands is a common epigenetic alteration associated with cancer. Global patterns of hypermethylation are tumor-type specific and nonrandom. The biological significance and the underlying mechanisms of tumor-specific aberrant promoter methylation remain unclear, but some evidence suggests that this specificity involves differential sequence susceptibilities, the targeting of DNA methylation activity to specific promoter sequences, or the selection of rare DNA methylation events during disease progression. Using restriction landmark genomic scanning on samples derived from tissue culture and in vivo models of T cell lymphomas, we found that MYC overexpression gave rise to a specific signature of CpG island hypermethylation. This signature reflected gene transcription profiles and was detected only in advanced stages of disease. The further inactivation of the Pten, p53, and E2f2 tumor suppressors in MYC-induced lymphomas resulted in distinct and diagnostic CpG island methylation signatures. Our data suggest that tumor-specific DNA methylation in lymphomas arises as a result of the selection of rare DNA methylation events during the course of tumor development. This selection appears to be driven by the genetic configuration of tumor cells, providing experimental evidence for a causal role of DNA hypermethylation in tumor progression and an explanation for the tremendous epigenetic heterogeneity observed in the evolution of human cancers. The ability to predict genome-wide epigenetic silencing based on relatively few genetic alterations will allow for a more complete classification of tumors and understanding of tumor cell biology.
Genetic and epigenetic alterations of the genome are common features of cancers. The relationship between these two types of alterations, however, remains unclear. One type of epigenetic modification—DNA methylation in promoter sequences of genes—is of particular interest, since tumor cells have different patterns of promoter methylation than normal cells. Previous studies on human tumor samples have suggested a link between genetic alterations and the induction of aberrant DNA methylation; however, this link has been difficult to rigorously assess because of the incredible genetic heterogeneity found in human cancer. In this study, a mouse model of T cell lymphoma was used to explore the relationship between genetic and epigenetic modifications experienced by tumor cells. By introducing defined genetic changes into preneoplastic T cells of mice, such as the overexpression of the MYC oncogene and the ablation of tumor suppressor genes, we could carefully evaluate how these genetic changes impacted promoter methylation profiles during development of lymphomas in vivo. We found that the introduction of different genetic insults resulted in unique and diagnostic profiles of promoter methylation. Understanding how these methylation signatures contribute to tumor progression could eventually have diagnostic, prognostic, and therapeutic value for human cancers.
Among the psychological difficulties seen in children of parents with substance use problems, the anxiety disorders are among the most chronic conditions. Although children of alcoholic parents often struggle with the effects of parental substance use problems long into adulthood, empirical investigations of the influence of parental substance use disorders on the course of anxiety disorders in adult offspring are rare. The purpose of this study was to examine prospectively the relationship between parental substance use disorders and the course of anxiety disorders in adulthood over the course of 12 years.
Data on 618 subjects were derived from the Harvard/Brown Anxiety Research Project (HARP), a longitudinal naturalistic investigation of the clinical course of multiple anxiety disorders. Kaplan-Meier survival estimates were used to calculate probabilities of time to anxiety disorder remission and relapse. Proportional hazards regressions were conducted to determine whether the likelihood of remission and relapse for specific anxiety disorders was lower for those who had a history of parental substance use disorders than for individuals without this parental history.
Adults with a history of parental substance use disorders were significantly more likely to be divorced and to have a high school level of education. History of parental substance use disorder was a significant predictor of relapse of social phobia and panic disorders.
These findings provide compelling evidence that adult children of parents with substance use disorders are more likely to have relapses of social phobia and panic disorders. Clinicians who treat adults with anxiety disorders should assess parental substance use disorders and dependence histories. Such information may facilitate treatment planning with regards to their patients' level of vulnerability to perceive scrutiny by others in social situations, and ability to maintain a long-term panic-free state.