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1.  Fetal lung and placental methylation is associated with in utero nicotine exposure 
Epigenetics  2014;9(11):1473-1484.
In utero smoke exposure has been shown to have detrimental effects on lung function and to be associated with persistent wheezing and asthma in children. One potential mechanism of IUS effects could be alterations in DNA methylation, which may have life-long implications. The goal of this study was to examine the association between DNA methylation and nicotine exposure in fetal lung and placental tissue in early development; nicotine exposure in this analysis represents a likely surrogate for in-utero smoke. We performed an epigenome-wide analysis of DNA methylation in fetal lung tissue (n = 85, 41 smoke exposed (48%), 44 controls) and the corresponding placental tissue samples (n = 80, 39 smoke exposed (49%), 41 controls) using the Illumina HumanMethylation450 BeadChip array. Differential methylation analyses were conducted to evaluate the variation associated with nicotine exposure. The most significant CpG sites in the fetal lung analysis mapped to the PKP3 (P = 2.94 × 10−03), ANKRD33B (P = 3.12 × 10−03), CNTD2 (P = 4.9 × 10−03) and DPP10 (P = 5.43 × 10−03) genes. In the placental methylome, the most significant CpG sites mapped to the GTF2H2C and GTF2H2D genes (P = 2.87 × 10−06 − 3.48 × 10−05). One hundred and one unique CpG sites with P-values < 0.05 were concordant between lung and placental tissue analyses. Gene Set Enrichment Analysis demonstrated enrichment of specific disorders, such as asthma and immune disorders. Our findings demonstrate an association between in utero nicotine exposure and variable DNA methylation in fetal lung and placental tissues, suggesting a role for DNA methylation variation in the fetal origins of chronic diseases.
PMCID: PMC4623268  PMID: 25482056
asthma; developmental biology; epigenomics; nicotine and DNA methylation; smoking
2.  A genome-wide survey of CD4+ lymphocyte regulatory genetic variants identifies novel asthma genes 
Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing.
We evaluated 6,706 cis-acting expression-associated variants (eSNP) identified through a genome-wide eQTL survey of CD4+ lymphocytes for association with asthma.
eSNP were tested for association with asthma in 359 asthma cases and 846 controls from the Childhood Asthma Management Program, with verification using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE)-qPCR and Chromatin-Immunoprecipitation (ChIP)-PCR in lung derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes.
Cis-acting eSNP demonstrated associations with asthma in both cohorts. We confirmed the previously-reported association of ORMDL3/GSDMB variants with asthma (combined p=2.9 × 108). Reproducible associations were also observed for eSNP in three additional genes: FADS2 (p=0.002), NAGA (p=0.0002), and F13A1 (p=0.0001). We subsequently demonstrated that FADS2 mRNA is increased in CD4+ lymphocytes in asthmatics, and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity.
Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes, and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.
PMCID: PMC4253878  PMID: 24934276
Asthma; CD4+; lymphocytes; regulatory variants; Expression Quantitative Trait Locus (eQTL); Haplotype; Integrative Genomics
3.  Expression Quantitative Trait Loci Information Improves Predictive Modeling of Disease Relevance of Non-Coding Genetic Variation 
PLoS ONE  2015;10(10):e0140758.
Disease-associated loci identified through genome-wide association studies (GWAS) frequently localize to non-coding sequence. We and others have demonstrated strong enrichment of such single nucleotide polymorphisms (SNPs) for expression quantitative trait loci (eQTLs), supporting an important role for regulatory genetic variation in complex disease pathogenesis. Herein we describe our initial efforts to develop a predictive model of disease-associated variants leveraging eQTL information. We first catalogued cis-acting eQTLs (SNPs within 100kb of target gene transcripts) by meta-analyzing four studies of three blood-derived tissues (n = 586). At a false discovery rate < 5%, we mapped eQTLs for 6,535 genes; these were enriched for disease-associated genes (P < 10−04), particularly those related to immune diseases and metabolic traits. Based on eQTL information and other variant annotations (distance from target gene transcript, minor allele frequency, and chromatin state), we created multivariate logistic regression models to predict SNP membership in reported GWAS. The complete model revealed independent contributions of specific annotations as strong predictors, including evidence for an eQTL (odds ratio (OR) = 1.2–2.0, P < 10−11) and the chromatin states of active promoters, different classes of strong or weak enhancers, or transcriptionally active regions (OR = 1.5–2.3, P < 10−11). This complete prediction model including eQTL association information ultimately allowed for better discrimination of SNPs with higher probabilities of GWAS membership (6.3–10.0%, compared to 3.5% for a random SNP) than the other two models excluding eQTL information. This eQTL-based prediction model of disease relevance can help systematically prioritize non-coding GWAS SNPs for further functional characterization.
PMCID: PMC4608673  PMID: 26474488
4.  Genome-wide interaction studies reveal sex-specific asthma risk alleles 
Human Molecular Genetics  2014;23(19):5251-5259.
Asthma is a complex disease with sex-specific differences in prevalence. Candidate gene studies have suggested that genotype-by-sex interaction effects on asthma risk exist, but this has not yet been explored at a genome-wide level. We aimed to identify sex-specific asthma risk alleles by performing a genome-wide scan for genotype-by-sex interactions in the ethnically diverse participants in the EVE Asthma Genetics Consortium. We performed male- and female-specific genome-wide association studies in 2653 male asthma cases, 2566 female asthma cases and 3830 non-asthma controls from European American, African American, African Caribbean and Latino populations. Association tests were conducted in each study sample, and the results were combined in ancestry-specific and cross-ancestry meta-analyses. Six sex-specific asthma risk loci had P-values < 1 × 10−6, of which two were male specific and four were female specific; all were ancestry specific. The most significant sex-specific association in European Americans was at the interferon regulatory factor 1 (IRF1) locus on 5q31.1. We also identify a Latino female-specific association in RAP1GAP2. Both of these loci included single-nucleotide polymorphisms that are known expression quantitative trait loci and have been associated with asthma in independent studies. The IRF1 locus is a strong candidate region for male-specific asthma susceptibility due to the association and validation we demonstrate here, the known role of IRF1 in asthma-relevant immune pathways and prior reports of sex-specific differences in interferon responses.
PMCID: PMC4159149  PMID: 24824216
5.  Pharmacogenomics: novel loci identification via integrating gene differential analysis and eQTL analysis 
Human Molecular Genetics  2014;23(18):5017-5024.
Nearly one-half of asthmatic patients do not respond to the most commonly prescribed controller therapy, inhaled corticosteroids (ICS). We conducted an expression quantitative trait loci (eQTL) analysis using >300 expression microarrays (from 117 lymphoblastoid cell lines) in corticosteroid (dexamethasone) treated and untreated cells derived from asthmatic subjects in the Childhood Asthma Management Program (CAMP) clinical trial. We then tested the associations of eQTL with longitudinal change in airway responsiveness to methacholine (LnPC20) on ICS. We identified 2484 cis-eQTL affecting 767 genes following dexamethasone treatment. A significant over-representation of lnPC20-associated cis-eQTL [190 single-nucleotide polymorphisms (SNPs)] among differentially expressed genes (odds ratio = 1.76, 95% confidence interval: 1.35–2.29) was noted in CAMP Caucasians. Forty-six of these 190 clinical associations were replicated in CAMP African Americans, including seven SNPs near six genes meeting criteria for genome-wide significance (P < 2 × 10−7). Notably, the majority of genome-wide findings would not have been uncovered via analysis of untreated samples. These results indicate that identifying eQTL after relevant environmental perturbation enables identification of true pharmacogenetic variants.
PMCID: PMC4140460  PMID: 24770851
6.  Peripheral CD5+ B Cells in Antineutrophil Cytoplasmic Antibody–Associated Vasculitis 
CD5+ B cells have been conceptualized as a possible surrogate for Breg cells. The aim of the present study was to determine the utility of CD5+ B cells as biomarkers in antineutrophil cytoplasmic antibody–associated vasculitis (AAV).
The absolute and relative numbers (percentages) of CD5+ B cells (explanatory variables) were measured longitudinally during 18 months in 197 patients randomized to receive either rituximab (RTX) or cyclophosphamide (CYC) followed by azathioprine (AZA) for the treatment of AAV (Rituximab in ANCA-Associated Vasculitis [RAVE] trial). Outcome variables included disease activity (status of active disease versus complete remission), responsiveness to induction therapy, disease relapse, disease severity, and, in RTX-treated patients, relapse-free survival according to the percentage of CD5+ B cells detected upon B cell repopulation.
CD5+ B cell numbers were comparable between the treatment groups at baseline. After an initial decline, absolute CD5+ B cell numbers progressively increased in patients in the RTX treatment arm, but remained low in CYC/AZA-treated patients. In both groups, the percentage of CD5+ B cells increased during remission induction and slowly declined thereafter. During relapse, the percentage of CD5+ B cells correlated inversely with disease activity in RTX-treated patients, but not in patients who received CYC/AZA. No significant association was observed between the numbers of CD5+ B cells and induction treatment failure or disease severity. The dynamics of the CD5+ B cell compartment did not anticipate disease relapse. Following B cell repopulation, the percentage of CD5+ B cells was not predictive of time to flare in RTX-treated patients.
The percentage of peripheral CD5+ B cells might reflect disease activity in RTX-treated patients. However, sole staining for CD5 as a putative surrogate marker for Breg cells did not identify a subpopulation of B cells with clear potential for meaningful clinical use. Adequate phenotyping of Breg cells is required to further explore the value of these cells as biomarkers in AAV.
PMCID: PMC4497572  PMID: 25332071
7.  Orchestrating high-throughput genomic analysis with Bioconductor 
Nature methods  2015;12(2):115-121.
Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors.
PMCID: PMC4509590  PMID: 25633503
8.  VariantAnnotation: a Bioconductor package for exploration and annotation of genetic variants 
Bioinformatics  2014;30(14):2076-2078.
Summary: VariantAnnotation is an R / Bioconductor package for the exploration and annotation of genetic variants. Capabilities exist for reading, writing and filtering variant call format (VCF) files. VariantAnnotation allows ready access to additional R / Bioconductor facilities for advanced statistical analysis, data transformation, visualization and integration with diverse genomic resources.
Availability and implementation: This package is implemented in R and available for download at the Bioconductor Web site ( The package contains extensive help pages for individual functions and a ‘vignette’ outlining typical work flows; it is made available under the open source ‘Artistic-2.0’ license. Version 1.9.38 was used in this article.
PMCID: PMC4080743  PMID: 24681907
9.  The Bioconductor channel in F1000Research  
F1000Research  2015;4:217.
Bioconductor ( is a rich source of software and know-how for the integrative analysis of genomic data. The Bioconductor channel in F1000Research provides a forum for task-oriented workflows that each cover a solution to a current, important problem in genome-scale data analysis from end to end, invoking resources from several packages by different authors, often combining multiple `omics data types, and demonstrating integrative analysis and modelling techniques.
PMCID: PMC4786899  PMID: 26998224
Bioconductor; bioinformatics; genomics; systems biology; R; statistical computing
10.  Effects of High vs Low Glycemic Index of Dietary Carbohydrate on Cardiovascular Disease Risk Factors and Insulin Sensitivity 
JAMA  2014;312(23):2531-2541.
Foods that have similar carbohydrate content can differ in the amount they raise blood glucose. The effects of this property, called the glycemic index, on risk factors for cardiovascular disease and diabetes are not well understood.
To determine the effect of glycemic index and amount of total dietary carbohydrate on risk factors for cardiovascular disease and diabetes.
Randomized crossover-controlled feeding trial conducted in research units in academic medical centers, in which 163 overweight adults (systolic blood pressure, 120–159 mm Hg) were given 4 complete diets that contained all of their meals, snacks, and calorie-containing beverages, each for 5 weeks, and completed at least 2 study diets. The first participant was enrolled April 1, 2008; the last participant finished December 22, 2010. For any pair of the 4 diets, there were 135 to 150 participants contributing at least 1 primary outcome measure.
(1) A high–glycemic index (65% on the glucose scale), high-carbohydrate diet (58% energy); (2) a low–glycemic index (40%), high-carbohydrate diet; (3) a high–glycemic index, low-carbohydrate diet (40% energy); and (4) a low–glycemic index, low-carbohydrate diet. Each diet was based on a healthful DASH-type diet.
The 5 primary outcomes were insulin sensitivity, determined from the areas under the curves of glucose and insulin levels during an oral glucose tolerance test; levels of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides; and systolic blood pressure.
At high dietary carbohydrate content, the low– compared with high–glycemic index level decreased insulin sensitivity from 8.9 to 7.1 units (−20%, P = .002); increased LDL cholesterol from 139 to 147 mg/dL (6%, P ≤ .001); and did not affect levels of HDL cholesterol, triglycerides, or blood pressure. At low carbohydrate content, the low– compared with high–glycemic index level did not affect the outcomes except for decreasing triglycerides from 91 to 86 mg/dL (−5%, P = .02). In the primary diet contrast, the low–glycemic index, low-carbohydrate diet, compared with the high–glycemic index, high-carbohydrate diet, did not affect insulin sensitivity, systolic blood pressure, LDL cholesterol, or HDL cholesterol but did lower triglycerides from 111 to 86 mg/dL (−23%, P ≤ .001).
In this 5-week controlled feeding study, diets with low glycemic index of dietary carbohydrate, compared with high glycemic index of dietary carbohydrate, did not result in improvements in insulin sensitivity, lipid levels, or systolic blood pressure. In the context of an overall DASH-type diet, using glycemic index to select specific foods may not improve cardiovascular risk factors or insulin resistance.
TRIAL REGISTRATION Identifier: NCT00608049
PMCID: PMC4370345  PMID: 25514303
11.  The Vitamin D Antenatal Asthma Reduction Trial (VDAART): Rationale, design, and methods of a randomized, controlled trial of vitamin D supplementation in pregnancy for the primary prevention of asthma and allergies in children 
Contemporary clinical trials  2014;38(1):37-50.
There is intense interest in the role of vitamin D in the development of asthma and allergies. However, studies differ on whether a higher vitamin D intake or status in pregnancy or at birth is protective against asthma and allergies. To address this uncertainty, the Vitamin D Antenatal Asthma Reduction Trial (VDAART) was developed. VDAART is a randomized, double-blind, placebo-controlled trial of vitamin D supplementation in pregnant women to determine whether prenatal supplementation can prevent the development of asthma and allergies in the women’s offspring. A secondary aim is to determine whether vitamin D supplementation can prevent the development of pregnancy complications, such as preeclampsia, preterm birth, and gestational diabetes. Women were randomized to the treatment arm of 4,000 IU/day of vitamin D3 plus a daily multivitamin that contained 400 IU of vitamin D3 or the placebo arm of placebo plus a multivitamin that contained 400 IU daily of vitamin D3. Women who were between the gestational ages of 10–18 weeks were randomized from three clinical centers across the United States – Boston Medical Center, Washington University in St. Louis, and Kaiser Permanente Southern California Region (San Diego, CA). Supplementation took place throughout pregnancy. Monthly monitoring of urinary calcium to creatinine ratio was performed in addition to medical record review for adverse events. Offspring are being evaluated quarterly through questionnaires and yearly during in-person visits until the 3rd birthday of the child. Ancillary studies will investigate neonatal T-regulatory cell function, maternal vaginal flora, and maternal and child intestinal flora.
PMCID: PMC4086903  PMID: 24614387
Vitamin D; asthma; allergy; randomized controlled trial; Deveopmental Origins; prenatal
Nature communications  2014;5:4753.
Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here, we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian program exhibits unique features, including a divergent group of rhythmic genes and metabolites compared to the basal state and a distinct periodicity and phase distribution. At the cellular level endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1-dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex reorganization of cellular and molecular circadian rhythms that are relevant to early events in lung injury.
PMCID: PMC4162491  PMID: 25208554
13.  Maternal Antibody at Delivery Protects Neonates From Early Onset Group B Streptococcal Disease 
The Journal of Infectious Diseases  2013;209(5):781-788.
Background. Further reduction in the group B streptococcal (GBS) disease burden in neonates in the United States awaits an additional prevention strategy, such as maternal immunization.
Methods. We performed a prospective, multicenter, case-control study of 33 mothers delivering neonates with early onset GBS infection (cases), and 99 age- and ethnicity-matched mothers colonized with the same capsular polysaccharide (CPS) types delivering healthy neonates (controls). Relative risk and absolute risk were calculated for early onset disease associated with concentrations of type Ia, III, or V CPS-specific antibody in maternal serum.
Results. For GBS types Ia and III, maternal CPS–specific antibody concentrations of ≥0.5 µg/mL were associated with a relative risk of approximately 0.1 (95% confidence intervals [CIs], .01–.74 and 0–.72, respectively; P = .02 for each), corresponding to a 90% risk reduction (by logistic regression). For type V, the relative risk was 0.3 (95% CI, .01–3.1), corresponding to a 70% risk reduction. By Bayesian modeling, the risk of early onset disease would decrease by 70% if maternal CPS-specific antibody concentrations for these 3 GBS types were ≥1 µg/mL.
Conclusions. Maternal CPS-specific antibody serum concentrations of ≥1 μg/mL at the time of delivery appear to protect most neonates from early onset GBS type Ia and III disease.
PMCID: PMC3923540  PMID: 24133184
Group B Streptococcus; neonate; neonatal sepsis; meningitis; glycoconjugate vaccine; immunization; serocorrelate; protective immunity
14.  Diet Type and Changes in Food Cravings following Weight Loss: Findings from the POUNDS LOST Trial 
Eating and weight disorders : EWD  2012;17(2):e101-e108.
Few well-controlled trials have evaluated the effects that macronutrient composition has on changes in food cravings during weight loss treatment. The present study, which was part of the POUNDS LOST trial, investigated whether the fat and protein content of four different diets affected changes in specific food cravings in overweight and obese adults. A sample of 811 adults were recruited across two clinical sites, and each participant was randomly assigned to one of four macronutrient prescriptions: (1) Low fat (20% of energy), average protein (15% of energy); (2) Moderate fat (40%), average protein (15%); (3) Low fat (20%), high protein (25%); (4) Moderate fat (40%), high protein (25%). With few exceptions, the type of diet that participants were assigned did not differentially affect changes in specific food cravings. Participants assigned to the high fat diets, however, had reduced cravings for carbohydrates at Month12 (p< .05) and fruits and vegetables at Month 24. Also, participants assigned to high protein diets had increased cravings for sweets at Month 6 (p< .05). Participants in all four dietary conditions reported significant reductions in food cravings for specific types of foods (i.e., high fat foods, fast food fats, sweets, and carbohydrates/starches; all ps< .05). Cravings for fruits and vegetables, however, were increased at Month 24 (p< .05). Calorically restricted diets (regardless of their macronutrient composition) yielded significant reductions in cravings for fats, sweets, and starches whereas cravings for fruits and vegetables were increased.
PMCID: PMC4189179  PMID: 23010779
Macronutrient composition; Caloric restriction; Food type; Fat; Carbohydrate; Protein
15.  Contribution of High Plasma Triglycerides and Low High-Density Lipoprotein Cholesterol to Residual Risk of Coronary Heart Disease After Establishment of Low-Density Lipoprotein Cholesterol Control 
The American journal of cardiology  2010;106(6):757-763.
To determine the relative contributions of triglycerides (TGs) and high-density lipoprotein (HDL) cholesterol in the residual risk of coronary heart disease (CHD) after the reduction of low-density lipoprotein (LDL) cholesterol to guideline-recommended levels, we conducted a hospital-based, case-control study with optimal matching in the strata of LDL cholesterol, gender, ethnicity, and age. The 170 cases and 175 controls were patients at Brigham and Women's Hospital (Boston, Massachusetts) from 2005 to 2008 who had an LDL cholesterol level <130 mg/dl. The cases had incident CHD, and the controls had diagnoses unrelated to CHD. The 170 cases and 175 controls had a mean LDL cholesterol level of 73 and 87 mg/dl, respectively. The association between TG and HDL cholesterol levels and CHD risk was assessed using conditional and unconditional logistic regression analysis. The models investigated accommodated the possibility of an interaction between lipid factors. The odds of CHD increased by approximately 20% per 23-mg/dl increase in TGs and decreased by approximately 40% per 7.5-mg/dl decrease in HDL cholesterol. High TGs and low HDL cholesterol interacted synergistically to increase the odds ratio to 10 for the combined greatest TG (≥190 mg/dl) and lowest HDL cholesterol quintiles (<30 mg/dl). High TG levels were more strongly associated with CHD when the HDL cholesterol was low than average or high; and low HDL cholesterol levels were more strongly associated with CHD when the TGs were high. TGs and HDL cholesterol were associated with CHD in patients with a LDL cholesterol level of ≤70 mg/dl, with a risk similar to, or greater than, those in the total group. In conclusion, high TG and low HDL cholesterol levels contribute strongly and synergistically to CHD when LDL cholesterol is well controlled. Thus, high TGs might have greater importance in patients with optimal rather than greater LDL cholesterol concentrations.
PMCID: PMC4102341  PMID: 20816113
16.  Prenatal Tobacco Smoke Exposure Is Associated with Childhood DNA CpG Methylation 
PLoS ONE  2014;9(6):e99716.
Smoking while pregnant is associated with a myriad of negative health outcomes in the child. Some of the detrimental effects may be due to epigenetic modifications, although few studies have investigated this hypothesis in detail.
To characterize site-specific epigenetic modifications conferred by prenatal smoking exposure within asthmatic children.
Using Illumina HumanMethylation27 microarrays, we estimated the degree of methylation at 27,578 distinct DNA sequences located primarily in gene promoters using whole blood DNA samples from the Childhood Asthma Management Program (CAMP) subset of Asthma BRIDGE childhood asthmatics (n = 527) ages 5–12 with prenatal smoking exposure data available. Using beta-regression, we screened loci for differential methylation related to prenatal smoke exposure, adjusting for gender, age and clinical site, and accounting for multiple comparisons by FDR.
Of 27,578 loci evaluated, 22,131 (80%) passed quality control assessment and were analyzed. Sixty-five children (12%) had a history of prenatal smoke exposure. At an FDR of 0.05, we identified 19 CpG loci significantly associated with prenatal smoke, of which two replicated in two independent populations. Exposure was associated with a 2% increase in mean CpG methylation in FRMD4A (p = 0.01) and Cllorf52 (p = 0.001) compared to no exposure. Four additional genes, XPNPEP1, PPEF2, SMPD3 and CRYGN, were nominally associated in at least one replication group.
These data suggest that prenatal exposure to tobacco smoke is associated with reproducible epigenetic changes that persist well into childhood. However, the biological significance of these altered loci remains unknown.
PMCID: PMC4070909  PMID: 24964093
17.  Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence 
Journal of Virology  2013;87(22):12270-12283.
Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates the maintenance of episomal viral genomes in latently infected cells. The two central components of episome persistence are DNA replication with each cell division and the segregation of DNA to progeny nuclei. LANA self-associates to bind KSHV terminal-repeat (TR) DNA and to mediate its replication. LANA also simultaneously binds to TR DNA and mitotic chromosomes to mediate the segregation of episomes to daughter nuclei. The N-terminal region of LANA binds histones H2A and H2B to attach to mitotic chromosomes, while the C-terminal region binds TR DNA and also associates with chromosomes. Both the N- and C-terminal regions of LANA are essential for episome persistence. We recently showed that deletion of all internal LANA sequences results in highly deficient episome maintenance. Here we assess independent internal LANA regions for effects on episome persistence. We generated a panel of LANA mutants that included deletions in the large internal repeat region and in the unique internal sequence. All mutants contained the essential N- and C-terminal regions, and as expected, all maintained the ability to associate with mitotic chromosomes in a wild-type fashion and to bind TR DNA, as assessed by electrophoretic mobility shift assays (EMSA). Deletion of the internal regions did not reduce the half-life of LANA. Notably, deletions within either the repeat elements or the unique sequence resulted in deficiencies in DNA replication. However, only the unique internal sequence exerted effects on the ability of LANA to retain green fluorescent protein (GFP) expression from TR-containing episomes deficient in DNA replication, consistent with a role in episome segregation; this region did not independently associate with mitotic chromosomes. All mutants were deficient in episome persistence, and the deficiencies ranged from minor to severe. Mutants deficient in DNA replication that contained deletions within the unique internal sequence had the most-severe deficits. These data suggest that internal LANA regions exert critical roles in LANA-mediated DNA replication, segregation, and episome persistence, likely through interactions with key host cell factors.
PMCID: PMC3807934  PMID: 24006437
18.  Systemic Steroid Exposure Is Associated with Differential Methylation in Chronic Obstructive Pulmonary Disease 
Rationale: Systemic glucocorticoids are used therapeutically to treat a variety of medical conditions. Epigenetic processes such as DNA methylation may reflect exposure to glucocorticoids and may be involved in mediating the responses and side effects associated with these medications.
Objectives: To test the hypothesis that differences in DNA methylation are associated with current systemic steroid use.
Methods: We obtained DNA methylation data at 27,578 CpG sites in 14,475 genes throughout the genome in two large, independent cohorts: the International COPD Genetics Network (ndiscovery = 1,085) and the Boston Early Onset COPD study (nreplication = 369). Sites were tested for association with current systemic steroid use using generalized linear mixed models.
Measurements and Main Results: A total of 511 sites demonstrated significant differential methylation by systemic corticosteroid use in all three of our primary models. Pyrosequencing validation confirmed robust differential methylation at CpG sites annotated to genes such as SLC22A18, LRP3, HIPK3, SCNN1A, FXYD1, IRF7, AZU1, SIT1, GPR97, ABHD16B, and RABGEF1. Functional annotation clustering demonstrated significant enrichment in intrinsic membrane components, hemostasis and coagulation, cellular ion homeostasis, leukocyte and lymphocyte activation and chemotaxis, protein transport, and responses to nutrients.
Conclusions: Our analyses suggest that systemic steroid use is associated with site-specific differential methylation throughout the genome. Differentially methylated CpG sites were found in biologically plausible and previously unsuspected pathways; these genes and pathways may be relevant in the development of novel targeted therapies.
PMCID: PMC3622442  PMID: 23065012
DNA methylation; glucocorticoids; chronic obstructive pulmonary disease
19.  Software for Computing and Annotating Genomic Ranges 
PLoS Computational Biology  2013;9(8):e1003118.
We describe Bioconductor infrastructure for representing and computing on annotated genomic ranges and integrating genomic data with the statistical computing features of R and its extensions. At the core of the infrastructure are three packages: IRanges, GenomicRanges, and GenomicFeatures. These packages provide scalable data structures for representing annotated ranges on the genome, with special support for transcript structures, read alignments and coverage vectors. Computational facilities include efficient algorithms for overlap and nearest neighbor detection, coverage calculation and other range operations. This infrastructure directly supports more than 80 other Bioconductor packages, including those for sequence analysis, differential expression analysis and visualization.
PMCID: PMC3738458  PMID: 23950696
20.  Isoniazid pharmacokinetics, pharmacodynamics and dosing in South African infants 
Therapeutic Drug Monitoring  2012;34(4):446-451.
There are limited data on isoniazid (INH) pharmacokinetics in infants and young children and, therefore, uncertainty on appropriate dosing.
Pharmacokinetic data were obtained from perinatally HIV-exposed South African infants ages 3–24 months receiving INH 10–20 mg/kg/day orally for Mycobacterium tuberculosis (TB) prophylaxis. INH pharmacokinetic parameters were characterized with a population pharmacokinetic approach. Dosing simulations were performed to evaluate weight-based INH doses in children based on N-acetyltransferase 2 enzyme (NAT2) genotype, age, maximum concentrations (Cmax) ≥ 3mg/L, and area under the curve (AUC0-24) ≥ 10.52 mg*hr/L.
In 151 infants (53% female, 48% HIV positive) receiving a mean INH dose of 14.5 mg/kg/day, mean (±SD) Cmax at 3, 6, and 23 months of age were 10.0 (3.5), 8.6 (2.6), and 9.3 (3.8) mg/L, respectively, mean (±SD) AUC0-24 were 53.6 (26.8), 42 (19.9), and 44 (30.7) mg*hr/L, respectively, and mean (±SD) half-life were 2.1 (0.7), 1.9 (0.6), and 1.8 (0.9) hours, respectively. A trimodal apparent oral clearance of INH as a function of NAT2 genotype was apparent as early as 3 months. INH was well tolerated. At an average INH dose of 14.5 mg/kg/day, 99% of infants ages 3–24 months have an INH Cmax ≥ 3 mg/L and 98% have an INH AUC0-24 ≥ 10.52 mg*hr/L.
INH at an average dose of 14.5 mg/kg once daily was well tolerated in infants and achieved INH Cmax values ≥ 3 mg/L and AUC0-24 values ≥ 10.52 mg*hr/L.
PMCID: PMC3397663  PMID: 22695364
isoniazid; pharmacokinetics; dosing; infants; children
21.  The Impact of Self-Identified Race on Epidemiologic Studies of Gene Expression 
Genetic epidemiology  2011;35(2):93-101.
Although population differences in gene expression have been established, the impact on differential gene expression studies in large populations is not well understood. We describe the effect of self-reported race on a gene expression study of lung function in asthma. We generated gene expression profiles for 254 young adults (205 non-Hispanic whites and 49 African Americans) with asthma on whom concurrent total RNA derived from peripheral blood CD4+ lymphocytes and lung function measurements were obtained. We identified four principal components that explained 62% of the variance in gene expression. The dominant principal component, which explained 29% of the total variance in gene expression, was strongly associated with self-identified race (P<10−16). The impact of these racial differences was observed when we performed differential gene expression analysis of lung function. Using multivariate linear models, we tested whether gene expression was associated with a quantitative measure of lung function: pre-bronchodilator forced expiratory volume in one second (FEV1). Though unadjusted linear models of FEV1 identified several genes strongly correlated with lung function, these correlations were due to racial differences in the distribution of both FEV1 and gene expression, and were no longer statistically significant following adjustment for self-identified race. These results suggest that self-identified race is a critical confounding covariate in epidemiologic studies of gene expression and that, similar to genetic studies, careful consideration of self-identified race in gene expression profiling studies is needed to avoid spurious association.
PMCID: PMC3718033  PMID: 21254216
ancestry; gene expression; population stratification; self-identified race
22.  Cigarette smoking behaviors and time since quitting are associated with differential DNA methylation across the human genome 
Human Molecular Genetics  2012;21(13):3073-3082.
The impact of cigarette smoking can persist for extended periods following smoking cessation and may involve epigenetic reprogramming. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to the latency between exposure and disease onset. Cross-sectional cohort data from subjects in the International COPD Genetics Network (n = 1085) and the Boston Early-Onset COPD study (n = 369) were analyzed as the discovery and replication cohorts, respectively. Genome-wide methylation data on 27 578 CpG sites in 14 475 genes were obtained on DNA from peripheral blood leukocytes using the Illumina HumanMethylation27K Beadchip in both cohorts. We identified 15 sites significantly associated with current smoking, 2 sites associated with cumulative smoke exposure, and, within the subset of former smokers, 3 sites associated with time since quitting cigarettes. Two loci, factor II receptor-like 3 (F2RL3) and G-protein-coupled receptor 15 (GPR15), were significantly associated in all three analyses and were validated by pyrosequencing. These findings (i) identify a novel locus (GPR15) associated with cigarette smoking and (ii) suggest the existence of dynamic, site-specific methylation changes in response to smoking which may contribute to the extended risks associated with cigarette smoking that persist after cessation.
PMCID: PMC3373248  PMID: 22492999
23.  Variable DNA Methylation Is Associated with Chronic Obstructive Pulmonary Disease and Lung Function 
Rationale: Chronic obstructive pulmonary disease (COPD) is associated with local (lung) and systemic (blood) inflammation and manifestations. DNA methylation is an important regulator of gene transcription, and global and specific gene methylation marks may vary with cigarette smoke exposure.
Objectives: To perform a comprehensive assessment of methylation marks in DNA from subjects well phenotyped for nonneoplastic lung disease.
Methods: We conducted array-based methylation screens, using a test-replication approach, in two family-based cohorts (n = 1,085 and 369 subjects).
Measurements and Main Results: We observed 349 CpG sites significantly associated with the presence and severity of COPD in both cohorts. Seventy percent of the associated CpG sites were outside of CpG islands, with the majority of CpG sites relatively hypomethylated. Gene ontology analysis based on these 349 CpGs (330 genes) suggested the involvement of a number of genes responsible for immune and inflammatory system pathways, responses to stress and external stimuli, as well as wound healing and coagulation cascades. Interestingly, our observations include significant, replicable associations between SERPINA1 hypomethylation and COPD and lower average lung function phenotypes (combined P values: COPD, 1.5 × 10−23; FEV1/FVC, 1.5 × 10−35; FEV1, 2.2 × 10−40).
Conclusions: Genetic and epigenetic pathways may both contribute to COPD. Many of the top associations between COPD and DNA methylation occur in biologically plausible pathways. This large-scale analysis suggests that DNA methylation may be a biomarker of COPD and may highlight new pathways of COPD pathogenesis.
PMCID: PMC3297093  PMID: 22161163
chronic obstructive pulmonary disease; epigenetics; DNA methylation; smoking
24.  Effect of Diet Composition on Energy Expenditure during Weight Loss: The POUNDS LOST Study 
Weight loss reduces energy expenditure, but the contribution of different macronutrients to this change is unclear.
We tested the hypothesis that macronutrient composition of the diet might affect the partitioning of energy expenditure during weight loss.
A sub-study of 99 participants from the POUNDS LOST trial had total energy expenditure (TEE) measured by doubly labeled water and resting energy expenditure (REE) measured by indirect calorimetry at baseline and repeated at 6 months in 89 participants. Participants were randomly assigned to one of 4 diets with either 15% or 25% protein and 20% or 40% fat.
TEE and REE were positively correlated with each other and with fat free mass and body fat, at baseline and 6 months. The average weight loss of 8.1±0.65 kg (LSmean±SE) reduced TEE by 120±56 kcal/d and REE by 136±18 kcal/d. A greater weight loss at 6 months was associated with a greater decrease in TEE and REE. Participants eating the high fat diet lost significantly more fat free mass (1.52±0.55 kg) than the low fat diet group (p<0.05). Participants eating the low fat diet had significantly higher measures of physical activity than the high fat group.
A greater weight loss was associated with a larger decrease in both TEE and REE. The low fat diet was associated with significant changes in fat free body mass and energy expenditure from physical activity compared to the high fat diet.
PMCID: PMC3289771  PMID: 21946707

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