Xinhui Wang

Genetic and epigenetic variations in inducible nitric oxide synthase promoter, particulate pollution, and exhaled nitric oxide levels in children

BACKGROUND Inducible nitric oxide synthase (iNOS; encoded by nitric oxide synthase isoform 2 [NOS2]) is the major enzyme for nitric oxide synthesis in airways. As such, measurement of fractional concentration of exhaled nitric oxide (Feno) provides an in vivo assessment of iNOS activity. Short-term exposure to air pollution, haplotypes, and DNA methylation in the NOS2 promoter has been associated independently with iNOS expression, Feno levels, or both. OBJECTIVE We aimed to examine the effects of ambient air pollutants, NOS2 promoter haplotypes, and NOS2 promoter methylation on Feno levels in children. METHODS We selected 940 participants in the Childrens Health Study who provided buccal samples and had undergone Feno measurement on the same day. DNA methylation was measured with a bisulfite-PCR Pyrosequencing assay. Seven single nucleotide polymorphisms captured the haplotype diversity in the NOS2 promoter. Average particulate matter with an aerodynamic diameter of 2.5 μm or less (PM(2.5)) and 10 μm (PM(10)) or less and ozone and nitrogen dioxide levels 7 days before Feno measurement were estimated based on air pollution data obtained at central monitoring sites. RESULTS We found interrelated effects of PM(2.5), NOS2 promoter haplotypes, and iNOS methylation on Feno levels. Increased 7-day average PM(2.5) exposure was associated with lower iNOS methylation (P = .01). NOS2 promoter haplotypes were globally associated with NOS2 promoter methylation (P = 6.2 × 10(-8)). There was interaction among 1 common promoter haplotype, iNOS methylation level, and PM(2.5) exposure on Feno levels (P(interaction) = .00007). CONCLUSION Promoter variants in NOS2 and short-term PM(2.5) exposure affect iNOS methylation. This is one of the first studies showing contributions of genetic and epigenetic variations in air pollution-mediated phenotype expression.

Prenatal Tobacco Smoke Exposure Is Associated with Childhood DNA CpG Methylation

Background 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. Objectives To characterize site-specific epigenetic modifications conferred by prenatal smoking exposure within asthmatic children. Methods 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. Results 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. Conclusions 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.

DNA Methylation in the Arginase–Nitric Oxide Synthase Pathway Is Associated with Exhaled Nitric Oxide in Children with Asthma

RATIONALE Genetic variation in arginase (ARG) and nitric oxide synthase (NOS) has been associated with exhaled nitric oxide (FeNO) levels in children. Little is known about whether epigenetic variation in these genes modulates FeNO. OBJECTIVES To evaluate whether DNA methylation in ARG and NOS genes is associated with FeNO. METHODS A subset of 940 participants in the Childrens Health Study were selected for this study. Children were eligible if they had FeNO measurements and buccal cells collected on the same day. CpG loci located in the promoter regions of NOS1, NOS2A, NOS3, ARG1, and ARG2 genes were analyzed. Multiple loci in each gene were evaluated individually and averaged together. DNA methylation was measured using a bisulfite-polymerase chain reaction pyrosequencing assay. Linear regression models were used to investigate the association between DNA methylation and FeNO and whether associations differed by asthma status. MEASUREMENTS AND MAIN RESULTS DNA methylation in ARG2 was significantly associated with FeNO. A 1% increase in average DNA methylation of ARG2 was associated with a 2.3% decrease in FeNO (95% confidence interval, -4 to -0.6). This association was significantly larger in children with asthma (%diff = -8.7%) than in children with no asthma (%diff = -1.6%; p(int) = 0.01). Differences in FeNO by asthma status were also observed for ARG1 (%diff(asthma) = -4.4%; %diff(non-asthma) = 0.3%; p(int) = 0.02). DNA methylation in NOS genes was not associated with FeNO. CONCLUSIONS DNA methylation in ARG1 and ARG2 is associated with FeNO in children with asthma and suggests a possible role for epigenetic regulation of nitric oxide production.

Particulate air pollutants, APOE alleles and their contributions to cognitive impairment in older women and to amyloidogenesis in experimental models.

Exposure to particulate matter (PM) in the ambient air and its interactions with APOE alleles may contribute to the acceleration of brain aging and the pathogenesis of Alzheimer’s disease (AD). Neurodegenerative effects of particulate air pollutants were examined in a US-wide cohort of older women from the Women’s Health Initiative Memory Study (WHIMS) and in experimental mouse models. Residing in places with fine PM exceeding EPA standards increased the risks for global cognitive decline and all-cause dementia respectively by 81 and 92%, with stronger adverse effects in APOE ɛ4/4 carriers. Female EFAD transgenic mice (5xFAD+/−/human APOE ɛ3 or ɛ4+/+) with 225 h exposure to urban nanosized PM (nPM) over 15 weeks showed increased cerebral β-amyloid by thioflavin S for fibrillary amyloid and by immunocytochemistry for Aβ deposits, both exacerbated by APOE ɛ4. Moreover, nPM exposure increased Aβ oligomers, caused selective atrophy of hippocampal CA1 neurites, and decreased the glutamate GluR1 subunit. Wildtype C57BL/6 female mice also showed nPM-induced CA1 atrophy and GluR1 decrease. In vitro nPM exposure of neuroblastoma cells (N2a-APP/swe) increased the pro-amyloidogenic processing of the amyloid precursor protein (APP). We suggest that airborne PM exposure promotes pathological brain aging in older women, with potentially a greater impact in ɛ4 carriers. The underlying mechanisms may involve increased cerebral Aβ production and selective changes in hippocampal CA1 neurons and glutamate receptor subunits.

Particulate Matter, DNA Methylation in Nitric Oxide Synthase, and Childhood Respiratory Disease

Background: Air pollutants have been associated with childhood asthma and wheeze. Epigenetic regulation of nitric oxide synthase—the gene responsible for nitric oxide production—may be affected by air pollutants and contribute to the pathogenesis of asthma and wheeze. Objective: Our goal was to investigate the association between air pollutants, DNA methylation, and respiratory outcomes in children. Methods: Given residential address and buccal sample collection date, we estimated 7-day, 1-month, 6-month, and 1-year cumulative average PM2.5 and PM10 (particulate matter ≤ 2.5 and ≤ 10 µm aerodynamic diameter, respectively) exposures for 940 participants in the Children’s Health Study. Methylation of 12 CpG sites in three NOS (nitric oxide synthase) genes was measured using a bisulfite-polymerase chain reaction Pyrosequencing assay. Beta regression models were used to estimate associations between air pollutants, percent DNA methylation, and respiratory outcomes. Results: A 5-µg/m3 increase in PM2.5 was associated with a 0.20% [95% confidence interval (CI): –0.32, –0.07] to 1.0% (95% CI: –1.61, –0.56) lower DNA methylation at NOS2A position 1, 0.06% (95% CI: –0.18, 0.06) to 0.58% (95% CI: –1.13, –0.02) lower methylation at position 2, and 0.34% (95% CI: –0.57, –0.11) to 0.89% (95% CI: –1.57, –0.21) lower methylation at position 3, depending on the length of exposure and CpG locus. One-year PM2.5 exposure was associated with 0.33% (95% CI: 0.01, 0.65) higher in average DNA methylation of 4 loci in the NOS2A CpG island. A 5-µg/m3 increase in 7-day and 1-year PM2.5 was associated with 0.6% (95% CI: 0.13, 0.99) and 2.8% (95% CI: 1.77, 3.75) higher NOS3 DNA methylation. No associations were observed for NOS1. PM10 showed similar but weaker associations with DNA methylation in these genes. Conclusions: PM2.5 exposure was associated with percent DNA methylation of several CpG loci in NOS genes, suggesting an epigenetic mechanism through which these pollutants may alter production of nitric oxide.

Childhood Air Pollutant Exposure and Carotid Artery Intima-Media Thickness in Young Adults

Background— Exposure to ambient air pollutants increases risk for cardiovascular health outcomes in adults. The contribution of childhood air pollutant exposure to cardiovascular health has not been thoroughly evaluated. Methods and Results— The Testing Responses on Youth study consists of 861 college students recruited from the University of Southern California in 2007 to 2009. Participants attended 1 study visit during which blood pressure, heart rate, and carotid artery intima-media thickness (CIMT) were assessed. Self-administered questionnaires collected information about health and sociodemographic characteristics, and a 12-hour fasting blood sample was drawn for lipid and biomarker analyses. Residential addresses were geocoded and used to assign cumulative air pollutant exposure estimates based on data derived from the U.S. Environmental Protection Agencys Air Quality System database. The associations between CIMT and air pollutants were assessed using linear regression analysis. Mean CIMT was 603 &mgr;m (±54 SD). A 2 standard deviation (SD) increase in childhood (aged 0–5 years) or elementary school (aged 6–12 years) O3 exposure was associated with a 7.8-&mgr;m (95% confidence interval, −0.3–15.9) or 10.1-&mgr;m (95% confidence interval, 1.8–18.5) higher CIMT, respectively. Lifetime exposure to O3 showed similar but nonsignificant associations. No associations were observed for PM2.5, PM10, or NO2, although adjustment for these pollutants strengthened the childhood O3 associations. Conclusions— Childhood exposure to O3 may be a novel risk factor for CIMT in a healthy population of college students. Regulation of air pollutants and efforts that focus on limiting childhood exposures continue to be important public health goals.

HDL anti-oxidant function associates with LDL level in young adults

OBJECTIVES The primary objective was to evaluate predictors of HDL anti-oxidant function in young adults. BACKGROUND High-density lipoprotein (HDL) cholesterol is considered a protective factor for cardiovascular disease (CVD). However, increased levels are not always associated with decreased cardiovascular risk. A better understanding of the importance of HDL functionality and how it affects CVD risk is needed. METHODS Fifty non-Hispanic white subjects from the Testing Responses on Youth (TROY) study were randomly selected to investigate whether differences in HDL anti-oxidant function are associated with traditional cardiovascular risk factors, including carotid intima media thickness (CIMT), arterial stiffness and other inflammatory/metabolic parameters. HDL anti-oxidant capacity was evaluated by assessing its ability to inhibit low-density lipoprotein (LDL) cholesterol oxidation by air using a DCF-based fluorescent assay and expressed as a HDL oxidant index (HOI). The associations between HOI and other variables were assessed using both linear and logistic regression. RESULTS Eleven subjects (25%) had an HOI ≥ 1, indicating a pro-oxidant HDL. Age, LDL, high sensitivity C-reactive protein (hsCRP), and paraoxonase activity (PON1), but not HDL, were all associated with HOI level in univariate linear regression models. In multivariate models that mutually adjusted for these variables, LDL remained the strongest predictor of HOI (0.13 increase in HOI per 1 SD increase in LDL, 95% CI 0.04, 0.22). Atherogenic index of plasma, pulse pressure, homocysteine, glucose, insulin, CIMT and measurements of arterial stiffness were not associated with HOI in this population. CONCLUSIONS These results suggest LDL, hsCRP and DBP might predict HDL anti-oxidant function at an early age.

Carotid artery intima-media thickness in college students: Race/ethnicity matters

OBJECTIVE Racial/ethnic differences in common carotid artery intima-media thickness (CIMT) and in risk factors associated with CIMT have been predominantly observed in middle-aged and older individuals. We aimed to characterize racial/ethnic differences CIMT and other cardiovascular risk factors in a healthy, young-adult population. METHODS College students were recruited as part of a study to characterize determinants of atherogenesis. Students were eligible if they were lifetime non-smokers, lived in the United States since six months of age, and attended high school in the United States. Blood pressure, heart rate, height, and weight were measured, B-mode carotid ultrasound was performed, questionnaires were administered and a 12-h fasting blood sample was collected. Associations between CIMT and other variables were assessed in 768 students aged 18-25 years using linear regression analysis. RESULTS In models adjusted for common cardiovascular risk factors, sex exhibited the strongest influence on CIMT, with men having 15.4 μm larger CIMT compared to women (95%CI 6.6, 24.2). Race/ethnicity was also strongly associated with CIMT. African Americans had 17.3 μm greater CIMT (95%CI -0.3, 34.8) compared to non Hispanic Whites, whereas Asians and Hispanic Whites had 14.3 (95%CI -24.3, -4.4) and 15.4 (95%CI -26.2, -4.7) μm smaller CIMT, respectively. BMI and systolic blood pressure were positively associated with CIMT. CONCLUSION The risk factors associated with atherogenesis later in life are already present and observable in college-aged young adults, so targeted campaigns to reduce life-long cardiovascular disease burden should be initiated earlier in life to improve public health.

Non-specific filtering of beta-distributed data

BackgroundNon-specific feature selection is a dimension reduction procedure performed prior to cluster analysis of high dimensional molecular data. Not all measured features are expected to show biological variation, so only the most varying are selected for analysis. In DNA methylation studies, DNA methylation is measured as a proportion, bounded between 0 and 1, with variance a function of the mean. Filtering on standard deviation biases the selection of probes to those with mean values near 0.5. We explore the effect this has on clustering, and develop alternate filter methods that utilize a variance stabilizing transformation for Beta distributed data and do not share this bias.ResultsWe compared results for 11 different non-specific filters on eight Infinium HumanMethylation data sets, selected to span a variety of biological conditions. We found that for data sets having a small fraction of samples showing abnormal methylation of a subset of normally unmethylated CpGs, a characteristic of the CpG island methylator phenotype in cancer, a novel filter statistic that utilized a variance-stabilizing transformation for Beta distributed data outperformed the common filter of using standard deviation of the DNA methylation proportion, or its log-transformed M-value, in its ability to detect the cancer subtype in a cluster analysis. However, the standard deviation filter always performed among the best for distinguishing subgroups of normal tissue. The novel filter and standard deviation filter tended to favour features in different genome contexts; for the same data set, the novel filter always selected more features from CpG island promoters and the standard deviation filter always selected more features from non-CpG island intergenic regions. Interestingly, despite selecting largely non-overlapping sets of features, the two filters did find sample subsets that overlapped for some real data sets.ConclusionsWe found two different filter statistics that tended to prioritize features with different characteristics, each performed well for identifying clusters of cancer and non-cancer tissue, and identifying a cancer CpG island hypermethylation phenotype. Since cluster analysis is for discovery, we would suggest trying both filters on any new data sets, evaluating the overlap of features selected and clusters discovered.

Epigenetic subgroups of esophageal and gastric adenocarcinoma with differential GATA5 DNA methylation associated with clinical and lifestyle factors.

Background Adenocarcinomas located near the gastroesophageal junction have unclear etiology and are difficult to classify. We used DNA methylation analysis to identify subtype-specific markers and new subgroups of gastroesophageal adenocarcinomas, and studied their association with epidemiological risk factors and clinical outcomes. Methodology/Principal Findings We used logistic regression models and unsupervised hierarchical cluster analysis of 74 DNA methylation markers on 45 tumor samples (44 patients) of esophageal and gastric adenocarcinomas obtained from a population-based case-control study to uncover epigenetic markers and cluster groups of gastroesophageal adenocarcinomas. No distinct epigenetic differences were evident between subtypes of gastric and esophageal cancers. However, we identified two gastroesophageal adenocarcinoma subclusters based on DNA methylation profiles. Group membership was best predicted by GATA5 DNA methylation status. We analyzed the associations between these two epigenetic groups and exposure using logistic regression, and the associations with survival time using Cox regression in a larger set of 317 tumor samples (278 patients). There were more males with esophageal and gastric cardia cancers in Cluster Group 1 characterized by higher GATA5 DNA methylation values (all p<0.05). This group also showed associations of borderline statistical significance with having ever smoked (p-value = 0.07), high body mass index (p-value = 0.06), and symptoms of gastroesophageal reflux (p-value = 0.07). Subjects in cluster Group 1 showed better survival than those in Group 2 after adjusting for tumor differentiation grade, but this was not found to be independent of tumor stage. Conclusions/Significance DNA methylation profiling can be used in population-based studies to identify epigenetic subclasses of gastroesophageal adenocarcinomas and class-specific DNA methylation markers that can be linked to epidemiological data and clinical outcome. Two new epigenetic subgroups of gastroesophageal adenocarcinomas were identified that differ to some extent in their survival rates, risk factors of exposure, and GATA5 DNA methylation.