Expert answer:What keywords or indicators in title, abstract can
Solved by verified expert:What keywords or indicators in title, abstract can be used to identify the article attached? Using the questions below to answer the questions included. No Plagiarism, on your own words SUMMARIZED IN 350 WORDS.After reading, reflecting, and evaluating the peer-reviewed journal article that you located, please answer the following questions: 350 WORDSWhat type of scholarly article did you select (research article, review article, theoretical article or meta-analysis)? What keywords or indicators in the title, abstract, or article helped you identify the type of scholarly article you selected?Provide a general overview of the article you selected. Summarize the main points based on the type of scholarly article you chose (e.g., for a research article, summarize research questions, population, findings, limitations, and recommendations for future study).Use proper APA formatting, including in-text citations.See and Use attached research article. Thank you!
nasal_dna_methylation_differentiates_corticosteroid_treatment_response_in_pediatric_asthma__a_pilot_study.pdf
Unformatted Attachment Preview
RESEARCH ARTICLE
Nasal DNA methylation differentiates
corticosteroid treatment response in pediatric
asthma: A pilot study
Xue Zhang1,2, Jocelyn M. Biagini Myers3,4, Veda K. Yadagiri2,3, Ashley Ulm2,3,
Xiaoting Chen5, Matthew T. Weirauch4,5,6, Gurjit K. Khurana Hershey3,4, Hong Ji2,3,4*
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
1 Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States
of America, 2 Pyrosequencing lab for genomic and epigenomic research, Cincinnati Children’s Hospital
Medical Center, Cincinnati, Ohio, United States of America, 3 Division of Asthma Research, Cincinnati
Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 4 Department of Pediatrics,
University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 5 Center for
Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United
States of America, 6 Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children’s
Hospital Medical Center, Cincinnati, Ohio, United States of America
* Hong.Ji@cchmc.org
Abstract
OPEN ACCESS
Citation: Zhang X, Biagini Myers JM, Yadagiri VK,
Ulm A, Chen X, Weirauch MT, et al. (2017) Nasal
DNA methylation differentiates corticosteroid
treatment response in pediatric asthma: A pilot
study. PLoS ONE 12(10): e0186150. https://doi.
org/10.1371/journal.pone.0186150
Editor: Koustubh Panda, University of Calcutta,
INDIA
Received: June 13, 2017
Accepted: September 26, 2017
Published: October 13, 2017
Copyright: © 2017 Zhang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: Array data have been
deposited to NCBI GEO (https://www.ncbi.nlm.nih.
gov/geo/) with the accession number GSE104087.
Funding: This work was supported by NIH/NIAID
R21AI119236 (HJ), NIH/NIAID R21AI101375 (HJ),
NIH R21HG008186 (MTW) and NIH/NIAID
2U19AI70235 (GKKH). REDCap was hosted at
Cincinnati Children’s and supported by the Center
for Clinical and Translational Science and Training
grant UL1-RR026314-01 NCRR/NIH.
Background
Treatment response to systemic corticosteroid in asthmatic children is heterogeneous and
may be mediated by epigenetic mechanism(s). We aim to identify DNA methylation (DNAm)
changes responsive to steroid, and DNAm biomarkers that distinguish treatment response.
Materials and methods
We followed 33 children (ages 5–18) presenting to the Emergency Department (ED) for
asthma exacerbation. Based on whether they met discharge criteria in 24 hours, participants were grouped into good and poor responders to steroid treatment. Nasal samples
were collected upon presentation to the ED (T0) and 18–24 hours later (T1). Genome-wide
DNAm was measured for both time points in 20 subjects, and compared between T0 and T1
in good and poor responders respectively. DNAm at T1 was also compared between two
responder groups. DNAm of selected CpGs was verified in the complete cohort, and expression of associated genes was examined. Interactions between DNAm, common single
nucleotide polymorphism (SNP) located at the CpG sites and treatment responses were
assessed.
Results
Three CpGs located in the OTX2 promoter showed responder-specific DNAm changes from
T0 to T1, in which DNAm decreased in good but not in poor responders. Good and poor
responders showed differential DNAm at T1 in 127 CpGs without and 182 CpGs with common SNP co-localization. Negative correlations between DNAm and gene expression were
observed at CpGs located within the LDHC promoter, suggesting an impact of DNAm on
PLOS ONE | https://doi.org/10.1371/journal.pone.0186150 October 13, 2017
1 / 19
DNA methylation variation and asthma treatment responses
Competing interests: Our authors do not have any
conflicts of interest or financial disclosures related
to this work, including declarations of financial
interest, to report.
Abbreviations: ACT, Asthma control test; DNAm,
DNA methylation; ED, Emergency department; ICS,
Inhaled corticosteroid; IPA, Ingenuity pathway
analysis; LOS, Length of stay; MAF, Minor allele
frequency; meQTL, Methylation quantitative trait
locus; SNP, Single nucleotide polymorphism; SVA,
Surrogate variable analysis.
gene regulation. Interactions between SNPs, DNAm and treatment response were
detected.
Conclusion
Acute systemic steroid treatment modifies nasal DNAm in good responders. Nasal DNAm,
dependent or independent of SNPs, can differentiate response to treatment in acute asthmatic children.
Introduction
Asthma is the leading cause of emergency care and hospitalization in children. The current
standard and most effective treatment for treating asthma exacerbation is systemic corticosteroid treatment. However, due to heterogeneity in asthma phenotypes and natural history [1,
2], 40–70% of asthma treatment showed absent or incomplete efficacy [3]. Heterogeneous
responses to current standard treatment regimens have been observed in children hospitalized
for acute asthma exacerbations, and nearly two thirds of children who currently have asthma
reported at least one attack in the previous 12 months. One potential explanation could be the
suboptimal management of asthma in the childhood age group [4]. Care practices other than
the standard treatments may improve asthma management; however, effective predictions of
responses to standard treatments are needed in prior.
Biomarkers that may be associated with differential responses to asthma treatments have
been researched in children presenting to the ED and hospitalized for exacerbation. As asthma
symptoms and responses to the standard treatment can change from episode to episode in the
same person, biomarkers that are more dynamic have drawn attention such as gene expression
[5]. Recently, DNA methylation has been increasingly explored due to its important role in the
regulation of gene expression. DNA methylation (DNAm) is the modification of cytosine by
adding a methyl group to the 5’ position of C. DNAm mostly occurs in the context of CpG
dinucleotides and represents an important epigenetic mechanism that regulates gene expression [6]. Several studies have successfully identified DNAm of certain nucleotides as a biomarker for asthma [7–12], and suggested that nasal DNAm marks may be useful biomarkers
of disease severity and treatment response [12]. Efforts have also been made to identify DNAm
markers for asthma that develops in childhood and persists into early adulthood [13] and
markers for temporal asthma transition [14]. Interactions between CpG methylation and
genetic variation, especially single nucleotide polymorphisms (SNPs) located at C or G that
can disrupt or create a CpG site (meSNPs or CpG-SNPs), are prevalent in the human genome
(~15%) [15, 16]; such interactions have been associated with several diseases including childhood asthma [17–21]. In the present study, we examined genome-wide DNAm using nasal
epithelial cells to comprehensively search for CpG sites that undergo differential methylation
changes in response to systemic corticosteroid treatment in good and poor steroid responder
groups, as well as to identify biomarkers for treatment response. We further validated these
findings by bisulfite pyrosequencing of a larger population, followed by determination of the
impact of DNAm on candidate gene expression and the impact of common meSNPs on
DNAm. We also evaluated associations between DNAm-modifying meSNPs (known as methylation quantitative trait locus or meQTLs) and treatment response. Our results provide novel
epigenetic biomarkers for treatment response to systemic steroid in asthmatic children and
potential therapeutic targets for alternative asthma therapies.
PLOS ONE | https://doi.org/10.1371/journal.pone.0186150 October 13, 2017
2 / 19
DNA methylation variation and asthma treatment responses
Materials and methods
Study population
This study was approved by the Institutional Review Board at Cincinnati Children’s Hospital
Medical Center (CCHMC # 2008–1384). Using inclusion and exclusion criteria previously
described [5], children aged 5–18 years who presented to the CCHMC ED with acute asthma
exacerbation and had an asthma diagnosis were recruited during March 2011-Oct 2014 and
written informed consents were obtained from parents or guardians of the minors. Information collected includes demographics, environmental exposures, asthma trigger information,
and personal and family allergy and asthma symptom history. Parent(s)/legal authorized representatives also provided current inhaled corticosteroid (ICS) controller medication (Asmanex1, Flovent1, Qvar1, Pulmicort1, Advair1, Dulera1, Symbicort1, etc.). Usage of nasal
steroids in the past 4 weeks was recorded. To assess baseline asthma symptom severity and
control, a respiratory symptom score was calculated (based on frequency of wheeze, cough,
shortness of breath, and chest tightness) [22] and the age-specific Asthma Control Test™
(ACT) score was collected [23]. Information regarding the hospital course was extracted from
medical charts. Enrolled patients were treated according to the CCHMC evidence-based treatment protocol for inpatient asthma exacerbation and discharged as previously defined [5]. As
previously described [5], they were classified as good and poor responders based on the time
required for them to clinically improve to the point where they achieve discharge criteria
(>24hrs or 24hrs).
Treatment protocol and definition of length of stay
Enrolled patients were treated according to the CCHMC evidence-based treatment protocol
for inpatient asthma exacerbation and discharged as previously defined [5]. Briefly, all patients
received 2mg/kg/day of prednisone while hospitalized and ICS were continued via mouthpiece. Clinical discharge criteria were met when: (1) oxygen saturations were greater than or
equal to 91% on room air for at least 6 hours; (2) no evidence of respiratory distress; and (3)
reached a time point at which the patient demonstrated sufficient clinical improvement while
receiving albuterol no more frequently than every 4 hours for a period of 8 hours (q4h x 2).
Length of stay (LOS) was calculated as the time the disposition was set to admit to the time the
subject met the clinical criteria for discharge. Based on our previous data [5], good responders
were defined as those with LOS 24 hours (short LOS) and poor responders as those with
LOS>24 hours (long LOS).
Nasal sample collection and DNA/RNA extraction
Nasal epithelial samples were collected at two time points from each subject: (1) upon presentation to the ED (T0) and (2) on the inpatient floor 18–24 hours after receiving systemic corticosteroids in the ED (T1). Nasal mucosa sampling was performed with a CytoSoft Brush
(Medical Packaging Corp, CA, USA), and the samples were immediately taken to the laboratory for processing. Nasal samples collected contained >90% epithelial cells, similar to our previous findings [24]. DNA and RNA were extracted with the AllPrep DNA/RNA Micro kit
(Qiagen, Hilden, Germany), according to the manufacturer’s protocols.
Illumina 450K array processing
For genome wide DNAm assay, we used 40 samples from 20 subjects as the discovery population. Genomic DNA from the nasal cells was bisulfite treated and assayed by the Illumina Infinium HumanMethylation450 BeadChip (Illumina). Quality of the array was assessed using
PLOS ONE | https://doi.org/10.1371/journal.pone.0186150 October 13, 2017
3 / 19
DNA methylation variation and asthma treatment responses
sample-independent and dependent internal control probes included on the array for staining,
extension, hybridization, specificity and bisulfite conversion. All the samples passed the QC
and were included in the discovery analysis. The signal intensities were then backgroundadjusted using out-of-band probes (noob), and normalized using functional normalization
signal
with R package “RnBeads”. Beta values were calculated as signal methylationmethylation
and used in
þ signalunmethylation
further analyses. CpG sites that were not detected in all samples at p = 0.01 level, and CpG sites
on the X and Y chromosomes were excluded.
Array data have been deposited to NCBI GEO (https://www.ncbi.nlm.nih.gov/geo/) with
the accession number GSE104087.
450K array SNP annotation and GREAT annotation
To examine whether reported sites from Illumina 450K Arrays overlap with any known polymorphisms, common SNPs from dbSNP 142 table with minor allele frequency (MAF) 1%
were extracted and used to annotate these sites. The majority of the reported array sites do not
contain strand-specific information and are CpG dinucleotides. These reported 1bp site along
with its consecutive downstream base were examined for overlapping with any known common SNPs. A small portion of the reported array sites have strand-specific information and
are non-CpG methylation sites (CHG, H = A, C, T). For these sites, only the reported 1bp site
was examined. When a site is found to be overlapping with polymorphisms, a binary indicator
along with allele information, rsID, and observed strand, are added to the reported array site
as annotations. In addition to the Illumina annotation for CpG sites on arrays, we also utilized
Genomic Regions Enrichment of Annotations Tool (GREAT) to search for genes in close
proximity with CpG sites [25].
Bisulfite pyrosequencing
For DNAm measurement, a total of 200ng genomic DNA was subjected to sodium bisulfite
treatment and purified using the EZ DNA methylation-Gold Kit (Zymo research, Irvine, CA,
USA) according to the manufacturer’s specifications. Pyrosequencing was carried out using
Pyro Gold reagents with a PyroMark vacuum prep workstation and a PyroMark Q96 MD
instrument (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The generated pyrograms were automatically analyzed using Pyro Q-CpG methylation analysis software (Qiagen, Valencia, CA, USA). 100% methylation control (SssI-treated human genomic
DNA) and 0% methylation control (human genomic DNA amplified by GenomePlex1 Complete WGA kit (Sigma, St. Louis, MO, USA)) were used in validating all assays. For SNP analysis, DNA was subjected to PCR amplification without bisulfite treatment. The allele frequency
of possible SNPs was calculated by the PyroMark MD 1.0 (Qiagen). Pyrosequencing assay
design and genomic coordinates are as documented in S1 Table.
Reverse transcription quantitative PCR (RT-qPCR)
Total RNA was reverse-transcribed to cDNA using the Superscript III kit (Life Technologies,
NY, USA) using random hexamers according to manufacturer’s instructions. Real-time quantitative PCR was performed using the SYBR Green Master Kit and LightCycler1 480 instrument (Roche Diagnostics, Indianapolis, IN, USA). PCR was carried out in triplicate from each
fraction and the mean Ct value of the triplicate reaction was normalized against the mean Ct
value of GAPDH. Primer sequences are described in S2 Table.
PLOS ONE | https://doi.org/10.1371/journal.pone.0186150 October 13, 2017
4 / 19
DNA methylation variation and asthma treatment responses
Ingenuity pathway analysis
To better understand the biological meaning of the methylation changes, genes associated with
identified differentially methylated CpG sites were extracted from 450K array annotation file
and was imported into Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood City,
CA) for pathway mapping, gene network detection, and upstream regulator identification. A
cutoff of 0.05 was used for statistical significance in IPA analysis.
Statistical analysis
The analyses were performed as outlined in Fig 1. To identify CpG sites with responder-specific T0-T1 changes, the association between DNAm (beta value from the array; see detailed
description in the supplementary information) and time were tested using linear regression in
poor and good responders respectively. To identify CpG sites with differential DNAm between
poor and good responders, we tested the association between beta value and responder status
with linear regression using data from T1. Age, gender, and race (dichotomized as black and
Fig 1. Analysis flowchart.
https://doi.org/10.1371/journal.pone.0186150.g001
PLOS ONE | https://doi.org/10.1371/journal.pone.0186150 October 13, 2017
5 / 19
DNA methylation variation and asthma treatment responses
non-black) were adjusted. To further correct for the batch effect and bias caused by unknown
confounders such as cell composition, we performed surrogate variable analysis (SVA) using
the R package “SVA”. Three significant SVs were detected. The associations described above
were then tested again with three SVs being included in the modeling. Only CpG sites identified in both sets of analyses were selected for further examination. For array analysis, nominal
significance was defined as p value 0.05, and genome-wide significance was defined as q
value 0.05 [26].
To verify the CpG sites identified from the array that showed responder-specific T0-T1
changes in DNAm, we assayed 33 pairs of samples with bisulfite pyrosequencing and calculated the percent of methylation. As T0 and T1 DNAm are paired by subjects, to better account
for between-subject random effects, we used mixed models, in which a responder time interaction was tested. Mixed models were also utilized to verify the CpG sites whose T1 DNAm
showed association with response to treatment. In these verification analyses, both T0 and T1
data were used with correlations between T0 and T1 being considered. We also calculated an
%
M value as M ¼ log2 1 methylation
, which was examined to ensure the validity of conclusions
methylation %
from models using beta values.
To determine genetic effects on DNAm, we clustered the beta values into full-, semi- and
non-methylated groups by examining the distribution of the beta values. The methylation
groups were then compared with the genotypes. To test the genetic association with the
responder status, we used an additive model in which genotypes were numerically coded
according to the number of minor allele. The genetic association was then tested using a logistic model.
To assess the relationship between gene expression and DNAm, we used the Spearman correlation. We also examined the association of LDHC expression with response to treatment,
and the responder-specific changes between T0 and T1 in OTX2 expression. As the expression
data exhibited log-normal distribution, the associations were tested using generalized estimating equation assuming a log-normal distribution of the data. Verification analyses were conducted using SAS 9.4 unless otherwise specified. P values 0.05 were designated statistically
significant.
Results
Study overview and population characteristics
Our discovery population consisted of 20 exacerbating asthmatics who were not exposed to
nasal steroid within the past 14 days (S3 Table). To identify CpG sites whose DNAm status
was associated with systemic corticosteroid treatment response, we assayed the genome-wide
nasal methylation using paired DNA samples collected at both T0 and T1 from these 20
patients (40 samples total). As false positive results would occur even after stringent QC, data
preprocessing, and complex analyses of 450K arrays, we further verified significant associations using bisulfite pyrosequencing in the complete cohort.
The discovery population was composed of 60% black males, and 45% were good responders. There were no statistically significant differences in age, sex, race or number of good or
poor responders between the discovery population and the 13 additional subjects (S3 Table).
Among the 13 additional subjects, 7 of them reported pr …
Purchase answer to see full
attachment
How it works
- Paste your instructions in the instructions box. You can also attach an instructions file
- Select the writer category, deadline, education level and review the instructions
- Make a payment for the order to be assignment to a writer
- Download the paper after the writer uploads it
Will the writer plagiarize my essay?
You will get a plagiarism-free paper and you can get an originality report upon request.
Is this service safe?
All the personal information is confidential and we have 100% safe payment methods. We also guarantee good grades