Structure and functional impact of seed region variant in MIR-499 gene family in bronchial asthma
Toraih et al. Respiratory Research
Structure and functional impact of seed region variant in MIR-499 gene family in bronchial asthma
Eman A. Toraih 0
Mohammad H. Hussein 3
Essam Al Ageeli 2
Eman Riad 6
Nouran B. AbdAllah 5
Ghada M. Helal 4
Manal S. Fawzy 1 7
0 Genetics Unit, Department of Histology and Cell Biology, Faculty of Medicine, Suez Canal University , Ismailia P.O. 41522 , Egypt
1 Department of Medical Biochemistry, Faculty of Medicine, Suez Canal University , Ismailia P.O. 41522 , Egypt
2 Department of Clinical Biochemistry (Medical Genetics), Faculty of Medicine, Jazan University , Jazan , Saudi Arabia
3 Pulmonologist, Ministry of Health , Cairo , Egypt
4 Department of Medical Biochemistry, Faculty of Medicine, Mansoura University , Mansoura , Egypt
5 Department of Pediatrics, Faculty of Medicine, Suez Canal University , Ismailia , Egypt
6 Department of Chest Diseases and Tuberculosis, Faculty of Medicine, Suez Canal University , Ismailia P.O. 41522 , Egypt
7 Department of Medical Biochemistry, Faculty of Medicine, Northern Border University , Arar, Kingdom of Saudi Arabia
Background: Small non-coding RNAs (microRNAs) have been evolved to master numerous cellular processes. Genetic variants within microRNA seed region might influence microRNA biogenesis and function. The study aimed at determining the role of microRNA-499 (MIR-499) gene family polymorphism as a marker for susceptibility and progression of bronchial asthma and to analyze the structural and functional impact of rs3746444 within the seed region. Methods: Genotyping for 192 participants (96 patients and 96 controls) in the discovery phase and 319 subjects (115 patients and 204 controls) in the replication phase was performed via Real Time-Polymerase Chain Reaction technology. Patients underwent the methacholine challenge test and biochemical analysis. Gene structural and functional analysis, target prediction, annotation clustering, and pathway enrichment analysis were executed. Predicted functional effect of rs37464443 SNP was analyzed. Results: miR-499 gene family is highly implicated in inflammation-related signaling pathways. Rs374644 (A > G) in MIR499A and MIR499B within the seed region could disrupt target genes and create new genes. The G variant was associated with high risk of developing asthma under all genetic association models (G versus A: OR = 3.27, 95% CI = 2.53-4.22; GG versus AA: OR = 9.52, 95% CI = 5.61-16.5; AG versus AA: OR = 2.13, 95% CI = 1.24-3.46; GG + AG versus AA: OR = 4.43, 95% CI = 2.88-6.82). GG genotype was associated with poor pre-bronchodilator FEV1 (p = 0. 047) and the worst bronchodilator response after Salbutamol inhalation, represented in low peaked expiratory flow rate (p = 0.035). Conclusions: miR-499 rs3746444 (A > G) polymorphism was associated with asthma susceptibility and bronchodilator response in Egyptian children and adolescents. Further functional analysis is warranted to develop more specific theranostic agents for selecting targeted therapy.
Asthma; Egyptians; miR-499a; miR-499b; Polymorphism; Airway hyper-responsiveness; qRT-PCR
Bronchial asthma is a chronic heterogeneous respiratory
disease that is characterized by airway inflammation,
recurring bronchial obstruction, and airway
]. Most common histopathological
features are inflammatory cell infiltration, sub-basement
fibrosis, smooth muscle hypertrophy, mucus
hypersecretion, injury to epithelial cells, and angiogenesis [
Treatment with anti-inflammatory drugs or
bronchodilators usually improves some of these features.
Nevertheless, therapeutic response relies on the interplay between
environmental exposure and genetic background [
Despite several advances over the past decades in
understanding the underlying mechanisms involved in the
disease, there are no current satisfactory strategies for
the cure or prevention of long-term decline in
pulmonary function [
]. Given the significant morbidity and
burden of childhood asthma worldwide, better
therapeutic modalities are mandatory to counteract the
development and progression of the disease. In prior studies,
a sizeable proportion of genetic influence existed,
ranging from 35 to 95% for asthma and 30 to 66% for
bronchial hyper-responsiveness [
]. Genome-wide association
studies provide evidence for multiple novel loci
associated with the disease. However, the exact maestro fine
tuning these putative genes is still uncovered.
In early 90’s, the presence of small non-coding RNAs
(ncRNAs) was discovered in the mammalian genome
]. These microRNAs are transcriped via specific
cellular machinery to form short single-stranded mature
RNAs of 19–24 nucleotides long. They function by
complementary base pairing with mRNA targets, leading to
its degradation or translational repression [
]. They are
estimated to modulate gene expression of 60% of
protein-coding genes, and to regulate many cellular
processes; including proliferation, apoptosis,
immunomodulation, stress response, and angiogenesis [
]. As a result
the focus of human genome studies has witnessed a shift
from mRNAs to ncRNAs as major key players in human
disorders. Currently, there are emerging opportunities
for targeting these disruptions of ncRNAs using novel
therapeutic approaches. Some strategies aimed to
increase the levels of abnormally down-regulated
microRNAs via epigenetic drugs as DNA demethylating
agents and histone deacetylase (HDAC) inhibitors or by
replacement of miRNAs using virus delivery systems
]. On the other hand, over-expressed microRNAs in
human diseases can be sequestered by anti-miRNA
oligonucleotides, miRNA sponges, miRNA masking and
small molecule inhibitors [
In silico analysis and surveying the literature revealed
the deregulation of microRNAs in various pulmonary
diseases (Fig. 1). Several lines of evidence suggest a key
role for hsa-microRNA-499a (miR-499a) in modulating
the immune response, cell proliferation, apoptosis,
neuromuscular regulation and neoangiogenesis [
Exploring gene targets of miR-499a by computational
tools identified inflammatory-related gene targets,
including IL-13 and Il-23, which represent important
mediators in asthma KEGG pathway [ID hsa05310]
(microRNA.org). A single nucleotide polymorphism
(SNP), rs3746444 (A > G), is located in the seed
sequence of miR-499a-3p, a region essential for
miRNAmediated silencing mechanism. SNPs within
mRNAbinding site of miRNAs may influence mRNA gene set,
target binding ability, or pre-miRNA maturation process,
which in turn, could alter the susceptibility to develop
human diseases. The rs3746444 MIR-499a SNP was
found to be associated with higher risk of rheumatoid
], coronary artery disease [
], and ankylosing Spondylitis [
]. Hence, the
current study was conducted to investigate the
association between rs3746444 polymorphism and
susceptibility to asthma disease in children and adolescents, and to
further assess computationally its impact on the clinical
outcome and bronchial hyper-responsiveness (BHR) in a
group of Egyptian asthmatic children and adolescents.
Structural gene analysis
Genetic analysis of MIR499A gene was performed using
Genecards.org, Ensembl.org, and NCBI. DNA and RNA
sequences were retrieved from miRBase.org. Multiple
sequence alignment and phylogenetic tree construction
were implemented to identify similarity regions in
different species by Ensembl.org and with its cluster gene
MIR499B by Rcoffee v11.0, a specific method for
noncoding RNA [
]. Analysis by PolymiRTS Database v3.0
(Polymorphism in microRNAs and their TargetSites) was
conducted to predict SNPs and INDELs in the whole gene
Variant calls in the chromosomal region were obtained from
Ensembl.org and UCSC via usegalaxy.org platform.
In silico target gene prediction
Next, list of computationally predicted and
experimentally validated gene targets (Additional file 1: Table S2)
were retrieved from multiple databases including
miRBase (http://www.mirbase.org/), TargetScanHuman
v6.2 (http://www.targetscan.org/), miRDB (http://mirdb.org
/miRDB/), miRNAMap v2.0 (http://mirnamap.mbc.nctu.
edu.tw/), and DIANA-TarBase v7.0 algorithm (http://diana.
index) databases. Result intersection and statistical
validation were performed as described previously [
Functional annotation clustering and pathway enrichment analysis
Functional analysis of the gene list was done via
DianamiRPath v3.0 software for gene ontology (GO) terms
and Kyoto encyclopedia of genes and genomes (KEGG)
]. Fisher’s exact test/hypergeometric
statistical test was applied at microT-CDS threshold of 0.8
and P value threshold at 0.05. Gene Ontology (GO)
system of classification was carried out to interpret the
target gene sets of miR-499a-3p, miR-499a-5p,
miR499b-3p, and miR-499b-5p based on their functional
characteristics. The attributes of targets were classified
and ranked in the context of biological process,
molecular function, or cellular component. Further comparison
of functional categories between miR-499a and
miR499b targets was conducted by miRpair2Go
webplatform using two combined miRNA target prediction
methods (TargetScan and miRanda) and moderate
hierarchial filtering level (http://compbio.uthsc.edu/miR2GO/
Predicted functional effect of rs37464443 SNP
Distribution of SNPs in miR-499 was analyzed via
miRdSNP database (http://mirdsnp.ccr.buffalo.edu/): a
database of disease-associated SNPs to identify the
spatial relationship of the miRNA with target sites on
the 3’UTR of human genes and to further explore the
molecular mechanism of gene deregulation at the
posttranscriptional level [
]. The predicted functional
impact of miR-499 rs3746444 variant was performed using
miRmut2Go (http://compbio.uthsc.edu/miR2GO) to
analyze the changes of target genes caused by miRNA
mutations and view the functional impacts of these
changes in the context of comparative functional GO
analysis using the same filters described in our prior
]. Advanced gene set enrichment analysis was
employed using the Gene Trail program with multiple
testing correction via false discovery rate (FDR)
estimation and significant level at 0.05 (https://genetrail2.
]. In addition, mfold RNAfold
(http://unafold.rna.albany.edu/?q=mfold) and KineFold
web-servers (http://kinefold.curie.fr/) were conducted
to predict the secondary structures of RNA sequence
in A and G alleles.
A total of 211 asthmatic patients and 300 controls (age
range 3 to 18 years old) were enrolled in the study. In
the discovery stage: the study participants were
composed of 96 patients and 96 controls. Patients were
obtained from the Pediatrics outpatient clinic of Suez
Canal University Hospital (SCUH), Ismailia, and Chest
and tuberculosis Department, Kasr Al-Ainy Hospital,
Giza. Whereas, in the replication phase: other
independent cohorts of 115 patients and 204 controls were
recruited from SCUH, Ismailia. They were diagnosed and
assessed according to the Global Initiative for Asthma
(GINA) guidelines [
]. A thorough clinical assessment
was performed for determining disease severity,
therapeutic history, and co-morbidities as previously
]. Controls had no history of wheezes, atopy,
or any other respiratory diseases. Chest X-ray was done
for participants to exclude concurrent chest disease.
Body mass index (BMI) percentile of patients and
controls was estimated and adjusted for age and sex using
an online pediatric calculator (http://www.quesgen.com/
]. Sexual maturity rating was
determined based on Tanner classification [
]. The study
was conducted in accordance with the guidelines in the
Declaration of Helsinki and had the approval of the Ethics
Committee of Faculty of Medicine, Suez Canal University.
Informed consent was obtained from participants’
Spirometry and methacholine challenge test
Baseline pulmonary function test assessment was done
using an electronic Spirometer (BTL-08 Spiro Pro
system; BTL) with a valve-spacer device following the
guidelines of the American Thoracic Society/European
Respiratory Society (ATS/ERS) [
]. Baseline lung
parameters were documented [
postbronchodilator forced Spirometry was performed 15 min
after administering a 400 μg dose of inhaled Salbutamol
(Ventolin; GlaxoSmithKline). BDRBASE, change in FEV1
as a percent of baseline forced expiratory volume at 1 s
(FEV1), was calculated with the following equation [=
((postbronchodilator FEV1 − prebronchodilator FEV1) /
prebronchodilator FEV1) × 100] [
bronchoconstriction provocation via methacholine
challenge test (MCT) was done to assess bronchial
hyperresponsiveness. Methacholine solution, mixed with
saline with the following gradient doses (0.06, 0.125, 0.25,
1, 2, 4, 8, 16 mg/ml), were aerosolized using a nebulizer
attached to an air compressor at 5 min interval.
Progressive increase in concentration was used, until the patient
encountered a significant worsening in lung function,
with a drop in FEV1 of 20% or more. BHR was
categorized according to the American Thoracic Society
guidelines with positive cutoff value defined as a PC20 below
8 mg/ml [
Blood samples were collected in EDTA Vacutainers.
Absolute eosinophil count (AEC) was calculated using the
Coulter Counter. Total IgE was measured by
enzymelinked immunosorbant assay (ELISA). Absolute
esinophilic count <0.1 × 103/μl and total IgE concentrations
<90 IU/ml were defined as normal [
Genotyping of seed region variant
Genomic DNA was purified from whole blood using
QIAamp DNA Blood Mini kit (Catalog No. 51104;
Qiagen) following the manufacturer’s protocol. Extracted
DNA purity and concentration were assessed by
NanoDrop ND-1000 (NanoDrop Technologies, Inc.
Wilmington, DE, USA). Genotyping for the hsa-miR-499a
(rs3746444) was assayed using Real-Time polymerase chain
reaction (RT-PCR) allelic discrimination technology. PCR
reactions were run blindly in duplicates in a 25-μl final
volume containing 20 ng genomic DNA, TaqMan Universal
PCR Master Mix, No UNG (4440043), and TaqMan SNP
Genotyping Assay Mix (assay ID C_2142612_30, Applied
Biosystems) with 100% concordance rate for genotype calls.
Appropriate controls were used in each reaction. PCR
amplification was done using StepOne™ Real-Time PCR System
(Applied Biosystems, USA) [
]. Allelic discrimination was
called by the SDS software version 1.3.1 (Applied
Statistical analysis was performed using PCORD v.5.0, R
programming language and the “Statistical Package for the
Social Sciences (SPSS) for windows” software, version 22.
Allele and genotype frequencies and carriage rate were
calculated as previously described [
]. The Hardy-Weinberg
equilibrium was estimated using the Online Encyclopedia
for Genetic Epidemiology (OEGE) software (http://
www.oege.org/software/hwe-mr-calc.shtml) and tested for
goodness of fit by chi square test. Genotype and allele
frequencies were compared between asthmatic patients and
control subjects using the chi-square test. Adjusted odds
ratios (OR) with a 95% confidence interval (CI) by logistic
regression analysis were calculated for multiple genetic
association models. Data distribution was checked by the
Kolmogorov-Smirnov test. Appropriate data presentation
and test were used for comparison between groups. A
twotailed P-value of 0.05 was considered statistically
significant. Both ordination and two-way agglomerative
hierarchal clustering techniques were applied to the data for
multivariate analysis [
Structural gene analysis and comparative genomics
Human miR-499a gene (MIR499A; ENSG 00000207635)
is located along the long arm of chromosome 20q11.22
spanning 122 bp long (Genomic coordinates at
20:34,990,376–34,990,497 on the forward strand;
according to the Human Genome Assembly GRCh38, release
annotation 108). MIR499A gene exists within intron 19 of
myosin, heavy chain 7B, cardiac muscle, beta MYH7B
gene and overlapping MIR499B gene (ENSG00000283441;
73 bp in length; 20: 34,990,400–34,990,472 on the reverse
strand) (Fig. 2). Similar orthologs are present in other
species; mouse MIR-499 was also mapped to intron 19 of the
Myh7b gene on chromosome 2 (Mus musculus;
2:155,622,880–155,622,958 (+); GRCm38). Whereas rat
MIR-499, existed on chromosome 3 (Rattus norvegicus;
3:151,138,862–151,138,926 (+); Rnor6.0). Multiple
sequence alignment showed MIR499A gene to display a
high level of conservation throughout 17 mammalian
species (Fig. 3).
MIR499A consists of a single exon that encodes for a
single transcript (MI0003183) of 122 bp long. Whereas, its
clustered MIR499B forms a shorter transcript of 73 bp long
from the reverse strand. Variant analysis of MIR499A
identified the presence of 30 non-coding transcript exon
variants (27 SNPs and 3 deletions) in MIR499A including 19
overlapping variants (17 SNPs and 2 deletions) existed in
MIR499B. All variants were rare except rs3746444 (A/G
alleles) at the position 20:34,990,448 (GRCh38) with minor
allele frequency (MAF) of 0.184. The rs3746444 variant
exists in the seed region of miR-499a-3p; AC[A/G]UCAC.
In silico target gene prediction
Hundreds of genes were predicted to be targeted by
miR-499a and miR-499b using multiple microRNA
databases. Though miRNAs form secondary hairpin
loop, with complementary sequences in their
structure; different gene sets were predicted to be
targeted by both mature forms synthesized from either
arm. A total of 1890 genes was predicted to be
influenced by miR-499a (919 genes by 3p, 810 by 5p,
and 161 genes by both). However, miR-499b was
involved in manipulating the transcription of 1528
genes (910 genes by 3p, 514 by 5p, and 104 genes
by both forms). Due to sequence similarity identified
between miR-499a and miR-499b, their mature forms
shared target genes; specifically 832 common genes
are targeted by both miR-499a-3p and miR-499b-3p,
whereas, 93 genes were the same for both 5p forms
Functional annotation clustering and pathway enrichment analysis
KEGG pathway enrichment analysis showed enrollment of
miR-499 gene targets in remodeling and
inflammationrelated signaling pathways; including mucus biosynthesis
and secretion, sphingolipid signaling, phosphatidylinositol
signaling cell adhesion (focal adhesion and adherence
junction pathways), fibrogenic and immune-modulator pathways
(TGF beta signaling and TNF signaling pathways) (Fig. 4b).
Functional clustering annotation of these gene sets identified
the most significant their molecular activities, biological
processes, pathways, and the cellular components where these
genes execute their functions (Fig. 5). Comparison between
the two microRNAs by miRpair 2GO explored the
functional similarity scores for gene ontology to be 0.676 for
biological process; 0.412 for molecular function, and 0.833 for
cellular components. These targets were significantly
clustered in four chromosomes; namely 67 genes on
chromosome 3, 47 genes on chromosome 12, 31 genes on
chromosome 16, and 30 genes on chromosome 19.
Predicted functional effect of rs3746444 SNP
In silico analysis identified the rs3746444 variant to
overlap 3 genes. It lies in the intron of MYH7B gene
(c.2103 + 138A > G in ENST00000262873 transcript or
c.2100 + 138A > G in ENST00000618182 transcript). In
addition, it represents the nucleotide number 73 (out of
122) of MIR499A on the forward strand (n.73A > G)
within the sequence of miR-499a-3p and the nucleotide
number 25 (out of 73) of MIR499B on the reverse strand
(n.25 T > C) within the sequence of miR-499b-5p
(Fig. 2d). Analyzing the secondary structure of
pre-miR499a hairpin loop with rs3746444 SNP (either A or G
alleles) via RNAfold and KineFold web-servers showed no
effect of the alleles on the folding pattern. The SNP does
not overlap any regulatory region or motif features.
However, being in the seed region at the 5′ end of
miR499a-3p generates the possibility of creating an altered
target gene set for that particular. Using PolymiRTS
Database 3.0 platform, identified the disruption of 667
(41.8%) genes of the miR-499a targets and creation of
new 763 genes when A allele is substituted by G at the
seed region sequence: AAC[A/G]UCACAGCAAGUCU
GUGCU. Comparing the two gene sets of each allele by
miRmut2Go web-server revealed low functional
similarity scores for the three GO domains; 0.378 for biological
process, 0.401 for molecular function, and 0.528 for
cellular component similarity scores, (Fig. 6). Further
enrichment analysis of the new gene list targeted by G
allele was significantly involved in two KEGG pathways
(glycan degradation and glycolysis/gluconeogenesis;
p = 0.016 and 0.031 respectively), 45 genes of them were
significantly located on a single chromosome
(p = 0.044).
Genotyping of rs3746444 polymorphism in the study groups
Baseline characteristics of patients and control groups in
both discovery and replication stages are illustrated in
Additional file 1: Table S1. Genotype distribution among the
studied groups was in agreement with Hardy Weinberg
equilibrium (p > 0.05). Analysis of the whole study
population (211 patients and 300 controls) revealed that the minor
allele frequency (MAF; G allele) in the control group was
0.34; the same allele was doubled in asthmatic cohort,
accounting for 0.63 (p < 0.001). Correspondingly, there was
higher frequency of GG genotype among asthmatic patients
(p < 0.001), Table 1. Both discovery and replication cohorts
demonstrated similar trends in genotype and allele
frequencies. Genetic association model analysis showed that
individuals with G variant were more likely to develop asthma
than non-carriers under all association models (G versus A:
OR = 3.27, 95% CI = 2.53–4.22; GG versus AA: OR = 9.52,
95% CI = 5.61–16.5; AG versus AA: OR = 2.13, 95%
CI = 1.24–3.46; GG + AG versus AA: OR = 4.43, 95%
CI = 2.88–6.82), Table 1. High significant proportions of
AG and GG genotypes were also observed in asthmatic
children and adolescents of both gender (p < 0.001) (Fig. 7).
Genotype distribution according to disease
characteristics of patients is shown in Table 2. There was no
significant association of MIR-499a genotypes with any
clinical or laboratory characteristics in the overall and
stratified analysis. However, children and adolescents
with early age at onset of asthma disease had higher
frequency of GG genotype (p = 0.036). Additionally, GG
homozygote patients have the lowest pre-bronchodilator
FEV1 (p = 0.047) and the worst bronchodilator response
after Salbutamol inhalation represented in low peaked
expiratory flow rate (PEFR) (p = 0.035) (Fig. 8). Similarly,
data exploration by multivariate analysis did not
demonstrate clustering of asthmatic patients according to their
genotypes or any other clinical variables (Additional file 2:
MicroRNAs are involved in various biological cellular
processes. Aberrant transcriptomic signatures of various
miRNAs have been detected in the airways and
circulation of asthmatic patients [
]. Sequence variations
within miRNA genes, especially in the mature miRNA
seed region, may have a profound impact on miRNA
biogenesis and function [
]. In 2009, an
inflammation-related MIR-499A was identified within
the intronic sequence of the myosin MYH7B gene .
Several lines of evidence suggest that miR-499a play
critical roles in orchestrating the immune response in
various human disorders [
11, 12, 37
analysis determined complement base pairing with
important cytokines as IL-13 and IL-23, both are
recognized effectors in asthma pathway. IL-13 signaling
results in mucin hypersecretion, airway remodeling and
fibrosis, and bronchial hyper-responsiveness [
induction regulates allergic asthma through modulation
of TH2 cell differentiation and eosinophilic infiltration
]. Functional enrichment clustering illustrated the
involvement of miR-499a-3a and miR-499a-3b in Toll-like
receptor (TLC) signaling pathways that are implicated in
the initial phase of the host defense against invading
pathogens and allergens leading to TH2 activation [
addition, miR-499a and miR-499b are involved in
Fcepsilon signaling pathway; cross-linking of IgE with FcεRI
receptors on mast cells, causes the release of leukotrienes
and histamines essential for immediate allergic reaction.
Other biological processes as gene expression, cellular
protein modification process, transcription initiation from
RNA polymerase II, and cellular component assembly are
significantly mediated by miR-499 gene family.
Using various computational tools, miR-499a and
miR499b were predicted to target key molecules in
asthma-related KEGG pathways. They could modulate 18
targets in mucin type O-biosynthesis pathway related to
polypeptide N-Acetylgalactosaminyltransferase (GALNT)
genes. Hyperglycosylated mucins are commonly found in
chronic inflammatory airway diseases such as asthma, for
which inhibition of mucin and their glycosylation could
contribute in controlling the disease [
]. The next most
significant pathway was fatty acid biosynthesis with 5
target genes. Recent studies demonstrated the vital role of
fatty acids in the formation of inflammatory mediators
relevant to the pathophysiology of asthma [
identified pathway that is highly impacted by miR-499
gene family is glycosphingolipid (GSL) biosynthesis.
βGlycosphingolipids have emerged as a family of potential
ligands for natural killer (NK) cells. Extensive infiltration
of NK cells in the airway bronchial mucosa is considered
one of the prominent driver in asthma development via
io ) ) ) ) )
eg a )6 .)8 .)6 .)6 .)8 .)2 .)6 .93 .)4 .)2 15 .)2 .)7 .)1 .)2 .78 .)1 .69 .)9 .78
rd thm 9= (02 (93 (93 (02 (97 46 (04 (54 (06 (97 1= (21 (14 (64 (21 (81 24 (33 (64 (35 (81
ee sA (n 02 83 83 02 67 .00 87 11 85 67 (n 41 84 35 41 01 .50 67 51 26 01
A G G A G
A A G A A A G A G
A G G A G
A A G A A A G A G
.0 .1 .
1 2 9
a ) ) ) ) ) ) ) ) ) b
tsahpm (003=n .(064831 .(714551 .)(32173 .(064831 .(045261 .7050 .(326134 .(743922 .(778362 .(045261 saeond
io ) ) ) ) ) ) eq rce
redg 211= .()161 .()408 .()431 .()161 .(7839 97 .(3465 .(6835 .(5069 .(5829 irengb IeBpM
ee (n 43 68 19 43 71 .00 51 62 21 71 -eW,ry
A G G A G P ed
A A G A A A G A G .) n
inducing the secretion of TH2 cytokines that results in
airway hyper-responsiveness and inflammation [
Lowering GSL levels in mast cells in an animal model of asthma
was found to ameliorate disease manifestations [
mature forms of miR-499a and miR-499b were predicted
to target about 40 and 60 genes in TGF-beta and TNF
signaling pathways, important mediators contributing in lung
tissue fibrosis and proliferation of smooth muscle cells
with subsequent airway remodeling [
]. We also
identified several targets for miR-499 gene family in adherence
junction and focal adhesion pathways. Tight junctions not
only seal the pulmonary epithelium and maintain the
structural integrity of the airway walls, but also act as key
regulators for epithelial cell proliferation and
differentiation. Dysfunction of this homeostasis could participate in
airway wall remodeling in asthma disease [
Within MIR499A and MIR499B genes, bioinformatics
analysis revealed the presence of a common
polymorphism at the position 20:34,990,448 within the seed region
of mature miR-499a-3p (rs3746444; AC[A/G]UCAC). In
the current study, we genotyped rs3746444
polymorphism in asthmatic children and adolescents compared to
controls. Genotype frequencies did not deviate from the
Hardy-Weinberg equilibrium in the study groups. G
variant was associated with increased susceptibility to
develop bronchial asthma under all genetic association
models. Despite the MAF (G) represented 0.26 in the
control group, it accounted for 0.59 among patients. The
same allele was found to be associated with poor
prebronchodilator FEV1 in asthmatic patients. In addition,
homozygotes for G had the worst bronchodilator
response after Salbutamol inhalation. That was consistent
with prior studies; G allele was also associated with
increased risk of developing of several autoimmune and
inflammatory diseases. miR-499a*G carriers showed
higher susceptibility to RA in Egyptians [
] and Iranian
], Behcet’s disease in Turks [
colitis in Japanese [
], ankylosing spondylitis [
idiopathic recurrent spontaneous abortion in Koreans [
and coronary artery disease [
]. In addition,
individuals with rs3746444*G allele showed a more active
phenotype of RA in Egyptian female patients  and a
severe course of multiple sclerosis [
Though our in silico data analysis revealed that the
substitution of A > G has no dramatic effect on the
folding pattern of the hairpin loop structure, each allele was
predicted to have different set of gene targets. A total of
1890 genes is influenced by mature miR-499a with
rs3746444*A variant. In contrast, rs3746444*G allele
could delete 41.8% of these genes and create new 763
targets. By conducting functional annotation clustering
and enrichment analysis, our results illustrated that gene
1 4 8 9 6 2
p o 1 4 7 1 3 6 0 3 5 3 9 1
c N 2 1 6 1 9 5 1 2 7 1 9 1
6 5 5 0 9 4 9 6 9 6
1 1 2 5 1 3 7 6 7 9 3
lea2bliilcaanndC iirrttssccaaeh l()rtaeuonb%m ,rsyaaaeeeng –il()re611hndC –l()tssc8ee211ondA reend laFeem laeM ftsaaohHm lrtttssaaeuub itsyeb isceeend ranbU lraRu ,ttsyaaaaeeeenong ≤l()ryyaE3 ()tyaLe3> ,irrttssyaaaeuhondm ttssyaeehhnoppm ittscaaohpAm i-ttscaaaonohpNm iiiir-ttssssvaeehnnpA iir-tssxccaEeeeuhndd tsyopmm ittssyyaeop>Dmmm lrttsscyauonopNmm lir/skveeee2eeRu>w iiiliittttscyvaonAm lrttscahonom llllrtceeonodW lllrrttycaePonod llrtcenonodU
T C T M G F P O R M A A S A
) )0 f
) ) ) ) ) ) ) ) 1 2 s
.2 7 .3 .8 .2 .9 .1 .4 – – n
3 6 3 2 9 7 8 1 5 5 io
(4 (4 (3 (4 (4 (2 (4 (5 (1 (2 ta
8 2 1 5 0 7 6 8 5 5 if
3 4 1 4 3 1 2 1 4 6 is
) ) t
2 5 i
i)tedun .)(839 .)(736 .)(554 .)(041 .)(832 .)(850 .)(938 .)(040 –(5121 –.(72423 ;lifyahm
n 5 3 8 3 0 1 1 4 6 8 FH
o 3 3 1 4 2 3 2 1 1 8 ,
le2liicanC irtsyaveehm ild rtaeeod rveee iiirtseobdm -rrsyyaeehpp lraom lirreeond ilrt/aeeoddm rveee ttsse IlI(t/aEogUm 6(E10C×)/L rssaaeeohnw IiliseeugndAN
ab ts M M S -o irw N B M S ab T A lau nG
T A C A L V o
sets for each allele only shared the same 37.8%, 40.1%,
and 52.8% for biological process, molecular function,
and cellular components, respectively. In particular, G
allele targets were significantly involved in two new
KEGG pathways (other glycan degradation and
glycolysis/gluconeogenesis). In the former pathway, miR-499
can target both aspartylglucosaminidase (AGA) gene
involved in the catabolism of N-linked oligosaccharides of
glycoproteins in the lysosomes, and galactosidase beta 1
(GLB1) gene, which plays functional roles in the
formation of extracellular elastic fibers and in the
development of connective tissue. In the other pathway for
glycolysis/gluconeogenesis, miR-499 mainly target
aldehyde dehydrogenase 1 family member A3
(ALDH1A3) and alcohol dehydrogenase 1 beta
polypeptide (ADH1B) genes, members of the enzyme
family which metabolize a wide variety of substrates as
multiple inositol-polyphosphate phosphatase 1, an
important second messenger in eukaryotic cells.
ALDH1A3 gene was found to be over-expressed more
than two times in asthmatic patients and 3.5 times
during exacerbations [
]. Further, in vivo functional
analysis is warranted to explore the mechanistic
regulation of these targets in the etiopathogenesis of
In some previous studies, computational methods
were devised to study the effects of air-borne dust
through high fidelity computational simulation of
different particle sizes over several breathing cycles [
modeling lung pressures [
], and developing 3D
Euler Lagrangian models to obtain the regional
deposition of the poly-dispersed drug Budesonide [
It would be a beneficial next step to assess the
pharmacogenomics effect of variants with the regional
deposition of dust particles and drug delivery to the
respiratory tract during respiration.
To the best of our knowledge, this is the first study
highlighting the role of miR-499 rs3746444 in
bronchial asthma disease. However, some limitations need
to be addressed. First, the small relative sample size
in the current study might underestimate the
synergistic effect of miR-499 SNP with environmental
exposure. Second, the impact of the SNP was not
correlated with the miRNA expression profile. Third,
functional assessment of targets involved in
bronchodilator response and methacholine challenge test is
mandatory to develop more efficient therapeutic
In conclusion, our study suggested that the rs3746444
(A > G) polymorphism in miR-499 gene family might
contribute to the susceptibility of asthma in children
and adolescents with bronchial asthma. Additional
studies, including larger cohorts with diverse ethnic
background, and functional tests are warranted to explore
the immunomodulatory mechanism of miR-499 genes in
bronchial asthma for developing more specific
Additional file 1: Table S1. Baseline characteristics of the study groups.
Table S2.. Predicted target gene sets for mature miR-499a and miR-499b.
(http://www.microrna.gr/microT-CDS). (ZIP 79 kb)
Additional file 2: Figure S1. Principal components analysis of asthmatic
patients. Ordination plot constructed using 211 patients strand and 20
clinical and laboratory variables. Samples are distributed along two axes.
Axis 1 explains 22.6% of variance among patients, whereas axis 2 resolves
11.3% of variance. Samples are scattered and colored according to their
genotype; AA (red), AG (green), and GG (blue). PCA did not reveal clustering
of patients according to their genotypes. (DOCX 95 kb)
The authors thank the Oncology Diagnostic Unit and the Center of
Excellence in Molecular and Cellular Medicine, Suez Canal University, Ismailia,
Egypt for providing the facilities for performing the research work as well as
we thank all participants who agree to participate in the current study.
No sources of funding were used for this work.
Availability of data and materials
All data generated or analyzed during this study are included in this
published article and its supplementary information files.
EAT, EA, NMB, GMH, MSF conceived and designed the experiments, MHH. ER
and NMB recruited the study samples and clinical patient data, EAT, MHH,
ER, NMB and MSF contributed parts of the experiments, EAT contributed to
the bioinformatic and the statistical analyses. All authors contributed the
reagents and materials needed for the current work, and all authors
contributed in writing, reading and approval of the final manuscript.
Ethics approval and consent to participate
The study was conducted in accordance with the guidelines in the
Declaration of Helsinki and it has been approved by the Medical Research
Ethics Committee of Faculty of Medicine, Suez Canal University. Written
informed consent was obtained from all participants.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Submit your next manuscript to BioMed Central
and we will help you at every step:
Our selector tool helps you to find the most relevant journal
1. Li S , Xie X , Song Y , Jiang H , Wu X , Su X , Yang L , Li M. Association of TLR4 (896A/G and 1196C/T) gene polymorphisms with asthma risk: a metaanalysis . Med Sci Monit . 2015 ; 21 : 3591 - 9 .
2. Hussein MH , Toraih EA , Aly NM , Riad E , Fawzy MS . A passenger strand variant in miR-196a2 contributes to asthma severity in children and adolescents: a preliminary study . Biochem Cell Biol . 2016 ; 94 : 347 - 57 .
3. Tang DD . Critical role of actin-associated proteins in smooth muscle contraction, cell proliferation, airway hyperresponsiveness and airway remodeling . Respir Res . 2015 ; 16 : 134 .
4. Nakajima H , Hirose K. Role of IL-23 and Th17 cells in airway inflammation in asthma . Immune Netw . 2010 ; 10 : 1 - 4 .
5. Hall SC , Agrawal DK . Vitamin D and bronchial asthma: an overview of data from the past 5 years . Clin Ther . 2017 ;
6. Ober C , Yao T-C. A 21stCentury the genetics of asthma and allergic disease perspective . Immunol Rev . 2011 ; 242 : 10 - 30 .
7. Ardekani AM , Naeini MM . The role of MicroRNAs in human diseases . Avicenna Journal of Medical Biotechnology . 2010 ; 2 : 161 - 79 .
8. Toraih EA , Mohammed EA , Farrag S , Ramsis N , Hosny S . Pilot study of serum MicroRNA-21 as a diagnostic and prognostic biomarker in Egyptian breast cancer patients . Mol Diagn Ther . 2015 ; 19 : 179 - 90 .
9. Fawzy MS , Hussein MH , Abdelaziz EZ , Yamany HA , Ismail HM , Toraih EA . Association of MicroRNA-196a2 Variant with Response to Short-Acting beta 2-Agonist in COPD: An Egyptian Pilot Study . PLoS One . 2016 ; 11 : 2 .
10. Esteller M. Non-coding RNAs in human disease . Nat Rev Genet . 2011 ; 12 : 861 - 74 .
11. Ji Q , Jiang Q , Yan W , Li X , Zhang Y , Meng P , Shao M , Chen L , Zhu H , Tian N. Expression of circulating microRNAs in patients with ST segment elevation acute myocardial infarction . Minerva Cardioangiol . 2015 ; 63 : 397 - 402 .
12. Toraih EA , Ismail NM , Toraih AA , Hussein MH , Fawzy MS . Precursor miR-499a variant but not miR-196a2 is associated with rheumatoid arthritis susceptibility in an Egyptian population . Mol Diagn Ther . 2016 ; 20 : 279 - 95 .
13. Song GG , Bae SC , Seo YH , Kim JH , Choi SJ , Ji JD , Lee YH . The association between susceptibility to inflammatory arthritis and miR-146a , miR -499 and IRAK1 polymorphisms. A meta-analysis . Z Rheumatol . 2015 ; 74 : 637 - 45 .
14. Chen W , Shao D , Gu H , Gong J , Zhang J . Hsa-mir-499 rs3746444 T/C polymorphism is associated with increased risk of coronary artery disease in a Chinese population . Acta Cardiol Sin . 2017 ; 33 : 34 - 40 .
15. Oner T , Yenmis G , Tombulturk K , Cam C , Kucuk OS , Yakicier MC , Dizman D , Sultuybek GK . Association of pre-miRNA-499 rs3746444 and pre-miRNA-146a rs2910164 polymorphisms and susceptibility to Behcet's disease . Genet Test Mol Biomarkers . 2015 ; 19 : 424 - 30 .
16. Xu HY , Wang ZY , Chen JF , Wang TY , Wang LL , Tang LL , Lin XY , Zhang CW , Chen BC . Association between ankylosing spondylitis and the miR-146a and miR- 499 polymorphisms. PLoS One . 2015 ; 10 : e0122055 .
17. Wilm A , Higgins DG , Notredame C. R-coffee: a method for multiple alignment of non-coding RNA . Nucleic Acids Res . 2008 ; 36 : e52 .
18. Bhattacharya A , Cui Y. miR2GO: comparative functional analysis for microRNAs . Bioinformatics . 2015 ; 31 : 2403 - 5 .
19. Vlachos IS , Zagganas K , Paraskevopoulou MD , Georgakilas G , Karagkouni D , Vergoulis T , Dalamagas T , Hatzigeorgiou AG . DIANA-miRPath v3 . 0: deciphering microRNA function with experimental support . Nucleic Acids Res . 2015 ; 43 : W460 - 6 .
20. Bhattacharya A , Ziebarth JD , Cui Y. PolymiRTS database 3.0: linking polymorphisms in microRNAs and their target sites with human diseases and biological pathways . Nucleic Acids Res . 2014 ; 42 : D86 - 91 .
21. Bruno AE , Li L , Kalabus JL , Pan Y , Yu A , Hu Z. miRdSNP: a database of disease-associated SNPs and microRNA target sites on 3'UTRs of human genes . BMC Genomics . 2012 ; 13 : 44 .
22. Keller A , Backes C , Al-Awadhi M , Gerasch A , Küntzer J , Kohlbacher O , Kaufmann M , Lenhof HP . GeneTrailExpress: a web-based pipeline for the statistical evaluation of microarray experiments . BMC Bioinformatics . 2008 ; 9 : 552 .
23. Global Initiative for Asthma. Global Strategy for Asthma Management and Prevention . Revised 2014 Vancouver, GINA, 2014 . Available from: www. ginasthma. org [Accessed 16 December 2016 ].
24. Fawzy MS , Alhadramy O , Hussein MH , Ismail HM , Ismail NM , Biomy NM , Toraih EA . Functional and structural impact of ATP-binding cassette transporter A1 R219K and I883M gene polymorphisms in obese children and adolescents . Mol Diagn Ther . 2015 ; 19 : 221 - 34 .
25. Marshall WA , Tanner JM . Variations in pattern of pubertal changes in boys and girls . Arch Dis Child . 1969 ; 44 : 291 - 303 .
26. Soriano JB , Alfageme I , Almagro P , Casanova C , Esteban C , Soler-Cataluña JJ , de Torres JP , Martinez-Camblor P , Miravitlles M , Celli BR , Marin JM . Distribution and prognostic validity of the new global initiative for chronic obstructive lung disease grading classification . Chest . 2013 ; 143 : 694 - 702 .
27. Hussein MH , Sobhy KE , Sabry IM , El Serafi AT , Toraih EA . Beta2-adrenergic receptor gene haplotypes and bronchodilator response in Egyptian patients with chronic obstructive pulmonary disease . Adv Med Sci . 2017 ; 62 : 193 - 201 .
28. Crapo RO , Casaburi R , Coates AL , Enright PL , Hankinson JL , Irvin CG , MacIntyre NR , McKay RT , Wanger JS , Anderson SD , Cockcroft DW , Fish JE , Sterk PJ . Guidelines for methacholine and exercise challenge testing-1999 . This official statement of the American Thoracic Society was adopted by the ATS Board of directors , July 1999 . Am J Respir Crit Care Med . 2000 ; 161 : 309 - 29 .
29. Pellegrino R , Viegi G , Brusasco V , Crapo RO , Burgos F , Casaburi R , Coates A , van der Grinten CPM , Gustafsson P , Hankinson J , et al. Interpretative strategies for lung function tests . Eur Respir J . 2005 ; 26 : 948 - 68 .
30. Demirjian M , Rumbyrt JS , Gowda VC , Klaustermeyer WB . Serum IgE and eosinophil count in allergic rhinitis-analysis using a modified Bayes' theorem . Allergol Immunopathol (Madr) . 2012 ; 40 : 281 - 7 .
31. Toraih E , Hussein MH , Badran DI . Beta2-adrenergic receptor gene polymorphisms in Egyptian patients with acute myocardial infarction . Advances in Molecular Biology . 2014 ; 2014 : 471635 .
32. Toraih EA , Fawzy MS , El-Falouji AI , Hamed EO , Nemr NA , Hussein MH , Abd el Fadeal NM . Stemness-related transcriptional factors and homing gene expression profiles in hepatic differentiation and cancer . Mol Med . 2016 ; 22
33. Fang C , Lu W , Li C , Peng X , Wang Y , Huang X , Yao Z , Cai N , Huang Y , Zhang X , Tan J. MiR- 3162 -3p is a novel MicroRNA that exacerbates asthma by regulating β-catenin . PLoS One . 2016 ; 11 : e0149257 .
34. Sun G , Yan J , Noltner K , Feng J , Li H , Sarkis DA , Sommer SS , Rossi JJ. SNPs in human miRNA genes affect biogenesis and function . RNA . 2009 ; 15 : 1640 - 51 .
35. Toraih EA , Fawz MS , Elgazzaz MG , Hussein MH , Shehata RH , Daoud HG . Combined genotype analyses of precursor miRNA196a2 and 499a variants with hepatic and renal cancer susceptibility a preliminary study . Asian Pac J Cancer Prev . 2016 ; 17 : 3369 - 75 .
36. van Rooij E , Quiat D , Johnson BA , Sutherland LB , Qi X , Richardson JA , Kelm RJ , Olson EN . A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance . Dev Cell . 2009 ; 17 : 662 - 73 .
37. Liu J , Liang X , Zhou D , Lai L , Xiao L , Liu L , Fu T , Kong Y , Zhou Q , Vega RB , et al. Coupling of mitochondrial function and skeletal muscle fiber type by a miR-499/Fnip1/AMPK circuit . EMBO Mol Med . 2016 ; 8 : 1212 - 28 .
38. Rael EL , Lockey RF . Interleukin-13 signaling and its role in asthma . World Allergy Organ J . 2011 ; 4 : 54 - 64 .
39. Peng J , Yang XO , Chang SH , Yang J , Dong C. IL-23 signaling enhances Th2 polarization and regulates allergic airway inflammation . Cell Res . 2010 ; 20 : 62 - 71 .
40. Liew FY , Patel M , Xu D : Toll-like receptor 2 signalling and inflammation . Ann Rheum Dis 2005 , 64 Suppl 4 : iv104 - 105 .
41. Oglesby IK , McElvaney NG , Greene CM . MicroRNAs in inflammatory lung disease-master regulators or target practice? Respir Res . 2010 ; 11 : 148 .
42. Pedersen JW , Bennett EP , Schjoldager KT , Meldal M , Holmér AP , Blixt O , Cló E , Levery SB , Clausen H , Wandall HH . Lectin domains of polypeptide GalNAc transferases exhibit glycopeptide binding specificity . J Biol Chem . 2011 ; 286 : 32684 - 96 .
43. Wendell SG , Baffi C , Holguin F. Fatty acids, inflammation, and asthma . J Allergy Clin Immunol . 2014 ; 133 : 1255 - 64 .
44. Adar T. Ilan Y : beta -glycosphingolipids as immune modulators . J Immunotoxicol . 2008 ; 5 : 209 - 20 .
45. Karman J , Tedstone JL , Gumlaw NK , Zhu Y , Yew N , Siegel C , Guo S , Siwkowski A , Ruzek M , Jiang C , Cheng SH . Reducing glycosphingolipid biosynthesis in airway cells partially ameliorates disease manifestations in a mouse model of asthma . Int Immunol . 2010 ; 22 : 593 - 603 .
46. Szymczak I , Wieczfinska J , Pawliczak R . Molecular background of miRNA role in asthma and COPD: an updated insight . Biomed Res Int . 2016 ; 2016 : 7802521 .
47. Davies DE. Epithelial barrier function and immunity in asthma . Ann Am Thorac Soc . 2014 ; 11 ( Suppl 5 ): S244 - 51 .
48. Hashemi M , Eskandari-Nasab E , Zakeri Z , Atabaki M , Bahari G , Jahantigh M , Taheri M , Ghavami S. Association of pre-miRNA-146a rs2910164 and premiRNA-499 rs3746444 polymorphisms and susceptibility to rheumatoid arthritis . Mol Med Rep . 2013 ; 7 : 287 - 91 .
49. Okubo M , Tahara T , Shibata T , Yamashita H , Nakamura M , Yoshioka D , Yonemura J , Kamiya Y , Ishizuka T , Nakagawa Y , et al. Association study of common genetic variants in pre-microRNAs in patients with ulcerative colitis . J Clin Immunol . 2011 ; 31 : 69 - 73 .
50. Jeon YJ , Choi YS , Rah H , Kim SY , Choi DH , Cha SH , Shin JE , Shim SH , Lee WS , Kim NK . Association study of microRNA polymorphisms with risk of idiopathic recurrent spontaneous abortion in Korean women . Gene . 2012 ; 494 : 168 - 73 .
51. Zhi H , Wang L , Ma G , Ye X , Yu X , Zhu Y , Zhang Y , Zhang J , Wang B . Polymorphisms of miRNAs genes are associated with the risk and prognosis of coronary artery disease . Clin Res Cardiol . 2012 ; 101 : 289 - 96 .
52. Fawzy MS , Toraih Eman A , Hamed EO , Hussein MH , Ismail HM . Association of MIR-499a expression and seed region variant (rs3746444) with cardiovascular disease in Egyptian patients . Acta Cardiol . 2017 ; https://doi. org/10.1080/00015385. 2017 . 1351243 .
53. El-Shal AS , Aly NM , Galil SM , Moustafa MA , Kandel WA . Association of microRNAs genes polymorphisms with rheumatoid arthritis in Egyptian female patients . Joint Bone Spine . 2013 ; 80 : 626 - 31 .
54. Kiselev I , Bashinskaya V , Kulakova O , Baulina N , Popova E , Boyko A , Favorova O . Variants of MicroRNA genes: gender-specific associations with multiple sclerosis risk and severity . Int J Mol Sci . 2015 ; 16 : 20067 - 81 .
55. Hershey G , Aronow B : Altered gene expression profiles in stable versus acute childhood asthma . US7 , 919 ,240B2 2011 . Available at https://www. google.com/patents/US7919240 (last accessed May 6th, 2017 ).
56. Kannan R , Guo P , Przekwas A . Particle transport in the human respiratory tract: formulation of a nodal inverse distance weighted Euler-Lagrangian transport and implementation of the wind-Kessel algorithm for an oral delivery . Int J Numer Method Biomed Eng . 2016 ; 32
57. Kannan R , Chen ZJ , Singh N , Przekwas A , Delvadia R , Tian G , Walenga R. A quasi-3D wire approach to model pulmonary airflow in human airways . Int J Numer Method Biomed Eng . 2017 ; 33
58. Ravi Kannan R , Przekwas AJ , Singh N , Delvadia R , Tian G , Walenga R . Pharmaceutical aerosols deposition patterns from a dry powder inhaler: Euler Lagrangian prediction and validation . Med Eng Phys . 2017 ; 42 : 35 - 47 .