Structure and functional impact of seed region variant in MIR-499 gene family in bronchial asthma
© The Author(s). 2017
Received: 15 May 2017
Accepted: 25 August 2017
Published: 8 September 2017
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.
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.
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).
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.
Bronchial asthma is a chronic heterogeneous respiratory disease that is characterized by airway inflammation, recurring bronchial obstruction, and airway hyper-responsiveness [1, 2]. Most common histopathological features are inflammatory cell infiltration, sub-basement fibrosis, smooth muscle hypertrophy, mucus hypersecretion, injury to epithelial cells, and angiogenesis [3, 4]. 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 .
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 non-coding RNA . Analysis by PolymiRTS Database v3.0 (Polymorphism in microRNAs and their TargetSites) was conducted to predict SNPs and INDELs in the whole gene (http://compbio.uthsc.edu/miRSNP/home.php) . 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 DIANA-microT-CDS v5.0 (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index), 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.imis.athena-innovation.gr/DianaTools/index.php?r=tarbase/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 Diana-miRPath v3.0 software for gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) pathways . 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, miR-499b-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 miR-499b targets was conducted by miRpair2Go web-platform using two combined miRNA target prediction methods (TargetScan and miRanda) and moderate hierarchial filtering level (http://compbio.uthsc.edu/miR2GO/help.php) .
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 post-transcriptional 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 work . 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.bioinf.uni-sb.de/) . 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 described . 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/BMIPedsCalc.php) . 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’ parents.
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 . Repeated post-bronchodilator 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] . Additionally, bronchoconstriction provocation via methacholine challenge test (MCT) was done to assess bronchial hyper-responsiveness. 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 [28, 29].
Blood samples were collected in EDTA Vacutainers. Absolute eosinophil count (AEC) was calculated using the Coulter Counter. Total IgE was measured by enzyme-linked 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 Biosystems).
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 two-tailed 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
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
Functional annotation clustering and pathway enrichment analysis
Predicted functional effect of rs3746444 SNP
Genotyping of rs3746444 polymorphism in the study groups
Genotype and allele frequencies of hsa-miR-499a seed region variant in asthma patients and controls
Crude OR (95%CI)
Adjusted OR (95%CI) a
(n = 96)
(n = 96)
AG + GG
(n = 115)
(n = 204)
AG + GG
(n = 211)
(n = 300)
AG + GG
Clinical and laboratory data of asthmatic patients, according to miR-499A rs3746444 genotypes
Total number (%)
Mean age, years
FH of asthma
Mean age at onset, years
3.82 ± 1.6
3.58 ± 1.9
3.25 ± 1.8
Asthma duration, years
5.55 ± 2.5
5.96 ± 2.3
6.23 ± 2.5
Daytime symptoms >2/week
Reliever use >2/week
Total IgE (IU/ml)
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 [34, 35]. 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]. Bioinformatics 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 . IL-23 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 [40, 41]. In addition, miR-499a and miR-499b are involved in Fc-epsilon 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 . Another identified pathway that is highly impacted by miR-499 gene family is glycosphingolipid (GSL) biosynthesis. β-Glycosphingolipids 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 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 . Both 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 pre-bronchodilator 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 people , Behcet’s disease in Turks , ulcerative colitis in Japanese , ankylosing spondylitis , idiopathic recurrent spontaneous abortion in Koreans , and coronary artery disease [51, 52]. 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 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 asthma.
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 strategies.
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 theranostic agents.
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.
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