The Study On The Relationship Between Levels Of Serum25-hydroxy Vitamin D And Its Receptor Gene Polymorphisms And Asthma In Children

Background:Bronchial asthma is a common chronic lung disease.It is also one of the most common chronic airway diseases affecting children’s health. The incidence is gradually rising, and brings heavy healthy and economic burden to the patient’s family and society,so doing some in-depth study of bronchial asthma will have profound social significance.Asthma is characterized by chronic airway inflammation, airway hyperresponsiveness and airway remodeling involving eosinophiles, macrophage, T lymphocyte and airway endothelial cells. It causes children with recurrent wheezing, shortness of breath, chest tightness and cough, and more and (or) in the early morning or at night.It seriously can appear status asthmaticu life-threatening that seriously affects the children’s physical and mental health. The pathogenesis of asthma is complex, closely related to immunity and genetic background. In recent years, the studies in domestic and foreign found that vitamin D (VitD) may play an important role in the etiology of asthma.We used to think that vitamin D had to adjust the action of bone metabolism and calcium balance, but now more and more epidemiology, genetics and animal model of experimental research shows that it also has strong immunomodulatory effect as a second steroid hormones, and is closely related to the inherent and adaptive immune responses.the study found that it can restrain the evolution of autoimmune diseases mediated by all sorts of Th1immune response, such as inflammatory bowel disease(IBD), rheumatoid arthritis, systemic lupus erythematosus, Multiple Sclerosis(MS), Type1Diabetes Mellitus(T1DM), etc., and may involved in the etiology of asthma. Related studies have shown that reducing the sun exposure is associated with the onset of asthma, and vitamin D are considered to be contact mediator between sun exposure with the onset of asthma, vitamin D deficiency and insufficiency has a higher morbidity in the world, even in the abundant sunlight exposure area, children are the high-risk groups, and serum25-hydroxy vitamin D(25-(OH)-VD) is the most abundant in human serum and the most stable VitD3metabolites, and the best indicator for judging the intake of VitD3content in body by skin or dietary. Therefore, there will has important clinical significance for testing their vitamin D levels in children asthma.Bronchial asthma is chronic type I allergic diseases mediated by the IgE, CD4+T cells play important role in regulating the pathogenesis of bronchial asthma.depending on the secretion of cytokines, it can be classified into two subgroup:Th1、Th2. The imbalances of Th1/Th2cells is now be thought the important basis for asthma morbidity. Thl as known as inflammatory T-cells, mainly.mediate cellular.immune and inflammatory response through the release of IFN-γ, IL-2and IL-3, and then play a role in negative regulation of asthma. Th2cells mainly involve in proliferation of B-cell, antibody production and type I allergy by releasing cytokines such as IL-4, IL-5, IL-13to involve in the onset of asthma and airway inflammation. The excessive activation of T cell(to Th2differentiation advantage) is the foundation of the onset of asthma. More and more studies confirmed that vitamin D plays an important role in immune regulation through a variety of immune cells such as mast cells, Dentritic Cells(DCS), regulatory T cells(Tregs) interact and their secretion of cytokines for the pathogenesis of asthma. Some studies indicate that vitamin D deficiency will influence regulatory T-cells function and break the balance between Thl and Th2in some allergic diseases. Camargo et al. showed that1,25-(OH)-VD inhibited Thl cells differentiation. Camargo et al. showed that1,25-(OH)-VD inhibited Th1cells differentiation.their experiments showed it reduced IL-4production during the initial T cells in vitro for polarization (Th)(early differentiation), had very good inhibitory effect for Th2cells differentiation and airway eosinophilia.Topilski et al.,in an murine model of asthma challenged witflammatory responses and reduced IL-4production in bronchoalveolar lavage fluid in the initial stage of induced asthma or completed Th2cells differentiation stage.1,25-(OH)-VD that can also play a role after differentiation of T cells showed that the effect of T cell migration is restrained, so they couldn’t successfully reach the inflammatory site to exert their effect. Influence mechanisms of1,25-(OH)-VD on asthma including two aspects:lowering IL-4and preventing the migration of effect T-cell.Pichler et al. showed that in CD4as well as CD8human cord blood cells, vitamin D not only inhibits IL-12-generated IFN-y production but also suppresses IL-4and IL-4-induced expression of IL-13. In contrast to these results. Aheu et al.on the animal experiments between equivalent human doses of1,25-(OH)-VD to prevent rickets with the onset of asthma, showed that vitamin D can promote antigen specific T lymphocyte proliferation, increase Th2cytokines secretion such as IL-4and IL-13,and also IgE. More of related experimental study needs to carry out on how does vitamin D effects Th2differentiation and its.specific mechanism.Affecting the level of serum vitamin D is closely related to genetic factors, besides factors such as the sun exposure, diet.A lot of gene polymorphism associated with vitamin D metabolism and its pathways have been found, such as cytochrome P4502r1(CYP2R1) gene, vitamin D receptor (VDR) gene, the GC-globulin (GC), cytochrome P450genes24Al (CYP24A1) gene mutations and so on.they can change vitamin D levels. The present study system is one of the most CYP2R1gene, VDR gene.Since Morrison reported relationship between VDR gene polymorphism and bone mineral density for the first time in1994, the correlation research between VDR gene polymorphism and diabetes, tuberculosis, bone metabolism, growth and development has been the hot topic for scholars. VDR is the main receptor which the biological activities of1,25(OH)2D3mediates, VDR gene was cloned in1988which maped to chromosome12q12-ql4,spaned60-70KB, consisted of nine coding exons and encoded a427-amino acid protein. Several studies have shown that VDR gene is very good candidate to observe the asthma, that the polymorphism is associated with asthma risk for children and adults. Five single nucleotide polymorphisms (SNPs) in the VDR gene have been well investigated by genetic associations studies,namely FokI (rs2228570), BsmI (rs1544410), Tru9I (rs757343), Apal (rs7975232) and TaqI (rs731236), which are respectively located in exon2, intron8,exon9and3’untranslated region (UTR). Rs2228570(Fokl) consists of a T to C change in the start codon of exon2,which results in amino acid codon change(ATG�'ACG). so when the C variant is present, the translation initiation site places afte three codes and the post-translational proteins is less3amino acids, with424amino acids, but greatly enhances the transcription activity;Rs7975232(Apal) is located in intron8of the VDR gene, due to the mutation(G�'T), leading to thymine to guanine;rs1544410(BsmI) is due to the mutation(G�'A), the polymorphism does not affect the amino acid sequence of the VDR, but protein synthesis was regulated by3’UTR region of the VDR gene.Rs1544410and rs7975232polymorphisms are also located in this region, its base change affects the stability of the mRNA, which influences the expression of VDR; Rs731236(TaqI) polymorphisms is located in exon9and leads to a silent codon change, with ATT and ATC. Though its change doesn’t make the VDR change the sequence of amino acids, it is associated with VDRmRNA stability and transcriptional activity,that will influence the expression of VDR. Rs757343(Tru9I) is located in intron8of the VDR gene, because of its mutation(G�'A), was first discovered and reported by the French Ye, etc in2000, Zajickova, etc once again proved the existence of the site in2003. The four SNPs (BsmI-ApaI-TaqI-Tru9I) showed significant Linkage disequilibrium (LD) with each other, as it has been shown in previous studies, especially BsmI-TaqI. There is a strong chain between BsmI and3’UTR. Most of the experiments conducted so far point to the fact that VDR gene may influence either its expression or its function which may cause alteration in the vitamin D activity. In addition, many of the VDR target genes are involved in the regulation of vitamin D metabolite concentrations via a classical endocrine feedback loop. Any defect in the VDR expression or function could influence concentrations of vitamin D metabolites.In recent years, the VDR gene polymorphism has been widely reported in some diseases such as rickets, cancer, diabetes, osteoporosis, and the relationship with asthma has caused more and more concern. Poon AH and colleagues addressed a study which involved two independent French Northeastern Quebec families found that VDR gene polymorphism was associated with asthma, rs2228570(FokI) affected the VDR transcription activity because of amino acid codon change(427ATG�'ACG) in exon2; Three domestic studies(Aaadi, Fang and Li etc) failed to show any association between VDR gene polymorphisms and asthma. Another study which951children from224pedigrees were analyzed for13SNPs in the VDR (including FokI, BsmI, TaqI) could not confirmed a preferential transmission of vitamin D receptor variants to children with asthma.But a study for VDR gene polymorphism in Tunisia children found that VDR variants(FokI, BsmI, TaqI)was significantly related to the increasing risk of developing childhood asthma between asthmatics and controls while ApaI had no difference. The study also found that the three3’SNPs (Bsml-ApaI-TaqI) showed significant LD with each other. These changes could influence the expression of mRNA and stability, thetranscription activity and expression of VDR,and then ultimately changed the phenotype of asthmatic children." The discrepancy among above studies may be a result of genetic heterogeneity and differences in environmental factors. There are different frequency of SNPs in different population groups. Asthma has been shown to represent a multiple genetic illness. The accumulated effects of multiple genes result in the genetic heterogeneity. Asthma is also affected by environmental factors, smoking (passive or active), dust, pets and professional exposure and so on. These factors are associated with divergence of experimental results. At present, the investigations concerned the correlation of VDR gene polymorphism (FokI、BsmI、ApaI、TaqI、Tru9I) and bronchial asthma are rare either in domestic and foreign. Objectives:To determine the role of25-(OH)-VD、Th1/Th2on the development and maintenance of asthma, to investigate the vitamin D receptor gene polymorphisms in children asthma, to provide some theoretical basis for pathophysiology, treatment and identification of susceptibility to asthma.Methods:1.40Follow-up children with asthma (clinical paracmasis) in clinic from Marlst,2012to Jun.30st,2013, were enrolled randomly to be asthma group.40healthy children were selected as the control group. The diagnostic standard accords with asthma formulated by Respiratory group, Academy of Pediatrics, Chinese Medical Association (2008). There are no difference in age and sex between two groups. All children enrolled are informed to sign the consent forms.2.3ml peripheral vein blood was drawn from each child with empty stomach in the early morning,1ml of each blood sample was prepared serum after preparation of blood smear. Other2ml of each one was extracted genomic DNA.3.Thelevels of25-(OH)-VD、IL-4. IL-5and IFN-γ in serum were detected by enzyme-labeled immunosorbent assay (ELISA). All experimental steps were carried out according to the instructions, including pipetting, seal plate, water bath, washer, enzyme, coloration, reading the OD value.4. Polymerase chain reaction (PCR), gene clone and DNA sequencing were used to analyse the vitamin D receptor polymorphisms (rs2228570、rs7975232、 rs1544410、rs731236、rs757343).5. Statistical analysis:statistical comparisons were performed using t or t’test and the χ2test. Differences with P values<0.05were considered significant. All statistical analysis was operated with SPSS13.0software.Result:1. The levels of25-(OH)-VD of serum in asthmatic children were lower remarkably than that of control group(t=7.498, P<0.001).2. The levels of IL-4of serum in asthmatic children were higher remarkably than those of control group(t=14.03, P<0.001).3.The levels of IL-5of serum in asthmatic children were higher remarkably than those of control group(t=6.7, P<0.001).4.The levels of IFN-y of serum in asthmatic children were lower remarkably than that of control group(t=6.4, P<0.001).5. Significant deviations from the genotypes (TT、TC. CC)of rs2228570were no found in the asthma and health group.(x2=0.854, P=0.652). There was no statistical difference in the distribution of alleles of vitamin D receptor(rs2228570),(x2=0.948, P=0.330). The OR for asthma respectively were0.824and1.130[95%CI,(0.556-1.220),(0.882.1.448)]for allele T and C.6. The mutant type(TT)of rs7975232is not found in both asthma group and health group. Significant deviations from the genotypes (GT, GG) were found in the asthma and health group.(x;2=7.218, P=0.007). The OR for asthma respectively were1.923and0.556[95%CI,(0.353-0.875).(0.353-0.875)]for wild type(GG)and the mutant type(GT).7. The mutant type(CC)of rs731236was not found in both asthma group and health-group. Significant deviations from the genotypes (TT. TC) were found in the asthma. arid. health group.(χ2=8.455, P=0.004). The" OR for asthma respectively were2.00and0.519[95%CI,(1.212-3.301).(0.323-0.833)]for wild type(TT)and the mutant type(TC).8. Significant deviations from the genotypes (TT、TC、CC)of rs11574114were found in the asthma and health group.(x2=7.632, P=0.022). The OR for asthma respectively were0.661and1.952[95%CI,(0.510-0.875).(1.276-2.986)]for allele T and C.9.The mutant type(AA)of rs1544410is not found in both asthma group and health group. Significant deviations from the genotypes (GG. GA) were found in the asthma and health group.(χ2=7.366, P=0.007). The OR for asthma respectively were0.586 and2.091[95%CI,(0.390-0.881)、(1.183-3.695)]for wild type(GG)and the mutant type(GA).10. Significant deviations from the genotypes (GG、GA、AA)of rs757343were found in the asthma and health group.(x2=6.651, P=0.036). The OR for asthma respectively were0.684and1.783[95%CI,(0.525-0.891)、(1.188-2.675)]for allele G and A.Conclusions:1. The levels of25-(OH)-VD of serum in asthma children were lower than those in control children,25-(OH)-VD maybe play a role in the pathophysiology of children asthma.2. Significant deviations from the genotypes of vitamin D receptor(rs7975232、 rs1544410、rs731236、rs11574114、rs757343) were found between the asthma and health groups, the polymorphisms may be associated to asthma. There were no statistical difference in the distribution of genotypes and alleles of vitamin D receptor(rs2228570), the polymorphisms may be not associated to asthma.3. The hypofunction of Th1cell immunity and the hyperfunction of Th2cell immunity, respectively induces the subregulation of IFN-γ and upregulation of IL-4and IL-5. The balance of different T-helper subsets maybe play a role in the pathophysiology of children asthma.