| Asthma is a complex disease triggered by the interaction of genetic predisposition and environmental factors. Recently, the first genome-wide association study (GWAS) of asthma in European subjects identified a novel region containing the ORM1-like3(ORMDL3) gene at chromosome17q21strongly associated with childhood asthma and ORMDL3transcript abundance.ORMDL3, located at17q21.1, belongs to the novel ORM gene family. In yeast this gene family includes two menbers:ORMDL1and ORMDL2, while in human it contains three menbers:ORMDL1, ORMDL2and ORMDL3. Breow et al. recently found in yeast Orm proteins as mediators of phingolipid homeostasis and suggested that sphingolipid misregulation might be related to the development of childhood asthma. Knockout of ORM genes in yeast resulted in slower growth and sensitivity to toxic compounds that can be rescued by transfection of human ORMDL3. With a total length of6560bp, including3exons, ORMDL3encodes a protein of153amino acids with4putative transmembrane domains anchored at the endoplasmic reticulum (ER). The gene expression is ubiquitous in human tissues, especially liver and peripheral blood lymphocytes. ORMDL3was recently found involved in Ca2+signaling and the unfolded protein response, so it may participate in signaling and facilitating ER-mediated inflammatory responses. ORMDL3is linked to interleukin17(IL-17) secretion:polymorphisms within the17q21locus increased ORMDL3and GSDMB gene expression and elevated IL-17secretion in cord blood. The17q21locus may affect T-cell development during maturation of the immune system and susceptibility to disease. Moreover,ORMDL3was found associated with type1diabetes, primary biliary cirrhosis, glioma, Crohn’s disease, allergic rhinitis, rheumatoid arthritis, and ankylosing spondylitis.Since ORMDL3was identified as a new asthma candidate gene, soon, the replication studies were carried out in different populations, and confirmed the relationship between ORMDL3and asthma. But all of the studies were carried out in childhood asthma, how about the relationship of ORMDL3and adult asthma? Whether genetic variants in chromosome17q21still linked to ORMDL3transcript abundance in peripheral blood leukocytes or not? How ORMDL3is regulatied? Or what’s the transcriptional regulation mechanism of ORMDL3? With these questions, we carried out our study in following studies.Part oneAssociation study of genetic variants in17q21and adult-onset asthma in a Chinese Han populationTo comfirm the relationship of17q21, containing ORMDL3, and adult-onset asthma, we recruited710adult-onset asthma patients from Qilu Hospital, Jinan.and656unrelated, random-sampled healthy controls who were matched to cases by age, ethnicity and geographic region, who underwent comprehensive medical screening at Qilu Hospital and were without symptoms or history of asthma or other pulmonary diseases or atopy. We selected5single nucleotide polymorphisms (SNPs) according to previous results and the linkage disequilibrium (LD) status in HCB to do case-control association study, they were:rs7216389, rs12603332, rs12936231, rs9303277, rs11557467. We found the5SNPs significantly associated with asthma (P<0.05), of which2, rs11557467and rs9303277, were strongly associated (P<0.001). Subjects carrying the G allele of rs11557467or the C allele of rs9303277showed increased risk of asthma (odds ratio [OR]1.27,95%confidence interval1.07-1.51, P=0.006, and OR1.27,1.07-1.49, P=0.005, respectively), even after adjusting for age and sex. As conpaired with TCCCG, the risk of asthma was lower for carriers of the haplotype CTGTT (OR0.81,0.67-0.97, P=0.02). That our results demonstrated that the genetic variants in17q21which containing ORMDL3gene was associated with adult-onset asthma in Chinese Han population.Part two Identifying the functional SNPs in17q21regionAccording to the ensemble database, ORMDL3contains2transcripts: ENST00000304046(ORMDL3-001) and ENST00000394169(ORMDL3-002). These two transcripts have same coding region, but different noncoding region, so they transale the same proteins. While, which transcript existed in human tissues? Besides, Moffatt et.al found that the genetic variants in chrosome17q21link to ORMDL3gene expression in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines from children in the asthma family, which suggested that the variants which regulating ORMDL3gene expression may be mediating susceptibility to asthma. So far, none of the variants that associated with asthma located in the coding region of ORMDL3, but one of the SNPs rsl2603332, that we selectied in part one, was located in the first intron of ORMDL3. It is known to all that the SNPs located in the intron may affect mRNA splicing. That the aim of this study was:first, to confirm which transcript was exited in different human tissues and second, to assess whether the SNP rs12603332, located in first intron of ORMDL3, affect ORMDL3gene splicing or not.We conducted this experiment in peripheral blood leukocytes in10samples, half of them containing the CC genotype and half the TT genotype in the rs12603332 site. Besides, we also collected human liver tissue, the tissue of esophageal cancer, tissue of carcinoma of large intestine, the tissue of lung cancer, each of them collected12samples, and total RNA was extracted from leukocytes by the TRIZOL reagent method (Invitrogen, Carlsbad, CA). According to the character of two transcripts, we designed2pairs of primers. One pair, located in the first and second exons, would amplify both transcripts. Another pair, located in the5’untranslated region of the second transcript, would yield products only when the second transcript was present. The results were similar that in human peripheral blood leukocytes, liver, esophageal cancer, carcinoma of large intestine and lung cancer, that they all express ORMDL3-001transcript. Only the first pair of primers yielded products for all cDNA samples. However, the second pair of primers yielded PCR products when genomic DNA was used as a template. Besides, both CC and TT homozygotes at rs12603332express ORMDL3-001transcript as well. That rs12603332did not affect ORMDL3gene mRNA splicing. And what is the mechanism of17q21associated with asthma? Which variant is the real functional SNP?As abnormal gene expression is an important mechanism in mediating susceptibility to asthma and other diseases, and the transcript abundances of genes may be directly modified by polymorphisms in regulatory elements. We detected the expression level of ORMDL3and GSDMB genes in asthma patients and healthy controls. To assess this question, we recruited61adult-onset asthma patients and70health controls from Qilu Hospital, Genomic DNA was extracted from peripheral blood leukocytes by a standard phenol-chloro-form method. Total RNA was extracted from leukocytes by the TRIZOL reagent method (Invitrogen, Carlsbad, CA). We performed real-time PCR for different genotype groups using peripheral leukocyte RNA. We found that ORMDL3and GSDMB gene expression was significantly increased in asthma patients. And he SNPs rs7216389, rs12603332, and rs12936231had striking effects on ORMDL3and GSDMB expression. The expression level was robustly increased in homozygotes for risk alleles as compared with those carrying1or2copies of the reference alleles, P<0.05. In this part of study, first, we found that only ORMDL3-001transcript was expressed in human peripheral blood leukocytes, liver, esophageal cancer, carcinoma of large intestine and lung cancer. And the allele of rs12603332does not affect ORMDL3gene splicing. Second, we found that as compared with healthy controls, ORMDL3and GSDMB gene expression was significantly increased in asthma patients in peripheral blood leukocytes. Genetic variants in chrosome17q21had striking effects on ORMDL3and GSDMB expression. Thus, the abnormal expression of ORMDL3might be one of the causes of asthma.Part three Characterization of the transcription factors regulating human ORMDL3geneAs the abnormal expression of ORMDL3might be one of the causes of asthma. And there are few studies about how it expressed or the transcriptional mechanism of ORMDL3is fairly unknown. Thus, we elucidate the mechanisms involved in human ORMDL3transcriptional regulation.First, we cloned the1.5-kb genomic DNA fragment containing the putative promoter region and evaluated its transcriptional activity in a luciferase reporter system by deletion analysis. The human ORMDL3proximal promoter elements were located-68bp downstream and the essential positive control elements were from-68to-42bp. Bioinformatics analysis predicted that the interval-64to-56bp contained signal transducer and activator of transcription6(STAT6) binding sites. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrated that STAT6bound to its binding site within the ORMDL3promoter.Thus, the human ORMDL3proximal promoter elements were located-68bp downstream and STAT6binding site, located at-64to-56region, was essential for the promote activity of ORMDL3gene. Part four STAT6directly regulates the expression of human ORMDL3geneAs the STAT6binding site, located at-64to-56region, was essential for the promote activity of ORMDL3gene. How transcriptional factor STAT6regulates ORMDL3gene expression? We transfected the ORMDL3expression vector or si-RNA for STAT6to Jurkat T cells, and found that STAT6overexpression or knockdown could trans-activate or trans-inhibit, respectively, the ORMDL3promoter that contains the STAT6-binding motif. Interleukin4(IL-4)/IL-13treatment increased ORMDL3promoter activity as well as endogenous ORMDL3expression. As Jin R. et al found p300bind to ORMDL3promoter and in coincidence it almost bind at the same region that STAT6bind we found in our study. That we performed immunoprecipitation (IP) and ChIP-Re-ChIP assays and the results indicated STAT6and p300exist in the same protein complex on the ORMDL3promoters. Thus, STAT6plays important roles in regulating the expression of human ORMDL3, which can shed light on its possible role in various human diseases.In this part of study, we provide an explanation for the association of ORMDL3and inflammation and asthma and describe the detailed signal transduction pathway: IL-4/IL-13up-regulation of ORMDL3expression depends on STAT6, which can bind to the ORMDL3gene promoter directly, along with the co-activator p300, and up-regulates the promoter activity. Targeting these pathway components with chemical inhibitors or molecular biology techniques might be effective treatment for asthma. |
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