Search

Introduction The chromosomal region 17q21 harbors the human orosomucoid‐like 3 (ORMDL3) gene and has been linked to asthma and other inflammatory diseases. ORMDL3 is involved in the unfolded protein response (UPR), lipid metabolism, and inflammatory reactions. We investigated the effects of ORMDL3 overexpression in RBL‐2H3 cells to determine the contribution of ORMDL3 to inflammatory disease development. Methods We generated ORMDL3 stably overexpressing RBL‐2H3 cells to assess degranulation, transcriptional upregulation of interleukin‐4 (IL‐4), tumor necrosis factor‐α (TNF‐α), monocyte chemoattractant protein‐1 (MCP‐1), and mitogen‐activated protein kinase (MAPK) phosphorylation via FcεRI. In addition, we examined the effects of ORMDL3 overexpression on thapsigargin (TG)‐mediated proinflammatory cytokine transcription and UPR by monitoring MAPK, protein kinase‐like endoplasmic reticulum kinase (PERK), and inositol‐requiring enzyme 1 (IRE1) phosphorylation. Results Overexpression of ORMDL3 enhanced IL‐4, TNF‐α, and MCP‐1 expression after FcεRI cross‐linking, whereas the sphingosine‐1‐phosphate (S1P) agonist FTY720 suppressed this enhancement. There was no significant difference in degranulation and MAPK phosphorylation via FcεRI‐mediated activation between vector‐transfected and ORMDL3‐overexpressing cells. ORMDL3 overexpression accelerated TG‐mediated PERK phosphorylation, while MAPK phosphorylation and proinflammatory cytokine expression showed no significant changes in ORMDL3‐overexpressing cells. Conclusions Our findings suggest that ORMDL3 plays an important role in regulating proinflammatory cytokine expression via the S1P pathway and selectively affects the UPR pathway in mast cells., The messenger RNA (mRNA) expression of TNF‐α, IL‐4, and MCP‐1 were significantly upregulated after engagement of FcεRI in orosomucoid‐like 3 (ORMDL3) overexpressing cells. FTY720 significantly reduced cytokine mRNA expression in ORMDL3‐overexpressing cells and inhibited the expression to a level that was comparable with that observed in vector‐transfected cells.

AimAngiogenesis plays a vital role in airway remodeling in chronic asthma. ORMDL3 has been identified to be closely associated with the development of asthma remodeling. This study was to investigate the mechanism of ORMDL3 in angiogenesis of chronic asthma.MethodsBALB/c mice were divided into three groups, including an asthmatic group (group A), a budesonide-treated group (group B), and a normal control group (group C). Hematoxylin and eosin and Masson staining were used to evaluate the pathological changes. Angiogenesis in lung tissue was examined by CD31 staining. The changes of ORMDL3, ERK1/2, and angiogenesis-associated MMP-9 and Vascular endothelial growth factor (VEGF) expression were examined. Furthermore, ORMDL3, MMP-9, and VEGF mRNA and protein levels were examined after transfection in BEAS-2B cells with the ORMDL3-overexpressed lentiviral vector.ResultsCompared with the control group, asthmatic mice indicated more severe airway angiogenesis with increased ORMDL3, ERK1/2, MMP-9, and VEGF expression. Budesonide alleviated airway angiogenesis, and CD31 expression was positive with the levels of ORMDL3, MMP-9, and VEGF (P < 0.01). After successful transfection in BEAS-2B cells with the ORMDL3-overexpressing lentiviral vector, VEGF, and MMP-9 expression were activated in vitro (P < 0.01).ConclusionIn conclusion, our study provides novel evidence that ORMDL3 promotes angiogenesis through upregulating VEGF and MMP-9 in chronic asthma.

Background Primary biliary cholangitis (PBC) is a classical autoimmune disease, which is highly influenced by genetic determinants. Many genome-wide association studies (GWAS) have reported that numerous genetic loci were significantly associated with PBC susceptibility. However, the effects of genetic determinants on liver cells and its immune microenvironment for PBC remain unclear. Results We constructed a powerful computational framework to integrate GWAS summary statistics with scRNA-seq data to uncover genetics-modulated liver cell subpopulations for PBC. Based on our multi-omics integrative analysis, 29 risk genes including ORMDL3, GSNK2B, and DDAH2 were significantly associated with PBC susceptibility. By combining GWAS summary statistics with scRNA-seq data, we found that cholangiocytes exhibited a notable enrichment by PBC-related genetic association signals (Permuted P ORMDL3 showed the highest expression proportion in cholangiocytes than other liver cells (22.38%). The ORMDL3+ cholangiocytes have prominently higher metabolism activity score than ORMDL3− cholangiocytes (P = 1.38 × 10–15). Compared with ORMDL3− cholangiocytes, there were 77 significantly differentially expressed genes among ORMDL3+ cholangiocytes (FDR ORMDL3+ cholangiocytes exhibited relatively high communications with macrophage and monocyte. Compared with ORMDL3− cholangiocytes, the VEGF signaling pathway is specific for ORMDL3+ cholangiocytes to interact with other cell populations. Conclusions To the best of our knowledge, this is the first study to integrate genetic information with single cell sequencing data for parsing genetics-influenced liver cells for PBC risk. We identified that ORMDL3+ cholangiocytes with higher metabolism activity play important immune-modulatory roles in the etiology of PBC. Graphical Abstract

Background Oroscomucoid 3 (ORMDL3) has been linked to susceptibility of childhood asthma and respiratory viral infection. Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of viral double-stranded RNA, a toll-like receptor 3 (TLR3) ligand and mimic of viral infection. Methods To investigate the functional role of ORMDL3 in the poly I:C-induced inflammatory response in airway epithelial cells, ORMDL3 knockdown and over-expression models were established in human A549 epithelial cells and primary normal human bronchial epithelial (NHBE) cells. The cells were stimulated with poly I:C or the Th17 cytokine IL-17A. IL-6 and IL-8 levels in supernatants, mRNA levels of genes in the TLR3 pathway and inflammatory response from cell pellets were measured. ORMDL3 knockdown models in A549 and BEAS-2B epithelial cells were then infected with live human rhinovirus (HRV16) followed by IL-6 and IL-8 measurement. Results ORMDL3 knockdown and over-expression had little influence on the transcript levels of TLR3 in airway epithelial cells. Time course studies showed that ORMDL3-deficient A549 and NHBE cells had an attenuated IL-6 and IL-8 response to poly I:C stimulation. A549 and NHBE cells over-expressing ORMDL3 released relatively more IL-6 and IL-8 following poly I:C stimulation. IL-17A exhibited a similar inflammatory response in ORMDL3 knockdown and over-expressing cells, but co-stimulation of poly I:C and IL-17A did not significantly enhance the IL-6 and IL-8 response. Transcript abundance of IFNB following poly I:C stimulation was not significantly altered by ORMDL3 knockdown or over-expression. Dampening of the IL-6 response by ORMDL3 knockdown was confirmed in HRV16 infected BEAS-2B and A549 cells. Conclusions ORMDL3 regulates the viral inflammatory response in airway epithelial cells via mechanisms independent of the TLR3 pathway.

Background Genetic variation has a key role in the development of asthma, but genetic influences may vary between different populations. In this study, we looked for evidence of association of key asthma SNPs, namely, rs1420101 and rs10192157 within the IL1RL1 gene, rs2305480 in GSDMB gene, and the rs3744246 polymorphism in the ORMDL3 gene, in the Algerian population. We included 266 unrelated subjects of an Algerian population in a case-control study, with 125 adult asthmatic and 141 healthy controls. DNA was extracted and genotypes determined by the Taqman PCR technique for characterization of the different genetic variants. Results The results show that there were no significant differences in allele frequencies for 3 of the chosen SNPs in the ORMDL3, GSDMB, and IL1RL1 genes between the asthmatic and control groups with respective P values of 0.922, 0.331, and 0.937. However the T allele of rs10192157 of the IL1RL1gene was associated with protection from asthma (P value=0.010). Conclusion These results indicate that there is no marked effect of rs3744246, rs2305480, and rs1420101 polymorphisms of the ORMDL3, GSDMB, and IL1RL1 genes on asthma risk in the Algerian population. However, a protective effect of the rs10192157 polymorphism of the IL1RL1 gene was found.

Background Methylation of DNA at CpG sites is an epigenetic modification and a potential modifier of disease risk, possibly mediating environmental effects. Currently, DNA methylation is commonly assessed using specific microarrays that sample methylation at a few % of all methylated sites. Methods To understand if significant information on methylation can be added by a more comprehensive analysis of methylation, we set up a quantitative method, bisulfite oligonucleotide-selective sequencing (Bs-OS-seq), and compared the data with microarray-derived methylation data. We assessed methylation at two asthma-associated genes, IL13 and ORMDL3, in blood samples collected from children with and without asthma and fractionated white blood cell types from healthy adult controls. Results Our results show that Bs-OS-seq can uncover vast amounts of methylation variation not detected by commonly used array methods. We found that high-density methylation information from even one gene can delineate the main white blood cell lineages. Conclusions We conclude that high-resolution methylation studies can yield clinically important information at selected specific loci missed by array-based methods, with potential implications for future studies of methylation-disease associations.

The main topic of the presented bachelor thesis is a sequence analysis of the ORMDL3 gene, which is identified as a potential risk factor for many diseases, the most discussed of which is childhood asthma. This gene is a member of the ORMDL gene family, which encodes transmembrane proteins anchored in the endoplasmic reticulum. In humans, ORMDL3 is located on chromosome 17, in region 17q21.1. In the theoretical part of the thesis the autoimmune diseases, on which origin and development the mutations in the ORMDL3 gene participate to a certain extent, are specified. These diseases include the aforementioned asthma, Crohn's disease, rheumatoid arthritis, insulin-dependent diabetes mellitus and primary biliary cirrhosis. Also, the Sanger sequencing method by which the samples were sequenced, and the modern NGS method are described here. The practical part of the thesis is focused on preparation of samples for Sanger sequencing, and evaluation of sequencing data. To amplify the desired region for the sequencing reaction, the PCR method was used. 20 anonymized DNA samples were prepared this way. For sequencing by Genseq s.r.o. 15 samples were sent. Furthermore, 20 provided samples were evaluated, 10 of which were from children from the České Budějovice region and 10 from children from the Karviná region. These samples were also anonymized. To analyze the sequences of individual DNA samples, freely available BioEdit program and the NCBI database was used. The sequences were read from both sides (forward and reverse), the found mutations were identified and the resulting data were plotted in graphs. After the sequences were read, a heterozygous mutation was present in 5 of the 15 samples. The same mutation was also present in the provided sequences, specifically in 8 out of 10 from České Budějovice and 3 out of 10 from Karviná. This mutation has not yet been described in the NCBI database.

Rationale: The 17q12-21.1 locus is one of the most highly replicated genetic associations with asthma. Individuals of African descent have lower linkage disequilibrium in this region, which could facilitate identifying causal variants.Objectives: To identify functional variants at 17q12-21.1 associated with early-onset asthma among African American individuals.Methods: We evaluated African American participants from SAPPHIRE (Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-Ethnicity) (n = 1,940), SAGE II (Study of African Americans, Asthma, Genes and Environment) (n = 885), and GCPD-A (Study of the Genetic Causes of Complex Pediatric Disorders-Asthma) (n = 2,805). Associations with asthma onset at ages under 5 years were meta-analyzed across cohorts. The lead signal was reevaluated considering haplotypes informed by genetic ancestry (i.e., African vs. European). Both an expression-quantitative trait locus analysis and a phenome-wide association study were performed on the lead variant.Measurements and Main Results: The meta-analyzed results from SAPPHIRE, SAGE II, and the GCPD-A identified rs11078928 as the top association for early-onset asthma. A haplotype analysis suggested that the asthma association partitioned most closely with the rs11078928 genotype. Genetic ancestry did not appear to influence the effect of this variant. In the expression-quantitative trait locus analysis, rs11078928 was related to alternative splicing of GSDMB (gasdermin-B) transcripts. The phenome-wide association study of rs11078928 suggested that this variant was predominantly associated with asthma and asthma-associated symptoms.Conclusions: A splice-acceptor polymorphism appears to be a causal variant for asthma at the 17q12-21.1 locus. This variant appears to have the same magnitude of effect in individuals of African and European descent.

Objective This study aimed to assess whether orosomucoid 1-like 3 (ORMDL3) expression and environmental and clinical factors are associated with wheezing episodes in preschool children. Methods Children diagnosed with wheezing episodes were classified according to their asthma predictive index (API) in the past year as follows: API+ (≥4 wheezing episodes), API− (1–3 wheezing episodes), and API0 (without wheezing). ORMDL3 expression was assessed by real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMCs). Receiver operating characteristic curve analysis of ORMDL3 expression and the API was performed for diagnosing wheezing episodes. Correlations between ORMDL3 expression and asthma risk factors were examined using Spearman’s correlation. Results PBMC ORMDL3 expression was higher in the API+ group compared with the API− and API0 groups. The area under the curve for ORMDL3 expression was 0.820 (95% confidence interval, 0.771–0.869). ORMDL3 expression was positively correlated with the API (r = 0.447), infantile eczema (r = 0.499), wheezing (r = 0.516), total immunoglobulin E (r = 0.208), and environmental factors, including Dermatophagoides pteronyssinus (r = 0.357), house dust mites (r = 0.112), dog fur (r = 0.226), and Aspergillus(r = 0.257). ORMDL3 expression was negatively correlated with amaranth (r = −0.122). Conclusions ORMDL3 expression in PBMCs is positively associated with the API and some asthma-related clinical and environmental risk factors in preschoolers.

Endoplasmic reticulum (ER) stress in beta cells induces a signaling network called the unfolded protein response (UPR), which plays a dual role in diabetes. A key regulator of ER-stress and UPR, the orosomucoid 1-like protein 3 (ORMDL3), has been shown to regulate airway remodeling through a major UPR protein, activating transcription factor 6 (ATF6), but the contribution of this regulatory axis to compensatory pancreatic beta cell proliferation in diabetes has not been studied. Here, we detected significantly lower levels of ORMDL3 mRNA in leukocytes of peripheral blood specimens from type 1 diabetes (T1D) children, compared to normal children. Moreover, these ORMDL3 levels in T1D children exhibited further decreases upon follow-up. ORMDL3 levels in islets from NOD mice, a mouse model for T1D in humans, showed a mild increase before diabetes onset, but a gradual decrease subsequently. In high glucose culture, beta cell proliferation, but not apoptosis, was increased by overexpression of ORMDL3 levels, likely mediated by its downstream factor ATF6. Mechanistically, ORMDL3 transcriptionally activated ATF6, which was confirmed in a promoter reporter assay. Together, our data suggest that ORMDL3 may increase beta cell proliferation through ATF6 as an early compensatory change in response to diabetes.

BACKGROUND: Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)-like protein isoform 3 (ORMDL3) gene across different populations. Although the ORM-homologues in yeast are well-known inhibitors of sphingolipid (SL) synthesis, it is still unclear whether and how mammalian ORMDL3 regulates SL metabolism and whether altered SL synthesis would be causally related to asthma risk. OBJECTIVE: To examine the in vivo role of ORMDL3 in SL metabolism and allergic asthma METHODS: Ormdl3-LacZ reporter mice, gene deficient Ormdl3(-/-) and overexpressing Ormdl3 (Tg/wt) mice were exposed to physiologically relevant aeroallergens such as house dust mite or Alternaria alternata to induce experimental asthma. Mass spectrometry based sphingolipidomics was performed, and airway eosinophilia, Th2 cytokine production, Ig synthesis, airway remodeling and bronchial hyperreactivity measured. RESULTS: HDM challenge significantly increased total sphingolipids in the lung of HDM-sensitized mice compared to controls. In Ormdl3(Tg/wt) mice, the allergen-induced increase in lung ceramides was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, total sphingolipids including ceramides were increased in Ormdl3(-/-) mice, while they were lowered in Ormdl3(Tg/wt) mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wildtype, Ormdl3(Tg/wt) and Ormdl3(-/-) mice, across several models of experimental asthma. CONCLUSION: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3. expression does not result in altered experimental asthma. CAPSULE SUMMARY: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma.

Three decades ago, store-operated Ca 2 + entry (SOCE) was identified as a unique mechanism for Ca 2 + entry through plasma membrane (PM) Ca 2 + -permeable channels modulated by the intracellular Ca 2 + stores, mainly the endoplasmic reticulum (ER). Extensive analysis of the communication between the ER and the PM leads to the identification of the protein STIM1 as the ER-Ca 2 + sensor that gates the Ca 2 + channels in the PM. Further analysis on the biophysical, electrophysiological and biochemical properties of STIM1-dependent Ca 2 + channels has revealed the presence of a highly Ca 2 + -selective channel termed Ca 2 + release-activated Ca 2 + channel (CRAC), consisting of Orai1 subunits, and non-selective cation channels named store-operated channels (SOC), including both Orai1 and TRPC channel subunits. Since the identification of the key elements of CRAC and SOC channels a number of intracellular modulators have been reported to play essential roles in the stabilization of STIM–Orai interactions, collaboration with STIM1 conformational changes or mediating slow Ca 2 + -dependent inactivation. Here, we review our current understanding of some of the key modulators of STIM1–Orai1 interaction, including the proteins CRACR2A, STIMATE, SARAF, septins, golli and ORMDL3.

Rationale: Polymorphisms on chromosome 17q21 confer the major genetic susceptibility to childhood-onset asthma. Risk alleles positively correlate with ORMDL3 (orosomucoid-like 3) expression. The locus influences disease severity and the frequency of human rhinovirus (HRV)–initiated exacerbations. ORMDL3 is known to regulate sphingolipid synthesis by binding serine palmitoyltransferase, but its role in inflammation is incompletely understood. Objectives: To investigate the role of ORMDL3 in cellular inflammation. Methods: We modeled a time series of IL1B-induced inflammation in A549 cells, using cytokine production as outputs and testing effects of ORMDL3 siRNA knockdown, ORMDL3 overexpression, and the serine palmitoyltransferase inhibitor myriocin. We replicated selected findings in normal human bronchial epithelial cells. Cytokine and metabolite levels were analyzed by analysis of variance. Transcript abundances were analyzed by group means parameterization, controlling the false discovery rate below 0.05. Measurements and Main Results: Silencing ORMDL3 led to steroid-independent reduction of IL6 and IL8 release and reduced endoplasmic reticulum stress after IL1B stimulation. Overexpression and myriocin conversely augmented cytokine release. Knockdown reduced expression of genes regulating host–pathogen interactions, stress responses, and ubiquitination: in particular, ORMDL3 knockdown strongly reduced expression of the HRV receptor ICAM1. Silencing led to changes in levels of transcripts and metabolites integral to glycolysis. Increased levels of ceramides and the immune mediator sphingosine-1-phosphate were also observed. Conclusions: The results show ORMDL3 has pleiotropic effects during cellular inflammation, consistent with its substantial genetic influence on childhood asthma. Actions on ICAM1 provide a mechanism for the locus to confer susceptibility to HRV-induced asthma.

Background Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. Objective We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3 Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Methods Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro . In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3 Zp3-Cre mice, which do not have a blood supply. Results Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3 Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Conclusion Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients.

Astma je resna kronična bolezen pljuč, ki povzroča vnetje dihal in je neznane etiologije. Sam razvoj astme je povezan z interakcijo številnih genov in vplivom okolja. V številnih študijah so z astmo povezali več kot 100 genov, ki pa se med različnimi populacijami razlikujejo. Raziskali smo dve kandidatni kromosomski regiji, 17q21 in 2q12. Za analizo 17q21 smo genotipizirali polimorfizem posameznega nukleotida (SNP) rs7216389, ter merili izražanje genov ORMDL3 in GSDMB, za analizo regije 2q12 pa smo genotipizirali SNP rs10206753 in merili izražanje genov IL1RL1 in IL18RAP. S tehniko verižne reakcije s polimerazo (PCR), ki ji je sledila reakcija polimorfizmov dolžin restrikcijskih encimov (RFLP) ali analiza talilne krivulje visoke ločljivosti (HRM), smo genotipizirali 384 vzorcev otrok z astmo z atopijskim in neatopijskim fenotipom ter 288 vzorcev zdravih kontrol. Analizirali smo povezavo izbranih SNP-jev z astmo ter vpliv izbranih SNP-jev na klinične parametre in izražanje genov ORMDL3, GSDMB, IL1RL1, ter IL18RAP. Ugotovili smo, da je SNP rs7216389 povezan s pojavom astme pri slovenskih otrocih (p=0,0031, OR=0,698). Astmatiki, atopijski in neatopijski, imajo višjo frekvenco alela T v primerjavi z zdravimi kontrolami (p=0,0005, OR=0,541). Farmakogenetska analiza SNP-ja rs7216389 je pokazala, da se atopijski in neatopijski astmatiki različno odzivajo na terapijo z inhalacijskimi glukokortikosteroidi. Atopijskim astmatikom z genotipom CC se vrednost dTI po terapiji poviša (p=0,0057, dTICC=7,00 , dTICT+CC=2,00), medtem ko se neatopijskim astmatikom zniža (p=0,0311, dTICC=-2,00 dTICT+CC=3,00). Pri vplivu genotipa na klinične parametre smo opazili tudi višjo vrednost eozinofilcev v krvi neatopijskih astmatikov z genotipom TT v primerjavi s posamezniki z genotipom CT ali CC, vendar je bila vrednost p na meji statistične značilnosti (p=0,0558). Ugotovili smo tudi, da SNP rs7216389 regulira izražanje gena ORMDL3, saj imajo astmatiki in atopijski astmatiki z genotipom TT višji nivo izražanja gena ORMDL3 v primerjavi s posamezniki z genotipom CT ali TT (p=0,0038 za astmatike, p=0,0133 za atopijske astmatike). Za območje 2q12 pa smo ugotovili, da SNP rs10206753 ni povezan s pojavom astme pri slovenskih otrocih. Smo pa ugotovili, da se vrednost dFEV1 po terapiji z antilevkotrieni v primerjavi s posamezniki z genotipom CT ali TT zviša pri astmatikih z genotipom CC (p=0,0002, 8,5/-2) in pri atopijskih astmatikih z genotipom CC (p=

Background Genome-wide association studies have identified the ORM (yeast)-like protein isoform 3 (ORMDL3) gene locus on human chromosome 17q to be a highly significant risk factor for childhood-onset asthma. Objective We sought to investigate in vivo the functional role of ORMDL3 in disease inception. Methods An Ormdl3-deficient mouse was generated and the role of ORMDL3 in the generation of allergic airways disease to the fungal aeroallergen Alternaria alternata was determined. An adeno-associated viral vector was also used to reconstitute ORMDL3 expression in airway epithelial cells of Ormdl3 knockout mice. Results Ormdl3 knockout mice were found to be protected from developing allergic airways disease and showed a marked decrease in pathophysiology, including lung function and airway eosinophilia induced by Alternaria. Alternaria is a potent inducer of cellular stress and the unfolded protein response, and ORMDL3 was found to play a critical role in driving the activating transcription factor 6–mediated arm of this response through Xbp1 and downstream activation of the endoplasmic reticulum–associated degradation pathway. In addition, ORMDL3 mediated uric acid release, another marker of cellular stress. In the knockout mice, reconstitution of Ormdl3 transcript levels specifically in the bronchial epithelium resulted in reinstatement of susceptibility to fungal allergen–induced allergic airways disease. Conclusions This study demonstrates that ORMDL3, an asthma susceptibility gene identified by genome-wide association studies, contributes to key pathways that promote changes in airway physiology during allergic immune responses., Graphical abstract

Genome-wide association studies linked increased ORMDL3 expression levels to several inflammatory diseases via single nucleotide polymorphisms. However, the pathophysiological mechanisms underlying this association are still poorly understood. ORMDL proteins have been shown to regulate de novo sphingolipid synthesis among other functions, a process important for proper innate immune responses and its imbalance was furthermore associated to asthma disease. This thesis was aimed to provide further inside into the ORMDL-SPT complex formation and to elucidate potential involvement of ORMDL3 in macrophage physiology using a transgenic mouse model. We herein demonstrated that a structural rearrangement under high sphingolipid content takes place, in order to release SPT activity. Moreover, ORMDL3 levels decreased cellular ceramide content in macrophages, and specifically impaired the de novo synthesis during activation leading to reduced autophagy and bacterial clearance. In addition, we explore the SNP-dependent regulation of ORMDL3 gene expression in human monocytes. Taken together, this thesis contributes to a better understanding of the implication of ORMDL3 in sphingolipid homeostasis and underlines its importance in macrophage physiology. Alguns estudis d’associació genòmica han relacionat un augment dels nivells d’expressió d’ORMDL3, causat per polimorfismes d’un sol nucleòtid, amb diverses malalties inflamatòries. Tot i així, els mecanismes fisiopatològics subjacents a aquesta associació no són del tot coneguts. Les proteïnes ORMDL estan involucrades en la síntesi d’esfingolípids de novo, un procés essencial per a la resposta immunitària de tipus innat i que ha estat associat amb l’asma. Aquesta tesi té l’objectiu d’aprofundir en la formació del complex ORMDL-SPT així com d’elucidar la potencial implicació d’ORMDL3 en la fisiologia dels macròfags utilitzant un model de ratolí transgènic. Els nostres resultats demostren un rearranjament estructural quan hi ha un alt contingut d’esfingolípids, per tal d’alliberar l’activitat de l’SPT. A més, els nivells d’ORMDL3 disminueixen el contingut cel·lular de ceramides i afecten específicament la síntesi de novo durant l’activació dels macròfags, produint una reducció en l’autofàgia i la liquidació bacteriana. Addicionalment, hem explorat la regulació d’ORMDL3 depenent de polimorfismes en monòcits humans. En conjunt, aquesta tesi contribueix a un coneixement més profund de la implicació d’ORMDL3 en la homeòstasi d’esfingolípids i remarca la seva importància en la fisiologia dels macròfags.

Background Gene variants on chromosome 17q12-21 are associated with an increased risk of childhood-onset asthma, a risk known to be modified by environmental tobacco smoke (ETS). Objectives To assess whether the association of rs2305480 on chromosome 17q12 in the GSDML gene with asthma-like symptoms in the first 4 years of life is modified by smoke exposure during fetal and early postnatal life. Methods We used data from two independent prospective cohort studies from fetal life onwards in the Netherlands. We genotyped rs2305480 and assessed maternal smoking during pregnancy and ETS exposure at the age of 2. Asthma-like symptoms, defined as any reported wheezing, shortness of breath or dry nocturnal cough, were reported by parents when the children were 1, 2, 3, and 4 years. Analyses were based on a total group of 4461 Caucasian children. Results The G risk-allele of rs2305480 was associated with asthma-like symptoms [overall odds ratio 1.17 (1.11, 1.24), 2.66E-9]. The effect of rs2305480 on asthma-like symptoms was stronger among children who were exposed to smoke during fetal life (P-interaction = 0.04). Smoke exposure in early postnatal life was also associated with an increased effect of the 17q12 single nucleotide polymorphism (SNP) on asthma-like symptoms (P-interaction = 5.06E-4). These associations were consistent in both cohorts. Conclusion A 17q12 variant, rs2305480, was associated with asthma-like symptoms in preschool children, and this association was modified by smoke exposure already during fetal life, and in infancy. Further investigation regarding SNPs in linkage disequilibrium with rs2305480 in relation to pathophysiological pathways is needed.

Rationale. Markers on chromosome 17q12 have been associated withchildhood asthma in genome-wide association studies. Objective.We investigated the association of a single nucleotide polymorphism(SNP) in GSDMB (rs7216389) on 17q12 with asthma presence andseverity in a population-based birth cohort study. Methods. Childrenwere followed from birth to age 8 years. Data on parentally-reportedsymptoms were collected using an interviewer-administered questionnaire at age 1, 3, 5 and 8 years. Atopy was assessed by skin testing at age 3, 5 and 8 years. Information on asthma/wheeze hospital admissions and severe asthma exacerbations was collected from child’s primary care medical record. Data were analyzed as a recessive genetic model, with T-allele homozygotes as the risk group. Results. Compared to C allele carriers, T-allele homozygotes of rs7216389 were significantly more likely to: wheeze at age 3, 5 and 8 years; have persistent wheeze (OR 1.69, 95% CI 1.05-2.71, p=0.03); have frequent episodes of wheezing and be on asthma medication. In a multiple logistic regression model adjusted for gender, atopic sensitisation and maternal smoking, T allele homozygotes were significantly more likely to be hospitalized (aOR 2.20 [1.22-3.99], p=0.0009) with Cox regression hazard ratio for T-allele homozygotes of 1.94 [1.13-3.33], p=0.016. Results of Cox regression analysis investigating the eff ect of genotype on the age of first severe exacerbation of asthma indicated an overall hazard ratio of severe asthma exacerbation among T-allele homozygotes of 1.53 [1.04-2.27], p=0.03. Conclusions. Th is is the fi rst population-based birth cohort study to confirm that the risk of childhood wheeze and severe asthma/wheeze exacerbations is increased among rs7216389 TT homozygotes.

Asthma is a clinically heterogeneous disorder, whose onset and progression results from a complex interplay between genetic susceptibility, allergens, and viral triggers. Sphingolipids and altered sphingolipid metabolism have emerged as potential key contributors to the pathogenesis of asthma. Orosomucoid-like 3 gene (ORMDL3) and the asthma susceptibility locus 17q21 have been strongly and reproducibly linked to childhood asthma, but how this gene is functionally linked to asthma is incompletely understood. ORMDL proteins play an integral role in sphingolipid homeostasis and synthesis, and asthma-associated ORMDL3 polymorphisms have been associated with early viral respiratory infections and increased risk of asthma. ORMDL proteins act as inhibitors of serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme for de novo sphingolipid synthesis, and decreased sphingolipid synthesis through SPT increases airway hyperreactivity, which is independent of allergy or inflammation. In allergic models of asthma, the sphingolipid mediators sphingosine-1-phosphate (S1P) and ceramide have been shown to be important signaling molecules for airway hyperreactivity, mast cell activation, and inflammation. This review will highlight how sphingolipids and altered sphingolipid metabolism may contribute towards the underlying mechanisms of childhood asthma.

BACKGROUND: Chromosome 17q21, harboring the orosomucoid 1-like 3 (ORMDL3) gene, has been consistently associated with childhood asthma in genome-wide association studies. OBJECTIVE: We investigated genetic variants in and around ORMDL3 that can change the function of ORMDL3 and thus contribute to asthma susceptibility. METHODS: We performed haplotype analyses and fine mapping of the ORMDL3 locus in a cross-sectional (International Study of Asthma and Allergies in Childhood Phase II, n=3557 total subjects, n=281 asthmatic patients) and case-control (Multicenter Asthma Genetics in Childhood Study/International Study of Asthma and Allergies in Childhood Phase II, n=1446 total subjects, n=763 asthmatic patients) data set to identify putative causal single nucleotide polymorphisms (SNPs) in the locus. Top asthma-associated polymorphisms were analyzed for allele-specific effects on transcription factor binding and promoter activity invitro and gene expression in PBMCs after stimulation ex vivo. RESULTS: Two haplotypes (H1 and H2) were significantly associated with asthma in the cross-sectional (P=9.9×10(-5) and P=.0035, respectively) and case-control (P=3.15×10(-8) and P=.0021, respectively) populations. Polymorphisms rs8076131 and rs4065275 were identified to drive these effects. For rs4065275, a quantitative difference in transcription factor binding was found, whereas for rs8076131, changes in upstream stimulatory factor 1 and 2 transcription factor binding were observed invitro by using different cell lines and PBMCs. This might contribute to detected alterations in luciferase activity paralleled with changes in ORMDL3 gene expression and IL-4 and IL-13 cytokine levels exvivo in response to innate and adaptive stimuli in an allele-specific manner. Both SNPs were in strong linkage disequilibrium with asthma-associated 17q21 SNPs previously related to altered ORMDL3 gene expression. CONCLUSION: Polymorphisms in a putative promoter region of ORMDL3, which are associated with childhood asthma, alter transcriptional regulation of ORMDL3, correlate with changes in TH2 cytokines levels, and therefore might contribute to the childhood asthma susceptibility signal from 17q21.

Calcium works as a second intracellular messenger in all cell types and its downstream signalling is a key pathway for many systemic functions. In the lungs, the majority of activating stimuli trigger intracellular calcium increase, which is indispensable for the normal functioning of the airways; thus, deregulation of this pathway leads to pathological conditions. This Thesis aims to understand the relationship of intracellular calcium homeostasis and chronic respiratory pathologies such as asthma. I have studied three different processes involved in calcium homeostasis and their role in asthma pathophysiology: 1) I have shown the genetic association of a defect in calcium entry via TRPV1 with wheezing and cough, which is one feature of asthma pathophysiology; 2) I have also demonstrated the product of the asthma associated ORMDL3 gene is a Ca++ homeostasis and UPR modulator; and 3) I have provided a new Ca++ dependent sorting mechanism for secretory cargoes that bind calcium., El Calci és un segon missatger intracel·lular en tots els tipus cel·lulars i la cascada de senyalització generada pel calci és una via de senyalització cel·lular clau per moltes funcions sistèmiques. En els pulmons, la majoria d’estímuls activadors produeixen un increment del calci intracel·lular, el qual és indispensable pel funcionament correcte de les vies respiratòries; i, per tant, una desregulació d’aquesta via de senyalització porta a diferents situacions patològiques. Aquesta Tesi té com a objectiu entendre la relació entre l’homeòstasi del calci intracel·lular i les malalties respiratòries cròniques, com per exemple, l’asma. Hem estudiat tres processos diferents implicats en l’homeòstasi del calci i el seu rol en la fisiopatologia de l’asma: 1) Hem demostrat que hi ha una associació genètica entre un defecte en l’entrada de calci via TRPV1 i un dels trets característics de l’asma, la tos; 2) també hem trobat que l’ORMDL3, que havia estat associat amb l’asma, és un modulador de l’homeòstasi del calci i de la UPR; i 3) hem aportat un nou mecanisme de classificació en el Golgi depenent de Ca++ per a proteïnes que uneixen calci i que seran secretades.

4 Abstract Mast cells are involved in variety of immunological processes, but they are mostly known for their role in allergy and asthma. As asthma and allergy are serious diseases with spreading tendency during last decades, mast cells are subject of intensive research. It is expected that studies of mast cell signalling pathways will contribute to our understanding of the nature of these diseases and help to design efficient treatment strategies. In an attempt to identify genes responsible for asthma disease, genome-wide screening methods have been currently applied. Using these methods, mutations in ORMDL3 (Orosomucoid1-like) protein were found out as a high risk asthma factor. ORMDL3 is a member of evolutionary conserved ORMDL family, comprising in mammals also of ORMDL1 and ORMDL2. Physiological function of these proteins is poorly understood and it has not been studied in mast cells. We decided to study the role of ORMDL proteins in mast cells. Lentiviral delivery system was optimised for generation of stable knock-downs (KD) of all three members of the ORMDL family in primary mast cells. The ORMDL gene expression was measured by improved qPCR (quantitative PCR) reaction buffers. We found that all ORMDL genes are expressed in mast cells in order ORMDL3 > ORMDL2 > ORMDL1. Next, we investigated the...

Asthma is a complex disease that is influenced by poorly understood genetic and environmental factors1. A genome-wide association study (GWAS) of 994 cases and 1243 controls from the UK and Germany found strong associations (P < 10−12) for SNPs at the 17q21 locus and childhood asthma using family and case-control panels2. The association between these SNPs and gene transcript levels in Epstein-Barr virus-transformed lymphoblastoid cell lines from the asthmatic children identified ORMDL3 as a candidate gene for asthma. One of the SNPs, rs7216389, located within a highly conserved region containing an element homologous to the proinflammatory transcription factor C/EBPb was associated to both asthma and ORMDL3 transcript levels with the highest degree of statistical significance in the initial study (uncorrected P = 9×10−11)2. The initial GWAS findings have subsequently replicated in a number of studies involving ethnically diverse populations and the variants were reported to contribute to early-onset asthma and interacting to early-life environmental tobacco smoke exposures3. A study published recently identified an association of rs7216389 variant with disease severity in early-onset asthma4. In the study, asthmatic cases were stratified according to asthma severity and they were classified into mild, moderate and severe asthmatics following national and international guidelines. Severe asthmatics were not recruited through severe asthma clinics; however, the study proposed rs7216389 is involved in early-onset severe asthma. Severe or “difficult/therapy-resistant” asthma refers to asthma that is poorly controlled in terms of persistent symptoms, episodic exacerbations and persistent and variable airway obstruction despite the use of high doses of inhaled corticosteroids, long-acting bronchodilators and short β2 agonists5. Studying individuals with an extreme phenotype can be very powerful when isolating the genetic determinants underlying a disease. Using this strategy we have consequently examined the role of rs7216389 in severe asthma. The case group consisted of 397 severe asthmatic adults identified through specialist severe asthma clinics at two UK centres, Royal Brompton Hospital, London and the Glenfield Hospital, Leicester. Asthma was defined using the international GINA (Global Initiative for Asthma: http://www.ginasthma.com) guidelines and the ATS criteria for refractory asthma5. For 226 subjects, the asthma age of onset was available. Childhood asthma-onset was present in 114 samples and adulthood asthma-onset in 112 subjects. The male to female ratio was 1:2, the mean age was 48.95 years (Standard Deviation 13.55) and mean IgE (kU/L) was 291.72 (Standard Deviation 456.09). We derived 1429 previously genotyped healthy UK adult controls from the 1958 British Birth Cohort study. The 1958 British birth cohort includes 17,638 males and females with sex ratio 1:1 enrolled in the Perinatal Mortality Survey at the time of their birth during 1 week in March 1958 across England, Wales and Scotland 6. A DNA collection was obtained during a follow-up in 2002 to 20047. Genome-wide genotyping data from the Illumina HumanHap550 Beadarray on 1430 subjects were deposited by the Wellcome Trust Sanger Institute8. Blood samples from cases were collected and DNA was extracted using whole blood DNA extraction protocols (Promega Wizard® Genomic DNA purification kit). TaqMan® SNP Genotyping Assays (Applied Biosystems 7300 Real-Time PCR System, 40 cycles of 10 min at 95 °C, 15 sec at 92 °C and 1 min at 60 °C) were used for the allelic discrimination (primer and probe sequences available upon request). Controls of known genotype were included. Deviation from Hardy Weinberg equilibrium (HWE) was calculated for the allele frequencies. Genotype and allele frequencies were compared between cases and controls by Fisher’s exact test and logistic regression. Associations between the genotypes and IgE were also examined by Kruskal-Wallis test. The genotyping success rate for rs7216389 was 97%. No significant deviation from HWE was detected (P > 0.05). The rs7216389 SNP was found to be significantly associated with severe adult asthma (OR 1.42, CI: 1.21-1.67, P = 1.8×10−5) (Table 1). In our study the frequency of the T allele in the asthmatic adults was 56%, which is lower than that reported by Moffatt et al. (62%)2. When the data was stratified according to the disease age of onset, a significant association between SNP rs7216389 and severe asthma was reported only in the childhood-onset asthmatics (OR 2.02, CI: 1.53-2.68, P=4.5×10−6) (Table 1). Interestingly, the frequency of the T allele in cases of childhood onset was 67%, similar to the figure reported by Moffatt et al.2 Adult-onset of the disease did not show any significant associations for this SNP (P=0.853) and minor allele frequency (T allele) was 49%, comparable to the control group (47%) (Table 1). No associations were found between genotypes and IgE levels. The results highlight the differences in the genetic components of childhood and adulthood asthma-onset. Table 1 Genotype frequencies and association test results for rs7216389 with severe asthma, childhood-onset and adult-onset asthma susceptibility. Our results confirm the role of the ORMDL3 genomic area as a locus conferring susceptibility to childhood asthma-onset of the most severe type of the disease. In combination with the recent published studies in ethnically diverse populations it highlights the importance of the ORMDL3 and other genes from the Chromosome 17q21 region in the development of this complex disease. Further studies are required to investigate the functional role of this polymorphism and its involvement in early-onset asthma that could contribute in elucidating the mechanisms underlying asthma and could be applied for therapeutic interventions.

A sequence variant (rs7216389-T) near the ORMDL3 gene on chromosome 17q21 was recently found to be associated with childhood asthma. We sought to evaluate the effect of rs7216389-T on asthma subphenotypes and its correlation with expression levels of neighboring genes. The association of rs7216389-T with asthma was replicated in six European and one Asian study cohort (N=4917 cases N=34 589 controls). In addition, we found that the association of rs7216389-T was confined to cases with early onset of asthma, particularly in early childhood (age: 0-5 years OR=1.51, P=6.89.10(-9)) and adolescence (age: 14-17 years OR=1.71, P=5.47.10(-9)). A weaker association was observed for onset between 6 and 13 years of age (OR=1.17, P=0.035), but none for adult-onset asthma (OR=1.07, P=0.12). Cases were further stratified by sex, asthma severity and atopy status. An association with greater asthma severity was observed among early-onset asthma cases (P=0.0012), but no association with sex or atopy status was observed among the asthma cases. An association between sequence variants and the expression of genes in the 17q21 region was assessed in white blood cell RNA samples collected from Icelandic individuals (n=743). rs7216389 associated with the expression of GSDMB and ORMDL3 genes. However, other sequence variants showing a weaker association with asthma compared with that of rs7216389 were more strongly associated with the expression of both genes. Thus, the contribution of rs7216389-T to the development of asthma is unlikely to operate only through an impact on the expression of ORMDL3 or GSDMB genes.

Single nucleotide polymorphisms (SNPs) at chromosome 17q21 confer an increased risk of early-onset asthma. The objective was to study whether 17q21 SNPs modify associations between early respiratory infections and asthma. Association analysis was conducted in 499 children (268 with asthma, median age 11 yrs) from the Epidemiological Study on the Genetics and Environment of Asthma (EGEA). The 12-yr follow-up data were used to assess persistent or remittent asthma in young adulthood. Respiratory infection before 2 yrs of age was assessed retrospectively. For the 12 17q21 SNPs studied, the odds ratios (OR) for association between infection and early-onset asthma (age at onsetor=4 yrs) were higher in carriers of risk genotypes (OR 3.42-6.36) than in noncarriers (OR 1.84-2.44; p-value for interaction 0.02-0.04 for five SNPs). Risk genotypes also increased the association between infection and childhood asthma that remits in adulthood (OR 4.84-7.16 in carriers and 1.74-2.25 in noncarriers; p-value for interaction 0.008-0.05 for 10 SNPs). In children with 17q21 risk genotypes and early-life environmental tobacco smoke (ETS) exposure, associations between infection and asthma were further enhanced. 17q21 genetic variants and early ETS exposure enhance the association between early respiratory infections and early-onset asthma and childhood asthma that remits in adulthood.

PURPOSE ORM1-like 3 (ORMDL3) belongs to a highly conserved protein family which is anchored as transmembrane protein in the endoplasmic reticulum. Gasdermin B (GSDMB) is adjacent to ORMDL3 on chromosome 17q21.2 and belongs to the gasdermin-domain containing the protein family (GSDM family). Recent reports suggest that GSDMB and ORMDL3 are associated with asthma in several populations. However, genetic association studies that examined the association of GSDMB and ORMDL3 gene variants with asthma showed conflicting results. To assess whether combined evidence shows the association between GSDMB/ORMDL3 polymorphism and asthma. METHODS A bibliographic search from MEDLINE identified 13 original articles using the search keywords 'GSDMB', 'ORMDL3', and 'asthma'. An updated literature-based meta-analysis involving 6,691 subjects with asthma, 9,281 control individuals, and 1,360 families were conducted. Meta-odds ratios (ORs) and 95% confidence intervals (CIs) based on the fixed effects model or the random effects model depended on Cochran's Q-statistic and I(2) values. Data from case-control and TDT studies were analyzed in an allelic model using the Catmap software. RESULTS We selected and identified 3 SNPs of ORMDL3 associated with asthma (rs8076131: OR=1.10; 95% CI, 1.02-1.20; P=0.012. rs12603332: OR=1.15; 95% CI, 1.05-1.25; P=0.002. rs3744246: OR=1.10; 95% CI, 1.02-1.17; P=0.008) and 1 SNP of GSDMB associated with asthma (rs7216389: OR=1.37; 95% CI, 1.27-1.47; P

Increased orosomucoid-like 3 (ORMDL3) expression levels, due to single nucleotide polymorphisms (SNPs), have been associated with several inflammatory diseases, including asthma and inflammatory bowel diseases. ORMDL proteins inhibit serine palmitoyltransferase (SPT), the first rate-limiting enzyme in de novo sphingolipid synthesis and alter cellular calcium homeostasis. Both processes are essential for immune response. The present study addresses ORMDL3 protein involvement in macrophage physiology using an overexpressing knock-in mouse model. Ceramide content was notably different in the bone-marrow-derived macrophages (BMDM) from the transgenic mouse model compared with the wild type (WT) macrophages. Our data revealed an alteration of de novo production of sphinganine upon BMDM activation in the transgenic mouse. Gene-expression analysis showed that alteration in ORMDL3 expression levels did not affect activation or macrophage polarization. Nevertheless, we studied phagocytosis and autophagy—crucial processes that are dependent on lipid membrane composition. Phagocytosis in transgenic macrophages was not affected by ORMDL3 overexpression, but we did find a reduction in toll-like receptor 4 (TLR-4)-mediated autophagy. Both genetic and functional studies have pointed to autophagy as an essential pathway involved in inflammation. We believe that our work provides new insights into the functional link between ORMDL3 expression and inflammatory diseases., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2010-16725, SAF2014-52228-R, and BES-2011-043839), Fondo de Investigación Sanitaria (Red HERACLES RD12/0042/0014), Unidad de Excelencia María de Maeztu, funded by the MINECO (MDM-2014-0370), and Fundació la Marató de TV3 (20134030), We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)