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Eukaryon

Examining the ORDML3 Gene and its relationship to asthma

Yuliya Zayats
Department of Biology
Lake Forest College
Lake Forest, Illinois 60045

Abstract

 

The purpose of my project is to research the significance and characteristics of the ORMDL3 gene, which is a gene involved in early onset childhood asthma. A mutated ORMDL3 protein exacerbates asthma symptoms by increasing the Unfolded Protein Response, decreasing de novo sphingolipid synthesis, and increasing eosinophil activity. Thus, a drug therapy that targets one of these three pathways impacted by ORMDL3 gene will help researchers discover an appropriate treatment for asthmatics. I propose that with the use of techniques such as the genetic engineering of the Cre LoxP mouse, immunohistochemistry, and in vitro flow chamber assay I can create a mouse model that is representative of human ORMDL2 rs7216389 SNP and that can be used to analyze the therapeutic effectiveness of the FTY720 immunosuppressant drug.

 

Phenotype

 

Have you ever been afraid of suddenly losing your ability to breathe? As a child, I frequently struggled with my asthma symptoms, fearing that any activity or environment would trigger the suffocating and terrifying disease. Currently, approximately 1 in 10 children are suffering from asthma, and the disease ranks as the number one chronic disease in children (“Asthma Statistics,” 2010). In fact, asthma is an increasing global problem with 300 million people suffering from asthma around the world, and by 2025, the number of people with asthma is expected to grow by 100 million. Also, Asthma is responsible for 1 in 250 deaths around the world. Thus, it is crucial to for us to become educated about asthma and what we can do alleviate its burden.

            Asthma is a chronic, respiratory disease that narrows and inflames the airways that carry air from the nose and mouth to the lung, which include the windpipe and smaller air passages called bronchi. The symptoms associated with asthma include shortness of breath, tightness in the chest, coughing, and wheezing (“Diseases & Conditions: Asthma,” 2015). Asthma has various different triggers, which include: airborne substances, such as pet dander or pollen, respiratory infections, physical activity, air pollutants like cigarette smoke, cold air, certain medications like aspirin or beta blockers, food preservatives, and stress. In addition, there are various risk factors that make it more likely for an individual to have asthma, such as having a close relative with asthma or an allergic condition, being overweight or a smoker, being exposed to secondhand smoke, exhaust fumes or polluting substances. Thus, Asthma impacts individuals with various different backgrounds, occupations, and lifestyles that live in various environments. However, the asthma phenotype is also significantly influenced by the individual’s genetics.

 

In the recent decades, the GWAS (genome wise association studies) over a hundred different chromosome loci or regions have been linked to risk of asthma and over and over 30 genes that have been associated with asthma and allergic phenotypes (Moffatt et al., 2007). GWAS studies use thousands of DNA genotype sequences of individuals that have asthmatic symptoms and compare those genotypes with the genotypes of individuals that do not express any asthma symptoms and have no history of asthma. Next, the differences between the nucleotide difference of the two groups are detected. Thus, GWAS studies can point to genes that may lead to asthma pathology. In particular, a meta-analysis of several GWAS studies that were conducted on various European and American populations have found consistent evidence that asthma is strongly linked to loci on chromosomes 5, 6, 13, and 17 (Cookson and Moffatt, 2000). For instance, chromosome loci 5q21 codes or gives instructions on how to make Interleukin 13 (IL-13), which is a signaling protein involved in regulating mucus secretion and antibody production. Several DNA single nucleotide variations called SNPs (single nucleotide polymorphisms) have been associated with increased airway bronchial mucus production and an increase in production of antibodies, which are known to exacerbate asthma symptoms. Another example of a gene that is involved in the asthma pathology would be a region of chromosome 6 because it codes for MHC (major histocompatibility complex) molecules, which are cell surface proteins that respond to allergic and inflammatory signals. Several SNPs that increase the sensitivity of MHC, which increases the responsiveness of bronchi to allergens, causing asthma symptoms. In the recent years, a new gene involved in childhood nonallergic asthma called ORMDL3 (orosomucoid like gene) has been discovered on chromosome 17q21 locus. GWAS studies have identified three SNPs (rs7216389, rs11650680, and rs3859192) that are strongly associated with asthma. The SNP rs7216389 has been most frequently researched. Thus, it is important to create a viable mouse model that will be used to study impact of ORMDL3 SNPs and analyze treatment that can help this phenotype.

 

The Molecular Function of the Gene Product

 

The ORMDL3 gene codes for the orosomucoid like sphingolipid biosynthesis regulator 3 (ORMDL3 protein), which is involved in sphingolipid synthesis, Unfolded Protein Response (UPR), and increasing eosinophil activity (Cantero-Recasens et al., 2010). The gene is located on chromosome locus 17q21, contains 6,591 base pairs, and has 4 exons (Online Mendelian Inheritance in Man, 2006). The sizes of exons 1, 2, 3, and 4 are 321, 190, 151, and 1644 base pairs, respectively. The ORMDL3 protein is approximately is 153-amino acids long, has 4 transmembrane regions, and is localized in the endoplasmic reticulum. A recent study by Shi et al. has recently identified the rs7216389 SNP to be one of most significant SNPs in ORMDL3 function (2015). The rs7216389 SNP that substitutes a T for C on the fourth ORMDL3 exon is predicted to give the protein a “gain of function” mutation, making it hyperactive. A study by Breslow et al. discovered that the ORDML3 protein is involved in the rate-liming step of sphingolipid synthesis (2010). More specifically, the ORMDL3 protein binds to and inhibits the serine palmitoyltransferase, which an enzyme that yields CoA, 3-dehydro-D-sphingamine and CO2 from L-serine and palmitoyl-CoA in the first step of sphingolipid synthesis. Since sphingolipids are molecules that are involved in cell recognition and signal transmission, ORMDL3 proteins serve as crucial regulators of signal transduction between cells. If sphingolipid metabolism is disrupted by the mutation of ORMDL3, and cell to cell signaling can be significantly impaired. In addition, ORMDL3 is involved in increasing the UPR, which is involved in alieving the cellular stress brought on by an access of misfolded proteins in the endoplasmic reticulum (ER). The UPR relives the stress caused by misfolded proteins by stopping protein translation, degrading misfolded proteins, and increasing the expression of chaperone proteins that are involved in refolding the misfolded proteins. When the ER is overwhelmed by many misfolded proteins, it releases calcium ions out into the cytoplasm, and the decreased concentration of calcium in the ER initiates the UPR response. However, in order prevent an overactive UPR and to maintain calcium homeostasis, the ER has a sacroendoplasmic reticulum calcium pump (SERCA) that pumps calcium ions back in the ER. A study by Cantero-Recasens et al. demonstrated that ORMDL3 protein also binds to SERCA and activates the pumping of calcium into the cell (2010). When ORMDL3 is mutated or overactive, SERCA become more inhibited and pumps less calcium into the ER, and the disrupted calcium concentration in the ER causes an increase in UPR. Thus, the absence ORMDL3 causes an increase in UPR even in the absence of misfolded on unfolded proteins, which may disrupt translation and cause the cell to undergo apoptosis.  

 

Since ORMDL3 is known to impact the organism’s immune response to asthma, recent studies have analyzed the impact of ORMDL3 expression in mouse eosinophil cells, which are cells white blood cells overexpressed during an asthma reaction and cells that have granule full of molecules that are used to kill parasites (“New research unveils role of ORMDL3 gene in asthma,” 2013). A study by Ha et al. used mouse bone marrow-derived eosinophil cells in order to demonstrate how the expression of ORMDL3 impacted the adhesion, rolling, shape change (2013). The researchers either prompted the knock-down of ORMDL3 by exposing the cells to siRNA specific to ORMDL3 or over expression of ORMDL3 by exposing the cells to IL-3 for 12 hours, which is a protein cytokine that induces immune system response and expression of ORMDL3. The researchers analyzed the effect of ORMDL3 expression on cell shape changes, cell rolling, and expression of CD48 (cluster of differentiation 48) marker, which is a protein that recruits and activated B-lymphocyte activation. The cell rolling was analyzed using the In vitro flow chamber assay that included vascular cell adhesion molecules (VCAM-1) that bind to eosinophils and showed that over expression of ORMDL3 caused increased eosinophil rolling, which would mean that ORMDL3 facilities the eosinophil movement throughout the immune system. Also, using immunohistochemistry, the results showed that overexpression of ORMDL3 causes the spreading and repolarization of the cytoskeleton, which facilitates eosinophil rolling. In addition, the immunohistochemistry experiments showed how ORMDL3 overexpression increases the expression of the CD48, which shows that ORMDL3 is also involved in recruiting B-lymphocytes to the site of infection. However, this study did not address how ORMDL3 may impact the immune system in vivo.

 

In order to examine the impact of ORMDL3 on the expression of genes and molecules associated with the respiratory system and inflammation, researchers created an ORMDL3 Cre LoxP mouse. First, fertilized mouse embryos were injected with pCA- GEN Lox RFP-H2B STOP Lox hORMDL3 linearized DNA (Miller et al., 2014). The linearized DNA included a CAG promoter that could selectively express ORMDL3 when the STOP H2B-mRFP would be inactivated. The fertilized eggs were injected in the surrogate mouse, which gave birth to transgenic hORMDL3zp3-Cre mice. By crossing these mice with another zp3- Cre mouse that had had the Cre recombinase protein that could alter the LoxP three sites, the offspring would have an inactivation of the STOP H2B-mRFP region that would enable the expression of ORMDL3. The analysis included the immunohistochemistry of the lung tissues, which revealed the ORMDL3 mice to have increased smooth muscle, subepithelial fibrosis, and mucus. In addition, the increased airway remodeling in the transgenic mice was demonstrated by quantifying Lung mRNA, Bronchial Epithelium mRNA, and bronchoaveolar lavage macrophages (BAL) using RT-PCR, and the hORMDL3zp3-Cr showed significantly higher level of mRNA when compared to the WT. However, it is unknown how responsiveness caused by ORMDL3 expression can be decreased through the use of pharmaceutical drugs and how this response can be used to treat the over responsiveness to allergens and infection seen in asthmatics.

A previous study by Oyeniran et al. intranasally administered either 25 µL of saline with or without 15 µL of house dust mite to the C57BL/6J mice for 5 days, which was meant to elicit an allergic lung inflammation that would increase ORMDL3 . However before receiving either administration, mice were first given either a control saline vehicle or a 0.3 mg/kg of FTY720 immunosuppressant drug intraperitoneally. The results showed that treating mice with FTY720 before exposure to the allergen decreased their levels of ORMDL3, and alleviated airway inflammation, hyperactivity, and mucus production. However, this study did not use an organism that would be genetically predisposed to be susceptible to asthma symptoms.

 

The Gap in Knowledge

The Cre LoxP mouse model discussed in the previous experiment does not use the ORMDL3 gene that is expressed in humans. More importantly, the models do not express the SNP rs7216389, which is linked to increased ORMDL3 activity in humans. In addition, previous studies do not address how the FTY720 immunosuppressant drug will impact mice that have the human ORMDL3 SNPs and how that treatment can relive hyperactive eosinophil pathology overtime. Thus, the aforementioned Cre LoxP model and FTY720 drug studies are not representative of human asthma pathology.

Experiment for the Future

Specific Aims

The aim of this study is to create a Cre LoxP mouse model that expresses both the high risk and low risk human ORMDL3 rs7216389 SNPs. In order to obtain the human SNP sequence, DNA samples from individuals with and without history of asthma will be extracted and used to create a linearized Cre LoxP DNA sequence that will be inserted into mouse embryos to create a transgenic mouse. Another aim is to analyze the impact of the FTY720 immunosuppressant drug on expression on the eosinophil cell shape, cell rolling, and expression of CD48 marker throughout the progression from adolescence to adulthood. I hypothesize that the FTY720 immunosuppressive drug will decrease eosinophil hyperactivity and responsiveness, which can be measured by cell rolling, cytoskeleton polarization and spreading, and CD48 marker expression.

 

Experimental Proposal

Diagram summarizing experimental methods

Zayats Fig 1

Creating a Cre LoxP mouse model

First, in order to create a CreLoxP mice that express either the T (high risk) or the C (low risk) human SNPs, I would recruit participants that reported having allergies or mild asthma as well as participants with no history of asthma and allergies. I would extract their DNA using primers specific for ORMDL3 gene. Next, I would send their DNA to University of Chicago and sequence it. After analyzing the sequence, I would pick two DNA sequences that have an rs7216389 SNP that has a homozygous T (high risk) and a homozygous C (low risk, normal). I would also make sure that the rest of their DNA contains no additional high risk SNP in order to avoid confounding variables. Next, I would design a Cre LoxP linearized DNA as was done in a previous study by Miller et al. The DNA would include the CAG promoter where translation of the protein would start, and it would include a LoxP site going in the same direction around the transcriptional stop sequence that is downstream of the CAG promoter. Also, downstream of the floxed transcriptional stop I would include ORMDL3 DNA that either had the high risk or the low risk SNP. Next, the Cre LoxP sequence would be injected into fertilized mouse embryos, and the embryos would be injected into the surrogate mouse that will give birth to transgenic mice. The transgenic mice will be bred and PCR and DNA sequencing will be used to make sure that the mice are homozygous for the desired Cre LoxP sequence. Next, the mice will be bred with mice that express Cre recombinase in order to create offspring that express both Cre recombinase and the Cre LoxP linearized DNA. Thus, the offspring will have a Cre recombinase that can delete the translational stop sequence between the two LoxP sites, which will cause the expression of the respective ORMDL3 sequences. Also, a wildtype mouse will be used a negative control to ensure that any eosinophil changes are due to ORMDL3 SNP expression.

Administering FTY720 and analyzing its impact on the immune system

 

Both the drug and the saline vehicle administration will be done as described by study Oyeniran et al. However, before any drug administration, a blood sample will be taken from the mice in order to extract the eosinophils for analysis of cell shape changes, cell rolling, and CD48 (cluster of differentiation 48) marker expression, which is a protein that recruits and activated B-lymphocyte activation. All of the three mice groups will receive either a control saline vehicle or a 0.3 mg/kg of FTY720 immunosuppressant drug intraperitoneally starting when they are 2 months old (adolescent stage). The drug or the saline will be administered daily for 60 days until they are 4 months old (adult stage). When the mice are 4 months old, the drug or vehicle administration will be stopped, and another blood sample will be taken from the mice for eosinophil analysis. Thus, the effect of human ORMDL3 SNP expression on the cell-shape changes, cell rolling, and expression of CD48 marker will be analyzed using the techniques previously described in the study by Ha et al. First, the peripheral blood mononuclear

cells (PBMCs) and eosinophils will be extracted from the donor by centrifuging the blood with Percoll. The eosinophils will be separated form PBMCs with the anti-CD16 beads that bind specifically to CD16 Fc receptors found on the eosinophil cells. Next, the eosinophil cells will be eluted from the column using the CD303/BDCA-2 kit using ice-cold solution that would prevent gene translation. The cell rolling will be analyzed using the In vitro flow chamber assay that included vascular cell adhesion molecules (VCAM-1) that bind to eosinophils cells. For immunohistochemistry analysis, the eosinophil cells will be fixed on a cover slip with 4% PFA and 1% Tween, and they will be blocked with 1% BSA and 1% NDS in order to ensure specific antibody binding. Next, the primary anti-rabbit antibodies will be added, which will be specific either to the cytoskeleton antigen or the CD48 marker. Next, an anti-mouse fluorescent secondary antibody, which will be specific to the primary antibody, will be added. A negative control in which no antibodies will be added to the cells will be used to make sure the cells have no autofluorescence. Fluorescent staining of the WT mouse eosinophils will serve as a negative control that shows typical wildtype eosinophil pathology. Thus, the immunohistochemistry techniques will be used to analyze the shape and structure of the cell cytoskeleton and polarization and the expression of the CD48 on eosinophil cells. Finally, the treated cells will be analyzed using the compound light microscope.

 

Possible outcomes, successes, and pitfalls of the experimental procedure

I predict that the high risk Cre LoxP mouse that did not receive any FTY720 will have the most cytoskeleton polarization and spreading, increase in CD48 marker, and increase cell rolling because those characteristics result from increased eosinophil activity, which resulted from the presence of the over reactive ORMDL3 and the absence of the immunosuppressive drug that mediates it. Of course, the low risk and the negative control wildtype mice that received the saline vehicle will have no change in cytoskeleton polarization, CD48 levels and cell rolling when compared to the analysis at two months. Using the FTY720 drug on low risk Cre LoxP and wild type mice will inhibit the normal functioning of the eosinophils and make it hypoactive, causing a decrease in the CD48 levels, cell rolling, and cytoskeleton polarization. However, the high risk Cre LoxP mouse that did receive the FTY720 drug is expected to have a decrease in the eosinophil hyperactivity and having cytoskeleton polarization, CD48 levels, and cell rolling that is similar to that of the low risk Cre LoxP mouse that didn’t receive the drug. Thus, the drug is expected to bring the eosinophil activity to a normal, healthy level that will not make the mouse prone to asthma. The strength of the experiment comes from using a mouse that expresses the human SNP rs7216389, which the SNP that has the most impact on ORMDL3 expression in humans. The mouse model will also be more representative of humans because it will express the human SNPs as opposed to the mouse SNPs. Also, another benefit of the study is that it will analyze the impact on the FTY720 immunosuppressant drug throughout the progression from adolescence to adulthood in the mouse model. However, the possible pitfalls of the study can be that the impact of the drug on the mouse may not be representative of the drug’s impact on human. If the results do not show the expected levels of cell rolling, cell-shape, and CD48 levels, then it can be concluded that there may a metabolic variable within the mouse model that may interfere with the expected results. Thus, the potential therapeutic benefits of the drug for human will not be elucidated. Also, the study does not analyze the impact of ORMDL3 on the lung pathology of the mice, which is necessary to see if the mouse model is truly a viable mouse model for the ORMDL3 gene.

 

Conclusion

This study will help determine the effect of SNP rs7216389 on the eosinophil pathology of the Cre LoxP mouse model. The research will focus on how the FTY720 immunosuppressant drug will impact the eosinophil pathology while interacting either with the high risk or low risk genetic make-up of the ORMDL3 genes over time. If the FTY720 drug therapy is proven to be viable, it can be used to develop a treatment for asthma that may potentially impact 300 million suffering from asthma worldwide.

 

References

Asthma Statistics. (2010, January). American Academy of Allergy, Asthma and Immunology. Retrieved on December 1, 2017, from http://www.aaaai.org/about-aaaai/newsroom/asthma-statistics

Breslow, D. K., Collins, S. R., Bodenmiller, B., Aebersold, R., Simons, K., Shevchenko, A., Ejsing, C. S., & Weissman, J. S. (2010), Orm family proteins mediate sphingolipid homeostasis. Nature 463, 1048-1053.

Cantero-Recasens, G., Fandos, C., Rubio-Moscardo, F., Valverde, M. A., & Vicente, R. (2010). The asthma-associated ORMDL3 gene product regulates endoplasmic reticulum-mediated calcium signaling and cellular stress. Human Molecular Genetics, 19(1), 111-121.

Cookson, W. O. & Moffatt, M. F. (2000). Genetics of asthma and allergic disease. Human Molecular Genetics, 9(16), 2359-2364.

Diseases & Conditions: Asthma. (2015, April). Mayo Clinic. Retrieved on December 1, 2017, from https://www.mayoclinic.org/diseases-conditions/asthma/symptoms-causes/syc-20369653

Ha, S. G., Ge, X. N., Bahaje, N. S., Kang, B. N., Rao, A., Rao, S. P., & Sriramarao, P. (2013). ORMDL3 promotes eosinophil trafficking and activation via regulation of integrins and CD48. Nature Communications, 4(2479). 

Moffatt, M. F, Kabesch, M., Liang, L., Dixon A. L., Strachan, D., Heath, S., & Cookson, W., O. (2007). Genetic variants regulating ORMDL3expression contribute to the risk of childhood asthma. Nature, 448, 470-473.

 

Miller, M., Rosenthal, P., Beppu, A., Mueller, J. L., Hoffman, H. M., Tam, A. B., Doherty, T. A., McGeough, M. D., Pena, C. A., Suzukawa, M., Niwa, M., & Broide, D. H. (2014). ORMDL3 Transgenic Mice Have Increased Airway Remodeling and Airway Responsiveness Characteristic of Asthma. The Journal of Immunology192(8), 3475-3487.

 

New research unveils role of ORMDL3 gene in asthma. (2013, September 24). News: Medical Life Sciences. Retrieved on December 1, 2017, from https://www.news-medical.net/news/20130924/New-research-unveils-role-of-ORMDL3-gene-in-asthma.aspx

Online Mendelian Inheritance in Man. (2006, April 24). ORM1-LIKE PROTEIN 3; ORMDL3. Retrieved on December 1, 2017, from https://www.omim.org/entry/610075?search=ORMDL3&highlight=ormdl3#1

Oyeniran, C., Sturgill, J. L., Hait, N. C., Huang, W. C., Avni, D., Maceyka, M., Newton, J., Allegood, J. C., Montpetit, A., Conrad, D. H., Milstien, S., & Spiegel, S. (2015). Aberrant ORM (yeast)–like protein isoform 3 (ORMDL3) expression dysregulates ceramide homeostasis in cells and ceramide exacerbates allergic asthma in mice. Journal of Allergy and Clinical Immunology, 136(4), 1035-1046.

 

Shi, H., Cheng, D., Yi, L., Huo, X., Zhang, K., & Zhen, G. (2015). Association between ORMDL3 polymorphism and susceptibility to asthma: a meta-analysis. Journal of Clinical and Experimental Medicine, 8(3), 3173-3183.

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