Conclusions
This webpage was produced as an assignment for Genetics 564, an undergraduate capstone at University of Wisconsin- Madison
Background
Figure 1. The general affected ADA SCID pathway
Severe Combined Immunodeficiency Disorder (SCID) is an immunological deficiency disorder disturbing T-, B- and NK-cell function and consequently compromising antibody responses[1]. Around 15 % SCID cases are due to a mutation in the Adenosine Deaminase (ADA1) enzyme. ADA1 deficiency disrupts adenosine metabolism & causes an accumulation of adenosine deoxyribonucleotides (dATPs) inhibiting ribonucleotide reductase activity essential for cell proliferation and DNA repair [2]. There is growing evidence that ADA-SCID is involved in various non-immunological defects. These include non-infective, adenosine metabolic disorders, such as neurocognitive, auditory, and pulmonary airway dysfunction[3][4]. It is still unclear how the adenosine metabolism disruption contributes to these pulmonary airway abnormalities. The goal is to investigate the cellular function of ADA1 in adenosine metabolism in the lungs.
Figure 2. Unknown ADA-SCID mechanisms in the pulmonary sytem
My primary goal is to determine the role of ADA1 deficiency in pulmonary dysfunction. A Danio rerio (zebrafish) model will be used to first study gills as they share the conserved respiratory function of lungs, have an ADA homolog (ada), produce many offspring frequently, and their transparent, externally developing embryos facilitate in-vivo modifications to observe potential differential gill development or inflammation. I will also use a Mus musculus (mouse) model because they share very similar lung physiology, have exhibited severe pulmonary inflammation in ADA-deficient lines[6], and I will be able to conduct a transfer impedance experiment to test for normal lung function[5]. I hypothesize that accumulated levels of adenosine increases cell signaling pathways responsible for hyper-constriction of the lungs and increased inflammation. My long-term goal: is to better understand the role ADA1 has on the lungs.
Aim 1: Determine conserved amino acid sequences in ADA1 necessary for normal pulmonary/gill function. Approach: I will use ENSEMBLE and ClustalWOmega to obtain and align homologous ADA1 protein sequences from model organisms to identify conserved regions of ADA1 that are only in organisms with lungs or gills. I will then use CRISPR-Cas9 to knock out these conserved ADA regions and search for individual zebrafish that have deficient gill function. Rationale: Identifying ADA knockout zebrafish with gill dysfunction will confirm the conserved, non-immunological, role of adenosine in respiration, also being the main role of the pulmonary system. Hypothesis: The ADA1 knockout zebrafish will show gill dysfunction due to the increased levels of adenosine.
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Figure 3. General overview of aim 1. Identify conserved amino acid sequences in model organisms' ADA1 homologs and identify sequence linked with pulmonary and gill function
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Aims 2:Use RNA-Seq on ADA1 knockout zebrafish to identify differentially expressed transcripts Approach: I will sequence isolated zebrafish RNA from both a control ada+/ada+ group and from the zebrafish of aim 1 that showed gill deficiencies. I will next Gene Ontology to identify clusters of gill development/functional genes that are lost or downregulated in the ada knockout group. Next, I will use CRISPR-Cas9 to knockout the mice homologs of the differentially expressed transcripts from aims 1 and test for signs of pulmonary defects through a transfer impedance test.Rationale: Online literature indicates ADA1 is linked to lung alveolus development and negative regulation of inflammatory response, two pathways affected in pulmonary dysfunction[7]. Identifying expression changes in these transcripts will demonstrate the role of adenosine in lung dysfunction.
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Figure 4. General overview of aim 2. Utilize RNA-seq to identify gene expression level changes to transcripts directly linked to gill function
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Aims 3: Identify novel protein interactors with ADA1 mutants. Approach: I will use In-Vivo SILAC labeling on wt+ vs ADA knockout mice from Aims 2 with a “heavy isotope” 13 Carbon Lysine diet fed to the ADA knockout group. Lysated protein isolates from both groups will be analyzed by high-resolution liquid chromatography-tandem mass spectrometry (nLC MS/MS) to identify the differences in protein ratios of the control and mutated groups. Rationale: Ubiquitination is known to have a regulatory role during periods of chronic inflammation in the lungs[9]. Assessing the protein ratios of ubiquitin in both wt+ vs ADA mutants could lead to a better understanding of the correlation between concentrations of adenosine and the positive anti-inflammatory responses ubiquitin brings to the lung cells. Hypothesis: I hypothesize that ubiquitin protein ratios will be significantly lower in the 13 Carbon Lysine metabolic diet of the ADA mutant mice group caused by indirect signaling of adenosine on a ubiquitin-like protein messenger.
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Figure 5. General overview of aim 3. Use SILAC to quantify protein expression level changes in genes directly related to pathways involved in regulating lung function
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Conclusion and future directions
ADA1 encodes for an enzyme extremely ubiquitous and essential for cellular replication, metabolism homeostasis, and regulation of the immune system. Although enzyme therapy approaches as well as stem cell transplant therapy have both shown success in effectively irradicating the ADA-SCID disease, it has yet to fully reverse defects found throughout the body such as cognitive, neurological, auditory, and pulmonary abnormalities. It is crucial to uncover the gaps of knowledge in these mechanisms in order to develop a cure that can sucessively bring patients to the best health conditions possible without persisting issues. With the advancement of ex vivo hematopoietic stem cell (HSC) gene therapy using market approved Strimvelis as well as advanced proteomic approaches such as Spiked-In SILAC quantification, increased disease elimination effeciency appears to be in the horizon. These technological strides will be possibly bring about new targeted approaches to close the unknown mechanisms once and for all in the ADA SCID disease linked to other vital organ functions.
ADA1 encodes for an enzyme extremely ubiquitous and essential for cellular replication, metabolism homeostasis, and regulation of the immune system. Although enzyme therapy approaches as well as stem cell transplant therapy have both shown success in effectively irradicating the ADA-SCID disease, it has yet to fully reverse defects found throughout the body such as cognitive, neurological, auditory, and pulmonary abnormalities. It is crucial to uncover the gaps of knowledge in these mechanisms in order to develop a cure that can sucessively bring patients to the best health conditions possible without persisting issues. With the advancement of ex vivo hematopoietic stem cell (HSC) gene therapy using market approved Strimvelis as well as advanced proteomic approaches such as Spiked-In SILAC quantification, increased disease elimination effeciency appears to be in the horizon. These technological strides will be possibly bring about new targeted approaches to close the unknown mechanisms once and for all in the ADA SCID disease linked to other vital organ functions.
References
[1] Whitte H, Thrasher A, Veys P, Kinnon C, Gaspar HB. Intrinsic defects of B cell function in X-linked severe combined immunodeficiency. Eur J Immunol. 2000;30:772–7.
[2] Immune Deficiency Foundation: The Story of David. https://primaryimmune.org/living-pi-explaining-pi-others/story-david
[3] Kathryn V. Whitmore1 and Hubert B. Gaspar,Adenosine Deaminase Deficiency More than just an Immunodeficicency Front Immunol. 2016; 7: 314.
[4]Komarow HD, Sokolic R, Hershfield MS, Kohn DB, Young M, Metcalfe DD, et al. Impulse oscillometry identifies peripheral airway dysfunction in children with adenosine deaminase deficiency. Orphanet J Rare Dis (2015)
[5]Bates JH1, Irvin CG Measuring lung function in mice: the phenotyping uncertainty principle. J Appl
[1] Whitte H, Thrasher A, Veys P, Kinnon C, Gaspar HB. Intrinsic defects of B cell function in X-linked severe combined immunodeficiency. Eur J Immunol. 2000;30:772–7.
[2] Immune Deficiency Foundation: The Story of David. https://primaryimmune.org/living-pi-explaining-pi-others/story-david
[3] Kathryn V. Whitmore1 and Hubert B. Gaspar,Adenosine Deaminase Deficiency More than just an Immunodeficicency Front Immunol. 2016; 7: 314.
[4]Komarow HD, Sokolic R, Hershfield MS, Kohn DB, Young M, Metcalfe DD, et al. Impulse oscillometry identifies peripheral airway dysfunction in children with adenosine deaminase deficiency. Orphanet J Rare Dis (2015)
[5]Bates JH1, Irvin CG Measuring lung function in mice: the phenotyping uncertainty principle. J Appl