Difference between revisions of "Team:ASIJ Tokyo/Description"

 
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                 <img src="https://static.igem.org/mediawiki/2018/6/6a/T--ASIJ_Tokyo--downarrow.png" height="180px">
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                 <h1 id="header"> PROJECT DESCRIPTION </h1>
 
                 <h1 id="header"> PROJECT DESCRIPTION </h1>
 
                 <hr>
 
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                 <div id="disease info">
 
                 <div id="disease info">
 
                     <h2>Alpha-1-Antitrypsin Deficiency: What is it?</h2>
 
                     <h2>Alpha-1-Antitrypsin Deficiency: What is it?</h2>
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                     <p> &emsp;&emsp;&emsp; The <b>SERPINA1</b> gene codes for the production of the protease Alpha-1 Antitrypsin in hepatocytes. A1AT is a protease inhibitor, and its function is mainly to protect delicate lung tissue from damage from other enzymes active in the area. Therefore, while produced in the liver, A1AT is secreted from the cells and makes its way to the lungs via the bloodstream. </p>
+
                     <p> &emsp;&emsp;&emsp; The <b>SERPINA1</b> gene codes for the production of the protease Alpha-1 Antitrypsin in hepatocytes. A1AT is a protease inhibitor whose function is mainly to protect delicate lung tissue from damage from other enzymes active in the area. Once produced in the liver, A1AT is secreted from the cells and makes its way to the lungs via the bloodstream. </p>
  
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                     <p id="slide2">  &emsp;&emsp;&emsp;The normal version of SERPINA1 is known as the <b>M allele</b> and the mutation that most often results in severe A1AT deficiency creates the <b>Z variant</b> of SERPINA1. The SERPINA1 gene is <i>codominant</i>, so those with two copies of the Z allele are affected most strongly, and this is the version of the gene we aimed to correct. As you can see, the mutation is only of a single base pair, and results in glutamine replacing lysine. The relative ease by which we could use CRISPR Cas9 to fix the mutation causing this disease was also a factor in selecting this disease for study.</p>
+
                     <p id="slide2">  &emsp;&emsp;&emsp;The normal version of SERPINA1 is known as the <b>M allele</b> and the mutation that most often results in severe A1AT deficiency creates the <b>Z variant</b> of SERPINA1. The SERPINA1 gene is <i>codominant</i>, so those with two copies of the Z allele are affected most strongly. This is the version of the gene we aimed to correct. As you can see, the mutation is only of a single base pair, and results in glutamine replacing lysine. The relative ease by which we could use CRISPR Cas9 to fix the mutation causing this disease was also a factor in selecting this disease for study.</p>
 
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                         <td><i>What the mutation looks like</i></td>
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                         <td><i>The E342K mutation in SERPINA1 that causes A1AT Deficiency</i></td>
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                         <td><i>The anti-trypsin trying to leave the cell</i></td>
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                         <td><i>Secretion of A1AT in cellsl</i></td>
 
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                         &emsp;&emsp;&emsp; The Z mutant version of A1AT is prone to <b>polymerization</b>, which renders the protein unable to be secreted from the cell producing it and builds up. However, if not polymerized and successfully secreted, the Z type A1AT is fully functional. As a result, those with the ZZ genotype usually only have <b>10%</b> of normal serum levels. As only a small fraction of needed levels of A1AT are available, the medical complications related to the disease ensue. These can include liver cirrhosis and chronic obstructive pulmonary disease, especially in the form of lung emphysema, all because A1AT cannot get to the lungs to protect the tissue and instead builds up in liver cells, causing damage.This, in turn, results in recurring respiratory infections, fatigue, unintentional weight loss, and rapid heart beat upon standing. Additionally the deficiency increases one's risk of a rare type of liver cancer. </p>
+
                         &emsp;&emsp;&emsp; The Z mutant version of A1AT is prone to <b>polymerization</b>, rendering the protein unable to be secreted from the cell producing it. This causes the protein to build up within the liver. However, if not polymerized and successfully secreted, the Z type A1AT is fully functional. As a result, those with the ZZ genotype usually only have <b>10%</b> of normal serum levels. As only a small fraction of needed levels of A1AT are available, the medical complications related to the disease ensue. This can include liver cirrhosis and chronic obstructive pulmonary disease, especially in the form of lung emphysema. These are all because A1AT cannot get to the lungs to protect the tissue and instead builds up in liver cells, causing damage. This in turn, results in recurring respiratory infections, fatigue, unintentional weight loss, and rapid heart beat upon standing. Additionally the deficiency increases one's risk of a rare type of liver cancer. </p>
 
                          
 
                          
 
                 <img src="https://static.igem.org/mediawiki/2018/6/60/T--ASIJ_Tokyo--infographic.png" id="infographic">       
 
                 <img src="https://static.igem.org/mediawiki/2018/6/60/T--ASIJ_Tokyo--infographic.png" id="infographic">       
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                         <p> &emsp;&emsp;&emsp; The exact prevalence of the ZZ genotype and associated disease is not known, but it is thought to be around 0.1% of the world population, though it varies by ethnic group. Northern Europeans, in particular, have particularly high instances of the mutation, while it is rare in Asian and African populations. The A1AT deficiency is the most common genetic cause of liver disease and the most common reason for liver transplant in children. Additionally, A1AT deficiency is often goes undiagnosed or misdiagnosed as asthma. Treatment options are also not convenient or cost-effective  - many patients require <b>weekly</b> intravenous augmentation therapy, which can cost up to <b>100,000</b> dollars a year - an astronomical amounts over a lifetime. These were also factors we considered when selecting this disease.  
+
                         <p> &emsp;&emsp;&emsp; The exact prevalence of the ZZ genotype and associated disease is not known, but it is thought to be around 0.1% of the world population, though it varies by ethnic group. Northern Europeans, in particular, have particularly high instances of the mutation, while it is rare in Asian and African populations. The A1AT deficiency is the most common genetic cause of liver disease and the most common reason for liver transplant in children. Additionally, A1AT deficiency often goes undiagnosed or misdiagnosed as asthma. Treatment options are also not convenient or cost-effective  - many patients require <b>weekly</b> intravenous augmentation therapy, which can cost up to <b>100,000</b> dollars a year - an astronomical amount over a lifetime. These were also factors we considered when selecting this disease.  
 
                 </p>
 
                 </p>
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<h2> CRISPR-Cas9 </h2>
 
−
<p> CRISPR-Cas9 is a technology that enables us to edit parts of the genome by removing, adding, or altering sections of DNA. As a high school team, it was a huge learning experience to work with CRISPR gene editing, an area of great interest in the field of biotechnology. As a natural mechanism in bacteria, the CRISPR-Cas9 system is used to respond to invading viruses to cut out parts of the virus DNA, but keep a bit of it behind to serve as memory, so that future recognition and defense can be carried out. This natural bacterial system was adapted by scientists to induce change in other organisms, such as in animals. In this process, an enzyme called Cas9 acts as a pair of molecular scissors that cuts the 2 strands of DNA at a specific location when recognizing a specific sequence called a PAM sequence. gRNA, a guide RNA, guides Cas9 to the right part of the genome, allowing for specific regions to be cut, and later replaced. In our experiment, we used the Zhang CRISPR protocol to edit our point mutation, using the U6 target gRNA expression vector, with the unmutated sequence of SERPINA1, to restore SERPINA1 to its original sequence in E.coli.</p>
 
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<div id="references">
 
<div id="references">
 
     <h3> Sources </h3>
 
     <h3> Sources </h3>
−
    <a href="http://webdev.med.upenn.edu/contribute/gastro/documents/AJGalpha1antitrypsindeficiency2008.pdf"> Liver Disease in Alpha 1-Antitrypsin Deficiency: A Review </a>
+
<div id="links">
−
  <br> <a href="https://watermark.silverchair.com/ajcpath130-0568.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAicwggIjBgkqhkiG9w0BBwagggIUMIICEAIBADCCAgkGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMZDFwiL4peHsEe0XrAgEQgIIB2nc3tuZxRHU5C_RtXPADhWk2guMncDaD5EDSD0M5UAN7Heond7IcUY8iz_N_B7J_47MQYmSq33dqMjNplMFsAzPJ2q1tIZE4i7_Pa-eYr6div5YWU4xsW4EKCEW7V_h2kzrHYGNHrW_nOFdWdZY7Iu15UOV9B8bXKz05yn5wXV07-dny_QIzUkYzrrQuOEmtHe5i_EGcKV3JAqFxZ0sGW80cWSisP8rqlRX5trpi3rHnLGRBUPdlepX57XxN4aWENNjghqUXDyCZ1PzlZ7fkcBmpmwYPQZEPbzl02O85FP3Tq8IhhAf-Q1rEqNO8Orni-WT1g5MSnPvN1BM2lxBGKXM6uR0PWowhXlPS_aP9iThw2Vp3kdeTsh0NYe3n61ZJf6uBYI0HZTQ--j2IoSTePvPY7Dl0UnAhemAUYG4gPmAgO2DQgvfC7HPxFt8Qg_77G0WZ8vZHkMnlNgz6y6siBddAUkqcasDQ3uR8lM3j004ncUa6renTphvChEj6nBcexi9G1_wiOZJd7s6kKrsX4n5kOzjkaXAXPu3LWdCXuW_JeDw9iV4IMgdc0DnCJusgMrgQi1CssfHM4NRzuQt8rMDd0pE90hSJWyX_ffEqmcESt82Bv-YvNTnApA"> Efficacy of the Detection of the α1-Antitrypsin
+
Alpha-1 Antitrypsin | NIH 3D Print Exchange. (n.d.). Retrieved October 18, 2018, from <a href="https://3dprint.nih.gov/discover/3dpx-004450" target="none"> https://3dprint.nih.gov/discover/3dpx-004450</a>
−
“Z” Deficiency Variant by Routine Serum Protein
+
 
−
Electrophoresis </a>
+
<br> Crystal, R. G. (1990). Alpha 1-antitrypsin deficiency, emphysema, and liver disease. Genetic basis and strategies for therapy. The Journal of Clinical Investigation, 85(5), 1343–1352.
−
    <br><a href="https://www.journal-of-hepatology.eu/article/S0168-8278(16)30083-6/pdf"> Update on alpha-1 antitrypsin deficiency: New therapies </a>
+
 
−
    <br><a href="http://www.jbc.org/content/261/21/9979.full.pdf"> Biosynthesis, Processing, and Secretion of M and Z Variant
+
<br>Genetics Home Reference. (2018, October 16). What are single nucleotide polymorphisms (SNPs)? Retrieved October 18, 2018, from <a href="https://ghr.nlm.nih.gov/primer/genomicresearch/snp0" target="none">https://ghr.nlm.nih.gov/primer/genomicresearch/snp</a>
−
Human al-Antitrypsin* </a>
+
 
 +
<br>Haq, I., Irving, J. A., Saleh, A. D., Dron, L., Regan-Mochrie, G. L., Motamedi-Shad, N., … Lomas, D. A. (2016). Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization. American Journal of Respiratory Cell and Molecular Biology, 54(1), 71–80.
 +
 
 +
<br>Jezierski, G., & Pasenkiewicz-Gierula, M. (n.d.). The effect of the Glu342Lys mutation in a-antitrypsin on its struc- ture, studied by molecular modelling methods*+. Retrieved from <a href="http://www.actabp.pl/pdf/1_2001/65-75s.pdf" target="none">http://www.actabp.pl/pdf/1_2001/65-75s.pdf</a>
 +
 
 +
<br>Lomas, D. A., Hurst, J. R., & Gooptu, B. (2016). Update on alpha-1 antitrypsin deficiency: New therapies. Journal of Hepatology, 65(2), 413–424.
 +
 
 +
<br>Lomas, D. A., & Mahadeva, R. (2002). Alpha1-antitrypsin polymerization and the serpinopathies: pathobiology and prospects for therapy. The Journal of Clinical Investigation, 110(11), 1585–1590.
 +
 
 +
<br>Lomas, D. A., & Parfrey, H. (2004). α1-Antitrypsin deficiency• 4: Molecular pathophysiology. Thorax, 59(6), 529–535.
 +
 
 +
<br>Mahadeva, R., Dafforn, T. R., Carrell, R. W., & Lomas, D. A. (2002). 6-mer Peptide Selectively Anneals to a Pathogenic Serpin Conformation and Blocks Polymerization: IMPLICATIONS FOR THE PREVENTION OF Z α 1 -ANTITRYPSIN-RELATED CIRRHOSIS. The Journal of Biological Chemistry, 277(9), 6771–6774.
 +
 
 +
<br>McNab, G. L., Ahmad, A., Mistry, D., & Stockley, R. A. (2007). Modification of Gene Expression and Increase in α1-Antitrypsin (α1-AT) Secretion After Homologous Recombination in α1-AT-Deficient Monocytes. Human Gene Therapy, 18(11), 1171–1177.
 +
 
 +
<br>SERPINA1 - SNPedia. (n.d.). Retrieved October 18, 2018, from <a href="https://www.snpedia.com/index.php/SERPINA1" target="none">https://www.snpedia.com/index.php/SERPINA1</a>
 +
 
 +
<br>snpdev. (n.d.). Submitted SNP(ss) Details: ss3218990554. Retrieved October 18, 2018, from <a href="https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ss.cgi?subsnp_id=3218990554" target="none">https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ss.cgi?subsnp_id=3218990554</a>
 +
 
 +
<br>Stoller, J. K., Lacbawan, F. L., & Aboussouan, L. S. (2006). Alpha-1 Antitrypsin Deficiency. In M. P. Adam, H. H. Ardinger, R. A. Pagon, S. E. Wallace, L. J. H. Bean, K. Stephens, & A. Amemiya (Eds.), GeneReviews®. Seattle (WA): University of Washington, Seattle.
 +
 
 +
<br>Slev, P. R., Williams, B. G., Harville, T. O., Ashwood, E. R., & Bornhorst, J. A. (2008). Efficacy of the Detection of the α1-Antitrypsin “Z” Deficiency Variant by Routine Serum Protein Electrophoresis. American Journal of Clinical Pathology, 130(4), 568–572.
 +
 
 +
<br>Song, C.-Q., Wang, D., Jiang, T., O’Connor, K., Tang, Q., Cai, L., … Xue, W. (2018). In Vivo Genome Editing Partially Restores Alpha1-Antitrypsin in a Murine Model of AAT Deficiency. Human Gene Therapy, 29(8), 853–860.
 +
 
 +
<br> Verbanac, K. M., & Heath, E. C. (1986). Biosynthesis, processing, and secretion of M and Z variant human alpha 1-antitrypsin. The Journal of Biological Chemistry, 261(21), 9979–9989.
 +
 
 +
<br> Yoshida, A., Lieberman, J., Gaidulis, L., & Ewing, C. (1976). Molecular abnormality of human alpha1-antitrypsin variant (Pi-ZZ) associated with plasma activity deficiency. Proceedings of the National Academy of Sciences of the United States of America, 73(4), 1324–1328.
 
     </p>
 
     </p>
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</div>
 
</div>
 
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Latest revision as of 00:02, 18 October 2018

PROJECT DESCRIPTION

Alpha-1-Antitrypsin Deficiency: What is it?

A1AT deficiency is an enzymatic liver disorder that results from a genetic mutation in the SERPINA1 gene.

    The SERPINA1 gene codes for the production of the protease Alpha-1 Antitrypsin in hepatocytes. A1AT is a protease inhibitor whose function is mainly to protect delicate lung tissue from damage from other enzymes active in the area. Once produced in the liver, A1AT is secreted from the cells and makes its way to the lungs via the bloodstream.

   The normal version of SERPINA1 is known as the M allele and the mutation that most often results in severe A1AT deficiency creates the Z variant of SERPINA1. The SERPINA1 gene is codominant, so those with two copies of the Z allele are affected most strongly. This is the version of the gene we aimed to correct. As you can see, the mutation is only of a single base pair, and results in glutamine replacing lysine. The relative ease by which we could use CRISPR Cas9 to fix the mutation causing this disease was also a factor in selecting this disease for study.

Mutation Anti-trypsin trying to leave the cell
The E342K mutation in SERPINA1 that causes A1AT Deficiency Secretion of A1AT in cellsl

    The Z mutant version of A1AT is prone to polymerization, rendering the protein unable to be secreted from the cell producing it. This causes the protein to build up within the liver. However, if not polymerized and successfully secreted, the Z type A1AT is fully functional. As a result, those with the ZZ genotype usually only have 10% of normal serum levels. As only a small fraction of needed levels of A1AT are available, the medical complications related to the disease ensue. This can include liver cirrhosis and chronic obstructive pulmonary disease, especially in the form of lung emphysema. These are all because A1AT cannot get to the lungs to protect the tissue and instead builds up in liver cells, causing damage. This in turn, results in recurring respiratory infections, fatigue, unintentional weight loss, and rapid heart beat upon standing. Additionally the deficiency increases one's risk of a rare type of liver cancer.

    The exact prevalence of the ZZ genotype and associated disease is not known, but it is thought to be around 0.1% of the world population, though it varies by ethnic group. Northern Europeans, in particular, have particularly high instances of the mutation, while it is rare in Asian and African populations. The A1AT deficiency is the most common genetic cause of liver disease and the most common reason for liver transplant in children. Additionally, A1AT deficiency often goes undiagnosed or misdiagnosed as asthma. Treatment options are also not convenient or cost-effective - many patients require weekly intravenous augmentation therapy, which can cost up to 100,000 dollars a year - an astronomical amount over a lifetime. These were also factors we considered when selecting this disease.

Sources