This web page was produced as an assignment for Genetics 564, an undergraduate course at the University of Wisconsin-Madison.
Future Research on CNGA3 and Achromatopsia
While there is a lot known about CNGA3 and its impact on vision, there is a lot still left to be researched in order to help patients with Achromatopsia. Gene therapies have been created that can reinstate a functional CNGA3 into the genome, however as a result of the damage that is caused to the retina because of having a mutant CNGA3, this gene therapy would not completely correct Achromatopsia in humans and would leave the retina weak. As a result I thought it would be important to explore what may be causing the damage in the retina, specifically cell death, as well as look at the conservation of CNGA3 and EMILIN1 (a binding partner that literature stated may be causing the cell death in the retina). For more in depth information on this click here.
Hypothesis: The CNGA3 Ion transport domain and the cNMP binding domain will be better conserved in species with eyes than without.
Approach:
Approach:
Results: Using Pfam and Clustal W2, I found that the domains in CNGA3 are in fact more highly conserved in species with eyes than without. However, the domains (and gene in general) are still pretty well conserved in species without eyes. I found this surprising because the function of the gene is centered around vision.
Figure 1: The figure on the left shows the results from pfam which were examined to look at the conservation in organisms with eyes vs. species without.
Figure 2: The figure on the right shows the clustal W2 results. And interesting results is that the amino acids that changed often changed in a manner that would result in a change in structure (i.e. polar to nonpolar).
Figure 2: The figure on the right shows the clustal W2 results. And interesting results is that the amino acids that changed often changed in a manner that would result in a change in structure (i.e. polar to nonpolar).
Hypothesis: EMILIN1 domains will be highly conserved in both species with and without eyes.
Approach:
Approach:
Results: Using Pfam and Clustal W2, I found that the domains in CNGA3 are more highly conserved in species with eyes than without. I found this to be very surprising because the function of EMILIN1 is not centered around vision. It is possible that the results appeared this way as a result of a lack of research, so more research could be done to identify homologs of EMILIN1 in species without eyes.
Figure 3: The figure on the left shows the results from pfam which were examined to look at the conservation in organisms with eyes. Organisms without eyes did not have homologs.
Figure 4: The figure on the right shows the clustal W2 results. This confirms how well conserved the amino acids are across species with eyes.
Figure 4: The figure on the right shows the clustal W2 results. This confirms how well conserved the amino acids are across species with eyes.
Aim #3: To determine if EMILIN1 being able to bind to CNGA3 is necessary for cell survival in the retina.
Hypothesis: Without EMILIN1 present, cell death will begin to occur due to cell adhesion being disrupted.
Approach:
Hypothesis: Without EMILIN1 present, cell death will begin to occur due to cell adhesion being disrupted.
Approach:
Expected Results: Using Pfam and Clustal W2, I found that the domains in CNGA3 are more highly conserved in species with eyes than without. I found this to be very surprising because the function of EMILIN1 is not centered around vision. It is possible that the results appeared this way as a result of a lack of research, so more research could be done to identify homologs of EMILIN1 in species without eyes.
Figure 5: This figure shows how staining can show where cell death is occurring within the zebrafish eye.
Conclusions:
If EMILIN1 is involved in cell death this will provide new insight and possibly fill the gap of information needed in order for gene therapies to be effective in clinical trials. If the Achromatopsia causing mutations in CNGA3 occurs in the domain that EMILIN1 binds to, EMILIN1 may not be able to bind which would then cause cell death.
Finding a successful gene therapy that could move to clinical trials in humans would be taking a huge step for people with Achromatopsia. Achromatopsia can influence how someone lives there life, effecting simple everyday tasks.
Finding a successful gene therapy that could move to clinical trials in humans would be taking a huge step for people with Achromatopsia. Achromatopsia can influence how someone lives there life, effecting simple everyday tasks.
References:
Information-
[1] Online Mendelian Inheritance in Man, OMIM®. Johns Hopkins University, Baltimore, MD. MIM Number: {216900}: {09/18/2012}: . World Wide Web URL: http://omim.org/entry/216900
[2] Claes E, Seeliger M, Michalakis S, Biel M, Humphrie P, Haverkamp S (2004) Morphological characterization of the retina of the CNGA3(-/-)Rho(-/-) mutant mouse lacking functional cones and rods. Invest Ophthalmol Vis Sci 45:2039-2048.
[3] Komáromy, András M., et al. "Gene therapy rescues cone function in congenital achromatopsia." Human molecular genetics 19.13 (2010): 2581-2593.
[4] Varsányi, Balázs, et al. "Optical coherence tomography of the macula in congenital achromatopsia." Investigative ophthalmology & visual science 48.5 (2007): 2249-2253.
[5] Selvakumar, Dakshnamurthy, et al. “CNGA3 is expressed in inner ear hair cells and binds to an intracellular C-terminus domain of EMILIN1” Biochem. J. (2012) 443, 463-476.
[6] The Gene Ontology Consortium. Gene ontology: tool for the unification of biology. Nat. Genet.. May 2000;25(1):25-9.
[7] Viringipurampeer, IA, et al. “Rip knockdown rescues photoreceptor cell death in blind pde6c zebra fish” Cell death and differentiation (2014), 1-11.
Images-
Zebrafish eye: http://www.ralf-dahm.com/index.php?id=67
Zebrafish: https://science.nichd.nih.gov/confluence/display/newsletter/2013/06/08/Four+NICHD+Postbacs+Receive+OITE+Poster+Award
Information-
[1] Online Mendelian Inheritance in Man, OMIM®. Johns Hopkins University, Baltimore, MD. MIM Number: {216900}: {09/18/2012}: . World Wide Web URL: http://omim.org/entry/216900
[2] Claes E, Seeliger M, Michalakis S, Biel M, Humphrie P, Haverkamp S (2004) Morphological characterization of the retina of the CNGA3(-/-)Rho(-/-) mutant mouse lacking functional cones and rods. Invest Ophthalmol Vis Sci 45:2039-2048.
[3] Komáromy, András M., et al. "Gene therapy rescues cone function in congenital achromatopsia." Human molecular genetics 19.13 (2010): 2581-2593.
[4] Varsányi, Balázs, et al. "Optical coherence tomography of the macula in congenital achromatopsia." Investigative ophthalmology & visual science 48.5 (2007): 2249-2253.
[5] Selvakumar, Dakshnamurthy, et al. “CNGA3 is expressed in inner ear hair cells and binds to an intracellular C-terminus domain of EMILIN1” Biochem. J. (2012) 443, 463-476.
[6] The Gene Ontology Consortium. Gene ontology: tool for the unification of biology. Nat. Genet.. May 2000;25(1):25-9.
[7] Viringipurampeer, IA, et al. “Rip knockdown rescues photoreceptor cell death in blind pde6c zebra fish” Cell death and differentiation (2014), 1-11.
Images-
Zebrafish eye: http://www.ralf-dahm.com/index.php?id=67
Zebrafish: https://science.nichd.nih.gov/confluence/display/newsletter/2013/06/08/Four+NICHD+Postbacs+Receive+OITE+Poster+Award