Download File - Achromatopsia

Achromatopsia is a retinal disorder that causes the cone structures in the eye to be dysfunctional,
resulting in colorblindness, loss of vision and light sensitivity. Several genes are associated with
Achromatopsia, including the CNGA3 gene, which are important for regulating the calcium channels
in the retina [1]. A few gene therapies have been successful, which reinstate a functional CNGA gene
into the genome [2,3], however these therapies have not been able to be moved to clinical use.
Achromatopsia gene therapies have not progressed because very little is known about how CNGA
functions in the eye. Interestingly, the CNGA3 gene is also seen in organisms without eyes despite the
proteins primary function revolving around vision [4]. It is also a mystery how well conserved the
regulation of CNGA3 via its binding partners are, in species with and without eyes. EMILIN1 is one
binding partner of CNGA3 that the functional conservation among species is unknown [5]. It is known
that EMILIN1 is involved in cell adhesion, which is essential to cell structure [6]. Zebrafish CNGA3
mutants show an increase in cell death in their eyes, yet it is unclear why this occurs [7]. I plan to take
a genomic and bioinformatics approach to determine where in the protein sequence CNGA3 and its
binding partner EMILIN1 are conserved across species with or without eyes. This information will
allow us to better understand the role of CNGA3 in eye development and possibly cell death.
The primary goal of this study is to gain a deeper understanding of the molecular function of CNGA3
and its binding partner EMILIN1. I will explore if they are conserved across species and if EMILIN1 is
necessary for cell survival. This will include a genomic analysis of both genes. I hypothesize that
CNGA3 will be less conserved in species without eyes and EMILIN1 will be well conserved in species
with and without eyes, while contributing to the ability for cells in the retina to survive.
Specific Aims #1: To determine how well conserved the ion transport and cNMP binding domains in
CNGA3 are in species with and without eyes. Approach: Using pfam and CLUSTAL W to examine
the differences in amino acids in the domain regions in the CNGA3 gene throughout species.
Hypothesis: The CNGA3 ion transport domain and the cNMP binding domain will be better
conserved in species with eyes than without eyes..
Specific Aims #2: To determine how well conserved the C1q and EMI domains in EMILIN1 are in
species with and without eyes.
Approach: Using pfam and CLUSTAL W to examine the differences in amino acids in the domain
regions in the EMILIN1 gene throughout species.
Hypothesis: I predict that both domains in EMILIN1 will be highly conserved in species with and
without eyes because they are involved in cell adhesion which is essential throughout the body.
Specific Aims #3: To determine if EMILIN1 is necessary for cell survival in the retina.
Approach: I will use string to confirm that EMILIN1 is a binding partner then do an RNAi experiment
using zebra fish to see if the absence of EMILIN1 proteins cell adhesion abilities, will cause cell death
to occur when CNGA3 is non-mutated.
Hypothesis: I hypothesize that without EMILIN1 present, cell death will begin to occur because cell
adhesion will be disrupted.
If EMILIN1 is involved in cell death this will provide new insight and possibly feill the gap of
information needed in order for gene therapies to be effective in clinical trials. If one of 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.
References:
[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.