Cone opsin gene variants in color blindness and other vision disorders
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The long wavelength sensitive (L) and middle wavelength sensitive (M) photopigments are encoded in a tandem array on the X chromosome. Due to their close proximity and high sequence homology, this locus is prone to unequal homologous recombination resulting in intermixing of the L and M opsin genes. This thesis explores the enormous variability in both L and M gene sequence and in total opsin gene copy number that results from this process. In a sample of 1022 normal trichromatic males, there were 89 unique L sequences and 31 unique M sequences. A majority of opsin gene arrays contained extra M opsin genes (55%) and a small number (5%) contained extra L opsin genes. Array structure was found to vary by race: African Americans were more likely to have extra L genes (10%); Caucasians to have extra M genes (64%); and Asians to have arrays without extra genes (54%). A second study of 335 women unselected for color vision phenotype was used to explore the frequency of extra cone types in the retina. Between 42% and 60% had opsin gene arrays indicative of having four cone types and between 2.4% and 11% had arrays indicative of five. The ability of the visual system to make use of these extra cone types to create a new chromatic channel is exceedingly rare though there may be a possibility of training women who have a fourth cone type to use it by creating artificial stimuli that specifically activate the extra cone in isolation of the other three. In a second project, knowledge of the gene mutations and rearrangements that lead to color vision deficiencies underpinned the creation of a genetic assay to find and classify color defects. That assay was tested on 1872 human subjects and its potential for use in clinical diagnosis was evaluated. Our results indicate that information about the underlying pathology of color blindness derived from genetic analyses can be extremely valuable in accurate diagnosis, and that a test incorporating gene analyses supplemented with behavioral testing approaches the ideal color vision test. While most opsin variants lead to normally functioning photopigments, certain variants that encode “toxic” sequences in exon three lead to serious eye diseases with symptoms that include cone dystrophy, diminished ERGs, color blindness, and pathologically high myopia. A virally-mediated gene therapy treatment aimed at curing these diseases was designed using the modified AAV2 capsid 7M8 that has enhanced tropism toward photoreceptors and an optimized expression cassette to drive high expression in cone cells. Initial work in a mouse model shows that this construct is capable of driving high and highly specific expression in cones following minimally invasive intravitreal injections. Further examination of the construct in mice using immunohistochemistry, spectral ERG, and behavioral testing will determine whether this is a viable approach to curing these diseases of the cones in human patients.