Effects of deleterious opsin gene variants on cone photoreceptor structure, function, and viability

Abstract

Specific L/M-opsin interchange variants have recently been linked to a variety of eye disorders that include diminished ERGs, color blindness, and pathologically high myopia. One disease-causing variant, designated LVAVA according to its defining amino acid sequence, has been linked to two distinct vision disorders. Males with LVAVA in one of the first two positions in the X-chromosome opsin gene array develop pathologically high myopia and have abnormal ERGs, while those with an LVAVA variant as their only X-chromosome opsin gene have extreme and uncorrectable vision loss. In this dissertation, retinal function was measured by ERG in two men with the latter, non-syndromic disease. While cones expressing the LVAVA opsin were physiologically compromised, residual function was maintained into adulthood. Furthermore, these cones disrupted the function of neighboring S cones that did not harbor the toxic variant. In order to directly test the relationship between the LVAVA variant and pathology, the non-syndromic disease was modeled in a targeted gene replacement mouse in which the endogenous <italic>Opn1mw</italic> gene was exchanged for an engineered <italic>Opn1lw<super>LVAVA</super></italic> gene. Because the presence of S-opsin in coexpressing mouse cones could interfere with the experiments, the <italic>Opn1sw</italic> genes were knocked out of both the LVAVA mice and the control mice that expressed a functional human opsin (LIAIS). ERG and histological experiments demonstrated that the mouse model faithfully recapitulated functional and morphological retinal defects associated with the human disease. Another variant, referred to as LIAVA, completely and selectively disrupts the function of cones that express it. Retinal function was evaluated by ERG in a subject who was previously reported to express an LIAVA variant in all of his L/M cones. The human data were compared to ERG and retinal morphology data collected from an <italic>Opn1lw<super>LIAVA</super> Opn1sw</italic><super>-/-</super> mouse model that exhibited a late-onset and relatively mild phenotype. While the results from mice demonstrated that the LIAVA amino acid sequence is directly harmful to cones, the source of dysfunction in human cones likely arises from aberrant splicing of the LIAVA messenger RNA due to the nucleotide haplotype of exon 3 associated with this amino acid combination. The S-opsin knockout mouse line that provided the genetic background for all mice used in the LVAVA and LIAVA studies was generated using targeted gene replacement technology, and the effects of removing S-opsin from cones that would otherwise coexpress both S and M/L-opsins were investigated. In addition to making the interpretation of data collected from the disease models more straightforward, characterizing the€ <italic>Opn1mw Opn1sw</italic><super>-/-</super> mice and the <italic>Opn1lw<super>LIAIS</super> Opn1sw</italic><super>-/-</super> mice provided insights into issues relating to visual adaptation, regulation of opsin gene expression, and cone viability.