Functional study of two biochemically unusual mutations in

purposeTo test, in living photoreceptors, two mutations, S248W and R1091x, in the

methods

resultsCyclase activity in retinas treated with either hWT or R1091x GUCY2D transgenes was similar but was undetectable in the S248W GUCY2D-treated retinas, which starkly contrasts their relative activities when heterologously expressed in human embryonic kidney (HEK293) cells. Rod and cone ERGs, absent in GCdKO, appeared in the hWT and R1091x GUCY2D-injected eyes, while the S248W mutant failed to restore scotopic ERG response and enabled only rudimentary photopic ERG response. The hWT and R1091x GUCY2D immunofluorescence was robust in the rod and cone outer segments, whereas the S248W was detectable only in the sparse cone outer segments and sporadic photoreceptor cell bodies. Robust RetGC1 expression was detected with immunoblotting in the hWT and R1091x-treated retinas but was marginal at best in the S248W GUCY2D retinas, despite the confirmed presence of the S248W GUCY2D transcripts.

conclusionsThe phenotype of S248W GUCY2D in living retinas did not correlate with the previously described normal biochemical activity of this mutant when heterologously expressed in non-photoreceptor cell culture. This result suggests that the S248W mutation contributes to LCA1 by hampering the expression, processing, and/or cellular transport of GUCY2D, rather than its enzymatic properties. In contrast, the effective restoration of rod and cone function by R1091x GUCY2D is paradoxical and does not explain the severe loss of vision typical for LCA1 associated with that mutant allele.