Determining consequences of retinal membrane guanylyl cyclase (RetGC1) deficiency in human Leber congenital amaurosis en route to therapy: residual cone-photoreceptor vision correlates with biochemical properties of the mutants

Abstract

The GUCY2D gene encodes retinal membrane guanylyl cyclase (RetGC1), a key component of the phototransduction machinery in photoreceptors. Mutations in GUCY2D cause Leber congenital amaurosis type 1 (LCA1), an autosomal recessive human retinal blinding disease. The effects of RetGC1 deficiency on human rod and cone photoreceptor structure and function are currently unknown. To move LCA1 closer to clinical trials, we characterized a cohort of patients (ages 6 months-37 years) with GUCY2D mutations. In vivo analyses of retinal architecture indicated intact rod photoreceptors in all patients but abnormalities in foveal cones. By functional phenotype, there were patients with and those without detectable cone vision. Rod vision could be retained and did not correlate with the extent of cone vision or age. In patients without cone vision, rod vision functioned unsaturated under bright ambient illumination. In vitro analyses of the mutant alleles showed that in addition to the major truncation of the essential catalytic domain in RetGC1, some missense mutations in LCA1 patients result in a severe loss of function by inactivating its catalytic activity and/or ability to interact with the activator proteins, GCAPs. The differences in rod sensitivities among patients were not explained by the biochemical properties of the mutants. However, the RetGC1 mutant alleles with remaining biochemical activity in vitro were associated with retained cone vision in vivo. We postulate a relationship between the level of RetGC1 activity and the degree of cone vision abnormality, and argue for cone function being the efficacy outcome in clinical trials of gene augmentation therapy in LCA1.

Evaluating the Role of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Domains in Binding Guanylyl Cyclase-activating Proteins (GCAPs)

“…The indication that binding sites for GCAP1 and GCAP2 are likely identical or overlapped also comes from the comparison of point mutations related to LCA in humans. Multiple mutations in GUCY2D gene found in LCA patients (15,16) can affect RetGC1 activity and regulation by GCAPs in profoundly different ways (16), but the effect of the tested mutations in each case was virtually identical for both GCAP1-and GCAP2-dependent regulation (16). This would be consistent with the two GCAPs having common or overlapping rather than distantly separated binding sites formed by remotely separated portions of the cyclase primary structure.…”

“…Some LCA-linked mutations inactivate RetGC1 and/or suppress its activation by GCAP1 and GCAP2 (16). Here we tested two LCA-linked point mutations in the KHD, W708R and I734T, found in LCA patients and assigned the highest estimated pathogenic probability (15).…”

“…Mutations Causing a Blinding Disorder Destroy GCAPRetGC1 Complex-Mutations in RetGC1 cause congenital LCA blindness through loss of photoreceptor function rather than photoreceptor degeneration (16). Considering that most of the cyclase activity in rods and nearly all of the activity in cones belongs to RetGC1 (11)(12)(13), blocking the cyclase activation by GCAPs could severely affect vision.…”

The R838S Mutation in Retinal Guanylyl Cyclase 1 (RetGC1) Alters Calcium Sensitivity of cGMP Synthesis in the Retina and Causes Blindness in Transgenic Mice

“…In animal models for the LCA1, vision can be sustainably restored by expression of a normal RetGC1 allele in dysfunctional rods and cones, when delivered via adeno-associated virus (54 -57). Consequently, such gene therapy is expected to be perspective in the treatment of a human GUCY2D LCA1 (31). In contrast, to suppress expression of a dominant disease-causing GUCY2D allele in CORD6, more complex approaches would likely be required, such as CRISPR-mediated destruction of the CORD6 allele or suppression of the CORD6-specific transcripts by short interference RNA.…”

“…RetGC also binds retinal degeneration 3 (RD3) protein (20 -21), which prevents cyclase activation by GCAPs (22)(23) and promotes accumulation of RetGC1 in the outer segments (21,24,25). RetGC1 (human gene GUCY2D) accounts for most of the cGMP synthesis in mammalian photoreceptors (26,27), therefore mutations in RetGC1 that disable the cyclase activity (28 -33) cause recessive blindness at birth, Leber congenital amaurosis type 1 (LCA1) (33), mostly a non-degenerative or partially degenerative (31,32) loss-of-function hereditary retinal disease. Different from the LCA1 type of severe congenital blindness linked to the RetGC1 gene, cone-rod dystrophy type 6 (CORD6), is a progressive early-onset loss of photoreceptors (34 -35).…”