Functional characterization of a novel GUCA1A missense mutation (D144G) in autosomal dominant cone dystrophy: A novel pathogenic GUCA1A variant in COD

PURPOSE: To elucidate the clinical phenotypes and pathogenesis of a novel missense mutation in guanylate cyclase activator A1A (GUCA1A) associated with autosomal dominant cone dystrophy (adCOD). METHODS: The members of a family with adCOD were clinically evaluated. Relevant genes were captured before being sequenced with targeted next-generation sequencing and confirmed with Sanger sequencing. Sequence analysis was made of the conservativeness of mutant residues. An enzyme-linked immunosorbent assay (ELISA) was implemented to detect the cyclic guanosine monophosphate (cGMP) concentration. Then limited protein hydrolysis and an electrophoresis shift were used to assess possible changes in the structure. Coimmunoprecipitation was employed to analyze the interaction between GCAP1 and retGC1. Immunofluorescence staining was performed to observe the colocalization of GCAP1 and retGC1 in human embryonic kidney (HEK)-293 cells. RESULTS: A pathogenic mutation in GUCA1A (c.431A>G, p.D144G, exon 5) was revealed in four generations of a family with adCOD. GUCA1A encodes guanylate cyclase activating protein 1 (GCAP1). D144, located in the EF4 loop involving calcium binding, was highly conserved in the species. GCAP1-D144G was more susceptible to hydrolysis, and the mobility of the D144G band became slower in the presence of Ca(2+). At high Ca(2+) concentrations, GCAP1-D144G stimulated retGC1 in the HEK-293 membrane to significantly increase intracellular cGMP protein concentrations. Compared with wild-type (WT) GCAP1, GCAP1-D144G had an increased interaction with retGC1, as detected in the coimmunoprecipitation assay. CONCLUSIONS: The newly discovered missense mutation in GUCA1A (p.D144G) might lead to an imbalance of Ca(2+) and cGMP homeostasis and eventually, cause a significant variation in adCOD.