Functional Characterization Of A Novel GUCA1A Missense Mutation(D144G) In Autosomal Dominant Cone Dystrophy:A Novel Pathogenic GUCA1A Variant In COD

Objective Cone dystrophy?COD?is a type of hereditary retinal disease with clinical and genetic heterogeneity.This study was elucidated the clinical phenotypes and pathogenesis of a novel missense mutation in guanylate cyclase activator A1A?GUCA1A?associated with autosomal dominant cone dystrophy.Methods Four generations of a family with adCOD were recruited in the Qingdao,China.There were 16 members in the family?7 affected,9 unaffected;3 males affected,4 females affected?.The members of a family with ad COD were clinically evaluated,and peripheral blood samples were collected to extract genomic DNA.Of 523 inheritable genetic vision system-related genes were captured before being sequenced by targeted next-generation sequencing,and the results were confirmed by Sanger sequencing.Sequence analysis was made of the conservativeness of mutant residues.The SWISS-MODEL was used to model the homology structure of the mutant GCAP1.Construction of prokaryotic and eukaryotic expression vectors of wild-type?WT?and mutant GUCA1A gene and retinal guanylate cyclase 1?retGC1?eukaryotic expression vector,and purification of WT and mutant GST-GCAP1 fusion proteins.Then limited protein hydrolysis,an electrophoresis shift and protein stability experiments were used to assess possible changes in the structure.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?co-IP?and GST-pull down were 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?-293cells.Results A pathogenic mutation in GUCA1A?c.431A>G,p.D144G,exon 5?was revealed in four generations of a family with adCOD,and the variant was co-segregated with the phenotype of this family.This mutation was not found in the 200 control individuals.GUCA1A encodes guanylate cyclase activating protein 1?GCAP1?.Multiple alignments of the GCAP1protein of different orthologous species indicated that D144 was located in the EF-4 loop involving calcium binding and was highly conserved in the species.The PolyPhen-2,SIFT,Provean,and Mutationtaster predicted that the p.D144G missense mutation could induce a"harmful change"in GUCA1A function?Table 3?.The homology structure of GCAP1-D144G showed that the hydrogen bonds between residue 144 and residue Phe140?Phenylalanine,Phe?or Gly147?Glycine,Gly?in GCAP1 were eliminated due to substitution from Aspartyl?Asp?to Glycine?Gly?.GCAP1-D144G was more susceptible to hydrolysis,and the mobility of the D144G band became slower in the presence of Ca²⁺.At high Ca²⁺concentrations,GCAP1-D144G stimulated retGC1 in the HEK-293 membrane to significantly increase intracellular cGMP protein concentrations.Compared with GCAP1-WT,GCAP1-144G had an increased interaction with retGC1,as detected in the co-IP assay and GST-pull down.GCAP1-D144G displayed colocalization with retGC1 in the HEK-293 cells.Conclusion This study illustrates a novel missense mutation in GUCA1A?c.431A>G,p.D144G,exon 5?in four generations of a family with adCOD.With functional prediction and analysis,we identified the pathogenic effect of GCAP1-D144G,which can lead to increased retGC1 activity and result in persistently high levels of cGMP.This may also represent a possible mechanism for the formation of adCOD.