Article

A novel GCAP1(N104K) mutation in EF-hand 3 (EF3) linked to autosomal dominant cone dystrophy.

Department of Biology, University of Utah, Salt Lake City, UT 84132, USA.
Vision research (impact factor: 2.29). 09/2008; 48(23-24):2425-32. DOI:10.1016/j.visres.2008.07.016 pp.2425-32
Source: PubMed

ABSTRACT The GUCA1A gene encodes a guanylate cyclase activating protein (GCAP1) that is involved in regulation of phototransduction in the vertebrate retina. We discovered a novel C312A transversion in exon 2 of the human GUCA1A gene, replacing Asn-104 (N104) in GCAP1 with Lys (K), in two affected members of a family with dominant cone dystrophy. The mutation N104K is located in the third EF-hand motif (EF3) shown previously to be instrumental in converting Ca2+-free GCAP1 to a GC inhibitor in the Ca2+-bound form. In one patient, rod ERGs were fairly stable over a 12-year-period whereas 30 Hz flicker ERG and single-flash cone ERGs declined. In both patients, double-flash ERGs showed that rod recovery from an intense test flash was significantly delayed. The EC(50) for GC stimulation shifted from approximately 250 nM in wild-type GCAP1 to approximately 800 nM in the GCAP1(N104K) mutant suggesting inability of the mutant to assume an inactive form under physiological conditions. The replacement of N104 by K in GCAP1 is the first naturally occurring mutation identified in the EF3 loop. The rod recovery delays observed in double-flash ERG of affected patients suggest a novel dominant-negative effect that slows GC stimulation.

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    Article: RNAi-Mediated Gene Suppression in a GCAP1(L151F) Cone-Rod Dystrophy Mouse Model.
    Li Jiang, Tansy Z Li, Shannon E Boye, William W Hauswirth, Jeanne M Frederick, Wolfgang Baehr
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    ABSTRACT: Dominant mutations occurring in the high-affinity Ca-binding sites (EF-hands) of the gene encoding guanylate cyclase-activating protein 1 (GCAP1) cause slowly progressing cone-rod dystrophy (CORD) in a dozen families worldwide. We developed a nonallele-specific adeno-associated virus (AAV)-based RNAi knockdown strategy to rescue the retina degeneration caused by GCAP1 mutations. We generated three genomic transgenic mouse lines expressing wildtype (WT) and L151F mutant mouse GCAP1 with or without a C-terminal GFP fusion. Under control of endogenous regulatory elements, the transgenes were expressed specifically in mouse photoreceptors. GCAP1(L151F) and GCAP1(L151F)-GFP transgenic mice presented with a late onset and slowly progressive photoreceptor degeneration, similar to that observed in human GCAP1-CORD patients. Transgenic expression of WT GCAP1-EGFP in photoreceptors had no adverse effect. Toward therapy development, a highly effective anti-mGCAP1 shRNA, mG1hp4, was selected from four candidate shRNAs using an screening assay. Subsequently a self-complementary (sc) AAV serotype 2/8 expressing mG1hp4 was delivered subretinally to GCAP1(L151F)-GFP transgenic mice. Knockdown of the GCAP1(L151F)-GFP transgene product was visualized by fluorescence live imaging in the scAAV2/8-mG1hp4-treated retinas. Concomitant with the mutant GCAP1-GFP fusion protein, endogenous GCAP1 decreased as well in treated retinas. We propose nonallele-specific RNAi knockdown of GCAP1 as a general therapeutic strategy to rescue any GCAP1-based dominant cone-rod dystrophy in human patients.
    PLoS ONE 01/2013; 8(3):e57676. · 4.09 Impact Factor
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Keywords

30 Hz flicker ERG
 
Ca2+-bound form
 
Ca2+-free GCAP1
 
dominant cone dystrophy
 
double-flash ERG
 
EF3 loop
 
exon 2
 
guanylate cyclase activating protein
 
GUCA1A gene encodes
 
human GUCA1A gene
 
intense test
 
mutation N104K
 
novel C312A transversion
 
novel dominant-negative effect
 
rod ERGs
 
rod recovery
 
rod recovery delays
 
single-flash cone ERGs
 
vertebrate retina
 
wild-type GCAP1