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Color fundus photographs of both eyes of patients carrying the mutation p.L84F in GUCA1A. (a), (b) Patient I:1 (75 y). Fundus photographs showing an evident disc pallor and arteriolar attenuation, area of macular atrophy with pigment migration, choriocapillaris atrophy surrounded this area, and the optic disc bilaterally. (c), (d) Patient II:1 (53 y). Fundus photographs showing bilateral temporal disc pallor, mild arteriolar attenuation, and bull’s eye pattern of maculopathy. (e), (f) Patient II:3 (47 y). Funduscopy showing mild temporal disc pallor, normal vessels, and perifoveal retinal pigment epithelium (RPE) alterations bilaterally. (g), (h) and (i), (j) Patients III:1 (25 y) and III:4 (12 y), respectively, presenting normal fundi.
Source publication
Here, we report two novel GUCA1A (the gene for guanylate cyclase activating protein 1) mutations identified in unrelated Spanish families affected by autosomal dominant retinal degeneration (adRD) with cone and rod involvement. All patients from a three-generation adRD pedigree underwent detailed ophthalmic evaluation. Total genome scan using singl...
Citations
... GCAP1 has been associated with autosomal dominant cone (COD) or cone-rod dystrophies (CORD) [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], a class of severe inherited retinal dystrophies (IRD) characterized by central vision loss, impaired color vision, and photophobia, due to photoreceptor degeneration [32]. Indeed, more than twenty point-mutations in the gene encoding for GCAP1 (GUCA1A) have been found to be linked to COD or CORD. ...
Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca²⁺. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00018-023-05022-0.
... The inheritance patterns of cone and cone-rod dystrophies include autosomal dominant (most common, 32%), autosomal recessive, and X-linked recessive inheritance [91,92]. Multiple genes have been identified, including AIPL1, CRX, GUCA1A, GUCY2D, PITPNM3, PROM1, PRPH2/RDS, RIMS1, SEMA4A, and UNC119 [93]. The autosomal dominant mutation of guanylate cyclase activator 1A (GUCA1A), which alters the structure and function of guanylyl cyclase-activating protein 1, is associated with both cone and cone-rod dystrophies [94], usually presenting between the third and fifth decade of life with mild photophobia and reduced central vision [17,95]. ...
... ffERG of pure cone dystrophies or early CORD shows normal rod responses with abnormal cone signals [97,98]. Cone dystrophy shows normal, preserved rod functions and severe cone dysfunction in ffERG [93]. ffERG can be particularly helpful in early stages when patients are asymptomatic with a normal fundus exam [90]. ...
Inherited retinal dystrophies encompass a diverse group of disorders affecting the structure and function of the retina, leading to progressive visual impairment and, in severe cases, blindness. Electrophysiology testing has emerged as a valuable tool in assessing and diagnosing those conditions, offering insights into the function of different parts of the visual pathway from retina to visual cortex and aiding in disease classification. This review provides an overview of the application of electrophysiology testing in the non-macular inherited retinal dystrophies focusing on both common and rare variants, including retinitis pigmentosa, progressive cone and cone-rod dystrophy, bradyopsia, Bietti crystalline dystrophy, late-onset retinal degeneration, and fundus albipunctatus. The different applications and limitations of electrophysiology techniques, including multifocal electroretinogram (mfERG), full-field ERG (ffERG), electrooculogram (EOG), pattern electroretinogram (PERG), and visual evoked potential (VEP), in the diagnosis and management of these distinctive phenotypes are discussed. The potential for electrophysiology testing to allow for further understanding of these diseases and the possibility of using these tests for early detection, prognosis prediction, and therapeutic monitoring in the future is reviewed.
... GCAP1 has been associated with autosomal dominant cone (COD) or cone-rod dystrophies (CORD) [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] , a class of severe inherited retinal diseases characterized by central vision loss, impaired color vision, and photophobia, resulting in photoreceptor degeneration 32 . Indeed, more than twenty pointmutations in the gene encoding GCAP1 (GUCA1A) have been found to be linked to COD or CORD. ...
Retinal dystrophies of genetic origin are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca ²⁺ . Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein (GCAP1) were compared in biological systems of increasing complexity ( in cyto, ex vivo , and in vivo). Analysis of protein biodistribution and direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases.
... To date, both GCAP1 and GCAP2 have been associated with retinal degeneration. Indeed, more than twenty mutations in the gene GUCA1A encoding for GCAP1 have been associated with autosomal dominant cones or cone-rod dystrophies (CORD) [19][20][21][22][23][24][25][26][27][28][29][30][31][32], while a relatively rare missense mutation found in GUCA1B, encoding for GCAP2, has been associated with retinitis pigmentosa (RP), in some cases with macular involvement [33,34]. Very recently, a study reported an association between GCAP3 and autosomal recessive primary congenital glaucoma [35], although the pathogenicity of the detected variant has been questioned [36]. ...
The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.301G>C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca2+-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 ms molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca2+ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.
... The gene coding for GCAP1, named GUCA1A, has been associated with autosomal dominant cone dystrophies (adCOD) [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25], a class of severe retinal degeneration diseases characterized by central vision loss, impaired color vision, and photophobia [26]. More than twenty point-mutations in GUCA1A have been found to be pathogenic and the highly heterogeneous phenotype seems to be strictly related to the specific amino acid substitution; indeed, different side chains at the same position can lead to dramatically different biochemical properties at the protein level [27]. ...
... A clear association between GUCA1A and adCOD was established over 20 years ago [15] and in the last years the number of point mutations found in the same gene has significantly raised [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Mutations are biochemically heterogeneous; therefore, a detailed molecular analysis is needed to infer general genotype-phenotype relations. ...
Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca2+, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca2+ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg2+ alone and Mg2+ and Ca2+. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC50 value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca2+. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca2+ conditions, as suggested by 2 μs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca2+ levels, which would ultimately lead to toxic accumulation of cGMP and Ca2+ in the photoreceptor outer segment, thus triggering cell death.
... 1,2 Cone dystrophy is a subgroup that reveals preserved rod functions and severe cone dysfunction in fullfield electroretinography (ffERG). 3 Histopathological studies have shown the shortening of photoreceptor outer segments and abnormal photoreceptor connections in these diseases. 4 Although their main pathological site is considered as photoreceptor outer segments, changes in choroidal structure and hemodynamics may also play a role in the pathogenesis of this group. ...
Purpose
This study aimed to evaluate macular perfusion using optical coherence tomography angiography (OCTA) in patients with cone dystrophy and to determine the associations between the quantitative data of OCTA and functional parameters.
Methods
The data of 36 eyes of 18 patients with cone dystrophy and 38 eyes of 19 healthy controls were analyzed. The superficial and deep capillary plexus (SCP and DCP) vessel densities (VD) of the macula, the foveal avascular zone (FAZ) and choriocapillary flow density values were obtained using OCTA. The associations between visual acuity (VA) and full-field electroretinography (ffERG) and the quantitative data of OCTA, and the associations between OCTA and ffERG were analyzed.
Results
: VD was significantly lower in all areas except the foveal area in SCP in the cone dystrophy group compared to the control group. VA was found to be associated with the VDs of the SCP and DCP except for that of the foveal SCP. VA was also associated with dark-adapted, light-adapted wave amplitudes.
Conclusion
OCTA quantitatively showed that macular perfusion was decreased in cone dystrophy compared to the healthy controls. In addition, there was an association between VA and ffERG parameters and quantitative data of OCTA.
... To date, more than twenty point mutations identified in GUCA1A, the gene coding for GCAP1, have been associated with various forms of autosomal dominant cone dystrophies (adCOD) [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24], a class of severe retinal degeneration diseases characterized by central vision loss, impaired color vision, and photophobia [25]. A thorough molecular investigation of the altered biochemical mechanisms seems to be a necessary step for deeply understanding the phenotype associated with each individual variant. ...
... Such constitutive activation of GC1 in the outer segment, already observed for other COD-associated variants [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24], may result in the dysregulation of the second messenger homeostasis, ultimately leading to the toxic and death-inducing accumulation of both cGMP and Ca 2+ [63]. However, the peculiar electrical response observed in this patient carrying a novel variant of GCAP1 suggests the transmission to downstream neurons to be compromised, a phenomenon that creates a virtual link from the GC1-GCAP1 transduction complex in the outer segment to that in the synaptic terminal. ...
Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+/cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1–cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers’ accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified.
... Several reports have described patients with COD/CORD showing a coloboma-like macular atrophy caused by pathogenic variants in several genes, such as NMNAT1, 51,52 ADAM9, 53 GUCA1A, 54 and GUCY2D. 55 In the present study, an intrachoroidal cavitation resembling coloboma-like macular atrophy was presented in three subjects bilaterally or unilaterally. ...
Purpose:
To determine the clinical and genetic characteristics of patients with GUCY2D-associated retinal disorder (GUCY2D-RD).
Methods:
Fifteen patients from 12 families with inherited retinal disorder (IRD) and harboring GUCY2D variants were ascertained from 730 Japanese families with IRD. Comprehensive ophthalmological examinations, including visual acuity (VA) measurement, retinal imaging, and electrophysiological assessment were performed to classify patients into three phenotype subgroups; macular dystrophy (MD), cone-rod dystrophy (CORD), and Leber congenital amaurosis (LCA). In silico analysis was performed for the detected variants, and the molecularly confirmed inheritance pattern was determined (autosomal dominant/recessive [AD/AR]).
Results:
The median age of onset/examination was 22.0/38.0 years (ranges, 0-55 and 1-73) with a median VA of 0.80/0.70 LogMAR units (ranges, 0.00-1.52 and 0.10-1.52) in the right/left eye, respectively. Macular atrophy was identified in seven patients (46.7%), and two had diffuse fundus disturbance (13.3%), and six had an essentially normal fundus (40.0%). There were 11 patients with generalized cone-rod dysfunction (78.6%), two with entire functional loss (14.3%), and one with confined macular dysfunction (7.1%). There were nine families with ADCORD, one with ARCORD, one with ADMD, and one with ARLCA. Ten GUCY2D variants were identified, including four novel variants (p.Val56GlyfsTer262, p.Met246Ile, p.Arg761Trp, p.Glu874Lys).
Conclusions:
This large cohort study delineates the disease spectrum of GUCY2D-RD. Diverse clinical presentations with various severities of ADCORD and the early-onset severe phenotype of ARLCA are illustrated. A relatively lower prevalence of GUCY2D-RD for ADCORD and ARLCA in the Japanese population was revealed.
Translational relevance:
The obtained data help to monitor and counsel patients, especially in East Asia, as well as to design future therapeutic approaches.
... Mutations in GUCA1A, encoding for GCAP1 result in aberrant GC regulation and in retinal degeneration, a set of progressive diseases involving cones, the macula, and in some cases rods, ultimately leading to blindness [11][12][13]. To date, 20 GCAP1 variants have been associated with retinal dystrophy [14][15][16][17][18][19][20][21][22][23][24][25], with one of the most recently discovered being the G86R mutation, located in the previously mentioned hinge region [26]. Biochemical experiments performed with the GC-GCAP1 reconstituted system in the presence of the G86R mutation showed that the complex forms with higher affinity than the WT and it is unable to decelerate the GC at high Ca 2+ -levels corresponding to the dark-adapted state [26]. ...
Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca2+-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca2+] and as an activator at low [Ca2+]. Recently, a novel missense mutation (G86R) was found in GUCA1A, the gene encoding for GCAP1, in patients diagnosed with cone-rod dystrophy. The G86R substitution was found to affect the flexibility of the hinge region connecting the N- and C-domains of GCAP1, resulting in decreased Ca2+-sensitivity and abnormally enhanced affinity for GC. Based on a structural model of GCAP1, here, we tested the hypothesis of a cation-π interaction between the positively charged R86 and the aromatic W94 as the main mechanism underlying the impaired activator-to-inhibitor conformational change. W94 was mutated to F or L, thus, resulting in the double mutants G86R+W94L/F. The double mutants showed minor structural and stability changes with respect to the single G86R mutant, as well as lower affinity for both Mg2+ and Ca2+, moreover, substitutions of W94 abolished “phase II” in Ca2+-titrations followed by intrinsic fluorescence. Interestingly, the presence of an aromatic residue in position 94 significantly increased the aggregation propensity of Ca2+-loaded GCAP1 variants. Finally, atomistic simulations of all GCAP1 variants in the presence of Ca2+ supported the presence of two cation-π interactions involving R86, which was found to act as a bridge between W94 and W21, thus, locking the hinge region in an activator-like conformation and resulting in the constitutive activation of the target under physiological conditions.
... To date, 19 GUCA1A missense and 3 in-frame deletion/insertion variants in heterozygous states have been reported as causes of AD-MD and AD-COD/CORD in HGMD Professional (2019.3) [14][15][16][17][18]20,[23][24][25][26][27][28][29][30][31][32][33] . Most reported variants (18/22, 81.8%) were concentrated within or around EF-hand domains 3 and 4, which are essential for cytosolic Ca 2+ /Mg 2+ binding 4-6 . ...
... In contrast, the other five variants (p.E17VfsX22, p.F42I, p.D68E, p.L80I, and p.Q184R) identified in the present study, which resulted in "uncertain significance" in the ACMG criteria, did not co-segregate with the disease. Previously, only five missense variants (p.L50I, p.L84F, p.G86R, p.E89K, and p.L176F) located outside EF-hand domains 3 and 4 have been reported as "pathogenic", 14,15,18,23,33 and one variant (p.L50I) has been concluded as "non-pathogenic" by subsequent studies 25,35 . Experimental studies have clarified that most GUCA1A variants within or around EF-hand domains 3 and 4 lead to the constitutive activation of RetGC by mechanisms of either a dominant-negative effect or gain of function, not haploinsufficiency 9,19,23,27,30,31,36 . ...
... All reported GUCA1A-associated phenotypes are classified into AD-MD or AD-COD/CORD with progressive macular atrophy 14,15,19,23,25,32 . In our study, the retinal atrophic areas were apparently more enlarged in older patients. ...
GUCA1A gene variants are associated with autosomal dominant (AD) cone dystrophy (COD) and cone-rod dystrophy (CORD). GUCA1A-associated AD-COD/CORD has never been reported in the Japanese population. The purpose of this study was to investigate clinical and genetic features of GUCA1A-associated AD-COD/CORD from a large Japanese cohort. We identified 8 variants [c.C50_80del (p.E17VfsX22), c.T124A (p.F42I), c.C204G (p.D68E), c.C238A (p.L80I), c.T295A (p.Y99N), c.A296C (p.Y99S), c.C451T (p.L151F), and c.A551G (p.Q184R)] in 14 families from our whole exome sequencing database composed of 1385 patients with inherited retinal diseases (IRDs) from 1192 families. Three variants (p.Y99N, p.Y99S, and p.L151F), which are located on/around EF-hand domains 3 and 4, were confirmed as “pathogenic”, whereas the other five variants, which did not co-segregate with IRDs, were considered “non-pathogenic”. Ophthalmic findings of 9 patients from 3 families with the pathogenic variants showed central visual impairment from early to middle-age onset and progressive macular atrophy. Electroretinography revealed severely decreased or non-recordable cone responses, whereas rod responses were highly variable, ranging from nearly normal to non-recordable. Our results indicate that the three pathogenic variants, two of which were novel, underlie AD-COD/CORD with progressive retinal atrophy, and the prevalence (0.25%, 3/1192 families) of GUCA1A-associated IRDs may be low among Japanese patients.
