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Accepted Manuscript
Genotype-functional-phenotype correlations in photoreceptor guanylate cyclase (GCE) encoded by GUCY2D
Dror Sharon, Hanna Wimberg, Yael Kinarty, Karl-Wilhelm Koch
PII:
S1350-9462(17)30082-4
DOI:
10.1016/j.preteyeres.2017.10.003
Reference:
JPRR 691
To appear in:
Progress in Retinal and Eye Research
Received Date: 21 August 2017
Revised Date:
16 October 2017
Accepted Date: 16 October 2017
Please cite this article as: Sharon, D., Wimberg, H., Kinarty, Y., Koch, K.-W., Genotype-functionalphenotype correlations in photoreceptor guanylate cyclase (GC-E) encoded by GUCY2D, Progress in
Retinal and Eye Research (2017), doi: 10.1016/j.preteyeres.2017.10.003.
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ACCEPTED MANUSCRIPT
Genotype-Functional-Phenotype Correlations in
Photoreceptor Guanylate Cyclase (GC-E) encoded by GUCY2D
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Dror Sharon a,b, Hanna Wimberg c, Yael Kinarty a,d, Karl-Wilhelm Koch c
Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem,
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91120, Israel
Hanse-Wissenschaftskolleg, Lehmkuhlenbusch 4, 27753 Delmenhorst, Germany
c
Department of Neuroscience, University of Oldenburg, D-26111 Oldenburg, Germany
d
Department of Medical Neurobiology, Institute for Medical Research Israel-Canada, the
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b
Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel
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Corresponding authors:
Dror Sharon, Ph.D., Department of Ophthalmology, Hadassah-Hebrew University Medical
Center, Jerusalem, 91120, Israel; E-mail: drorsh@ekmd.huji.ac.il
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Karl-Wilhelm Koch, Ph.D., Department of Neuroscience, University of Oldenburg, D-26111
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Oldenburg, Germany; E-mail: karl.w.koch@uni-oldenburg.de
ACKNOWLEDGEMENTS
We would like to thank the Hanse-Wissenschaftskolleg (Delmenhorst, Germany) for the
generous scholarship to DS.
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ABSTRACT
The GUCY2D gene encodes for the photoreceptor guanylate cyclase GC-E that synthesizes
the intracellular messenger of photoreceptor excitation cGMP and is regulated by intracellular
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Ca2+-sensor proteins named guanylate cyclase-activating proteins (GCAPs). Over 140
disease-causing mutations have been described so far in GUCY2D, 88% of which cause
autosomal recessive Leber congenital amaurosis (LCA) while heterozygous missense
mutations cause autosomal dominant cone-rod degeneration (adCRD). Mutations in GUCY2D
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are one of the major causes of all LCA cases and are the major cause of adCRD. A single
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amino acid, arginine at position 838, is likely to be the most sensitive one in GC-E as four
single mutations and two complex mutations were reported to affect R838.
The biochemical effect of 45 GC-E variants was studied showing a clear genotypephenotype correlation: LCA-causing mutations either show reduced ability or complete
inability to synthesize cGMP from GTP, while CRD-causing mutations are functional, but
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shift the Ca2+-sensitivity of the GC-E – GCAP complex.
Eight animal models of retinal guanylate cyclase deficiency have been reported
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including knockout (KO) mouse and chicken models. These two models were used for gene
augmentation therapy that yielded promising results.
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Here we integrate the available information on the genetics, biochemistry and
phenotype that is related to GUCY2D mutations. These data clearly show that mutation type
(missense versus null) and localization (dimerization domain versus other protein domains)
are correlated with the pattern of inheritance, impact on enzymatic function and retinal
phenotype. Such clear correlation is unique to GUCY2D while mutations in many other retinal
disease genes show variable phenotypes and lack of available biochemical assays.
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Keywords: cyclic GMP, Genotype-phenotype correlation, Guanylate cyclase, GUCY2D,
Mutation, Photoreceptor, Retinal diseases
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List of abbreviations:
AAV, adeno- associated virus; ad, autosomal dominant; ar, autosomal recessive; BSP, bone
spicule-like pigmentation; CCD, cyclase catalytic domain; CD, cone dystrophy; cGMP, cyclic
guanosine monophosphate; CNG, cyclic nucleotide-gated; CRD, cone-rod dystrophy; CSNB,
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congenital stationary night blindness; DD, dimerization domain; ECD, extracellular domain;
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ERG, electroretinography; GCAP, guanylate cyclase-activating proteins; GTP, guanosine 5’triphosphate; IRD, inherited retinal disease; IS, inner segment; IUPHAR , International Union
of Basic and clinical Pharmacology; JMD, juxtamembrane domain; KHD, kinase homology
domain; KO, knock-out; LCA, Leber congenital amaurosis; LS, leader sequence; NGS, next
generation sequencing; OS, outer segment; PDE, phosphodiesterases; RP, retinitis
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pigmentosa; RPE, retinal pigment epithelium; TM, transmembrane domain;
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1.
Introduction
2.
Guanylate cyclases in humans
2.1
Guanylate cyclase function
2.2
Genes encoding GCs
2.3
Regulation and function of GC-E
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Contents
2.3.1 Guanylate cyclase-activating proteins (GCAPs)
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2.3.2 Activation by the S100B protein
2.3.3 GC-E trafficking in photoreceptor cells involves the RD3 protein
2.5
Evolution of GC-E
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Functional domains of GC-E
Genetics, Biochemistry and clinical aspects of GUCY2D / GC-E
3.1
Disease-causing mutations in GUCY2D
3.2
Biochemical analysis of missense mutations
3.3
Clinical assessment of patients with GUCY2D mutations
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3.
2.4
3.3.1 Clinical assessment of biallelic LCA1 patients
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3.3.2 Clinical assessment of parents of LCA1 patients
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3.3.3 Phenotypic data of adCRD patients with heterozygous mutations in
GUCY2D
3.4
4.
Genotype-phenotype correlation
Animal models with mutations in retinal guanylate cyclase
4.1
Animal models for LCA1
4.1.1 Mouse models
4.1.2 Chicken models
4.1.3 Zebrafish models
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4.2
Animal models for adCRD
4.2.1 A transgenic mouse model
4.2.2 Pig models
Potential therapies for GUCY2D-associated phenotypes
6.
Future perspectives
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4.2.3 A zebrafish model
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1.
Introduction
Inherited retinal diseases (IRDs) are usually characterized by heterogeneity both at the
clinical and genetic levels (Hartong et al., 2006; Rivolta et al., 2002). The most common IRD
phenotype is retinitis pigmentosa (RP, MIM #268000) that is characterized by a rod > cone
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pattern of dysfunction often leading to marked visual loss (Berson, 1993). Leber congenital
amaurosis (LCA- MIM #204000) is a congenital retinal dysfunction characterized by
blindness or severe visual impairment at birth or within the first month of life, congenital
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nystagmus and extinguished or severely impaired electroretinography (ERG) amplitudes.
Cone-rod dystrophy (CRD) on the other hand is characterized by a cone > rod pattern, often
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exhibiting macular involvement and accompanied by loss of visual acuity (Hamel, 2007;
Roosing et al., 2014). RP is estimated to affect approximately 1 in about 3500 individuals
(Bundey and Crews, 1984; Bunker et al., 1984; Haim, 2002; Rosenberg, 2003; Sharon and
Banin, 2015), while the prevalence of LCA and CRD is much lower [estimated as 1 in 40,000
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(Hamel, 2007; Koenekoop, 2004)].
The unexpected discovery of a missense mutation in a rod-specific gene, rhodopsin, as
the cause of autosomal dominant RP (adRP ) (Dryja et al., 1990), in which cones degenerate
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as well, opened the gate for the discovery of complicated and unexpected disease mechanisms
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in IRDs. Different mutations in rhodopsin, for example, were later on reported to cause RP in
an autosomal recessive (AR) pattern of inheritance (Rosenfeld et al., 1992), or even a nondegenerative disease, congenital stationary night blindness (CSNB) (Dryja et al., 1993). Since
IRDs are considered as one of the most heterogeneous groups of diseases in humans, rare and
highly interesting inheritance patterns were identified, such as biallelic and triallelic digenic
inheritance patterns (Kajiwara et al., 1994; Katsanis et al., 2001). The current number of
genes in which mutations can cause IRDs (including syndromic forms) is estimated at 300
(RETNET website at https://sph.uth.edu/retnet/) and is growing rapidly, mainly due to recent
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use of next generation sequencing (NGS) techniques in which large sets of genes are being
analyzed simultaneously. A recent review contains a comprehensive description of the genetic
techniques used to identify and analyze IRD genes (Broadgate et al., 2017).
As more genes were identified to cause IRDs, a relatively large proportion were found
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to either cause multiple phenotypes or multiple inheritance patterns. Out of the 112 autosomal
genes that are listed in RETNET (as of July 22, 2016) that are known to cause non-syndromic
IRDs (RP, LCA, and CRD), 16% were reported to cause disease in both dominant and
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recessive inheritance patterns and 46% were reported to cause multiple phenotypes.
Interestingly, 16 of the genes (14.3%) cause multiple diseases in multiple inheritance patterns,
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usually with no clear genotype-phenotype correlation and no available functional assay to
predict the mutation effect on protein function. The GUCY2D gene, encoding for retinal
guanylate cyclase, is a unique member of this group. Different GUCY2D mutations cause
either arLCA1 or adCRD and characterization of patients with GUCY2D mutations has been
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reported. In addition, functional expression and biochemical analyses of representative
mutations from each group allows us to establish genotype-phenotype correlation in vitro and
in mouse models. In this review, we will describe and summarize the large number of
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reported GUCY2D mutations, the functional analysis of some of these mutations, the
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established genotype-phenotype correlation, and the development of animal models and gene
augmentation therapy for GUCY2D. A few previous excellent reviews on the photoreceptor
guanylate cyclase were recently published (Boye, 2016; Koch and Dell'Orco, 2013; Sharma
and Duda, 2014; Wen et al., 2014) and the current one aims to integrate recent genetic,
functional and clinical studies for a broader understanding of mutation effect on protein
function and disease type.
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2.
Guanylate cyclases in humans
2.1 Guanylate cyclase function
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Guanosine 3’, 5’ –cyclic monophosphate (cGMP) is an important second messenger
with many cellular functions like smooth muscle relaxation and blood pressure regulation,
intestinal fluid and electrolyte homeostasis, skeletal growth and sensory physiology, and
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phototransduction (Kuhn, 2016; Stryer, 1991). Downstream effectors of cGMP in signaling
pathways are mainly cyclic nucleotide-gated (CNG) ion channels, cGMP dependent protein
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kinases and cGMP-dependent phosphodiesterases (PDE) that hydrolyze cGMP. Intracellular
cGMP levels are controlled by the PDE enzyme which hydrolyze cGMP to 5’GMP upon
activation and guanylate cyclases (GCs- also known as guanylyl cyclases) which catalyze
cGMP synthesis (Beavo and Brunton, 2002). GCs exist in membrane-bound and soluble
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2.2 Genes encoding GCs
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forms, and are found in many species.
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The human genome contains five functional genes encoding membrane-bound forms
of GCs. These include NPR1 and NPR2 encoding the natriuretic peptide receptors GC-A and
GC-B, that act to decrease blood volume by stimulating natriuresis and diuresis in the kidney
(Potter et al., 2006) The third gene (GUCY2C) codes for the guanylin GC receptor GC-C that
regulates fluid and electrolyte balance in the intestine and the heat- stable enterotoxin peptides
secreted by pathogenic bacteria in the intestine (Nakazato, 2001). Two members of the
membrane-bound GCs (GUCY2D and GUCY2F) encode GC-E and GC-F, respectively and
are expressed in photoreceptor cells. Two other genes (GUCY1A2 and GUCY1B3) encode the
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alpha and beta subunits of the soluble GC (Garbers, 1990). In addition, three pseudogenesGUCY2EP, GUCY2GP, and GUCY1B2 are present in the human genome.
Numerous names have been used in the literature to describe the major retinal
guanylate cyclase, including GC-E, photoreceptor GC, ROS-GC1, RET-GC1, and GC1
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(Table 1). A recent report (Kuhn, 2016) recommended using GUCY2D as the gene name and
GC-E as the protein name [see also the International Union of Basic and clinical
Pharmacology (IUPHAR)] website at: http://www.guidetopharmacology.org] and we will use
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these terms throughout the manuscript. A list of the currently used nomenclature of retinal
photoreceptor guanylate cyclases and their corresponding genes is presented in Table 1.
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The expression of GC-E was studied in different species using immunolabeling and
found to be localized mainly in photoreceptor outer segments and to a lesser extent in the
plexiform layers (Cooper et al., 1986; Dizhoor et al., 1994; Liu et al., 2001). The protein was
much more concentrated in the outer segments of cones than rods (Cooper et al., 1986; Liu et
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al., 2001). Immunolabeling of the plexiform layers suggests that GC-E is not unique to the
photoreceptor outer segments, and may also play a role at retinal synapses (Cooper et al.,
1986; Duda et al., 2002).
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The second photoreceptor-specific GC encoded in the human genome by GUCY2F
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was cloned from human, rat and bovine cDNA libraries (Goraczniak et al., 1997; Lowe et al.,
1995; Yang et al., 1995). The protein was named ROS-GC2, retGC2 or GC-F, but in contrast
to GC-E had never been purified from native retinal sources. In situ hybridization and
immunogold labeling demonstrated its expression in photoreceptor cells (Lowe et al., 1995;
Yang and Garbers, 1997). Studies in bovine and mouse photoreceptor cells revealed a lower
expression level of GC-F in comparison to GC-E, but very similar Ca2+-sensitive activation
profiles mediated by GCAP1 and GCAP2 (Helten et al., 2007; Peshenko et al., 2011b). The
GUCY2F gene is located on the X- chromosome (Yang et al., 1996) and so far no human
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retinal disease has been linked to a mutation in this gene, but somatic mutation in the
GUCY2F gene had been correlated to human cancers (Wood et al., 2006).
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2.3 Regulation and function of GC-E
Photoreceptor GCs differ from other membrane-bound receptor GCs by the mode of
activation and regulation. They are not activated by extracellular ligands (as we know so far),
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but instead are regulated by intracellular Ca2+-sensor proteins named guanylate cyclaseactivating proteins (GCAPs- see section 2.3.1; Fig. 1A and Fig. 2). Photoreceptor GCs are key
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enzymes in photoreceptor physiology, since they synthesize the intracellular messenger of
photoreceptor excitation cGMP [for recent reviews on that topic see (Koch and Dell'Orco,
2015; Korenbrot, 2012; Palczewski, 2012)]. GC-E and GC-F double-knockout mice (see
section 4.1) do not show any photoresponse, their rods and cones degenerate and the
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intracellular transport of some phototransduction proteins is impaired (Baehr et al., 2007),
demonstrating the critical role of balanced cGMP synthesis. The cyclic nucleotide cGMP
keeps CNG channels in the plasma membrane open in the dark allowing ions, including Ca2+,
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to enter the outer segment (Fig. 1A). Ca2+ efflux by the Na+/ Ca2+,K+ exchanger removes Ca2+
from the cytosol thereby establishing a steady-state Ca2+ concentration below 1 µM. The
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intracellular Ca2+-concentration in mice and amphibian outer segments ranging from 250 to
500 nM (Gray-Keller and Detwiler, 1996; Nakatani et al., 2002; Woodruff et al., 2002), and
human photoreceptor outer segment will probably have a value within this range. Illumination
of rod and cone cells causes PDE activation mediated by the G protein transducin, a decrease
in cGMP concentration, closure of CNG channels and decrease in cytoplasmic Ca2+
concentration to below 100 nM. The reduced Ca2+ concentration activates several feedback
loops including a control mechanism by GCAP1 and GCAP2 (Fig. 1A and Fig. 2).
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2.3.1 Guanylate cyclase-activating proteins (GCAPs)
The human genome contains three genes (GUCA1A, GUCA1B, GUCA1C) encoding
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GCAP1, GCAP2, and GCAP3 respectively. GCAP1 was initially identified as a 21-kDa
protein from bovine rod outer segments (Gorczyca et al., 1994), its gene was cloned from
cDNA libraries (Palczewski et al., 1994) and immunohistochemistry showed its expression in
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both rods and cones (Frins et al., 1996; Gorczyca et al., 1995). Further, Dizhoor et al. (1994)
reported that a 24-kDa protein regulated recombinant human GC-E in a Ca2+-dependent
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manner (Dizhoor et al., 1994). Gorczyca and colleagues (Gorczyca et al., 1995) isolated and
cloned a Ca2+-sensor protein that had ca. 50% identity with GCAP1 and therefore named this
protein GCAP2. Cloning, sequencing and expression of the 24 kDa protein identified earlier
by Dizhoor et al. (1994) demonstrated its identity with GCAP2 (Dizhoor et al., 1995). The
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GCAP proteins contain four EF-hand Ca2+ -binding motifs (Dizhoor et al., 1995; Frins et al.,
1996; Gorczyca et al., 1995), but the first EF-hand is non-functional as a Ca2+ -binding site.
Instead, it probably acts as or is part of an interaction region for the target GC in GCAP1 and
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GCAP2 (Ermilov et al., 2001; Hwang et al., 2004). GCAPs are therefore able to sense the
reduction in Ca2+ concentration and undergo a conformational change in their Ca2+-free/
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Mg2+-bound state, which stimulates GC-E activity (Peshenko and Dizhoor, 2007). The
resulting faster rate of cGMP synthesis allows GC-E to regenerate cGMP (Fig. 1A and Fig.
2).
GCAP1 and GCAP2 make distinct contributions to regulation of GCs in rods (Mendez
et al., 2001) and differ in their activating and conformational properties leading to a concept
of a gradual step-by-step activation of the target GCs (Koch and Dell'Orco, 2013; Wen et al.,
2014). This Ca2+-relay mechanism of activation is best characterized by the difference in
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Ca2+-dependent regulation of photoreceptor GCs shown in Fig. 2. GCAP1 is active at higher
cytoplasmic Ca2+ than GCAP2 enabling a gradual response of the photoreceptor cell to
oscillating levels of Ca2+ (Hwang et al., 2003; Makino et al., 2012; Mendez et al., 2001;
Peshenko et al., 2011b). The differential response characteristics of GCAP activation by
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changing Ca2+-concentration (Fig. 2) is finally caused by their specific Ca2+sensitivities and
their different Ca2+-binding properties in combination with their target interaction. The latter
point becomes apparent, when CRD-causing mutants in GC-E exhibit a shift of the Ca2+-
GCAP1 or GCAP2 (see also section 3.2. below).
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dependent activation curve to higher free Ca2+-concentration (Fig. 2), when activated by wt
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GCAPs bind GC-E in the presence and absence of Ca2+. The drop in cytoplasmic Ca2+
after illumination triggers different conformational changes in GCAP1 and GCAP2
(Kollmann et al., 2012; Robin et al., 2015), leading to an activation of GC-E and a subsequent
increase of cGMP synthesis in the cyclase catalytic center. Target sites of GCAP1 and
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GCAP2 on GC-E are currently a matter of debate. Arguments and experimental results of this
issue are presented in section 2.4.
About 18 different point mutations have been reported in GUCA1A to cause
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adCD/CRD/maculopathy (Downes et al., 2001; Jiang et al., 2005; Jiang et al., 2008;
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Kitiratschky et al., 2009; Nishiguchi et al., 2004; Sokal et al., 2005; Sokal et al., 1998; Wilkie
et al., 2001). Patients suffering from a GUCA1A-related disease experience a decrease in
visual acuity and abnormal or impaired color vision. Fundus changes are located in the
macular region. Peripheral vision appears normal in cone dystrophy and maculopathy
patients, since it is mediated by rod photoreceptors. Progressive loss of rod and cone vision on
the other hand is observed in patients suffering from CRD. Functional in vitro studies
revealed some common properties of mutated GCAP1, the most prominent of which was an
abnormal Ca2+ sensitivity profile. GCAP1 mutants are constitutively active under
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physiological Ca2+-concentrations leading to excess cGMP production and an imbalance of
the Ca2+/cGMP homeostasis (Fig. 1C). A comparative discussion of these functional aspects
of GCAP1 mutants has been covered by reviews (Behnen et al., 2010; Hunt et al., 2010) and
was included more recently in a set of comprehensive biochemical and biophysical
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characterizations of newly described GUCA1A mutations (Marino et al., 2015; Vocke et al.,
2017).
GCAP3 is more closely related to GCAP1 than to GCAP2 and its function seems to be
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nearly identical to GCAP1. So far, there had been no thorough study on its biochemical
properties. It seems a mystery that, GCAP3 being similar to GCAP1, is expressed in cones
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and has similar properties, but no mutation has been found to correlate with a retinal disease.
However, the crystal structure of nonmyristoylated human GCAP3 has served as a homology
model of GCAP1 offering insights into retinal disease-causing mutations in GCAP1 (Stephen
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et al., 2006).
2.3.2 Activation by the S100B protein
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The ubiquitous S100B protein (also known as Ca2+-dependent GCAP or CD-GCAP in
short) binds to the C-terminus of GC-E (Duda et al., 1996b) and activates GC-E in cones at
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increasing Ca2+-concentrations, a process that is not involved in phototransduction in the
outer segment, but rather modulation of neural transmission to ON-bipolar cells (Wen et al.,
2012). S100B was found to be expressed in mouse photoreceptor outer segments (Wen et al.,
2012). Electroretinography (ERG) recordings in a mouse knockout model for S100B indicate
a possible modulation of the transmission of neural signals to cone ON-bipolar cells by the
Ca2+-dependent catalytic activity of GC-E (Duda et al., 2002; Wen et al., 2012).
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2.3.3 GC-E trafficking in photoreceptor cells involves the RD3 protein
Photoreceptor cells are composed of the outer segments (OS), the inner segments (IS),
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a cilium connecting OS and IS and a synaptic terminal. The OS contain the proteins of the
phototransduction process and OS membranes undergo a rapid turnover. The machinery for
protein production and posttranslational modification is located in the IS of these cells. Newly
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synthesized proteins have to undergo a long journey from the endoplasmic reticulum and find
their way through the cilium to reach their site of action in the OS. Since the OS are renewed
photoreceptors is imperative.
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continuously (Young, 1976), effective protein transport from the IS to the OS in the
In general, protein transport in photoreceptor cells is not well understood. So far, the
identification of targeting motifs from photoreceptor OS proteins was possible for just a few
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candidates like rhodopsin (Tam et al., 2000). Such a signaling sequence has not been found
for GC-E and therefore, a co-transport with other proteins is hypothesized (Karan et al.,
2011). In 2003, the RD3 gene was identified. It encodes a 23-kDa protein with unknown
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retinal function (Lavorgna et al., 2003). It was initially thought to be part of subnuclear
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protein complexes involved in transcription and splicing (Friedman et al. 2006). A nonsense
mutation in RD3 leading to protein truncation was reported to be the cause of LCA12
(Friedman et al., 2006). Mutations in rd3 were found in a naturally occurring mouse strain in
1969 (Chang et al., 2002; Chang et al., 1993) which became recognized as a model for retinal
degeneration. Photoreceptors of rd3 mice appear normal at birth, but can degenerate nearly
completely within 8 weeks and ERG amplitudes diminish within the same time scale.
However, depending on the genetic background degeneration can occur in less than 8 weeks
or in more than 5 months (Friedman et al., 2006; Peshenko et al., 2016).
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At present, two functions for RD3 in photoreceptors are described: inhibition of GC-E
catalytic activity and promoting GC-E transport to its target site in the outer segments (Fig.
3). RD3 inhibits the basal activity of GC-E as well as GCAP stimulated activity at low Ca2+
concentrations in a nanomolar range (Peshenko et al., 2011a). This mechanism may be needed
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to block GC-E activity and an uncontrolled cGMP production in the IS, which otherwise
could lead to an uncontrolled activation of cGMP targets (e.g. Protein kinase G, CNG-
channels) and a disturbance in Ca2+ homeostasis of the cell. Additional work demonstrated the
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need of RD3 for GC-E to exit the endoplasmic reticulum (ER) into vesicles in non-
photoreceptor cells leading to the hypothesis that RD3 is involved in the upward transport and
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incorporation of GC-E into the disc membranes (Azadi et al., 2010). These studies were
conducted in cultured COS-7 cells. Immunofluorescence experiments showed colocalization
of RD3 with GC-E and GC-F in rods and cones of normal mice and a lack of GC staining in
the rd3 mouse. Further GCAP1 and GCAP2 showed an reduced expression level and
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mislocalization in the RD3 lacking retina (Azadi et al., 2010).
Recently, a process for RD3/GC-E/GCAP1 interaction and the transport of this
complex to the photoreceptor OS was described. In addition, the binding efficiency of LCA1-
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causing GC-E mutants towards RD3 has been analyzed. Among the studied GC-E mutants, 10
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showed a significantly reduced binding (Zulliger et al., 2015), indicating that a disturbed GCE – RD3 interaction prevents the correct assembly of the RD3/GC-E/GCAP1 complex that is
necessary for the transport out of the ER to the OS. For GC-E trafficking a vesicular
mechanism is proposed. It was shown that the transport of the RD3/GC-E/GCAP1 complex
involves members of the RAB protein family including RAB5 and RAB11 (Zulliger et al.,
2015).
At present the process of GC-E trafficking is not completely understood, but different
research groups are focusing on this topic. Recent in vivo studies on GC-E trafficking in mice
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indicate that both functions of RD3 are equally important for photoreceptor survival. In a
mouse model, the GC-E p.R838S mutant still reaches the outer segments although the
apparent affinity of RD3 for the GC-E/GCAP1 complex was reduced (Dizhoor et al., 2016).
For another GC-E mutant p.S248W it was reported that RD3 can still bind and suppress GC-E
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catalytic activity in vitro but p.S248W expression in photoreceptors is very low (Boye et al.,
2016). Probably a less effective GC-E – RD3 interaction contributes to disease development
in case of guanylate cyclase mutations.
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In the next years new insights will be revealed, which may help to better understand
retinal diseases due to a disturbance in protein transport involving the RD3 protein.
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Interestingly, patients with LCA1 (due to GUCY2D mutations) and LCA12 (due to RD3
mutations) show a very similar phenotype whereas LCA12 is even more severe (Preising et
al., 2012), indicating a broader range of action and additional targets for RD3. A
comprehensive review on the molecular properties and the role of RD3 in GC-E trafficking
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was published elsewhere (Molday et al., 2014).
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2.4 Functional domains of GC-E
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Native GC-E was first purified from bovine, toad and frog rod outer segments as a
110-115 kDa membrane bound protein (Hayashi and Yamazaki, 1991; Koch, 1991). Twodimensional gel electrophoresis indicated up to five GC variants in the purified samples
(Hayashi and Yamazaki, 1991), but the molecular basis for this polymorphism was not
resolved. Subsequent isolation and characterization of cDNA clones from human and bovine
retina cDNA libraries led to a predicted amino acid sequence with a molecular mass of 110120 kDa and with homology to known membrane bound guanylate cyclases (Goraczniak et
al., 1994; Shyjan et al., 1992).
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The topology of GC-E has been further analyzed by sequence alignments with other
receptor GCs and experimental approaches (Bereta et al., 2010; Duda et al., 1996a;
Goraczniak et al., 1994; Lange et al., 1999; Laura et al., 1996; Ma et al., 2010; Peshenko et
al., 2015a, b). It contains seven defined domains: a leader sequence (LS), an extracellular
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domain (ECD), a transmembrane domain (TM), a juxtamembrane domain (JMD), a kinase
homology domain (KHD), a dimerization domain (DD), and a cyclase catalytic domain
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(CCD). Each of the domains has a unique and partially characterized function as follows:
LS (amino acids M1-S51) is an N-terminal signal peptide that is probably targeting the
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protein to the endoplasmic reticulum. LS is removed post-translationally by proteolysis as
revealed by direct amino acid sequencing of the isolated protein (Koch et al., 1994; Margulis
et al., 1993).
ECD (A52-E462) is located on the extracellular side of the cone outer segment membrane or
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in the lumen of the disc membranes in rod outer segments. It shows only a very limited
sequence homology to the receptor GCs, indicating a lack of a binding site for peptide ligands
like natriuretic peptides (Duda et al., 1996a; Goraczniak et al., 1994; Margulis et al., 1993;
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Shyjan et al., 1992). The ECD is N-glycosylated harboring mainly mannose, N-
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acetylglucosamine and sialic acid as terminal carbohydrates (Koch et al., 1994).
TM (P463-V487) is an α–helical transmembrane domain spanning the membrane once.
JMD (R488 - K603) was identified as a unique part of the amino acid sequence in
photoreceptor GCs (Lange et al., 1999; Rätscho et al., 2010). It extends C-terminally from the
TM region and bears no sequence homology to peptide receptor GCs. The domain contains
two short GCAP1 –associated regions: a GCAP1 transducer site (M496- V507) and a GCAP1
binding region (V554- I573) identified by peptide competition and mutagenesis studies
(Lange et al., 1999). A recent report shows that GCAP1 and GCAP2 bind to overlapping
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binding sites within the region R488-R851 covering the JMD, KHD and most of the DD in a
mutually exclusive manner (Peshenko et al., 2015b). However, employing the biosensor
technique backscattering interferometry for label free interaction analysis (Sulmann et al.,
affinities to different regions in the cytoplasmic part of GC-E.
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2017), Sulmann et al. (2017) reported that GCAP1 and GCAP2 target with submicromolar
KHD (A604-N815) is more conserved among the membrane-bound GCs and is named for its
similarity to tyrosine kinases. It harbors a binding site for ATP (Tucker et al., 1997) that
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allosterically enhances the catalytic activity of the enzyme. This domain contains also two
stretches of amino acids that were predicted to interact with GCAP1 (Krylov and Hurley,
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2001). A functional KHD is essential for cGMP synthesis (Bereta et al., 2010) and it contains
serine residues that undergo autophosphorylation, which is neither triggered by light nor
signal transduction (Aparicio and Applebury, 1996; Bereta et al., 2010).
DD (also known as the linker domain- I816-P859) aids the enzyme in adopting an optimal
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monomer−monomer arrangement for catalysis (Peshenko et al., 2015a; Ramamurthy et al.,
2001; Yu et al., 1999; Zägel et al., 2013). It has a predicted coiled-coil structure and bears
sequence homology to a diverse set of signaling proteins including other membrane bound
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receptor GCs (Anantharaman et al., 2006). It serves as a regulatory module in receptor GCs
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by transducing the activating signal from ligand binding to the catalytic center (Saha et al.,
2009).
CCD (S860-S1103) contains the catalytic center that synthesizes cGMP from GTP. This
domain has the highest sequence homology among all GCs and the three-dimensional
structure of the GC catalytic domain of the green algae Clamydomonas reinhardtii showed
the dimeric formation of the catalytic center (Winger et al., 2008). This active catalytic site is
formed from two head-to-tail oriented catalytic domain monomers (Liu et al., 1997; Tucker et
al., 1999) and represents a general architectural motif of cyclase enzymes: cyclization of
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nucleotides occurs via a base-catalyzed reaction with inversion of the stereochemical
configuration at the α–phosphorus atom (Koch et al., 1990). It is obvious from these studies
that any mismatch in the correct orientation of the dimeric catalytic interface would lead to an
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impaired cGMP synthesis.
2.5 Evolution of GC-E
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The evolution of GC genes is likely to be influenced not only by the evolution of other
genes that encode retinal photoreceptors transduction pathway proteins but also by the
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complex and long evolution of the eye (Korenbrot, 2012). A comprehensive molecular
evolutionary analysis of 199 GC proteins revealed multiple lineage-specific expansions of GC
genes in the genomes of many eukaryotes (Biswas et al., 2009). A few regions showing the
most extensive level of conservation, and mainly KHD and DD, were used to correctly build
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the evolutionary trees. With the focus on the human GC-E protein sequence, we performed a
sliding window analysis comparing it to six orthologous sequences in various organisms (Fig.
4A). The analysis shows an overall preservation along evolution with a few regions showing
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long stretches of amino acid similarity (Fig. 4B) which are located mainly in the KHD, DD,
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and CC domains. The longest highly conserved regions were found between amino acids 829
and 1032 harboring the whole dimerization domain and part of the catalytic domain (Fig. 4B).
3.
Genetics, Biochemistry and clinical aspects of GUCY2D / GC-E
IRD mutations affecting the GC-E/GCAP complex seem to be restricted to the
GUCA1A and the GUCY2D genes (to date only one exception is known). The exception is the
p.G157R substitution in GCAP2 (GUCA1B) reported to cause ad retinal degeneration (Sato et
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al., 2005). Disease-causing mutations in the GUCA1B gene seem to be rare occasions based
on screening tests on heterogeneous groups of patients (Kitiratschky et al., 2011; Payne et al.,
1999).
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3.1 Disease-causing mutations in GUCY2D
The human GUCY2D gene maps to chromosome 17p13.1 (Oliveira et al., 1994).
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Using homozygosity mapping, the LCA1 locus has also been identified on chromosome
17p13.1 in consanguineous families of North African origin (Camuzat et al., 1995). Genetic
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and physical mapping of this region (Perrault et al., 1996) revealed an expressed sequence tag
(EST) that corresponds to the human gene encoding GC-E (synonymously assigned as retGC)
that was considered as an excellent candidate, leading to the identification of three diseasecausing mutations in GUCY2D as the cause of arLCA1 (Perrault et al., 1996). This study was
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followed by dozens of reports of different types of mutations causing arLCA1 (Table 2).
Genetic linkage analysis of a large four-generation British family with adCRD
revealed strong linkage to an 8 cM locus on chromosome 17 (the CORD6 locus) (Kelsell et
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al., 1997). Although GUCY2D has been mentioned by the authors as a candidate gene for this
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disease, the clinical differences between LCA, CRD, and cone dystrophy (CD) which were
linked to this region prompted the authors to suggest that these diseases are caused by
mutations in different genes (Kelsell et al., 1997). Only one year later, the same group
reported of two heterozygous missense GUCY2D mutations as the cause adCRD (Kelsell et
al., 1998). In the original British family that was used to map CORD6 (Kelsell et al., 1997),
the authors identified the p.E837D variant, however their re-evaluation of the genetic data
revealed a double mutation in this family (Kelsell et al., 1998). This study was also followed
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by a large number of reports on the association between GUCY2D mutations and adCD/CRD/
maculopathy (Table 2).
To obtain a comprehensive analysis of genotype-phenotype correlations in GUCY2D,
we tabulated the information on GUCY2D disease-causing mutations reported in the literature
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(Table 2 and Fig. 5). A total of 144 GUCY2D mutations have been reported thus far, most of
which (82 mutations- 57%) are missense. The vast majority of mutations (127- 88%) were
reported to cause arLCA1 while 13 mutations were reported to cause adCRD. Three
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additional mutations were reported to cause other phenotypes (ad central areolar choroidal
dystrophy, CACD, by one mutation and autosomal recessive early-onset retinitis pigmentosa
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by two mutations). The adCRD-causing mutations are all missense which are located in exon
13 affecting the GC-E dimerization domain (see section 2.4).
Mutations in GUCY2D appear to be a relatively common cause for both arLCA1 and
adCRD. Many groups have studied the frequency of GUCY2D mutations in LCA cohorts and
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the prevalence ranges from 0% to 21.2% with an average of 8% index cases with biallelic
mutations (Table 3). GUCY2D cases were relatively common in French cohorts (including
patients mainly from France and North Africa) (Hanein et al., 2004; Perrault et al., 2000) with
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alleles.
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only five relatively common founder mutations found in about one-half of GUCY2D mutated
In addition, GUCY2D mutations appear to be a major cause of adCD/CRD in various
populations. A survey of 52 index cases revealed 12 (23%) with GUCY2D mutations, and
hence GUCY2D mutations are the most common cause for this phenotype in this cohort (Kohl
et al., 2012). Interestingly, mutations in GUCA1A were reported in this study to be the 3rd
cause of this phenotype (8%), and therefore mutations in the GC/GCAP complex are
responsible for two thirds of solved adCRD cases in this cohort (Kohl et al., 2012). Similarly,
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GUCY2D mutations were shown to be the most common cause of adCRD in other cohorts
with an average of 18% of cases (Table 3).
Amino acid 838 seems to be the most sensitive GC-E position as four single mutations
and two complex mutations (affecting two or three amino acids) were reported to affect this
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amino acid. Haplotype analysis of six chromosomes carrying the p.R838C mutation showed
that only two carried a shared haplotype (Payne et al., 2001), indicating that Arg838 is a
mutation hotspot, as is also supported by the different mutations affecting the same stretch of
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nucleotides encoding amino acids 837-839. This is expected to yield de-novo mutations in
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recent generations, as is evident for the p.R838H mutation (Mukherjee et al., 2014).
3.2 Biochemical analysis of missense mutations
A total of 45 variants (21 reported to cause LCA1, 7 causing CRD, 2 nonpathogenic, 1
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variant of uncertain pathogenicity, and 14 artificial) have been so far evaluated for the
biochemical effect on the protein (Table 4). The effect of each variant on the protein was
assessed by all or some of the following functional properties (Table 4): enzyme activity at
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concentrations.
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the basal level and upon activation with GCAP1 and GCAP2, and sensitivity to Ca2+
The expression level of some of the mutant proteins was studied in heterologous
expression systems and found to be unaltered (Duda et al., 1999a; Peshenko et al., 2010;
Zägel and Koch, 2014). Therefore, protein expression level does not seem to take a role in
GUCY2D disease mechanism. The other functional properties vary between different mutant
proteins as detailed below and shown in Table 4. In general, LCA1-causing mutations either
show reduced ability or complete inability to synthesize cGMP from GTP (Duda et al., 1999a;
Duda et al., 1999b; Rozet et al., 2001). Furthermore, some mutations are also likely to result
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in misfolding and subsequent degradation in the endoplasmatic reticulum (Rozet et al., 2001),
while CRD-causing mutations are functional, but shift the Ca2+-sensitivity curve (Fig. 1C and
Fig. 2). Functional studies were performed using heterologous expression systems and a
variable set of techniques, which differ in some cases when comparing the results from
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different labs. We will point to these differences in cases where it seems necessary in the
following paragraph.
The effect of the studied mutations is described below and divided by the affected
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protein domain:
Mutations in the LS domain: One LCA1-causing mutation (p.M1?), one variant of uncertain
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pathogenicity (p.L41F), and one polymorphism were studied in this domain (Table 4). The
first is a start-loss mutation (p. M1?) reported in two families (Hanein et al., 2004; Perrault et
al., 2000) in which the initiation codon is altered. Such mutations can have different effects on
the protein: if there is a nearby ATG, it can be used as an alternative initiation codon, but if
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there is none- there might be no protein expression at all. Since no potential alternative ATG
codon is available in the sequence of GUCY2D- this is likely to be a null mutation.
Nonetheless, the effect of p.M1? has been studied enzymatically and found to have a reduced
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basal activity (about 13% of the wt protein). However, a missense variant (p.L41F) as well as
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a nonpathogenic variant (p.W21R) in this domain that were studied using the same conditions
showed normal basal activity (Rozet et al., 2001). The LS domain is assumed to be required
for the correct targeting of the protein to the outer segments, mutations in this region might
prevent the correct cellular localization and / or misfolding of the mutant protein without
affecting the ability of the mutant protein to synthesize cGMP. Thus, mutations in this domain
might lead to protein degradation.
Mutations in the ECD domain: Deletion of the ECD domain has no impact on the basal and
GCAP-mediated GC activity (Duda et al., 1996a; Laura et al., 1996). Six missense mutations
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have been studied in this domain (Table 4A), four of which were analyzed for basal activity
and displayed normal function. Four of the mutations were studied for GCAP stimulation, and
three displayed reduced stimulation of both GCAP1 and GCAP2, while one (p.S248W)
showed enhanced activity with normal Ca2+ sensitivity. The slight increase of activity in the
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p.S248W mutant would not dramatically change the synthesis profile of the cGMP pool
(Jacobson et al., 2013). To better understand the disease mechanism of the p.S248W mutant,
it has been investigated in living photoreceptors (Boye et al., 2016). The mutant protein did
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not show in vitro any noticeable change in activity, which does not explain its contribution to
the LCA1 phenotype (Jacobson et al., 2013). However, in vivo expression studies in GC-E
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and GC-F KO mice failed to show detectable GC-E activity and an immunofluorescence
staining of photoreceptors produced only sporadic staining. This indicates that the p.S248W
mutation contributes to LCA1 by hampering the processing or the cellular transport of GC-E
(Boye et al., 2016).
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The results obtained for ECD missense mutations (p.C105Y, p.R313C, and p.L325P)
showing normal basal, but reduced GCAP-stimulated activity, are not in-line with those
obtained by deletion of the whole ECD which showed no disabling consequence on basal or
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GCAP-mediated activity. This discrepancy can be explained by other possible molecular
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consequences of this missense mutations, including misfolding and impaired trafficking, that
have not been investigated and deserve further attention (Tucker et al., 2004). Correct
trafficking of GC-E from the inner to the outer segment requires an interaction with the RD3
protein (Azadi et al., 2010). Reduced or abolished binding of RD3 to LCA1 mutants including
p.L41F (present in LS) and p.Y351C (localized in the ECD) were reported recently (Zulliger
et al., 2015).
Mutations in the JMD domain: One missense mutation (p.F565) and one nonpathogenic
missense variant (p.P575L) were studied. The p.F565S substitution is located in the GCAP1
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binding and/or regulatory region and severely affects the basal activity and the heterologously
expressed mutant lacks sensitivity to GCAP1 regulation, consistent with this position’s
critical role in binding to and regulating GCAP1 (Duda et al., 1999b; Lange et al., 1999). Due
to these defects, restoration of the dark state will take much more time because less cGMP is
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produced. When the cytoplasmic cGMP is strongly reduced, CNG-channels will nearly
permanently close leading to slower dark state restoration (Fig. 1B). The enzyme activity is
disturbed without destroying the catalytic core since the p.F565S mutant can still be activated
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by S100B (Duda et al. 1999). It is not surprising though that the nonpathogenic variant
(p.P575L) did not affect these protein features, however a shift of the GC-E activity profile to
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lower Ca2+ concentrations has been observed (Zägel and Koch, 2014).
Mutations in the KHD domain: The two missense mutations in the KHD that were studied
(Table 4A- p.D639Y and p.R768W) show similar consequences as the one in the JMD; the
activity is largely reduced or nearly absent, any GCAP-mediated activation is lost (Jacobson
dark state restoration.
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et al., 2013; Peshenko et al., 2010). Similar to the JMD mutation, this will result in a slowed
Loss of GCAP-mediated activity control caused by mutations in different regions
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(JMD and KHD) might indicate that binding of GCAPs to the target GC is flexible with rapid
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on- and off-rates being consistent with apparent affinities of the interaction around 1 µM. It
might also point to a three-dimensional structure of the intracellular GC domains that are
folded in a form, which would allow many contacts between domains. Such a threedimensional structure would create functional significant interfaces between secondary
structural regions.
Mutations in the DD domain: Due to the finding that mutations in this domain cause adCRD
and not arLCA1, mutations in this domain were studied by many groups, with a total of 9
studied pathogenic mutations and 7 artificial variants (usually studied to better explain the
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effect of the pathogenic mutations). The importance of the DD as a regulatory module or
signaling helix (Anantharaman et al., 2006; Saha et al., 2009) has been demonstrated in
different cyclases and it comes as no surprise that retinal disease-causing mutations in this
region affect GC-E activity. A very short stretch of two amino acids (positions 837 and 838)
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is particularly striking, since several missense mutations were found in patients. All these
mutations correlate with CRD, cause sometimes normal, but mainly a decreased basal
activity, and have an effect on the Ca2+-sensitive regulation by GCAPs. Interestingly,
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activation by GCAPs is not completely absent, but shifted to higher free Ca2+-concentration
(p.E837D being the exception as there is a normal response to GCAP1, but increased
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stimulation by GCAP2, Table 4C). The shifted Ca2+ sensitivity would lead to a constitutively
active GC-E and a failure to inactivate GC-E at high [Ca2+]. The uncontrolled cGMP
production would disturb the Ca2+-feedback in photoreceptor cells having consequences on
the Ca2+-cGMP homeostasis (Fig. 1C).
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The p.E837D mutation was found to cause adCRD in two combinations: as a double
mutation with p.R838S (Payne et al., 2001) (ER mutation) and as a triple mutation with
p.R838C and p.T839M (Payne et al., 2001; Perrault et al., 1998) (ERT mutation). Both
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combinations show a higher sensitivity for GCAP1 (Ramamurthy et al., 2001), indicated by a
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lower GCAP1 concentration needed for half maximal activation of the GC-E (K1/2 values for
wt GC-E: 9.1 µM, ER: 2.5 µM, ERT: 5.3 µM). The sensitivity for GCAP2, on the other hand,
remained normal. In addition, the maximal stimulation of the double mutant by GCAP1
remains rather normal while it is reduced by more than 50% in the triple mutation (Duda et
al., 2000; Ramamurthy et al., 2001). The basal and TritonX100/Mn2+ stimulated activity for
the double mutant is normal and for the triple mutant reduced by the half. The Ca2+ sensitivity
of the two mutants is shifted. Significantly higher Ca2+ concentrations are needed to bring
GC-E activity down, compared to the wt. They even retain 15 - 30% of activity at
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concentrations above 10 µM. This effect is also dominant in co-expression with the wt
cyclase (Table 4D). Studies of p.E837D alone showed normal functional properties (other
than an increased stimulation by GCAP2), indicating that p.E837D alone is not the cause of
adCRD (Duda et al., 1999a).
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The arginine amino acid at position 838 in GC-E was found to be altered by 6
different mutations and the function of 5 of which has been studied. These mutations usually
showed reduced basal activity and GCAP stimulation (Table 4A), and more importantly, all
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showed a shift in Ca2+ sensitivity which is more likely to cause the CRD phenotype. All R838
mutants need much higher Ca2+ levels to reduce GC-E activity leading to a constantly
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activated enzyme. The double mutation in amino acids 847 and 848 shows similar properties.
The analysis of each mutation separately (Table 4C) gave similar results, indicating that both
affect protein function.
The two missense mutations causing arLCA1, p.I816S and p.R822P, are at the border
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of the DD, which is described as a linker region that functions as a regulatory module (Saha et
al., 2009). This region is supposed to form a coiled-coil domain facilitating the dimerization
of the enzyme, which is particularly important for the correct matching interface of the CCD
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dimer. A change of amino acids at the KHD-DD border might have different consequences
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than in the center of the coiled-coil helix structure. In addition, the I>S substitution makes the
residue more polar. More dramatic seems to be the R>P substitution, since P is known as
helix breaker, which might lead to a severe disruption of the coiled-coil structure. The
p.R822P mutant shows nearly no activity in the presence of GCAP1 or GCAP2.
Mutations in the CCD mutations: The borders between the different domains seem to
represent borders between the two disease forms: LCA1 and CRD. This can be clearly seen in
the occurrence of LCA1 mutations in the CCD domain and CRD mutations in the DD
domain. All mutations in the CCD severely compromise GC activity. In most cases, no
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functional enzyme is expressed indicating a damaged catalytic core. Nearly similar severe
effects were observed with the mutants p.P858S and p.L954P, although some residual activity
was detected (Tucker et al., 2004).
An interesting aspect of GC-E biochemical analyses are studies in which either two
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mutants were co-expressed (aiming to mimic the genotype in human patients with arLCA1
who are compound heterozygous for two different mutations) or a mutant GC-E was
expressed along with wt GC-E (either to mimic the genotype of heterozygous adCRD patients
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or to mimic the phenotype of carriers of arLCA1 mutations). These data are summarized in
Table 4D.
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Co-transfection of HEK293 cells with wt and p.P858S or p.L954P mutants resulted in
a 50% decrease of GCAP2 stimulated cyclase activity, suggesting that heterodimers are
inactive or poorly active (Tucker et al., 2004). This dominant-negative effect for recessive
mutations could be shown in a heterozygous carrier of the p.L954P mutation, who showed
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cone ERG abnormalities (see chapter 3.3.2 for more details) (Koenekoop et al., 2002).
A recent analysis of mutants yielded interesting insights regarding the effect of two
mutations identified in patients with LCA1 (Boye et al., 2016). The biochemical properties of
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these two mutations, p.S248W and p.R1091*, were studied in cell culture by heterologous
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expression (Jacobson et al., 2013), but resulted in nearly normal enzyme characteristics that
could not explain the clinical phenotype. Using a different method of analysis, in which
cDNA constructs of the mutants were delivered in the subretinal space via adeno-associated
virus (AAV) to a mouse model lacking endogenous cyclase activity (GC-E and GC-F double
knockout) yielded different results. While p.S248W failed to express properly in the mouse
retina, p.R1091* was expressed in a wt-like pattern, leaving the open question, why the
p.R1091* mutation correlates with severe loss of vision in human LCA1 patients (Boye et al.,
2016).
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In summary, heterozygous mutations causing adCRD are limited to the dimerization
domain (DD), show reduced basal activity, and affect mainly Ca2+ sensitivity properties: GCE activity is still detectable at Ca2+ concentrations above 10 µM, and higher [Ca2+] are needed
to reduce its activity. In some mutations, GCAP activation or sensitivity for GCAPs increases
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while in others it decreases. The effect of these mutations on RD3 interaction is lacking for
most mutants thus far.
Can we draw a general conclusion about how CRD mutations lead to a shift or
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disturbance in Ca2+-sensitive activity regulation of GC-E? Localization of the mutations in the
DD suggests an influence of this region on the catalytic domain. Constitutive activation of
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GC-E mediated by Ca2+-bound GCAPs indicates a stabilization of the dimeric interface at the
catalytic substrate (GTP) binding site or in other words, a stabilization of the enzymatic
transition state. Interestingly, this stabilization of the transition state can be achieved by wt
GCAPs interacting with GC-E mutants or by GCAP mutants interacting with wt GC-E. In the
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photoreceptors, this will lead to a permanent production of cGMP and persistent opening of
CNG-channels. The resulting overall high [Ca2+] is toxic for cell physiology and may trigger
photoreceptor cell degeneration.
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LCA1 mutations, on the other hand, can be found in all GC domains, and in particular
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all catalytic domain mutations (except R1091*) lead to complete loss of GC-E function. The
effect of mutations in KHD, JMD and CD are more severe compared to CRD mutations in
DD, since stimulation by Ca2+-free Mg2+-bound GCAPs is either very low or not detectable.
LCA1 mutations in the ECD show no drastic impact on biochemical properties, and might be
pathogenic due to disturbed interaction with other proteins or disturbed transport from IS OS.
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3.3 Clinical assessment of patients with GUCY2D mutations
3.3.1 Clinical assessment of biallelic LCA1 patients
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LCA is a congenital retinal phenotype that is diagnosed at birth or during the first
months of life. Babies with LCA suffer from blindness or greatly impaired vision, usually
have normal fundus appearance, and unrecordable or severely abnormal ERG amplitudes.
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Clinical variability among LCA patients is considered to be small compared to other, noncongenital inherited retinal phenotypes. An initial clinical comparison between different
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groups of patients with LCA reported minor differences between the RPE65 and GUCY2D
LCA groups (Perrault et al., 1999), a result that would later be disputed. No differences were
obtained in age of onset (at birth), mode of onset, fundus appearance (normal at birth
followed by salt and pepper and typical RP appearance later in life), and ERG (non-
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recordable). Some differences, however, were noticed in refraction (severe hyperopia in the
GUCY2D group), visual field (non-recordable), and visual acuity (more severe in the
GUCY2D group). In addition, patients with LCA1 mutations suffered from severe
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photophobia (Perrault et al., 1999). This study was further expanded by the same group who
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performed a comprehensive genetic and clinical analysis of LCA patients (including fundus
images and visual fields to characterize disease phenotype) that revealed two major groups of
patients that can be distinguished by the early retinal phenotype (Hanein et al., 2004). Patients
with GUCY2D mutations belong to the cone-rod group with photophobia, hypermetropia, and
severe involvement of both rods and cones leading to early peripheral and macular
degeneration of the retina with bone spicule- like pigmentation (BSPs) in the periphery,
retinal atrophy including the macular region, very thin blood vessels, and optic disc pallor.
With the presence of hyperopia (higher than +7), the visual acuity was reduced to counting
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fingers or light perception. A similar clinical analysis (Pasadhika et al., 2010), which
benefited from the availability of more advanced in vivo imaging techniques, revealed a
relatively preserved retinal structure with visible photoreceptor inner/outer segment juncture
in patients with GUCY2D mutations, a structural feature with important therapeutic
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implications.
A unique and comprehensive clinical analysis of 11 patients ages 6 months to 37 years
(Jacobson et al., 2013) also revealed valuable features en route to therapy. Using in vivo
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analyses of retinal architecture (by autofluorescence and optical coherence tomography) intact
rod photoreceptors were observed in all patients. The thickness of the outer nuclear layer
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(ONL) in the cone-exclusive fovea fell within normal limits in some patients whereas, in
others, abnormalities in foveal cones were evident. Moreover, substantial rod function was
detectable in the majority of GUCY2D-LCA1 patients with no correlation to the extent of
cone vision or age. Visual adaptation studies revealed measurable cone function in some
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patients and abnormal light adaptation of rods (Jacobson et al., 2013).
In summary, retinal disease and structure of GUCY2D-LCA1 patients is different from
all other subtypes of LCA studied in detail to date. For example, LCA due to RPE65
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mutations is primarily a rod disease and involves progressive and severe retinal degeneration
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while LCA1 due to GUCY2D mutations is primarily a cone disease, but has rod involvement.
Preservation of retinal structure makes GUCY2D LCA1 patients good candidates for gene
augmentation therapy as discussed in chapter 5.
3.3.2 Clinical assessment of parents of LCA1 patients
An interesting question regarding the effect of GUCY2D mutation on retinal function
is whether individuals who carry heterozygous recessive mutations develop any mild signs of
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retinal disease. Aiming to answer this question, clinical analysis of parents of LCA1 patients
has been reported (Galvin et al., 2005; Koenekoop et al., 2002), with a total of four parents
who are heterozygous for identified GUCY2D mutations (ages 40-55 years). Some of the
patients reported mild and recent visual impairment (including difficulties driving at night and
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photoaversion during the day) with best-corrected visual acuity that was normal or subnormal
and with normal appearance of the retina. ERG showed reduced (80%-89% of lower normal
limit) and delayed flicker cone responses as well as slightly reduced (86%-100%) but not
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delayed single-flash light-adapted responses. Dark-adapted rod responses were subnormal
(Koenekoop et al., 2002).
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These findings are consistent with a mild cone-rod dysfunction and are in-line (but of
course much milder) with the GUCY2D-related LCA1 phenotype. It is interesting to note that
co-transfection of the p.L954P mutation with a wt protein (which is expected to result in a
carrier state) resulted in a 50% decrease of GCAP2 stimulated cyclase activity (Table 4D),
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suggesting a dominant-negative effect for arLCA1 mutations (Tucker et al., 2004). This effect
might explain the mild phenotype identified in individuals who are heterozygous for p.L954P
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(Galvin et al., 2005).
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3.3.3 Phenotypic data of adCRD patients with heterozygous mutations in GUCY2D
Most individuals who are heterozygous for dominant mutations in GUCY2D are
clinically diagnosed with CD or CRD, while in a few cases the diagnosis was determined as
macular degeneration (also known as maculopathy). Since these three phenotypes overlap in
their clinical appearance and might represent different disease stages (for example- rod
function will be more preserved in younger patients and therefore they will be initially
diagnosed with CD but this will later in life be revised to CRD), the phenotype that better
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describes the disease in most cases is adCRD. Some common clinical features were reported
in patients with heterozygous dominant GUCY2D mutations: Disease onset in most patients is
during childhood deteriorating to very low visual function (counting fingers or worse) by the
5th decade. Most patients have myopia, photophobia, and nystagmus. Their fundus appearance
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is initially normal in the periphery but the macular appearance is usually abnormal with large
variability among patients (from subtle RPE changes to gross macular atrophy), central or
paracentral scotoma with normal peripheral field of vision, and decreased cone function by
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ERG with normal or abnormal rod function (Garcia-Hoyos et al., 2011; Ito et al., 2004; Jiang
et al., 2015; Kelsell et al., 1998; Kitiratschky et al., 2008; Lazar et al., 2014; Mukherjee et al.,
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2014; Smith et al., 2007; Xiao et al., 2011; Xu et al., 2013; Yoshida et al., 2006; Zhao et al.,
2013).
In an interesting attempt to compare the retinal phenotype of two groups of patients,
Zobor and colleagues (Zobor et al., 2014) clinically evaluated 5 patients with a heterozygous
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GUCA1A mutation (c.451C.T, p.L151F) and nine patients with various heterozygous
GUCY2D mutations affecting the amino acid R838 (p.R838C, p.R838G, and p.R838H); all
suffered from autosomal dominant CD/CRD. In both groups, a generalized cone dysfunction
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was noted with a tendency for more rod involvement and a more severe phenotype in patients
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from the GUCY2D group.
3.4 Genotype-phenotype correlation
The major striking genetic difference between arLCA1 and adCRD caused by
GUCY2D mutations, is that while individuals who are heterozygous for LCA1 mutations are
unaffected (as described in chapter 3.3.3.3, some might have a very mild phenotype or show
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nonsymptomatic reduced retinal function), those heterozygous for CRD mutations are
affected.
In general, one can expect that GUCY2D mutations associated with arLCA1 are null
mutations including frameshift, nonsense, and splicing mutations that are likely to be
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degraded by the nonsense-mediated mRNA decay surveillance or missense mutations that
would lead to a non-functional protein, which could be a misfolded or unstable protein that is
degraded or a protein that is stable but shows no cyclase activity. Individuals who are
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heterozygous for such mutations will have 50% of normal wt GC-E activity, which is enough
for a normal photoreceptor function (this phenomenon is known as haplosufficiency). On the
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other hand, CRD mutations are likely to be missense and produce mutated proteins affecting
the function of the wt proteins. Since GC-E is active in a dimer form, a few scenarios for
homodimer versus heterodimer formation are possible:
A. In normal, heterozygous “carriers” of LCA1- null mutations- only wt proteins are
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present but their amount is half of the normal amount and all formed dimers are therefore
active.
B. In normal heterozygotes for LCA1-missense mutations in which a mutant protein is
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expressed, there are two options:
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1. The mutant and wt proteins cannot form dimers and wt-wt dimerization allows
production of 50% of the active enzyme.
2. The mutant and wt proteins can form dimers and therefore 25% of the dimers are
wt-wt, 50% wt-mut, 25% mut-mut. While the wt-wt form is fully active, wt-mut dimers might
have some activity, and mut-mut dimers are non-active. This still allows enough production
of functional enzymes.
C. In affected individuals carrying heterozygous adCRD mutations- both the wt and
mutant alleles produces each 50% of the total protein amount. Upon dimerization, there are
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three options: 25% wt-wt dimers, 50% wt-mut dimers, and 25% mut-mut dimers. While wt-wt
dimers are fully active, wt-mut dimers and mut-mut dimers have a different Ca2+sensitivity
profile and mut-mut dimers are constitutively active. This combination of both wt dimers and
mutant dimers is likely to result in a non-congenital disorder that shows progression over
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time. The delayed age of onset allows one to see the “true” nature of the GUCY2D-disease,
which is more cone > rod phenotype. In addition, based on functional assays, the mut-mut
dimers might induce a dysregulation of the Ca2+-sensitive cyclase activation profile.
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It should be noted that homozygosity for complete loss-of-function mutations are
expected to mimic a condition of constant light exposure during photoreceptor development
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and therefore biallelic null mutations lead to a more severe LCA phenotype (Perrault et al.,
2000).
Animal models with mutations in retinal guanylate cyclase
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4.
Seven animal models have been reported so far to harbor mutations in GUCY2D as
summarized in Table 5. This topic has been previously reviewed (Boye, 2016) and will be
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only briefly mentioned here.
4.1 Animal models for LCA1
4.1.1 Mouse models
A KO mouse model for GUCY2D showed a CD phenotype due to rapidly
degenerating cones and morphologically normal rods that show reduced a- and b-wave
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amplitudes (Yang et al., 1999). The rods have a normal dark current despite the absence of
functional GC-E and no compensatory increase in the expression of GC-F (Yang et al., 1999).
The findings of Peshenko and colleagueas (Peshenko et al., 2011b), who showed that the
turnover rate (kcat) of nonstimulated GC activity in mice is 7-fold higher for GC-F than for
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GC-E provide an explanation for the recording of a normal dark current, which apparently can
be sustained by the resting GC-F activity. Both GCAP1 and GCAP2 proteins were found to
be down-regulated in the retina of this animal model (Coleman et al., 2004). The KO mouse
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phenotype does not fully recapitulate the human phenotype in which biallelic null mutations
cause LCA1.
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Consistent with its lack of association to a human disease, KO of GUCY2F yielded
mice with normal retinal structure and function by ERG, and normal expression of
phototransduction proteins (Baehr et al., 2007). However, GC-E and GC-F double KO mice
(GCdko) (Baehr et al., 2007) yielded mice lacking ERG responses, suggesting that no GC
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other than GC-E and GC-F is involved in rod or cone phototransduction in the mouse retina.
The rod and cone physiology of GCdko resembles recessive LCA1 in human patients, but is
actually not an equivalent of human LCA1, since a knock-out of both GCs has not been
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reported in patients. Phototransduction proteins (including PDE6, GCAP1, and GCAP2) were
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undetectable in the rod outer segments in the double KO model, although rhodopsin and the
alpha-subunit of transducin were mostly unaffected. Cone outer segment membranes were
destabilized and either did not express cone proteins (cone transducin, cone PDE, and G
protein-coupled receptor kinase 1) or expressed them at low levels (cone pigments). The
mRNA transcripts of these proteins were normal expression levels, indicating that downregulation occurs at the posttranslational level. These results are likely to demonstrate an
intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated
phototransduction proteins (Baehr et al., 2007).
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Transgenic expression of GCAP1 mutants that are calcium-insensitive and
constitutively active in the Gucy2e KO mice revealed a partial rescue of the retinal phenotype
(Olshevskaya et al., 2012). In contrast, expression of the mutants in the Gucy2f KO mice led
to rapid retinal degeneration. These results in combination with electrophysiological
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recordings of different gene knock-out models were interpreted as GCAP1 being the
preferential target to GC-E due to limited access of GCAP1 to GC-F.
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4.1.2 Chicken models
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A naturally occurring retinal degeneration is the rd chicken (or GUCY1*B chicken)
was reported to produce blindness at hatch (Semple-Rowland et al., 1998) with normal retina
at the age of 1-day. Pathology appears only 7–10 days after hatching in photoreceptors that
are located in the central retina, and then progress to peripheral regions. At 115 days, very
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few cone photoreceptors remain in the central retina, and by 6–8 months, the photoreceptor
cell layer is degenerated. In addition, cGMP levels since embryonic day 18 were significantly
reduces as early as embryonic day 18 (Semple-Rowland et al., 1998), GCAP1 was
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downregulated (Semple-Rowland et al., 1996), and GC-E was absent (Semple-Rowland et al.,
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1998). These finding led to the identification of a deletion/insertion mutation (insertion of 81
bp and a deletion of exons 4-7) causing expression of a nonstable protein that is likely to be
degraded (Semple-Rowland et al., 1998). The phenotype of the rd chicken mimics the human
LCA1 phenotype and was therefore used as an animal model for gene therapy (see section 5).
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4.1.3 Zebrafish models
Two different morpholino-modified oligonucleotides, one that blocks translation and
one that blocks splicing, were used to study the effect of Gucy2f (the zebrafish ortholog of
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GUCY2D) knockdown on retinal function and structure in zebrafish (Stiebel-Kalish et al.,
2012). Knockdown fish had significantly lower vision, as well as loss and shortening of cone
and rod outer segments. The authors concluded that Gucy2f knockdown in zebrafish can be
4.2.1 A transgenic mouse model
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4.2 Animal models for adCRD
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used as a model for LCA1
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A transgenic mouse model, in which the GC-E p.R838S mutation was selectively
expressed in rod photoreceptors resulted in early-onset and rapidly progressive decline in
visual responses from the targeted rods, leading to progressive degeneration of rods between 1
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and 6 months of age (Dizhoor et al., 2016).
4.2.2 Pig models
A transgenic pig line including 22 animals was produced by expressing the GUCY2D-
dominant mutant allele- E837D/R838S using a lentiviral vector under the control of the conespecific promoter arrestin-3. Visual tests and histological analyses at ages 3 months and 1
year showed a variable phenotype characterized by decreased cone function and
disorganization of cones (Kostic et al., 2013), therefore reflecting the human clinical situation.
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Although pigs can be used as more reliable animal models for gene augmentation therapy, the
described models for adCRD is transgenic and therefore is limited in the ability to be used as
an efficient test animal for this treatment.
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4.2.3 A zebrafish model
The effect of transgenic expression of mutant GC-E (p.E837D and p.R838S on a
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single allele) on retinal morphology and function was evaluated (Collery et al., 2013).
Although no difference in visual function was identified in 5-day-old larvae, transgenic
mimicking the human phenotype.
Potential therapies for GUCY2D-associated phenotypes
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5.
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expression resulted in aberrant cone morphology and a reduced cone density, therefore
It is a very exciting time in the field of inherited retinal disease research. This includes
advances in treatment modalities that are not specific to the causative gene (i.e. stem cell
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therapy and retinal implants) which will not be covered in this review. The area with perhaps
the greatest momentum is gene-based therapy (Lipinski et al., 2013). Most of these
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approaches are based on subretinal injection of adeno- associated virus (AAV) that is packed
with the gene of interest and are currently being studied in animal models. A few (including
CHM, CNGA3, CNGB3, RPE65, and XLRS) have been tested in human subjects and some of
which show both promising results as well as some limitations that have been reviewed
elsewhere (Barnard et al., 2014; Dalkara and Sahel, 2014; Pierce and Bennett, 2015; Trapani
et al., 2014).
Two proof of concept studies addressing GUCY2D gene therapy in models of LCA1
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were reported in 2006. The first was performed on the GUCY1*B chicken model (section
4.1.2), which is homozygous for a null mutation, is blind at hatching and serves as a model
for LCA1. An in ovo treatment strategy was used by injecting a lentivirus vector carrying the
bovine Gucy2d gene into 7 embryos, prior to the appearance of retinal pathology. Six of the
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injected animals showed improved visual function, ERG responses, and a slower pace of
retinal degeneration compared to wt animals (Williams et al., 2006). The authors noted that
although gene transfer was at a very early stage and was successful at delivering the gene to
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the retina, it did not prevent the process of retinal degeneration, but rather a slower
degeneration process was evident and ultimately, the rescue effects were transient.
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The first report of GUCY2D-related gene augmentation therapy in mammalians was
also reported in 2006 in the KO mouse model (section 4.1.1). In this study, an AAV5 vector
by using AAV5-mediated transfer of the bovine cDNA (the same used in the chicken study)
to the post-natal KO retina at the age of 3 weeks followed by ERG and immunocytochemical
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analyses 5 weeks post injection. The treatment effort failed to restore cone ERG responses or
prevent cone degeneration but it restored the light-driven translocation of cone arrestin in
transduced cone cells (Haire et al., 2006). In these two studies the injected virus included the
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bovine Gucy2d sequence (probably since this sequence was historically used in biochemical
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GC-E functional assays) rather than the species-specific cDNA.
To overcome this potential obstacle, AAV5 and AAV8 containing murine Gucy2e
were subretinally delivered to the same KO mouse model at P14 and P25 (Boye et al., 2010).
Using two promoters (either a photoreceptor specific or a ubiquitous promoter), expression
was verified in both rods and cones, leading to long-term improvement in photopic ERG
responses (~45% of normal) lasting for 3 months as well as improved visual behavior (Boye
et al., 2010; Boye et al., 2011). The same KO mouse was injected with AAV8 vector
containing the human rhodopsin kinase promoter and the human GUCY2D gene (Mihelec et
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al., 2011). This lead to a long-term effect including an appropriate localization of the
transgene product to the outer segments of rods and cones, increased levels and appropriate
localization of cone alpha-transducin, higher cone ERG function, improvement in conemediated visual function, and a significant improvement in rod ERG. The slightly higher
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ERG amplitudes seen in the latter study were likely attributed to the earlier treatment time
point applied in this study (P10) (Boye et al., 2012).
A recent proof of concept experiment used the Nrl(-/-) Gucy2e(-/-) mouse, an all-cone
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model deficient in GC-E (Boye et al., 2015). AAV5 treatment was used to deliver using
human GUCY2D to the subretinal space. The treatment fully restored cone function, cone-
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mediated visual behavior, and guanylate cyclase activity, and preserved cones for at least 6
months. Retinal function could be restored to levels above that in Nrl(-/-) controls, probably
due to to increased cyclase activity in treated Nrl(-/-) Gucy2e(-/-) mice relative to Nrl(-/-)
controls. In addition, delivery of a candidate clinical vector, AAV5-GRK1-GUCY2D, which
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was shown to be suitable in primate photoreceptors (Boye et al., 2015), restored retinal
function that persists for at least 6 months. This study therefore provides strong support for
patients.
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clinical application of a gene therapy targeted to the cone-rich, central retina of LCA1
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As discussed in chapter 3.3.1, LCA1 patients exhibit relatively good preservation of
retinal structure and slow progression of disease over time, which makes GUCY2D an
excellent candidate for gene therapy with a relatively wide window for treatment. Moreover,
the successful AAV treatment in the KO mouse model is a proof-of-concept for this treatment
modality. In addition, a recent study demonstrated a relatively intact postgeniculate white
matter pathway in LCA1 patients, which is encouraging for the prospect of recovery of visual
function with gene augmentation therapy (Aguirre et al., 2017).
An important aspect of developing gene therapies is to determine the appropriate
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outcome measures for clinical trials. A recent study (Jacobson et al., 2017) involving 28
LCA1 patients (Ages 2-59 years) showed that conventional measures, such as visual acuity,
will need to be complemented by other methods such as chromatic full-field sensitivity testing
that provides a photoreceptor-based subjective outcome, and optical coherence tomography to
Future perspectives
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6.
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assess photoreceptor structure in the fovea and the superior retina.
The genetics of GUCY2D-related phenotypes is well-established with a relatively
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large number of LCA and CRD-causing mutations showing a clear genotype-phenotype
correlation. The major advances in understanding GC-E are expected to be achieved by
studying the biochemical aspects of GC-E and by developing treatment modalities for LCA1
patients. For a large number of mutations, the scientific community lacks key parameters of
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GC enzyme function including regulation by affinity for GCAPs and Ca2+sensitive regulation
by GCAPs. Using established cell culture techniques for heterologous expression of GC-E
will allow the comparison of results from different labs. It should be noted, however, that cell
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culture analyses lack important parameters that regulate GC-E in vivo. Therefore, parallel
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studies on transgenic animals (mainly mice) will be needed to explore the effects of mutations
in an animal model before the models can be employed for test in gene therapy approaches. It
is clear that better understanding of the pathogenicity of GC-E mutations will allow the
development of more efficient therapies.
Enzyme parameters obtained for GC-E variants can further be introduced in kinetic
models of phototransduction using computational approaches. A similar approach has been
successfully applied in the investigation of disease causing mutations of GCAP1 to simulate
rod and cone photoresponses under disease conditions (Dell'Orco et al., 2014).
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So far, we have no high resolution structural information of the GC multiprotein
complex involving a GC dimer, GCAPs and RD3. Such information is needed to understand
in better detail protein-protein interfaces and possible impacts of mutations on conformational
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changes that trigger enzyme function.
With proof of concept for gene augmentation therapy now established in GC-E
deficient mouse models, and verification that and AAV5 vector carrying the GRK1 promoter
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is capable of driving transgene exclusively in rods and cones of a clinically relevant species
(non-human primate), it is expected that the next step would be a phase I/II clinical trial for
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testing safety concerns followed by phase III for testing treatment efficacy. One should take
into account the long-term results obtained in RPE65-treated LCA patients, showing that gene
therapy does not stop the process of photoreceptor degeneration. However, because LCA1 is a
more stationary disease, and because the therapy will be aimed directly at the disease target
(photoreceptors) rather than a neighboring epithelial layer, treatment might yield better long-
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term results. Applying the same treatment strategy to cure adCRD due to a heterozygous
GUCY2D mutation is unlikely to be efficient since the mutated allele produced an abnormal
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protein that affect GC-E biochemical properties. Knocking down the mutant allele (without
interfering the wt allele) is therefore needed to prevent the effect of the mutant allele,
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assuming a haplosufficieny mechanism in which 50% of the wt protein amount is sufficient
for normal photoreceptor function.
Although a large number of LCA and CRD patients were found to harbor pathogenic
mutations in GUCY2D, it is likely that many additional patients were misdiagnosed and were
not screened for mutations in this gene. Misdiagnosis can occur for both LCA cases (who
might not be available for clinical assessment at a young age and therefore might be
diagnosed with RP or a cone-dominated disease) and CRD cases (who might be diagnosed
with CD, maculopathy, and Stargardt disease). Moreover, clinical variability among patients
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who carry mutations in the same gene or even carry the same mutation, can lead to different
clinical diagnoses, for example RP/CRD versus LCA (Avila-Fernandez et al., 2007; Perrault
et al., 2005; Ugur Iseri et al., 2010). Therefore, regardless the retinal phenotype of a patient,
genetic testing should include GUCY2D for both recessive and dominant inheritance patterns
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as well as isolate cases. Indeed, next generation sequencing techniques that are now available
for either a specific gene-panel or the whole exome, revealed unexpected genetic findings and
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should be preferred over screening a specific single gene for mutations.
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Wood, L.D., Calhoun, E.S., Silliman, N., Ptak, J., Szabo, S., Powell, S.M., Riggins, G.J.,
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Velculescu, V.E., 2006. Somatic mutations of GUCY2F, EPHA3, and NTRK3 in human
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Xiao, X., Guo, X., Jia, X., Li, S., Wang, P., Zhang, Q., 2011. A recurrent mutation in
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leads to leber congenital amaurosis-1. J Biol Chem 290, 3488-3499.
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FIGURE LEGENDS
Figure 1: Dark and light state of the signaling cascade in rod cells. (A) Situation in wt
photoreceptor cells: in the dark PDE is not activated by the G protein transducin (T).
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Guanylate cyclases (GC-E and GC-F) are mainly inactive due to interaction with Ca2+-
saturated GCAPs, but residual synthesis of cGMP keeps a fraction of the CNG-channels open.
Na+ and Ca2+ enter the cell via the CNG-channel. Ca2+ is extruded by the exchanger.
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Photoactivation (lower part indicated “Light”) of rhodopsin (Rh to Rh*) catalyzes GDP/GTP
exchange at the G protein transducin (T) triggering the hydrolysis of cGMP by the activated
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PDE and subsequent channel closure. Closed CNG-channels prevent influx of Ca2+ leading to
resynthesis of cGMP by GCs, which is under control of a negative Ca2+-feedback involving
GCAPs that sense the decrease of cytoplasmic Ca2+. (B) Hypothetical cellular consequences
of biallelic GUCY2D mutations. LCA1-causing mutations in GC-E lead to a drastically
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reduced level of cGMP keeping almost all of the CNG-channels closed. Very low cGMP
synthesis might arise from GC-F, which is present in much lower amounts. Operation of the
Na+/Ca2+,K+ exchanger extrudes the cytoplasmic Ca2+. Lowered levels of Ca2+ will not trigger
EP
increased synthesis of cGMP, when no activation by GCAPs occurs. Affected cells would
show no photoresponse. (C) Heterozygous CRD-causing mutations can have several
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consequences leading to a disturbance of the Ca2+-cGMP homeostasis, but not to a complete
suppression of cGMP synthesis. A shift in the Ca2+-sensitive regulation of GCs by GCAPs
can even lead to constitutive activation and synthesis of cGMP in the dark and light sufficient
to keep a higher percentage of CNG-channels open.
Figure 2: Idealized GC-E activation curves as a function of Ca2+. Activation of GC-E by
GCAP1 (green line) and GCAP2 (black) occurs in different ranges of free Ca2+-concentration.
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The black to grey bar in the back indicates the changes in cytoplasmic Ca2+ during
illumination. LCA1-causing GC-E mutants have either a drastically reduced or no measurable
activity (blue interrupted line). CRD mutants show a distortion in the GCAP-mediated
activation causing a shift in the Ca2+-sensitivity (red dotted curve). This shift can be larger
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and in some reported cases the activity level does not return to the basal level (symbolized by
the orange interrupted curve). Curves were created according to several reports in the
literature (see main text). Please note that the shape of the curve observed in mutants can
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deviate from the idealized one (see for example (Dizhoor et al., 2016))
Figure 3: RD3 function in photoreceptor cells. Hypothesis of GC-E regulation by RD3 during
maturation and transport to the outer segments.
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Figure 4: Evolutionary analysis of the photoreceptor GC protein. (A) Schematic
representation of the protein is presented in the upper panels. An amino acid sliding window
(length of 30 amino acids) comparing the human protein sequence to selected orthologs
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(macaca, dog, cow, mouse, frog and zebrafish) is shown. X-axis: amino acid number; Y-axis:
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percentage of amino acid identity in a 30 amino acid window. (B) Schematic representation of
the protein is presented in the upper panels. A summary of the data in mammalian sequences
(red) versus all eight sequences (blue), with a cumulative sliding window analysis of data
points that are above the average percentage of amino acid identity for each studied sequence
(chimp 99%, macaca 98%, dog 87%, cow 86%, rat 85%, mouse 85%, frog 57%, zebrafish
51%). Accession numbers are as follows: human- NP_000171.1, chimp- XP_003315414.1,
macaca- XP_001111670.1, dog- NP_001003207.1, cow- NP_776973.2, rat- NP_077356.1,
mouse- NP_032218.2, frog- XP_002942678.2, zebrafish- NP_571941.1
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Figure 5: The distribution of GUCY2D mutations along the different protein domains. (A)
Schematic representation of the protein and its domains. (B) Location of the different
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GUCY2D mutations (LCA-1causing mutations in red, CRD-causing mutations in blue) along
the protein. (C) A multiple sequence alignment of GUCY2D proteins in different species
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showing the CRD-rich area.
Figure 6: Localization of GUCY2D mutations that have been analyzed biochemically.
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LCA1-causing mutations are highlighted in red, CRD-causing mutations in blue.
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Table 1: Nomenclature of retinal guanylate cyclases
Nomenclature used in
Other names used in the literature
this review
GUCY2D (human)
LCA1, RCD2, CORD6, GUCY2D-1
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Gene
Gucy2e (mouse)
Gucy2f (zebrafish)
GUCY2F
Gucy2f (mouse)
ROS-GC1, RET-GC2, GC2
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Protein GC-F
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Gene
photoreceptor GC, ROS-GC1, RET-GC1, GC1
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Protein GC-E
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Table 2A: Reported GUCY2D mutations (accession number NM_000180.3).
Exon /
Intron
Consequence at protein
level (aa)
Mutation
Type
Diagnosis
Reference
c.3G>C
2
p.M1I (M1?)
Misstart
arLCA
(Hanein et al., 2004; Perrault et al., 2000)
c.52_99dup
2
p.G18_L33dup
Inframe insertion
arLCA
(Hanein et al., 2004)
c.91dup
2
p.R31Pfs*288
Frameshift
arLCA
c.124_129del
2
p.L44_L45del
Inframe deletion
arLCA
c.226_239del
2
p.A76Rfs*238
Frameshift
arLCA
c.232_243del
2
p.R78_A81del
Inframe deletion
arLCA
c.238_252del
2
p.A80_L84del
Inframe deletion
arLCA
(Stone, 2007)
c.289G>C
2
p.E97Q
Missense
arLCA
(Stone, 2007)
c.308A>T
2
p.E103K
Missense
arLCA
(Li et al., 2011; Stone, 2007; Xu et al., 2016)
c.314G>A
2
p.C105Y
Missense
arLCA
(Dharmaraj et al., 2000b)
c.380C>T
2
p.P127L
Missense
arLCA
(Astuti et al., 2016)
c.387C>A
2
p.N129K
Missense
arLCA
(Hanein et al., 2004; Perrault et al., 2000)
c.389del
2
p.P130Lfs*36
Frameshift
arLCA
(Coppieters et al., 2010; Hanein et al., 2004; Perrault et al., 1996)
c.448T>C
2
p.W150R
Missense
arLCA
(Stone, 2007)
c.449G>A
2
p.W150*
Nonsense
arLCA
(Xu et al., 2016)
c.450G>A
2
p.W150*
Nonsense
arLCA
(Stone, 2007)
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Variant
(nucleotide)
2
p.E196V
c.622del
2
p.R208Gfs*8
c.693dup
2
c.722-1G>T
(Hanein et al., 2004; Perrault et al., 2000)
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c.587A>T
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p.Q156*
p.A174_L175insHA
(Stone, 2007)
Nonsense
arLCA
(Stone, 2007)
Inframe insertion
arLCA
(Stone, 2007)
Missense
arLCA
(Coppieters et al., 2010)
Frameshift
arLCA
(Hanein et al., 2004; Perrault et al., 1996)
p.K232Efs*87
Frameshift
arLCA
(Stone, 2007; Wiszniewski et al., 2011)
i2
IVS2-1G>T
Splicing
arLCA
(Stone, 2007)
c.726del
3
p. I243Sfs*3
Frameshift
arLCA
(Xu et al., 2016)
c.743C>T
3
p.S248L
Missense
arLCA
(Safieh et al., 2016)
c.743C>G
3
p.S248W
Missense
arLCA
(Jacobson et al., 2013)
c.752T>C
3
p.L251P
Missense
arLCA
(Xu et al., 2016)
c.763G>A
3
p.E255K
Missense
arLCA
(Stone, 2007)
c.779T>C
3
p.L260P
Missense
arLCA
(Astuti et al., 2016)
EP
2
2
(Jacobson et al., 2013; Stone, 2007)
AC
C
c.466C>T
c.518_523dup
(Hanein et al., 2004)
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3
p.Y283*
Nonsense
arLCA
(Stone, 2007)
c.934A>C
3
p.T312P
Missense
arLCA
(Stone, 2007)
c.935C>T
3
p.T312M
Missense
arLCA
(Galvin et al., 2005; Li et al., 2011; Stone, 2007; Xu et al., 2016)
c.937C>T
c.974T>C
3
3
p.R313C
p.L325P
Missense
Missense
arLCA
arLCA
(Hanein et al., 2004; Perrault et al., 2000)
(Dharmaraj et al., 2000b)
c.994delC
3
p.R332Afs*63
Frameshift
arLCA
c.995G>C
3
p.R332P
Missense
arLCA
c.1026+1G>A
c.1052A>G
i3
4
IVS3+1G>A
p.Y351C
Splicing
Missense
arLCA
arLCA
c.1093C>T
4
p.R365W
Missense
arLCA
(Stone, 2007)
c.1214_1216delinsCAA
4
p.L405_D406delinsPN
Missense
arLCA
arLCA
c.1343C>A
4
p.S448*
Nonsense
(Li et al., 2011; Xu et al., 2016)
(Hanein et al., 2004; Perrault et al., 2000; Stone, 2007; Wiszniewski et
al., 2011; Zernant et al., 2005)
c.1401dup
5
p.L468Sfs*89
Frameshift
arLCA
(Khan et al., 2014a)
c.1433_1442dup
5
p.F482Gfs*78
Frameshift
arLCA
(Jacobson et al., 2013; Lotery et al., 2000; Stone, 2007)
c.1445del
5
p.F482Sfs*6
Frameshift
arLCA
(Xu et al., 2016)
c.1514T>C
6
p.L505P
Missense
arLCA
(Xu et al., 2016)
c.1561C>T
6
p.R521*
c.1573del
7
p.Q525Rfs*38
c.1633C>T
7
p.Q545*
c.1668+2T>C
i7
IVS7+2T>C
c.1694T>C
8
p.F565S
c.1749+1G>T
i8
IVS8+1G>T
c.1760T>G
9
c.1762C>T
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c.849C>G
(Srilekha et al., 2015)
(Xu et al., 2016)
(Li et al., 2011)
(Hanein et al., 2004)
arLCA
(Astuti et al., 2016)
arLCA
(Khan et al., 2014b)
Nonsense
arLCA
(Jacobson et al., 2013; Stone, 2007)
Splicing
arLCA
arLCA
Splicing
arLCA
(Li et al., 2011; Xu et al., 2016)
p.L587R
Missense
arEORP
(Avila-Fernandez et al., 2007)
9
p.R588W
Missense
arLCA
(Stone, 2007)
c.1805_1829del
9
p.R602Pfs*27
Frameshift
arLCA
(Hanein et al., 2004; Perrault et al., 2000)
c.1877C>T
9
p.S626F
Missense
arLCA
(Astuti et al., 2016)
c.1915G>T
9
p.D639Y
Missense
arLCA
(Peshenko et al., 2010)
EP
Missense
(Hanein et al., 2004; Perrault et al., 2000)
(Astuti et al., 2016; Coppieters et al., 2010; Hanein et al., 2004; Perrault
et al., 1996; Perrault et al., 1998; Yzer et al., 2006)
AC
C
TE
D
Nonsense
Frameshift
c.1919G>A
9
p.W640L
Missense
arLCA
(Li et al., 2011; Xu et al., 2016)
c.1956+1G>T
int9
IVS9+1G>T
Splicing
arLCA
(Chen et al., 2013; Xu et al., 2016)
c.1956+2T>A
int9
IVS9+2T>A
Splicing
arLCA
(Hanein et al., 2004; Perrault et al., 2000)
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c.1957-1G>T
int9
IVS9-1G>T
Splicing
arLCA
(Hanein et al., 2004; Perrault et al., 2000)
c.1957-1G>TA
int9
IVS9-1G>A
Splicing
arLCA
(Perrault et al., 2000)
c.1957G>A
10
p.G653R
Missense
arLCA
(Astuti et al., 2016)
(Khan et al., 2014a; Li et al., 2009; Lotery et al., 2000)
10
p.R660*
Nonsense
arLCA
10
p.R660G
Missense
arLCA
c.1979G>A
10
p.R660Q
Missense
arLCA
c.2015G>A
10
p.C672Y
Missense
arLCA
c.2113+2T>C
int10
IVS10+2T>C
Splicing
arLCA
c.2113+2 _3insT
int10
IVS10+2_3insT
Splicing
arLCA
(Hosono et al., 2015)
c.2122T>C
11
p.W708R
Missense
arLCA
(Stone, 2007)
c.2129C>T
11
p.A710V
Missense
arLCA
(Gradstein et al., 2016)
c.2132C>T
11
p.P711L
Missense
11
p.E712V
Missense
arLCA
arLCA
(Coppieters et al., 2010)
c.2135_2136delinsTC
(Li et al., 2011; Xu et al., 2016)
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c.1978C>T
c.1978C>G
(Li et al., 2011)
(Lotery et al., 2000; Stone, 2007; Xu et al., 2016)
M
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SC
(Chen et al., 2013; Xu et al., 2016)
(Xu et al., 2016)
(Astuti et al., 2016)
11
p.D728N
Missense
c.2200_2201delinsGC
11
p.I734A
Missense
arLCA
(Li et al., 2011; Xu et al., 2016)
c.2201C>T
11
p.I734T
Missense
arLCA
(Stone, 2007)
c.2234del
11
p.P745Lfs*39
Frameshift
arLCA
(Astuti et al., 2016)
c.2237A>G
11
p.Y746C
Missense
arLCA
(Lotery et al., 2000)
c.2248G>T
11
p.E750*
Nonsense
arLCA
(Stone, 2007; Wiszniewski et al., 2011)
c.2283del
12
p.S762Afs*22
Frameshift
arLCA
c.2285del
12
p.S762Tfs*22
Frameshift
arLCA
(Maria et al., 2015)
(Khan et al., 2014a)
c.2302C>T
12
p.R768W
Missense
arLCA
(Astuti et al., 2016; Hanein et al., 2004; Jacobson et al., 2013; Li et al.,
2011; Peshenko et al., 2010; Simonelli et al., 2007; Stone, 2007;
Wiszniewski et al., 2011; Xu et al., 2016; Yzer et al., 2006)
c.2303G>A
12
p.R768Q
Missense
arLCA
(Booij et al., 2005; Wiszniewski et al., 2011)
c.2317A>C
12
p.M773L
Missense
arLCA
(Lotery et al., 2000)
c.2351T>G
12
p.M784R
Missense
arLCA
(Li et al., 2011; Xu et al., 2016)
c.2383C>T
12
AC
C
EP
TE
D
c.2182G>A
arLCA
p.R795W
Missense
arLCA
(Pasadhika et al., 2010)
c.2384G>A
12
p.R795Q
Missense
arLCA
(Simonelli et al., 2007; Stone, 2007)
c.2413-1G>C
I12
IVS12-1G>C
Splicing
arLCA
(Xu et al., 2016)
c.2447T>G
13
p.I816S
Missense
arLCA
(Rezaie et al., 2007)
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13
p.M820Dfs*10
Frameshift
arLCA
(Stone, 2007)
c.2465G>C
13
p.R822P
Missense
arLCA
(Jacobson et al., 2013; Stone, 2007)
c.2476C>T
13
p.Q826*
Nonsense
arLCA
(Chen et al., 2013; Xu et al., 2016)
(Kelsell et al., 1998)
c.2511G>C
13
p.E837D
Missense
adCRD
c.2511_2512delinsCA
13
p.E837_R838delinsDS
Missense
adCRD
c.2511_2516delinsCTGCAT
13
p.E837_T839delinsDCM
Missense
adCRD
RI
PT
c.2456dup
(Payne et al., 2001)
(Payne et al., 2001; Perrault et al., 1998)
(Boulanger-Scemama et al., 2015; Huang et al., 2016; Ito et al., 2004;
Jiang et al., 2015; Kelsell et al., 1998; Kitiratschky et al., 2008; Kohl et
al., 2012; Lazar et al., 2014; Oishi et al., 2016; Payne et al., 2001; Sun et
al., 2015; Udar et al., 2003; Van Ghelue et al., 2000; Watson et al., 2014)
13
p.R838C
Missense
adCRD
c.2512C>G
13
p.R838G
Missense
adCRD
c.2513G>A
13
p.R838H
Missense
adCRD
(Kitiratschky et al., 2008; Kohl et al., 2012)
(Garcia-Hoyos et al., 2011; Huang et al., 2016; Ito et al., 2004; Jiang et
al., 2015; Kitiratschky et al., 2008; Kohl et al., 2012; Mukherjee et al.,
2014; Payne et al., 2001; Udar et al., 2003; Xiao et al., 2011)
c.2513G>C
13
p.R838P
Missense
adCRD
(Garcia-Hoyos et al., 2011; Jiang et al., 2015; Xu et al., 2013)
c.2516del
13
p.T839Rfs*27
Frameshift
arLCA
(Stone, 2007; Wiszniewski et al., 2011)
c.2521G>A
13
p.E841K
Missense
adCRD
(Lazar et al., 2014)
c.2538G>C
13
p.K846N
Missense
adCRD
(Lazar et al., 2014)
c.2540_2542delinsTCC
13
p.Q847_K848delinsLQ
(Yoshida et al., 2006)
c.2545A>G
13
p.T849A
Missense
adCRD
adCRD
(Jiang et al., 2015; Zhao et al., 2013)
c.2563C>T
13
p.G855*
Nonsense
arLCA
(El-Shanti et al., 1999)
c.2572C>T
13
p.P858S
Missense
arLCA
c.2576+1G>A
i13
IVS13+1G>A
c.2577-2A>C
i13
IVS13-2A>C
c.2595del
14
c.2598G>C
TE
D
M
AN
U
SC
c.2512C>T
Missense
arLCA
Splicing
arLCA
(Coppieters et al., 2010)
p.Lys866Argfs*14
Frameshift
arLCA
(Jacobson et al., 2013)
14
p.K866D
Missense
arLCA
(Coppieters et al., 2010)
c.2714T>C
14
p.L905P
Missense
arLCA
(Hosono et al., 2015)
AC
C
EP
Splicing
(Dharmaraj et al., 2000b)
(Stone, 2007)
c.2743A>G
14
p.I915V
Missense
arLCA
(Wiszniewski et al., 2011)
c.2744_2749delinsCCATTC
14
p.I915_G917delinsTIR
Missense
adCRD
(Ito et al., 2004)
c.2747T>C
14
p.I916T
Missense
adCRD
(Boulanger-Scemama et al., 2015; de Castro-Miro et al., 2014)
c.2785G>T
15
p.D929Y
Missense
arLCA
(Stone, 2007)
c.2798T>C
15
p.V933A
Missense
adCACD
(Hughes et al., 2012)
79
ACCEPTED MANUSCRIPT
Missense
Missense
arLCA
arLCA
(Hanein et al., 2004)
(Yzer et al., 2006)
c.2837C>T
15
p.A946V
c.2846T>C
15
p.I949T
Missense
arCRD
(Ugur Iseri et al., 2010)
c.2849C>T
15
p.A950V
Missense
arLCA
c.2861T>C
15
p.L954P
Missense
arLCA
(Astuti et al., 2016; Stone, 2007)
(Dharmaraj et al., 2000b; Galvin et al., 2005; Koenekoop et al., 2002;
Pasadhika et al., 2010)
c.2896delC
15
p.R966Afs*12
Frameshift
arLCA
c.2899del
15
p.H967Ifs*11
Frameshift
arLCA
c.2927G>T
15
p.R976L
Missense
arLCA
c.2939A>T
15
p.H980L
Missense
arLCA
arLCA
c.2944delG
15
p.G982Vfs*39
Frameshift
c.2944+1delG
i15
IVS15+1delG
Splicing
c.2951G>A
16
p.C984Y
Missense
arLCA
(Stone, 2007)
c.2983C>T
16
p.R995W
Missense
arLCA
(Hanein et al., 2004; Perrault et al., 1996)
c.2996T>C
16
p.F999S
Missense
arLCA
(Stone, 2007)
c.3020C>T
16
p.S1007L
Missense
arLCA
c.3025A>T
16
p.M1009L
c.3034A>C
16
p.T1012P
c.3043+4A>T
i16
IVS16+4A>T
c.3043+5G>A
I16
IVS16+5G>A
RI
PT
p.A934P
SC
15
M
AN
U
c.2800G>C
arLCA
(Stone, 2007)
(Lotery et al., 2000)
(Hanein et al., 2004; Perrault et al., 2000)
(Jacobson et al., 2013; Stone, 2007)
(Hanein et al., 2004; Jacobson et al., 2013; Pasadhika et al., 2010;
Perrault et al., 2005; Stone, 2007; Tucker et al., 2004)
(Galvin et al., 2005)
arLCA
Missense
arLCA
(Chen et al., 2013; Xu et al., 2016)
Splicing
arLCA
arLCA
(Hanein et al., 2004; Perrault et al., 2005)
TE
D
Missense
(Li et al., 2011; Stone, 2007)(Xu et al., 2016)
(Hanein et al., 2004; Perrault et al., 1996; Perrault et al., 2000; Stone,
2007)
EP
Splicing
(Verma et al., 2013)
17
p.H1019_L1022delinsTV
Missense
c.3056A>C
17
p.H1019P
Missense
arLCA
(Hanein et al., 2004; Perrault et al., 2005; Xu et al., 2016)
c.3067_3075dup
17
p. S1023_V1025dup
Inframe insertion
arLCA
(Verma et al., 2013)
(Hanein et al., 2004; Perrault et al., 2000)
AC
C
c.3055_3064delinsACAG
arLCA
(Dharmaraj et al., 2000b)
c.3078_3079delGA
17
p.I1027Sfs*44
Frameshift
arLCA
c.3078_3083dup
17
p.I1027_L1028dup
Inframe insertion
arLCA
(Li et al., 2011; Xu et al., 2016)
c.3106C>T
17
p.Q1036*
Nonsense
arLCA
c.3118C>G
17
p.R1040G
Missense
arLCA
(Hanein et al., 2004; Perrault et al., 2000)
(Lotery et al., 2000)
c.3118C>T
17
p.R1040*
Nonsense
arLCA
(Hanein et al., 2004; Verma et al., 2013)
c.3224G>C
c.3233_3236dup
i18
IVS18+1G>C
IVS18+1G>C
Splicing
arLCA
(Jacobson et al., 2013)
19
p.H1079Qfs*54
Frameshift
arEORP
(Perrault et al., 2005)
80
ACCEPTED MANUSCRIPT
c.3271C>T
19
p.R1091*
Nonsense
arLCA
(Jacobson et al., 2013; Stone, 2007)
Table 2B: GUCY2D variants with uncertain pathogenicity.
Exon
Consequence at
protein level (aa)
Mutation
Type
Diagnosis
c.121C>T
2
p.L41F
Missense
arLCA
Reference
RI
PT
Variant
(nucleotide)
(Hanein et al., 2004; Perrault et al., 2000)
c.974T>G
3
p.L325R
Missense
arLCA
c.1312C>T
4
p.R438C
Missense
arLCA
(Simonelli et al., 2007)
c.1618C>T
7
p.R540C
Missense
arLCA
(Hanein et al., 2004; Perrault et al., 2000)
c.1720C>T
8
p.R574C
Missense
arLCA
(Song et al., 2011)
c.2834A>G
15
p.H945R
Missense
arLCA
(Zernant et al., 2005)
c.3206del
18
p.Pro1069Argfs*37
Frameshift
iRP
(Booij et al., 2005)
M
AN
U
SC
(Li et al., 2011)
AC
C
EP
TE
D
In some cases the mutation nomenclature has been edited for a uniform appearance in this table. In rare cases, errors in nomenclature were corrected
and are shown here in the correct form. The effect of each mutation on the protein sequence was determined using Mutalyzer
(http://www.lovd.nl/mutalyzer/).
The table includes only heterozygous mutations in families with ad inheritance and biallelic mutations in families with ar inheritance. Heterozygous
variants identified in arLCA patients with no mutation identified on the counter allele are listed in part B of the table.
81
ACCEPTED MANUSCRIPT
Table 3: Frequency of GUCY2D mutations in arLCA and adCD/CRD
arLCA
Reference
(Perrault et al., 2000)
118
21
18%
(Lotery et al., 2000)
(Dharmaraj et al., 2000a)
(Sitorus et al., 2003)
(Hanein et al., 2004)
(Booij et al., 2005)
(Zernant et al., 2005)
(Yzer et al., 2006)
(Yzer et al., 2006)
(Vallespin et al., 2007)
(Henderson et al., 2007)
(Stone, 2007)
(Simonelli et al., 2007)
(Li et al., 2009)
(Coppieters et al., 2010)
(Henderson et al., 2007)
(Li et al., 2011)
(Chen et al., 2013)
(Verma et al., 2013)
(Xu et al., 2016)
(Astuti et al., 2016)
TOTAL
176
100
21
179
35
205
58
58
42
59
642
95
37
91
59
87
41
30
159
64
2756
11
2
0
37
4
24
6
6
0
9
11
2
1
7
2
9
2
3
16
9
217
6.3%
2%
0%
21%
11%
12%
10%
10%
0%
15%
2%
2%
3%
8%
3%
10%
5%
10%
10%
14%
8%
Analyzed
families
With
heterozygous
GUCY2D
mutation
%
(Garcia-Hoyos et al., 2011)
22
2
9%
(Kitiratschky et al., 2008)
27
11
41%
(Kohl et al., 2012)
(Lazar et al., 2015)
(Payne et al., 2001)
(Boulanger-Scemama et al., 2015).
TOTAL
52
24
91
17
233
12
6
6
5
42
23%
25%
6%
29%
18%
RI
PT
%
SC
With biallelic
GUCY2D
mutation/s
AC
C
EP
TE
D
Analyzed
families
adCD/CRD
M
AN
U
Reference
Only cohorts including at least 17 index cases were included in this table.
82
ACCEPTED MANUSCRIPT
Table 4A: The effect of missense mutations on GC-E biochemical properties
LCA
R838C
CRD
R838H
CRD
R838S
CRD
DD
R838S
CRD
CRD
CRD
~ 30%
125%
50%
~ 50%
125%
<10%
No activity
50% [Mn2+]
15% [Mn2+]
No activity
No activity
No activity
2%
6%
Normal
200%
50%
23%
25%
43%
20%
50%
35%
~ 500%
130%
Normal
50%
~115%
~30%
Normal
Normal
Normal
Not
reported
No
No
~ 50%
Normal
Normal
33%
~ 85%
7%
5-fold increase
in affinity
Not reported
IC50 ~1.62.0 x higher
IC50 ~1.4x
higher
No
No
Yes
No
IC50 ~2x
higher
IC50 ~2x
higher
2.5-fold
shift to
higher Ca2+
83
RD3 binding
efficiency
RI
PT
R822P
E837D
R838S
E837D
R838C
T839M
~ 30%
SC
LCA
LCA
GCAP2
stimulation
M
AN
U
KHD
D639Y
R768W
Normal
Normal
GCAP1
stimulation
TE
D
F565S
Y351C
GC basal
activity
13%
Normal
Normal
EP
JMD
Disease
LCA
LCA
LCA
LCA
LCA
LCA
LCA
LCA
ECD
Mutation
M1?
C105Y
N129K
S248W
R313C
L325P
AC
C
GC
domain
LS
Ca2+ sensitivity
Constitutively
IC50 shift
active
Reduced
Reduced
Reduced
Reference
(Rozet et al., 2001)
(Tucker et al., 2004)
(Rozet et al., 2001)
(Jacobson et al., 2013)
(Rozet et al., 2001)
(Tucker et al., 2004)
(Zulliger et al., 2015)
(Duda et al., 1999b; Zulliger et
al., 2015)
(Peshenko et al., 2010)
(Jacobson et al., 2013; Peshenko
et al., 2010; Zulliger et al.,
2015)
(Jacobson et al., 2013)
Yes
(Ramamurthy et al., 2001)
Yes
No
No
Yes
Yes
Yes
(Ramamurthy et al., 2001)
(Duda et al., 2000)
(Duda et al., 1999a)
(Tucker et al., 1999; Wilkie et
al., 2000)
(Wilkie et al., 2000)
(Peshenko et al., 2010; Wilkie et
al., 2000)
Yes
Yes, partially
Binding of
GCAP1/R838
S complex is
reduced
(Dizhoor et al., 2016)
ACCEPTED MANUSCRIPT
R976
LCA
H980L
LCA
R995W
LCA
M1009L
H1019P
LCA
LCA
Q1036*
R1091*
LCA
LCA
Nearly no
activity
No
activity
No
activity
No
activity
No
activity
No
activity
No
activity
20 - 25%
~ 5%
Yes
Reduced
Nearly no
activity
Nearly no
activity
No activity
No activity
Reduced
Reduced
Reduced
RI
PT
LCA
~ 5%
Ic50 3-5x
higher
SC
L954P
>300%
No activity
No activity
M
AN
U
LCA
LCA
>200%
TE
D
P858S
A934P
Increased
Decrease
d
Reduced
Normal
EP
CRD
AC
C
CCD
Q847L
K848Q
84
Reduced
Reduced
Reduced
(Zägel et al., 2013)
(Tucker et al., 2004; Zulliger et
al., 2015)
(Zulliger et al., 2015)
(Tucker et al., 2004; Zulliger et
al., 2015)
(Rozet et al., 2001)
(Jacobson et al., 2013)
(Rozet et al., 2001; Zulliger et
al., 2015)
(Rozet et al., 2001; Zulliger et
al., 2015)
(Rozet et al., 2001; Zägel and
Koch, 2014; Zulliger et al.,
2015)
(Rozet et al., 2001)
(Jacobson et al., 2013)
ACCEPTED MANUSCRIPT
Table 4B: The effect of other variants on GC-E biochemical properties
Variant
LS
L41F
GC
basal
activity
Normal
LS
W21R
Normal
JMD
P575L
Normal
GCAP1
stimulation
GCAP2
stimulation
Ca2+ sensitivity
RD3 binding efficiency
Reduced
70%
75%
Reference
RI
PT
GC
domain
IC50 0.5x lower
AC
C
EP
TE
D
M
AN
U
SC
W21R and P575L are nonpathogenic. L41F is of uncertain pathogenicity.
85
(Rozet et al., 2001; Zulliger et al., 2015)
(Rozet et al., 2001)
(Zägel and Koch, 2014)
ACCEPTED MANUSCRIPT
Table 4C: The effect of artificial missense variants on GC-E biochemical properties
GCAP1
stimulation
~ 50%
GCAP2
stimulation
~ 50%
Reference
(Tucker et al., 2004)
(Duda et al., 2011)
Normal
Normal
Normal
Normal
Normal
~ 50%
27%
60%
60%
60% left
Normal
Normal
30%
55 - 65%
55 - 65%
55 - 65%
>150%
Q847L*
Increased
200%
150%
K848Q*
P858S**
Higher
Increased
Increased
20%
Higher
30%
D885A
D929A
P1069R
No activity
No activity
No activity
No activity
No activity
No activity
No activity
No activity
No activity
Normal
Constitutively
active
Constitutively
active
Increased
Constitutively
active
EP
TE
D
DD
CCD
Ca2+
sensitivity
RI
PT
KHD
C105Y
L325P**
WTAPEL
L708714del
W708A
T709A
P711A
E712A
E837D
R838A
GC basal
activity
Normal
AC
C
*- A complex mutation Q747L and K848Q has been reported to cause CRD.
**- Co-expression
***- A complex mutation P858S and L954P has been reported to cause CRD.
86
(Duda et al., 2011)
(Duda et al., 2011)
(Duda et al., 2011)
(Duda et al., 2011)
(Duda et al., 1999a)
(Wilkie et al., 2000)
SC
ECD
Variant
M
AN
U
GC
domain
(Zägel et al., 2013)
(Zägel and Koch, 2014)
(Tucker et al., 2004)
(Ramamurthy et al., 2001)
(Ramamurthy et al., 2001)
(Zägel and Koch, 2014)
ACCEPTED MANUSCRIPT
Table 4D: The effect of co-expression of two variants on GC-E biochemical properties
DD
GCAP2
stimulation
~ 50%
~ 60%
~ 55%
(Mn/Tx100
stimulated)
L954P
wt
~ 60%
~ 55%
(Mn/Tx100
stimulated)
Higher
sensitivity
Ca2+
sensitivity
R838C
wt
E837D
R838C
T839M
wt
Normal
Higher
sensitivity
E837D
R838S
wt
Normal
No dominant
effect
Higher
sensitivity
87
General
effect
No
dominant
negative
effect
Dominant
negative
RI
PT
GCAP1
stimulation
~ 50%
SC
wt
GC basal
activity
Normal
~ 90%
~ 70%
M
AN
U
CCD
P858S
Variant
2
L325P
wt
wt
TE
D
DD
GC
domain
ECD
EP
KHD
Variant
1
C105Y
D639Y
R768W
AC
C
GC
domain
ECD
Shifted,
~15%
activity
>10µm ca2+
Shifted,
~30%
activity
>10µm ca2+
Reference
(Tucker et al., 2004)
(Peshenko et al., 2010)
(Peshenko et al., 2010)
(Tucker et al., 2004)
Dominant
negative
(Tucker et al., 2004)
Dominant
positive
(GCAP1)
Dominant
Positive
(GCAP1)
(Tucker et al., 1999)
Dominant
positive
(GCAP1)
(Ramamurthy et al.,
2001)
(Ramamurthy et al.,
2001)
ACCEPTED MANUSCRIPT
Table 5: Animal models for retinal guanylate cyclases
Animal
Mutated region
Mutant name
Mouse
GC-1 KO
Portion of exon 5 was
replaced with a neo cassette
Method
Targeted
Mouse
GC-2 KO
GCdko
Targeted
ERG responses are absent. Outer
(Baehr et al., 2007)
RI
PT
Targeted
(Baehr et al., 2007)
segments form but degenerate
R838S RetGC1
A double mutation: E837D /
R838S
GUCY2DE837D/R838S
Chicken
Null (insertion of 81 bp and
deletion of exons 4-7)
rd or GUCY1*B
Zebrafish
Morpholinos
Zebrafish
Transgene
Block translation
and splicing
p.E837D and
p.R838S on a
single allele
Progressive loss of visual function and
progressive retinal degeneration
Transgenic
Variable phenotype characterized by
decreased cone function and
disorganization of cones
Spontaneous Normal retinal morphology during the
first 7 – 10 days post-hatching. The
retina is non-functional by ERG at this
early stage when retinal morphology is
normal
Targeted
Dysfunction but also to photoreceptor
degeneration
Transgenic
Aberrant cone morphology and a
reduced cone density
AC
C
EP
TE
D
Pig
Transgenic
SC
Mouse
Reference
(Coleman et al., 2004;
Yang et al., 1999)
M
AN
U
Mouse
Portion of exon 2 was
replaced with a neo cassette
Gucy2e and Gucy2f double
KO
R838S in Gucy2e
Phenotype
CD- rods remain viable and
responsive to light but show reduced
a- and b-wave amplitudes
Normal ERG and retinal structure
88
(Dizhoor et al., 2016)
(Kostic et al., 2013)
(Semple-Rowland et al.,
1998)
(Stiebel-Kalish et al.,
2012)
(Collery et al., 2013)
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights
•
GUCY2D mutations cause different retinal phenotypes with different inheritance
RI
PT
patterns.
Biochemical analysis of GC-E variants shows a clear genotype-phenotype correlation.
•
Cone-rod degeneration mutations are clustered in the dimerization domain.
•
Amino acid 838 is the most sensitive GC-E position affected by six different
SC
•
AC
C
EP
TE
D
M
AN
U
mutations.
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