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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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ACCEPTED MANUSCRIPT Genotype-Functional-Phenotype Correlations in Photoreceptor Guanylate Cyclase (GC-E) encoded by GUCY2D a RI PT Dror Sharon a,b, Hanna Wimberg c, Yael Kinarty a,d, Karl-Wilhelm Koch c Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, SC 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 M AN U b Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel TE D Corresponding authors: Dror Sharon, Ph.D., Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, 91120, Israel; E-mail: firstname.lastname@example.org EP Karl-Wilhelm Koch, Ph.D., Department of Neuroscience, University of Oldenburg, D-26111 AC C Oldenburg, Germany; E-mail: email@example.com ACKNOWLEDGEMENTS We would like to thank the Hanse-Wissenschaftskolleg (Delmenhorst, Germany) for the generous scholarship to DS. ACCEPTED MANUSCRIPT 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 RI PT 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 SC are one of the major causes of all LCA cases and are the major cause of adCRD. A single M AN U 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 TE D shift the Ca2+-sensitivity of the GC-E – GCAP complex. Eight animal models of retinal guanylate cyclase deficiency have been reported EP including knockout (KO) mouse and chicken models. These two models were used for gene augmentation therapy that yielded promising results. AC C 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. 1 ACCEPTED MANUSCRIPT Keywords: cyclic GMP, Genotype-phenotype correlation, Guanylate cyclase, GUCY2D, Mutation, Photoreceptor, Retinal diseases RI PT 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, SC congenital stationary night blindness; DD, dimerization domain; ECD, extracellular domain; M AN U 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 AC C EP TE D pigmentosa; RPE, retinal pigment epithelium; TM, transmembrane domain; 2 ACCEPTED MANUSCRIPT 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 RI PT Contents 2.3.1 Guanylate cyclase-activating proteins (GCAPs) SC 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 M AN U 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 TE D 3. 2.4 3.3.1 Clinical assessment of biallelic LCA1 patients EP 3.3.2 Clinical assessment of parents of LCA1 patients AC C 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 3 ACCEPTED MANUSCRIPT 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 AC C EP TE D M AN U SC 5. RI PT 4.2.3 A zebrafish model 4 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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 TE D (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 EP as well, opened the gate for the discovery of complicated and unexpected disease mechanisms AC C 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 5 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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, M AN U 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 TE D 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 EP reported GUCY2D mutations, the functional analysis of some of these mutations, the AC C 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. 6 ACCEPTED MANUSCRIPT 2. Guanylate cyclases in humans 2.1 Guanylate cyclase function RI PT 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 SC phototransduction (Kuhn, 2016; Stryer, 1991). Downstream effectors of cGMP in signaling pathways are mainly cyclic nucleotide-gated (CNG) ion channels, cGMP dependent protein M AN U 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 EP 2.2 Genes encoding GCs TE D forms, and are found in many species. AC C 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 7 ACCEPTED MANUSCRIPT 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 RI PT (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 SC 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. M AN U 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 TE D 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). EP The second photoreceptor-specific GC encoded in the human genome by GUCY2F AC C 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 8 ACCEPTED MANUSCRIPT 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). RI PT 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), SC 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 M AN U 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 TE D 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+, EP 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 AC C 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). 9 ACCEPTED MANUSCRIPT 2.3.1 Guanylate cyclase-activating proteins (GCAPs) The human genome contains three genes (GUCA1A, GUCA1B, GUCA1C) encoding RI PT 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 SC 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 M AN U 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 TE D 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 EP 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/ AC C 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 10 ACCEPTED MANUSCRIPT 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 RI PT 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). SC dependent activation curve to higher free Ca2+-concentration (Fig. 2), when activated by wt M AN U 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 TE D 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 EP adCD/CRD/maculopathy (Downes et al., 2001; Jiang et al., 2005; Jiang et al., 2008; AC C 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 11 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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 TE D et al., 2006). 2.3.2 Activation by the S100B protein EP 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 AC C 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). 12 ACCEPTED MANUSCRIPT 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), RI PT 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 SC 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. M AN U 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 TE D 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 EP retinal function (Lavorgna et al., 2003). It was initially thought to be part of subnuclear AC C 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). 13 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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 TE D 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- EP causing GC-E mutants towards RD3 has been analyzed. Among the studied GC-E mutants, 10 AC C 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 14 ACCEPTED MANUSCRIPT 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 RI PT 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. SC 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. M AN U 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 TE D was published elsewhere (Molday et al., 2014). EP 2.4 Functional domains of GC-E AC C 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). 15 ACCEPTED MANUSCRIPT 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 RI PT domain (ECD), a transmembrane domain (TM), a juxtamembrane domain (JMD), a kinase homology domain (KHD), a dimerization domain (DD), and a cyclase catalytic domain SC (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 M AN U 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 TE D 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; EP Shyjan et al., 1992). The ECD is N-glycosylated harboring mainly mannose, N- AC C 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 16 ACCEPTED MANUSCRIPT 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. RI PT 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 SC 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, M AN U 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 TE D 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 EP receptor GCs (Anantharaman et al., 2006). It serves as a regulatory module in receptor GCs AC C 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 17 ACCEPTED MANUSCRIPT 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 RI PT impaired cGMP synthesis. 2.5 Evolution of GC-E SC 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 M AN U 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 TE D 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 EP long stretches of amino acid similarity (Fig. 4B) which are located mainly in the KHD, DD, AC C 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 18 ACCEPTED MANUSCRIPT 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). RI PT 3.1 Disease-causing mutations in GUCY2D The human GUCY2D gene maps to chromosome 17p13.1 (Oliveira et al., 1994). SC 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 M AN U 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 TE D 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 EP al., 1997). Although GUCY2D has been mentioned by the authors as a candidate gene for this AC C 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 19 ACCEPTED MANUSCRIPT 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 RI PT (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 SC additional mutations were reported to cause other phenotypes (ad central areolar choroidal dystrophy, CACD, by one mutation and autosomal recessive early-onset retinitis pigmentosa M AN U 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 TE D 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 AC C alleles. EP 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, 20 ACCEPTED MANUSCRIPT 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 RI PT 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 SC nucleotides encoding amino acids 837-839. This is expected to yield de-novo mutations in M AN U 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 TE D 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 AC C concentrations. EP 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 21 ACCEPTED MANUSCRIPT 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 RI PT 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 SC protein domain: Mutations in the LS domain: One LCA1-causing mutation (p.M1?), one variant of uncertain M AN U 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 TE D 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 EP basal activity (about 13% of the wt protein). However, a missense variant (p.L41F) as well as AC C 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 22 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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). TE D 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 EP GCAP-mediated activity. This discrepancy can be explained by other possible molecular AC C 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 23 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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. TE D 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 EP (JMD and KHD) might indicate that binding of GCAPs to the target GC is flexible with rapid AC C 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 24 ACCEPTED MANUSCRIPT 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) RI PT 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, SC 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 M AN U 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). TE D 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 EP combinations show a higher sensitivity for GCAP1 (Ramamurthy et al., 2001), indicated by a AC C 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 25 ACCEPTED MANUSCRIPT 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). RI PT 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 SC 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 M AN U 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 TE D 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 EP dimer. A change of amino acids at the KHD-DD border might have different consequences AC C 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 26 ACCEPTED MANUSCRIPT 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 RI PT 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 SC or to mimic the phenotype of carriers of arLCA1 mutations). These data are summarized in Table 4D. M AN U 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 TE D 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 EP these two mutations, p.S248W and p.R1091*, were studied in cell culture by heterologous AC C 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). 27 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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 TE D 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. EP LCA1 mutations, on the other hand, can be found in all GC domains, and in particular AC C 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. 28 ACCEPTED MANUSCRIPT 3.3 Clinical assessment of patients with GUCY2D mutations 3.3.1 Clinical assessment of biallelic LCA1 patients RI PT 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. SC Clinical variability among LCA patients is considered to be small compared to other, noncongenital inherited retinal phenotypes. An initial clinical comparison between different M AN U 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- TE D 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 EP photophobia (Perrault et al., 1999). This study was further expanded by the same group who AC C 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 29 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U (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 TE D 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 EP mutations is primarily a rod disease and involves progressive and severe retinal degeneration AC C 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 30 ACCEPTED MANUSCRIPT 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 RI PT 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 SC delayed single-flash light-adapted responses. Dark-adapted rod responses were subnormal (Koenekoop et al., 2002). M AN U 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), TE D 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 EP (Galvin et al., 2005). AC C 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 31 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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., M AN U 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 TE D 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 EP was noted with a tendency for more rod involvement and a more severe phenotype in patients AC C 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 126.96.36.199, some might have a very mild phenotype or show 32 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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 TE D 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 EP expressed, there are two options: AC C 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 33 ACCEPTED MANUSCRIPT 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 RI PT 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. SC 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 M AN U and therefore biallelic null mutations lead to a more severe LCA phenotype (Perrault et al., 2000). Animal models with mutations in retinal guanylate cyclase TE D 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 AC C EP 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 34 ACCEPTED MANUSCRIPT 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 RI PT 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 SC phenotype does not fully recapitulate the human phenotype in which biallelic null mutations cause LCA1. M AN U 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 TE D 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 EP reported in patients. Phototransduction proteins (including PDE6, GCAP1, and GCAP2) were AC C 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). 35 ACCEPTED MANUSCRIPT 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 RI PT 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. SC 4.1.2 Chicken models M AN U 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 TE D 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 EP downregulated (Semple-Rowland et al., 1996), and GC-E was absent (Semple-Rowland et al., AC C 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). 36 ACCEPTED MANUSCRIPT 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 RI PT 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 M AN U 4.2 Animal models for adCRD SC used as a model for LCA1 TE D 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 AC C EP 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. 37 ACCEPTED MANUSCRIPT 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. RI PT 4.2.3 A zebrafish model The effect of transgenic expression of mutant GC-E (p.E837D and p.R838S on a SC 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 TE D 5. M AN U 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 EP 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 AC C 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 38 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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. M AN U 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 TE D 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 EP bovine Gucy2d sequence (probably since this sequence was historically used in biochemical AC C 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 39 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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- M AN U 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 TE D 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. EP clinical application of a gene therapy targeted to the cone-rich, central retina of LCA1 AC C 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 40 ACCEPTED MANUSCRIPT 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 SC 6. RI PT assess photoreceptor structure in the fovea and the superior retina. The genetics of GUCY2D-related phenotypes is well-established with a relatively M AN U 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 TE D 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 EP culture analyses lack important parameters that regulate GC-E in vivo. Therefore, parallel AC C 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). 41 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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- TE D 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 EP 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, AC C 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 42 ACCEPTED MANUSCRIPT 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 RI PT as well as isolate cases. 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Impaired association of retinal degeneration-3 with guanylate cyclase-1 and guanylate cyclase-activating protein-1 AC C EP TE D leads to leber congenital amaurosis-1. J Biol Chem 290, 3488-3499. 71 ACCEPTED MANUSCRIPT 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). RI PT 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. SC 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 M AN U 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 TE D 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 AC C 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. 72 ACCEPTED MANUSCRIPT 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 RI PT 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 M AN U SC 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. TE D 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 EP (macaca, dog, cow, mouse, frog and zebrafish) is shown. X-axis: amino acid number; Y-axis: AC C 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 73 ACCEPTED MANUSCRIPT 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 RI PT 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 SC showing the CRD-rich area. Figure 6: Localization of GUCY2D mutations that have been analyzed biochemically. AC C EP TE D M AN U LCA1-causing mutations are highlighted in red, CRD-causing mutations in blue. 74 ACCEPTED MANUSCRIPT 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 RI PT Gene Gucy2e (mouse) Gucy2f (zebrafish) GUCY2F Gucy2f (mouse) ROS-GC1, RET-GC2, GC2 AC C EP TE D Protein GC-F M AN U Gene photoreceptor GC, ROS-GC1, RET-GC1, GC1 SC Protein GC-E 75 ACCEPTED MANUSCRIPT 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) RI PT 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) SC M AN U c.587A>T TE D 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) 76 ACCEPTED MANUSCRIPT 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 M AN U SC RI PT 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) 77 ACCEPTED MANUSCRIPT 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) RI PT c.1978C>T c.1978C>G (Li et al., 2011) (Lotery et al., 2000; Stone, 2007; Xu et al., 2016) M AN U 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) 78 ACCEPTED MANUSCRIPT 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.