Naturally Occurring Animal Models with Outer Retina Phenotypes

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Vision Research 49 (2009) 2636–2652

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Vision Research

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Naturally occurring animal models with outer retina phenotypes

Wolfgang Baehr *, Jeanne M. Frederick

John A. Moran Eye Center, Department of Ophthalmology and Visual Sciences, 65 Mario Capecchi Dr., Salt Lake City, UT 84132, USA

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Article history: Naturally occurring and laboratory generated animal models serve as powerful tools with which to inves- Received 9 February 2009 tigate the etiology of human retinal degenerations, especially retinitis pigmentosa (RP), Leber congenital Received in revised form 7 April 2009 amaurosis (LCA), cone dystrophies (CD) and macular degeneration (MD). Much progress has been made in elucidating gene defects underlying disease, in understanding mechanisms leading to disease, and in designing molecules for translational research and gene-based therapy to interfere with the progression Keywords: of disease. Key to this progress has been study of naturally occurring murine and canine retinal degen- Vertebrate animal models for retinal eration mutants. This article will review the history, phenotypes and gene defects of select animal models degeneration with outer retina (photoreceptor and retinal pigment epithelium) degeneration phenotypes. Rod and cone photoreceptors Retinoid cycle Ó 2009 Elsevier Ltd. All rights reserved. Leber congenital amaurosis Retinitis pigmentosa Cone/rod dystrophy

1. Introduction many unrelated genes (den Hollander, Roepman, Koenekoop, & Cremers, 2008). Macular degeneration (MD) is a disorder of the Gene defects linked to retinal dystrophies in human and animal macula and RPE causing decreased central visual acuity. Break- models cover a wide range of phenotypes, from non-progressive down of the RPE interferes with phagocytosis and the retinoid cy- night blindness to early or late-onset retinal degeneration, cle, causing thinning of the retina and new blood vessel growth. inherited in dominant, recessive, autosomal or X-linked fashion MD becomes increasingly frequent in people beyond age 50, affect- (for a comprehensive list of retina dystrophy genes, see Ret- ing 25% of people aged 75 years and older (Brown et al., 2005; [email protected]). Many genes affecting vision are Stone, 2007). expressed in either photoreceptors where phototransduction oc- In most cases, there is only rudimentary understanding of the curs or the retinal pigmented epithelium (RPE) where the chromo- pathobiology leading to retinal degeneration, and no safe therapy phore of visual pigments is recycled. Mutations in photoreceptor or cure. Historically, naturally occurring animal models (e.g., mu- genes leading to blindness involve a large variety of gene products, tant mice, cats, dogs) have been especially useful in determining including components of the phototransduction cascade, the reti- biochemical mechanisms and phenotypes. For example, the retinal noid cycle, structural proteins, transcription factors, and vesicular degeneration (rd) mouse, to our knowledge the first published ver- transport. Retinitis pigmentosa typically affects rod photoreceptors tebrate photoreceptor degeneration model, was described in 1924 first, producing night blindness and attenuated peripheral vision, (Keeler, 1924). The last fifteen years have seen an explosion in lab- then progresses to involve cones and central vision as well (Har- oratory generated mouse models by transgenic or gene replace- tong, Berson, & Dryja, 2006). As the disease progresses, peripheral ment technologies, but naturally or spontaneously occurring vision is gradually lost and cones are also affected. Signs and symp- models are still being discovered at The Jackson Laboratory. While toms often first appear in childhood, but severe visual problems of- the mouse has become a standard animal model for retinitis pig- ten do not develop until early adulthood. Multiple RP subtypes mentosa and cone dystrophies (Chang et al., 2005; Dalke & Graw, exist, and in the United States, an estimated 100,000–200,000 peo- 2005), emphasis has shifted to larger animal models (e.g., dogs or ple have some form of RP. In Leber congenital amaurosis, rod and monkeys) since the eyes of these animals are more human-like cone photoreceptor function is absent or severely compromised and are more amenable to pre-clinical gene-based therapies. In re- at birth, as evidenced by extinguished or barely detectable phot- cent years, significant progress has been made with viral gene opic and scotopic electroretinograms (ERGs). As with RP, LCA is replacement therapy particularly in canine models with retinal highly heterogeneous and at least eleven LCA subtypes link to as degeneration (Acland et al., 2001, 2005a). To illustrate the utility of animal models for understanding mechanisms that lead to reti-

* Corresponding author. Fax: +1 801 585 1515. nopathy, we summarize current ﬁndings for animal retinal dystro- E-mail address: [email protected] (W. Baehr). phies and examine the underlying genetic defects.

Fig. 1. N-terminal regions of the wt (top) and nob2 (middle, bottom) a1F channel subunit. The amino acid sequences starts at the translocation initiator, M, in exon 1. The area shaded in blue is replaced by the transposon sequence shown in red. The middle sequence shows the truncation after residue 25 (adapted from (Doering et al., 2008). In the bottom sequence, the stop codon was removed by alternative splicing. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

2+ 2. Cacna1f (CaV1.4 or a1F Ca channel subunit): nob2 mouse NA1F leads to block of synaptic transmission from photoreceptors to bipolar cells. The human CACNA1F gene is also associated with Voltage activated Ca2+ channels are multimeric complexes com- Aland Island eye disease characterized by fundus hypopigmenta-

posed of a pore-forming a1 subunit and several regulatory sub- tion and decreased visual acuity (Wutz et al., 2002). units. a1F, encoded by the CACNA1F gene, is a retina-specific 2+ member of the L-type family of Ca channel a1 subunits medi- 3. Cep290 (centrosomal protein of 290 kDa): rd16 mouse, rdAc ating glutamate release at mammalian retinal ribbon synapses. Abyssinian cat 2+ In the dark, a1F is open and active producing Ca influx into the synaptic terminal and glutamate release. Light hyperpolarizes CEP290, also known as nephrocystine 6 (NPHP6), localizes to photoreceptors, an event which closes a1F and reduces gluta- the centrosome (microtubule organizing center) of dividing mate release (recent review: (Morgans et al., 2005). cells, and in photoreceptors, to the connecting cilium in rods and cones. In humans, loss-of-function mutations in the Identified at The Jackson Laboratory, nob2 mice were shown to CEP290 gene have been associated with Joubert syndrome have a reduced rod b-wave (as distinguished from nob mice which (affecting the controls of balance and coordination in the brain) have no b-wave) (Chang, Heckenlively, et al., 2006). The nob2 rod a- (Sayer et al., 2006; Valente et al., 2006), nephronophthisis wave is unaffected, while cone b-waves are reduced. The pheno- (medullary cystic kidney disease), and hypomorphic mutations type resembles incomplete X-linked congenital stationary night- with Leber Congenital Amaurosis (LCA10) (disabling rod and blindness (CSNB2) in humans caused by CACNA1F null alleles cone function) (den Hollander et al., 2006). (Bech-Hansen et al., 1998). The OPL of the nob2 retina is disorganized, i.e. horizontal cells were observed to extend dendritic pro- The rd16 mouse is a spontaneous mutant discovered at The Jack- cesses into the photoreceptor layer and mGluR6 of bipolar cell son Laboratory (Chang, Khanna, et al., 2006). An in-frame deletion dendrites could be immunodetected in the ONL (Chang, Hecken- of 298 amino acids (exons 35–39) of Cep290 (Fig. 2) causes a rap- lively, et al., 2006; Bayley & Morgans, 2007). idly progressing degeneration that is complete by 6 weeks postna- The nob2 gene defect was identified as a transposon (ETn) inser- tally. Scotopic and photopic ERG a-wave amplitudes, diminished tion in exon 2 of the Cacna1f gene located on the X-chromosome by PN18, are absent at PN28. CEP290-LCA in humans and rd16 mice 2+ and encoding the a1F subunit of the voltage-dependent Ca chan- show abnormal olfactory function, and abnormal transport of nel a1F. This insertion produces a null allele and translational stop membrane-associated Ga-olf (Gnal) and Gc13 (Gng13), both of after codon 26. However, the phenotype of a laboratory generated which are subunits of the olfactory G protein (McEwen et al., Cacna1f knockout mouse (G305ter, complete loss of the rod b- 2007). Despite loss of vision and olfaction, cilia of photoreceptors wave) was more severe than that of the nob2 mouse (Mansergh and olfactory sensory neurons (OSN) appear normal. Rhodopsin et al., 2005). Doering et al. (2008) discovered by RNA analysis that and arrestin mislocalized to the rd16 ONL suggesting a role for the nob2 retina actually expresses two mRNA species, one with the CEP290 in membrane protein transport. stop codon at position 33 and a second mRNA in which the stop CEP290 (2,479 amino acids) has multiple motifs, including was removed, presumably by alternative splicing occurring within coiled-coil domains (an a-helical heptad repeat of amino acids Etn transposon. This RNA produced a a1F protein in which a 22-res- from 1 to 7 where 1 and 4 are hydrophobic and 5 and 7 predomi- idue segment (pos. 10–31) was replaced by 24-residue section nantly polar) and an ATP/GTP binding loop, among other motifs deriving from Etn (Fig. 1). As a consequence, the mutant a1F lost (Chang, Khanna, et al., 2006). CEP290 was shown to be associated its ability to interact with filamins A and B (Doering et al., 2008). with RPGR-interacting protein (RPGRIP), dynactin subunits, kine- The nob2 and human CSNB2 phenotype is restricted to the ret- sin-II subunits KIF3A and KAP3, centrin-1, periciliary membrane ina and is non-progressive, consistent with expression of a1F in protein 1 (PCM1) (Chang, Khanna, et al., 2006) and other centro- photoreceptor synapses. Mechanistically, loss of functional CAC- somal proteins, e.g., CP110 (Tsang et al., 2008). CEP290 is part of

Fig. 2. The murine Cep290 gene. In the rd16 mouse, exons 35–39 are deleted, thereby removing 298 amino acids in the C-terminal half of CEP290. The deletion joins exons 34 and 40 in-frame. In the rdAc cat gene, a single nucleotide polymorphism (SNP) generates a new splice site (blue) (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.), resulting in the truncation of cat CEP290 by 159 residues. The protein has multiple coiled-coil motifs (shaded in gray). 2638 W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652

Table 1 Naturally occurring animal models. The animal models are ordered alphabetically by their mutant genes. Column 1, gene symbols; column 2, the gene product; column 3, commonly used trivial names for the animal model; column 4, gene defects; column 5, animal phenotypes; column 6, human phenotypes with mutations in orthologous genes; column 7, references. Abbreviations used: RP, retinitis pigmentosa; LCA, Leber’s congenital amaurosis; CSNB, congenital stationary night blindness; ar, autosomal recessive; ad, autosomal dominant; X, X-linked; D, deletion.

Gene Protein Animal Gene defect Animal phenotype Human disease References model

Cacna1f VDCC a1F nob2 mouse insertion of Reduced rod b-wave, disorganized Incomplete X-linked CSNB (CSNB2) Chang,

subunit (CaV1.4) Etn transposon OPL, non-progressive Heckenlively, into X2 et al. (2006) CEP290 Centrosomal rd16 mouse Dexons 35– ERG absent at PN28, abnormal Joubert syndrome nephronophthisis LCA Chang, Khanna, protein 290 kDa 39) olfaction et al. (2006) CEP290 Centrosomal rdAc cat IVS50 + 9T > G Progressive retina atrophy, complete Menotti- protein 290 kDa degeneration at 3–5 years Raymond et al. (2007) Chx10 C. elegans ceh- Chx10 ocular Y176ter Abnormal eye development (ocular Microphthalmia Burmeister et al. 10 homeo retardation, retardation (or), rudimentary eyes, (1996) and Ferda domain or optic nerve absent et al. (2000) containing homologue Cngb3 Cone CNG Alaskan D all exons Day blindness at 8–12 weeks, cone Total color blindness (ACHM3) Sidjanin et al. channel b Malamute dystrophy (2002) subunit dog Cngb3 Cone CNG German D262 N Day blindness at 8–12 weeks, cone Total color blindness (ACHM3) Sidjanin et al. channel b shorthaired dystrophy (2002) subunit pointer Crb1 Drosophila rd8 mouse Dnt3481 in Slowly progressing retinal RP, LCA Mehalow et al. Crumbs exon 9 degeneration with relatively stable (2003) homolog 1 ERGs Gnat2 Cone transducin cpﬂ3 mouse D200 N in At PN28, near normal scotopic ERGs, Recessive achromatopsia (rod Chang, Dacey, a subunit exon 6 reduced cone b-wave amplitudes; at monochromacy) et al. (2006) and 9 months, undetectable cone ERGs Kohl et al. (2002) Gnb1 Transducin b- rd4+/ inverted Rd4 rd4+/ mouse: recessive Kitamura et al. subunit mouse chromosome 4 RP (2006) breakpoint in rd4/ mouse: lethal intron 2 Gnb3 Cone transducin rge chicken DD153 Cone ERG reduced at hatch, but still Tummala et al. b-subunit (retinopathy present at one year. Scotopic and (2006) globe photopic b-waves oscillatory enlarged) potentials absent Grm6 Metabotropic nob3 mouse S207P Lack of scotopic and photopic b-waves CSNB1B Maddox et al. glutamate (2008) and Dryja receptor 6 et al. (2005) (mgluR6) Grm6 Metabotropic nob4 mouse IVS1ins65nt Lack of scotopic and photopic b-waves CSNB1B Pinto et al. (2007) glutamate and Dryja et al. receptor 6 (2005) (mgluR6) Gucy2e Guanylate rd chicken Dexons 4–7 No ERG measurable at hatch; slowly LCA Semple-Rowland cyclase 1 (GUCY1B) progressing degeneration et al. (1998) Mertk Mertk gene (c- RCS rat Dexon 2 Retinal degeneration Recessive RP cone–rod dystrophy D’Cruz et al. mer proto- (2000), Gal et al. oncogene (2000), and tyrosine kinase) McHenry et al. (2004) Mfrp Membrane-type rd6 mouse Dexon 4 Discrete subretinal spots across the Human null alleles: nanophthalmos, Kameya et al. frizzled-related retina; progressive retinal characterized by extreme hyperopia and (2002) and protein (Mfrp) degneration late-onset retinal macular degeneration Sundin et al. (2005) Myo7a Myosin VIIA sh1 mouse Sh1 R502P Sh1/sh1 mice show circling, head- Human Null alleles: Usher syndrome Liu et al. (1998) (shaker-1 Sh16J R241P tossing, deafness, and hyperactivity Type 1B and Weil et al. mouse) phenotypes (1995) Nr2e3 Photoreceptor- rd7 mouse Dexon 4–5 Retinal degeneration characterized by Enhanced S-cone syndrome Akhmedov et al. speciﬁc Nuclear whorls and rosettes in the ONL. (2000) Receptor (PNR) Nyx Nyctalopin nob1 mouse 85 bp deletion No rod b-wave, normal rod a-wave X-linked CSNB1A (no b-wave) Gregg et al. and cone ERG (2003) Pde6a Rod PDE6 a rcd3 X15Dnt1939 Arrest in outer segment formation; Null alleles: autosomal recessive RP Huang et al. subunit Cardigan progressive retinal atrophy (PRA). (1995) and Welsh corgi Petersen-Jones et al. (1999) PDE6b Rod PDE b rd1 mouse (r Y347ter Failure to elaborate normal outer Null alleles: autosomal recessive RP Pittler et al. subunit mouse) segments, and rapid degeneration (1993) following eye opening at P12. Pde6b Rod PDE b rd10 mouse R560C in exon Measurable scotopic ERG at P18–P30, (Chang et al., subunit 13 later onset of RP than in the rd1 2002) mouse; rods are gone by P30–35, cones die later. (continued on next page) W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652 2639

Table 1 (continued) Gene Protein Animal Gene defect Animal phenotype Human disease References model Pde6b Rod PDE b rcd1 Irish W807ter Progressive retinal atrophy (PRA) (Suber et al. subunit setter (1993)) Pde6b Rod PDE b Sloughi dog C816ins8nt Progressive retinal atrophy (PRA) Dekomien et al. subunit (2000) Prph2 Peripherin/Rds rd2 mouse 9.2 kb No outer segments, no scotopic or Null alleles: recessive RP. Missense Sanyal and Jansen (rds mouse) insertion in photopic ERG response. ONL mutations cause dominant macular (1981) and Travis exon 2 after disappears completely after about dystrophy, digenic RP with ROM1, and et al. (1989) codon 229 1 year dominant adult vitelliform macular dystrophy Rd3 RD3 rd3 mouse R107ter Rod/cone degeneration similar to Friedman et al. human LCA (2006) Rho Rhodopsin English T4R Retinal degeneration that mimics adRP Kijas et al. (2002) mastiff dog dominant RP in humans with T4 K RHO mutations Rpe65 Retinal pigment Swedish D4 bp exon 5 Night blindness, much reduced day LCA Aguirre et al. epithelium Briard- (485delAAGA) vision, but normal fundi; slowly (1998) and Veske protein of BeagleDog progressing degeneration et al. (1999) 65 kDa Rpe65 Retinal pigment rd12 mouse R44ter After 5 months, white spots visible LCA Pang et al. (2005) epithelium throughout the fundus by protein of ophthalmoscopy;. Scotopic ERG 65 kDa severely attenuated; photopic responses recordable. Rpgr RPGR (Retinitis XLPRA1 D5bpin Normal retinal morphology until early XLRP (RP3) Zhang et al. Pigmentosa Siberian ORF15 adulthood. After age 6 months, severe (2002) GTPase husky anomalies develop. Regulator Rpgr RPGR XLPRA2 D2bpin Early onset degeneration; XLRP Zhang et al. ORF15 disorganized outer segments during (2002) photoreceptor development Rpgrip RPGR- Dachshund Insertion of 44 Recessive cone/rod dystrophy LCA Lheriteau et al. interacting nt in exon 2 (2009) and protein Mellersh et al. (2006) Tub Tubby protein rd5 mouse G18,626T Late onset obesity, retinal/cochlear Chang et al. (TUB) tubby splice site degeneration, reduced fertility, insulin (2002) mouse mutation resistance DX12

centrosomal and microtubule-associated protein complexes, which include rab8 (a small GTPase involved in vesicular transport) and components of the BBSome, a stable complex of at least seven BBS proteins involved in membrane protein trafﬁcking (Kim, Krishnaswami, & Gleeson, 2008; Loktev et al., 2008). Abyssinian Cat. Another naturally occurring Cep290 mutant is the long-studied Abyssinian retinal degeneration cat model (rdAc).

Progressive retina atrophy (PRA) in the Abyssinian cat was re- Fig. 3. The Chx10/Vsx2 gene. In the orJ mouse, a nonsense codon in exon 3 truncates corded by Swedish researchers almost 30 years ago (Narfstrom, the transcription factor in the center of the Hox domain. OAR (named after the 1983). The retina phenotype of the mutant cat resembles a slowly initials of otp, aristaless, and rax) is a 14-aa motif identified within the C-terminal developing recessive RP, with reduced ERG a-wave amplitudes at region of several paired-like homeodomain-containing proteins. 7 months, and complete photoreceptor degeneration at 3–5 years of age. The CEP290 gene defect was recently identified as a SNP encodes VSX2/CHX10, a transcription factor expressed in retinal of intron 50 (Table 1), generating a new splice site, a 4 bp insertion progenitor cells during development (optic cup stage) (Liu et al., and a CEP protein that is shortened by 159 amino acids (Menotti- 1994). Expression of VSX2/CHX10 is lost in postmitotic cells. A stop Raymond et al., 2007). codon was identified in exon 3 of the Chx10 gene (Fig. 3) truncating CHX10 in the homeodomain (Burmeister et al., 1996). No 4. Chx10/Vsx2 (ceh10 homeodomain-containing homolog/visual CHX10 protein is detectable in orJ, therefore or J is a Chx10 null al- J system homeobox 2): or mouse lele. In human CHX10 null alleles, R20Q and R200P missense mutations were located in the homeodomain (HOX) within the DNA CHX10 (now called Vsx2) is a homeodomain-containing tran- recognition helix (Ferda et al., 2000). The mouse Or J and human scription factor that is expressed exclusively in retinal progeni- CHX10 null phenotypes are virtually orthologous. tors, and plays a critical role in the formation of the neural retina. Recessive CHX10 mutations were shown to segregate 5. Cngb3 (cone cyclic nucleotide-gated (CNG) channel with microphthalmia in human patients. b-subunit): cone degeneration (cd) dogs The mouse mutant orJ (ocular retardation J) is characterized by abnormal eye development (TRUSLOVE, 1962). Or J mice are blind, CNG cation channels are located in the photoreceptor plasma with rudimentary eyes, cataracts, and no optic nerve. The or J gene membrane. CNG channels are heterotetrameric complexes 2640 W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652

tion of the large extracellular domain containing multiple EGF-like and laminin-AG-like repeats is unknown. The rd8 mutation causes a frameshift that truncates the protein. The truncated protein re- tains the EGF-like and Laminin-like repeats, but lacks the transmembrane and cytoplasmic domains of CRB1 protein encoded by exon 12 (Fig. 5). Homozygous rd8 mice show a slowly progressing retinal degeneration with relatively stable ERGs. Histology at 5 weeks of age Fig. 4. The CNGB3 gene and protein. The cd phenotype of the German Shorthaired shows areas of retinal folding that correspond to the white retinal pointer is caused by a missense mutation (D262 N) in exon 6 (TM domain 2). The CNGB3 protein has six TM domains, a pore region (P), and a cNMP binding site. In spots (Chang et al., 2002). Contrasting with other retinal degener- the Alaskan Malamute, the entire CNGB3 structural gene is deleted (the canine ation mutants, the rd8 degeneration is focal, i.e., sharply demar- CNGB3 is presently unavailable, therefore the defect is modeled on the mouse cated. A Crb1/ mouse also showed a relatively mild retinal CNGB3 gene). degeneration phenotype consisting of lesions disrupting interaction between photoreceptors and Mueller cells (van de Pavert et al., 2004). comprised of a- and b-subunits, termed CNG3A and CNG3B in cones. Each subunit contains cGMP binding domains. Homo- 7. Gnat2 (cone transducin a subunit): cpfl3 mouse meric CNGA3 forms functional channels, while CNG3B does 2+ + not. The function of CNG channels is to regulate Ca /Na influx c Cone transducin (cT, or Gt ) is the heterotrimeric G protein of the depending on the light- or dark-state of photoreceptors (corre- cone phototransduction cascade, composed of cTa (Gnat2), cTb sponding to low or high cytoplasmic cGMP, respectively). (Gnb2), and cTc (Gngt2) subunits. Its role is analogous to that Cone degeneration in the Alaskan Malamute was initially of rod transducin in the rod phototransduction cascade. The pri- termed hemeralopia (Rubin, Bourns, & Lord, 1967), and later chan- mary function of cTa is to exchange GDP with GTP upon receptor ged to cone degeneration (cd)(Gropp et al., 1996). The cone disease activation, and to activate PDE to accelerate cGMP hydrolysis. is recessive and resembles human achromatopsia caused by CNGB3 The structure of cTa is predicted to be very similar to rod Ta null alleles (total color blindness, ACHM3). The Alaskan Malamute (Noel, Hamm, & Sigler, 1993) showing two main domains, the defect was identified as a deletion of the entire structural gene (all helical domain and the GTPase domain, flanking the GDP/GTP 18 exons) of the canine Cngb3 gene (Sidjanin et al., 2002). In an- binding cleft. In human patients, GNAT2 null alleles cause auto- other breed affected by cd (German Shorthaired pointer), a mis- somal recessive achromatopsia (color blindness, rod monochro- sense mutation (D262 N) was detected in a conserved region of macy) (Kohl et al., 2002; Rosenberg et al., 2004). CNGB3 encoded by exon 6 (Sidjanin et al., 2002), generating a null The cpfl3 mouse was identified by ERG testing as a novel achro- allele (Fig. 4). Affected dogs develop day blindness at 8–12 weeks matopsia model (Chang, Dacey, et al., 2006). Cpfl3/cpfl3 mice re- of age when retina development is complete. Cone function is vealed near normal scotopic ERGs, but cone ERGs were 25% of detectable in very young cd puppies, perhaps due to the presence normal b-wave amplitudes at 4 weeks, and were undetectable at of the channel-forming CNGA3 subunit. Cone ERG responses are 9 months of age. The defect appears as a missense mutation extinguished in adult cd dogs, although the affected retina remains (D200 N) in exon 6 of the Gnat2 gene, encoding cone transducin a normal throughout life, as judged by ophthalmoscopy. subunit (Fig. 6). D200 is in a highly conserved area of transducin The cone CNG cation channel (stoichiometry B3 B3 A3 A3) and may affect the GTPase activity of cone transducin (Kerov et al., (Peng, Rich, & Varnum, 2004) is open in the dark when the photo- 2005). Using an AAV5 vector expressing mouse Gnat2 cDNA under transduction cascade is silent and cGMP is high. The channel closes control of the human red–green opsin promoter, the cpfl3 pheno- in light when cGMP is degraded by cGMP-specific phosphodiester- type could be rescued: postnatal injection into the subretinal space ase (PDE6). In the absence of a functional CNGB3 subunit, a normal of adult Gnat2cpfl3 mouse eyes demonstrated that light-adapted ERG channel cannot be assembled, cone phototransduction is disabled, amplitudes were at levels comparable to the wild-type controls. and the photoreceptor cannot hyperpolarize. Another consequence Most treated eyes continued to have recordable light-adapted ERG of CNG3B deletion is downregulation of cone Tc (Gngt2) and cone signals at 6 and 7 months of age (Alexander et al., 2007). Tb (Gnb3) which were undetectable in predegenerate cd AM retina (Akhmedov et al., 1998), presumably as a consequence of traffick- 8. Gnb1 (transducin b-subunit): rd4 mouse ing problems.

Transducin (T, or Gt) is the heterotrimeric G protein of the rod 6. Crb1 (crumbs homolog 1): rd8 mouse phototransduction cascade, composed of Ta (Gnat1), Tb (Gnb1)

CRB1 is a highly conserved transmembrane protein homologous to Drosophila crumbs, a protein important for maintaining api- co-basal cell polarity. CRB1 is expressed mostly in brain and retina. Nonsense or frameshift mutations in the human CRB1 gene lead to various forms of human retinal dystrophy, including retinitis pigmentosa and LCA (den Hollander et al., 1999). The rd8 mouse was isolated at The Jackson Laboratory by re- peated backcrossing of rd6 mice with C57BL/6 mice on the basis of a ‘large retinal spots’ phenotype (Mehalow et al., 2003). The rd8 retina phenotype occurs due to a single base pair deletion in exon 9 of the mouse Crb1 gene (nt3481). CRB1 is a 1406-residue Fig. 5. The Crb1 gene and protein. An exon 9 frameshift leads to a truncation removing the TM domain (gray). The extracellular domain of CRB1 has laminin G protein containing a 37-amino acid cytoplasmic domain with (globular) motifs, multiple EGF-like domains (not shown), and two cysteine-rich PDZ motifs involved in organizing a macromolecular protein scaf- regions at the C- and N-termini (yellow). ‘D’ depicts the C-terminal deletion. Motifs fold (Gosens, den Hollander, Cremers, & Roepman, 2008). The func- were determined with Motif Scan at http://hits.isb-sib.ch/cgi-bin/PFSCAN. W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652 2641

Fig. 8. The chicken GNB3 gene and the retinopathy globe enlarged (rge) gene defect. The defect was determined to be a deletion of 3 bp in exon 6, deleting a single amino acid (D163).

(Sondek, Bohm, Lambright, Hamm, & Sigler, 1996). It consists primarily of a seven-bladed b-propeller which contains seven Fig. 6. The murine Gnat2 gene and protein. The location of the Cpfl3 mutation is structurally similar WD repeats. WD repeats are commonly indicated. Underneath, cartoon of the cTa protein. The numbers refer to amino acids predicted to be in contact with GTPcS in the nucleotide binding cleft (Noel et al., found in other proteins, and consist of 40 amino acids ending 1993). The mutant residue in cone Ta, D200 N, is situated within the GTP binding in conserved Trp (W) and Asp (D) residues. cleft. Blue box, helical domain (HD). Yellow, b-sheets forming the GTPase domain. Rge is a naturally occurring autosomal recessive retinal disorder leading to blindness. The Rge chicken arose spontaneously in a Brit- ish chicken flock (Tummala et al., 2006). The ERG responses are reduced at hatch, but still measurable at one year. Notably, the scotopic and photopic b-waves lack oscillatory potentials (Mon- tiani-Ferreira, Shaw, Geller, & Petersen-Jones, 2007). The rge retina shows early OPL disorganization and endoplasmic reticulum mislocalization (Tummala et al., 2006). Older birds develop globe Fig. 7. The Rd4 gene defect. The Rd4 mouse chromosome 4 carries a large inversion enlargement and cataracts. encompassing most of the chromosome. The proximal breakpoint is in the The rge defect was identified as a 3 bp deletion in exon 6 (Fig. 8) centromere, the distal breakpoint is in intron 2 of the Gnb1 gene. eliminating one amino acid (D153), one of two highly conserved aspartic acids in the third of the seven WD repeats. Normal and and Tc (Gngt1) subunits. Transducin is activated by a conforma- mutant transcript levels are similar, but the mutant GNB3 protein tional change in rhodopsin after light absorption. Activated Ta is 70% reduced. Modelling of the mutant GNB3 protein predicts with GTP bound, in turn, activates cGMP phosphodiesterase that b-sheets in propellers 1 and 5 are abolished by the deletion (PDE6) which degrades cGMP, the second-messenger in photo- of D153, thereby weakening the structure (Tummala et al., 2006). receptors. The Tb and Tc subunits form a tight complex that is membrane-associated using a farnesyl anchor attached to the 10. Grm6 (metabotropic glutamate receptor 6 (mGluR6): nob3, C-terminus of Tc. The TbTc crystal structure shows that Tb nob4 mice mainly consists of a seven-bladed propeller formed by seven WD repeats (flanked by conserved W and D residues). The metabotropic glutamate receptors are a family of G protein- The Rd4+/ mouse was found in stock carrying the inversion coupled receptors responsive to L-glutamate. mGluR6 is a mem- In(4)56Rk, which was induced in a DBA/2 J male (Roderick, Chang, ber of group III family of metabotropic glutamate receptors Hawes, & Heckenlively, 1997). The inversion is very large and expressed in ON-bipolar cells. It couples to a downstream G o encompasses nearly all of chromosome 4. The homozygous Rd4 protein termed Ga , but the molecular identities of the target mutation is lethal, but heterozygotes survive and exhibit autoso- enzyme and the synaptic cation channel are unknown. mal dominant retinal degeneration. The histological phenotype is The nob3 mouse mutant was discovered at The Jackson Labora- severe, the outer nuclear layer is reduced at 10 days of age, and ab- tory on the basis of its ERG phenotype (Maddox et al., 2008). The sent at 6 weeks. Kitamura et al. (2006) demonstrated that the dis- nob4 mutant was generated by ENU chemical mutagenesis and tal breakpoint of the inverted Rd4 chromosome 4 is localized in the screening of several generations of mutant mice (Pinto et al., second intron of the Gnb1 gene encoding Tb, whereas the proximal 2007). Both mutants have similar ERG and visual abnormalities breakpoint lies within the centromere (Fig. 7). The second intron is mimicking autosomal recessive congenital stationary night blind- upstream of ATG in exon 3, and therefore no protein is produced in ness (CSNB). The nob3 and nob4 retinal morphologies are normal. the mutant since the promoter region is lost due to the inversion. The most outstanding phenotype is lack of scotopic and photopic Tb in complex with Tc regulates the activity of transducin. In con- b-waves; a-waves differ only slightly from those of WT. trast to Ta (Gnat1), which is expressed only in rods, Tb is expressed Nob3 and nob4 have defects in the Grm6 gene encoding metab- in brain, liver and other tissues, explaining the lethal effect of the otropic glutamate receptor 6 (mgluR6) located in bipolar cell den- homozygous Rd4 mutation. In contrast to the severe effect of the dritic terminals. Gmr6(nob3) carries a C ? T transition at position Gnb1 null allele, Gnat1 null mice exhibit a very slowly progressing 648 of intron 1, a change that creates a new donor splice site and retinal degeneration (Calvert et al., 2000). In human, no CNB1 a new short exon (exon 1a in Fig. 9B). The new exon derails the mutations associated with disease have been identified to date. ORF of the downstream exon truncating the mGluR6(nob3) protein. Nob4 carries a missense mutation (S185P) in exon 2 9. Gnb3 (cone tranducin b-subunit): retinopathy globe enlarged (Fig. 9A). S185 is located in the glutamate binding domain of (rge) chicken mgluR6, most likely affecting the trafficking of mGluR6(S185P) to ON-bipolar cell terminals and stability of the protein. Both mutants Cone transducin is encoded by Gnat2 (a-subunit), Gnb3 (b-sub- are undetectable by immunoblot, therefore both mutations gener- unit), and Gngt2 (c-subunit). The b-subunit is ubiquitously ate null alleles. Aside from absent b-waves, the null mutations expressed, but at particularly high levels in cones. A human mostly affect the ON-bipolar cell pathway leading to a reduction GNB3 splice variant is associated with hypertension, obesity in visual function (nice review: McCall & Gregg, 2008). The nob4 and diabetes. The structure of chicken GNB3 is likely very close phenotype is identical to that of the laboratory generated Grm6 to bovine GNB1 which has been determined by crystallography knockout (Masu et al., 1995). Different sets of mutations in GRM6 2642 W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652

Fig. 9. The Grm6 gene and nob3/nob4 gene defects. The Nob3 mutant is generated by a SNP in intron 1 (C648T) that produces a new splice site. As a result, a new exon (X1a) is created, as shown in B (red sequence). Grm6(nob4) contains a missense mutation in exon 2 (S185P). The blue boxes 1–7 indicate the seven TM domains of mGluR6. have been described in human recessive CSNB (Dryja et al., 2005), from that of the Gucy2e knockout mouse in which rod photorecep- but human and mouse phenotypes are very similar. tors still function (Yang et al., 1999). This distinction is explained by the ability of a closely related GC (Gucy2f) in mouse rods to 11. Gucy2D (guanylate cyclase 1): rd (GUCY1B) chicken compensate for the loss of Gucy2e (Baehr et al., 2007). GUCY2F apparently has lost this ability in chicken and human rods. Membrane guanylate cyclases (GCs) synthesize cGMP, the sec- ondary messenger of phototransduction. Photoreceptors con- 12. Mertk (receptor tyrosine kinase expressed in monocytes, tain two GCs, here termed GC1 and GC2. GC1 and GC2 are epithelia, and reproductive tissues): RCS (Royal College of activated by two GC-activating proteins, termed GCAP1 and Surgeons) rat GCAP2. GCAPs are Ca2+ sensors sensitive to changes in free [Ca2+] in the cytoplasm of photoreceptors, and activate GCs in MERTK is expressed in monocytes and tissues of epithelial and low free [Ca2+]. This mechanism leads to restoration of dark reproductive origin. Murine Mertk is homologue of the human cGMP levels and opening of CNG channels in the final step of c-mer receptor tyrosine kinase proto-oncogene (Graham et al., recovery from photobleaching. 1995). Mertk encodes a transmembrane protein with two extracellular fibronectin and two immunoglobulin-like domains, and The rd (GUCY1B) chicken is a naturally occurring blind mutant an intracellular tyrosine kinase domain. The MERTK polypep- discovered in a Rhode Island Red flock about 30 years ago (re- tide presumably plays a role in adhesion of the RPE to photore- viewed in Semple-Rowland and Cheng (1999), Ulshafer, Allen, ceptor outer segments. Null mutations in the human MERTK Dawson, and Wolf (1984). As a hallmark, photoreceptors of the gene are associated with recessive RP (Gal et al., 2000) and rd/rd retina appeared normal at hatch, but without a measurable cone–rod dystrophy (McHenry et al., 2004). electroretinogram. Degeneration of the rods and cones begins approximately at P7, and progresses relatively slowly over many The long-studied RCS rat is an animal model for recessive RP months. The genetic defect (Fig. 10) is a large deletion of parts (Bourne, Campbell, & Tansley, 1938). This rat strain was developed the extracellular domain, the transmembrane domain and parts by Richard Sidman and John Dowling from stock obtained from Ar- of the kinase-like domain of GC1 (GUCY1B in chicken), generating nold Sorsby of the Royal College of Surgeons, London (hence the a null allele (Semple-Rowland & Lee, 2000; Semple-Rowland, Lee, name) (Dowling & Sidman, 1962; Reading & Sorsby, 1966). Con- Van Hooser, Palczewski, & Baehr, 1998). In the absence of GC, genic RCS strains were later established (LaVail, Sidman, & Ger- cGMP levels in the mutant retina were very low and unable to sus- hardt, 1975). The genetic defect renders the RPE unable to tain phototransduction, which presumably leads to permanent clo- phagocytose rod outer segments, a circadian-controlled disk shed- sure of the CNG channels and elimination of the dark current ding process that is vital for rod and cone photoreceptor outer seg- (constitutive hyperpolarization). Vector-mediated expression of ment renewal. The RPE can bind, but is unable to ingest, shed outer bovine GC-E in the photoreceptors of rd chickens restored photore- segment membrane. The rdy (retinal dystrophy) locus was identi- ceptor function in mutant animals demonstrating that the rd phe- fied by positional cloning and gene sequencing revealed a deletion notype is indeed caused by deletion of GC-E (Williams et al., 2006). of the second coding exon in the receptor tyrosine kinase mertk The rd chicken phenotype closely mimics human LCA type 1 (Fig. 11)(D’Cruz et al., 2000). Exon 3 is out of frame leading to caused by a GUCY2D null allele (Perrault et al., 2000), but is distinct frameshift and a stop codon after codon 19. The consequences of Mertk null allele include subretinal accumulation cellular debris, and rod degeneration followed by cone degeneration as is typical for recessive RP. Numerous RPE transplantation experiments have been con- ducted in the RCS rat since the early 1980s. Anatomical rescue (thickness of outer nuclear layers) has been very encouraging, although functional rescue (full-field ERGs) has been very mixed (review: Da Cruz, Chen, Ahmado, Greenwood, & Coffey, 2007). Fig. 10. The gene defect of the retinal degeneration (rd) chicken, gene symbol AAV-MERTK delivery to an RCS rat retina resulted in correction of GUCY1B). Exons 4–7 were deleted and replaced by a 81 bp fragments with high the phagocytosis defect, increased sensitivity of treated eyes to sequence similarity to exon 9. The deletion removes the single transmembrane domain of guanylate cyclase 1 (Gucy2e in mouse). The defect is modeled on the low intensity light, and photoreceptor preservation (Vollrath mouse Gucy2e gene (the chicken GC1 gene has not been cloned and sequenced). et al., 2001). W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652 2643

Fig. 11. The Mertk gene and protein. In the RCS rat, exon 2 is deleted causing a frameshift, deleting all of the molecule after codon 19. MERTK has a protein kinase domain (yellow), immunoglobulin-like domains (IG), and ﬁbronectin (FN) domains.

13. Mfrp (membrane-type frizzled–related protein): rd6 mouse signalling. C1QTNF5 has a leader sequence and is likely secreted into the interphotoreceptor matrix (Won et al., 2008). Both MFRP Mfrp is a recently identified gene encoding a frizzled-related and C1QTNF5 are associated with human retina disease (nanoph- protein with a cysteine-rich domain, and a binding domain for thalmos, a rare disorder of eye development characterized by ex- the WNT glycoproteins, which have important roles in embryo- treme hyperopia (farsightedness) and late-onset retinal macular genesis. Mfrp is expressed in brain and RPE, its precise function degeneration) (Shu et al., 2006; Sundin et al., 2005). is unknown. 14. Myo7a (myosin VIIa): Shaker-1 (sh1) mouse The rd6 mouse was discovered in an inbred mouse strain, derived from offspring of irradiated mice. A congenic line was established at Myosin VIIa is a molecular motor composed of two heavy chains The Jackson Laboratory (Hawes et al., 2000). Ophthalmoscopic (250 kDa) that move towards the plus-end of actin filaments (or examination of rd6/rd6 mice showed discrete subretinal spots across barbed end, the site where the filaments grow). Myosin VIIa is the retina, appearing by 8–10 weeks postnatally. The spots were involved in light-dependent transport of RPE melanosomes caused by large subretinal cells filled with membraneous and lipo- from the cell body to the apical processes. fuscin-like material, a phenotype that resembles the human condi- tion retinitis punctata albescens. Rd6 outer segments appear The shaker-1 mouse is a model for Usher syndrome 1B (USH1B), disorganized and shorten progressively. The rd6 ONL is about half the most common form of blindness and deafness in humans (Weil thickness at 2 months of age (Won et al., 2008). et al., 1995). Premature stop codons in the human MYO7A gene Rd6 mice carry a donor splice mutation in the Mfrp gene caused cause cytoskeletal abnormalities, including abnormal organization by a 4 bp insertion in intron 3, resulting in skipping of exon 4 (aa of microtubules in the cilium of photoreceptor cells, nasal cilia 91–140) without frameshift (Kameya et al., 2002)(Fig. 12). The cells, sperm cells, as well as widespread degeneration of the organ mutant Mfrp gene is a null allele (exon 4 contains a portion of of Corti (Weil et al., 1995). the transmembrane domain (TM)). Interestingly, the Mfrp gene is The original shaker-1 mutation (sh1) was found as a naturally expressed as a dicistronic message with C1qtnf5 (complement – occurring mutant on the Balb/C background (Lord & Gates, 1929) C1q tumor necrosis factor-related protein 5), producing an mRNA and maintained at The Jackson Laboratory. Sh1/sh1 mice show cir- with two open reading frames. MFRP (584 amino acids) has a (pre- cling, head-tossing, deafness, and hyperactivity phenotypes, dicted) N-terminal transmembrane domain and C-terminal cys- mainly due to inner ear dysfunction. The sh1 gene was shown to teine-rich frizzled-like domain (CRD) possibly involved in Wnt encode a mutant form of the myosin VIIa motor carrying a missense mutation in the myosin head (Gibson et al., 1995). The mutation corresponds to R241P on the myosin 7a isoform 1 (Fig. 13) near a putative actin binding site. A second mutation, sh16J (R241P, Fig. 11), arose on the C57BL background (Gibson et al., 1995). Defective melanosome distribution in the retinal pigment epithelium (RPE) of shaker-1 mice can be observed (Liu, Ondek, & Williams, 1998). Myosin VIIA is also thought to facilitate opsin transport in photoreceptors, however the sh1 retina does not degenerate (Liu, Udovichenko, Brown, Steel, & Williams, 1999). Fig. 12. The Mfrp gene (rd6). Exon 4 (red) is skipped in the rd6 mouse. The Mfrp is flanked by C1qtnf5, such that the 30-UTR of Mfrp also is the 50-UTR of C1qtnf5 Williams and collaborators showed in a Myo7a null mouse consisting of two exons. A bicistronic mRNA is transcribed where C1qtnf5 mRNA is (4626SB allele, generated by ENU chemical mutagenesis) that in- part of the 30UTR of Mfrp. The MFRP protein has a transmembrane domain (TM), an gested ROS membranes fail to clear normally during phagocytosis extracellular cysteine-rich frizzled domain (fz) and two CUB domain profiles by the RPE (Gibbs, Kitamoto, & Williams, 2003). Absence of Myo7a, (named after initials of Cir, Cis, uEGF, and bone morphogenetic protein) (Stohr, however, does not block phagocytosis. Berger, Frohlich, & Weber, 2002).

Fig. 13. The mouse Myo7a gene and protein. Missense mutations sh16 J (exon 7) and sh1 (exon 13) are located in the myosin head region. A third mutation, Q720ter, was generated by ENU mutagenesis. R and E in the protein schematic are arginine- and glutamic acid-rich regions. Myth4 are predicted microtubule-binding domains, Ferm domains participate in the linkage of cytoplasmic proteins to the membrane. 2644 W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652

15. Nr2e3 (nuclear receptor subfamily 2, group E, member 3): rd7 mouse

Nuclear receptors are transcription factors which act as ligand- inducible transcription regulators controlling the activity of specific gene networks during development and differentiation (Wurtz et al., 1996). NR2E3 is preferentially expressed in rods, where it acts in concert with other transcription factors to reg- Fig. 15. The mouse Nyx gene and protein. The 85 bp deletion in exon 3 truncates the protein, eliminating a predicted GPI anchor at the C-terminal region. ulate photoreceptor-specific gene expression. Rd7 mice display recessive retinal degeneration characterized The nob gene, located on the X chromosome, was shown to en- by whorls and rosettes in the ONL. Rosettes form early, around code a novel protein termed nyctalopin, a small leucine-rich glyco- P13, but disappear eventually, around 16 months (Akhmedov protein (SLRPs) (Gregg et al., 2003). It has an N-terminal leader et al., 2000). Rosette-formation requires the presence of cones, sequence and is predicted to be GPI-anchored, but biochemistry since transgenic ablation of cones prevents the phenotype (Chen showing this is absent. The nyx sequence shows several LRRs (leu- & Nathans, 2007). Onset of retinal degeneration is relatively late, cine-rich repeats) which are 20–29 residue sequence motifs pres- rod and cone ERGs are still 50% of normal at 16 months of age. ent in many proteins that participate in protein–protein Recently it was shown that expression of the phenotype depends interactions. The mouse nyx gene contains three exons, most of on genetic modifiers present in some strains (Haider et al., 2008). the protein is encoded by exon 3. The nob gene defect consists of The rd7 gene was identified as a photoreceptor-specific nuclear an 85-bp deletion in exon 3 (Fig. 15). This deletion causes a frame- receptor NR2E3 (Akhmedov et al., 2000), also known as PNR shift that adds 170 foreign C-terminal amino acids to the 188-res- (Kobayashi et al., 1999). On the RNA level, the genetic defect was idue N-terminal portion of nyctalopin, likely eliminating protein identified as a deletion of exons 4 and 5 (Fig. 14)(Akhmedov function. et al., 2000); a gene analysis revealed that exons 4 and 5 are si- The nyx gene is expressed in the ONL, INL, and GCL (Gregg et al., lenced by multiple mutations, including a nonsense codon, and 2003), and nyctalopin can be immunolocalized to the OPL (photo- skipped by alternative splicing (Haider, Naggert, & Nishina, receptor/bipolar cell synapse) and the INL (rod bipolar cells) (Mor- 2001). Exons 4 and 5 encode a ligand-binding domain (LBD) typical gans, Ren, & Akileswaran, 2006). Transgenic expression of of nuclear hormone receptors (Wurtz et al., 1996), but no ligand nyctalopin-EYFP under the control of the murine GABA q1 pro- has been identified. Exons 1–3 encode the DNA binding domain C moter rescued the nob phenotype. The fusion protein was detected containing two Zinc-finger motifs. Nearly simultaneously with exclusively in the OPL, at the depolarizing bipolar cell dendritic ter- identification of the rd7 gene, mutations in the human NR2E3 gene minals, and colocalizing with metabotropic glutamate receptor 6 were shown to cause recessive ‘‘enhanced S-cone syndrome” (Gregg et al., 2007). (ESCS), characterized by 30x elevated S-cone sensitivity (Haider et al., 2000) and increased number of cones (Milam et al., 2002). Correspondingly, the rd7/rd7 retina shows a significant increase 17. PDE6a (PDE6a-subunit): rcd3 Cardigan Welsh corgi dog in cones expressing S-opsin (2–3-fold) (Haider et al., 2001). The expression pattern of NR2E suggests that it may serve as a repres- Cyclic GMP phosphodiesterase 6 (PDE6), the target enzyme in sor for cone cell proliferation (Haider et al., 2001), likely in concert the phototransduction cascade, is comprised of two catalytic with other transcription factors (Cheng et al., 2004). subunits (PDE6a and PDE6b) and two identical inhibitory subunits (PDE6c). PDE6ab activity is controlled by the transducin 16. Nyx (Nyctalopin): nob mouse a-subunit charged with GTP which displaces PDEc from its inhibitory site during the activation phase. Activation produces Nyctalopin (nyctalopia = nightblindness) is a small leucine-rich hydrolysis of cytoplasmic cGMP, closure of CNG-gated cation glycoprotein of unknown function, belonging to a larger family channels and hyperpolarization of the photoreceptor. Muta- of leucine-rich proteoglycans. Its closest relatives by sequence tions in the human PDE6A gene causing recessive RP in humans are chondroadherins (31% identity), glycoprotein 5 (31%) and are relatively common (Dryja, Rucinski, Chen, & Berson, 1999; synleurin (28%). Huang et al., 1995). The nob (no rod b-wave) mouse was identified by ERG in 1990 Progressive retinal atrophy (PRA) in the Cardigan Welsh corgi and is now recognized as a model for complete X-linked congenital was recorded first in the 1970s (Keep, 1972). Mutant puppies fail stationary nightblindness (CSNB1A). The rod a-wave and the cone to develop normal rod ERGs and exhibited reduced cone a-wave ERG responses are normal, suggesting functioning photoreceptors amplitudes (Tuntivanich et al., 2009). Elaboration of outer segment with no morphological abnormalities (Pardue, McCall, LaVail, membrane stagnates and photoreceptors eventually degenerate. Gregg, & Peachey, 1998). The nob ERG phenotype is similar to that The rcd3 defect consists of a 1-bp deletion in exon 15 of the PDE6A / / gene encoding the PDEa subunit (Fig. 16)(Petersen-Jones, Entz, & observed with Grm6 (mGluR6) and Gnao1 (Go alpha subunit) knockout mice (Dhingra et al., 2000; Masu et al., 1995). Sargan, 1999). The deletion produces a frameshift at codon 616 that truncates the PDEa subunit. Twenty-eight foreign residues are added at the C-terminus by the frameshift. If produced, the truncated protein would lack part of the catalytic domain and the farnesylated C-terminus. The hydrophobic farnesyl chain acts as membrane anchor tethering PDE6a to the disk membrane. The result of the truncation is an inactive PDE, most likely increasing cytoplasmic cGMP to toxic levels and leading to apoptosis. The PRA phenotype in this animal is similar to that of the rcd1 Irish setter carrying a null mutation in the PDE6b gene, described Fig. 14. The Nr2e3 gene and protein. Multiple mutations, including a nonsense as arrest in photoreceptor development and early onset photore- mutation, silence exons 4 and 5 that encode part of a ligand-binding domain. ceptor cell degeneration (Suber et al., 1993). Mouse models with W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652 2645

eny of Brueckner’s mice were analyzed by several research groups (reviewed by Farber, Flannery, and Bowes-Rickman (1994), Pittler, Keeler, Sidman, and Baehr (1993)), who determined that the degeneration of partially differentiated photoreceptor cells was probably unrelated to Keeler’s rodless retina, and therefore war- ranted a formal gene designation, retinal degeneration (gene symbol, rd). Reduced photoreceptor PDE activity with a resultant increase in cGMP levels (Farber & Lolley, 1974), mapping of the Fig. 16. The canine PDE6a gene. The rcd3 PDE6a gene defect is a deletion of 1 nt in rd gene to mouse chromosome 5 (Sidman & Green, 1965), and link- exon 15 (CAT domain, red). Also shown are two missense mutations (V685 M, age of Pde6b to rd (Danciger, Bowes, Kozak, LaVail, & Farber, 1990) D670G) generated by ENU mutagenesis in exons 18 and 19 of the mouse (canine and mouse PDE6a gene structures are identical). GAF1 and GAF2 contain noncat- focused attention on PDE6B as the causative gene. The Pde6b iden- alytic cGMP binding sites. Fa denotes farnesylation of the C-terminal cysteine. tity of the rd gene was established by subtractive cloning (Bowes et al., 1990) and by the identification of a nonsense mutation in exon 7 (codon 347) of the Pde6b gene (Pittler & Baehr, 1991) Pde6a mutations (V685 M, D670G) were recently generated by (Fig. 18). Determination as to whether r and rd were different ENU chemical mutagenesis (Sakamoto, McCluskey, Wensel, Nagg- genes was achieved when Steve Pittler extracted DNA from a ert, & Nishina, 2009). microscope slide (Fig. 17)ofKeeler’s vintage-1924 r/r retinal tissue. Amplification and sequencing of r exon 7 unambiguously established that r and rd were allelic, carrying the same nonsense muta- 18. PDE6b (PDE6 b-subunit): rd1/rodless mouse; rd10 mouse tion (Y347ter) (Pittler et al., 1993). Consequently, we assume that r mice survived WWII unknowingly and were re-discovered as rd PDE6b and PDE6a are the catalytic subunits of rod PDE6. mice (now classified as rd1). Domain structures of the two subunits are identical. The N-ter- The rd1/rodless phenotype consists of arrested photoreceptor minal half of each protein carries two non-catalytic cGMP bind- development at P8–10, failure to elaborate normal outer segment ing sites, termed GAF domains (Martinez, Beavo, & Hol, 2002), structure, and rapid degeneration following eye opening at P12. whereas the C-terminal half harbours the catalytic domain. C- By P17, 98% of rod nuclei have degenerated but cones are still pres- terminal cysteines are isoprenylated, a modification required ent (Carter-Dawson, LaVail, & Sidman, 1978). At 18 months, only for membrane association and vesicular transport. A functional about 1.5% of cones remain (Carter-Dawson et al, 1979). Transient PDE is critical for normal phototransduction. In human, PDE6B rescue of the degeneration phenotype by a bovine Pde6b gene con- null alleles are associated with recessive RP (McLaughlin, Ehr- struct under control of the opsin promoter demonstrated that the hart, Berson, & Dryja, 1995), and a H258 N missense mutation Pde6b defect produces the rd1 phenotype (Lem et al., 1992). Gene with dominant congenital stationary nightblindness (CSNBAD2) therapy with rAAV expressing mouse PDEb (Bennett et al., 1996) (Gal, Orth, Baehr, Schwinger, & Rosenberg, 1994). and HIV expressing HA-tagged PDEb (Takahashi, Miyoshi, Verma, Rodless, rd1 mouse. Over 80 years ago, Clyde Keeler first de- & Gage, 1999) were encouraging, although less successful. Single- scribed a naturally occurring mouse model of retinal degeneration stranded oligonucleotides were directed for an innovative, targeted (Fig. 17)(Keeler, 1924). Some of his mice lacked rod photorecep- gene repair in the rd1 mouse, but repair was estimated at less than tors (rodless, gene symbol r), a phenotype that segregated as an 1% (Andrieu-Soler et al., 2007). autosomal recessive trait. Keeler viewed this phenotype as a failure The rd10 mutant mouse, first reported in 2002 (Chang et al., of development because PN6 retinas were normal histologically. 2002), carries a missense mutation (R560C) in exon 13 of the Pde6b Keeler’s entire r colony, and all other known r stocks, were lost gene (Fig. 18)(Chang et al., 2007). A second missense mutation was by the end of World War II (Keeler, 1966). identified in exon 16 (L659P) of the nmf137 strain which arose after Wild mice caught near Basel, Switzerland. were shown to have ENU chemical mutagenesis. Both strains show autosomal recessive retinal degeneration by ophthalmoscopy (Brueckner, 1951). Prog- inheritance patterns. The nmf137 strain showed retinal degeneration at P21 but has not been further characterized. Both mutations are in the catalytic domain of PDEb probably diminishing PDE activity. Retinal degeneration in rd10/rd10 mice begins later than in the rd1 mouse (measurable scotopic ERG at P18–P30) but eventually progresses similarly as in the rd1 mouse: rods have disap- peared by P30–35, cones die later. PDE b immunoreactivity was detectable at reduced levels at P10 (it is undetectable in rd1 retina). Interestingly, dark-rearing rd10 mice delayed degeneration for at least a week. A recent study with rd10 mice showed that photoreceptor cell death is accompanied by dendritic retraction of bipolar and horizontal cells (Gargini, Terzibasi, Mazzoni, & Strettoi, 2007), consistent with retinal remodelling observed in degeneration models (Jones et al., 2003). Gene therapy using an AAV serotype 5 vector expressing PDE6b under the control of the chicken b-actin promoter showed significant delay of retinal degeneration of the rd10 phenotype (Pang et al., 2008).

19. PDE6b (PDE6 b-subunit): rcd1 Irish setter and Sloughi dog Fig. 17. Transverse section of rodless mouse retina. Left, a histological section generated by Clyde E. Keeler, re-photographed in 1993. The section, gifted to The rcd1 Irish setter, a prominent show dog, was the second ani- Richard Sidman in the 1960s, was used for DNA extraction and PCR ampliﬁcation (Pittler et al., 1993). Right, camera lucida drawing of rodless retina published by mal species in which a retinal degeneration was recognized early Keeler (1924). (1930s) (Parry, 1953). The recessive rod/cone dysplasia leads to a 2646 W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652

Fig. 18. The mouse Pde6b gene and protein. The rd1 Pde6b gene carries a proviral insertion in intron 1 and a disease-causing stop codon in exon 7. Exon 21 mutations found in rcd1 Irish setter and Sloughi dog are shown in blue. Relevant PDE6B domains: GAF domains (black), catalytic domain (red) and prenylated C-terminus (yellow). GAF domains are cyclic GMP binding sites, named after proteins that contain them: cGMP-specific and -regulated cNMP PDEs, Adenylyl cyclase, and E. coli transcription factor FhlA. rapidly progressing loss of photoreceptors after P13 (Aguirre et al., pletely. Heterozygous mice have disorganized outer segments 1982). First signs include arrest in photoreceptor development (no likely due to haploinsufficiency. Prph2 cDNA was identified by sub- outer segment formation), rod degeneration and later, cone degen- tractive cDNA cloning and the gene product was shown to be a tet- eration followed by inner retina remodelling – a phenotype typical raspanning glycoprotein localized to the periphery of disc for human recessive RP. Nearly all rods have degenerated by membranes (Travis, Brennan, Danielson, Kozak, & Sutcliffe, 1989). 5 months, and cone degeneration follows by about 1 year. cGMP The genetic defect was shown to consist of a 9.2 kb insertion of for- levels increase 10-fold over those of age-matched controls, which eign DNA into exon 2 (after N229) of the Prph2 gene (Ma et al., is reminiscent of the rd1 mouse phenotype. The rcd1 defect was 1995; Travis et al., 1989), an insert that is contained in the rd2 determined to be a nonsense amber mutation at codon 807 mRNA (Fig. 19). The insertion causes a frameshift and truncation (W807ter) (Clements, Gregory, Peterson-Jones, Sargan, & Bhattach- removing 117 C-terminal residues including the 4th TM domain arya, 1993; Suber et al., 1993) leading to a truncation of the PDEb and a sorting signal required for transport to the outer segment. subunit (Fig. 18). The truncation removes 49 amino acids of the C- The entire insertion is contained in the mutant rd2 mRNA. Final terminal region, including the geranylgeranylated C-terminus that evidence that the cloned gene represented Prph2 was provided mediates PDE membrane association. Although the PDEa subunit by transgenic rescue of the rd2 phenotype using a mouse opsin is unaffected and detectable by immunoblot, no PDE activity is promoter/Prph2 cDNA construct (Travis, Groshan, Lloyd, & Bok, measurable (Suber et al., 1993). This mutation is unidentified in 1992). Several efforts have been directed towards gene replace- other dog breeds. ment therapy since mutations in the PRPH2 gene are associated Sloughi dog. Blindness in Sloughi dogs was ascribed to an 8-bp with dominant retinitis pigmentosa, dominant macular dystrophy, insertion after codon 816 truncating the protein similarly as ob- digenic retinitis pigmentosa with ROM1, and dominant adult vitelli- served in the rcd1 Irish setter (Fig. 18)(Dekomien, Runte, Godde, form macular dystrophy. Subretinal injection of recombinant AAV & Epplen, 2000). This mutation cosegregates with disease status expressing Peripherin/rds resulted in stable generation of outer in a large pedigree. segment discs (Ali et al., 2000; Sarra et al., 2001).

20. Prph2 (peripherin/rds): rd2 mouse 21. Rd3: rd3 mouse

Peripherin/rds is a tetraspanning integral membrane protein Mice designated as rd3 were originally collected in Switzerland required for structural stability of outer segment membrane. Per- in 1969, and shown to have retinal degeneration at The Jackson ipherin/rds is related in sequence and origin to Rom1, another Laboratory (Chang et al., 2002). Photoreceptors start to degenerate tetraspanning integral membrane protein with which it forms at P14–21, and degeneration is complete at 16 weeks. The rd3 gene heterotetrameric complexes (Goldberg & Molday, 1996). (three exons, ﬁrst exon in 50-UTR) was identiﬁed by positional cloning (Friedman et al., 2006) and shown to encode a 195-residue The retinal degeneration slow (rds) mouse is a naturally occur- (22 kDa) soluble protein of unknown function. The rd3 mutation ring Prph2 knockout (Van Nie, Ivanyi, & Demant, 1978). This mu- results in a stop codon in exon 3 after codon 106 (Fig. 20). A donor tant was named ‘‘retinal degeneration slow” because its rate of splice site mutation at the end of exon 2 was observed in two sib- degeneration was much slower relative to that of the rd mouse. lings with LCA (Friedman et al., 2006). Rd2 mice are unable to form outer segments (Sanyal & Jansen, 1981), and reveal no scotopic or photopic ERG response (for review, see Goldberg (2006)). Although the degeneration begins at 22. Rho (rhodopsin): English mastiff dog PN14, it takes nearly a full year for the ONL to disappear com- Rhodopsin (Rho) is a G-protein coupled receptor (GPCR) present in rod photoreceptors. It is a heptaspanning transmembrane

Fig. 19. The Prph2 gene and rds mutation. The gene product is a glycoprotein with four TM domains. A large insert of foreign DNA into exon 2 produces the Prph2 null allele. The red bar designates a sorting signal required for transport to the OS (Tam, Fig. 20. The rd3 gene and proteins. The RD3 protein has no recognizable motifs. The Moritz, & Papermaster, 2004). mutation truncates the protein by 89 residues. W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652 2647

protein that uses 11-cis-retinal as chromophore. Rho is activated by light, an event that initiates the phototransduction cascade which results in visual perceptions by the CNS. Numer- ous mutations in the human RHO gene are known to cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration affecting rods first, then cones, and eventually leading to blindness. To identify English Mastiff dogs affected by PRA, roughly 500 dogs were screened and 29 affected were identified (Kijas, Miller, Pearce-Kelling, Aguirre, & Acland, 2003). Genetic analysis revealed a point mutation in exon 1 producing an amino acid change (T4R) Fig. 21. The Rpe65 gene. The Briard gene defect is a 4 nt deletion in exon 5. The rd12 mouse carries a stop codon in exon 3 (canine and murine Rpe65 genes structures (Kijas et al., 2002). T4 is located at the intradiscal (extracellular) are identical). side, and is predicted to affect the N-glycosylation of N2. Subse- quent biochemical analysis showed that Rho(T4R) was unglycosy- lated at N2, whereas glycosylation at N15 was unaffected (Zhu responses are severely attenuated while photopic responses are et al., 2004). The mutant opsin localized normally to the rod outer recordable. The gene defect was identified as a stop codon in exon segments indicating that biosynthesis and vesicular transport was 3, truncating RPE65 at codon 44 (R44ter) (Fig. 21). Phenotypically, / unaffected in T4R/+ dogs. Light-activated Rho(T4R) lost its chromo- Rpe65 and rd12 mice are very similar. phore faster and was less resistant to heat denaturation. Zhu et al. In recent years, much emphasis was directed towards gene proposed that the deleterious effect of the T4R mutation may be replacement therapy to develop treatments in Rpe65 null dogs mediated by T4R-opsin rather the Rho(T4R). Structurally, the and mice, with encouraging and significant success. Particularly mutation was suggested to affect mainly the ‘‘plug” at the intradi- large animal models like the Briard Beagle were considered very scal side of Rho which may protect the chromophore of rhodopsin attractive to test the efficacy of gene-based therapies in human pa- from access by water (Zhu et al., 2004). tients. As proof of principle, one eye of a Briard dog, called ‘‘Lance- Affected dogs exhibit a slowly progressing retinal degeneration lot”, blind since birth, was injected intraocularly with AAV2/2 virus that mimics dominant RP in humans with T4 K RHO mutations, expressing RPE65. Lancelot and other Briards showed significant including a slowed time course of rod recovery after light exposure. improvement of ERG responses that were stable for more than Mutant dogs are clinically normal for the first several months of 3 years (Acland et al., 2001, 2005b). A breakthrough was recently life before disease onset, and therefore present an animal model achieved in a phase 1 trial with human LCA2 patients using recom- for adRP ideally suited for gene therapy. Cideciyan et al. showed binant AAV-RPE65 virus (Maguire et al., 2008). While normal vi- that clinical photography caused retinal degeneration in the exact sion was not yet achieved, progress towards this goal has been montage pattern on the retina as the circular light flashes. Addi- made. tionally, degeneration rate correlated with light-dosage: moderate doses of light caused slow degeneration while higher doses of light 24. Rpgrip (RPGR-interacting protein): Miniature Longhaired accelerated retinal degeneration (Cideciyan et al., 2005). Dachshund

23. Rpe65 (RPE protein of 65 kDa): Swedish Briard/Briard RPGRIP interacts with RPGR through the C-terminal RID (RPGR- Beagle, rd12 mouse interacting domain). RPGRIP and RPGR are localized to the photoreceptor cilium in mouse, and RPGRIP is required for correct RPE65, a 65 kDa microsomal protein of the retinal pigment epi- localization of RPGR. Both proteins are involved in intraflagellar thelium (RPE), isomerizes all-trans-retinol to 11-cis-retinol transport through the cilium. Mutations in the RID domain of using retinyl esters as substrates (Jin, Li, Moghrabi, Sun, & Tra- human RPGRIP gene are associated with LCA. vis, 2005; Moiseyev et al., 2003). Without RPE65, the visual pig- The Miniature Longhaired Dachshund (MLDH) is the first canine ment chromophore (11-cis-retinal) cannot be regenerated by cone–rod dystrophy model for which the mutation has been char- the retinoid cycle, visual pigments are largely nonfunctional, acterized (Lheriteau et al., 2009). The MLDH is a model for reces- and photoreceptor cells degenerate. sive cone–rod dystrophy, a rare disease typically characterized by The Briard dog, originally described as being affected by con- early loss of cone photoreceptors. In homozygous MLDH, the genital stationary night blindness (CSNB), manifests visual abnor- 30 Hz cone flicker was barely detectable at 2 months (Turney malities of variable degree (Narfstrom, Wrigstad, & Nilsson, et al., 2007), and the cone ERGs was reduced at 6 months of age, 1989). Affected dogs are congenitally night blind, have much re- followed by rod b-wave reduction at a later stage (Lheriteau duced day vision, but have normal-appearing fundi. The gene de- et al., 2009). At 40 weeks, both rod and cone ERGs are unrecord- fect was identified as a 4-bp deletion (485delAAGA) in the mRNA able. A thinning of the ONL was followed by complete disappear- encoding RPE65 (Aguirre et al., 1998; Veske, Nilsson, Narfstrom, ance of the inner retina at 10 years of age (Lheriteau et al., 2009). & Gal, 1999). The deletion produces a frameshift and premature The genetic defect was identified as a 44 bp insertion in exon 2, termination of the polypeptide chain after codon 153 in exon 5 close to the donor splice site. The insertion alters the reading frame (Fig. 21). The mutant protein includes 52 RPE65-unrelated amino leading to a premature stop in exon 3 (Mellersh et al., 2006). The acids from residue 153 onward. insertion contains a stretch of 29 A flanked by 15 bp perfect Clinical features of the canine disease are quite similar to those repeats. described in human. Multiple mutations in this gene are associated with severe, early onset recessive LCA in humans (LCA-2 or RPE65- 25. RPGR (Retinitis pigmentosa GTPase Regulator): XLPRA1, LCA) (den Hollander et al., 2008; Thompson & Gal, 2003). XLPRA2 in dogs The rd12 mouse is a naturally occurring Rpe65 null mutant that was discovered in a single male mouse (Pang et al., 2005). Homo- The Rpgr gene, located on the X chromosome, generates multi- zygous rd12 mice develop white spots visible throughout the fun- ple splice variants of unknown function. The Rpgr-ORF15 vari- dus by ophthalmoscopy after 5 months of age. Scotopic ERG ant is most important for photoreceptor function. Multiple 2648 W. Baehr, J.M. Frederick / Vision Research 49 (2009) 2636–2652

Fig. 22. The canine Rpgr gene has a complex splicing pattern. The transcript containing the RCC1 (sequence similarity to regulator of chromatin condensation) domain encoded by exons 1–11, and the exon ORF14/15 is shown underneath the gene structure. Mutations causative for XLPRA1 and XLPRA2 are both located in ORF14/15 within a 100-bp interval. The ‘‘constitutive” transcript consisting of exons 1–13 and 16–19 is not depicted.

mutations in the ORF14/15 exon are associated with human X- structures show that the side chain of K330 intercalates linked RP (RP3). In mouse, RPGR is expressed in connecting cilia between the two phosphate groups (Santagata et al., 2001). This of rods and cones suggesting a function related to ciliary struc- interaction is abolished by activation of Gaq and PLCb which ture or intraflagellar transport. hydrolyses PIP2. The GPCR and the ligand of this cascade have not been identified. Subsequently TUB translocates to the The phenotype of the canine disease, ‘‘X-linked progressive ret- nucleus where it may be involved in gene regulation (reviewed inal atrophy” (XLPRA), is similar to human RP3, an X-linked form of in Carroll, Gomez, and Shapiro (2004)). retinitis pigmentosa, caused by mutations in the orthologous human gene. The original XLPRA was identified in Siberian Huskies, The tubby mouse arose spontaneously in a mouse colony at The a naturally occurring mutant. XLPRA has been renamed XLPRA1 Jackson Laboratory (Chang et al., 2002; Coleman & Eicher, 1990). to distinguish it from a second disease, XLPRA2, mapping to the The tubby phenotype is characterized by late onset obesity, reti- same gene (Zhang et al., 2002), but exhibiting a distinct phenotype. nal/cochlear degeneration, reduced fertility, and insulin resistance. XLPRA2 was identified in a mixed breed (mongrel) dog and could The combination of these phenotypes resembles Usher Syndromes not be traced to a specific breed (Zangerl, Johnson, Acland, & Agu- (retinal and cochlear degenerations), Bardet-Biedl and Alstrom} syn- irre, 2007). The XLPRA1 gene defect is a 5-bp deletion in the ORF15 dromes (obesity and neurosensory deficits). In the tubby mouse, exon of the Rpgr gene, resulting in a frameshift followed immedi- retinal degeneration begins around P21. The ERG responses are ately by a stop and removal of 230 C-terminal residues (Fig. 22). never normal, and are extinct at around 6 months of age. Obesity The XLPRA2 gene defect is a 2-bp deletion in ORF15, resulting in is observed relatively late, beginning at about age 3 months Fig. 23. a frameshift and the addition of 34 foreign amino acids. Both muta- The tub gene was identified by positional cloning (Kleyn et al., tions are located within a 100-bp interval in ORF15 (Zhang et al., 1996; Noben-Trauth, Naggert, North, & Nishina, 1996). TUB is ex- 2002). Functions of the constitutive variant of RPGR (consisting pressed in hair cells of the organ of Corti, in retinal ganglion cells, of exons 1–19) and the ORF15 splice variant (lacking exons 16– and inner segments of photoreceptors. The phenotype arose by 19) are unknown. mutation of a donor splice site at the 30 end of exon 11, abolishing XLPRA1 photoreceptors show normal morphology until early splicing of intron 11 (Fig. 24). Retaining intron 11 resulted in trun- adulthood. After age 6 months, the photoreceptor layer develops se- cation of 44 native amino acids of the TUB C-terminus, and addi- vere anomalies and retinal degeneration ensues. As is typical for RP, tion of 20 foreign amino acids. The tubby phenotype is identical cones appear to survive longer. In XLPRA2, disease phenotype is more severe, retina development is aberrant, and outer segments are highly disorganized and disoriented during photoreceptor development (Beltran, Hammond, Acland, & Aguirre, 2006). Scotopic and photopic ERG responses in XLPRA1 are stable for more than 1 year, but decline significantly by 2.5 years (Zhang et al., 2002). In contrast, XLPRA2 scotopic ERG responses are absent by 1 year of age.

26. Tub (tubby protein or TUB): tubby (rd5) mouse

The function of the TUB protein, a member of the tubby-like Fig. 24. The tubby gene defect is a G > T conversion of the ﬁrst nucleotide of intron protein (TULP) family, is unknown. TUB is anchored to the cyto- 11 (pos. 18,626 of the murine Tub gene, numbering starting with ATG), abolishing solic side of the plasma membrane by its afﬁnity to membrane- the donor splice site and deleting exon 12. NLS, putative nuclear localization signals

associated phosphatidylinositol(4,5)bisphosphate (PIP2). Crystal (K39KKR, R301KRKKsK). PIP2, phosphatidylinositol(4,5)bisphosphate binding site.

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