NR2E3 mutations in enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP)

MUTATION UPDATE

Human Mutation OFFICIAL JOURNAL

NR2E3 Mutations in Enhanced S-Cone Sensitivity Syndrome (ESCS), Goldmann-Favre Syndrome (GFS), Clumped Pigmentary Retinal Degeneration (CPRD), and Retinitis Pigmentosa (RP)

www.hgvs.org

Daniel F. Schorderet1–3 and Pascal Escher1,2 1

Institute for Research in Ophthalmology, Sion, Switzerland; 2Department of Ophthalmology, University of Lausanne, Lausanne, Switzerland;

3

Ecole Polytechnique Fe´de´rale de Lausanne, Lausanne, Switzerland

Communicated by Mark H. Paalman Received 1 May 2009; accepted revised manuscript 13 July 2009. Published online 22 July 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/humu.21096

Background ABSTRACT: NR2E3, also called photoreceptor-specific nuclear receptor (PNR), is a transcription factor of the nuclear hormone receptor superfamily whose expression is uniquely restricted to photoreceptors. There, its physiological activity is essential for proper rod and cone photoreceptor development and maintenance. Thirtytwo different mutations in NR2E3 have been identified in either homozygous or compound heterozygous state in the recessively inherited enhanced S-cone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), and clumped pigmentary retinal degeneration (CPRD). The clinical phenotype common to all these patients is night blindness, rudimental or absent rod function, and hyperfunction of the ‘‘blue’’ S-cones. A single p.G56R mutation is inherited in a dominant manner and causes retinitis pigmentosa (RP). We have established a new locus-specific database for NR2E3 (www.LOVD.nl/eye), containing all reported mutations, polymorphisms, and unclassified sequence variants, including novel ones. A high proportion of mutations are located in the evolutionarily-conserved DNA-binding domains (DBDs) and ligand-binding domains (LBDs) of NR2E3. Based on homology modeling of these NR2E3 domains, we propose a structural localization of mutated residues. The high variability of clinical phenotypes observed in patients affected by NR2E3-linked retinal degenerations may be caused by different disease mechanisms, including absence of DNA-binding, altered interactions with transcriptional coregulators, and differential activity of modifier genes. Hum Mutat 30:1475–1485, 2009. & 2009 Wiley-Liss, Inc. KEY WORDS: NR2E3; PNR; nuclear hormone receptor; transcription factor; retina; retinal degeneration

Additional Supporting Information may be found in the online version of this article. Correspondence to: Pascal Escher, Institute for Research in Ophthalmology, Av.

Grand-Champsec 64, CH-1950 Sion, Switzerland. E-mail: [email protected]

Nuclear receptor, class 2, subfamily E, member 3 (NR2E3; MIM] 604485), also called photoreceptor-specific nuclear receptor (PNR), is a member of the nuclear hormone receptor superfamily of ligand-modulated transcription factors [Kobayashi et al., 1999]. The NR2E3 gene is located on chromosome 15q22.32 and spans over 7.5 kb (Fig. 1). A mature 2.2-kb mRNA is transcribed from the eight exons of the NR2E3 gene and translated into a polypeptidic chain of 410 amino acids, with an apparent molecular weight of 45 kDa [Chen et al., 1999; Haider et al., 2000]. NR2E3 shares an evolutionarily-conserved structural organization with all nuclear receptors (Supp. Fig. S1) [Mangelsdorf et al., 1995]. In the N-terminus, the highly variable A/B domain comprises a ligand-independent activator function (AF-1). The most conserved C domain forms a DNA-binding domain (DBD) of about 70 amino acids, consisting of two Cys4 zinc fingers, where four Cys residues coordinate the binding of a Zn atom (Fig. 2B). In vitro, unique so-called P- and D-boxes located in the first and second Cys4 zinc fingers, respectively, mediate binding to a DR1 response element; i.e., a direct repeat of the core motif AGGTCA spaced by one nucleotide [Kobayashi et al., 1999]. The DR1 consensus sequence for NR2E3 has been determined as 50 -(A/G)AG(A/G)TCAAA(A/G)(A/G)TCA-30 [Chen et al., 2005]. In vivo, binding of NR2E3 to response elements located in the promoters of target genes is not fully elucidated. The DBD is prolonged into a carboxy-terminal extension (CTE), also called a T/A box. No structural data is available for the flexible D domain, also called hinge domain, that links the DBD to the ligand-binding domain (LBD). The hinge domain contains a nuclear localization signal that may overlap on the DBD. The C-terminal LBD, or E/F domain, consists of 12 a-helices that fold into a conserved hydrophobic pocket, where a yet elusive natural ligand could bind to [Wolkenberg et al., 2006] (Fig. 2C). In addition to this liganddependent activator function AF-2, the LBD is also essential for homo- and heterodimerization. At a cellular level, NR2E3 expression is uniquely restricted to the outer nuclear layer of the neurosensory retina [Bookout et al., 2006; Haider et al., 2000], transcripts being present in putative immature human rods on the foveal edge at fetal week 11.7 [Bumsted O’Brien et al., 2004]. NR2E3 also accumulates in rod precursors of the developing mouse retina [Chen et al., 2005], where developmental studies have delineated a transcription factor cascade regulating photoreceptor development. The orthodenticle-like cone-rod homeobox transcription factor

& 2009 WILEY-LISS, INC.

p.R76W p.R76Q p.Y81C p.P276RfsX59 p.Q350X p.G88V p.L263P p.F71del p.L345KfsX2 p.Q350R p.R97H p.A256V p.N65_C67del p.L336P p.L353V p.R104W p.A256E p.G56R p.R334G p.P365P p.R104Q p.W234S p.V49M p.R311Q p.R385P p.E121K p.T161HfsX18 p.V41AfsX23 p.R309G p.M407K

A/B

DBD

hinge

LBD

ATG Exon

1

TAG 2

3 4

c.119-2A>C c.119-3C>G

5

6

c.361G>A c.481delA

7

c.701G>A c.747+1G>C

c.145G>A c.226C>T c.166G>A c.227G>A c.194_202del9 c.242A>G c.211_213del3

c.263G>T c.290G>A c.310C>T c.311G>A

0.5 kb

8

c.767C>A c.767C>T c.788T>C c.827_843del17 c.925C>G c.932G>A

c.1000C>G c.1007T>C c.1034_1038del5 c.1048C>T c.1049A>G c.1057C>G c.1095C>G

c.1154G>C c.1220T>A c.1101+1G>A

Figure 1.

Genomic organization of the human NR2E3 gene. NR2E3 is located on chromosome 15q22.32, corresponding to sequence NC_000015.8, nt 69,889,948–69,897,654. Coding sequences in the exons are shown by black boxes with the ATG translation initiation codon and the TAG stop codon indicated above. The 50 - and 30 -UTR are shown by white boxes. Exons 1 to 8 have a size of 308 bp, 127 bp, 104 bp, 222 bp, 176 bp, 247 bp, 106 bp, and 708 bp, respectively. According to the reference cDNA sequence NM_014249.2, where the 11 A of the ATG translation initiation codon is located at nt 191, exon 1 contains nt 1–118, exon 2 nt 119–245, exon 3 nt 246–349, exon 4 nt 350–571, exon 5 nt 572–747, exon 6 nt 748–994, exon 7 nt 995–1100, and exon 8 nt 1101–1233, respectively, of the coding sequence. Intronic sequences are shown as lines, and the size of introns 1–7 are 621 bp, 156 bp, 85 bp, 159 bp, 877 bp, 376 bp, and 3434 bp, respectively. All NR2E3 mutations are listed as they occur in the respective exons. Above the genomic sequence the NR2E3 protein structure is schematically drawn with the different functional domains. Mutations at protein level are indicated above.

(CRX; MIM] 602225) starts to be expressed at embryonic day 12.5 (E12.5) in retinal progenitor cells and commits them to the photoreceptor lineage [Chen et al., 1997; Freund et al., 1997; Furukawa et al., 1997]. The expression of the neural retina basic motif-leucine zipper transcription factor (NRL; MIM] 162080) starts at E14.5 and determines the rod lineage [Liu et al., 1996; Mears et al., 2001]. NRL binds to an enhancer region of the Nr2e3 gene and activates transcription in synergism with CRX [Oh et al., 2008]. Nr2e3 transcripts start to be detected at E16.5 and peak at postnatal day 6 (P6), concomitant with the onset of rhodopsin expression [Cheng et al., 2004; Oh et al., 2008]. In the absence of NR2E3, an about two-fold increase in short wavelength-sensitive (S)-opsin (Opn1sw) expressing cones (‘‘blue’’ cones) is observed, indicating that NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells [Haider et al., 2001, 2006]. A derepression of additional cone-specific genes, e.g., cone transducin (Gnat2), cone phosphodiesterases (Pde6c, Pde6h) cone arrestin (Arr3), but not M-opsin (Opn1mw), probably occurs in these cells from P14 on, in accordance with the reported in vitro repressor function of NR2E3 [Chen et al., 1999, 2005; Corbo and Cepko, 2005; Haider et al., in press; Kobayashi et al., 1999]. However, a majority of photoreceptors represent a morphologically hybrid cell type expressing both rod and cone genes, providing circumstantial evidence that NR2E3 is essential to suppress cone-specific gene expression in mature rods [Chen et al., 2005; Cheng et al., 2004; Corbo and Cepko, 2005; Peng et al., 2005]. Cone-specific gene expression is also suppressed in transgenic mice ectopically expressing NR2E3 under the control of the Crx promoter in photoreceptor precursor cells; instead of cones, nonfunctional rod-like photoreceptors are generated [Cheng et al., 2006]. With respect to Opn1sw regulation, the nuclear receptors NR1A2b (thyroid hormone receptor b2) and NR1F2 (retinoid-related orphan receptor b) probably act as

1476

HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

additional transcriptional repressors or activators, respectively [Ng et al., 2001; Srinivas et al., 2006]. NR2E3 acts as a transcriptional activator of several rod-specific genes, including rhodopsin [Cheng et al., 2004, 2006; Peng et al., 2005]. To achieve transcriptional regulation, NR2E3 physically interacts with the DBD of CRX [Peng et al., 2005]. CRX/NRL/NR2E3-mediated transcriptional activation of the rhodopsin gene is further enhanced by NR1D1 (also called Rev-erba) that interacts with NR2E3 through its LBD [Cheng et al., 2004]. The dual function of NR2E3 in mature rods appears to be regulated by the E3 small ubiquitin-related modifier (SUMO) ligase PIAS3 (protein inhibitor of activated Stat3) [Onishi et al., 2009]. PIAS3-dependent SUMOylation of NR2E3 converts it into a potent repressor of cone-specific gene expression. By blocking SUMOylation in photoreceptors, cells with morphological and molecular features of cones are observed, but no rod-specific markers. Consistent with the photoreceptor-specific expression of this transcription factor, mutations in NR2E3 are solely associated with retinal degenerations. NR2E3 mutations have first been associated with the recessively inherited enhanced short wavelength (S-) cone sensitivity syndrome, also called enhanced S-cone syndrome (ESCS; MIM] 268100) [Haider et al., 2000]. All patients presented with night blindness from early in life and are characterized by unique fullfield and spectral electroretinographic (ERG) findings [Jacobson et al., 1990; Marmor et al., 1990]. In response to blue and red stimuli, a hyperfunction of S-cones (‘‘blue’’ cones) was detected, with impaired M- and L-cone functions. The rod system sensitivity was severely reduced, with similar ERG waveforms under scotopic and photopic conditions. Further analyses of the patients ERG suggested that an increased number of S-cones caused the observed gain-of-function of S-cones [Hood et al., 1995]. Histological analysis of a postmortem retina from a

A

C

* *

B

Figure 2.

Structural analysis of NR2E3. A: Schematic representation of potential NR2E3-mediated transcriptional activity. NR2E3 (blue) has a modular domain structure with a DBD and a LBD. NR2E3 binds as a dimer and through its DBD photoreceptor-specific promoter regions (white), to which NRL (green) and CRX (dark green) also bind. NR2E3 intrinsically acts as a transcriptional repressor. SUMOylation of the NR2E3 LBD (stars) is necessary to repress cone-specific target genes (red bars), possibly by binding of corepressors in complex with histone deacetylases (HDAC) (purple). B: Homology modeling of the NR2E3 DBD. In the N-terminus of the first Cys4 Zn-finger, two b-sheets (green) are predicted to span from G56 to H58, and I61 to A63, respectively. In the C-terminus of the first Cys4 Zn-finger, an a-helix spans from N65 to R77 (orange). The a-helix of the second Cys4 Zn-finger (red) lies perpendicular to the first one and is predicted to extend from Q101 to A111. The C-terminal extension of the DBD is formed by an a-helical structure starting at D116 (dark red). Each Cys4 Zn-finger coordinates one Zn atom (white ball). The NR2E3 DBD was aligned on the RXRa DBD (PDB_1BYA) using SWISS-Model and DeepView 4.0 [Arnold et al., 2006]. C: Homology modeling of the NR2E3 LBD. The LBD is predicted to form a hydrophobic pocket formed by 12 a-helices (h) and 2 b-sheets (s). An additional a-helix, h0 (dark blue), is predicted to form in the C-terminus of the hinge region. Localization of the a-helices and the b-sheets are indicated in Supp. Figure S1. The NR2E3 LBD was aligned on the RXRa LBD (PDB_1LBD) using SWISS-Model and DeepView 4.0 [Arnold et al., 2006].

77-year-old ESCS patient later confirmed an absence of rods and an increase by approximately two-fold of the number of cones, 92% of which were S-cones [Milam et al., 2002]. Additional clinical findings with an important variability and onset between patients, even inside a same family, including: cystoid maculopathy; retinal degeneration in the region of the vascular arcades, ranging from yellow flecks to clumped or nummular pigment deposition; relative ring scotomas; macular retinoschisis; disorganized retinal lamination; rosette formation in the outer nuclear layer; subfoveal neovascularization; and reduced visual acuity; posterior subcapsular cataracts [Audo et al., 2008; Cideciyan et al., 2003; Greenstein et al., 1996; Hayashi and Kitahara, 2005; Jacobson et al., 1990, 2004; Jurklies et al., 2001; Marmor et al., 1990; Nakamura et al., 2004; Pachydaki et al., 2009; Vaclavik et al., 2008; Yamamoto et al., 1999]. The Goldmann-Favre syndrome (GFS; MIM] 268100) shared several clinical features with ESCS. GFS was initially described as an autosomal recessive vitreoretinal degeneration characterized by night blindness, pigmentary degeneration, macular and peripheral retinoschisis, posterior subcapsular cataract, markedly abnormal and undetectable electroretinograms, and degenerative vitreous changes, such as liquefaction, strands, or bands [Favre, 1958]. Because appropriate ERG analyses demonstrated a relatively

enhanced S-cone function in GFS patients, it was concluded that ESCS and GFS were not distinct entities but simply two identifiable phenotypes in a wide spectrum of clinical expression of a same retinal degeneration [Jacobson et al., 1991]. Consistently, NR2E3 mutations were also identified in the patients affected by GFS [Bernal et al., 2008; Chavala et al., 2005; Marmor, 2006; Pachydaki et al., 2009; Sharon et al., 2003]. Patients affected by a rare retinal degeneration termed clumped pigmentary retinal degeneration (CPRD), showed some clinical signs reminiscent of ESCS and GFS [To et al., 1996]. CPRD was recessively inherited, patients did report night blindness early in life and showed numerous clumped pigment deposits throughout the midperipheral fundus, instead of pigment deposits with a bone-spicule-type found in retinitis pigmentosa (RP). ERG analyses, however, showed a reduction in night and peripheral vision similar to that of RP patients. NR2E3 mutations were identified in several patients affected by CPRD [Bernal et al., 2008; Sharon et al., 2003]. Additionally, a number of patients initially diagnosed with autosomal recessive RP (arRP) also carried NR2E3 mutations [Bandah et al., 2009; Bernal et al., 2008; Gerber et al., 2000]. A distinct c.166G4A (p.G56R) mutation located in the first Cys4 zinc finger of the NR2E3 gene was identified as a causal HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

1477

mutation for autosomal dominant RP (adRP), and termed RP37 (MIM] 611131) [Coppieters et al., 2007; Escher et al., 2009; Gire et al., 2007]. The phenotype corresponded to that seen in classic adRP, with progressive degeneration of rods and subsequent involvement of cones. Some more ESCS-like clinical findings were however present, namely the presence of mixed retinal deposits of both spicule-like and clumped/nummular type. The present mutation update includes the many new NR2E3 mutations that have been reported in recent years, as well as new polymorphisms and unclassified sequence variants [Audo et al., 2008; Bandah et al., 2009; Bernal et al., 2008; Coppieters et al., 2007; Lam et al., 2007; Pachydaki et al., 2009]. Functional studies of mutant alleles, homology modeling of NR2E3, animal models of NR2E3 diseases, and clinical remarks are also presented. To facilitate future NR2E3 mutation updates, a locus-specific mutation database has been established.

Variants in the NR2E3 gene Mutations So far, a total of 33 different mutations of NR2E3 have been identified in a group of 209 patients belonging to 83 independent families (Tables 1 and 2). Mutations were defined as sequence variants segregating with the disease within the families and that were absent in unaffected control individuals. Mutations occur in

Table 1. Region Intron 1 Intron 1 Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 3 Exon 3 Exon 3 Exon 3 Exon 4 Exon 4 Exon 5 Intron 5 Exon 6 Exon 6 Exon 6 Exon 6 Exon 6 Exon 6 Exon 7 Exon 7 Exon 7 Exon 7 Exon 7 Exon 7 Exon 7 Intron 7 Exon 8 Exon 8

all exons of NR2E3, except the ATG-containing exon 1, and in several exon-intron boundaries (Fig. 1). There are 22 missense mutations (67%), five deletions (15%), four splice-site changes (12%), one nonsense mutation (3%), and 1 silent mutation (3%); i.e., p.P365P, that presumably creates a cryptic splice donor site yielding a truncated p.P365fsX10 protein [Wright et al., 2004]. With the exception of mutations leading to truncated NR2E3 proteins, all disease-linked mutations are located in the evolutionarily-conserved DBD and LBD of NR2E3 (Supp. Fig. S1). A majority of missense mutations occur at amino acid residues that are strictly conserved in vertebrates and even echinoderms (Supp. Fig. S1). Fifteen mutations (45.5%) are present in a homozygous state in 96 patients from 46 independent families. The remaining 114 patients from 44 independent families are compound heterozygotes for NR2E3 mutations. The most frequent mutations are the c.119–2A4C (p.V41AfsX23), c.932G4A (p.R311Q), and c.166G4A (p.G56R) mutations. The c.119–2A4C mutation is carried by 23 patients (from 14 families) in a homozygous state, and in a compound heterozygous state with 12 different NR2E3 mutations by another 21 patients (from 19 families). Fifty-one patients (from 18 families) are homozygous for the c.932G4A mutation and another 14 patients (from 13 families) are heterozygous carriers with eight different NR2E3 mutations. The adRP-linked c.166G4A (p.G56R) mutation is present in a heterozygous state in 83 patients from eight independent families, accounting for approximately 1 to 2% of

Mutations in the NR2E3 Gene DNA sequence changea c.119–2A4C c.119–3C4G c.145G4A c.166G4A c.194_202del9d c.211_213del3 c.226C4T c.227G4A c.242A4G c.263G4T c.290G4A c.310C4T c.311G4A c.361G4A c.481delA c.701G4A c.74711G4C c.767C4A c.767C4T c.788T4C c.827_843del17 c.925C4G c.932G4A c.1000C4G c.1007T4C c.1034_1038del5 c.1048C4T c.1049A4G c.1057C4G c.1095C4G c.1101-1G4A c.1154G4C c.1220T4A

Protein changeb

Functional domain

p.V41AfsX23 n.d. p.V49M p.G56R p.N65_C67del p.F71del p.R76W p.R76Q p.Y81C p.G88V p.R97H p.R104W p.R104Q p.E121K p.T161HfsX18 p.W234S n.d. p.A256E p.A256V p.L263P p.P276RfsX59 p.R309G p.R311Q p.R334G p.L336P p.L345X p.Q350X p.Q350R p.L353V p.P365P n.d. p.R385P p.M407K

n.a. n.a. DBD DBD DBD DBD DBD DBD DBD DBD DBD DBD DBD DBD Hinge LBD n.a. LBD LBD LBD LBD LBD LBD LBD LBD LBD LBD LBD LBD LBD n.a. LBD LBD

Predicted effect/in vitro studiesc Skipping of exon 2 Skipping of exon 2 No DNA binding

No DNA binding No DNA binding No No No No

DNA DNA DNA DNA

binding binding binding binding

No LBD Impaired repression Skipping of exon 6

Truncated LBD Impaired repression

Truncated LBD Truncated LBD

Aberrant splicing Skipping of exon 8 Impaired repression Impaired repression

First description Haider et al. [2000] Audo et al. [2008] Audo et al. [2008] Coppieters et al. [2007] Haider et al. [2000] Pachydaki et al. [2009] Haider et al. [2000] Haider et al. [2000] Audo et al. [2008] Wright et al. [2004] Haider et al. [2000] Haider et al. [2000] Hayashi et al. [2005] Haider et al. [2000] Wright et al. [2004] Haider et al. [2000] Bandah et al. [2009] Sharon et al. [2003] Lam et al. [2007] Wright et al. [2004] Sharon et al. [2003] Haider et al. [2000] Haider et al. [2000] Hayashi et al. [2005] Wright et al. [2004] Bernal et al. [2008] Nakamura et al. [2004] Pachydaki et al. [2009] Wright et al. [2004] Wright et al. [2004] Audo et al. [2008] Haider et al. [2000] Haider et al. [2000]

n.d., not determined; n.a., not appropriate. a Mutations are numbered in accordance to the GenBank entry NM_014249.2, where 11 corresponds to the A of the ATG translation initiation codon; i.e., nucleotide 191. For intronic sequences, human genomic sequence NG_009113.1 was used. b Amino acid changes are numbered in accordance to the SwissProt entry Q9Y5X4. c The predicted effects and in vitro studies are discussed in the main text. d This mutation was initially reported as p.C67_G69del.

1478

HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

Table 2.

Genotypes of Patients Affected by NR2E3-Linked Retinal Degenerations

Allele1 (nt)a

Allele 2 (nt)a

Allele1 (aa)b

Allele 2 (aa)b

c.119–2A4C

c.119–2A4C

p.V41AfsX23

p.V41AfsX23

c.119–3C4G c.145G4A c.226C4T c.227G4A c.263G4T c.290G4A c.310C4T c.481delA c.925C4G c.932G4A

c.119–3C4G c.145G4A c.226C4T c.227G4A c.263G4T c.290G4A c.310C4T c.481delA c.925C4G c.932G4A

n.d. p.V49M p.R76W p.R76Q p.G88V p.R97H p.R104W p.T161HfsX18 p.R309G p.R311Q

n.d. p.V49M p.R76W p.R76Q p.G88V p.R97H p.R104W p.T161HfsX18 p.R309G p.R311Q

c.1034_1038del5 c.1048C4T c.1101–G4A c.1220T4A c.119–2A4C

c.1034_1038del5 c.1048C4T c.1101–G4A c.1220T4A c.194_202del9

p.L345X p.Q350X n.d. p.M407K p.V41AfsX23

p.L345X p.Q350X n.d. p.M407K p.N65_C67del

c.119–2A4C c.119–2A4C c.119–2A4C c.119–2A4C c.119–2A4C

c.242A4G c.290G4A c.310C4T c.481delA c.767C4A

p.V41AfsX23 p.V41AfsX23 p.V41AfsX23 p.V41AfsX23 p.V41AfsX23

p.Y81C p.R97H p.R104W p.T161HfsX18 p.A256E

c.119–2A4C c.119–2A4C c.119–2A4C c.119–2A4C

c.767C4T c.788T4C c.827_843del17 c.932G4A

p.V41AfsX23 p.V41AfsX23 p.V41AfsX23 p.V41AfsX23

p.A256V p.L263P p.P276RfsX59 p.R311Q

c.119–2A4C c.119–2A4C c.166G4A

c.1007T4C c.1095C4G Wild-type

p.V41AfsX23 p.V41AfsX23 p.G56R

p.L336P p.P365P Wild-type

c.166G4A c.194_202del9

c.932G4A c.932G4A

p.G56R p.N65_C67del

p.R311Q p.R311Q

c.211_213del3 c.311G4A c.481delA c.701G4A c.74711G4C c.925C4G c.932G4A c.932G4A

c.932G4A c.1000C4G c.1057C4G Unknown c.932G4A c.932G4A c.1049A4G Unknown

p.F71del p.R104Q p.T161HfsX18 p.W234S n.d. p.R309G p.R311Q p.R311Q

p.R311Q p.R334G p.L353V Unknown p.R311Q p.R311Q p.Q350R Unknown

Family/Patient 1/4 1/2 2/2 1/1 1/1 1/1 4/8 1/1 1/1 1/2 1/1 1/1 2/2 1/1 1/1 1/3 1/3 1/2 1/1 5/6 1/1 1/1 1/1 1/1 1/1 1/2 1/1 2/3 2/2 1/27 1/1 1/5 1/3 1/1 1/1 1/2 1/1 1/1 1/2 1/1 1/1 1/1 1/1 2/3 1/1 1/1 1/1 1/1 1/1 1/1 2/2 1/1 1/1 3/45 3/22 1/11 1/3 1/2 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

Ethnic origin Syrian/Lebanese Ashkenazi Jewish German Cuban Saudi Arabia Palestinian

Spanish

German British

Italian Ashkenazi Jewish Japanese Saudi Arabia Spanish Jewish-Italian Chinese Arab Emirates Spanish

Iberic Crypto-Jews Moroccan Jewish Palestinian Spanish Japanese Spanish/Italian Ashkenazi Jewish

British/American German British/Amerindian

British/Italian Western European Ashkenazi Jewish

British German Belgian/French Caucasian Swiss Jewish-American Jewish-American Ashkenazi Jewish

Japanese German British/German Ashkenazi Jewish Italian Ashkenazi Jewish

Clinical diagnostic ESCS ESCS ESCS ESCS ESCS ESCS ESCS GFS CPRD CPRD ESCS ESCS ESCS ESCS ESCS ESCS ESCS ESCS ESCS ESCS ESCS ESCS ESCS ESCS ESCS GFS GFS GFS CPRD arRP arRP arRP arRP ESCS ESCS ESCS ESCS GFS ESCS ESCS ESCS ESCS ESCS CPRD ESCS ESCS CPRD ESCS ESCS ESCS CPRD ESCS ESCS adRP adRP adRP adRP ESCS ESCS CPRD GFS ESCS ESCS ESCS ESCS ESCS GFS ESCS

Reference Haider et al. [2000] Wright et al. [2004] Wright et al. [2004] Wright et al. [2004] Khan et al. [2007] Audo et al. [2008] Bandah et al. [2009] Rayborn et al. [2008] Sharon et al. [2003] Bernal et al. [2008] Audo et al. [2008] Audo et al. [2008] Haider et al. [2000] Haider et al. [2000] Wright et al. [2004] Haider et al. [2000] Vaclavik et al. [2008] Audo et al. [2008] Galantuomo et al. [2008] Wright et al. [2004] Nakamura et al. [2002] Khan et al. [2007] Bernal et al. [2008] Iannaccone et al. [2009] Wang et al. [2009] Chavala et al. [2005] Bernal et al. [2008] Pachydaki et al. [2009] Sharon et al. [2003] Gerber et al. [2000] Bandah et al. [2009] Bandah et al. [2009] Bernal et al. [2008] Nakamura et al. [2004] Audo et al. [2008] Haider et al. [2000] Wright et al. [2004] Sharon et al. [2003] Audo et al. [2008] Sharon et al. [2003] Wright et al. [2004] Wright et al. [2004] Wright et al. [2004] Sharon et al. [2003] Lam et al. [2007] Wright et al. [2004] Sharon et al. [2003] Wright et al. [2004] Wright et al. [2004] Audo et al. [2008] Sharon et al. [2003] Wright et al. [2004] Wright et al. [2004] Coppieters et al. [2007] Gire et al. [2007] Escher et al. [2009] Escher et al. [2009] Escher et al. [2009] Bandah et al. [2009] Sharon et al. [2003] Pachydaki et al. [2009] Hayashi et al. [2005] Wright et al. [2004] Wright et al. [2004] Bandah et al. [2009] Wright et al. [2004] Pachydaki et al. [2009] Wright et al. [2004]

ESCS, enhanced S-cone syndrome; CPRD, clumped pigmentary retinal degeneration; GFS, Goldmann-Favre syndrome; arRP, autosomal recessive retinitis pigmentosa; adRP, autosomal dominant RP; Family/Patient, number of independent families and number of affected patients; nt, nucleotide; aa, amino acid; n.d., not determined; n.a., not appropriate. a Mutations are numbered in accordance to the GenBank entry NM_014249.2, where 11 corresponds to the A of the ATG translation initiation codon; i.e., nucleotide 191. For intronic sequences, human genomic sequence NG_009113.1 was used. b Amino acid changes are numbered in accordance to the SwissProt entry Q9Y5X4. The initial clinical diagnostic is mentioned for each group of patients. HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

1479

Table 3. Region Intron 1 Intron 1 Intron 1 Exon 2 Intron 2 Exon 3 Exon 3 Exon 4 Exon 4 Exon 4 Exon 5 Intron 5 Exon 6 Exon 6 Exon 6 Exon 7

Polymorphisms in the NR2E3 Gene as Reported in the Literature DNA sequence changea c.119–28T4C c.119–27dup16 c.119–27del16 c.210C4T c.24518C4T c.285C4T c.333G4T c.419A4G c.488T4C c.505C4T c.694G4A c.747190C4T c.864T4A c.882G4A c.904G4A c.1005C4A

Protein changeb n.d. n.d. n.d. p.F70F n.d. p.A95A p.A111A p.E140G p.M163T p.L169L p.V232I n.d. p.G288G p.T294T p.V302I p.G335G

Functional domain n.a. n.a. n.a. DBD n.a. DBD DBD Hinge Hinge Hinge LBD n.a. LBD LBD LBD LBD

First report Haider et al. [2000] Coppieters et al. [2007] Haider et al. [2000] This study Sharon et al. [2003] Audo et al. [2008] Haider et al. [2000] Haider et al. [2000] Haider et al. [2000] Haider et al. [2000] Haider et al. [2000] Bernal et al. [2008] Audo et al. [2008] Haider et al. [2000] Haider et al. [2000] This study

n.d., not determined; n.a., not appropriate. Mutations are numbered in accordance to the GenBank entry NM_014249.2, where 11 corresponds to the A of the ATG translation initiation codon; i.e., nucleotide 191. For intronic sequences, human genomic sequence NG_009113.1 was used. b Amino acid changes are numbered in accordance to the SwissProt entry Q9Y5X4. a

Table 4.

Unclassified Sequence Variants in the NR2E3 Gene

Region

DNA sequence changea

Exon 1 Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 6 Exon 6 Exon 6 3´-UTR

c.67G4C c.131C4T c.188C4A c.195C4T c.205G4A c.230G4A c.859G4A c.874C4T c.971A4G c.12301362_368del5c

Protein changeb

Functional domain

First report

p.A23P p.S44L p.A63D p.N65N p.G69S p.R77Q p.G287S p.R292W p.K324R n.d.

AF1 AF1/DBD DBD DBD DBD DBD LBD LBD LBD n.a.

This study Bernal et al. [2008] Coppieters et al. [2009] Bernal et al. [2008] This study Coppieters et al. [2007] Bernal et al. [2008] This study Bernal et al. [2008] Bernal et al. [2008]

n.d., not determined; n.a., not appropriate. a Mutations are numbered in accordance to the GenBank entry NM_014249.2, where 11 corresponds to the A of the ATG translation initiation codon; i.e., nucleotide 191. For intronic sequences, human genomic sequence NG_009113.1 was used. b Amino acid changes are numbered in accordance to the SwissProt entry Q9Y5X4. c Mutation written as reported by authors.

adRP cases [Gire et al., 2007]. In contrast to the adRP families and the patients carrying the c.119–2A4C mutation, who are reportedly of Western European/Near Eastern/Caucasian origin, the c.932G4A mutation is also present in the Japanese and Chinese populations, suggesting a mutational hotspot [Nakamura et al., 2002; Wang et al., in press].

Polymorphisms and Unclassified Sequence Variants Table 3 lists 14 polymorphisms; i.e., sequence variants not segregating with disease within families and present in control individuals. We detected two novel polymorphisms during our systematic screening of patients affected with retinal degenerations. The p.F70F polymorphism was found in three RP patients and two controls of Swiss origin, and two controls of Algerian origin. In one Algerian control, p.F70F was present together with the previously reported p.G288G polymorphism [Audo et al., 2008]. The other novel polymorphism, p.G335G, was detected in a control of Swiss origin. Two missense polymorphisms, p.E140G and p.M163T, are located in the poorly conserved hinge domain (Supp. Fig. S1) [Haider et al., 2000]. We detected these two reported polymorphisms in three affected and three unaffected individuals altogether, originating from three apparently unrelated

1480

HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

Swiss families. Two additional missense polymorphisms located in the LBD are conservative amino acid changes; i.e., p.V232I and p.V302I [Haider et al., 2000]. We detected the p.V232I polymorphism in one RP patient and one unrelated control of Algerian origin. Additional noncurated polymorphisms are listed in the public dbSNPs database (www.ncbi.nlm.nih.gov/projects/ SNP). Table 4 lists seven reported unclassified sequence variants and three novel ones. The p.A23P is present in a heterozygous state in an RP patient and his two unaffected daughters of Swiss origin. The p.A63D mutation occurred in a heterozygous state in a patient affected with cone-rod dystrophy, as well as in his unaffected mother [Coppieters et al., 2009]. The grandmother was a homozygote carrier of the mutation and affected with a RP-like retinal degeneration. The p.G69S variant is present in a Swiss family partially affected by cone-rod dystrophy. A p.R77Q mutation was present in a heterozygous state in an unaffected control person [Coppieters et al., 2007]. The p.G287S variant had been reported in a heterozygous state in a Spanish RP patient [Bernal et al., 2008], and we detected it in a heterozygous state in a French patient out of a consanguineous marriage, with both paternal and maternal history of mental retardation. The p.R292W was present in a heterozygous state in a patient affected

with Steinert disease. The additional sequence variants, i.e., p.S44L, p.N65N, and p.K324R, including the previously reported c.24518C4T polymorphism (Table 3), were found in Spanish patients, but not in control individuals [Bernal et al., 2008]. All new sequence variants were detected by direct sequencing of the coding sequence and the exon-intron boundaries as previously reported [Escher et al., 2009].

Databases To group all sequence variant information on NR2E3-linked retinal degenerations, we created a new public database at www.LOVD.nl/eye. With the aim to have the most complete and up-to-date information publicly available, all clinicians and researchers can submit online new sequence variants and patients carrying new or known sequence variants. The database is powered by Leiden Open Variation Database (LOVD) v.2.0 and hosted by the Leiden University Medical Center. New data will be checked by the Mutalyzer program to ensure a nomenclature following Human Genome Variation Society (HGVS) recommendations, and will be curated by P.E. [den Dunnen and Antonarakis, 2000; Wildeman et al., 2008]. Sequence variant information includes exon-intron location, DNA change, RNA change, protein change, protein domain, predicted effect, and first description of the variant. Additional patient information includes original clinical diagnosis, pathogenicity of the sequence variant, mode of inheritance, ethnic origin, and the number of families and patients reported so far. Sequence variant tables can be displayed as a listing of unique sequence variants or only nonpathogenic sequence variants. Patient information appears by displaying all sequence variants contained in the database. The database is fully searchable for all above-mentioned information. Additionally, hyperlinks to relevant gene and disease information from the Entrez, OMIM, and HGMD databases are also included.

Biological Relevance Functional Studies of NR2E3 Mutant Alleles NR2E3 mutant proteins have been mainly studied in cell-based reporter assays. Consistent with its physiological function on cone-specific gene expression, the NR2E3 wild-type LBD fused to the Gal4 DBD acted as transcriptional repressor [Chen et al., 1999, 2005; Kobayashi et al., 1999, 2008]. Transcriptional repression was lost in presence of the LBD mutations p.R385P and p.M407K [Chen et al., 2005; Fradot et al., 2007]. However, the DBD mutant p.E121K, as well as the LBD mutants p.W234S, p.R309G and p.R311Q showed a repressive activity comparable to that of the wild-type protein, suggesting that the loss of transcriptional inhibition is not a necessary cause of NR2E3-linked retinal degenerations [Fradot et al., 2007]. However, by using full-length proteins, transcriptional repression of an M-opsin or a S-opsin promoter fragment was markedly reduced in presence of the p.W234S mutation, but only slightly in presence of p.R311Q [Escher et al., 2009; Peng et al., 2005]. The corepressor atrophin was also unable to enhance NR2E3-mediated repression of an M-opsin promoter fragment in presence of the p.R311Q mutation [Escher et al., 2009]. Remarkably, the repressive effect of NR2E3 on cyclin D1 by a cell cycle-dependent corepressor of NR2E3, RetCoR, was attenuated in the presence of the p.R311Q mutation [Takezawa et al., 2007]. Taken together, these data suggest that retinal degenerations induced by p.R311Q might be caused by

impaired corepressor binding. The DBD-mutants p.R76W and p.R97H also showed reduced transcriptional repression, presumably because of impaired DNA-binding [Peng et al., 2005]. The adRP-linked p.G56R mutant protein was unable to bind DNA and exhibited a dominant negative activity in presence of the NR2E3 wild-type protein [Escher et al., 2009]. Interestingly, repression of M- and S-opsin promoter fragments was further enhanced in presence of the p.G56R mutation vs. wild-type protein, suggesting an interference of this DBD mutant with CRX/NRL-mediated transcriptional activation. Finally, the most common splice-site mutation c.119–2A4C was tested in a functional in vitro splicing assay [Bernal et al., 2008]. The predicted skipping of exon 2 did occur, yielding in a premature termination codon and a truncated p.V41AfsX23 mutant protein. Whether this truncated protein is translated or the mutated transcript undergoes nonsense-mediated mRNA decay remains to be determined.

Mouse Models of NR2E3 Diseases The retinal degeneration 7 (rd7) mouse is a spontaneous null mutation of the Nr2e3 gene by L1 retrotransposon insertion, and has been instrumental to determine in vivo the essential role of NR2E3 in proper photoreceptor cell fate determination, differentiation, and maintenance [Akhmedov et al., 2000; Chen et al., 2006]. Consistent with the recessive mode of inheritance of human ESCS, GFS and CRPD, heterozygote mice were normal, and a slow, but progressive degeneration of both the rod and cone systems was only observed in rd7/rd7 mice [Akhmedov et al., 2000; Haider et al., 2001]. Starting at P12, the presence of waves, whorls, and rosettes was observed in the outer nuclear layer of the retina [Haider et al., 2001]. At P16, shortly after eye opening at P14, white retinal spots were present over the entire retina [Haider et al., 2001]. The number of retinal spots decreased by 5 months of age and completely disappeared by 16 months [Akhmedov et al., 2000]. This was consistent with the disappearance of rosettes, suggesting an association of white spots with rosettes. The presence of retinal folds was presumably caused by an increased number of S-cones in rd7/rd7 retinas [Haider et al., 2001]. Consistent with a spatial hindrance in presence of an abnormally high number of cones, no retinal folds were observed in transgenic rd7/rd7 mice, where the cones were selectively deleted by expression of the diphtheria toxin [Chen and Nathans, 2007]. By ERG testing, at 5 months of age, the scotopic b-wave amplitudes were reduced, but photopic ERGs were still normal using weak stimulus flashes [Akhmedov et al., 2000; Haider et al., 2006]. By using white stimuli at high intensities, the scotopic ERG a-wave was reduced by about 50% at 6 weeks, indicating impaired rod functions [Ueno et al., 2005]. However, in these experimental conditions, normal S-cone ERGs were recorded in up to 6-monthold mice, questioning whether the rd7/rd7 mouse was a complete functional model of recessive human NR2E3 diseases, or if some species differences between human and mouse exist. Retinal degeneration had progressed by 12 months of age, and by 16 months, the outer nuclear layer and outer segments were reduced by 50%. Additionally, ERG amplitudes were reduced, retinal vessels were attenuated, and mottled retinal pigments were present [Akhmedov et al., 2000; Haider et al., 2001]. Mice with a targeted disruption of the Nr2e3 gene (Nr2e3/ mice) recapitulated most of the phenotype and the gene expression profile of rd7/rd7 mice, but the retinal defects were not fully penetrant in this mixed C57BL/6J/129Sv genetic HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

1481

background [Webber et al., 2008]. Equally, retinal spotting in rd7/ rd7 mice was fully penetrant only in the C57BL/6J genetic background, but not in the original mixed 77-2C2a one [Akhmedov et al., 2000]. Retinal degeneration and S-cone proliferation were also suppressed in F2 mice resulting from crossings of rd7/rd7 C57BL/6J mice with inbred CAST/EiJ, AKR/J, and NOD.NON-H2nbl strains [Haider et al., 2008]. These mouse studies therefore provided circumstantial evidence of modifier genes that modulate and rescue NR2E3 deficiencies. In Nrl/ mice, where Nr2e3 is not expressed, a complete loss of rod function and an increase in S-cone function was also observed [Mears et al., 2001]. In Nr1a2b/ mice that lack the thyroid hormone receptor b2, no M-cones developed, but an increase in S-opsin expressing cones was observed, suggesting a role of NR1A2b in the repression of the S-opsin gene [Ng et al., 2001]. The Nrl/ and Nr1a2b/ mice are therefore instructive in the elucidation of transcriptional regulation by NR2E3.

Clinical and Diagnostic Relevance Clinical Diagnosis of Recessive NR2E3-Linked Retinal Degenerations All patients affected with NR2E3-linked recessive retinal degenerations, i.e., ESCS, GFS and CRPD, consistently present with night blindness from early in life [Jacobson et al., 1990; Marmor, 2006; Marmor et al., 1990]. Fundus examination in adult patients typically reveals mid-peripheral nummular pigment clumping along the vascular arcades, and the subtle pigmentary changes with white dots observed in young patients may represent an early stage of the disease process [Khan et al., 2007; Sharon et al., 2003; Wang et al., in press]. The loss of retinal lamination and the rosette formation in the ONL can be monitored during disease progression by spectral domain optical coherence tomography [Wang et al., in press]. Full-field electroretinogram (ERG) testing reveals the following characteristic findings: (1) no rod responses are recorded; (2) the waveforms of the scotopic maximal response are similar to those of the transient photopic response, except for reduced amplitudes in the a- and b-waves; (3) the amplitude of the a-wave in the transient photopic cone ERG is larger than the amplitude of the photopic 30-Hz flicker cone ERG; (4) the b-wave in the transient photopic cone ERG is large and prolonged, in contrast to a peaking b-wave in healthy subjects; (5) S-cone-specific ERG testing, using a blue stimulus (445 nm) on an orange background (620 nm) detects a remarkably high amplitude, i.e., 60 to 70 mV; and (6) L- and M-cone function is significantly reduced [Audo et al., 2008; Jacobson et al., 1990; Marmor, 2006; Marmor et al., 1990; Wang et al., in press].

Genotype–Phenotype Relationships A clear genotype–phenotype correlation is only observed for the adRP-linked c.166G4A (p.G56R) mutation [Coppieters et al., 2007; Escher et al., 2009; Gire et al., 2007]. All patients showed the typical progressive degeneration of rods and subsequent degeneration of cones. Though, the retinal pigment deposits were of both spicule-like and clumped/nummular type, the latter being also observed in recessive NR2E3-linked retinal degenerations [Coppieters et al., 2007]. No genotype–phenotype correlation could be established among the recessively inherited NR2E3-linked retinal degenerations. Strikingly, homozygous carriers of the p.R311Q mutation

1482

HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

have been diagnosed with ESCS [Bernal et al., 2008; Haider et al., 2000; Iannaccone et al., 2009; Khan et al., 2007; Nakamura et al., 2002; Wright et al., 2004], GFS [Bernal et al., 2008; Chavala et al., 2005; Pachydaki et al., 2009], CPRD [Sharon et al., 2003], and arRP [Bandah et al., 2009; Gerber et al., 2000]. The original cohort of patients diagnosed with arRP was not available for ERG analyses, but the reported clinical data was very similar to ESCS [Gerber et al., 2000; Weleber, 2002]. Additional patients were diagnosed with arRP based on the presence of clumped pigment deposits, but these were not of the bone-spicule type characteristic of RP [Bandah et al., 2009; Bernal et al., 2008]. All p.R311Qpatients presented with the symptoms consistently found in recessive NR2E3-linked retinal degenerations as described above. Clumped pigment deposits were shown to involve the macula in one 60-year-old patient, but not in his contemporary first cousin, in line with the previously observed high intrafamilial variability [Jurklies et al., 2001; Pachydaki et al., 2009]. These two patients had no cystoid maculopathy or macular schisis, whereas a 20-yearold patient suffered from cystoid maculopathy, subretinal hemorrhage, and subfoveal neovascularization [Nakamura et al., 2002]. Macular schisis was found to be often associated with poor visual acuity, providing a clinical basis for the high variability in visual acuity with no correlation with age observed in patients [Audo et al., 2008]. Notably, a beneficial effect of the p.R311Q mutation was uncovered in presence of the adRP-linked p.G56R mutation, resulting in a more ESCS-like clinical phenotype in the compound heterozygous patients [Escher et al., 2009]. A phenotypic variability comparable to p.R311Q homozygote patients could also be observed in patients homozygous for the c.119–2A4C mutation [Audo et al., 2008; Bandah et al., 2009; Bernal et al., 2008; Khan et al., 2007; Rayborn et al., 2008; Sharon et al., 2003]. Patients homozygous for the p.R309Q or p.V49M mutations, or compound heterozygous for either the p.R104Q/p.R334G or the p.V41AfsX23/p.A256V mutations, showed some residual rod function, suggesting a milder form of ESCS [Audo et al., 2008; Galantuomo et al., 2008; Hayashi et al., 2005; Lam et al., 2007].

Differential Diagnosis The clinical symptoms common to ESCS, GFS, and CRPD are early-onset night blindness and rudimental rod function. These symptoms are also present in other retinal degenerations, e.g., congenital stationary night blindness (CSNB; MIM]s 163500, 257270, 300071, 310500, 610427, 610444, and 610445(, fundus albipunctatus (MIM] 136880), retinitis punctata albescens (MIM] 136880), and crystalline corneoretinal Bietti dystrophy (MIM] 210370). However, the ERGs from CSNB patients are different from those affected by ESCS [Jurklies et al., 2001]. In fundus albipunctatus, dark adaptation is markedly prolonged. The progression of retinal degeneration is electrophysiologically measurable in retinitis punctata albescens and in crystalline corneoretinal Bietti dystrophy. CSNB patients can be screened for mutations in the NYX (nyctalopin) (MIM] 300278), GRM6 (mGluR6: metabotropic glutamate receptor receptor 6) (MIM] 604096), CACNA1F (L-type Ca21 channel Cav1.4 a1 subunit) (MIM] 300110), RHO (rhodopsin) (MIM] 180380), PDE6B (rod cGMP phosphodiesterase b subunit) (MIM] 180072), GNAT1 (rod transducin a subunit) (MIM] 139330), and CABP4 (Ca21binding protein 4) (MIM] 608965) genes. Mutations in RLBP1 (cellular retinaldehyde-binding protein) (MIM] 180090) cause retinitis punctata albescencs and fundus albipunctatus, the latter also being caused by mutations in RDS (peripherin 2)

(MIM] 179605) and RDH5 (11-cis retinol dehydrogenase 5) (MIM] 601617). Finally, patients affected by crystalline corneoretinal Bietti dystrophy can be screened for mutations in the CYP4V2 gene (MIM] 608614). The cystic maculopathy and retinoschisis observed in many ESCS and GFS patients are also a hallmark of X-linked retinoschisis (MIM] 312700). However, the mode of inheritance is different and patients carry mutations in the RS1 (retinoschisin) gene. The nummular pigments observed in ESCS, GFS, and CRPD patients have also been reported in Bardet-Biedl syndrome (MIM] 209900) and in RP with preserved para-arteriolar RPE (RP12) (MIM] 600105). This latter retinal degeneration is caused by mutations in the CRB1 (crumbs homolog-1) gene (MIM] 604210).

Molecular Diagnostic Strategies Because there is no genotype–phenotype correlation in recessively inherited NR2E3 diseases, the molecular diagnostic cannot be restricted to specific mutations. The molecular analysis of the NR2E3 gene should include DNA sequencing of all exons and exon-intron boundaries, also with respect to the high proportion of compound heterozygote patients. The molecular analysis of adRP patients may be restricted to exon 2, where the c.166G4A mutation is located, assuming that potential additional mutations are also located in the first Cys4 finger. No NR2E3 mutations were detected in seven ESCS patients [Audo et al., 2008; Bandah et al., 2009; Haider et al., 2000; Khan et al., 2007; Wright et al., 2004] and in about 50% of the CRPD patients [Sharon et al., 2003]. Sequencing of the NRL gene detected a heterozygous mutation in one ESCS patient [Wright et al., 2004], and one family affected by CRPD [Nishiguchi et al., 2004]. NRL mutations are thus rare in ESCS and CRPD patients, but screening should be performed in patients lacking NR2E3 mutations. Of note, screening the NR1A1 and NR1D1 genes has not revealed any mutation in ESCS patients [Acar et al., 2003; Bandah et al., 2009; Wright et al., 2004].

Future Prospects Screening patients affected by ESCS, GFS, and CRPD identified in NR2E3 a common genetic origin for these recessive retinal degenerations. The identification of the adRP-linked p.G56R mutation added NR2E3 to a list of over 45 genes causing RP [Hartong et al., 2006]. Therefore, genetic counseling absolutely needs to consider the possibility of both dominant and recessive inheritance of novel mutations. This was recently exemplified in a family where the p.G56R and the p.R311R mutations cosegregated [Escher et al., 2009]. At a molecular level, the mechanisms by which mutations located in the LBD of NR2E3 cause similar clinical phenotypes as those located in the DBD and by which distinct mutations in the DBD cause different clinical phenotypes, remain elusive, but strongly suggest the existence of different disease mechanisms. Disease mechanisms may include: (1) absence of protein because of frameshift mutation or aberrant splicing [Bernal et al., 2008]; (2) absence of DNA binding in presence of mutations located in the DBD [Escher et al., 2009]; (3) differential interaction with corepressors, as suggested for the p.R311Q mutation [Escher et al., 2009; Takezawa et al., 2007]; (4) differential interaction with other transcription factors such as CRX and NRL; (5) altered ligand binding, whenever a natural NR2E3 ligand exists [Wolkenberg et al., 2006]; and, (6) impaired posttranslational modifications, e.g., SUMOylation that converts NR2E3 into a repressor [Onishi

et al., 2009]. To elucidate these potential disease mechanisms, additional developmental studies in appropriate mouse models have to be performed to ascertain the exact physiologic functions of NR2E3 in rod vs. cone photoreceptors. So far, there is no treatment to restore vision in NR2E3-linked diseases. Nevertheless, acute macular retinoschisis with an acuteonset visual acuity loss has been successfully treated by the oral carbonic anhydrase inhibitor acetazolamide in a 48-year-old patient homozygous for the p.R311Q mutation [Iannaccone et al., 2009]. However, other patients did not respond to this established treatment for cystoid macular edema in RP patients [Audo et al., 2008]. Possible treatments for NR2E3-diseases include somatic gene therapy by viral NR2E3 gene transfer, modulation of NR2E3 mutant protein activity by pharmacological agents, and differential activation of potential modifier genes that are suggested by the high variability in clinical phenotypes of NR2E3-linked recessive retinal degenerations.

Acknowledgments The collaboration of Dr. Jacopo Celli (Leiden University Medical Center, Leiden, The Netherlands) in establishing the locus-specific database for NR2E3 is greatly acknowledged. We thank the affected individuals and their families for participation in this study, and Mrs. Ce´line Agosti, Martine Emery, Tatiana Favez, and Nathalie Voirol from the IRO sequencing core facility for expert technical assistance. We thank Drs. Stephen H. Tsang and Nan-Kai Wang (Columbia University) for discussing data before publication. We acknowledge the help of Drs. Neena Haider (University of Nebraska Medical Center), Edwin Stone (University of Iowa), Samuel Jacobson (University of Pennsylvania), and Alan Wright (MRC, Edinburgh) in ascertaining reported mutations. This work was supported by the Swiss National Foundation Grant 3100A0-122269/1 and the Gottfried-und-Julia-Bangerter-Rhyner-Stiftung (both to P.E. and D.F.S.).

References Acar C, Mears AJ, Yashar BM, Maheshwary AS, Andreasson S, Baldi A, Sieving PA, Iannaccone A, Musarella MA, Jacobson SG, Swaroop A. 2003. Mutation screening of patients with Leber congenital amaurosis or the enhanced S-cone syndrome reveals a lack of sequence variations in the NRL gene. Mol Vis 9:14–17. Akhmedov NB, Piriev NI, Chang B, Rapoport AL, Hawes NL, Nishina PM, Nusinowitz S, Heckenlively JR, Roderick TH, Kozak CA, Danciger M, Davisson MT, Farber DB. 2000. A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse. Proc Natl Acad Sci USA 97:5551–5556. Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISS-MODEL Workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. Audo I, Michaelides M, Robson AG, Hawlina M, Vaclavik V, Sandbach JM, Neveu MM, Hogg CR, Hunt DM, Moore AT, Bird AC, Webster AR, Holder GE. 2008. Phenotypic variation in enhanced S-cone syndrome. Invest Ophthalmol Vis Sci 49:2082–2093. Bandah D, Merin S, Ashhab M, Banin E, Sharon D. 2009. The spectrum of retinal diseases caused by NR2E3 mutations in Israeli and Palestinian patients. Arch Ophthalmol 127:297–302. Bernal S, Solans T, Gamundi MJ, Hernan I, de Jorge L, Carballo M, Navarro R, Tizzano E, Ayuso C, Baiget M. 2008. Analysis of the involvement of the NR2E3 gene in autosomal recessive retinal dystrophies. Clin Genet 73:360–366. Bookout AL, Jeong Y, Downes M, Yu RT, Evans RM, Mangelsdorf DJ. 2006. Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network. Cell 126:789–799. Bumsted O’Brien KM, Cheng H, Jiang Y, Schulte D, Swaroop A, Hendrickson AE. 2004. Expression of photoreceptor-specific nuclear receptor NR2E3 in rod photoreceptors of fetal human retina. Invest Ophthalmol Vis Sci 45:2807–2812. Chavala SH, Sari A, Lewis H, Pauer GJ, Simpson E, Hagstrom SA, Traboulsi EI. 2005. An Arg311Gln NR2E3 mutation in a family with classic Goldmann-Favre syndrome. Br J Ophthalmol 89:1065–1066. HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

1483

Chen S, Wang QL, Nie Z, Sun H, Lennon G, Copeland NG, Gilbert DJ, Jenkins NA, Zack DJ. 1997. Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron 19:1017–1030. Chen F, Figueroa DJ, Marmorstein AD, Zhang Q, Petrukhin K, Caskey CT, Austin CP. 1999. Retina-specific nuclear receptor: a potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Muller glial cells. Proc Natl Acad Sci USA 96:15149–15154. Chen J, Rattner A, Nathans J. 2005. The rod photoreceptor-specific nuclear receptor Nr2e3 represses transcription of multiple cone-specific genes. J Neurosci 25:118–129. Chen J, Rattner A, Nathans J. 2006. Effects of L1 retrotransposon insertion on transcript processing, localization and accumulation: lessons from the retinal degeneration 7 mouse and implications for the genomic ecology of L1 elements. Hum Mol Genet 15:2146–2156. Chen J, Nathans J. 2007. Genetic ablation of cone photoreceptors eliminates retinal folds in the retinal degeneration 7 (rd7) mouse. Invest Ophthalmol Vis Sci 48:2799–2805. Cheng H, Khanna H, Oh EC, Hicks D, Mitton KP, Swaroop A. 2004. Photoreceptorspecific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors. Hum Mol Genet 13:1563–1575. Cheng H, Aleman TS, Cideciyan AV, Khanna R, Jacobson SG, Swaroop A. 2006. In vivo function of the orphan nuclear receptor NR2E3 in establishing photoreceptor identity during mammalian retinal development. Hum Mol Genet 15:2588–2602. Cideciyan AV, Jacobson SG, Gupta N, Osawa S, Locke KG, Weiss ER, Wright AF, Birch DG, Milam AH. 2003. Cone deactivation kinetics and GRK1/GRK7 expression in enhanced S cone syndrome caused by mutations in NR2E3. Invest Ophthalmol Vis Sci 44:1268–1274. Coppieters F, Leroy BP, Beysen D, Hellemans J, De Bosscher K, Haegeman G, Robberecht K, Wuyts W, Coucke PJ, De Baere E. 2007. Recurrent mutation in the first zinc finger of the orphan nuclear receptor NR2E3 causes autosomal dominant retinitis pigmentosa. Am J Hum Genet 81:147–157. Coppieters F, He´on E, Cima I, Glavac D, Hawlina M, Mersy E, Hooghe S, Leroy BP, De Baere E. 2009. Exploring the role of genes of the retinal transcription network in retinal degeneration. Invest Ophthalmol Vis Sci 50:E-Abstract 3745. Corbo JC, Cepko CL. 2005. A hybrid photoreceptor expressing both rod and cone genes in a mouse model of enhanced S-cone syndrome. PLoS Genet 1:e11. den Dunnen JT, Antonarakis SE. 2000. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 15:7–12. Escher P, Gouras P, Roduit R, Tiab L, Bolay S, Delarive T, Chen S, Tsai C-C, Hayashi M, Zernant J, Merriam JE, Mermod N, Allikmets R, Munier FL, Schorderet DF. 2009. Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family. Hum Mutat 30:342–351. Favre M. 1958. A propos de deux cas de de´ge´ne´rescence hyaloide´ore´tinienne. [Two cases of hyaloid-retinal degeneration] Ophthalmologica 135:604. Fradot M, Lorentz O, Wurtz J-M, Sahel J-A, Le´veillard T. 2007. The loss of transcriptional inhibition by the photoreceptor-cell specific nuclear receptor (NR2E3) is not a necessary cause of enhanced S-cone syndrome. Mol Vis 13: 594–601. Freund CL, Gregory-Evans CY, Furukawa T, Papaioannou M, Looser J, Ploder L, Bellingham J, Ng D, Herbrick JA, Duncan A, Scherer SW, Tsui LC, LoutradisAnagnostou A, Jacobson SG, Cepko CL, Bhattacharya SS, McInnes RR. 1997. Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor. Cell 91: 543–553. Furukawa T, Morrow EM, Cepko CL. 1997. Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell 91:531–541. Galantuomo MS, Zucca I, Banfi S, Ziviello C, Carboni G, Forma G, Titi Y, Fossarello M. 2008. A NR2E3 mutation (R309G) associated with a mild form of enhanced S-cone syndrome. Invest Ophthalmol Vis Sci 49:E-Abstract 2186. Gerber S, Rozet JM, Takezawa SI, dos Santos LC, Lopes L, Gribouval O, Penet C, Perrault I, Ducroq D, Souied E, Jeanpierre M, Romana S, Fre´zal J, Ferraz F, Yu-Umesono R, Munnich A, Kaplan J. 2000. The photoreceptor cell-specific nuclear receptor gene (PNR) accounts for retinitis pigmentosa in the CryptoJews from Portugal (Marranos), survivors from the Spanish Inquisition. Hum Genet 107:276–284. Gire AI, Sullivan LS, Bowne SJ, Birch DG, Hughbanks-Wheaton D, Heckenlively JR, Daiger SP. 2007. The Gly56Arg mutation in NR2E3 accounts for 1-2% of autosomal dominant retinitis pigmentosa. Mol Vis 13:1970–1975. Greenstein VC, Zaidi Q, Hood DC, Spehar B, Cideciyan AV, Jacobson SG. 1996. The enhanced S cone syndrome: an analysis of receptoral and post-receptoral changes. Vision Res 36:3711–3722. Haider NB, Jacobson SG, Cideciyan AV, Swiderski R, Streb LM, Searby C, Beck G, Hockey R, Hanna DB, Gorman S, Duhl D, Carmi R, Bennett J, Weleber RG, Fishman GA, Wright AF, Stone EM, Sheffield VC. 2000. Mutation of a nuclear

1484

HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate. Nat Genet 24:127–131. Haider NB, Naggert JK, Nishina PM. 2001. Excess cone cell proliferation due to lack of a functional NR2E3 causes retinal dysplasia and degeneration in rd7/rd7 mice. Hum Mol Genet 10:1619–1626. Haider NB, Demarco P, Nystuen AM, Huang X, Smith RS, McCall MA, Naggert JK, Nishina PM. 2006. The transcription factor Nr2e3 functions in retinal progenitors to suppress cone cell generation. Vis Neurosci 23:917–929. Haider NB, Zhang W, Hurd R, Ikeda A, Nystuen AM, Naggert JK, Nishina PM. 2008. Mapping of genetic modifiers of Nr2e3 (rd7/rd7) that suppress retinal degeneration and restore blue cone cells to normal quantity. Mamm Genome 19:145–154. Haider NB, Mollema N, Gaule M, Yuan Y, Sachs AJ, Nystuen AM, Naggert JK, Nishina PM. Nr2e3-directed transcriptional regulation of genes involved in photoreceptor development and cell-type specific phototransduction. Exp Eye Res (in press). [http://dx.doi.org/10.1016/j.exer.2009.04.006] Hartong DT, Berson EL, Dryja TP. 2006. Retinitis pigmentosa. Lancet 368:1795–1809. Hayashi T, Gekka T, Goto-Omoto S, Takeuchi T, Kubo A, Kitahara K. 2005. Novel NR2E3 mutations (R104Q, R334G) associated with a mild form of enhanced S-cone syndrome demonstrate compound heterozygosity. Ophthalmology 112:2115. Hayashi T, Kitahara K. 2005. Optical coherence tomography in enhanced S-cone syndrome: large macular retinoschisis with disorganized retinal lamination. Eur J Ophthalmol 15:643–646. Hood DC, Cideciyan AV, Roman AJ, Jacobson SG. 1995. Enhanced S cone syndrome: evidence for an abnormally large number of S cones. Vision Res 35:1473–1481. Iannaccone A, Fung KH, Eyestone ME, Stone EM. 2009. Treatment of adult-onset acute macular retinoschisis in enhanced S-cone syndrome with oral acetazolamide. Am J Ophthalmol 147:307–312. Jacobson SG, Marmor MF, Kemp CM, Knighton RW. 1990. SWS (blue) cone hypersensitivity in a newly identified retinal degeneration. Invest Ophthalmol Vis Sci 31:827–838. Jacobson SG, Roma´n AJ, Roma´n MI, Gass JD, Parker JA. 1991. Relatively enhanced S cone function in the Goldmann-Favre syndrome. Am J Ophthalmol 111:446–453. Jacobson SG, Sumaroka A, Aleman TS, Cideciyan AV, Schwartz SB, Roman AJ, McInnes RR, Sheffield VC, Stone EM, Swaroop A, Wright AF. 2004. Nuclear receptor NR2E3 gene mutations distort human retinal laminar architecture and cause an unusual degeneration. Hum Mol Genet 13:1893–1902. Jurklies B, Weismann M, Kellner U, Zrenner E, Bornfeld N. 2001. [Clinical findings in autosomal recessive syndrome of blue cone hypersensitivity]. Ophthalmologe 98:285–293. Khan AO, Aldahmesh M, Meyer B. 2007. The enhanced S-cone syndrome in children. Br J Ophthalmol 91:394–396. Kobayashi M, Takezawa S, Hara K, Yu RT, Umesono Y, Agata K, Taniwaki M, Yasuda K, Umesono K. 1999. Identification of a photoreceptor cell-specific nuclear receptor. Proc Natl Acad Sci USA 96:4814–4819. Kobayashi M, Hara K, Yu RT, Yasuda K. 2008. Expression and functional analysis of Nr2e3, a photoreceptor-specific nuclear receptor, suggest common mechanisms in retinal development between avians and mammals. Dev Genes Evol 218:439–444. Lam BL, Goldberg JL, Hartley KL, Stone EM, Liu M. 2007. Atypical mild enhanced S-cone syndrome with novel compound heterozygosity of the NR2E3 gene. Am J Ophthalmol 144:157–159. Liu Q, Ji X, Breitman ML, Hitchcock PF, Swaroop A. 1996. Expression of the bZIP transcription factor gene Nrl in the developing nervous system. Oncogene 12:207–211. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM. 1995. The nuclear receptor superfamily: the second decade. Cell 83:835–839. Marmor MF, Jacobson SG, Foerster MH, Kellner U, Weleber RG. 1990. Diagnostic clinical findings of a new syndrome with night blindness, maculopathy, and enhanced S cone sensitivity. Am J Ophthalmol 110:124–134. Marmor MF. 2006. A teenager with nightblindness and cystic maculopathy: enhanced S cone syndrome (Goldmann-Favre syndrome). Doc Ophthalmol 113:213–215. Mears AJ, Kondo M, Swain PK, Takada Y, Bush RA, Saunders TL, Sieving PA, Swaroop A. 2001. Nrl is required for rod photoreceptor development. Nat Genet 29:447–452. Milam AH, Rose L, Cideciyan AV, Barakat MR, Tang WX, Gupta N, Aleman TS, Wright AF, Stone EM, Sheffield VC, Jacobson SG. 2002. The nuclear receptor NR2E3 plays a role in human retinal photoreceptor differentiation and degeneration. Proc Natl Acad Sci USA 99:473–478. Nakamura M, Hotta Y, Piao CH, Kondo M, Terasaki H, Miyake Y. 2002. Enhanced S-cone syndrome with subfoveal neovascularization. Am J Ophthalmol 133:575–577. Nakamura Y, Hayashi T, Kozaki K, Kubo A, Omoto S, Watanabe A, Toda K, Takeuchi T, Gekka T, Kitahara K. 2004. Enhanced S-cone syndrome in a Japanese family with a nonsense NR2E3 mutation (Q350X). Acta Ophthalmol Scand 82:616–622.

Ng L, Hurley JB, Dierks B, Srinivas M, Salto C, Vennstrom B, Reh TA, Forrest D. 2001. A thyroid hormone receptor that is required for the development of green cone photoreceptors. Nat Genet 27:94–98. Nishiguchi KM, Friedman JS, Sandberg MA, Swaroop A, Berson EL, Dryja TP. 2004. Recessive NRL mutations in patients with clumped pigmentary retinal degeneration and relative preservation of blue cone function. Proc Natl Acad Sci USA 101:17819–17824. Oh EC, Cheng H, Hao H, Jia L, Khan NW, Swaroop A. 2008. Rod differentiation factor NRL activates the expression of nuclear receptor NR2E3 to suppress the development of cone photoreceptors. Brain Res 1236:16–29. Onishi A, Peng GH, Hsu C, Alexis U, Chen S, Blackshaw S. 2009. Pias3-dependent SUMOylation directs rod photoreceptor development. Neuron 61:234–246. Pachydaki SI, Klaver CC, Barbazetto IA, Roy MS, Gouras P, Allikmets R, Yannuzzi LA. 2009. Phenotypic features of patients with NR2E3 mutations. Arch Ophthalmol 127:71–75. Peng GH, Ahmad O, Ahmad F, Liu J, Chen S. 2005. The photoreceptor-specific nuclear receptor Nr2e3 interacts with Crx and exerts opposing effects on the transcription of rod versus cone genes. Hum Mol Genet 14:747–764. Rayborn ME, Bonilha VL, Fishman GA, Hollyfield JG. 2008. Photoreceptor analysis in the retina of a donor with Goldmann-Favre syndrome. Invest Ophthalmol Vis Sci 49:E-Abstract 2996. Sharon D, Sandberg MA, Caruso RC, Berson EL, Dryja T. 2003. Shared mutations in NR2E3 in enhanced S-cone syndrome, Goldmann-Favre syndrome, and many cases of clumped pigmentary retinal degeneration. Arch Ophthalmol 121:1316–1323. Srinivas M, Ng L, Liu H, Jia L, Forrest D. 2006. Activation of the blue opsin gene in cone photoreceptor development by retinoid-related orphan receptor beta. Mol Endocrinology 20:1728–1741. Takezawa S, Yokoyama A, Okada M, Fujiki R, Iriyama A, Yanagi Y, Ito H, Takada I, Kishimoto M, Miyajima A, Takeyama K, Umesono K, Kitagawa H, Kato S. 2007. A cell cycle-dependent co-repressor mediates photoreceptor cell-specific nuclear receptor function. EMBO J 26:764–774.

To KW, Adamian M, Jakobiec FA, Berson EL. 1996. Clinical and histopathologic findings in clumped pigmentary retinal degeneration. Arch Ophthalmol 114:950–955. Ueno S, Kondo M, Miyata K, Hirai T, Miyata T, Usukura J, Nishizawa Y, Miyake Y. 2005. Physiological function of S-cone system is not enhanced in rd7 mice. Exp Eye Res 81:751–758. Vaclavik V, Chakarova C, Bhattacharya SS, Robson AG, Holder GE, Bird AC, Webster AR. 2008. Bilateral giant macular schisis in a patient with enhanced Scone syndrome from a family showing pseudo-dominant inheritance. Br J Ophthalmol 92:299–300. Wang NK, Fine H, Chang S, Chou CL, Cella W, Tosi J, Lin CS, Nagasaki T, Tsang SH. Cellular origin of fundus autofluorescence in patients and mice with defective NR2E3 gene. Br J Ophthalmol (in press). [http://dx.doi.org/10.1136/bjo.2008.153577] Webber AL, Hodor P, Thut CJ, Vogt TF, Zhang T, Holder DJ, Petrukhin K. 2008. Dual role of Nr2e3 in photoreceptor development and maintenance. Exp Eye Res 87:35–48. Weleber RG. 2002. Infantile and childhood retinal blindness: a molecular perspective (The Franceschetti Lecture). Ophthalmic Genet 23:71–97. Wildeman M, van Ophuizen E, den Dunnen JT, Taschner PE. 2008. Improving sequence variant descriptions in mutation databases and literature using the Mutalyzer sequence variation nomenclature checker. Hum Mutat 29:6–13. Wolkenberg SE, Zhao Z, Kapitskaya M, Webber AL, Petrukhin K, Tang YS, Dean DC, Hartman GD, Lindsley CW. 2006. Identification of potent agonists of photoreceptor-specific nuclear receptor (NR2E3) and preparation of a radioligand. Bioorg Med Chem Lett 16:5001–5004. Wright AF, Reddick AC, Schwartz SB, Ferguson JS, Aleman TS, Kellner U, Jurklies B, Schuster A, Zrenner E, Wissinger B, Lennon A, Shu X, Cideciyan AV, Stone EM, Jacobson SG, Swaroop A. 2004. Mutation analysis of NR2E3 and NRL genes in enhanced S-cone syndrome. Hum Mutat 24:439–450. Yamamoto S, Hayashi M, Takeuchi S. 1999. Electroretinograms and visual evoked potentials elicited by spectral stimuli in a patient with enhanced S-cone syndrome. Jpn J Ophthalmol 43:433–437.

HUMAN MUTATION, Vol. 30, No. 11, 1475–1485, 2009

1485