Molecular Vision 2007; 13:1970-1975 <http://www.molvis.org/molvis/v13/a222/>
Received 24 May 2007 | Accepted 30 September 2007 | Published 17 October 2007
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The Gly56Arg mutation in NR2E3 accounts for 1-2% of autosomal dominant retinitis pigmentosa

Anisa I. Gire,1 Lori S. Sullivan,1 Sara J. Bowne,1 David G. Birch,2 Dianna Hughbanks-Wheaton,2 John R. Heckenlively,3 Stephen P. Daiger1,4
 
 

1Human Genetics Center, School of Public Health, and 4Department of Ophthalmology and Visual Science, The University of Texas Health Science Center at Houston; 2Retina Foundation of the Southwest, Dallas, TX; 3Kellogg Eye Center, University of Michigan, Ann Arbor, MI

Correspondence to: Stephen P. Daiger, The University of Texas Health Science Center, Human Genetics Center, School of Public Health, 1200 Herman Pressler Drive, Houston, TX 77030; Phone: (713) 500-9829; FAX: (713) 500-0900; email: stephen.p.daiger@uth.tmc.edu


Abstract

Purpose: Mutations in the orphan nuclear receptor gene NR2E3 have been found to cause both recessive and dominant retinopathies. The purpose of this study was to determine the prevalence of the recently described Gly56Arg mutation in a well-characterized cohort of families with autosomal dominant retinitis pigmentosa (adRP).

Methods: A cohort of 215 families with adRP which have already been screened for mutations in 13 of the other known adRP genes was used to determine the frequency of the Gly56Arg mutation. The 92 families without a disease-causing mutation in a known gene were tested for the presence of the Gly56Arg mutation using direct DNA sequencing. An additional set of 100 normal controls (200 chromosomes) was also screened by DNA sequencing.

Results: The Gly56Arg mutation was found in three of the 92 adRP families studied and was not found in unaffected control samples.

Conclusions: The Gly56Arg mutation in NR2E3 accounts for approximately 1-2% of adRP, making it one of the more common single mutations in adRP.


Introduction

Autosomal dominant retinitis pigmentosa (adRP) is a heterogeneous disease that can be caused by mutations in at least 18 different genes [1] (and see RetNet). Fifteen of the 18 genes have been identified and the others have been localized by linkage mapping, but are still unknown. In all cases, the disease is characterized by progressive retinal degeneration, but the course and severity can vary depending on the underlying causative gene, the particular mutation, or on factors within a family that may be genetic or environmental, or both. Screening of adRP families for mutations in the known genes results in identification of the disease-causing mutation in approximately 58% of cases (Figure 1) [2,3], suggesting that there are additional genes left to find as well as "hidden" mutations in the known genes that are currently undetected.

One gene, NR2E3, was recently added to the list of adRP-causing genes [4,5], although it had already been known to cause autosomal recessive enhanced S-cone syndrome (ESCS) [6]. The NR2E3 gene consists of eight coding exons spanning 7.7 kb and encodes a retinal nuclear receptor protein, 410 amino acids in length, that acts as a transcriptional regulator, activating rod-specific genes in concert with cone-rod otx-like photoreceptor homeobox transcription factor (CRX), neural retina lucine zipper transcription factor (NRL), and other proteins, as well as repressing the transcription of cone-specific genes in differentiating rod photoreceptors [7-9]. Mutations in other transcriptional regulators such as CRX and NRL have been shown to cause both recessive [10,11] and dominant [12-15] forms of retinal degeneration, so a dual role for NRL is not surprising.

So far, only a single missense mutation, Gly56Arg (c.356G>A), has been found in patients with adRP. This single mutation has been found in at least four putatively unrelated families - three families reported by Bouayed-Tiab et al. [4] and a single family described by Leroy et al. [5]. A different heterozygous missense mutation (Ala63Asp) has been reported to cause dominant cone-rod dystrophy in a single family [4]. Both mutations are located in exon 2 of the gene, in the first zinc-finger of the DNA binding domain.


Methods

Autosomal dominant retinitis pigmentosa cohort and controls

The cohort of adRP patients used in this study is described in detail in Sullivan 2006 [2] and an additional 15 families were added subsequent to that publication. Briefly, this is a set of 215 families with a high likelihood of having the autosomal dominant form of retinitis pigmentosa based on pedigree analysis and calculations of the relative likelihoods of dominant, recessive, and X-linked RP. Each proband has been screened previously for mutations in the complete coding regions of CA4, CRX, FSCN2, IMPDH1, NRL, PRPF31, RDS, RHO, ROM1, and RP9 and in mutation hotspots of RP1, PRPF3, and PRPF8. Of the 215 families in the cohort, likely disease-causing mutations have been identified in 123 families [2,3] (and unpublished); the remaining 92 families were tested in this study. These families are largely of Caucasian origin. A set of 100 unrelated normal control samples, obtained from the Centre d'Etude du Polymorphisme Humain (CEPH) [16], was also tested.

This study was performed in accordance with the Declaration of Helsinki and informed consent was obtained from all participants. The research was approved by the Committee for Protection of Human Subjects, University of Texas, Houston and by the respective human subjects review boards at each participating academic institution.

Mutation testing

Patient DNA samples were screened for the Gly56Arg mutation by PCR amplification and sequencing of a 120 bp fragment of exon 2. The following PCR primers were used for both DNA amplification and DNA sequencing: Ex2 forward primer 5'-AGA TGA GCC TCC GCC G-3'; Ex2 reverse primer 5'-GAG CCC CTC GCT CCA G-3'.

PCR amplification and sequencing was performed as previously described [2]. In general, 30-50 ng of genomic DNA was amplified with AmpliTaq Gold (Applied Biosystems, Foster City, CA) in a 12.5 μl reaction volume for 35 cycles. PCR products were treated with ExoSapIt (USB, Cleveland, OH) and sequenced bidirectionally with BigDye v1.1 (Applied Biosystems). Sequencing reactions were purified with sephadex columns (Princeton Separations, Adelphia, NJ) and run on an ABI 3100-Avant Genetic Analyzer (Applied Biosystems). Sequence analysis was done using SeqScape software (Applied Biosystems).


Results

Prevalence of the NR2E3 Gly56Arg mutation in adRP

Mutation analysis of exon 2 of the NR2E3 gene was performed in 92 probands with adRP. Of the 92 probands studied, three were found to be heterozygous for the Gly56Arg (c.356G>A) mutation. In 2 of the 3 families, a single additional affected family member was available for testing and in both cases the mutation cosegregated with disease. The mutation was not found in over 100 normal control samples. The frequency of this mutation in the adRP cohort is 1.5% (3/215), making it one of the more common mutations causing adRP (Table 1). No other variants were observed in the region that was sequenced, either in affected patients or control samples.

RFS029

The propositus (III:1) was diagnosed with retinitis pigmentosa at age 40 (Figure 2A). She reports that she first became aware of vision loss at age 28. Visual acuity at age 40 was 20/40 OD and 20/50 OS. Her Goldmann visual field diameter to a spot size V4e was 10° in each eye with no peripheral islands. Absolute dark-adapted threshold in the macula was elevated 2.7 log units above the upper limit of normal. Her rod electroretinogram (ERG) was non-detectable. Full-field rod ERGs were non-detectable. Cone responses to 31 Hz flicker were 1.1 μV compared to a lower age-matched limit of normal of 38 μV.

RFS179

The propositus (III:2) is a 61-year-old female who was referred to the Retina Foundation of the Southwest with retinitis pigmentosa (Figure 2B). She reports that she was night blind as a child, lost color vision at around 40, and could not see beyond age 45. Her visual acuity was LP in each eye. Absolute dark-adapted threshold in the macula was elevated 5.2 log units above the upper limit of normal. Full-field ERGs were non-detectable (<0.1 V) to ISCEV standard stimuli despite selective amplification at the stimulus frequency and extensive computer averaging.

Her son (IV:2) was initially diagnosed at 12 years of age and reports that he has always been night blind. He became aware of substantial field loss by age 20, had cataracts removed at 22, and has taken 20,000 IU of vitamin A palmitate for the past 12 years. At age 34 he retained 24/40 acuity OD and 20/25 acuity OS. His visual field (Humphrey spot size 5) measured less than 10° in each eye. Absolute dark-adapted threshold in the macula was elevated 2.5 log units above the upper limit of normal. Full-field rod ERGs were non-detectable. Cone responses to 31 Hz flicker were barely detectable (0.13 μV).

RFS268

The propositus (III:1) is a 31-year-old female who was diagnosed with retinitis pigmentosa as a teenager (Figure 2C). She was night blind from at least age 6 years and was declared legally blind at age 25. At age 31, acuity OD was 20/50 and acuity OS was 20/63. Her Humphrey visual field (spot size 3) retained values within the central 20°. Absolute dark-adapted threshold in the macula was elevated 3.1 log units above the upper limit of normal. Full-field rod ERGs were non-detectable. Cone responses to 31 Hz flicker measured 9.5 mV compared to a lower age-matched limit of normal of 38 μV.

Her daughter (IV:2) was age eight years at the time of her referral to the Retina Foundation of the Southwest. Her visual acuity was 20/16 OD and 20/32 OS. Her central 30-2 Humphrey fields showed a mean defect of 8 dB with no absolute scotomas. Absolute dark-adapted threshold in the macula was elevated 2.3 log units above the upper limit of normal. Fullfield rod ERGs were non-detectable. Cone responses to 31 Hz flicker measured 24.6 mV compared to a lower age-matched limit of normal of 41 mV.


Discussion

AdRP is genetically complex, with at least 18 known causative genes and an unknown fraction of genes still undiscovered. The Gly56Arg mutation in NR2E3 appears to be a relatively frequent cause of adRP, with a frequency of approximately 1.5% in our adRP cohort. Fourteen mutations in 6 of the other adRP genes have frequencies of 1% or more [2] and, together with the NR2E3 mutation, account for almost 30% of adRP cases in Americans of European origin and Europeans (Table 1).

Phenotypic date were available from 5 affected individuals from the 3 families with NR2E3-associated dominant retinitis pigmentosa. Each patient was diagnosed with typical retinitis pigmentosa by an ophthalmologist specializing in retinal disease. Each patient came from a pedigree that was unambiguously autosomal dominant. In general, the phenotypes are remarkable for the severity of rod loss in early childhood. No patient, including one tested at eight years of age, retained a detectable rod ERG. All reported night blindness in childhood and severe field constriction by age 21. At least within this limited cohort, the phenotype and natural history of progression seem fairly homogeneous.

The mechanisms by which mutations in retinal transcription factors such as NR2E3 cause retinal degenerations are slowly being elucidated. The three genes identified so far - CRX, NRL, and NR2E3 - are all part of a complex pathway that controls the differentiation of postmitotic photoreceptor precursors.

Mutations in CRX are known to cause dominant cone-rod dystrophy [12,13] or dominant, recessive, or de novo Leber congenital amaurosis (LCA) [10,14,15,17]. Mutations in NRL can cause autosomal dominant or recessive retinitis pigmentosa [11,18]. Mutations in NR2E3 can cause recessive enhanced S-cone syndrome [6], dominant cone-rod dystrophy [5], or dominant retinitis pigmentosa [4,5]. Thus there is a range of clinical phenotypes associated with mutations in each of these retinal transcription factors.

CRX is expressed in all photoreceptor precursors, including cells destined to be both rods and cones [19]. The CRX protein is a homeobox-containing transcription factor, binding to target DNA sequences via the paired homeodomains near the N-terminus [19] and activating transcription through sequences found in the WSP-domain and the OTX-tail, located near the C-terminus [20]. CRX targets include a large number of photoreceptor genes such as the opsins, arrestin, and PDE6A [19,21].

A subset of all photoreceptor precursor cells expresses NRL, a basic leucine zipper (bZip) transcription factor and a member of the Maf family of transcription factors [22]. The C-terminal bZip domain binds to cis-regulatory sequence elements called negative regulatory elements (NREs) [23], while an N-terminal transactivating domain controls transcriptional activation. NRL appears to be essential for rod differentiation and has been found to control the transcription of the majority of rod-specific genes [24]. Transcriptional activation is regulated by phosphorylation of specific residues and possibly additional posttranslational modifications [25].

Expression of NRL is necessary to commit precursor cells to becoming rod photoreceptors [26]. NR2E3 is thought to interact with NRL, to both suppress cone-specific gene expression and to activate a subset of rod-specific genes [7,8,27]. NR2E3 is a member of a third class of transcription factors, the nuclear hormone receptors. These proteins contain zinc finger domains that bind to specific DNA target sequences and a ligand-binding domain, responsible for regulation of the protein's activity. NR2E3 is an "orphan" nuclear receptor protein, as its ligand has not been identified.

Mutations in any one of these genes can have a variety of consequences. Complete loss of protein function, for any of the three, leads to failure of the photoreceptor cells to differentiate properly. In the case of CRX, which functions early in the developmental pathway, protein loss leads to a retina completely lacking in photoreceptor outer segments. In humans, this manifests as severe LCA, and is seen in patients with recessive CRX mutations [10]. For NRL and NR2E3, complete loss of protein function leads to the inability to suppress cone genes and activate rod genes, resulting in the failure of rod development. In humans, this results in enhanced S-cone syndrome in patients with recessive mutations in NR2E3 [6] and a similar phenotype in the rare patients who have two NRL mutations [11].

Mutations that do not result in a complete loss of functional protein have more complex phenotypes and phenotype/genotype correlations often cannot be made. Recent studies of NRL suggest that a number of pathogenic mutations result in aberrant phosphorylation, which in turn leads to altered transcriptional activity [25]. Such gain-of function mutations in NRL, in general, appear to lead to the phenotype of dominant RP. CRX appears to be a more complicated story, with dominant mutations causing RP, cone-rod dystrophy, and LCA. In vitro studies have shown that while the mutations all clearly appear to interfere with CRX's ability to regulate transcription, there is still no explanation for the significant differences in phenotypes [28]. The recent discovery of dominant mutations in NR2E3 causing RP and cone-rod dystrophy (CORD) raises the same questions, since both reported mutations are only 7 amino acids apart in the zinc finger region, presumably both affect DNA binding, and yet each causes distinctly different phenotypes.


Acknowledgements

The authors thank Elizabeth Cadena for invaluable technical assistance. Supported by grants from the Foundation Fighting Blindness, The William Stamps Farish Fund, the Gustavus and Louise Pfeiffer Research Foundation, the Hermann Eye Fund, and National Eye Institute grants EY007142 and EY005235.


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