Molecular Vision 2003; 9:14-17 <>
Received 23 December 2002 | Accepted 13 January 2003 | Published 24 January 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

Ceren Acar,1,2 Alan J. Mears,1 Beverly M. Yashar,1,3 Anjali S. Maheshwary,1 Sten Andreasson,4 Alfonso Baldi,5 Paul A. Sieving,6 Alessandro Iannaccone,7 Maria A. Musarella,8 Samuel G. Jacobson,9 Anand Swaroop1,3

Departments of 1Ophthalmology and Visual Sciences and 3Human Genetics, University of Michigan, Ann Arbor, MI; 2Department of Biology, Hacettepe University, Ankara, Turkey; 4Department of Ophthalmology, University of Lund, Lund, Sweden; 5Department of Biochemistry, Section of Molecular Pathology, II Università di Napoli, Naples, Italy; 6National Eye Institute, Bethesda, MD; 7Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN; 8Department of Ophthalmology, SUNY Downstate Medical Center, Brooklyn, NY; 9Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA

Correspondence to: Anand Swaroop, Ph.D., Department of Ophthalmology and Visual Sciences, W. K. Kellogg Eye Center, University of Michigan, 1000 Wall Street, Ann Arbor, MI, 48105; Phone: (734) 763-3731; FAX: (734) 647-0228; email:


Purpose: To determine if mutations in the retinal transcription factor gene NRL are associated with retinopathies other than autosomal dominant retinitis pigmentosa (adRP).

Methods: Genomic DNA was isolated from blood samples obtained from 50 patients with Leber Congenital Amaurosis (LCA), 17 patients with the Enhanced S-Cone Syndrome (ESCS), and a patient with an atypical retinal degeneration that causes photoreceptor rosettes with blue cone opsin. The 5' upstream region (putative promoter), untranslated exon 1, coding exons 2 and 3, and exon-intron boundaries of the NRL gene were analyzed by direct sequencing of the PCR-amplified products.

Results: Complete sequencing of the NRL gene in DNA samples from this cohort of patients revealed only one nucleotide change. The C->G transversion at nucleotide 711 of NRL exon 3 was detected in one LCA patient; however, this change did not alter the amino acid (L237L).

Conclusions: No potential disease causing mutation was identified in the NRL gene in patients with LCA, ESCS, or the atypical retinal degeneration. Together with previous studies, our results demonstrate that mutations in the NRL gene are not a major cause of retinopathy. To date, only missense changes have been reported in adRP patients, and sequence variations are rare. It is possible that the loss of NRL function in humans is associated with a more complex clinical phenotype due to its expression in pineal gland in addition to rod photoreceptors.


The neural retina leucine zipper (NRL) gene encodes a member of the Maf-subfamily of bZIP (basic region leucine zipper) transcription factors [1]. Multiple isoforms of NRL are generated by differential phosphorylation and their expression appears to be restricted to the rod photoreceptors [2] or pineal gland (A.J. Mears, unpublished data). NRL is shown to regulate the expression of several phototransduction genes [3-5]. Stringent regulation of rhodopsin is achieved by NRL in synergy with the cone-rod homeobox transcription factor CRX [6,7], whereas the regulation of the gene for rod-specific cGMP phosphodiesterase β-subunit involves NRL and SP4 [8]. Since mutations in phototransduction genes lead to retinal diseases (RetNet), it was anticipated that sequence changes in photoreceptor-specific transcription factors would also cause retinopathies. For example, mutations in CRX have been identified in a broad spectrum of retinal diseases, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis (LCA) [9-13]. NRL mutation screenings have so far revealed five missense mutations in the NRL gene in autosomal dominant retinitis pigmentosa (adRP) and one potential mutation in a patient with simplex RP [14-18]. Based on in vitro rhodopsin promoter activity assays, it was hypothesized that the first reported NRL mutation (S50T) results in a gain of function leading to higher activity of the mutant protein [15].

Though no loss of function mutation has been identified in the human NRL gene, the targeted deletion of Nrl in mouse (Nrl-/-) provided considerable insights into its in vivo function [19]. The retinas of Nrl-/- mice showed a dramatic phenotype with a complete loss of rod function (as measured by electroretinography) and rod-specific phototransduction proteins (as revealed by RNA and protein expression studies). Instead, there was enhanced S-cone (short wavelength / UV-sensitive) function, associated with increased expression of S-opsin and many cone-specific proteins. This phenotype showed similarity to the rd7 mouse retina [20,21] and the human retinal disease, Enhanced S-cone Syndrome (ESCS) [22,23]; both of these are associated with mutations in the transcription factor NR2E3 [20,24]. ESCS is associated with night blindness and heightened sensitivity to short wavelength light, originally hypothesized [22] and recently proven to be mediated by an increase in S-cones [25]. Sensitivity to long (red) and medium (green) wavelength light is altered to varying degrees and the extent of visual loss due to degeneration varies considerably within the ESCS phenotype [22,23,25]. It has, therefore, been proposed that NR2E3 plays a crucial role in photoreceptor differentiation. In the Nrl-/- mouse retina, the Nr2e3 transcript was undetectable and the temporal expression data suggested that Nrl is upstream of Nr2e3 in the transcriptional hierarchy associated with photoreceptor cell fate determination [19].

Based on NRL's synergistic interaction with CRX, the phenotype of the Nrl-/- mouse retina, and its involvement in regulating phototransduction proteins, we hypothesized that loss of function mutations in the human NRL gene might result in severe retinopathy and/or congenital blindness, such as in the disease category of LCA. In addition, since NRL is upstream of NR2E3 in transcriptional hierarchy in the retina, we postulated that sequence variations in NRL may modify clinical manifestation of ESCS patients carrying NR2E3 mutations. We also tested the hypothesis that NRL may be abnormal in an unusual retinal degeneration (proven not to be ESCS) that shows extensive rod loss but photoreceptor rosettes with blue cone opsin immunoreactivity, as determined by postmortem retinal histopathology in one of two affected siblings [26]. The finding in rd7 and Nrl-/- mice of photoreceptor rosettes and increased S cones warranted inclusion of this atypical retinopathy in the cohort screening.

Here, we report the results of direct sequencing of the 5' upstream region, untranslated exon 1, the coding exons 2 and 3, and exon-intron boundary regions of NRL in a cohort of 68 patients with the above-mentioned retinopathies.


Mutation screening, described in this report, included unrelated patients with the following retinopathies: LCA (50 patients), ESCS (17 patients), and an atypical retinal degeneration (1 patient). These patients were of different races and ancestry. A majority of LCA patients had been screened for mutations in RPE65, GUCY2D, and CRX, and no disease-causing mutations were identified ([27] and authors' unpublished data). Of the 17 ESCS patients, 13 had been screened for mutations in the NR2E3 gene; all ESCS patients, screened so far, have at least one allelic and potentially disease-causing variation in the NR2E3 gene [24,25]. However, phenotypic variations in affected patients prompted us to evaluate a possible modifier effect of NRL. The atypical retinal degeneration patient was the affected sibling of an eye donor (Case 2 in reference [26]) and had no previous molecular investigations.

Peripheral blood samples were obtained from the subjects after informed consent, and the research procedures were in accordance with institutional guidelines and the Declaration of Helsinki. Genomic DNA was isolated form the peripheral blood using standard methods. The 5' upstream region (potential promoter region) and the three exons of NRL were PCR-amplified using LA Taq polymerase (Panvera-Takara, Madison, WI) and sequenced using the following primers, designed from the human genomic sequence (GenBank Accession number AL136295): 5' upstream region (-603 to -1; forward primer: 5'-TATTTGGGTGGCCTCAGAAG-3', reverse primer: 5'-AGAGGGGGTTCTAGGTGAGC-3', forward nested sequencing primer: 5'-CTCCCAAGCTGGATTAGCAA-3'), exon 1 (forward and sequencing primer: 5'-CTCAGAGAGCTGGCCCTTTA-3', reverse primer: 5'-AGAGGGGGTTCTAGGTGAGC-3'), exon 2 (forward and sequencing primer: 5'-CCATGTGCTCCAGACCTCTC-3', reverse primer: 5'-CTCTCTTGGGCAGTCCTCCT-3'), exon 3 (forward primer: 5'-GGGGATCCCAGAGACGAG-3', reverse and sequencing primer: 5'-AAGGCGCTCTGGTAACGAT-3'). PCR products were purified either with Qiaquick columns (Qiagen, Valencia, CA) or with the MultiScreen PCR system (Millipore, Bedford, MA). Products were sequenced by the University of Michigan DNA sequencing core using reagents (BigDye version 1) and automated sequencers (models 373, 377 and 3700) from Applied Biosystems (Foster City, CA), according to the manufacturer's protocols. Sequence variation was confirmed by manual sequencing with the 33P-ThermoSequenase Cycle Sequencing Kit (Amersham Pharmacia Biotech, Piscataway, NJ).


DNA samples from 68 index patients were used for PCR with four primer sets that amplified 603 base pairs upstream of the transcription start site (putative promoter region), the three exons (including the complete coding region of 237 amino acids) and the exon-intron boundary regions of NRL. This comprehensive sequence analysis did not reveal any sequence change in 67 of the 68 samples. In one LCA patient, a C->G transversion was detected at nucleotide position 711 of the NRL coding sequence. This change was, however, in the wobble base and did not alter the amino acid residue (L237L).


In vitro studies and the identification of potential "hypermorphic" missense mutations associated with adRP in humans have implicated NRL as a major regulator of rod photoreceptor genes. Targeted disruption in mouse suggests an essential role for Nrl in the terminal differentiation of rod photoreceptors. In its absence, at least some of the rod precursors appear to develop into S-cones. With such a critical role in both photoreceptor development and function, the human NRL gene is an excellent candidate for retinopathies, such as LCA, ESCS, and the atypical retinal degeneration with S-cone rosettes. The lack of sequence changes in our cohort of patients suggests that mutations in NRL do not account for a significant proportion of these cases. It should, however, be noted that we can not rule out the possibility of intronic sequence variations, which result in splicing errors. Together with previously published studies, the mutation screenings of the NRL gene in 677 index patients with retinopathies (18 adRP, 23 arRP, and 12 LCA [14]; 200 adRP [15,16]; 130 adRP and 37 simplex RP [17]; 189 adRP [18]; this study) have identified only six potential disease-causing mutations in three codons (S50T: four apparently-related families in the UK [16]; S50L: two patients; S50P, P51T, P51L, and G122E). None of these changes are observed in control individuals. All of these mutations appear to originate independently and alter the putative transactivation domain of the NRL protein.

We identified only one nucleotide substitution in the NRL gene in our study and this does not result in an amino acid change (L237L). Only two nucleotide changes (in addition to the disease-causing mutations) in the coding region of NRL have been reported previously but were erroneously described as L155L and V245V [17]. These two silent polymorphisms were actually L208L and L237L, the latter being the same as identified in this study. As illustrated, even these are not commonly observed in the population. A relative lack of polymorphic amino acid changes and/or single nucleotide variations in NRL is rather remarkable, particularly in light of the frequency of haplotype variations observed in human genes [28]. A comprehensive analysis of single nucleotide variations from 313 human genes has identified on average one single nucleotide polymorphism (SNP) every 185 bases (SNPs per kbp: 3.4 in the coding regions, 5.3 in the 5' untranslated regions, 5.9 in 5' upstream region, 6.5 in the exon-intron boundaries, and 7.0 in the 3' untranslated regions) [28]. Thus, one would predict identification of 8-9 SNPs in the NRL sequence analyzed in this study (a total of 1702 bp). The discovery of a single SNP in our study is significantly different from the expected 8-9 SNPs (P<0.01).

Although the photoreceptor phenotype of Nrl-/- mice suggested there may be human retinal diseases other than adRP that may be caused by NRL gene mutations, not a single mutation in our cohort of patients was observed. It is possible that the function(s) of NRL in mouse and human retina is different. Though both species have rod-dominant retinas the photoreceptor topology is distinct and humans have three different cone types [29]. In humans, additional transcription factors may compensate for the loss of NRL and the adRP clinical phenotype can be accounted by a dominant gain of function mutation [15]. Conversely, it is plausible that, in humans, NRL may have alternative functions and the loss of NRL would result in a more complex phenotype. For example, in mice, we detect the expression of Nrl in the pineal gland and the loss of Nrl radically affects pineal gene expression and circadian behavior (A.J. Mears, unpublished data). Continued analysis of NRL mutations in both animal models and human patients will therefore be necessary to gain further insight into the specific roles of this important retinal transcription factor.


We thank Debra Breuer, Elaine deCastro, Suja Hiriyanna, Leigh Gardner, Mirna Mustapha-Chaib, Adam Reddick, Jindan Yu, and Sepideh Zareparsi for their advice and assistance. This research was supported by grants from National Institutes of Health (EY11115, EY13385, EY13729), Foundation Fighting Blindness (Owings Mills, MD), Research to Prevent Blindness (New York, NY), Daniel Matzkin Research Fund (Philadelphia, PA), Elmer and Sylvia Sramek Charitable Foundation (Chicago, IL), Scientific and Technical Research Council of the Turkish Republic (TUBITAK), and the State Planning Organization of Turkey (DPT; Grant number 00K120660).


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