Helicoid Subretinal Fibrosis Associated With a Novel Recessive NR2E3 Mutation p.S44X

Objectives  To describe a unique pattern of helicoid subretinal fibrosis associated with a novel recessive NR2E3 mutation and to highlight how examination of the proband's affected relative allowed appropriate genetic testing.

Design  Interventional family study (ophthalmic examination and candidate gene testing).

Results  The proband (mother), who complained of poor vision since early childhood, had bilateral helicoid subretinal fibrosis mostly involving the macula. Two children were symptomatic; one had ophthalmic findings similar to her mother while the second had macular retinoschisis, retinal pigment epithelium changes, and refractive accommodative esotropia. The father and third child were asymptomatic and had unremarkable ophthalmic examination findings. Based on the findings in the second symptomatic child, NR2E3 analysis was performed, which revealed homozygosity for a novel mutation, p.S44X, in all 3 affected individuals and heterozygosity for the mutation in both asymptomatic individuals.

Conclusion  Helicoid subretinal fibrosis is another potential phenotypic manifestation of recessive NR2E3 mutation.

Clinical Relevance  Examination of affected relatives can be helpful in guiding molecular genetic testing for hereditary eye disease when the proband's diagnosis is unclear.

The gene NR2E3 encodes a ligand-dependent evolutionarily conserved transcription factor important for regulation of photoreceptor development and function and uniquely expressed in the outer nuclear layer of the neurosensory retina.^1,2 The 410–amino acid protein NR2E3 has 4 functional domains: the ligand-independent A/B domain, the DNA-binding domain, a flexible D domain (hinge), and the ligand-binding domain.³ The DNA-binding domain begins at position 45 and is the most evolutionarily conserved region, consisting of 70 amino acids arranged in 2 Cys₄ zinc fingers. Mutation in NR2E3 as a cause of human disease was first reported in recessive enhanced S-cone syndrome (ESCS), a retinal degeneration associated with increased S-cone function at the expense of other photoreceptors.¹ Patients with ESCS often have night blindness, decreased acuity, macular retinoschisis, and nummular pigmentary disturbance at the level of the retinal pigment epithelium; in addition, affected children can have refrac tive accommodative esotropia.⁴ Since the original description of NR2E3 mutation as the cause for recessive ESCS,¹ mutations in NR2E3 have been associated with a variety of retinal phenotypes such as recessive Goldmann-Favre syndrome, recessive clumped pigmentary retinal degeneration, recessive retinitis pigmentosa, dominant retinitis pigmentosa, dominant ESCS, and dominant and recessive retinal disease within a single family.^5-15 In this report, we describe a phenotype of helicoid subretinal fibrosis that was found to be associated with a novel recessive NR2E3 mutation in a consanguineous family. In addition, we highlight how appropriate molecular genetic testing became apparent only after examination of the proband's affected daughter, who had a phenotype consistent with childhood NR2E3-related disease.

During an ophthalmic evaluation for poor vision since childhood, the proband (the mother) was found to have a unique pattern of subretinal fibrosis. Two of her 3 children also had poor vision. She was encouraged to bring her immediate family for complete ophthalmic examination and potential genetic testing. After institutional board approval and family informed consent, the entire immediate family (both parents and 3 children) underwent ophthalmic examination and venous blood sampling for candidate gene testing and possible linkage analysis. Ancillary diagnostic testing (ocular coherence tomography [RTVue Scanner; Optovue, Fremont, California], fluorescein angiography, and electroretinography¹⁶) was performed on the proband only as the parents did not consent for ancillary testing beyond fundus photography on their 2 symptomatic children.

Genomic DNA was extracted from whole blood anticoagulated with EDTA using the Puregene DNA Extraction Kit (catalog number D-5000; Gentra Systems, Minneapolis, Minnesota) according to the manufacturer's instructions. DNA was quantified spectrophotometrically and stored in aliquots at −20°C until required. For candidate gene analysis, polymerase chain reaction amplification was performed on a thermocycler (DNA Engine Tetrad; MJ Research Inc, Waltham, Massachusetts) in a total volume of 25 μL, containing 10 ng of DNA, 50mM potassium chloride, 10mM TRIS–hydrochloric acid (pH 9.0), 1.5mM magnesium chloride, 0.1% Triton X-100, 0.25mM of each deoxyribonucleotide triphosphate, 0.8μM of each primer, and 0.5 U of Taq polymerase (Qiagen, Hilden, Germany). For polymerase chain reaction, an initial denaturation step at 95°C for 10 minutes was followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing for 30 seconds at 60°C, and extension at 72°C for 30 seconds followed by a final extension step of 72°C for 10 minutes. All exons and their intronic boundaries for the candidate gene were sequenced using an ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Piscataway, New Jersey) following the manufacturer's instructions. Sequence analysis was performed using the SeqManII module of the Lasergene software package (DNASTAR Inc, Madison, Wisconsin) using normal sequence for comparison.

The mother and 2 of her 3 children had a history of poor vision since early childhood. There was no other significant history or eye or medical disease. The parents were first cousins. The family pedigree is shown in Figure 1. The asymptomatic father (I.1) and asymptomatic child (II.3) had unremarkable ophthalmic examination findings. Results of general medical assessment by an internist (for the parents) and by a pediatrician (for the children) were unremarkable.

The mother (I.2), a 25-year-old woman with poor vision especially at night since early childhood, had a best-corrected visual acuity of 20/60 OD and 20/200 OS. Ophthalmic examination findings were significant for left esotropia of approximately 20 prism diopters at near and bilateral subretinal fibrosis in a helicoid pattern mostly involving the macula and without evidence for active inflammation or exudates (Figure 2A and B). No retinoschisis or retinal elevation was seen. Cycloplegic refraction (cyclopentolate hydrochloride, 1%) revealed mild myopia consistent with her current glasses. Results of testing for sarcoidosis (angiotensin-converting enzyme levels, chest radiography), tuberculosis (purified protein derivative skin testing, chest radiography), toxoplasmosis (antibody levels), Brucella (antibody levels), and syphilis (rapid plasma reagin) were negative. Fluorescein angiography showed staining of the fibrotic lesions and attenuation of the surrounding retinal pigment epithelium but no evidence for uveitis, retinitis, or neovascularization. Electroretinography revealed nonspecific depressed (<25%) rod and cone function and delayed implicit times. Ocular coherence tomography confirmed the clinical impression of subretinal fibrosis (Figure 2G).

The older symptomatic child (II.1) was a 7-year-old girl with decreased vision (especially at night) and strabismus since approximately 2 years of age. With her current glasses (+3.00 −2.00 × 180 OD and +5.00 −2.00 × 180 OS), visual acuity was 20/400 OD and 20/50 OS. She was orthotropic at near with her glasses. Ophthalmic examination was significant for retinal findings: bilateral helicoid fibrosis mostly involving the macula and fine stippling of the retinal pigment epithelium (Figure 2C and D). No retinoschisis or retinal elevation was seen. Cycloplegic refraction (following administration of cyclopentolate, 1%) was +7.75 −2.00 × 180 OD and +7.75 −2.00 × 180 OS. There was no visual improvement with the new glasses. Results of testing for sarcoidosis (angiotensin-converting enzyme levels, chest radiography), tuberculosis (purified protein derivative skin testing, chest radiography), toxoplasmosis (antibody levels), Brucella (antibody levels), cytomegalovirus (antibody levels), and syphilis (rapid plasma reagin) were negative.

The younger symptomatic child (II.2) was a 5-year-old girl who had decreased vision and strabismus since approximately 2 years of age. Visual acuity was counting fingers at 3 ft OD and 20/60 OS. There was a right esotropia of 40 prism diopters at near and a right afferent pupillary defect. Ophthalmic examination findings were significant for macular retinoschisis greater in the right eye than in the left eye and fine retinal pigment epithelium stippling in both eyes (Figure 2E and F). Cycloplegic refraction (cyclopentolate, 1%) was +7.00 OD and +7.25 −0.75 × 180 OS. She was prescribed her full hyperopic correction, which improved vision to 20/40 OS and decreased the esotropia to 10 prism diopters at near. Based on the macular retinoschisis, fine retinal pigment epithelium stippling, and refractive accommodative esotropia, NR2E3-related disease was suspected.⁴

Based on the clinical findings of individual II.2, which were suggestive of childhood NR2E3 mutation,⁴NR2E3 sequencing was performed for all family members. The designed primers used for amplification of the coding region of NR2E3 are listed in the Table. Homozygosity for a stop mutation was identified in exon 2 (p.S44X, c.131C>A) (GenBank AF121129.1) in the proband and her 2 affected children (Figure 2H). The unaffected husband and child were heterozygous for the mutation. This mutation is predicted to disrupt the highly conserved DNA-binding domain and was not found in an online locus-specific database for NR2E3 variants (www.LOVD.nl/eye; accessed September 23, 2009).¹⁷ A recessive p.S44L missense variant was previously described in a patient with autosomal recessive retinitis pigmentosa; however, with further analysis of that family, the variant did not cosegregate with the phenotype.¹⁵ The previously reported mutation that disrupts NR2E3 in a position closest to p.S44 is a splice mutation (c.119-2A>C, p.41AfsX23) that also disrupts the DNA-binding domain and has been reported in association with ESCS, Goldmann-Favre disease, clumped pigmentary retinal degeneration, and autosomal recessive retinal dystrophy.^1,5,6,15

Retinal findings in this consanguineous family were associated with a novel recessive NR2E3 mutation (p.S44X). Two affected members had a unique pattern of helicoid subretinal fibrosis without evidence for posterior uveitis. NR2E3 analysis for the family was performed for the family following examination of the third affected individual, who had a phenotype suggestive of childhood NR2E3-related disease (macular retinoschisis, retinal pigment epithelium changes, and refractive accommodative esotropia).⁴ This report expands the phenotypic spectrum of recessive NR2E3 mutation and illustrates how examination of relatives can be useful in establishing a diagnosis for hereditary eye disease of unclear etiology.

The helicoid pattern of subretinal fibrosis seen in the proband and her daughter are unique in our experience. Subretinal fibrosis can occur in a variety of infectious, inflammatory, and autoimmune conditions.^18-21 However, in such situations, the fibrosis is not typically helicoid and there is usually clinical evidence for posterior uveitis or medical disease. Serpiginous choroiditis, an idiopathic chronic progressive helicoid peripapillary choroidopathy, is perhaps the previously reported condition that most resembles the phenotype of patients I.2 and II.1. However, serpiginous choroiditis typically has evidence of inflammatory activity at the margins of the lesions, large areas of geographic atrophy, and involvement that remains contiguous with the peripapillary area, and is not familial.²¹

NR2E3 accumulates in rod precursors of the developing mouse retina and is involved in a transcription factor cascade that regulates photoreceptor development.²² In the absence of the protein, an almost 2-fold increase in S-cone development occurs^22,23 but most photoreceptors still have a hybrid cell type expressing both rod and cone genes²²; this is consistent with the concept that background interacting genetic and/or environmental modifiers influence the final observed phenotype associated with NR2E3 mutation.¹⁴ The retinal degeneration that is part of NR2E3 mutation may be related to the inability of cone cells to survive in the absence of rod photoreceptors and/or because a normal complement of NR2E3 protein is required for photoreceptor maintenance.²⁴ Although features of the standard electroretinogram¹⁶ can be diagnostic for ESCS (a similar, simplified, and delayed waveform response to flashes under photopic and scotopic conditions and a delayed 30-Hz flicker amplitude lower than that of the photopic a wave), they are less helpful when amplitudes are very depressed in more advanced disease. In such situations, more sophisticated techniques that isolate S-cone function can be useful if such testing is available. It is unclear why subretinal fibrosis would be associated with a homozygous p.S44X NR2E3 mutation; however, it is now well established that NR2E3 mutation is associated with a variety of different retinal phenotypes.^1,4-15,17 In the current family, fibrosis may have been a reactive response from the retinal pigment epithelium, the level at which the typical nummular deposits of ESCS occur. Macrophages may have played a role, as a murine model suggests that macrophages contribute to pigmentary changes in ESCS.²⁵

Our report documents helicoid subretinal fibrosis as another potential phenotypic manifestation of recessive NR2E3 mutation. NR2E3-related disease in this family would not have been suspected had we not examined the youngest affected relative, highlighting how examination of other affected family members can be useful when a given patient has apparent hereditary eye disease but an unclear diagnosis.

Correspondence: Arif O. Khan, MD, Division of Pediatric Ophthalmology, King Khaled Eye Specialist Hospital, PO Box 7191, Riyadh 11462, Saudi Arabia (arif.khan@mssm.edu).

Submitted for Publication: July 9, 2009; final revision received September 23, 2009; accepted October 13, 2009.

Financial Disclosure: None reported.