Author Affiliations: Associated Retinal Consultants,William Beaumont Hospital, Royal Oak, Mich (Drs Wu, Drenser, Trese, and Capone,and Ms Dailey), and Department of Ophthalmology, Chang Gung Memorial Hospital,and College of Medicine, Chang Gung University, Tao-Yuan, Taiwan (Dr Wu).
To correlate the ophthalmic findings of patients with pediatric vitreoretinopathies with mutations occurring in the Norrie disease gene (NDP).
One hundred nine subjects with diverse pediatric vitreoretinopathies and 54 control subjects were enrolled in the study. Diagnoses were based on retinal findings at each patient's first examination. Samples of DNA from each patient underwent polymerase chain reaction amplification and direct sequencing of the NDP gene.
Eleven male patients expressing mutations in the NDP gene were identified in the test group, whereas the controls demonstrated wild-type NDP. All patients diagnosed as having Norrie disease had mutations in the NDP gene. Four of the patients with Norrie disease had mutations involving a cysteine residue in the cysteine-knot motif. Four patients diagnosed as having familial exudative vitreoretinopathy were found to have noncysteine mutations. One patient with retinopathy of prematurity had a 14-base deletion in the 5′ untranslated region (exon 1), and 1 patient with bilateral persistent fetal vasculature syndrome expressed a noncysteine mutation in the second exon.
Mutations disrupting the cysteine-knot motif corresponded to severe retinal dysgenesis, whereas patients with noncysteine mutations had varying degrees of avascular peripheral retina, extraretinal vasculature, and subretinal exudate.
Patients exhibiting severe retinal dysgenesis should be suspected of carrying a mutation that disrupts the cysteine-knot motif in the NDP gene.
Many pediatric vitreoretinopathies, including Norrie disease (ND),1- 3 familial exudative vitreoretinopathy (FEVR),4,5 Coats disease,6 and retinopathy of prematurity (ROP),7- 9 have been associated with mutations occurring in the ND gene (NDP). The common pathology in these diseases is an aberration of retinal development demonstrating varying degrees of peripheral avascular retina,abnormal vascularization with retinal neovascularization, subretinal exudation,an abnormal vitreous composition and vitreoretinal interface, and retinal detachment. Disease classification based on visual dysfunction (rather than retinal findings) and associated systemic findings often lead to improper diagnoses. To our knowledge, a detailed evaluation of retinal findings and associated NDP mutations from a pediatric database with diversified retinal pathology has not been performed, which prompted this prospective analysis of our patient database.
The NDP gene is located on the short arm of chromosome X at position p11.4. The gene product, norrin, is a small secreted protein with a cysteine-knot motif.10- 12 It is a member of the mucinlike subgroup of 10-membered cysteine-knot proteins.The cysteine-knot motif is highly conserved in many growth factors (eg, transforming growth factor β, human chorionic gonadotropin, nerve growth factor, and platelet-derived growth factor). Norrin has 2 primary domains: a signal peptide that directs localization of the molecule and a cysteine knot that provides the structural conformation required for receptor binding and activation of signal transduction. Norrin acts as a ligand in a Wnt receptor–β-catenin signal transduction pathway that plays a regulatory role in retina development and is necessary for regression of hyaloid vessels in the eye.10,13,14 Frizzled (FZ) gene receptors are coupled to the β-catenin canonical signaling pathway, which results in activation of Wnt target genes.10 A Wnt receptor frizzled 4 (Fzd4) knockout mouse model demonstrates the importance of this pathway in vasculogenesis and normal retinal development.10 Computer modeling of norrin highlights the role of the cysteine residues and their disulfide bonds in the structural conformation of norrin and in its function.Mutations not affecting cysteine residues may alter protein folding and compromise pathway activation to varying degrees, but their effect on norrin structure and function is not as clear. Untranslated regions (UTRs) have regulatory functions that control the expression and stability of norrin.15- 17 Mutations in these regions have also been associated with vitreoretinopathy.8,9
The purpose of this study was to correlate retinal findings with mutations in the NDP gene in pediatric vitreoretinopathies.Retinal findings were documented and diagnoses were assigned on the basis of these findings. Diagnoses, retinal phenotypes, and NDP gene mutations were then evaluated. This report discusses our findings regarding the norrin protein and its effect on retinal development.
Patients referred to our practice were recruited to the study through a protocol approved by the internal review board at William Beaumont Hospital and consented to participation. From November 1, 2003, through February 28,2006, 109 pediatric patients with various vitreoretinopathies were prospectively enrolled in this study. Inclusion criteria consisted of a clinical diagnosis of ND, FEVR (using the criteria listed in Table 1), persistent fetal vasculature syndrome (PFVS), Coats disease,or stage 4 or 5 ROP. Fifty-four patients referred to our clinic for consultation but not found to have retinal findings consistent with ND, FEVR, ROP, PFVS,and Coats disease were used as control subjects. Ethnicity was similar in both groups (predominantly white subjects in the study group, which also included 1 Asian and 1 Hispanic subject, and all white subjects in the control group).Before enrollment, patients underwent dilated fundus examination at our clinic or had fundus photographs forwarded when examination in our clinic was not possible. Detailed birth, medical, and family histories were obtained at the first visit.
Participants provided a blood sample from which genomic DNA was isolated using the manufacturer's recommended product protocol (Puregene; Gentra Systems,Inc, Minneapolis, Minn). Polymerase chain reaction (Herculase; Stratagene,La Jolla, Calif) was performed to amplify the NDP gene.Five sets of forward and reverse primer pairs were used for site-specific amplification as previously described.8,18 Exon 3 was amplified with single polymerase chain reaction; other DNA segments were amplified with multiplex polymerase chain reaction. The amplification conditions involved an initial warming at 98°C for 1 minute followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 1 minute. The final extension at 72°C was for 10 minutes. Purified DNA served as a template for direct DNA sequencing using a thermocycle sequencing kit (Dye Terminator Cycle Sequencing Quick Start; Beckman Coulter, Inc, Fullerton, Calif). Direct sequencing was performed using an autosequencer (CEQ8000; Beckman Coulter, Inc). The sequencing data were compiled with the use of a data processing software suite (Beckman Coulter, Inc), which produced 4-color plots. The sequencing information was compared with the GenBank database using BLAST software (available at http://www.ncbi.nlm.nih.gov/blast).
One hundred nine patients with diverse pediatric vitreoretinopathies were enrolled. Included were 5 patients with ND, 52 with FEVR (34 male and 18 female), 15 with PFVS (4 unilateral and 11 bilateral), 4 with Coats disease,and 33 with ROP (stage 4 or 5). The mutations identified in 11 of these patients,all male, are shown in Table 2.
Of these 11 patients, 5 were diagnosed as having ND on the basis of ophthalmoscopic examination results. When first examined by us, 4 of the patients (patients 3, 6, 8, and 9 in Table 2)had a characteristic fundus structure believed to be specific for ND (Figure 1).19 We found a stalk attached to the posterior aspect of the lens with variably sized footplates; the sphere of the retina to which it was attached had an exudative appearance with a yellow color. Unbranched retinal vessels could be seen coursing through the tissue, which was assumed to be dysplastic retina. The peripheral retina demonstrated a variable area of avascular attached retina with underlying areas of pigment change. The fifth patient (patient 10) had an extremely dystrophic retina that was associated with total bilateral retinal detachments at birth.
Norrie disease manifestations in patients 3 (A), 6 (B), and 8 (C). Severe retinal dysgenesis is evident with severely atrophic underlying pigment changes.
The first patient with ND (patient 3) was found to have a point mutation (TGC>TAC) in exon 3 at codon 65. This substitution caused a cysteine-to-tyrosine mutation. Cysteine 65 is the second cysteine in the highly conserved cysteine-knot motif; its alteration interfered with the formation of the disulfide bridge between the β2 and β4 strands of norrin (Figure 2B). The patient's mother was confirmed to be a carrier of ND. Another patient with ND (patient 6) expressed a point mutation (TGC>CGC)in exon 2. This changed the cysteine at residue 39 to arginine, which altered the first cysteine residue in the cysteine-knot motif and in turn interfered with the formation of the disulfide bridge between the β1 and β3strands of norrin (Figure 2B). The mother of patient 6 was also confirmed to be a carrier of ND. Two brothers with ND (patients 8 and 9) shared mutations in exon 3 at codon 95, where a point mutation (TGC>TGA) altered the cysteine codon to a termination codon. Theoretically,the resulting protein would lack the last 38 amino acids of the wild-type Norrie protein, which contains cysteine residues involved in the cysteine knot (Figure 2A). The final patient with ND (patient 10) had a 12-base insertion in the 5′ UTR (exon 1)that involved a CT dinucleotide repeat region. This repeat plays a role in transcriptional regulation and affects the translatability of the transcripts.16,17
Norrin, a product of the Norrie disease (NDP) gene, is a small secreted protein with a cysteine-knot motif. A, Amino acid sequence of norrin. Highlighted C's indicate cysteine residues involved in disulfide bonds; underlined C’s, cysteine residues not directly involved in disulfide bonds.B, Secondary structure of the NDP gene. The roman numerals label the cysteine residues involved in disulfide bonds (connecting lines) and in the tertiary structure of norrin.
Four patients with FEVR (Figure 3)had mutations in the NDP gene. All of them exhibited some degree of retinal detachment or exudation (stage 2B or higher). The visual acuities correlated most closely with the location of exudate (involving the macula vs extramacular) rather than the extent of the detachment. One patient (patient 1) had a point mutation (CGG>TGG) in exon 3, which altered amino acid 121 from an arginine to a tryptophan. Another patient (patient 2) expressed a mutation (CAC>CGC) in exon 2, which changed the histidine at codon 42 to an arginine. The third patient with FEVR (patient 5) had a mutation (CTC>ATC)in exon 3 at codon 61, which changed leucine to isoleucine. The last patient with FEVR (patient 7) had a mutation in the 3′ UTR involving nucleotide position *717. All of the amino acid changes were predicted to affect the secondary structure to various degrees but not to interfere with the cysteine bonds required for the active domain (the cysteine knot). Charge changes in amino acid substitutions alter the isoelectric point of norrin, which may compromise its activity under physiologic conditions. All of the patients had typical manifestations of FEVR, with peripheral retinal vascular anomalies or absent peripheral vascularization of the retina.
Manifestations of familial exudative vitreoretinopathy. A-D, Patient 1, right eye, stage 4B (A and B), and left eye, stage 2B (C and D). E and F, Left eye of patient 7, stage 4B.
Persistent fetal vascular syndrome represents a failure of regression of the primary hyaloid system. This is supported by findings in Ndp knockout animal models that demonstrate failure of the primary hyaloid artery and associated structures to regress.14 Bilateral cases of PFVS can often be difficult to distinguish from ND or FEVR. One patient diagnosed as having bilateral PFVS (patient 4) had a mutation (AGG>AGC) in exon 2 at amino acid 41. The G-to-C transversion changed arginine to serine.The mother of patient 4 was confirmed to be a carrier of PFVS. Patient 4 also had glucose-6-phosphate dehydrogenase deficiency, which was diagnosed after birth. On the basis of enzyme activity, the deficiency was considered by his pediatrician to be severe. The effect of the mutation in the NDP gene in conjunction with increased oxidative stress was unknown but may have exacerbated norrin dysfunction, thereby worsening the vascular dysgenesis seen in this patient.20
One patient with ROP (patient 11) was found to have a mutation in the NDP gene consisting of a 14-kilobase CT dinucleotide repeat deletion in the 5′ UTR (exon 1) after nucleotide 8. None of the patients with ROP had mutations in the NDP coding region sequence or 3′ UTR. Dipyrimidine repeats in the 5′ UTR are responsible for transcription regulation and efficiency of translation, and this insertion may alter gene regulation.16,17 None of the controls had NDP gene mutations or polymorphisms.
The cysteine residues responsible for the cysteine-knot formation are found at positions 39, 65, 69, 96, 126, and 128 (Figure 2).21 Mutations affecting these amino acids interfere with the folding and stability of norrin by disrupting key disulfide bonds in the molecule. In our patients, mutations involving cysteine residues corresponded with severe retinal dysgenesis, and these patients were diagnosed as having ND. Mutations in noncysteine residues of the norrin protein showed abnormal vascular and retinal development and phenotypes consistent with FEVR. The 5′ UTR contains elements that regulate gene expression,16,17 and the 3′ UTR is responsible for localizing the messenger RNA in the intracellular microenvironment.22 Mutations in these regions may alter regulatory and/or co-localizing elements that regulate norrin and its translation. Clinical presentation and retinal findings correlate well with diagnoses and genotypes.
Norrie disease and FEVR are known to be allelic. However, the retinal phenotype of ND is more dysgenic than that of FEVR. Patients with ND manifest severe retinal dysgenesis, which is generally detected within the first 3months of life. Familial exudative vitreoretinopathy is initially detected at various ages, and the retinal structure is significantly different from that found in ND. Four (80%) of our 5 patients with ND had mutations that directly altered the cysteine-knot motif. Mutations of cysteine residues within this domain would be expected to dramatically affect the tertiary structure of norrin and result in severely compromised receptor binding. Inability to activate the Wnt receptor–β-catenin pathway results in early abrogation of neurosensory and vascular development.10,14 The exception has a 12-dinucleotide CT repeat insertion in the 5′ UTR and is thought to alter the transcription regulation of the NDP gene and the translation efficiency of norrin, thereby affecting gene expression and regulation. A patient with retinal detachments associated with ROP (patient 11) has a deletion in the 5′ UTR of the NDP gene. Loss of the CT repeat may also affect the regulation of transcription and translation.
The patients with X-linked FEVR in our study (stage 2B or greater) had noncysteine mutations in the NDP gene. These mutations are predicted to cause secondary structural changes that result in suboptimal folding of the norrin protein. Receptor binding may be compromised but should still occur to varying degrees. Suboptimal pathway activation would be predicted and is consistent with the varying degrees of retinal dysgenesis seen in patients with FEVR.
The retinal phenotype correlated well with the genotype findings in our patients. The most consistent correlation was seen with the diagnosis of ND and the mutations that disrupted the cysteine-knot motif. The correlation between noncysteine mutations and the other diagnoses was less evident. Presumably,noncysteine mutations result in suboptimal norrin folding to varying degrees.Further studies of norrin may elucidate whether and how these changes alter receptor binding and signal transduction activation. Exogenous environmental or systemic factors may also play a role in disease presentation. These results highlight the importance of the tertiary structure of proteins and their effect on structural changes of specialized tissues.
Correspondence: Kimberly A. Drenser, MD,PhD, Associated Retinal Consultants, 632 William Beaumont Medical Bldg, 3535W 13 Mile Rd, Royal Oak, MI 48073 (firstname.lastname@example.org).
Submitted for Publication: June 27, 2006; final revision received August 17, 2006; accepted September 21, 2006.
Financial Disclosure: None.
Funding/Support: This study was supported in part by Taiwan Merit Scholarship TMS-094-1-B-001.
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