Retinal Phenotype Genotype Correlation of Pediatric Patients

OPHTHALMIC MOLECULAR GENETICS Retinal Phenotype–Genotype Correlation of Pediatric Patients Expressing Mutations in the Norrie Disease Gene

Wei-Chi Wu, MD, PhD; Kimberly Drenser, MD, PhD; Michael Trese, MD; Antonio Capone, Jr, MD; Wendy Dailey, BS

Objective: To correlate the ophthalmic findings of pa- exudative vitreoretinopathy were found to have noncys- tients with pediatric vitreoretinopathies with mutations teine mutations. One patient with retinopathy of prema- occurring in the Norrie disease gene (NDP). turity had a 14-base deletion in the 5Ј untranslated region (exon 1), and 1 patient with bilateral persistent fetal Methods: One hundred nine subjects with diverse pe- vasculature syndrome expressed a noncysteine muta- diatric vitreoretinopathies and 54 control subjects were tion in the second exon. enrolled in the study. Diagnoses were based on retinal findings at each patient’s first examination. Samples of Conclusion: Mutations disrupting the cysteine-knot mo- DNA from each patient underwent polymerase chain re- tif corresponded to severe retinal dysgenesis, whereas pa- action amplification and direct sequencing of the NDP tients with noncysteine mutations had varying degrees gene. of avascular peripheral retina, extraretinal vasculature, and subretinal exudate. Results: Eleven male patients expressing mutations in the NDP gene were identified in the test group, whereas Clinical Relevance: Patients exhibiting severe retinal the controls demonstrated wild-type NDP. All patients dysgenesis should be suspected of carrying a mutation diagnosed as having Norrie disease had mutations in the NDP gene. Four of the patients with Norrie disease had that disrupts the cysteine-knot motif in the NDP gene. mutations involving a cysteine residue in the cysteine- knot motif. Four patients diagnosed as having familial Arch Ophthalmol. 2007;125:225-230

ANY PEDIATRIC VITREO- The NDP gene is located on the short retinopathies, includ- arm of chromosome X at position p11.4. ing Norrie disease The gene product, norrin, is a small se- (ND),1-3 familial exu- creted protein with a cysteine-knot mo- dative vitreoretinopa- tif.10-12 It is a member of the mucinlike sub- thy (FEVR),4,5 Coats disease,6 and reti- group of 10-membered cysteine-knot M 7-9 nopathy of prematurity (ROP), have proteins. The cysteine-knot motif is highly been associated with mutations occur- conserved in many growth factors (eg, ring in the ND gene (NDP). The common transforming growth factor ␤, human cho- pathology in these diseases is an aberra- rionic gonadotropin, nerve growth fac- tion of retinal development demonstrat- tor, and platelet-derived growth factor). ing varying degrees of peripheral avascu- Norrin has 2 primary domains: a signal lar retina, abnormal vascularization with peptide that directs localization of the mol- retinal neovascularization, subretinal exu- ecule and a cysteine knot that provides the dation, an abnormal vitreous composi- structural conformation required for re- tion and vitreoretinal interface, and reti- ceptor binding and activation of signal Author Affiliations: Associated nal detachment. Disease classification transduction. Norrin acts as a ligand in a Retinal Consultants, William based on visual dysfunction (rather than Wnt receptor–␤-catenin signal transduc- Beaumont Hospital, Royal Oak, retinal findings) and associated systemic tion pathway that plays a regulatory role Mich (Drs Wu, Drenser, Trese, findings often lead to improper diag- in retina development and is necessary for and Capone, and Ms Dailey), noses. To our knowledge, a detailed evalu- regression of hyaloid vessels in the and Department of ation of retinal findings and associated eye.10,13,14 Frizzled (FZ) gene receptors are Ophthalmology, Chang Gung ␤ Memorial Hospital, and College NDP mutations from a pediatric database coupled to the -catenin canonical sig- of Medicine, Chang Gung with diversified retinal pathology has not naling pathway, which results in activa- University, Tao-Yuan, Taiwan been performed, which prompted this pro- tion of Wnt target genes.10 A Wnt recep- (Dr Wu). spective analysis of our patient database. tor frizzled 4 (Fzd4) knockout mouse

(REPRINTED) ARCH OPHTHALMOL / VOL 125, FEB 2007 WWW.ARCHOPHTHALMOL.COM 225

©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 model demonstrates the importance of this pathway in ings regarding the norrin protein and its effect on reti- vasculogenesis and normal retinal development.10 Com- nal development. puter modeling of norrin highlights the role of the cysteine residues and their disulfide bonds in the structural METHODS conformation of norrin and in its function. Mutations not affecting cysteine residues may alter protein folding and compromise pathway activation to varying degrees, but PATIENT ENROLLMENT their effect on norrin structure and function is not as clear. Patients referred to our practice were recruited to the study Untranslated regions (UTRs) have regulatory functions 15-17 through a protocol approved by the internal review board at that control the expression and stability of norrin. Mu- William Beaumont Hospital and consented to participation. tations in these regions have also been associated with From November 1, 2003, through February 28, 2006, 109 pe- vitreoretinopathy.8,9 diatric patients with various vitreoretinopathies were prospec- The purpose of this study was to correlate retinal find- tively enrolled in this study. Inclusion criteria consisted of a ings with mutations in the NDP gene in pediatric vitreo- clinical diagnosis of ND, FEVR (using the criteria listed in retinopathies. Retinal findings were documented and di- Table 1), persistent fetal vasculature syndrome (PFVS), Coats agnoses were assigned on the basis of these findings. disease, or stage 4 or 5 ROP. Fifty-four patients referred to our Diagnoses, retinal phenotypes, and NDP gene muta- clinic for consultation but not found to have retinal findings tions were then evaluated. This report discusses our find- 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 sub- Table 1. Clinical Classification of Familial jects in the control group). Before enrollment, patients under- Exudative Vitreoretinopathy went dilated fundus examination at our clinic or had fundus photographs forwarded when examination in our clinic was not Stage Clinical Features possible. Detailed birth, medical, and family histories were ob- 1 Avascular retina without extraretinal vascularization tained at the first visit. 2 Avascular retinal periphery with extraretinal vascularization A Without exudate GENETIC ANALYSIS B With exudate 3 Partial retinal detachment; subtotal, fovea spared A Primarily exudative Participants provided a blood sample from which genomic DNA B Primarily tractional was isolated using the manufacturer’s recommended product 4 Partial retinal detachment; fovea involved protocol (Puregene; Gentra Systems, Inc, Minneapolis, Minn). A Primarily exudative Polymerase chain reaction (Herculase; Stratagene, La Jolla, Calif) B Primarily tractional was performed to amplify the NDP gene. Five sets of forward 5 Total retinal detachment and reverse primer pairs were used for site-specific amplifica- A Open funnel tion as previously described.8,18 Exon 3 was amplified with single B Closed funnel polymerase chain reaction; other DNA segments were amplified with multiplex polymerase chain reaction. The amplifica-

Table 2. Mutations of the ND Gene Found Among 109 Patients With Pediatric Vitreoretinopathies

Patient No./ Hearing Novel Age at First Examination† Diagnosis Impairment Region Mutation Mutation 1/4 y FEVR No Exon 3 c.765CϾT No p.Arg121ϾTrp 2/3 y FEVR No Exon 2 c.533AϾG No p.His42ϾArg 3/4 mo ND No Exon 3 c.602GϾA No p.Cys65ϾTyr 4/3 mo PFVS No Exon 2 c.531GϾC Yes p.Arg41ϾSer 5/3 mo FEVR No Exon 3 c.589CϾA Yes p.Leu61ϾIle 6/2 mo ND No Exon 2 c.523TϾC No p.Cys39ϾArg 7/8 mo FEVR No 3Ј UTR c.*717TϾCYes 8/After birth‡ ND Yes Exon 3 c.693CϾA Yes p.Cys95Ͼstop 9/After birth‡ ND No Exon 3 c.693CϾA Yes p.Cys95Ͼstop 10/1 mo ND No Exon 1 c.3_4insCTCTCTCTCTCC No 11/5 mo ROP No Exon 1 c.9_22del No

Abbreviations: FEVR, familial exudative vitreoretinopathy; ND, Norrie disease; PFVS, persistent fetal vasculature syndrome; ROP, retinopathy of prematurity; UTR, untranslated region. †All of the patients are male. None of the patients exhibited mental retardation. ‡Patients 8 and 9 are brothers.

(REPRINTED) ARCH OPHTHALMOL / VOL 125, FEB 2007 WWW.ARCHOPHTHALMOL.COM 226

Figure 1. Norrie disease manifestations in patients 3 (A), 6 (B), and 8 (C). Severe retinal dysgenesis is evident with severely atrophic underlying pigment changes.

tion conditions involved an initial warming at 98°C for 1 minute followed by 35 cycles of denaturation at 95°C for 30 seconds, A 1020 30 annealing at 53°C for 30 seconds, and extension at 72°C for 1 MRKHV LAASFSMLSLL VIM GDTDSKTDSSFIMDSDPRR C minute. The final extension at 72°C was for 10 minutes. Puri- 40 5060 70 fied DNA served as a template for direct DNA sequencing us- MR HHY VD SI SH PKLLAREGHSQASRSEPLY C SS KMVL C C ing a thermocycle sequencing kit (Dye Terminator Cycle Se- 8090 100 110 quencing Quick Start; Beckman Coulter, Inc, Fullerton, Calif). VFSSTVLKQPFRSSHC C C RPQSKLKALRLRSGGMRLTT C Direct sequencing was performed using an autosequencer 120 130 (CEQ8000; Beckman Coulter, Inc). The sequencing data were ATYRYILSC HC EEC NS compiled with the use of a data processing software suite (Beck- man Coulter, Inc), which produced 4-color plots. The sequenc- B ing information was compared with the GenBank database using BLAST software (available at http://www.ncbi.nlm.nih.gov /blast).

RESULTS II 65 V 126 One hundred nine patients with diverse pediatric vitreoretinopathies were enrolled. Included were 5 patients with IV 96 VI 128 ND, 52 with FEVR (34 male and 18 female), 15 with PFVS I 39 (4 unilateral and 11 bilateral), 4 with Coats disease, and

33 with ROP (stage 4 or 5). The mutations identified in NCIII 69 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 yel- low color. Unbranched retinal vessels could be seen cours- Figure 2. Norrin, a product of the Norrie disease (NDP ) gene, is a small secreted protein with a cysteine-knot motif. A, Amino acid sequence of ing through the tissue, which was assumed to be dys- norrin. Highlighted C ’s indicate cysteine residues involved in disulfide bonds; plastic retina. The peripheral retina demonstrated a underlined C ’s, cysteine residues not directly involved in disulfide bonds. variable area of avascular attached retina with underly- B, Secondary structure of the NDP gene. The roman numerals label the cysteine residues involved in disulfide bonds (connecting lines) and in the ing areas of pigment change. The fifth patient (patient tertiary structure of norrin. 10) had an extremely dystrophic retina that was associated with total bilateral retinal detachments at birth. The first patient with ND (patient 3) was found to have tif and in turn interfered with the formation of the disul- a point mutation (TGCϾTAC) in exon 3 at codon 65. This fide bridge between the ␤1 and ␤3 strands of norrin substitution caused a cysteine-to-tyrosine mutation. Cys- (Figure 2B). The mother of patient 6 was also confirmed teine 65 is the second cysteine in the highly conserved cys- to be a carrier of ND. Two brothers with ND (patients 8 teine-knot motif; its alteration interfered with the forma- and 9) shared mutations in exon 3 at codon 95, where a tion of the disulfide bridge between the ␤2 and ␤4 strands point mutation (TGCϾTGA) altered the cysteine codon of norrin (Figure 2B). The patient’s mother was con- to a termination codon. Theoretically, the resulting pro- firmed to be a carrier of ND. Another patient with ND (pa- tein would lack the last 38 amino acids of the wild-type tient 6) expressed a point mutation (TGCϾCGC) in exon Norrie protein, which contains cysteine residues in- 2. This changed the cysteine at residue 39 to arginine, which volved in the cysteine knot (Figure 2A). The final patient altered the first cysteine residue in the cysteine-knot mo- with ND (patient 10) had a 12-base insertion in the 5Ј UTR

(REPRINTED) ARCH OPHTHALMOL / VOL 125, FEB 2007 WWW.ARCHOPHTHALMOL.COM 227

C D

E F

Figure 3. 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.

(exon 1) that involved a CT dinucleotide repeat region. altered amino acid 121 from an arginine to a trypto- This repeat plays a role in transcriptional regulation and phan. Another patient (patient 2) expressed a mutation affects the translatability of the transcripts.16,17 (CACϾCGC) in exon 2, which changed the histidine at Four patients with FEVR (Figure 3) had mutations codon 42 to an arginine. The third patient with FEVR in the NDP gene. All of them exhibited some degree of (patient 5) had a mutation (CTCϾATC) in exon 3 at retinal detachment or exudation (stage 2B or higher). The codon 61, which changed leucine to isoleucine. The last visual acuities correlated most closely with the location patient with FEVR (patient 7) had a mutation in the 3Ј of exudate (involving the macula vs extramacular) rather UTR involving nucleotide position *717. All of the amino than the extent of the detachment. One patient (patient acid changes were predicted to affect the secondary struc- 1) had a point mutation (CGGϾTGG) in exon 3, which ture to various degrees but not to interfere with the cys-

(REPRINTED) ARCH OPHTHALMOL / VOL 125, FEB 2007 WWW.ARCHOPHTHALMOL.COM 228

©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 teine bonds required for the active domain (the cysteine (80%) of our 5 patients with ND had mutations that di- knot). Charge changes in amino acid substitutions alter rectly altered the cysteine-knot motif. Mutations of cys- the isoelectric point of norrin, which may compromise teine residues within this domain would be expected to its activity under physiologic conditions. All of the pa- dramatically affect the tertiary structure of norrin and re- tients had typical manifestations of FEVR, with periph- sult in severely compromised receptor binding. Inabil- eral retinal vascular anomalies or absent peripheral vas- ity to activate the Wnt receptor–␤-catenin pathway re- cularization of the retina. sults in early abrogation of neurosensory and vascular Persistent fetal vascular syndrome represents a fail- development.10,14 The exception has a 12-dinucleotide CT ure of regression of the primary hyaloid system. This is repeat insertion in the 5Ј UTR and is thought to alter the supported by findings in Ndp knockout animal models transcription regulation of the NDP gene and the trans- that demonstrate failure of the primary hyaloid artery and lation efficiency of norrin, thereby affecting gene expres- associated structures to regress.14 Bilateral cases of PFVS sion and regulation. A patient with retinal detachments can often be difficult to distinguish from ND or FEVR. associated with ROP (patient 11) has a deletion in the One patient diagnosed as having bilateral PFVS (patient 5Ј UTR of the NDP gene. Loss of the CT repeat may also 4) had a mutation (AGGϾAGC) in exon 2 at amino acid affect the regulation of transcription and translation. 41. The G-to-C transversion changed arginine to serine. The patients with X-linked FEVR in our study (stage The mother of patient 4 was confirmed to be a carrier of 2B or greater) had noncysteine mutations in the NDP gene. PFVS. Patient 4 also had glucose-6-phosphate dehydro- These mutations are predicted to cause secondary struc- genase deficiency, which was diagnosed after birth. On tural changes that result in suboptimal folding of the nor- the basis of enzyme activity, the deficiency was consid- rin protein. Receptor binding may be compromised but ered by his pediatrician to be severe. The effect of the mu- should still occur to varying degrees. Suboptimal path- tation in the NDP gene in conjunction with increased oxi- way activation would be predicted and is consistent with dative stress was unknown but may have exacerbated the varying degrees of retinal dysgenesis seen in pa- norrin dysfunction, thereby worsening the vascular dys- tients with FEVR. genesis seen in this patient.20 The retinal phenotype correlated well with the geno- One patient with ROP (patient 11) was found to have type findings in our patients. The most consistent cor- a mutation in the NDP gene consisting of a 14-kilobase relation was seen with the diagnosis of ND and the mu- CT dinucleotide repeat deletion in the 5Ј UTR (exon 1) tations that disrupted the cysteine-knot motif. The after nucleotide 8. None of the patients with ROP had correlation between noncysteine mutations and the other mutations in the NDP coding region sequence or 3Ј UTR. diagnoses was less evident. Presumably, noncysteine mu- Dipyrimidine repeats in the 5Ј UTR are responsible for tations result in suboptimal norrin folding to varying de- transcription regulation and efficiency of translation, and grees. Further studies of norrin may elucidate whether this insertion may alter gene regulation.16,17 None of the and how these changes alter receptor binding and sig- controls had NDP gene mutations or polymorphisms. nal transduction activation. Exogenous environmental or systemic factors may also play a role in disease presentation. These results highlight the importance of the COMMENT tertiary structure of proteins and their effect on structural changes of specialized tissues. The cysteine residues responsible for the cysteine-knot formation are found at positions 39, 65, 69, 96, 126, and 128 Submitted for Publication: June 27, 2006; final revision (Figure 2).21 Mutations affecting these amino acids inter- received August 17, 2006; accepted September 21, 2006. fere with the folding and stability of norrin by disrupting Correspondence: Kimberly A. Drenser, MD, PhD, As- key disulfide bonds in the molecule. In our patients, mu- sociated Retinal Consultants, 632 William Beaumont tations involving cysteine residues corresponded with se- Medical Bldg, 3535 W 13 Mile Rd, Royal Oak, MI 48073 vere retinal dysgenesis, and these patients were diag- ([email protected]). nosed as having ND. Mutations in noncysteine residues Financial Disclosure: None. of the norrin protein showed abnormal vascular and reti- Funding/Support: This study was supported in part by nal development and phenotypes consistent with FEVR. Taiwan Merit Scholarship TMS-094-1-B-001. The 5Ј UTR contains elements that regulate gene expres- 16,17 Ј sion, and the 3 UTR is responsible for localizing the REFERENCES messenger RNA in the intracellular microenvironment.22 Mutations in these regions may alter regulatory and/or co- 1. Schuback DE, Chen ZY, Craig IW, Breakefield XO, Sims KB. Mutations in the Nor- localizing elements that regulate norrin and its transla- rie disease gene. Hum Mutat. 1995;5:285-292. tion. Clinical presentation and retinal findings correlate 2. Meindl A, Berger W, Meitinger T, et al. Norrie disease is caused by mutations in well with diagnoses and genotypes. an extracellular protein resembling C-terminal globular domain of mucins. Nat Genet. 1992;2:139-143. Norrie disease and FEVR are known to be allelic. How- 3. Berger W, van de Pol D, Warburg M, et al. Mutations in the candidate gene for ever, the retinal phenotype of ND is more dysgenic than Norrie disease. Hum Mol Genet. 1992;1:461-465. that of FEVR. Patients with ND manifest severe retinal 4. Shastry BS, Hejtmancik JF, Plager DA, Hartzer MK, Trese MT. Linkage and can- dysgenesis, which is generally detected within the first didate gene analysis of X-linked familial exudative vitreoretinopathy. Genomics. 1995;27:341-344. 3 months of life. Familial exudative vitreoretinopathy is 5. Chen ZY, Battinelli EM, Fielder A, et al. A mutation in the Norrie disease gene initially detected at various ages, and the retinal struc- (NDP) associated with X-linked familial exudative vitreoretinopathy. Nat Genet. ture is significantly different from that found in ND. Four 1993;5:180-183.

(REPRINTED) ARCH OPHTHALMOL / VOL 125, FEB 2007 WWW.ARCHOPHTHALMOL.COM 229

©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 6. Black GC, Perveen R, Bonshek R, et al. Coats’ disease of the retina (unilateral necessary for regression of hyaloid vessels. Invest Ophthalmol Vis Sci. 2004; retinal telangiectasis) caused by somatic mutation in the NDP gene: a role for 45:2384-2390. norrin in retinal angiogenesis. Hum Mol Genet. 1999;8:2031-2035. 15. Sims KB. NDP-related retinopathies. http://www.geneclinics.org/profiles/norrie 7. Talks SJ, Ebenezer N, Hykin P, et al. De novo mutations in the 5Ј regulatory re- /details.html#gcID1397. Accessed June27, 2006. gion of the Norrie disease gene in retinopathy of prematurity. J Med Genet. 2001; 16. Kenyon JR, Craig IW. Analysis of the 5Ј regulatory region of the human Norrie’s 38:e46 http://jmg.bmjjournals.com/cgi/content/full/38/12/e46. Accessed Febru- disease gene: evidence that a non-translated CT dinucleotide repeat in exon one ary 13, 2006. has a role in controlling expression. Gene. 1999;227:181-188. 8. Hutcheson KA, Paluru PC, Bernstein SL, et al. Norrie disease gene sequence vari- 17. Davuluri RV, Suzuki Y, Sugano S, Zhang MQ. CART classification of human 5Ј ants in an ethnically diverse population with retinopathy of prematurity. Mol Vis. UTR sequences. Genome Res. 2000;10:1807-1816. 2005;11:501-508. 18. Shastry BS, Hejtmancik JF, Trese MT. Identification of novel missense muta- 9. Hiraoka M, Berinstein DM, Trese MT, Shastry BS. Insertion and deletion mutations in the Norrie disease gene associated with one X-linked and four spo- tions in the dinucleotide repeat region of the Norrie disease gene in patients with radic cases of familial exudative vitreoretinopathy. Hum Mutat. 1997;9: advanced retinopathy of prematurity. J Hum Genet. 2001;46:178-181. 396-401. 10. Xu Q, Wang Y, Dabdoub A, et al. Vascular development in the retina and inner 19. Drenser KA, Fecko A, Dailey W, Trese MT. A characteristic phenotypic retinal ap- ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell. pearance in Norrie disease. Retina. In press. 2004;116:883-895. 20. Dani C, Cecchi A, Bertini G. Role of oxidative stress as physiopathologic factor 11. Berger W. Molecular dissection of Norrie disease. Acta Anat (Basel). 1998;162: in the preterm infant. Minerva Pediatr. 2004;56:381-394. 95-100. 21. Mintz-Hittner HA, Ferrell RE, Sims KB, et al. Peripheral retinopathy in offspring 12. Black G, Redmond RM. The molecular biology of Norrie’s disease. Eye. 1994;8: of carriers of Norrie disease gene mutations. Possible transplacental effect of 491-496. abnormal Norrin. Ophthalmology. 1996;103:2128-2134. 13. Robitaille J, MacDonald ML, Kaykas A, et al. Mutant frizzled-4 disrupts retinal 22. Kislauskis EH, Li Z, Singer RH, Taneja KL. Isoform-specific 3Ј-untranslated se- angiogenesis in familial exudative vitreoretinopathy. Nat Genet. 2002;32: quences sort ␣-cardiac and ␤-cytoplasmic actin messenger RNAs to different 326-330. cytoplasmic compartments [published correction appears in J Cell Biol. 1993; 14. Ohlmann AV, Adamek E, Ohlmann A, Lutjen-Drecoll E. Norrie gene product is 123(6)(pt 2):following 1907]. J Cell Biol. 1993;123:165-172.

Call for Papers

Archives of Ophthalmology will publish articles on medical education and ophthalmology in conjunction with a JAMA theme issue on the same topic in September 2007. Manuscripts received by April 1, 2007, will have the best chance for consideration for this theme issue.

(REPRINTED) ARCH OPHTHALMOL / VOL 125, FEB 2007 WWW.ARCHOPHTHALMOL.COM 230