Secondary Logo

Journal Logo

Brief Communication

A novel missense mutation of NDP in a Chinese family with X-linked familial exudative vitreoretinopathy

Liu, Hong Yana,*; Huang, Jiaa; Wang, Rui Lib; Wang, Yuec; Guo, Liang Jiea; Li, Taoa; Wu, Donga; Wang, Hong Dana; Guo, Qian Nana; Dong, Dao Quand

Author Information
Journal of the Chinese Medical Association: November 2016 - Volume 79 - Issue 11 - p 633-638
doi: 10.1016/j.jcma.2016.08.002


    1. Introduction

    Familial exudative vitreoretinopathy (FEVR) is a genetically-heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and severe visual impairment. The most prominent characteristics of the disease result from the incomplete and aberrant vascularization of the peripheral retina, retinal blood-vessel differentiation,1 or both. The latter can lead to various complications, such as retinal neovascularization and exudates, vitreous hemorrhage, vitreoretinal traction, ectopia of the macula, and retinal folds and detachments. The clinical signs in affected individuals can be diverse, ranging from hardly detectable vascular anomalies in the peripheral retina in asymptomatic individuals to bilateral retinal detachments leading to blindness. Patients with mild symptoms show little or no change in visual acuity. Fundus fluorescein angiography (FFA) examination reveals a small area of no vascular perfusion around the retinal periphery, a common feature in all affected individuals among the family. FEVR is a typical Mendel single-gene disease that was first described by Criswick and Schepens in 1969,2 and has since become a well-recognized and extensively studied condition.

    To date, mutations in genes encoding Norrin [encoded by Norrie disease pseudoglioma (NDP)] for X-linked recessive form,3 and Frizzled 4 (FZD4), low density lipoprotein receptor related protein 5 (LRP5), Tetraspanin-12 (TSPAN12), and zinc finger protein 408 (ZNF408) for autosomal dominant (AD) form have been shown to cause FEVR.4–7 A few families with LRP5 and TSPAN12 autosomal recessive (AR) inheritance related to FEVR have also been documented.8,9

    Each of these encoded proteins is a component of the Norrin/β-catenin signaling pathway (also referred to as the Norrin / Frizzled-4 pathway).10 The NDP locus maps to chromosome Xp11.4, spans 28kb, and comprises three exons; however, only exons 2 and 3 of NDP are translated into a 133 amino-acid protein Norrin. Norrin acts as a ligand for the LRP5, FZD4, and TSPAN12 coreceptors that activate canonical Wnt signaling.10 Wnt signaling plays an important role in eye organogenesis and angiogenesis.10 Mutations in NDP disrupts the Wnt signaling pathway, directly leading to FEVR.

    Here we describe a large typical four-generation FEVR family with a total of 40 family members. All seven affected patients were male. Mutation analysis identified a novel mutation in NDP that caused the X-linked FEVR.

    2. Methods

    2.1. Participants

    The FEVR family was from the northern area of the Henan province in China (Fig. 1). The proband (V9) was born in 1997 after normal pregnancy. He was diagnosed with FEVR at 10 years of age. During genetic consulting, we found that the family had seven members with similar symptoms. These family members were then examined at the Eye Institute in the People's Hospital of Henan Province. The clinical diagnosis of FEVR was made based on the following criteria: (1) positive family history with seven male-only affected individuals; (2) ophthalmic examination confirming bilateral vitreous opacity, retina surrounding no vascular zone, merging heterotopy of macula, retinal fold, and retinal detachment; and (3) absence of history of premature labor and oxygen uptake.

    Fig. 1
    Fig. 1:
    Pedigree of the familial exudative vitreoretinopathy (FEVR) family reported in this study. The affected FEVR patients (black square), asymptomatic heterozygous mutation carriers (dot).

    Informed consent was obtained from all individuals tested after explanation of the nature and possible consequences of the study, and the research adhered to the tenets of the Declaration of Helsinki. Ethical approval was obtained from the People's Hospital of Henan Provincial Ethics Committee.

    Peripheral venous blood (EDTA-K2 anticoagulant; 5 mL per individual) was collected from 11 family members, including four patients (IV1, IV3, IV4, and V9) and seven unaffected individuals (III2, III14, IV11, IV13, IV14, V4, V8, and V10).

    2.2. Genetic analysis

    Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures using a Qiagen Blood kit (QIAGEN, Germantown, MD, USA). The two exons (exons 2 and 3) and the corresponding intron–exon boundaries of the NDP gene were amplified by polymerase chain reaction (PCR). Primer sequences and annealing temperatures are listed in Table 1. Each 25-μL PCR amplification reaction contained 1X buffer, 150 ng of genomic DNA, 0.2 mM of each dNTP, 2-U Taq polymerase, 1 mM of forward and reverse primers, and 1.5-mM MgCl2. PCR products were analyzed in 1.5% agarose gels. Amplified products were excised and purified using QIA quick PCR Purification kit (QIAGEN, Germantown, MD, USA). Sequencing was performed using an ABI Big Dye terminator cycle sequencing kit (v3.1) on an ABI 3730 Genetic analyzer (Applied Biosystems, Foster City, CA, USA). The proband (V9) was sequenced first for mutation identification. The sequencing results were compared with the human reference sequence from the University of California, Santa Cruz (UCSC) 2013 Human Genome Assembly. A missense mutation was found in exon 3 of NDP. To confirm this mutation, the exon 3 of NDP from the other individuals (affected individuals IV1, IV3, and IV4; unaffected individuals III2, III14, IV11, IV13, IV14, V4, V8, and V10) were then amplified and sequenced.

    Table 1
    Table 1:
    Primers and annealing temperatures used for NDP amplification.

    3. Results

    3.1. Clinical examination

    This family had seven blind patients. Six patients, including the proband V9 and III3, IV1, IV3, IV4, and VI1, were checked for existing symptoms. All patients showed weak eyesight. Spotlight reaction was weak and progressive with blindness at the age of 3–4 years old. All patients had signs of a cataract. III3, IV1, IV3, and IV4 affected individuals also had a sign of glaucoma; the vision of those patients was sharp decline after the transient high intra-ocular pressure with a severe headache. Eye examination for the proband V9 (17 years old): vision oculus dexter (OD) showed light perception with best-corrected visual acuity (BCVA): 11.0/–2.50 × 10° → counting fingers (CF)/30 cm, vision oculus sinister (OS): 20/400, BCVA: –4.00/–2.00 × 70° → 20/60. Intraocular pressure: OD: 9 mmHg; OS: 13 mmHg. Opacity was found in both eye lenses, the cortex, the gray of the posterior capsule, and gray flocculent in the vitreous body. Fundus: retinal vascular abnormalities, expansion, and straight peripheral in both eyes. There was retina fold in the posterior pole of right eye, and macular ectopic, vitreous retinal adhesion along with tractional hole formation in the left eye. Ocular ultrasound: retina fold in the right eye, unsmooth peripheral wall of eyeball (suspicious hole) in the left eye. Both eyes presented with vitreous opacity. FFA test: the various vascular branches of posterior pole retina, the temporal peripheral retinal showing nonperfusion zones along with abnormal new blood vessels and fluorescence leakage. The fundus examination was consistent with the FEVR. The diagnosis was FEVR oculus uterque (OU), complicated cataract OU, high myopia and amblyopia OD, and retina fold OD. Patient VI1 was a 2-year-old young child, full-term normal delivery, no history of oxygen uptake, eye exam: vision OD showed light perception, vision OS: 0.1, only distinguish the first line of visual chart. The other detailed clinical manifestations of affected individuals are showed in Table 2.

    Table 2
    Table 2:
    The ophthalmology manifestations of affected individuals.

    3.2. Mutation analysis

    A missense mutation c.310A>C in exon 3 of NDP was detected in the proband (V9) and his three uncles (IV1, IV3, IV4). This mutation resulted in a lysine to glutamine substitution at position 104 (p. Lys104Gln) (Fig. 2). The proband's mother (IV13), aunt (IV11), and his grandmother (III2) were confirmed as carriers. The unaffected individuals (III14, IV11, IV14, V4, and V8) were not detected for this mutation. This missense mutation was not observed in normal Chinese humans of the 1000 Genomes Project.

    Fig. 2
    Fig. 2:
    Partial nucleotide sequences of NDP exon 3. An affected male shows a missense mutation c. 310A>C (upper panel). The arrow indicates nucleotide change that causes substitution of lysine residue (AAG) with glutamine (CAG) at position 104 of NDP (p.Lys104Gln). Middle panel shows the mutation in heterozygous state from a carrier. Lower panel is a normal NDP sequence. NDP = Norrie disease gene.

    This missense mutation position is located in the C-terminal cystine knot-like domain of Norrin. As can be seen in Fig. 3, the 104 codon lysine is a highly conserved amino acid across different species.

    Fig. 3
    Fig. 3:
    Protein sequence alignment of human NDP with its orthologues. Conserved amino acid residues are shaded. The position of the missense mutations p.K104Q is indicated. NDP = Norrie disease gene.

    4. Discussion

    In this work, we report a large family with a history of typical X-linked FEVR. A novel mutation (c.310A>C, P. Lys104Gln) in the NDP was found in four hemizygous-affected male individuals and three heterozygous-unaffected female individuals. This missense mutation is located in the C-terminal cystine knot-like domain of Norrin. Mutations affecting this domain appear to cause more severe phenotypes.11 Comparative analysis shows that the 104 codon lysine is a highly conserved amino acid across different species. It suggests that any mutation at this codon may lead to a deleterious effect.

    Previously, the same missense mutation was reported in one patient with less severe Norrie disease.12 This result suggests that X-linked FEVR and Norrie disease can share the codon mutation in the same gene. There are some similarities in clinical symptoms between X-linked FEVR and Norrie disease, such as retinal traction, retinal folds, and retinal detachment. In typical Norrie disease, the characteristic manifestation is bilateral congenital blindness within the 1st year of life. However, in this family, the vision for the seven affected individuals was normal at birth, and blindness in both eyes did not occur at the same time. Two additional distinctions are that 40% of Norrie patients also develop progressive sensorineural deafness and 50% of patients have mental retardation in early childhood.13 In contrast, the affected individuals of this family were found to have neither mental retardation nor hearing abnormality. Finally, the severe deterioration of the eye in X-linked FEVR is blindness. However, deterioration of the eye in Norrie disease is continuous, and atrophy of the eye globe is a severe characteristic. Therefore, this family can be confirmed as FEVR rather than Norrie disease. Our results suggest that X-linked FEVR and Norrie disease may be allele-dependent.

    More than 100 nucleotide variants have been reported for NDP. Most of the mutations cause Norrie disease; only a small percentage of mutations were reported in X-linked FEVR (Table 3). To date, seven NDP mutations (p.R38C, p.R41K, p.K58N, p.R74C, p.C96Y, p.R121W, and p.R121Q) have been suggested to be associated with X-linked FEVR and Norrie disease. Interestingly, Allen et al22 reported a Syrian family with the R74C NDP mutation; one affected individual IV-33 was diagnosed with X-linked FEVR, whereas another affected individual IV-31 was diagnosed with Norrie disease. The same mutation clearly can lead to two different phenotypes or diseases. This heterozygous phenotype has also been observed in many other datasets. Obviously, epigenetic and other unidentified factors are also involved in determining the phenotypic expression of X-linked FEVR and Norrie disease. Of course, NDP variations play a key role in X-linked FEVR and Norrie disease, and examining other genetic variations may help explain the difference between X-linked FEVR and Norrie disease.

    Table 3
    Table 3:
    NDP sequence missense mutations that are likely to be familial exudative vitreoretinopathy (FEVR).


    We thank our patients and their family members for their participation in this study. This study was supported in part by the Foundation and Cutting-edge Research Projects of Henan Province Science and Technology Department (No. 162102310174), Oversea Training Projects for Medical Academic Leaders of Henan Province (No. 2014089), Medical Science and Technology Research Projects of Henan Provincial Health Bureau (No. 201403180), and the Scientific and Technological Projects of the Technology Bureau of Jinshui District (No. 38).


    1. Benson WE. Familial exudative vitreoretinopathy. Trans Am Ophthalmol Soc. 1995;93:473-521.
    2. Criswick VG, Schepens CL. Family exudation vitreoretinopathy. Am J Ophthalmol. 1969;68:578-594.
    3. Chen ZY, Battinelli EM, Fielder A, Bundey S, Sims K, Breakefield XO, et al. A mutation in the Norrie disease gene (NDP) associated with X-linked familial exudative vitreoretinopathy. Nat Genet. 1993;5:180-183.
    4. Robitaille J, MacDonald ML, Kaykas A, Sheldahl LC, Zeisler J, Dubé MP, et al. Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy. Nat Genet. 2002;32:326-330.
    5. Toomes C, Bottomley HM, Jackson RM, Towns KV, Scott S, Mackey DA, et al. Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q. Am J Hum Genet. 2004;74:721-730.
    6. Poulter JA, Ali M, Gilmour DF, Rice A, Kondo H, Hayashi K, et al. Mutations in TSPAN12 cause autosomal-dominant familial exudative vitreoretinopathy. Am J Hum Genet. 2010;86:248-253.
    7. Collin RW, Nikopoulos K, Dona M, Gilissen C, Hoischen A, Boonstra FN, et al. ZNF408 is mutated in familial exudative vitreoretinopathy and is crucial for the development of zebrafish retinal vasculature. Proc Natl Acad Sci U S A. 2013;110:9856-9861.
    8. Jiao X, Ventruto V, Trese MT, Shastry BS, Hejtmancik JF. Autosomal recessive familial exudative vitreoretinopathy is associated with mutations in LRP5. Am J Hum Genet. 2004;75:878-884.
    9. Poulter JA, Davidson AE, Ali M, Gilmour DF, Parry DA, Mintz-Hittner HA, et al. Recessive mutations in TSPAN12 cause retinal dysplasia and severe familial exudative vitreoretinopathy (FEVR). Invest Ophthalmol Vis Sci. 2012;53:2873-2879.
    10. Clevers H. Eyeing up new Wnt pathway players. Cell. 2009;139:227-229.
    11. Drenser KA, Fecko A, Dailey W, Trese MT. A characteristic phenotypic retinal appearance in Norrie disease. Retina. 2007;27:243-246.
    12. Meindl A, Lorenz B, Achatz H, Hellebrand H, Schmitz-Valckenberg P, Meitinger T. Missense mutations in the NDP gene in patients with a less severe course of Norrie disease. Hum Mol Genet. 1995;4:489-490.
    13. Riveiro-Alvarez R, Trujillo-Tiebas MJ, Gimenez-Pardo A, Garcia-Hoyos M, Cantalapiedra D, Lorda-Sanchez I, et al. Genotype-phenotype variations in five Spanish families with Norrie disease or X-linked FEVR. Mol Vis. 2005;11:705-712.
    14. Kondo H, Qin M, Kusaka S, Tahira T, Hasebe H, Hayashi H, et al. Novel mutations in Norrie disease gene in Japanese patients with Norrie disease and familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci. 2007;48:1276-1282.
      15. Royer G, Hanein S, Raclin V, Gigarel N, Rozet JM, Munnich A, et al. NDP gene mutations in 14 French families with Norrie disease. Hum Mutat. 2003;22:499.
        16. Shastry BS, Hejtmancik JF, Trese MT. Identification of novel missense mutations in the Norrie disease gene associated with one X-linked and four sporadic cases of familial exudative vitreoretinopathy. Hum Mutat. 1997;9:396-401.
          17. Yang H, Li S, Xiao X, Guo X, Zhang Q. Screening for NDP mutations in 44 unrelated patients with familial exudative vitreoretinopathy or Norrie disease. Curr Eye Res. 2012;37:726-729.
            18. Drenser KA, Dailey W, Capone A, Trese MT. Genetic evaluation to establish the diagnosis of X-linked familial exudative vitreoretinopathy. Ophthalmic Genet. 2006;27:75-78.
              19. Boonstra FN, van Nouhuys CE, Schuil J, de Wijs IJ, van der Donk KP, Nikopoulos K, et al. Clinical and molecular evaluation of probands and family members with familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci. 2009;50:4379-4385.
                20. Fuentes JJ, Volpini V, Fernández-Toral F, Coto E, Estivill X. Identification of two new missense mutations (K58N and R121Q) in the Norrie disease (ND) gene in two Spanish families. Hum Mol Genet. 1993;2:1953-1955.
                  21. Wu WC, Drenser K, Trese M, Capone A Jr, Dailey W. Retinal phenotype-genotype correlation of pediatric patients expressing mutations in the Norrie disease gene. Arch Ophthalmol. 2007;125:225-230.
                    22. Allen RC, Russell SR, Streb LM, Alsheikheh A, Stone EM. Phenotypic heterogeneity associated with a novel mutation (Gly112Glu) in the Norrie disease protein. Eye (Lond). 2006;20:234-241.
                    23. Berger W, van de Pol D, Warburg M, Gal A, Bleeker-Wagemakers L, de Silva H, et al. Mutations in the candidate gene for Norrie disease. Hum Mol Genet. 1992;1:461-465.
                      24. Meindl A, Berger W, Meitinger T, van de Pol D, Achatz H, Dörner C, et al. Norrie disease is caused by mutations in an extracellular protein resembling C-terminal globular domain of mucins. Nat Genet. 1992;2:139-143.
                        25. Dickinson JL, Sale MM, Passmore A, FitzGerald LM, Wheatley CM, Burdon KP, et al. Mutations in the NDP gene: contribution to Norrie disease, familial exudative vitreoretinopathy and retinopathy of prematurity. Clin Exp Ophthalmol. 2006;34:682-688.
                          26. Torrente I, Mangino M, Gennarelli M, Novelli G, Giannotti A, Vadalà P, et al. Two new missense mutations (A105T and C110G) in the norrin gene in two Italian families with Norrie disease and familial exudative vitreoretinopathy. Am J Med Genet. 1997;72:242-244.
                            27. Jia HY, Yang QS, Wang NL. X-linked familial exudative vitreoretinopathy caused by a novel missense mutation G113D in NDP gene. Ophthalmol CHN. 2013;22:389-392.
                              28. Shastry BS, Hejtmancik JF, Plager DA, Hartzer MK, Trese MT. Linkage and candidate gene analysis of X-linked familial exudative vitreoretinopathy. Genomics. 1995;7:341-344.
                                29. Kellner U, Fuchs S, Bornfeld N, Foerster MH, Gal A. Ocular phenotypes associated with two mutations (R121W, C126X) in the Norrie disease gene. Ophthalmic Genet. 1996;17:67-74.
                                  30. Johnson K, Mintz-Hittner HA, Conley YP, Ferrell RE. X-linked exudative vitreoretinopathy caused by an arginine to leucine substitution (R121L) in the Norrie disease protein. Clin Genet. 1996;50:113-115.

                                    Chinese; familial exudative vitreoretinopathy; mutation; Norrie disease pseudoglioma; X-linked

                                    © 2016 by Lippincott Williams & Wilkins, Inc.