# Familial Exudative Vitreoretinopathy-Related Disease-Causing Genes and Norrin/β-Catenin Signal Pathway: Structure, Function, and Mutation Spectrums

Authors: Hongtao Xiao; Yuna Tong; Yuxuan Zhu; Min Peng
Journal: Journal of Ophthalmology
 (2019)

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## Abstract

Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by incomplete vascularization/abnormality of peripheral retina. Four of the identified disease-causing genes of FEVR wereNDP, FZD4, LRP5, and TSPAN12, the protein coded by which were the components of the Norrin/β-catenin signal pathway. In this review, we summarized and discussed the spectrum of mutations involving these four genes. By the end of 2017, the number of FEVR causing mutations reported for NDP, FZD4, LRP5, and TSPAN12 was, respectively, 26, 121, 58, and 40. Three most frequently reported mutations were c. 362G > A (p.R121Q) of NDP, c. 313A > G (p.M105V), and c.1282_1285delGACA (p.D428SfsX2) of FZD4. Mutations have a tendency to cluster in some “hotspots” domains which may be responsible for protein interactions.


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## Body

## 1. Introduction

Familial exudative vitreoretinopathy (FEVR), described first by Criswick and Schepens in 1969 [1], is a hereditary ocular disorder characterized by incomplete vascularization/abnormality of peripheral retina. Incomplete and aberrant vascularization leads to various complications, including retinal neovascularization and exudates, retinal fold and detachments, vitreous hemorrhage, and macular ectopia, ultimately leading to total blindness.FEVR is genetically heterogeneous and can be inherited as a dominant, recessive, or X-linked trait. The dominant form is the most common mode of inheritance. So far, mutations in at least 9 genes have been attributed to the development of FEVR includingNDP, FZD4, LRP5, TSPAN12, ZNF408, KIF11, RCBTB1, CTNNB1, and JAG1 [2–10]. The proteins encoded by the first four genes are cooperative in the Norrin/β-catenin signaling pathway (also named as Norrin/Frizzled-4 pathway) and showed intense interaction with each other [11]. So, this review specially focused on the mutation spectrums of these genes.The mechanisms ofNDP, FZD4, LRP5, and TSPAN12 in retinal vascular had been intensively investigated during the past years. The Ndp knockout mouse exhibited superficial retinal vasculature development delay and was unable to form deep retinal vasculature [12]. Similarly, FZD4 played a central role in vascular development in the eye and ear. Knockout of Fz4 has been shown to affect vascular development both in retinal and in inner ear and cause retinal stress [13, 14]. Compared with Fzd4 or Ndp knockout mice, Lrp5 knockout mice showed many milder vascular defects, in which attenuated retinal vessels and capillaries lacking lumen structure was observed [15, 16]. Afterwards, Tspan12 was verified to cause vascular defect and affect neural cells through association with Norrin/β-catenin but not Wnt/β-catenin signaling. Formation of microaneurisms, aberrant fenestration, and delayed hyaloid vessel regression was reported in Tspan12 knockout mice [11].In the Norrin/β-catenin pathway, Norrin (coded by NDP) worked as a ligand, while Frizzled-4 (FZD4) acted as the receptor of Norrin, in concert with low-density lipoprotein receptor-related protein-5 (LRP5) as coreceptor. Norrin binds to FZD4 and its coreceptor LRP5, forming a ternary complex. Together with the auxiliary component tetraspanin-12 (TSPAN12), this complex initiates downstream β-catenin signaling. Specifically, FZD4-bound Dishevelled and phosphorylated LRP5 recruited Axin to the plasma membrane, resulting in the suppression of β-catenin phosphorylation/degradation. The cytoplasmic levels of β-catenin consequently increased. Subsequently, β-catenin was translocated to the nucleus where it interacts with the T-cell factor/lymphoid enhancing factor, family of transcription factors, to initiate RNA transcription and elongation, as shown in Figure 1 [17–19]. This signaling pathway shared many similarities with the canonical Wnt/β-catenin pathway except that Norrin substituted Wnt as the ligand and traspanin-12 had been linked to the Norrin/β-catenin signaling pathway. Norrin/Frizzled-4 signaling plays an important role in retinal vascular growth, remodeling, and maintenance [20].Figure 1
A schematic of the Norrin/β-catenin signal pathway. When Norrin was bond to the receptor complex FZD4/LRP5/TSPAN12, Dishevelled and Axin would be recruited to FZD5 and LRP5. Consequently, β-catenin escaped from the degradation complex and entered nucleus to initiate gene transcription collaborated with T-cell factor/lymphoid-enhancing factor.Prior to this review, a great many mutations inNDP, FZD4, LRP5, and TSPAN12 had been reported by different study groups from different countries as disease-causing mutation of FEVR. Although most of the mutations were documented for once by one study group, some mutations seemed to be more common than others. Here, we presented the comprehensive list of currently known mutations in NDP, FZD4, LRP5, and TSPAN12 associated with FEVR and discussed their coding consequences. This aims in facilitating the construction of a complete spectrum of mutations that occur in the above four genes. We discuss about each gene mutation individually and then highlight how they disturb the protein interactions.


## 2. Materials and Methods

The current review article aimed to analyze the studies on FEVR caused byNDP, FZD4, LRP5, and TSPAN12 gene mutations to find the spectrum of these four genes. For this review study, an extensive search in PubMed and Web of Science up to December 30, 2017, was conducted independently by two individuals (Tong and Zhu) using the following search terms: “Familial exudative vitreoretinopathy” and “mutation”. To avoid losing relevant information, no limitations were set in the search. Furthermore, the related studies and the references of literatures were manually screened for additional potential eligible studies.Mutations in NDP can result in Norrie disease and X-linked exudative vitreoretinopathy. Some earlier reports investigated Norrie disease (ND) and FEVR together. In addition, loss-of-function mutations in the LRP5 gene either cause osteoporosis pseudoglioma syndrome (OPPG) or FEVR depending on the functional severity of mutation. These distinct clinical entities share some common pathological features such as abnormal retinal blood vessel growth that may result in retinal detachment. So, we read the relevant articles of the candidates carefully to make sure the probands on whom the mutations were found were definitely diagnosed as FEVR. Then, we recorded the mutations related to FEVR and excluded those caused ND and OPPG. A total of 433 potentially relevant articles were identified, but only 41 studies involving FEVR patients caused by NDP, FZD4, LRP5, and TSPAN12 gene mutations were included in this review.


## 3. Results

### 3.1.NDP Mutations and Norrin Structure

TheNDP gene locus mapped to chromosome Xp11.4 and comprised three exons. However, the first exon corresponds to the untranslated region of the gene that has regulatory functions, and only exons 2 and 3 of encode a secreted protein of 133 amino acids called Norrin or Norrie disease protein. Norrin consists of two major parts: a signal peptide at the amino-terminus of the protein that directs its localization and a region containing a typical motif of six cystines forming a cystine-knot. The cystine-knot motif is highly conserved in many growth factors as transforming growth factor-β, human chorionic gonadotropin, nerve growth factor, and platelet derived growth factor [21]. Cystine residues and their disulfide bonds in the cystine-knot play important structural and functional roles. Among 10 Frizzled family members, Norrin specifically binds to the transmembrane FZD4 with high affinity, forming a Norrin/FZD4 complex with LRP5 and TSPAN12 coreceptors to activate the Norrin/β-catenin signaling pathway [22]. Norrin was also reported to play a major role in controlling retinal vascular growth and architecture both in the developing eye and in adult vasculature.Twenty-six nucleotide variants have been identified forNDP in patients with FEVR. These include 21 missense changes, 4 deletions, and 1 insertion resulting frame shift [2, 23–31] (Table 1 and Figure 2). Most of the mutations were found in single or only a few patients, while several mutations are generally more common. By far, the most prevalent mutation was c.362G > A (p.R121Q), distributed in Spanish, Mexican, Indian, Chinese, and Italian. It is noteworthy that although probands containing c.11_12delAT (p.H4RfsX21), c.170C > G (p.S57X), and c.310A > C (p.K104Q) were definitely diagnosed as FEVR following explicit criteria, these three mutations were also reported to cause Norrie disease by other researches [28, 32, 33]. The ocular features and retinal changes observed in Norrie disease are similar to those observed in cases of FEVR. Not all the Norrie disease patients have mental retardation and develop a progressive sensorineural hearing loss; it is really difficult to distinguish Norrie disease from FEVR.Table 1
Spectrum ofNDP gene mutations among patients with familial exudative vitreoretinopathy.
Studies
No. of patients
No. of mutations
DNA variant
Coding effect
Location of the amino residue
Mutant phenotypes
Country of origin
Chen et al. [2]
30
1
c.370C>T
p.L124F
Norrin dimer interface
Retina detached
UK
Riveiro-Alvarez et al. [30]
45
1
c.362G>A
p.R121Q
Norrin dimer interface
Congenital blindness, phthisis bulbi
Spain
Dickinson et al. [23]
13
1
c.307C>G
p.L103V
Norrin-FZD4 interface
Not mentioned
Australia
Hiroyuki et al. [34]
62
3
c.53T>A
p.I18K
Signal domain
Peripheral avascularization, neovascularization
Japan
c.162G>C
p.K54N
Deductive Norrin-LRP5 interface
Retinal detachment and macular traction with temporal avascularization
c.344G>T
p.R115L
Deductive Norrin-LRP5 interface
Retinal detachment
Pelcastre et al. [35]
127
3
c.361C>T
p.R121W
Norrin dimer interface
On-perfusion in peripheral retina
Mexico
c.362G>A
p.R121Q
Norrin dimer interface
Retinal detachment
Musada et al. [36]
110
8
c.11_12delAT
p.H4RfsX21
Signal domain
Bilateral total retinal detachment
India
c.69delC
p.D23EfsX9
Signal domain
Pigmentation and vitreoretinal traction
c.142_145delATCA
p.I48VfsX55
Premature termination
Bilateral leukocoria and total retinal detachment
c.148C>G
p.H50D
Deductive Norrin-LRP5 interface
Straightening of the blood vessel, macular dragging
c.170C>G
p.S57X
Norrin-FZD4 interface
Retinal detachments and retrolental membranes
c.338G>A
p.G113D
Near deductive Norrin-LRP5 interface
Avascular peripheral retina, straightening of the blood vessels, and dye leakage
c.362G>A
p.R121Q
Norrin dimer interface
Retinal detachments with retrolental membranes
c.376T>C
p.C126R
Norrin dimer interface
Bilateral total retinal detachment
Liu Y. L. et al. [37]
40
1
c.310A>C
p.K104Q
Norrin-FZD4 interface
Weak eyesight, retinal vascular abnormalities
China
Tang et al. [31]
100
5
c.196G>A
p.E66K
Cystine-knot motif
Macular dragging
China
c.203A>C
p.H68P
Cystine-knot motif
Ectopic macular
c.281A>T
p.H94L
Norrin dimer interface
Peripheral avascular zone and retinal exudates
c.362G>A
p.R121Q
Norrin dimer interface
Retinal fold, retinal detachment
c.334delG
p.G113AfsX149
Premature termination
Bilateral tractional retinal detachment
Iarossi et al. [24]
8
2
c.362G>A
p.R121Q
Norrin dimer interface
Falciform fold, partial traction
Italian
c.313G>C
p.A105F
Norrin-FZD4 interface
Macula-involving retinal detachment
Rao et al. [29]
31
3
c.127C>A
p.H43N
Norrin-FZD4 interface
Complete retinal detachment
China
c.52_53ins32bp
p.S29fs
Premature termination
Complete retinal detachment
c.195C>G
p.C65W
Cystine-knot motif, form disulfide bond with C126
Complete retinal detachmentFigure 2
Schematic diagram of the Norrin protein shows the location of the mutations within the protein domains. Superscript number means the reported times of the same or different mutations at a certain site. The color of the mutations which were reported more than one time was recolored as orange. The opacity varied with the reported frequency of the mutations.It was demonstrated from the three-dimensional structure of Norrin that two-monomer Norrins formed a homodimer in the crystal. The Norrin monomer contained exclusiveβ strands with two β-hairpins on one side and one β-hairpin on the other side. Crystal structures of Norrin in complex with the extracellular domain of FZD4 showed that two β-hairpins in Norrin (β1-β2 and β5-β6) interacted with three loops in FZD4 cystine-rich domain (FZD4-CRD) [38, 39]. There were 19 mutations located in domains from C39 to C65 and C96 to C126, which covered two β-hairpins (β1-β2 and β5-β6) and loops between them, namely, 73% of the mutations (19/26) concentrated in the interacting domains with FZD4-CRD.Specifically, 9 mutations were located in the Norrin dimer interface which was formed fromβ2 and β4 sheets of one monomer and β2′ of another monomer (Table 1). Three mutations were reported from the cystine-knot motif, one of which (C65W) obviously impaired intermolecular disulfide bond-forming. Five mutations disturbed the hydrogen bonds or hydrophobic contacts between Norrin and FZD4 CRD in the Norrin-FZD4 CRD interface [38, 40]. Four mutations clustered on the edge of the Norrin molecule in the β1-β2 and β3-β4 loop regions were inferred as LRP5 binding sites because they did not affect Fz4 binding yet reduced the ability of Norrin to activate the TCF reporter [39]. The residues in the interaction interface are well defined and overlap with disease-associated mutations in NDP. The level of signaling activity of K104Q, R121Q, and L124F was between 20% and 80% of the wide-type Norrin, suggesting that even a modest decrement in Norrin/Fz4 signaling may have a significant phenotypic effect in humans [14, 41]. It is of no surprise that the mutations located in β1-β2 and β5-β6 obstructed the formation of two β-hairpins and the interactions between Norrin and FZD4.


### 3.2.FZD4 Mutations and FZD4-CRD Structure

TheFZD4 gene is located on chromosome 11q14.2, and its mRNA consists of two exons coding for 537 amino acid protein called FZD4 or Frizzled-4 protein. FZD4 acted as the receptor for Wnt and Norrin along with LRP5, which has a pivotal role in various cellular processes including cell fate determination, control of cell polarity, and malignant transformation. The FZD4 contains a ∼120-residue N-terminal extracellular cystine-rich domain(CRD), seven helix transmembrane domains, three extracellular and three intracellular loops, and a C terminal cytoplasmic domain [42, 43]. The cystine-rich domain is indispensable to Wnts or Norrin and is conserved among Frizzled family members [22, 39]. The FZD4 carboxyl cytoplasmic region contains juxtamembrane KTXXXW motif which is responsible for association with Dishevelled to activate downstream signaling [44, 45].In this update, we summarized a total of 121 mutations already reported in patients with FEVR in the literatures consisting of 70 missense mutations, 19 nonsense mutations, and 30 insertions or deletions that lead to either frame shifts or in-frame deletions; a single base change resulted in 2 amino acids extension and a whole-gene deletion [7, 24, 28, 29, 46–71] (Table 2 and Figure 3). No splice mutations have been reported for FZD4, and the mutations seem to cluster in two specific “hotspots”. Although the mutations span in whole FZD4 gene, 49% (59 of 121 mutations) and 13% (16 of 121 mutations) of them have a tendency to bunch in the N terminal extracellular domain and C terminal intracellular domain, respectively.Table 2
Spectrum ofFZD4 gene mutations among patients with familial exudative vitreoretinopathy.
Studies
No. of patients
No. of mutations
DNA variant
Coding effect
Location of the amino residue
Mutant phenotype
Country of origin
Zhang et al. [65]
49
5
c.134G>A
p.C45Y
CRD domain, no plasma membrane localization, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
China and USA
c.173A>G
p.Y58C
CRD domain, failed to bind Norrin, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
c.610T>C
p.C204R
CRD domain, failed to bind Norrin, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
c.678G>A
p.W226X
Transmembrane 1, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
c.1488G>A
p.W496X
C-terminal intracellular domain, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
Drenser et al. [48]
123
5
c.97C>T
p.P33S
Signal sequence
2-stage FEVR, rhegmatogenous retinal detachment
USA
c.349T>C
p.C117R
CRD domain, conserved cystine residue
4B stage FEVR
c.502C>T
p.P168S
CRD domain
2-stage FEVR, rhegmatogenous retinal detachment
c.542G>A
p.C181Y
CRD domain, conserved cystine residue
4B stage FEVR
c.1513C>T
p.Q505X
Immediately downstream from KTxxxW motif
4B stage FEVR
Qin et al. [56]
56
2
c.1005G>C
p.W335C
Highly conserved across all members of the FZD family
Bilateral retinal folds
Japan
c.1024A>G
p.M342V
Intracellular loop 2, function not shown
Bilateral dragged disc
Robitaille et al. [7]
27
2
c.1479_1484del
p.M493_W494del
Failed to activate calcium/calmodulin-dependent protein kinase II and protein kinase C
Bilateral retinal detachment
Canada
c.1501_1502delCT
p.L501fsX533
No membrane accumulation, failed to activate calcium/calmodulin-dependent protein kinase II and protein kinase C
Not mentioned
Kondo et al. [51]
24
4
c.313A>G
p.M105V
CRD domain
Bilateral vitreous opacity, retinal exudates, macular ectopia, falciform retinal fold
Japan
c.957G>A
p.W319X
Transmembrane domain
Falciform retinal fold, chronic retinal detachment
c.1250G>A
p.R417Q
Intracellular loop 3
Falciform retinal fold, posterior synechiae, chronic retinal detachment
c.1463G>A
p.G488D
Transmembrane domain
Falciform retinal folds
Dailey et al. [47]
421
11
c.40 Del/inser
Unknown
Not mentioned
Not mentioned
USA
c.97C>T
p.P33S
Signal sequence, reduced Wnt reporter activity
Not mentioned
c.151T>A
p.S51T
CRD domain
Not mentioned
c.169G>T
p.G57C
CRD domain
Not mentioned
c.349T>C
p.C117R
CRD domain
Not mentioned
c.502C>T
p.P168S
CRD domain, reduced Wnt reporter activity
Not mentioned
c.542G>A
p.C181Y
CRD domain
Not mentioned
c.758G>A
p.R253H
Transmembrane domain
Not mentioned
c.1074A>C
p.K358N
Transmembrane domain
Not mentioned
c.1513C>T
p.Q505X
Immediately downstream from KTxxxW motif
Not mentioned
c.1589G>A
p.G530E
C-terminal
Not mentioned
Fei et al. [49]
61
3
c.C205T
p.H69Y
CRD domain
Not mentioned
China
c.G400T
p.E134X
CRD domain, failed to activateβ-catenin reporter
Peripheral avascular zone, dragged disc
c.1506delAC
p.T503fs
Failed to activateβ-catenin reporter
Total retinal detachment
Yang et al. [69]
56
5
c.313A>G
p.M105V
CRD domain
Increased branching of peripheral vessels, retinal detachment, Avascular zone, Retrolenticular fibrotic mass, neovascularization
China
c.631T>C
p.Y211H
Linker upstream of transmembrane 1
Temporal dragging of optic disc, peripheral fibrous proliferation
c.1282-1285delGACA
p.D428SfsX2
Intracellular loop 3
Straightening of temporal arcades, temporal dragging of optic disc, peripheral fibrous proliferation
c.1482G>A
p.W494X
Transmembrane domain
Retrolenticular fibrotic mass, lens dislocation, brushlike peripheral, avascular zone, neovascularization, peripheral fibrous proliferation
c.1513C>T
p.Q505X
Immediately downstream from KTxxxW motif
Temporal dragging of optic disc, f alciform retinal fold, branching of peripheral vessels, avascular zone, peripheral exudates
Nallathambi et al. [55]
75
3
c.97C>T
p.P33S
Signal sequence, reduced Wnt reporter activity
Peripheral lattice degeneration, atrophic holes, macular ectopia, bilateral peripheral avascular zone
India
c.244_251del8ins27
p.F82fsX135
CRD domain
Macular ectopia, terminal branching, peripheral avascular zone
c.610T>C
p.C204R
CRD domain
Temporal peripheral avascular zone, terminal branching, tractional retinal detachment
Seo et al. [59]
51
9
c.160C>T
p.Q54X
CRD domain
1B stage FEVR
Korea
c.313A>G
p.M105V
CRD domain
1B,1A, 2B stage FEVR
c.456C>G
p.N152K
CRD domain
1B, 2B stage FEVR
c.470T>C
p.M157T
CRD domain
1B stage FEVR
c.539_540delAG
p.E180VfsX9
CRD domain
1B, 3A stage FEVR
c.676T>A
p.W226R
Linker upstream of transmembrane 1
1B, 3A stage FEVR
c.1210_1211delTT
p.L404VfsX54
Transmembrane domain
1A stage FEVR
c.1282_1285delGACA
p.D428SfsX2
Intracellular loop 3
1A stage FEVR
Whole gene deletion
No protein
No protein
2B stage FEVR
Musada et al. [66]
110
7
c.313A>G
p.M105V
CRD domain
Diagnosed with FEVR, symptoms not mentioned
Indian
c.341T>G
p.I114S
CRD domain
Diagnosed with FEVR, symptoms not mentioned
c.470T>C
p.M157T
CRD domain
Diagnosed with FEVR, symptoms not mentioned
c.1282_1285delGACA
p.D428SfsX2
Intracellular loop 3
Diagnosed with FEVR, symptoms not mentioned
c.1286_1290delAGTTA
p.K429RfsX28
Intracellular loop 3
Diagnosed with FEVR, symptoms not mentioned
c.1395_1396insT
p.R466SfsX6
Extracellular loop
Diagnosed with FEVR, symptoms not mentioned
c.1613A>C
p.X538SextX2
C-terminus
Diagnosed with FEVR, symptoms not mentioned
Jia et al. [50]
48
12
c.39-49delCCCGGGGGCG
p.P14fsX57
Signal sequence, Truncated protein
Avascular retina, dragged macula
China
c.65G>A
p.G22E
Signal sequence, loss of activity
Nystagmus, retrolental fibroplasia, retinal detachment
c.205C>T
p.H69Y
CRD domain, loss of activity
Avascular retina, fibrous proliferation, and dragged macula
c.313A>G
p.M105V
CRD domain, loss of activity
Retinal vascular tortuosity, exudates, and avascularization
c.538G>A
p.E180K
CRD domain, loss of activity
Not mentioned
c.710C>G
p.T237R
Linker upstream of transmembrane 1, loss of activity
Preretinal fibrosis, peripheral nonperfusion
c.757C>T
p.R253C
Transmembrane domain, loss of activity
Peripheral avascularization and typical scalloped border
c.983T>C
p.F328S
Transmembrane domain, loss of activity
Retinal folds and persistent hyperplastic primary vitreous
c.1015G>A
p.A339T
Intracellular loop 2, loss of activity
Dragged discs, retinal elevation with hemorrhage
c.1408G>A
p.D470N
Transmembrane domain, loss of activity
Dragged discs, retinal folds, and macular ectopia
c.1472C>A
p.S491X
Transmembrane domain, truncated protein
Dragged discs, macular ectopia, and pigment changes
c.1488G>A
p.W496X
C-terminus, truncated protein
Not mentioned
Peachey et al. [70]
1
1
c.1026A>G
p.M342V
Intracellular loop 2
Straightening of the retinal vessels, peripheral avascular areas
Japanese
Tang et al. [60]
100
14
c.107G>A
p.G36D
Signal sequence
Not mentioned
China
c.133T>C
p.C45R
CRD domain
Avascular zone, increasing of peripheral vessels, straightening of vessels
c.133T>A
p.C45S
CRD domain
Not mentioned
c.134G>A
p.C45Y
CRD domain
Not mentioned
c.158G>C
p.C53S
CRD domain
Macular dragging, Avascular zone, increasing of peripheral vessels, straightening of vessels
c.223G>A
p.A75T
CRD domain
Not mentioned
c.268T>C
p.C90R
CRD domain
Not mentioned
c.313A>G
p.M105V
CRD domain
Not mentioned
c.957G>A
p.W319X
Transmembrane domain
Avascular zone, increasing of peripheral vessels
c.975_978delCACT
p.T326fsX356
Transmembrane domain
Avascular zone, neovascularization, increasing of peripheral vessels, SV
c.1034_1054delCTTATTTCCACATTGCAGCCT
p.S345_A351del
Intracellular loop 2
Avascular zone, increasing of peripheral vessels, straightening of vessels
c.1282_1285delGACA
p.D428SfsX2
Intracellular loop 3
Not mentioned
c.1475delG
p.G492fsX512
Intracellular loop 3
Avascular zone, neovascularization, increasing of peripheral vessels, straightening of vessels,vessels exudates
c.1498delA
p.T500fsX512
Truncated protein
Not mentioned
Nikopoulos et al. [68]
16
5
c.118G>C
p.E40Q
Signal sequence
Not mentioned
Netherlands
c.611G>A
p.C204Y
CRD domain
Deformation of posterior retina, ectopia of the macula, stretched retinal vessels, retinal detachment
c.856G>T
p.E286X
Extracellular loop
Few abnormal temporal retinal branches, avascular peripheral fundus
c.1282_1285del
p.D428SfsX2
Intracellular loop 3
Macular ectopia. Haemorrhagic and exudative areas present in the retina
c.1573G>C
p.G525R
C-terminus
Macular ectopia and peripheral, retinal detachment
Kondo et al. [25]
1
1
c.1250G>A
p.R417Q
Intracellular loop 3
retrolental fibroplasia, falciform retinal fold
Japanese
Toomes et al. [69]
40
8
c.107G>A
p.G36D
Signal sequence
Unable to obtain detailed clinical notes
UK
c.314T>C
p.M105T
CRD domain
Macula-off rhegmatogenous retinal detachment, inadequate vascularization
c.469A>G
p.M157V
CRD domain
Macular folds and retinal detachments
c.957delG
p.W319fsX323
Transmembrane domain
Peripheral retinal fold
c.1490C>T
p.S497F
C-terminus
Disc-dragging
c.1498delA
p.T500fsX512
KTxxxW domain
Small myopic optic disc, diffuse nonspecific pigmentary changes
c.1501_1502delCT
p.L501fsX533
KTxxxW domain
Bilateral cicatrized tractional retinal detachments
c.1513C>T
p.Q505X
Immediately downstream from KTxxxW motif
Temporal sector of retina with deficient vascularization
Robitaille et al. [57]
68
11
c.316 T>C
p.C106G
CRD domain
Dragging of the retina, macular fold
Canadian
c.470T>A
p.M157K
CRD domain
Peripheral pigmentary, total retinal detachment, nonperfusion with leucocoria
c.633delC
p.Y211fsX
Linker upstream of transmembrane 1
Haemangiomatous lesion with exudation and peripheral avascular retina
c.1282_1285del
p.D428SfsX2
Intracellular loop 3
Left macula dragged
c.1463G>T
p.G488V
Transmembrane domain
c.1508insC
p.T503fsX31
KTxxxW motif
Bilateral dragging of the macula with peripherally straightened, avascular retina
c.313A>G
p.M105V
CRD domain
Bilateral dragging of the macula, retina detachment
c.678G>A
p.W226X
Linker upstream of transmembrane 1
Large elevated tight fold, large falciform fold
c.1448G>A
p.W496X
C-terminal intracellular domain
Tractional retinal detachment
c.1479_1484del
p.M493_W494del
Transmembrane domain
Not mentioned
c.341T>C
p.I114T
CRD domain
Not mentioned
Robitaille et al. [67]
5
2
c.1479_1484del
p.M493_W494del
Transmembrane domain
Absence of retinal vasculature, hypoplastic iris with posterior synechiae
Canadian
c.341T>C
p.I114T
CRD domain
Falciform retinal folds, small atrophic retinal hole
Boonstra et al. [46]
83
4
c.668T>A
p.M223K
Linker upstream of transmembrane 1
Diagnosed with FEVR, symptoms not mentioned
Netherlands
c.957G>A
p.W319X
Transmembrane domain
Diagnosed with FEVR, symptoms not mentioned
c.1333A>C
p.T445P
Transmembrane domain
Diagnosed with FEVR, symptoms not mentioned
c.1448G>A
p.W496X
C-terminus, truncated protein
Diagnosed with FEVR, symptoms not mentioned
Iarossi et al. [24]
8
3
c.277C>T
p.Q93X
CRD domain
Large avascular area, falciform retinal fold
Italian
c.542G>A
p.C181Y
CRD domain
Stage 3 and stage 2 FEVR
c.611G>T
p.C204F
CRD domain
Stage 4A FEVR
Rao et al. [29]
31
2
c.1282_1285delGACA
p.D428SfsX2
Intracellular loop 3
Complete retinal detachment
China
c.227delA
p.E76fs
CRD domain
Falciform retinal detachment.
Murken et al. [53]
1
1
c.1474delG
p.G492fsX
Intracellular loop 3
Peripheral avascular zone and macular dragging
Mexico
Schatz and Khan [58]
3
1
c.349T>C
p.C117R
CRD domain, forms a disulfide bond with Cys158
Mild temporal avascularity, mild peripheral temporal avascularity
SwedenThe 120-residue N-terminal extracellular cystine-rich domain (CRD) domain, connected to the first transmembrane helix by a 50-amino-acid linker, was crucial to ligand recognition. In the CRD domain, mutations at C45, M105, and M157 were three most frequently reported mutations, for 4, 9, and 4 times by different studies, respectively. One of these mutations, C45Y, was found to disrupt protein folding, resulting FZD4 being stuck in the cytoplasm with no membrane location [71]. It was supposed that the disulfide bond between Cys45 and Cys106 was imperative to protein transportation and functional activity. It was also visible from the crystal structure of FZD4-CRD that five disulfide bridges (Cys45–Cys106, Cys53–Cys99, Cys90–Cys128, Cys117–Cys158, and Cys121–Cys145) stabilized the α helices [38].Two crystal structures of Norrin/FZD4-CRD complex and a FZD4 transmembrane domain had been registered in the Protein Data Bank [38, 40, 70]. The structures showed that one FZD4-CRD coupled a Norrin monomer with no interactions between the two FZD4-CRDs. Three loops between α helices were responsible for binding to the β-hairpins in Norrin [38]. The C-terminal tail of FZD4-CRD also made contribution to Norrin recognition. Residues V45, M59, L61, and L124 of Norrin and F96, M105, I110, M157, and M159 FZD4-CRD constituted a hydrophobic core at the binding interface [40]. Based on this, it is speculated that FEVR-related mutations at M105 and M157 may interrupt the binding of Norrin to FZD4. Biophysical analysis of Norrin and FZD4 demonstrated that the linker region of FZD4 contributes to a high-affinity interaction with Norrin and signaling [71]. Mutation C181Y in this domain not only destroyed the disulfide bond but also interrupted the binding of Norrin. The FZD4 transmembrane domain structure showed mutations in key positions (M309L, C450I, C507F, and S508Y) of the ΔCRD-FZD4 structure which led to aberrant downstream signaling. However, no disease-causing mutation had been reported in abovementioned four amino residuals.The FZD4-mediated membrane recruitment of the cytoplasmic effector Dishevelled is a critical step in Wnt/β-catenin signaling. Considerable domains on FZD4 were identified as critical sites for recruitment of Dishevelled. A conserved motif (KTxxxW) located two amino acids after the seventh transmembrane domain was firstly verified to be crucial for membrane relocalization and phosphorylation of Dishevelled [44, 45]. The interaction between FZD4 and Dishevelled was further found to be pH- and charge-dependent [72]. Several amino residuals in intracellular loops 1, 2, and 3 and the flanking region near to intracellular loop 3 were also important for the intracellular location of Dishevelled while the mutant impaired the binding of Dishevelled [73–77]. Research based on FZD6 also showed that the linker domain, especially some conserved cystines, between the CRD domain and seven transmembrane core was imperative for Dishevelled recruitment [78]. One potential mechanism for FZD4 activation would be a Wnt/Norrin-induced movement of the seventh transmembrane domain to expose the key FZD4-Dishevelled interaction site [79]. Although 21% (26 of 121 mutations) of the mutations aggregated in the third intracellular loop and C terminal intracellular domain, it was not clear how the mutations affect the interaction between FZD4 and Dishevelled.


### 3.3.LRP5 Mutations and LRP5/LRP6 Structure

LRP5 gene, localized on human chromosome 11q13.2, consists of 23 exons and encodes 1615 amino acid single-pass transmembrane protein. LRP5 is a member of the low-density lipoprotein receptor family and belongs to a subfamily consisting of its mammalian homolog LRP6 and the Drosophila protein arrow. LRP5 and LRP6 share 73% identity in their extracellular domains. The LRP5/6 protein contains three domains including an extracellular domain, one transmembrane domain, and a cytoplasmic domain. The LRP5/6 ectodomain contains four β-propeller motifs (composed of six YWTD repeats) at the amino terminal end that alternate with four epidermal growth factor- (EGF-) like repeats (YWTD-EGF domain). These are followed by three low-density-lipoprotein receptor-like ligand-binding domains. LRP5 can act synergistically with FZD4 or other members of the Frizzled family to bind Wnts or Norrin, forming a functional ligand-receptor complex that triggers canonical Wnt/β-catenin or the Norrin/β-catenin signaling pathway and induce the transcription of target genes subsequently [80, 81].Thus far, 58 causative mutations identified in patients with FEVR have been reported forLRP5, of which 46 mutations are missense changes, 6 frame shift mutations resulted by deletions, insertion, and duplication, 2 introduce premature stop codons, and 4 changes affect splicing [28, 29, 31, 46, 56, 59, 69, 69, 82–85] (Table 3 and Figure 4). Mutations located in first, second, and third YWTD-EGF domain accounted for 12% (7 of 58 mutations), 38% (22 of 58 mutations), and 17% (10 of 58 mutations) of all the mutations, respectively. Thus, it can be seen causative mutations have a trend of clustering in the second YWTD-EGF domain since this segment is composed of only about 300 amino acids, accounting for less than 20% of whole LRP5 protein. Five of the included mutations (c.1828G>A, c.731C>G, c.1042C>T, c.1058G>A, and c.1481G>A) were also reported as causative mutation for OPPG [86], which was characterized as blindness and decreased bone density. But FEVR and OPPG were two different diseases because of the distinct pathogenesis of visual loss. OPPG patients often presented with blindness in the neonatal period and the symptoms initiated during early childhood. Inconformity of these results may was due to omission of bone density and definite pathogenesis of visual loss.Table 3
Spectrum ofLRP5 gene mutations among patients with familial exudative vitreoretinopathy.
Studies
No. of patients
No. of mutations
DNA variant
Coding effect
Location of the amino residue
Mutant phenotype
Country of origin
Toomes et al. [68]
32
6
c.518C>T
p.T173M
Firstβ-propeller motif
Abnormal retinal vasculature and retinal fold
USA
c.3502T>C
p.Y1168H
Low-density-lipoprotein receptor-like ligand binding domains
Total retinal detachment and retinoschisis
c.3840delA
p.R1270fsX1438
Premature termination
Not mentioned
c.4081T>G
p.C1361G
Low-density-lipoprotein receptor-like ligand binding domains
Classic features of FEVR
c.4119_4120insC
p.K1374fsX1549
Premature termination
Not mentioned
c.4488 + 2T>G
Splice-donor mutation
Premature termination
Undetermined
Qin et al. [56]
56
9
c.433C>T
p.L145F
Firstβ-propeller motif
Bilateral retrolental fibroplasias and total retinal detachment
Japan
c.803_812del
p.G268fsX272
Premature termination
Bilateral dragged macula
c.1330C>T
p.R444C
Secondβ-propeller motif
Severe falciform retinal fold
c.1564G>A
p.A522T
Secondβ-propeller motif
Tractional retinal detachment, severe macular ectopia along with peripheral fibrovascular mass
c.1604C>T
p.T535M
Secondβ-propeller motif
Bilateral retinal folds followed by total retinal detachment
c.1828G>A
p.G610R
Second epidermal growth-like factor
Bilateral dragged macula
c.1850T>G
p.F617C
Second epidermal growth-like factor
Bilateral retinal folds followed by total retinal detachment
c.2392A>G
p.T798A
Thirdβ-propeller motif
Bilateral peripheral avascular retinas
c.3361A>G
p.N1121D
Fourthβ-propeller motif
Unilateral falciform retinal fold with bilateral retinal avascularization
Boonstra et al. [46]
83
2
c.1532A>C
p.D511A
Secondβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
Netherlands
c.2413C>T
p.R805W
Thirdβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
Nikopoulos et al. [28]
16
4
c.1321G>A
p.E441K
Secondβ-propeller motif
Not mentioned
Netherlands
c.2978G>A
p.W993X
EGF-like domain following the third “β-propeller” module
Not mentioned
c.3758G>T
p.C1253F
EGF-like domain following the third “β-propeller” module
Not mentioned
c.4489-1G>A
Splice defect
Not applicated
Not mentioned
Yang et al. [69]
49
6
c.891-892delTC
p.R298LfxX2
Premature termination
Retrolenticular fibrotic mass, retinal detachment, microcornea, flat anterior chamber
China
c.2484C>G
p.I828M
Thirdβ-propeller motif
Retrolenticular fibrotic mass, stretched ciliary process
c.2626G>A
p.G876S
Third epidermal growth like factor
Retrolenticular fibrotic mass, stretched ciliary process
c.3361A>G
p.N1121D
Fourthβ-propeller motif
Temporal dragging of optic disc, retrolenticular fibrotic mass
c.4025G>A
p.R1342Q
Low-density-lipoprotein receptor-like ligand binding domains
Microcornea, retrolenticular fibrotic mass, avascular zone
c.4087G>A
p.D1363N
Low-density-lipoprotein receptor-like ligand binding domains
Increased branching of peripheral vessels, retrolenticular fibrotic mass
Fei et al. [82]
2
2
c.1264G>A
p.A422T
Secondβ-propeller motif
Not mentioned
China
c.1619T>C
p.L540P
Second epidermal growth like factor
Not mentioned
Seo et al. [59]
51
4
c.731C>G
p.T244R
Firstβ-propeller motif
3A/2B stage FEVR
Korea
c.1330C>T
p.R444C
Secondβ-propeller motif
2A stage FEVR
c.1833dupG
p.C612VfsX25
Premature termination
1B/4A stage FEVR
c.4098C>G
p.D1366E
Low-density-lipoprotein receptor-like ligand binding domains
3B stage FEVR
Zhang et al. [85]
4
4
c.C1042T
p.R348W
First epidermal growth-like factor
Not mentioned
China
c.G1141A
p.D381N
Secondβ-propeller motif
Not mentioned
c.C1870T
p.R624W
Second epidermal growth-like factor
Not mentioned
c.A4550G
p.Y1517C
Cytoplasmic tail
Not mentioned
Tang et al. [31]
100
10
c.1058G>A
p.R353Q
First epidermal growth-like factor
Ilateral retrolenticular fibrotic mass and total retinal detachment
China
c.1183C>T
p.R395W
Secondβ-propeller motif
Falciform retinal fold
c.1318A>T
p.I440F
Secondβ-propeller motif
Retinal fold
c.1582G>A
p.E528K
Secondβ-propeller motif
Peripheral vascular deficiencies
c.1942G>A
p.V648I
Second epidermal growth-like factor
Rhegmatogenous retinal detachment
c.2738G>T
p.C913F
Third epidermal growth-like factor
Retinal fold and macular dragging
c.4087G>C
p.D1363H
Low-density-lipoprotein receptor-like ligand binding domains
Falciform retinal fold
c.4733C>T
p.T1578M
Cytoplasmic tail
Retinal fold
c.92-2A>C
Splice site mutation
Premature termination
Ilateral retrolenticular fibrotic mass and total retinal detachment
c.4488 + 2T>G
Splice site mutation
Premature termination
Retinal folds
Rao et al. [29]
31
5
c.4205G>A
p.G1402D
Transmembrane domain
Falciform fold
China
c.2237G>C
p.R746P
Thirdβ-propeller motif
Peripheral avascular zone
c.2618A>T
p.K873M
Thirdβ-propeller motif
Peripheral avascular zone
c.1384C>T
p.R462X
Secondβ-propeller motif
Complete retinal detachment
c.2817_2827+1del12bp
p.N940fs
Premature termination
Complete retinal detachment
Liu et al. [83]
10
5
c.542T>G
p.M181R
Firstβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
China
c.1197G>T
p.R399S
Secondβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
c.1481G>A
p.R494Q
Secondβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
c.1507G>A
p.G503R
Secondβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
c.2626G>A
p.G876S
Third epidermal growth-like factor
Diagnosed with FEVR, symptoms not mentioned
Pefkianaki et al. [84]
1
1
c.2234C>T
p.A745V
Thirdβ-propeller motif
Extensive exudative retinopathy and shallow retinal detachment
USAFigure 3
Schematic diagram of the Frizzled-4 protein shows the locations of the mutations. A whole gene deletion and a deletion/insertion (c.40 del/inser) with unknown protein change are not shown. Superscript number means the reported times of the same or different mutations at a certain site. The color of the mutations which were reported more than one time was recolored as orange. The opacity varied with the reported frequency of the mutations.In the crystal of the first two YWTD-EGF structure of LRP6, each of the two EGF domains packs tightly against the bottom surface of the preceding YWTDβ-propellers [87]. Extensive interface interactions was observed between the first β-propellers and second β-propellers, and the first EGF domain also interacts with the second β-propellers, which was critical to maintain the stability and orientation of LRP6’s first two YWTD-EGF domains.Early studies revealed that the interaction of LRP6 with Wnt-Fzd4 was mediated by the first two propeller domains [88], while other researchers pointed out that a single LRP6 might engage two different Wnt proteins simultaneously. LRP5/6 binds to different Wnts via different regions or multiple domains together [89]. The four β-propeller domains in LRP5/6 share a relatively low identity among them, indicating the functional differences among these YWTD propellers. Ke et al. demonstrated that Norrin interacted with β-propeller domain 1 (BP1) and β-propeller domain 2 (BP2) but not BP3-4 of LRP6. However, the binding sites of Norrin with LRP5 remain unclear. From these two perspectives, the mutations accumulated in the second YWTD-EGF domain may destroy the stable structure of first two β-propellers or interrupted their interaction with Norrin or Fzd4.


### 3.4.TSPAN12 Gene, Protein, and Spectrum

TheTSPAN12 gene is located on chromosome 7q31 and encodes for a 305 amino acid transmembrane protein. TSPAN12 is a member of the tetraspanin family that shares certain specific structural features that distinguishes them from other proteins that pass the membrane four times. Both the N and C terminals of TSPAN12 were inside the cell membrane, and it has an unusually long C-terminal intracellular tail of approximately 60 amino acids. It contains four transmembrane domains connected by two extracellular loops (ECL-1 and ECL-2) and an intracellular loop. The ECL-1 is smaller compared to the ECL-2.TSPAN12 was discovered to associate selectively with Norrin/β-catenin signaling but not with Wnt/β-catenin signaling. It acted as the fourth important component of Norrin/FZD4/LRP5 complex. Signaling reduction could be rescued by TSPAN12 overexpression although direct binding with Norrin and FZD4 was not detected. However, another study reported that TSPAN12 interacted with Norrin and FZD4 via its extracellular loops and enhanced the FZD4 ligand selectivity for NDP [90]. Thus, TSPAN12 was postulated to elicit physiological levels of signaling that was required for normal retinal angiogenesis by promoting FZD4 multimerization cooperated with Norrin and facilitating selective ligand recognition [11].We summarized 40 currently known mutations in TSPAN12 identified in patients affected with FEVR and discussed their coding consequences [6, 24, 29, 31, 54, 58, 59, 68, 83, 91–96] (Table 4 and Figure 5). All types of mutations were identified, including 22 missense mutation, 4 nonsense mutations, 9 splice-site mutations, 3 deletions, and 2 insertions. Mutations at residues T49, L140, C189, and L233 were reported more than one time. It was reported that L233P strongly impaired the TSPAN12 activity, while T49M mildly impaired the activity. Unfortunately, the authors did not investigate the signaling defect strength of L140X and C189Y/R. In all of the mutations, 38% (15 in 40 mutations) of them were located in the ECL-2 domain. These mutations were highly consistent with the biochemical results. TSPAN12 is anchored to the Norrin receptor complex via an interaction of the LEL with FZD4. The ECL-2 domain of TSPAN12 is essential for enhancing Norrin-induced FZD4 signaling. TSPAN12 can also alleviate the defects of FZD4 M105V, a mutation that destabilizes the NDP/FZD4 interaction [90].Table 4
Spectrum ofTSPAN12 gene mutations among patients with familial exudative vitreoretinopathy.
Studies
No. of patients
No. of mutations
DNA variant
Coding effect
Location of the amino residue
Mutant phenotypes
Country of origin
Savarese et al. [94]
1
1
c.668T>C
p.L223P
Transmembrane domain
No sign of neovascularization
Pakistan
Poulter et al. [93]
58
5
c.67-1G>C
p.L23GfsX66
Transmembrane domain, premature termination
Bilateral retinal folds
Mexican and Pakistan
c.146C>T
p.T49M
First extracellular loop
Bilateral congenital cataract, large retinal fold
c.285 + 1g>a
p.R50DfsX12
Premature termination
Bilateral congenital cataract, large retinal fold
c.413A>G
p.Y138C
Second extracellular loop
Peripheral retina avascularity
c.668T>C
p.L223P
Transmembrane domain
Bilateral retinal folds, funnel retinal detachments
Poulter et al. [6]
70
7
c.68T>G
p.L23X
Transmembrane domain
Bilateral retinal folds and unilateral, persistent hyperplastic primary vitreous
USA, UK, Britain, Japan, Australia
c.149 + 3a>g
Splice-site mutation
Premature termination
Unilateral retinal fold
c.218_219insGCTGTTT
p.F73LfsX119
Premature termination
Macula ectopia, with a large retinal fold
c.302T>A
p.L101H
Transmembrane domain
Lassic signs of FEVR
c.361-5_361-1delaccag
Splice-site mutation
Premature termination
Bilateral temporal retinal avascularity
c.419T>A
p.L140X
Second extracellular loop
Bilateral retinal folds
c.629T>G
p.M210R
Bilateral macular traction
Bilateral macular traction
Nikopoulos et al. [68]
43
2
c.709G>C
p.A237P
Transmembrane domain
Avascular peripheral retina
Netherlands
c.562G>C
p.G188R
Second extracellular loop
Avascular peripheral retina
Yang et al. [96]
49
3
c.146C>T
p.T49M
First extracellular loop, conserved residue
Falciform retinal folds
China
c.313T>C
p.C105R
Transmembrane domain, conserved residue
Midperipheral retina, an avascular zone on the peripheral retina
c.601delC
p.L201FfsX14
Conserved residue
Inferotemporal dragging of the optic disc and macula
Gal et al. [91]
64
1
c.542G>T
p.C181F
Second extracellular loop, form disulfide bonds
Bilateral visual impairment, various ocular abnormalities
Israel
Xu et al. [95]
85
3
c.177delC
p.Y59fsX67
Premature termination
Falciform retinal folds
China
c.C254T
p.T85M
Intracellular loop
Pigment deposit, dragged disc
c.566G>A
p.C189Y
Second extracellular loop, form disulfide bonds
Bilateral retinal folds
Kondo et al. [92]
90
2
c.419T>A
p.L140X
Second extracellular loop
Abnormal retinal vessels with vitreous degeneration
Japan
c.734T>C
p.L245P
C-terminal cytoplasmic tail
Retinal fold resulting
Seo et al. [59]
51
1
c.56T>G
p.L19R
Transmembrane domain
3A stage FEVR
Korea
Ganeswara Rao Musada et al. [2016]
110
3
c.125T>C
p.V42A
First extracellular loop
Diagnosed with FEVR, symptoms not mentioned
India
c.334G>A
p.V112I
Second extracellular loop
Diagnosed with FEVR, symptoms not mentioned
c.479G>A
p.C160Y
Second extracellular loop
Diagnosed with FEVR, symptoms not mentioned
Tang et al. [31]
100
8
c.2T>C
p.M1T
N-terminal domain
Not mentioned
China
c.464G>C
p.R155T
Second extracellular loop
Not mentioned
c.438-439insT
p.T147YfsX12
Premature termination
Total retinal detachment and massive vitreous proliferation
c.655delC
p.Q219NfsX5
Premature termination
Total retinal detachment
c.916-918 + 3delTAAAAA
p.∗306Eext∗35
Elongated protein
Peripheral avascular retina
c.150-1G>A
Splice acceptor mutations
Not applicated
Not mentioned
c.285 + 1G>A
Splice acceptor mutations
Not applicated
Not mentioned
c.469-1G>A
Splice acceptor mutations
Not applicated
Not mentioned
Iarossi et al. [24]
8
1
c.67-2A>G
Defective splicing
Not applicated
Falciform retinal fold
Italia
Rao et al. [29]
31
1
c.345T>G
p.Y115X
Second extracellular loop
Falciform folds, complete retinal detachment
China
Liu et al. [83]
10
1
c.566G>A
p.C189Y
Second extracellular loop
China
Schatz and Khan [58]
3
1
c.565T>C
p.C189R
Second extracellular loop, affects cystine residues forming
Total retinal detachment
SwedenFigure 4
Schematic representation of LRP5 protein shows the location of the mutations within the protein domains. Four splice site mutations are not shown. Superscript number means the reported times of the same or different mutations at a certain site. The color of the mutations which were reported more than one time was recolored as orange. The opacity varied with the reported frequency of the mutations.


## 3.1.NDP Mutations and Norrin Structure

TheNDP gene locus mapped to chromosome Xp11.4 and comprised three exons. However, the first exon corresponds to the untranslated region of the gene that has regulatory functions, and only exons 2 and 3 of encode a secreted protein of 133 amino acids called Norrin or Norrie disease protein. Norrin consists of two major parts: a signal peptide at the amino-terminus of the protein that directs its localization and a region containing a typical motif of six cystines forming a cystine-knot. The cystine-knot motif is highly conserved in many growth factors as transforming growth factor-β, human chorionic gonadotropin, nerve growth factor, and platelet derived growth factor [21]. Cystine residues and their disulfide bonds in the cystine-knot play important structural and functional roles. Among 10 Frizzled family members, Norrin specifically binds to the transmembrane FZD4 with high affinity, forming a Norrin/FZD4 complex with LRP5 and TSPAN12 coreceptors to activate the Norrin/β-catenin signaling pathway [22]. Norrin was also reported to play a major role in controlling retinal vascular growth and architecture both in the developing eye and in adult vasculature.Twenty-six nucleotide variants have been identified forNDP in patients with FEVR. These include 21 missense changes, 4 deletions, and 1 insertion resulting frame shift [2, 23–31] (Table 1 and Figure 2). Most of the mutations were found in single or only a few patients, while several mutations are generally more common. By far, the most prevalent mutation was c.362G > A (p.R121Q), distributed in Spanish, Mexican, Indian, Chinese, and Italian. It is noteworthy that although probands containing c.11_12delAT (p.H4RfsX21), c.170C > G (p.S57X), and c.310A > C (p.K104Q) were definitely diagnosed as FEVR following explicit criteria, these three mutations were also reported to cause Norrie disease by other researches [28, 32, 33]. The ocular features and retinal changes observed in Norrie disease are similar to those observed in cases of FEVR. Not all the Norrie disease patients have mental retardation and develop a progressive sensorineural hearing loss; it is really difficult to distinguish Norrie disease from FEVR.Table 1
Spectrum ofNDP gene mutations among patients with familial exudative vitreoretinopathy.
Studies
No. of patients
No. of mutations
DNA variant
Coding effect
Location of the amino residue
Mutant phenotypes
Country of origin
Chen et al. [2]
30
1
c.370C>T
p.L124F
Norrin dimer interface
Retina detached
UK
Riveiro-Alvarez et al. [30]
45
1
c.362G>A
p.R121Q
Norrin dimer interface
Congenital blindness, phthisis bulbi
Spain
Dickinson et al. [23]
13
1
c.307C>G
p.L103V
Norrin-FZD4 interface
Not mentioned
Australia
Hiroyuki et al. [34]
62
3
c.53T>A
p.I18K
Signal domain
Peripheral avascularization, neovascularization
Japan
c.162G>C
p.K54N
Deductive Norrin-LRP5 interface
Retinal detachment and macular traction with temporal avascularization
c.344G>T
p.R115L
Deductive Norrin-LRP5 interface
Retinal detachment
Pelcastre et al. [35]
127
3
c.361C>T
p.R121W
Norrin dimer interface
On-perfusion in peripheral retina
Mexico
c.362G>A
p.R121Q
Norrin dimer interface
Retinal detachment
Musada et al. [36]
110
8
c.11_12delAT
p.H4RfsX21
Signal domain
Bilateral total retinal detachment
India
c.69delC
p.D23EfsX9
Signal domain
Pigmentation and vitreoretinal traction
c.142_145delATCA
p.I48VfsX55
Premature termination
Bilateral leukocoria and total retinal detachment
c.148C>G
p.H50D
Deductive Norrin-LRP5 interface
Straightening of the blood vessel, macular dragging
c.170C>G
p.S57X
Norrin-FZD4 interface
Retinal detachments and retrolental membranes
c.338G>A
p.G113D
Near deductive Norrin-LRP5 interface
Avascular peripheral retina, straightening of the blood vessels, and dye leakage
c.362G>A
p.R121Q
Norrin dimer interface
Retinal detachments with retrolental membranes
c.376T>C
p.C126R
Norrin dimer interface
Bilateral total retinal detachment
Liu Y. L. et al. [37]
40
1
c.310A>C
p.K104Q
Norrin-FZD4 interface
Weak eyesight, retinal vascular abnormalities
China
Tang et al. [31]
100
5
c.196G>A
p.E66K
Cystine-knot motif
Macular dragging
China
c.203A>C
p.H68P
Cystine-knot motif
Ectopic macular
c.281A>T
p.H94L
Norrin dimer interface
Peripheral avascular zone and retinal exudates
c.362G>A
p.R121Q
Norrin dimer interface
Retinal fold, retinal detachment
c.334delG
p.G113AfsX149
Premature termination
Bilateral tractional retinal detachment
Iarossi et al. [24]
8
2
c.362G>A
p.R121Q
Norrin dimer interface
Falciform fold, partial traction
Italian
c.313G>C
p.A105F
Norrin-FZD4 interface
Macula-involving retinal detachment
Rao et al. [29]
31
3
c.127C>A
p.H43N
Norrin-FZD4 interface
Complete retinal detachment
China
c.52_53ins32bp
p.S29fs
Premature termination
Complete retinal detachment
c.195C>G
p.C65W
Cystine-knot motif, form disulfide bond with C126
Complete retinal detachmentFigure 2
Schematic diagram of the Norrin protein shows the location of the mutations within the protein domains. Superscript number means the reported times of the same or different mutations at a certain site. The color of the mutations which were reported more than one time was recolored as orange. The opacity varied with the reported frequency of the mutations.It was demonstrated from the three-dimensional structure of Norrin that two-monomer Norrins formed a homodimer in the crystal. The Norrin monomer contained exclusiveβ strands with two β-hairpins on one side and one β-hairpin on the other side. Crystal structures of Norrin in complex with the extracellular domain of FZD4 showed that two β-hairpins in Norrin (β1-β2 and β5-β6) interacted with three loops in FZD4 cystine-rich domain (FZD4-CRD) [38, 39]. There were 19 mutations located in domains from C39 to C65 and C96 to C126, which covered two β-hairpins (β1-β2 and β5-β6) and loops between them, namely, 73% of the mutations (19/26) concentrated in the interacting domains with FZD4-CRD.Specifically, 9 mutations were located in the Norrin dimer interface which was formed fromβ2 and β4 sheets of one monomer and β2′ of another monomer (Table 1). Three mutations were reported from the cystine-knot motif, one of which (C65W) obviously impaired intermolecular disulfide bond-forming. Five mutations disturbed the hydrogen bonds or hydrophobic contacts between Norrin and FZD4 CRD in the Norrin-FZD4 CRD interface [38, 40]. Four mutations clustered on the edge of the Norrin molecule in the β1-β2 and β3-β4 loop regions were inferred as LRP5 binding sites because they did not affect Fz4 binding yet reduced the ability of Norrin to activate the TCF reporter [39]. The residues in the interaction interface are well defined and overlap with disease-associated mutations in NDP. The level of signaling activity of K104Q, R121Q, and L124F was between 20% and 80% of the wide-type Norrin, suggesting that even a modest decrement in Norrin/Fz4 signaling may have a significant phenotypic effect in humans [14, 41]. It is of no surprise that the mutations located in β1-β2 and β5-β6 obstructed the formation of two β-hairpins and the interactions between Norrin and FZD4.


## 3.2.FZD4 Mutations and FZD4-CRD Structure

TheFZD4 gene is located on chromosome 11q14.2, and its mRNA consists of two exons coding for 537 amino acid protein called FZD4 or Frizzled-4 protein. FZD4 acted as the receptor for Wnt and Norrin along with LRP5, which has a pivotal role in various cellular processes including cell fate determination, control of cell polarity, and malignant transformation. The FZD4 contains a ∼120-residue N-terminal extracellular cystine-rich domain(CRD), seven helix transmembrane domains, three extracellular and three intracellular loops, and a C terminal cytoplasmic domain [42, 43]. The cystine-rich domain is indispensable to Wnts or Norrin and is conserved among Frizzled family members [22, 39]. The FZD4 carboxyl cytoplasmic region contains juxtamembrane KTXXXW motif which is responsible for association with Dishevelled to activate downstream signaling [44, 45].In this update, we summarized a total of 121 mutations already reported in patients with FEVR in the literatures consisting of 70 missense mutations, 19 nonsense mutations, and 30 insertions or deletions that lead to either frame shifts or in-frame deletions; a single base change resulted in 2 amino acids extension and a whole-gene deletion [7, 24, 28, 29, 46–71] (Table 2 and Figure 3). No splice mutations have been reported for FZD4, and the mutations seem to cluster in two specific “hotspots”. Although the mutations span in whole FZD4 gene, 49% (59 of 121 mutations) and 13% (16 of 121 mutations) of them have a tendency to bunch in the N terminal extracellular domain and C terminal intracellular domain, respectively.Table 2
Spectrum ofFZD4 gene mutations among patients with familial exudative vitreoretinopathy.
Studies
No. of patients
No. of mutations
DNA variant
Coding effect
Location of the amino residue
Mutant phenotype
Country of origin
Zhang et al. [65]
49
5
c.134G>A
p.C45Y
CRD domain, no plasma membrane localization, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
China and USA
c.173A>G
p.Y58C
CRD domain, failed to bind Norrin, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
c.610T>C
p.C204R
CRD domain, failed to bind Norrin, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
c.678G>A
p.W226X
Transmembrane 1, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
c.1488G>A
p.W496X
C-terminal intracellular domain, failed to mediate Norrin induction of theseβ-catenin target genes
Not mentioned
Drenser et al. [48]
123
5
c.97C>T
p.P33S
Signal sequence
2-stage FEVR, rhegmatogenous retinal detachment
USA
c.349T>C
p.C117R
CRD domain, conserved cystine residue
4B stage FEVR
c.502C>T
p.P168S
CRD domain
2-stage FEVR, rhegmatogenous retinal detachment
c.542G>A
p.C181Y
CRD domain, conserved cystine residue
4B stage FEVR
c.1513C>T
p.Q505X
Immediately downstream from KTxxxW motif
4B stage FEVR
Qin et al. [56]
56
2
c.1005G>C
p.W335C
Highly conserved across all members of the FZD family
Bilateral retinal folds
Japan
c.1024A>G
p.M342V
Intracellular loop 2, function not shown
Bilateral dragged disc
Robitaille et al. [7]
27
2
c.1479_1484del
p.M493_W494del
Failed to activate calcium/calmodulin-dependent protein kinase II and protein kinase C
Bilateral retinal detachment
Canada
c.1501_1502delCT
p.L501fsX533
No membrane accumulation, failed to activate calcium/calmodulin-dependent protein kinase II and protein kinase C
Not mentioned
Kondo et al. [51]
24
4
c.313A>G
p.M105V
CRD domain
Bilateral vitreous opacity, retinal exudates, macular ectopia, falciform retinal fold
Japan
c.957G>A
p.W319X
Transmembrane domain
Falciform retinal fold, chronic retinal detachment
c.1250G>A
p.R417Q
Intracellular loop 3
Falciform retinal fold, posterior synechiae, chronic retinal detachment
c.1463G>A
p.G488D
Transmembrane domain
Falciform retinal folds
Dailey et al. [47]
421
11
c.40 Del/inser
Unknown
Not mentioned
Not mentioned
USA
c.97C>T
p.P33S
Signal sequence, reduced Wnt reporter activity
Not mentioned
c.151T>A
p.S51T
CRD domain
Not mentioned
c.169G>T
p.G57C
CRD domain
Not mentioned
c.349T>C
p.C117R
CRD domain
Not mentioned
c.502C>T
p.P168S
CRD domain, reduced Wnt reporter activity
Not mentioned
c.542G>A
p.C181Y
CRD domain
Not mentioned
c.758G>A
p.R253H
Transmembrane domain
Not mentioned
c.1074A>C
p.K358N
Transmembrane domain
Not mentioned
c.1513C>T
p.Q505X
Immediately downstream from KTxxxW motif
Not mentioned
c.1589G>A
p.G530E
C-terminal
Not mentioned
Fei et al. [49]
61
3
c.C205T
p.H69Y
CRD domain
Not mentioned
China
c.G400T
p.E134X
CRD domain, failed to activateβ-catenin reporter
Peripheral avascular zone, dragged disc
c.1506delAC
p.T503fs
Failed to activateβ-catenin reporter
Total retinal detachment
Yang et al. [69]
56
5
c.313A>G
p.M105V
CRD domain
Increased branching of peripheral vessels, retinal detachment, Avascular zone, Retrolenticular fibrotic mass, neovascularization
China
c.631T>C
p.Y211H
Linker upstream of transmembrane 1
Temporal dragging of optic disc, peripheral fibrous proliferation
c.1282-1285delGACA
p.D428SfsX2
Intracellular loop 3
Straightening of temporal arcades, temporal dragging of optic disc, peripheral fibrous proliferation
c.1482G>A
p.W494X
Transmembrane domain
Retrolenticular fibrotic mass, lens dislocation, brushlike peripheral, avascular zone, neovascularization, peripheral fibrous proliferation
c.1513C>T
p.Q505X
Immediately downstream from KTxxxW motif
Temporal dragging of optic disc, f alciform retinal fold, branching of peripheral vessels, avascular zone, peripheral exudates
Nallathambi et al. [55]
75
3
c.97C>T
p.P33S
Signal sequence, reduced Wnt reporter activity
Peripheral lattice degeneration, atrophic holes, macular ectopia, bilateral peripheral avascular zone
India
c.244_251del8ins27
p.F82fsX135
CRD domain
Macular ectopia, terminal branching, peripheral avascular zone
c.610T>C
p.C204R
CRD domain
Temporal peripheral avascular zone, terminal branching, tractional retinal detachment
Seo et al. [59]
51
9
c.160C>T
p.Q54X
CRD domain
1B stage FEVR
Korea
c.313A>G
p.M105V
CRD domain
1B,1A, 2B stage FEVR
c.456C>G
p.N152K
CRD domain
1B, 2B stage FEVR
c.470T>C
p.M157T
CRD domain
1B stage FEVR
c.539_540delAG
p.E180VfsX9
CRD domain
1B, 3A stage FEVR
c.676T>A
p.W226R
Linker upstream of transmembrane 1
1B, 3A stage FEVR
c.1210_1211delTT
p.L404VfsX54
Transmembrane domain
1A stage FEVR
c.1282_1285delGACA
p.D428SfsX2
Intracellular loop 3
1A stage FEVR
Whole gene deletion
No protein
No protein
2B stage FEVR
Musada et al. [66]
110
7
c.313A>G
p.M105V
CRD domain
Diagnosed with FEVR, symptoms not mentioned
Indian
c.341T>G
p.I114S
CRD domain
Diagnosed with FEVR, symptoms not mentioned
c.470T>C
p.M157T
CRD domain
Diagnosed with FEVR, symptoms not mentioned
c.1282_1285delGACA
p.D428SfsX2
Intracellular loop 3
Diagnosed with FEVR, symptoms not mentioned
c.1286_1290delAGTTA
p.K429RfsX28
Intracellular loop 3
Diagnosed with FEVR, symptoms not mentioned
c.1395_1396insT
p.R466SfsX6
Extracellular loop
Diagnosed with FEVR, symptoms not mentioned
c.1613A>C
p.X538SextX2
C-terminus
Diagnosed with FEVR, symptoms not mentioned
Jia et al. [50]
48
12
c.39-49delCCCGGGGGCG
p.P14fsX57
Signal sequence, Truncated protein
Avascular retina, dragged macula
China
c.65G>A
p.G22E
Signal sequence, loss of activity
Nystagmus, retrolental fibroplasia, retinal detachment
c.205C>T
p.H69Y
CRD domain, loss of activity
Avascular retina, fibrous proliferation, and dragged macula
c.313A>G
p.M105V
CRD domain, loss of activity
Retinal vascular tortuosity, exudates, and avascularization
c.538G>A
p.E180K
CRD domain, loss of activity
Not mentioned
c.710C>G
p.T237R
Linker upstream of transmembrane 1, loss of activity
Preretinal fibrosis, peripheral nonperfusion
c.757C>T
p.R253C
Transmembrane domain, loss of activity
Peripheral avascularization and typical scalloped border
c.983T>C
p.F328S
Transmembrane domain, loss of activity
Retinal folds and persistent hyperplastic primary vitreous
c.1015G>A
p.A339T
Intracellular loop 2, loss of activity
Dragged discs, retinal elevation with hemorrhage
c.1408G>A
p.D470N
Transmembrane domain, loss of activity
Dragged discs, retinal folds, and macular ectopia
c.1472C>A
p.S491X
Transmembrane domain, truncated protein
Dragged discs, macular ectopia, and pigment changes
c.1488G>A
p.W496X
C-terminus, truncated protein
Not mentioned
Peachey et al. [70]
1
1
c.1026A>G
p.M342V
Intracellular loop 2
Straightening of the retinal vessels, peripheral avascular areas
Japanese
Tang et al. [60]
100
14
c.107G>A
p.G36D
Signal sequence
Not mentioned
China
c.133T>C
p.C45R
CRD domain
Avascular zone, increasing of peripheral vessels, straightening of vessels
c.133T>A
p.C45S
CRD domain
Not mentioned
c.134G>A
p.C45Y
CRD domain
Not mentioned
c.158G>C
p.C53S
CRD domain
Macular dragging, Avascular zone, increasing of peripheral vessels, straightening of vessels
c.223G>A
p.A75T
CRD domain
Not mentioned
c.268T>C
p.C90R
CRD domain
Not mentioned
c.313A>G
p.M105V
CRD domain
Not mentioned
c.957G>A
p.W319X
Transmembrane domain
Avascular zone, increasing of peripheral vessels
c.975_978delCACT
p.T326fsX356
Transmembrane domain
Avascular zone, neovascularization, increasing of peripheral vessels, SV
c.1034_1054delCTTATTTCCACATTGCAGCCT
p.S345_A351del
Intracellular loop 2
Avascular zone, increasing of peripheral vessels, straightening of vessels
c.1282_1285delGACA
p.D428SfsX2
Intracellular loop 3
Not mentioned
c.1475delG
p.G492fsX512
Intracellular loop 3
Avascular zone, neovascularization, increasing of peripheral vessels, straightening of vessels,vessels exudates
c.1498delA
p.T500fsX512
Truncated protein
Not mentioned
Nikopoulos et al. [68]
16
5
c.118G>C
p.E40Q
Signal sequence
Not mentioned
Netherlands
c.611G>A
p.C204Y
CRD domain
Deformation of posterior retina, ectopia of the macula, stretched retinal vessels, retinal detachment
c.856G>T
p.E286X
Extracellular loop
Few abnormal temporal retinal branches, avascular peripheral fundus
c.1282_1285del
p.D428SfsX2
Intracellular loop 3
Macular ectopia. Haemorrhagic and exudative areas present in the retina
c.1573G>C
p.G525R
C-terminus
Macular ectopia and peripheral, retinal detachment
Kondo et al. [25]
1
1
c.1250G>A
p.R417Q
Intracellular loop 3
retrolental fibroplasia, falciform retinal fold
Japanese
Toomes et al. [69]
40
8
c.107G>A
p.G36D
Signal sequence
Unable to obtain detailed clinical notes
UK
c.314T>C
p.M105T
CRD domain
Macula-off rhegmatogenous retinal detachment, inadequate vascularization
c.469A>G
p.M157V
CRD domain
Macular folds and retinal detachments
c.957delG
p.W319fsX323
Transmembrane domain
Peripheral retinal fold
c.1490C>T
p.S497F
C-terminus
Disc-dragging
c.1498delA
p.T500fsX512
KTxxxW domain
Small myopic optic disc, diffuse nonspecific pigmentary changes
c.1501_1502delCT
p.L501fsX533
KTxxxW domain
Bilateral cicatrized tractional retinal detachments
c.1513C>T
p.Q505X
Immediately downstream from KTxxxW motif
Temporal sector of retina with deficient vascularization
Robitaille et al. [57]
68
11
c.316 T>C
p.C106G
CRD domain
Dragging of the retina, macular fold
Canadian
c.470T>A
p.M157K
CRD domain
Peripheral pigmentary, total retinal detachment, nonperfusion with leucocoria
c.633delC
p.Y211fsX
Linker upstream of transmembrane 1
Haemangiomatous lesion with exudation and peripheral avascular retina
c.1282_1285del
p.D428SfsX2
Intracellular loop 3
Left macula dragged
c.1463G>T
p.G488V
Transmembrane domain
c.1508insC
p.T503fsX31
KTxxxW motif
Bilateral dragging of the macula with peripherally straightened, avascular retina
c.313A>G
p.M105V
CRD domain
Bilateral dragging of the macula, retina detachment
c.678G>A
p.W226X
Linker upstream of transmembrane 1
Large elevated tight fold, large falciform fold
c.1448G>A
p.W496X
C-terminal intracellular domain
Tractional retinal detachment
c.1479_1484del
p.M493_W494del
Transmembrane domain
Not mentioned
c.341T>C
p.I114T
CRD domain
Not mentioned
Robitaille et al. [67]
5
2
c.1479_1484del
p.M493_W494del
Transmembrane domain
Absence of retinal vasculature, hypoplastic iris with posterior synechiae
Canadian
c.341T>C
p.I114T
CRD domain
Falciform retinal folds, small atrophic retinal hole
Boonstra et al. [46]
83
4
c.668T>A
p.M223K
Linker upstream of transmembrane 1
Diagnosed with FEVR, symptoms not mentioned
Netherlands
c.957G>A
p.W319X
Transmembrane domain
Diagnosed with FEVR, symptoms not mentioned
c.1333A>C
p.T445P
Transmembrane domain
Diagnosed with FEVR, symptoms not mentioned
c.1448G>A
p.W496X
C-terminus, truncated protein
Diagnosed with FEVR, symptoms not mentioned
Iarossi et al. [24]
8
3
c.277C>T
p.Q93X
CRD domain
Large avascular area, falciform retinal fold
Italian
c.542G>A
p.C181Y
CRD domain
Stage 3 and stage 2 FEVR
c.611G>T
p.C204F
CRD domain
Stage 4A FEVR
Rao et al. [29]
31
2
c.1282_1285delGACA
p.D428SfsX2
Intracellular loop 3
Complete retinal detachment
China
c.227delA
p.E76fs
CRD domain
Falciform retinal detachment.
Murken et al. [53]
1
1
c.1474delG
p.G492fsX
Intracellular loop 3
Peripheral avascular zone and macular dragging
Mexico
Schatz and Khan [58]
3
1
c.349T>C
p.C117R
CRD domain, forms a disulfide bond with Cys158
Mild temporal avascularity, mild peripheral temporal avascularity
SwedenThe 120-residue N-terminal extracellular cystine-rich domain (CRD) domain, connected to the first transmembrane helix by a 50-amino-acid linker, was crucial to ligand recognition. In the CRD domain, mutations at C45, M105, and M157 were three most frequently reported mutations, for 4, 9, and 4 times by different studies, respectively. One of these mutations, C45Y, was found to disrupt protein folding, resulting FZD4 being stuck in the cytoplasm with no membrane location [71]. It was supposed that the disulfide bond between Cys45 and Cys106 was imperative to protein transportation and functional activity. It was also visible from the crystal structure of FZD4-CRD that five disulfide bridges (Cys45–Cys106, Cys53–Cys99, Cys90–Cys128, Cys117–Cys158, and Cys121–Cys145) stabilized the α helices [38].Two crystal structures of Norrin/FZD4-CRD complex and a FZD4 transmembrane domain had been registered in the Protein Data Bank [38, 40, 70]. The structures showed that one FZD4-CRD coupled a Norrin monomer with no interactions between the two FZD4-CRDs. Three loops between α helices were responsible for binding to the β-hairpins in Norrin [38]. The C-terminal tail of FZD4-CRD also made contribution to Norrin recognition. Residues V45, M59, L61, and L124 of Norrin and F96, M105, I110, M157, and M159 FZD4-CRD constituted a hydrophobic core at the binding interface [40]. Based on this, it is speculated that FEVR-related mutations at M105 and M157 may interrupt the binding of Norrin to FZD4. Biophysical analysis of Norrin and FZD4 demonstrated that the linker region of FZD4 contributes to a high-affinity interaction with Norrin and signaling [71]. Mutation C181Y in this domain not only destroyed the disulfide bond but also interrupted the binding of Norrin. The FZD4 transmembrane domain structure showed mutations in key positions (M309L, C450I, C507F, and S508Y) of the ΔCRD-FZD4 structure which led to aberrant downstream signaling. However, no disease-causing mutation had been reported in abovementioned four amino residuals.The FZD4-mediated membrane recruitment of the cytoplasmic effector Dishevelled is a critical step in Wnt/β-catenin signaling. Considerable domains on FZD4 were identified as critical sites for recruitment of Dishevelled. A conserved motif (KTxxxW) located two amino acids after the seventh transmembrane domain was firstly verified to be crucial for membrane relocalization and phosphorylation of Dishevelled [44, 45]. The interaction between FZD4 and Dishevelled was further found to be pH- and charge-dependent [72]. Several amino residuals in intracellular loops 1, 2, and 3 and the flanking region near to intracellular loop 3 were also important for the intracellular location of Dishevelled while the mutant impaired the binding of Dishevelled [73–77]. Research based on FZD6 also showed that the linker domain, especially some conserved cystines, between the CRD domain and seven transmembrane core was imperative for Dishevelled recruitment [78]. One potential mechanism for FZD4 activation would be a Wnt/Norrin-induced movement of the seventh transmembrane domain to expose the key FZD4-Dishevelled interaction site [79]. Although 21% (26 of 121 mutations) of the mutations aggregated in the third intracellular loop and C terminal intracellular domain, it was not clear how the mutations affect the interaction between FZD4 and Dishevelled.


## 3.3.LRP5 Mutations and LRP5/LRP6 Structure

LRP5 gene, localized on human chromosome 11q13.2, consists of 23 exons and encodes 1615 amino acid single-pass transmembrane protein. LRP5 is a member of the low-density lipoprotein receptor family and belongs to a subfamily consisting of its mammalian homolog LRP6 and the Drosophila protein arrow. LRP5 and LRP6 share 73% identity in their extracellular domains. The LRP5/6 protein contains three domains including an extracellular domain, one transmembrane domain, and a cytoplasmic domain. The LRP5/6 ectodomain contains four β-propeller motifs (composed of six YWTD repeats) at the amino terminal end that alternate with four epidermal growth factor- (EGF-) like repeats (YWTD-EGF domain). These are followed by three low-density-lipoprotein receptor-like ligand-binding domains. LRP5 can act synergistically with FZD4 or other members of the Frizzled family to bind Wnts or Norrin, forming a functional ligand-receptor complex that triggers canonical Wnt/β-catenin or the Norrin/β-catenin signaling pathway and induce the transcription of target genes subsequently [80, 81].Thus far, 58 causative mutations identified in patients with FEVR have been reported forLRP5, of which 46 mutations are missense changes, 6 frame shift mutations resulted by deletions, insertion, and duplication, 2 introduce premature stop codons, and 4 changes affect splicing [28, 29, 31, 46, 56, 59, 69, 69, 82–85] (Table 3 and Figure 4). Mutations located in first, second, and third YWTD-EGF domain accounted for 12% (7 of 58 mutations), 38% (22 of 58 mutations), and 17% (10 of 58 mutations) of all the mutations, respectively. Thus, it can be seen causative mutations have a trend of clustering in the second YWTD-EGF domain since this segment is composed of only about 300 amino acids, accounting for less than 20% of whole LRP5 protein. Five of the included mutations (c.1828G>A, c.731C>G, c.1042C>T, c.1058G>A, and c.1481G>A) were also reported as causative mutation for OPPG [86], which was characterized as blindness and decreased bone density. But FEVR and OPPG were two different diseases because of the distinct pathogenesis of visual loss. OPPG patients often presented with blindness in the neonatal period and the symptoms initiated during early childhood. Inconformity of these results may was due to omission of bone density and definite pathogenesis of visual loss.Table 3
Spectrum ofLRP5 gene mutations among patients with familial exudative vitreoretinopathy.
Studies
No. of patients
No. of mutations
DNA variant
Coding effect
Location of the amino residue
Mutant phenotype
Country of origin
Toomes et al. [68]
32
6
c.518C>T
p.T173M
Firstβ-propeller motif
Abnormal retinal vasculature and retinal fold
USA
c.3502T>C
p.Y1168H
Low-density-lipoprotein receptor-like ligand binding domains
Total retinal detachment and retinoschisis
c.3840delA
p.R1270fsX1438
Premature termination
Not mentioned
c.4081T>G
p.C1361G
Low-density-lipoprotein receptor-like ligand binding domains
Classic features of FEVR
c.4119_4120insC
p.K1374fsX1549
Premature termination
Not mentioned
c.4488 + 2T>G
Splice-donor mutation
Premature termination
Undetermined
Qin et al. [56]
56
9
c.433C>T
p.L145F
Firstβ-propeller motif
Bilateral retrolental fibroplasias and total retinal detachment
Japan
c.803_812del
p.G268fsX272
Premature termination
Bilateral dragged macula
c.1330C>T
p.R444C
Secondβ-propeller motif
Severe falciform retinal fold
c.1564G>A
p.A522T
Secondβ-propeller motif
Tractional retinal detachment, severe macular ectopia along with peripheral fibrovascular mass
c.1604C>T
p.T535M
Secondβ-propeller motif
Bilateral retinal folds followed by total retinal detachment
c.1828G>A
p.G610R
Second epidermal growth-like factor
Bilateral dragged macula
c.1850T>G
p.F617C
Second epidermal growth-like factor
Bilateral retinal folds followed by total retinal detachment
c.2392A>G
p.T798A
Thirdβ-propeller motif
Bilateral peripheral avascular retinas
c.3361A>G
p.N1121D
Fourthβ-propeller motif
Unilateral falciform retinal fold with bilateral retinal avascularization
Boonstra et al. [46]
83
2
c.1532A>C
p.D511A
Secondβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
Netherlands
c.2413C>T
p.R805W
Thirdβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
Nikopoulos et al. [28]
16
4
c.1321G>A
p.E441K
Secondβ-propeller motif
Not mentioned
Netherlands
c.2978G>A
p.W993X
EGF-like domain following the third “β-propeller” module
Not mentioned
c.3758G>T
p.C1253F
EGF-like domain following the third “β-propeller” module
Not mentioned
c.4489-1G>A
Splice defect
Not applicated
Not mentioned
Yang et al. [69]
49
6
c.891-892delTC
p.R298LfxX2
Premature termination
Retrolenticular fibrotic mass, retinal detachment, microcornea, flat anterior chamber
China
c.2484C>G
p.I828M
Thirdβ-propeller motif
Retrolenticular fibrotic mass, stretched ciliary process
c.2626G>A
p.G876S
Third epidermal growth like factor
Retrolenticular fibrotic mass, stretched ciliary process
c.3361A>G
p.N1121D
Fourthβ-propeller motif
Temporal dragging of optic disc, retrolenticular fibrotic mass
c.4025G>A
p.R1342Q
Low-density-lipoprotein receptor-like ligand binding domains
Microcornea, retrolenticular fibrotic mass, avascular zone
c.4087G>A
p.D1363N
Low-density-lipoprotein receptor-like ligand binding domains
Increased branching of peripheral vessels, retrolenticular fibrotic mass
Fei et al. [82]
2
2
c.1264G>A
p.A422T
Secondβ-propeller motif
Not mentioned
China
c.1619T>C
p.L540P
Second epidermal growth like factor
Not mentioned
Seo et al. [59]
51
4
c.731C>G
p.T244R
Firstβ-propeller motif
3A/2B stage FEVR
Korea
c.1330C>T
p.R444C
Secondβ-propeller motif
2A stage FEVR
c.1833dupG
p.C612VfsX25
Premature termination
1B/4A stage FEVR
c.4098C>G
p.D1366E
Low-density-lipoprotein receptor-like ligand binding domains
3B stage FEVR
Zhang et al. [85]
4
4
c.C1042T
p.R348W
First epidermal growth-like factor
Not mentioned
China
c.G1141A
p.D381N
Secondβ-propeller motif
Not mentioned
c.C1870T
p.R624W
Second epidermal growth-like factor
Not mentioned
c.A4550G
p.Y1517C
Cytoplasmic tail
Not mentioned
Tang et al. [31]
100
10
c.1058G>A
p.R353Q
First epidermal growth-like factor
Ilateral retrolenticular fibrotic mass and total retinal detachment
China
c.1183C>T
p.R395W
Secondβ-propeller motif
Falciform retinal fold
c.1318A>T
p.I440F
Secondβ-propeller motif
Retinal fold
c.1582G>A
p.E528K
Secondβ-propeller motif
Peripheral vascular deficiencies
c.1942G>A
p.V648I
Second epidermal growth-like factor
Rhegmatogenous retinal detachment
c.2738G>T
p.C913F
Third epidermal growth-like factor
Retinal fold and macular dragging
c.4087G>C
p.D1363H
Low-density-lipoprotein receptor-like ligand binding domains
Falciform retinal fold
c.4733C>T
p.T1578M
Cytoplasmic tail
Retinal fold
c.92-2A>C
Splice site mutation
Premature termination
Ilateral retrolenticular fibrotic mass and total retinal detachment
c.4488 + 2T>G
Splice site mutation
Premature termination
Retinal folds
Rao et al. [29]
31
5
c.4205G>A
p.G1402D
Transmembrane domain
Falciform fold
China
c.2237G>C
p.R746P
Thirdβ-propeller motif
Peripheral avascular zone
c.2618A>T
p.K873M
Thirdβ-propeller motif
Peripheral avascular zone
c.1384C>T
p.R462X
Secondβ-propeller motif
Complete retinal detachment
c.2817_2827+1del12bp
p.N940fs
Premature termination
Complete retinal detachment
Liu et al. [83]
10
5
c.542T>G
p.M181R
Firstβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
China
c.1197G>T
p.R399S
Secondβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
c.1481G>A
p.R494Q
Secondβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
c.1507G>A
p.G503R
Secondβ-propeller motif
Diagnosed with FEVR, symptoms not mentioned
c.2626G>A
p.G876S
Third epidermal growth-like factor
Diagnosed with FEVR, symptoms not mentioned
Pefkianaki et al. [84]
1
1
c.2234C>T
p.A745V
Thirdβ-propeller motif
Extensive exudative retinopathy and shallow retinal detachment
USAFigure 3
Schematic diagram of the Frizzled-4 protein shows the locations of the mutations. A whole gene deletion and a deletion/insertion (c.40 del/inser) with unknown protein change are not shown. Superscript number means the reported times of the same or different mutations at a certain site. The color of the mutations which were reported more than one time was recolored as orange. The opacity varied with the reported frequency of the mutations.In the crystal of the first two YWTD-EGF structure of LRP6, each of the two EGF domains packs tightly against the bottom surface of the preceding YWTDβ-propellers [87]. Extensive interface interactions was observed between the first β-propellers and second β-propellers, and the first EGF domain also interacts with the second β-propellers, which was critical to maintain the stability and orientation of LRP6’s first two YWTD-EGF domains.Early studies revealed that the interaction of LRP6 with Wnt-Fzd4 was mediated by the first two propeller domains [88], while other researchers pointed out that a single LRP6 might engage two different Wnt proteins simultaneously. LRP5/6 binds to different Wnts via different regions or multiple domains together [89]. The four β-propeller domains in LRP5/6 share a relatively low identity among them, indicating the functional differences among these YWTD propellers. Ke et al. demonstrated that Norrin interacted with β-propeller domain 1 (BP1) and β-propeller domain 2 (BP2) but not BP3-4 of LRP6. However, the binding sites of Norrin with LRP5 remain unclear. From these two perspectives, the mutations accumulated in the second YWTD-EGF domain may destroy the stable structure of first two β-propellers or interrupted their interaction with Norrin or Fzd4.


## 3.4.TSPAN12 Gene, Protein, and Spectrum

TheTSPAN12 gene is located on chromosome 7q31 and encodes for a 305 amino acid transmembrane protein. TSPAN12 is a member of the tetraspanin family that shares certain specific structural features that distinguishes them from other proteins that pass the membrane four times. Both the N and C terminals of TSPAN12 were inside the cell membrane, and it has an unusually long C-terminal intracellular tail of approximately 60 amino acids. It contains four transmembrane domains connected by two extracellular loops (ECL-1 and ECL-2) and an intracellular loop. The ECL-1 is smaller compared to the ECL-2.TSPAN12 was discovered to associate selectively with Norrin/β-catenin signaling but not with Wnt/β-catenin signaling. It acted as the fourth important component of Norrin/FZD4/LRP5 complex. Signaling reduction could be rescued by TSPAN12 overexpression although direct binding with Norrin and FZD4 was not detected. However, another study reported that TSPAN12 interacted with Norrin and FZD4 via its extracellular loops and enhanced the FZD4 ligand selectivity for NDP [90]. Thus, TSPAN12 was postulated to elicit physiological levels of signaling that was required for normal retinal angiogenesis by promoting FZD4 multimerization cooperated with Norrin and facilitating selective ligand recognition [11].We summarized 40 currently known mutations in TSPAN12 identified in patients affected with FEVR and discussed their coding consequences [6, 24, 29, 31, 54, 58, 59, 68, 83, 91–96] (Table 4 and Figure 5). All types of mutations were identified, including 22 missense mutation, 4 nonsense mutations, 9 splice-site mutations, 3 deletions, and 2 insertions. Mutations at residues T49, L140, C189, and L233 were reported more than one time. It was reported that L233P strongly impaired the TSPAN12 activity, while T49M mildly impaired the activity. Unfortunately, the authors did not investigate the signaling defect strength of L140X and C189Y/R. In all of the mutations, 38% (15 in 40 mutations) of them were located in the ECL-2 domain. These mutations were highly consistent with the biochemical results. TSPAN12 is anchored to the Norrin receptor complex via an interaction of the LEL with FZD4. The ECL-2 domain of TSPAN12 is essential for enhancing Norrin-induced FZD4 signaling. TSPAN12 can also alleviate the defects of FZD4 M105V, a mutation that destabilizes the NDP/FZD4 interaction [90].Table 4
Spectrum ofTSPAN12 gene mutations among patients with familial exudative vitreoretinopathy.
Studies
No. of patients
No. of mutations
DNA variant
Coding effect
Location of the amino residue
Mutant phenotypes
Country of origin
Savarese et al. [94]
1
1
c.668T>C
p.L223P
Transmembrane domain
No sign of neovascularization
Pakistan
Poulter et al. [93]
58
5
c.67-1G>C
p.L23GfsX66
Transmembrane domain, premature termination
Bilateral retinal folds
Mexican and Pakistan
c.146C>T
p.T49M
First extracellular loop
Bilateral congenital cataract, large retinal fold
c.285 + 1g>a
p.R50DfsX12
Premature termination
Bilateral congenital cataract, large retinal fold
c.413A>G
p.Y138C
Second extracellular loop
Peripheral retina avascularity
c.668T>C
p.L223P
Transmembrane domain
Bilateral retinal folds, funnel retinal detachments
Poulter et al. [6]
70
7
c.68T>G
p.L23X
Transmembrane domain
Bilateral retinal folds and unilateral, persistent hyperplastic primary vitreous
USA, UK, Britain, Japan, Australia
c.149 + 3a>g
Splice-site mutation
Premature termination
Unilateral retinal fold
c.218_219insGCTGTTT
p.F73LfsX119
Premature termination
Macula ectopia, with a large retinal fold
c.302T>A
p.L101H
Transmembrane domain
Lassic signs of FEVR
c.361-5_361-1delaccag
Splice-site mutation
Premature termination
Bilateral temporal retinal avascularity
c.419T>A
p.L140X
Second extracellular loop
Bilateral retinal folds
c.629T>G
p.M210R
Bilateral macular traction
Bilateral macular traction
Nikopoulos et al. [68]
43
2
c.709G>C
p.A237P
Transmembrane domain
Avascular peripheral retina
Netherlands
c.562G>C
p.G188R
Second extracellular loop
Avascular peripheral retina
Yang et al. [96]
49
3
c.146C>T
p.T49M
First extracellular loop, conserved residue
Falciform retinal folds
China
c.313T>C
p.C105R
Transmembrane domain, conserved residue
Midperipheral retina, an avascular zone on the peripheral retina
c.601delC
p.L201FfsX14
Conserved residue
Inferotemporal dragging of the optic disc and macula
Gal et al. [91]
64
1
c.542G>T
p.C181F
Second extracellular loop, form disulfide bonds
Bilateral visual impairment, various ocular abnormalities
Israel
Xu et al. [95]
85
3
c.177delC
p.Y59fsX67
Premature termination
Falciform retinal folds
China
c.C254T
p.T85M
Intracellular loop
Pigment deposit, dragged disc
c.566G>A
p.C189Y
Second extracellular loop, form disulfide bonds
Bilateral retinal folds
Kondo et al. [92]
90
2
c.419T>A
p.L140X
Second extracellular loop
Abnormal retinal vessels with vitreous degeneration
Japan
c.734T>C
p.L245P
C-terminal cytoplasmic tail
Retinal fold resulting
Seo et al. [59]
51
1
c.56T>G
p.L19R
Transmembrane domain
3A stage FEVR
Korea
Ganeswara Rao Musada et al. [2016]
110
3
c.125T>C
p.V42A
First extracellular loop
Diagnosed with FEVR, symptoms not mentioned
India
c.334G>A
p.V112I
Second extracellular loop
Diagnosed with FEVR, symptoms not mentioned
c.479G>A
p.C160Y
Second extracellular loop
Diagnosed with FEVR, symptoms not mentioned
Tang et al. [31]
100
8
c.2T>C
p.M1T
N-terminal domain
Not mentioned
China
c.464G>C
p.R155T
Second extracellular loop
Not mentioned
c.438-439insT
p.T147YfsX12
Premature termination
Total retinal detachment and massive vitreous proliferation
c.655delC
p.Q219NfsX5
Premature termination
Total retinal detachment
c.916-918 + 3delTAAAAA
p.∗306Eext∗35
Elongated protein
Peripheral avascular retina
c.150-1G>A
Splice acceptor mutations
Not applicated
Not mentioned
c.285 + 1G>A
Splice acceptor mutations
Not applicated
Not mentioned
c.469-1G>A
Splice acceptor mutations
Not applicated
Not mentioned
Iarossi et al. [24]
8
1
c.67-2A>G
Defective splicing
Not applicated
Falciform retinal fold
Italia
Rao et al. [29]
31
1
c.345T>G
p.Y115X
Second extracellular loop
Falciform folds, complete retinal detachment
China
Liu et al. [83]
10
1
c.566G>A
p.C189Y
Second extracellular loop
China
Schatz and Khan [58]
3
1
c.565T>C
p.C189R
Second extracellular loop, affects cystine residues forming
Total retinal detachment
SwedenFigure 4
Schematic representation of LRP5 protein shows the location of the mutations within the protein domains. Four splice site mutations are not shown. Superscript number means the reported times of the same or different mutations at a certain site. The color of the mutations which were reported more than one time was recolored as orange. The opacity varied with the reported frequency of the mutations.


## 4. Discussion

FEVR causingNDP, FZD4, LRP5, and TSPAN12 mutations was reported from 15 countries including USA, UK, China, Spain, India, Australia, Mexico, Japan, Netherlands, Italy, Canada, Korea, Sweden, Pakistan, and Israel. Top three countries with the largest number of reported mutations about NDP, FZD4, LRP5, and TSPAN12 genes were China, Netherlands, and Japan. The number of reported mutations did not completely match the population, since the three most populous countries were China, India, and USA. One of the major reasons contributing to this phenomenon might be the number of research groups was more in China, Netherlands, and Japan than that in other regions. Although most of the mutations were reported by only one study just once, some specific mutations were more common than others. For example, mutations of NDP at c.362G (p.R121) was independently reported by 5 different studies and distributed in Spanish, Indian, Mexican, Chinese, and Italian. FZD4 c. 313A>G (p.M105V) was reported for 8 times by 8 different research groups. Thus, it is significant to investigate the structure and function changes of the coding protein which resulted by the widely reported mutations.Although the mutations scattered widely through the whole genes, they have an inclination to distribute in certain areas. From the point of view of the coding proteins, the mutations concentrated at the N-terminal and C-terminal domains of Norrin. There were 19 mutations located in domains from C39 to C65 and C96 to C126, which covered the twoβ-hairpins (β1-β2 and β5-β6) and loops between and was crucial for binding with FZD4-CRD, namely, 73% of the mutations (19/26) concentrated in the interacting domains with FZD4-CRD. In terms of FZD4, 49% (59 of 121 mutations) of the mutations were positioned in the extracellular domain, which played a significant role in ligand recognition, while 13% (16 of 121 mutations) of the mutations were positioned in the intracellular domain which recruited Dishevelled to activate downstream signaling. The sum of mutations from the two domains accounted for 61% of total reported mutations. The tendency of mutations accumulating in certain domains was more obvious in regard to LRP5 protein. More than a third of reported mutations (38%, 22/58) were found from the second YWTD-type β-propeller domain and EGF domain, which were comprised of approximately 300 amino acids, accounting for less than 20% of whole LRP5 protein. But whether the second YWTD-EGF domains interacted with Norrin and FZD4 directly or not remained unknown. As far as TSPAN12 was concerned, it seemed that the mutations were intensively located in the ECL-2 domain (38%, 15/40). A recent study revealed that the large extracellular loop of TSPAN12 is required for enhancing Norrin-induced FZD4 signaling. In conclusion, the “hotspots” where mutations clustered were highly consistent with the domains participating protein interactions.Overall, mutations inNDP, FZD4, LRP5, and TSPAN12 genes explained up to ∼50% of all FEVR cases worldwide [97]. Besides the four genes we reviewed in this review, ZNF408, KIF11, RCBTB1, CTNNB1, and JAG1 were also reported to be the disease-causing genes of FEVR. The proteins encoded by NDP, FZD4, LRP5, TSPAN12, and CTNNB1 genes participate in the Norrin/β-catenin pathway, the signaling which is critical for retinal angiogenesis by controlling retinal vascular growth and architecture. The connection of proteins coded by ZNF408, KIF11, and RCBTB1 genes with the Norrin/β-catenin pathway was still unclear. A comprehensive spectrum covering other four causative genes (ZNF408, KIF11, RCBTB1, and CTNNB1) and further investigation on the biochemical functions of their coding proteins will undoubtedly facilitate thorough understanding of the pathogenic mechanism of FEVR.Pathogenic mutations inNDP and FZD4 lead to a number of retina-related diseases including FEVR, Norrie disease, persistent hyperplastic primary vitreous, advanced stage of retinopathy of prematurity, and Coats disease. These diseases can be diagnosed according to their unique symptoms which can be distinguished from FEVR [98]. The common characteristic of these NDP and FZD4 related diseases was defects in the vascularization of the retina. Further study on the role of the Norrin/β-catenin pathway in the retinal vascular may promote the understanding of the mechanism of the pathogenic mutations [12]. Furthermore, other sprouting angiogenesis associated components will in some way help provide in-depth insight about these retina-related diseases.Figure 5
Schematic diagram of the TSPAN12 protein shows the location of the 31 known mutations within the protein domains. Nine splice-site mutations besides one mutation (c.916-918 + 3delTAAAAA) resulting in protein extension and one mutation (c.67-1G>C) resulting in frame shift are not shown in this diagram. Superscript number means the reported times of the same or different mutations at a certain site. The color of the mutations which were reported more than one time was recolored as orange. The opacity varied with the reported frequency of the mutations.



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*Source: 5782536-2019-11-16.xml*