Role of NDP- and FZD4-Related Novel Mutations Identified in Patients with FEVR in Norrin/β-Catenin Signaling Pathway

Mutations in NDP and FZD4 have been closely related to a series of retinal diseases including familial exudative vitreoretinopathy (FEVR). Our study was designed to identify novel NDP and FZD4 mutations by whole exome sequencing (WES) in a cohort of patients with a definitive diagnosis of FEVR and explore the underlying molecular mechanism. During 2016, we investigated fifty nonconsanguineous families with affected individuals exhibiting FEVR phenotype and WES identified one recently reported mutation: NDP c.127C>A (p.H43N), and five novel mutations: NDP c.129_131del (p.44del), NDP c.320_353del (p.R107Pfs), NDP c.321delG (p.L108Cfs), NDP c.377G>T (p.C126F), and FZD4 c.314T>G (p.M105R) that cosegragated with the abnormal fundus vascular manifestations in six families. All the mutations were perceived to be pathogenic or likely pathogenic according to the standards and guidelines from the American College of Medical Genetics and Genomics (ACMG) and predicted to be deleterious by a series of bioinformatics analyses. We systematically performed functional analyses on the six mutations utilizing the Topflash reporter assay, where all NDP and FZD4 mutants revealed at least 50% loss of wild-type activity. Immunoprecipitation finally demonstrated that the six mutations could degrade the Norrin-Frizzled-4 pair-binding effect to varying degrees. Finally, our study underscores the correlation between the FEVR phenotype and genotype in NDP and FZD4, extending the mutation spectrum, allowing a reliable assessment of FEVR recurrence and improving genetic counseling. Further, our findings provide essential evidence for the follow-up study of animal models and drug targets by Topflash assays and immunoprecipitation.


Introduction
Familial exudative vitreoretinopathy (FEVR) is a rare inheritable retinal-vascular disorder characterized by retinal avascularity and first described by Criswick and Schepens in 1969 [1]. Typically, the visual problems of FEVR have been featured by secondary complications such as macular and vascular dragging, tractional retinal detachment, retinal folds, ocular neovascularization, and vitreous hemorrhage [2,3].
To our knowledge, numerous investigations have been conducted to date into the pathological mechanism of FEVR, where the canonical Wnt signaling network has been demonstrated to play a pivotal role during retinal organogenesis and angiogenesis [6,10]. The Norrie disease protein (NDP), Norrin, an unconventional Wnt ligand, coordinated with coreceptors: Frizzled-4 (FZD4), LRP5, and TSPAN12, has been elucidated to induce the Norrin/β-catenin canonical signaling pathway, one of the main drivers for retinal angiogenesis in the mammalian eye [6,[11][12][13][14][15]. As Xu et al. described, Norrin with a cysteine-knot motif could bind specifically with the cysteine-rich domain (CRD) of Frizzled-4 and potently activate the classical Wnt signaling pathway [13]. Thus, mutations of NDP and FZD4 in cysteine-knot motif and CRD, respectively, are a good model for elucidating the high affinity of the Norrin-Frizzled-4 pair. Mutations in NDP have been associated with X-linked FEVR (MIM#305390), and mutant FZD4 is linked to an autosomal dominant form of FEVR (MIM#133780). The similar clinical phenotype presented by NDP and FZD4 mutants has also led us to inquire the mechanism of the Norrin-Frizzled-4 pair involved in retinal vasculogenesis.
In our study, we successfully detected one reported mutant and five novel mutants of NDP and FZD4 in fifty Chinese patients with FEVR during 2016 by whole exome sequencing (WES). Then, we showed that FEVR-associated Norrin and Frizzled-4 mutations inhibited the canonical signaling cascade and the possible mechanism: the decrement of Norrin-Frizzled-4 pair affinity, which might implicate the significant phenotypic effect of the patients.  2.3. 3D Modeling of NDP and FZD4 Missense Changes. We adopted University of California, San Francisco (UCSF) chimera software to mutate the residue identified in our study according to three-dimensional structure of Norrin and Frizzled-4 (PDB: 5pqc/5pqe).

Construction of Expression Plasmids.
Wild-type NDP cDNA was purchased from Kelei Biological Technology Co., Ltd. (Shanghai, China) and amplified using forward 5 ′ -CCGctcgagCGGATGAGAAAACATGTACTAGCTG-3 ′ and reverse primer 5 ′ -CGggatccCGGGAATTGCATTCCT CGCAGTGA-3 ′ . Then, we subcloned wild-type NDP cDNA into pEGFP-N1 vector (BD Biosciences) using XhoI and BamHI sites. All NDP mutants were introduced into the wild-type NDP cDNA by primer-mediated PCR mutagenesis. It should be noted that for deletion frameshift mutations (NDP c.321del/320_353del), in order not to affect the subsequent expression of EGFP, the reverse primer was designed as follows: 5 ′ -CGggatccGGAATT GCATTCCTCGCAGTGA-3 ′ .
The recombinant plasmids containing NDP-EGFP fusion constructs were verified by direct DNA sequencing and then amplified and purified for transfection (Qiagen Inc., Valencia, CA). Construction of wild-type and mutant pCMV-3xFlag-FZD4 recombinant plasmids was described as above using HindIII and BamHI sites. The corresponding primers were as follows: forward 5′-aagcttATGGCCTGGCG GGGCGCA-3′ and reverse primer 5′-CggattcCGTACC ACAGTCTCACTG-3′.

Generation of Norrin-Conditioned
Medium. HEK 293T cells were seeded at a density of 0:8 × 10 6 in six 10 cm 2 plates and transfected with mutant or wild-type NDP-EGFP fusion constructs. Sixteen hours later, the medium was removed and replaced with fresh medium. After thirty-six hours, the medium was neutralized by adding sterile 1 M HEPES pH 8.0 and conditioned for additional one day. The medium was concentrated in Amicon Ultra 3K Millipore.

Native Polyacrylamide Gel Electrophoresis. Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent
Biotechnologies, Inc., Eden Prairie, USA) was used to isolated membrane proteins. A NativePAGE Preparation Kit (Sangon Biotech, Shanghai, China) was adopted to make up a standard 8% polyacrylamide gel, which was loaded (without SDS and reducing reagent) with 8 μl of corresponding conditioned medium for each construct tested. Electrophoresis was carried out using as described by Qin et al. [18].
2.9. Immunoprecipitation. We transfected HEK 293T cells from a 60 mm 2 plate with wild-type FZD4-3xFlag constructs or mutants, which were subsequently incubated with 3 ml of the mutant or wild-type Norrin conditional medium on ice for one hour. Subsequent immunoprecipitation was performed using GFP monoclonal antibody (1 : 1000, Beyotime AF0159) and Flag monoclonal antibody (1 : 1000, Beyotime AF5051) as described by Xu et al. [13].  (Figures 3(a) and 3(b)). The expression of βcatenin was also detected after the transfection of the corresponding wild-type and mutant plasmids to HEK 293T cells, where the obvious reduction was also consistent with the FEVR presentations (Figures 3(c) and 3(d)). The attenuation of the activity of the Norrin/β-catenin signaling strongly proved that the six mutations were pathogenic.

Defective Norrin and Frizzled-4 Binding. Norrin and
Frizzled-4 are a high-affinity ligand-receptor pair, which could be used to isolate each other from the plasma membrane [13]. Norrin harbors the cysteine knot domain, which is cysteine-rich and involved in extracellular signaling transduction. Five NDP-related mutations were located in the domain, where most reported pathogenic mutations also resided (Figure 4(a)). Moreover, the specificity of Norrin-Frizzled-4 binding has been assessed systematically, and the CRD of Frizzled-4 is the only domain which Norrin binds to. FZD4 p.M105R was located in the CRD functional domain (Figure 4(a)). To investigate the mechanism of 3 BioMed Research International  I 1  I 2   II 1  II 2 II 3 II 4 II 5 II 6 III 1 III 2 I2 NDP c.321del (heterozygous)

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(e)

Discussion
In our study, we identified one reported mutation: It is critical to unravel the defective mechanism of FEVR-linked mutations identified in FEVR patients with genetic heterogeneity. Therefore, to assess if the missense mutations were important for Norrin/β-catenin signaling, we first utilized bioinformatics tools to predict to be deleterious preliminarily. Finally, by Topflash assays and immunoprecipitation, we strongly proved that the Norrin and Frizzled-4 binding sensitivity of the six mutations was severely compromised and further impaired the Norrin/βcatenin signaling, confirming the link between the mutants in NDP and FZD4 and abnormal retinal presentation. Previous studies have demonstrated the variable impaired Norrin signal induced by different mutations observed regardless of the disease phenotype [13,18,22,23]. Thus, the finding that the NDP and FZD4 mutations match the significant phenotypic effects could sufficiently evidence the presumed haploinsufficiency. It has been elucidated that NDP p.H43R and NDP H43Q are associated with Norrie (MIM#310600), an inherited retinal disorder of highly overlapping ocular manifestations with FEVR [24,25]. Notably, in our study, NDP p.H43N was found to be related to FEVR, which was a novel mutation when we con-ducted the research in 2016, was later reported by Rao et al. in May 2017, and was less characterized in the literature [19]. Thus, the reasons for different diseases caused by different substitutions of the amino acid at the same position also need to be explored. As our knowledge, Norrin function depends on three pairs of cysteines, forming the highly conserved disulfide bonds: Cys 65 -Cys 126 , Cys 69 -Cys 128 , and Cys 39 -Cys 96 , for which any substitution could possibly compromise signaling and Norrin-Frizzled-4 CRD binding, consistent with the significant decrement of signaling and binding activity induced by NDP p.C126F [21]. As for NDP p.R107Pfs and NDP p.L108Cfs, the subsequent amino acids' substitutions might imply the conformational changes of the β sheet of Norrin and dimerization, which still need further investigation to clarify. However, there are some discrepancies between the binding defects of FZD4 M105V to Norrin, located at the same position as FZD4 p.M105R and reported by Xu et al. and Qin et al., where the former describe a normal production at plasma membrane and the latter detect obvious binding defects [13,18].
So far, there have been at least nine genes attributed to the progression of FEVR including NDP [26], FZD4 [27], LRP5 [28], TSPAN12 [29], ZNF408 [30], KIF11 [31], RCBTB1 [32], CTNNB1 [33], and JAG1 [34]. Our previous results also provided insight into the relationship between a novel splicing mutation (c.734+1G>A) in CTNNB1 and a 27-year-old Chinese pregnant woman with a severe intellectual disability and FEVR [35]. Human FEVR displays variable ocular defects and serves as an excellent model to explore Wnt signaling, especially causing mutations in NDP, FZD4, LRP5, and TSPAN12. To date, the Norrin-Frizzled-4 signaling system has been clarified to play a central role in retinal vascular development and blood-brain barrier plasticity [13,36]. Luhmann et al. showed that NDP-knockout mice could occur in the regression of the hyaloid vascular alongside the delay of the superficial retinal vasculature and the failure of the deep retinal vasculature [37]. Similarly, FZD4-knockout mice have been shown defective in vasculature in the retina and inner ear [13]. Xia et al. verify that LRP5-knockout mice develop capillary network incompletely and capillaries lacking lumen structure [38]. Afterwards, the formation of microaneurysms, aberrant fenestration, and delayed hyaloid vessel regression could be observed in the TSPAN12-knockout mice [12]. Subsequently, further research on the role of Norrin/β-catenin in the retinal vascular induced by the mutations detected in our study would be carried out using animal models.    Figure 3: Topflash reporter assay and Western blotting. (a, b) Failure of NDP and FZD4 mutants in the activation of Norrin/β-catenin signaling (72.5% reduction for NDP p.H43N, 70% reduction for NDP p.44del, 71.2% reduction for NDP p.R107Pfs, 70.5% reduction for NDP p.L108Cfs, 73% reduction for NDP p.C126F, and 65% reduction for FZD4 p.M105R). The experiment was carried out in triplicate at the same time and repeated three times. (c, d) Western blotting showed the expression level of β-catenin was decreased, similar to the Topflash reporter assay. Asterisks indicated significant differences from the positive control by pairwise Student's test (P < 0:05).

Conclusions
Pathogenic mutations in NDP and FZD4 could lead to a series of retina-related diseases such as FEVR, Norrie, and retinopathy of prematurity (MIM#133780), which could be distinguished according to their unique characteristics. Furthermore, our present work represents a step in representing points along the FEVR phenotypic spectrum. Taken together, these findings would allow a reliable assessment of FEVR recurrence and improve genetic counseling. We believe that our findings would provide essential evidence for the follow-up study of animal models and drug targets, as well as for understanding the underlying molecular mechanism of FEVR [39].

Data Availability
The data used to support the findings of this study are included within the article.