Molecular Vision 2016; 22:917-932 <>
Received 03 April 2016 | Accepted 28 July 2016 | Published 30 July 2016

Novel mutations in FZD4 and phenotype–genotype correlation in Chinese patients with familial exudative vitreoretinopathy

Miao Tang, Xiaoyan Ding, Jiaqing Li, Andina Hu, Miner Yuan, Yu Yang, Zongyi Zhan, Zijing Li, Lin Lu

The first two authors contributed equally to this study.

State Key Laboratory of Ophthalmology, Retina Division, Zhongshan Ophthalmic Center, Sun Yat-sen University, 510060, Guangzhou, Guangdong, China

Correspondence to: Lin Lu, State Key Laboratory of Ophthalmology, Retina Division, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xianlie S Rd, Guangzhou, China 510060; Phone: 0086-20-87330373; FAX: 0086-20-87333271; email:


Purpose: To identify novel mutations in the frizzled 4 (FZD4) gene in patients with familial exudative vitreoretinopathy (FEVR) in southern China and to delineate the mutation-associated clinical manifestations.

Methods: Clinical data and genomic DNA were collected from 100 probands and their family members. The coding regions of FZD4 were screened for mutations with PCR and Sanger sequencing. Cosegregation analysis was used to verify suspected variants, and clinical symptoms in the probands were analyzed.

Results: Fourteen causative heterozygous mutations in FZD4 in 21 unrelated probands were noted, in 21.0% of the index patients (21/100). Four novel missense mutations (C45R, C45S, C53S, and C90R) and three novel deletion mutations (T326fsX356, G492fsX512, and S345_A351del) with a high possibility of pathogenicity were detected. None of these mutations were found in current online databases and 150 ethnically matched control subjects without retinopathy. The majority of the mutations in FZD4 were identified in probands with retinal folds (15/21) and ectopic macula (5/21). No mutations in FZD4 were found in probands with complete tractional retinal detachment in infancy or with mild asymptomatic FEVR in adulthood.

Conclusions: Seven novel mutations found in this study have broadened the spectrum of mutations in FZD4 known to cause FEVR, providing a deeper understanding of this disease. The results show that mutations in FZD4 are associated with the phenotypes of retinal folds or ectopic macula in FEVR but might not be associated with extreme severe bilateral FEVR during infancy, at least in southern Chinese patients.


Familial exudative vitreoretinopathy (FEVR, OMIM 133780) is a hereditary disorder that can cause severe visual impairment at a young age. The primary pathological process of FEVR involves a deficiency in the peripheral retina vascularization. By itself, this usually causes no clinical symptoms; the visual problems associated with FEVR mainly result from secondary complications due to retinal ischemia [1], such as vitreous hemorrhage, vitreoretinal traction with deformation of the posterior retina, retinal folding, and retinal detachment throughout childhood and adulthood. FEVR exhibits highly variable clinical features among patients from the same family, and there may even be asymmetric manifestation in one individual. Severely affected patients may be registered blind during their infancy, whereas mildly affected individuals may not even be aware of any symptoms, and in such cases, the disease is diagnosed only with fluorescein angiography (FFA).

FEVR is genetically heterogeneous and is inherited in different modes. Autosomal dominant FEVR (adFEVR, OMIM 133780) is most common, and mutations in the frizzled class receptor 4 (FZD4), low-density lipoprotein receptor protein 5 (LRP5; Gene ID 4041, OMIM 603506), and tetraspanin 12 (TSPAN12; Gene ID 23554, OMIM 613138) genes [2-4] are involved with adFEVR. In recent studies, the zinc finger protein 408 (ZNF408; Gene ID 79797, OMIM 616454) and KIF11kinesin family member 11 (KIF11; Gene ID 3832, OMIM 148760) genes were suggested to be associated with adFEVR [5-7]. LRP5 and TSPAN12 have also been associated with autosomal recessive FEVR (arFEVR, OMIM 601813) [8,9]. Mutations in the Norrie disease protein (NDP; Gene ID 4693, OMIM 300658) gene are involved with X-linked recessive FEVR (OMIM 305390) [10]. The proteins encoded by the FZD4, LRP5, TSPAN12, and NDP genes are involved in the highly conserved Wnt/β-catenin and Norrin/β-catenin signaling pathways, which play an important role in eye development and angiogenesis [11-13].

The FZD4 gene is located on chromosome 11q14.2 and encodes a seven-transmembrane protein of 537 amino acids, which acts as a coreceptor for the Wnt and Norrin ligands along with LRP5 [13]. To date, more than 50 mutations in FZD4 have been described in FEVR (see Appendix 1). Pedigrees in previous studies exhibited high interfamilial variability, and genotype–phenotype correlations have not yet been established. To better understand the molecular characterization of mutations in FZD4 in southern Chinese patients with FEVR, mutation screening of FZD4 and analysis of clinical features were performed in 100 unrelated individuals and their families in the present study.


Patients and pedigrees

Written informed consent in accordance with the guidelines of the Declaration of Helsinki was obtained from the participating individuals or their guardians before the clinical data and DNA samples were collected. All procedures were approved by the Internal Review Board of the Zhongshan Ophthalmic Center at Sun Yat-sen University. Diagnosis of FEVR was based on criteria previously described [14]. One hundred patients were recruited and had a final diagnosis of FEVR after comprehensive ophthalmic examinations, including slit-lamp, color photography, and fluorescein angiography. Seventy-six cases were confirmed with fluorescein angiography. Twenty-four patients under 2 years old were not available for FFA; therefore, the diagnosis of FEVR was based on typical fundus changes and a positive family history. Among the 24 children without FFA, 18 were diagnosed based on typical fundus change and family history (confirmed with FFA). Another six patients without a positive family history were diagnosed based on typical fundus change, including retinal folds (three children) or total tractional retinal detachment in infancy (three children). Patients with a gestational age of less than 38 weeks or with a neonatal birthweight of less than 2,000 g were excluded, to eliminate the possible presence of retinopathy of prematurity. Moreover, all possible cases of persistent fetal vasculature, microphthalmia, and unclassified chorioretinal dysplasia were ruled out. Data collected from medical records included gender, gestational age at birth, birthweight, age at diagnosis, family history, clinical presentation, and angiographic findings. Fundus examinations, color photography, and fluorescein angiography were performed for family members. One hundred fifty ethnically matched individuals without history of ocular diseases were recruited as controls. Comprehensive fundus examinations were performed, and no signs of retinal diseases were observed.

Genetic analysis

Tissue samples (blood or buccal swabs) were obtained from each participant for genomic DNA extraction using the standard protocols of the QIAGEN QIAamp DNA Blood Kit (Qiagen, Hilden, Germany) and the Epicenter Master Pure Complete DNA and RNA Purification Kit (Illumina, Madison, WI). For mutational analysis, the coding exons of FZD4 and their adjacent intronic sequences were amplified with PCR (primers listed in Appendix 2). The annealing temperature was 59 °C. A total of 50 ng of DNA was amplified in a reaction volume of 20 μl. The amplification conditions were as follows: 95 °C for 30 s, annealing temperature for 30 s, and 72 °C for 1 min. The resulting amplicons were analyzed with Sanger sequencing. Direct sequencing was performed with an ABI BigDye Terminator Cycle Sequencing Kit using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The reference sequence of FZD4 (GenBank NC_000011.9 for gDNA, NM_012193.2 for mRNA) was used for the identification of variants.

Assessment of missense mutations

Sequence alignments for novel FZD4 missense mutations were performed using the online COBALT program. The following protein sequences were used: Homo sapiens frizzled 4 (GenBank NP_036325), Mus musculus frizzled 4 (GenBank NP_032081), Bos taurus frizzled 4 (GenBank NP_001193198), Rattus norvegicus frizzled 4 (GenBank NP_072145), Gallus gallus frizzled 4 (GenBank NP_989430), Xenopus laevis frizzled 4 (GenBank NP_001083922), Danio rerio frizzled 4 (GenBank NP_001292398), Homo sapiens frizzled 1 (GenBank NP_003496), Homo sapiens frizzled 2 (GenBank NP_001457), Homo sapiens frizzled 3 (GenBank NP_059108), Homo sapiens frizzled 5 (GenBank NP_003459), Homo sapiens frizzled 6 (GenBank NP_001158087), Homo sapiens frizzled 7 (GenBank NP_003498), Homo sapiens frizzled 8 (GenBank NP_114072), Homo sapiens frizzled 9 (GenBank NP_003499), and Homo sapiens frizzled 10 (GenBank NP_009128). Online protein prediction programs were used to estimate the pathogenicity of the missense changes (SIFT, PolyPhen-2, and Align-GVGD).

Cloning sequencing

The target fragments covering the mutation sites were amplified with PCR. The PCR products were gel purified by using the Universal DNA Purification Kit (TIANGEN, Beijing, China) and cloned into pMD19-T (Takara BIO, Shiga, Japan) according to the manufacturer’s instructions. Escherichia coli DH5α was transformed with the resulting plasmids and cultured in Luria-Bertani (LB) agar plates with ampicillin at 37 °C for 12–14 h. A single colony was placed in 5 ml of liquid LB medium with ampicillin and grown overnight in the 37 °C incubator shaker for 12–14 h. The plasmids were isolated from the suspension with the TIANprep Mini Plasmid Kit (TIANGEN), and Sanger sequencing was used to confirm the mutant and wild-type alleles.


All analyses were performed using SPSS software version 16.0. The independent samples Student t test was used for the normally distributed continuous variables, while the Kruskal–Wallis test was used for the non-normally distributed continuous variables. The Pearson chi-square test and Fisher’s exact test were used for the categorical variables. A p value of less than 0.05 was considered statistically significant.


Fourteen heterozygous mutations in FZD4, including seven novel and seven known mutations, were identified in 21 of the 100 individuals with FEVR (Table 1). Among the 21 individuals with mutations in FZD4, 16 had a positive familial history of FEVR, and five were sporadic cases. Of the seven novel mutations, four were missense (C45R, C45S, C53S, and C90R), and three were deletion mutations (T326fsX356, G492fsX512, and S345_A351del; Figure 1).

Chromatograms of these novel mutations are shown in Figure 2. None of these novel mutations were detected in 150 ethnically matched control subjects. These variants were absent in current online databases of human sequence variants, including the 1000 Genomes Project, dbSNP, ExAC databases and the human gene mutation database (HGMD). All four novel missense changes affected evolutionarily conserved residues (Figure 3).

Deletion mutations

Three novel deletion mutations (T326fsX356, G492fsX512, and S345_A351del) were detected in exon 2 of FZD4. Mutant and wild-type alleles of these heterozygous deletion mutations were confirmed with cloning sequencing (Figure 2H–J). The T326fsX356 mutation resulted from a 4 bp heterozygous deletion of bases 975–978 (975_978delCACT). This mutation was identified in two unrelated individuals with a family history of FEVR following cosegregation analysis (Families 3 and 7). This mutation caused a frameshift resulting in 30 incorrect amino acids after codon 325 and a premature termination at codon 356. The proband in Family 3 was a 4-year-old boy with bilateral retinal folds. Peripheral avascularization with a scalloped border was observed in both eyes of his mutation-carrying grandfather, father, and elder sister, who were without any visual symptoms. The proband’s affected paternal aunt, who exhibited a characteristic dragged disc in her right eye and retinal avascularization in both eyes, was not available for mutation analysis (Family 3, Figure 4; Table 2). The proband in Family 7 was a 14-year-old girl with tractional retinal detachment in her left eye. Peripheral retinal avascularization and remarkable retinal exudates were observed in her right eye. Her mother and brother, who had bilateral peripheral avascularization and retinal exudates but who were completely asymptomatic, carried the same mutation (Family 7, Figure 4; Table 2).

The second mutation resulted from a 1 bp heterozygous deletion of base 1475 (1475delG), which caused 20 incorrect amino acids after codon 491 and introduced a premature stop at codon 512 (G492fsX512). This mutation was found in a proband as sporadic FEVR (Family 25, Figure 4; Table 2). He was a 13-year-old boy who displayed retinal folding in the left eye with poor visual acuity (hand movement only) from infancy. His parents and sister underwent a full examination, and no retinopathy or mutation was observed.

A 21 bp heterozygous deletion (1034_1054delCTTATTTCCACATTGCAGCCT) was identified in an 11-year-old boy with asymmetric manifestation (Family 4, Figure 4; Table 2). This mutation caused an in-frame deletion of seven amino acids, from Ser345 to Ala351 (S345_A351del). The proband displayed tractional retinal detachment in his right eye, resulting in vision limited to light perception. His left eye suffered milder visual impairment due to an ectopic macula. His affected father and paternal uncle were clinically healthy, only with bilateral myopia, and FEVR was diagnosed only with fluorescein angiography. Moreover, the proband’s mutation-carrying paternal aunt had undergone a full ophthalmic examination and showed no sign of retinopathy. Intriguingly, Y221H, a reported pathogenic mutation, was identified in the proband’s unaffected mother and sister [15].

Missense mutations

A recurrent mutation, C45Y, previously reported in a patient from Southern California in the United States [16], was detected in a 6-year-old boy with retinal folding in his right eye and an ectopic macula in his left eye (Family 29, Figure 4; Table 2). His affected but asymptomatic father carried the same mutation. Furthermore, two novel mutations altering the same codon (C45R and C45S) were found in two of the southern Chinese pedigrees. The C45R mutation was detected in an 8-year-old boy with bilateral retinal folds. His father and elder brother carried the same mutation. Straightening and increased branching of peripheral vessels with bilateral avascular retina were noted on FFA of the proband’s father, while ectopic macula (right eye) and vessel exudates (left eye) were identified in the proband’s elder brother (Family 26, Figure 4; Table 2). The C45S mutation was detected in a 7-month-old infant girl with retinal folding in her right eye (Figure 5D). Her affected mother and asymptomatic elder sister carried the same mutation. The proband’s mother showed retinal folding and a secondary cataract in her right eye with poor visual acuity since childhood (Family 43, Figure 4; Table 2).

Novel missense mutation C53S was identified in an 8-year-old boy with bilateral dragged discs. Peripheral retinal avascular zone and neovascularization with exudates were observed in both eyes. His mutation-carrying mother and sister had normal vision acuity, and FFA showed typical peripheral non-perfusion area in their eyes (Family 12, Figure 4; Table 2).

Mutation A75T was identified in a 26-year-old female patient. She had blurred vision in the right eye caused by vitreous hemorrhage from retinal neovascularization. Her left eye had normal visual acuity with typical peripheral retinal avascularization and neovascularization (Family 42, Figure 4; Table 2). Although the A75T variant has been reported as SNV (rs764357846) in NCBI, the minor allele frequency of this change was 0.01273% in the East Asian population (data from the ExAC database). Moreover, in the in silico analysis, all three programs predicted the variant to be deleterious. Thus, we considered it a rare disease-causing variant.

Another novel missense mutation, C90R, was detected in a 3-year-old female proband with retinal folding in her right eye (Family 30, Figure 4; Table 2). Her mutation-carrying mother was clinically normal, but peripheral retinal avascularization with vascular tortuosity in both eyes was observed with FFA. Her healthy father did not carry this mutation.

The four novel missense mutations (C45R, C45S, C53S, and C90R) were detected in exon 1 of FZD4. The cysteines at codons 45, 53, and 90 are located in the N-terminal extracellular cysteine-rich domain (CRD), which acts as the binding site for Wnt or Norrin [11,17]. These amino acids are highly conserved across all members of the Frizzled protein family, as well as in the FZD4 protein in other vertebrate species. These mutations were predicted to be pathogenic in the in silico analysis (Table 1).

Other variants

Variants without strong evidence of pathogenicity were considered as being of unknown significance. The Y221H variant was identified in a singleton case, and the proband’s healthy son did not carry the mutation (Appendix 3). This variant was previously reported as a pathogenic mutation in a Chinese singleton case [15]. However, the Y221H variant was identified in two unaffected members from the pedigree that harbored the S345_A351del mutation (Family 4, Figure 4). Y221H was not found in the control group of this study. The protein prediction programs SIFT and Align-GVGD predicted this variant to be deleterious, while PolyPhen-2 predicted it to be benign (Appendix 4). Biological assays are needed to determine the mutation’s pathogenicity. A novel nonsynonymous variant, I437T, was identified in a 43-year-old female patient with a positive familial history of FEVR, but this change did not segregate with the disorder in her family (Appendix 3). Codon 437 is located in the sixth transmembrane domain of FZD4. The I437T variant changed a hydrophobic isoleucine residue into a hydrophilic threonine residue. It was predicted to be deleterious by bioinformatics analyses (Appendix 4) and was not found in the control subjects and current online databases. The patient’s right eye showed an ectopic macula and atrophic retinal holes, and visual acuity was limited to finger counting. Both eyes showed typical deficient vascularization and exudates of the peripheral retina. Fluorescein angiography confirmed that her mother also had FEVR, with bilateral retinal avascularization and exudates; however, after DNA sequencing, her mother did not harbor the same variants (I437T). Thus, we considered it less likely to be pathogenic mutation.

In addition to these mutations, we identified five previously reported disease-causing mutations: G36D [18], M105V [15,19-22], W319X [22,23], D428fsX429 [15,20,21], and T500fsX512 [18]. The G36D missense mutation was identified in an 8-year-old boy with retinal folding in his right eye (Family 33, Figure 4; Table 2). The W319X nonsense mutation was detected in a 15-year-old boy with an ectopic macula in his right eye (Family 27, Figure 4; Table 2). The T500fsX512 deletion mutation was found in a 12-year-old boy with retinal folding in his right eye (Family 28, Figure 4; Figure 5B; Table 2). Two probands harbored the same M105V mutation (Families 36 and 44, Figure 4; Table 2), and six unrelated families carried the same D428fsX429 mutation (Families 14, 31, 37, 38, 39, and 41; Figure 4; Table 2). M105V was previously reported among ethnically diverse probands with variable symptoms and is considered a FEVR-causing mutation with high prevalence [15,19-22].

Ocular findings in family members

Overall, 100 probands and their family members were included in this study. In 64 pedigrees, screening in both parents was performed with fundus examination and FFA. The FEVR phenotype, mild or moderate, was identified in 53 families; however, no FEVR-associated retinopathies were identified in 11 families (data not shown).

Other first- or second-degree relatives were also checked if available. A total of 75 individuals from 53 families were diagnosed with FEVR. Importantly, among the 75 relatives, 69 (69/75) were clinically asymptomatic. An avascular zone and other peripheral retinopathy were observed only with careful ophthalmic examination and confirmed with FFA. These 69 individuals were not aware of the existence of this disease. Only 13 had visual dysfunction (data not shown).

Phenotype–genotype correlations based on Trese’s staging system

The staging system of FEVR was recently updated by Trese’s group [14], based on the widely used staging system Trese addressed in 1998 [24]. In this study, 100 probands were staged according to the Trese system based on the highest stage of disease in either eye. Thus, patients with macula-uninvolving folds were identified as stage 3, those with macula-involving folds were identified as stage 4, and patients with ectopic macula were sub-grouped in stage 1 or 2, depending on the existence of neovascularization. Patients with active vitreous hemorrhage secondary to retinal neovascularization were identified as stage 2 (Table 3). As a result, mutations in FZD4 were seen in six patients with mild FEVR (stages 1–2) and in 15 patients with severe FEVR (stages 4 and 5). No distinct phenotype–genotype correlation was observed.

Phenotype–genotype correlations based on modified classification system

To delineate the possible genotype–phenotype correlation, all probands were categorized into four subgroups according to the new classification system, which was modified from the criteria described by Pendergast et al. [24] and Kashani et al. [14]. In this new classification system, not only the severity of the avascular area and its complications but also the age of onset was considered. Group I included individuals with asymptomatic FEVR confirmed only with FFA with bilateral peripheral retinopathy in adulthood (mild). Group II included patients with active vitreous hemorrhage due to retinal neovascularization or rhegmatogenous retinal detachment secondary to peripheral retinal degeneration in childhood or adulthood (moderate). Group III included patients with retinal folds or an ectopic macula in at least one eye, which typically occurred in infancy (called severe). Group IV included patients with FEVR with severe tractional total retinal detachment in at least one eye in infancy (extremely severe; Table 4).

In this study, with this new classification system, significant statistical differences were observed among the four groups in average age at diagnosis (p<0.001) and gender ratio (p=0.012). The more severe the phenotype, the younger the average age at diagnosis, and a higher prevalence of male probands was observed. Group IV had the youngest average age at diagnosis (2.0±2.3, range from 1 month to 7 years old), mostly in the first year of the life. However, the mean age at diagnosis for the probands in Group I was 44.0±15.1 years old (range from 12 years old to 61 years old); 82.3% of the probands in Group IV were male, while 40% in Group I. Most of the cases in Group I were registered as other diseases (uveitis in 1 case, central retinal venous occlusion in two cases, diabetic retinopathy in one case, age related macular degeneration in one case, macular hole in one case, macular edema in one case, and pathologic myopia in three cases) but were eventually confirmed with FFA (Table 4).

Interestingly, 20 of 21 patients with disease-causing mutations in FZD4 were categorized in Group III FEVR (15 retinal folds and five with an ectopic macula). For the 24 children without FFA (13 cases in Group III, 11 cases in Group IV), causative mutations in FZD4 were identified in three children with severe FEVR (Group III). Only one patient with a mutation in FZD4 was categorized in Group II due to vitreous hemorrhage secondary to retinal neovascularization. No mutations in FZD4 were found in ten patients in Group I or in 17 patients in Group IV (Table 4).


Mutation screening of genes known to cause FEVR would provide invaluable information for diagnosis and genetic counseling, especially for patients without a family history of the disorder or with atypical presentations. To our knowledge, the present study used one of the largest cohorts of patients with FEVR, with 100 families. Fourteen causative heterozygous mutations in FZD4 in 21 unrelated probands were noted, accounting for 21.0% of the index patients (21/100). The percentage of FEVR caused by mutations in FZD4 in this study is slightly higher when compared to data from Western countries, such as 20.0% (8/40) in the United Kingdom [18], 14.1–14.3% (13/92 and 9/63) in the United States [16,25], and 17.6% (12/68) in Canada [21]. However, data from Asian populations revealed a more varied range. For example, a comprehensive molecular screening of FEVR-associated genes in the Korean population indicated mutations in FZD4 accounted for 25.5% (13/51) of FEVR cases [20]. In northern China, Jia et al. reported an even higher rate of 31.3% (15/48) [19]. However, in eastern China, Fei et al. recently reported only 4.9% (3/61) of families with FEVR in Han Chinese had causative mutations in FZD4 [26]. Thus, the current study provides new evidence of the high prevalence of mutations in FZD4 in FEVR in East Asia. One of the reasons for the differences is a possible ethnic difference in FEVR genotypes between Asian and Western populations. Another possible reason is that the diagnostic methods varied among the studies. Among the 100 pedigrees in the current study, diagnosis in the majority of the probands and family members was confirmed with FFA, which is considered the golden standard in the diagnosis of FEVR.

In this study, the novel in-frame deletion mutation S345_A351del was identified in one pedigree (Family 4, Figure 4). In this family, all affected relatives and one healthy family member harbored the same mutation, which suggested nonpenetrance in mutations in FZD4. Nonpenetrance was also observed in previously reported FEVR pedigrees with mutations in FZD4 and other known genes [3,5,23]. Moreover, two novel disease-causing mutations were predicted to result in truncation of the FZD4 mRNA (T326fsX356 and G492fsX512). Both mutations were located in the large exon 2 of FZD4, which encodes 442 amino acids. The proportions of the mRNA truncated were 33.8% for the T326fsX356 variant and 4.8% for the G492fsX512 variant, respectively. The novel missense mutations found in this study—C45R, C45S, C53S, and C90R—are located in the highly conserved cysteine-rich domain of frizzled 4. According to a previous study, Cys45 forms a disulfide bond with Cys106, Cys53 forms a disulfide bond with Cys99, and Cys90 forms a disulfide bond with Cys128 [27]. The substitutional residues damage these disulfide bonds and may affect the function of the cysteine-rich domain, which is critical for ligand binding.

Intriguingly, a recurrent mutation, D428fsX429, was identified in six unrelated probands. All individuals were from separate areas in southern China. There was no evidence of consanguinity. Among the six families, cosegregation analysis revealed that all affected individuals carried the same mutation, while unaffected individuals did not. As D428fsX429 was frequently detected in this study, we checked the occurrence in the control subjects, and none carried the mutation. This mutation was previously reported in a Caucasian fe male singleton case [21], a Chinese female [15,20], and two Korean patients [15,20]. The minor allele frequency of this change was far lower than 0.05% (0.001498%) according to the ExAC database, and it was considered pathogenic in the NCBI ClinVar database. Thus, we consider D428fsX429 a disease-causing mutation in the six families. All probands with the D428fsX429, in previous studies and the present study, showed retinal folds or ectopic macula as the initial symptoms.

In this study, probands showed highly variable clinical features. By using the traditional staging system, six probands with causative mutations in FZD4 were classified with mild FEVR (stages 1–2, two cases in stage 1 and four cases in stage 2) while 15 of 21 probands with causative mutations were classified as severe FEVR (stages 3–5, 13 cases in stage 4 and two cases in stage 5). No obvious phenotype–genotype correlation was observed, which is consistent with previous reports [19,21]. However, with this modified system, in 100 unrelated probands, we found that mutations in FZD4 were not found in Group I FEVR (mild cases, manifested with bilateral asymptomatic retinopathy) or Group IV FEVR (extreme severe cases, manifested with chorioretinal dysplasia in infancy). Interestingly, most mutations in FZD4 (95.2%, 20/21) were identified in the probands in Group III FEVR (severe cases, manifested with retinal folding or ectopic macula); only one mutation in FZD4 was noted in a proband with Group II FEVR (moderate cases, manifested with vitreous hemorrhage or rhegmatogenous retinal detachment; Table 4). These results showed that mutation in FZD4 is most likely one of the genetic etiology of retinal folds or ectopic macula due to FEVR. It is hypothesized that mutations in FZD4 exert only moderate effects on retinal vascularization delay. Patients who carried the mutations were less likely to present with blindness immediately after birth. The underlying molecular mechanisms should be investigated in future studies.

In summary, this study offers insights into the type, distribution, and frequency of FEVR-causing mutations in FZD4. The discovery of seven novel mutations with potential pathogenicity has broadened the spectrum of mutations in FZD4 underlying FEVR. Further discovery of novel causative genes may contribute to our understanding of the molecular mechanisms of retinal angiogenesis, which is crucial for FEVR and other retinal vascular diseases. It will also enable the accurate diagnosis of asymptomatic individuals and better genetic counseling of patients. Our emerging understanding of FZD signaling offers hope that pharmaceutical agents that selectively target this pathway will be developed to treat phenotypic characteristics of these diseases in the future.

Appendix 1. FZD4 mutations associated with familial exudative vitreoretinopathy

Appendix 2. Sequence of oligonucleotide primers used in the this study

Appendix 3. Pedigree.

Appendix 4. FZD4 variants with unknown significance identified in 100 probands with familial exudative vitreoretinopathy


This study was supported by the Fundamental Research Funds of State Key Laboratory of Ophthalmology and National Natural Science Foundation of China (81,341,028), research funds of Sun Yat-sen University (15ykjc22d), Science and Technology Program of Guangdong Province, China (2013B020400003) and Science and Technology Program Guangzhou, China (15,570,001). No authors have any financial/conflicting interests to disclose. Involved in design and conduct of the study (L.L., D.X.); collection (M.T., J.L., Y.Y.), management (A.H., M.Y.), analysis (M.T., Z.Z.), and interpretation of the data (D.X., L.L.); and preparation and review of the manuscript (M.T., X.D., L.L., A.H., M.Y., Y.Y., Z.Z., Z.L.).


  1. Gow J, Oliver GL. Familial exudative vitreoretinopathy. An expanded view. Arch Ophthalmol. 1971; 86:150-5. [PMID: 5571414]
  2. Toomes C, Bottomley HM, Jackson RM, Towns KV, Scott S, Mackey DA, Craig JE, Jiang L, Yang Z, Trembath R, Woodruff G, Gregory-Evans CY, Gregory-Evans K, Parker MJ, Black GC, Downey LM, Zhang K, Inglehearn CF. Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q. Am J Hum Genet. 2004; 74:721-30. [PMID: 15024691]
  3. Nikopoulos K, Gilissen C, Hoischen A, van Nouhuys CE, Boonstra FN, Blokland EA, Arts P, Wieskamp N, Strom TM, Ayuso C, Tilanus MA, Bouwhuis S, Mukhopadhyay A, Scheffer H, Hoefsloot LH, Veltman JA, Cremers FP, Collin RW. Next-generation sequencing of a 40 Mb linkage interval reveals TSPAN12 mutations in patients with familial exudative vitreoretinopathy. Am J Hum Genet. 2010; 86:240-7. [PMID: 20159111]
  4. Robitaille J, MacDonald ML, Kaykas A, Sheldahl LC, Zeisler J, Dube MP, Zhang LH, Singaraja RR, Guernsey DL, Zheng B, Siebert LF, Hoskin-Mott A, Trese MT, Pimstone SN, Shastry BS, Moon RT, Hayden MR, Goldberg YP, Samuels ME. Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy. Nat Genet. 2002; 32:326-30. [PMID: 12172548]
  5. Collin RW, Nikopoulos K, Dona M, Gilissen C, Hoischen A, Boonstra FN, Poulter JA, Kondo H, Berger W, Toomes C, Tahira T, Mohn LR, Blokland EA, Hetterschijt L, Ali M, Groothuismink JM, Duijkers L, Inglehearn CF, Sollfrank L, Strom TM, Uchio E, van Nouhuys CE, Kremer H, Veltman JA, van Wijk E, Cremers FP. ZNF408 is mutated in familial exudative vitreoretinopathy and is crucial for the development of zebrafish retinal vasculature. Proc Natl Acad Sci USA. 2013; 110:9856-61. [PMID: 23716654]
  6. Hu H, Xiao X, Li S, Jia X, Guo X, Zhang Q. KIF11 mutations are a common cause of autosomal dominant familial exudative vitreoretinopathy. Br J Ophthalmol. 2016; [PMID: 26472404]
  7. Robitaille JM, Gillett RM, LeBlanc MA, Gaston D, Nightingale M, Mackley MP, Parkash S, Hathaway J, Thomas A, Ells A, Traboulsi EI, Heon E, Roy M, Shalev S, Fernandez CV, MacGillivray C, Wallace K, Fahiminiya S, Majewski J, McMaster CR, Bedard K. Phenotypic overlap between familial exudative vitreoretinopathy and microcephaly, lymphedema, and chorioretinal dysplasia caused by KIF11 mutations. JAMA Ophthalmol. 2014; 132:1393-9. [PMID: 25124931]
  8. Poulter JA, Davidson AE, Ali M, Gilmour DF, Parry DA, Mintz-Hittner HA, Carr IM, Bottomley HM, Long VW, Downey LM, Sergouniotis PI, Wright GA, MacLaren RE, Moore AT, Webster AR, Inglehearn CF, Toomes C. Recessive mutations in TSPAN12 cause retinal dysplasia and severe familial exudative vitreoretinopathy (FEVR). Invest Ophthalmol Vis Sci. 2012; 53:2873-9. [PMID: 22427576]
  9. Jiao X, Ventruto V, Trese MT, Shastry BS, Hejtmancik JF. Autosomal recessive familial exudative vitreoretinopathy is associated with mutations in LRP5. Am J Hum Genet. 2004; 75:878-84. [PMID: 15346351]
  10. Chen ZY, Battinelli EM, Fielder A, Bundey S, Sims K, Breakefield XO, Craig IW. A mutation in the Norrie disease gene (NDP) associated with X-linked familial exudative vitreoretinopathy. Nat Genet. 1993; 5:180-3. [PMID: 8252044]
  11. Xu Q, Wang Y, Dabdoub A, Smallwood PM, Williams J, Woods C, Kelley MW, Jiang L, Tasman W, Zhang K, Nathans J. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell. 2004; 116:883-95. [PMID: 15035989]
  12. Junge HJ, Yang S, Burton JB, Paes K, Shu X, French DM, Costa M, Rice DS, Ye W. TSPAN12 regulates retinal vascular development by promoting Norrin- but not Wnt-induced FZD4/beta-catenin signaling. Cell. 2009; 139:299-311. [PMID: 19837033]
  13. Warden SM, Andreoli CM, Mukai S. The Wnt signaling pathway in familial exudative vitreoretinopathy and Norrie disease. Semin Ophthalmol. 2007; 22:211-7. [PMID: 18097984]
  14. Kashani AH, Brown KT, Chang E, Drenser KA, Capone A, Trese MT. Diversity of retinal vascular anomalies in patients with familial exudative vitreoretinopathy. Ophthalmology. 2014; 121:2220-7. [PMID: 25005911]
  15. Yang H, Li S, Xiao X, Wang P, Guo X, Zhang Q. Identification of FZD4 and LRP5 mutations in 11 of 49 families with familial exudative vitreoretinopathy. Mol Vis. 2012; 18:2438-46. [PMID: 23077402]
  16. Salvo J, Lyubasyuk V, Xu M, Wang H, Wang F, Nguyen D, Wang K, Luo H, Wen C, Shi C, Lin D, Zhang K, Chen R. Next-generation sequencing and novel variant determination in a cohort of 92 familial exudative vitreoretinopathy patients. Invest Ophthalmol Vis Sci. 2015; 56:1937-46. [PMID: 25711638]
  17. Dann CE, Hsieh JC, Rattner A, Sharma D, Nathans J, Leahy DJ. Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains. Nature. 2001; 412:86-90. [PMID: 11452312]
  18. Toomes C, Bottomley HM, Scott S, Mackey DA, Craig JE, Appukuttan B, Stout JT, Flaxel CJ, Zhang K, Black GC, Fryer A, Downey LM, Inglehearn CF. Spectrum and frequency of FZD4 mutations in familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci. 2004; 45:2083-90. [PMID: 15223780]
  19. Jia LY, Li XX, Yu WZ, Zeng WT, Liang C. Novel frizzled-4 gene mutations in chinese patients with familial exudative vitreoretinopathy. Arch Ophthalmol. 2010; 128:1341-9. [PMID: 20938005]
  20. Seo SH, Yu YS, Park SW, Kim JH, Kim HK, Cho SI, Park H, Lee SJ, Seong MW, Park SS, Kim JY. Molecular Characterization of FZD4, LRP5, and TSPAN12 in Familial Exudative Vitreoretinopathy. Invest Ophthalmol Vis Sci. 2015; 56:5143-51. [PMID: 26244290]
  21. Robitaille JM, Zheng B, Wallace K, Beis MJ, Tatlidil C, Yang J, Sheidow TG, Siebert L, Levin AV, Lam WC, Arthur BW, Lyons CJ, Jaakkola E, Tsilou E, Williams CA, Weaver RG, , Jr Shields CL, Guernsey DL. The role of Frizzled-4 mutations in familial exudative vitreoretinopathy and Coats disease. Br J Ophthalmol. 2011; 95:574-9. [PMID: 21097938]
  22. Kondo H, Hayashi H, Oshima K, Tahira T, Hayashi K. Frizzled 4 gene (FZD4) mutations in patients with familial exudative vitreoretinopathy with variable expressivity. Br J Ophthalmol. 2003; 87:1291-5. [PMID: 14507768]
  23. Boonstra FN, van Nouhuys CE, Schuil J, de Wijs IJ, van der Donk KP, Nikopoulos K, Mukhopadhyay A, Scheffer H, Tilanus MA, Cremers FP, Hoefsloot LH. Clinical and molecular evaluation of probands and family members with familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci. 2009; 50:4379-85. [PMID: 19324841]
  24. Pendergast SD, Trese MT. Familial exudative vitreoretinopathy. Results of surgical management. Ophthalmology. 1998; 105:1015-23. [PMID: 9627651]
  25. Drenser KA, Dailey W, Vinekar A, Dalal K, Capone A, , Jr Trese MT. Clinical presentation and genetic correlation of patients with mutations affecting the FZD4 gene. Arch Ophthalmol. 2009; 127:1649-54. [PMID: 20008721]
  26. Fei P, Zhu X, Jiang Z, Ma S, Li J, Zhang Q, Zhou Y, Xu Y, Tai Z, Zhang L, Huang L, Yang Z, Zhao P, Zhu X. Identification and functional analysis of novel FZD4 mutations in Han Chinese with familial exudative vitreoretinopathy. Sci Rep.. 2015; 5:16120 [PMID: 26530129]
  27. Smallwood PM, Williams J, Xu Q, Leahy DJ, Nathans J. Mutational analysis of Norrin-Frizzled4 recognition. J Biol Chem. 2007; 282:4057-68. [PMID: 17158104]