Mutations in TSPAN12 gene causing familial exudative vitreoretinopathy.

BACKGROUND: To report newly found TSPAN12 mutations with a unique form of familial exudative vitreoretinopathy (FEVR) and find out the possible mechanism of a repeated novel intronic variant in TSPAN12 led to FEVR. RESULTS: Nine TSPAN12 mutations with a unique form of FEVR were detected by panel-based NGS. MINI-Gene assay showed two splicing modes of mRNA that process two different bands A and B, and mutant-type shows replacement with the splicing mode of Exon11 hopping. Construction of wild-type and mutant TSPAN12 vector showed the appearance of premature termination codons (PTC). In vitro expression detection showed significant down-regulated expression level of TSPAN12 mRNAs and proteins in cells transfected with mutant vectors compared with in wild-type group. On the contrary, translation inhibitor CHX and small interfering RNA of UPF1 (si-UPF1) significantly increased mRNA or protein expression of TSPAN12 in cells transfected with the mutant vectors. CONCLUSIONS: Nine mutations in TSPAN12 gene are reported in 9 FEVR patients with a unique series of ocular abnormalities. The three novel TSPAN12 mutations trigger NMD would cause the decrease of TSPAN12 proteins that participate in biosynthesis and assembly of microfibers, which might lead to FEVR, and suggest that intronic sequence analysis might be a vital tool for genetic counseling and prenatal diagnoses.

Deciphering a crucial dimeric interface governing Norrin dimerization and the pathogenesis of familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disease that could cause blindness. It has been established that Norrin forms dimers to activate ß-catenin signaling, yet the core interface for Norrin dimerization and the precise mechanism by which Norrin dimerization contributes to the pathogenesis of FEVR remain elusive. Here, we report an NDP variant, c.265T>C (p.Phe89Leu), that interrupted ß-catenin signaling by disrupting Norrin dimerization. Structural and functional analysis revealed that the Phe-89 of one Norrin monomer interacts with Pro-98, Ser-101, Arg-121, and Ile-123 of another, forming two core symmetrical dimerization interfaces that are pivotal for the formation of a "hand-by-arm" dimer. Intriguingly, we proved that one of the two core symmetrical interfaces is sufficient for dimerization and activation of ß-catenin signaling, with a substantial contribution from the Phe-89/Pro-98 interaction. Further functional analysis revealed that the disruption of both dimeric interfaces eliminates potential binding sites for LRP5, which could be partially restored by over-expression of TSPAN12. In conclusion, our findings unveil a core dimerization interface that regulates Norrin/LRP5 interaction, highlighting the essential role of Norrin dimerization on ß-catenin signaling and providing potential therapeutic avenues for the treatment of FEVR.

Characterization of a novel heterozygous frameshift variant in NDP gene that causes familial exudative vitreoretinopathy in female patients.

Familial exudative vitreoretinopathy (FEVR) is a severe inherited disease characterized by defective retinal vascular development. With genetic and clinical heterogeneity, FEVR can be inherited in different patterns and characterized by phenotypes ranging from moderate visual defects to complete vision loss. This study was conducted to unravel the genetic and functional etiology of a 4-month-old female FEVR patient. Targeted gene panel and Sanger sequencing were utilized for genetic evaluation. Luciferase assays, western blot, quantitive real-time PCR, and immunocytochemistry were performed to verify the functional defects in the identified candidate variant. Here, we report a 4-month-old girl with bilateral retinal folds and peripheral avascularization, and identified a novel frameshift heterozygous variant c.37dup (p.Leu13ProfsTer13) in NDP. In vitro experiments revealed that the Leu13ProfsTer13 variant led to a prominent decrease in protein levels instead of mRNA levels, resulting in compromised Norrin/ß-catenin signaling activity. Human androgen receptor assay further revealed that a slight skewing of X chromosome inactivation could partially cause FEVR. Thus, the pathogenic mechanism by which heterozygous frameshift or nonsense variants in female carriers cause FEVR might largely result from a loss-of-function variant in one X chromosome allele and a slightly skewed X-inactivation. Further recruitment of more FEVR-affected females carrying NDP variants and genotype-phenotype correlation analysis can ultimately offer valuable information for the prognosis prediction of FEVR.

Severe isolated exudative vitreoretinopathy caused by biallelic FZD4 variants.

Familial exudative vitreoretinopathy (FEVR) is linked to disruption of the Norrin/Frizzled-4 signaling pathway, which plays an important role in retinal angiogenesis. Severe or complete knock-down of proteins in the pathway also causes syndromic forms of the condition. Both heterozygous and biallelic pathogenic variants in the FZD4 gene, encoding the pathway's key protein frizzled-4, are known to cause FEVR. However, it is not clear what effect different FZD4 variants have, and whether extraocular features should be expected in those with biallelic pathogenic FZD4 variants. Biallelic FZD4 variants were found in a young boy with isolated, severe FEVR. His parents were heterozygous for one variant each and reported normal vision. In-vitro studies of the two variants, demonstrated that it was the combination of the two which led to severe inhibition of the Norrin/Frizzled-4 pathway. Our observations demonstrate that biallelic FZD4-variants are associated with a severe form of FEVR, which does not necessarily include extraocular features. In addition, variants causing severe FEVR in combination, may have no or minimal effect in heterozygous parents as non-penetrance is also a major feature in dominant FZD4-FEVR disease. This underscores the importance of genetic testing of individuals and families with FEVR.

Novel truncating variants in CTNNB1 cause familial exudative vitreoretinopathy.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is an inheritable blinding disorder with clinical and genetic heterogeneity. Heterozygous variants in the CTNNB1 gene have been reported to cause FEVR. However, the pathogenic basis of CTNNB1-associated FEVR has not been fully explored. METHODS: Whole-exome sequencing was performed on the genomic DNA of probands. Dual-luciferase reporter assay, western blotting and co-immunoprecipitation were used to characterise the impacts of variants. Quantitative real-time PCR, EdU (5-ethynyl-2'-deoxyuridine) incorporation assay and immunocytochemistry were performed on the primary human retinal microvascular endothelial cells (HRECs) to investigate the effect of CTNNB1 depletion on the downstream genes involved in Norrin/ß-catenin signalling, cell proliferation and junctional integrity, respectively. Transendothelial electrical resistance assay was applied to measure endothelial permeability. Heterozygous endothelial-specific Ctnnb1-knockout mouse mice were generated to verify FEVR-like phenotypes in the retina. RESULTS: We identified two novel heterozygous variants (p.Leu103Ter and p.Val199LeufsTer11) and one previously reported heterozygous variant (p.His369ThrfsTer2) in the CTNNB1 gene. These variants caused truncation and degradation of ß-catenin that reduced Norrin/ß-catenin signalling activity. Additionally, knockdown (KD) of CTNNB1 in HRECs led to diminished mRNA levels of Norrin/ß-catenin targeted genes, reduced cell proliferation and compromised junctional integrity. The Cre-mediated heterozygous deletion of Ctnnb1 in mouse endothelial cells (ECs) resulted in FEVR-like phenotypes. Moreover, LiCl treatment partially rescued the defects in CTNNB1-KD HRECs and EC-specific Ctnnb1 heterozygous knockout mice. CONCLUSION: Our findings reinforced the current pathogenesis of Norrin/ß-catenin for FEVR and expanded the causative variant spectrum of CTNNB1 for the prenatal diagnosis and genetic counselling of FEVR.

A comprehensive functional analysis on the pathogenesis of novel TSPAN12 and NDP variants in familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder; however, the known FEVR-associated variants account for approximately only 50% cases. Currently, the pathogenesis of most reported variants is not well studied, we aim to identify novel variants from FEVR-associated genes and perform a comprehensive functional analysis to uncover the pathogenesis of variants that cause FEVR. Using targeted gene panel and Sanger sequencing, we identified six novel and three known variants in TSPAN12 and NDP. These variants were demonstrated to cause significant inhibition of Norrin/ß-catenin pathway by dual-luciferase reporter assay and western blot analysis. Structural analysis and co-immunoprecipitation revealed compromised interactions between missense variants and binding partners in the Norrin/ß-catenin pathway. Immunofluorescence and subcellular protein extraction were performed to reveal the abnormal subcellular trafficking. Additionally, over-expression of TSPAN12 successfully enhanced the Norrin/ß-catenin signaling activity by strengthening the binding affinity of mutant Norrin with FZD4 or LRP5. Together, these observations expanded the spectrum of FEVR-associated variants for the genetic counseling and prenatal diagnosis of FEVR, as well providing a potential therapeutic strategy for the treatment of FEVR.

Five novel dysfunctional variants in the TSPAN12 gene in familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is an inheritable vitreoretinal disease characterized by incomplete retinal vascular development, which often leads to multiple retinal complications and causes severe vision loss in children. We reported the TSPAN12 variants' frequency in a cohort of FEVR and five novel TSPAN12 variants and related clinical features in six Chinese families. Seven hundred thirty-four families' genetic in-house data were reviewed. Whole-exome sequencing (WES) was performed in all probands; Sanger sequencing was conducted in the family members. Five novel variants from six families were noted, and clinical data were collected. Luciferase assays were applied to test the activity of the Norrin/ß-catenin signal caused by the mutant TSPAN12 genes. The frequency of TSPAN12 variants in FEVR is 8.79% (50/569). Five novel variants in TSPAN12 were identified in six families, including two missense variants, c.476G > A(p.Cys159Tyr) and c.81T > G(p.Ser27Arg), two frameshift variants, c.628_629insA(p.Met210Asnfs*42) and c.251delG(p.Gly84Glufs*3) and one nonsense, c.352G > T(p.Glu118*). Low vision, high myopia, nystagmus, and leukocoria are the common symptom at the first presentation. All variants were also predicted as pathogenic in silico. Moreover, the luciferase assay demonstrated that all variants caused severely compromised Norrin/ß-catenin signaling activity. In conclusion, the frequency of TSPAN12 variants in FEVR was 8.79% in our cohort. Five novel variants of TSPAN12 were identified. Moreover, we demonstrated the dysfunction of mutant variants via the downregulation of Norrin/ß-catenin signaling. These findings expanded the genetic and clinical spectrum of FEVR with TSPAN12 variants.

[The role of endothelin-1 in the pathogenesis of familial exudative vitreoretinopathy]. / Rol' endotelina-1 v patogeneze semeinoi ekssudativnoi vitreoretinopatii.

Familial exudative vitreoretinopathy (FEVR) is a rare hereditary disease characterized by pathological retinal vascularization with a progressive and variable course. The mechanisms of disease progression remain unclear. One substance that plays an important role in the pathogenesis of retinal vascular diseases is endothelin (ET). It was found that tissue hypoxia enhances the expression of the gene encoding ET-1, and ET-1 can be locally produced in the eye. PURPOSE: The study evaluates the possible role of endothelin-1 in the pathogenesis of FEVR. MATERIAL AND METHODS: The study included 85 patients with FEVR aged from 1 months to 17 years who were examined in Helmholtz National Medical Research Center of Eye Diseases. The concentration of ET-1 was evaluated in 19 patients with FEVR in the blood serum (n=17), lacrimal fluid (n=18) and 16 patients from the control group. RESULTS: The median of ET-1 in the lacrimal fluid in patients with FEVR was 13.74 pg/mL, respectively, which exceeded the same indicator of the control group 4.66 pg/mL by 2.5 times (p<0.001). The median of ET-1 in the blood serum exceeded the control group by 2.4 times (21.61 pg/mL and 9.21 pg/mL, respectively, p<0.001). CONCLUSIONS: An increase in the concentration of ET-1 in the lacrimal fluid and blood serum of patients with FEVR in comparison with the control group indicates its involvement in the pathogenesis of the disease.

A mouse model for kinesin family member 11 (Kif11)-associated familial exudative vitreoretinopathy.

During mitosis, Kif11, a kinesin motor protein, promotes bipolar spindle formation and chromosome movement, and during interphase, Kif11 mediates diverse trafficking processes in the cytoplasm. In humans, inactivating mutations in KIF11 are associated with (1) retinal hypovascularization with or without microcephaly and (2) multi-organ syndromes characterized by variable combinations of lymphedema, chorioretinal dysplasia, microcephaly and/or mental retardation. To explore the pathogenic basis of KIF11-associated retinal vascular disease, we generated a Kif11 conditional knockout (CKO) mouse and investigated the consequences of early postnatal inactivation of Kif11 in vascular endothelial cells (ECs). The principal finding is that postnatal EC-specific loss of Kif11 leads to severely stunted growth of the retinal vasculature, mildly stunted growth of the cerebellar vasculature and little or no effect on the vasculature elsewhere in the central nervous system (CNS). Thus, in mice, Kif11 function in early postnatal CNS ECs is most significant in the two CNS regions-the retina and cerebellum-that exhibit the most rapid rate of postnatal growth, which may sensitize ECs to impaired mitotic spindle function. Several lines of evidence indicate that these phenotypes are not caused by reduced beta-catenin signaling in ECs, despite the close resemblance of the Kif11 CKO phenotype to that caused by EC-specific reductions in beta-catenin signaling. Based on prior work, defective beta-catenin signaling had been the only known mechanism responsible for monogenic human disorders of retinal hypovascularization. The present study implies that retinal hypovascularization can arise from a second and mechanistically distinct cause.

Ocular manifestations of Chinese patients with copy number variants in the NDP gene.

Purpose: Familial exudative vitreoretinopathy (FEVR) and Norrie disease (ND) are genetic disorders that can be caused by mutations in the NDP gene and affect retinal vasculature growth and development. This study aimed to describe the copy number variations (CNVs) in the NDP gene in Chinese FEVR families and the associated phenotypes. Methods: This study recruited 651 FEVR families. SeqCNV was used to analyze the CNVs in the families without mutations in known FEVR-associated genes. Multiplex ligation-dependent probe amplification and semiquantitative multiplex PCR were performed to verify the NDP CNVs. The probands and family members underwent complete ocular examinations. Results: NDP CNVs were identified in four patients from three unrelated families, accounting for 15% of the patients with NDP mutations and 0.46% of the entire FEVR cohort. Exon 2 deletions were detected in two families, and whole gene deletion was identified in one family. The affected individuals were born blind with total retinal detachment. Conclusions: The findings confirm that CNVs are a common NDP mutation type. The CNV-associated phenotype is congenital blindness with total retinal detachment. Antenatal genetic analyses and fetal ultrasound can facilitate early diagnosis and interventions in patients with NDP mutations.

Whole exome sequencing revealed novel pathogenic variants in Vietnamese patients with FEVR.

Background: Familial exudative vitreoretinopathy (FEVR) is a rare inherited disorder marked by incomplete retinal vascularization associated with exudation, neovascularization, and tractional retinal detachment. FEVR is genetically heterogeneous and is caused by variants in six genes: FZD4, LRP5, NDP, TSPAN12, ZNF408, and CTNNB1. In addition, the phenotypic overlap between FEVR and other disorders has been reported in patients harboring variants in other genes, such as KIF11, ATOH7, and RCBTB1. Purpose: To identify pathogenic variants in Vietnamese pediatric patients diagnosed with FEVR and to investigate the clinical findings in correlation with each causative gene. Methods: A total of 20 probands underwent ocular examinations with fundoscopy (ophthalmoscopy) or fluorescein angiography. Genomic DNA was extracted from the peripheral blood of the probands and their family members. Multiplex ligation-dependent probe amplification (MLPA) was employed to detect copy number variants of FEVR-causing genes. Short variants were screened by whole-exome sequencing (WES) and then validated by Sanger sequencing. Results: Fluorescein angiography showed retinal vascular anomalies in all patients. Other ocular abnormalities commonly found were strabismus, nystagmus, exudation, and retinal detachment. Genetic analysis identified 12 different variants in the FZD4, NDP, KIF11, and ATOH7 genes among 20 probands. Four variants were novel, including FZD4 c.169G>C, p.(G57R); NDP c.175-3A>G, splicing; KIF11 c.2146C>T, p.(Q716*) and c.2511_2515del, p.(N838Kfs*17). All patients with the KIF11 variant showed signs of microcephaly and intellectual disability. The patient with Norrie syndrome and their family members were found to have a deletion of exon 2 in the NDP gene. Conclusions: This study sheds light on the genetic causes of ocular disorders with the clinical expression of FEVR in Vietnamese patients. WES was applied as a comprehensive tool to identify pathogenic variants in complex diseases, such as FEVR, and the detection rate of pathogenic mutations was up to 60%.

Structure and function of the retina of low-density lipoprotein receptor-related protein 5 (Lrp5)-deficient rats.

Loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), result in familial exudative vitreoretinopathy (FEVR), osteoporosis-pseudoglioma syndrome (OPPG), and Norrie disease. CRISPR/Cas9 gene editing was used to produce rat strains deficient in Lrp5. The purpose of this study was to validate this rat model for studies of hypovascular, exudative retinopathies. The retinal vasculature of wildtype and Lrp5 knockout rats was stained with Giffonia simplifolia isolectin B4 and imaged by fluorescence microscopy. Effects on retinal structure were investigated by histology. The integrity of the blood-retina barrier was analyzed by measurement of permeability to Evans blue dye and staining for claudin-5. Retinas were imaged by fundus photography and SD-OCT, and electroretinograms were recorded. Lrp5 gene deletion led to sparse superficial retinal capillaries and loss of the deep and intermediate plexuses. Autofluorescent exudates were observed and are correlated with increased Evans blue permeability and absence of claudin-5 expression in superficial vessels. OCT images show pathology similar to OCT of humans with FEVR, and retinal thickness is reduced by 50% compared to wild-type rats. Histology and OCT reveal that photoreceptor and outer plexiform layers are absent. The retina failed to demonstrate an ERG response. CRISPR/Cas9 gene-editing produced a predictable rat Lrp5 knockout model with extensive defects in the retinal vascular and neural structure and function. This rat model should be useful for studies of exudative retinal vascular diseases involving the Wnt and norrin pathways.

Hypoxia tolerance in the Norrin-deficient retina and the chronically hypoxic brain studied at single-cell resolution.

The mammalian CNS is capable of tolerating chronic hypoxia, but cell type-specific responses to this stress have not been systematically characterized. In the Norrin KO (NdpKO ) mouse, a model of familial exudative vitreoretinopathy (FEVR), developmental hypovascularization of the retina produces chronic hypoxia of inner nuclear-layer (INL) neurons and Muller glia. We used single-cell RNA sequencing, untargeted metabolomics, and metabolite labeling from 13C-glucose to compare WT and NdpKO retinas. In NdpKO retinas, we observe gene expression responses consistent with hypoxia in Muller glia and retinal neurons, and we find a metabolic shift that combines reduced flux through the TCA cycle with increased synthesis of serine, glycine, and glutathione. We also used single-cell RNA sequencing to compare the responses of individual cell types in NdpKO retinas with those in the hypoxic cerebral cortex of mice that were housed for 1 week in a reduced oxygen environment (7.5% oxygen). In the hypoxic cerebral cortex, glial transcriptome responses most closely resemble the response of Muller glia in the NdpKO retina. In both retina and brain, vascular endothelial cells activate a previously dormant tip cell gene expression program, which likely underlies the adaptive neoangiogenic response to chronic hypoxia. These analyses of retina and brain transcriptomes at single-cell resolution reveal both shared and cell type-specific changes in gene expression in response to chronic hypoxia, implying both shared and distinct cell type-specific physiologic responses.

Identification of a novel mutation in KIF11 with functional analysis in a cohort of 516 familial patients with exudative vitreoretinopathy.

Purpose: To identify a novel mutation in KIF11 with clinical and functional analysis among 516 familial patients with exudative vitreoretinopathy (FEVR). Methods: Next-generation sequencing was performed on 516 patients with FEVR between January 2015 and October 2017. Clinical data were collected from patient charts, including sex, age at presentation, visual acuity if available, axial length, stage, and systemic clinical findings. Protein and mRNA levels were detected with western blotting and real-time quantitative PCR, respectively. Mass spectrometry was used to analyze the interacting protein of KIF11. Results: In total, 304 of 516 patients were identified with at least one mutation in FEVR causative genes. Mutations in KIF11 were identified in 14.47% of all carriers. The novel mutation p. H718L accounted for the greatest proportion (12/44; 27.30%) among all mutations in KIF11. Fundus presentations in these 12 individuals varied from the avascular zone of the peripheral retina to total retinal detachment. The p. H718L mutation can reduce the proliferation of human retinal endothelial cells (HRECs) compared to the wild type. The mRNA level of vascular endothelial growth factor-α, transforming growth factor-α, metalloproteinase-1, and angiopoietin-like 4 were depressed in the KIF11 (p. H718L) groups under hypoxia stimuli. Mass spectrometry results demonstrated that eukaryotic elongation factor 2 (EEF2) was an interacting protein of KIF11 and that the p. H718L mutation can attenuate the binding activity. Conclusions: Patients with the most frequent KIF11 mutation p. H718L showed typical FEVR presentations in this cohort. The mutation in KIF11 likely plays a role in the proliferation of HRECs, and the p. H718L mutation can reduce the proliferation.

Exome sequencing revealed Notch ligand JAG1 as a novel candidate gene for familial exudative vitreoretinopathy.

PURPOSE: Familial exudative vitreoretinopathy (FEVR) is a blindness-causing retinal vascular disease characterized by incomplete vascularization of the peripheral retina and by the absence or abnormality of the second/tertiary capillary layers in the deep retina. Variants in known FEVR disease genes can only explain about 50% of FEVR-affected cases. We aim to identify additional disease genes in patients with FEVR. METHODS: We applied exome sequencing analysis in a cohort of 49 FEVR families without pathogenic variants in known FEVR genes. Functions of the affected proteins were evaluated by reporter assay. Knockout mouse models were generated by endothelial-specific Cre line. RESULTS: Three novel rare heterozygous variants in Notch ligand JAG1 were identified in FEVR families-c.413C>T p. (A138V), c.1415G>A p. (R472H), and c.2884A>G p. (T962A)-and verified by Sanger sequencing analysis. Notch reporter assay revealed that mutant JAG1 proteins JAG1-A138V and JAG1-T962A lost almost all of their activities, and JAG1-R472H lost approximately 50% of its activity. Deletion of Jag1 in mouse endothelial cells resulted in reduced tip cells at the angiogenic front and retarded vessel growth, reproducing FEVR-like phenotypes. CONCLUSION: Our data suggest that JAG1 is a novel candidate gene for FEVR and pinpoints a potential target for therapeutic intervention.

Novel variants in familial exudative vitreoretinopathy patients with KIF11 mutations and the Genotype-Phenotype correlation.

Familial exudative vitreoretinopathy (FEVR) is an inherited disease characterized by abnormal development of retinal vasculature. KIF11 mutations were identified to be associated with FEVR in recent years. The purpose of this study was to investigate novel variants and describe associated ocular and extraocular phenotypes in FEVR patients with KIF11 mutations. Herein, 417 probands with clinical diagnosis of FEVR were enrolled. Genetic testing and ophthalmic examinations were performed in all subjects, and the genotype-phenotype correlation was analyzed. Overall, KIF11 mutation was identified in nine probands (9/417, 2.2%) among the patients with FEVR phenotype. There were six males and three females whose median age was six months (range: four months to six years old) at first visit. Among the detected mutations, five (55.6%) were frameshift, two (22.2%) were missense, one (11.1%) nonsense, and one (11.1%) splicing. Seven of these KIF11 mutations were detected as novel. Four (4/9, 44.4%) of the mutations were de novo. Clinical examinations showed that: four probands presented with bilateral falciform retinal fold; two with bilateral tractional retinal detachment; one was observed tractional retinal detachment in one eye and retinal fold in the other eye; one had falciform retinal fold in one eye and chorioretinal atrophy in the other eye; one exhibited rhegmatogenous retinal detachment in the left eye. Six of the probands were detected to have microcephaly. In conclusion: Most (5/9,55.6%) of the causative mutations were frameshift, and nearly half (4/9, 44.4%) of the mutations were de novo. Most (8/9, 88.9%) patients with KIF11 mutations showed typical ocular manifestations of severe FEVR. Majority (6/9, 66.7%) of the probands had a KIF11 mutation and were detected to have microcephaly. Seven of these harbored KIF11 mutations detected to be novel.

The spectrum of genetic mutations in patients with asymptomatic mild familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a disease exhibits a wide range of clinical signs, ranging mild peripheral retinal vascular anomalies to severe retinal detachments. Individuals with mild FEVR are frequently asymptomatic with good visual function and are often undiagnosed. However, little is known about the genetic characters of the cohort. The purpose of this study was to investigate the clinical characteristics and genetic spectrum of in patients with asymptomatic mild FEVR. Herein, sixty-two patients (124 eyes) with asymptomatic mild FEVR were studied in a case series. Comprehensive ophthalmic examinations and genetic testing were performed in all patients. Clinical examinations showed that the avascular zone was seen in all 124 eyes and was the most common abnormality observed. Increased vessel branching and straightened peripheral vessel branches were found in 122 (98.4%) eyes. Late-phase angiographic posterior and peripheral leakage (LAPPEL) was observed in 80 (64.5%) eyes and V-shape degeneration was noted in 36 (29.0%) eyes. Other manifestations including extensive anastomoses, retinal ridges, and extraretinal neovascularization, which were detected in 30 (24.2%), 10 (8.1%) and 2 (1.6%) eyes respectively. Overall, pathogenic mutations were identified in 48.4% (30/62) of individuals with asymptomatic mild FEVR. Mutations in FZD4, LRP5, TSPAN12, and KIF11 were detected in 21.0% (13/62), 12.9% (8/62), 12.9% (8/62), and 1.6% (1/62) of our patients respectively. Ten novel mutations were found. In conclusion: Pathogenic mutations in the known FEVR-associated genes were detected in nearly half (48.4%) of the asymptomatic mild FEVR cohort. Among these mutations, FZD4 was predominant, appearing in 21.0% of all individuals. Patients with asymptomatic mild FEVR should receive timely examinations, lifelong monitoring, and some of them need preventive therapy and treatment. Additionally, we discovered 10 novel variants, which may enable a deeper understanding of this disease.

Identification of novel variants in the FZD4 gene associated with familial exudative vitreoretinopathy in Chinese families.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR, OMIM 133780) is a severe hereditary retinal disease characterized by incomplete retinal vascular development and pathological neovascularization. It has been reported that variants in nine genes are associated with FEVR, but they can only explain approximately 50% of FEVR patients, suggesting that other FEVR-associated variants or genes remain to be discovered. METHODS: Whole-exome sequencing (WES) was carried out to analyse genomic DNA samples from the probands of 68 families with FEVR. Sanger sequencing was used to verify all identified variants. Western blot analysis was utilized to detect the expression of the variant mutant proteins. A luciferase assay was conducted to test the receptor activity of the mutant FZD4 proteins in Norrin-ß-catenin signaling. RESULTS: Seven heterozygous FZD4 variants were found to cause FEVR in seven families, including six missense variants and one deletion variant: c.182C>T (p.T61I), c.205C>T (p.H69Y), c.217_234del (p.73T_78Qdel), c.264C>A (p.Y88X), c.344G>T (p.G115V), c.678G>A (p.W226X) and c.1310T>C (p.I437T). Among these variants, c.205C>T (p.H69Y) and c.678G>A (p.W226X) are known FEVR-causing variants, while the other five variants are novel pathogenic variants. CONCLUSION: Our study revealed the cause of FEVR in seven Chinese families and identified five novel pathogenic variants in FZD4, which expanded the mutation spectrum of FEVR in the Chinese population. These findings also provided further support for using WES in the clinical diagnosis of FEVR.

Frizzled 4 regulates ventral blood vessel remodeling in the zebrafish retina.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a rare congenital disorder characterized by a lack of blood vessel growth to the periphery of the retina with secondary fibrovascular proliferation at the vascular-avascular junction. These structurally abnormal vessels cause leakage and hemorrhage, while the fibroproliferative scarring results in retinal dragging, detachment and blindness. Mutations in the FZD4 gene represent one of the most common causes of FEVR. METHODS: A loss of function mutation resulting from a 10-nucleotide insertion into exon 1 of the zebrafish fzd4 gene was generated using transcription activator-like effector nucleases (TALENs). Structural and functional integrity of the retinal vasculature was examined by fluorescent microscopy and optokinetic responses. RESULTS: Zebrafish retinal vasculature is asymmetrically distributed along the dorsoventral axis, with active vascular remodeling on the ventral surface of the retina throughout development. fzd4 mutants exhibit disorganized ventral retinal vasculature with discernable tubular fusion by week 8 of development. Furthermore, fzd4 mutants have impaired optokinetic responses requiring increased illumination. CONCLUSION: We have generated a visually impaired zebrafish FEVR model exhibiting abnormal retinal vasculature. These fish provide a tractable system for studying vascular biology in retinovascular disorders, and demonstrate the feasibility of using zebrafish for evaluating future FEVR genes identified in humans.

Symmetry of folds in FEVR: A genotype-phenotype correlation study.

Familial exudative vitreoretinopathy (FEVR) is a hereditary retinal vascular disorder. Among the various clinical phenotypes of this disease, retinal folds are the primary and typical feature of FEVR. However, little is known about the clinical characteristics, genetic spectrum, or potential phenotype-genotype correlation of retinal folds. Herein, we describe and analyze the clinical and genetic characteristics of retinal folds in FEVR. Eighty-nine patients with unilateral or bilateral retinal folds were included in this study. Clinical examinations showed that the retinal folds were bilateral in 37 patients (41.6%). Various retinal abnormalities were noted in the fellow eyes in the remaining 52 patients with unilateral folds. Most of the folds were located temporally (98.4%, 124/126), and were complete (97.6%, 123/126). 67.5% (60/89) probands were genetic confirmed FEVR. 25 novel pathogenic mutations (7 in FZD4, 7 in LRP5, 1 in NDP, 4 in TSPAN12, and 6 in KIF11) were identified in 25 families. Overall, 87.5% (14/16) and 73.7%(14/19) patients with LRP5 and FZD4 mutations were with unilateral folds, respectively.Nevertheless, only 25% (2/8), 36.4%(4/11) and 16.7%(1/6) patients with NDP, TSPAN12, and KIF11 mutations were with unilateral folds. Moreover, 85.7%(12/14),100% (6/6) and 100%(8/8) of the patients with mutated TSPAN12, KIF11, and NDP genes presented with symmetry in disease staging, while 55% and 64.7% of patients with FZD4 and LRP5 mutation displayed symmetry in staging. In conclusion, the majority of retinal folds extended completely and radially in the temporal peripheral retina. Patients with causative mutations in NDP, TSPAN12, or KIF11 were more likely to have bilaterally symmetrical severe retinopathy. In contrast, patients with LRP5 and FZD4 mutations displayed a relatively milder but broader spectrum of phenotypes and a higher frequency of asymmetry.