Identification of Novel FZD4 Mutations in Familial Exudative Vitreoretinopathy and Investigating the Pathogenic Mechanisms of FZD4 Mutations.

Purpose: The purpose of this study is to report five novel FZD4 mutations identified in familial exudative vitreoretinopathy (FEVR) and to analyze and summarize the pathogenic mechanisms of 34 of 96 reported missense mutations in FZD4. Methods: Five probands diagnosed with FEVR and their family members were enrolled in the study. Ocular examinations and targeted gene panel sequencing were conducted on all participants. Plasmids, each carrying 29 previously reported FZD4 missense mutations and five novel mutations, were constructed based on the selection of mutations from each domain of FZD4. These plasmids were used to investigate the effects of mutations on protein expression levels, Norrin/ß-catenin activation capacity, membrane localization, norrin binding ability, and DVL2 recruitment ability in HEK293T, HEK293STF, and HeLa cells. Results: All five novel mutations (S91F, V103E, C145S, E160K, C377F) responsible for FEVR were found to compromise Norrin/ß-catenin activation of FZD4 protein. After reviewing a total of 34 reported missense mutations, we categorized all mutations based on their functional changes: signal peptide mutations, cysteine mutations affecting disulfide bonds, extracellular domain mutations influencing norrin binding, transmembrane domain (TM) 1 and TM7 mutations impacting membrane localization, and intracellular domain mutations affecting DVL2 recruitment. Conclusions: We expanded the spectrum of FZD4 mutations relevant to FEVR and experimentally demonstrated that missense mutations in FZD4 can be classified into five categories based on different functional changes.

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.

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.

Defective EMC1 drives abnormal retinal angiogenesis via Wnt/ß-catenin signaling and may be associated with the pathogenesis of familial exudative vitreoretinopathy.

Endoplasmic reticulum (ER) membrane protein complex (EMC) is required for the co-translational insertion of newly synthesized multi-transmembrane proteins. Compromised EMC function in different cell types has been implicated in multiple diseases. Using inducible genetic mouse models, we revealed defects in retinal vascularization upon endothelial cell (EC) specific deletion of Emc1, the largest subunit of EMC. Loss of Emc1 in ECs led to reduced vascular progression and vascular density, diminished tip cell sprouts, and vascular leakage. We then performed an unbiased transcriptomic analysis on human retinal microvascular endothelial cells (HRECs) and revealed a pivotal role of EMC1 in the ß-catenin signaling pathway. Further in-vitro and in-vivo experiments proved that loss of EMC1 led to compromised ß-catenin signaling activity through reduced expression of Wnt receptor FZD4, which could be restored by lithium chloride (LiCl) treatment. Driven by these findings, we screened genomic DNA samples from familial exudative vitreoretinopathy (FEVR) patients and identified one heterozygous variant in EMC1 that co-segregated with FEVR phenotype in the family. In-vitro expression experiments revealed that this variant allele failed to facilitate the expression of FZD4 on the plasma membrane and activate the ß-catenin signaling pathway, which might be a main cause of FEVR. In conclusion, our findings reveal that variants in EMC1 gene cause compromised ß-catenin signaling activity, which may be associated with the pathogenesis 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.

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.

Refractive status and optical components in premature infants with and without retinopathy of prematurity: A 4- to 5-year cohort study.

This study was aimed to investigate the characteristics of refractive parameters in premature infants and children aged 3-8 years with mild retinopathy of prematurity (ROP) and to explore the effects of premature delivery and mild ROP on the development of refractive status and ocular optical components. Premature infants who underwent ocular fundus oculi screening in our hospital between January 2009 and February 2011 were included and divided into the ROP group and the non-ROP group. Full-term infants were the controls. The results of the annual ocular examination conducted between 2014 and 2018 were analysed, and the refractive status, optical components, and developmental trends were compared among the three groups. The total follow-up time was 4-5 years. The prevalence of myopia and astigmatism was high in the ROP group (P < 0.05). In the non-ROP group, the prevalence of myopia was also higher than that in the control group. The prevalence of myopia increased with age in the ROP and non-ROP groups, while the prevalence of astigmatism remained unchanged. In the ROP group, the corneal refractive power was the largest, the lens was the thickest and the ocular axis was the shortest; in the control group, the corneal refractive power was the smallest, the lens was the thinnest, and the ocular axis was the longest. These parameters in the non-ROP group were between those in the two groups mentioned above (P < 0.05). The corneal refractive power was relatively stable at 3-8 years old in the three groups. The change in lens thickness was small in both the ROP group and the non-ROP group (P = 0.75, P = 0.06), and the lens became thinner in the control group (P < 0.001). The length of the ocular axis increased in the three groups. Preterm infants are more likely to develop myopia than full-term infants, and children with ROP are more likely to develop both myopia and astigmatism. Thicker lenses were the main cause of the high prevalence of myopia in premature infants with or without ROP.

CTNND1 variants cause familial exudative vitreoretinopathy through the Wnt/cadherin axis.

Familial exudative vitreoretinopathy (FEVR) is a hereditary disorder that can cause vision loss. CTNND1 encodes a cellular adhesion protein p120-catenin (p120), which is essential for vascularization with unclear function in postnatal physiological angiogenesis. Here, we applied whole-exome sequencing to 140 probands of FEVR families and identified 3 candidate variants in the human CTNND1 gene. We performed inducible deletion of Ctnnd1 in the postnatal mouse endothelial cells (ECs) and observed typical phenotypes of FEVR with reactive gliosis. Using unbiased proteomics analysis combined with experimental approaches, we conclude that p120 is critical for the integrity of adherens junctions (AJs) and that p120 activates Wnt signaling activity by protecting ß-catenin from glycogen synthase kinase 3 beta-ubiqutin-guided (Gsk3ß-ubiquitin-guided) degradation. Treatment of CTNND1-depleted human retinal microvascular ECs with Gsk3ß inhibitors LiCl or CHIR-99021 enhanced cell proliferation. Moreover, LiCl treatment increased vessel density in Ctnnd1-deficient mouse retinas. Variants in CTNND1 caused FEVR by compromising the expression of AJs and Wnt signaling activity. Genetic interactions between p120 and ß-catenin or α-catenin revealed by double-heterozygous deletion in mice showed that p120 regulates vascular development through the Wnt/cadherin axis. In conclusion, variants in CTNND1 can cause FEVR through the Wnt/cadherin axis.

A novel frameshift variant in the TSPAN12 gene causes autosomal dominant FEVR.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is an inherited blinding eye disease with abnormal retinal vascular development. We aim to broaden the variant spectrum of FEVR and provide a basis for molecular diagnosis and genetic consultation. METHODS: We recruited five FEVR patients from one large Chinese family. Whole-exome sequencing (WES) and Sanger sequencing were applied to sequence, analyze, and verify variants on genomic DNA samples. Immunocytochemistry, western blot, qPCR, and luciferase assay were performed to test the influence of the variant on the protein expression and activity of the Norrin/ß-catenin pathway. RESULTS: We identified a novel heterozygous frameshift variant c.533dupC (p.D179Rfs*6) in Tetraspanin 12 (TSPAN12) gene that is related to FEVR. This variant caused degradation of the entire TSPAN12 protein, which failed to activate Norrin/ß-catenin signaling, possibly causing FEVR. CONCLUSION: Our study revealed a novel frameshift variant D179Rfs*6 in TSPAN12 that is inherited in an autosomal dominant manner. We found that D179Rfs*6 caused a failure to activate Norrin/ß-catenin signaling. This finding broadens the variant spectrum of TSPAN12 and provides invaluable information for the molecular diagnosis of FEVR.

Identification of Two Novel Variants in the LRP5 Gene that Cause Familial Exudative Vitreoretinopathy.

Background: Familial exudative vitreoretinopathy (FEVR, OMIM 133780) is a severe inherited eye disease characterized by abnormal development of the retinal vasculature. Variants in the reported genes account for ∼50% of total FEVR cases. However, the pathogenesis of the other 50% of FEVR cases remains unclear. Therefore, it is crucial to identify novel variants responsible for the pathogenesis of FEVR. Aims: To find causative variants responsible for FEVR in two Han Chinese families. Materials and Methods: We recruited two families with FEVR patients and applied exome sequencing on the genomic DNA samples from the probands. Sanger sequencing was performed for variant validation. Western blot analysis and luciferase assays were performed to test the expression levels and activity of the mutant proteins. Results: We identified two novel missense variants in the LRP5 gene (NM_002335), c.1176 C > A (p.Asp392Glu) and c.2435 A>C (p.Asp812Ala), both inherited in an autosomal dominant manner. Both variants significantly reduced Norrin/ß-catenin signaling activity without affecting the expression of the LRP5 protein. Conclusion: This study expands the variant spectrum of the LRP5 gene for FEVR, providing valuable information for prenatal counseling and molecular diagnosis of FEVR.

Heterozygote loss-of-function variants in the LRP5 gene cause familial exudative vitreoretinopathy.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is an inherited ocular disease with clinical manifestations of aberrant retinal vasculature. We aimed to identify novel causative variants responsible for FEVR and provided evidence for the genetic counselling of FEVR. METHODS: We applied whole-exome sequencing (WES) on the genomic DNA samples from the probands and performed Sanger sequencing for variant validation. Western blot analysis and luciferase assays were performed to test the expression levels and the activity of mutant proteins. RESULTS: We identified one novel heterozygous nonsense variant, and three novel heterozygous frameshift variants including c.1801G>T (p.G601*), c.1965delC (p.H656Tfs*41), c.4445delC (p.S1482Cfs*17), and c.4482delC (p.P1495Rfs*4), which disabled the function of LRP5 on the Norrin/ß-catenin signalling. Overexpression of variant-carrying LRP5 proteins resulted in down regulation of the protein levels of ß-catenin and the Norrin/ß-catenin signalling target genes c-Myc and Glut1. CONCLUSION: Our study showed that four inherited LRP5 variants can cause autosomal dominant FEVR via down regulation of Norrin/ß-catenin signalling and expanded the spectrum of FEVR-associated LRP5 variants.

Whole-Exome Sequencing Reveals Novel NDP Variants in X-Linked Familial Exudative Vitreoretinopathy.

PURPOSE: To investigate causative variants in three Chinese families affected with familial exudative vitreoretinopathy (FEVR). METHODS: Three unrelated Chinese families were recruited in this study. The three probands and their family members experienced a comprehensive age-appropriate eye examination and genetic analysis. Luciferase assay was performed to evaluate impacts of variants on Norrin/ß-catenin signaling activity. RESULTS: Here we report two novel NDP variants associated with FEVR in three families, including c.17T>C (p.Leu6Pro) in family 1 and c.58G>A (p.Gly20Arg) in family 2 and 3. These two variants were co-segregated with the disease phenotypes within each family. In addition, both variants resulted in compromised Norrin/ß-catenin signaling activity. CONCLUSION: Our study identified two FEVR-associated pathogenic variants in NDP, which expanded the variant spectrum and provided information for the genetic diagnosis of FEVR.

CD146 as a promising therapeutic target for retinal and choroidal neovascularization diseases.

Blood vessel dysfunction causes several retinal diseases, including diabetic retinopathy, familial exudative vitreoretinopathy, macular degeneration and choroidal neovascularization in pathological myopia. Vascular endothelial growth factor (VEGF)-neutralizing proteins provide benefits in most of those diseases, yet unsolved haemorrhage and frequent intraocular injections still bothered patients. Here, we identified endothelial CD146 as a new target for retinal diseases. CD146 expression was activated in two ocular pathological angiogenesis models, a laser-induced choroid neovascularization model and an oxygen-induced retinopathy model. The absence of CD146 impaired hypoxia-induced cell migration and angiogenesis both in cell lines and animal model. Preventive or therapeutic treatment with anti-CD146 antibody AA98 significantly inhibited hypoxia-induced aberrant retinal angiogenesis in two retinal disease models. Mechanistically, under hypoxia condition, CD146 was involved in the activation of NFκB, Erk and Akt signalling pathways, which are partially independent of VEGF. Consistently, anti-CD146 therapy combined with anti-VEGF therapy showed enhanced impairment effect of hypoxia-induced angiogenesis in vitro and in vivo. Given the critical role of abnormal angiogenesis in retinal and choroidal diseases, our results provide novel insights into combinatorial therapy for neovascular fundus diseases.

Whole-Exome Sequencing Reveals Novel TSPAN12 Variants in Autosomal Dominant Familial Exudative Vitreoretinopathy.

Background: Familial exudative vitreoretinopathy (FEVR), a group of rare inherited retinal vascular disorders, is the major cause of vision loss in juveniles. At present, the diagnosis of FEVR remains difficult due to its clinical and genetic heterogeneities. Aims: To identify the causative genetic variants in two unrelated FEVR-affected families: one Indian family and one Chinese Han family. Materials and Methods: Five affected patients from two families were recruited for this study. Whole-exome sequencing was applied to the probands, and Sanger sequencing was performed for validation. Stringent whole-exome sequence data analyses were performed to evaluate all of the identified pathogenic variants. Results: Two novel variants in the TSPAN12 gene, were identified: a missense variant c.437 T > G (p.Leu146Arg); and a nonsense variant c.477 C > A (p.Cys159*). Both variants cosegregated with the disease in the investigated FEVR-affected families. Additionally, both variants inactivated the ability of TSPAN12 protein to enhance Norrin/ß-catenin signaling. Conclusion: This study expands the mutational spectrum of TSPAN12 for FEVR.

Refractive status and optical components of premature babies with or without retinopathy of prematurity at 7 years old.

BACKGROUND: This study aimed to investigate the refractive status and optical components of premature babies with or without retinopathy of prematurity (ROP) at 7 years old and to explore the influence of prematurity and ROP on the refractive status and optical components. METHODS: From January 2009 to February 2011, premature babies receiving fundus photographic screening (FPS) were recruited and divided into non-ROP group and ROP group. Full-term babies matched in age were recruited as controls. Auto-refractometer was employed to detect the corneal refractive power, corneal radius (CR) of curvature and corneal astigmatism, A-scan ultrasonography was performed to detect the anterior chamber depth (ACD), lens thickness (LT), vitreous thickness (VITR) and ocular axial length (AL), and retinoscopy was done following cycloplegia with 1% cyclopentolate in these babies at 7 years old. These parameters were compared among groups, and the correlations of gestational age and birth weight with the refractive status and optical components were further evaluated. RESULTS: Of 126 subjects, a total of 252 eyes were evaluated in this study, including 50 eyes of 25 subjects in ROP group (pre-threshold stage 1-3), 110 eyes of 55 subjects in non-ROP group and 92 eyes of 46 subjects in control group. The incidence of myopia was the highest in ROP group (9/50, 18%), followed by non-ROP group (11/110; 10%) and control group (6/92; 6.52%). The incidence of hyperopia was the highest in control group (21/92; 22.83%), followed by ROP group (8/50; 16%) and non-ROP group (10/110; 9.09%). The incidence of astigmatism was the highest in ROP group (18/50; 36%), followed by non-ROP group (25/110; 22.73%) and control group (12/92; 13.04%). The corneal astigmatism (-1.58, -1.11, -0.86 DC, P<0.01) and the mean degree of astigmatism (1.38, 1.17, 0.64 DC, P<0.05) in ROP group and non-ROP group were significantly higher than those in control group. The corneal refractive power in ROP group was more potent as compared to non-ROP group and control group (43.98, 43.16, 42.99 D, P<0.05); the corneal curvature in ROP group was significantly higher than that in non-ROP group and control group (7.87, 7.71, 7.67 mm, P<0.05); the ocular AL in ROP group and non-ROP group was significantly shorter than that in control group (2.41, 22.47, 22.78 mm, P<0.05). The LT in ROP group and non-ROP group was markedly thicker than that in control group (4.48, 4.45, 4.37 mm, P>0.05); the ACD in ROP group and non-ROP group was markedly deeper than in control group (3.16, 3.12, 3.21 mm, P>0.05). The gestational age was negatively related to corneal astigmatism (r=-0.208, P=0.013) and astigmatism (r=-0.226, P=0.004), but positively associated with ocular AL (r=0.252, P=0.005). The birth weight was negatively associated with corneal astigmatism (r=-0.30, P<0.001), astigmatism (r=-0.267, P=0.001), corneal refractive power (r=-0.255, P=0.001) and corneal curvature (r=0.242, P=0.001), but positively to ocular AL (r=0.243, P=0.001) and spherical equivalent refraction (SER) (r=0.151, P=0.028). CONCLUSIONS: (I) Premature babies with or without ROP are susceptible to myopia and astigmatism; (II) low birth weight, prematurity and ROP synergistically influence the development of refractive status and optical components, resulting in myopia and astigmatism; (III) premature babies with or without ROP have increased corneal curvature and LT, which are related to the higher incidence of myopia and astigmatism.

Refractive state and optical compositions of preterm children with and without retinopathy of prematurity in the first 6 years of life.

This study aimed to investigate the refractive state and optical compositions of preterm children with and without mild retinopathy of prematurity (ROP) and explore the influence of prematurity and mild ROP on the development of refractive state and optical compositions.Preterm children who received fundus screening were recruited, and divided into ROP group and non-ROP group. Term children matched in age were also recruited as controls. Several correspondence indicators were measured before and after ciliary muscle paralysis with 1% cyclopentanone.A total of 250 eyes from 126 patients were included for analysis. The incidence of myopia was the highest in ROP group. The incidence of hyperopia was the highest in control group. The incidence of astigmatism was the highest in ROP group. The corneal astigmatism and mean astigmatism in ROP group and non-ROP group were significantly higher than in control group. Corneal refraction in ROP was markedly higher than in non-ROP group and control group; corneal curvature in ROP group increased significantly as compared with non-ROP group and control group (P < .05). The axial eye length in ROP group and non-ROP group reduced significantly as compared with control group (P < .05). Gestational age had negative relationships with corneal astigmatism (P = .019) and astigmatism (P = .001) and positive relationship with axial eye length (P = .005). Birth weight had negative relationships with corneal astigmatism (P = .001), astigmatism (P < .001), corneal refraction (P = .001), and corneal curvature (P = .001) and positive relationships with axial eye length (P = .001) and spherical equivalent refraction (P = .039). The incidence of myopia increased and that of hyperopia reduced in children over age. In children aged 3 to 4 years, the anterior chamber depth, lens thickness, vitreous thickness, and axial eye length significantly increased as compared with those aged 5 years (P < .05); the vitreous thickness and axial eye length in children aged 5 years increased significantly as compared with those aged 6 years (P < .05).This study shows that preterm children with and without mild ROP are more likely to develop myopia and astigmatism, and low birth weight, prematurity, and ROP may simultaneously affect the development of optical compositions, leading to myopia and astigmatism.