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.

Frameshift variants in the C-terminal of CTNNB1 cause familial exudative vitreoretinopathy by AXIN1-mediated ubiquitin-proteasome degradation condensation.

The ß-catenin has two intrinsically disordered regions in both C- and N-terminal domains that trigger the formation of phase-separated condensates. Variants in its C-terminus are associated with familial exudative vitreoretinopathy (FEVR), yet the pathogenesis and the role of these variants in inducing abnormal condensates, are unclear. In this study, we identified a novel heterozygous frameshift variant, c.2104-2105insCC (p.Gln703ProfsTer33), in CTNNB1 from a FEVR-affected family. This variant encodes an unstable truncated protein that was unable to activate Wnt signal transduction, which could be rescued by the inhibition of proteasome or phosphorylation. Further functional experiments revealed the propensity of the Gln703ProfsTer33 variant to form cytoplasmic condensates, exhibiting a lower turnover rate after fluorescent bleaching due to enhanced interaction with AXIN1. LiCl, which specifically blocks GSK3ß-mediated phosphorylation, restored signal transduction, cell proliferation, and junctional integrity in primary human retinal microvascular endothelial cells over-expressed with Gln703ProfsTer33. Finally, experiments on two reported FEVR-associated mutations in the C-terminal domain of ß-catenin exhibited several functional defects similar to the Gln703ProfsTer33. Together, our findings unravel that the C-terminal region of ß-catenin is pivotal for the regulation of AXIN1/ß-catenin interaction, acting as a switch to mediate nucleic and cytosolic condensates formation that is implicated in the pathogenesis 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.

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.

Secure attachment in infancy predicts context-dependent emotion expression in middle childhood.

Attachment security has been linked to healthy socioemotional development, but less is known about how secure attachment in infancy relates to emotional functioning in middle childhood, particularly across multiple contexts. The present study examined associations between secure attachment in infancy and children's context-dependent emotion expression during a parent-child interaction at age 9 (N = 78) among families with Child Protective Services involvement (i.e., children at risk for emotion dysregulation). The results indicated that children classified as securely attached in infancy exhibited less task-incongruent affect (i.e., less positive affect during a distressing discussion, less negative affect during a positive discussion) and a greater decrease in negative affect from a distressing discussion to a positive discussion than children classified as insecurely attached. In addition, secure children were rated as more appropriate in their emotion expression than insecure children. The present study highlights attachment as a promising intervention target for children at risk for emotion dysregulation. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

The ER membrane protein complex subunit Emc3 controls angiogenesis via the FZD4/WNT signaling axis.

The endoplasmic reticulum (ER) membrane protein complex (EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell (EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of blunt-ended, aneurysm-like tip endothelial cells (ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells (HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of ß-catenin signaling using RNA sequencing, real-time quantitative PCR (RT-qPCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride (LiCl) treatment remarkably enhanced ß-catenin signaling and cell proliferation of HRECs. Additionally, LiCl partially reversed the angiogenesis defects in Emc3-cKO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/ß-catenin signaling.

Lactamization of sp(2) C-H Bonds with CO2 : Transition-Metal-Free and Redox-Neutral.

The first direct use of carbon dioxide in the lactamization of alkenyl and heteroaryl C-H bonds to synthesize important 2-quinolinones and polyheterocycles in moderate to excellent yields is reported. Carbon dioxide, a nontoxic, inexpensive, and readily available greenhouse gas, acts as an ideal carbonyl source. Importantly, this transition-metal-free and redox-neutral process is eco-friendly and desirable for the pharmaceutical industry. Moreover, these reactions feature a broad substrate scope, good functional group tolerance, facile scalability, and easy product derivatization.

PEG-derivatized octacosanol as micellar carrier for paclitaxel delivery.

In this study, PEG-derivatized octacosanol copolymer was successfully developed to improve the anti-tumor activity and eliminate toxicity of the commercial formulation of paclitaxel (PTX). MPEG2K-C28, the conjugation of monomethoxy Poly(ethylene glycol) 2000 and octacosanol, was readily soluble in aqueous solution and self-assembled to form micelles with small sizes (< 20 nm) that are efficient in encapsulating PTX with a drug loading of 9.38 ± 0.18% and an encapsulation efficiency of 93.90 ± 2.12%. Meanwhile, octacosanol is very safe for humans and amazingly exhibits antitumor activity through inhibition activity of matrix metalloproteinases (MMPs) and translocation of the transcription factor (nuclear factor-kappa B, NF-κB) to the nucleus, which may be able to promote synergistic effects with PTX. A sustained and slower in vitro release behavior was observed in the (PTX micelles) than that of Taxol. PTX micelles exhibited more potent cytotoxicity than Taxol in the 4T1 breast cancer cell line. More interestingly, MPEG2K-C28 selectively inhibited the growth of 4T1 cells rather than the normal cells (HEK293 and L929 cell lines), indicating the antitumor activity of octacosanol remained after conjugation with MPEG. Acute toxicity evaluations indicated that MPEG2K-C28 was a safe drug carrier. Pharmacokinetic study revealed that PTX micelles improved the T1/2 and AUC of PTX (compared with Taxol) from 1.910 ± 0.139 h and 13.999 ± 1.109 mg/l × h to 2.876 ± 0.532 h and 76.462 ± 8.619 mg/l × h in vivo, respectively. The maximal tolerated dose (MTD) for PTX micelles (ca. 120 mg PTX/kg) in mice was significantly higher than that for Taxol (ca. 20mg PTX/kg). PTX micelles exhibited slightly better antitumor activity than Taxol but safer in 4T1 breast cancer model in vivo. The cell apoptosis in the immunofluorescent studies and the cell proliferation in the immunohistochemical studies also proved the results. In conclusion, MPEG2K-C28 is a simple, safe and effective drug delivery carrier for PTX, and has some therapeutic effects in 4T1 cells in vitro. PTX micelles showed significant antitumor activity in vivo with low systemic toxicity in 4T1 breast cancer. MPEG2K-C28 micelles entrapping PTX deserve more studies in the future.

Wave energy technology in China.

This paper traces the research stages of China's study of wave energy technology, summarizing the findings and deficiencies of each stage from oscillating water column, through onshore oscillating buoy to floating Duck. It also highlights the major innovations in China's new floating Duck device.