Catenin alpha 1 mutations cause familial exudative vitreoretinopathy by overactivating Norrin/beta-catenin signaling Xianjun Zhu, … , Weiquan Zhu, Zhenglin Yang J Clin Invest. 2021. https://doi.org/10.1172/JCI139869. Research In-Press Preview Angiogenesis Genetics Graphical abstract Find the latest version: https://jci.me/139869/pdf 1 Catenin alpha 1 mutations cause familial exudative vitreoretinopathy by over- 2 activating Norrin/beta-catenin signaling 3 Xianjun Zhu1,2,6,7,8, Mu Yang1,2,8, Peiquan Zhao3,8, Shujin Li1,2,8, Lin Zhang1, Lulin Huang1, Yi 4 Huang1, Ping Fei3, Yeming Yang1, Shanshan Zhang1, Huijuan Xu1, Ye Yuan1, Xiang Zhang3, 5 Xiong Zhu1, Shi Ma1, Fang Hao1, Periasamy Sundaresan4, Weiquan Zhu5, Zhenglin Yang1,2,6,7,* 6 1Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial Peo- 7 ple's Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan, 8 China; 9 2Research Unit for Blindness Prevention of Chinese Academy of medical Sciences 10 (2019RU026), Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, 11 Chengdu, Sichuan, China; 12 3Department of Ophthalmology, Xinhua Hospital, School of Medicine, Shanghai Jiaotong Uni- 13 versity, Shanghai, China; 14 4Department of Genetics, Aravind Medical Research Foundation, Aravind Eye Hospital, Madu- 15 rai, Tamil Nadu, India; 16 5Department of Molecular Medicine, School of Medicine, University of Utah, Salk Lake City, 17 Utah, USA. 18 6Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu, 19 610072, China. 20 7These authors jointly directed this work. 21 8These authors contributed equally to this work. 22 23 Conflict of interest: The authors have declared that no conflict of interest exists. 24 *Correspondence should be addressed to: Zhenglin Yang, Sichuan Provincial Key Laboratory for 25 Human Disease Gene Study, Sichuan Provincial People's Hospital, University of Electronic Sci- 26 ence and Technology of China, 27 32 The First Ring Road West 2, Chengdu, Sichuan, 610072, China 28 Email: [email protected]. 29 Phone: 86-28-87393375 30 Fax: 86-28-87393596 31 32 1 1 Abstract 2 Familial exudative vitreoretinopathy (FEVR) is a severe retinal vascular disease that causes 3 blindness. FEVR has been linked to mutations in several genes associated with inactivation of 4 the Norrin/β-catenin signaling pathway, but these account for only approximately 50% of cases. 5 We report that mutations in CTNNA1 (α-catenin) cause FEVR by overactivating the β-catenin 6 pathway and disrupting cell adherens junctions. Three heterozygous mutations in CTNNA1 7 (p.F72S, p.R376Cfs*27 and p.P893L) were identified by exome-sequencing. We further demon- 8 strated that FEVR-associated mutations led to overactivation of Norrin/β-catenin signaling due to 9 impaired protein interactions within the cadherin/catenin complex. The clinical features of FEVR 10 were reproduced in mice lacking Ctnna1 in vascular endothelial cells (ECs) or with overac- 11 tivated β-catenin signaling by an EC-specific gain-of-function allele of Ctnnb1. In isolated 12 mouse lung endothelial cells, both CTNNA1-P893L and F72S mutants failed to rescue either the 13 disrupted F-ACTIN arrangement or VE-Cadherin and CTNNB1 distribution. Moreover, we dis- 14 covered that compound heterozygous Ctnna1 F72S and a deletion allele could cause similar phe- 15 notype. Furthermore, a LRP5 mutation, which activates Norrin/β-catenin signaling, was identi- 16 fied in a FEVR family and the corresponding knock-in mice exhibited partial FEVR-like pheno- 17 type. Our study demonstrates that precise regulation of β-catenin activation is critical for retinal 18 vascular development and provides new insights into the pathogenesis of FEVR. 19 20 21 22 2 1 Clinical Perspective 2 What Is New? 3 • We identified three heterozygous mutations in CTNNA1 in familial exudative vitreoreti- 4 nopathy (FEVR) patients, and these mutations resulted in overactivation of Norrin/β- 5 catenin signaling and disruption of the cadherin/catenin complex. 6 • Clinical features of FEVR were reproduced in mice lacking Ctnna1 in vascular endothe- 7 lial cells and in mice with an endothelial-cell-specific gain-of-function Ctnnb1 allele. 8 • In a large Indian family with FEVR, we identified an LRP5 mutation (p.P848L) that re- 9 sults in overactivation of Norrin/β-catenin signaling, and we observed clinical features of 10 FEVR in the retina of Lrp5P847L knock-in mice. 11 • The precise regulation of β-catenin activation is critical for normal retinal vascular devel- 12 opment. 13 What Are the Clinical Implications? 14 • Because both in- and overactivation of Norrin/β-catenin signaling can cause defective an- 15 giogenesis, careful monitoring during drug treatment targeting β-catenin is warranted. 16 • The cadherin/catenin complex has the potential to be a therapeutic target for other neo- 17 vascular diseases affecting the blood–brain barrier, which contribute to altered brain 18 function and intellectual disability. 19 20 3 1 Introduction 2 3 Familial exudative vitreoretinopathy (FEVR) is a severe inherited retinal disorder characterized 4 by the incomplete vascularization of the peripheral retina and by the absence or abnormality of 5 the second/tertiary capillary layers in the deep retina (1). These pathological changes are be- 6 lieved to result in neovascularization and exudate, vitreous hemorrhaging, traction from the vitre- 7 ous membranes, displacement (ectopia) of the macula, and the folding and detachment of the ret- 8 ina (2). FEVR is genetically heterogeneous, and its clinical features can be highly variable; fam- 9 ily members with the same mutation can present without symptoms or can exhibit a range of 10 clinical features, including complete blindness (3). 11 12 FEVR can be inherited as an autosomal-dominant, autosomal-recessive, or X-linked disorder. 13 Mutations in 11 genes and one locus have been identified as causing FEVR: LRP5 (low-density 14 lipoprotein receptor-related protein 5) (4, 5); FZD4 (frizzled 4) (3); NDP (norrin, previously 15 known as Norrie disease pseudoglioma) (6); TSPAN12 (tetraspanin-12) (7-9); ZNF408 (zinc fin- 16 ger protein 408) (2); KIF11 (kinesin family member 11) (10); CTNNB1 (catenin beta 1) (11, 12); 17 ATOH7 (atonal homolog 7) (13); RCBTB1 (RCC1 and BTB domain-containing protein 1) (14); 18 EVR3 (exudative vitreoretinopathy 3) on chromosome 11p12-13 (15); ILK (integrin-linked ki- 19 nase) (16) and JAG1 (jagged canonical Notch ligand 1) (17). In addition to FEVR, mutations in 20 LRP5 are associated with osteopenia and osteoporosis (18), whereas mutations in NDP cause 21 Norrie disease, which features intellectual disability (ID) and cognitive impairments (19). Muta- 22 tions in KIF11 are also associated with microcephaly, with or without chorioretinopathy, 23 lymphedema, or ID (20), and mutations in CTNNB1 with syndromic ID (21). Thus, FEVR can 24 feature in syndromes that cause intellectual impairment or disability. 4 1 Previous studies have indicated that the pathogenesis of FEVR may involve disrupted Nor- 2 rin/β-catenin pathway signaling (7, 22-25). Normally, FZD4 and LRP5 form a complex with 3 TSPAN12 in the plasma membrane of endothelial cells (ECs) (7, 22-25). Norrin is an extracellu- 4 lar ligand that binds to FZD4 on ECs with the aid of LRP5 and TSPAN12 to activate down- 5 stream β-catenin/Wnt pathway signaling (7, 22-25). Therefore, mutations in any of the genes en- 6 coding these five proteins (NDP, FZD4, LRP5, TSPAN12, and β-catenin) might lead to FEVR by 7 inactivating the Norrin/β-catenin pathway and altering the expression of its downstream target 8 genes (7, 22-25). However, it remains unclear how mutations in the ZNF408, KIFF11, ATOH7, 9 and RCBTB1 genes and EVR3 on chromosome 11p12-13 contribute to the pathogenesis of 10 FEVR. Mutations in the 11 genes and one locus currently associated with FEVR can explain 11 only approximately 50% of FEVR cases (26, 27), so we performed a whole-exome sequencing 12 (WES) analysis of 49 FEVR families who do not carry mutations in these genes. From this se- 13 quence analysis, we identified three heterozygous mutations in α-catenin (CTNNA1) of the cad- 14 herin/catenin complex associated with the phenotypes of FEVR. CTNNA1 is a core member of 15 the cadherin/catenin complex, which integrates adherens junctions (AJs) with the actin cytoskel- 16 eton and promotes intercellular adhesion (28, 29). CTNNA1 has been reported as a putative tu- 17 mor suppressor in myeloid leukemia (30), glioblastoma (31), skin cancer (32) and gastric cancer 18 (33, 34). Saskens et al. identified mutations in CTNNA1 that cause butterfly-shaped pigment dys- 19 trophy (35). Very recently, a missense CTNNA1 mutation was identified in an age-related macu- 20 lar degeneration (AMD) patient (36). Furthermore, Alexander et al. identified four mutations in 21 CTNNA1 that cause macular pattern dystrophy (37). These findings together introduced a new 22 perspective that CTNNA1 might affect retina development. Using a range of approaches, includ- 23 ing cell biological assays and genetically modified mouse models, we demonstrate in this paper 5 1 that the mutant forms of CTNNA1 identified in our WES analysis disrupt conformation of the 2 cadherin/catenin complex and induce inappropriate overactivation of the Norrin/β-catenin path- 3 way in vascular ECs that causes FEVR. 6 1 Results 2 WES of FEVR families identified mutations in CTNNA1 3 We used WES to identify potentially novel genetic mutations in 49 families (14 unaffected in- 4 dividuals and 86 individuals affected with FEVR) with an autosomal-dominant form of FEVR 5 without mutations in known FEVR genes. Variants identified by WES with a frequency of < 6 0.005 in the dbSNP138, Exome Variant Server, ExAC, and gnomAD databases and absent in 7 1,000 sequenced controls were filtered. These filtered variants were then annotated with ANNO- 8 VAR software (see Methods for detailed information) (Table S1).