Pathogenic mutations of human phosphorylation sites affect protein-protein interactions.

Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis.

Clinical sequencing of the retinitis pigmentosa gene RPGR in over 1,000 cases of vision loss.

RPGR pathogenic variants are the major cause of X-linked retinitis pigmentosa. Here, we report the results from 1,033 clinical DNA tests that included sequencing of RPGR. A total of 184 RPGR variants were identified: 78 pathogenic or likely pathogenic, 14 uncertain, and 92 likely benign or benign. Among the pathogenic and likely pathogenic variants, 23 were novel, and most were frameshift or nonsense mutations (87%) and enriched (67%) in RPGR exon 15 (ORF15). Identical pathogenic variants found in different families were largely on different haplotype backgrounds, indicating relatively frequent, recurrent RPGR mutations. None of the 16 mother/affected son pairs showed de novo mutations; all 16 mothers were heterozygous for the pathogenic variant. These last two observations support the occurrence of most RPGR mutations in the male germline.

Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina.

Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1-CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1-CRB2 interactions. We observed no or a mild loss of CRB1-CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.

Rapid Variant Pathogenicity Analysis by CRISPR Activation of CRB1 Gene Expression in Patient-Derived Fibroblasts.

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding genetic disorders caused by pathogenic variants in genes expressed in the retina. In this study, we sought to develop a method for rapid evaluation of IRD gene variant pathogenicity by inducing expression of retinal genes in patient-derived fibroblasts using CRISPR-activation (CRISPRa). We demonstrate CRISPRa of CRB1 expression in fibroblasts derived from patients with retinitis pigmentosa, enabling investigation of pathogenic mechanisms associated with specific variants. We show the CRB1 c.4005 + 1G>A variant caused exon 11 skipping in CRISPR-activated fibroblasts and retinal organoids (ROs) derived from the same RP12 patient. The c.652 + 5G>C variant was shown to enhance exon 2 skipping in CRISPR-activated fibroblasts and differentially affected CRB1 isoform expression in fibroblasts and ROs. Our study demonstrates an accessible platform for transcript screening of IRD gene variants in patient-derived fibroblasts, which can potentially be applied for rapid pathogenicity assessments of any gene variant.

Genotype-phenotype associations in CRB1 bi-allelic patients: a novel mutation, a systematic review and meta-analysis.

PURPOSE: The goal of the study was to search for novel bi-allelic CRB1 mutations, and then to analyze the CRB1 literature at the genotypic and phenotypic levels. APPROACH: We screened various variables such as the CRB1 mutation types, domains, exons, and genotypes and their relation with specific ocular phenotypes. An emphasis was given to the bi-allelic missense and nonsense mutations because of their high prevalence compared to other mutation types. Finally, we quantified the effect of various non-modifiable factors over the best-corrected visual acuity oculus uterque (BCVA OU) using multivariate linear regression models and identified genetic interactions. RESULTS: A novel bi-allelic missense in the exon 9 of CRB1; c.2936G > A; p.(Gly979Asp) was found to be associated with rod-cone dystrophy (RCD). CRB1 mutation type, exons, domains, and genotype distribution varied significantly according to fundus characteristics, such as peripheral pigmentation and condition, optic disc, vessels, macular condition, and pigmentation (P < 0.05). Of the 154 articles retrieved from PubMed, 96 studies with 439 bi-allelic CRB1 patients were included. Missense mutations were significantly associated with an absence of macular pigments, pale optic disc, and periphery pigmentation, resulting in a higher risk of RCD (P < 0.05). In contrast, homozygous nonsense mutations were associated with macular pigments, periphery pigments, and a high risk of LCA (P < 0.05) and increased BCVA OU levels. We found that age, mutation types, and inherited retinal diseases were critical determinants of BCVA OU as they significantly increased it by 33% 26%, and 38%, respectively (P < 0.05). Loss of function alleles additively increased the risk of LCA, with nonsense having a more profound effect than indels. Finally, our analysis showed that p.(Cys948Tyr) and p.(Lys801Ter) and p.(Lys801Ter); p.(Cys896Ter) might interact to modify BCVA OU levels. CONCLUSION: This meta-analysis updated the literature and identified genotype-phenotype associations in bi-allelic CRB1 patients.

Research progress of RP1L1 gene in disease.

Retinitis pigmentosa 1-like 1 (RP1L1) is a component of photoreceptor cilia. Pathogenic variants in RP1L1 cause photoreceptor diseases, suggesting that RP1L1 plays an important role in photoreceptor biology, although its exact function is unknown. To date, RP1L1 variants have been associated with occult macular dystrophy (cone degeneration) and retinitis pigmentosa (rod degeneration). Here, we summarize the reported RP1L1-associated photoreceptor pathogenic mutations. The association between RP1L1 and other diseases (mainly several tumors) is also summarized and RP1L1 is included in a wider range of diseases. Finally, it is necessary to further explore the influence mechanism of RP1L1 gene on the health of photoreceptors and how it participates in the occurrence and development of tumors.

Fine-tuning FAM161A gene augmentation therapy to restore retinal function.

For 15 years, gene therapy has been viewed as a beacon of hope for inherited retinal diseases. Many preclinical investigations have centered around vectors with maximal gene expression capabilities, yet despite efficient gene transfer, minimal physiological improvements have been observed in various ciliopathies. Retinitis pigmentosa-type 28 (RP28) is the consequence of bi-allelic null mutations in the FAM161A, an essential protein for the structure of the photoreceptor connecting cilium (CC). In its absence, cilia become disorganized, leading to outer segment collapses and vision impairment. Within the human retina, FAM161A has two isoforms: the long one with exon 4, and the short one without it. To restore CC in Fam161a-deficient mice shortly after the onset of cilium disorganization, we compared AAV vectors with varying promoter activities, doses, and human isoforms. While all vectors improved cell survival, only the combination of both isoforms using the weak FCBR1-F0.4 promoter enabled precise FAM161A expression in the CC and enhanced retinal function. Our investigation into FAM161A gene replacement for RP28 emphasizes the importance of precise therapeutic gene regulation, appropriate vector dosing, and delivery of both isoforms. This precision is pivotal for secure gene therapy involving structural proteins like FAM161A.

Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

A Precision Therapy Approach for Retinitis Pigmentosa 11 Using Splice-Switching Antisense Oligonucleotides to Restore the Open Reading Frame of PRPF31.

Retinitis pigmentosa 11 is an untreatable, dominantly inherited retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 PRPF31. The expression level of PRPF31 is linked to incomplete penetrance in affected families; mutation carriers with higher PRPF31 expression can remain asymptomatic. The current study explores an antisense oligonucleotide exon skipping strategy to treat RP11 caused by truncating mutations within PRPF31 exon 12 since it does not appear to encode any domains essential for PRPF31 protein function. Cells derived from a patient carrying a PRPF31 1205C>A nonsense mutation were investigated; PRPF31 transcripts encoded by the 1205C>A allele were undetectable due to nonsense-mediated mRNA decay, resulting in a 46% reduction in PRPF31 mRNA, relative to healthy donor cells. Antisense oligonucleotide-induced skipping of exon 12 rescued the open reading frame with consequent 1.7-fold PRPF31 mRNA upregulation in the RP11 patient fibroblasts. The level of PRPF31 upregulation met the predicted therapeutic threshold of expression inferred in a non-penetrant carrier family member harbouring the same mutation. This study demonstrated increased PRPF31 expression and retention of the nuclear translocation capability for the induced PRPF31 isoform. Future studies should evaluate the function of the induced PRPF31 protein on pre-mRNA splicing in retinal cells to validate the therapeutic approach for amenable RP11-causing mutations.

Investigating the Impact of Dimer Interface Mutations on Norrin's Secretion and Norrin/ß-Catenin Pathway Activation.

Purpose: This study aimed to investigate the impact of 21 NDP mutations located at the dimer interface, focusing on their potential effects on protein assembly, secretion efficiency, and activation of the Norrin/ß-catenin signaling pathway. Methods: The expression level, secretion efficiency, and protein assembly of mutations were analyzed using Western blot. The Norrin/ß-catenin signaling pathway activation ability after overexpression of mutants or supernatant incubation of mutant proteins was tested in HEK293STF cells. The mutant norrin and wild-type (WT) FZD4 were overexpressed in HeLa cells to observe their co-localization. Immunofluorescence staining was conducted in HeLa cells to analyze the subcellular localization of Norrin and the Retention Using Selective Hook (RUSH) assay was used to dynamically observe the secretion process of WT and mutant Norrin. Results: Four mutants (A63S, E66K, H68P, and L103Q) exhibited no significant differences from WT in all evaluations. The other 17 mutants presented abnormalities, including inadequate protein assembly, reduced secretion, inability to bind to FZD4 on the cell membrane, and decreased capacity to activate Norrin/ß-catenin signaling pathway. The RUSH assay revealed the delay in endoplasmic reticulum (ER) exit and impairment of Golgi transport. Conclusions: Mutations at the Norrin dimer interface may lead to abnormal protein assembly, inability to bind to FZD4, and decreased secretion, thus contributing to compromised Norrin/ß-catenin signaling. Our results shed light on the pathogenic mechanisms behind a significant proportion of NDP gene mutations in familial exudative vitreoretinopathy (FEVR) or Norrie disease.

Genotype-Phenotype of CRB1-Associated Early-Onset Retinal Dystrophy: Novel Insights on Retinal Architecture and Therapeutic Window for Clinical Trials.

Purpose: The purpose of this study was to investigate the genotypic and phenotypic characteristics of CRB1-associated early onset retinal dystrophy (CRB1-eoRD) and retinal architecture by swept-source optical coherence tomography (SS-OCT). Methods: Eleven probands with CRB1-eoRD were recruited. Clinical information, genetic analysis, and comprehensive ophthalmic examinations including SS-OCT and SS-OCT angiography (SS-OCTA) were conducted. Results: A total of 81.8% (9/11) of CRB1-eoRD presented as Leber congenital amaurosis (LCA). Common clinical manifestations included coin-like yellow-white retinal spots (20/22, 90.9%) and para-arteriolar retinal pigment epithelial retention (12/22, 54.5%). Nineteen different CRB1 variants were detected in our case series, including 12 missense, 3 frameshifts, 3 nonsense, and 1 splicing. Of them, 12 variants had been reported, and 7 were novel. SS-OCT showed thinner central macula (the LCA group, P < 0.0001), thicker total retina (P < 0.0001), thinner outer retina (P < 0.05), and thicker inner retina (P < 0.0001) compared with the healthy control. The inner/outer (I/O) retina thickness ratio of CRB1-eoRD was 3.0, higher than the healthy control of 1.2 and other inherited retinal diseases (IRDs) of 2.2 (P < 0.0001 and P = 0.0027, respectively). SS-OCTA revealed an increased vascular density and perfusion area of the superficial vascular complex and deep vascular complex in CRB1-eoRD. Conclusions: LCA emerges as a frequently occurring phenotype in CRB1-eoRD. The unique features of SS-OCT and SS-OCTA are illustrated, and the novel biomarker, I/O ratio, may facilitate early diagnosis. The insights gained from this study hold significant value in determining the treatment window for potential forthcoming CRB1 gene therapy.

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.

Protective roles of peroxiporins AQP0 and AQP11 in human astrocyte and neuronal cell lines in response to oxidative and inflammatory stressors.

In addition to aquaporin (AQP) classes AQP1, AQP4 and AQP9 known to be expressed in mammalian brain, our recent transcriptomic analyses identified AQP0 and AQP11 in human cortex and hippocampus at levels correlated with age and Alzheimer's disease (AD) status; however, protein localization remained unknown. Roles of AQP0 and AQP11 in transporting hydrogen peroxide (H2O2) in lens and kidney prompted our hypothesis that up-regulation in brain might similarly be protective. Established cell lines for astroglia (1321N1) and neurons (SHSY5Y, differentiated with retinoic acid) were used to monitor changes in transcript levels for human AQPs (AQP0 to AQP12) in response to inflammation (simulated with 10-100 ng/ml lipopolysaccharide [LPS], 24 h), and hypoxia (5 min N2, followed by 0 to 24 h normoxia). AQP transcripts up-regulated in both 1321N1 and SHSY5Y included AQP0, AQP1 and AQP11. Immunocytochemistry in 1321N1 cells confirmed protein expression for AQP0 and AQP11 in plasma membrane and endoplasmic reticulum; AQP11 increased 10-fold after LPS and AQP0 increased 0.3-fold. In SHSY5Y cells, AQP0 expression increased 0.2-fold after 24 h LPS; AQP11 showed no appreciable change. Proposed peroxiporin roles were tested using melondialdehyde (MDA) assays to quantify lipid peroxidation levels after brief H2O2. Boosting peroxiporin expression by LPS pretreatment lowered subsequent H2O2-induced MDA responses (∼50%) compared with controls; conversely small interfering RNA knockdown of AQP0 in 1321N1 increased lipid peroxidation (∼17%) after H2O2, with a similar trend for AQP11 siRNA. Interventions that increase native brain peroxiporin activity are promising as new approaches to mitigate damage caused by aging and neurodegeneration.

Intraocular pressure across the lifespan of Tg-MYOCY437H mice.

Transgenic C57BL/6 mice expressing human myocilinY437 (Tg-MYOCY437H) are a well-established model for primary open-angle glaucoma (POAG). While the reduced trabecular meshwork (TM) cellularity due to severe endoplasmic reticulum (ER) stress has been characterized as the etiology of this model, there is a limited understanding of how glaucomatous phenotypes evolve over the lifespan of Tg-MyocY437H mice. In this study, we compiled the model's intraocular pressure (IOP) data recorded in our laboratory from 2017 to 2023 and selected representative eyes to measure the outflow facility (Cr), a critical parameter indicating the condition of the conventional TM pathway. We found that Tg-MYOCY437H mice aged 4-12 months exhibited significantly higher IOPs than age-matched C57BL/6 mice. Notably, a decline in IOP was observed in Tg-MYOCY437H mice at 17-24 months of age, a phenomenon not attributable to the gene dosage of mutant myocilin. Measurements of the Cr of Tg-MYOCY437H mice indicated that the age-related IOP reduction was not a result of ongoing TM damage. Instead, Hematoxylin and Eosin staining, immunohistochemistry analysis, and transmission electron microscopic examination revealed that this reduction might be induced by degenerations of the non-pigmented epithelium in the ciliary body of aged Tg-MYOCY437H mice. Overall, our findings provide a comprehensive profile of mutant myocilin-induced ocular changes over the Tg-MYOCY437H mouse lifespan and suggest a specific temporal window of elevated IOP that may be ideal for experimental purposes.

AAV-RPGR Gene Therapy Rescues Opsin Mislocalisation in a Human Retinal Organoid Model of RPGR-Associated X-Linked Retinitis Pigmentosa.

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGRORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGRORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial.

A systematic review of inherited retinal dystrophies in Pakistan: updates from 1999 to April 2023.

BACKGROUND: Inherited retinal degenerations (IRDs) are a group of rare genetic conditions affecting retina of the eye that range in prevalence from 1 in 2000 to 1 in 4000 people globally. This review is based on a retrospective analysis of research articles reporting IRDs associated genetic findings in Pakistani families between 1999 and April 2023. METHODS: Articles were retrieved through survey of online sources, notably, PubMed, Google Scholar, and Web of Science. Following a stringent selection criterion, a total of 126 research articles and conference abstracts were considered. All reported variants were cross-checked and validated for their correct genomic nomenclature using different online resources/databases, and their pathogenicity scores were explained as per ACMG guidelines. RESULTS: A total of 277 unique sequence variants in 87 distinct genes, previously known to cause IRDs, were uncovered. In around 70% cases, parents of the index patient were consanguineously married, and approximately 88.81% of the detected variants were found in a homozygous state. Overall, more than 95% of the IRDs cases were recessively inherited. Missense variants were predominant (41.88%), followed by Indels/frameshift (26.35%), nonsense (19.13%), splice site (12.27%) and synonymous change (0.36%). Non-syndromic IRDs were significantly higher than syndromic IRDs (77.32% vs. 22.68%). Retinitis pigmentosa (RP) was the most frequently observed IRD followed by Leber's congenital amaurosis (LCA). Altogether, mutations in PDE6A gene was the leading cause of IRDs in Pakistani families followed by mutations in TULP1 gene. CONCLUSION: In summary, Pakistani families are notable in expressing recessively inherited monogenic disorders including IRDs likely due to the highest prevalence of consanguinity in the country that leads to expression of rare pathogenic variants in homozygous state.

CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut.

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.

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.