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.

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.

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.

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.

[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.

Genetic and clinical characteristics of ZNF408-related familial exudative vitreoretinopathy.

OBJECTIVE: To analyze the clinical and genetic characteristics of zinc finger protein 408 (ZNF408)-related familial exudative vitreoretinopathy (FEVR) in a Chinese cohort. METHODS: Ninety families from Chongqing and 16 families from Xinjiang were selected according to fundus lesion characteristics. Peripheral venous blood was collected from patients and their families; genomic DNA was extracted for whole exome sequencing. Relationships between genotype and phenotype in patients with ZNF408-related FEVR were analyzed. RESULTS: ZNF408 variants were detected in three patients (2.83%, 3/106). ZNF408 variants in these three probands were all missense mutations at novel sites. One proband had a ZNF408 and LRP5 double-gene variant, and two probands had ZNF408 single-gene variants. Patients with double-gene variants did not display more severe clinical manifestations. CONCLUSIONS: This study expands the spectrum of known ZNF408 variants and confirms that ZNF408 variants can cause FEVR. Most variants detected in this study have not been reported in the literature and are suspected pathogenic variants of FEVR. In patients with FEVR, phenotype and genotype do not necessarily display a direct one-to-one relationship.

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.

Identification of Five Novel Variants in the TSPAN12 Gene in Chinese Families With Familial Exudative Vitreoretinopathy.

Purpose: To report the novel causative variants in five Chinese families with familial exudative vitreoretinopathy (FEVR). Methods: Five unrelated Chinese families diagnosed with FEVR were enrolled in this study. Ocular examinations and genetic analysis were performed on the probands and family members. Luciferase assay was performed to evaluate the variants' impacts on Norrin/ß-catenin signaling activity. Results: Five novel variants, including two frameshifts, c.518delA (p.Glu173Glyfs*42) and c.719delT (p.Leu240Profs*21), two missenses, c.482G>T (p.Gly161Val) and c. 614G>C (p. Gly205Ala), and one nonsense, c.375G>A (p.Trp125*), were identified in the TSPAN12 gene in this study. All the variants were co-segregated within each family and were predicted as pathogenic in silico. The luciferase assay showed all variants lead to various degrees of compromised Norrin/ß-catenin signaling activity. Conclusions: Our study expanded the variant spectrum and provided information for the genetic testing of FEVR by showing five novel FEVR-associated pathogenic variants in TSPAN12. Translational Relevance: Our study expanded the spectrum of FEVR-associated TSPAN12 variants and further supported the inclusion of TSPAN12 gene in the evaluation of cases concerning for FEVR.

An SNX31 variant underlies dominant familial exudative vitreoretinopathy-like pathogenesis.

Familial exudative vitreoretinopathy (FEVR) is a complex hereditary eye disorder characterized by incomplete development of the retinal vasculature, which thereby affects retinal angiogenesis. But the genetic factors contributing to FEVR's development or pathogenesis remain elusive. In a Chinese family with FEVR with 19 members, by using whole-exome sequencing, we identified a candidate disease-causing DNA variant in sorting nexin 31 (SNX31) (c.963delG; p. Trp321Cys), which results in a frameshift mutation. We studied the biochemical mechanism of this mutation and determined that it is deficient in ß1-integrin binding and stability. The SNX31 c.963delG point mutation mouse model (SNX31m/m) was constructed with CRISPR/Cas9 technology. At 2-4 months of age, SNX31m/m mice showed fundus phenotypes similar to FEVR-like changes, including vascular leakage and retinal atrophy. Moreover, we found that VEGF and apoptotic pathways were involved in these ocular phenotypes. Hence, our study extended the FEVR mutation spectrum to include SNX31. These findings expanded our understanding of the molecular pathogenesis of FEVR and may facilitate the development of methods for the diagnosis and prevention of FEVR.

Novel Exon 7 Deletions in TSPAN12 in a Three-Generation FEVR Family: A Case Report and Literature Review.

Familial exudative vitreoretinopathy (FEVR) is a severe clinically and genetically heterogeneous disease that is characterized by vascular disorder. FEVR exhibits strikingly variable clinical phenotypes, ranging from asymptomatic to total blindness. In this case, we present a patient who was first treated as having high myopia and retinopathy but was finally diagnosed with FEVR caused by the heterozygous deletion of exon 7 in TSPAN12 with the aid of whole genome sequencing (WGS). Typical vascular changes, including vascular leakage and an avascular zone in the peripheral retina, were observed in the proband using fundus fluorescein angiography (FFA), and the macular dragging was shown to be progressing in the follow-up visit. Furthermore, the proband showed unreported TSPAN12-related phenotypes of FEVR: ERG (full-field electroretinogram) abnormalities and retinoschisis. Only mild vascular changes were exhibited in the FFA for the other three family members who carried the same deletion of exon 7 in TSPAN12. This case expands our understanding of the phenotype resulting from TSPAN12 mutations and signifies the importance of combining both clinical and molecular analysis approaches to establish a complete diagnosis.

Familial exudative vitreoretinopathy (FEVR) in a child with novel microarray-defined deletion of 11q14 previously diagnosed as retinopathy of prematurity (ROP).

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a rare inherited disease characterized by abnormal retinal angiogenesis that leads to incomplete vascularization of the peripheral retina and ischemia. The disease demonstrates complex genetics and can be inherited in an autosomal recessive, autosomal dominant, or X-linked recessive fashion. All presently identified pathogenic genetic variants account for about 50% of all FEVR cases worldwide. Genetic testing can confirm the diagnosis. MATERIALS AND METHODS: Case report. CASE: A 7-year-old female who was born prematurely at 33 weeks gestation and was thought to have progression of bilateral retinopathy of prematurity (ROP) was referred to a pediatric-retina specialist for management. Upon initial examination under anesthesia with multimodal imaging, the diagnosis of FEVR was suspected. Genetic testing identified a FZD4 variant involving a novel complex interchromosomal rearrangement involving chromosomes 2 and 11 associated with microarray-defined deletion of 11q14. The patient was conceived via IVF and has a fraternal twin without FEVR. This is the first report of familial exudative vitreoretinopathy associated with this combination of genetic findings. CONCLUSION: Autosomal dominant FEVR involves abnormalities in several genes, including FZD4 at the chromosome 11q. We recommend that patients with microarray-defined deletions of 11q have careful review of the allelic deletions in Chromosome 11 to determine if FZD4 is included because a loss of function variant of a single copy of FZD4 is sufficient to cause the FEVR phenotype. It is essential to differentiate FEVR from other pediatric retinal diseases in children, including ROP, persistent fetal vasculature, and Coats disease.

A novel splicing variant of VCAN identified in a Chinese family initially diagnosed with familial exudative vitreoretinopathy.

BACKGROUND: Wagner vitreoretinopathy (WVR) is a rare autosomal dominant vitreoretinopathy caused by pathogenic variants in the VCAN gene. The aim of this study was to report a novel splicing variant in VCAN identified in a three-generation Chinese family initially diagnosed with familial exudative vitreoretinopathy and to describe the patients' clinical features. METHODS: Four affected individuals from a three-generation family underwent detailed ophthalmic examinations, including best-corrected visual acuity by Snellen E chart, slit-lamp biomicroscopy, indirect ophthalmoscopy under pupil dilatation, ocular B-ultrasonography, optical coherence tomography scans, and fundus autofluorescence. Targeted next-generation sequencing was performed to identify variants of the disease-causing gene for the proband, followed by co-segregation analysis using Sanger-DNA sequencing. Reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out to verify the effects of a variant on VCAN pre-mRNA splicing in the lymphocytes from the patients. RESULTS: We detected a novel heterozygous variant c.4004-4_c.4004-3delinsCA of VCAN in all four affected individuals. RT-PCR revealed that the novel variant caused an abnormal splicing in exon 8 of the VCAN and imbalanced versican transcripts. All four patients presented vitreous syneresis and bilateral retinal detachment occurring at different ages. The patients also showed different extents of visual defects and diverse clinical manifestations, including cataract, iris-lens synechiae, inverted papillae, and ectopic foveas. CONCLUSIONS: Our results expand the mutation spectrum of VCAN and further confirm that the splicing sites for exon 8 are mutation hot spots. Patients with WVR may present high phenotype variation; therefore, molecular analysis is very important for precise diagnosis of patients with inherited vitreoretinopathy.

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.

Unrecognized ROPER in a child with a novel pathogenic variant in ZNF408 gene.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a rare hereditary disorder characterized by abnormal or incomplete retinal angiogenesis commonly inherited in an autosomal dominant fashion. Up to 50% of FEVR cases are linked to known genetic mutations affecting retinal vasculature development. PURPOSE: To report a case, a novel pathogenic variant of the ZNF408 gene associated with a case of FEVR in a premature male. MATERIALS AND METHODS: Case report. RESULTS: A 10-month-old male who was born prematurely at 34 weeks' gestation in the Dominican Republic was referred for persistent avascular retina. The baby was treated with bilateral intravitreal ranibizumab injections for retinopathy of prematurity (ROP) with the presence of plus disease. Fundus examination several months after treatment revealed the absence of tortuosity of the vessels with avascular periphery; fluorescein angiography (FA) confirmed peripheral avascularity and demonstrated irregular sprouts of vascularization in the absence of neovascularization. We performed genetic testing under the suspicion of FEVR and results identified a heterozygous mutation in the ZNF408 gene on chromosome 11, c.1307 C > T. CONCLUSION: FEVR is an important differential diagnosis in premature infants with retinopathy, as clinical presentation can overlap with common findings in ROP. Maintaining high suspicion for the disease is especially critical in cases with findings unusual for ROP. FEVR in the presence of prematurity has been well described, falling under the proposed term ROPER. Genetic testing is key to confirm diagnosis.

NDP-related retinopathies: clinical phenotype of female carriers.

BACKGROUND/AIMS: Norrin cysteine knot growth factor (NDP) located on the X chromosome, was previously reported to cause Norrie disease and familial exudative vitreoretinopathy (FEVR), which are blindness-causing ocular disorders, in males. In this study, we aimed to explore the clinical characteristics of female carriers with NDP mutations. METHODS: Twelve female carriers from 11 unrelated families with pathogenic NDP mutations were recruited. Clinical data were collected from the NDP carriers. Comprehensive ocular examinations, including best corrected visual acuity, slit lamp examination, fundus photography and fundus fluorescein angiography (FFA) were evaluated. Targeted gene or whole exome sequencing was performed in the probands, and Sanger sequencing was performed to confirm NDP mutations in female carriers. RESULTS: Of the 12 females, 1 (1/12, 8.3%) presented with decreased visual acuity and 11 (11/12, 91.7%) were asymptomatic. Based on the FFA, peripheral vascular changes were noted in 66.7% (16/24) of the eyes of 75.0% (9/12) of the carriers. A total of 33.3% (8/24) had typical FEVR phenotype, 33.3% (8/24) had mild vascular abnormalities and 33.3% (8/24) was unremarkable. In addition, predominant changes such as telangiectatic endings (66.7%), anomalous circumferential vessel (37.5%), supernumerary vascular branching (33.3%), fluorescein leakage (29.2%), avascular area (8.3%), retina fold (8.3%) and peripheral straightening of retinal vessels (33.3%) were noted. CONCLUSION: Although NDP-related retinopathy is an X-linked recessive disorder, most of the female carriers of NDP exhibited clinical features of FEVR. Thus, timely examinations and lifelong monitoring should be conducted in the NDP female carriers.

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.

Mutation spectrum in a cohort with familial exudative vitreoretinopathy.

PURPOSE: To expand the mutation spectrum of patients with familial exudative vitreoretinopathy (FEVR) disease. PARTICIPANTS: 74 probands (53 families and 21 sporadic probands) with familial exudative vitreoretinopathy (FEVR) disease and their available family members (n = 188) were recruited for sequencing. METHODS: Panel-based targeted screening was performed on all subjects. Before sanger sequencing, variants of LRP5, NDP, FZD4, TSPAN12, ZNF408, KIF11, RCBTB1, JAG1, and CTNNA1 genes were verified by a series of bioinformatics tools and genotype-phenotype co-segregation analysis. RESULTS: 40.54% (30/74) of the probands were sighted to possess at least one etiological mutation of the nine FEVR-causative genes. The etiological mutation detection rate was 37.74% (20/53) in family-attainable probands while 47.62% (10/21) in sporadic cases. The diagnosis rate of patients in the early-onset subgroup (≤5 years old, 45.4%) is higher than that of the children or adolescence-onset subgroup (6-16 years old, 42.1%) and the late-onset subgroup (≥17 years old, 39.4%). A total of 36 etiological mutations were identified in this study, comprising 26 novel mutations and 10 reported mutations. LRP5 was the most prevalent mutant gene among the 36 mutation types with a percentage of 41.67% (15/36). Followed by FZD4 (10/36, 27.78%), TSPAN12 (5/36, 13.89%), NDP (4/36, 11.11%), KIF11 (1/36, 2.78%), and RCBTB1 (1/36, 2.78%). Among these mutations, 63.89% (23/36) were missense mutations, 25.00% (9/36) were frameshift mutations, 5.56% (2/36) were splicing mutations, 5.56% (2/36) were nonsense mutations. Moreover, the clinical pathogenicity of these variants was defined according to American College of Medical Genetics (ACMG) and genomics guidelines: 41.67% (15/36) were likely pathogenic variants, 27.78% (10/36) pathogenic variants, 30.55% (11/36) variants of uncertain significance. No etiological mutations discovered in the ZNF408, JAG1, and CTNNA1 genes in this FEVR cohort. CONCLUSIONS: We systematically screened nine FEVR disease-associated genes in a cohort of 74 Chinese probands with FEVR disease. With a detection rate of 40.54%, 36 etiological mutations of six genes were authenticated in 30 probands, including 26 novel mutations and 10 reported mutations. The most prevalent mutated gene is LRP5, followed by FZD4, TSPAN12, NDP, KIF11, and RCBTB1. In total, a de novo mutation was confirmed. Our study significantly clarified the mutation spectrum of variants bounded up to FEVR disease.

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.

FZD4 in a Large Chinese Population With Familial Exudative Vitreoretinopathy: Molecular Characteristics and Clinical Manifestations.

Purpose: The purpose of this study was to establish a genotype-phenotype correlation of familial exudative vitreoretinopathy (FEVR) caused by FZD4 gene mutations. Methods: Six hundred fifty-one probands and their family members were recruited based on a clinical diagnosis of FEVR between 2015 and 2021 at Zhongshan Ophthalmic Center. Ocular examinations were performed in all participants. Targeted gene panel sequencing and whole-exome sequencing were performed in the probands, and Sanger sequencing was used to verify the mutations and segregation analysis was performed in the family members. Results: Fifty-one FZD4 mutations (24 novels and 27 known) were detected in 84 families. Of these 168 eyes with FEVR, the eyes at stages 1, 2, 3, 4, and 5 were 29 (17.3%), 15 (8.9%), 19 (11.3%), 55 (32.7%), and 12 (7.1%), respectively. Exact stage of 38 (22.6%) eyes could not be determined. The FEVR phenotypes were more severe in the probands than the phenotypes in the family members (P < 0.001). The families were divided into two groups, probands that inherited the variant from the mother, and probands that inherited the variant from the father. In addition, the FEVR stage differences between these two groups were different (P < 0.05). Despite the mutations being located in different domains of FZD4, no significant differences were identified among the domains in terms of FEVR staging, retinal folds, retinal detachment, temporal midperipheral vitreoretinal interface abnormality, and foveal hypoplasia. Conclusions: The FZD4 probands had severer phenotype than the family members, and the FEVR stage difference was greater between the probands and mothers than that between the probands and fathers.