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.

PCDH12 variants are associated with basal ganglia anomalies and exudative vitreoretinopathy.

PCDH12 is a member of the non-clustered protocadherins that mediate cell-cell adhesion, playing crucial roles in many biological processes. Among these, PCDH12 promotes cell-cell interactions at inter-endothelial junctions, exerting essential functions in vascular homeostasis and angiogenesis. However, its exact role in eye vascular and brain development is not completely understood. To date, biallelic loss of function variants in PCDH12 have been associated with a neurodevelopmental disorder characterized by the typical neuroradiological findings of diencephalic-mesencephalic junction dysplasia and intracranial calcifications, whereas heterozygous variants have been recently linked to isolated brain calcifications in absence of cognitive impairment or other brain malformations. Recently, the phenotypic spectrum associated with PCDH12 deficiency has been expanded including cerebellar and eye abnormalities. Here, we report two female siblings harboring a novel frameshift homozygous variant (c.2169delT, p.(Val724TyrfsTer8)) in PCDH12. In addition to the typical diencephalic-mesencephalic junction dysplasia, brain MRI showed dysmorphic basal ganglia and thalamus that were reminiscent of a tubulin-like phenotype, mild cerebellar vermis hypoplasia and extensive prominence of perivascular spaces in both siblings. The oldest sister developed profound and progressive monocular visual loss and the eye exam revealed exudative vitreoretinopathy. Similar but milder eye changes were also noted in her younger sister. In summary, our report expands the clinical (brain and ocular) spectrum of PCDH12-related disorders and adds a further line of evidence underscoring the important role of PCDH12 in retinal vascular and brain development.

Whole-Gene Deletions of FZD4 Cause Familial Exudative Vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is an inherited disorder characterized by abnormalities in the retinal vasculature. The FZD4 gene is associated with FEVR, but the prevalence and impact of FZD4 copy number variation (CNV) on FEVR patients are unknown. The aim of this study was to better understand the genetic features and clinical manifestations of patients with FZD4 CNVs. A total of 651 FEVR families were recruited. Families negative for mutations in FEVR-associated genes were selected for CNV analysis using SeqCNV. Semiquantitative multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification were conducted to verify the CNVs. Four probands were found to carry whole-gene deletions of FZD4, accounting for 5% (4/80) of probands with FZD4 mutations and 0.6% (4/651) of all FEVR probands. The four probands exhibited similar phenotypes of unilateral retinal folds. FEVR in probands with CNVs was not more severe than in probands with FZD4 missense mutations (p = 1.000). Although this is the first report of FZD4 CNVs and the associated phenotypes, the interpretation of FZD4 CNVs should be emphasized when analyzing the next-generation sequencing data of FEVR patients because of their high prevalence.

Novel FZD4 and LRP5 mutations in a small cohort of patients with familial exudative vitreoretinopathy (FEVR).

Purpose: To report novel mutations in the FZD4 and LRP5 genes, associated with familial exudative vitreoretinopathy (FEVR), and to correlate with clinical features of 7 FEVR patients.Methods: In this retrospective case series, 7 patients who had undergone genetic panel testing and carried a diagnosis of FEVR were identified. Comprehensive ophthalmic examination and direct DNA sequencing of FEVR-associated genes were performed in all patients. Identified sequence variants were analyzed in silico.Results: Eight mutations were identified amongst the 7 patients, that included 4 FZD4 mutations and 4 LRP5 mutations. Four novel mutations were identified, two in FZD4 (c.615delC, p.Y206MfsX34) and (c.964A>T, p.I322F), and two in LRP5 (c.2585A>T, p.D862V) and (c.1412 + 1 G > A, splice donor). A broad phenotypic spectrum was noted and no clear genotypic-phenotypic correlation was observed.Conclusion: These findings expand the mutation spectrum of FZD4 and LRP5.

Missense variants in CTNNB1 can be associated with vitreoretinopathy-Seven new cases of CTNNB1-associated neurodevelopmental disorder including a previously unreported retinal phenotype.

BACKGROUND: CTNNB1 (MIM 116806) encodes beta-catenin, an adherens junction protein that supports the integrity between layers of epithelial tissue and mediates intercellular signaling. Recently, various heterozygous germline variants in CTNNB1 have been associated with human disease, including neurodevelopmental disorder with spastic diplegia and visual defects (MIM 615075) as well as isolated familial exudative vitreoretinopathy without developmental delays or other organ system involvement (MIM 617572). From over 40 previously reported patients with CTNNB1-related neurodevelopmental disorder, many have had ocular anomalies including strabismus, hyperopia, and astigmatism. More recently, multiple reports indicate that these abnormalities are associated with the presence of vitreoretinopathy. METHODS: We gathered a cohort of three patients with CTNNB1-related neurodevelopmental disorder, recruited from both our own clinic and referred from outside providers. We then searched for a clinical database comprised of over 12,000 exome sequencing studies to identify and recruit four additional patients. RESULTS: Here, we report seven new cases of CTNNB1-related neurodevelopmental disorder, all harboring de novo variants, six of which were previously unreported. All patients but one presented with a spectrum of ocular abnormalities and one patient, who was found to carry a missense variant in CTNNB1, had notable vitreoretinopathy. CONCLUSIONS: Our findings suggest ophthalmologic screening should be performed in all patients with CTNNB1 variants.

Role of NDP- and FZD4-Related Novel Mutations Identified in Patients with FEVR in Norrin/ß-Catenin Signaling Pathway.

Mutations in NDP and FZD4 have been closely related to a series of retinal diseases including familial exudative vitreoretinopathy (FEVR). Our study was designed to identify novel NDP and FZD4 mutations by whole exome sequencing (WES) in a cohort of patients with a definitive diagnosis of FEVR and explore the underlying molecular mechanism. During 2016, we investigated fifty nonconsanguineous families with affected individuals exhibiting FEVR phenotype and WES identified one recently reported mutation: NDP c.127C>A (p.H43N), and five novel mutations: NDP c.129_131del (p.44del), NDP c.320_353del (p.R107Pfs), NDP c.321delG (p.L108Cfs), NDP c.377G>T (p.C126F), and FZD4 c.314T>G (p.M105R) that cosegragated with the abnormal fundus vascular manifestations in six families. All the mutations were perceived to be pathogenic or likely pathogenic according to the standards and guidelines from the American College of Medical Genetics and Genomics (ACMG) and predicted to be deleterious by a series of bioinformatics analyses. We systematically performed functional analyses on the six mutations utilizing the Topflash reporter assay, where all NDP and FZD4 mutants revealed at least 50% loss of wild-type activity. Immunoprecipitation finally demonstrated that the six mutations could degrade the Norrin-Frizzled-4 pair-binding effect to varying degrees. Finally, our study underscores the correlation between the FEVR phenotype and genotype in NDP and FZD4, extending the mutation spectrum, allowing a reliable assessment of FEVR recurrence and improving genetic counseling. Further, our findings provide essential evidence for the follow-up study of animal models and drug targets by Topflash assays and immunoprecipitation.

Novel mutation in CTNNB1 causes familial exudative vitreoretinopathy (FEVR) and microcephaly: case report and review of the literature.

Purpose: Neonatal retinal folds and/or vitreoretinal traction can be signs of isolated ocular or syndromic disorders. Etiologies include retinopathy of prematurity, perinatal infections or inherited vitreoretinal disorders such as familial exudative vitreoretinopathy (FEVR) or Norrie disease. We present the clinical and genetic findings of a two-month-old infant with microcephaly, mild motor developmental delay, and FEVR, who required urgent surgical interventions.Methods: The patient underwent an initial examination under anesthesia (EUA) with fluorescein angiography (FA) and subsequent medical and surgical treatments. Genetic testing was undertaken to identify the etiology.Results: Examination at 2 months of age demonstrated microcephaly with a head circumference smaller than the 1st percentile. Family history was negative for microcephaly or retinal disease. Anterior segment eye exam was normal OU. There were bilateral macular folds involving the fovea and extending from the disc to the temporal periphery. FA demonstrated bilateral incomplete vascularization of the retina most notable nasally. Indirect laser was applied to ischemic retina OU. Scleral buckling procedures were performed OU as well as a vitrectomy in the left eye. Follow-up examinations demonstrated the stable appearance of the folds and attached retinas OU. Genetic testing identified a novel dominant heterozygous c.2046_2047del [p.Phe683Glnfs*9] mutation in CTNNB1, predicted to result in a frameshift causing a truncated protein.Conclusions: CTNNB1 mutations are an uncommon cause of FEVR with microcephaly.

A mouse model for kinesin family member 11 (Kif11)-associated familial exudative vitreoretinopathy.

During mitosis, Kif11, a kinesin motor protein, promotes bipolar spindle formation and chromosome movement, and during interphase, Kif11 mediates diverse trafficking processes in the cytoplasm. In humans, inactivating mutations in KIF11 are associated with (1) retinal hypovascularization with or without microcephaly and (2) multi-organ syndromes characterized by variable combinations of lymphedema, chorioretinal dysplasia, microcephaly and/or mental retardation. To explore the pathogenic basis of KIF11-associated retinal vascular disease, we generated a Kif11 conditional knockout (CKO) mouse and investigated the consequences of early postnatal inactivation of Kif11 in vascular endothelial cells (ECs). The principal finding is that postnatal EC-specific loss of Kif11 leads to severely stunted growth of the retinal vasculature, mildly stunted growth of the cerebellar vasculature and little or no effect on the vasculature elsewhere in the central nervous system (CNS). Thus, in mice, Kif11 function in early postnatal CNS ECs is most significant in the two CNS regions-the retina and cerebellum-that exhibit the most rapid rate of postnatal growth, which may sensitize ECs to impaired mitotic spindle function. Several lines of evidence indicate that these phenotypes are not caused by reduced beta-catenin signaling in ECs, despite the close resemblance of the Kif11 CKO phenotype to that caused by EC-specific reductions in beta-catenin signaling. Based on prior work, defective beta-catenin signaling had been the only known mechanism responsible for monogenic human disorders of retinal hypovascularization. The present study implies that retinal hypovascularization can arise from a second and mechanistically distinct cause.

Exome sequencing revealed Notch ligand JAG1 as a novel candidate gene for familial exudative vitreoretinopathy.

PURPOSE: Familial exudative vitreoretinopathy (FEVR) is a blindness-causing retinal vascular disease characterized by incomplete vascularization of the peripheral retina and by the absence or abnormality of the second/tertiary capillary layers in the deep retina. Variants in known FEVR disease genes can only explain about 50% of FEVR-affected cases. We aim to identify additional disease genes in patients with FEVR. METHODS: We applied exome sequencing analysis in a cohort of 49 FEVR families without pathogenic variants in known FEVR genes. Functions of the affected proteins were evaluated by reporter assay. Knockout mouse models were generated by endothelial-specific Cre line. RESULTS: Three novel rare heterozygous variants in Notch ligand JAG1 were identified in FEVR families-c.413C>T p. (A138V), c.1415G>A p. (R472H), and c.2884A>G p. (T962A)-and verified by Sanger sequencing analysis. Notch reporter assay revealed that mutant JAG1 proteins JAG1-A138V and JAG1-T962A lost almost all of their activities, and JAG1-R472H lost approximately 50% of its activity. Deletion of Jag1 in mouse endothelial cells resulted in reduced tip cells at the angiogenic front and retarded vessel growth, reproducing FEVR-like phenotypes. CONCLUSION: Our data suggest that JAG1 is a novel candidate gene for FEVR and pinpoints a potential target for therapeutic intervention.

Frizzled 4 regulates ventral blood vessel remodeling in the zebrafish retina.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a rare congenital disorder characterized by a lack of blood vessel growth to the periphery of the retina with secondary fibrovascular proliferation at the vascular-avascular junction. These structurally abnormal vessels cause leakage and hemorrhage, while the fibroproliferative scarring results in retinal dragging, detachment and blindness. Mutations in the FZD4 gene represent one of the most common causes of FEVR. METHODS: A loss of function mutation resulting from a 10-nucleotide insertion into exon 1 of the zebrafish fzd4 gene was generated using transcription activator-like effector nucleases (TALENs). Structural and functional integrity of the retinal vasculature was examined by fluorescent microscopy and optokinetic responses. RESULTS: Zebrafish retinal vasculature is asymmetrically distributed along the dorsoventral axis, with active vascular remodeling on the ventral surface of the retina throughout development. fzd4 mutants exhibit disorganized ventral retinal vasculature with discernable tubular fusion by week 8 of development. Furthermore, fzd4 mutants have impaired optokinetic responses requiring increased illumination. CONCLUSION: We have generated a visually impaired zebrafish FEVR model exhibiting abnormal retinal vasculature. These fish provide a tractable system for studying vascular biology in retinovascular disorders, and demonstrate the feasibility of using zebrafish for evaluating future FEVR genes identified in humans.

A start codon mutation of the TSPAN12 gene in Chinese families causes clinical heterogeneous familial exudative vitreoretinopathy.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a severe clinically and genetically heterogeneous retinal disorder characterized with failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. The purpose of this study was to investigate the molecular mechanisms by which the start codon mutation of the TSPAN12 causes difference in clinical manifestations between individuals in the same family. METHODS: Next-generation sequencing (NGS)-based target capture sequencing was performed in proband with a diagnosis of FEVR and their normal visual acuity family members. Cosegregation analysis of the candidate causative variant was performed in additional family members by using Sanger sequencing. Complete fundus examination, fundus fluorescein angiography (FFA), and family history collection were performed in all family members. Potential candidate causative variants were verified with reference to guidelines and standards from the American College of Medical Genetics and Genomics. RESULTS: We identified a novel heterozygous missense mutation (c.1A>G, p.M1V) localized in the start codon of the TSPAN12 and was detected as a potentially disease-causing variant for the proband. Retrospective analysis of clinical data, fundus examination, and FFA showed that the mutant carrier presented peripheral retinal vascular anomalies in early stages, and visual acuity did not show significant effects. However, the proband who carried this mutation and his cousin showed typical high-stage FEVR fundus changes coupled with a sharp decline in vision. CONCLUSIONS: We report a novel start codon mutation (c.1A>G, p.M1V) in the TSPAN12 that causes clinically heterogeneous manifestations. Our results expand the mutation spectrums of TSPAN12, and will be valuable for disease diagnosis, prognosis, genetic counseling, and enriching our understanding of the role of the tetraspanin-12 protein in the pathogenesis of FEVR.

Symmetry of folds in FEVR: A genotype-phenotype correlation study.

Familial exudative vitreoretinopathy (FEVR) is a hereditary retinal vascular disorder. Among the various clinical phenotypes of this disease, retinal folds are the primary and typical feature of FEVR. However, little is known about the clinical characteristics, genetic spectrum, or potential phenotype-genotype correlation of retinal folds. Herein, we describe and analyze the clinical and genetic characteristics of retinal folds in FEVR. Eighty-nine patients with unilateral or bilateral retinal folds were included in this study. Clinical examinations showed that the retinal folds were bilateral in 37 patients (41.6%). Various retinal abnormalities were noted in the fellow eyes in the remaining 52 patients with unilateral folds. Most of the folds were located temporally (98.4%, 124/126), and were complete (97.6%, 123/126). 67.5% (60/89) probands were genetic confirmed FEVR. 25 novel pathogenic mutations (7 in FZD4, 7 in LRP5, 1 in NDP, 4 in TSPAN12, and 6 in KIF11) were identified in 25 families. Overall, 87.5% (14/16) and 73.7%(14/19) patients with LRP5 and FZD4 mutations were with unilateral folds, respectively.Nevertheless, only 25% (2/8), 36.4%(4/11) and 16.7%(1/6) patients with NDP, TSPAN12, and KIF11 mutations were with unilateral folds. Moreover, 85.7%(12/14),100% (6/6) and 100%(8/8) of the patients with mutated TSPAN12, KIF11, and NDP genes presented with symmetry in disease staging, while 55% and 64.7% of patients with FZD4 and LRP5 mutation displayed symmetry in staging. In conclusion, the majority of retinal folds extended completely and radially in the temporal peripheral retina. Patients with causative mutations in NDP, TSPAN12, or KIF11 were more likely to have bilaterally symmetrical severe retinopathy. In contrast, patients with LRP5 and FZD4 mutations displayed a relatively milder but broader spectrum of phenotypes and a higher frequency of asymmetry.