A novel TSPAN12 mutation causing retinitis pigmentosa-like appearance of familial exudative vitreoretinopathy

This issue of IJO features an interventional study discussing the clinical profile, management, and outcomes of pediatric macula-off rhegmatogenous retinal detachment (RRD) secondary to familial exudative vitreoretinopathy (FEVR).[1] FEVR is a rare inherited disorder of retinal angiogenesis, characterized by phenotypic and genotypic heterogenicity.[2] Phenotypic expression may range from the presence of temporal avascular peripheral retina in an asymptomatic patient to extensive subretinal exudation, florid fibrovascular proliferation, retinal folds, tractional retinal detachment (RD) or RRD, or severe retinal dysplasia at birth resulting in severe visual dysfunction and even total blindness.[2] Treatment options include observation, laser photocoagulation or cryotherapy, anti-vascular endothelial growth factor (anti-VEGF) injection and scleral buckle, and/or pars plana vitrectomy (PPV), depending upon the stage of disease at presentation.[2] Management of RRD in FEVR is complicated by its presentation in younger age as well as by the underlying abnormal vitreoretinal interface, which is more adherent and difficult to completely separate from the retina, resulting in postoperative pre-retinal proliferation and recurrent RD.[3] Unique surgical problems are associated with these RRDs, especially in the presence of associated falciform retinal folds.[3] Where peripheral breaks with falciform folds can be supported on a scleral buckle, posterior retinal breaks in eyes with falciform folds necessitating PPV and large posterior relaxing retinectomies result in particularly dismal outcomes.[3] Due to phenotypic variability, improper and delayed diagnosis is the biggest hurdle in the management of FEVR. When presenting in the neonatal period, it can masquerade other pediatric retinal disorders like retinopathy of prematurity (ROP) and persistent fetal vasculature (PFV).[2] A small subset of premature infants who exhibit retinal findings more characteristic of FEVR than ROP have been described as ROPER (ROP vs. FEVR) by Gologorsky et al.[4] These eyes behave more like FEVR, with a less predictable and more progressive course of disease in long term.[4] FEVR can mimic PFV, especially if associated with "knifelike retinal folds "extending from the optic disk radially to the peripheral retina and anteriorly to the ciliary processes.[2] It is quite possible for a patient to have advanced FEVR findings in one eye and grossly normal other eye, further creating confusion with PFV. The stalk in PFV, unlike the falciform fold in FEVR, is not typically a retinal fold, but a hyaloid stalk of persistent vascular tissue that extends from the optic nerve to the posterior lens surface.[5] Accurate diagnosis of FEVR is best done by careful clinical examination coupled with fundus fluorescein angiography (FFA) and genetic testing wherever possible. FFA enhances the diagnostic sensitivity because subtle vascular changes in the periphery or even in the posterior pole can easily be overlooked by a routine fundus examination.[2] Wide-field retinal imaging is particularly helpful to detect asymptomatic family members. Because of phenotypic variations, genetic testing may become essential in some cases to establish the diagnosis. The inheritance pattern of FEVR may be autosomal dominant (AD; most common), autosomal recessive (AR), or X-linked recessive (XL-R), and the common genes implicated so far include NDP, FZD4, LRP5, TSPAN12, ZNF408 and KIIF11.[267] The first four genes result in mutations in the "Norrin/b-catenin signaling pathway," which plays a pivotal role in vascular morphogenesis in the eye.[2] KIF11, localized to the spindle microtubules, is involved in the mitotic progression and growth of the retinal vessels.[6] The ZNF408 protein has been implicated in abnormal retinal vascularization and trunk vascularization in zebrafish.[7] While almost all patients with NDP mutations are reported to have a severe presentation, milder phenotypes ranging from stage 2 to stage 5 have been seen in eyes with LRP5 mutations.[8] Disease phenotype is reportedly more bilaterally symmetrical in patients with KIFF11, TSPAN12, and NDP gene mutations compared to patients with LRP5 and FZD4 mutations.[9] Widespread chorioretinal degeneration may additionally be present in cases with KIFF11 and NDP mutations, along with neurodevelopmental delay and microcephaly.[10] Genetic testing in FEVR is important for diagnosis, better understanding of the disease, and prognostication. Knowledge of the gene mutation can also facilitate the molecular prenatal analysis of fetal DNA; this along with antenatal ultrasound can be used to predict FEVR in at-risk babies.[11] Timely intervention in the early neonatal period itself can prevent total blindness in some at-risk babies. FEVR, a clinically and genetically heterogeneous disorder, requires a high index of suspicion, careful examination, timely management, and long-term follow-up. Increased genetic testing in these patients will further improve our understanding of the disease. Awareness and education of parents as well is of paramount importance to reduce the risk of bilateral total blindness due to FEVR.

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Although mutations in three genes (LRP5, FZD4, and NDP) are known to cause FEVR, these account for only a fraction of FEVR cases. The proteins encoded by these FEVR genes form part of a signaling complex that activates the Norrin-beta-catenin signaling pathway. Recently, through a large-scale reverse genetic screen in mice, Junge and colleagues identified an additional member of this signaling complex, Tspan12. Here, we report that mutations in TSPAN12 also cause autosomal-dominant FEVR. We describe seven mutations identified in a cohort of 70 FEVR patients in whom we had already excluded the known FEVR genes. This study provides further evidence for the importance of the Norrin-beta-catenin signaling pathway in the development of the retinal vasculature and also indicates that more FEVR genes remain to be identified.

Objective To observe and analyze the genotype and clinical phenotype in 34 families of familial exudative vitreoretinopathy associated with (FEVR) gene variation. Methods Cohort study. Thirty-four FEVR families, in which the patients and both of their parents were all found to have FEVR-related gene mutations (proband 34 cases, 67 eyes; parents 68 cases, 136 eyes), were included in the study. These patients were identifIed from 722 FEVR patients through genetic screening, which diagnosed in Department of Ophtalmology of Xinhua Hospital and Tianjin Medical University Eye Hospital from January 2010 to December 2018. The probands and their parents underwent a comprehensive ophthalmological examination appropriate to their age, including BCVA, intraocular pressure, axial length, slit lamp examination, indirect ophthalmoscopy, FFA or color fundus photography or wide field color fundus photography. According to the severity of the disease, the clinical manifestations were divided into severe phenotype and mild phenotype. Thirty-four normal healthy people over 40 years old were included as the control group. The peripheral blood samples of FEVR family members and control group members were collected, and the genes known to be involved in FEVR, such as FZD4, LRP5, NDP, TSPAN12, ZNF408 and KIF11, were analyzed by next generation sequencing molecular genetics. The data were statistically analyzed by SPSS. The counting data was expressed in numbers or rates, and tested by Kruskal-Wallis test and χ2 test to find out the existence of significant difference. Results In 67 eyes of the 34 probands, 48 eyes (71.64%) were classified into severe phenotype and 19 eyes (28.36%) were mild phenotype. In 136 eyes of 68 parents of the proband patients, 76 eyes (55.88%) were normal, 60 eyes (44.12%) were classified into mild phenotype, and no severe phenotype was found. A total of 65 variants of FEVR-related genes were detected in the 34 probands, of which LRP5 mutation was the most common (64.61 %), followed by FZD4 (12.31%), NDP (10.77%), TSPAN12 (6.15%), ZNF408 (4.62%) and KIF11 (1.54%). Missense mutations were the most common variant in FEVR-related genes. However, the results of correlation analysis indicated that there was no significant correlation between the type of mutation and the severity of clinical phenotype (H=1.775, P=0.620). Among the 65 mutation types, 21 types have been previously identified and 44 were novel in this study. Thirty-nine eyes of 20 cases had only one single pathogenic mutation gene but with multiple mutation sites, 26 eyes of 13 cases carried 2 relevant pathogenic mutation genes, and 2 eyes in one case had 3 pathogenic mutation genes. The mutation frequencies of LRP5, NDP, ZNF408, FZD4, TSPAN12 and KIF11 genes in probands were significantly higher than those in control group, and the difference was statistically significant. The total mutation frequencies of LRP5, NDP, ZNF408, FZD4, TSPAN12 and KIF11 genes in proband group were significantly higher than those in control group (χ2=64.702, P <0.001). Conclusions In the FEVR families, the most frequent mutations were those in LRP5, followed by FZD4, NDP, TSPAN12,ZNF408 and KIF11. Missense mutation is the most common type of FEVR-related gene mutation, but there is no significant correlation between the clinical phenotype and gene variation type. Most of the probands were with severe clinical phenotype, while most of the parents with FEVR pathogenic gene mutation showed normal or mild manifestations. Key words: Retinal diseases/genetics; Genotype; Mutation; Familial exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disease characterized by defects in the development of periphery retinal vessels. However, the clinical phenotypes of FEVR vary widely from asymptomatic to complete blindness. We analyzed patients from three Chinese families and one sporadic patient with FEVR to investigate the clinical features and disease-causing mutations. Ocular phenotypes included increased ramification of the peripheral retinal vessels, a peripheral avascular zone, inferotemporal dragging of the optic disc and macula, and retinal folds. Peripheral blood DNA samples were obtained from patients with FEVR and their family members. Primers were designed to amplify the coding exons and adjacent intronic regions of the FEVR-causing genes FZD4, LRP5, NDP and TSPAN12. By polymerase chain reactions, each amplicon was subjected to direct Sanger sequencing analysis. Potential pathogenic changes of the sequence variants were analyzed by the orthologous protein sequence alignment and computational prediction software. We identified five LRP5 mutations: three novel heterozygous mutations-p.M181R, p.R399S and p.G503R and two known mutations that were never reported in FEVR patients: p.R494Q and p.G876S. All five mutations involved highly conserved residues and were predicted to be damaging by SIFT and PolyPhen-2. None was present in 500 normal individuals. To assess the pathogenesis of these mutations, wild-type and all five mutant LRP5 proteins were assayed for the ability to activate the Norrin/β-catenin pathway by established luciferase reporter assays, and all mutants failed to activate the pathway. This study extends the genetic database of the FEVR disease in China and provides a basis for molecular diagnosis of the disease.

Familial exudative vitreoretinopathy (FEVR) is a rare inherited slowly progressive disorder characterized by failure of vascularization of the peripheral retina and poor vascular differentiation. Retinitis pigmentosa (RP) is a group of clinically and genetically heterogeneous inherited retinal disorders characterized by diffuse progressive dysfunction of predominantly rod photoreceptors with subsequent involvement of cone photoreceptors. A 13-year-old boy presented with nyctalopia and low vision for 10 years. Fundus evaluation showed retinal pigmentary changes in the mid-periphery. The fundus fluorescein angiogram showed straightening of retinal vessels, vascular leakage, and avascularity of the peripheral retina, suggestive of FEVR. Full-field electroretinogram (FFERG) showed grossly reduced scotopic and photopic responses in both eyes, pointing toward retinitis pigmentosa (RP). To the best of our knowledge, this case is the second one internationally, and the first in India to show the coexistence of FEVR and RP in the same patient.

Objective To analysis the genotype and phenotype of hereditary retinal diseases (HRD) which are easily misdiagnosed as amblyopia. Methods A case-control study was designed.The patients with HRD who were misdiagnosed as amblyopia in Ningxia Eye Hospital from January to December, 2017 were recruited in this study.The clinical medical history and ophthalmic examinations of patients and their family members were recorded, and family maps were drawed.Peripheral venous blood (5 ml) from each patient and their family members was collected, and genomic DNA was extract.The target sequence capture sequencing technology was used to detect the genetic testing in serum of the patient, and the pathogenic mutation site was determined by Sanger sequencing and co-segregation verification.Genetic testing results with related ophthalmic examination were considered together to analyze the relationship between genotype and phenotype.This study followed the Declaration of Helsinki.Written informed consent was obtained from each subject or the guardian prior to entering study cohort.This study protocol was approved by Ethic Committee of People's Hospital of Ningxia Hui Autonomous Region Hospital (No.2016018). Results Twenty-two patients with HRD were enrolled in the study, including 10 Stargardt disease (STGD), 8 cases of cone dystrophy (COD) or cone and rod dystrophy (CRD), and 5 cases of familial exudative vitreoretinopathy(FEVER). Nine patients were detected to have pathogenic mutations, and the positive rate was 40.9%, of which 4 patients with STGD carried mutation gene, including ABCA4 and PROM1 genes; mutations in RPGR, PROM1 and GUCY2D genes were detected in 3 patients with COD or CRD; TSPAN12 gene mutation were detected in 2 patients with FEVER.Eleven mutation sites were detected, 4 of which were newly discovered mutation sites.All of the patients in 9 HRD families developed symptoms during adolescence.At the early stage of the disease, there was severe damage to the eyesight, but the fundus was normal or only slightly abnormal.As the disease progressed, the fundus changes were characteristic, and there were clinical phenotypic overlap between some diseases.All family genotypes and clinical phenotypes were co-separated. Conclusions The main pathogenic gene of STGD is ABCA4 gene, and PROM1 gene can also cause partial STGD; COD and CRD have similar clinical manifestations, and the pathogenic genes also cross each other, and the genetic pattern is diverse; FEVER caused by mutation of TSPAN12 gene is autosomal dominant, and the mutation type has missense mutation and frameshift mutation.HRDs lack typical early clinical signs, and genetic diagnosis can provide pre-symptomatic diagnosis. Key words: Hereditary retinal disease; Genotype; Phenotype

Correlating Changes in the Macular Microvasculature and Capillary Network to Peripheral Vascular Pathologic Features in Familial Exudative Vitreoretinopathy

While Inherited Retinal Diseases (IRDs) are typically considered rare diseases, Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie Disease (ND) are more rare than retinitis pigmentosa. We wanted to determine if multigenic protein-altering variants are common in FEVR subjects within a set of FEVR-related genes. The potential occurrence of protein-altering variants in two different genes has been documented in a very small percentage of patients, but potential multigenic contributions to FEVR remain unclear. Genes involved in these orphan pediatric retinal diseases are not universally included in available IRD targeted-sequencing panels, and cost is also a factor limiting multigenic-sequence-based testing for these rare conditions. To provide an accurate solution at lower cost, we developed a targeted-sequencing protocol that includes seven genes involved in Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie disease. Seventy-six DNA samples from persons refered to clinic with possible FEVR and some close relatives were sequenced using a novel Oakland-ERI orphan pediatric retinal disease panel (version 2) providing 900 times average read coverage. The seven genes involved in FEVR/ND were: NDP (ChrX), CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), ZNF408 (Chr11). A total of 33 variants were found that alter protein sequence, with the following relative distribution: LRP5 13/33 (40%), FZD4 9/33 (27%), ZNF408 6/33 (18%), (KIF11 3/33 (9%), NDP 1/33 (3%), CTNNB1 1/33 (3%). Most protein-altering variants, 85%, were found in three genes: FZD4, LRP5, and ZNF408. Four previously known pathogenic variants were detected in five families and two unrelated individuals. Two novel, likely pathogenic variants were detected in one family (FZD4: Cys450ter), and a likely pathogenic frame shift termination variant was detected in one unrelated individual (LRP5: Ala919CysfsTer67). The average number of genes with protein-altering variants was greater in subjects with confirmed FEVR (1.46, n = 30) compared to subjects confirmed unaffected by FEVR (0.95, n = 20), (p = 0.009). Thirty-four percent of persons sequenced had digenic and trigenic protein-altering variants within this set of FEVR genes, which was much greater than expected in the general population (3.6%), as derived from GnomAD data. While the potential contributions to FEVR are not known for most of the variants in a multigenic context, the high multigenic frequency suggests that potential multigenic contributions to FEVR severity warrant future investigation. The targeted-sequencing format developed will support such exploration by reducing the testing cost to $250 (US) for seven genes and facilitating greater access to genetic testing for families with this very rare inherited retinal disease.

Genetic Testing for Inherited Retinal Disease

Using Molecular Diagnostics for Inherited Retinal Dystrophies: The 6 "I"s That Are Necessary to Diagnose 2 Eyes Genetically.

Objective To identify mutations in NDP, FZD4, LRP5, TSPAN12 in Chinese families with familial exudative vitreoretinopathy (FEVR) and observe the clinical features. Methods Retrospective case series study. The 9 patients (18 eyes) and 5 normal members from 4 unrelated families were included in the study. The patients medical history and family history were collected in detail. All patients underwent best corrected visual acuity (BCVA), slit-lamp biomicroscopy, fundus colorized photography, fundus fluorescein angiography (FFA). Genomic DNA were collected from all the patients. Mutations were detected by directly sequencing to the whole coding region and exon-intron boundaries of NDP, FZD4, LRP5 and TSPAN12 gene. Polyphen and SIFT programs were used to predict the effects on the structure and functional properties of mutant protein. Results There were two affected individuals in the family 2 carried LRP5 gene mutation [c.1330C> T(p.R444C )] in exon 6 by sequence analysis. A score of 0.882 was acquired by Polyphen program analysis. And the missense change was predicted to be pathogenic by SIFT. Fundus changes of the proband showed angioplasia, tortuosity of peripheral vessels. And temporal dragging of the optic disc, peripheral avascular zone, neovascularization were found in FFA. Brush-like and straight of peripheral vessels were found in Ⅰ1. No variant was found in NDP, FZD4 and TSPAN12 gene. Conclusion Our study supports the gene mutation c.1330C> T (p.R444C) of LRP5 is pathogenesis of FEVR. Patients with the same mutation could have variable phenotypic characteristics. Key words: Retinal diseases/genetics; Retinal diseases/etiology; Genes; Mutation; Sequence analysis

Introduction Familial Exudative Vitreoretinopathy (FEVR) is a heritable retinal vascular disease characterized by incomplete vascularization of the peripheral retina resulting in ischemia. Fifty percent of FEVR cases 10 are due to known pathogenic genetic variants, and disease phenotype can vary greatly. FEVR is a clinical diagnosis, however, genetic testing can play a key role in screening for FEVR in genetically susceptible populations, thus leading to early treatment and improved patient outcomes. Case A 2-year-old male with no known past ocular or medical history was diagnosed with FEVR upon examination under anesthesia and multimodal retinal imaging. Genetic testing identified a Jagged 1 (JAG1) variant of uncertain significance, 15 which has been linked to FEVR in recent studies. Despite close follow-up and treatment, the patient experienced a funnel retinal detachment in the right eye approximately one year after diagnosis. Discussion This case in conjunction with recent literature suggests that JAG1 variants are likely associated with FEVR. Further investigations are necessary to identify the frequency of JAG1 variants among patients with FEVR. Robust understanding of FEVR’s heterogenous genetic profile will lead to improved treatment modalities 20 and patient outcomes.

PurposeTo report a rare clinical finding of preretinal granules associated with atypical familial exudative vitreoretinopathy (FEVR) and perform a review of the literature. ObservationsAn asymptomatic 18-year-old male was referred for unilateral peripheral avascular retina evaluation in association with presumed FEVR. He was first noted to have white preretinal granules on fundus examination at five years of age. The lesions remained unchanged over the subsequent years. Genetic testing did not reveal a pathogenic or likely pathogenic variant in a known FEVR gene. A review of the literature revealed five other cases of FEVR with similar findings. Conclusions and ImportanceLiterature review suggests preretinal granules may present rarely in FEVR. Negative genetic screening of known FEVR genes in our patient with atypical FEVR suggests either a molecularly distinct etiology supporting the rarity of this association with FEVR or, alternatively, the presence of granules in developmental retinal vascular anomalies that are not specific to FEVR. Future study and genetic testing is necessary to better understand the cause of these preretinal granules and the clinical manifestations of FEVR.

To report a case of Familial Exudative Vitreoretinopathy (FEVR) where both the mother and the daughter had the same genetic mutation in TSPAN 12, and the daughter had treatable retinal detachment at birth. This is an interventional case report. A 28-year-old primi gravida, a known case of FEVR presented to our OPD in the first trimester. Targeted Imaging for Fetal Anomalies (TIFFA scan) ruled out any anomalies. A planned near-term but early elective delivery was done at the 36th week of gestational age, and the child was brought for early neonatal screening for FEVR at ten days of age. The birth weight was 3.6 kg. Fundus evaluation showed bilateral disc and arcade drag due to a highly vascular temporal peripheral tractional retinal detachment. Diagnosis of bilateral FEVR - Stage 3A retinal detachment was made. A bilateral lens-sparing vitrectomy with endolaser was performed to mitigate the traction onto the retina. The child did well postoperatively. Genetic testing of the mother and the child showed the same TSPAN12 (c.738G>A) gene mutation. Surgical success in FEVR can hinge on the timely screening of the new family member. Progression of FEVR to more advanced stages may be prevented with laser photocoagulation and prompt surgery whenever indicated. It is imperative to educate FEVR patients about the risk to the next generation and the importance of good antenatal monitoring, early delivery, and immediate postnatal ophthalmic fundoscopic examination of newborns.

AimRetinopathy of prematurity (ROP) is a vasoproliferative eye disease in preterm infants. Based on its phenotypic similarities with familial exudative vitreo retinopathy (FEVR), the present study was conducted to screen...