TSPAN12 Research Articles

Tetraspanin-enriched microdomains play an important role in pathogenesis in the protozoan parasite Entamoeba histolytica.

Tetraspanins (TSPANs) are a family of highly conserved proteins present in a wide variety of eukaryotes. Although protein-protein interactions of TSPANs have been well established in eukaryotes including parasitic protists, the role they play in parasitism and pathogenesis remains largely unknown. In this study, we characterized three representative members of TSPANs, TSPAN4, TSPAN12, and TSPAN13 from the human intestinal protozoan Entamoeba histolytica. Co-immunoprecipitation assays demonstrated that TSPAN4, TSPAN12 and TSPAN13 are reciprocally pulled down together with several other TSPAN-interacting proteins including TSPAN binding protein of 55kDa (TBP55) and interaptin. Blue native-PAGE analysis showed that these TSPANs form several complexes of 120-250 kDa. Repression of tspan12 and tspan13 gene expression led to decreased secretion of cysteine proteases, while repression of tspan4 led to a four-fold increase in the activity of cysteine proteases in crude extracellular vesicles (EVs) fraction. Meanwhile, strains overexpressing HA-tagged TSPAN12 and TSPAN13 demonstrated reduced adhesion to collagen. Altogether, this study reveals that the TSPANs, especially TSPAN12 and TSPAN13, are engaged with complex protein-protein interactions and are involved in the pathogenicity-related biological functions such as protease secretion and adhesion, offering insights into the potential regulatory mechanisms of tetraspanins in protozoan parasites.

Familial Exudative Vitreoretinopathy With and Without Pathogenic Variants of Norrin/β-Catenin Signaling Genes

PurposeTo determine the clinical characteristics of familial exudative vitreoretinopathy (FEVR) associated with or without pathogenic variants of the Norrin/β-catenin genes. DesignThis was a multicenter, cross-sectional, observational, and genetic study. SubjectsTwo-hundred eighty-one probands with FEVR were studied. MethodsWhole exome sequence and/or Sanger sequence was performed for the Norrin/β-catenin genes, the FZD4, LRP5, TSPAN12 and NDP genes on blood collected from the probands. The clinical symptoms of the probands with or without the pathogenic variants were assessed as well as differences in the inter Norrin/β-catenin genes. ResultsOne-hundred eight probands (38.4%) had 88 different pathogenic or likely pathogenic variants in the genes: 24 with the FZD4, 42 with the LRP5, 10 with the TSPAN12, and 12 with the NDP gene. Compared with the 173 probands without pathogenic variants, the 108 variant-positive probands had characteristics of familial predisposition (63.9% vs 37.6%, P <0.0001), progression during infancy (75.0% vs 53.8%, P = 0.0004), asymmetrical severity between the two eyes (50.0% vs 37.6%, P = 0.0472), and non-syndromic characteristics (10.2% vs 17.30%, P = 0.1185). The most frequent stage at which the more severe eye conditions was present was at Stage 4 in both groups (40.7% vs 34.7%). However, the advanced Stages of 3 to 5 in the more severe eye was found more frequently in probands with variants than in those without variants (83.3% vs 58.4%, P <0.0001). Patients with rhegmatogenous retinal detachments progressed from Stage 1 or 2 were found less frequently in the variant-positive probands (8.3% vs 17.3%, P = 0.0346). Eight probands with NDP variants had features different from probands with typical Norrin/β-catenin gene variants including the sporadic, symmetrical, and systemic characteristics consistent with Norrie disease. ConclusionsThe results showed that the clinical characteristics of FEVR of patients with variants in the Norrin/β-catenin genes are different from those with other etiologies. We recommend that clinicians who diagnose a child with FEVR perform genetic testing so that the parents can be informed on the prognosis of the vision and general health in the child.

Mutations in TSPAN12 gene causing familial exudative vitreoretinopathy

BackgroundTo 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.ResultsNine 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.ConclusionsNine 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.

De novel heterozygous copy number deletion on 7q31.31-7q31.32 involving TSPAN12 gene with familial exudative vitreoretinopathy in a Chinese family.

To investigate the genetic and clinical characteristics of patients with a large heterozygous copy number deletion on 7q31.31-7q31.32. A family with familial exudative vitreoretinopathy (FEVR) phenotype was included in the study. Whole-exome sequencing (WES) was initially used to locate copy number variations (CNVs) on 7q31.31-31.32, but failed to detect the precise breakpoint. The long-read sequencing, Oxford Nanopore sequencing Technology (ONT) was used to get the accurate breakpoint which is verified by quantitative real-time polymerase chain reaction (QPCR) and Sanger Sequencing. The proband, along with her father and younger brother, were found to have a heterozygous 4.5 Mb CNV deletion located on 7q31.31-31.32, which included the FEVR-related gene TSPAN12. The specific deletion was confirmed as del(7)(q31.31q31.32)chr7:g.119451239_123956818del. The proband exhibited a phase 2A FEVR phenotype, characterized by a falciform retinal fold, macular dragging, and peripheral neovascularization with leaking of fluorescence. These symptoms led to a significant decrease in visual acuity in both eyes. On the other hand, the affected father and younger brother showed a milder phenotype. The heterozygous CNV deletion located on 7q31.31-7q31.32 is associated with the FEVR phenotype. The use of long-read sequencing techniques is essential for accurate molecular diagnosis of genetic disorders.

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/β-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/β-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/β-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.

To report the novel causative variants in five Chinese families with familial exudative vitreoretinopathy (FEVR). 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/β-catenin signaling activity. 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/β-catenin signaling activity. 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. 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.

Mutation spectrum in a cohort with familial exudative vitreoretinopathy

PurposeTo expand the mutation spectrum of patients with familial exudative vitreoretinopathy (FEVR) disease.Participants74 probands (53 families and 21 sporadic probands) with familial exudative vitreoretinopathy (FEVR) disease and their available family members (n = 188) were recruited for sequencing.MethodsPanel‐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.Results40.54% (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.ConclusionsWe 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.

Commentary: Familial exudative vitreoretinopathy-The masquerade in pediatric retinal disorders.

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.

Whole exome sequencing revealed 14 variants in NDP, FZD4, LRP5, and TSPAN12 genes for 20 families with familial exudative vitreoretinopathy

BackgroundFamilial exudative vitreoretinopathy (FEVR) is a complex form of blindness-causing retinal degeneration. This study investigated the potential disease-causing variants in 20 Chinese families with FEVR.MethodsAll available family members underwent detailed ophthalmological examinations, including best-corrected visual acuity and fundus examination. All probands and most family members underwent fluorescein fundus angiography. Twenty probands underwent whole exome sequencing; 16 of them also underwent copy number variant and mitochondrial genome analysis. Bioinformatics analysis and Sanger sequencing of available family members were used to confirm the disease-causing gene variant.ResultsTwenty families were diagnosed with FEVR based on clinical symptoms, fundus manifestations, and fundus fluorescein angiography. Whole exome sequencing revealed 14 variants in NDP, FZD4, LRP5, and TSPAN12 genes among the 13 families. These variants were predicted to be damaging or deleterious according to multiple lines of prediction algorithms; they were not frequently found in multiple population databases. Seven variants had not previously been reported to cause FEVR: c.1039T>G p.(Phe347Val) in the FZD4 gene; c.1612C>T p.(Arg538Trp) and c.3237-2A>C in the LRP5 gene; and c.77T>A p.(Ile26Asn), c.170dupT p.(Leu57Phe fsTer60), c.236T>G p.(Met79Arg) and c.550dupA p.(Arg184Lys fsTer16) in the TSPAN12 gene. We did not detect any variants in the remaining seven families.ConclusionsThese results expand the spectrum of variants in the NDP, FZD4, LRP5, and TSPAN12 genes and provide insights regarding accurate diagnosis, family genetic counseling, and future gene therapy for FEVR.

Ocular phenotype and genetical analysis in patients with retinopathy of prematurity

BackgroundRetinopathy of prematurity (ROP) is a multifactorial retinal disease, involving both environmental and genetic factors; The purpose of this study is to evaluate the clinical presentations and genetic variants in Chinese patients with ROP.MethodsA total of 36 patients diagnosed with ROP were enrolled in this study, their medical and ophthalmic histories were obtained, and comprehensive clinical examinations were performed. Genomic DNA was isolated from peripheral blood of ROP patients, polymerase chain reaction and direct sequencing of the associated pathogenic genes (FZD4, TSPAN12, and NDP) were performed.ResultsAll patients exhibited the clinical manifestations of ROP. No mutations were detected in the TSPAN12 and NDP genes in all patients; Interestingly, three novel missense mutations were identified in the FZD4 gene (p.A2P, p.L79M, and p.Y378C) in four patients, for a detection rate of 11.1% (4/36).ConclusionsThis study expands the genotypic spectrum of FZD4 gene in ROP patients, and our findings underscore the importance of obtaining molecular analyses and comprehensive health screening for this retinal disease.

Loss of Endothelial TSPAN12 Promotes Fibrostenotic Eosinophilic Esophagitis via Endothelial Cell–Fibroblast Crosstalk

Eosinophilic esophagitis (EoE) can progress to fibrostenosis by unclear mechanisms. Herein, we investigated gene dysregulation in fibrostenotic EoE, its association with clinical parameters and specific pathways, and thefunctional consequences. Esophageal biopsies from subjects with EoE were collected across 11 Consortium of Eosinophilic Gastrointestinal Disease Researchers sites (n= 311) and 2 independent replication cohorts (n= 83). Inclusion criteria for fibrostenotic EoE were endoscopic rings, stricture, and/or a history of dilation. Endoscopic, histologic, and molecular features were assessed by the EoE Endoscopic Reference Score, EoE Histology Scoring System, EoE Diagnostic Panel, and RNA sequencing. Esophageal endothelial TSPAN12 expression and functional effects on barrier integrity and gene expression were analyzed invitro. TSPAN12 was the gene most correlated with fibrostenosis (r=-0.40, P < .001). TSPAN12 was lower in fibrostenotic EoE and correlated with EoE Endoscopic Reference Score, EoE Diagnostic Panel, and EoE Histology Scoring System (r= 0.34-0.47, P < .001). Lower TSPAN12 associated with smaller esophageal diameter (r= 0.44, P= .03), increased lamina propria fibrosis (r=-0.41, P < .001), and genes enriched in cell cycle-related pathways. Interleukin (IL)-13 reduced TSPAN12 expression in endothelial cells. Conversely, anti-IL-13 therapy increased TSPAN12 expression. TSPAN12 gene silencing increased endothelial cell permeability and dysregulated genes associated with extracellular matrix pathways. Endothelial cell-fibroblast crosstalk induced extracellular matrix changes relevant to esophageal remodeling. Patients with fibrostenotic EoE express decreased levels of endothelial TSPAN12. We propose that IL-13 decreases TSPAN12, likely contributing to the chronicity of EoE by promoting tissue remodeling through fibroblast-endothelial cell crosstalk.

Familial Exudative Vitreoretinopathy (FEVR); Pathophysiology, Findings, Diagnosis, and Treatment

Familial exudative vitreoretinopathy (FEVR) is an inherited disease occurring due to defective retinal angiogenesis. FEVR patients have an avascular peripheral retina which, depending on the degree of ischemia, causes the secondary complications of the disease (retinal neovascularization, exudation, hemorrhage, and retinal detachment) Mutations in the NDP, FZD4, LRP5, TSPAN12 and ZNF08 genes have been shown to contribute to FEVR. Cases can be inherited in an autosomal dominant, autosomal recessive, or X-linked manner, or can affect individuals with no family history. Examination with wide-field fluorescein angiography is essential and can identify the disease in its earlier stages, enabling the timely treatment, in addition to helping identify asymptomatic family members. The current treatment modalities involve laser photocoagulation of the avascular peripheral retina for neovascular complications and vitreoretinal surgery for retinal detachment. Studies are ongoing to better understand the pathogenesis of FEVR and to improve treatment

Whole-Exome Sequencing Reveals Novel TSPAN12 Variants in Autosomal Dominant Familial Exudative Vitreoretinopathy.

Background: Familial exudative vitreoretinopathy (FEVR), a group of rare inherited retinal vascular disorders, is the major cause of vision loss in juveniles. At present, the diagnosis of FEVR remains difficult due to its clinical and genetic heterogeneities. Aims: To identify the causative genetic variants in two unrelated FEVR-affected families: one Indian family and one Chinese Han family. Materials and Methods: Five affected patients from two families were recruited for this study. Whole-exome sequencing was applied to the probands, and Sanger sequencing was performed for validation. Stringent whole-exome sequence data analyses were performed to evaluate all of the identified pathogenic variants. Results: Two novel variants in the TSPAN12 gene, were identified: a missense variant c.437 T > G (p.Leu146Arg); and a nonsense variant c.477 C > A (p.Cys159*). Both variants cosegregated with the disease in the investigated FEVR-affected families. Additionally, both variants inactivated the ability of TSPAN12 protein to enhance Norrin/β-catenin signaling. Conclusion: This study expands the mutational spectrum of TSPAN12 for FEVR.

Cohort study of genotype and clinical phenotype in 34 families with familial exudative vitreoretinopathy

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

Differential Expression Analysis on Schizophrenia Dataset Suggests Pseudogene RNU6-505P as under Selective Pressure

Schizophrenia is one of the 15 leading causes of disability worldwide. About 1% of the global population has schizophrenia, with 10% of premature mortality chance. Schizophrenia is therefore associated with significant health, social and economic concerns. In this context, thalamus and striatum areas play important roles as much in schizophrenia as processing information before reaching the conscious thought: step happening soon in the creativity action. Creativity is defined by psychological scientists as the generation of ideas or products that are both original and valuable. Creativity relies on imagination and this fundamental human ability remains understudied in comparison to other important psychological phenomena. It is natural to ask whether the gene expression profiling of samples from schizophrenic patients could highlight the activity of some genes specific to humans. Microarray analysis of the dataset GSE25673 revealed that the pseudogene RNU6-505P is expressed differentially in schizophrenic samples and correlates to CYP26A1, ARHGAP18, TSPAN12, HEY2 and TMEM132A genes. Ontological analysis showed that the RNU6-505P pseudogene is involved in brain development and certain neurological pathologies. Evolutionary analysis showed that the AGA 3-nucleotide sequence of RNU6-505P has been under positive selective pressure. Finally, the 1-nucleotide mutation prediction test revealed that variations on the AGA nucleotides could be fatal to the RNA structure of the sequence. We conclude that differential expression of the RNU6-505P pseudogene can be valid to diagnose schizophrenia and the RNU6-505P pseudogene may have a relevant function in the cerebral development and in the divergent evolution of humans from apes.

Analysis of genotype and phenotype of hereditary retinal diseases which are easily misdiagnosed as amblyopia

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

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.

Genetic variants of TSPAN12 gene in patients with retinopathy of prematurity.

Genetic susceptibility to retinopathy of prematurity (ROP) has been reported. However, no virulence genes are currently known for ROP. This study aimed to assess FZD4, LRP5, TSPAN12, and NDP, which are known virulence genes involved in familial exudative vitreoretinopathy, an ailment that shares some symptoms with ROP. After approval from the parents of diseased infants, blood samples from 29 Han patients with ROP were collected for genomic DNA extraction. Direct sequencing was used to assess the four candidate genes, namely FZD4, LRP5, TSPAN12, and NDP. Finally, genetic mutations were screened. Changes of three nucleotide sequences were found in the four candidate genes; notably, a c.954G>A hybrid mutation in the TSPAN12 gene was predicted to cause protein structure and function alterations. The molecular pathogenesis of ROP is complex, and likely involves the c.954G>A mutation in TSPAN12.

Detection of FZD4, LRP5 and TSPAN12 Genes Variants in Malay Premature Babies with Retinopathy of Prematurity.

Purpose:To determine the mutational analyses of familial exudative vitreoretinopathy (FEVR)-causing genes in Malay patients with retinopathy of prematurity (ROP) to obtain preliminary data for gene alterations in the Malay community.Methods:A comparative cross-sectional study involving 86 Malay premature babies (ROP = 41 and non-ROP = 45) was performed from September 2012 to December 2014. Mutation analyses in (FEVR)-causing genes (NDP, FZD4, LRP5, and TSPAN12) were performed using DNA from premature babies using polymerase chain reaction (PCR) and direct sequencing. Sequencing results were confirmed with PCR-Restriction Fragment Length Polymorphism (RFLP).Results:We found variants of FZD4, LRP5, and TSPAN12 in this study. One patient from each group showed a non-synonymous alteration in FZD4, c.502C>T (p.P168S). A synonymous variant of LRP5 [c.3357G>A (p.V1119V)] was found in 30 ROP and 28 non-ROP patients. Two variants of TSPAN12, c.765G>T (p.P255P) and c.*39C>T (3′UTR), were also recorded (29 and 21 in ROP, 33 and 26 in non-ROP, respectively). Gestational age and birth weight were found to be significantly associated with ROP (P value < 0.001 and 0.001, respectively).Conclusion:Analysis of data obtained from the ROP Malay population will enhance our understanding of these FEVR-causing gene variants. The c.3357G>A (p.V1119V) variant of LRP5, and c.765G>T (p.P255P) and c.*39C>T variants of TSPAN12 could be common polymorphisms in the Malay ethnic group; however, this requires further elucidation. Future studies using larger groups and higher numbers of advanced cases are necessary to evaluate the relationship between FEVR-causing gene variants and the risk of ROP susceptibility in Malaysian infants.

Clinical features and identification of gene mutation in families with familial exudative vitreoretinopathy

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