ObjectiveTo reveal the pathogenic mutation in a three-generation Chinese family with autosomal dominant familial exudative vitreoretinopathy (FEVR). MethodsThree patients and a healthy spouse from the index family with FEVR were recruited. The proband was a 5 years old boy. His mother and grandpa were presented with typical FEVR presentations, while his father with normal ocular fundus. DNA was extracted from peripheral blood samples taken from all four participants. All coding and exon-intron boundary regions of five targeted genes, including NDP, FZD4, LRP5, TSPAN12 and ZNF408 were amplified with polymerase chain reaction and sequenced using direct sequencing. In silico analyses were applied to determine the conservation of the mutation site, pathogenic effect and the potential protein crystal structural changes caused by the mutation. ResultsFZD4 c.478G > A, a susceptible mutation was found after four high frequency mutation sites which MAF values were higher than 0.001 was filtered among 5 single nucleotide variations detected in four participants, leading to the residue 160 changing from glutamate to lysine (p.E160K). Co-segregation analysis between genotypes and phenotypes revealed FZD4 p.E160K as the disease-causing mutation for this family. Conservational analysis suggested that this mutation site was highly conserved among all tested species. Functional analysis predicated that this mutation may be a damaging mutation. Crystal structural analysis also indicated that this mutation could lead to the elimination of the hydrogen bond between residue 160 and asparagine at residue 152, thus altering the tertiary structure of the protein and further impairing the protein function. ConclusionOur study demonstrates FZD4 p.E160K as a novel pathogenic mutation for FEVR.
The rapid development of genetic diagnosis-related technologies has paved a wide road for gene therapy. Different gene therapy clinical trials for retinal disorders, including gene-replacement therapy, anti-neovascular gene therapy and opotogenetic gene therapy, have been developed and achieved fruitful results, which have gradually confirmed the efficacy and safety of adeno-associated virus (AAV)-mediated gene therapy for recessive retinal diseases. In recent years, novel gene editing technologies also shows great potential to treat dominant retinal disease, or recessive retinal disease when the therapeutic gene fragments are too long to fit into the AAV vectors. These results make it possible for most of the patients with inherited retinal diseases to be treated by the safe and effective AAV-mediated gene therapy, which will also benefit Chinese patients soon.
Familial exudative vitreoretinopathy (FEVR) is a rare inherited disorder of retinal angiogenesis, including autosomal dominant, autosomal recessive, or X-linked forms. Zinc finger protein 408 (ZNF408) was recently found to be associated with FEVR. Cell transfection showed that it was a dominant negative regulator of FEVR pathogenesis. Knocking down ZNF408 in zebrafish by antisense morpholino oligonucleotides indicated it involved in retinal blood vessel development. Understanding the protein structure, gene localization, basic functions and the role of ZNF408 in retinal development will contribute to uncover the pathogenesis of FEVR.
Objective To analyze the BEST1 gene mutations and clinical features in patients with multifocal vitelliform retinopathy (MVR). Methods This is a retrospective case series study. Five MVR families with MVR, including 9 patients and 10 healthy family members were recruited. Clinical evaluations were performed in all MVR patients and their family members, including best-corrected visual acuity (BCVA), intraocular pressure (IOP), refraction, slit-lamp examination, 90 D preset lens examination, gonioscopy, color fundus photography, optical coherence tomography (OCT), fundus autofluorescence (AF), ultrasound biomicroscopy (UBM) and axial length measurement. Electro-oculogram (EOG) was performed in 12 eyes and visual field were performed in 13 eyes. Peripheral blood samples were collected in all subjects to extract genomic DNA. Coding exons and flanking intronic regions of BEST1 were amplified by polymerase chain reaction and analyzed by Sanger sequencing. Results Among the 5 MVR families, 3 probands from three families had family history, including 1 family had autosomal dominant inheritance pattern. Two patients from 2 families were sporadic cases. Screening of BEST1 gene identified four mutations, including three missense mutations (c.140G>T, p.R47L; c.232A>T, p.I78F; c.698C>T, p.P233L) and 1 deletion mutation (c.910_912del, p.D304del). Two mutations (p.R47L and p.I78F) were novel. The BCVA of affected eyes ranged from hand motion to 1.0. The mean IOP was (30.39±11.86) mmHg (1 mmHg=0.133 kPa). The mean refractive diopter was (-0.33±1.68) D. Twelve eyes had angle-closure glaucoma (ACG) and 4 eyes had angle closure (AC). EOG Arden ratio was below 1.55 in all patients. The mean anterior chamber depth was (2.17±0.29) mm. Visual field showed defects varied from paracentral scotoma to diffuse defects. The mean axial length was (21.87±0.63) mm. All MVR patients had multifocal vitelliform lesions in the posterior poles of retina. ACG eyes demonstrated pale optic disc with increased cup-to-disc ratio. OCT showed retinal edema, extensive serous retinal detachment and subretinal hyper-reflective deposits which had high autofluorescence in AF. The genetic testing and clinical examination were normal in 10 family members. Conclusions MVR patients harbored heterozygous mutation in the BEST1 gene. Two novel mutations (p.R47L and p.I78F) were identified. These patients had clinical features of multifocal vitelliform retinopathy and abnormal EOG. Most patients suffered from AC/ACG.
Familial exudative vitreoretinopathy (FEVR) is a hereditary disease with high geneticheterogeneity, including autosomal dominant inheritance, autosomal recessive inheritance, snd X-linked recessive inheritance. So far, six genes have been found to be associated with FEVR: Wnt receptor fizzled protein (FZD4), Norrie disease (NDP), co-receptor low-densitylipoprotein receptor-related protein 5 (LRP5), and tetrasin 12 (TSPANI2), zinc finger protein408 (ZNF408), kinesin family member 11 (KIF11) gene. Among them, FZD4, NDP, LRPS, TSPANI2 and other four genes play an important role in the Norrin/Frizzled 4 signaling pathway. In retinal capillary endothelial cells, Norrin specifically controls the occurrence of ocular capillaries by activating the Norrin/Frizzled 4 signaling pathway. ZNF408 and KIF11 are newly discovered pathogenic genes related to FEVR in the past 5 years. ZNF408 encodes the transcription factor that plays an important role in retinal angiogenesis. KIF11 plays a role in eye development and maintenance of retinal morphology and function.
ObjectiveTo identify mutations in NDP, FZD4, LRP5, TSPAN12 in Chinese families with familial exudative vitreoretinopathy (FEVR) and observe the clinical features.MethodsRetrospective 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.ResultsThere 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.ConclusionOur 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.
Hereditary ocular fundus disease is an important cause of irreversible damage to patients' visual acuity. It has attracted much attention due to its poor prognosis and lack of effective clinical interventions. With the discovery of a large number of hereditary ocular fundus genes and the development of gene editing technology and stem cell technology, gene and stem cell therapy emerged as the new hope for curing such diseases. Gene therapy is more directed at early hereditary ocular fundus diseases, using wild-type gene fragments to replace mutant genes to maintain existing retinal cell viability. Stem cell therapy is more targeted at advanced hereditary ocular fundus diseases, replacing and filling the disabled retinal cell with healthy stem cells. Although gene and stem cell therapy still face many problems such as gene off-target, differentiation efficiency, cell migration and long-term efficacy, the results obtained in preclinical and clinical trials should not be underestimated. With the emergence of various new technologies and new materials, it is bound to further assist gene and stem cell therapy, bringing unlimited opportunities and possibilities for the clinical cure of hereditary ocular fundus diseases.
Objective To observe the gene mutation and clinical phenotype of patients with retinitis pigmentosa (RP) and cone rod dystrophy (CORD). Methods Thirty-seven patients with RP and 6 patients with CORD and 95 family members were enrolled in the study. The patient’s medical history and family history were collected. All the patients and family members received complete ophthalmic examinations to determine the phenotype, including best corrected visual acuity, slit lamp microscope, indirect ophthalmoscopy, color fundus photography, optical coherence tomography, full-field electroretinogram, and fluorescein fundus angiography. DNA was abstracted from patients and family members. Using target region capture sequencing combined with next-generation sequencing to screen the 232 candidate pathogenic mutations. Polymerase chain reaction and direct sequencing were used to confirm the pathogenic pathogenic mutations and Co-segregation is performed among members in the family to determine pathogenic mutation sites. The relationship between genotype and clinical phenotype of RP and CORD was analyzed. Results Of the 37 patients with RP, 13 were from 6 families, including 4 families with autosomal dominant inheritance, 2 families with autosomal recessive inheritance, and 3 in 6 families were detected pathogenic gene mutations. 24 cases were scattered RP. Six patients with CORD were from four families, all of which were autosomal recessive. Of the 43 patients, 21 patients were detected the pathogenic gene mutation, and the positive rate was 48.8%. Among them, 15 patients with RP were detected 10 pathogenic gene mutations including USH2A, RP1, MYO7A, C8orf37, RPGR, SNRNP200, CRX, PRPF31, C2orf71, IMPDH1, and the clinical phenotype included 10 typical RP, 2 cases of RPSP, 3 cases of Usher syndrome type 2 and 6 cases of CORD patients were all detected pathogenic gene mutations, including 2 cases of ABCA4, 2 mutations of RIMS1 gene, 1 case of CLN3 gene mutation, and 1 case of CRB1 and RPGR double gene mutation. Conclusions RP and CORD are clinically diverse in genotype and clinically phenotypically similar. For patients with early RP and CORD, clinical phenotype combined with genetic analysis is required to determine the diagnosis of RP and CORD.