Allele-specific antisense oligonucleotides for the treatment of BEST1-related dominantly inherited retinal diseases: An in vitro model.

Retinal dystrophies are a common health problem worldwide that are currently incurable due to the inability of retinal cells to regenerate. Inherited retinal diseases (IRDs) are a diverse group of disorders characterized by progressive vision loss caused by photoreceptor cell dysfunction. The eye has always been an attractive organ for the development of novel therapies due to its independent access to the systemic pathway. Moreover, anti-sense oligonucleotides (ASOs), which facilitate manipulation of unwanted mRNAs via degradation or splicing, are undergoing rapid development and have been clinically deployed for the treatment of several diseases. The primary aim of this study was to establish a reliable in vitro model utilizing induced photoreceptor-like cells (PRCs) for assessing the efficacy and safety of ASOs targeting the BEST1 gene. Despite advances in gene therapy, effective treatments for a broad range of IRDs remain limited. An additional aim was to develop an in vitro model for evaluating RNA-based therapeutics, specifically ASOs, for the treatment in IRDs. Firstly, a cell culture model was established by induction of PRCs from dermal fibroblasts via direct programming. The induced PRCs were characterized at both the transcriptomic and protein level. Then, a common single nucleotide polymorphism (SNP) was identified in the BEST1 gene (rs1800007) for targeting with ASOs. ASOs were designed using the GapmeR strategy to target multiple alleles of this SNP, which is potentially suitable for a large proportion of the population. The efficacy and possible off-target effects of these ASOs were also analyzed in the induced PRC model. The findings show that the selected ASOs achieved allele-specific mRNA degradation with virtually no off-target effects on the global transcriptome profile, indicating their potential as safe and effective therapeutic agents. The presented in vitro model is a valuable platform for testing personalized IRD treatments and should inspire further research on RNA-based therapeutics. To the best of our knowledge this study is the first to test RNA-based therapeutics involving the use of ASOs in an induced PRC model. Based on the present findings, it will be possible to establish an ex vivo disease model using dermal fibroblast samples from affected individuals. In other words, the disease model and the ASOs that were successfully designed in this study can serve as a useful platform for the testing of personalized treatments for IRDs.

Clinical and genetic features in autosomal recessive bestrophinopathy in Chinese cohort.

PURPOSE: To provide a genotype and phenotype characterization of the BEST1 mutation in Chinese patients with autosomal recessive bestrophinopathy (ARB) through multimodal imaging and next-generation sequencing (NGS). METHODS: Seventeen patients from 17 unrelated families of Chinese origin with ARB were included in a retrospective cohort study. Phenotypic characteristics, including anterior segment features, were assessed by multimodal imaging. Multigene panel testing, involving 586 ophthalmic disease-associated genes, and Sanger sequencing were performed to identify disease-causing variants. RESULTS: Among 17 ARB patients, the mean follow-up was 15.65 months and average onset age was 30.53 years (range: 9-68). Best corrected visual acuity ranged from light perception to 0.8. EOG recordings showed a typically decreased Arden ratio in 12 patients, and a normal or slightly decreased Arden ratio in two patients. Anterior features included shallow anterior chambers (16/17), ciliary pronation (16/17), iris bombe (13/17), iridoschisis (2/17), iris plateau (1/17), narrow angles (16/17) and reduced axial lengths (16/17). Sixteen patients had multiple bilateral small, round, yellow vitelliform deposits distributed throughout the posterior pole, surrounding the optic disc. Initial diagnoses included angle-closure glaucoma (four patients), Best disease (three patients), and central serous chorioretinopathy secondary to choroidal neovascularization (CNV) (one patient), with the remainder diagnosed with ARB. Fourteen patients underwent preventive laser peripheral iridotomy, four of whom also received combined trabeculectomy and iridotomy in both eyes for uncontrolled intraocular pressure. One patient received intravitreal conbercept for CNV. Overall, 15 distinct disease-causing variants of BEST1 were identified, with 14 (82.35%) patients having missense mutations. Common mutations included p. Arg255-256 and p. Ala195Val (both 23.68%), with the most frequent sites in exons 7 and 5. CONCLUSIONS: This study provides a comprehensive characterization of anterior segment and genetic features in ARB, with a wide array of morphological abnormalities. Findings are relevant for refining clinical practices and genetic counseling and advancing pathogenesis research.

Autosomal recessive bestrophinopathy combined with neurofibromatosis type 1 in a patient.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a multisystem genetic disorder that may affect multiple systems of the body. Autosomal recessive bestrophinopathy (ARB) is a rare retinal dystrophy caused by autosomal recessively mutations in bestrophin 1 (BEST1) gene. So far, we have not retrieved any case report of the same patient with both NF1 and BEST1 gene mutations. CASE PRESENTATION: An 8-year-old female patient with café-au-lait spots, freckling on skin presented to our ophthalmology clinic for routine ophthalmological examination. Her best corrected visual acuity (BCVA) was 20/20 in both eyes. Slit-lamp examination of both eyes revealed few yellowish-brown dome-shaped Lisch nodules over the iris surface. Fundus examination was notable for bilateral confluent yellowish subretinal deposits at macula, few yellow flecks at temporal retina, and cup-to-disc ratio of 0.2. Optical coherence tomography (OCT) revealed subretinal fluid (SRF) involving the fovea, elongated photoreceptor outer segments and mild intraretinal fluid (IRF) at bilateral macula. Fundus autofluorescence demonstrated hyperautofluorescence in the area corresponding to the subretinal deposits. Whole-exome sequencing and Sanger sequencing were used to investigate genetic mutation in the patient and her parents. A BEST1 gene heterozygous missense c.604 C > T (p.Arg202Trp) was identified in the patient and her mother. Also, the patient carries an NF1 nonsense mutation c.6637 C > T (p.Gln2213*) with the mosaic generalized phenotype. There were no visual impairments or obvious neurological, musculoskeletal, behavioral or other symptoms in this patient, so she was managed conservatively and advised to follow up regularly for a long time. CONCLUSIONS: ARB and NF1, which are caused by two different pathogenic gene mutations, have rarely coexisted in the same patient. The discovery of pathogenic gene mutations may play a crucial role in more accurate diagnostics and genetic consultations for individuals and their families.

Variants of BEST1 and CRYBB2 cause a complex ocular phenotype comprising microphthalmia, microcornea, cataract, and vitelliform macular dystrophy: case report.

BACKGROUND: Best vitelliform macular dystrophy (BVMD), caused by pathogenic variants of the BEST1 gene, has not been reported in association with cataracts and ocular malformations. We reported a case with a complex ocular phenotype comprising microphthalmia, microcornea, cataract, and vitelliform macular dystrophy. CASE PRESENTATION: A six-year-old girl manifested photophobia and a poor visual behavior. A thorough ophthalmic examination revealed the patient to have bilateral microphthalmia, microcornea, congenital cataract, and Best vitelliform macular dystrophy (BVMD). Whole exome sequencing (WES) identified one variant in the BEST1 and one variant in CRYBB2 genes: c.218 T > G p.(Ile73Arg) and c.479G > C p.(Arg160Pro). The first variant was inherited from the proband's father, who was diagnosed with subclinical BVMD, while the second was a de novo variant. A minigene assay showed that c.218 T > G in BEST1 did not affect pre-mRNA splicing. CONCLUSIONS: This case suggests that the complex ocular phenotype comprising BVMD and congenital cataract with microphthalmia cannot be explained by variation in one gene but is caused by variants in BEST1 and CRYBB2. This case highlights the importance of general clinical evaluation and comprehensive genetic testing for diagnosing complex eye diseases.

RPE-Directed Gene Therapy Improves Mitochondrial Function in Murine Dry AMD Models.

Age-related macular degeneration (AMD) is the most common cause of blindness in the aged population. However, to date there is no effective treatment for the dry form of the disease, representing 85-90% of cases. AMD is an immensely complex disease which affects, amongst others, both retinal pigment epithelium (RPE) and photoreceptor cells and leads to the progressive loss of central vision. Mitochondrial dysfunction in both RPE and photoreceptor cells is emerging as a key player in the disease. There are indications that during disease progression, the RPE is first impaired and RPE dysfunction in turn leads to subsequent photoreceptor cell degeneration; however, the exact sequence of events has not as yet been fully determined. We recently showed that AAV delivery of an optimised NADH-ubiquinone oxidoreductase (NDI1) gene, a nuclear-encoded complex 1 equivalent from S. cerevisiae, expressed from a general promoter, provided robust benefit in a variety of murine and cellular models of dry AMD; this was the first study employing a gene therapy to directly boost mitochondrial function, providing functional benefit in vivo. However, use of a restricted RPE-specific promoter to drive expression of the gene therapy enables exploration of the optimal target retinal cell type for dry AMD therapies. Furthermore, such restricted transgene expression could reduce potential off-target effects, possibly improving the safety profile of the therapy. Therefore, in the current study, we interrogate whether expression of the gene therapy from the RPE-specific promoter, Vitelliform macular dystrophy 2 (VMD2), might be sufficient to rescue dry AMD models.

Genetic and clinical features of BEST1-associated retinopathy based on 59 Chinese families and database comparisons.

Variants in BEST1 are one of the most common cause of retinopathy mainly involving the retinal pigment epithelium with both dominant and recessive traits. This study aimed to describe the characteristics of potential pathogenic variants (PPVs) in BEST1 and their associated clinical features. Variants in BEST1 were collected from our in-house exome sequencing data and systematically evaluated by in silico prediction tools as well as genotype-phenotype analysis. The pathogenicity features of the BEST1 variants were further assessed through database comparison among the in-house data, Genome Aggregation Database from the general population, and all previously published literature. The clinical information of the in-house patients was summarized. The PPVs in BEST1 were identified in 66 patients from 59 families, including 32 families with Best vitelliform macular dystrophy (BVMD) and 27 families with autosomal recessive bestrophinopathy (ARB). These PPVs included 31 missense variants, seven truncation variants, one in-frame deletion, and a known 3-untranslated region variant. All the truncations detected in our study were exclusively involved in ARB but not BVMD. Among the 31 missense variants, 18 missenses associated with BVMD in the dominant trait were clustered in four hotspot regions with statistically significant differences from the recessive missenses. Except for distinct macular changes, there were no statistically significant differences among the other associated clinical features between BVMD and ARB, including peripheral retinopathy, high hyperopia, and angle-closure glaucoma. In conclusion, BEST1-associated dominant retinopathy was preferentially caused by missense variants located in important functional regions. Truncations were most likely benign in heterozygous status. Future studies are expected to elucidate the mystery of the same missense variants contributing to both BVMD and ARB.

A novel compound heterozygous BEST1 gene mutation in two siblings causing autosomal recessive bestrophinopathy.

PURPOSE: To describe the clinical features, imaging characteristics, and genetic test results associated with a novel compound heterozygous mutation of the BEST1 gene in two siblings with autosomal recessive bestrophinopathy. METHODS: Two siblings underwent a complete ophthalmic examination, including dilated fundus examination, fundus photography, fundus autofluorescence imaging, spectral-domain optical coherence tomography, fluorescein angiography, electroretinography, and electrooculography. A clinical diagnosis of autosomal recessive bestrophinopathy was established based on ocular examination and multimodal retinal imaging. Subsequently, clinical exome sequencing consisting of a panel of 6670 genes was carried out to confirm the diagnosis and assess genetic alterations in the protein-coding region of the genome of the patients. The identified mutations were tested in the two affected siblings and one of their parents. RESULTS: Two siblings (a 17-year-old female and a 15-year-old male) presented with reduced visual acuity and bilaterally symmetrical subretinal deposits of hyperautofluorescent materials in the posterior pole, which showed staining in the late phase of fluorescein angiogram. Spectral-domain optical coherence tomography demonstrated hyperreflective subretinal deposits and subretinal fluid accumulation. Both patients shared two mutations in the protein-coding region of the BEST1 gene, c.103G > A, p.(Glu35Lys) and c.313C > A, p.(Arg105Ser) (a novel disease-causing mutation). Sanger sequencing confirmed that the unaffected mother of the proband was carrying p.(Glu35Lys) variant in a heterozygous state. CONCLUSIONS: We have identified and described the phenotype of a novel disease-causing mutation NM_004183.4:c.313C > A, p.(Arg105Ser) in a heterozygous state along with a previously reported mutation NM_004183.4:c.103G > A, p.(Glu35Lys) of the BEST1 gene in two related patients with autosomal recessive bestrophinopathy.

Branch retina vein occlusion combined with angle-closure glaucoma is associated with a mutation in BEST1: a case report.

BACKGROUND: It is rare for a patient to be diagnosed with branch retina vein occlusion (BRVO), angle-closure glaucoma (ACG) and autosomal recessive bestrophinopathy (ARB). ARB is strongly associated with ACG. Although glaucoma is a significant risk factor for RVO, there is a plausible relationship between ACG and BRVO. To discuss correlation of these diseases is necessary. CASE PRESENTATION: The genetic testing and medical treatment of a patient with ocular fundus diseases and ACG were recorded. We present a 47-year-old male patient with BRVO who was diagnosed with angle-closure glaucoma and a homozygous mutation of c.140G > A (p.R47H) in BEST1. Intravitreal ranibizumab was administered in combination with three antiglaucomatous eyedrops to lower intraocular pressure (IOP) in the right eye. One month later, BCVA improved to 0.3. IOP was controlled at 13 mmHg. CONCLUSIONS: ACG was likely combined to ARB, while there's a plausible relationship between ACG and BRVO.

Development of retinal bullae in dogs with progressive retinal atrophy.

OBJECTIVE: To report the development of focal bullous retinal detachments (bullae) in dogs with different forms of progressive retinal atrophy (PRA). PROCEDURES: Dogs with three distinct forms of PRA (PRA-affected Whippets, German Spitzes and CNGB1-mutant Papillon crosses) were examined by indirect ophthalmoscopy and spectral domain optical coherence tomography (SD-OCT). Retinal bullae were monitored over time. One CNGB1-mutant dog was treated with gene augmentation therapy. The canine BEST1 gene coding region and flanking intronic sequence was sequenced in at least one affected dog of each breed. RESULTS: Multiple focal bullous retinal detachments (bullae) were identified in PRA-affected dogs of all three types. They developed in 4 of 5 PRA-affected Whippets, 3 of 8 PRA-affected Germans Spitzes and 15 of 20 CNGB1-mutant dogs. The bullae appeared prior to marked retinal degeneration and became less apparent as retinal degeneration progressed. Bullae were not seen in any heterozygous animals of any of the types of PRA. Screening of the coding region and flanking intronic regions of the canine BEST1 gene failed to reveal any associated pathogenic variants. Retinal gene augmentation therapy in one of the CNGB1-mutant dogs appeared to prevent formation of bullae. CONCLUSIONS: Retinal bullae were identified in dogs with three distinct forms of progressive retinal atrophy. The lesions develop prior to retinal thinning. This clinical change should be monitored for in dogs with PRA.

GABA Release from Astrocytes in Health and Disease.

Astrocytes are the most abundant glial cells in the central nervous system (CNS) mediating a variety of homeostatic functions, such as spatial K+ buffering or neurotransmitter reuptake. In addition, astrocytes are capable of releasing several biologically active substances, including glutamate and GABA. Astrocyte-mediated GABA release has been a matter of debate because the expression level of the main GABA synthesizing enzyme glutamate decarboxylase is quite low in astrocytes, suggesting that low intracellular GABA concentration ([GABA]i) might be insufficient to support a non-vesicular GABA release. However, recent studies demonstrated that, at least in some regions of the CNS, [GABA]i in astrocytes might reach several millimoles both under physiological and especially pathophysiological conditions, thereby enabling GABA release from astrocytes via GABA-permeable anion channels and/or via GABA transporters operating in reverse mode. In this review, we summarize experimental data supporting both forms of GABA release from astrocytes in health and disease, paying special attention to possible feedback mechanisms that might govern the fine-tuning of astrocytic GABA release and, in turn, the tonic GABAA receptor-mediated inhibition in the CNS.

Impaired Bestrophin Channel Activity in an iPSC-RPE Model of Best Vitelliform Macular Dystrophy (BVMD) from an Early Onset Patient Carrying the P77S Dominant Mutation.

Best Vitelliform Macular dystrophy (BVMD) is the most prevalent of the distinctive retinal dystrophies caused by mutations in the BEST1 gene. This gene, which encodes for a homopentameric calcium-activated ion channel, is crucial for the homeostasis and function of the retinal pigment epithelia (RPE), the cell type responsible for recycling the visual pigments generated by photoreceptor cells. In BVMD patients, mutations in this gene induce functional problems in the RPE cell layer with an accumulation of lipofucsin that evolves into cell death and loss of sight. In this work, we employ iPSC-RPE cells derived from a patient with the p.Pro77Ser dominant mutation to determine the correlation between this variant and the ocular phenotype. To this purpose, gene and protein expression and localization are evaluated in iPSC-RPE cells along with functional assays like phagocytosis and anion channel activity. Our cell model shows no differences in gene expression, protein expression/localization, or phagocytosis capacity, but presents an increased chloride entrance, indicating that the p.Pro77Ser variant might be a gain-of-function mutation. We hypothesize that this variant disturbs the neck region of the BEST1 channel, affecting channel function but maintaining cell homeostasis in the short term. This data shed new light on the different phenotypes of dominant mutations in BEST1, and emphasize the importance of understanding its molecular mechanisms. Furthermore, the data widen the knowledge of this pathology and open the door for a better diagnosis and prognosis of the disease.

Autosomal Recessive Bestrophinopathy: Clinical Features, Natural History, and Genetic Findings in Preparation for Clinical Trials.

PURPOSE: To investigate the clinical course, genetic findings, and phenotypic spectrum of autosomal recessive bestrophinopathy (ARB) in a large cohort of children and adults. DESIGN: Retrospective case series. PARTICIPANTS: Patients with a detailed clinical phenotype consistent with ARB, biallelic likely disease-causing sequence variants in the BEST1 gene, or both identified at a single tertiary referral center. METHODS: Review of case notes, retinal imaging (color fundus photography, fundus autofluorescence, OCT), electrophysiologic assessment, and molecular genetic testing. MAIN OUTCOME MEASURES: Visual acuity (VA), retinal imaging, and electrophysiologic changes over time. RESULTS: Fifty-six eyes of 28 unrelated patients were included. Compound heterozygous variants were detected in most patients (19/27), with 6 alleles recurring in apparently unrelated individuals, the most common of which was c.422G→A, p.(Arg141His; n = 4 patients). Mean presenting VA was 0.52 ± 0.36 logarithm of the minimum angle of resolution (logMAR), and final VA was 0.81 ± 0.75 logMAR (P = 0.06). The mean rate of change in VA was 0.05 ± 0.13 logMAR/year. A significant change in VA was detected in patients with a follow-up of 5 years or more (n = 18) compared with patients with a follow-up of 5 years or less (n = 10; P = 0.001). Presence of subretinal fluid and vitelliform material were early findings in most patients, and this did not change substantially over time. A reduction in central retinal thickness was detected in most eyes (80.4%) over the course of follow-up. Many patients (10/26) showed evidence of generalized rod and cone system dysfunction. These patients were older (P < 0.001) and had worse VA (P = 0.02) than those with normal full-field electroretinography results. CONCLUSIONS: Although patients with ARB are presumed to have no functioning bestrophin channels, significant phenotypic heterogeneity is evident. The clinical course is characterized by a progressive loss of vision with a slow rate of decline, providing a wide therapeutic window for anticipated future treatment strategies.

BEST1 gene therapy corrects a diffuse retina-wide microdetachment modulated by light exposure.

Mutations in the BEST1 gene cause detachment of the retina and degeneration of photoreceptor (PR) cells due to a primary channelopathy in the neighboring retinal pigment epithelium (RPE) cells. The pathophysiology of the interaction between RPE and PR cells preceding the formation of retinal detachment remains not well-understood. Our studies of molecular pathology in the canine BEST1 disease model revealed retina-wide abnormalities at the RPE-PR interface associated with defects in the RPE microvillar ensheathment and a cone PR-associated insoluble interphotoreceptor matrix. In vivo imaging demonstrated a retina-wide RPE-PR microdetachment, which contracted with dark adaptation and expanded upon exposure to a moderate intensity of light. Subretinal BEST1 gene augmentation therapy using adeno-associated virus 2 reversed not only clinically detectable subretinal lesions but also the diffuse microdetachments. Immunohistochemical analyses showed correction of the structural alterations at the RPE-PR interface in areas with BEST1 transgene expression. Successful treatment effects were demonstrated in three different canine BEST1 genotypes with vector titers in the 0.1-to-5E11 vector genomes per mL range. Patients with biallelic BEST1 mutations exhibited large regions of retinal lamination defects, severe PR sensitivity loss, and slowing of the retinoid cycle. Human translation of canine BEST1 gene therapy success in reversal of macro- and microdetachments through restoration of cytoarchitecture at the RPE-PR interface has promise to result in improved visual function and prevent disease progression in patients affected with bestrophinopathies.

Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases.

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

The molecular mechanism of synaptic activity-induced astrocytic volume transient.

KEY POINTS: Neuronal activity causes astrocytic volume change via K+ uptake through TREK-1 containing two-pore domain potassium channels. The volume transient is terminated by Cl- efflux through the Ca2+ -activated anion channel BEST1. The source of the Ca2+ required to open BEST1 appears to be the stretch-activated TRPA1 channel. Intense neuronal activity is synaptically coupled with a physical change in astrocytes via volume transients. ABSTRACT: The brain volume changes dynamically and transiently upon intense neuronal activity through a tight regulation of ion concentrations and water movement across the plasma membrane of astrocytes. We have recently demonstrated that an intense neuronal activity and subsequent astrocytic AQP4-dependent volume transient are critical for synaptic plasticity and memory. We have also pharmacologically demonstrated a functional coupling between synaptic activity and the astrocytic volume transient. However, the precise molecular mechanisms of how intense neuronal activity and the astrocytic volume transient are coupled remain unclear. Here we utilized an intrinsic optical signal imaging technique combined with fluorescence imaging using ion sensitive dyes and molecular probes and electrophysiology to investigate the detailed molecular mechanisms in genetically modified mice. We report that a brief synaptic activity induced by a train stimulation (20 Hz, 1 s) causes a prolonged astrocytic volume transient (80 s) via K+ uptake through TREK-1 containing two-pore domain potassium (K2P) channels, but not Kir4.1 or NKCC1. This volume change is terminated by Cl- efflux through the Ca2+ -activated anion channel BEST1, but not the volume-regulated anion channel TTYH. The source of the Ca2+ required to open BEST1 appears to be the stretch-activated TRPA1 channel in astrocytes, but not IP3 R2. In summary, our study identifies several important astrocytic ion channels (AQP4, TREK-1, BEST1, TRPA1) as the key molecules leading to the neuronal activity-dependent volume transient in astrocytes. Our findings reveal new molecular and cellular mechanisms for the synaptic coupling of intense neuronal activity with a physical change in astrocytes via volume transients.

Familial autosomal recessive bestrophinopathy: identification of a novel variant in BEST1 gene and the specific metabolomic profile.

BACKGROUND: Autosomal recessive bestrophinopathy (ARB) is a retinal degenerative disorder caused by BEST1 mutations with autosomal recessive inheritance. We aim to map a comprehensive genomic and metabolomic profile of a consanguineous Chinese family with ARB. METHODS: Ophthalmic examinations were performed on the affected patients with ARB. The proband was screened for potential causative mutations in a panel with 256 known retinal disease genes by using target capture sequencing. The related mutation was further validated and segregated in the family members by Sanger sequencing. In silico prediction tools were used for pathogenicity assessment. A UHPLC-MS/MS metabolomic analysis was performed to explore the disease-associated metabolic feature. RESULTS: The affected patients from this family were characterized by low vision, the presence of subretinal fluid, macular edema, and hyperopia with coincidental angle closure. DNA sequencing identified a novel missense mutation in the BEST1 gene c.646G > A (p.Val216Ile) of the proband. Sanger sequencing further confirmed the mutation. The missense mutation was co-segregation across the pedigree and predicted to be deleterious by SIFT (0.017). The blood metabolic profiles were highly similar among all family members probably because of the same lifestyle, habitat and genomic background. However, ARB patients presented a significant deregulation of metabolites, such as citric acid, L-Threonic acid, and eicosapentaenoic acid. CONCLUSIONS: We identified a novel disease-associated variant in the BEST1 gene as well as a disease-specific metabolic feature in familial ARB. Our findings helped improve the understanding of ARB mechanisms.

Disease-causing mutations associated with bestrophinopathies promote apoptosis in retinal pigment epithelium cells.

PURPOSE: Best vitelliform macular dystrophy (BVMD) and autosomal recessive bestrophinopathy (ARB) are two kinds of bestrophinopathies which are caused by BEST1 mutations and characterized by accumulation of lipofuscin-like materials on the retinal pigment epithelium cell-photoreceptor interface. In the past two decades, research about the pathogenesis of bestrophinopathies was mainly focused on the anion channel and intracellular Ca2+ signaling, but seldom concentrated on the function of retinal pigment epithelium (RPE) cells. In this study, we explored the possible effect of the three BEST1 mutations p.V143F, p.S142G, and p.A146T on the apoptosis in human fetal RPE cells. METHODS: Wild-type plasmid and mutant plasmids BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T were transfected to human fetal RPE cells. The molecules caspase-3, phospho-Bcl-2, BAX, PARP, and AIF associated with apoptosis were determined by quantitative PCR and Western blot. Apoptotic rate and active Caspase-3 staining were analyzed by flow cytometry. RESULTS: Caspase-3 and PARP expression were significantly increased in BEST1-pcDNA3.1 p.S142G and p.A146T group. Flow cytometry showed that the apoptosis rates were significantly increased in the BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T group compared with the wild-type group. CONCLUSIONS: For the first time, we found that the three mutations promoted RPE cell apoptosis. Furthermore, the results indicated that BEST1 mutations p.S142G and p.A146T may contribute apoptosis of RPE cells by targeting Caspase 3. Our observations suggested that the apoptosis of RPE cells may be one of the mechanisms in bestrophinopathies, which may provide a new potential therapeutic target for the treatment of this disease.

Clinical Heterogeneity in Autosomal Recessive Bestrophinopathy with Biallelic Mutations in the BEST1 Gene.

Autosomal recessive bestrophinopathy (ARB) has been reported as clinically heterogeneous. Eighteen patients (mean age: 22.5 years; 15 unrelated families) underwent ophthalmological examination, fundus photography, fundus autofluorescence, and optical coherence tomography (OCT). Molecular genetic testing of the BEST1 gene was conducted by the chain-terminating dideoxynucleotide Sanger methodology. Onset of symptoms (3 to 50 years of age) and best-corrected visual acuity (0.02-1.0) were highly variable. Ophthalmoscopic and retinal imaging defined five phenotypes. Phenotype I presented with single or confluent yellow lesions at the posterior pole and midperiphery, serous retinal detachment, and intraretinal cystoid spaces. In phenotype II fleck-like lesions were smaller and extended to the far periphery. Phenotype III showed a widespread continuous lesion with sharp peripheral demarcation. Single (phenotype IV) or multifocal (phenotype V) vitelliform macular dystrophy-like lesions were observed as well. Phenotypes varied within families and in two eyes of one patient. In addition, OCT detected hyperreflective foci (13/36 eyes) and choroidal excavation (11/36). Biallelic mutations were identified in each patient, six of which have not been reported so far [c.454C>T/p.(Pro152Ser), c.620T>A/p.(Leu207His), c.287_298del/p.(Gln96_Asn99del), c.199_200del/p.(Leu67Valfs*164), c.524del/p.(Ser175Thrfs*19), c.590_615del/p.(Leu197Profs*26)]. BEST1-associated ARB presents with a variable age of onset and clinical findings, that can be categorized in 5 clinical phenotypes. Hyperreflective foci and choroidal excavation frequently develop as secondary manifestations.

Mutation-Dependent Pathomechanisms Determine the Phenotype in the Bestrophinopathies.

Best vitelliform macular dystrophy (BD), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and the autosomal recessive bestrophinopathy (ARB), together known as the bestrophinopathies, are caused by mutations in the bestrophin-1 (BEST1) gene affecting anion transport through the plasma membrane of the retinal pigment epithelium (RPE). To date, while no treatment exists a better understanding of BEST1-related pathogenesis may help to define therapeutic targets. Here, we systematically characterize functional consequences of mutant BEST1 in thirteen RPE patient cell lines differentiated from human induced pluripotent stem cells (hiPSCs). Both BD and ARB hiPSC-RPEs display a strong reduction of BEST1-mediated anion transport function compared to control, while ADVIRC mutations trigger an increased anion permeability suggesting a stabilized open state condition of channel gating. Furthermore, BD and ARB hiPSC-RPEs differ by the degree of mutant protein turnover and by the site of subcellular protein quality control with adverse effects on lysosomal pH only in the BD-related cell lines. The latter finding is consistent with an altered processing of catalytic enzymes in the lysosomes. The present study provides a deeper insight into distinct molecular mechanisms of the three bestrophinopathies facilitating functional categorization of the more than 300 known BEST1 mutations that result into the distinct retinal phenotypes.

The E3 ubiquitin ligase, NEDD4L (NEDD4-2) regulates bestrophin-1 (BEST1) by ubiquitin-dependent proteolysis.

The bestrophin family comprises well-known Ca2+-activated chloride channels (CaCC) that are expressed in a variety tissues including the brain, eye, gastrointestinal tract, and muscle tissues. Among the family members, bestrophin-1 (BEST1) is known to exist mainly in retinal pigment epithelium cells, but we recently reported that BEST1 mediates Ca2+-activated Cl- currents in hippocampal astrocytes. Despite its critical roles in physiological processes, including tonic γ-aminobutyric acid release and glutamate transport, the mechanisms that regulate BEST1 are poorly understood. In this study, we identified NEDD4L (NEDD4-2), an E3 ubiquitin ligase, as a binding partner of BEST1. A series of deletion constructs revealed that the WW3-4 domains of NEDD4L were important for interaction with BEST1. We observed that BEST1 underwent ubiquitin-dependent proteolysis and found that the conserved lysine370 residue in the C-terminus of BEST1 was important for its ubiquitination. Finally, we demonstrated that NEDD4L inhibited whole cell currents mediated by BEST1 but not by the BEST1(K370R) mutant. Collectively, our data demonstrated that NEDD4L played a critical role in regulating the surface expression of BEST1 by promoting its internalization and degradation.