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.

Induced Pluripotent Stem Cell Modeling of Best Disease and Autosomal Recessive Bestrophinopathy.

PURPOSE: To understand the pathophysiology of Best disease (BD) and autosomal recessive bestrophinopathy (ARB) by establishing an in vitro model using human induced pluripotent stem cell (iPSC). MATERIALS AND METHODS: Human iPSC lines were generated from mononuclear cells in peripheral blood of one ARB patient, one autosomal dominant BD patient, and two normal controls. Immunocytochemistry and reverse transcriptase polymerase chain reaction in iPSC lines were conducted to demonstrate the pluripotent markers. After the differentiation of iPSC into functional retinal pigment epithelium (RPE), morphological characteristics of the RPE were evaluated using confocal microscopy and immunocytochemistry. The rates of fluid flow across iPSC-RPE monolayer were measured to compare apical to basal fluid transports by RPE. RNA sequencing was performed on iPSC-RPE to identify the differences in gene expression profiles, and specific gene sets were tested using Gene Set Enrichment Analysis. RESULTS: Morphological characteristics, gene expression, and epithelial integrity of ARB iPSC were comparable to those of BD patient or normal control. Fluid transport from apical to basal was significantly decreased in ARB iPSC-RPE compared with BD iPSC-RPE or control iPSC-RPE. Gene Set Enrichment Analysis confirmed that ARB iPSC-RPE exhibited significant enrichments of epithelial-mesenchymal transition gene set and TNF-α signaling via NF-κB gene set compared to control iPSC-RPE or BD iPSC-RPE. CONCLUSION: A human iPSC model of ARB showed a functional deficiency rather than anatomical defects. ARB may be caused by RPE dysfunction following BEST1 mutation.

Multicolor Imaging Characteristics of Best's Vitelliform Macular Dystrophy.

A 40-year-old woman presented with vitellieruptive stage of Best's vitelliform macular dystrophy (BVMD) in the right eye and pseudohypopyon stage in the left eye. She underwent comprehensive ophthalmic examination and fundus imaging using multicolor (MC) imaging technology of Spectralis (Heidelberg Engineering, Heidelberg, Germany) spectral-domain-optical coherence tomography system. Composite MC imaging revealed larger area of retinal pigment epithelium atrophy in vitellierruptive stage of the disease in the right eye compared to color fundus photograph. Retinal elevation in the pseudohypopyon stage was better delineated on composite MC and blue reflectance images in the left eye. Subretinal lipofuscin was best seen in green reflectance and short-wave autofluorescence images. The present case reports the MC imaging features of BVMD.

Generation of Best disease-derived induced pluripotent stem cell line (FRIMOi006-A) carrying a novel dominant mutation in BEST1 gene.

Best disease, also known as Best vitelliform macular dystrophy, is an autosomal dominant form of macular degeneration. Here, we have generated an induced pluripotent stem cell (iPSC) line derived from a Best disease patient carrying a new dominant mutation in the BEST1 gene. Skin fibroblasts were reprogrammed to iPSCs by the non-integrative Sendai-virus method. The iPSC line has been characterized preserving the BEST1 mutation, expressing the pluripotency markers and being capable to differentiate to endoderm, mesoderm and ectoderm in vitro.

Nanomedicine-based Curcumin Approach Improved ROS Damage in Best Dystrophy-specific Induced Pluripotent Stem Cells.

Best dystrophy (BD), also termed best vitelliform macular dystrophy (BVMD), is a juvenile-onset form of macular degeneration and can cause central visual loss. Unfortunately, there is no clear definite therapy for BD or improving the visual function on this progressive disease. The human induced pluripotent stem cell (iPSC) system has been recently applied as an effective tool for genetic consultation and chemical drug screening. In this study, we developed patient-specific induced pluripotent stem cells (BD-iPSCs) from BD patient-derived dental pulp stromal cells and then differentiated BD-iPSCs into retinal pigment epithelial cells (BD-RPEs). BD-RPEs were used as an expandable platform for in vitro candidate drug screening. Compared with unaffected sibling-derived iPSC-derived RPE cells (Ctrl-RPEs), BD-RPEs exhibited typical RPE-specific markers with a lower expression of the tight junction protein ZO-1 and Bestrophin-1 (BEST1), as well as reduced phagocytic capabilities. Notably, among all candidate drugs, curcumin was the most effective for upregulating both the BEST1 and ZO-1 genes in BD-RPEs. Using the iPSC-based drug-screening platform, we further found that curcumin can significantly improve the mRNA expression levels of Best gene in BD-iPSC-derived RPEs. Importantly, we demonstrated that curcumin-loaded PLGA nanoparticles (Cur-NPs) were efficiently internalized by BD-RPEs. The Cur-NPs-based controlled release formulation further increased the expression of ZO-1 and Bestrophin-1, and promoted the function of phagocytosis and voltage-dependent calcium channels in BD-iPSC-derived RPEs. We further demonstrated that Cur-NPs enhanced the expression of antioxidant enzymes with a decrease in intracellular ROS production and hydrogen peroxide-induced oxidative stress. Collectively, these data supported that Cur-NPs provide a potential cytoprotective effect by regulating the anti-oxidative abilities of degenerated RPEs. In addition, the application of patient-specific iPSCs provides an effective platform for drug screening and personalized medicine in incurable diseases.

Establishment of an induced pluripotent stem cell line (FDEENTi002-A) from a patient with Best's disease carrying c.888C > A mutation in BEST1 gene.

Best's disease (BD) is an inherited retinal degenerative disease caused by mutations in BEST1 gene. A human induced pluripotent stem cell (iPSC) line has been generated with integration-free Sendai virus method from peripheral blood mononuclear cells (PBMCs) of a BD patient carrying c.888C > A mutation in BEST1 gene. This cell line may serve as a model for the study of pathogenesis of BD.

Underdeveloped RPE Apical Domain Underlies Lesion Formation in Canine Bestrophinopathies.

Canine bestrophinopathy (cBest) is an important translational model for BEST1-associated maculopathies in man that recapitulates the broad spectrum of clinical and molecular disease aspects observed in patients. Both human and canine bestrophinopathies are characterized by focal to multifocal separations of the retina from the RPE. The lesions can be macular or extramacular, and the specific pathomechanism leading to formation of these lesions remains unclear. We used the naturally occurring canine BEST1 model to examine factors that underlie formation of vitelliform lesions and addressed the susceptibility of the macula to its primary detachment in BEST1-linked maculopathies.

Generation of induced pluripotent stem cells from a patient with Best Dystrophy carrying 11q12.3 (BEST1 (VMD2)) mutation.

Best disease (BD), also termed Best vitelliform macular dystrophy (BVMD), is a juvenile-onset form of macular degeneration and central visual loss. In this report, we generated an induced pluripotent stem cell (iPSC) line, TVGH-iPSC-012-04, from the peripheral blood mononuclear cells of a female patient with BD by using the Sendai virus delivery system. The resulting iPSCs retained the disease-causing DNA mutation, expressed pluripotent markers and could differentiate into three germ layers. We believe that BD patient-specific iPSCs provide a powerful in vitro model for evaluating the pathological phenotypes of the disease.

Adult-Onset Vitelliform Macular Dystrophy caused by BEST1 p.Ile38Ser Mutation is a Mild Form of Best Vitelliform Macular Dystrophy.

Adult-onset vitelliform macular dystrophy (AVMD) is a common and benign macular degeneration which can be caused by BEST1 mutation. Here, we investigated the clinical characteristics associated with a newly identified BEST1 mutation, p.Ile38Ser and confirmed the associated physiological functional defects. The 51-year-old patient presented bilateral small subretinal yellow deposits. Consistent with AVMD, the corresponding lesions showed hyperautofluorescence, late staining in fluorescein angiography, and subretinal hyper-reflective materials in spectral-domain optical coherence tomography. Genetic analysis demonstrated that the patient presented with a heterozygous p.Ile38Ser BEST1 mutation. Surface biotinylation and patch clamp experiments were performed in transfected HEK293T cells. Although, the identified BEST1 mutant maintains normal membrane expression, p.Ile38Ser mutant showed significantly smaller currents than wild type (WT). However, it showed larger currents than other BEST1 mutants, p.Trp93Cys, causing autosomal dominant best vitelliform macular dystrophy (BVMD), and p.Ala195Val, causing autosomal recessive bestrophinopathy (ARB). The cells expressing both WT and each BEST1 mutant showed that the functional defect of p.Ile38ser was milder than that of p.Trp93Cys, whereas combination of p.Ala195Val with WT showed good current. We identified and described the phenotype and in vitro functions of a novel BEST1 mutation causing AVMD. AVMD induced by p.Ile38Ser BEST1 mutation is a mild form of BVMD.

Subretinal Hyperreflective Material Imaged With Optical Coherence Tomography Angiography.

PURPOSE: The range of subretinal hyperreflective material (SHRM) seen in macular disease includes type 2 macular neovascularization, fibrosis, exudation, vitelliform material, and hemorrhage. The prognostic significance of SHRM has been evaluated retrospectively in clinical trials, but discriminating SHRM subtypes traditionally requires multiple imaging modalities. The purpose of this study is to describe optical coherence tomography angiography (OCTA) flow characteristics and artifacts that might help to distinguish SHRM subtypes. DESIGN: Validity analysis. METHODS: Patients with age-related macular degeneration (AMD), myopia, pachychoroid disease, and macular dystrophy, manifesting SHRM on optical coherence tomography (OCT), were recruited. Clinical chart review and multimodal imaging established the SHRM subtype. All patients underwent OCTA. OCT and OCTA images were examined together for (1) intrinsic flow, (2) retinal projection onto the anterior SHRM surface (strong, weak, absent), (3) retinal projection through SHRM onto retinal pigment epithelium (RPE), and (4) masking of choriocapillaris flow. RESULTS: Thirty-three eyes of 25 patients were included (type 2 neovascularization ×3; fibrosis ×4; exudation ×10; hemorrhage ×5; vitelliform ×17). Mean age per eye was 76 years (standard deviation: 12). Intrinsic flow was strongest in type 2 neovascularization. Subretinal fibrosis showed limited flow in residual large-caliber vessels and branches. Flow was not detected within foci of exudation, hemorrhage, or vitelliform lesions. Retina-SHRM surface projection was strongest onto smooth-surfaced SHRM and weaker onto exudation. Retinal projection was weakest on the surface of vitelliform lesions. Retina-RPE projection was masked by dense hemorrhage and vitelliform material. In compound SHRM, OCTA distinguished between vascular and avascular components. CONCLUSION: Optical coherence tomography angiography can distinguish vascular from avascular SHRM components. OCTA artifacts may distinguish certain avascular SHRM components.

Novel BEST1 Mutations and Special Clinical Features of Best Vitelliform Macular Dystrophy.

PURPOSE: To describe the clinical features and to analyze BEST1 mutations in patients with Best vitelliform macular dystrophy (BVMD). METHODS: Thirteen individuals affected by BVMD from 6 unrelated Chinese families were studied. Complete ophthalmological examinations were performed, and the BEST1 gene was screened in all participants and 100 controls. Follow-ups were arranged within 12 months. RESULTS: All 6 probands showed typical fundus appearances of BVMD. One of the 6 had a history of angle closure glaucoma, and another showed a unilateral focal choroidal excavation on optical coherence tomography. One patient experienced progression of the macular lesion during the follow-up. The asymptomatic mutation carriers had normal fundus but abnormal Arden ratios on electro-oculograms. Besides 4 previously reported mutations (p.Ser16Phe, p.Ser144Asn, p.Glu292Lys, p.Glu300Lys), 2 novel disease-causing mutations (p.Thr307Asp, p.Arg47His) were identified, of which p.Arg47His has been reported in adult-onset vitelliform macular dystrophy. CONCLUSIONS: Our findings have expanded the mutational and clinical spectrum of BVMD in a Chinese population. Clinical presentations of angle closure glaucoma and/or focal choroidal excavation may be related to BVMD, underlining the necessity of multimodal studies and risk assessment of angle closure glaucoma in BEST1 mutation carriers. BVMD and adult-onset vitelliform macular dystrophy may share a common etiology in some cases.

Quantitative Fundus Autofluorescence in Best Vitelliform Macular Dystrophy: RPE Lipofuscin is not Increased in Non-Lesion Areas of Retina.

Since the lipofuscin of retinal pigment epithelial (RPE) cells has been implicated in the pathogenesis of Best vitelliform macular dystrophy, we quantified fundus autofluorescence (quantitative fundus autofluorescence, qAF) as an indirect measure of RPE lipofuscin levels. Mean non-lesion qAF was found to be within normal limits for age. By spectral domain optical coherence tomography (SD-OCT) vitelliform lesions presented as fluid-filled subretinal detachments containing reflective material. We discuss photoreceptor outer segment debris as the source of the intense fluorescence of these lesions and loss of anion channel functioning as an explanation for the bullous photoreceptor-RPE detachment. Unexplained is the propensity of the disease for central retina.

Contribution of Ion Channels in Calcium Signaling Regulating Phagocytosis: MaxiK, Cav1.3 and Bestrophin-1.

Mutations in the BEST1 gene lead to a variety of retinal degenerations including Best's vitelliforme macular degeneration. The BEST1 gene product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE). It is likely that mutant bestrophin-1 impairs functions of the RPE which support photoreceptor function and will thus lead to retinal degeneration. However, the RPE function which is influenced by bestrophin-1 is so far not identified. Previously we showed that bestrophin-1 interacts with L-type Ca²âº channels of the CaV1.3 subtype and that the endogenously expressed bestrophin-1 is required for intracellular Ca²âº regulation. A hallmark of Best's disease is the fast lipofuscin accumulation occurring already at young ages. Therefore, we addressed the hypothesis that bestrophin-1 might influence phagocytosis of photoreceptor outer segments (POS) by the RPE. Here, siRNA knock-down of bestrophin-1 expression as well as inhibition of L-type Ca²âº channel activity modulated the POS phagocytosis in vitro. In vivo CaV1.3 expression appeared to be diurnal regulated with a higher expression rate in the afternoon. Compared to wild-type littermates, Ca V 1.3 (-/-) mice showed a shift in the circadian POS phagocytosis with an increased activity in the afternoon. Thus we suggest that mutant bestrophin-1 leads to an impaired regulation of the POS phagocytosis by the RPE which would explain the fast lipofuscin accumulation in Best patients.

Two novel mutations in the bestrophin-1 gene and associated clinical observations in patients with best vitelliform macular dystrophy.

The purpose of the current study was to investigate the 11 bestrophin-1 (BEST1) exons in patients with best vitelliform macular dystrophy (BVMD), and to characterize the associated clinical features. Complete ophthalmic examinations were conducted on two families, and two family members were diagnosed with BVMD. Genomic DNA was extracted from the leukocytes of peripheral blood collected from the patients and their family members, in addition to 100 unrelated control subjects recruited from the same population. The polymerase chain reaction was used to amplify a total of 11 exons of the BEST1 gene, which were directly sequenced. Ophthalmic examinations, including best-corrected visual acuity, slit-lamp examination, fundus examination, fundus photography and fluorescein angiography imaging, as well as anterior segment analysis with Pentacam and optical coherence tomography, were conducted. The patients exhibited yellowish lesions in the macular area. A heterozygous mutation c.910_912delGAT (p.304del Asp) in exon 7 was identified in Case 1. A heterozygous BEST1 missense mutation c.685T>G (p.Trp229Gly) in exon 5 was identified in Case 2, but not in any of the unaffected family members or normal controls. Although BEST1 gene mutations and polymorphisms have previously been reported in various ethnic groups, the current study identified, for the first time to the best of our knowledge, two novel BEST1 gene mutations in patients with BVMD.

Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium.

PURPOSE: Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1. METHODS: Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp. RESULTS: Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1(W93C). Examination of [Ca2+]i following ATP stimulation demonstrated that the expression of Best1W93C impaired intracellular Ca2+ signaling. CONCLUSIONS: These data indicate that Best1 activity strongly influences electrophysiology and Ca2+ signaling in RPE cells, and that a common BVMD mutation disrupts both of these parameters. Our findings support the hypothesis that Best1 functions as an anion channel in human RPE.

A case of acute exudative polymorphous vitelliform maculopathy: follow-up and wide-field spectral-domain optical coherence tomography.

PURPOSE: To present a case of an HIV-positive patient with acute exudative polymorphous vitelliform maculopathy (AEPVM) and evaluate the presence of specific spectral-domain optical coherence tomography (SD-OCT) findings. METHODS: Case report. RESULTS: We reviewed the AEPVM cases reported in the literature and compared those to our patient to determine if there is a correspondence between the etiology that leads to the onset of AEPVM and clinical and SD-OCT findings. CONCLUSIONS: Acute exudative polymorphous vitelliform maculopathy is a disease that involves the outer retinal layers with lipofuscin deposits and serous detachment of the neuroepithelium with or without intraretinal cysts. Not much is known about the etiology and pathogenesis, and not many cases have been described. A review of the few clinical cases reported in the literature does not show a specific correspondence between etiology and SD-OCT findings.

Application of evolutionary based in silico methods to predict the impact of single amino acid substitutions in vitelliform macular dystrophy.

Recent developments in high-throughput discovery and genotyping have generated a tremendous amount of information about the existence of single amino acid polymorphisms (SAPs). Detailed understanding of the SAPs that affect protein structure and function can provide us valuable insight into disease genotype-phenotype correlations. Functional variants of biological importance are likely to be missed in large-scale analysis. Over the past decade, numerous efforts are underway in understanding and characterizing the potential consequences of variants in assessing the risk associated with vitelliform macular dystrophy (VMD). Yet, in spite of this success, we conducted a first SAP analysis via evolutionary-based in silico pipeline to unravel functional SAPs from a pool, containing both functional and neutral ones. Furthermore, based on the prediction scores, a ranking system was developed to prioritize the functional SAPs in order to minimize the number of SAPs screened for further genotyping.

The role of bestrophin-1 in intracellular Ca(2+) signaling.

Mutations in the BEST1 gene lead to a variety of retinal degenerations, among them Best's vitelliforme macular degeneration. To clarify the mechanism of the disease, the understanding of the function of BEST1 gene product, bestrophin-1, is mandatory. In overexpression studies bestrophin-1 appeared to function as a Ca(2+)-dependent Cl channel. On the other hand, bestrophin-1 is able to participate in intracellular Ca(2+) signaling. Endogenously expressed bestrophin-1 largely localized to the cytosolic compartment close to the basolateral membrane of the retinal pigment epithelium (RPE) as it can be shown using differential centrifugation, immunohistochemistry, and transmission electron microscopy. To elucidate a cytosolic function of bestrophin-1, we explored the store-operated Ca(2+) entry in short-time cultured porcine RPE cells. Depletion of cytosolic Ca(2+)stores by SERCA inhibition led to activation of Orai-1 Ca(2+) channels. This resulted in an influx of extracellular Ca(2+) into the cell which was reduced when bestrophin-1 expression was knocked down using siRNA techniques. Quantification of Ca(2+) which can be released from cytosolic Ca(2+) stores revealed that after reduction of bestrophin-1 expression less Ca(2+) is stored in ER Ca(2+) stores. Thus, bestrophin-1 functions as an intracellular Cl channel which helps to accumulate and to release Ca(2+) from stores by conducting the counterion for Ca(2+).