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

ABSTRACT: 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-? signaling via NF-?B 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.

SUBMITTER: Lee JH

PROVIDER: S-EPMC7471084 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Lee Ji Hwan JH Oh Jin Ok JO Lee Christopher Seungkyu CS

Yonsei medical journal 20200901 9

<h4>Purpose</h4>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).<h4>Materials and methods</h4>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 demonstrat ...[more]

PMID: 32882766

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Project description:BackgroundTo describe ocular manifestations, imaging characteristics, and genetic test results of autosomal recessive bestrophinopathy (ARB). The study design is an observational case series.MethodsForty-eight eyes of 24 patients diagnosed with ARB underwent complete ophthalmic examinations including refraction, anterior and posterior segment examination, enhanced depth imaging optical coherence tomography (EDI-OCT), fluorescein angiography (FA), electroretinography (ERG), and electrooculography (EOG). Optical coherence tomography angiography (OCTA) and BEST1 gene sequencing were performed in selected patients.ResultsThe age at onset was 4-35 years (mean: 18.6 years). The male-to-female ratio was 0.45. All patients were hyperopic, except one with less than one diopter myopia. EOG was abnormal in 18 cases with near-normal ERGs. Six patients did not undergo EOG due to their young age. Eighteen patients (75%) had a thick choroid on EDI-OCT, of which three had advanced angle-closure glaucoma, 15 patients were hyperopic, and eight of them had more than four diopters hyperopia in both eyes. Macular retinoschisis was observed in 46 eyes of 23 patients (95%) with cysts mostly located in the inner nuclear layer (INL) to the outer nuclear layer (ONL). Of the 18 patients who underwent FA, mild peripheral leakage was seen in eight eyes of four patients (22%). Subfoveal choroidal neovascularization (CNV) was seen in three eyes of two patients (6%) that responded well to intravitreal bevacizumab (IVB). Seven mutations of the bestrophin-1 (BEST1) gene were found in this study; however, only two of them (p.Gly34 = and p.Leu319Pro) had been previously reported as the cause of ARB based on ClinVar and other literature studies.ConclusionsARB can be presented with a wide spectrum of ocular abnormalities that may not be easily diagnosed. Pachychoroid can occur alongside retinal schisis and may be the underlying cause of angle-closure glaucoma in ARB. Our study also expands the pathogenic mutation spectrum of the BEST1 gene associated with ARB.

Project description:Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients' genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with iPSC.

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

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

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