Mutations In Bestrophin-1 Research Articles

Evaluating BEST1 mutations in pluripotent stem cell-derived retinal pigment epithelial cells.

Bestrophin-1 (BEST1) is a calcium-activated chloride channel (CaCC) predominantly expressed at the basolateral membrane of the retinal pigment epithelium (RPE). Over 250 mutations in the BEST1 gene have been documented to cause at least five retinal degenerative disorders, commonly termed bestrophinopathies, to which no treatment is currently available. Therefore, understanding the influences of BEST1 disease-causing mutations on the physiological function of BEST1 in RPE is critical for deciphering the pathology of bestrophinopathies and developing therapeutic strategies for patients. However, this task has been impeded by the rarity of BEST1 mutations and limited accessibility to native human RPE cells. Here, we describe a pluripotent stem cell (PSC)-based pipeline for reproducibly generating RPE cells expressing endogenous or exogenous mutant BEST1, which provides us with a powerful "disease-in-a-dish" approach for studying BEST1 mutations in physiological environments.

Bestrophinopathies: perspectives on clinical disease, Bestrophin-1 function and developing therapies.

Bestrophinopathies are a group of clinically distinct inherited retinal dystrophies that typically affect the macular region, an area synonymous with central high acuity vision. This spectrum of disorders is caused by mutations in bestrophin1 (BEST1), a protein thought to act as a Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE) of the eye. Although bestrophinopathies are rare, over 250 individual pathological mutations have been identified in the BEST1 gene, with many reported to have various clinical expressivity and incomplete penetrance. With no current clinical treatments available for patients with bestrophinopathies, understanding the role of BEST1 in cells and the pathological pathways underlying disease has become a priority. Induced pluripotent stem cell (iPSC) technology is helping to uncover disease mechanisms and develop treatments for RPE diseases, like bestrophinopathies. Here, we provide a comprehensive review of the pathophysiology of bestrophinopathies and highlight how patient-derived iPSC-RPE are being used to test new genomic therapies in vitro.

Generation of a human induced pluripotent stem cell line, BRCi005-A, derived from a Best disease patient with BEST1 mutations.

Best Disease is an inherited retinal dystrophy that results in progressive and irreversible central vision loss caused by mutations of BESTROPHIN1 (BEST1). We established human induced pluripotent stem cells (iPSCs) from a Best disease patient with mutations R218H and A357V in the BEST1 gene. The generated iPSCs showed pluripotency markers and three-germ layer differentiation ability in vitro. A genetic analysis revealed mutations of R218H and A357V in the iPSCs. This iPSC line will be useful for elucidating the pathomechanisms of and drug discovery for Best disease.

Mutationsabhängige Mechanismen und deren Bedeutung für zielgerichtete Behandlungsstrategien am Beispiel von Bestrophin 1 und den Bestrophinopathien

Bestrophin 1 (BEST1) encodes an integral membrane protein localized in the basolateral aspect of the retinal pigment epithelium. Mutations in BEST1 are associated with distinct retinal dystrophies, the so-called "bestrophinopathies", often causing visual impairment, even in early childhood. The clinical entities of the bestrophinopathies can be distinguished by phenotypic characteristics and mode of inheritance of the respective gene defect. While the autosomal dominant inheritance pattern with one altered copy of BEST1 is common, heterozygous carriers of the autosomal recessive bestrophinopathy are generally but not consistently symptom-free. This review highlights the significance of understanding the underlying molecular mechanisms that contribute to disease pathogenesis of autosomal dominant and autosomal recessive bestrophinopathies. This knowledge is deemed crucial and needs to be considered in future planning of treatment strategies.

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.

Novel therapies for Bestrophinopathies

AbstractBestrophinopathies are a group of clinically distinct diseases caused by mutations in Bestrophin1 (BEST1). BEST1 is expressed exclusively in the retinal pigment epithelial (RPE) cells of the eye, where it functions as an ion channel. Mutations in the BEST1 gene affect the function of the RPE leading to the death of overlying retinal cells and subsequent vision loss.The dominant and recessive nature of Bestrophinopathies may necessitate different approaches to prevent or ameliorate these diseases. Potential therapeutic options for Bestrophinopathies focus on restoring correct gene expression, though viral gene therapy and gene editing, or by targeting nonsense mutations through translational readthrough‐inducing drugs. We are using induced pluripotent stem‐derived RPE created from patients with Bestrophinopathies to investigate these potential therapies in vitro.

Diffuse Outer Layer Opacification: A Novel Finding in Patients With Autosomal Recessive Bestrophinopathy.

Purpose:Autosomal recessive bestrophinopathy (ARB) is a rare inherited retinal dystrophy resulted from mutations in bestrophin-1 (BEST1) which affect functioning of the retinal pigment epithelium (RPE). Descriptions of disease findings in patients with ARB to date have focused only on macular changes. In this case series, we report previously undescribed mid-peripheral retinal changes occurring in 4 patients with ARB.Design:Case series.Methods:A single-center, retrospective review of medical records from Mayo Clinic patients with ARB was performed. Imaging reviewed include fundus photography, fundus autofluorescence, spectral domain optical coherence tomography (OCT), and fluorescein angiography. Demographic information and disease progression were noted.Results:4 affected patients from 3 families were identified. All 4 patients were female, and mean age was 12.5 years (range 5–19 years). Diffuse mid-peripheral whitening was consistently noted on fundus photography. Concomitant OCT imaging demonstrated areas of hyperreflectivity in the photoreceptor outer segment layer in areas corresponding to whitening seen on fundus photography. In 1 patient who was followed for 12 years, this finding persisted. Subretinal fluid was also consistently present. Other pathologic imaging findings observed in each patient were in agreement with previous reports of ARB.Conclusions:This is the first descriptive report of pathologic findings occurred beyond the posterior pole in patients with ARB. These mid-peripheral retinal changes potentially imply that the entirety of the RPE is affected by mutations in BEST1, as also suggested by previous electro-oculogram (EOG) findings. Such implications will be important when developing treatment trials, as past trials have focused only on the posterior pole of the RPE.

Novel Missense Mutations in BEST1 Are Associated with Bestrophinopathies in Lebanese Patients.

To identify Bestrophin 1 (BEST1) causative mutations in six Lebanese patients from three families, of whom four had a presumed clinical diagnosis of autosomal recessive bestrophinopathy (ARB) and two showed a phenotype with a single vitelliform lesion, patients were subjected to standard ophthalmic examinations. In addition, BEST1 exons and their flanking regions were amplified and sequenced by Sanger sequencing. Co-segregation and detailed bio-informatic analyses were performed. Clinical examination results were consistent with ARB diagnosis for all index patients showing multifocal vitelliform lesions and a markedly reduced light peak in the electrooculogram, including the two patients with a single vitelliform lesion. In all cases, most likely disease-causing BEST1 mutations co-segregated with the phenotype. The ARB cases showed homozygous missense variants (M1, c.209A>G, p.(Asp70Gly) in exon 3, M2, c.1403C>T; p.(Pro468Leu) in exon 10 and M3, c.830C>T, p.(Thr277Met) in exon 7), while the two patients with a single vitelliform lesion were compound heterozygous for M1 and M2. To our knowledge, this is the first study describing mutations in Lebanese patients with bestrophinopathy, where novel biallelic BEST1 mutations associated with two phenotypes were identified. Homozygous mutations were associated with multifocal lesions, subretinal fluid, and intraretinal cysts, whereas compound heterozygous ones were responsible for a single macular vitelliform lesion.

The Y227N mutation affects bestrophin-1 protein stability and impairs sperm function in a mouse model of Best vitelliform macular dystrophy.

ABSTRACTHuman bestrophin-1 (BEST1) is an integral membrane protein known to function as a Ca2+-activated and volume-regulated chloride channel. The majority of disease-associated mutations in BEST1 constitute missense mutations and were shown in vitro to lead to a reduction in mutant protein half-life causing Best disease (BD), a rare autosomal dominant macular dystrophy. To further delineate BEST1-associated pathology in vivo and to provide an animal model useful to explore experimental treatment efficacies, we have generated a knock-in mouse line (Best1Y227N). Heterozygous and homozygous mutants revealed no significant ocular abnormalities up to 2 years of age. In contrast, knock-in animals demonstrated a severe phenotype in the male reproductive tract. In heterozygous Best1Y227N males, Best1 protein was significantly reduced in testis and almost absent in homozygous mutant mice, although mRNA transcription of wild-type and knock-in allele is present and similar in quantity. Degradation of mutant Best1 protein in testis was associated with adverse effects on sperm motility and the capability to fertilize eggs. Based on these results, we conclude that mice carrying the Best1 Y227N mutation reveal a reproducible pathologic phenotype and thus provide a valuable in vivo tool to evaluate efficacy of drug therapies aimed at restoring Best1 protein stability and function.

BEST1 protein stability and degradation pathways differ between autosomal dominant Best disease and autosomal recessive bestrophinopathy accounting for the distinct retinal phenotypes.

Mutations in bestrophin-1 (BEST1) are associated with distinct retinopathies, notably three forms with autosomal dominant inheritance and one condition with an autosomal recessive mode of transmission. The molecular mechanisms underlying their distinct retinal phenotypes are mostly unknown. Although heterozygous missense mutations in BEST1 reveal dominant-negative effects in patients with autosomal dominant Best disease (BD), heterozygous mutations associated with autosomal recessive bestrophinopathy (ARB) display no disease phenotype. Here we show that the recessive mutations trigger a strong and fast protein degradation process in the endoplasmic reticulum (ER), thereby favoring a decreased stoichiometry of mutant versus normal BEST1 subunits in the assembly of the homo-pentameric BEST1 chloride channel. In contrast, dominant mutations escape ER-associated degradation and are subjected to a slightly delayed post-ER degradation via the endo-lysosomal degradation pathway. As a result, increased formation of a non-functional BEST1 channel occurs due to a roughly equimolar incorporation of normal and mutant BEST1 subunits into the channel complex. Taken together, our data provide insight into the molecular pathways of dominantly and recessively acting BEST1 missense mutations suggesting that the site of subcellular protein quality control as well as the rate and degree of mutant protein degradation are ultimately responsible for the distinct retinal disease phenotypes in BD and ARB.

BESTROPHIN1 mutations cause defective chloride conductance in patient stem cell-derived RPE.

Bestrophin1 (BEST1) is expressed in human retinal pigment epithelium (RPE) and mutations in the BEST1 gene commonly cause retinal dysfunction and macular degeneration. BEST1 is presumed to assemble into a calcium-activated chloride channel and be involved in chloride transport but there is no direct evidence in live human RPE cells to support this idea. To test whether BEST1 functions as a chloride channel in living tissue, BEST1-mutant RPE (R218H, L234P, A243T) were generated from patient-derived induced pluripotent stem cells and compared with wild-type RPE in a retinal environment, using a biosensor that visualizes calcium-induced chloride ion flux in the cell. Calcium stimulation elicited chloride ion export in normal RPE but not in RPE derived from three patients with BEST1 mutations. These data, along with three-dimensional modeling, provide evidence that BEST1 assembles into a key calcium-sensing chloride channel in human RPE.

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.

Differential effects of Best disease causing missense mutations on bestrophin-1 trafficking

Mutations in bestrophin-1 (Best1) cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited retinal degenerative disease. Best1 is a homo-oligomeric anion channel localized to the basolateral surface of retinal pigment epithelial (RPE) cells. A number of Best1 mutants mislocalize in Madin-Darby canine kidney (MDCK) cells. However, many proteins traffic differently in MDCK and RPE cells, and MDCK cells do not express endogenous Best1. Thus, effects of Best1 mutations on localization in MDCK cells may not translate to RPE cells. To determine whether BVMD causing mutations affect Best1 localization, we compared localization and oligomerization of Best1 with Best1 mutants V9M, W93C, and R218C. In MDCK cells, Best1 and Best1(R218C) were basolaterally localized. Best1(W93C) and Best1(V9M) accumulated in cells. In cultured fetal human retinal pigment epithelium cells (fhRPE) expressing endogenous Best1, Best1(R218C) and Best1(W93C) were basolateral. Best1(V9M) was intracellular. All three mutants exhibited similar fluorescence resonance energy transfer (FRET) efficiencies to, and co-immunoprecipitated with Best1, indicating unimpaired oligomerization. When human Best1 was expressed in RPE in mouse eyes it was basolaterally localized. However, Best1(V9M) accumulated in intracellular compartments in mouse RPE. Co-expression of Best1 and Best1(W93C) in MDCK cells resulted in basolateral localization of both Best1 and Best1(W93C), but co-expression of Best1 with Best1(V9M) resulted in mislocalization of both proteins. We conclude that different mutations in Best1 cause differential effects on its localization and that this effect varies with the presence or absence of wild-type (WT) Best1. Furthermore, MDCK cells can substitute for RPE when examining the effects of BVMD causing mutations on Best1 localization if co-expressed with WT Best1.

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Bestrophin-1, an integral membrane protein encoded by the BEST1 gene, is localized predominantly to the basolateral membrane of the retinal pigment epithelium. Mutations in the BEST1 gene have been associated with Best vitelliforme macular dystrophy (BMD), a central retinopathy with autosomal dominant inheritance and variable penetrance. Over 120 disease-causing mutations are known, the majority of which result in amino acid substitutions within four mutational hot-spot regions in the highly conserved N-terminal half of the protein. Although initially thought to impair Cl⁻ channel function, the molecular pathology of BEST1 mutations is still controversial. We have analyzed the subcellular localization of 13 disease-associated BEST1 mutant proteins in polarized MDCK II cells, an established model of apical to basolateral protein sorting. Immunostaining demonstrated that nine of the 13 mutant proteins failed to integrate into the cell membrane. The defective proteins were predominantly retained in the cytoplasm, whereas wild-type bestrophin-1 revealed cell membrane localization. Functional analysis of I⁻ fluxes in HEK-293 cells showed that all mutants exhibited a significant reduction in anion conductance. Our data indicate that defective intracellular trafficking could be a common cause of BMD accompanied by impaired anion conductance, representing a loss of anion channel function that is probably due to mistargeting of mutant protein.

Missense Mutations in a Retinal Pigment Epithelium Protein, Bestrophin-1, Cause Retinitis Pigmentosa

Bestrophin-1 is preferentially expressed at the basolateral membrane of the retinal pigmented epithelium (RPE) of the retina. Mutations in the BEST1 gene cause the retinal dystrophies vitelliform macular dystrophy, autosomal-dominant vitreochoroidopathy, and autosomal-recessive bestrophinopathy. Here, we describe four missense mutations in bestrophin-1, three that we believe are previously unreported, in patients diagnosed with autosomal-dominant and -recessive forms of retinitis pigmentosa (RP). The physiological function of bestrophin-1 remains poorly understood although its heterologous expression induces a Cl--specific current. We tested the effect of RP-causing variants on Cl- channel activity and cellular localization of bestrophin-1. Two (p.L140V and p.I205T) produced significantly decreased chloride-selective whole-cell currents in comparison to those of wild-type protein. In a model system of a polarized epithelium, two of three mutations (p.L140V and p.D228N) caused mislocalization of bestrophin-1 from the basolateral membrane to the cytoplasm. Mutations in bestrophin-1 are increasingly recognized as an important cause of inherited retinal dystrophy.

Bestrophin-2 Is Involved in the Generation of Intraocular Pressure

The bestrophin family of proteins has been demonstrated to generate or regulate Ca2+-activated Cl(-) conductances. Mutations in bestrophin-1 (Best1) cause several blinding eye diseases, but little is known about other bestrophin family members. This study involved disruption of the Best2 gene in mice. The mouse Best2 gene was disrupted by replacing exons 1, 2, and part of exon 3 with a Lac Z. The expression profile of Bestrophin-2 (Best2) was examined using RT-PCR, X-gal staining, and immunohistochemistry. Intraocular pressure (IOP) was measured by anterior chamber cannulation. RT-PCR of mouse tissues revealed Best2 mRNA in eye, colon, nasal epithelia, trachea, brain, lung, and kidney. X-gal staining, confirmed expression in colon epithelia and in the eye, in the nonpigmented epithelia (NPE). Best2 was not expressed in RPE cells. Best2 protein was observed only in NPE and colon epithelia. The absence of Best2 had no obvious deleterious effect on the mice. However, the Best2-/- mice were found to have significantly (P < 0.02) diminished IOP with respect to the Best2+/+ and Best2+/- littermates. The Best2-/- and Best2+/- mice responded better to the carbonic anhydrase inhibitor brinzolamide than did their Best2+/+ littermates, although the beta-blocker timolol brought IOP to the same level, regardless of genotype. Best2 plays a role in the generation of IOP by regulating formation of aqueous humor, and inhibition of Best2 function represents an attractive new avenue for regulating IOP in individuals with glaucoma.